Pcsk9 vaccine

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology and biotechnology. Presented is an immunogen for inducing an immune response to the PCSK9 protein, containing the PCSK9 antigen peptide specified in a group consisting of the disclosed SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 314, 318 and 319, containing the amino acid sequence TRFHRQ, and SEQ ID NO: 182, 183, 184, 185, 186, 187, 188, 317, 401, 402 and 403, containing the amino acid sequence SIPWNLE, wherein the above PCSK9 peptide is conjugated with an immunogenic carrier specified in CRM197 or a Qbeta viral-like particle (VLP). Described is a composition for inducing the immune response to the PCSK9 protein containing at least two immunogens in immunologically effective amounts, wherein the first immunogen represents the above immunogen. Disclosed is a composition for inducing the immune response to the PCSK9 protein containing at least two immunogens in the immunologically effective amounts, wherein the first immunogen is specified in a group of immunogens containing the amino acid sequence SIPWNLE, and wherein the second immunogen is specified in a group of immunogens containing the amino acid sequence TRFHRQ; and wherein the composition can additionally contain at least one adjuvant. What is also presented is a pharmaceutical composition for preventing, treating or relieving PCSK9-related disorders containing: the above immunogen in a therapeutically effective amount, or one of the above compositions, and a pharmaceutically acceptable excipient.

EFFECT: invention enables preparing the effective vaccine for the disorders related to a reaction of the PCSK9 protein and its LDLR receptor.

24 cl, 19 dwg, 6 tbl, 9 ex

 

The SCOPE of the INVENTION

The present invention relates to providing new immunogens containing antigenic PCSK9 peptide, preferably associated with immunogenic carrier for the prevention, treatment and relief of PCSK9-related disorder. The invention also relates to methods of manufacturing these medicines, their immunogenic compositions and pharmaceutical compositions and their use in medicine.

PRIOR art

Proprotein-convertase subtilisin/Kexin 9 type (called hereinafter "PCSK9"), also known as neural apoptosis-adjustable convertase 1 ("NARC-i"), is a proteinase K-like subtilase identified as 9thmember of the family of PCSK mammals; see Seidah et al., 2003, PNAS 100:928-933. Gene PCSK9 is located on human chromosome R-P34.3. PCSK9 is expressed in cells capable of proliferation and differentiation, including, for example, hepatocytes, mesenchymal cells of the kidneys, putting the cells of the ileum and the large intestine epithelium and embryonic neurons brain bubble.

Initial PCSK9 synthesis presented in the form of an inactive precursor of an enzyme, or zymogen, with a molecular weight of approximately 72 kDa, which is autocatalytically, intramolecular processing in endoplasmatic the om reticulum ("ER") to activate its functional properties. The sequence of the gene for human PCSK9, which has a length of approximately 22, etc., ad with 12 exons, coding 692-amino-acid protein that can be found, for example, under the Depository No. NP_777596.2. Nucleic acid sequence of PCSK9 human, mouse and rat were deposited; see, for example, GenBank access number: AH 127530 (also ACH), AH and AH, respectively.

Human PCSK9 is a secretory protein that is expressed mainly in the kidney, liver and intestine. It has three domains: the inhibitory prodomain (amino acids 1-152; including the signal sequence of amino acids 1-30), catalytic domain (amino acids 153-448) and C-terminal domain of length 210 residues (amino acids 449-692), which is rich in cysteine residues. PCSK9 is synthesized in the form of zymogen, which is autocatalytically splitting between predomina and catalytic domain in the endoplasmic reticulum. Prodomain remains associated with the Mature protein after cleavage and the complex is secreted. Sistemi-rich domain may play a role similar to that of the P-processing domains other Furin/Kexin/Subtilisin-like serine proteases, which seems necessary for folding and regulation of activated proteases. Mutations in PCSK9 are associated with abnormal levels of cholesterol LDL, low density the values (LDL-c) in plasma (Horton et al., 2006 Trends. Biochem. Sci, 32(2):71-77).

PCSK9, which is attributed to the role in the differentiation of hepatocytes and nerve cells (Seidah et al., above), highly expressed in fetal liver and to a large extent involved in the homeostasis of cholesterol.

Highly desirable to identify compounds and/or agents effective in the treatment of cardiovascular diseases. Lower levels of LDL cholesterol, as has been demonstrated in clinical trials, directly associated with the incidence of coronary attacks; Law et al., 2003 BMJ 326: 1423-1427. Moreover, it was recently shown that a moderate lifetime decrease levels of LDL cholesterol in plasma is essentially correlated with a significant reduction in the incidence of coronary attacks; Cohen et al., above. This was found even in populations with a high prevalence of risk factors for cardiovascular disease, not associated with lipids; above.

Consequently, great importance is the identification of a therapeutic agent, providing control of the levels of LDL cholesterol.

Accordingly crucial to the manufacture of a medicinal product which inhibits or counteracts the activity of PCSK9 and the appropriate role of PCSK9 plays in various therapeutic conditions.

Expression or increase regulation of PCSK9 is associated with elevated levels of the LDL cholesterol in plasma, and the inhibition or absence of expression of PCSK9 is associated with low levels of LDL cholesterol in plasma. Interestingly, lower levels of LDL cholesterol associated with variations in PCSK9 sequence, provided protection against coronary artery disease; Cohen, 2006 N. Engl. J. Med. 354: 1264-1272.

SUMMARY of the INVENTION

The present invention relates to an immunogen containing the antigenic PCSK9 peptide and possibly immunogenic carrier.

The invention also relates to methods of obtaining such antigenic PCSK9 peptide, possibly associated with immunogenic carrier.

The invention also relates to immunogenic compositions containing such antigenic PCSK9 peptide, possibly associated with immunogenic carrier may contain one or more adjuvants, preferably one or two adjuvant.

Another aspect of the invention relates to pharmaceutical compositions containing the antigenic PCSK9 peptide according to the invention, or immunogenic composition, as well as medical applications such compositions.

In particular, the invention relates to the antigenic PCSK9 peptide of the invention or immunogenic or pharmaceutical composition for use as a medicine, preferably for the treatment, mitigation or prevention of PCSK9-wired the x disorders.

In particular, the invention relates to the antigenic PCSK9 peptide of the invention or immunogenic or pharmaceutical composition for use as a medicine, preferably for the treatment, mitigation or prevention of diseases associated with high cholesterol levels.

Antigenic PCSK9 peptides according to the invention are particularly suitable for the treatment of patients-people with high cholesterol LDL or condition associated with elevated LDL cholesterol, for example, lipid disorder (e.g., hyperlipidemia, hyperlipidemia type I, type II, type III, type IV or V type, secondary hypertriglyceridemia, hypercholesterolemia, familial hypercholesterolemia, xanthomata deficit holesterinesterzy) or at risk of their development. Antigenic PCSK9 peptide of the invention is also suitable for the treatment of patients-people with atherosclerotic condition (e.g., atherosclerosis), coronary artery disease, cardiovascular disease, and patients at risk of developing these disorders, for example, due to the presence of one or more risk factors (e.g. hypertension, Smoking, diabetes, obesity, or hyperhomocysteinemia).

In another aspect of the present invention proposed the use of antigenic PCSK9 peptide on which obreteniyu or immunogenic compositions or pharmaceutical compositions in the manufacture of a medicine for the treatment of Alzheimer's disease.

In one of the embodiments of the antigenic PCSK9 peptide or immunogenic composition or pharmaceutical composition are administered together with another agent.

A BRIEF DESCRIPTION of GRAPHIC MATERIALS:

Fig.1: Structure PDB human PCSK9 associated with the domain EGF-And LDL-R (3BPS) showing 5 peptide sequences in PCSK9 (peptide 1-5), selected for their involvement in the interaction between these two proteins.

Fig.2: Mice were immunized with peptides from VR_9.1 to VR_9.9, conjugated with the VLP using alum with CpG adjuvant, or antibody-based test and answer full-size recombinant human PCSK9 were measured by titration of the sera in ELISA analysis. The results are presented in the form of reciprocal titers for each of 6 mice per group, with reciprocal titers, measured as the serum dilution giving an optical density reading of 0.5.

Fig.3: or antibody-based test answers on a full-size recombinant murine PCSK9 protein, as shown in Fig.2.

Fig.4: Levels of cholesterol in plasma was measured in the sera of vaccinated mice (in the same samples that were used for antibody assays in Fig.2 and 3)

Fig.5: plasma Samples used in Fig.2-4, were tested at various dilutions on their ability to inhibit the interaction between recombinant PCSK9 and the extracellular domain of the LDL receptor, as measured Ana is Isom FRET.

Fig.6: Dilution of plasma from vaccinations peptides VR_9 5 and VR_9.6 in the analysis of FRET, showing dose-dependent inhibition of the interaction between PCSK9 and LDL receptor.

Fig.7: Complex PCSK9 (tape) and EGF-A (filled area) from PDB:3BPS. Potential areas of PCSK9, which can interact with the LDLR domains other than EGF-A, is shown by the ellipse.

Fig.8: the complex PCSK9 (tape) and EGF-A (filled area) with amino acids corresponding to the peptides VR_13/14 (a) and VR_15/16(B) and VR_9.5(C).

Fig.9 and 10: Responses of antibodies in plasma from mice vaccinated with peptides VR_9.5 and VR_9.10 no VR_9.16. Antibodies to mouse PCSK9 were measured using ELISA analysis of serial dilution of plasma using a full-sized mouse PCSK9 protein. Individual titration curves are shown for 8 mice per group, where ELISA responses of plasma of mice immunized with unconjugated the VLP, shown as a control.

Fig.11: Serum or antibody-based test answers on a full-sized human PCSK9 protein, induced in mice BALB/c and C57BL/6, vaccinated or peptide VR_9.5, or VR_9.10, conjugated with the VLP (using alum +/-CpG as adjuvant) or CRM197 (using TiterMax adjuvant). Antibodies to human PCSK9 were measured by titration of the sera in ELISA analysis. The results are presented as the log of reciprocal titers, op is edelenyi at an optical density of 1.0 for each of the 8 mice in the group.

Fig.12: Serum or antibody-based test answers on a full-sized mouse PCSK9 protein, induced in mice BALB/c and C57BL/6, vaccinated, as described for Fig.11. The results are presented as the log of reciprocal titers determined at an optical density of 0.5 for each of the 8 mice in the group.

Fig.13: Total cholesterol levels measured in serum samples from vaccinated BALB/c mice (the same samples used for antibody assays in Fig.11 and 12).

Fig.14: Total cholesterol levels measured in serum samples from vaccinated mice C57BL/6 (same sample used for the analysis of antibodies in Fig.11 and 12).

Fig.15: or antibody-based test answers on a full-sized human PCSK9, induced in BALB/c mice immunized with peptides VR_9.5 or VR 9.17 on VR,9.35, conjugated with the VLP, using alum plus CpG as adjuvant. Antibodies to human PCSK9 were measured by titration of the sera in ELISA analysis. The results are presented as the log of reciprocal titers determined at an optical density of 1.0 for each of the 8 mice and the group.

Fig.16: or antibody-based test answers on a full-sized mouse PCSK9, induced in BALB/c mice immunized as described for Fig, 15, Antibodies to mouse PCSK9 were measured by titration of the sera in ELISA analysis. The results are presented as the log of reciprocal titers defined when the optical p is h 0.5 for each of the 8 mice in the group.

Fig.17: Total cholesterol levels measured in serum samples from vaccinated BALB/c mice (the same samples used for antibody assays in Fig.15 and 16.

Fig.18: Complex PCSK9 (tape) and EGF-A (filled area) with areas of PCSK9 containing amino acid sequences that are associated with mutations of acquisition (gain) or loss of function loss-of-function) and/or surface proteins, exposing epitopes indicated by ellipses.

DETAILED description of the INVENTION

Antigenic PCSK9 peptide of the invention

The present invention relates to an immunogen containing the antigenic PCSK9 peptide, possibly associated with immunogenic carrier.

In one of the embodiments of the antigenic PCSK9 peptide is a plot of PCSK9 containing from 4 to 20 amino acids and, when administered to a subject, is able to reduce LDL-cholesterol in the blood of the specified entity. Preferably, the specified subject is a mammal, preferably human. Preferably, the specified antigenic PCSK9 peptide is able to reduce LDL-cholesterol by at least 2%, 5%, 10%, 20%, 30% or 50%.

In one of the embodiments of the antigenic PCSK9 peptide is a plot of PCSK9, which is involved in the interaction of PCSK9 with the LDL receptor.

In one of the embodiments of the antigenic PCSK9 peptide is a plot of PCSK9, which is involved wowzamediaserver PCSK9 with the LDL receptor, containing from 4 to 20 amino acids and, when administered to a subject, is able to reduce LDL-cholesterol in the blood of the specified entity. Preferably, the specified subject is a mammal, preferably human. Preferably, the specified antigenic PCSK9 peptide is able to reduce LDL-cholesterol by at least 2%, 5%, 10%, 20%, 30% or 50%.

In one of the embodiments of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587 and 588.

In one of the embodiments of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:1-312, 330-398, 421, 423, 424, 426 and 428-588.

In one of the embodiments of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 24, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397 and 398.

In one of the embodiments of the antigenic PCSK9 peptide is a plot of PCSK9, which may participate in the interaction with the EGF domain is A receptor for LDL. Examples of such plots is shown in Fig.1.

In one of the embodiments of the antigenic PCSK9 peptide is a plot of PCSK9, which may participate in the interaction with the EGF domain-A receptor LDL containing from 4 to 20 amino acids and, when administered to a subject, is able to reduce LDL-cholesterol in the blood of the specified entity. Preferably, the specified subject is a mammal, preferably human. Preferably, the specified antigenic PCSK9 peptide is able to reduce LDL-cholesterol by at least 2%, 5%, 10%, 20%, 30% or 50%.

In one of the embodiments of the antigenic PCSK9 peptide is a peptide containing from 5 to 13, preferably from 6 to 8 consecutive amino acids fragment of PCSK9 SEQ ID NO:1.

In the bottom of the embodiments of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22. 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45.

