High-affinity human anti-pcsk9 antibodies

FIELD: medicine.

SUBSTANCE: presented invention refers to immunology. Disclosed are versions of a human antibody or an antigen-binding fragment of the human antibody, which specifically bind and inhibit human proprotein convertase subtilisin/kexin type 9 (hPCSK9). Each version is characterised by the fact that it contains 6 CDRs of heavy and light chains. Described are: an isolated coding nucleic acid, and expression vector and a method for producing the antibody with the use of the above vector. Presented is a pharmaceutical formulation based on the antibody for treating a disease or a condition which can be relieved, improved, suppressed or prevented by means of the anti-PCSK9 antibody.

EFFECT: using the invention provides the new versions of the antibodies able to reduce serum low-density lipoprotein cholesterol accompanied by no considerable changes or no effect on the hepatic function according to measured alanine-aminotransferase and aspartate-aminotransferase.

8 cl, 14 dwg, 28 tbl, 17 ex

 

Field of the invention

The present invention relates to human antibodies and antigen-binding fragments of human antibodies that specifically bind human paraproteinemias of subtilisin/Kexin type 9 (PCSK9), and to therapeutic methods of using these antibodies.

Prior art

Paraproteinemias of subtilisin/Kexin type 9 (PCSK9) refers to propertycommercial included in the subfamily of proteinases To a family of secretory subtilase. The encoded protein is synthesized in soluble form known as a zymogen that undergoes autocatalytic intramolecular transformation in the endoplasmic reticulum. Available evidence suggests that PCSK9 promotes LDL cholesterol in plasma by stimulating the collapse of the LDL receptor, which mediates the endocytosis of LDL in the liver, which is the main channel of LDL clearance from circulation. The structure of the PCSK9 protein indicates the presence of a signal sequence followed by prodomain, catalytic domain containing conservative triad residues (D186, H226 and S386) and C-terminal domain. It is synthesized in the form of a soluble precursor of 74 kDa, which undergoes autocatalytic decay in AR with the formation of predomina 14 kDa and a catalytic fragment of 60 kDa. Was demonstrated the necessary�nce the presence of autocatalytic activity for secretion. After the collapse of prodomain remains tightly associated with the catalytic domain.

Antibodies to PCSK9 are described, for example, in applications WO 2008/057457, WO 2008/057458, WO 2008/057459, WO 2008/063382, WO 2008/125623 patent US 2008/0008697.

BRIEF description of the INVENTION

In the first aspect of the invention are fully human monoclonal antibodies (mAb) and their antigen-binding fragments that specifically bind and neutralize the activity of human PCSK9 (hPCSK9).

In one implementation of the present invention includes an antibody or antigen-binding fragment of an antibody which specifically binds hPCSK9 and is characterized by at least one of the following features:

(i) the ability to reduce total serum cholesterol by no less than 25-35% and maintain a reduced level for a period of not less than 24-day period compared with the level before the introduction, the preferred reduction in total cholesterol level is not less than approximately 30-40%;

(ii) the ability to reduce serum LDL cholesterol by no less than approximately 65-80% and maintain a reduced level for a period of not less than 24-day period compared with the level prior to the introduction;

(iii) the ability to reduce the level of serum triglycerides of not less than approximately 25-40% compared with the level prior to the introduction;

(iv) don't bring� to decrease the level of serum HDL cholesterol or reduces serum HDL cholesterol no more than 5% compared with the level prior to the introduction.

In one implementation of the present invention includes an antibody or antigen-binding fragment of an antibody which specifically binds hPCSK9 and is characterized by at least one of the following features:

(i) the ability to reduce serum LDL cholesterol by no less than approximately 40-70% and maintain a reduced level for a period of not less than 60-day period compared with the level prior to the introduction;

(ii) the ability to reduce the level of serum triglycerides of not less than approximately 25-40% compared with the level prior to the introduction;

(iii) does not reduce serum HDL cholesterol or reduces serum HDL cholesterol no more than 5% compared with the level prior to the introduction.

In one implementation, the antibody or a fragment thereof characterized as binding an epitope comprising amino acid residue 238 from hPCSK9 (SEQ ID no: 755). In one more specific implementation of the antibody or a fragment thereof bind an epitope that includes one or more amino acid residues 238, 153, 159 and 343 of hPCSK9 (SEQ ID no: 755). In a more specific implementation of the antibody or a fragment thereof characterized as binding an epitope not containing the amino acid residue at positions 192, 194, 197 and/or 237 of the sequence SEQ ID no: 755.

In one implementation, the antibody or a fragment thereof described�Xia as a binding epitope, comprising amino acid residue 366 from hPCSK9 (SEQ ID no: 755). In one more specific implementation of the antibody or a fragment thereof bind an epitope that includes one or more amino acid residues 147, 366 and 380 of SEQ ID no: 755. In a more specific implementation of the antibody or a fragment thereof characterized as binding an epitope not containing the amino acid residue at positions 215 and/or 238 of the sequence SEQ ID no: 755.

In one implementation, the antibody or a fragment thereof characterized as having enhanced binding affinity (KD) for hPCSK9 at pH 5.5 compared with KDat a pH of 7.4, as the results of measurement of surface plasmon resonance. In one specific implementation, the antibody or a fragment thereof is shown not less than 20-fold, at least 40-fold or at least 50-fold higher affinity to PCSK9 at acidic pH compared to neutral pH, as shown by the data of surface plasmon resonance.

In one implementation, the antibody or a fragment thereof characterized as not having an increased binding affinity for PCSK9 at acidic pH compared to neutral pH, as the results of measurement of surface plasmon resonance. In one specific implementation, the binding is weaker at acidic pH, and T1/2shorter than at neutral pH.

In another implementation�of the antibody or antigen-binding fragment bind to human PCSK9, man with GOF-mutation D374Y, cynomolgus macaque, rhesus monkeys, mice, rats and hamster.

In one implementation, the antibody or antigen-binding fragment associated with PCSK9 humans and apes, but not associated with PCSK9 mice, rats and hamster.

mAb can be full-sized (e.g., antibody IgG1 or IgG4) or can include only an antigen-binding portion (e.g., Fab fragment, F(ab')2or scFv) and can be modified with the aim to affect functionality, e.g., to remove residual effector functions (Reddy et al. (2000) J. Immunol. 164:1925-1933).

In one implementation of the present invention includes an antibody or antigen-binding fragment of an antibody containing the variable region of the heavy chain (HCVR) selected from the group of SEQ ID№: 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, 522, 526, 530, 546, 550, 554, 570, 574, 578, 594, 598, 602, 618, 622, 626, 642, 646, 650, 666, 670, 674, 690, 694, 698, 714, 718, 722, 738 and 742, or a substantially similar sequence having a sequence identity of at least 90%, at least 95%, at least 98% or at least 99%. In one implementation of the HCVR amino-acid sequence is selected from the group of SEQ ID№: 50, 66, 70, 74, 90, 94, 122, 138, 142, 218, 234, 238, 242, 258, 262, 314, 330 and 334. In a more specific implementation of the HCVR contains SEQ ID �: 90 or 218.

In one implementation, the antibody or a fragment thereof further contains a variable region light chain (LCVR) selected from the group of SEQ ID№: 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, 504, 514, 524, 528, 538, 548, 552, 562, 572, 576, 586, 596, 600, 610, 620, 624, 634, 644, 648, 658, 668, 672, 682, 692, 696, 706, 716, 720, 730, 740 and 744, or a substantially similar sequence having a sequence identity of at least 90%, at least 95%, at least 98% or at least 99%. In one implementation of the LCVR amino-acid sequence is selected from the group of SEQ ID№: 58, 68, 72, 82, 92, 96, 130, 140, 144, 226, 236, 240, 250, 260, 264, 322, 332 and 336. In a more specific implementation of the LCVR contains SEQ ID no: 92 or 226.

In a specific implementation, the antibody or a fragment thereof comprises a pair of sequences of the HCVR and LCVR (HCVR/LCVR) selected from the group including SEQ ID№: 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, 502/504, 506/514, 522/524, 526/528, 530/538, 546/548, 550/552, 554/562, 570/572, 574/576, 578/586, 594/596, 598/600, 602/610, 618/620, 622/624, 626/634, 642/644, 646/648, 650/658, 666/668, 670/672, 674/682, 690/692, 694/696, 698/706, 714/716, 718/720, 72/730, 738/740 and 742/744. In one implementation of the HCVR and LCVR are selected from pairs of amino acid sequences SEQ ID№: 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 122/130, 138/140, 142/144, 218/226, 234/236, 238/240, 242/250, 258/260, 262/264, 314/322, 330/332 and 334/336. In a more specific implementation of a pair HCVR/LCVR contains SEQ ID nos: 90/92 or 218/226.

In a second aspect, the present invention provides an antibody or antigen-binding fragment of an antibody containing the domain CDR3 (HCDR3) in the heavy chain selected from the group including SEQ ID№: 8, 32, 56, 80, 104, 128, 152, 176, 200, 224, 248, 272, 296, 320, 344, 368, 392, 416, 440, 464, 488, 512, 536, 560, 584, 608, 632, 656, 680, 704 and 728, or a substantially similar sequence having a sequence identity of at least 90%, at least 95%, at least 98% or at least 99%; and domain CDR3 (LCDR3) in the light chain selected from the group including SEQ ID№: 16, 40, 64, 88, 112, 136, 160, 184, 208, 232, 256, 280, 304, 328, 352, 376, 400, 424, 448, 472, 496, 520, 544, 568, 592, 616, 640, 664, 688, 712 and 736, or a substantially similar sequence having a sequence identity of at least 90%, at least 95%, at least 98% or at least 99%. In one implementation of a pair of sequences of the HCVR/LCVR are SEQ ID№: 56/64, 80/88, 128/136, 224/232, 248/256 or 320/328. In a more specific implementation of the HCDR3/LCDR3 contains SEQ ID no: 80/88 or 224/232.

In the further implementation of the present invention provides an antibody or a fragment thereof, further comprising a domain CDR1 (HCDR1) the heavy chain selected from the group including�sponding SEQ ID no: 4, 28, 52, 76, 100, 124, 148, 172, 196, 220, 244, 268, 292, 316, 340, 364, 388, 412, 436, 460, 484, 508, 532, 556, 580, 604, 628, 652, 676, 700 and 724, or a substantially similar sequence having a sequence identity of at least 90%, at least 95%, at least 98% or at least 99%; domain CDR2 (HCDR2) of the heavy chain, selected from the group including SEQ ID№: 6, 30, 54, 78, 102, 126, 150, 174, 198, 222, 246, 270, 294, 318, 342, 366, 390, 414, 438, 462, 486, 510, 534, 558, 582, 606, 630, 654, 678, 702 and 726, or a substantially similar sequence having a sequence identity of at least 90%, at least 95%, at least 98% or at least 99%; domain CDR1 (HCDR1) light chain selected from the group including SEQ ID№: 12, 36, 60, 84, 108, 132, 156, 180, 204, 228, 252, 276, 300, 324, 348, 372, 396, 420, 444, 468, 492, 516, 540, 564, 588, 612, 636, 660, 684, 708 and 732, or a substantially similar sequence having a sequence identity of at least 90%, at least 95%, at least 98% or at least 99%; and domain CDR2 (HCDR2) light chain selected from the group including SEQ ID№: 14, 38, 62, 86, 110, 134, 158, 182, 206, 230, 254, 278, 302, 326, 350, 374, 398, 422, 446, 470, 494, 518, 542, 566, 590, 614, 638, 662, 686, 710 and 734, or a substantially similar sequence having a sequence identity of at least 90%, at least 95%, at least 98% or at least 99%. In one implementation of the CDR sequences of the heavy and light chain are SEQ ID№: 52, 54, 56, 60, 62, 64; 76, 78, 80, 84, 86, 88; 124, 126, 128, 132, 134, 136; 220, 222, 224, 228, 230, 232; 244, 246, 248, 252, 254, 256; and 316, 318, 320, 324, 326, 328. In a more specific OS�westline CDR sequences of the heavy and light chain are SEQ ID no: 76, 78, 80, 84, 86, 88; or 220, 222, 224, 228, 230, 232.

In a related implementation of the present invention provides an antibody or antigen-binding antibody fragment that contains a domain that specifically binds hPCSK9, where the antibody or its fragment contains domains of the heavy and light chain CDR that are in pairs of sequences of the heavy and light chains selected from the group including SEQ ID№: 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, 502/504, 506/514, 522/524, 526/528, 530/538, 546/548, 550/552, 554/562, 570/572, 574/576, 578/586, 594/596, 598/600, 602/610, 618/620, 622/624, 626/634, 642/644, 646/648, 650/658, 666/668, 670/672, 674/682, 690/692, 694/696, 698/706, 714/716, 718/720, 722/730, 738/740 and 742/744. In one implementation of the CDR sequences contained within HCVR and LCVR selected from the pairs of amino acid sequences SEQ ID№: 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 122/130, 138/140, 142/144, 218/226, 234/236, 238/240, 242/250, 258/260, 262/264, 314/322, 330/332 and 334/336. In a more specific implementation of the CDR sequences contained within HCVR and LCVR selected from the pairs of amino acid sequences SEQ ID nos: 90/92 or 218/226.

In one implementation of the present invention provides the complete monoclonal antibody human, or er� the antigen-binding fragment, which specifically binds and neutralizes the activity of hPCSK9, wherein the antibody or its fragment demonstrates one or more of the following:

(i) the ability to reduce total serum cholesterol by no less than 25-35% and maintain a reduced level for a period of not less than 24-day period compared with the level before the introduction, the preferred reduction of total serum cholesterol is not less than approximately 30-40%;

(ii) the ability to reduce serum LDL cholesterol by no less than approximately 65-80% and maintain a reduced level for a period of not less than 24-day period compared with the level prior to the introduction;

(iii) the ability to reduce the level of serum triglycerides of not less than approximately 25-40% compared with the level prior to the introduction;

(iv) does not reduce serum HDL cholesterol or reduces serum HDL cholesterol no more than 5% compared with the level prior to the introduction;

(v) binds an epitope containing amino acid residue 238 of hPCSK9 (SEQ ID no: 755);

(vi) exhibits increased binding affinity (KD) for hPCSK9 at pH 5.5 compared with KDat a pH of 7.4, as shown by measurements using surface plasmon resonance, and this increased affinity is not less �eat 20-50-fold increase in affinity;

(vii) binds to human PCSK9, a person with GOF-mutation D374Y, cynomolgus macaque, rhesus monkeys, mice, rats and hamster;

(viii) contains the sequence of CDR3 of the heavy and light chain containing SEQ ID no: 80 and 88;

(ix) contains the CDR sequences of SEQ ID no: 90 and 92.

In one implementation of the present invention provides the complete monoclonal human antibody, or antigen-binding fragment that specifically binds and neutralizes the activity of hPCSK9, wherein the antibody or its fragment demonstrates one or more of the following:

(i) the ability to reduce serum LDL cholesterol by no less than approximately 40-70% and maintain a reduced level for a period of not less than 60-day period compared with the level prior to the introduction;

(ii) the ability to reduce the level of serum triglycerides of not less than approximately 25-40% compared with the level prior to the introduction;

(iii) does not reduce serum HDL cholesterol or reduces serum HDL cholesterol no more than 5% compared with the level prior to the introduction;

(iv) binds to an epitope containing amino acid residues 366 hPCSK9 (SEQ ID no: 755);

(v) exhibits increased binding affinity for PCSK9 at acidic pH compared to neutral pH, as the results of measurement of surface plasma�tion of resonance;

(vi) binds to PCSK9 humans and apes, but does not bind to PCSK9 mouse, rat and hamster;

(vii) contains a sequence of CDR3 of the heavy and light chain containing SEQ ID no: 224 and 232; and

(viii) contains the CDR sequences of SEQ ID no: 218 and 226.

In the third aspect of the present invention provides nucleic acid molecules that encode the antibody anti-PCSK9 or fragments thereof. The present invention also encompasses recombinant expression vectors carrying the nucleic acid of the present invention, and the host cell, in which are included such vectors, and methods for the production of antibody by culturing host cells under conditions which permit the production of antibodies and obtaining antibodies produced.

In one implementation of the present invention provides an antibody or a fragment thereof containing a HCVR encoded by a nucleic acid sequence selected from the group including SEQ ID№: 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, 521, 525, 529, 545, 549, 553, 569, 573, 577, 593, 597, 601, 617, 621, 625, 641, 645, 649, 665, 669, 673, 689, 693, 697, 713, 717, 721, 737 and 741, or a substantially similar sequence with a homology of at least 90%, at least 95%, at least 98% or at least 99%. In one implementation of the HCVR is encoded followers�the nost nucleic acid, selected from the group including SEQ ID№: 49, 65, 69, 73, 89, 93, 121, 137, 141, 217, 233, 237, 241, 257, 261, 313, 329 and 333. In a more specific implementation HCVR encoded by a nucleic acid sequence selected from the group including SEQ ID no: 89 and 217.

