High-affinity human angiopoietin-2 antibodies

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology. What is presented is a recovered human antibody or its antigen-binding fragment, which specifically binds to human angiopoietin-2 (hAng-2), but does not substantially binds to hAng-1 characterised by the presence of CDR variable heavy and light chain domains. What is described is a pharmaceutical composition on the basis of a therapeutically effective amount of the antibody. Disclosed are: variants of the recovered antibody or its antigen-binding fragment in producing drug preparation for treating a patient suffering various diseases including a tumour. Described are the versions of the methods of treating various diseases.

EFFECT: using this invention provides the antibody highly specific to human angiopoietin-2 (hAng-2) with an affinity constant of approximately 10-11, which does not substantially bind to hAng-1, that can find application in treating various diseases related to hAng-2 hyperactivity.

19 cl, 35 tbl, 3 dwg, 13 ex

 

Area of technology

The present invention relates to antibodies and antigen-binding fragments that are specific for angiopoietin-2 (Ang-2).

The level of technology

Angiogenesis is a biological process by which new blood vessels form.

Aberrant angiogenesis is associated with several painful conditions, including, for example, proliferative retinopathy, rheumatoid arthritis and psoriasis. In addition, it is well known that angiogenesis is critical for the growth and maintenance of tumor. Angiopoietin-2 (Ang-2) is a ligand for the receptor Tie-2 (Tie-2), and, as has been demonstrated, it plays a role in angiogenesis. Ang-2 is also denoted in the art as a ligand of Tie-2 (USA 5643755; Yancopoulos et al., 2000, Nature 407:242-248).

Antibodies and other peptide inhibitors that bind to Ang-2, referred to in US 6166185; 7521053; 7205275; 2006/0018909 and 2006/0246071. There is a need in the art to create new Ang-2-modulating agents, including Ang-2 antibodies that can be used for treating diseases and pathological conditions caused by or burdened by angiogenesis.

Brief description of the invention

The present invention proposes human antibodies that bind to human Ang-2. The authors present izobreteny� due to various obvious information and research understood the need for the establishment of Ang-2 inhibitors, are not referenced and are not antagonists related molecule Ang-1. For example, previous studies have demonstrated or suggested useful role of Ang-1 in hemostasis (see, for example, Li et al., 2001, Thrombosis and Haemostasis 85:191-374) and in the protection of the adult vascular system from infiltration of plasma (see, for example, Thurston et al., 2000, Nature Medicine 6:460-463; Thurston et al., 1999, Science 286:2511-2514). Thus, the authors present invention, it is clear that in certain anti-angiogenic therapeutic cases, it may be useful to keep the activity of Ang-1. Accordingly, the present invention provides antibodies that are specific contact Ang-2, but essentially do not bind to Ang-1. The present invention also includes antibodies that block the interaction between Ang-2 and its receptor Tie-2, but essentially do not block the interaction between Ang-1 and Tie-2. The antibodies of the invention are used among other things for inhibiting angiogenesis-stimulating activity of Ang-2 and for treating diseases and disorders caused by or associated with the process of angiogenesis.

The antibodies of the invention can be full-sized (for example, the antibody is IgG1 or IgG4) or can include only an antigen-binding portion (for example, a Fab fragment, F(ab')2or scFv), and can be modified to impact on the functionality of, for example, with the exception of residual �ffectornah functions.

In one embodiment, the invention includes an antibody or antigen-binding fragment of an antibody comprising a variable plot heavy chain (HCVR) having the 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 essentially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In one embodiment, the invention includes an antibody or antigen-binding fragment of an antibody comprising a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 42, 66, 162, 210, 266 and 434.

In one embodiment, the invention includes an antibody or antigen-binding fragment of an antibody comprising a variable area light chain (LCVR) having the amino acid sequence selected from the group consisting of SEQ ID NO: 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 essentially similar sequence having at least 90%, at least 95%, at m�re, 98% or at least 99% sequence identity. In one embodiment, the invention includes an antibody or antigen-binding fragment of an antibody comprising a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 20, 44, 68, 164, 212, 274 and 442.

In specific embodiments, the antibody or antigen-binding fragment includes a pair of amino acid sequences HCVR and LCVR (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 one embodiment, the antibody or a fragment thereof include HCVR and LCVR selected from pairs of amino acid sequences SEQ ID NO.: 18/20, 42/44, 66/68, 162/164, 210/212, 266/274 and 434/442.

In the following aspect, the invention provides an antibody or antigen-binding antibody fragment comprising a domain of the heavy chain CDR3 (HCDR3) having the 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 essentially similar sequence having at least 90%, at least 95%, at least 98% �whether at least 99% sequence identity; and domain light chain CDR3 (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 essentially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity to the sequence.

In specific embodiments, the antibody or antigen-binding fragment of an antibody include a pair of amino acid sequences 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 fragment of an antibody include a pair of amino acid sequences HCDR3/LCDR3 selected from the group consisting of SEQ ID NO: 8/16, 32/40, 56/64, 152/160, 200/208, 272/280 and 440/448. Non-limiting examples of anti-Ang-2 antibodies containing these pairs HCDR3/LCDR3 are antibodies, designated H1 H685, H1 H690, H1 H691, H1 H696, H1 H706, H1 M724 and H2M744, respectively.

In the following embodiment, the invention includes an antibody or a fragment thereof, further comprising 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 essentially similar sequence having, at m�re, 90%, at least 95%, at least 98% or at least 99% sequence identity; domain heavy chain CDR2 (HCDR2) having the 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 essentially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a 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 essentially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a 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 essentially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% identity to the sequence.

Specific non-limiting typical antibodies and antigen-binding fragments of the invention include domains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, selected from the group consisting of: (i) SEQ ID NO: 4, 6, 8, 12, 14 and 16 (e.g.�measures H1 H685); (ii) SEQ ID NO: 28, 30, 32, 36, 38 and 40 (for example, H1 H690); (iii) SEQ ID NO: 52, 54, 56, 60, 62 and 64 (for example, H1 H691 ); (iv) SEQ ID NO: 148, 150, 152, 156, 158 and 160 (for example, H1 H696); (v) SEQ ID NO: 196, 198, 200, 204, 206 and 208 (for example, H1 H706); (vi) SEQ ID NO: 268, 270, 272, 276, 278 and 280 (for example, H1 M724); and (vii) SEQ ID NO: 436, 438, 440, 444, 446 and 448 (for example, H2M744).

In a related embodiment, the invention includes an antibody or antigen-binding fragment of an antibody that binds to specific Ang-2, where the antibody or fragment include the domains of the heavy and light chain CDR (I. e., CDR1, CDR2 and CDR3) contained in the sequences of variable domains of the heavy and light chain 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 a fragment thereof include CDR sequences contained within HCVR and LCVR selected from pairs of amino acid sequences SEQ ID NO.: 18/20, 42/44, 66/68, 162/164, 210/212, 266/274 and 434/442.

In another aspect, the invention provides nucleic acid molecules encoding the anti-Ang-2 antibodies or fragments thereof. The invention also covers the recombinant expression vectors carrying nuclein�s acid according to the invention, and the host cell, in which shall be entered such vectors, and methods for the production of antibodies by culturing cells under conditions enabling the production of antibodies and extraction of the resulting antibodies.

In one embodiment, the invention provides an antibody or a fragment thereof comprising a HCVR 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 essentially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In one embodiment, the antibody or a fragment thereof include a HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 17, 41, 65, 161, 209, 265 and 433.

In one embodiment, the invention provides an antibody or a fragment thereof, comprising a 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 essentially identical sequence�scenic spots, having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In one embodiment, the antibody or a fragment thereof include a LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 19, 43, 67, 163, 211, 273 and 441.

In one embodiment, the invention provides an antibody or antigen-binding fragment of an antibody comprising a 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 essentially identical sequence having at least 90%, at least 95%at least 98% or at least 99% sequence identity; and a 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 essentially similar sequence having at least 90%, at least 95%at least 98% or at least 99% sequence identity. In one embodiment, the antibody or a fragment thereof include sequence HCDR3 and LCDR3 encoded by a pair of nucleic acid sequences selected from the group consisting of SEQ ID NO: 7/15, 31/39, 55/63, 151/159, 199/207, 271/279 and 439/447.

The following is embodied�and, antibody or a fragment thereof further comprise: 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 essentially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; a 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 essentially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a 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 essentially identical sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a 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 essentially similar sequence having at least 90%, at least 95%, at least 98 or, at least 99% identity to the sequence.

In one embodiment, the antibody or a fragment thereof include CDR sequences of heavy and light chain encoded by the nucleic acid sequences SEQ ID NO: 17 and 19; SEQ ID NO: 41 and 43; SEQ ID NO: 65 and 67; 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.

The invention encompasses anti-Ang-2 antibodies having a modified glycosylation profile. In some applications can be used with modification to remove glycosylation sites. For example, the present invention encompasses modified versions of any of the antibodies provided herein, where a modified version of deprived fukanaga component that is present on the oligosaccharide chains of the chain, for example, to increase the functions of antibody-dependent cellular cytotoxicity (ADCC) (see Shield et al. (2002) JBC 277:26733). In other applications, can be carried out modification of galactosylceramide for the purpose of modification of complement-dependent cytotoxicity (CDC).

In another aspect, the present invention provides a pharmaceutical composition comprising a recombinant human antibody or a fragment thereof, which are specific contact Ang-2, and a pharmaceutically acceptable carrier or diluent. In a related aspect, the invention describes a composition which is a combinatio�Yu inhibitor of Ang-2 and the second therapeutic agent. In one embodiment, the inhibitor of Ang-2 is an antibody or a fragment thereof. In one embodiment, the second therapeutic agent is any agent that mainly combined with an inhibitor of Ang-2. Typical agents which may advantageously be combined with an inhibitor of Ang-2, include without limitation any agent that inhibits or reduces angiogenesis, other anti-cancer therapeutic agents, anti-inflammatory agents, inhibitors of cytokines, inhibitors of growth factors, protivogerpeticheskiy factors, non-steroidal anti-inflammatory drug (NSAID), antiviral agents and antibiotics.

In another aspect, the invention provides methods for inhibiting the activity of Ang-2 with the use of anti-Ang-2 antibody or antigen-binding fragment of the antibody according to the invention, where therapeutic methods include the introduction of a therapeutically effective amount of a pharmaceutical composition comprising the antibody or antigen-binding fragment of the antibody according to the invention. The breach exposed to treatment represents any disease or pathological condition which is improved, reduced, inhibited or prevented by removal, inhibition or reduction in the activity of Ang-2. Preferably, the anti-Ang-2 antibody or fragment and�of tetela according to the invention are used for treatment of any disease or pathological condition, caused, associate or continuing with the process of angiogenesis. In specific embodiments of the invention, the anti-Ang-2 antibodies or their antigen-binding sites are used to treat cancer. In the context of anticancer therapies, anti-Ang-2 antibodies of the invention or antigen-binding sites may be administered alone or in combination with other anti-cancer therapeutic antibodies, chemotherapeutic agents and/or radiation therapy. In other embodiments of the present invention, the anti-Ang-2 antibodies or their antigen-binding fragments are used to treat one or more ocular disorders, for example, age-related macular degeneration, diabetic retinopathy, etc., and/or one or more inflammatory or infectious diseases.

Other embodiments will become apparent in the review of the detailed description.

Brief description of the drawings

Fig.1 is an alignment of the last 88 C-terminal amino acids of human Ang-2 (residues 409-496 SEQ ID NO:518) corresponding to amino-acid sequence of human Ang-1 (SEQ ID NO:531). Residues that differ between hAng-1 and hAng-2, marked with white font and black shading. Asterisks (*) indicates amino acids hAng-2, which, as has been demonstrated by analysis of crystalline PT�structure, interact with a human Tie-2. Cm. Barton et al., Nat. Struct. Mol. Biol. 73:524-532 (2006). Triangles (▲^) denotes the position of the amino acids interacting with Tie-2, which differ from hAng-2 and hAng-1.

Fig.2 (Panels A-C) depicts the results of Western blot analyses that illustrate the degree to which Ang-2 binding molecules inhibit or not able to inhibit stimulated with Ang-1 phosphorylation of Tie-2.

Fig.3 is a summary of the binding experiment with a point mutant Ang-2FD-mFc Example 13, showing amino acid substitutions that result in a more than five-fold decrease in T1/2dissociation (shown shaded circles •) relative to wild-type tested for different antibodies and peptides (peptibodies).

Detailed description

Before the present invention will be further described, it should be understood that this invention is not limited to the described specific methods and experimental conditions, as such methods and conditions may vary. You should also understand that used in this document terminology necessary only for the purpose of describing particular embodiments and is not intended to limit the scope of the present invention, which will be limited only by the appended claims invent�Oia.

If not defined otherwise, all technical and scientific terms used herein have the same meaning attached to them by ordinary specialist in the field to which the present invention belongs. When used herein, the term "about" when used in relation to specific lists the values, indicates that the value may vary relative to the values given for not more than 1%. For example, when used herein, the expression "approximately 100" includes 99 and 101 and all the values between them (for example, 99,1, 99,2, 99,3, 99,4, etc.).

Although any methods and materials similar or equivalent to those described herein can also be used in practice or testing of the present invention, the following preferred methods and materials.

Definition

When used herein, the term "angiopoietin-2" or "Ang-2" until it is determined that it is derived from a species different from the person (for example, "mouse Ang-2", "monkey Ang-2," etc.), refers to human Ang-2 or biologically active fragment (for example, a protein fragment of Ang-2, which is able to induce angiogenesis in vitro or in vivo). Human Ang-2 is encoded by the nucleic acid sequence presented in SEQ IDNO:517, and has the amino acid sequence of SEQ ID NO:518. The amino acid sequence of mouse and monkey proteins Ang-2 is available from the database of the NCBI protein sequences under Registration numbers NP_031452 and BAE89705.1, respectively.

The term "angiopoietin-1 or Ang-1" until it is determined that it is derived from a species different from the person (for example, "mouse Ang-1", "monkey Ang-1", etc.), refers to human Ang-1 or its biologically active fragment. Human Ang-1 has the amino acid sequence represented in the database of NCBI protein sequences under Registration number AAB50557. The term "Tie-2 (also referred to in the prior art as "TEK") until it is determined that it is derived from a species different from human (e.g. murine Tie-2", "monkey Tie-2," etc.), refers to human Tie-2 or biologically active fragment. Human Tie-2 has the amino acid sequence represented in the database of NCBI protein sequences under Registration number AAA61130.

The term "antibody" when used herein is intended to refer to molecules of immunoglobulins, comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as their multimer (for example, IgM). To�each heavy chain comprises a variable plot heavy chain (designated herein as HCVR or V H) and a constant phase of the heavy chain. Const plot heavy chain comprises three domains, CH1, CH2 and CH3. Each heavy chain comprises a variable area light chain (designated herein as LCVR or VL) and a constant phase light chain. Const plot light chain includes one domain (CL1). Sections VHand VLcan be further subdivided into areas of hypervariability, called plots, which are responsible for the complementarity of binding (CDR), alternated with areas that are more conservative, called frame regions (FR). Each VHand VLis composed of three CDRs and four FR, arranged from N-Terminus to C-Terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the invention, the FR-sections of the anti-Ang-2 antibody (or an antigen-binding site) may be identical to the human germline sequences, or may be naturally or artificially modified. A consensus amino acid sequence may be determined based on the parallel analysis of two or more CDR.

When used herein, the term "antibody" also includes antigen-binding fragments of the full-size antibody molecules. When used herein, the terms "and�of then-binding site of the antibody, "antigen-binding fragment" of an antibody and the like, include any natural derived enzyme, synthetic or genetically engineered polypeptide or glycoprotein, which binds to specific antigen with the formation of the complex. The antigen-binding antibody fragments can be isolated, for example, from the full-size antibody molecules using any suitable standard methods, such as proteolytic hydrolysis or methods of genetic engineering, including the manipulation and expression of DNA encoding the variable and not necessarily constant domains of the antibody. Such a DNA is known and publicly available, for example, from commercial sources, DNA libraries (including, for example, phage libraries of antibodies), or can be synthesized. DNA can be sequenced, and her manipulation is performed using chemical methods or by methods of molecular biology, for example, for the location of one or more variable and/or constant domains in a suitable configuration or to the introduction of codons, creating cysteine residues, modifications, insertions or deletions of amino acids, etc.

Non-limiting examples of antigen-binding fragments include (i) Fab fragments; (ii) F(ab')2fragments; (iii) Fd fragments; (iv) Fv fragments; (v) odnosima�Ichnya Fv molecules (scFv); (vi) dAb fragments; and (vii) the minimum discriminating components consisting of amino acid residues that mimic the hypervariable site of an antibody (for example, allocated to the sector responsible for the complementarity of binding (CDR)). When used herein, the term "antigen-binding fragment" also covers other engineered molecules, such as diamela, Triatel, tetrathele and Minitel.

The antigen-binding fragment of the antibody will also include at least one variable domain. Variable domain may be of any size or amino acid composition and typically will include at least one CDR that is adjacent or located in one frame with one or more frame sequences. In the antigen-binding fragments containing the domain VHassociated with the domain VLdomains VHand VLcan be relative to each other in a suitable location. For example, a variable phase may be dimeric and may contain dimers VH-VHVH-VLor VL-VL. Alternatively, the antigen-binding antibody fragment may contain a monomer domain VHor VL.

In specific embodiments, the antigen-binding antibody fragment can contain, at �'ere, a single variable domain that is covalently linked to at least one constant domain. Non-limiting typical configuration variable and constant domains that can be detected within the antigen-binding fragment of the antibody of the present invention include: (i) VH-CH1; (ii) 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 variable and constant domains, including any of the typical configurations listed above, the variable and constant domains can be linked either directly with each other, or may be connected by full or partial hinge or linker section. The hinge area is at least 2 (for example, 5, 10, 15, 20, 40, 60 or more) amino acids, which result in obtaining flexible or semi-flexible connection between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, the antigen-binding antibody fragment of the present invention may include� C) an homodimer or heterodimer (or other multimer) any of the configurations of variable and constant domains, listed above, in a non-covalent bond with each other and/or with one or more monomer domains VHor VL(for example, via disulfide bonds(s)).