In one of the embodiments of the antigenic PCSK9 peptide is a peptide containing from 5 to 15, preferably from 6 to 8 consecutive amino acids fragment of PCSK9 SEQ ID NO:4S.

In one of the embodiments of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 and 101.

In one of the embodiments of the antigenic PCSK9 peptide is a peptide containing from 5 to 14, preferably from 6 to 8 consecutive amino acids fragment of PCSK9 SEQ ID NO:102.

In one of the embodiments of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144 145 and 146.

In one of the embodiments of the antigenic PCSK9 peptide is a peptide containing from 5 to 13, preferably from 6 to 8 consecutive amino acids fragment of PCSK9 SEQ ID NO:147.

In one of the embodiments of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180 and 181.

In one of the embodiments of the antigenic PCSK9 peptide is a peptide, aderrasi from 5 to 13, preferably from 6 to 8 consecutive amino acids fragment of PCSK9 SEQ ID NO:182.

In one of the embodiments of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and 226.

In one of the embodiments of the antigenic PCSK9 peptide is a peptide containing from 5 to 13, preferably from 6 to 8 consecutive amino acids fragment of PCSK9 SEQ ID NO:330.

In one of the embodiments of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358 and 359.

In the preferred embodiment of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:19, 56, 63, 109, 153, 165, 184, 186, 187, 188, 332 and 424.

In the preferred embodiment of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:19, 56, 63, 109, 153 and 184.

In the preferred embodiment of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:56, 184, 186, 187, 188 and 332.

In the most preferred embodiment of the antigenic PCSK9 peptide is a peptide having the sequence of SEQ ID NO:56.

In a more preferred embodiment of the antigenic PCSK9 peptide is a peptide having the sequence of SEQ ID NO:184 or 187.

In the most preferred embodiment of the antigenic PCSK9 peptide of t is made by a peptide having the sequence of SEQ ID NO:184.

In the most preferred embodiment of the antigenic PCSK9 peptide is a peptide having the sequence of SEQ ID NO:332.

In one of the embodiments of the antigenic PCSK9 peptide is selected from the plot of PCSK9, which may participate in the interaction with other than the domain of the EGF-A, plot of the LDL receptor. Examples of such plots is shown in Fig.7 and 8.

In one of the embodiments of the antigenic PCSK9 peptide is a plot of PCSK9, which may participate in the interaction with other than the domain of the EGF-A, plot of the LDL receptor containing from 4 to 20 amino acids and, when administered to a subject, is able to reduce LDL-cholesterol in the blood of the specified entity. Preferably, the specified subject is a mammal, preferably human. Preferably, the specified antigenic PCSK9 peptide is able to reduce LDL-cholesterol by at least 2%, 5%, 10%, 20%, 30% or 50%.

In one of the embodiments of the antigenic PCSK9 peptide is a peptide containing from 5 to 12, preferably from 6 to 8 consecutive amino acids fragment of PCSK9 SEQ ID NO:227.

In one of the embodiments of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO: 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 and 262.

In one of the embodiments of the antigenic PCSK9 peptide presented yet a peptide, containing from 5 to 13, preferably from 6 to 8 consecutive amino acids fragment of PCSK9 SEQ ID NO:263.

In one of the embodiments of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306 and 307.

In one of the embodiments of the antigenic PCSK9 peptide is a peptide containing from 5 to 13, preferably from 6 to 8 consecutive amino acids fragment of PCSK9 SEQ ID NO:360.

In one of the embodiments of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397 and 398.

In one of the embodiments of the antigenic PCSK9 peptide is selected from the plot of predomina PCSK9 (SEQ ID NO:329).

In one of the embodiments of the antigenic PCSK9 peptide is a plot of predomina PCSK9 containing from 4 to 20 amino acids and, when administered to a subject, is able to reduce LDL-cholesterol in the blood of the specified entity. Preferably, the specified subject is a mammal, preferably human. Preferably, the specified antigenic PCSK9 peptide is able to reduce LDL-cholesterol by at least 2%, 5%, 10%, 20%, 30% or 50%.

In one of the embodiments of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:308, 309, 310, 311 and 312.

which one of the embodiments of the antigenic PCSK9 peptide is a peptide, containing from 5 to 12, preferably from 6 to 8 consecutive amino acids fragment of PCSK9 SEQ ID NO:309.

In one of the embodiments of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:309, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462 and 463.

In one of the embodiments of the antigenic PCSK9 peptide is a peptide containing from 5 to 12, preferably from 6 to 8 consecutive amino acids fragment of PCSK9 SEQ ID NO:508.

In one of the embodiments of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542 543.

In one of the embodiments of the antigenic PCSK9 peptide is a peptide containing from 5 to 13, preferably from 6 to 8 consecutive amino acids fragment of PCSK9 SEQ ID NO:310.

In one of the embodiments of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:310, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506 and 507.

In one of the embodiments of the antigenic PCSK9 peptide is a peptide containing from 5 to 13, preferably from 6 to 8 consecutive amino acids fragment of PCSK9 SEQ ID NO:544.

In one of the embodiments of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:544, 545, 546, 547, 548, 549, 550, 551, 552, 55, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587 and 588.

In the preferred embodiment of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:312, 421, 422, 423, 426, 427, 428, 445, 482, 525 and 563.

In a more preferred embodiment of the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO:445, 482, 525 and 563.

In the most preferred embodiment of the antigenic PCSK9 peptide is a peptide having the sequence of SEQ ID NO:445.

Such antigenic PCSK9 peptides can be used separately or in combination, preferably in the conjugation with immunogenic carrier, for the induction of antibodies against PCSK9 for a subject to treat, prevent or facilitate PCSK9-related disorder.

Specialist in the art it is obvious what methods can be used to confirm whether a particular design under the scope of the present invention. Such methods include, but are not limited to, the methods described in the Examples section of this application, as well as the following.

The ability of the antigenic PCSK9 peptide of the invention to induce antibodies against PCSK9 can be measured in mice using the test described in example 3 of this application. The ability of autoantibodies induced by antigenic PCSK9 peptide of the invention, to reduce the level is ü circulating in plasma cholesterol can be measured in mice using the test described in example 3. The ability of autoantibodies induced by antigenic PCSK9 peptide of the invention, to inhibit the interaction between PCSK9 and LDL receptor can be measured directly, using the test described in example 3 (analysis of FRET (resonance energy transfer fluorescence), or indirectly by measuring the increase regulation of LDL receptors on the cell surface, which is a consequence of blocking PCSK9-mediated down-regulation (and also described in the relevant literature or by using cell lines in vitro, or by measuring the levels of LDL receptors in the liver biopsies of animals expressing antibodies (for example, by Western blotting)).

The term "biological activity of the antigenic PCSK9 peptide", when used in this application refers to the ability of the antigenic PCSK9 peptides according to the invention to induce antibodies against PCSK9 to the patient.

Preferably, the specified antigenic PCSK9 peptide when administered to a subject, is able to reduce LDL-cholesterol in the blood of the specified entity. Preferably, the specified subject is a mammal, preferably human. Preferably, the specified antigenic PCSK9 peptide is able to reduce LDL-cholesterol by at least 2%, 5%, 10%, 20%, 30% or 50%.

In one of waples the deposits antigenic PCSK9 peptides according to the present izobreteniya have such a size they mimic the plot, selected from the full domain of PCSK9, which is found native epitope. In a particular embodiment of the antigenic PCSK9 peptides according to the invention have a length of less than 100 amino acids, preferably less than 75 amino acids, more preferably less than 50 amino acids, even more preferably less than 40 amino acids.Antigenic PCSK9 peptides according to the invention have a length of typically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids, preferably from 4 to 20 amino acids, for example, from 6 to 12, from 6 to 8 or 9 to 12 amino acids.

Specific examples of antigenic PCSK9 peptides according to the invention proposed in the sequence listing and include peptides ranging in length from 5 to 17 amino acids.

Antigenic peptides of the invention include the amino acid sequence originating from a plot of PCSK9 mammal, preferably human PCSK9 (SEQ ID NO:399) or mouse PCSK9 (SEQ ID NO:400), more preferably human PCSK9, such a happening area PCSK9 or corresponds to amino acid sequences found in nature PCSK9, or meet PCSK9 variant, i.e., amino acid sequence found in nature PCSK9, in which a small number of amino acids has been substituted, added or deleterule, but which retains essentially the same immunological properties. In addition, such an origin is odasi plot PCSK9 can be further modified by amino acids, especially at the N - and C-ends. to provide conformational constraints antigenic PCSK9 peptide and/or to ensure binding of the antigenic PCSK9 peptide to the immunogenic carrier after an appropriate chemistry.

Antigenic PCSK9 peptides described in this application, cover functionally active variants of peptides derived from the amino acid sequence of PCSK9 in which amino acids have been delegated, inserted, or replaced without significantly reducing their immunological properties, i.e. such functionally active variants of the peptides retain substantial biological activity of the antigenic PCSK9 peptide. Typically, these functionally active variants of the peptides have an amino acid sequence homologous, preferably highly homologous, the amino acid sequence selected from the group consisting of SEQ ID NO:1-312, 330-398 and 420-588.

In one of the embodiments of such functionally active variants of the peptides exhibit at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity with the amino acid sequence selected from the group consisting of SEQ ID NO:1-312, 330-398 and 420-588.

The similarity of the sequences of polypeptides, which is also referred to as sequence identity, usually measured with the use of software to analyze pic is of egovernance. Software for analysis of protein aligns similar sequence using the criteria of similarity related to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as "Gap" and "Bestfit" which can be used with default parameters to determine the homology of sequences or sequence identity between closely related polypeptides, such as homologous polypeptides from different organisms or between a wild-type protein and its muteena. See, for example, the GCG version 6.1. Polypeptide sequences can also be compared using FASTA. applying the parameters to the default or recommended parameters, the program GCG version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions with the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000)). An alternative algorithm when comparing sequences according to the invention with a database containing a large number of sequences from different organisms, is a computer program BLAST, especially blastp or tbiastn using the default settings. See, for example, Altscul et al., J. Mol. Biol. 215:403-410 (1990); Altschu! et al., Nucleic Acids Res. 25:3389-402 (1997).

Functionally active variants include naturally occurring functionally active variants such as allelic variants and species variants, and not naturally occurring functionally active variants which can be obtained, for example, methods of mutagenesis or direct synthesis.

Functionally active variant differs from any of the peptides presented in SEQ ID NO:1-312, 330-398 and 420-588 about, for example, 1, 2, 3, 4 or 5 amino acid residues and yet retains the biological activity of the antigenic PCSK9. If this comparison requires alignment the sequences align with the maximum homology. Site variations can be presented anywhere in the peptide, while biological activity is essentially the same peptide presented in SEQ ID NO:1-312, 330-398 and 420-588.

Guide concerning how to make phenotypically silent amino acid substitutions is represented in Bowie et al., Science, 247: 1306-1310 (1990), which teaches that there are two main strategies to study the stability of amino acid sequence to change.

The first strategy uses a tolerance to amino acid substitutions in the course of natural selection in evolution. When comparing amino acid sequences in different species can identify the SQL amino acid position, who were conservative among species. These conserved amino acids, apparently, important for the function of the protein. In Alicia from this, the amino acid positions where substitutions have been made by natural selection, indicate provisions which are not critical for the function of the protein. Thus, provisions that allow for amino acid replacement can be modified, while maintaining the specific immunogenic activity of the modified peptide.

The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify areas that are critical to the function of the protein. For example, you can use the site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science, 244: 1081-1085 (1989). The obtained variants of the peptides can then be tested against specific biological activity of the antigenic PCSK9.

According to Bowie and others, these two strategies have found that proteins are unexpectedly resistant to amino acid substitutions. The authors also indicate which amino acid changes, apparently, are valid in some amino acid positions in the protein. For example, the most concealed or internal (in the tertiary structure of the protein) amino acid residues require nonpolar side chains, while some is that the characteristics of the surface or exterior of the side chains are usually conservative.

Kinds of introduction of mutations in the amino acids of a protein are well known to specialists in this area, See, for example, Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994); T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, N. Y. (1989)).

Mutations can also be entered using commercially available kits, such as "QuikChangeTM Site-Directed Mutagenesis Kit" (Stratagene), directly or peptide synthesis. Obtain functionally active variant for antigenic PCSK9 peptide by replacement of amino acids, which essentially does not affect the function specified antigenic PCSK9 peptide, can be performed by a specialist in this field of technology.

The type of amino acid substitutions which can be made in one of the peptides according to the invention is a conservative amino acid substitution. "Conservative amino acid substitution" is a substitution in which the amino acid residue is replaced with another amino acid residue having a group of the side chain R) with similar chemical properties (e.g. charge or hydrophobicity). In General, a conservative amino acid substitution essentially does not change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other conservative substitutions, the percent identity of the sequences of the Il is the degree of similarity can be adjusted upwards to correct the conservative nature of the substitution. The ways of making this adjustment are well known to specialists in this field of technology. See, for example, Pearson, Methods Mol. Biol. 243:307-31 (1994).

Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanin, valine, leucine and isoleucine; 2) alifaticheskie hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) basic side chains: lysine, arginine and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine and methionine. Preferred groups for conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine.

Alternatively, a conservative replacement is any change having a positive value in RAM matrix logarithmic likelihood (log-likelihood matrix), described in gonnet on et al., Science 256:1443-45 (1992). "Moderately conservative" replacement is any change having a positive value in RAM matrix logarithmic likelihood.

Functionally active variant of the peptide can also be selected with the use of the Finance method of hybridization. Briefly, DNA having high homology with a whole or part of a nucleic acid sequence that encodes intercouse peptide such as SEQ ID NO:1-312, 330-398 and 420-588, is used to produce functionally active peptide. Therefore, the antigenic PCSK9 peptide of the invention also includes peptides which are functionally equivalent to one or more than one of the peptides SEQ ID NO:1-312, 330-398 and 420-588 and which is encoded by a nucleic acid molecule, which hybridizes with nucleic acid that encodes any of SEQ ID NO:1-312, 330-398 and 420-588 or its complement. The person skilled in the art can easily determine the sequence of nucleic acids that encode the peptides according to the invention, using a publicly available tables codons. As such, these nucleic acid sequence not represented in this application.