In one implementation, the antibody or a fragment thereof further comprises a LCVR encoded by a nucleic acid sequence selected from the group including SEQ ID№: 99, 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, 503, 513, 523, 527, 537, 547, 551, 561, 571, 575, 585, 595, 599, 609, 619, 623, 633, 643, 647, 657, 667, 671, 681, 691, 695, 705, 715, 719, 729, 739 and 743, or a substantially similar sequence with a homology of at least 90%, at least 95%, at least 98% or at least 99%. In one implementation of the LCVR is encoded by a nucleic acid sequence selected from the group including SEQ ID№: 57, 67, 71, 81, 91, 95, 129, 139, 143, 225, 235, 239, 249, 259, 263, 321, 331 and 335. In a more specific implementation LCVR encoded by a nucleic acid sequence selected from the group including SEQ ID no: 91 and 225.

In one implementation of the present invention provides an antibody or antigen-binding fragment of an antibody containing HCDR3 domain encoded by a nucleotide sequence selected from the group including SEQ ID№: 7, 31, 55, 79, 103, 127, 151, 175, 199, 223, 24, 271, 295, 319, 343, 367, 391, 415, 439, 463, 487, 511, 535, 559, 583, 607, 631, 655, 679, 703 and 727, or a substantially similar sequence with a homology of at least 90%, at least 95%, at least 98% or at least 99%; and a LCDR3 domain encoded by a nucleotide sequence selected from the group including SEQ ID№: 15, 39, 63, 87, 111, 135, 159, 183, 207, 231, 255, 279, 303, 327, 351, 375, 399, 423, 447, 471, 495, 519, 543, 567, 591, 615, 639, 663, 687, 711 and 735, or a substantially similar sequence with a homology of at least 90%, at least 95%, at least 98% or at least 99%. In one implementation, the sequence of HCDR3 and LCDR3 are encoded by nucleic acid sequence SEQ ID№: 55/63, 79/87, 127/135, 223/231, 247/255 and 319/327 respectively. In a more specific implementation of a pair of sequences HCDR3 and LCDR3 is encoded by the nucleic acid sequence SEQ ID no: 79/87 and 223/231.

In yet another implementation, the antibody or a fragment thereof further comprises a HCDR1 domain encoded by a nucleotide sequence selected from the group including SEQ ID№: 3, 27, 51, 75, 99, 123, 147, 171, 195, 219, 243, 267, 291, 315, 339, 363, 387, 411, 435, 459, 483, 507, 531, 555, 579, 603, 627, 651, 675, 699 and 723, or a substantially similar sequence with a homology of at least 90%, at least 95%, at least 98% or at least 99%; HCDR2 domain encoded by a nucleotide sequence selected from the group including SEQ ID№: 5, 29, 53, 77, 101, 125, 149, 173, 197, 221, 245, 269, 293, 317, 341, 365, 389, 413, 437, 461, 485, 509, 533, 557, 581, 605, 629, 653, 677, 701 and 725, or�significantly similar sequence with a homology of at least 90%, at least 95%, at least 98% or at least 99%; LCDR1 domain encoded by a nucleotide sequence selected from the group including SEQ ID№: 11, 35, 59, 83, 107, 131, 155, 179, 203, 227, 251, 275, 299, 323, 347, 371, 395, 419, 443, 467, 491, 515, 539, 563, 587, 611, 635, 659, 683, 707 and 731, or a substantially similar sequence with a homology of at least 90%, at least 95%, at least 98% or at least 99%; LCDR2 domain encoded by a nucleotide sequence selected from the group including SEQ ID№: 13, 37, 61, 85, 109, 133, 157, 181, 205, 229, 253, 277, 301, 325, 349, 373, 397, 421, 445, 469, 493, 517, 541, 565, 589, 613, 637, 661, 685, 709 and 733, or a substantially similar sequence with a homology of at least 90%, at least 95%, at least 98% or at least 99%. In one implementation of the CDR sequences of the heavy and light chains are encoded by nucleic acid sequences SEQ ID№: 51, 53, 55, 59, 61, 63; 75, 77, 79, 83, 85, 87; 123, 125, 127, 131, 133, 135; 219, 221, 223, 227, 229, 231; 243, 245, 247, 251, 253, 255; and 315, 317, 319, 323, 325, 327. In a more specific implementation of the CDR sequences of the heavy and light chains are encoded by nucleic acid sequences SEQ ID№: 75, 77, 79, 83, 85, 87; and 219, 221, 223, 227, 229, 231.

In a fourth aspect the present invention provides an isolated antibody or antigen-binding fragment of antibodies that specifically bind hPCSK9 containing HCDR3 and LCDR3, where HCDR3 contains the amino acid sequence of the formula X1-X2-X3-X4-X5 -X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20(SEQ ID no: 747), where X1- Ala, X2Is Arg or Lys, X3- Asp, X4- Ser or Ile, X5Is Asn or Val, X6Is Leu or Trp, X7Is Gly or Met, X8Is Asn or Val, X9Is Phe or Tyr, X10- Asp, X11Is Leu or Met, X12Is Asp or absent, X13- Tyr or absent, X14- Tyr or absent, X15- Tyr or absent, X16- Tyr or absent, X17Is Gly or absent, X18- Met or absent, X19Is Asp or absent, and X20- Val or absent; and LCDR3 contains the amino acid sequence of the formula X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID no: 750), where X1- Gln or Met, X2- Gln, X3- Tyr or Thr, X4Is Tyr or Leu, X5- Thr or Gln, X6- Thr, X7- Pro, X8Is Tyr or Leu and X9- Thr.

In yet another implementation, the antibody or a fragment thereof further comprising a HCDR1 sequence of the formula X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID no: 745), where X1- Gly, X2- Phe, X3- Thr, X4- Phe, X5- Ser or Asn, X6- Ser or Asn, X7- Tyr or H and X8- Ala or Trp; a HCDR2 sequence of the formula X1-X2-X3-X4-X5-X6-X7 -X8(SEQ ID no: 746), where X1- Ile, X2- Ser or Asn, X3- Gly or Gln, X4Is Asp or Ser, X5- Gly, X6- Ser or Gly, X7- Thr or Glu, and X8- Thr or Lys; a LCDR1 sequence of the formula X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12(SEQ ID no: 748), where X1- Gln, X2- Ser, X3- Val or Leu, X4- Leu, X5- H or Tyr, X6Is Arg or Ser, X7- Ser or Asn, X8Is Asn or Gly, X9- Asn, X10Is Arg or Asn, X11Is Asn or Tyr, and X12Is Phe or absent; a LCDR2 sequence of the formula X1-X2-X3(SEQ ID no: 749), where X1- Trp or Leu, X2- Ala or Gly, and X3- Ser. Fig.1 shows an alignment of sequences of the variable regions of heavy and light chain for 316P and 300N mAb.

In the fifth aspect of the present invention provides an antibody anti-human PCSK9 or antigen-binding fragment of an antibody containing the variable region of the heavy chain (HCVR) encoded by segments of the nucleotide sequence derived from the sequences of germ line VH, DHand JHand variable region light chain (LCVR) encoded by segments of the nucleotide sequence derived from the sequences of germ line VKand JKwhere embryonic lines are gene segment VH3-23, g�nny segment D H7-27, gene segment JH2, gene segment VK4-1 and gene segment JK2; or (b) gene segment VH3-7, gene segment, DH2-8, gene segment JH6, gene segment VK2-28 and gene segment JK4.

In the sixth aspect of the present invention provides an antibody or antigen-binding fragment that bind to PCSK9 protein of SEQ ID no: 755, where the binding of an antibody or its fragment with a variant PCSK9 protein is less than 50% of communication between the antibody or its fragment with a PCSK9 protein of SEQ ID no: 755. In a particular implementation of the antibody or a fragment thereof is contacted with a variant PCSK9 protein having a binding affinity (KDless than 50%, less than 60%, 70%, 80%, 90% or 95%, compared to binding to PCSK9 (SEQ ID no: 755).

In one implementation variant PCSK9 protein contains at least one mutation at position 238 of SEQ ID no: 755. In a more specific implementation of the mutation is D238R. In one implementation, the binding affinity of the antibody or antibody fragment for the variant PCSK9 protein is not less than 90% less in comparison with the native protein SEQ ID no: 755, where the variant protein contains a mutation at residue 238. In one implementation, the binding affinity of the antibody or antibody fragment for the variant PCSK9 protein is not less than 80% less in comparison with the native protein sequence SEQ ID no: 755, where vari�HT protein contains a mutation of one or more residues 153, 159, 238 and 343. In a more specific implementation of mutation is one of S153R, E159R, D238R and D343R.

In one implementation variant PCSK9 protein contains at least one mutation at position 366 of SEQ ID no: 755. In a more specific implementation of the mutation is E366K. In one implementation, the binding affinity of the antibody or antibody fragment for the variant PCSK9 protein is not less than 95% less in comparison with the native protein sequence SEQ ID no: 755, where the variant protein contains a mutation at residue 366. In one implementation, the binding affinity of the antibody or antibody fragment for the variant PCSK9 protein at least 70%, 80% or 90% less in comparison with the native protein sequence SEQ ID no: 755, where the variant protein contains a mutation of one or more residues 147, 366 and/or 380. In a more specific implementation of mutation is one of S147F, E366K and V380M.

The present invention includes antibody anti-PCSK9 with modified glycosylation structure. In some applications it may be useful to remove undesirable glycosylation sites, for example, to remove falosny fragment to enhance the function of antibody-dependent cell-induced cytotoxicity (ADCC) (see Shield et al. (2002) JBC 277:26733). In other applications can be carried out modification of galactosylceramide to change the complement-dependent cytotoxicity (CDC).

In CE�mom aspect of the present invention provides a pharmaceutical composition, comprising a recombinant human antibody or a fragment thereof which specifically binds hPCSK9 and a pharmaceutically acceptable carrier. In one implementation of the present invention provides a composition representing a combination of an antibody or antigen-binding fragment of the antibody of the present invention and a second therapeutic agent. The second therapeutic agent may be any agent that positive effect is combined with the antibody or its fragment of the present invention, for example, an agent that is able to cause impaired synthesis of cholesterol at the cellular level by inhibiting 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors, for example, from among such as cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, etc.; able to inhibit the capture of cholesterol and/or reabsorption of bile acids; able to increase the catabolism of lipoproteins (e.g., Niacin); and/or activators of the transcription factor LXR, which plays a role in removing cholesterol, for example, 22-hydroxycholesterol.

In the eighth aspect of the present invention provides methods for inhibiting the activity of hPCSK9 with an antibody anti-PCSK9 or an antigen-binding region of the antibody of the present invention, where therapeutic meth�dy provide for the introduction of a therapeutically effective amount of the pharmaceutical composition, containing the antibody or antigen-binding fragment of the antibody of the present invention. Subject to the treatment of the disorder is any disease or condition which is improved, facilitated, inhibited, or prevented by eliminating, inhibiting or reducing the activity of PCSK9. Specific groups that may be treated using therapeutic methods of the present invention, include patients who have shown the LDL apheresis, patients with mutations that activate PCSK9 (mutations with increased functions in the future, "GOF"), patients with heterozygous genetic hypercholesterolemia (heFH); patients with primary hypercholesterolemia who cannot tolerate statins or statin uncontrolled; and patients with risk of developing hypercholesterolemia who may receive preventive treatment. Other indications include dyslipidemia associated with secondary causes, such as diabetes II type, cholestatic liver disease (primary biliary cirrhosis), nephrotic syndrome, hypothyroidism, obesity; and prevention and treatment of atherosclerosis and cardiovascular diseases.

In a particular implementation of the method of the present invention, the anti-hPCSK9 antibody or antibody fragment of the present invention are used to nigeriaonline levels of total cholesterol, non-HDL cholesterol, LDL cholesterol and/or apolipoprotein B (apolipoprotein B100).

The antibody or antigen-binding fragment of the present invention can be used individually or in combination with a second agent, e.g., an inhibitor of HMG-CoA a reductase and/or other drugs that reduce the level of lipids.

Additional implementation include the use described above antibody or antigen-binding fragment of the antibody for alleviating or inhibiting a disease or condition mediated by PCSK9.

The present invention provides use of an antibody or antigen-binding fragment of an antibody in accordance with the description above in the manufacture of medicinal drugs used for alleviating or inhibiting a disease or condition mediated by PCSK9. In a particular implementation, in which PCSK9 mediated disease or condition is hypercholesterolemia, hyperlipidemia, HDL cholesterol apheresis, heterozygous familial hypercholesterolemia, intolerance to statins, the uncontrollability statins, the risk of developing hypercholesterolemia, dyslipidemia, cholestatic liver disease, nephrotic syndrome, hypothyroidism, obesity, atherosclerosis and cardiovascular diseases.

Other implementation will be obvious�DNAME when reading the following detailed description.

BRIEF DESCRIPTION of FIGURES

Fig.1. Table comparison of sequences of the variable regions of the heavy chain (A) and light chain (B) antibodies and CDR H1H316P and H1M300N.

Fig.2. The concentration of antibodies in the serum in time. 316P 5 mg/kg (□); 300N 5 mg/kg (σ); 316P 15 mg/kg (ν); 300N 15 mg/kg (●).

Fig.3. The level of total serum cholesterol as the percent change compared to control buffer. Control (T); 316P 5 mg/kg (ν); 300N 5 mg/kg (σ); 316P 15 mg/kg (□); 300N 15 mg/kg (Δ).

Fig.4. The level of serum LDL cholesterol in percentage changes compared with the control buffer. Control (T); 316P 5 mg/kg (ν); 300N 5 mg/kg (σ); 316P 15 mg/kg (□); 300N 15 mg/kg (Δ).

Fig.5. The level of serum LDL cholesterol, normalized to the control buffer. Control buffer (T); 316P 5 mg/kg (ν); 300N 5 mg/kg (σ); 316P 15 mg/kg (□); 300N 15 mg/kg (Δ).

Fig.6. The level of serum HDL cholesterol in percentage changes compared with the control buffer. Control (T); 316P 5 mg/kg (ν); 300N 5 mg/kg (σ); 316P 15 mg/kg (□); 300N 15 mg/kg (Δ).

Fig.7. The level of serum triglyceride in percentage changes compared with the control buffer. Control buffer (T); 316P 5 mg/kg (ν); 300N 5 mg/kg (σ); 316P 15 mg/kg (□); 300N 15 mg/kg (Δ).

Fig.8. The level of serum LDL cholesterol in the percent change from baseline after single subcutaneous administration. 316P 5 mg/kg (ν); 300N 5 mg/kg (●).

Fig.9. The concentration of na�Itel in serum over time after single subcutaneous injection. 316P 5 mg/kg (●); 300N 5 mg/kg (σ).

Fig.10. Western blot for the LDL receptor mice in total liver homogenates. Samples were taken 24 hours after administration of the FSB (strips 1-3), 5 mg/kg 316P (4-6 strips) or 5 mg/kg unspecific hPCSK9 MABs (bars 7-8) and 4 hours after administration of 1.2 mg/kg hPCSK9-mmh (all strips).

Fig.11. The effect of 316P on the level of serum LDL cholesterol in micePCSK9hu/hu. Control buffer ( ); 316P 1 mg/kg (); 316P 5 mg/kg (); 316P 10 mg/kg ().

Fig.12. Serum pharmacokinetic profile of anti-hPCSK9 MABs in mice C57BL/6. A single dose of control I mAb (λ) at 10 mg/kg; 316P (σ) at 10 mg/kg and 300N (ν) at 10 mg/kg.

Fig.13. Serum pharmacokinetic profile of anti-hPCSK9 MABs in heterozygous mice hPCSK9. A single dose of 10 mg/kg: Control I mAb (λ); 316P (σ) and 300N (ν).

Fig.14. The effect of 316P on the level of serum LDL cholesterol in Syrian hamsters treated with normal food. Control buffer (●); 316P 1 mg/kg (ν); 316P 3 mg/kg (σ); 316P 5 mg/kg (τ).

DETAILED description of the INVENTION

Before proceeding to the description of the present method, it should be noted that the present invention is not limited to particular methods and experimental conditions outlined, as such methods and conditions may vary. It should also be understood that use�supported in this document terminology is intended only to describe specific accomplishments and is not restrictive since the scope of the present invention is limited only by the appended claims of the invention.

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning, which is well known to ordinary experts in the field of the present invention. Although in practice or test the present invention can be used any methods and materials similar or identical to those described herein methods and materials the following is a description of preferred methods and materials.

Definitions

The term "human paraproteinemias of subtilisin/Kexin type 9" or "hPCSK9" as used herein means hPCSK9 with the nucleic acid sequence shown in SEQ ID no: 754, and the amino acid sequence of SEQ ID no: 755, or its biologically active fragment.

As used herein, the term "antibody" is intended to refer to immunoglobulin molecules, composed of four polypeptide chains, two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain contains a variable region heavy chain ("HCVR" or "VH") and a constant region of the heavy chain (containing domains CH1, CH2 and CH3). Each light chain contains�it variable region light chain ("LCVR", or "VL") and a constant region light chain. Region VH and VL can be divided into the areas of hypervariability, called complementarity determining regions (CDR), interspersed with areas with higher levels of conservatism, called spanning regions (FR). Each region VH and VL is composed of three CDR and four FR located from amino-terminal end to the carboxy-terminal end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

It is also possible substitution of one or more CDR residues or skip one or more CDR. In the scientific literature describes antibodies that bind you can do without one or two of CDR. In the work Padlan et al. (1995 FASEB J. 9:133-139) were analyzed area contact between antibodies and their antigens on the basis of the published crystal structures, and the conclusion was that only about a third CDR residues actually comes in contact with the antigen. In this work also found a lot of antibodies, in which one or two CDRs do not contain amino acids that are in contact with the antigen (see also, Vajdos et al. 2002 J Mol Biol 320:415-428).