As a full-sized molecules are antibodies, antigen-binding fragments can be monospecificity or multispecificity (for example, bispecific). Multispecificity the antigen-binding fragment of an antibody typically will include at least two different variable domain, where each variable domain capable of specific binding to separate the antigen or with different epitopes on the same antigen. Any multispecificity the format of the antibody, including typical bispecific formats antibodies disclosed herein can be adapted for use in the context of the antigen-binding fragment of the antibody of the present invention using standard methods available in the art.

Constant site of an antibody is important for the ability of the antibody to fix complement and mediate cell-mediated cytotoxicity. Thus, the isotype of the antibody can be selected on the basis of whether it was appropriate for the antibodies mediating cytotoxicity.

When used herein, it is understood that the term "human antibody" includes�AET antibodies, contains variable and constant portions extracted from the sequences of the human germline immunoglobulin. Human antibodies of the invention may include amino acid residues not encoded by human germline sequences of immunoglobulins (for example, mutations introduced nonspecific or sitespecifically mutagenesis in vitro or by somatic mutation in vivo), for example, in the areas of CDR and especially in CDR3. However, when used in this document, it is understood that the term "human antibody" includes antibodies in which CDR sequences derived from the germline of the other mammalian species such as mice, were transplanted in the human frame of the sequence.

When used herein, it is understood that the term "recombinant human antibody" includes all human antibodies that receive, Express, create, or isolated using recombinant methods, such as antibodies expressed using a recombinant expression vector, transfitsirovannykh in the host (described below), antibodies isolated from a recombinant combinatorial library of human antibodies (described below), antibodies isolated from an animal (e.g. a mouse), which are Tran�gene for human immunoglobulin genes (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies obtained, expressed, created or selected by any other method that includes splicing sequences of human immunoglobulin genes with other DNA sequences. Such recombinant human antibodies contain variable and constant sections, sequences obtained from human germline immunoglobulin. In some embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, if you are using transgenic relative to the human sequence immunoglobulin animals, somatic mutation in vivo), and, thus, the amino acid sequence of plots of VHand VLrecombinant antibodies are sequences that though descended from and related to human germline sequences of the VHand VL, cannot exist in vivo in embryonic repertoire of human antibodies in vivo.

Human antibodies can exist in two forms that are associated with the pivot heterogeneity. In one form, the immunoglobulin molecule consists of a stable design of the four chains of approximately 150-160 kDa, in which the dimers are supported VM�CTE with the cooperation of the disulfide bonds of the heavy chain. In the second form, the dimers are not linked by disulfide bonds between the chains, and form a molecule with a molecular weight of about 75-80 kDa, consisting of covalently linked light and heavy chains (semi-antibody). These forms are very difficult to separate even after affinity purification.

The frequency of occurrence of the second form in a variety of intact IgG isotypes associated in particular with the isotype hinge site of an antibody. A single amino acid substitution in the hinge portion of the hinge of human IgG4 can greatly reduce the frequency of occurrence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to a level that typically occurs with use of the hinge, human IgG1. The present invention encompasses antibodies that contain one or more mutations in the hinge area, CH2 or CH3 plot may be appropriate, for instance, production of antibodies to improve the yield of the desired form of the antibody.

When used herein, means that the selected antibody" means an antibody that is essentially free of other antibodies having excellent antigenic specificnosti (for example, the selected antibody that is specific binds to human Ang-2 or a fragment of human Ang-2, essentially free of antibodies that bind specific�Xia antigens, non-human Ang-2). The term "specific binds" or the like means that the antibody or antigen-binding fragment form with antigen complex that is relatively stable under physiological conditions. Specific binding can be characterized by a KDapproximately 1×10-8M or less. Methods to determine whether specific bind two molecules, are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. The selected antibody that is specific binds to human Ang-2 may, however, have cross-reactivity with other antigens, such as molecules of Ang-2 from other species. In addition, the selected antibody may be substantially free of other cellular material and/or chemicals.

When used herein, it is understood that "neutralizing" or "blocking" antibody refers to an antibody, the binding of which Ang-2 inhibits 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 the antibody, neutralizing or blocking Ang-2, does not have to be complete, provided that it is detected when using an appropriate�Lisa. Typical assays for the detection of inhibition of Ang-2 are described elsewhere in this document.

Fully human anti-Ang-2 antibodies disclosed herein can include one or more amino acid substitutions, insertions and/or deletions in the framework and/or in areas CDR of the variable domains of the heavy and light chain compared to the corresponding germline sequences. Such mutations can easily be identified by comparing the amino acid sequences disclosed herein, with germline sequences available, for example, from publicly available databases of sequences of antibodies. The present invention includes antibodies and antigen-binding fragments that are derived from any of amino acid sequences disclosed herein, where one or more amino acids within one or more frame and/or parcels CDR mutated back to the corresponding germline residues or conservative amino acid substitution (natural or artificial) of the corresponding germline residue (such replacement sequence denoted herein as "embryonic reverse mutation"). The person skilled in the art, having the original sequence variable regions thee�Eloi and light chains, disclosed herein, can easily obtain a variety of antibodies and antigen-binding fragments that include one or more individual embryonic reverse mutation or a combination of both. In some embodiments, each of the residues of a frame and/or CDR sites within domain VHand/or VLmutated back to the germline sequence. In other embodiments, only specific residues are mutated back to the germline sequence by, for example, only the mutated residues are detected within the first 8 amino acids FR1 or within the last 8 amino acids FR4, or only the mutated residues found in CDR1, CDR2 or CDR3. In addition, the antibodies of the present invention can contain any combination of two or more embryonic reverse mutations inside the frame and/or CDR plots, T. e., where specific individual residues are mutated back to the germline sequence, while some other residues that differ from the germline sequence are retained. After receipt, the antibodies and antigen-binding fragments that contain one or more embryonic reverse mutations, can be easily tested for one or more target properties, such as improved binding specificity, improved affinity tie�tion, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc., Antibodies and antigen-binding fragments received by this General method covered by this invention.

The present invention also includes anti-Ang-2 antibodies comprising variants of any of the amino acid sequences of the HCVR, LCVR, and/or CDR disclosed herein and containing one or more conservative substitutions. For example, the present invention includes anti-Ang-2 antibody containing the amino acid sequence HCVR, LCVR, and/or CDR, including, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions relative to any of the amino acid sequences of the HCVR, LCVR, and/or CDRs disclosed herein. In one embodiment, the antibody includes a HCVR having the amino acid sequence of SEQ ID NO: 18 with 8 or less conservative amino acid substitutions. In another embodiment, the antibody includes a HCVR having the amino acid sequence of SEQ ID NO: 18 with 6 or less conservative amino acid substitutions. In another embodiment, the antibody includes a HCVR having the amino acid sequence of SEQ ID NO: 18 with 4 or less conservative amino acid substitutions. In other�the first embodiment, the antibody includes a HCVR having the amino acid sequence of SEQ ID NO: 18 with 2 or less conservative amino acid substitutions. In one embodiment, the antibody includes a LCVR having the amino acid sequence of SEQ ID NO: 20 comprising 8 or less conservative amino acid substitutions. In another embodiment, the antibody includes a LCVR having the amino acid sequence of SEQ ID NO: 20 comprising 6 or less conservative amino acid substitutions. In another embodiment, the antibody includes a LCVR having the amino acid sequence of SEQ ID NO: 20 comprising 4 or less conservative amino acid substitutions. In another embodiment, the antibody includes a LCVR having the amino acid sequence of SEQ ID NO: 20 comprising 2 or less conservative amino acid substitutions.

When used herein, the term "surface plasmon resonance" refers to an optical phenomenon that involves the analysis of interactions in real time by detecting changes in protein concentrations within a biosensor matrix, for example using the BIAcore system™ (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).

When used herein, it is understood that the term "KD"refers to the dissociation constant at equilibrium the specific interaction of antibody-antigen.

The term "apito�" refers to an antigenic determinant, which interacts with a specific antigen-binding site in the variable site of a molecule of antibody, known as Pratap. One antigen may contain more than one epitope. Thus, different antibodies can contact different areas of the antigen and may have different biological effects. The epitopes can be either conformational or linear. Conformational epitope obtained using the spatially combined amino acids from various segments of the linear polypeptide chain. Linear epitope is the epitope obtained with the neighboring amino acid residues in the polypeptide chain. In certain circumstances, the epitope may include components of saccharides, phosphoryl groups, or sulfanilic groups on the antigen.

The term "substantial identity" or "substantially identical" in relation to nucleic acid or its fragment indicates that when a nucleic acid is optimally aligned in the appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there will be a the nucleotide sequence identity in at least about 95%, and more preferably at least approximately 96%, 97%, 98% or 99% nucleotide bases according to the measurements using a well known algorithm� sequence identity, such as FASTA, BLAST or Gap, as discussed below. The nucleic acid molecule and has substantial identity to a reference nucleic acid molecule may, in certain cases, to encode a polypeptide having essentially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned, such as by using the programs GAP or BESTFIT using the parameters of the passing masses default share at least 95% sequence identity, yet more preferably at least 98%, or 99% sequence identity. Preferably, the provisions of residues that are not identical differ by conservative amino acid substitutions. "Conservative amino acid substitution" is a substitution in which the amino acid residue is replaced with another residue having a side chain (R group) with similar chemical properties (e.g. charge or hydrophobicity). In General, a conservative amino acid substitution is essentially not change the functional properties of the protein. In cases where two or more amino acid sequences differ from each�nservative replacements, the percentage of sequence identity or similarity measure can be adjusted upwards to correct for the purpose of obtaining conservative nature of the substitution. Ways of implementing this regulation, the well-known to experts in this field. 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 and threonine; (3) amesterdam 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: cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternatively, a conservative substitution is any replacement for having a positive value in the PAM250 matrix logarithmic likelihood disclosed in Gonnet et al. (1992) Science 256: 1443-1445. "Moderately conservative" replacement is any replacement for having a positive value in the PAM250 matrix logarithmic plausible�of Adobe.

The similarity of the sequences of polypeptides, which is also referred to as sequence identity, usually measured, using a software package of sequence analysis. The software package of protein analysis finds similar sequences using similarity measures that assess to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For example, the GCG program package 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 from different species of organisms or between a wild type protein and its mutant form. See, for example, the GCG Version 6.1. Polypeptide sequences can also be compared using FASTA using the default settings or use the recommended settings in the program of the GCG Version 6.1. FASTA (for example, FASTA2 and FASTA3) provides alignment and percent identity to the sequence of plots of the best overlap between the target and search sequences (Pearson (2000) above). Other preferred algorithm to compare the sequence according to the invention with a database containing a large number of consistently�TEI from different organisms, is the computer program BLAST, especially BLASTP or TBLASTN, using the default settings. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

Obtaining human antibodies

Methods of generating monoclonal antibodies, comprising a fully human monoclonal antibody known in the art. Any such known methods can be used in the context of the present invention for obtaining human antibodies that are specific bind to human Ang-2 and which have one or more of the antigen-binding and/or functional characteristics of any typical anti-Ang-2 antibodies disclosed herein.

Using VELOCIMMUNE technology™, or any other known method of generating monoclonal antibodies, initially secrete high-affinity chimeric antibodies to Ang-2 containing human variable plot and a murine constant plot. According to the experimental section below, the antibodies are characterized and selected for the purpose of obtaining the desired characteristics, including affinity, selectivity, epitope, etc. Murine constant plots to replace human target constant plot of generating fully human antibodies according to the invention, for example, wild �IPA or modified IgG1 or IgG4. While selected constant phase may vary according to application-specic characteristics of high-affinity antigen binding and target specificity are variable in the plot.

Bioequivalence

Anti-Ang-2 antibodies and fragments of antibodies of the present invention encompass proteins having amino acid sequences that differ from the sequences described antibodies, but which retain the ability to bind to human Ang-2. Such variant antibodies and fragments of antibodies include one or more insertions, deletions or substitutions of amino acids compared to the parent sequence, but exhibit a biological activity that is substantially equivalent activity described antibodies. Similarly, DNA sequences encoding the anti-Ang-2 antibody of the present invention encompass sequences that comprise one or more insertions, deletions or substitutions of nucleotides compared to the disclosed sequence, but that encode an anti-Ang-2 antibody or antibody fragment that is essentially bioekvivalentna anti-Ang-2 antibody or the antibody fragment according to the invention. Examples of such variant amino acid and DNA sequences disclosed above.

The two antigen-binding protein or antibody� are considered bioequivalent, if they are pharmaceutical equivalents or pharmaceutical alternatives, the rate and extent of absorption which does not show a significant difference when administered at the same molar dose under similar experimental conditions as in a single dose and repeated doses. Some antibodies will be considered as equivalents or pharmaceutical alternatives if they are equivalent in their degree of absorption, but not in the rate of absorption and yet may be considered bioequivalent, such as differences in the rate of absorption are intentional and are reflected in the labeling, not being essential in achieving effective concentrations of the drug, for example, with regular use, and are considered to be insignificant from a medical point of view for a particular investigational medicinal product.

In one embodiment, the two antigen-binding protein are bioequivalent, if there were no clinically significant differences in safety, purity and effectiveness.

In one embodiment, the two antigen-binding protein to be bioequivalent if the patient can switch one or more times between the reference product and the biological product without the expected increased risk of side effects, including clinically significant changes and�monogenetic or weakening of the efficiency versus continued therapy without such a switch.

In one embodiment, the two antigen-binding protein to be bioequivalent if they both act through a common mechanism or mechanisms of action for the condition or conditions to the extent to which these mechanisms are known.

Bioequivalence may be demonstrated using the methods of in vivo and in vitro. Measurement bioequivalence include, for example, (a) in vivo test in humans or other mammals for which measure the concentration of the antibody or its metabolites as a function of time in the blood, plasma, serum or other biological fluid; (b) in vitro test, which correlates with reasonably predictable data in vivo bioavailability to humans; (c) in vivo test in humans or other mammals, which have a corresponding explicit pharmacological effect of the antibody (or the target) is measured as a function of time; and (d) well-controlled clinical trial that evaluates the safety, efficacy or bioavailability or bioequivalence of the antibody.

Biosimilar versions of anti-Ang-2 antibodies of the invention can be designed, for example, by implementing the various substitutions of residues or sequences, or deletions of internal residues or sequences that are not necessary for biological activity. For example, octadecylamine, not essential for biological activity, can be deleterows or replaced by other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation.

Biological and therapeutic characteristics of antibodies

In General, antibodies of the present invention bind to human Ang-2 with KDconstituting less than 100 PM, as a rule, with KDconstituting less than 50 PM, and in some embodiments, with KDconstituting less than 40 PM, according to the measurement by means of binding to the antigen or immobilized on a solid support or is in the liquid phase.

In addition, some typical anti-Ang-2 antibodies of the invention may exhibit one or more of the following characteristics: (1) the ability to bind to human Ang-2, but not with mouse Ang-2; (2) the ability to bind to human Ang-2 and murine Ang-2; (3) the ability to bind to human Ang-2, but not with human Ang-1, -3 or -4; (4) the ability to bind to human Ang-2, but not with mouse Ang-1, -3 or -4; (5) the ability to bind to human Ang-2 and human Ang-1, -3 or -4; (6) the ability to bind to human Ang-2 and murine Ang-1, -3 or -4; (7) ability to block binding of human Ang-2 with human Tie-2; (8) W�ü blocking the binding of human Ang-2 from murine Tie-2; (9) the ability to block the binding of murine Ang-2 with human Tie-2; (10) the ability to block the binding of murine Ang-2 from murine Tie-2; (11) ability to block binding of human Ang-1 with human Tie-2; (12) ability to block binding of human Ang-1 from murine Tie-2; (13) the ability to block the binding of murine Ang-1 with human Tie-2; (14) the ability to block the binding of murine Ang-1 from murine Tie-2; (15) the ability to inhibit phosphorylation of human Tie-2, human induced Ang-2; (16) the ability to inhibit the phosphorylation of murine Tie-2, human induced Ang-2; (17) the ability to inhibit phosphorylation of human Tie-2, induced murine Ang-2; (18) the ability to inhibit the phosphorylation of murine Tie-2, induced murine Ang-2; (19) the ability to inhibit phosphorylation of human Tie-2, which is induced by human Ang-1; (20) the ability to inhibit the phosphorylation of murine Tie-2, which is induced by human Ang-1; (21) the ability to inhibit phosphorylation of human Tie-2, induced murine Ang-1; (22) the ability to inhibit the phosphorylation of murine Tie-2, induced murine Ang-1; (23) the ability to inhibit in vivo angiogenesis in experimental models (for example, angiogenesis, induced cylinder of Matrices, containing�them cells MCF-7, implanted subcutaneously naked mice); and/or (24) the ability to inhibit or reduce tumor size in a mouse model of xenotransplantation.

The present invention also includes antibodies that bind with high affinity with the design, including fibronectin-like domain of Ang-2, but devoid of N-terminal supercoiled domain of Ang-2 (such structures are denoted in this document as "Ang-2FD"). Typical Ang-2FD designs include human Ang-2FD (SEQ ID NO;519), murine Ang-2FD (SEQ ID NO:520) and monkey Ang-2FD (SEQ ID NO:521). Human, mouse and monkey designs Ang-2FD can be Monomeric or dimeric. Design Ang-2FD can also include other than Ang-2, the amino acid sequence, such as human or mouse Fc domain attached to molecules Ang-2FD. Another typical design Ang-2FD denoted herein "hBA2" (or human "fold-Ang2"), which is a tetramer of human fibrinopeptide domains Ang-2, associated with each other through human or murine Fc domains with the formation of loop-like configuration. Typically, hBA2 consists of two dimers Ang-2, where each dimer Ang-2 contains two fibroadenomatoid domain of Ang-2 that are associated with each other by the Fc-domain. Typical components of hBA2 include polypeptides, denoted by�military hBA2-hIgG1 (SEQ ID NO:522) and hBA2-mIgG2a (SEQ ID NO:523). Suddenly discovered that some anti-Ang-2 antibodies of the present invention are associated with the structures of Ang-2FD with much higher affinity than known control antibody for Ang-2 (see Examples herein).