The rigidity of hybridization to a nucleic acid that encodes a peptide, polypeptide or protein that is functionally active variant is, for example, 10% formamide, 5 × SSPE, 1 × denhardt's solution and 1 × DNA salmon sperm (low stiffness). More preferred conditions are 25% formamide, 5 × SSPE, 1 × denhardt's solution and 1 × DNA salmon sperm (conditions of moderate stringency), even more preferred conditions are 50% formamide, 5 × SSPE, 1 × solution of Denham is GTA and 1 × DNA salmon sperm (high stiffness). However, the rigidity of hybridization influenced by some other factors, in addition to the above concentration of formamide, and the specialist in the art may accordingly select these factors to achieve the same stiffness.

The nucleic acid molecule encoding a functionally active variant, can also be selected by the method of gene amplification, such as PCR (polymerase chain reaction) using the portion of the molecule of nucleic acid, DNA that encodes a desired peptide, polypeptide or protein, for example, any of the peptides presented in SEQ ID NO:1-312, 330-398 and 420-588, as a probe.

In one embodiment of the invention the peptide according to the invention originates from a natural source and isolated from a mammal such as a human, Primate, cat, dog, horse, mouse or rat, preferably from a human source. The peptide according to the invention, thus, can be isolated from cells or tissues using standard methods of protein purification.

Alternatively, the peptides according to the invention can be chemically synthesized or produced using methods of recombinant DNA.

For example, the peptide according to the invention can be synthesized using solid phase procedures well known in the art. Suitable syntheses can be performed using T-boc or F-oc" procedures. Cyclic peptides can be synthesized using solid-phase procedures using well-known F-moc procedures and polyamide resin in a fully automated apparatus. Alternatively, specialists in the art will know the necessary laboratory methods for performing the process manually. Methods and procedures for solid-phase synthesis is described in "Solid Phase Peptide Synthesis: A Practical Approach" by E. Atherton and R. C. Sheppard, published by IRL at Oxford University Press (1989) and "Methods in Molecular Biology", Vol.35: Peptide Synthesis Protocols (ed. M. W. Pennington and B. M. Dunn), chapter 7, pp91-171 by D. Andreau et al.

Alternatively, polynucleotide encoding the peptide according to the invention may be put into expressing a vector that can Express in a suitable expression system using methods well known in the field, followed by separation or purification of expressed peptide, polypeptide or protein of interest. A variety of expression systems in bacteria, yeast, plants, mammals and insects available in the art and can be used any such expression system. In some cases, polynucleotide encoding the peptide according to the invention can be transferred in a cell-free translational system.

Antigenic PCSK9 peptides according to the invention can also contain peptides that arise as a result of existence which multiple genes alternative transcription events, events, alternative RNA splicing and alternative translational and post-translational events. The peptide can Express C systems, such as cultured cells, which leads essentially to the same post-translational modifications, as in the case when the peptide is expressed in a native cell, or in systems which result in the modification or omission of post-translational modifications, such as glycosylation or cleavage, present when the expression of molecules in the cell.

Antigenic PCSK9 peptide of the invention can be obtained in the form of a fused protein that contains other non-PCSK9 or not-PCSK9-occurring amino acid sequence, such as amino acid linkers or signal sequences, or immunogenic carriers, as defined in this application as well as ligands useful for protein purification, such as glutathione-3-transferase, his-tag tag, and staphylococcal protein A. In fused protein may be present in more than one antigenic PCSK9 peptide of the invention. The heterologous polypeptide can be fused, for example, N - end, or the end of the peptide according to the invention. The peptide according to the invention can also be obtained in the form of a fused protein containing the homologous amino acid sequence, i.e., other PCSK9 and the and PCSK9-occurring sequence.

Antigenic PCSK9 peptides according to the invention can be linear or conformational limited. Used in this application with respect to the peptide, the term "conformationally constrained" means a peptide in which a three-dimensional structure is maintained essentially in the same spatial location over time. Conformationally restricted molecules can have improved properties such as increased affinity, metabolic stability, membrane permeability or solubility.

In addition, such conformationally restricted peptides, is expected to present the epitope antigenic PCSK9 in conformation similar to the conformation of their native loop, thus inducyruya more sensitive antibody against PCSK9 to recognize the intact, native native PCSK9 molecule or antibody against PCSK9 with high affinity to recognize own PCSK9 molecule. Methods of conformational constraints is well known in the art and include, without limitation, the formation of bridges and cyclization.

There are several approaches known from the prior art, for the introduction of conformational constraints in the linear peptide. For example, the formation of bridges between two adjacent amino acids in the peptide leads to a local conformational change, flexibility is limited to p is compared with the conventional peptides. Some possibilities for the formation of such bridges include the introduction of lactams and piperazinone (see review Giannis and. Kolter, Angew. Chem. int. Ed., 1993,32: 1244).

Used in this application with respect to the peptide, the term "cyclic" refers to a structure including an intramolecular bond between two non-adjacent amino acids or amino acid analogues. Cyclization can be achieved by covalent or non-covalent linkages. Intramolecular communication include, but are not limited to, communications of the skeleton from the skeleton, the side chain skeleton, the side chain to side chain, the side chain with a terminal group, "end to end". Methods of cyclization include, without limitation, the formation of amide bond between the N-terminal residue and the C-terminal residue of the peptide, the formation of a disulfide bond between the side chains of the non-contiguous amino acids or amino acid analogues; the formation of amide bond between the side chains of residues Lys and Asp/Glu; the formation of ester bonds between serine residues and residues Asp/Glu; the formation of lactam communication, for example, between a group of the side chain of one amino acid or its analog with N-terminal amine aminoanisole residue and education lysinoalanine and dityrosine links. Can also be used carbon options disulfide bonds, for example, Attila or Atil the flanged connection (J. Peptide Sc., 2008, 14, 898-902), and alkylation reaction with an appropriately polyamidine electrophilic reagent, such as di-, tri - or tetrachloroethane (PNAS, 2008, 105(40), 15293-15298; ChemBioChem, 2005, 6, 821-824). For the introduction of conformational constraints in peptides can also be used in a variety of modified analogues of Proline (Zhang et al., J. Med Chem., 1996,39: 2738-2744; Pfeifer and Robinson, Chem. Comm., 1998, 1977-1978). Chemistry, which can be used for the cyclization of peptides according to the invention, leads to peptides, cyklinowanie communication, including, but not limited to, the following communication: lactam, gidrazonami, Aksinya, thiazolidinone, thioester or sulfonate connection.

Another approach in the design of conformationally restricted peptides, which are described in US 10/114918, is to attach a short amino acid sequence of interest; to the matrix with obtaining cyclic conformationally constrained peptide. Such cyclic peptides are not only structurally stable their matrix, and thus provide a three-dimensional conformation, which could mimic conformational epitopes of the native protein, such as viruses and parasites, or on its own proteins (autologous mammalian proteins, such as PCSK9), but they are also more stable than linear peptides to proteolyticus the th degradation in serum. In the US 10/114918 also described the synthesis of conformationally restricted cross-linked peptides by synthetic amino acids to bind skeleton with correspondingly spaced amino acids to stabilize Nadterechny structure of peptides. Crosslinking can be achieved through an amide linkage of the primary amino groups of orthogonally protected (2S,3R)-3-aminopropanoic balance with appropriately located carboxyl group of the side chain of glutamate. This approach was adopted upon receipt of conformationally restricted tetrapeptide repeats of the CS protein, where at least one Proline was replaced with (2S,3R)-3-aminopropanol and, in order to introduce the carboxyl group of the side chain, glutamate was included as a substitution of alanine.

Strategy stitching also include the application of metathesis reaction with the closing of the loop Grubbs (Grubbs) with the formation of "fixed" ("stapled") peptides designed to mimic alpha-helical conformation (Angew. Int. Ed. Engl., 1998, 37, 3281; JACS, 2000, 122, 5891); application polyfunctionalized saccharides; application tryptamines communication (Chemistry Her. J., 2008, 24, 3404-3409); the application of "click" reactions of azides and alkynes, which can be included either in the form of amino acid residue side chain, either located within the skeleton of the peptide sequence (Drug Dic. Today, 2003, 8(24), 1128-1137). From the literature it is also known that metal ions can stabilize the restricted conformation of linear peptides by sekvestirovanija specific residues, such as histidine, which is koordinaciyi with metal cations (Angew. Int. Ed. Engl., 2003, 42, 421). Similarly, the functionalization of linear peptide sequence unnatural acid and amine functional group, or polyamines and polyacidic functional group can be used to provide access to cyklinowanie structures after activation and formation of amide linkages.

According to one of embodiments of the antigenic PCSK9 peptide of conformationally restricted by intramolecular covalent bonds two non-contiguous amino acids of the antigenic PCSK9 peptide with each other, for example N - and C-terminal amino acids. According to another embodiment of the antigenic PCSK9 peptide of the invention conformationally restricted through formation of covalent bonds with the skeleton molecule. According to an additional embodiment of the antigenic PCSK9 peptide is simply limited, i.e., attached to the one end (the C - or N-end), or through another amino acid, is not located either at one end, to frame the molecule. According to another embodiment of the antigenic PCSK9 peptide is doubly limiting the, i.e. attached and the C-and N-ends to frame the molecule. According to another embodiment of the antigenic peptide is limited by the cyclization of a matrix effect heterochiral diprolene unit (D-Pro-L-Pro) (Spath et al., 1998, Helvetica Chimica Acta 81, p 1726-1738).

Frame (also called "platform") can be any molecule that is able to reduce, through the formation of covalent bonds, the number of conformations that can be antigenic PCSK9 peptide. Examples of conformationally restricted frames include proteins and peptides, for example, lipocalin-related molecules, such as beta-barrel-containing thioredoxin and thioredoxin-like proteins, nucleases (e.g., Mcasa A), protease (e.g. trypsin), protease inhibitors (e.g., Eglin C), antibodies or their structurally rigid fragments, fluorescent proteins such as GFP (green fluorescent protein) or YFP (yellow fluorescent protein), conotoxins, loop parts of the domain, fibronectin type III, CTL-A4 and virus-like particles (the VLP).

Other suitable platform molecules include carbohydrates, such as sepharose. The platform can be a linear or circular molecule, for example, closed with loop. Platform as a rule, is heterologous relative to the antigenic PCSK9 peptide. Assume that such conformation is restricted peptides associated with the platform, are more resistant to proteolytic degradation than the linear peptide.

According to a preferred embodiment of the frame is an immunogenic carrier, as defined in this application. In another embodiment the antigenic PCSK9 peptide is simply limited to the immunogenic carrier. In another embodiment the antigenic PCSK9 peptide is doubly restricted to the immunogenic carrier. Thus, the antigenic PCSK9 peptide forms a conformationally restricted loop structure, which proved to be particularly suitable structure as an intracellular molecule recognition.

Antigenic PCSK9 peptides according to the invention can be modified for ease of conjugation with the platform, for example, by adding a terminal cysteine at one or both ends and/or by adding a linker sequence, such as a double glycine head or tail plus terminal cysteine, linker, ending with a lysine residue or any other linker, known to experts in the art to perform such functions. Detailed information about such linkers disclosed below. You can also use biorthogonol chemistry (such as "click"-reaction described above) to link the full peptide sequence with the carrier, thus avoiding any who regiochemistry and chemoselective restrictions. Can also be used with rigid linkers, such as those described in Jones et al. Angew. Chem. Int. Ed. 2002, 41:4241-4244, which are known to cause enhanced immune response. In another embodiment the antigenic PCSK9 peptide is attached to the polyvalent matrix, which itself is connected with the carrier, thus increasing the density of the antigen (see below). Polyvalent matrix can be an appropriately functionalized polymer or oligomer, such as (but not limited to) oligopotent or oligochitosan (see Fig.19).

Immunogenic carrier according to the invention

In one of the embodiments of the present invention antigenic PCSK9 peptide of the invention relates to immunogenic molecule-media education immunogenic protocols for vaccination, preferably, where the molecule-carrier is not related molecule native PCSK9.

The term "immunogenic carrier" in this application includes those substances which have the property independently to cause immunogenic response in an animal host and which can be Malente associated with the peptide, polypeptide or protein either directly through the formation of peptide or ester links between free carboxyl, amino or hydroxyl groups in the peptide, polypeptide or protein and the relevant groups on immunogen the m matter-carrier, or, alternatively, by binding through a standard bifunctional linking group, or a fused protein.

The types of media used in immunogen of the present invention, are well known to the person skilled in the art. Examples of such immunogenic carriers are: serum albumins such as bovine serum albumin (BSA); globulins; thyroglobulin; hemoglobins; hemocyanine (in particular hemocyanin lymph snails [KLH]); polylysine; polyglutamine acid; copolymers of lysine-glutamic acid; copolymers containing lysine or ornithine; liposomal carriers; purified protein derivative of tuberculin (PPD); inactivated bacterial toxins and toxoids, such as tetanus and diphtheria toxins (TT and DT) or a fragment of TT, CRM197 (a non-toxic, but identical antigenic variant of diphtheria toxin)other point mutants DT, such as CRM176, CRM228, CRM 45 (Uchida et al J. Biol. Chem. 218; 3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM103 and CRM107 and other mutations described Nicholls and Co. in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992, a deletion or mutation of Glu-148 Asp, Gln or Ser, and/or Ala-158 to Gly, and other mutations described in US 4709017 or US 4950740; mutation of at least one or more Lys residues 516, Lys 526, Phe 530 and/or Lys 534 and other mutations described in US 5917017 or US 6455673; or the fragment described in US 5843711, pneumococci the initial pneumolysin (Kuo et al (1995) Infect Immun 63; 2706-13), including ply, detoxified enduring some way, for example, dPLY-GMBS (WO 04081515, PCT/EP2005/010258) or dPLY-formalin, PhtX, including PhtA, PhtB, PhtD, PhtE (sequence PhtA, PhtB, PhtD or PhtE disclosed in WO 00/37105 or WO 00/39299) and merge Pht proteins, for example, PhtDE merge PhtBE merge, Pht A-E (WO 01/98334, WO 03/54007, WO 2009/000826), OMPC (meningococcal outer membrane protein is usually extracted from N. meningitidis of serogroup IN - ER), PorB (from N. meningitidis), PD (Haemophilus influenzae protein D - see, for example, EP 0594610) or immunologically functional equivalents, synthetic peptides (EP 0378881, EP 0427347), heat shock proteins (WO 93/17712, WO 94/03208), pertussis proteins (WO 98/58668, EP 0471177), cytokines, lymphokines, growth factors and hormones (WO 91/01146), artificial proteins, containing multiple human CD4+T-cell epitopes from various pathogen-derived antigens (Falugi et al (2001) Eur J Immunol 31; 3816-3824), such as protein N19 (Baraldoi et al (2004) Infect immun 72; 4884-7). pneumococcal surface protein PspA (WO 02/091998), protein capture iron (WO 01/72337), toxin a or b from C. difficile (WO 00/61761).