The remains of the CDR, not in contact with the antigen can be determined on the basis of previous studies (e.g., residues H60-H65 in CDRH2 is often not required), the CDR regions of Kabat beyond the CDR Scotia, using molecular modeling�ing and/or empirically. If CDR or its remains are missed, they are usually replaced by the amino acid occupying a similar position in another sequence of the human antibody or consensus of such sequences. The provisions for substitution of CDR and amino acids can also be selected empirically. Empirical substitution may be conservative or non-conservative.

As used herein, the term "human antibody" is intended to refer to antibodies to variable and constant regions derived from immunoglobulin sequences of the germline of the lines person. Described in the present invention monoclonal antibodies (mAb) may include amino acid residues not encoded by immunoglobulin germline sequences of human (for example, mutations caused by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDR regions, especially in the CDR3 region. However, as used herein, the term "human antibody" does not imply the inclusion of mAb, in which the base sequence of the human CDR grafted sequence derived from the germ lines of another mammalian species, e.g. mouse.

The term "binds specifically" or the like means that the antibody or antigen-binding FR�gment forms a complex with the antigen, which is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant not less than about 1×10-6M or less (i.e., smaller KDindicates stronger binding). Methods of determining specific binding of molecules known to experts in the field and include, for example, equilibrium dialysis, surface plasmon resonance, etc. an Isolated antibody that specifically binds hPCSK9, however, may exhibit cross-reactivity to other antigens, for example, PCSK9 molecules from other species. In addition, multispecific antibodies (e.g., bespecifically) that bind to hPCSK9 and one or more additional antigens are nonetheless considered antibodies that "specifically bind" hPCSK9 in accordance with the terminology of this document.

As used herein, the term antibody "high affinity" refers to a mAb with an affinity of binding to hPCSK9 not less than 10-10M; preferably 10-11M; even more preferably 10-12M, according to the results of measurement by surface plasmon resonance, e.g., BIACORE™, or the results of the measurement of affinity in solution by enzyme immunoassay (ELISA).

Under used � this document the term “slowly dissociating”, “'koff” or “kd” is meant an antibody complex which hPCSK9 dissociates with a rate constant of 1×10-3with-1or lower, preferably 1×10-4with-1or below, measured by surface plasmon resonance, e.g., BIACORE™.

As used herein, the term "antigen-binding region" of an antibody (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specific binding to hPCSK9. The antibody fragment may include a Fab fragment, a fragment F(ab')2the Fv fragment, a dAb fragment, a fragment containing CDR or an isolated CDR.

Specific implementation, the antibody or antibody fragments of the present invention can konjugierte with therapeutic agent ("immunoconjugate"), for example, cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioactive isotope.

As used herein, the term "isolated antibody" is intended to refer to antibodies that are substantially free of other mAb with different antigenic specificity (e.g., an isolated antibody that specifically binds hPCSK9, substantially free from mAb that specifically bind antigens that are different from hPCSK9). An isolated antibody that SP�civicism way binds hPCSK9, however, may exhibit cross-reactivity to other antigens, for example, PCSK9 molecules from other species.

As used herein, the term "neutralizing antibody" (or an "antibody that neutralizes the activity of PCSK9") is intended to refer to antibodies, the binding of which PCSK9 leads to inhibition of at least one biological activity of PCSK9. This inhibition of the biological activity of PCSK9 can be assessed by measuring one or more indicators of biological activity of PCSK9 using one or more standard methods of analysis in vitro or in vivo, are known in the art (see examples below).

As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows for the analysis of biospecific interactions in real-time by detecting changes in protein concentrations within a biosensor matrix, for example using the BIACORE system™ (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N. J.).

As used herein, the term "KD"is intended to refer to the equilibrium dissociation constants of the specific interaction of antibody-antigen.

The term "epitope" refers to a region of the antigen to which the binding with the antibody. The epitopes may be structural or functional�bubbled. Functional epitopes are typically only a subset of structural epitopes and contain such residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e. consisting of branched chain amino acids. In some realizations, the epitopes may include the determinants representing a chemically active surface groupings of molecules such as amino acids, side chains of sugars, phosphoryl group or sulfonylurea groups, and in some realizations may have specific three dimensional structural characteristics and/or specific charge characteristics.

The term "substantial identity" or "essentially identical" in relation to nucleic acid or its fragment means that under the optimal alignment insertions and deletions of the corresponding nucleotides nucleic acid (or its complementary chain) is observed identity of nucleotide sequences is not less than about 90% and, more preferably, not less 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, e.g., FASTA, BLAST or GAP, as discussed below.

Applied to polypeptides, the term "substantial similarity" or "substantially similar" means, Thu� two peptide sequences, when optimally aligned, for example, using the programs GAP or BESTFIT considering the weight passes by default show a sequence identity of at least 90%, and even more preferably at least 95%, 98% or 99%. Preferably, the position of non-identical residues differ by conservative amino acid substitution. "Conservative amino acid substitution" is a substitution in which one amino acid residue is replaced with another amino acid residue having a side chain (R) with similar chemical properties (e.g. charge or hydrophobicity). In General, a conservative amino acid substitution will not lead to significant changes in the functional properties of the protein. In case two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to take into account the conservative nature of the substitution. Methods such adjustments are well known to specialists in this field. Cm. 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 hydroxylated side chains: serine and threonine; 3) the side chain amide group�Oh: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) side chains with the properties of the base: lysine, arginine and histidine; 6) side chains with the properties of acids: aspartate and glutamate; and 7) sulfur-containing side chains: cysteine and methionine. Preferred groups for conservative substitution of amino acids are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a negative value in the logarithmic PAM250 matrix of probabilities is described in Gonnet et al. (1992) Science 256: 1443 45. "Moderately conservative" replacement is any change that leads to a nonnegative value in the logarithmic matrix likelihood PAM250.

The similarity of the sequences of polypeptides is typically determined using a software for sequence analysis. Program for analysis of proteins matches similar sequences using indices of similarity, asked for various substitutions, deletions and other modifications, including conservative amino acid substitution. For example, GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters �La determining homology or sequence identity between closely related polypeptides, for example, the homologous polypeptides of different species of organisms or between a source protein and its mutant. See, for example, GCG Ver. 6.1. Polypeptide sequences can also be compared using FASTA programs included in the GCG Ver. 6.1, using the default settings or recommended settings. FASTA (e.g., FASTA2 and FASTA3) provides alignment and determines the percentage identity of sequences in regions of greatest overlap between the target and the studied sequences (see above Pearson (2000)). Other preferred matching algorithm to the sequence of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, which uses the default settings. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403 410 and Altschul et al. (1997) Nucl Acids Res. 25:3389 402.

In a specific implementation, the antibody or the antibody fragment used in the method of the present invention can be monospecifičeskoj, bespecifically or multispecificity. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains that are specific against several epitopes of the target polypeptides. P�the emer format cooking especifismo antibodies, which can be used in the context of the present invention may be the use of the first immunoglobulin domain (Ig) CH3 and the second Ig domain CH3, where the first and second Ig domains CH3 differ from each other by at least one amino acid, and where the distinction of not less than one amino acid reduces the binding especificacao antibody to protein A as compared with bespecifically antibody in which amino acid such distinction does not exist. In one implementation, the first Ig domain CH3 binds to protein A, and the second domain contains a mutation that reduces or blocks the binding of protein A, for example, modification H95R (according to the nomenclature of exons IMGT; H435R by EU nomenclature). Second CH3 may also contain Y96F modification (by IMGT; Y436F EU). The second sub-CH3 may be additional modifications: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M and V422I EU) in the case of IgG1 mAb; N44S, K52N, and V82I (IMGT; N384S, K392N and V422I EU) in the case of IgG2 mAb; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I EU) in the case of IgG4 mAb. Variations on the format bespecifically antibodies described above, belong to the scope of the present invention.

The definition of "therapeutically effective amount" means an amount which provides the desired effect for which it is introduced. The exact number will W�hanging from the goals of treatment can be assessed by a specialist in the field on the basis of known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

Obtaining human antibodies

Methods of generating human antibodies in transgenic mice are well-known (see, e.g., US 6596541, Regeneron Pharmaceuticals, VELOCIMMUNE™). VELOCIMMUNE technology™ involves the production of transgenic mice with the genome containing the variable regions of heavy and light chains of human rights, which are functionally linked to the endogenous loci of the constant region of the mouse, so in response to antigenic stimulation, the mouse produces an antibody containing the variable region of a human and a constant region of the mouse. DNA encoding variable regions of heavy and light chain antibodies, isolated and functionally associated with the DNA encoding the constant heavy and light chains of a human. Then the DNA is expressed in a cell that is able to Express the full human antibody. In a particular implementation of such a cell is a cell of the SSS.

The antibodies can be used therapeutically to block the ligand-receptor interaction or inhibiting receptor interaction components instead of destruction of cells by fixing complement and participating in complement-dependent cytotoxicity (CDC) or destruction of cells by antibody-dependent cell-induced cytotoxicity (ADCC). Constant region �Titel, thus, it is important to ensure the ability of the antibody to fix complement and mediate cell-induced cytotoxicity. Therefore, the isotype of the antibody can be selected in dependence from that, is it desirable that the antibody was posredovano cytotoxicity.

Antibodies may exist in two forms that are associated with the heterogeneity of hinge region. In one form of the antibody molecule contains stable chutyrehzveznuyu design weighing approximately 150-160 kDa in which the dimers are held linking the heavy chains by disulfide bridge. In the second form, the dimers are not linked megamachine disulfide bonds and forms a molecule with an approximate weight of 75-80 kDa, comprising covalently articulated light and heavy chains (half antibody). These forms were extremely difficult to separate, even after affinity purification.

The frequency of manifestations of the second form in various isotypes of intact IgG caused, inter alia, structural differences associated with the isotype hinge region of the antibody. The replacement of one amino acid in the hinge region of human IgG4 can significantly reduce the proportion of the second form (Angal et al. 1993 Molecular Immunology 30:105) to levels typically found by using the hinge region of human IgG1. Within the scope of the present invention includes antibodies having one �whether multiple mutations in the hinge region, fields CH2 or CH3, which may be desirable, for example, when producing antibodies to increase the yield of the desired form of the antibody.

Usually the mouse VELOCIMMUNE™ stimulated interest antigen, and mouse expressing the antibodies, are lymphatic cells (e.g. b cells). Lymphatic cells may be fused with myeloma cells to obtain immortal cell lines of hybridomas, and such lines of cells of hybridomas are screened and selection to identify those cell lines of hybridomas that produce antibodies of interest to the antigen. DNA encoding variable regions of heavy and light chain antibodies, can be isolated and associated with the desired isotype constant regions of heavy and light chains. Such protein antibodies can be produced in a cell, for example, in the cell of the SSS. In an alternative embodiment, the DNA that encodes the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains, can be isolated directly from antigen-specific lymphocytes.

Originally isolated chimeric antibodies with high affinity, having a variable region of a human and a constant region of the mouse. As described below, the antibodies are characterized and selected according to the properties, including affinity, selectivity, epitope, etc. To�instantie the field mouse are replaced by the desired constant regions of a human, in order to obtain fully human antibodies described in the present invention, for example, nematanthus or modified IgG1 or IgG4 (for example, SEQ ID no: 751, 752, 753). While selected constant region may vary depending upon the particular implementation, the properties of the high affinity of antigen-binding and the desired specificity are determined by the variable region.

Epitope mapping and related technologies

For the screening of antibodies that bind to a particular epitope (e.g., antibodies that block binding of IgE specific receptor, with high affinity), it is possible to make the standard cross-blocking, as described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY). Other methods include scanning mutants of alanine, peptide blots (Reineke (2004) Methods Mol Biol 248:443-63), and analysis of the cleavage of peptides. In addition, there can be used methods such as cutting epitope, epitope extraction and chemical modification of antigens (Tomer (2000) Protein Science 9: 487-496).

As used herein, the term "epitope" refers to the site of antigen that react B and/or T cells. The epitopes of B-cells can be formed as a continuous sequence of amino acids, amino acids and a myriad of bystanders as a result of the folding of Bel�and tertiary structure. Epitopes formed by a continuous sequence of amino acids, generally preserved when exposed to denaturing solvents whereas epitopes resulting from the folding of protein tertiary structure, are destroyed in the processing of denaturing solvents. The epitope, usually formed by at least three, and usually at least five or 8-10 amino acids in a very specific spatial conformation.

The profile definition with modifications (MAP), also known as the determination of the profile of antibodies based on the structure of the antigen (ASAP) is a method of classification of large numbers of monoclonal antibodies (mAb) to the same antigen according to the similarities of the binding profile of each antibody to chemically or ènzimatičeski modified antigen surfaces (US 2004/0101920). Each category can reflect the unique epitope that is either clearly different from the epitope presented in any other category, or partially overlaps with it. This technology allows you to quickly separate genetically identical mAb and, thus, focus on the identification of genetically different mAb. In the case of applications for screening of hybridomas, MAP may facilitate the identification of rare clones of hybridomas producing mAb with the desired characteristics�I. MAP can be used to sort mAb anti-PCSK9 present invention, in the group mAb that bind different epitopes.

In various realizations hPCSK9 antibody or antigen-binding fragment of the antibody binds the epitope with a catalytic domain that is approximately 153 425 SEQ ID no: 755); more specifically, an epitope from about 153 to about 250 or from about 250 to about 425; more specifically, the antibody or antibody fragment of the present invention binds an epitope within the fragment from about 153 to about 208, about 200 to about 260, from about 250 to about 300, from about 275 to about 325, from about 300 to about 360, from about 350 to about 400, and/or from about 375 to about 425.

In various realizations antibody anti-hPCSK9 or antigen-binding fragment of the antibody binds the epitope with the domain of propeptide (residues 31 - 152 SEQ ID no: 755); more specifically, an epitope from about residue 31 to about residue 90 or residue from about 90 to about residue 152; more specifically, the antibody or antibody fragment of the present invention binds an epitope within the fragment from about 31 to about 60, from about 60 to about 90, from about 85 to about 110, from about 100 to about 130, from about 125 to about 150, from about 135 to about 152, and/or from about 140 to about 152.

In some realizations antibody anti-hPCSK9 or anti�n-binding fragment of an antibody binds an epitope in the C-terminal domain (residues 426 - according to 692 of SEQ ID no: 755); more specifically, an epitope from about residue 426 to about residue 570 or from the residue of about 570 to about residue 692; more specifically, the antibody or antibody fragment of the present invention binds an epitope within the fragment from about 450 to about 500, from about 500 to about 550, from about residue 550 to about 600, and/or from about 600 to about 692.

In some realizations, the antibody or fragments of the antibodies bind to an epitope that includes more than one of these epitopes in the catalytic, propeptide or C-terminal domain, and/or within two or three different domains (for example, the epitopes in the catalytic and C-terminal domains or propeptide and catalytic domains, or in propeptide, catalytic and C-terminal domains).

In some realizations, the antibody or a fragment thereof in contact with the epitope hPCSK9 containing amino acid residue 238 of hPCSK9 (SEQ ID no: 755). Experimental results (table 27) show that in the case of mutations D238 KDmAb 316P showed >400-fold reduction of binding affinity (~1×10-9M to ~410×10-9M) and T1/2decreased >30-fold (from ~37 to ~1 minute). In the specific implementation used mutation D238R. In a specific implementation, the antibody or a fragment thereof in contact with the epitope hPCSK9 containing od�n or more amino acid residues 153, 159, 238 and 343.

As shown below, mutation of amino acid residues 153, 159 or 343 resulted from 5 - to 10-fold reduction of affinity or similar reduction in T1/2. In a specific implementation, the mutation was S153R, E159R and/or D343R.

In some realizations, the antibody or a fragment thereof in contact with epitope hPCSK9 containing amino acid residue 366 of hPCSK9 (SEQ ID no: 755). Experimental results (table 27) show that in the case of mutations E affinity mAb 300N showed about 50-fold lower (~0,7×10-9M to ~36×10-9M), with a similar decrease in T1/2(from ~120 to ~2 minutes). In the specific implementation used mutation E366K.

The present invention includes anti-PCSK9 antibodies that bind to the same epitope as any of the specific examples of the antibodies described herein. Similarly, the present invention includes anti-PCSK9 antibodies that compete for binding to the fragment of PCSK9 or PCSK9 with any of the specific examples of the antibodies described herein.

Easy to install, connects to whether or not the antibody to the same epitope as that of the control anti-PCSK9 antibody is, or competes for binding with it, using standard methods known to experts in the field. For example, to determine whether associated antibody test with t�m same epitope, that and reference anti-PCSK9 antibody of the present invention, the reference antibody is allowed to bind to PCSK9 protein or peptide under saturated conditions. Then evaluates the ability of a test antibody to contact the PCSK9 molecule. If the test antibody is able to communicate with after binding of PCSK9 under conditions of saturation control with anti-PCSK9 antibody, it can be concluded that the test antibody binds to the epitope that is different from the control anti-PCSK9 antibody. On the other hand, if the test antibody is not able to contact the PCSK9 molecule after binding under saturated conditions with a control anti-PCSK9 antibody, then the test antibody can bind to the same epitope as the epitope bound control anti-PCSK9 antibody of the present invention.