High-affinity binding in the context of the binding of anti-Ang-2 antibodies with dimeric structure of human or murine Ang-2FD, means that the anti-Ang-2 antibody binds to human or murine dimer Ang-2FD with KDconstituting less than 300 PM. For example, anti-Ang-2 antibodies that bind with high affinity to human or murine dimer Ang-2-FD, include antibodies that bind to human or murine dimer Ang-2-FD KDconstituting 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, according to the measurements at 25°C using analysis of surface plasmon resonance.

High-affinity binding in the context of the binding of anti-Ang-2 antibody binding by design monkey Ang-2FD means that the anti-Ang-2 antibody binds to monkey dimer Ang-2FD with KDconstituting less than 500 PM. For example,�t-Ang-2 antibody, which bind with high affinity with monkey dimer Ang-2-FD, include antibodies that bind to monkey dimer Ang-2-FD KDconstituting 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, less than 80 PM, according to the measurements at 25°C using analysis of surface plasmon resonance.

High-affinity binding in the context of the binding of anti-Ang-2 antibodies with Monomeric structure of human Ang-2FD, means that the anti-Ang-2 antibody binds to human monomer Ang-2FD with KDconstituting less than 40 PM. For example, anti-Ang-2 antibodies that bind with high affinity monomer with human Ang-2-FD, include antibodies that bind to human monomer Ang-2-FD KDconstituting 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, less than 0.6 nm, according to the measurements at 25°C using surface analysis of plasma�tion of the resonance.

High-affinity binding in the context of the binding of anti-Ang-2 antibodies by design hBA2, means that the anti-Ang-2 antibody binds to hBA2 with KDconstituting less than 80 PM. For example, anti-Ang-2 antibodies that bind with high affinity with hBA2 include antibodies that bind to hBA2 with KDconstituting 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, according to the measurements at 25°C using analysis of surface plasmon resonance.

The present invention includes antibodies that bind to Ang-2, but essentially do not bind to Ang-1. When used herein, an antibody "substantially not associated with Ang-1", if the antibody when tested for binding to Ang-1 by analysis of surface plasmon resonance, and a full-sized human Ang-1 wild-type in a concentration of about 25 nm was injected over the surface of the captured antibodies a flow rate of about 60 μl/min for about 3 minutes at 25°C, and demonstrates the KDmore than about 1 nm, for example, KDmore than about 5 nm, more than about 10 nm, more than about 50 nm, more h�m to about 100 nm, more than about 150 nm, more than about 200 nm, more than about 250 nm, more than about 300 nm, more than about 350 nm, more than about 400 nm, more than about 450 nm, more than about 500 nm, or more. (See for example, Example 4). In addition, an antibody essentially not associated with Ang-1", if the antibody is not able to demonstrate any binding to Ang-1, when tested in this assay, or its equivalent.

The present invention also includes antibodies that block the interaction between Ang-2 and Tie-2, but essentially does not inhibit binding of Ang-1 to Tie-2. When used herein, an antibody essentially blocks the binding of Ang-1, Tie-2, in the case when the antibody is pre-mixed with Ang-1 antigen in a ratio of about 100:1 (antibody:antigen) and incubated at 25°C for about 60 minutes and then balanced mixture tested for binding to Tie-2 by using surface plasmon resonance on a surface covered with a Tie-2 (5 μl/min for 5 min at 25°C), the amount of Ang-1, associated with Tie-2, is at least 50% of the amount of Ang-1 that is associated with Tie-2, in the presence of extraneous control molecules. (See for example, Example 6). For example, if the number of Ang-1 that is associated with Tie-2, after pre-incubation with the antibody is at least about 50%, at least approx�RNO 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% of the amount of Ang-1 that binds to Tie-2, after pre-incubation with someone's master molecule in the above experimental conditions, after which it is considered the antibody essentially blocks the binding of Ang-1 to Tie-2."

In addition, the present invention includes antibodies that block or substantively weaken the biological activity of Ang-2 (for example, Ang-2-mediated phosphorylation of Tie-2; Ang-2-induced angiogenesis; etc.), but do not block or essentially weaken the corresponding biological activity of Ang-1 (for example, Ang-1-mediated phosphorylation of Tie-2; Ang-1-induced angiogenesis; etc.). Analyses and tests used to determine whether the antibody is one or more of the above characteristics, it will be easy understandable and easy to implement in practice by experts in the field and/or can be fully installed from the present description. For example, experimental procedures are described in detail below, can be used to determine whether associated or not this antichaos Ang-2 and/or Ang-1; blocks or not the binding of Ang-2 and/or Ang-1, Tie-2; inhibits or not Ang-2 and Ang-1-mediated phosphorylation of Tie-2; etc.

Mapping of epitopes and related technologies

To screen for antibodies that bind to a specific epitope (for example, those which block binding of IgE to its high-affinity receptor), can be carried out regular analysis of cross-blocking, such as that described in "Antibodies," Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY). Other methods include alanine scanning mutagenesis, peptide blots (Reineke (2004) Methods Mol Biol 248:443-63), or analysis of peptide cleavage. In addition, methods can be used, such as cutting epitopes, the selection of epitopes and chemical modification of antigens (Tomer (2000) Protein Science 9: 487-496).

The term "epitope" refers to a site on an antigen to which the answer is B and/or T-cells. B-cell epitopes can be formed both from contiguous amino acids, and not a sequence of amino acids contiguous with the tertiary structure of the protein. Epitopes formed from contiguous amino acids, typically remain exposed upon denaturation solvents, whereas epitopes formed tertiary structure, as a rule, are lost in the processing of denaturing� solvents. An epitope typically includes at least 3 or more, and more often at least 5 or 8-10 amino acids in a unique spatial conformation.

Profiling method with Modifications (Modification-Assisted Profiling) (MAP), also known as Profiling of Antibodies Based on the Structure (Antigen Structure-based Antibody Profiling) (ASAP) is a method that categorizes a large number of monoclonal antibodies (mAb) directed against the same antigen according to the similarity of the profile of the binding of each antibody with chemically and enzymatically modified surface antigens (USA 2004/0101920). Each category can reflect the unique epitope or significantly different or partially overlapping with an epitope characterized by another category. This technology allows quick filtering of genetically identical antibodies, so that the characterization can be focused on genetically distant antibodies. When applied to hybrid screening, MAP may facilitate identification of rare hybrid clones that produce mAb having target characteristics. MAP can be used to sort the anti-Ang-2 antibodies of the invention in the group of antibodies binding to different epitopes.

Anti-Ang-2 antibodies can bind to epitope within the N-terminal dominas double helix or within the C-terminal fibrinogenolysis domain (FD). In the preferred embodiments of the present invention, the anti-Ang-2 antibodies and antigen-binding fragments bind to epitope within FD.

Amino acids inside FD Ang-2, which interacts with Tie-2 were established by analysis of the crystal structure. See Barton et al., Nat. Struct. Mol. Biol. 13:524-532 (May 2006). With regard to antibodies that block the binding of Ang-2, Tie-2, but essentially does not inhibit the binding of Ang-1 to Tie-2 (for example, H1 H685P, see Examples 5 and 6 below), the epitope to which these antibodies bind may include one or more amino acids Ang-2 (a) interact with Tie-2 and (b) are not identical to the corresponding amino acid in Ang-1. (see Figure 1). Thus, the epitope bound by such antibodies, preferring Ang-2, may include one or more of the following amino acids hAng-2 (SEQ ID NO:518): S-417; K-432; I-434; N-467; F-469; Y-475; or S-480. For example, the authors of the present invention found that antibodies that interact with amino acids F-469, Y-475 and S-480 of Ang-2 (SEQ ID NO:518), preferably interact with Ang-2 compared with Ang-1, and this is the preferred binding may have therapeutic benefit. Thus, the present invention includes anti-Ang-2 antibodies, which are specific contact with human angiopathies-2 (hAng-2), but essentially do not bind to hAng-1, where antibodies bind to epitope� to hAng-2 (SEQ ID NO:518), comprising amino acids F-469, Y-475 and S-480. Thus, the present invention includes anti-Ang-2 antibodies, which are specific contact with human angiopathies-2 (hAng-2), but essentially do not bind to hAng-1, where antibodies bind to an epitope on hAng-2 (SEQ ID NO:518), including amino acids F-469, Y-475 and S-480.

The present invention includes anti-Ang-2 antibodies that bind to the same epitope that any specific typical antibody described herein (for example, H1 H685, H1 H690, H1 H691, H1H696, H1 H706, H1 M724 and/or H2M744). Similarly, the present invention also includes anti-Ang-2 antibodies that compete for binding to Ang-2 with any of the specific typical antibodies described herein (for example, H1 H685, H1 H690, H1 H691, H1 H696, H1 H706, H1 M724 and/or H2M744).

Specialist using conventional methods known in the art can easily determine whether the associated antibody to the same epitope as that of the reference anti-Ang-2 antibody, or competes for binding to it with the reference anti-Ang-2 antibody. For example, to determine communicates whether the test antibody to the same epitope as that of the reference anti-Ang-2 antibody of the invention, the reference antibody is given the opportunity of binding to Ang-2 protein or peptide under conditions of saturation. Then evaluates the ability of a test antibody to contact molecules�th Ang-2. If the test antibody is able to bind to Ang-2 after bonding under conditions of saturation with the reference anti-Ang-2 antibody, it can be concluded that the test antibody binds to an epitope different from the one associated with the reference anti-Ang-2 antibody. On the other hand, if the test antibody is not able to contact the molecule Ang-2 after bonding under conditions of saturation of the reference anti-Ang-2 antibody, then the test antibody can bind to the same epitope, which binds the reference anti-Ang-2 antibody of the invention. Then there might be additional ordinary experiments (for example, analysis of peptide mutations and binding) to confirm that in fact, the observed absence of binding of the test antibody is due to the binding to the same epitope that of the reference antibody, or if the lack of observed binding is responsible steric blocking (or other phenomenon). Experiments of this type can be carried out using ELISA, RIA, Biacore, flow cytometry or any other quantitative or qualitative analysis, available in the art. According to some embodiments of the present invention, two antibodies bind to the same (or overlapping) the epitope, for example if, 1-, 5-, 10-, 20- or a 100-fold excess of one �Titel inhibits binding of the other, at least 50% but preferably 75%, 90% or even 99% on measurements in the competitive analysis of binding (see, for example, Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, it is believed that the two antibodies bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody, thus reduce or eliminate the binding of the other. It is believed that two antibodies have "overlapping epitopes", if only a portion of the amino acid mutations that reduce or eliminate binding of one antibody, thus reduce or eliminate the binding of the other.

To determine whether competes antibody for binding with a reference anti-Ang-2 antibody, carry out the above-described methodology binding in two steps: in the first step of a reference antibody to allow you to track molecule Ang-2 under conditions of saturation, followed by assessment of binding of the test antibody molecule with Ang-2. In the second step, the test antibody is given the opportunity to contact the molecule Ang-2 under conditions of saturation, followed by assessment of binding of the reference antibody molecule with Ang-2. if both actions are only the first antibody (saturating) is able to bind to a molecule Ang-2, we conclude that the test antibody and the reference antibody compete� for binding to Ang-2. The person skilled in the art it is clear that an antibody that competes for binding with a reference antibody may not necessarily contact the same epitope as that of the reference antibody, but may sterically block the binding of a reference antibody by binding to an overlapping or adjacent epitope.

Species selectivity and species cross reactivity

According to some embodiments of the invention, the anti-Ang-2 antibodies bind to human Ang-2, but not Ang-2 from other species. Alternatively, the anti-Ang-2 antibodies of the invention in some embodiments are associated with human Ang-2 and Ang-2 from one or more species that is non-human. For example, Ang-2 antibodies of the invention can bind to human Ang-2 and can contact or not to contact in the case one or more of Ang-2 mice, rats, Guinea pigs, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, monkey-graboid, marmoset, rhesus or chimpanzees.

Immunoconjugate

The invention encompasses anti-Ang-2 monoclonal antibody conjugated to a therapeutic component ("immunoconjugate"), such as cytotoxin, a chemotherapeutic drug, immunomodulate agent or a radioisotope. The cytotoxic agent� include any agent, which adversely affect the cells. Examples of suitable cytotoxic agents, and chemotherapeutic agents for education immunoconjugates known in the art, see, for example, WO 05/103081).

Polyspecific antibodies

The antibodies of the present invention can be monospecificity, bispecific or polyspecific antibodies. Polyspecific mAb can be specific for different epitopes of a single polypeptide-target or may contain the antigen-binding domains for more than one polypeptide target. See, for example, Tutt et al. (1991) J. Immunol. 147:60-69. Anti-Ang-2 antibodies of the present invention or part thereof may be connected or can co-expressed with another functional molecule, for example, with another peptide or protein with the formation of polyspecific molecules. For example, an antibody or a fragment thereof can be functionally linked (for example, using chemical condensation, genetic linkage, noncovalent Association or otherwise) to one or more molecular components, such as another antibody or antibody fragment, to obtain bispecific or polyspecific antibody with a second binding specificity.

Typical bispecific format of antibodies that can be used in the context of the present invention, includes ISP�Lituanie first C H3-domain immunoglobulin (Ig) and second (CH3-domain immunoglobulin Ig, where the first and second CH3-Ig domains are different from each other, at least one amino acid, and where at least one amino acid difference reduces the binding bispecific antibodies with Protein A as compared with bispecific antibody devoid of amino acid differences. In one embodiment, the first CH3-Ig domain binds to Protein A, and the second CH3-Ig domain contains a mutation that reduces or eliminates binding to Protein A, such as modification H95R (in the numbering of exons IMGT; H435R in EU numbering). The second CH3 may further include a modification Y96F (by IMGT; Y436F by EU). Additional modifications that can be found in the second CH3, include: D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L358M, N384S, K392N, V397M and V422I by EU) in the case of IgGI antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU) in the case of IgG4 antibodies. Variations of format bispecific antibodies described above are carried out within the framework of the present invention.

Therapeutic composition and introduction

In the invention proposes a therapeutic composition comprising an anti-Ang-2 antibodies or their antigen-binding fragments of the present invention. Therapeutically� song about the present invention can additionally include one or more pharmaceutically acceptable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and so on (in this document are collectively referred to as "pharmaceutically acceptable carriers or diluents"). A set of appropriate formulations can be found in the guide, known to all chemists pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These compounds include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lepidosauria (cationic or anionic) media (such as LIPOFECTIN™), DNA conjugates, anhydrous paste suction, emulsion oil-in-water and water-in-oil emulsion carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See, also Powell et al. "Compendium of excipients for parenteral formulations" PDA, 1998, J Pharm Sci Technol 52:238-311.

The dose of the antibody may vary depending on the age of the object, which carry out the introduction, target disease, conditions, route of administration, and so on. The preferred dose is usually calculated according to body weight or surface area of the body. When the antibody of the present invention is used for the treatment of a pathological condition or disease associated with the activity of Ang-2 in the adult patient, it may be advantageous nutrion�th the introduction of the antibodies of the present invention, typically, in a single dose of about 0.01 to about 20 mg/kg body weight, more preferably, about 0.02 to about 7, from about 0.03 to about 5, or about 0.05 to about 3 mg/kg of body weight. Depending on the severity of the pathological condition may be governed by the frequency and duration of treatment. Effective dosages and schedules for the introduction of Ang-2 antibodies may be determined empirically; for example, through periodic evaluations, you can track the progress of the patient and accordingly is regulated dose. In addition, interspecies scaling of dosages can be carried out using methods well known in the art (for example, considered rough et al., 1991, Pharmaceut. Res. 8:1351).

There are various delivery systems, and they can be used to introduce the pharmaceutical compositions according to the invention, for example, encapsulation in liposomes, microparticles, microcapsules, the use of recombinant cells capable of expressing the mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al. 1987, J. Biol. Chem. 262:4429-4432). Methods of administration include, in particular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural or oral routes of administration. The composition may be administered by any standard, for example, by infusion or basnayaka, by absorption through epithelial or Muko-mucous lining (eg, oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other biologically active agents. Administration may be systemic or local.

The pharmaceutical composition of the present invention can be delivered, for example, subcutaneously or intravenously using a standard needle and syringe. In addition, in relation to subcutaneous delivery, a delivery device with an injector in the form of the handle provides easy application in the form of delivery of the pharmaceutical compositions of the present invention. Such a delivery device with an injector in the form of a pen can be re-used or can be a single use device. Reusable delivery device with an injector in the form of a pen, as a rule, uses a replaceable cartridge that contains a pharmaceutical composition. After you have entered all pharmaceutical composition inside the cartridge, and the cartridge is empty, it can be easy or deleted or replaced with a new cartridge, which contains a pharmaceutical composition. A delivery device with an injector in the form of a pen can then be reused. The disposable delivery device with an injector in the form of a pen is not replaceable cartridge. In contrast, disposable �the delivery device with an injector in the form of a pen, already pre-filled with a pharmaceutical composition, which is located inside the tank inside the unit. Once removed from the tank pharmaceutical composition, the entire device is discarded.

Numerous reusable delivery device with an injector in the form of a pen and with autoinjection find application in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include in particular AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), is a few listed. Examples of disposable delivery device with an injector in the form of a handle having application in the subcutaneous delivery of a pharmaceutical composition of the present invention include, in particular, SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (EIi Lilly).