In the preferred embodiment of the immunogenic carrier according to the invention is a CRM197.

In another embodiment the immunogenic carrier is a virus-like particle (the VLP). preferably recombinant virus-like particle.

Used in this application, the term "virus-like particle" refers to the structure of the e, reminiscent of the viral particle, but which, as has been demonstrated, is not pathogenic. In General, virus-like particles devoid of at least part of the viral genome. In addition, virus-like particles can often be obtained in large quantities by heterologous expression and can be easily cleaned. Virus-like particle according to the invention may contain nucleic acid that is different from their genome. A typical and preferred embodiment of virus-like particles according to the present invention is a viral capsid, such as viral capsid of the corresponding virus, bacteriophage or RNA-phage.

Used in this application, the term "virus-like particle of a bacteriophage" refers to a virus-like particle, resembling the structure of the bacteriophage, which dereplication and noncommunicable and devoid of at least the gene or genes encoding the replication apparatus of the bacteriophage and typically also lacking the gene or genes encoding the protein or proteins responsible for attaching the virus to the host or penetration into it. This definition should, however, also include virus-like particle of a bacteriophage in which the aforementioned gene or genes are still present, but inactive and therefore also lead to the formation of rereplicating, reinfects is the R virus-like particle of a bacteriophage.

Capsid structure, which is formed through the self-Assembly 180 abietinic envelope protein of RNA phage and possibly containing RNA owner, referred to in this application as "the VLP envelope protein of RNA phage". Specific examples are the VLP envelope proteins Qbeta, MS2, RR or AR. In the specific case of shell protein Qbeta, for example, the VLP can be collected either exclusively of subunits WED Qbeta (formed as a result of gene expression CF Qbeta containing, for example, the stop codon TAA, preventing the expression of the longer protein A1 through suppression, see Kozlovska, T. M., et al., Intervirology 39: 9-15 (1996)), or optionally contain subunit protein A1 in the Assembly of the capsid. As a rule, the percentage of protein A1 Qbeta relatively WED Qbeta in the Assembly of the capsid will be limited to ensure the formation of the capsid. Examples of the VLP, suitable as immunogenic carriers in the context of the present invention include, but are not limited to, the VLP Qbeta, MS2, RR, AR and other envelope proteins of bacteriophage capsid and core proteins of hepatitis b virus (Ulrich, et al., Virus Res. 50: 141-182 (1998)), measles virus (Warnes, et al., Gene 160: 173-178 (1995)), virus Sindbis, rotavirus (U.S. patent No. 5071651 and 5374426), FMD virus (Twomey, et al., Vaccine 13: 1603-1610, (1995)), norovirus (Jiang, X., et al., Science 250: 1580-1583 (1990); Matsui, S. M., et al., J Clin. Invest. 87: 1456-1461 (1991)), the retroviral GAG protein (patent PCT application no WO 96/30523), protein pl retrotransposon That, the surface protein of hepatitis b virus (WO 92/11291), human papilloma virus (WO 98/15631), virus polyoma person (Sasnauskas K., et al., Biol. Chem. 380 (3): 381-386 (1999); Sasnauskas K, et al., Generation of recombinant virus-like particles of different polyomaviruses in yeast. 3rd Interational Workshop "Virus-like particles as vaccines". Berlin, September 26-29 (2001)), RNA phages, Ty, fr-phage, GA-phage, AP 205-phage and, in particular, Qbeta-phage, virus chlorotic spots cow pea mosaic virus, cow peas, human papilloma virus (HPV), a virus of a papilloma of cattle, pig parvovirus, parvovirus, such as B19, pig parvovirus (PPV) and dogs (CPV), caliciviruses (such as norovirus. haemorrhagic disease rabbit [RHDV]), the VLP measles antigen hepadnavirus animals, filamentous/rod-shaped plant viruses, including, but not limited to, tobacco mosaic virus (TMV), virus X potato (PVX), mosaic virus papaya (PapMV), alfalfa mosaic virus (AIMV) and grass mosaic virus by Johnson (JGMV), insect viruses such as virus flock house (FHV) and retrovirus, polyomavirus, such as polyomavirus mice (MPyV), pantropic virus of mice (MPtV), the BK virus (BKV) and JC virus (JCV).

As will be obvious to experts in this field, the VLP, which are used as the immunogenic carrier according to the invention is not limited to any specific form. The particle can be synthesized chem is Cesky or through a biological process, which can be natural or unnatural. As an example, this embodiment includes a virus-like particle or a recombinant form. In a more specific embodiment of the VLP may contain or alternatively consists of recombinant polypeptides of any virus which are known to form the VLP. Virus-like particle may further comprise, or alternatively consist of one or more fragments of such polypeptides, and variants of such polypeptides. Variants of the polypeptide can have, for example, at least 80%, 85%, 90%, 95%, 97% or 99% identity at the amino acid level with the wild-type counterparts. Options VLPs suitable for use in the present invention may be from any organism, until they can form a "virus-like particle" and can be used as an "immunogenic carrier," as defined in this application.

Preferred VLPs according to the invention include the capsid protein or the surface antigen of HBV (HBcAg and HBsAg, respectively) or recombinant proteins or their fragments, and shell BelCCI RNA-phage or recombinant proteins, or fragments thereof, more preferably the envelope protein Qbeta or recombinant proteins, or fragments thereof.

In one of the embodiments of the immunogenic carrier, ispolzuemyi in combination with the antigenic Pat the house PCSK9 according to the invention, is a protein HBcAg. Examples of HBcAg protein that can be used in the context of the present invention can be easily determined by a person skilled in the art. Examples include, but are not limited to, core protein of HBV, described in Yuan et al., (J. Virol. 73: 10122-10128 (1999)) and in WO 00/198333, WO 00/177158, WO 00/214478, WO 00/32227, WO 01/85208, WO 02/056905, WO 03/024480 and WO 03/024481. HBcAgs suitable for use in the present invention may be from any organism, until they can form a "virus-like particle" and can be used as an "immunogenic carrier," as defined in this application.

Options HBcAg, special interest, which can be used in the context of the present invention are variants in which one or more constantly present cysteine residues were either delegated or replaced. In the art it is well known that free cysteine residues can be involved in various chemical reactions, including disulfide exchange reaction with chemicals or metabolites that, for example, are introduced or formed in combination therapy with other substances, or direct oxidation and reaction with nucleotides under the influence of UV light. Thus, it can be obtained paccione adducts, especially given the traveler the fact, what HBcAgs have a strong tendency to bind nucleic acids. Toxic adducts, thus, will be distributed between many species, which individually may be present in low concentration, but reaches toxic levels, when considered together. In connection with the above, one of the advantages of using HBcAgs in vaccine compositions that have been modified to remove constantly present cysteine residues, is that the number of sites that can reach toxic species, when joined by the antigens or antigenic determinants, will be reduced or these sites are generally liquidated.

In addition, protestirovanny form of HBcAg, devoid of N-terminal leader sequence of the protein precursor of measles antigen of hepatitis b virus, can also be used in the context of this invention, especially when HBcAg is produced under conditions where the processing does not occur (for example, when expression in bacterial systems).

Other HBcAg variants according to the invention include: 1) polypeptide sequence at least 80%, 85%, 90%, 95%, 97% or 99% identical to one of the HBcAg amino acid sequence of wild-type or subfragment using known computer programs, 2) mutants with C-terminal shortening, what with the mutants, have 1, 5, 10, 15, 20, 25, 30, 34 or 35 amino acids were deleted from the C-end, 2) mutants with N-terminal shortening, including mutants, whose 1, 2, 5, 7, 9, 10, 12, 14, 15 or 17 amino acids have been deleted from the N-end, 3) mutants, abbreviated as N-end and C-end, include HBcAgs, which 1, 2, 5, 7, 9, 10, 12, 14, 15 or 17 amino acids have been deleted from the N-Terminus and 1, 5, 10, 15, 20, 25, 30, 34 or 35 amino acids were deleted from the C-end.

Other options HBcAg protein within the scope of the invention are variants, modified to increase immunogenic presentation of a foreign epitope, where one or more of the four arginine repeats have been delegated, but in which is stored the C-terminal cysteine (see, for example, WO 01/98333), and chimeric, shortened at the C-end of HBcAg, such as described in WO 02/14478, WO 03/102165 and WO 04/053091.

In another embodiment the immunogenic carrier used in combination with the antigenic PCSK9 peptide of the invention, is a protein of HBsAg. Proteins HBsAg, which can be used in the context of the present invention can be easily determined by a person skilled in the art. Examples include, but are not limited to, HBV surface proteins, are described in US 5792463, WO 02/10416 and WO 08/020331. HBsAgs, suitable for use in the present invention may be from any organism, until they are able to form viral h is stico" and can be used as an "immunogenic carrier", as defined in this application.

In yet another embodiment the immunogenic carrier used in combination with the antigenic peptide or the PCSK9 polypeptide according to the invention, is a protein shell Qbeta.

Found that the protein shell Qbeta organized around the capsid during expression in E. coli (Kozlovska TM. et al., GENE 137: 133-137 (1993)). The resulting capsid or virus-like particles demonstrated icosahedral agopton capsid structure with a diameter of 25 nm and T=3 quasi-symmetry. In addition, there was obtained the crystal structure of phage Qss. The capsid contains 180 copies of a protein shell, which are connected with covalent pentamers and hexamers via disulfide bridges (Golmohammadi, R. et al., Structure 4: 5435554 (1996)), leading to the remarkable stability of the capsid shell protein Qbeta. Capsid protein Qbeta also shows unusual resistance to organic solvents and denaturing agents. The high stability of the capsid shell protein Qbeta is a good sign, particularly for its application in the immunization or vaccination mammals and humans according to the present invention.

Examples of envelope proteins Qbeta, which can be used in the context of the present invention can be easily determined by a person skilled in the art. Examples were detailed description is found in WO 02/056905, WO 03/024480, WO 03/024481 (incorporated by reference in its entirety) and include, but are not limited to, amino acid sequences disclosed in the database PIR (Protein Information Resource), no access VCBPQbeta refers to WED Qbeta; no access AAA refers to a protein A1 Qbeta; and their variants, including variants of proteins in which the N-terminal methionine derived; form A1 Qbeta, shortened at the C-end that are 100, 150 or 180 amino acids, variants of proteins, which has been modified by removal of the lysine residue in the deletion or replacement, or by adding lysine residue resulting from the replacement or insertion (see, for example, Qbeta-240, Qbeta-243, Qbeta-250, Qbeta-251 and Qbeta-259, described in WO 03/024481, incorporated by reference in its entirety), and variants showing at least 80%, 85%, 90%, 95%, 97% or 99% identity with any of measles-containing proteins Qbeta described above. Variants of envelope proteins Qbeta, suitable for use in the present invention may be from any organism, until they can form a "virus-like particle" and can be used as immunogenic carriers" as defined in this application.

Antigenic PCSK9 peptides according to the invention can be linked to immunogenic carriers by chemical conjugation or by the expression of genetically POPs the data partners merger. Linking does not necessarily have to be direct, but may occur through linker sequences. In General, when antigenic peptides or fused, conjugated, or otherwise attached to an immunogenic carrier, spacer elements or linker sequence is usually added to one or both ends of antigenic peptides. Such linker sequences typically contain sequences recognized by the proteasome, a protease from endosomes or other vesicular compartment cells.

In one of the embodiments of the peptides of the present invention are expressed in the form of fused proteins with immunogenic carrier. The fusion peptide may be exercised by inserting in the primary sequence of the immunogenic carrier or by fusion with the N - or C-end immunogenic carrier. In the future, when it comes to slit proteins peptide to the immunogenic carrier, treated or merge with any of the ends of the sequence, subunit, or internal insertion of the peptide within the sequence of media. The merger, as discussed later, may be carried out by insertion of the antigenic peptide in the sequence of media, by replacing parts of the sequence of the carrier of antigenic peptide, or through a combination of deletions, substitutions, or getting the K.

When the immunogenic carrier is a the VLP, subunit chimeric antigenic peptide-the VLP will usually be capable of self-Assembly in the VLP. The VLP, exposing epitopes, merged with their subunits, also referred to in this application chimeric VLPs. For example, in EP 0421635 described IN the application of chimeric hepadnavirus crustal antigenic particles for the presentation of foreign peptide sequences in the virus-like particle.

Flanking amino acid residues can be added to any end of the sequence of the antigenic peptide to be merged with any end of the sequence of the VLP subunit, or for the insertion of this peptide sequence in the sequence of the VLP subunit. Residues of glycine and serine are especially preferred amino acids used in the flanking sequences, added to the peptide to be merged. Residues of glycine provide additional flexibility, which can reduce the potentially destabilizing effect of the merger alien sequence with the sequence of the VLP subunit.