To determine competes whether the antibody upon binding with a control anti-PCSK9 antibody, the above methodology linking was used in two variants: In the first case, the reference antibody is allowed to contact the PCSK9 molecule under saturated conditions, followed by assessment of binding of the control antibody to PCSK9 molecule. In the second case, the subject antibody is allowed to contact the PCSK9 molecule under saturated conditions, followed by assessment of binding of the control antibody to PCSK9 molecule. If in both cases we�Ko first (saturating) antibody is able to contact the PCSK9 molecule, it is concluded that the test antibody and the reference antibody compete for binding to PCSK9. The specialist will be obvious that an antibody that competes for binding with the control antibody does not necessarily contact the same epitope as that of the control antibody, but may sterically block the control antibody by binding to an overlapping or adjacent epitope.

Two antibodies bind the same or overlapping epitope if each competitive inhibits (blocks) binding of another antibody to the antigen. That is 1-, 5-, 10-, 20- or a 100-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% according to the analysis of competitive binding (see, e.g., Junghans et al., Cancer Res. 1990 50: 1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or inhibit the binding of one antibody also reduce or inhibit the binding of the other. Two antibodies have overlapping epitopes, if some amino acid mutations that reduce or inhibit the binding of one antibody also reduce or block the binding of another.

Then you can spend additional standard experiments (for example�EP, peptide mutation or analysis of binding) to confirm whether, in fact, a lack of binding of the test antibody to the fact that the test antibody is contacted with the same epitope as that of the control antibody, or a marked lack of binding is due to steric blocking (or another phenomenon). Experiments of this kind can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative analysis of antibody binding, available to specialists in the field.

In a particular implementation of the invention includes anti-PCSK9 antibody or antigen-binding fragment of the antibody that bind to PCSK9 protein sequence SEQ ID no: 755, wherein the binding between the antibody or its fragment with a PCSK9 and a variant PCSK9 protein is less than 50% of the binding between the antibody or fragment and the protein PCSK9 sequence SEQ ID no: 755. In one particular implementation variant PCSK9 protein contains at least one mutation of a residue in a position selected from the group including 153, 159, 238 and 343. In a more specific implementation of one mutation is S153R, E159R, D238R and D343R. In another specific implementation variant PCSK9 protein contains at least one mutation of a residue in a position selected from the group include�her 366. In one particular implementation variant PCSK9 protein contains at least one mutation of a residue in a position selected from the group including 147, 366 and 380. In a more specific implementation of mutation is one of S147F, E366K and/or V380M.

Immunoconjugate

Within the scope of the present invention includes anti-PCSK9 monoclonal antibody human, anywhereman with therapeutic agent ("immunoconjugate"), for example, cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioactive isotope. To the cytotoxic agents include any substances which are damaging to cells. Examples are suitable for application of cytotoxic agents and chemotherapeutic agents for education immunoconjugates known to experts in the field, see, e.g., WO 05/103081.

Bespecifically

The antibodies of the present invention can be monospecifičeskoj, bespecifically or multispecificity. Multispecificity mAb can be specific to different epitopes of one target polypeptide or may contain antigen-binding domains specific for multiple target polypeptides. See, for example, Tutt et al. (1991) J. Immunol. 147:60-69. mAb anti-human PCSK9 can be linked to another functional molecule, or to be expressed, n�example, with another peptide or protein. For example, an antibody or a fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent Association or otherwise) with one or more molecular structures, for example, another antibody or antibody fragment, with the formation of especificacao or multispecific antibodies with additional specificity of binding.

Example of the format of especifismo antibodies that can be used in the context of the present invention may be the use of the first immunoglobulin domain (Ig) CH3 and the second Ig domain CH3, where the first and second Ig domains CH3 differ from each other by at least one amino acid, and where the distinction of not less than one amino acid reduces the binding especificacao antibody to protein A as compared with bespecifically antibody in which amino acid such distinction does not exist. In one implementation, the first Ig domain CH3 binds to protein A, and the second domain contains a mutation that reduces or blocks the binding of protein A, for example, modification H95R (according to the nomenclature of exons IMGT; H435R by EU nomenclature). Second CH3 may also contain Y96F modification (by IMGT; Y436F EU). The second sub-CH3 may be additional modifications: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M N384S, K392N, V397M and V422I EU) in the case of IgG1 antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N and V422I EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I EU) in the case of in the case of IgG4 antibodies. Variations on the format bespecifically antibodies, described above, are within the scope of the present invention.

Bioequivalente

Anti-PCSK9 antibodies and fragments of antibodies of the present invention include proteins containing amino acid sequences that differ from those described mAb, but retain the ability to bind to human PCSK9. Such variant mAb and fragments of the antibodies contain one or more additions, deletions or substitutions of amino acids compared with the parent sequence, but exhibit biological activity that is substantially equivalent to the activity described mAb. Similarly, DNA sequences encoding an anti-PCSK9 antibodies of the present invention include sequences that contain one or more additions, deletions or substitutions of nucleotides compared to the disclosed sequence, but they encode anti-PCSK9 antibody or antibody fragment, which largely bioekvivalentnaanti-PCSK9 antibody or antibody fragment of the present invention. Examples of such variant amino acid sequences of posledovatelnostei DNA are discussed above.

Two antigen-binding protein or antibody are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show significant difference when administered at the same molar dose under similar experimental conditions, as when a single dose or in multiple doses. Some antibodies will be considered to be equivalent or pharmaceutical alternatives if they are equivalent in their degree of absorption, but not on the velocity of such removals, but, nevertheless, may be considered bioequivalent because such differences in the rate of absorption, intentionally made and reflected in the labeling, are not essential to achieve effective drug concentrations in the body when, for example, prolonged use and are considered minor from a medical point of view for a particular investigational drug. In one implementation of the two antigen-binding protein to be bioequivalent if they are no clinically meaningful differences in safety, purity and potency.

In one implementation of the two antigen-binding protein to be bioequivalent, if the patient can be entered and biological control product pooche�one, one or more times, without the expected increase risk of side effects, including clinically significant changes in immunogenicity or reduce efficiency, compared with continuous therapy in one product.

In one implementation, two antigen-binding protein to be bioequivalent if they both act by the same mechanism or mode of action in this disease or conditions to the extent that such mechanisms are known.

Bioequivalence may be demonstrated and techniquesin vivoandin vitro. The methods of measurement of bioequivalence include, for example, (a) the testing ofin vivoin humans or other mammals in which the concentration of the antibody or its metabolites measured in blood, plasma, serum or other biological fluids depending on time; (b) the testing ofin vitrothat correlate with bioavailability for humansin vivoand with a reasonable degree of certainty predict them; (c) the testing ofin vivoin humans or other mammals in which an appropriate acute pharmacological effect of the antibody (or its target) is measured depending on time; and (d) well-controlled clinical trials, in which is installed the safety, efficiency, bioavailability or bioe�violentest antibodies.

Bioequivalent variants of anti-PCSK9 antibodies of the present invention may be constructed, for example, through the introduction of various substitutions of residues or sequences, or deletions of terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues, are not having significant values for biological activity, can be deleted or substituted with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges in the course of renaturation.

The group of patients

The present invention offers therapeutic methods for the treatment of the person in need of the application of the composition of the present invention. Despite the fact that changes in lifestyle and treatment with standard drugs often provide success in reducing the level of cholesterol, not all patients are able to achieve the recommended target levels of cholesterol in the framework of such approaches. Different States, for example, familial hypercholesterolemia (FH), are sustainable in terms of reducing the level of LDL's, despite the aggressive use of standard therapy. Homozygous and heterozygous familial hypercholesterolemia (hoFH, heFH) is a condition that is associated with premature vascular �therosclerosis. However, in patients diagnosed with hoFH often there is no response to standard drug therapy, and the treatment is very limited. In particular, treatment with statins, which reduce LDL-X through inhibition of cholesterol synthesis and enhance the function of LDL receptors in the liver, may have a negligible effect on patients with no LDL receptors, or their functioning is impaired. It has recently been shown that patients with confirmed genotype hoFH treated with the maximum dose of statins, it is noted an average reduction in LDL-X just less than about 20%. Add to this scheme the treatment ezetimibe in the dose of 10 mg/day resulted in a total reduction in LDL-27%, which is still far from the optimal rate. Similarly, many patients are resistant to statins, poorly controlled latinboy therapy or unable to tolerate statin treatment; in the General case, such a patient cannot achieve control over the level of cholesterol in terms of alternative treatment. There is a huge unmet medical need for new treatment methods that are able to take into account the shortcomings of existing treatment options.

Specific groups that may be treated using a therapeutic method�in the present invention, include patients who have shown the LDL apheresis, patients with mutations that activate PCSK9 ("GOF"), hereditary heterozygous hypercholesterolemia (heFH); patients with primary hypercholesterolemia who cannot tolerate statins or statin uncontrolled; and patients with risk of developing hypercholesterolemia who may receive preventive treatment.

Therapeutic applications and formulations of drugs

In the present invention are therapeutic drugs, including antibody anti-PCSK9 or their antigen-binding fragments included within the scope of the present invention. The introduction of therapeutic agents in accordance with the present invention will be carried out in suitable media, shaping and other substances that are included in the products, to ensure more effective dissemination, delivery, portability, etc. a Variety of appropriate formulations can be found in the forms known to all specialists in the field of pharmaceutical chemistry: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Such drugs include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, emulsions of oil in water and water in oil emulsions Carbowax (carton s�ringlike different molecular weight), semi-solid gels, and semi-solid mixtures containing Carbowax. Cm. also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

The dose can be changed depending on the age and weight of the object receiving treatment, diseases targeted by the treatment, conditions, method for administration, etc. When using the antibodies of the present invention for the treatment of various illnesses and diseases associated with PCSK9, including hypercholesterolemia, disorders associated with low density lipoprotein (LDL) and apolipoprotein B, and lipid metabolism, etc. in adult patients, it is advisable to introduce the antibody of the present invention intravenously, usually in a single dose of approximately 0.01 to 20 mg per kg of body weight, more preferably from about 0.02 to 7 mg, from about 0.03 to 5 mg, or from about 0.05 to 3 mg per kg of body weight. Depending on the severity of the condition, you can adjust the frequency and duration of treatment.

There are various delivery systems, and they can be used for the introduction of pharmaceutical product of the present invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). The methods of introduction, among others, include Chris�one, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral. The drug may be administered by any suitable method, for example, by infusion or bolus injection, by absorption through epithelial or skin-mucous membranes (e.g., oral mucosa, mucosa of the rectum and intestines, etc.) and can be used in combination with other biologically active agents. Administration may be systemic or local.

Pharmaceutical drug can 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, SS. 353-365; Lopez-Berestein, ibid., SS. 317-327; see generally ibid.).

In certain situations the pharmaceutical drug can be delivered in a controlled release system. In one implementation can be used in the pump (see Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201). In another implementation can be used polymeric materials; see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974). In yet another implementation, the controlled release system can be placed in the immediate vicinity of the purpose of the drug, and therefore need only a certain fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, vol. 2, SS. 115-138, 1984).

To inject prep�rather may include dosage forms for intravenous, subcutaneous, transcutaneous and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared using known methods. For example, the injectable preparations can be prepared in particular by dissolving, suspending or emulsifying the above-described antibody or its salt in a sterile aqueous medium or in an oil environment, which are commonly used for injection. As the aqueous medium for injections are available, for example, saline, an isotonic solution containing glucose and other auxiliary substances etc. that can be used in combination with the appropriate solubilizers agent such as alcohol (in particular ethanol), polyvalent alcohol (e.g., propylene glycol, polyethylene glycol), nonionic surface-active compound [e.g., Polysorbate 80, VAT-50 (the adduct of polyoxyethylene (50 mol) and hydrogenated castor oil], etc. as oil media are used, for example, sesame oil, soybean oil, etc., which can be used in combination with solubilizers agent, such as benzyl benzoate, benzyl alcohol, etc. thus Prepared the drug for injection is preferably packaged in appropriate ampoules. The pharmaceutical composition of the present invention also can enter�mails subcutaneously or intravenously with a standard needle and syringe. In addition, as for subcutaneous injection, in the case of application of the pharmaceutical preparation of the present invention it is possible to use the pen. The autoinjector can be reusable or disposable. In the reusable insulin pen usually uses a replaceable cartridge, which contains the pharmaceutical composition. After all, the pharmaceutical composition inside the cartridge was introduced, and the cartridge is empty, used toner cartridge, you can easily throw it away and replace it with a new cartridge, which contains the pharmaceutical composition. After that, the autoinjector can be reused. In one syringe-pen replacement cartridge is not provided. In contrast, single-use syringe-pen comes already filled with a pharmaceutical composition, which is placed in the tank inside the unit. Once in the tank remains the drug compound, the entire device is discarded.

For subcutaneous administration of a pharmaceutical composition of the present invention can be applied to various designs of reusable syringes, pens and devices for automatic injection. Examples include, without limitation, 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). Examples of disposable syringes pens that can be used for subcutaneous administration of a pharmaceutical composition of the present invention, among others, include the insulin pen SOLOSTAR™ (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly).

Pharmaceutical preparations for oral or parenteral use described above are preferably prepared in dosage forms with a single dose, selected in accordance with the dosage of active ingredients. To those dosage forms with a single dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. the Amount of a specific antibody contained in a single dose, is generally from 5 to 500 mg per dosage form; especially in the form of an injection, it is preferable that the amount of a specific antibody was from about 5 to about 100 mg and about 10 to about 250 mg for other dosage forms.

In the invention offers therapeutic methods in which the antibody or antibody fragment of the present invention is used for the treatment of hypercholesterolemia associated with a variety of diseases involving hPCSK9. The antibody anti-PCSK9 or fragments thereof described in the present invention is particularly effective for the treatment� hypercholesterolemia or similar breaches. Combination therapy can include an anti-PCSK9 antibody of the present invention, for example, with one or more agents that (1) induce the violation of the synthesis of cholesterol at the cellular level by inhibiting 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors, e.g., cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin; (2) inhibit the capture of cholesterol and/or reabsorption of bile acids; (3) increase the catabolism of lipoproteins (e.g., Niacin); and activators of the transcription factor LXR, that plays a role in the removal of cholesterol, for example, 22-hydroxycholesterol, or fixed combinations, for example, ezetimibe plus simvastatin; a statin with sequestrants bile acids on ion-exchange resin (eg, cholestyramine, colestipol, colesevelam), a fixed combination of Niacin plus a statin (e.g., Niacin with lovastatinom); or with other agents that reduce the concentration of lipids, for example, ethyl esters of omega-3 fatty acids, (for example, omacor).

EXAMPLES

The examples below are designed to give professionals full details and description of how to formulate and use the methods and preparations of the present invention, and are not intended to limit the scope of what the inventors rassmotrev�Ute as the subject of his invention. Efforts were made to ensure the accuracy of indicators used, but it is necessary to take into account some experimental errors and deviations. Unless otherwise stated, under molecular weight refers to the average molecular weight, temperature is in degrees Celsius and pressure is atmospheric or close to this level.

Example 1. Obtaining human antibodies to human PCSK9.

VELOCIMMUNE mice™ were immunized human PCSK9, and immune response of antibodies was measured using antigen-specific immunoassay using serum taken from these mice. B cells expressing anti-hPCSK9, taken from the spleen of immunized mice, which demonstrated elevated titers of anti-hPCSK9 antibodies were fused with cells of the mouse myeloma with the formation of the hybrid. The hybridomas are screened and selection to identify those cell lines that Express hPCSK9-specific antibodies using assays described below. The analysis allowed to identify several cell lines that produce chimeric anti-hPCSK9 antibodies, denoted as H1M300, H1M504, H1M505, H1M500, H1M497, H1M498, H1M494, H1M309, H1M312, H1M499, H1M493, H1M496, H1M503, H1M502, H1M508, H1M495 and H1M492.

PCSK9-specific antibodies also were isolated directly from antigen-immunized B cells without fusion to myeloma cells, as description�but in US patent 2007/0280945A1. Variable regions of heavy and light chains are cloned to obtain a fully human anti-hPCSK9 antibodies, denoted as H1H313, H1H314, H1H315, H1H316, H1H317, H1H318, H1H320, H1H321 and H1H334. Were established stable CHO cell line expressing such antibodies.

Example 2. An analysis of the use of genes

For analysis of the structure of the produced mAb was cloned and sequenced nucleic acids encoding variable regions of antibodies. The predicted amino acid sequence of variable regions was confirmed by N-terminal amino acid sequencing. On the basis of nucleic acid sequence and the predicted amino acid sequence to mAb for each chain of the antibody was determined by the degree of use of the genes.