For the treatment of eye disorders antibodies and antigen-binding fragments of the invention may be administered, for example, using eye drops, podklyuchilas injection, podchinyonnogo implant, injection into the vitreous body of the implant in the vitreous body, subtenon injections injections or subtenon injections they�of landata.

The pharmaceutical composition may also be delivered in a vesicle, in particular a liposome (see Langer 1990 Science 249:1527-1533; Treat et al. (1989) in Liposomes in therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see, mostly ibid.).

In some embodiments, the pharmaceutical composition can be delivered in the system of controlled release. In one embodiment, can be used in the delivery of the injection pump (see Langer, supra; Sefton, 1987 CRC Crit. Ref. Biomed. Eng, 14:201). In another embodiment, can be used polymeric materials. In another embodiment, the system controlled release may be placed close to the target composition, thus requiring a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138).

Injectable preparations may include metered form for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, etc., These injectable preparations can be obtained using well-known methods. For example, the injectable preparations may be obtained, for example, by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or in an oil environment, the standard used for injection. As the aqueous medium for injections used�t, for example, physiological saline, an isotonic solution containing glucose and other additional agents, etc., which can be used in combination with the corresponding solubilization agent. such as alcohol (e.g. ethanol), polisport (for example, propylene glycol, polyethylene glycol), nonionic surface active substance [for example, Polysorbate 80, HCO-50 (polyoxyethylene adduct (50 mol) hydrogenated castor oil)], and so on as the oil medium is used, for example, sesame oil, soybean oil, etc., which can be used in combination with solubilizers agent such as benzyl benzoate, benzyl alcohol, etc., Therefore, receive a solution for injection, preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteral use described above. get in dosage forms with a single dose, appropriate to add a dose of active ingredients. Dosed forms in a single dosage include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc., Contained amount of the above-mentioned antibodies, typically is from about 5 to about 500 mg per dosage form in a single dose; especially in the form of solution for injection, preferably stabilisation antibody contained in amounts of from about 5 to about 100 mg and about from about 10 to 250 mg for other dosage forms.

Therapeutic applications of antibodies

Antibodies according to the invention is used, among other things, to treat, prevent and/or ameliorate any disease or disorders associated with the activity of Ang-2, including diseases or disorders associated with angiogenesis. The antibodies or antigen-binding sites of the present invention can be used to treat, for example, primary and/or metastatic tumors, which appeared in the brain and the meninges, in the oral part of the pharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive tract, muscles, bones, skin and appendages, connective tissue, spleen, immune system, blood-forming cells in the bone marrow, liver and urinary tract, and in a special sense organs such as eyes. In some embodiments, the antibodies and antigen-binding fragments of the invention are used to treat one or more of the following cancers: počečnokletočnyj carcinoma, pancreatic carcinoma, breast cancer, cancer brodaty, malignant glioma, osteosarcoma, colorectal cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial carcinoma, thyroid cancer Il� melanoma.

Antibodies and antigen-binding fragments of the present invention may also be used to treat one or more ocular disorders. Typical ocular disorders that can be treated using the antibodies or antigen-binding fragments of the invention include, for example, age-related macular degeneration, diabetic retinopathy and other ocular disorders associated with choroidal neovascularization, leak vessels, oedema and inflammation of the retina. In addition, the anti-Ang-2 antibodies of the invention may be administered as an adjuvant in glaucoma surgery to prevent early hemo - and lymphangiogenesis and involvement of macrophages to filter the pillow after glaucoma surgery and to improve clinical outcome.

In other embodiments of the present invention, the antibody or antigen-binding fragments are used for treatment, hypertension, diabetes (including insulin-dependent diabetes mellitus), psoriasis, arthritis (including rheumatoid arthritis), asthma, sepsis, kidney disease and edema associated with injury, stroke or tumor.

It was demonstrated that the expression of Ang-2 correlates with 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). �sootvetstvenno, anti-Ang-2 antibodies of the present invention can be used to treat, prevent or ameliorate one or more inflammatory or infectious diseases. Typical infectious diseases that can be subjected to the treatment, prevention or suppression with the introduction of anti-Ang-2 antibodies of the invention include, in particular: malaria (Plasmodium falciparum infection), viral hemorrhagic fevers (for example, dengue fever), Rickettsia infection, toxic shock syndrome, sepsis, hepatitis C, infection with Bartonella bacilliformis, leishmaniasis, mycobacterial infection and infection with Epstein-Barr.

Combined therapy

Combination therapy can include an anti-Ang-2 antibody of the invention and, for example, another antagonist of Ang-2 (for example, anti-Ang-2 antibody, Patiala (peptibody), or CovX-body, such as CVX-060 (see, U.S. 7521425)). Anti-Ang-2 antibodies of the invention may also be administered together with another anti-angiogenic agent, such as, for example, the VEGF antagonist (for example, VEGF-Trap, see, for example, U.S. 7087411 (also designated herein as "VEGF-inhibiting protein"), anti-VEGF antibody (such as bevacizumab), a small molecule kinase inhibitor of VEGF receptor (e.g. sunitinib, sorafenib or pazopanib), anti-DLL4 antibody (e.g. anti-DLL4 antibody, as disclosed in U.S. 2009/0142354, so�e as REGN421), etc.) or together with a receptor antagonist of epidermal growth factor (EGFR) (for example, anti-EGFR antibody or small molecule inhibitor of EGFR activity). Other agents that can usefully be administered in combination with anti-Ang-2 antibodies of the invention include inhibitors of cytokines, including small molecule inhibitors of cytokines and antibodies that bind to 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 with their respective receptors. The present invention also includes a therapeutic combination comprising any of the anti-Ang-2 antibodies specified herein and an inhibitor of one or more of VEGF, DLL4, EGFR, or any of the above cytokines, where the inhibitor is an aptamer, an antisense molecule, a ribozyme, miRNAs, Patiala, nanotesla or a fragment of an antibody (for example, Fab fragment; an F(ab')2-fragment; Fd-fragment; Fv fragment; scFv; a dAb fragment; or other engineered molecules, such as diamela, Triatel, tetrathele, Minitel and minimal recognition elements). Anti-Ang-2 antibodies of the invention may also be administered in combination with antiviral agents, antibiotics, analgesics, corticosteroids and/or NSAID. Anti-Ang-2 antibodies of the invention may also be administered as part of a regimen that also includes radiation therapy and / or standard chemotherapy. When combined with one or more additional agents, anti-Ang-2 antibodies of the invention may be administered prior to administration of the other agent (other agents), at the same time (for example, in the same composition or in separate compositions) or sequentially with the introduction of another agent (other agents).

Diagnostic use of antibodies

Anti-Ang-2 antibodies of the present invention can also be used for detection and/or measurement of Ang-2 in the sample, for example, for diagnostic purposes. For example, anti-Ang-2 antibody or a fragment thereof can be used for diagnosing a pathological condition or disease characterized by aberrant expression (for example, over-expression, low expression, no expression, etc.) Ang-2. Typical diagnostic tests for Ang-2 may include, for example, the contact of the sample obtained from the patient, with an anti-Ang-2 antibody according to the invention, where the anti-Ang-2 antibody labeled with apparently detected by using a label or reporter molecule. Alternatively, unlabeled anti-Ang-2 antibody can be used in diagnostic applications in combination with a secondary antibody, which itself marked with an apparently detected markers. Detectable label or reporter molecule can be a radioisotope, such as3H,14C,32, 35S or125I; a fluorescent or chemiluminescent component, such as a fluorescein isothiocyanate or rhodamine; or an enzyme, such as alkaline phosphatase, β-galactosidase, horseradish peroxidase or luciferase. Specific typical tests that can be used for detection or measurement of Ang-2 in a sample include enzyme-linked immunoassay (ELISA), radioimmunoassay (RIA), analysis using cell sorting device with the activation of fluorescence (FACS).

EXAMPLES

The following examples are used to provide the person skilled in the art a complete disclosure and description of how to make or apply the methods and compositions according to the invention, and are not intended to limit the scope of what the authors regard as their invention. Efforts have been made to ensure accuracy in respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be taken into account. Unless indicated otherwise, parts are parts are mass, molecular weight is weight average molecular weight, temperature is expressed in degrees Celsius, and the pressure is atmospheric or close to it.

Example 1. The generation of human antibodies to human Ang-2

Human Ang-2-antigen enter�whether directly, with an adjuvant to stimulate the immune system, mouse VELOCIMMUNE® containing DNA encoding the variable portions of heavy and Kappa light chain of human immunoglobulin. Antibody immune response was measured using Ang-2-specific immunoassay. When he reached the target of the immune response, splenocytes were harvested and fused with mouse myeloma cells to maintain their viability and formation of the hybrid cell lines. Hybrid cell lines were skanirovali and selectively to identify cell lines that produce Ang-2-specific antibodies. Using this method you receive multiple anti-Ang-2 chimeric antibodies (I. e., antibodies possessing human variable domains and mouse constant domains); typical antibodies generated in this way, designated as follows: H1 M724, H1 M727, H1 M728, H2M730, H1 M732, H1 M737, H2M742, H2M743, H2M744, H1 M749, H2M750 and H1 M810.

Anti-Ang-2 antibodies also were isolated directly from antigen-positive B cells without fusion to myeloma cells, as described in US 2007/0280945A1. Using this method resulted in several human anti-Ang-2 antibodies (I. e., antibodies possessing human variable domains and human constant domains); typical antibodies generated in this way meant as a way�m; H1 H685, H1 H690, H1 H691, H1 H693, H1 H694, H1 H695, H1 H696, H1 H704, H1 H706 and H1 H707.

Biological properties of typical anti-Ang-2 antibodies generated in accordance with the methods of this Example are described in the Examples below.

Example 2. Analysis using variable gene

To analyze the structure of the obtained antibodies were cloned and sequenced nucleic acids encoding the variable portions of the antibodies. On the basis of nucleic acid sequence and predicted amino acid sequence of the antibodies identified the use of a gene for each variable part of the heavy chain (HCVR) and a variable segment light chain (LCVR) (table 1).

td align="center"> 3-33
Table 1
AntibodyHCVRLCVRID antibodies
VHDHJHVKJKHCVR/LCVR SEQ ID NO
H1H6853-133-1643-201 2/10
H1H6903-234-433-11426/34
H1H6913-94-1763-20450/58
H1H6933-234-431-12174/82
H1H6943-156-641-5198/106
H1H6953-335-1263-155122/130
H1H6963-114-1741-164146/154
H1H7046-641-165170/178
H1H7063-333-331-161194/202
H1H7073-333-333-204218/226
H1M7243-333-351-174266/274
H1M7271-183-362-282338/346
H1M7283-76-1941-51290/298
H2M7303-76-13 41-51362/370
H1M7323-151-741-173242/250
H2M7423-235-552-284386/394
H2M7433-232-841-124410/418
H2M7441-184-451-124434/442
H1M7493-335-543-151314/322
H2M7503-336-641-16 4458/466
H1M8103-233-331-121482/490

Table 2 presents the pair of the amino acid sequences of the variable regions of the light chain of the anti-Ang-2 antibodies and their corresponding identifiers antibodies. Denote N, P and G denote an antibody containing heavy and light chains with identical CDR sequences, but with variations in the sequence of the areas that lie outside the CDR sequences (for example, in frame areas). Thus, the options are N, P and G specific antibodies have identical CDR sequences within the variable regions of heavy and light chains, but contain modifications inside the frame sections.

Table 2
NameHCVR/LCVR
SEQ ID NO
NameHCVR/LCVR
SEQ ID NO
NameHCVR/LCVR
SEQ ID NO
H1H685N2/10H1H685P 18/20H1H685G22/24
H1H690N26/34H1H690P42/44H1H690G46/48
H1H691N50/58H1H691P66/68H1H691G70/72
H1H693N74/82H1H693P90/92H1H693G94/96
H1H694N98/106H1H694P114/116H1H694G118/120
H1H695N122/130H1H695P138/140H1H695G142/144
H1H696N146/154H1H696P162/164H1H696G166/168
H1H704N170/178 H1H704P186/188H1H704G190/192
H1H706N194/202H1H706P210/212H1H706G214/216
H1H707N218/226H1H707P234/236H1H707G238/240
H1M724N266/274H1M724P282/284H1M724G286/288
H1M727N338/346H1M727P354/356H1M727G358/360
H1M728N290/298H1M728P306/308H1M728G310/312
H2M730N362/370H2M730P378/380H2M730G382/384
H1M32N 242/250H1M732P258/260H1M732G262/264
H1M737N506/X*H1M737P514/X*H1M737G516/X*
H2M742N386/394H2M742P402/404H2M742G406/408
H2M743N410/418H2M743P426/428H2M743G430/432
H2M744N434/442H2M744P450/452H2M744G454/456
H1M749N314/322H1M749P330/332H1M749G334/336
H2M750N458/466H2M750P474/476H2M750G 478/480
H1M810N482/490H1M810P498/500H1M810G502/504
* LCVR amino acid sequence of H1 M737 not presented.

Control constructs used in the following examples

Various control structures (anti-Ang-2 antibodies and anti-Ang-2 Patiala) were included in the following experiments for comparative purposes. Control structures are identified as follows: Control I: human anti-Ang-2 antibody containing the variable domains of the heavy and light chain having the amino acid sequence of the corresponding domains "Ab536(THW), as presented in U.S. 2006/0018909 (see also Oliner et al., 2004, Cancer Cell 6:507-516); Control II: Patiala that binds to human Ang-2 having the amino acid sequence of "2XCon4(C), as presented in U.S. 7205275, (see also Oliner et al., 2004, Cancer Cell 6:507-516); Control III: Patiala that binds to human Ang-2 having the amino acid sequence of L1-7", as shown in U.S. 7138370; Control IV: human anti-Ang-2 antibody containing the variable portions of heavy and light chain having the amino acid sequence corresponding Domino� "3.19.3", as presented in U.S. 2006/0246071; and Control V: human anti-Ang-2 antibody containing the variable portions of heavy and light chain having the amino acid sequence of the corresponding domains "MEDI1/5", as shown in WO 2009/097325. (Not all control design was used in each Example). In the tables presented below, the notation "Ab" and "Pb" are included to identify antibody and peptidergic controls, respectively (T. e., Control I=Ab; Control II=Pb; Control III=Pb; Control IV=Ab; and Control V=Ab).

Example 3. Determination of antigen-binding affinity

Dissociation constants in equilibrium (the value of KDto associate the selected purified Ang-2 antibodies with dimeric fibrinogenolysis domain of human (SEQ ID NO: 519), mouse (Mus musculus; SEQ ID NO: 520) and monkey (Macca fascicularis; SEQ ID NO: 521) Ang-2 (Ang-2FD), conjugated with human IgG1 (SEQ ID NO:528), were determined using surface kinetics in real-time using analysis of surface plasmon resonance using a biosensor. The antibody was captured on the antibody surface polyclonal goat antibodies to mouse IgG, goat antibody to human K (Southern Biotech, Birmingham, AL) or surface antibody polyclonal goat antibody to human IgG (Jackson Immuno Research Lab, West Grove, PA), established through direct �myocardinal touch chip (BIACORE™ CM5 with the formation of the captured antibody surface. Different concentrations (in the range from 50 nm up to 6.25 nm) of protein were injected at a rate of 100 μl/min over the captured antibody surface for 90 seconds. The binding and dissociation of antigen-antibody tracked in real time at room temperature. Kinetic analysis was performed to calculate KD and half-life of dissociation of the complex antigen/antibody. The results are summarized in Table 3 below.

Table 3
AntibodyDimeric human Ang-2FDDimeric murine Ang-2FDDimeric monkey Ang-2FD
KD(PM)T1/2(min)KD(PM)T1/2(min)KD(PM)T1/2(min)
H1M724N17942,76941673025,7
H1M728N137 56509,9158069,5
H2M730N21047--84236,6
H1M732N48435,5170021,4733024,1
H1M737N25134,51740a 6.3381016
H2M742N2953861030,8617028,5
H2M743N154167882195,2234169,2
H2M744Nto 98.9109,1143 223,1500281,7
H2M749N16542,952925,5150040,9
H2M750N36232,2--147023

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

Table 4
AntibodyDimeric human Ang-2FDDimeric murine Ang-2FDDimeric monkey Ang-2FD
KD(PM)T1/2(min)KD(PM)T1/2(min)KD(PM)T1/2(min)
H1H685P 71,4229,4148128,799,4177,1
H1H690P79to 126.191,3105,255,6195,2
H1H691P22038,5220to 43.829041
H1H693P50037,144663,7117017,6
H1H694P126265,6237166,535685,6
H1H695P245147347to 124.244084,1
H1H696P28940237,635436,6
H1H704P33186,148461,9818of 33.5
H1H706P20150,43574716453,3
H1H707P26226,632834,428322,3
H1H724N11510718584239173
H1H728N16281576020200077
H1H730N2346297,1 90340087
H1H732N3865752951439118
H1H742N186652765868393
H1H743N88,225412423396,5780
H1H744N114127158115346164
H1H749N11810917796407143
H1H750N164127218121199 244
Control I (Ab)33934,8339of 47.153727,1

Additional binding experiments were performed using selected anti-Ang-2 antibodies at two different temperatures to further assess cross-species affinity. Each selected antibody or control design has captured a flow rate of 40 μl/min for 1 minute on surface antibody goat polyclonal antibody to human Kappa chain created through direct chemical condensation with chip BIACORE™ with the formation of the captured antibody surface. Human, monkey and murine Ang-2FD-Fc at a concentration of 25 nm or of 0.78 nm injectively over the captured antibody surface at a flow rate of 60 μl/min for 3 minutes, and the dissociation of antigen-antibody tracked in real-time for 20 minutes or at 25°C, or at 37°C.

The results are summarized in Tables 5 (binding at 25°C) and 6 (binding at 37°C), below.