In a particular embodiment of the invention the immunogenic carrier is a the VLP HBcAg. Slit proteins are antigenic peptide or N-end HBcAg (Neyrinck, S. et al., Nature Med. 5: 11571163 (1999)), or with inserts in the so-called primary immunodominant in the Astok (MIR) have been described (Pumpens, P. and Grens, E., Intervirology 44: 98114 (2001)), WO 01/98333) and represents a specific embodiment of the invention. Naturally occurring variants of HBcAg with divisions in MIR have also been described (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001)), and fusion with the N - or C-end, and insert in position MIR corresponding to the site of deletions compared with wt HbcAg, represent an additional embodiment of the invention. Merge with C-end have also been described (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001)). Specialist in the art can easily find advice on how to construct fused proteins using classical molecular biology techniques. Vectors and plasmids encoding the fused protein HBcAg and HBcAg and useful for the expression of fused protein HBcAg and HbcAg, have been described (Pumpens, P. and Grens, E. Intervirology 44: 98-114 (2001), Neyrinck, S. et al., Nature Med. 5: 1157-1163 (1999)) and can be used in the practical implementation of the invention. An important factor to optimize the effectiveness of self-Assembly and the exposure of the epitope to be inserted in MIR HbcAg is the choice of the site of insertion and the number of amino acids to be deleted from a sequence of HBcAg within MIR (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001); EP 0421635; U.S. patent No. 6231864) after pasting, or, in other words, what amino acids of HBcAg must be replaced by a new epitope. For example, was described replacement of amino acids 76-80, 79-81, 79-80, 75-85 80-81 or HBcAg alien epitopes (Pumpens, P and Grens, E., Intervirology 44: 98-114 (2001); EP 0421635; U.S. No. 6231864, WO 00/26385). HBcAg arginine contains a long tail (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001)), which is dispensable for capsid Assembly and capable of binding nucleic acids (Pumpens, P. and Grens, E., intervirology 44: 98-114 (2001)). HbcAg, either containing or not containing such arginine tail, both represent an embodiment of the invention.

In another specific embodiment of the invention the immunogenic carrier is a the VLP of RNA phage, preferably Qbeta. The major envelope proteins of RNA-phages spontaneously gather in VLPs after expression in bacteria and in particular in E. coli. Design fused protein, where the antigenic peptides were fused with the C-end of the truncated form of the protein A1 Qbeta or embedded within the protein A1, have been described (Kozlovska, T. M., et al., Intervirology, 39: 9-15 (1996)). Protein A1 is formed by the suppression of the stop codon UGA and has a length of 329.to. or 328.K. if we take into account the removal of N-terminal methionine. Cleavage of N-terminal methionine alanine before (the second amino acid of the encoded gene CF Qbeta) usually observed in E. coli, and in the case of N-ends of the envelope proteins Qbeta. Part of the A1 gene, 3' amber-codon UGA encodes elongation CF, which has a length of 195 amino acids. Insert antigenic peptide between position 72 and 73 extend CF leads to additional embodiments of the invention (Kozlvska, T. M., et al., Intervirology 39: 9-15 (1996)). Merge antigenic peptide on the C-end shortened from the C-end of protein A1 Qbeta leads to additional preferred embodiments of the invention. For example, Kozlovska, etc. (Intervirology, 39: 9-15 (1996)) describe the fusion protein A1 Qbeta, where the epitope is fused on the C-end elongation WED Qbeta, shortened in position 19.

As described in Kozlovska, etc. (Intervirology, 39: 9-15 (1996)), Assembly of particles, exposing the slit epitopes, usually requires the presence of both merge A1 protein antigen, and wt CF for the formation of mosaic particles. However, embodiments containing virus-like particles and, in particular, VLPs envelope protein of RNA phage Qbeta, which consist only of the VLP subunits, with antigenic peptide, attached to them, are also within the scope of the present invention.

Getting mosaic particles can be done in different ways. In Kozlovska et al., Interviroiogy, 39: 9-15 (1996) described three methods that can be used in the practical implementation of the invention. In the first approach, the effective exposure fused epitopes on VLPs is mediated by expression of a plasmid that encodes a protein Qbeta an with a stop codon UGA between CF and extension CF the strain of E. coli containing a plasmid encoding the cloned suppressor tRNA UGA, which leads to the translation of the UGA codon in Trp (plasmid RM (Smiley C. K., et al., Gene 134:33-40 (1993))). In another approach the stop codon of the gene CF modify in the UAA, and a second plasmid expressing the fusion protein A1-antigen, is subjected to cotransformation. The second plasmid encodes resistance to another antibiotic, and the replication origin is compatible with the first plasmid. In the third approach, CF and the fusion protein A1-antigen encode bicistronic method, functionally associating with a promoter such as the Trp promoter, as described in Fig.1 in Kozlovska et al., Interviroiogy, 39: 9-15 (1996).

Other VLPs suitable for fusion of antigens or antigenic determinants described in WO 03/024481 and include the bacteriophage fr, RNA phage MS-2 capsid protein of human papillomavirus, The retrotransposon, yeast, and retrovirus-like particles, H1V2 Gag, mosaic virus, cow peas, the VLP VP2 of parvovirus, HBsAg (US 4722840, EP 0020416 B1). Examples of chimeric VLPs suitable for the practical implementation of the invention, also described in Interviroiogy 39: 1 (1996). Other examples of VLPs provided for use in the invention are: HPV-1, HPV-6, HPV-11, HPV-16, HPV-18, HPV-33, HPV-45, CRPV, COPY, HIV GAG, mosaic virus tobacco. Virus-like particles SV-40, polyomavirus, adenovirus, herpes simplex virus, rotavirus, and norovirus.

For any recombinante expressed antigenic PCSK9 peptide according to the invention, associated or not associated with immunogenic carrier, nucleic acid, to the which encodes the specified peptide or protein also forms an aspect of the present invention, as expressing a vector containing a nucleic acid, a host cell containing expressing vector (stand-alone or integrated in the chromosome). The recombinant method of obtaining a peptide or protein by the expression in the above cell host and discharge from her immunogen is an additional aspect of the invention. The present invention does not include a full-sized native PCSK9 molecule or a full-sized native DNA sequence encoding it.

In another embodiment, the peptide according to the invention is chemically related to the immunogenic carrier using methods well known in the art. Conjugation can occur with the aim of ensuring the free movement of peptides through a single point konjugierte (for example, N-terminal or C-terminal point) or in the form of a conformationally restricted (locked down) structure where both ends of the peptides conjugated to either immunogenic protein carrier or frame structure, such as the VLP. Conjugation is carried out by means of conjugation reactions, known to experts in the art, for example, through cysteine residues, lysine residues or other carboxyl group, widely known as points of conjugation, such as glutamic acid or aspartic acid. Thus, voltage is emer, for direct covalent binding of omage to use carbodiimide, glutaraldehyde or (N-[y-maleimidomethyl]operations ester using conventional commercially available heterobifunctional linkers, such as CDAP and SPDP (using the manufacturer's instructions). Examples of conjugation of peptides, especially cyklinowanie peptides with protein carrier through acylhydrazines peptide derivatives described in WO 03/092714. After the reaction, the combination of the immunogen can be easily isolated and purified by the method of dialysis, a method, gel filtration, fractionation method, etc. Peptides ending with the cysteine residue (preferably, a linker outside cyklinowanie plot), can be conjugated to a protein carrier through maleimide chemistry.

When the immunogenic carrier is a the VLP, separate antigenic peptide, or having the identical amino acid sequence, or another amino acid sequence, may be subjected to combined with one molecule of the VLP, which preferably leads to a repetitive and ordered structure representing several antigenic determinants oriented way, as described in WO 00/32227, WO 03/024481, WO 02/056905 and WO 04/007538.

In the preferred embodiment of the antigenic PCSK9 peptide is associated with the VLP via chemical is someone stapling, typically and preferably by using heterobifunctional cross-linker. In the art there are several heterobifunctional cross-linkers. In some embodiments heterobifunctional cross-linker contains a functional group that can react with the first sites of accession, i.e., the amino group of the side chain lysine residues the VLP or the VLP subunit, and another functional group that can react with a second preferred site of accession, i.e., a cysteine residue, merged with antigenic peptide and may also be provided for response through recovery. The first stage of the procedure, usually called derivatization, is a reaction of the VLP with the cross-linker. The product of this reaction is an activated the VLP, also called activated media. In the second stage, unreacted cross-linker is removed using conventional methods such as gel filtration or dialysis. In the third stage, the antigenic peptide is subjected to interaction with activated the VLP, and this stage is usually referred to as the stage combinations. Unreacted antigenic peptide can be removed in the fourth stage, for example by dialysis. In the art there are several heterobifunctional cross-linkers. They include pre is respectful cross-linkers SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SlAB Suifo-SMPB, Sulfo-SMCC, SVSB, SIA and other cross-linkers, such as those available from Pierce Chemical Company (Rockford, IL, USA) and having one functional group reactive towards amino groups and one functional group reactive against cysteine residues. All of the above cross-linkers lead to the formation of thioester linkages.

Another class of cross-linkers suitable for the practical implementation of the invention, are characterized by introduction of a disulfide bond between the antigenic peptide and the VLP when combined. Preferred cross-linkers belonging to this class include, for example, SPDP and Sulfo-LC-SPDP (Pierce). The degree of derivatization the VLP with the cross-linker may be affected by various experimental conditions such as concentration of each of the partners of the reaction, an excess of one reagent over the other, pH, temperature and ionic strength. The degree of combination, i.e., the number of antigenic peptide to the VLP subunit, can be adjusted by varying the experimental conditions described above to match the requirements of the vaccine.

Another way of binding antigenic peptide to the VLP is a binding lysine residue on the surface of the VLP with a cysteine residue on antigenic peptide In some embodiments may require the merger of the amino acid linker containing a cysteine residue, as in the showing customers joining or part thereof for binding antigenic peptide to the VLP. In General, flexible amino acid linkers are preferred. Examples of the amino acid linker is selected from the group consisting of: (a) CGG; (b) N-terminal gamma 1-linker; (C) N-terminal gamma 3-linker; (d) hinge regions Ig; (d) N-terminal glycine of linkers; (f) (G) kC (G) n, where n=0-12 and k=0-5; (g) N-terminal glycine-serine linkers; (h) (G) kC (G) m (S) i (GGGGS) n, where n=0-3, k=0-5, m=0-10, i=0-2; (I) GGC; (K) GGC-NH2; (l) C-terminal gamma 1-linker; (m) C-terminal gamma 3-linker; (n) C-terminal glycine of linkers; (o) (G)nC(G)k with n=0-12 and k=0-5; (p) C-terminal glycine-serine linkers; (R) (G)m(S)t (GGGGS)n(G)oC (G) k, where n=0-3, k=0-5, m=0-10, t=0-2, and o=0-8. Other examples of amino acid linkers are the hinge region of immunoglobulins, glycine-serine linkers (GGGGS)n and glycine linkers (G)n, which are all optionally contain a cysteine residue as second site connection and possibly additional glycine residues. As a rule, preferable examples of the above amino acid linkers represent the N-terminal gamma 1: CGUKTHTSPP; C-terminal gamma 1: DKTHTSPPCG; N-terminal gamma 3: CGGPKPSTPPGSSGGAP; C-terminal gamma 3: PKPSTPPGSSGGAPGGCG; N-terminal glycine linker: GCGGGG and C-terminal glycine linker: GGGGCG.

Other amino acid linkers, especially suitable for the practical implementation of the invention, when a hydrophobic antigenic peptide is associated with the VLP, predstavlyaet a CGKKGG or CGDEGG for N-terminal linkers, or GGKKGC and GGEDGC for C-terminal linkers. For C-terminal linkers terminal cysteine may amitirova on the C-end.

In some embodiments of the present invention GGCG, GGC or GGC-NH2("NH2means amidation) linkers at the C-end of the peptide or CGG at its N-end are preferred as amino acid linkers. In General, the residues of glycine would be built between the major amino acids and cysteine is used as the second site of accession, in order to avoid possible steric hindrances from larger amino acids in the reaction combinations. In another embodiment of the invention, the amino acid linker GGC-NH2merged with With the end of the antigenic peptide.

The cysteine residue present on the antigenic peptide, must be in its reduced state to interact with heterobifunctional cross-linker for activated the VLP, i.e., a free cysteine or a cysteine residue with a free sulfhydryl group must be available. When the cysteine residue to function Kakashi binding site is in the oxidized form, for example, if it is the formation of a disulfide bridge requires the restoration of this disulfide bridge, for example, using DTT, TCER or β-mercaptoethanol. Low concentrations at which vitela compatible with the combination, as described in WO 02/05690, higher concentrations inhibit the reaction mix, as known to the expert, in this case, the reducing agent must be removed or its concentration is reduced before the combination, for example, by dialysis, gel filtration or HPLC with reversed phase.

The binding of the antigenic peptide to the VLP using heterobifunctional cross-linker in accordance with the methods described above, makes possible the combination of antigenic peptide to the VLP oriented way. Other methods of binding antigenic peptide to the VLP include the ways in which the antigenic peptide is bound to the VLP using the carbodiimide EDC, and NHS.

When using other methods antigenic peptide attached to the VLP using homobifunctional cross-linker such as glutaraldehyde, DSGBM [PEO] 4, BS3 (Pierce Chemical Company, Rockford, IL, USA) or other known homobifunctional cross-linkers with functional groups reactive towards amine groups or carboxyl groups of the VLP.

Other methods of binding the VLP with antigenic peptide include methods where the VLP biotinylated, and antigenic peptide is expressed in the form of streptavidin-fused, Baka, or how, where and antigenic peptide, and the VLP biotinylated, for example as described in WO 00/23955. In this case, the antigenic peptide may be linked to what davidina or Avidya by adjusting the ratio of the antigenic peptide to streptavidin Hakim way to available binding sites available for binding the VLP, which is added at the next stage. Alternatively, all components can be mixed in one-step reaction. Other ligand-receptor pairs, where a soluble form of the receptor and ligand are available and capable of cross-linking with the VLP or antigenic peptide can be used as binding agents for binding antigenic peptide to the VLP. Alternatively, either the ligand or the receptor may be merged with the antigenic peptide and thus to mediate binding the VLP, chemically bound or fused with either receptor or ligand, respectively. Fusion can also be accomplished by insertion or substitution.

One or more antigen molecules can be attached to one subunit of the capsid or the VLP envelope proteins of RNA-phages, preferably through the exposed lysine residues of the VLP RNA-phages, if it is sterically possible. A special characteristic of the VLP envelope protein of RNA phage and, in particular, the VLP protein shell QP represents, therefore, the ability to bind multiple antigens for subunit. This allows you to create dense antigenic matrix.