Table 1
AntibodyThe variable region of the heavy chainVariable region light chain
VHDJHVKJK
H1H3133-131-26 43-153
H1H3143-333-341-52
H1H3153-333-344-11
H1H3163-237-2724-12
H1H3173-131-2641-61
H1H3184-593-1061-91
H1H3201-182-262-301
H1H3212-51-762-28 4
H1H3342-56-662-284
H1M3003-72-862-284
H1M5043-302-862-284
H1M5053-302-862-284
H1M5002-55-562-284
H1M4971-182-262-302
H1M4983-212-241-52
H1M494 3-115-1263-204
H1M3093-216-1341-51
H1M3123-216-1341-51

H1M4993-216-1341-51
H1M4933-216-1341-51
H1M4963-136-1943-153
H1M5031-182-262-281
H1M502 3-136-1343-153
H1M5083-136-1343-153
H1M4953-94-1761-93
H1M4923-233-323-204

Example 3. Determination of antigen-binding affinity

Equilibrium dissociation constants (KD) for binding hPCSK9 with mAb derived from cell lines of hybridomas described above were determined by surface kinetics by the method of plasmon resonance on the surface of the biosensor in real time (BIACORE™ T100). Each antibody was captured at a flow rate of 4 µl/min for 90 seconds on the surface anti-mouse polyclonal antibody goat IgG formed by direct chemical bonding on the chip of the BIACORE™, with the formation of the surface captured by the antibodies. Human PCSK9-mmh at a concentration of 50 nm or 12.5 nm was coated on the surface�rnost captured antibody at a flow rate of 50 μl/min for 300 seconds then monitored the dissociation of antigen-antibody for 15 minutes at 25°C or 37°C (KD=RM; T1/2=min).

Table 2
Antibody25°C37°C
KDT1/2KDT1/2
H1M300399170151032

H1M30929,97461537326
H1M3120,22515568432392
H1M49346,54921522341
H1M4948701142350 30
H1M495440222750019
H1M496254257421118
H1M49720,15801480290
H1M498640030750014
H1M4991062253582316
H1M500140091601015
H1M50278,3958411151
H1M503510118188030
H1M5043470 35112006
H1M50527404292006
H1M508138572442139
H1M510107068396010

Equilibrium dissociation constants (KD) for binding hPCSK9 with mAb, obtained by direct allocation of splenocytes, were determined by surface kinetics by the method of plasmon resonance on the surface of the biosensor in real time (BIACORE™ T100). Each selected antibody was captured at a flow rate of 2 µl/min for 6 minutes on the surface of the anti-human polyclonal antibody goat IgG formed by direct chemical bonding on the chip of the BIACORE™, with the formation of the surface captured by the antibodies. Human PCSK9-mmh at a concentration of 50 nm or 12.5 nm was coated on the surface of the captured antibody at a flow rate of 70 μl/min for 5 minutes, then monitored the dissociation of antigen-antibody for 15 minutes at 25°C or 37°C (KD=RM; T1/2=min).

Table 3
Antibody25°C37°C
KDT1/2KDT1/2
H1H313P24423078060
H1H314P399065356043
H1H315P12915141335
H1H316P37742108011
H1H317P304001371860070
H1H318P97259169028
H1H320P 7712819308
H1H321P865106336023
H1H334P375046159008

The rate of dissociation (kd) to separate the labeled mAb for rhesus monkeys (Macaca mulata) PCSK9 (mmPCSK9; SEQ ID no: 756) (mmPCSK9-mmh) at 25°C was determined in accordance with the above-described methods.

Table 4
Antibodykd (1/s)T1/2(min)
H1H313POf 2.92×10-5396
H1H318POf 3.69×10-33
H1H334P8,06×10-31
H1H315PTo 2.29×10-451
H1H316PTo 2.29×10-4 51
H1H320POf 3.17×10-436

NMOf 1.52×10-476
NMTo 5.04×10-423
NMOf 6.60×10-5175
NM8,73×10-5132
NMOf 4.45×10-5260

Example 4. The effect of pH on the affinity of binding to the antigen

The effects of pH on the binding affinity of the antigen for produced by cells of the SNO fully human anti-hPCSK9 MABs was assessed using methods described above. Subjects mAb is a fully human versions NNR ("R") (HCVR/LCVR SEQ ID nos:90/92; CDR sequences of SEQ ID no:76/78/80 and 84/86/88) and H1M300N ("300N") (HCVR/LCVR SEQ ID no:218/226; CDR sequences of SEQ ID no:220/222/224 and 228/230/232). hPCSK9-mmh was placed on the surface of the anti-myc mAb at high density (approximately 35 to 45 resonance units) (RU) or at low density (approximately 5 to 14 RU). To�each antibody at a concentration of 50 nm in HBST (pH 7.4 or pH 5.5) was injected onto the surface of the captured hPCSK9 a flow rate of 100 ál/min for 1.5 minutes at 25°C, the dissociation of antigen-antibody was monitored for 10 minutes. Control I: anti-hPCSK9 MABs SEQ ID no:79/101 (WO 2008/063382) (KD=RM; T1/2=min).

Table 5
AntibodyThe surface of the high density hPCSK9The surface low density hPCSK9
pH 7.4pH 5.5PH 7.4pH 5.5
ToT1/2kdT1/2kdT1/2kdT1/2
R19174144833394518858
300N65507118026 310119138013
Control I2000029NDNDNDNDNDND

Antigen-binding properties R and 300N at pH 7.4 or pH 5.5 was determined using a modified analysis BIACORE™, as described above. Briefly, mAb was immobilizovana on the touch chip BIACORE™ CM5 through a combination with an amine. Different concentrations of labeled myc-myc-his hPCSK9, murine PCSK9 (mPCSK9, SEQ ID no:757), hPCSK9 with a point mutation with the appearance of new pathological functions (GOF) D374Y (hPCSK9(D374Y)), PCSK9 (mfPCSK9, SEQ ID no:761) (mfPCSK9) cynomolgus macaque (Masasa fascicularis), rat (Rattus norvegicus) PCSK9 (rPCSK9, SEQ ID no:763) and his-tagged PCSK9 (maPCSK9, SEQ ID no:762) (maPCSK9) the Syrian Golden hamster (Mesocricetus auratus) in the range from 11 to 100 nm were injected at the surface of the antibody at a flow rate of 100 ál/min for 1.5 minutes, and the dissociation of antigen-antibody tracked in real time for 5 minutes at 25°C (table 6) or at 37°C (table 7). Control II: aHTH-hPCSK9 MABs SEQ ID no:67/12 (WO 2009/026558). NS: in experimental conditions, binding was not observed (KD=RM; T1/2=min).

5
Table 6
The effect of pH at 25°C
Antigen316P
a pH of 7.4pH 5.5
KDT1/2KDT1/2
hPCSK9-mmh1260362239
mPCSK9-mmh4460106311
hPCSK9(D347Y)-mmh24901516613
mfPCSK9-mmh142042823
maPCSK9-h83508878
rPCSK9-mmh241002349
300N
hPCSK9-mmh11007631005
mPCSK9-mmhNBNBNBNB
hPCSK9(D347Y)-mmh13104690303
mfPCSK9-mmh217031385000,4
maPCSK9-hNBNBNBNB
rPCSK9-mmhNBNBNBNB
Control I
hPCSK9-mmh3310014174031
mPCSK9-mmh NBNBNBNB
hPCSK9(D347Y)-mmh7100011732030
mfPCSK9-mmh3620000,2672003
maPCSK9-hNBNBNBNB
rPCSK9-mmhNBNBNBNB
Control II
hPCSK9-mmh1432662212

mPCSK9-mmh3500113312
hPCSK9(D347Y)-mmh19115549mfPCSK9-mmh1022621263
maPCSK9-h65003NDND
rPCSK9-mmh2240021065

Table 7
The effect of pH at 37°C
Antigen316P
a pH of 7.4pH 5.5
KDT1/2KDT1/2
hPCSK9-mmh4000914211
mPCSK9-mmh122003136003
hPCSK9(D347Y)-mmh 415605
mfPCSK9-mmh377011445
maPCSK9-h217002NDND
rPCSK9-mmh5510023991
300N
hPCSK9-mmh247020119001
mPCSK9-mmhNBNBNBNB
hPCSK9(D347Y)-mmh261014280001
mfPCSK9-mmh217031385000,4
maPCSK9-h NBNBNBNB
rPCSK9-mmhNBNBNBNB
Control I
hPCSK9-mmh459000,1113003
mPCSK9-mmhNBNBNBNB

hPCSK9(D347Y)-mmh1690000,4270003
mfPCSK9-mmh5000000,653600,3
maPCSK9-hNBNBNBNB
rPCSK9-mmhNBNBNB NB
Control II
hPCSK9-mmh284872044
mPCSK9-mmh86803893
hPCSK9(D347Y)-mmh2515748326
mfPCSK9-mmh18012721465
maPCSK9-h88300,5NDND
rPCSK9-mmh3020012331

Example 5. Binding of anti-hPCSK9 MABs to hPCSK9 with a point mutation D374Y

The affinity of binding of some anti-hPCSK9 MABs to hPCSK9 with a point mutation with the appearance of new pathological functions (GOF) D374Y (hPCSK9(D374Y)-mmh) was determined using methods described above. Each antibody was captured at SC�grow flow 40 µl/min for 8-30 seconds on the surface anti-mouse polyclonal antibody goat IgG, formed by direct chemical bonding on the chip of the BIACORE™, with the formation of the surface captured by the antibodies. Human PCSK9(D374Y)-mmh in various concentrations from 1,78 nm to 100 nm was coated on the surface of the captured antibody at a flow rate of 50 μl/min for 5 minutes, then monitored the dissociation of human PCSK9(D374Y)-mmh - antibody for 15 minutes at 25°C. Control III: anti-hPCSK9 MABs SEQ ID no: 49/23 (WO 2009/026558) (KD=RM; T1/2=min).

Table 8
AntibodyKDT1/2
316P178014
300N106049
Control I2360025
Control II66216
Control III1020126

Example 6. The specificity of binding of anti-hPCSK9 MABs

316P, 300N and monitoring I anti-hPCSK9 MABs were fixed on aminomethane anti-hFc CM5 chip on a BIACORE™2000. IU�enny (myc-myc-his) human PCSK9, human PCSK1 (hPCSK1) (SEQ ID no: 759), human PCSK7 (hPCSK7) (SEQ ID no: 760), or murine PCSK9 was applied (100 nm) on the surface of the captured mAb) and kept for binding at 25°C for 5 minutes. Registered changes in RU. Results: 300N and control I was associated only with hPCSK9 and 316P associated with hPCSK9 and mPCSK9.

The specificity of binding of anti-hPCSK9 MABs were determined using ELISA. In short, the anti-hPCSK9 antibody was coated on the surface of 96-well plates. Human PCSK9-mmh, mPCSK9-mmh, maPCSK9-h, hPCSK1-mmh or hPCSK7-mmh at a concentration of 1.2 nm was added to coated wells plates and incubated at room temperature for 1 hour. Associated with plansyou protein PCSK then determined by HRP-conjugarea anti-His antibody. The results show that 316P binds human, mouse and hamster PCSK9, while 300N and control (I only link between hPCSK9. None of the anti-hPCSK9 MABs did not show significant binding to hPCSK1 or hPCSK7.

Example 7. The cross-reactivity of anti-hPCSK9 MABs

The cross-reactivity of anti-hPCSK9 MABs with mmPCSK9, mfPCSK9, mPCSK9, maPCSK9 or rPCSK9 was determined using BIACORE™ 3000. Anti-hPCSK9 MABs were fixed on the surface of the anti-hFc, which is formed by reaction of the direct chemical combination on the chip of the BIACORE™. The purified labeled hPCSK9, hPCSK9(D374Y), mmPCSK9, mfPCSK9, mPCSK9, maPCSK9 or rPCSK9, each in a concentration of from 1.56 nm to 50 nm, deposited on a surface�knosti antibodies at 25°C or 37°C. Determined the binding between 316P, 300N, control I, control II and control III and protein PCSK9 (KD=RM; T1/2=min).

Table 9
316P mAb
Antigen37°C25°C
KDT1/2KDT1/2
hPCSK9-mmh1800958036
hPCSK9(D374Y)-mmh42004169015
mmPCSK9-mmh18002155092
mfPCSK9-mmh18001152060
mPCSK9-mmh47003230011
maPCSK9-h19000168105
rPCSK9-mmh375001145002

Table 10
MAb 300N
Antigen37°C25°C
KDT1/2KDT1/2
hPCSK9-mmh240022740110
hPCSK9(D374Y)-mmh22001490065
mmPCSK9-mmh16002661079
mfPCSK9-mmh380011150045
mPCSK9-mmhNBNBNBNB
maPCSK9-hNBNBNBNB
rPCSK9-mmhNBNBNBNB

Table 11
Control I mAb
Antigen37°C25°C
KDT1/2KDT1/2
hPCSK9-mmh22600022750016
hPCSK9(D374Y)-mmhNDND2360025
mmPCSK9-mmh4200003 2910002
mfPCSK9-mmh14300102490014
mPCSK9-mmhNBNBNBNB
maPCSK9-hNBNBNBNB
rPCSK9-mmhNBNBNBNB

Table 12
Control II mAb
Antigen37°C25°C
KDT1/2KDT1/2
hPCSK9-mmh9116261372
hPCSK9(D374Y)-mmh93 9066216
mfPCSK9-mmh3325226546
mPCSK9-mmh47003230011
maPCSK9-h608000,4250002
rPCSK9-mmh14100169003

Table 13
Control III mAb
Antigen37°C25°C
KDT1/2KDT1/2
hPCSK9-mmh380378490450
hPCSK9(D374Y)-mmh6601000126
mfPCSK9-mmh110750340396
mPCSK9-mmh335001109004
maPCSK9-h780107210067
rPCSK9-mmhNBNB332002

Example 8. The inhibition of binding between hPCSK9 and domains hLDLR

The ability of some anti-hPCSK9 MABs to block binding to hPCSK9 with an extracellular domain of the human LDLR full length (hLDLR-ecto SEQ ID no: 758), domain, hLDLR EGF-A (amino acids 313-355 SED ID no: 758), or domains, hLDLR EGF-AB (amino acids 314-393 SEQ ID no: 758) (LDLR Genbank room NM_000527) were determined using a BIACORE™ 3000. Briefly, proteins hLDLR-ecto, EGF-A-hFc or EGF-AB-hFc were fixed on the CM5 chip by combining with an amine to form a surface receptor or receptor fragment. Some anti-hPCSK9 MABs at a concentration of 62.5 nm (a 2.5-fold excess compared to the Antiga�ω) is pre-mixed with 25 nm hPCSK9-mmh, followed by incubation for 40 minutes at 25°C, in order to ensure the achievement of the equilibrium of the antibody-antigen binding with the formation of the equilibrium solutions. The equilibrium solutions were applied to the surface of the receptors or receptor fragments at a speed of 2 μl/min for 40 minutes at 25°C. was Determined by changes in EN the binding of anti-hPCSK9 MABs with hLDLR-ecto, EGF-A-hFc or EGF-AB-hFc. The results show that H1H316P and H1M300N blocked the binding hPCSK9-mmh with hLDLR-ecto, domain, hLDLR EGF-a and domains hLDLR EGF-AB; H1H320P blocked the binding hPCSK9-mmh with hLDLR-ecto and domain hLDLR EGF-A; and H1H321P blocked the binding hPCSK9-mmh domain hLDLR EGF-A.

The ability of mAb to block the binding hPCSK9 with hLDLR-ecto, domain, hLDLR EGF-domains a or hLDLR EGF-AB was also assessed by enzyme-linked immunosorbent assay based on ELISA. Briefly, hLDLR-ecto, hLDLR EGF-A-hFc or hLDLR EGF-AB-hFc, each 2 µg/ml, were applied to 96-well plate in PBS-buffer over night at 4°C, and nonspecific binding sites blocked with BSA treatment. This tablet was used for the measurement of free hPCSK9-mmh PCSK9 in solution-mmh, previously in equilibrium with various concentrations of mAb anti-hPCSK9. The same number of hPCSK9-mmh (500 PM) was pre-mixed with different doses of the antibody in the concentration range from 0 to ~50 nm in serial dilutions, then held a one-hour incubation at room temperature for �of stijene equilibrium binding of the antibody-antigen. After reaching equilibrium, samples of the solutions were transferred to covered with a receptor or receptor fragments tablets. After one hour of binding, the plates were washed, and associated hPCSK9-mmh were detected using HRP-conjugated anti-myc antibody. The values of IC50(PM) was defined as the amount of antibody required for 50% reduction hPCSK9-mmh associated with applied on the tablet with the receptor or receptor fragments. The results show that specific mAb functional block PCSK9 binding to three receptors at neutral pH of 7.2) and acidic pH (5,5).

Table 14
AbpH 7,2pH 5.5
The surface tablet
hLDLR-ectoEGF-AEGF-ABhLDLR-ectoEGF-AEGF-AB
316P<125<125<125<125<125<125
144146<1251492538447
Control I->100000>100000->100000>100000
Control II288510274411528508
Control III3036353917427871073

The ability of mAb to block the binding of mutant hPCSK9 GOF-hPCSK9(D374Y)-mmh domain hLDLR EGF-A or a domain hLDLR EGF-AB (values of IC50in the field of PM) was also determined using the above enzyme-linked immunosorbent assay based on ELISA, for which we used a fixed amount of 0.05 nm hPCSK9(D374Y)-mmh.

Table 15
pH 7,2pH 5.5
The surface tablets
EGF-AEGF-ABEGF-AEGF-AB
316P20313911231139
300N13514234633935
Control I>100000>100000>100000>100000
Control II7257129118
Control III537427803692

The ability of mAb to block the binding mmPCSK9 or mPCSK9 domain hLDLR-ecto, domain, hLDLR EGF-A or a domain hLDLR EGF-AB (values of IC50in the field of PM) was evaluated at neutral pH using the above enzyme-linked immunosorbent assay based on ELISA, for which the ISP�litovali fixed number of 1 nm mmh-labeled mmPCSK9 or 1 nm mPCSK9.