Table 5
Binding at 25°C
Anti�lo Dimeric human Ang-2FD-mFcDimeric murine Ang-2FD-hFcDimeric monkey Ang-2FD-hFc
KD(Molar)T1/2(min)KD(Molar)T1/2(min)KD(molar)T1/2(min)
H1H685P1,17 E-11227is 6.51 E-112082,20 E-11275
H1H744N1,16 E-10233,85 E-10332,44 E-1024
Control I (Ab)1,07 E-09151,07 E-09151,03 E-094
Control IV (Ab)1,27 E-112694,02 E-11289342

Table 6
Binding at 37°C
AntibodyDimeric human Ang-2FD-mFcDimeric murine Ang-2FD-hFcDimeric monkey Ang-2FD-hFc
KD(Molar)T1/2(min)KD(Molar)T1/2(min)KD(Molar)T1/2(min)
H1H685P2,70 E-11609,39 E-11647,21 E-1165
H1H744N1,05 E-10182.15 E-10263,20 E-1011
Control I (Ab)------3,90 E-1012--- ---
Control IV (Ab)9,91 E-121845,40 E-111194,74 E-11107

In another experiment determine the values of KDfor selected purified antibodies that bind to human "bow-Ang-2" tetrameric structure ("hBA2") (using the methods described above). hBA2 is composed of two dimers, each dimer contains two fibroadenomatoid domain of Ang-2 that are associated with each other by using a human Fc domain. Amino acid sequence of the dimeric components of hBA2 is presented in SEQ ID NO:522. The results are summarized in Table 7 below.

Table 7
AntibodyhBA2
KD(PM)T1/2(min)
H1H685P11,9587,2
H1H690P17,9299,3
H1H691P1060,6
H1H693P29928,7
H1H694P68,4111,3
H1H695P40,1254,3
H1H696P11151,5
H1H704P93,9117,7
H1H706P79,163,9
H1H707P75,2to 51.4
H1H724N23,3323
H1H728Nof 41.8185
H1H730Nto 55.9152
H1H732N13273
H1H742N72,187
H1H743N9,711118
H1H744N17,2 442
H1H749N32,5235
H1H750N36,9284
Control I (Ab)8357,5

In another experiment, determine the values of KDfor selected purified antibodies that bind to human Ang-2 wild-type (hAng-2-WT; SEQ ID NO: 518) and fibrinogenolysis domain of human Ang2 (hAng-2FD) (as described above). The results are summarized in Table 8 below.

33,48
Table 8
AntibodyMonomeric hAng-2FDhAng-2-WT
KD(nm)T1/2(min)KD(PM)T1/2(min)
H1M724N1,7517,433,1568
H1M728N1,1733,933,8 725
H2M730N2,0624,4to 49.2519
H1M732N6,1318,7131333
H1M737N2,8213,159,3282
H2M742N4,8118,067,9437
H2M743N0,399156,714,32366
H2M744N0,475to 89.328,9846
H2M749N1,3827,949479
H2M750N4,42a 21.5of 40.8991
H1H685P0,578/td> 5547,61000
H1H691P110,5719,1684,6
H1H690P0,59425,1612,41568
H1H693P44,80,61425100
H1H694Pto 7.899,85158209,7
H1H695P1,1250,59to 31.11770,7
H1H696P38,40,20of 40.3642,7
H1H704P0,393,3136,2of 747.6
H1H706P111,0227,4 of 661.9
H1H707P145-77,1217,4
H1H724N2,413,3422,6895
H1H728N1,185,8643566
H1H730N2,843,4447,5534
H1H732N2640,22202264
H1H742N4862,2944,9666
H1H743Nof 2.35of 33.039,483927
H1H744N1,0242,1430,8837
H1H749N1,1312,51833
H1H750Nat 0.78730,209,54442
Control I (Ab)44,50,0347,6512
Control II (Pb)90-of 44.7334,8

Additional experiments were performed to measure the binding properties of the selected anti-Ang-2 antibodies of the monomer hAng-2FD at 25°C and at 37°C. Each selected antibody or control design has captured a flow rate of 40 μl/min for 1 minute on surface antibody polyclonal goat antibody to human IgG, developed through direct chemical condensation with chip BIACORE™ with the formation of the captured antibody surface. Human Ang-2FD at a concentration of 500 nm 7.8 nm injectively over the captured antibody surface at a flow rate of 60 μl/min for 3 minutes, and the dissociation of antigen-antibody tracked in real-time for 20 minutes or at 25°C or at 37°C.

The results sum�initialized in Tables 9 (25°C) and 10 (37°C), is presented below. N/D=not determined.

Table 9
Linking monomer hAng-2FD at 25°C
ka (Mc-1)kd (-1)KD(Molar)
H1H685Pof 2.44 E+057,96 E-053,36 E-10145 minutes
H1H744N2,92 E+051,24 E-044,24 E-1093 minutes
Control I (Ab)4,00 E+055,10 E-021,28 E-0714 seconds
Control II (Pb)Stationary stateStationary state9,00 E-08Stationary state
Control III (Pb)5,40 E+056,30 E-021,17 E-07 11 seconds
Control IV (Ab)2,84 E+053.56 E-021,25 E-0719 seconds

Table 10
Linking monomer hAng-2FD at 37°C
ka (Mc-1)ka (Mc-1)ka (Mc-1)KD(Molar)
H1H685P4,06 E+051,39 E-043,42 E-1083 minutes
H1H744N3,86 E+055,48 E-041,42 E-0921 minutes
Control I (Ab)Stationary stateStationary state1,51 E-07Stationary state
Control II (Pb)N/DN/DN/DN/D
Control III (Pb)Stationary stateStationary state2,94 E-07Stationary state
Control IV (Ab)Stationary stateStationary state9,40 E-08Stationary state

As demonstrated in this Example, some of the anti-Ang-2 antibodies generated according to the methods of Example 1 was associated with the structures of Ang-2 with equivalent or higher affinity than that of the controls. For example, antibodies H1 H685, H1 H690, H1 H724 and H1 H744 contacted dimeric human Ang-2-FD KD71,4, 79, 115 and 114 PM, respectively, whereas the Control antibody (I contacted dimeric human Ang-2-FD KD339 RM (see Table 4). Similarly, antibodies H1 H685, H1 H690, H1 H724 and H1 H744 been associated with human BA2 (a construct containing tetrameric fibrinogenolysis domain of Ang-2) with KD11,9, 17,9, 23,3 and 17.2 PM, respectively, whereas the Control antibody (I contacted hBA2 with KD83 PM (see Table 7).

Thus, compared with the control structures, many of the antibodies according to the invention showed increased binding to Ang-2. Antibody H1 H685P demonstrated properties with�VA particularly strong binding to Ang-2 compared to control.

Example 4. Preferred binding to Ang-2 compared with Ang-1

The binding experiment (plasmon resonance analysis) was performed to test whether the selected contact with both antibodies, Ang-2 and Ang-1, or they are preferably associated only with Ang-2. Each selected antibody or control design has captured a flow rate of 40 μl/min for 1 minute on surface antibody polyclonal goat antibody to human IgG, developed through direct chemical condensation with chip BIACORE™ with the formation of the captured antibody surface. A full-sized human Ang-1 or Ang-2 wild-type at a concentration of 25 nm or of 0.78 nm were injected over the captured antibody surface at a flow rate of 60 μl/min for 3 minutes, and the dissociation of antigen-antibody tracked in real-time for 20 minutes or at 25°C, or at 37°C.

The results of these experiments are summarized in Tables 11-14 below. N/D=not determined. "No binding" means that not watched the detected binding to specific experimental conditions used in these experiments.

Table 11a
Linking to hAng-2-WT at 25°C
ka (Mc-1)kd (-1)KD(Molar)T½ (min)
H1H685P6,59 E+051.60 E-052,42 E-11722
H1H744N7,65 E+052,57 E-053,35 E-11450
Control I (Ab)4.74 in E+052,26 E-054,76 E-11512
Control II (Pb)7,73 E+053,45 E-054,47 E-11335
Control III (Pb)3,29 E+051,98 E-056,01 E-11584
Control IV (Ab)3,80 E+06to 2.74 E-047,22 E-1142

Table 11b
Linking to hAng-2-WT at 25°C
ka (Mc-1)kd (-1)KD(Molar)T½ (min)
H1H685P1,15 E+058,50 E-067,39 E-111359
Control II (Pb)8,30 E+045,41 E-056,52 E-10213
Control V (Ab)1,12 E+052,66 E-052,73 E-10434

Table 12a
Linking hAng-1-WT at 25°C
ka (Mc-1)kd (-1)KD(Molar)T½ (min)
H1H685PNo bindingNo bindingNo bindingNo binding
H1H744N4,10 E+053,81 E-059,30 E-11303
Control I (Ab)4,55 E+052,49 E-055,47 E-11464
Control II (Pb)a 4.53 E+053,54 E-057,82 E-11326
Control III (Pb)No bindingNo bindingNo bindingNo binding
Control IV (Ab)6,60 E+05of 1.11 E-041,68 E-10105

Table 12b
Linking hAng-1-WT at 25°C
ka (Mc-1)kd (-1)KD(Molar)T½ (min)
H1H685P No bindingNo bindingNo bindingNo binding
Control II (Pb)3,04 E+052,51 E-058,26 E-11460
Control V (Ab)2,75 E+056,68 E-052,43 E-10173

Table 13a
Linking to hAng-2-WT at 37°C
ka (Mc-1)kd (-1)KD(Molar)T½ (min)
H1H685P8,54 E+053,76 E-054,40 E-11707
H1H744N7,01 E+052,43 E-043,47 E-1048

Table 13b
Linking to hAng-2-WT at 37°C
ka (Mc-1)kd (-1)KD(Molar)T½ (min)
H1H685Pto 1.36 E+052,16 E-051,59 E-10535
Control II (Pb)3,79 E+041,17 E-043,09 E-0999
Control V (Ab)9,42 E+047,92 E-05to 8.41 E-10146

tr>
Table 14a
Linking hAng-1-WT at 37°C
ka (Mc-1)kd (-1)KD(Molar)T½ (min)
H1H685PNo bindingNo bindingNo bindingNo binding
H1H744N1,47 E+065,20 E-053,12 E-11222
Control III (Pb)No bindingNo bindingNo bindingNo binding

Table 14b
Linking hAng-1-WT at 37°C
ka (Mc-1)kd (-1)KD(Molar)T½ (min)
H1H685PNo bindingNo bindingNo bindingNo binding
Control II (Pb)with 2.81 E+054,35 E-051,55 E-10266
Control V (Ab)4,42 E+055,47 E-051,24 E-10211

These �esult demonstrate that H1 H685P is unique among the antibodies tested in this experiment is that it binds with high affinity with Ang-2, but not associated with Ang-1. The only other structure that demonstrates binding to Ang-2, but not Ang-1, this Control III. However, it should be emphasized that Control III is Patiala and that all other antibodies tested in this experiment, was associated with both Ang-2 and Ang-1. Selectivity in binding Ang-2 can give H1 H685P therapeutic efficacy, which do not possess antibodies that bind to both Ang-2 and Ang-1.

Example 5. The inhibition of binding of Ang-2 with human Tie-2

Tie-2 is a natural receptor of Ang-2. Anti-Ang-2 antibodies were tested for their ability to block the binding of Ang-2 with human Tie-2 (hTie-2). hTie-2-mFc (a chimeric construct consisting of the human Tie-2, conjugated mouse IgG; SEQ ID NO:525) was applied as a coating on 96-well plates at a concentration of 2 μg/ml and incubated overnight, followed by washing four times in the buffer for washing (PBS with 0.05% of Tween-20). The tablet is 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. In a separate tablet purified anti-Ang-2 antibodies in the starting end�ations of 50 nm are sequentially diluted three times in the wells. Human, mouse or squirrel monkey Ang-2FD. conjugated with human IgG (Ang-2FD-hFc) was added to a final concentration of 2 nm, 8 nm, or 2 nm, respectively, and were incubated for one hour at room temperature.

A mixture of antibody/Ang-2FD-Fc was then added to the tablet containing hTie-2-mFc, and were incubated for one hour at room temperature. Detection of Ang-2FD-hFc associated with protein hTie-2-mFc was determined using horseradish Peroxidase (HRP), conjugated to human IgG antibody (Jackson lmmuno Research Lab, West Grove, PA) and displayed using standard colorimetric reaction using tetramethylbenzidine (TMB) substrate (BD Biosciences, San Jose, CA). The absorption was read at OD450) for 0.1 sec. the percentage of blocking the binding of Ang-2FD-hFc with hTie-2-mFc using 16,67 nm selected anti-Ang-2 antibodies, are presented in Table 15.

Table 15
AntibodyThe percentage of blocking the binding of Ang-2FD with Tie-2
Human
Ang-2FD-hFc
Mouse
Ang-2FD-hFc
Monkey
Ang-2FD-hFc
H1M724N99,596,6 95,2
H1M728N98,583,997,1
H2M730Nto 98.955,097,3
H1M732N97,7of 90.995,8
H1M737N99,195,490,5
H2M742N99,698,694,1
H2M743N99,698,495,1
H2M744N99,598,495,5
H2M749N99,597,397,4
H2M750N99,453,797,4
Control I (Ab)94,590,296,9

In a similar experiment�mobile, selected purified anti-Ang-2 antibodies, cloned human IgG1, were tested for their ability to block the binding of Ang-2FD with hTie-2 (as described above). The percentage of blocking the binding of Ang-2FD-hFc with hTie-2-mFc using 16,67 nm selected anti-Ang-2 antibodies, are presented in Table 16. NT: not tested.

Table 16
AntibodyThe percentage of blocking the binding of Ang-2FD with Tie-2
Human
Ang-2FD-hFc
Murine Ang-2FD-hFcMonkey
Ang-2FD-hFc
H1H685P93,897,162,2
H1H690P97,298,099,6
H1H691P97,496,799,8
H1H693P73,963,6NT
H1H694P79,836,0NT
H1H695P98,497,6NT
H1H696P98,294,9the 99.2
H1H704P97,0of 41.8NT
H1H706P97,195,999,8
H1H707P95,193,8NT
H1H724N96,697,196,6
H1H744N97,997,696,2
Control I (Ab)97,382,298,5

In another experiment, the selected purified anti-Ang-2 antibodies were tested for their ability to block the binding 20 PM biotinylating hBA2 with hTie-2 (as described above). For this experiment, human Tie-2, conjugated with histidinol tag (hTie-2-His; SEQ ID NO:526), used similarly with hTie-2-mFc, describing how�about above. The antibody concentration ranging from 5 nm, consistently bred three times. The value of the IC50(Inhibiting Concentration) were generated by calculating the amount of antibody required to block 50% of the signal resulting from the binding of Biotin-hBA2 with Tie-2. The average value of the IC50for each antibody was calculated based on two separate experiments. The results are summarized in Table 17. NB: not observed block at a concentration of 5 nm.

Table 17
AntibodyBiotin - hBA2
Average IC50(PM)
H1M724N9,72
H1M728N14,05
H2M730N14,60
H1M732N82,17
H1M737N13,01
H2M742N9,65
H2M743N11,01
H2M744Nof 11.43
H2M749N6,43
H2M750N8,83
Control I (Ab)30,23
Control II (Pb)the 7.75
Control III (Pb)16,49

In a similar experiment, selected purified anti-Ang-2 antibodies, cloned human IgG1, were tested for their ability to block the binding of biotinylating hBA2 with hTie-2 (as described above). The results are presented in Table 18. NB: not observed block at a concentration of 5 nm.

Table 18
AntibodyBiotin - hBA2
IC50(PM)
H1H685P20
H1H690P17
H1H691P13
H1H693PNB
H1H694PNB
H1H695P59
H1H696P22
H1H704P 56
H1H706P8
H1H707P22
H1H724N4
H1H744N25

This Example illustrates that some of the anti-Ang-2 antibodies generated according to the methods of Example 1, blocked the interaction between fibrinogenolysis domain of Ang-2 and its receptor (TIE-2) to an equivalent or greater degree than the control antibody. For example, each of the antibodies H1 H690, H1 H691, H1 H695, H1 H696, H1 H704, H1 H706, H1 H707, H1 H724 and H1 H744 caused more than 95% of blocking human, mouse and monkey designs Ang-2FD with the receptor TIE-2, similar to the results observed for the control structures (see Table 16).

Example 6: Inhibition of binding of full-Ang-2 and Ang-1 with human Tie-2

Tie-2 is the receptor for Ang-1, as well as for Ang-2. Thus, in this Example were measured and compared the ability of some anti-Ang-2 antibodies to block the binding of Ang-2 Ang-1 with human Tie-2.

The ELISA experiments presented in this Example was carried out similarly to the experiments of Example 5. Briefly, hTie-2-mFc (a chimeric construct consisting of the human Tie-2, conjugated mouse IgG; SEQ ID NO:525) caused in the wee�e coating on 96-well plates at a concentration of 2 μg/ml and incubated overnight, followed by washing four times in the buffer for washing (PBS with 0.05% of Tween-20). The tablet is 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. In a separate tablet purified anti-Ang-2 antibodies and control design in a starting concentration of 300 nm were successively dissolved in three times in the wells. A full-sized human protein Ang-2 Ang-1, conjugated with 6X histidinol tag (R&D Systems, Minneapolis, MN) was added to a final concentration of 0.6 nm and incubated for one hour at room temperature. A mixture of antibody/antigen was then added to the tablet containing hTie-2-mFc, and were incubated for one hour at room temperature. Detection of Ang-2-His or Ang-1-His-related protein hTie-2-mFc was determined using horseradish Peroxidase (HRP), conjugated with Penta-His antibody (Qiagen, Valencia, CA) and displayed using standard colorimetric reaction using tetramethylbenzidine (TMB) substrate (BD Biosciences, San Jose, CA).