VLPs or capsid shell protein Q exhibit a certain number of lysine residues on its surface, with a certain topology: three residues l is Zina, directed inside the capsid and interacting with RNA, and the other four lysine residues facing outward from the capsid. These specific properties contribute to attach antigens to the outer surface of the particles and not to the inner surface of particles, where lysine residues interact with RNA. VLPs envelope proteins of other RNA-phage also have a certain number of lysine residues on its surface and a certain topology of these lysine residues.

In another embodiment of the present invention, the first site of accession is a lysine residue and/or the second site of accession contains a sulfhydryl group or a cysteine residue. In yet another embodiment of the present invention, the first site of accession is a lysine residue and a second site accession represents a cysteine residue. In other embodiments of the invention, the antigen or antigenic determinant is bound via a cysteine residue with lysine residues the VLP envelope protein of RNA phage and, in particular, with the VLP protein shell Qbeta.

Another advantage of VLPs derived from RNA-phages is their high yield expression in bacteria, which makes possible the production of large quantities of material at an affordable price. In addition, the use of VLPs as the media makes possible the formation of stable an Hennig matrices and conjugates, accordingly, different densities of antigen. In particular, the use of VLPs of RNA-phages and thus, in particular, the use of the VLP envelope protein of RNA phage Qbeta allows to obtain very high epitope density.

According to the embodiment of the present invention antigenic PCSK9 peptide described in this application, is connected, preferably chemically cross-linked with CRM197, either directly or through one of the peptide linkers described in this application, to obtain the immunogen. In one of the embodiments of the antigenic PCSK9 peptide described in this application is linked to CRM197 via chemical crosslinking, as described in this application, and preferably by using heterobifunctional cross-linker, as described above.

Preferred heterobifunctional cross-linkers for use with CRM197 are BAANS (N-hydroxysuccinimidyl ester bromoxynil acid), SMPH (Succinimidyl-6-[β-maleimidopropionamide]hexanoate), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SlAB, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and other cross-linkers, such as those available from Pierce Chemical Company (Rockford, IL, USA). In the preferred embodiment of the present invention heterobifunctional cross-linker is a BAANS or SMPH.

Alternatively, a suitable cross-linkers, which makes possible the introduction of a disulfide bond between the antigenic peptide and CRM197, can also be used is carried out in the context of the present invention. Preferred cross-linkers belonging to this class include, for example, SPDP and Sulfo-LC-SPDP (Pierce).

In a particular embodiment, when the sequence of the antigenic PCSK9 peptide described in this application, contains cysteine, then the specified antigenic PCSK9 peptide may be covalently linked to CRM197 directly through the specified cysteine.

In some embodiments of the invention the immunogenic composition according to the invention can contain a mixture of immunogenic conjugates, i.e., immunogenic carriers, associated with one or more antigenic PCSK9 peptides according to the invention. Thus, these immunogenic composition can comprise immunogenic carriers, which differ in amino acid sequence. For example, can be obtained vaccine compositions containing the VLP "wild-type" and the modified protein of the VLP, in which one or more amino acid residues have been modified (for example, delegated, inserted, or replaced). Alternatively, it may be used the same immunogenic carrier, but is associated with antigenic PCSK9 peptides with different amino acid sequences.

Therefore, the invention also relates to a method for producing immunogen according to the invention, including: 1) ensuring the antigenic PCSK9 peptide according to the invention, 2) ensuring the immunogenic carrier is according to the invention, preferably the VLP, and 3) the Union of the specified antigenic PCSK9 peptide and the immunogenic carrier. In one of the embodiments of the specified stage of the Association is carried out by chemical crosslinking, preferably through heterobifunctional cross-linker.

In one of the embodiments of the present invention antigenic PCSK9 peptide described in this application is associated with a molecule immunogenic carrier. In one of the embodiments specified immunogenic carrier selected from the group consisting of any immunogenic carrier, as described in this application. In another embodiment of the specified immunogenic carrier selected from the group consisting of: serum albumin, such as bovine serum albumin (BSA); globulins; thyroglobulin; hemoglobins; hemocyanine (in particular, hemocyanin lymph snails [KLH]) and virus-like particles (VLPs). In the preferred embodiment of the specified immunogenic carrier is a diphtheria toxoid, CRM197 mutant of diphtheria toxin, tetanus toxoid, hemocyanin lymph snails or virus-like particle (VLPs). In yet another preferred embodiment of the specified immunogenic carrier is a DT, CRM197, or the VLP selected from the group consisting of the VLP HBcAg, the VLP HBsAg, the VLP Qbeta, the VLP PP7, the VLP PPV, the VLP of the norovirus, or any option, described in this application. Another pre is respectful embodiment of the specified immunogenic carrier represents the VLP of bacteriophage, such as the VLP Qbeta selected from the group consisting of CF Qbeta; A1 Qbeta, Qbeta-240, Qbeta-243, Qbeta-250, Qbeta-251 and Qbeta-259 (disclosed in WO 03/024481) or PP7.

In another preferred embodiment of the specified immunogenic carrier is a CRM197.

In one of the embodiments specified immunogenic carrier covalently linked to an antigenic PCSK9 peptide described in this application, either directly or through a linker. In one of the embodiments specified immunogenic carrier linked to antigenic PCSK9 peptide described in this application, through the expression of the fused protein, as described in this application. In another embodiment the antigenic PCSK9 peptide described in this application relates to immunogenic carrier, preferably the VLP by chemical crosslinking, as described in this application, and preferably using heterobifunctional cross-linker. Some heterobifunctional cross-linkers known in the art. In some embodiments heterobifunctional cross-linker contains a functional group that interacts with the first sites of accession, i.e., the amino group of the side chain lysine residues the VLP or the VLP subunit, and an additional functional group which can interact with the second preferred site of accession, i.e., a cysteine residue, merged with antigenic peptide made available for reaction by reduction.

Antigenic PCSK9 peptide of the invention containing linker

In one of the embodiments of the present invention antigenic PCSK9 peptide described in this application further comprises either at its N end, or at its C-end, or at both the N-end and C-end of the linker, which is able to interact with customers joining immunogenic carrier in chemical reactions stitching. In one of the embodiments of the antigenic PCSK9 peptide described in this application, further comprises at its C-end of the linker having the formula (G)nC (G)nSC or (G)nK, preferably (G)nC, where n is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, preferably from the group consisting of 0, 1, 2, 3, 4 and 5, more preferably from the group consisting of 0, 1, 2 and 3, most preferably n is 0 or 1 (if n is 0, the formula represents a cysteine). Preferably, the antigenic PCSK9 peptide described in this application, further comprises at its C-end of the linker having the formula GGGC, GGC, GC or C.

In another embodiment of the present invention antigenic PCSK9 peptide described in this application, further comprises at its N-end linker having the formula C(G)n, CS(G)nor K(G)npreferably C(G)nwhere n represents an integer selected from the group sotoyama is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, preferably from the group consisting of 0, 1, 2, 3, 4 and 5, more preferably from the group consisting of 0, 1, 2 and 3, most preferably n is 0 or 1 (if n is 0, the formula represents a cysteine). Preferably, the antigenic PCSK9 peptide described in this application, further comprises at its N-end linker having the formula CGGG, CGG CG or C.

In another embodiment the antigenic PCSK9 peptide described in this application, further comprises at its C-end of the linker having the formula (G)nC (G)nSC or (G)nK, preferably (G)nC, where n is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, preferably from the group consisting of 0, 1, 2, 3, 4 and 5, more preferably from the group consisting of 0, 1, 2 and 3, most preferably n is 0 or 1 (if n is 0, the formula represents a cysteine), and at its N-end of the linker, having the formula C(G)n, CS(G)nor K(G)npreferably C(G)nwhere n is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, preferably from the group consisting of 0, 1, 2, 3, 4 and 5, more preferably from the group consisting of 0, 1, 2 and 3, most preferably n is 0 or 1 (if n is 0, the formula represents a cysteine). Preferably, the antigenic PCSK9 peptide described in this application,further comprises at its N-end of the linker, those having the formula: CGGG, CGG CG or C, and at its C-end of the linker having the formula GGGC, GGC, GC or S. Preferably, the antigenic PCSK9 peptide described in this application, further comprises at its N-end cysteine, and at its C-end of cysteine.

Typical representatives of these antigenic PCSK9 peptides, optionally containing such linker are disclosed in SEQ ID NO:313, 314, 315, 316, 317, 322, 323, 324, 325, 326, 327, 328, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418 and 419.

Typical representatives of these antigenic PCSK9 peptides, optionally containing such linker are disclosed in SEQ ID NO:313, 314, 315, 316, 317, 322, 323, 324, 325, 326, 327 and 328.

Preferred antigenic PCSK9 peptide containing a linker are disclosed in SEQ ID NO:317, 322, 323, 324, 401, 402, 403, 413, 414, 415 and 416.

Preferred antigenic PCSK9 peptide containing a linker are disclosed in SEQ ID NO:317, 322, 323 and 324.

The most preferred antigenic PCSK9 peptide containing a linker are disclosed in SEQ ID NO:317, 322, 402 and 413.

In one of the embodiments of the antigenic PCSK9 peptide is cyklinowanie. In one of the embodiments cyklinowanie antigenic PCSK9 peptide is attached to an immunogenic carrier. In one of the embodiments specified cyklinowanie antigenic PCSK9 peptide is attached to an immunogenic carrier via covalent bonding. In one of the embodiments specified cyklinowanie antigenic PCSK9 peptide is attached to immunogenic but is Italy through covalent binding of one of the side chains of amino acids with the carrier. In one of the embodiments of cysteine, GC or CC-fragment, containing different number of glycine residues and one cysteine residue, add to cyklinowanie PCSK9 peptides to provide covalent binding to immunogenic carrier through the added cysteine.

In one of the embodiments of the antigenic PCSK9 peptide is cyklinowanie and contains cysteine, (G)nC - or(G)nslice, where n is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, preferably from the group consisting of 0, 1, 2, 3, 4 and 5, more preferably from the group consisting of 0, 1, 2 and 3, most preferably n is 0 or 1 (if n is 0, the formula represents a cysteine).

Non-limiting examples of such conformationally restricted antigenic PCSK9 peptides are the peptides with SEQ ID NO:318, 319, 320 and 321. Preferred cyklinowanie peptide is a peptide with SEQ ID NO:318.

Examples of conjugatively antigenic PCSK9 peptide with a carrier or cage, described above, are within the scope of the present invention, and form different embodiments using different linkers, which are listed below:

Peptide - GGGGGC - frame, peptide - GGGGC - frame, peptide - GGGC - frame, peptide - GGC - frame, peptide - GC - frame, the peptide - To - frame, the peptide - GGGGGK - frame, peptide - GGGGK - frame, Peptide - GGGK - frame, PE is the Chida - GGK - frame, Peptide - GK - frame, Peptide -, K - frame, Peptide - GGGGSC - frame, Peptide - GGGSC - frame, Peptide - GGSC - frame, Peptide - GSC - frame, Peptide - SC - frame, Frame - CSGGGG - Peptide, Frame - CSGGG - Peptide, Frame - CSGG - Peptide, Frame - CSG - Peptide, Frame - CS - Peptide, Frame - KGGGG - Peptide, Frame - KGGG - Peptide, Frame - KGG - Peptide, Frame - KG - Peptide, Frame - To - Peptide.

In one embodiments, the peptide consists of any of the antigenic PCSK9 peptides described in this application, and the frame consists of any of the immunogenic carriers described in this application, preferably the VLP.

A typical combination of conjugatively using different linkers and doubly constrained peptides presented below, where the media can be a identical monomer of media or different monomer media. (In the example below, GC-linker can be substituted for any GK-linker or GSC-linker described above or any other well-known experts in the field of technology):

Media - CGGGGG - Peptide - GGGGGC - carrier, Carrier-CGGGG - Peptide - GGGGC - carrier, Carrier - CGGGG - Peptide-GGGGC - carrier, Carrier - CGGG - Peptide - GGGC - carrier, Carrier - CG - Peptide - GC - carrier, Carrier - CG - Peptide - carrier, Carrier - s - Peptide - s - media.

In one embodiments, the peptide consists of any antigenic PCSK9 peptide described in this application, and the carrier state is t from any immunogenic carrier, described in this application, preferably the VLP.

In one of the embodiments of the invention relates to an immunogen containing the antigenic PCSK9 peptide consisting of or consisting essentially of the amino acid sequence selected from the group consisting of SEQ ID NO:1-312, 330-398 and 420-588 where specified antigenic PCSK9 peptide further comprises at its C-end or at its N end of cysteine which is chemically cross-stitched with immunogenic carrier through thioester linkages. In the preferred embodiment of the specified immunogenic carrier selected from the group consisting of DT (diphtheria toxin), TT (tetanus toxoid) or fragment of TT, PD (Haemophilus influenzae protein D), CRM197, other point mutants DT, such as CRM176, CRM228, CRM45, CRM9, CRM102, CRM103 and CRM107. Preferably, the specified immunogenic carrier is a CRM197.

In one of the embodiments of the invention relates to an immunogen containing the antigenic PCSK9 peptide consisting of or consisting essentially of the amino acid sequence selected from the group consisting of 1-312, 330-398 and 420-588 where specified antigenic PCSK9 peptide further comprises at its C-end or at its N end of cysteine which is chemically cross-linked to immunogenic carrier via a thioester communication using SMPH (Succinimidyl-6-[β-maleimidopropionamide]hexanoate) or BAANS (N-hydroxy who Succinimidyl ester bromoxynil acid) as cross-linker. In the preferred embodiment of the specified immunogenic carrier selected from the group consisting of DT (diphtheria toxin), TT (tetanus toxoid) or fragment of TT, PD (Haemophilus influenzae protein D), CRM197, other point mutants DT, such as CRM176, CRM228, CRM 45, CRM 9, CRM102, CRM103 and CRM107. Preferably, the specified immunogenic carrier is a CRM 197.