Table 16
1 nm mmPCSK9-mmh1 nm mPCSK9-mmh
hLDLR-ectoEGF-AEGF-ABEGF-AEGF-AB
316P<250<250<250<250<250
300N255256290>33000>33000

The ability of mAb to block the binding hPCSK9, mmPCSK9, rPCSK9, maPCSK9, mfPCSK9 or mPCSK9 domain hLDLR EGF-A (value of IC50in the field of PM) was evaluated at neutral pH (7,2) (table 17) or acidic pH (5.5 and table 18) using the above enzyme-linked immunosorbent assay based on ELISA, which used a fixed number of 0.5 nm hPCSK9-mmh, 1 nm mmPCSK9-mmh, 1 nm rPCSK9-mmh, 1 nm maPCSK9-h, 0.3 nm mfPCSK9-mmh or 1 nm mPCSK9-mmh.

Table 17
mmPCSK9rPCSK9maPCSK9mfPCSK9mPCSK9
316P<125<250266234975305
300N182460>100000>100000473>100000
Control I->100000>100000>100000>100000>100000
Control II1468325722038361855
Control III249293>100000245572>100000

Table 18
hPCSK9mmPCSK9rPCSK9maPCSK9mPCSK9
316P<125<250428801299991
300N2233704100000100000100000
Control I>10000>100000100000100000100000
Control II154<2501164083392826
Control III390376>100000414>100000

Also determined the ability 316P and control I block the binding hPCSK9 with hLDLR. Briefly said�I, through a combination with an amine recombinant hLDLR or hLDLR-EGFA-mFc was immobilizovana chips on the BIACORE™ CM5. The mixture of antigen-antibody of 100 nm hPCSK9-mmh and R, control I mAb or nonspecific hPCSK9 MABs (each a CR 250 nm) were incubated at room temperature for 1 hour, then applied to the surface or hLDLR hLDLR-EGFA a flow rate of 10 μl/min for 15 minutes at 25°C. were Recorded changes in RU due to binding between the free hPCSKS-mmh mixed with hLDLR or hLDLR-EGFA. Binding hPCSK9 with hLDLR or hLDLR-EGFA completely blocked R and 300N, but not the control I mAb.

Example 9. Epitope mapping

To determine the specificity of binding with the epitope has prepared a group of three chimeric protein PCSK9-mmh, in which specific domains of human PCSK9 domains have been replaced by mouse PCSK9. Chimeric protein #1 contained the murine prodomain PCSK9 (amino acid residues 1-155 of SEQ ID no:757), followed by human catalytic domain of PCSK9 (residues 153-425 SEQ ID no:755) and murine C-terminal domain of PCSK9 (residues 429-694 SEQ ID no:757) (mPro-hCat-mC-term-mmh). Chimeric protein #2 contained human prodomain PCSK9 (amino acid residues 1-152 of SEQ ID no:755), followed by murine catalytic domain of PCSK9 (residues 156-428 SEQ ID no:757) and murine C-terminal domain of PCSK9 (residues 429-694 SEQ ID no:757) (hPro-mCat-mC-term-mmh). Chimeric protein #3 contains murine prodomain and murine PCSK9 catalytic domain of PCSK9 which�should contain human C-terminal domain of PCSK9 (residues 426-692 SEQ ID no:755) (mPro-mCat-hC-term-mmh). In addition, hPCSK9 was obtained with a point mutation D374Y (hPCSK9(D374Y)-mmh).

The specificity of binding of the mAb with the studied proteins hPCSK9-mmh, murine PCSK9-mmh, chimeric proteins #1, #2 and #3, as well as hPCSK9(D374Y)-mmh checked as follows: mAb was coated on 96-hole tablet overnight at 4°C, then the tablet was added to each of the subjects of proteins (1,2 nm). After 1 hour of binding at room temperature the plate was washed and the bound test protein was determined using a polyclonal HRP-conjugated antibodies anti-myc (++ = OD >1,0; + = OD 0,4-1,0; - = OD <0,4).

Table 19
AntibodyhPCSK9mPCSK9Chimeric proteinhPCSK9(D374Y)
#1#2#3
H1M300++-+++-++
H1M309++--- ++++
H1M312++---++++
H1M492++----+
H1M493++---++++
H1M494++--+++++
H1M495++---++++
H1M496++---++++
H1M497 ++--+++++
H1M498++---+++
H1M499++---++++
H1M500++-++--++
H1M502++---++++
H1M503++--++-++

H1M504++- ---+
H1M505++-+++-++
H1M508++---++++
H1H318P++-++--++
H1H334P++-++--++
H1H316P++++++++++++
H1H320P++--++ -++
Control I++---++++

The binding specificity 316P, 300N and control anti-hPCSK9 MABs with hPCSK9-mmh, mPCSK9-mmh, mmPCSK9-mmh, mfPCSK9-mmh, rPCSK9-mmh, chimeric proteins #1, #2 and #3, as well as with hPCSK9(D374Y)-mmh checked, as described above, but protein concentration was 1.7 nm (- = OD <0,7; + = OD 0,7-1,5; ++ = OD >1,5).

Table 20
316P300NControl IControl IIControl III
hPCSK9-mmh++++++++++
mPCSK9-mmh++--++++
mmPCSK9-mmh++++++++ ++
mfPCSK9-mmh++++++++++
rPCSK9-mmh++--+++
Chimeric protein No. 1++++-++++
Chimeric protein No. 2++++-++++
Chimeric protein No. 3+++++++++
hPCSK9(D374Y)++++++++++

Similar results for some mAb were obtained using analysis of binding BIACORE™. In short, 316P, 300N or control I mAb was fixed on the amino�knit the chip CM 5 with anti-hFc CM5, and 100 nm of each protein was applied to the surface with fixed mAb. Determined changes EN as a result of binding of each protein with the surface of the mAb.

Table 21
AntibodyhPCSK9mPCSK9Chimeric protein
#1#2#3
316P500505529451467
300N320132437610
Control I6574369

To further assess the binding specificity 316P, which cross-reacts with mPCSK9-mmh, was developed by cross-competitive ELISA analysis, to evaluate the binding specificity of the domain. Briefly, mAb, specific to hee�Arnim proteins #1, #2 or #3, first applied to the surface of 96-well plates over night at a concentration of 1 μg/ml To each well was then added human PCSK9-mmh (2 μg/ml) followed by incubation for 1 hour at room temperature. 316P (1 μg/ml) was added and incubated for another hour at room temperature. Associated with plansyou S then determined by HRP-conjugarea anti-hFc polyclonal antibody. Despite the fact that the binding 316P with hPCSK9-mmh did not depend on the presence of mAb, specific for the chimeric protein #2 or chimeric protein #3, tying 316P with hPCSK9-mmh greatly reduced because of the presence of antibodies specific for the chimeric protein #1.

Example 10. Evaluation of the profile of binding to the antigen on the basis of the BIACORE™

Profiles of binding of the antibodies was also determined to 316P, 300N, control I, II and III mAb using BIACORE™1000. In short, hPCSK9-mmh was fixed on the surface of the anti-myc. The first anti-hPCSK9 mAb (50 μg/ml) was applied to the surface associated with PCSK9 for 10 minutes at a flow rate of 10 μl/min at 25°C. Then struck a second anti-hPCSK9 mAb (50 μg/ml) to the surface fixed to the first mAb for 10 minutes at a flow rate of 10 μl/min at 25°C. was Evaluated by the ability of the first mAb to block the binding of the second mAb, which were expressed in percentage of inhibition.

Table 22
The first mAbSecond mAb
316P300NControl IControl IIControl III
316P1001012799101
300N771001282-2
Control I61210069
Control II91102-61003
Control III7310-121100

Example 11. Increased gripping LDL antibodies anti-hPCSK9

The ability of anti-hPCSK9 MABs took�pay capture LDL in vitrowas determined using the cell lines of hepatocellular carcinoma in human liver (HepG2). The HepG2 cells were seeded on 96-well plates at 9×104cells per well in complete DMEM and incubated at 37°C, 5% CO2for 6 hours to obtain monolayers of HepG2. Added human PCSK9-mmh at a concentration of 50 nm in lipoprotein-deficient medium (LPDS) and the test mAb at various concentrations ranging from 500 nm to 0.98 nm in the environment of LPDS. Data were expressed as the values of the IC50for each experiment (IC50= concentration of the antibody in which there is an increase in the capture of LDL by 50%). In addition, it was also experimentally demonstrated that 316P and 300N were able to completely reverse the inhibitory effect hPCSK9 to capture the LDL, while for the control I mAb or H1M508 anti-hPCSK9 MABs treatment inhibitory effect was achieved by about 50%.

Table 23
AntibodyIC50(nm)
316P21,30
300NOf 22.12
Control I>250
H1M508>250

JV�the ability of anti-hPCSK9 MABs to draw inhibitory effect on seizure LDL PCSK9 protein in different species of mammals also tested on cell lines HepG2, as described above. Briefly, HepG2 cells were incubated overnight with serial dilutions of antibody in the medium of LPDS (starting at a concentration of 500 nm) and 50 nm hPCSK9-mmh, mfPCSK9-mmh, mPCSK9-mmh, rPCSK9-mmh or maPCSK9-h. The HepG2 cells also were incubated overnight with serial dilutions of antibody in LPDS (from 50 nm) and 1 nm hPCSK9(D374Y). As shown in table 24, while 316P was able to completely reverse the inhibitory effect on LDL all tested protein PCSK9, 300N was able to pay only the inhibitory effect hPCSK9, hPCSK9(D374Y) and mfPCSK9 to capture the LNP. Values are expressed in nm IC50.

Table 24
316P300NControl IControl IIControl III
hPCSK9-mmh14,112,6>50013,412,4
hPCSK9(D374Y)-mmh2,11,1>500,70,6
mfPCSK9-mmh 14,713,4>50014,213,6
mPCSK9-mmhOf 21.2>500>50019>500
rPCSK9-mmh27,7>500>50021,9>500
maPCSK9-h14,4>500>50029,512,7

Example 12. Neutralization of the biological effects of hPCSK9 in vivo

To assess the biological effect of neutralizing PCSK9 were sverkhekspressiya hPCSK9 in mice C57BL/6 by hydrodynamic delivery (HDD) of DNA constructs encoding full length hPCSK9-mmh. 4 mice (C57BL/6) received an injection idle vector/saline (control), and 16 mice received an injection of 50 µg of a mixture of hPCSK9-mmh-DNA/saline in the tail vein in a volume equal to 10% of their body mass. On day 7 after HDD shipping hPCSK9 led to a 1.6-fold increase in total cholesterol, a 3.4-fold increase in LDL cholesterol (LDL's) and 1.9-fold increase of C�and without HDL (relative to control). The hPCSK9 levels in serum on day 7 were always above 1 μg/ml, as data showed quantitative ELISA.

Introduction H1M300N on day 6 after HDD in 3 experimental groups (1, 5 or 10 mg/kg) (n=4 per group) by intraperitoneal injection (I/b) led to a significant smoothing of the level of serum cholesterol. 18 hours after the introduction of total cholesterol decreased by 9.8%, 26.3% and 26,8%, LDL-fell by 5.1%, 52.3% and 56,7% and cholesterol without HDL decreased by 7.4% to 33.8% and 28.6% in the groups treated with 1, 5 or 10 mg/kg H1M300N, respectively.

Example 13. Pharmacokinetic and chemical study of the serum in monkeys

The pharmacokinetic study (FC) was performed on intact male cynomolgus macaque (Macaca fascicularis) with body weight in the range of 5-7 kg and aged 3 to 5 years.

The distribution by groups.Monkeys were divided into 5 treatment groups: Treatment group 1 (n=3) received a control buffer (10 mm sodium phosphate, pH 6, 1 ml/kg); treatment group 2 (n=3) received 1 ml/kg 316P (5 mg/ml); treatment group 3 (n=3) received 1 ml/kg 300N (5 mg/ml); treatment group 4 (n=3) received 1 ml/kg 316P (15 mg/ml); and treatment group 5 (n=3) received 1 ml/kg 300N (15 mg/ml). All drugs were injected through the IV bolus with subsequent flushing with 1 ml of saline. The amount of total dose (ml) was calculated according to the most recent indicator of body weight (each animal were weighed twice in per�od acclimatization and once a week throughout the study. On day 1 was administered a single dose of the test mAb or control buffer.

Care for the animals.Animals were kept under conditions of controlled temperature and humidity. Target temperature and relative humidity lying in the interval from 18 to 29°C and 30-70%, respectively. Automatic lighting system was provided by a 12-hour diurnal cycle. The dark cycle can be interrupted for activities related to research, or maintenance of the premises. Animals were placed in individual cells, which is consistent with the Law on the protection of the rights of animals and the recommendations in the Guide for the care and use of laboratory animals (National Research Council 1996).

Diet and nutrition.Animals received food twice a day in accordance with the standard procedures of work SNBL USA. Animals were not given food, if it was required by a specific procedure (e.g., before the blood sampling for biochemical analysis of serum, fence urine, or if the procedure was carried out, which required the introduction of sedatives). The composition of food products has passed the standard procedure of analysis for the presence of pollutants, and it was determined that it was consistent with the results of the manufacturer.

Scheme of the experiment.The required number of animals were selected from the animal house SNBL USA. The health of the animals was analyzed wind�nary staff, and performed biochemical analysis of serum, hematological examination and screening coagulation. For study b took 16 healthy males. 15 males were distributed among the specific research groups, and the remaining animal was kept as a spare. For the distribution of animals in groups to research used stratified randomizearray scheme which includes checking the level of serum cholesterol (based on the two fences for acclimatization).

The acclimatization period.Pre-quarantine the animals are acclimatized to the conditions of the study areas at least 14 days prior to the introduction of drugs. Data on the stage of acclimatization was going in all animals, including the spare. All animals were assessed for abnormal behavior that could affect the results of the study. After 1 day emergency animal was returned to the vivarium.

Blood.Blood sampling was performed by venipuncture from a peripheral vein at a fixed in the minds of animals. Where possible, blood was taken single image, and then divided into the required portions.

FC research. Blood samples (1.5 ml) were collected into test tubes to separate the serum (SST) prior to the introduction, after 2 minutes, 15 m�chickpeas, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, and then every 24 hours. Serum samples for storage was transferred into two vials and stored at -60°C or below.

Serum samples were analyzed using the optimized procedure, ELISA (ELISA). In short, tablets for micrometrology initially covered hPCSK9-mmh. Then the subjects mAb 316P or 300N was applied on the tablet hPCSK9-mmh. Fixed 316P or 300N were detected using biotinylated murine anti-hIgG4 with subsequent binding to NeutrAvidin-HRP. As a standard used in various concentrations 316P or 300N in the range from 100 to 1.56 ng/ml. as the zero standard (0 mg/ml) used a one percent serum monkeys (analytical matrix) in the absence of 316P or 300N. The results are shown in Fig.2, indicate a dose-dependent increase in the level 316P and 300N in serum. The parameters of FC were analyzed using the software WinNonlin (accompanimental analysis, model 201 - /bolus).

Table 25
Pharmacokinetic parameters316P300N
5 mg/kg 15 mg/kg5 mg/kg15 mg/kg
Tmax(h)0,4280,1054,020,428
Cmax(ág/ml)1845272261223
T1/2(h)83184215366

Biochemical analysis of the serum.Blood samples were collected before injection, 12 hours, 48 hours, and then every 48 hours for clinical biochemical analysis, in particular, lipid profiles (cholesterol, LDL, HDL, triglycerides). With the exception of the sample taken 12 hours after injection, before the blood sampling, all animals received no food during the night. Sample volume was approximately 1 ml. the Parameters of the chemical composition was determined using an automatic analyzer Olympus. Measured parameters (code Xybion): albumin (ALB); alkaline phosphatase (ALP); alanineaminotransferase (ALT); aspartatamino (AST), total bilirubin (TBIL); calcium (Ca); total cholesterol (TCho), creatine kinase (CK), creatinine (CRN); gamma glutaminase�inasa (GGT); glucose (GLU); inorganic phosphorus (IP); total protein (TP); triglycerides (TRIG); urea nitrogen blood (BUN); globulin (GLOB); the ratio of albumin/globulin (A/G); chloride (Cl), potassium (K), sodium (Na); LDL and HDL cholesterol. Any remaining serum was stored at -20°C or below and removed the waste in no earlier than one week after analysis.