The absorption was read at OD450in a period of 0.1 sec. The value of the IC50(Inhibiting Concentration) were generated by calculating the amount of antibody required to block 50% of the signal resulting from the binding of human Ang-2 Ang-1 to Tie-2. The results, expressed as IC50presented in Table 19, columns (1) and (2). The degree to which antibodies or control� - to-date designs block the interaction of hAng-2/Tie-2 regarding interactions hAng-1/Tie-2, reflected in the fold difference of the IC50presented in column (3); that is, a greater number in column (3) indicates a greater ability to block the interaction of hAng-2/Tie-2 in comparison with the interaction of hAng-1/Tie-2.

Table 19
Antibody(1)
Block hAng-2 WT with Tie-2 IC50(M)
(2)
Block hAng-1 WT with Tie-2 IC50(M)
(3)
Fold difference in IC50, blocking hAng-1, compared with IC50* blocking h-Ang-2
H1H685P1,294 E-10>3,000 E-07>2318
H1H744N7,871 E-111,872 E-072378
Control I (Ab)9,372 E-116,171 E-08658
Control II (Pb)3,096 E-115,509 E-111,8
Control III (Pb)1,626 E-10>1,000 E-06>6150
Control IV (Ab)1,476 E-104,252 E-0928,8
* Calculated by dividing hAng-1 blocker IC50(column 2) hAng-2-block IC50(column 1).

To further assess the ability of selected anti-hAng-2 antibodies to block the binding of Ang-1, Tie-2, an experiment was conducted using surface plasmon resonance. In this experiment design with a full-length extracellular domain of human Tie-2 (hTie-2-mFc-ecto) condensible the N-end chip, BIACORE™ to create a surface with a coating in the form of the receptor. Selected anti-hAng-2 antibody and control structures in an amount of 1 μm (100-fold excess relative to the antigen) is pre-mixed with 10 nm of hAng-1-WT, followed by incubation for 60 minutes at 25°C to give the possibility of binding of the antibody-antigen to achieve equilibrium with the formation of the equilibrium solutions. The equilibrium solutions were injected over the surfaces of the receptor at 5 μl/min for 5 minutes at 25°C. was Determined changes in resonance units (EP), caused by the binding of hAng-1-WT with hTie-2-mFc. Extraneous design with Patiala, not communicating with hAng-1 included in this experiment to establish 0% source lock�tion, and the design of human Tie-2-mFc was used as a positive control block. The amount of Ang-1 that is associated with Tie-2 after pre-incubation with the antibody, expressed as a percentage of the amount of Ang-1 that is associated with Tie-2 after pre-incubation of the negative control, are presented in Table 20. (A greater number of binding Ang-1, Tie-2 means a lower degree of blocking antibodies).

Table 20
AntibodyRE (average)The percentage of binding of the negative control
Negative control (outside Patiala)169100
hTie-2-mFc7142
H1H685P13781
H1H744N5734
H1H691P11769
H1H706P14083
H1H724N 5734
Control I (Ab)4828
Control II (Pb)4828
Control III (Pb)16095

The above experiment was repeated using different amounts blockers Ang-2 and controls. Specifically, the design with a full-length extracellular domain of human Tie-2 (hTie-2-mFc-ecto) condensible the N-end chip, BIACORE™ to create a surface with a coating in the form of the receptor. Selected anti-hAng-2 antibody and control structures (50 or 150 nm) was mixed with hAng-2-WT (25 nm), followed by incubation for 60 minutes at 25°C to allow binding of the antibody-antigen with the achievement of equilibrium. The equilibrium solutions were injected over the surfaces of the receptor at 10 μl/min for 5 minutes at 25°C. To evaluate the ability of selected anti-hAng-2 antibodies to block the binding of Ang-1-WT with hTie-2, a similar procedure was performed except that the antibodies were tested at three concentrations (50, 100, or 1000 nm) and incubated with 10 nm of hAng-1-WT. Determined changes in resonance units (EP), caused by the binding of Ang-2-WT or hAng-1-WT with hTie-2-mFc. Stranger and�titulo, not communicating with any angiopoietin included in these experiments to establish 0% source block, and the design of human Tie-2-mFc was used as a positive control block. The results are summarized in Tables 21 (h.Ang-1 was applied to the surface hTie-2) and 22 (hAng-2 was applied to the surface hTie-2).

Table 21
(hAng-1 WT)
The amount of antibody or control
50 nm100 nm1000 nm
AntibodySpecific associated REThe percentage of binding of the OTP. counter.Specific associated REThe percentage of binding of the OTP. counter.Specific associated REThe percentage of binding of the OTP. counter.
Negative control (outside antibody)316100307100276 100
hTie-2-mFc70223913-470
H1H685P2999529195289105
Control II (Pb)82,541,3-10
Control V (Ab)15048114372911

Table 22
(hAng-2 WT)
The amount of antibody or control
50 nm150 nm
AntibodySpecific associated RESpecific associated REThe percentage of binding of the OTP. counter.
Negative control (outside antibody)281100278100
hTie-2-mFc97358230
H1H685P124,3124,3
Control II (Pb)103,6103,6
Control V (Ab)124,3124,3

The results obtained from these experiments are consistent with previous results that demonstrated that H1 H685P preferably binds to Ang-2 compared with Ang-1 (see Example 4). Specifically, the results from this Example demonstrate that some anti-Ang-2 antibodies (for example, H1H685P and H1 H706P) is not blocked in a significant article�penalties linking human Ang-1 with human Tie-2, despite the fact that in other experiments it was demonstrated that these antibodies significantly blocked the interaction between Ang-2 and Tie-2 (see Example 5, table 16). In addition, in these experiments, none of the control structures, with the exception of Monitoring III, Patiala, did not demonstrate the same degree preferred binding/blocking Ang-2 compared with Ang-1 as a typical anti-Ang-2 antibodies of the present invention, such as H1 H685P.

Example 7. Inhibition of Ang-2-mediated phosphorylation of Tie-2 using anti-Ang-2 antibodies

The authors of the present invention have demonstrated that the expression of Ang-2 may be induced in human endothelial cells from umbilical vein (HUVEC) using a transcription factor FOXO1 (Daly et al. 2006 PNAS 103:15491). In addition, the inventors have demonstrated that infection of HUVEC adenovirus encoding FOXO1, it results in the expression and secretion of Ang-2, with subsequent activation of the phosphorylation of Tie-2 (Daly et al. 2006 PNAS 103:15491).

Anti-Ang-2 antibodies were tested for their ability to inhibit the phosphorylation of Tie-2. Briefly, 7×105HUVEC (Vec Technologies, Rensselaer, NY) were seeded in 6 cm culture dishes in 3.5 ml of Complete MCDB131 medium (Vec Technologies, Rensselaer, NY). The next day cells were washed with Opti-MEM (Invitrogen Corp., Carlsbad, CA) and added to 2 ml of Opti-MEM. Recombinant adeno�the virus, coding or green fluorescent protein (GFP; control), or human FOXO1 (Daly et al. 2004 Genes Dev. 18:1060) was added to the cells at a concentration of 10-fight/cell and incubated for four hours. Then the cells were washed with MCDB131 and added 2 ml of MCDB131 containing anti-Ang-2 antibody at a concentration of 0.5 μg/ml. twenty-four hours after infection the cells were literally and subjected Tie-2 - immunoprecipitation, as described by Daly et al., Proc. Natl. Acad. Sci. USA 103: 15491-15496 (2006). Immunoglobulin collected pellets with protein A/G (Santa Cruz Biotechnology, Santa Cruz, CA) for one hour. Pellets were washed with cold lysing buffer and were resuspended in SDS buffer for sample for analysis by Western blot with antibodies specific for phosphotyrosine (Millipore, Billerica, MA) or Tie-2. Signals were detected using HRP-conjugated secondary antibodies and ECL reagents (GE Healthcare, Piscataway, NJ). The x-ray film were scanned and evaluated quantitatively signals of phospho-Tie-2 and Tie-2 using the software ImageJ. The ratio of phospho-Tie-2/Tie-2 was used to determine the % inhibition for each anti-Ang-2 antibody (e. The percentage of inhibition=Reduction of phospho-Tie-2/Tie-2 compared to control). For example, it is believed that the decrease in the phosphorylation of Tie-2 to the level observed in a control sample, corresponding to 100% inhibition. Rel�relative inhibition (+, ++, +++) for each anti-Ang-2 antibodies, tested according to the observed percentage inhibition(25-50%, 50-75%, 75-100%, respectively), are presented in Table 23.

Table 23
AntibodyThe inhibition of phosphorylation of Tie-2
H1H685P+++
H1H690P+++
H1H691P+++
H1H693P+++
H1H694P++
H1H695P+++
H1H696P+++
H1H704P+++
H1H706P+++
H1H707P+++
H1M724N+++
H1M728N++
H1M732N++
H1M742N++
1M743N +++
H1M744N+++
H1M749N++
H1M750N+++
Control I (Ab)+
Control II (Pb)+++

As demonstrated in this Example, anti-Ang-2 antibodies generated according to the methods of Example 1 inhibited the phosphorylation of Tie-2 to a greater degree than the Control antibody I. Particularly strong inhibition was observed with antibodies H1 H685, H1 H690, H1 H691, H1 H693, H1 H695, H1 H696, H1 H704, H1 H706, H1 H707, H1 M724, H1 M744 H1 and M750.

Example 8. Inhibition of Ang-1-mediated phosphorylation of Tie-2

As shown in the previous Example, Ang-2 may mediate the phosphorylation of Tie-2. Ang-1 is also able to stimulate the phosphorylation of Tie-2. In the present Example was evaluated by the ability of the selected anti-Ang-2 antibodies to block Ang-1-mediated phosphorylation of Tie-2.

Cells EA.hy926 (Edgell et al., Proc. Natl. Acad. Sci. USA 80:3734-3737 (1983)) were seeded with a density of 5×106cells on 10 cm Cup in 10 ml of DMEM containing 10% FBS, HAT, L-glutamine and penicillin/streptomycin. After 24 hours, the cells withstand without serum for 1 hour in an environment with 10 ml of DMEM + 1 mg/ml BSA. Then the cells �has stimulirovalo for 10 minutes using 500 ng/ml recombinant human Ang-1 (R& D Systems) in the presence or extraneous isotype control antibody ("9E10") in the amount of 400 nm, or in the presence of anti-Ang-2 antibody H1 H685P, or in the presence of control agents (Control I, Control II, Control IV or Control V) in concentrations in the range from 10 to 400 nm.

After incubation the cells were literally and Tie-2 were subjected to immunoprecipitation as described in Daly et al., Proc. Natl. Acad. Sci. USA 703:15491-15496 (2006). Immune complexes were collected by incubation in the presence of granules with protein A/G (Santa Cruz Biotechnology, Santa Cruz, CA) for 60 min, the Pellets were washed with cold lysing buffer, and associated proteins were suirable by heating in SDS-sample buffer. Then the samples were subjected to Western blot analysis with monoclonal antibodies against Tie-2, or against phosphotyrosine (clone 4G10, Millipore, Billerica, MA). The results are presented in Figure 2.

Signals were detected using HRP-conjugated secondary antibodies and ECL reagents (GE Healthcare, Piscataway, NJ). The x-ray film were scanned and evaluated quantitatively signals of phospho-Tie-2 and Tie-2 using the software ImageJ. The ratio of phospho-Tie-2/Tie-2 was used to determine the % inhibition for each antibody or Patiala. The percentage of inhibition=reduction of phospho-Tie-2/Tie-2 in comparison with the control sample (400 nm isotype control antibody).

In presence�and control antibody 9E10, Ang-1 is strongly activated the phosphorylation of Tie-2 (Figure 2, panel A, compare lanes 2 and 3 vs. lanes 1). All control agents that were tested, significantly inhibited the phosphorylation of Tie-2, with complete inhibition observed at 50 nm for Control II (Figure 2, panel B - track 17), 100 nm for Control IV (Figure 2, panel A-track 11), and 200 nm for Control I (Figure 2, panel B - track 24) and Control V (Figure 2, panel C - track 9). In contrast, H1 H685P did not possess significant inhibitory effect even at 400 nm (Figure 2, panel A - lanes 4-8). These results provide additional confirmation of the specificity of H1 H685P for Ang-2 compared with Ang-1.

Example 9. Inhibition of tumor growth using anti-Ang-2 antibodies

The influence of selected purified anti-Ang-2 antibodies on tumor growth was determined using two cell lines.

PC3 (cell line prostate cancer person)Briefly, 5×106PC3 cells in 100 μl Matrigel deficit growth factor (BD Biosciences) were injected subcutaneously into the side of 6-8-week-old male Nude NCr mice (Taconic, Hudson, NY). Once the tumor size reached an average of about 200 mm3, the mice were divided randomly into groups of treatment. The mice in each treatment group was injected with anti-Ang-2 antibody, Fc-protein or a control structure in a concentration of 10 mg/kg by vnutribruchinno�Oh injections twice a week for about three weeks (table 24) or in concentrations of 2.5, of 12.5, or 25 mg/kg by subcutaneous injection twice a week for about three weeks (table 25). Tumor size was measured twice a week throughout the course of the experiment and the tumor mass was measured when cutting the tumor at the conclusion of the experiment. Mean values (mean +/- standard deviation) in tumor weight and growth were calculated for each treatment group. The percentage mass loss and tumor growth was calculated based on comparison with measurements for Fc protein. The results are summarized in Tables 24 and 25.

Table 24
AntibodyThe average weight of tumor (g)% reduction in tumor weightThe average tumor growth (mm3)% reduction of tumor growth
Fc-protein0,66±0,26-509±213-
Control I (Ab)0,47±0,2329300±24241
H1H724N0,55±0,0717/td> 392±16923
H1H744N0,43±0,2035259±21249
H1H685P0,44±0,1233305±14340
H1H691P0,59±0,0711485±1415
H1H706P0,52±0,1421329±12535

Table 25
AntibodyThe average tumor growth (mm3)% reduction of tumor growth
Fc-protein1031±485-
Control II (Pb)
(2.5 mg/kg)
356±19665
Control II (Pb)
(12.5 mg/kg)
360±16265
Control II (Pb)
(25 mg/kg)
527±21849
H1H685P
(2.5 mg/kg)
308±27470
H1H685P
(12.5 mg/kg)
550±15047
H1H685P
(25 mg/kg)
413±20860

As presented above, antibodies H1 H744N and H1 H685P showed particularly marked antitumor activity in a mouse model of PC3 tumors compared to control.

The results of similar experiments using a mouse model of PC3 tumors and various experimental antibodies (dosed at 2 mg/kg, twice weekly), are presented in Tables 26 and 27.

Table 26
AntibodyThe average weight of tumor (g)% Reduction in tumor weightThe average tumor growth (mm3)% Reduction of tumor growth
Fc-protein0,626±0,156-356±93 -
Control I (Ab)0,347±0,09345250±14530
H2M742N0,407±0.076 mm35220±10238
H2M743N0,372±0,12241179±16950

Table 27
AntibodyThe average weight of tumor (g)% Reduction in tumor weightThe average tumor growth (mm3)% reduction of tumor growth
Fc-protein0,552±0,211-473±202-
H1M749Nthe 0.383±0,27531220±26154
H1M750N0,348±of 0.12837227±195/td> 52

COLO 205 (colorectal Cell line human adenocarcinoma)Briefly, 2×106cells COLO 205 in 100 ál serum-free medium were injected subcutaneously into the side of 6-8-week-old male Nude NCr mice (Taconic, Hudson, NY). Once the tumor size reached an average of about 150 mm3, the mice were divided randomly into groups of treatment. The mice in each treatment group was injected with anti-Ang-2 antibody or Fc-protein at a concentration of 4 mg/kg by intraperitoneal injection twice a week for about two weeks. Tumor size was measured twice a week throughout the course of the experiment and the tumor mass was measured when cutting the tumor at the conclusion of the experiment. Mean values (mean +/- standard deviation) in tumor weight and growth were calculated for each treatment group. "AVG. Tumor Growth" refers to the average growth from the time of initiation of treatment (when the tumor size was approximately 150 mm3). The percentage mass loss and tumor growth was calculated based on comparison with measurements for Fc protein. The results are summarized in Table 28.

% Reduction in tumor weight
Table 28
AntibodyThe average weight of tumor (g)The average tumor growth (mm3)% Reduction of tumor growth
Fc-protein0,847±0,180-731±249-
Control I (Ab)0,503±0,09041367±12150
Control II (Pb)it was 0.608±0,08528492±8233
H1M724Nof 0.531±0,10337336±12554
H2M742N0,576±0,05732427±9242
H2M744N0,491±0,05142409±16244
H1M749N0,603±0,14229449±16939

Similar ex�eriment conducted to evaluate the effect of H1 H685P, specifically, on tumor growth of COLO 205. Briefly, 2×106cells COLO 205 in 100 ál serum-free medium were implanted subcutaneously in the posterior part of the right side of 9 to 11-week-old male mice SCID CB17. When the tumor size reached ~125 mm3, the mice were divided randomly into 5 groups (n=7-8 mice/group) and treated twice a week with the help Fc-protein (15 mg/kg), H1 H685P (5 or 25 mg/kg) or Control II (5 or 25 mg/kg) during the time period of 19 days. Tumor size was measured twice a week throughout the course of the experiment and the tumor mass was measured when cutting the tumor at the end of the experiment. The average values of mass and tumor growth since the start of treatment was calculated for each group. The percentage reduction in tumor weight and growth was calculated based on comparison with the control group Fc. The results are presented in Table 29.

Table 29
AntibodyThe concentration of antibodiesThe average weight of tumor (g)% Reduction in tumor weightThe average tumor growth (mm3)% Reduction of tumor growth
Fc-protein25 mg/kg 0.800 to±0,108-675±93-
Control II (Pb)5 mg/kg0,481±0,09140288±8557
Control II (Pb)25 mg/kg0,393±0,13651267±15560
H1H685P5 mg/kg0,458±0,12543370±11445
H1H685P25 mg/kg0,430±0,13946295±16056

As with murine tumor model PC3, some of the antibodies according to the invention, including H1 H685P, showed essentially antitumor activity in murine tumor models COLO 205 that were at least equivalent antitumor activities, which demonstrated the control of a molecule.