In one of the embodiments of the invention relates to an immunogen containing the antigenic PCSK9 peptide consisting of or consisting essentially of the amino acid sequence selected from the group consisting of SEQ ID NO:1-312, 330-398 and 420-588 where specified antigenic PCSK9 peptide further comprises at its C-end of cysteine which is chemically cross-linked to immunogenic carrier via a thioester communication using SMPH (Succinimidyl-6-[β-maleimidopropionamide]hexanoate) or BAANS (N-hydroxysuccinimidyl ester bromoxynil acid) as cross-linker, where this link is located between the lysine residue CRM 197 and the cysteine residue of the specified antigenic peptide.

A composition containing antigenic PCSK9 peptide of the invention

The present invention also relates to compositions, in particular to immunogenic compositions, also referred to as "the immunogenic compositions containing the antigenic PCSK9 peptide according to the invention preferably relates the output with immunogenic carrier, and possibly at least one adjuvant. Such immunogenic compositions in particular, when prepared in the form of pharmaceutical compositions, are useful for the prevention, cure or relief of PCSK9-related disorder.

In some embodiments considered immunogenic composition according to the invention contains antigenic PCSK9 peptide may contain a linker containing an amino acid sequence selected from SEQ ID NO:1-328, 330-398 and 401-588 and functionally active variants. In some embodiments specified antigenic PCSK9 peptide is linked to immunogenic carrier, preferably with DT, CRM 197 or the VLP, preferably with the VLP HBcAg, HBsAg, Qbeta, PP7, PPV or norovirus.

In the preferred embodiment considered immunogenic composition according to the invention contains antigenic PCSK9 peptide may contain a linker containing an amino acid sequence selected from SEQ ID NO:1-328, 330-398 and 401-588 and functionally active variants associated with the VLP, preferably with the VLP Qbeta.

In the preferred embodiment considered immunogenic composition according to the invention contains antigenic PCSK9 peptide may contain a linker containing an amino acid sequence selected from SEQ ID NO:1-328, 330-398 and 401-588 and functionally active variants associated with CRM197.

Russ is trevena immunogenic composition, containing antigenic PCSK9 peptide according to the invention, can be prepared in the form of the drug in different ways, as described in more detail below.

In some embodiments considered immunogenic composition comprises one type of antigenic PCSK9 peptide, for example, the immunogenic composition comprises a population of antigenic PCSK9 peptides, essentially all of which have the same amino acid sequence. In other embodiments considered immunogenic composition comprises two or more different antigenic peptides PCSK9, for example, the immunogenic composition comprises a population of antigenic PCSK9 peptides, where the members of this population may differ in amino acid sequence. Consider immunogenic composition may contain from two to about 20 different antigenic PCSK9 peptides, for example, considered immunogenic composition may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-15 or 15-20 different antigenic PCSK9 peptides, each of which has an amino acid sequence that differs from amino acid sequences of other antigenic PCSK9 peptides.

In other embodiments considered immunogenic composition comprises multimediabay antigenic PCSK9 polypeptide, as described above. Used in this application, the terms "immunogenic composition, with the holding antigenic PCSK9 peptide or immunogenic composition according to the invention" or "the immunogenic composition" refers to an immunogenic composition, containing any one kind (multimediabay or not), or several types of antigen(s) of the peptide(s) PCSK9 associated or not associated with immunogenic carrier. If two or more peptides use associated with the carrier, the peptide can be associated with one and the same molecule carrier or individually associated with molecules-carriers and then combined with obtaining immunogenic composition.

Another aspect of the invention relates to methods of producing immunogen according to the invention comprising the combination of antigenic PCSK9 peptide to the immunogenic carrier. In one of the embodiments of the specified combination is chemical.

Adjuvants

In some embodiments considered immunogenic composition comprises at least one adjuvant. Suitable adjuvants include adjuvants suitable for use in mammals, preferably in humans. Examples of known suitable adjuvants that can be used in humans include, but are not necessarily limited to, alum, aluminum phosphate, a hydrate of aluminum oxide, MF59 (4,3% wt./about. squalene, 0.5% wt./about. Polysorbate 80 (Tween 80), 0.5% wt./about. sorbitrate (Span 85)), CpG-containing nucleic acid (where the cytosine demetilirovanny), QS21 (saponify adjuvant), MPL (monophosphorylated A), 3DMPL (3-0-describeany MPL), extracts from Aquilla, ISCOMS (see, for example, the R, Sjölander et al. (1998) J. Leukocyte Biol. 64:713; WO 90/03184, WO 96/11711, WO 00/48630, WO 98/36772, WO 00/41720, WO 06/134423 and WO 07/026190), mutants of LT/CT, poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A, TiterMax classic, TiterMax Gold, interleukins, etc., For applications in veterinary medicine, including but not limited to experiments on animals, you can use beta-blockers, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-norbornyl-L-alanyl-D-isoglutamine (CGP 11637, known as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyrisperidone)-ethylamine (CGP A known as MTP-PE) and RIBI, which contains three components extracted from bacteria, monophosphorylated And trehalose of dimycolate and cell wall skeleton (MPL+TDM+CWS) in the emulsion 2% squalene/Tween 80.

Other typical adjuvants to increase the effectiveness of the composition include, but are not limited to; (1) preparations of emulsions of oil-in-water (with or without other specific immunostimulating agents such as muramylpeptide (see below) or components of bacterial cell walls), such as for example (a) MF59™ (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell &Newman, Plenum Press 1995), containing 5% squalene, 0.5% of Tween 80 (polyoxyethylenesorbitan monooleate) and 0.5% Span 85 (sorbitan trioleate) (possibly containing muramyldipeptide, covalently associated with dipalmitoyl-phosphatidate what aluminum (MTP-PE)), prepared in the form of submicron particles using microfluidizer, (b) SAF, containing 10% squalene, 0.4% of Tween 80, 5% pluronic-blocked polymer 1.121 and thr-MDP, either treated microfluidizer in submicron emulsion or processed on the vortex with obtaining emulsions with particles of larger size and (b) adjuvant system RIBS™ (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% squalene, 0.2% of Tween 80, and one or more components of the bacterial cell wall, such as monophosphorylated A (MPL), trehalose of dimycolate (TDM) and cell wall skeleton (CWS), preferably MPL+CWS (DETOX™); (2) can be used saponine adjuvants, such as QS21, STIMULON™ (Cambridge Bioscience, Worcester, MA), Abisco® (Isconova, Sweden) or Iscornatrix® (Commonwealth Serum Laboratories, Australia) or particles formed from them, such as ISCOMs (immunostimulating complexes), which ISCOMS may be devoid of additional detergent, for example, WO 00/07621; (3) complete adjuvant's adjuvant (CFA) and incomplete adjuvant's adjuvant (IFA); (4) cytokines, such as interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO 99/44636), and so on), interferons (e.g. gamma interferon), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), and so on; (5) monophosphorylated A (MPL) or 3-D-describeany MPL (3dMPL), for example, GB-2220221, EP-A-0689454, perhaps in the actual absence of alum when used with pneumococcal saccharides, n is the sample, WO 00/56358; (6) combinations of 3dMPL with, for example, QS21 and/or oil emulsions in water, for example, EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) oligonucleotides containing CpG-motifs [Krieg Vaccine 2000, 19, 618-622; Krieg Curropin Mol Ther2001 3:15-24; Roman et al., Nat. Med., 1997, 3, 849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Davis et al., J. Immunol, 1998, 160, 870-876, Chu et al., J. Exp. Med, 1997, 186, 1623-1631; Lipford et al., Ear. J. Immunol., 1997, 27, 2340-2344; Moldoveami et al., Vaccine, 1988, 16, 1216-1224, Krieg et al., Nature, 1995, 374, 546-549; Klinman et al., PNAS USA, 1996, 93, 2879-2883; Ballas et al., J. Immunol, 1996, 157, 1840-1845; Cowdery et al., J. Immunol, 1996, 156, 4570-4575; Halpern et al. Cell Immunol, 1996. 167, 72-78; Yamamoto et al., Jpn. J. Cancer Res., 1988, 79, 866-873; Stacey et al., J. Immunol, 1996, 157,2116-2122; Messina et al., J. Immunol, 1991. 147, 1759-1764; Yi et al., J. Immunol, 1996, 157,4918-4925; Yi et al., J. Immunol, 1996, 157, 5394-5402; Yi et al., J. Immunol, 1998, 160, 4755-4761; and Yi et al., J. Immunol, 1998, 160, 5898-5906; International patent application WO 96/02555, WO 98/16247, WO 98/18810, WO 98/40100, WO 98/55495, WO 98/37919 and WO 98/52581], i.e. containing at least one CG dinucleotide, where the cytosine demetilirovanny: (8) a simple polyoxyethylene ether or complex polyoxyethylene ether, for example, WO 99/52549; (9) polyoxyethylenesorbitan ester surfactant in combination with octoxynol (WO 01/21207) or polyoxyethylenated-essential or clonifine surfactant in combination with at least one additional non-ionic surface-active agent, such as an octoxynol (WO 01/21152); (10) a saponin and an immunostimulating oligonucleotide (e.g. a CpG-oligonucleotide) (WO 0/62800); (11) an immunostimulant and a particle of metal salt, for example, WO 00/23105; (12) a saponin and an emulsion oil in water, for example, WO 99/11241; (13) a saponin (e.g., QS21)+3dMPL+IM2 (possibly +a Sterol), for example, WO 98/57659; (14) other substances that act as immunostimulating agents to enhance the effectiveness of the composition, such as muramylpeptide, including N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-norbornyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyrisperidone)-ethylamine MTP-PE), (15) ligands for toll-like receptors (TLR), natural or synthesized (e.g., as described in the Kanzler et al 2007, Nature Medicine 13, p.1552-9), including TLR3 ligands, such as poly-I:C and similar compounds such as Hiltonol and Ampligen.

In a particular embodiment of the specified adjuvant is an immune-stimulating oligonucleotide and more preferably CpG oligonucleotide. CpG oligonucleotide as used in this application refers to immunostimuliruyushhim CpG-oligodeoxynucleotide (one CpG), and therefore these terms are used interchangeably, unless otherwise noted. Immunostimulatory CpG-oligodeoxynucleotide contain one or more immunostimulatory CpG motifs, which are neetilirovannye cytosine-goinnovate dinucleotides, possibly defined in the s preferred contexts grounds. The methylation status immunostimulating CpG-motif, usually refers to the cytosine residue in the study. Immune-stimulating oligonucleotide containing at least one demetilirovanny CpG dinucleotide, is an oligonucleotide that contains a 5'-demetilirovanny cytosine associated phosphate bond with the 3'-guanine, and which activates the immune system by binding to Toll-like receptor 9 (TLR-9). In another embodiment of the immune-stimulating oligonucleotide may contain one or more methylated CpG dinucleotides, which can activate the immune system through TLR9, but not as much as if CpG motif(s) was(and is) demetilirovanny(and), Immunostimulatory CpG-oligonucleotides may contain one or more palindromes, which, in turn, can cover a CpG dinucleotide. CpG oligonucleotides have been described in many issued patents, published patent applications and other publications, including U.S. patents№№6194388; 6207646; 6214806; 6218371; 6239116; and 6339068.

Were identified various classes of immunostimulatory CpG-oligonucleotides. They are referred to as a, b, C and R class and are described in more detail below. The methods of the invention encompass the use of these different classes of immunostimulatory CpG-oligonucleotides.

Any of the classes may be subject of modification, which is valitsimet its effectiveness. E. modification can be a substitution of the 5'-terminal nucleotide halogen; examples of such substitutions include, but are not limited to, substitution of the bromine-uridine or iodine-uridine. E. modification can also include the substitution of ethyl-uridine for 5'-terminal nucleotide.

Immunostimulatory CpG-oligonucleotides "And class" functionally characterized by their ability to induce high levels of interferon-alpha (IFN-α) plasmocytoid dendritic cells (pDC) and induction of activation MK-cells, at the same time with minimal effects on the activation of b-cells. Structurally this class usually has stabilized poly-G sequence at the 5'- and 3'-ends. He also has palindrome fosfodiesterazu CpG dinucleotide-containing a sequence of at least 6 nucleotides, for example, but not necessarily, it contains one of the following review of palindromes: GACGTC, AGCGCT or AACGTT described by Yamamoto et al.Yamamoto S et al. J. Immunol 148:4072-6 (1992). Immunostimulatory CpG-oligonucleotides And class and typical sequences of this class have been described in the patent application U.S. serial No. 09/672126 and published PCT application PCT/US00/26527 (WO 01/22990), which both filed September 27, 2000

In one of the embodiments of the CpG-oligonucleotide "And class" according to the invention has the following sequence of nucleic acids' 5' GGGGACGACGTCGTGGGGGGG 3'

Some non-limiting examples of oligonucleotides a-class include:

5' G*G*G_G_A_C_G_A_C_G_T_C_G_T_G_G*G*G*G*G*G 3';

where * refers to phosphorothioates communication, and _ refers to a phosphodiester bond.

The sequence of the CpG oligonucleotide In class according to the invention described above and disclosed in the published patent applications PCT PCT/US 95/01570 and PCT/US97/19791 and in patents US 6,194,388, 6,207,646, 6,214,806, 6,218,371, 6,239,116 and 6,339,068. Typical sequences include, but are not limited to, the sequence described in these applications and patents.

In one of the embodiments of the CpG-oligonucleotide "In class" according to the invention has the following nucleic acid sequence:

5' TCGTCGTTTTTCGGTGCTTTT 3' (SEQ ID NO:589), or

5' TCGTCGTTTTTCGGTCGTTTT 3' (SEQ ID NO:590), or

5' TCGTCGTTTTGTCGTTTTGTCGTT 3' (SEQ ID NO:591), or

5' TCGTCGTTTCGTCGTTTTGTCGTT 3' (SEQ ID NO:592), or

5' TCGTCGTTTTGTCGTTTTTTTCGA 3' (SEQ ID NO:593).