The results for samples up to 105 days post-dose are shown in Fig.3-7. There was a decrease of total cholesterol and LDL-X animals treated 316P and 300N, regardless of the dose within 24 hours after the first dose. Total serum cholesterol fell hard and fast (~35%, Fig.3). Strong reduction of ~80% was observed for LDL-X (Fig.4-5) on day 6. In animals that received a dose of 15 mg/kg 300N, the reduction in total cholesterol (reduction of ~10-15%) and LDL-X (reduction ~40%) lasted not less than 80 days of the study. Furthermore, HDL cholesterol was elevated in animals that received 316P at 15 mg/kg (Fig.6). Animals that received a higher dose (15 mg/kg), or 316P or 300N, also showed a decrease in triglycerides during the study (Fig.7). 316P showed maximum inhibition of LDL levels-up to 80% compared with baseline. The duration of the period of inhibition was dependent on the dose, not less than 60% suppression (compared with baseline levels of LDL-X) lasted about 18 days (dose 5 mg/kg) and approximately 45 days (dose of 15 mg/kg. 300N demonstrates a clear pharmacodynamic profile that is different from 316P. Suppression of LDL-the introduction of 300N was maintained for a significantly longer period of time when comparable doses (50% of inhibition of lipoprotein-X within 28 days after dose 5 mg/kg and 50% of inhibition of lipoprotein-X in approximately 90 days after the dose of 15 mg/kg). According to the measurement of ALT and AST were observed noticeable or measurable changes in the liver. All animals receiving anti-PCSK9 antibody in the study demonstrated a rapid reduction of LDL-and total cholesterol.

A similar effect of reducing LDL-X under the influence of 316P and 300N were observed in cynomolgus rhesus, which received a single subcutaneous (s/C) injection of 5 mg/kg 316P or 5 mg/kg 300N (Fig.8). And 316P and 300N dramatically inhibit the levels of lipoprotein-X and support the effect of reducing LDL-X for about 15 and 30 days, respectively (Fig.8). Pharmacodynamic effect (about 40% inhibition of lipoprotein-X) apparently correlates with functional levels of antibodies in the serum of monkeys (Fig.9). Along with the reduction of antibody levels below 10 μg/ml inhibition of lipoprotein-X is also reduced. In addition, 300N showed a significantly higher level of half-life from circulation compared to 316P, which means longer for the observed inhibition of lipoprotein-X.

Table 26
Pharmacokinetic parameters316P300N
Tmax(h)6084
Cmax(ág/ml)4663
T1/2(h)64286

Example 14. Decreasing the breakdown of LDL receptor antibodies anti-hPCSK9

To assess the biological impact of PCSK9 on the level of activity of repatriation and subsequent effects on serum levels of lipoprotein-X, hPCSK9 was administered intravenously to mice expressing hPCSK9, but not mPCSK9 (mousePCSK9hu/hu). In particular, micePCSK9hu/huvia the tail vein injected with PBS (control) or 1.2 mg/kg hPCSK9-mmh. Six hours after the introduction of hPCSK9 was observed a 1.4-fold increase (relative to baseline) in total cholesterol and 2.3-fold increase in serum LDL-H. analysis of the levels of repatriation LDL in a separate cohort (n=3) animals 4 hours after the introduction of hPCSK9 showed a significant decline recorded LDL receptors in the liver homogenates.

To assess the biological vozdeistviyami-hPCSK9 levels of repatriation LDL and subsequent influence on serum levels of lipoprotein-X, 316P and nonspecific to hPCSK9 mAb injected micePCSK9hu/huin equivalent dose (5 mg/kg/b) 20 hours before mentioned the introduction of the protein hPCSK9-mmh. Four hours after the introduction of hPCSK9 mice were sacrificed and took samples of eight types of tissues (liver, brain, lungs, kidneys, heart, ileum, adrenal and pancreas), in which the levels of LDL receptors was determined by Western-blotting. Changes in the levels of LDL receptor was observed only in the liver. Compared with the control dosage FSB introduction 316P significantly blocked PCSK9 mediated increase of total cholesterol and LDL cholesterol (LDL-X=2,49 mg/DL baseline and 3.1 mg/DL 6 hours after PCSK9; which corresponds to an increase of 25% compared to 135% in the presence of carrier). Prior administration of nonspecific to hPCSK9 MABs blocked the increase in LDL-X approximately 27% compared to pure FSB (LDL-X=4,1 mg/DL compared with those observed for the FSB to 5.6 mg/DL). Analysis of the levels of LDL receptors in a separate cohort of mice (n=3 per group) showed a significant decrease in the levels of LDL receptors with the introduction of PCSK9, which were blocked 316P, but not blocked nonspecific to hPCSK9 MABs (Fig.10).

The effect of different doses of 316P also evaluated for micePCSK9hu/huwith elevated LDL levels and elevated levels of hPCSK9. MousePCSK9hu/huwnac�Les transferred on a diet with a high content of carbohydrates per 8 weeks which resulted in ~2-fold increased levels of lipoprotein-X and hPCSK9. The mice were injected either 316P or nonspecific to hPCSK9 MABs, each in a dose of 1 mg/kg, 5 mg/kg or 10 mg/kg. Serum was collected after 24 hours and then analyzed the levels of LDL-H. 316P effectively reduce the levels of LDL-X dose-dependent manner (Fig.11). In addition, 316P in the dose of 10 mg/kg in 24 hours quickly reduce the levels of LDL's to the original (before the diet) (data not shown).

Example 15. Studies of FC in mice

The study, FC was performed on C57BL/6 aged 6 weeks and heterozygous mice hPCSK9 aged 11-15 weeks. A single injection of control I, 316P or 300N, each in a dose of 10 mg/kg, conducted n/a. serum Samples were measured for levels of hIgG at time 0 hours (before selection), 6 hours day 1, 3, 6, 10, 14, 21, 28, 35, 42 and 56, a total of 12 points in time using techniques of capture anti-hFc and detection of anti-hFc using a sandwich ELISA (Fig.12 and 13). All mAb were reached Tmaxafter about 3 days at appropriate levels Cmaxabout 47-115 μg/ml for C57BL/6 and 55-196 µg/ml for heterozygous mice hPCSK9. On day 56 the levels of mAb control (I was about 12 μg/ml, and the levels were 300N approximately 11 μg/ml, whereas levels 316P were somewhat less than 0.02 μg/ml in mice C57BL/6. On day 56 in heterozygous mice hPCSK9 levels control mAb (I was about 29 μg/ml, whereas levels 300N and 316P was� below the detected limit (BQL) of 0.02 μg/ml.

Example 16. Binding of anti-hPCSK9 MABs to mutant/variant hPCSK9

To further assess the binding between hPCSK9 and anti-hPCSK9 MABs received 21 variant hPCSK9 protein, each of which contained a single point mutation, as well as two variant hPCSK9 protein, each of which contained a double mutation. Each selected antibody was kept on the surface F(ab')2anti-hIgG formed by direct chemical combination with the chip BIACORE™ for the formation of the surface bound antibodies. Each mmh-labeled variant hPCSK9 at various concentrations from 100 nm to 25 nm is then deposited on the surface of the fixed antibody at a flow rate of 60 μl/min for 240 seconds, and the dissociation variant hPCSK9 antibodies was monitored in real time for 20 minutes at 25°C. NS: in these experimental conditions, the binding was not observed (KD=M×10-9; T1/2=min; WT=native).

td align="center"> 36,0
Table 27
316P300NControl IControl IIControl III
KD T1/2KDT1/2KDT1/2KDT1/2KDT1/2
Native1,00370,6912030,6160,103330,60481
P70A1,42321,688019,0160,241680,90325
S127R2,40361,8711025,0180,262880,55550
D129G1,27361,408822,9180,192570,75445
S147F1,29329,072421,1150,221780,231468
S153R5,6440,5614136,6170,093223,3360
E159R6,9650,829431,7160,083502,9768
T162R 0,98430,5814029,0170,093220,48362
D192ROf 1.35280,7511930,2150,09326NBNB
R194E0,38710,65129Of 31.4160,07389NBNB
E197R1,42270,6711530,2170,09339NBNB
R215H086 411,039837,8170,65490,74272
R215E0,90431,817744,0164,48120,78276
F216L1,83320,99121Of 21.215Of 1.35390,33880
R237E2,48151,0310929,6150,07481To 5.8943
D238R410 10,7812325,9190,241440,141273
A341RA 1.54210,3419028,7180,083400,88200
D343ROf 7.8861,188927,0160,084024,1366
R357H6,2630A 6.536626,4130,63165Of 1.91896
E366K2,9213228,8180,46690,38808
D374Y2,04150,668325,0170,082851,02161
V380M0,48632,822825,9170,151770,35711
P70A, S147F1,1834A 7.872423,5180,231640,79348
E366K, V380M3,3312125,5180,59600,52551

The results show that in the case of mutation of residue D238 affinity binding R with hPCSK9 was decreased by a factor of >400 times, with KDapproximately 1×10-9M to 410×10-9M and T1/2shortened by about 30 times, with a 37 to 1 minute, which indicates the binding R with the epitope on hPCSK9 containing D238 from hPCSK9 (SEQ ID no: 755). In addition, the analysis techniques BIACORE™ shows that the affinity of binding of 316P and T1/2decreased by about 5 to 10 times, if the mutation occurred at residues 153, 159 or 343. Specifically, KDdecreased from about 1×10-9M to about 5-8×10-9M, if there is any mutation of residues S153, E159 or D343; while T1/2decreased from about 37 minutes to about 4-6 minutes.

Binding 300N with hPCSK9 declined about 50 times, if the mutation of the residue at position 366, which led to the decrease of KDwith approximately 0.7×10-9M to about 36×10-9M and shorter T1/2with approximately 120 to 2 minutes. These results indicate that the binding of 300N with the epitope on hPCSK9 containing E366 from hPCSK9 (SEQ ID no: 755). In addition, the analysis techniques BIACORE™ shows that the affinity of the con�of ivania 300N and T 1/2decreased from 2 to >10 times, if the mutation occurred on the balance or 380 147. In particular, KDdecreased from approximately 0,69×10-9M to about 2-9×10-9M, if there is any mutation of residues S147 or V380; while T1/2was reduced from about 120 minutes to about 24-66 minutes. Compared to 316P linking 300N with hPCSK9 not be relinquished due to a mutation at residue 238.

In contrast, the control antibody I showed no change in the affinity of binding or T1/2as a result of any mutations in subjects positions; a control antibody II showed reduced 40-fold affinity upon mutation of residue 215 (R215E) (~0.1 x 10-9to ~4,5×10-9), and T1/2it was about 27 times shorter (~333 to 12 minutes); whereas the control antibody III showed a lower affinity upon mutation of residue 237 (KDdecreased from ~0,6×10-9to ~of 5.9×10-9and T1/2dropped from ~481 ~43 minutes).

The binding specificity 316P, 300N and control anti-hPCSK9 MABs with options hPCSK9-mmh was tested using the immunoassay-based ELISA. Anti-PCSK9 mAb was coated on 96-hole tablet overnight at 4°C. Each mmh-labeled variant hPCSK9 in the lysate supernatants of transient transfections of CHO-k1 were applied to coated tablet antibodies at different concentrations in the range from 0 to 5 nm. After 1 hour of binding at room temperature PLA�shetu were washed and bound option hPCSK9 was determined using a polyclonal HRP-conjugated antibodies anti-myc (- = OD < 0,7; + = OD 0,7-1,5; ++ = OD >1,5).

Table 28
hPCSK9 or option316P300NControl IControl IIControl III
hPCSK9(WT)++++++++++
hPCSK9(S127R)++++++++++
hPCSK9(D129G)++++++++++
hPCSK9(S153R)++++++++++
hPCSK9(R215H)++++++++++
hPCSK9(F216L) ++++++++++
hPCSK9(R237E)++++++++++

hPCSK9(D238R)-++++++++
hPCSK9(A341R)++++++++++
hPCSK9(D343R)++++++++++
hPCSK9(R357H)++++++++++
hPCSK9(E159R)++++++++++
hPCSK9(T162R) ++++++++++
HPCSK9(D192R)++++++++-
hPCSK9(R194E)++++++++-
hPCSK9(E197R)++++++++-
hPCSK9(R215E)++++++++++
hPCSK9(P70A)++++++++++
hPCSK9(S147F)++++++++++
hPCSK9(E366K)+++ ++++++
hPCSK9(V380M)++++++++++
hPCSK9(P70A, S147F)++++++++++
hPCSK9(E366K, V380M)+++++++++

Example 17. The effect of 316P on normolipidemia and hyperlipidemics hamsters

The ability of anti-PCSK9 mAb 316P to reduce the level of serum LDL-experienced normolipidemia or hyperlipidemics Golden Syrian hamsters (Mesocricetus auratus). Male Syrian hamsters aged 6-8 weeks, weighing in the range of 80-100 grams were acclimatized for 7 days before the start of the study. All animals were fed a diet with standard food or hyperlipidemics diet, during which food was added 0.1% cholesterol and 10% coconut oil. 316P mAb was administered to the hamsters in the form of a single subcutaneous injection at doses of 1, 3 or 10 mg/kg normolipidemic hamsters in doses of 3, 10 or 30 m�/kg for hyperlipidemics hamsters. Serum samples were collected from all groups after 24 hours and 7, 14 and 22 days after administration, conducting the analysis of the levels of serum lipids at that time and comparing it with the original levels in samples taken 7 days before the introduction of the mAb. Total cholesterol in the circulatory system and of lipoprotein-X in normolipidemic hamsters was significantly reduced dose-dependent manner compared with the introduction of media. As shown in Fig.14, the introduction of 316P effectively reduce the levels of LDL-X almost 60% in seven days after administration of the highest tested doses (10 mg/kg). A similar effect of reducing cholesterol with the introduction of 316P at hyperlipidemics hamsters were observed.

1. The human antibody or antigen-binding fragment of a human antibody that specifically binds human paraproteinemias of subtilisin/Kexin type 9 ("hPCSK9"), where the antibody or antigen-binding fragment contains the CDR sequences of heavy and light chain represented by the sequences SEQ ID NO.:76, 78, 80, 84, 86, 88 or SEQ ID NO: 220, 222, 224, 228, 230, 232.

2. The antibody or antigen-binding fragment according to claim 1 comprising a pair of amino acid sequences of variable region of the heavy chain/variable region light chain (HCVR/LCVR), represented by the sequences SEQ ID nos: 90/92 or 218/226.

3. An isolated nucleic acid molecule, coding�I antibody or antigen-binding fragment according to claim 1 or 2.

4. Expression vector to obtain the antibody according to claim 1 comprising the nucleic acid molecule of claim 3.

5. The method of producing the antibody anti-human PCSK9 or antigen-binding fragment of the antibody according to claim 1, comprising the stages of introducing the expression vector according to claim 4 in an isolated host cell, growing the cell under conditions permitting production of the antibody or its fragment, and obtaining the antibody or its fragment, produced in this way.

6. Pharmaceutical composition for treating a disease or condition that is amenable to mitigation, enhancement, suppression or prevention using the PCSK9 antagonist, including an antibody or antigen-binding fragment according to claim 1 or 2 in a therapeutically effective amount and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, further comprising a second therapeutic agent, wherein the second therapeutic agent selected from the group consisting of one of the inhibitors of 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors, statin, an inhibitor of cholesterol or capture the reabsorption of bile acids, the agent that increases the catabolism of lipoproteins and activator of transcription factor LXR.

8. The pharmaceutical composition according to claim 6, where PCSK9 mediated disease or condition selected from the group consisting of hypercholesterolemia, hyperlipidemia, �the Perizzites HDL, heterozygous genetic hypercholesterolemia, intolerance of statins, statin uncontrolled, the risk of hypercholesterolemia, dyslipidemia, cholestatic liver disease, nephrotic syndrome, hypothyroidism, obesity, atherosclerosis and cardiovascular diseases.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention refers to biochemistry, particularly to monoclonal antibodies binding to c-Met and able to inhibit both ligand-dependent, and ligand-independent c-Met activation. The above antibodies, as well as a composition containing them can be used for producing a therapeutic agent for treating cancer.

EFFECT: invention enables specifically inhibiting both ligand-dependent, and ligand-independent c-Met activation, particularly inhibiting c-Met dimerisation.

34 cl, 111 dwg, 4 tbl, 27 ex

FIELD: medicine.

SUBSTANCE: presented invention refers to immunology. There are described versions of antibodies or antigen-binding fragments binding to human 4-1BB. One of the versions is characterised by the presence of respective 3 CDR light chain sites and 3 CDR of heavy chain sites. The other version is characterised by the presence of the heavy and light chain with respective amino acid sequences. There are described versions of a pharmaceutical composition for reducing tumour growth or for treating cancer in an individual, as well as methods for reducing the tumour growth or treating cancer in the individual using the versions of antibodies or antigen-binding fragments in a therapeutically effective amount. What is described is a method of treating cancer with using a combination of the antibody and an immunotherapeutic agent. There are disclosed: versions of coding nucleic acids, an expression vector and a host cell containing the antibody expression vector. What is disclosed is a method for producing the antibody with using the cell.

EFFECT: invention provides the new agonist anti-human 4-1BB (also called CD137 or TNFRSF9) antibodies, which recognise an epitope within the amino acid residues K115, C121, R134, R154, V156 of the antigen, have Kd affinity measured by the BIACORE method and approximated to nM, eg 0,4 nM or 8 nM (for the anti-IgG1 antibody format) that can find application in the therapy of cancer and cancerous diseases.

19 cl, 8 dwg, 11 tbl, 9 ex

FIELD: chemistry.