Example 10. Inhibition of tumor growth and perfusion using combinations�AI anti-Ang-2 antibody and a VEGF inhibitor

To determine the effects of combination anti-Ang-2 antibody together with a VEGF inhibitor, exerted on the growth of the xenograft COLO 205, 2×106cells were implanted subcutaneously in the posterior part of the right side of 6-8-week-old female SCID mice. When tumors reached an average size of ~350 mm3, the mice were divided randomly into 4 groups (n=6 mice/group) and treated with: human Fc protein (7.5 mg/kg), H1 H685P (5 mg/kg), VEGF Trap (see U.S. 7087411) (2.5 mg/kg) or with a combination of H1 H685P + VEGF Trap. Mouse had in total 3 doses for 10 days of processing. Tumor size was measured twice a week during the entire course of the experiment. The mean values of tumor growth since the start of treatment (mean +/- standard deviation) was calculated for each treatment group. The percentage reduction of tumor growth and the growth was calculated based on comparison with the control group Fc. The results are presented in Table 30. It should be noted that in groups of VEGF Trap and H1 H685P + VEGF Trap the average tumor size was smaller by the end of the process than at the beginning, i.e. the observed tumor regression.

Table 30
AntibodyThe average tumor growth (mm3)% Reduction in growth swollen�
Fc-protein366±65-
H1H685P74±7780
VEGF Trap-62±44117
H1H685P + VEGF Trap-221±131160

The results of this experiment demonstrate that the combination of H1 H685P + VEGF Trap causes a reduction of tumor growth, which is higher than the percentage of reduction of tumor growth induced by each component individually.

To provide additional evidence of the effectiveness of the combination, assessed the effects of combination H1H685P + VEGF Trap exerted on the growth of tumors MMT, of 0.5×106the MMT cells subcutaneously implanted in the back part of the right side of 6-8-week-old female SCID mice. When tumors reached an average size of ~400 mm3, the mice were divided randomly into 4 groups (n=11 mice/group) and treated with: human Fc protein (17,5 mg/kg), H1 H685P (12.5 mg/kg), VEGF Trap (5 mg/kg) or with a combination of H1 H685P + VEGF Trap. Group Fc and H1 H685P received 3 doses over a period of 9 days. The group of VEGF Trap and the combined group received 4 doses for 12 days. Tumor size was measured twice a week throughout the course e�of speriment and tumor mass was measured when cutting the tumor at the conclusion of the experiment (because of their large size tumors from groups Fc and H1 H685P collected three days before the collection of tumors from the group of VEGF Trap and combination group). Mean values (mean +/- standard deviation) of tumor growth from the beginning of the treatment and the tumor mass was calculated for each group. The percentage reduction in tumor weight and growth was calculated based on comparison with the control group Fc. The results are presented in Table 31.

Table 31
AntibodyThe average weight of tumor (g)% Reduction in tumor weightThe average tumor growth (mm3)% Reduction of tumor growth
Fc-protein1,591±0,265-1337±273-
H1H685P1,409±0,314111135±30615
VEGF Trap0,889±0,14144536±17960
H1H685P + VEGF Trap0,599±0,06662215±9284

These results�you confirm the enhancing effect of inhibition of the tumor, rendered H1 H685P + VEGF Trap, relative to treatment with a single agent.

To determine whether the combination of H1 H685P + VEGF Trap a stronger effect on the function of tumor vessels than the individual agents used microlitres (image processing system VisualSonics' Vevo 770) to assess changes in tumor perfusion. Tumor COLO 205 were grown to ~125 mm3and then the mice were treated for 24 hours with the help of H1 H685P, VEGF Trap, or with a combination of both agents. After treatment, the tumor vessel perfusion was determined on the basis of getting a flat contrast-enhanced ultrasound image on the basis of the analysis of the curve of the "wash-in", which characterizes the amount of contrast agent that is part of the tumor. Mean values (mean +/- standard deviation) of tumor perfusion was calculated for each group. The percentage reduction was calculated based on comparison with the control group Fc. The results are presented in Table 32.

Table 32
AntibodyRelative tumor perfusion% Reduction in tumor perfusion
Fc-protein8,09±2,16-
H1H685P6.32 per±2,8122
VEGF Trapof 6.99±1,3614
H1H685P + VEGF Trap2,46±0,3470

According to the enhanced effect of a combination treatment provided to perfusion, anti-CD31 staining of tumor sections showed a stronger effect of combination on the density of tumor blood vessels (data not shown). The increased effect of a combination of H1 H685P + VEGF Trap function on tumor vascular system, provides a potential explanation for the increased effects of combination therapy provided on tumor growth.

Example 11. Inhibition of tumor growth using a combination of anti-Ang-2 antibody and a chemotherapeutic agent

To test the effect of H1 H685P in combination with a chemotherapeutic agent exerted on tumor growth of 2.5×106tumor cells COLO 205 were implanted subcutaneously in the posterior part of the right side of 8-9-week-old male SCID mice. When tumors reached an average size of ~150 mm3(day 17 after implantation), mice were divided randomly into 4 groups (n=5 mice/group) and treated following�m follows: the first group was treated subcutaneously (sc) with 15 mg/kg hFc and intraperitoneally (ip) by using media 5-FU; the second group was treated subcutaneously (sc) with 15 mg/kg H1 H685P; the third group was treated ip with 75 mg/kg 5-FU; the fourth group was treated with a combination of 15 mg/kg H1 H685P sc with 75 mg/kg 5-FU ip. Mice received a total of three treatments, which are administered every 3-4 days. Tumor size was measured twice a week during the entire course of the experiment. Mean values (mean +/- standard deviation) of tumor growth from the beginning of treatment and up to 38 days was calculated for each group. The percentage reduction of tumor growth was calculated based on comparison with the control group. The results are presented in Table 33.

Table 33
ProcessingThe average tumor growth (mm3)% reduction of tumor growth
Fc-protein + 5-FU media574±110-
H1H685P405±8029
5-FU313±6045
H1H685P + 5-FU175±7870

The results of the experimental�that demonstrate the combination of H1 H685P and 5-FU caused a stronger reduction of tumor growth of each one of the agents, in their individual introduction.

Example 12. Anti-Ang-2 antibodies weaken ocular angiogenesis in vivo

In this Example evaluated the effects of anti-Ang-2 antibodies exerted on the vascularization of the retina in a mouse model.

In one set of experiments used the wild type mice. In another set of used mice expressing human Ang-2 instead of mouse Ang-2 wild-type (designated as "mouse hu-Ang-2").

Mice at the age of two days (P2) were injected subcutaneously or via Fc or control using the selected anti-Ang-2 antibodies in doses of 12.5 mg/kg Three days later (P5), the mice were sacrificed and the eyeballs were removed and fixed in 4% PFA for 30 minutes. The retina was sectionlevel, stained with lectin-1 from Griffonia simplicifolia for 3 hours or over night at 4°C for visualization of the vascular system, and mounted on glass slides. Images were obtained using a microscope camera Nikon Eclipse 80i and analyzed using the software Adobe Photoshop CS3, Fovea 4.0 and Scion 1.63.

Measured retinal area covered surface of the vascular system, and used to read the activity of the antibody. Reducing the size of the vascular region in mice treated with antibodies compared to mice, treated Fc-controls, are presented in Table 34. The percentage reduction of the vascular region reflects the anti-angiogenic effect of the antibody. (N/D=not determined).

Table 34
% Reduction in vascular area relative to Fc-control
AntibodyMouse wild-typeMouse hAng-2
H1H685P39,7N/D
H1H690P30,7of 41.5
H1H691P30,4N/D
H1H696Pto 31.1N/D
H1H724N32,233,2
H1H744N35,850,5
Control I (Ab)26,935,6

As shown in this Example, the selected anti-Ang-2 antibodies of the present invention essentially ing�was biovale ocular angiogenesis in vivo, thus reflecting the likely anti-angiogenic potential of these antibodies in other therapeutic contexts.

Example 13. Amino acids Ang-2, are important for antibody binding

To further characterize the binding between hAng2 and anti-hAng2 mAb of the invention, generated seven variants of proteins hAng2-FD-mFc, each of which contained a single point mutation. Selection of amino acids for mutations was based on the difference in the sequence between hAng-2 and hAng-1 in the plot, which interacts with hTie-2 (Figure 1). Specifically, amino acids inside fibrinogenolysis domain (FD) Ang-2, which are believed to interact with Tie-2, based on the analysis of the crystal structure, but which differ from the corresponding amino acids in Ang-1, were subjected to individual mutation corresponding to residues hAng-1. The results of this example identify amino acid residues hAng-2, interact with antibodies, preferring binding to Ang-2. That is, if a particular residue (or residues) hAng-2 change to an appropriate balance of hAng-1, binding of antibodies that prefer binding to Ang-2, essentially decreases, it can be concluded that the antibody interacts with this particular residue(s) hAng-2.

In this experiment, each of the seven mutant proteins hAng-2FD-mFc were taken (-147-283 EN) on the surface of the anti-mising�-Fc, created by direct chemical condensation with chip BIACORE™. Then each Ang-2 antibody (or Patiala, depending on the case) in the amount of 100 nm were injected over the captured surface protein hAng-2FD with mFc-tag a flow rate of 50 μl/min for 180 sec, and the dissociation variant hAng2-FD-mFc and antibodies were monitored in real-time for 20 min at 25°C. the Results are summarized in Tables 35a-35d and the Figure 3.

Table 35a
The mutated amino acid[1]hAng-2H1H685PH1H744N
REKD(M)T½ (min)REKD(M)T½ (min)
WT[2]210,702,23 E-1119882133,98 E-11904
S-417-I127,653,05 E-111809127 5,12 E-111590
K-432-N152,681,40 E-1144681374,87 E-111690
I-434-M235,951.79 E-1136002133,18 E-112589
N-467-G152,25at 9.38 E-1267621397,72 E-111011
F-469-L101,161,38 E-0841801,95 E-10237
Y-475-H181,531,96 E-102892473,06 E-10136
S-480-P161,132,05 E-10289228 2,25 E-112129

hurdle of 48.80
Table 35b
The mutated amino acid[1]hAng-2Control I (Ab)Control II (Pb)
REKD(M)T½ (min)REKD(M)T½ (min)
WT[2]195,254,69 E-1054,3367,444,29 E-1039,86
S-417-I142,965,79 E-1032,8149,991,88 E-1036,38
K-432-N189,693,49 E-1051,7563,211,39 E-1042,34
I-434-M282,10a 4.64 E-1089,151,36 E-1057,09
N-467-G180,904,61 E-1044,6660,941,54 E-1046,97
F-469-L173.01 in1,05 E-0925,1346,732,40 E-1036,20
Y-475-H170,051,15 E-081,8574,791,40 E-1054,12
S-480-P181,322,98 E-0913,3671,901.79 E-1045,45

Table 35c
The mutated amino acid[1]hAng-2Control III (Pb)Control V (Ab)
R� KD(M)T½ (min)REKD(M)T½ (min)
WT[2]80,332,07 E-11170,03214,487,97 E-1048,43
S-417-I57,135,31 E-11114,81126,452,40 E-09of 29.17
K-432-N79,222,88 E-11200,94149,148.48 to E-1075,59
I-434-M116,222.15 E-1062,77214,752,23 E-0931,76
N-467-G74,648,90 E-11109,07146,771,11 E-0955,66
F-469-L72,66to 2.74 E-1066,11131,961,37 E-081,46
Y-475-H76,216,87 E-094,11260,932,66 E-1093,22
S-480-P77,932,78 E-0911,69177,103,47 E-0910,33

Table 35d
The mutated amino acid[1]hAng-2Negative control
(outside antibody)
REKD(M)T½ (min)
WT[2]0,81N/BN/B
S-417-I-1,21N/BN/B
K-432-N -0,38N/BN/B
I-434-M-1,31N/BN/B
N-467-G-1,09N/BN/B
F-469-L0,32N/BN/B
Y-475-Hto -0.20N/BN/B
S-480-P-0,52N/BN/B
[1] the Numbering of amino acids is based on the numbering of amino acids in SEQ ID NO:518.
[2] WT=design Ang-2FD-mFc wild-type.
N/B=binding was observed.

For the purposes of the present invention, it is believed that anti-Ang-2 antibody interacts with a specific amino acid residue of Ang-2 in the case when the residue is subjected to mutagenesis to an appropriate balance of Ang-1, then T1/2dissociation is at least 5-times less than T1/2dissociation observed for the wild-type design, the experimental conditions used in this PR�least. In view of this definition, the antibody H1 H685P, apparently, is unique among the tested antibodies in that it interacts with F469, Y475 and S480. Since H1 H685P is also unique because of its strong preferential binding to Ang-2 compared with Ang-1, we can conclude that F469, Y475 S480 and include epitope, which allows immunological differences between Ang-2 and Ang-1. Other antibodies/Patiala tested in this experiment, apparently, interact mostly with one or two of these residues; T. e., H1H744N and Control I interact with Y475; Control III interacts with Y475 and S480; and Control V interacts with F469. Interestingly, Control II, which, as has been demonstrated, blocks the binding of both Ang-1 and Ang-2, Tie-2 with equal force, it does not interact with any of Ang-2-specific amino acids identified in this experiment.

The scope of the present invention is not limited to specific embodiments described herein. In fact, on the basis of the above description, the person skilled in the art will be apparent various modifications of the invention in addition to those described herein. It is implied that such modifications are within the framework of the attached claims.

1. Selected human antibody or�, the antigen-binding fragment, which specific binds to human angiopoietin-2 (hAng-2), but in essence is not associated with the hAng-1, where the antibody or antigen-binding fragment comprises hypervariable sites (CDR) of the variable segment of the heavy chain (HCVR) having the amino acid sequence of SEQ ID NO:18, and sections of the CDR of the variable segment light chain (LCVR) having the amino acid sequence of SEQ ID NO:20.

2. The selected antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment binds to an epitope on hAng-2 (SEQ ID NO:518), comprising at least one amino acid selected from F-469, Y-475 and S-480.

3. The selected antibody or antigen-binding fragment according to claim 2, where the antibody or antigen-binding fragment binds to an epitope on hAng-2 comprising amino acids F-469, Y-475 and S-480.

4. The selected antibody or antigen-binding fragment according to any of claims.1-3, where the antibody or antigen-binding fragment binds to hAng-2 with KDconstituting less than about 80 PM, less than about 40 PM, less than about 20 PM or less than about 10 PM according to the measurements using analysis of surface plasmon resonance.

5. The selected antibody or antigen-binding fragment according to any of claims.1-3, where the antibody or antigen-binding fragment binds to hAng-1 with KDconstituting �more than about 1 nm, more than about 10 nm, more than about 50 nm or more than about 100 nm according to the measurements using analysis of surface plasmon resonance.

6. The selected antibody or antigen-binding fragment according to any of claims.1-3, where the antibody or antigen-binding fragment does not show any binding with hAng-1 when tested using analysis of surface plasmon resonance.

7. The selected antibody or antigen-binding fragment according to claim 2, where the antibody or antigen-binding fragment: (a) binds to an epitope on hAng-2 comprising amino acids F-469, Y-475 and S-480; (b) binds to hAng-2 with KDconstituting less than about 80 PM according to the measurements using analysis of surface plasmon resonance; and (c) does not show any binding with hAng-1 when tested using analysis of surface plasmon resonance.

8. The selected antibody or antigen-binding fragment according to claims.1-3 or 7, where the antibody or antigen-binding fragment comprises CDR-1 heavy chain (HCDR1), having the amino acid sequence of SEQ ID NO:4, HCDR-2 having the amino acid sequence of SEQ ID NO:6, HCDR-3 having the amino acid sequence of SEQ ID NO:8, CDR-1 light chain (LCDR-1) having the amino acid sequence of SEQ ID NO:12, LCDR-2 having the amino acid sequence of SEQ ID NO:14, and LCDR-3, having�th amino acid sequence of SEQ ID NO:16.

9. The selected antibody or antigen-binding fragment according to claim 8, wherein the antibody or antigen-binding fragment comprises HCVR having the amino acid sequence of SEQ ID NO:18, and a LCVR having the amino acid sequence of SEQ ID NO:20.

10. The selected antibody or antigen-binding fragment according to any of claims.1-3 or 7, where the antibody or antigen-binding fragment inhibits the binding of hAng-2 c hTie-2, but essentially blocks the binding of hAng-2 c hTie-2.

11. Pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment according to any of claims.1-3 or 7 and a pharmaceutically acceptable carrier or diluent, where the pharmaceutical composition is used in the treatment of a patient having a tumor, hypertension, diabetes, asthma, sepsis, kidney disease, Eden or ocular disease associated with angiogenesis.

12. Pharmaceutical composition according to claim 11, further comprising the antagonist of the growth factor vascular endothelial (VEGF).

13. Pharmaceutical composition according to claim 12, wherein the VEGF antagonist is selected from anti-VEGF antibody, a small molecule kinase inhibitor of VEGF receptor and VEGF-inhibiting fused protein.

14. The selected antibody or antigen-binding fragment according to any of claims.1-3 or 7 for use in treating a patient having a tumor.

15. The selected antibody or �antigen-binding fragment according to any of claims.1-3 or 7 for use in treating a patient, having hypertension, diabetes, asthma, sepsis, kidney disease, Eden or ocular disease associated with angiogenesis.

16. A dedicated antibody or antigen-binding fragment according to any of claims.1-3, or 7 in the manufacture of a medicament for use in treating a patient having a tumor.

17. A dedicated antibody or antigen-binding fragment according to any of claims.1-3, or 7 in the manufacture of a medicament for use in treating a patient with hypertension, diabetes, asthma, sepsis, kidney disease, Eden or ocular disease associated with angiogenesis.