SUBSTANCE: invention refers to biotechnology. What is presented is nucleic acid coding protein possessing acetyl-CoA-carboxylase activity making up the deficiency of acetyl-CoA-carboxylase in yeast, wherein a nucleotide sequence is specified in nucleic acid, which contains a nucleotide sequence: (a) coding protein consisting of the amino acid sequence SEQ ID NO:2; (b) hybridised in the hard conditions with nucleic acid complementary to SEQ ID NO:1; (c) SEQ ID NO:1; and (d) hybridised in the hard conditions with nucleic acid consisting of the complementary nucleic sequence coding protein SEQ ID NO:2; wherein SEQ ID NO:1 and 2 are disclosed in the description. There are also described: acetyl-CoA-carboxylase (SEQ ID NO:2) increasing the host-specific arachidonic acid content; a recombinant vector containing the above nucleic acid; and a cell transformed by the above vector for producing the fatty acid composition rich in arachidonic acid. What is presented is a method for producing the fatty acid composition involving culturing the above cell and collecting the fatty acid composition from the transformed cell culture.

EFFECT: invention enables producing the fatty acid composition rich in arachidonic acid in the host cell.

11 cl, 8 dwg, 5 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to molecular biology and medicine. Presented is using an oligonucleotide having a length of 10-30 nucleotides, which is specifically targeted on a natural antisense transcript of sirtuin 1 (SIRT1), for preventing or treating a sirtuin 1 (SIRT1)-related disease or disorder, wherein the above oligonucleotide increases sirtiun 1 (SIRT1) expression, and wherein the natural antisense transcript has a nucleotide sequence with any of SEQ ID NO:2-5.

EFFECT: presented is using the oligonucleotide for preventing or treating the sirtuin 1 (SIRT1)-related disease or disorder.

19 cl, 20 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: claimed inventions deal with a modified protein, nucleic acid, coding such protein, a vector, containing nucleic acid, and a carrier for biotin binding, which such protein is immobilised on. The characterised modified biotin-binding protein is obtained by the introduction of a mutation from one to several amino acid residues into a sequence, represented in SEQ ID NO:2, or an amino acid sequence, identical to the said sequence by 98% or more, and the presence of the biotin-binding activity, where at least one residue, selected from the group, consisting of residues from 1) to 4), presented below, is substituted with the residue of acidic amino acid or residue of neutral amino acid; 1) residue of arginine in position 104 SEQ ID NO: 2; 2) residue of lysine in position 141 SEQ ID NO: 2; 3) residue of lysine in position 26 SEQ ID NO: 2 and 4) residue of lysine in position 73 SEQ ID NO: 2.

EFFECT: claimed inventions make it possible to obtain the biotin-binding protein and can be applied for biotin binding.

14 cl, 6 dwg, 11 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to biotechnology, namely to novel IL-17-inhibiting polypeptides, corresponding to fused proteins, to compositions and their application for medicinal purposes. Polypeptide contains amino acid sequence, which is selected from group, consisting of GVTLFVALYD YKAFWPGDLS FHKGEKFQIL RTSDGDWWEA RSLTTGETGY IPSNYVAPVD SIQ (SEQ ID NO: 39), GVTLFVALYD YKAFWPGDIS FHKGEKFQIL RTSDGEWWVA RSLTTGEEGY IPSNYVAPVD SIQ (SEQ ID NO: 57) or GVTLFVALYD YKAFWPGDIS FHKGEKFQIL RTSDGEWWIA RSLTTGEEGY IPSNYVAPVD SIQ (SEQ ID NO: 107); amino acid sequence, which has, at least, 80%, preferably, at least, 90%, more preferably, at least, 95% identity of amino acid sequence with SEQ ID NO: 39, SEQ ID NO:57 or SEQ ID NO: 107; fragment or functional derivative of SEQ ID NO: 39, SEQ ID NO: 57 or SEQ ID NO: 107, obtained due to substitution, addition and/or removal of not more than 5 amino acids.

EFFECT: invention makes it possible to bind IL-17 with high specificity and affinity.

33 cl, 17 dwg, 3 tbl, 12 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to biotechnology, gene and protein engineering. There are engineered plasmids pCLm4/hygro-14D5 and pCHm2-14D5. The plasmids provide the synthesis in eukaryotic cells of polypeptides with light and heavy chain properties of a chimeric antibody, which are combined into the chimeric antibody ch14D5 class IgG1/kappa. The producing chimeric antibody ch14D5 recovered from the culture fluid of CHO-K1 cells transfected by plasmids pCLm4/hygro-14D5 and pCHm2-14D5 having a molecular weight of about 150 kDa. The antibody consists of two identical light chains, a constant portion of which corresponds to human kappa antibodies, and two identical heavy chains, a constant portion of which corresponds to human IgGI antibodies; having an amino acid sequence SEQ ID NO: 1 and SEQ ID NO: 2.

EFFECT: antibody is targeted to tick-borne encephalitis and provides the acute prevention of mice against tick-borne encephalitis.

3 cl, 7 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and can be used for recombinant production of human tissue factor (hTF). Constructed is a plasmid pHYP-10ETFCS6 having a length of 5,912 b.p. with a physical map presented on Fig. 2, for expression in a bacterium of the genus Escherichia, which is a precursor of the mutant [C245S] hTF containing an inseparable N-terminal leader peptide containing a deca-histidine cluster and an enterokinase identification sequence fused in a frame with a sequence coding the above mutein fused in the frame with the sequence coding the additional inseparable C-terminal peptide containing the deca-histidine cluster. A method for producing the precursor of the mutein[C245S]hTF contains culturing the producing bacterium in a nutrient medium, recovering inclusion bodies, solubilising the precursor protein, performing a metal chelator chromatography in the denaturation environment, re-folding and diafiltration of the protein solution. A method for producing the mature mutein[C245S] hTF involves detecting the N-terminal leader peptide from the above mutein precursor with using enterokinase and recovering the target protein.

EFFECT: invention enables increasing the level of biosynthesis and yield of pro-coagulation active hTF.

9 cl, 5 dwg, 1 tbl, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a completely human monoclonal antibody, which binds insulin-like growth factor-II (IGF-II) and has a cross responsiveness to IGF-I, as well as its antigen-binding fragment. There are disclosed a nucleic acid molecule coding an antibody according to the invention, a vector and a host cell for the expression of the antibody according the invention. There are described a pharmaceutical composition, as well as conjugates for treating and diagnosing malignant tumour, using the antibody according to the invention in preparing the therapeutic agent and a method for determining IGF-II and IGF-I levels in a patient's sample.

EFFECT: present invention can find further application in cancer therapy.

16 cl, 27 ex, 18 tbl

FIELD: medicine.

SUBSTANCE: invention relates to field of immunology. Claimed is isolated antibody to ICOS protein of people with increased effector function. Also described are cell and method of obtaining antibody in accordance with claimed invention, pharmaceutical composition, method of treating autoimmune disease or disorder, transplant rejection and malignancy of human T-cells, as well as method of depletion of ICOS-expressing T-cells, method of destroying germ centre structure in secondary lymphoid organ of primates, methods of depleting B-cells of germ centre of secondary lymphoid organ and circulating B-cells, which have undergone class switching, in primates.

EFFECT: invention can be further applied in therapy of diseases, mediated by T-cells.

33 cl, 21 dwg, 3 tbl

FIELD: medicine.

SUBSTANCE: presented invention refers to immunology. There are described versions of antibodies or antigen-binding fragments binding to human 4-1BB. One of the versions is characterised by the presence of respective 3 CDR light chain sites and 3 CDR of heavy chain sites. The other version is characterised by the presence of the heavy and light chain with respective amino acid sequences. There are described versions of a pharmaceutical composition for reducing tumour growth or for treating cancer in an individual, as well as methods for reducing the tumour growth or treating cancer in the individual using the versions of antibodies or antigen-binding fragments in a therapeutically effective amount. What is described is a method of treating cancer with using a combination of the antibody and an immunotherapeutic agent. There are disclosed: versions of coding nucleic acids, an expression vector and a host cell containing the antibody expression vector. What is disclosed is a method for producing the antibody with using the cell.

EFFECT: invention provides the new agonist anti-human 4-1BB (also called CD137 or TNFRSF9) antibodies, which recognise an epitope within the amino acid residues K115, C121, R134, R154, V156 of the antigen, have Kd affinity measured by the BIACORE method and approximated to nM, eg 0,4 nM or 8 nM (for the anti-IgG1 antibody format) that can find application in the therapy of cancer and cancerous diseases.

19 cl, 8 dwg, 11 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biotechnology and immunology. What is presented is a hybridoma cell line FP12H3-C2 deposited under No. DSM ACC2750 producing the anti-beta-amyloid antibody. Presented are methods for preparing an antibody, including a humanised antibody with using a hybridoma line according to the invention, as well as diagnostic techniques for a beta-amyloid associated disease or condition in a patient or a liability to such disease or condition, a method for determining a degree of the tissue involvement into amyloidogenic plaques, a method for monitoring minimal residual signs of the disease into a patient following treating with the antibody or its active fragment, a method for prediction of sensitivity in the patient treated with the antibody or its active fragment.

EFFECT: present invention can find further application in diagnosing and therapy of beta-amyloid related diseases, such as Alzheimer disease.

9 cl, 4 dwg, 5 tbl, 17 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a completely human monoclonal antibody, which binds insulin-like growth factor-II (IGF-II) and has a cross responsiveness to IGF-I, as well as its antigen-binding fragment. There are disclosed a nucleic acid molecule coding an antibody according to the invention, a vector and a host cell for the expression of the antibody according the invention. There are described a pharmaceutical composition, as well as conjugates for treating and diagnosing malignant tumour, using the antibody according to the invention in preparing the therapeutic agent and a method for determining IGF-II and IGF-I levels in a patient's sample.

EFFECT: present invention can find further application in cancer therapy.

16 cl, 27 ex, 18 tbl

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and immunology. There are presented optimised genes of light and heavy chains of Infliximab, an anti-tumour necrosis factor alpha (TNF-alpha) antibody, as well as a cell line VKPM-N-131, and a method for antibody biosynthesis. Nucleotide sequences of the genes coding the light and heavy chains of Infliximab are optimised in order to provide the content of codones most specific for mammals; the G/C content is expected to make 50-60% of the total composition; the absence of expanded tracts of a degenerate composition and the absence of RNA secondary structures.

EFFECT: Chinese hamster ovary cell line (CHO) produced by transfection by expression structures containing the genetic sequences according to the invention, enables producing at least 50 mg/l of the monoclonal antibody Infliximab.

4 cl, 3 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biotechnology and represents anti-nerve growth factor (NGF) antibodies. The present invention also discloses a pharmaceutical composition for relieving pain associated with a disease or a condition, wherein pain progression or persistence is mediated by NGF, containing the above antibodies, as well as a kit for treating a HGF-related disease, such as e.g. osteoarthritis, nucleic acids coding a heavy or light chain of the antibody, an expression vector, a host cell for preparing the above antibodies, a method for expressing the above anti-NGF antibodies, as well as using the above antibodies in managing pain and for preparing a therapeutic agent for managing pain associated with the disease or condition, wherein pain progression or persistence is mediated by NGF.

EFFECT: present invention enables producing the anti-NGF antibodies characterised by high stability in vivo.

16 cl, 7 dwg, 13 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: present invention refers to immunology. Presented is an antibody able to bind to an amplified epidermal growth factor receptor (EGFR) and to de2-7 EGFR, a truncated version of EGFR, and characterised by sequences of variable domains. There are also disclosed a kit for diagnosing a tumour, an immunoconjugate, pharmaceutical compositions and methods of treating a malignant tumour based on using the antibody according to the invention, as well as a single-cell host to form the antibody according to the present invention.

EFFECT: invention can find further application in diagnosing and treating cancer.

43 cl, 98 dwg, 20 tbl, 26 ex

FIELD: medicine.

SUBSTANCE: invention refers to immunology. Presented are anti-Dickkopf 1 (anti-Dkk-1) antibodies and their functional fragments specified among the antibodies: 1) containing CDR1 VH containing the amino acid sequence SSYAIS, SYAIS or GFTFSSY; CDR2 VH containing the amino acid sequence SVSGTGLGFGTYYPDSVKG or SVSGTGLGFGTY; and CDR3 VH, containing the amino acid sequence TSLENYAFDY or SLENYAFDY; and CDR1 VL containing the amino acid sequence RASESVDDFGISFIN; CDR2 VL containing the amino acid sequence AGSKQGS; and CDR3 VL containing the amino acid sequence QQLKEVPPT; and 2) the antibodies disclosed in Table 4 presented in the application materials. Described are: nucleic acids coding the above antibodies or their functional fragments; expression vectors containing the above nucleic acids; and cells used for expression of the above antibodies or their functional fragments and containing the above expression vectors. Presented is a method for producing the antibody or its functional fragment involving the stage of culturing the above expression cell. Disclosed is a composition possessing Dkk-1 binding activity, containing the antibody or its functional fragment in a therapeutically effective amount and a pharmaceutically acceptable excipient, thinner or carrier.

EFFECT: invention enables extending the range of products for treating the diseases associated with Dkk-1 and LRP5/6 excessive reaction, which cause Wnt activation.

14 cl, 14 dwg, 14 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology. Presented are variants of anti-CD20 modified antibody or its antigen-binding fragment. Each of the variants is characterised by the fact that it contains a variable light and heavy chain domain, and induces a higher apoptosis level as compared to anti-B-Ly1 chimeric antibody. There are presented: a mixture of antibodies, wherein at least 20% of oligosaccharides in Fc domain have a branched chain and are not fucosylated, as well as a pharmaceutical composition for producing a therapeutic agent for a malignant haematological or autoimmune disease by using the antibodies or the mixture of antibodies. Described are: an expression vector, a based host cell, variants of coding polynucleotides, as well as a method for producing the antibody in the cell.

EFFECT: using these inventions provides the new antibodies with the improved therapeutic properties, including with increased binding of Fc receptor, and with the increased effector function that can find application for treating the malignant haematological or autoimmune disease.

32 cl, 3 ex, 9 tbl, 26 dwg

FIELD: medicine.

SUBSTANCE: present invention refers to immunology. Presented is a molecule of bispecific single-chain antibody containing a first binding domain able to bind to epitope of CD3-epsilon-chain of human and Callithrix jacchus (tamarin), Saguinus oedipus (cotton-top tamarin) and Saimiri sciureus (squirrel monkey), and a second binding domain able to bind to an antigen specified in a group consisting of: PSCA, CD19, C-MET, endosialin, EGF-like domain 1 EpCAM coded by exon 2, FAP-alpha or IGF-IR (or IGF-1R) or a human and/or a primate. The epitope CD3e contains an amino acid sequence disclosed in the description. Disclosed are a nucleic acid coding the above molecule of the bispecific single-chain antibody, an expression vector, a host cell and a method for producing the antibody, as well as the antibody produced by the method. Described is a based pharmaceutical composition containing the molecule of the bispecific single-chain antibody and a method for preventing, treating or relieving cancer or an autoimmune antibody. Presented is using the above molecule of the bispecific single-chain antibody for making the pharmaceutical composition for preventing, treating or relieving cancer or the autoimmune disease.

EFFECT: using the invention provides the clinical improvement in relation to T-cell redistribution, reducing it, and the improved safety profile.

23 cl, 74 dwg, 17 tbl, 33 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, more specifically to biospecific antibodies, and can be used in medicine. Constructed is an antibody containing one of the following groups of six hypervariable region (HVR) sequences: (a) HVR-L1 containing the sequence NIAKTISGY; (b) HVR-L2, containing the sequence WGSFLY; (c) HVR-L3 containing the sequence HYSSPP; (d) HVR-H1 containing the sequence NIKDTY; (e) HVR-H2 containing the sequence RIYPTNGYTR; and (f) HVR-H3 containing the sequence WGGDGFYAMD; or (a) HVR-L1 containing the sequence NIAKTISGY; (b) HVR-L2 containing the sequence WGSFLY; (c) HVR-L3 containing the sequence HYSSPP; (d) HVR-H1 containing the sequence NISGTY; (e) HVR-H2 containing the sequence RIYPSEGYTR; and (f) HVR-H3 containing the sequence WVGVGFYAMD. The produced antibody specifically binds human epidermal growth factor receptor 2 (HER2) and vascular endothelial growth factor (VEGF) The invention also refers to a recovered Fab fragment of the above antibody, a polynucleotide coding it, to an expression vector, a host cell, a method for producing it, as well as to using it for treating HER2-mediated diseases.

EFFECT: present invention enables producing the bispecific high-affinity antibody able to bind VEGF and HER2 simultaneously.

14 cl, 65 dwg, 16 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry, particularly to a recovered monoclonal anti-LOXL2 antibody or its antigen-binding fragment, as well as conjugates containing them. What is disclosed is a pharmaceutical composition for treating a disease related to angiogenesis, fibrosis, tumour or metastases, containing the above recovered monoclonal anti-LOXL2 antibody or its antigen-binding fragment in an effective amount. The invention also refers to a method for determining and/or measuring LOXL2 in a biological sample, to a method for inhibiting LOXL2 activity, to a method for reducing growth of LOXL2-expressing tumour in an individual in need thereof, to a method for inhibiting angiogenesis in the individual in need thereof, to a method for suppressing a fibrous disease in the individual in need thereof, as well as to a method for monitoring an individual's response to an anti-LOXL2 therapy with using the above antibody or its antigen-binding fragment and the above conjugate.

EFFECT: invention enables treating the LOXL2-related diseases effectively.

28 cl, 6 dwg, 6 tbl, 9 ex

Up!