18. A method of treating tumors, comprising administering in need of such treatment the patient selected antibody or antigen-binding fragment according to any of claims.1-3 or 7.

19. A method of treating diseases or disorders selected from hypertension, diabetes, asthma, sepsis, kidney disease, edema or ocular disease, comprising administering in need of such treatment the patient selected antibody or antigen-binding fragment according to any of claims.1-3 or 7.



 

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FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. What is presented is an antibody representing a neutralising VEGFR-2/KDR antibody with its hypervariable regions being identical to the hypervariable regions of TTAC 0001 of VEGFR-2/KDR antibody fused with a binding domain of angiopoietin 2 which is Tie-2 ligand for treating cancer by angiogenesis inhibition. A DNA coding the above antibody, an expression vector containing the above DNA, and a CHO host cell transformed by the above vector for preparing the antibody are also described. What is also presented is a method for preparing the antibody involving: host cell incubation, and the antibody recovery from a culture fluid of CHO cell. What is described is a pharmaceutical composition for treating an angiogenesis-related disease, containing an effective amount of the above antibody and at least one pharmaceutically acceptable carrier.

EFFECT: invention enables preparing the VEGFR-2/KDR antibody fused with the binding domain of angiopoietin 2 which may be used for effective treatment of a disease related to excessive angiogenesis.

13 cl, 10 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: invention relates to the field of immunology, namely to enzyme-immunoassay, in particular to a method of detecting forms of vascular endothelial growth factor (VEGF) with a size more than 110 amino acids in a biological sample. The method includes the following stages: contact and incubation of the biological sample with an uptake reagent, immobilised on a solid substrate, where the uptake reagent contains a monoclonal antibody, which recognises and specifically binds with residues, in quantity more than 110, from human VEGF; separation of the biological sample from the immobilised uptake reagents; contact of the immobilised molecular complex of the reagent of the uptake-target with detected antibody, which binds with VEGF domains, responsible for binding with KDR and/or FLT1 receptor, or which binds with an epitope in VEGF1-110; measurement of the level of VEGF110+, bound with reagents of the uptake, with application of means of detection for the detected antibody. Set of immune assay reagents for detection of VEGF110+ forms in the biological sample. An antibody 5C3, obtained from hybridoma 5C3.1.1 with a depositary number PTA-7737, with the said antibody 5C3 binding VEGF110+ forms, including VEGF121+. Hybridoma 5C3.1.1, deposited in ATCC with the depositary number PTA-7737, to obtain the monoclonal antibody 5C3.

EFFECT: application of the claimed invention makes it possible to increase accuracy of detecting VEGF isoforms, which must not include isoform VEGF110 and must obligatory include isoform VEGF121.

25 cl, 3 dwg, 2 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to immunology. Described are antibodies against VEGF, one on which contains complementary regions with amino acid sequences SEQ ID NO:1, 2, 3, 4, 6 and 7, another contains complementary regions with amino acid sequences SEQ ID NO:1, 2, 3, 5, 6 and 7, disclosed in description. Also described are polynucleotides, coding said antibodies; espression vectors, containing said polynucleotides, and host cells, intended for obtaining antibodies in accordance with the claimed invention. Claimed is method of obtaining antibodies against VEGF, which includes expression of vector in host cell and separation of antibody. Disclosed is method of obtaining immunocongugate of antibody against VEGF, which includes conjugation of antibody with drug or cytotoxic agent. Described is method of VEGF identification, which includes identification of complex VEGF-antibody against VEGF in biological sample. In addition, described are compositions for treatment of VEGF-associated disease, one of which contains efficient quantity of antibody against VEGF, and another - efficient quantity of polynucleotide, coding said antibody. Also disclosed are methods of: 1) treating tumour, cancer or VEGF-associated cell proliferative disease; 2) inhibition if angiogenesis in subject and 3) inhibition of vascular permeability; consisting in introduction to subject of efficient quantity of antibody against VEGF in accordance with claimed invention.

EFFECT: invention makes it possible to obtain antibodies against VEGF and apply them for treatment of VEGF-associated diseases.

41 cl, 16 dwg, 2 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers immunology and medicine. What is presented is an antibody for the recovery of the central nervous system, comprising an antigen-binding site that specifically binds to human Nogo A polypeptide or human NiG described by (SEQ ID NO: 2 and 3, respectively, presented in the description), wherein the antigen-binding site comprises: CDR-H1-6A3 (SEQ ID NO:8), CDR-H2-6A3 (SEQ ID NO:9) and CDR-H3-6A3 (SEQ ID NO:10); and CDR-L1-6A3 (SEQ ID NO:11), CDR-L2-6A3 (SEQ ID NO:12) and CDR-L3-6A3 (SEQ ID NO:13). There are also described a polynucleotide coding the above antibody; an expression vector comprising the above polynucleotide; and a host cell specified in bacterium, yeast or mammalian cell line comprising myeloma, hybridoma, or immortalised B-cell for producing the antibody according to the present invention. A pharmaceutical composition for the CNS recovery comprising an effective amount of the above antibody mixed with at least one acceptable carrier or solvent is also described. Using the polynucleotide, the expression vector or the host cell for the above pharmaceutical composition is also described. The invention enables producing the human Nogo A or NiG antibody effective in treating CNS injuries.

EFFECT: what is presented is a method for producing the above antibody involving the polynucleotide or vector expression in the host cell.

16 cl, 11 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: antibody against angiopoietin-2 (Ang-2) is presented; with that, the above antibody includes a variable area of heavy chain SEQ ID No: 7 (MEDI5) and a variable area of light chain, which has been chosen from SEQ ID No:3 (MEDI1); SEQ ID No:4 (MEDI2); SEQ ID No:5 (MEDI3); and SEQ ID No:6 (MEDI4). Nucleic acid coding the above antibody has been developed. The invention describes a composition for being used in inhibition of angiogenesis of cancer tumour of a mammal, which requires it; the above composition contains the extracted antibody in therapeutically effective amount and an excipient.

EFFECT: proposed variant antibodies have increased stability at storage, out of which they have been obtained, and are capable of competing with Tie-2 for binding with Ang-2.

12 cl, 17 dwg, 15 tbl, 13 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes an antibody that specifically binds heparin-binding EGF-like growth factor (HB-EGF) and its antigen-binding fragment. Invention describes a nucleic acid molecule, an expressing vector, a host cell and a method for obtaining an antibody or its antigen-binding fragment, as well as use of antibody or its antigen-binding fragment for obtaining pharmaceutical composition for diagnostics, prevention or treatment of hyperproliferation disease, methods and sets for diagnostics and prevention or treatment of the state associated with HB-EGF expression. This invention can be further found in therapy of diseases determined with or related to HB-EGF expression.

EFFECT: improving efficiency of composition and treatment method.

34 cl, 43 dwg, 28 ex, 12 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to compound of formula:

double line between N and C represents double bond, X is absent, a Y stands for H; W stands for C=O; each of R1, R2, R3, R4 stands for H; R5 is selected from groups OR15, where R15 has the same determination as R; optionally R5 stands for binding group or is selected from groups: polypyrrole, polyindolyl, polyimidazolyle, polypyrrole-imidazolyle, polypyrrole-indolyl or polyimidazole-indolyl unit, optionally bound to binding groups; R6 stands for OR or optionally, R6 stands for binding group; Z is selected from groups (CH2)n, where n stands for 1, 2 or 3, CR15R16, where each of R15 and R16 independently stands for H or linear alkyl, having from 1 to 10 carbon atoms; R stands for H or linear or branched alkyl, having from 1 to 3 carbon atoms, optionally substituted with group -COR11; R11 stands for H or -OR14; and R14 stands for H or linear or branched alkyl, having from 1 to 3 carbon atoms; each of R1, R2, R3, R4, R1', R2', R3' and R4' stands for H, optionally any of R1, R2, R3, R4, R1', R2', R3' or R4' stands for binding group, Z is selected from groups (CH2)n, where n stands for 1, 2 or 3; R6 stands for OR, or optionally R6 stands for binding group; A and A′ stand for O, D and D', similar or different, and independently represent linear or branched alkyls, having from 1 to 10 carbon atoms; L is absent or stands for phenyl group, where said phenyl group representing L, is optionally substituted, where substituent is represented by binding group or is selected from OR7, NR8R9, NRCOR' or OCOR11; R and R' independently represent H or linear or branched alkyl, having from 1 to 10 carbon atoms, optionally substituted with halogen or group -COR7; R7, R8, R9 and R11 independently represent H or linear or branched alkyl, having from 1 to 10 carbon atoms, or polyethylene glycol unit (-OCH2CH2)n, where n stands for integer number from 1 to 10; on condition that said compound has not more than one binding group, which provides bond with cell-binding agent due to covalent bond, which possess anti-proliferative activity.

EFFECT: obtaining novel compounds.

24 cl, 59 dwg, 9 tbl, 40 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to organic chemistry, namely to a heterocyclic compound of formula and to its pharmaceutically acceptable salts, stereoisomers and isomers, wherein T: N, U: N, X: CR3 and Y: N; or T: CR6, U: CR4, X: CR3 and Y: N; or T: CR6, U: N, X: NR3 and Y: C; or T: O, U: N, X: CR3 and Y: C; or T: NR6, U: N, X: CR3 and Y: C; and R1, R2 and R5: H, heteroaryl substituted by 1-2 substitutes; or T: CR6, U: N, X: CR3 and Y: N; or T: N, U: CR4, X: CR3 and Y: N; and R1 and R2: H, heteroaryl substituted by 1-2 substitutes; R5: heteroaryl substituted by 1-2 substitutes; R3: H, bridging (C7-C10)cycloalkyl; (C1-C8)alkyl optionally substituted by 1 substitute; (C3-C10)cycloalkyl optionally substituted by 1 substitute; (C6-C8)cycloalkenyl substituted by two (C1-C6)alkyl; (C6)aryl optionally substituted by 1-2 substitutes; heteroaryl optionally substituted by (C1-C6)alkyl; heterocyclyl optionally substituted by (C1-C6)alkyl or heteroaryl; or R3: -A-D-E-G, wherein: A: a bond or (C1-C6)alkylene; D : (C1-C2)alkylene optionally substituted by (C1-C6)alkyl, bridging (C6-C10)cycloalkylene optionally substituted by (C1-C6)alkyl, (C3-C10)cycloalkylene optionally substituted by 1-2 substitutes, (C4-C6)cycloalkenylene optionally substituted by (C1-C6)alkyl, (C6)arylene, heteroarylene or heterocyclylene optionally substituted by one (C1-C6)alkyl; E: a bond, -Re-, -Re-C(O)-Re-, -Re-C(O)O-Re-, -Re-O-Re-, -Re-S(O)2-Re-, -Re-N(Ra)-Re-, -Re-N(Ra)C(O)-Re-, -Re-C(O)N(Ra)Re-, -Re-N(Ra)C(O)ORe- or -Re-N(Ra)S(O)2-Re-; wherein in each case, E is bound to either a carbon atom, or a nitrogen atom in D; G: H, -N(Ra)(Rb), halogen, -ORa, S(O)2Ra, -CN, -C(O)N(Ra)(Rb), -N(Ra)C(O)Rb, -C(O)Ra, -CF3, N(Ra)S(O)2Rb, -(C1-C6)alkyl optionally substituted by 1-3 substitutes; -(C3-C6)cycloalkyl optionally substituted by CN; -heteroaryl optionally substituted by 1-2 halogens, CN, -C(O)NH2 or -CF3; -heterocyclyl optionally substituted by 1-5 substitutes, -(C6-C10)aryl optionally substituted by 1-3 substitutes; wherein in a fragment containing -N(Ra)(Rb), nitrogen, Ra and Rb can form a ring so that -N(Ra)(Rb) represents (C3-C6)heterocyclyl optionally substituted by 1 substitute, wherein said (C3-C6)heterocyclyl is bound through nitrogen; R4 and R6: H, (C1-C4)alkyl optionally substituted by -OH, -COOH; (C3-C8)cycloalkyl, phenyl, optionally substituted by -SO2CH3 or -NHSO2CH3, halogen or -J-L-M-Q; wherein: J: (C2-C6)alkenylene; L: a bond; M: a bond; Q: -C(O)ORa; Ra and Rb: H, (C1-C4)alkyl optionally substituted by cyano, -CF3 or cyclopropane; (C6)aryl optionally substituted by halogen or -O(C1-C4)alkyl; and Re: a bond, (C1-C4)alkylene or (C3)cycloalkylene. Besides, the invention refers to specific compounds, a pharmaceutical composition based on the compound of formula I, using the compound of formula I for treating and using the compounds of formulas 2-6

for preparing the compound of formula I.

EFFECT: prepared are the new compounds effective in treating a condition mediated by Jak1, Jak3 or Syk protein kinase activity.

51 cl, 34 tbl, 44 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to crystalline citrate of 9E-15-(2-pyrrolidin-1-yl-ethoxy)-7,12,25-trioxa-19,21,24-triazatetracyclo[18.3.1.1(2,5).1(14,18)]hexacosa-1(24),2,4,9,14,16,18(26),20,22-nonaene (Compound I), a based pharmaceutical composition, a method of treating using the compound and using it for treating proliferative diseases.

EFFECT: compound

exists in the single polymorphic form and has the improved properties of stability; it is non-hygroscopic and insoluble in organic solvents.

15 cl, 8 tbl, 23 dwg, 12 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology. What is presented is a composition of an antibody for treating HER2-positive cancer containing an active antibody presented by an antibody characterised by the fact that it has a variable light and heavy chain domain, and its acidic variants, namely: glycosilated, deaminated variants, as well as a variant with a reduced disulphide bond, a syalylated variant and a irreducible variant. A number of acidic variants makes less than approximately 25%. There are described a pharmaceutical composition containing this composition, for treating HER2-positive cancer and a method for preparing the composition involving the evaluation of the acidic variants and verification of the fact that their number makes at least than approximately 25%.

EFFECT: using the invention provides the new composition, wherein the antibody and its acidic variants have the pharmacokinetic parameters that can find application in treating HER2-positive cancer.

14 cl, 17 dwg, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry and represents using sulphated hyaluronic acid for preparing a therapeutic agent for local administration for treating inflammatory/irritating skin diseases specified in dermatitis, atopic dermatitis, photocontact dermatitis, rash, vitiligo, eczema, psoriasis, all skin irritations related to activation of anti-inflammatory cytokines, such as IL-1, IL-2, IL-7, IL-8, IL-9 and TNF, wherein hyaluronic acid has a molecular weight falling within the ranges of 10000 D to 50000 D, 150000 D to 250000 D and 500000 D to 750000 D, and a sulphatation degree equal to 1.

EFFECT: invention provides stimulating the immune system protein synthesis for eliciting the immune response.

8 cl, 33 ex, 15 dwg, 3 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to chemotherapy of cancer and represents a composition for hepatic cancer in individuals and containing a complex of formula [M(RCS3)2(RCS2)], wherein M represents 188Re having an activity of more than 3.7 GBq and a lipophilic organic phase emulsified with a water phase; as well as a method for preparing the above composition.

EFFECT: invention provides higher stability and efficacy of the composition for treating hepatic cancer in the individuals.

17 cl, 1 dwg, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine, namely to oncology, and can be used for treatment of cerebral glioma. For this purpose combination of cannabinoids-tetrahydrocannabinol (THC) and cannabidiol (CBD) and non-cannabinoid chemotherapeutic agent is claimed. THC and CBD ratio constitutes from 5:1 to 1:5.

EFFECT: combination of anti-tumour medications provides efficient reduction of brain tumour volume due to selective death of transformed cells.

14 cl, 2 tbl, 8 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to specific compounds or to their therapeutically acceptable salt presented in the patent claim and representing sulphonyl benzamide derivatives. The invention also refers to a pharmaceutical composition inhibiting the activity of anti-apoptotic proteins of the family Bcl-2, containing an excipient and an effective amount of a specific sulphonyl benzamide derivative.

EFFECT: sulphonyl benzamide derivatives inhibiting the activity of anti-apoptotic Bcl-2 proteins.

2 cl, 3 tbl, 558 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a pharmaceutical composition for treating bladder cancer. The above composition contains an effective amount of valrubicin and dimethyl sulphoxide, as well as polyethoxylated castor oil or one or more substances specified in trimethyl chitosan, mono-N-carboxymethyl chitosan, N-diethylmethyl chitosan, sodium caprylate, cytochalasin B, IL-1, polycarbophil, Carbopol 934P, N-sulphate-N,O-carboxymethyl chitosan, Zonula occludens toxin, 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine, and represents a dosage form for intra-bladder administration by instillation. The invention also refers to liposomal pharmaceutical compositions containing valrubicin, and methods of treating bladder cancer involving administering the above compositions.

EFFECT: invention reduces bladder irritation and increases the clinical effectiveness in bladder cancer.

12 cl, 3 dwg, 3 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: cyclophosphane 200 mg/kg is injected into experimental animals carrying developed ascytis. Then, a DNA preparation is administered intraperitoneally every hour after administering cyclophosphane for 12 hours. The DNA preparation consists of a mixture of preparations of fragmented human DNA and cross-linked human DNA processed in nitrogen mustard.

EFFECT: method provides the effective eradication of cancer stem cells by the synergism of the cytostatic and DNA preparation.

3 cl, 5 dwg

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to ophthalmopathy, and can be used in treating endothelial-epithelial corneal dystrophy. That is ensured by de-epithelisation, 12-o'clock corneal incision and splitting, administration of a biologically active substance into a stromal pocket of the cornea. The biologically active substance is poludan-activated autoplasma. Before the epithelisation is completed, a soft contact lens is placed on the cornea. The operation is followed by instillations of ciprofloxacin, diclofenac 6 times a day and infusions of cornegel.

EFFECT: method provides reducing the intraoperative injuries and the length of patients' treatment, including by local (intrastromal) autocytokinotherapy.

2 ex

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