In any of these sequences, all communication can be phosphorothioate communication. In another embodiment, in any of these sequences, one or more ties can be phosphodieterase, preferably between "C" and "G" CpG motif with obtaining semi CpG oligonucleotide. In any of these sequences, 5' T can be substituted ethyl-uridine or halogen; examples of the halogen substitutions include, but are not limited to, substitution of the bromine-uridine or iodine-uridine.

Some non-limiting examples of oligonucleotides In class

include;

5' T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*G*C*T*T*T*T 3', or

5' T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T 3', or

5' T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T 3', or

5' T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T 3', or

5' T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*T*T*T*C*G*A 3',

where * refers to phosphorothioates connection.

Immunostimulatory CpG-oligonucleotides "class" functionally characterized by their ability to activate b-cells and NK-cells and to induce IFN-α. Structurally this class typically includes a plot with one or more immunostimulating CpG motifs In class, and GC-rich palindrome or allopolyploidy plot, which allows the molecules to form secondary (e.g., stem-loop) or tertiary (e.g., dimeric) patterns. Some of these oligonucleotides have a traditional "stimulating" CpG sequence, and "GC-rich" or "neutralizing B-cell" motif. Oligonucleotides with these combined motives exert Immunostimulatory effects that are somewhere between the effects associated with traditional CpG-oligonucleotides In class (i.e., a strong induction of the activation of b-cells and activation of dendritic cells (DC), and effects associated with CpG And one class (i.e., a strong induction of the activation of IFN-α and NK-cells, but relatively weak induction of activated b cells and DC). Krieg AM et A1. (1995) Nature 374:546-9; Ballas ZK et al. (1996) J Immunol 157:1840-5; Yamamoto S et al. (1992) J Immnol 148:4072-6.

Immunostimulatory Oligonucleotides From class with a combined motif can be either fully stabilized (for example, all phosphorothioate), chimeric (fosfomifira Central region), or semi-flexible (for example, fosfomifira communication within a CpG motif) skeletons. This class has been described in the patent application US 10/224,523, filed August 19, 2002.

One incentive domain or motif CpG-oligonucleotide With a class defined by the formula: 5' X1DCGHX23'. D represents a nucleotide other than S. is vicosin. G represents guanine. N represents a nucleotide other than g X1and X2represent any sequence of nucleic acids of length from 0 to 10 nucleotides. X1may include CG, in this case, preferably T immediately precedes this CG. In some embodiments DCG is a TCG. X1has a length of preferably from 0 to 6 nucleotides. In some embodiments X2contains no poly-G or poly-A motifs. In other embodiments immune-stimulating oligonucleotide has a poly-T sequence at the 5'-end or 3'end. As used in this application, "poly-a or poly-T" refers to the segment of four or more consecutive a or T, respectively, for example, 5' AAAA 3' or 5' TTTT 3'. How to use the about this application, "poly-G end" refers to the segment of four or more consecutive G, for example, 5' GGGG 3', located at the 5'-end or 3'-end of nucleic acids. As used in this application, "poly-G oligonucleotide" refers to an oligonucleotide having the formula 5' X1X2GGGX3X43', where X1, X2, X3and X4are nucleotides, and preferably at least one of X3and X4is a G. Some of the preferred designs for the domain, stimulating b-cells, according to this formula contain TTTTTCG, TCG, TTCG, TTTCG, TTTTCG, TCGT, TTCGT, TTTCGT, TCGTCGT.

The second motif CpG-oligonucleotide With a class called either P or N and is located 5' relative to the X1or directly from the 3' relative to the X2.

N is a sequence, a neutralizing b-cells, which begins with a trinucleotide CGG and has a length of at least 10 nucleotides. Motive, neutralizing b cell includes at least one CpG sequence in which CG is preceded or followed by G (Krieg AM et al. (1998) Proc Natl Acad Sd USA 95:12631-12636) or is a CG-containing DNA sequence, in which CG methylated. Neutralizing motifs or sequences have some degree immunostimulatory capacity, when the Pris is contained in another nastyalien motive, but when present in the context of other immunostimulatory motifs that serve to reduce immunostimulating potential of these other motives.

R is a GC-rich palindrome-containing sequence length of at least 10 nucleotides.

As used in this application, "palindrome" and equivalent to "palindrome sequence" refers to the inverted repeat, i.e., a sequence such as ABCDEE'D C B A', where a and a', and b' and so on, are bases capable of forming the usual base pairs Watson and Crick.

As used in this application, "GC-rich palindrome" refers to the palindrome having a composition of at least two-thirds of G and C. In some embodiments GC-rich domain is preferably 3' relative to the domain, stimulating b-cells". Thus, in the case of GC-rich palindrome length of 10 bases specified string contains at least 8 G and C. In the case of GC-rich palindrome length of 12 bases palindrome also contains at least 8 G and C. In the case of a 14-dimensional GC-rich palindrome at least ten bases of palindrome are G and C. In some embodiments of the GC-rich palindrome consists exclusively of G and C.

In some embodiments of the GC-rich palindrome has the composition is based is the first of at least 81% G and C. Thus, in the event of such GC-rich palindrome length of 10 bases specified palindrome consists exclusively of G and C. In the case of such GC-rich palindrome length of 12 bases and preferably at least ten bases (83%) of the palindrome was a G and C. In some preferred embodiments of the GC-rich palindrome length of 12 bases consists exclusively of G and C. In the case of a 14-dimensional GC-rich palindrome at least twelve foundations (86%) palindrome are G and C. In some preferred embodiments of the GC-rich palindrome length of 14 bases consists exclusively of G and S. of the GC-rich palindrome can be neetilirovannyj or can be methylated.

In General, this domain has at least 3 s and G, more preferably 4 each, and most preferably 5 or more each. The number of C and G in this domain does not need to be identical. Preferably, C and G were located so that they mogl to form semicomplementary duplex, or a palindrome, such as CCGCGCGG. It can be interrupted And or T, but it is preferable that semicompetent was at least partially preserved, as, for example, in the motives CGACGTTCGTCG or CGGCGCCGTGCCG. When complementarity is not saved, it is preferable that complementary base pairs are represented and a TG. In the preferred embodiment there is no more than 3 consecutive bases that are not part of the palindrome, preferably not more than 2 and most preferably only 1. In some embodiments of the GC-rich palindrome includes at least one CGG trimer, at least one trimer CCG or at least one tetramer CGCG. In other embodiments of the GC-rich palindrome is not CCCCCCGGGGGG or GGGGGGCCCCCC, CCCCCGGGGG or GGGGGCCCCC.

At least one of G in GC-rich region can be replaced by inosine (I). In some embodiments, R includes more than one I.

In some embodiments immune-stimulating oligonucleotide has one of the following formulas: 5' NX1DCGHX23', 5' XiDCGHX2N 3', 5' PX1DCGHX23', 5' X1DCGHX2P 3', 5' X1DCGHX2PX33', 5' X1DCGHPX33', 5' DCGHX2PX33', 5' TCGHX2PX33', 5' DCGHPX33' or 5'DCGHP 3'.

In the invention proposed other immunostimulatory oligonucleotides defined by the formula 5' N1PyGN2P 3'. N1represents any sequence of length from 1 to 6 nucleotides. PY is a pyrimidine. G represents guanine. N2represents any sequence of length from 0 to 30 nucleotides. R is a GC-rich palindrome-containing sequence length less than the least 10 nucleotides.

N1and N2may contain more than 50% pyrimidines and more preferably more than 50% of T. N1may include CG, in this case, preferably T immediately precedes this CG In some embodiments N1PyG is a TCG, and most preferably TCGN2where N2not a G.

N1PyGN2P may include one or more nucleotides inosine (I). With a or G in Ni can be replaced by inosine, but the Cpl is preferred compared to the IpG. For substitutions by inosine, such as IpG, the optimum activity can be achieved using a "semi" or Chimera skeleton, where the relationship between IG or CI is fosfomifira. N1 may include at least one CI, TCI-, IG -, or TIG-motive.

In some embodiments N1PyGN2is a sequence selected from the group consisting of TTTTTCG, TCG, TTCG, TTTCG, TTTTCG, TCGT, TTCGT, TTTCGT and TCGTCGT.

In one of the embodiments of the CpG-oligonucleotide "class" according to the invention has the following nucleic acid sequence:

5' TCGCGTCGTTCGGCGCGCGCCG 3' (SEQ ID NO:594), or

5' TCGTCGACGTTCGGCGCGCGCCG 3' (SEQ ID NO:595), or

5' TCGGACGTTCGGCGCGCGCCG 3' (SEQ ID NO:596), or

5' TCGGACGTTCGGCGCGCCG 3' (SEQ ID NO:597), or

5' TCGCGTCGTTCGGCGCGCCG 3' (SEQ ID NO:598), or

5' TCGACGTTCGGCGCGCGCCG 3' (SEQ ID NO:599), or

5' TCGACGTTCGGCGCGCCG 3' (SEQ ID NO:600), or

5' TCGCGTCGTTCGGCGCCG 3' (SEQ ID NO:601), or

5' TCGCGACGTTCGGCGCGCGCCG 3' (SEQ ID NO:602), or

5' TCGTCGTTTTCGGCGCGCGCC 3' (SEQ ID NO:603), or

5' TCGTCGTTTTCGGCGGCCGCCG 3' (SEQ ID NO:604), or

5' TCGTCGTTTTACGGCGCCGTGCCG 3' (SEQ ID NO:605), or

5' TCGTCGTTTTCGGCGCGCGCCGT 3' (SEQ ID NO:606).

In any of these sequences, all communication can be phosphorothioate communication. In another embodiment, in any of these sequences, one or more ties can be phosphodieterase, preferably between "C" and "G" CpG motif with obtaining semi CpG oligonucleotide.

Some non-limiting examples of oligonucleotides C-class include:

5' T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3', or

5' T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3', or

5' T*C_G*G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3', or

5' T*C_G*G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3', or

5' T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3', or

5' T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3', or

5' T*C_G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3', or

5' T*SS*SS*T*SS*T*T*SS*WITH*WITH*WITH*WITH*WITH*WITH 3', or

5' T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3', or

5' T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G 3', or

5' T*C*G*T*C*G*T*T*PT*C*G*G*C*G*G*C*C*G*C*C*G 3', or

5' T*C*G*T*C_G*T*T*T*T*A*C_G*G*C*G*C*C_G*T*G*C*C*G 3', or

5' T*C_G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3',

where * refers to phosphorothioates communication, and _ refers to a phosphodiester bond.

In any of these sequences, 5' T possible to replace ethyl-uridine or halogen, examples of the halogen substitutions include, but are not limited to, those substitution of the bromine-uridine or iodine-uridine.

Immunostimulatory CpG-oligonucleotides "P class" have been described in WO 2007/095316 and is characterized by the fact that h is about contain the duplex-forming sections, such as, for example, perfect or imperfect palindromes on the 5'- and 3'-ends, or near them, giving them the opportunity to form a more ordered structure, such as concatamer. These oligonucleotides, called oligonucleotides, R-class, able in some cases to induce very high levels of secretion of IFN-α compared to the s-class. Ophonekitd R-class is capable of spontaneously self-organize in concatamer in vitro and/or in vivo. He being bound to any specific theory for the mode of action of these molecules, one possible hypothesis is that this property gives the oligonucleotides P-class ability to much more efficient crosslinking of TLR9 in some immune cells inducyruya picture of immune activation that differs from the previously described classes of CpG-oligonucleotides.

In one of the embodiments of the CpG oligonucleotide for use in the present invention is a CpG oligonucleotide P class containing the 5' domain of TLR activation and at least two palindromic plot, where one palindromic the plot is a 5'-palindromic plot of length at least 6 nucleotides and is linked to the 3'-palindromes pipe of a length of at least 8 nucleotides directly or through a spacer, where the oligonucleotide includes at least one YpR-dinucleotide. In one and the embodiments specified oligonucleotide is not T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G. In one of the embodiments of the CpG-oligonucleotide P class includes at least one demetilirovanny CpG dinucleotide. In another embodiment the domain of TLR activation is a TCG, TTCG, TTTCG, TYpR, TTYpR, TTTYpR, UCG, UUCG, UUUCG, TTT or TTTT. In yet another embodiment the domain of TLR activation is within 5'-palindromes plot. In another embodiment the domain of TLR activation is directly 5' relative to the 5'-palindromes plot. In another embodiment the 5'-palindromic section has a length of at least 8 nucleotides. In another embodiment the 3'-palindromic section has a length of at least 10 nucleotides. In another embodiment the 5'-palindromic section has a length of at least 10 nucleotides. In another embodiment the 3'-palindromic section includes demetilirovanny CpG dinucleotide. In another embodiment the 3'-palindromic plot includes two neetilirovannyj dinucleotide CpG. In another embodiment the 5'-palindromic section includes demetilirovanny CpG dinucleotide. In another embodiment the 5'-palindromic plot includes two neetilirovannyj dinucleotide CpG. In another embodiment the 5'- and 3'-palindrome areas are of importance to the stability of the duplex is at least 25. In another embodiment the 5'-and 3'-palindrome areas are of importance to the stability of the duplex is at least 30. In another embodiment the 5'- and 3'-palindrome areas are set consistently the tee duplex of at least 35. In another embodiment the 5'- and 3'-palindrome areas are of importance to the stability of the duplex of at least 40. In another embodiment the 5'- and 3'-palindrome areas are of importance to the stability of the duplex is at least 45. In another embodiment the 5'- and 3'-palindrome areas are of importance to the stability of the duplex is at least 50. In another embodiment the 5'- and 3'-palindrome areas are of importance to the stability of the duplex is at least 55. In another embodiment the 5'- and 3'-palindrome areas are of importance to the stability of the duplex is at least 60. In another embodiment the 5'- and 3'-palindrome areas are of importance to the stability of the duplex is at least 65.

In one of the embodiments of the two palindromic area are directly related. In another embodiment two palindromic plot are connected by 3'-3' linkages. In another embodiment two palindromic area overlap by one nucleotide. In another embodiment two palindromic area overlap by two nucleotides. In another embodiment two palindromic area do not overlap. In another embodiment two palindromic area bound by the spacer. In one embodiments the spacer is a nucleic acid having a length of from 1 to 50 nucleotides. In another embodiment, the spacer is a nucleic acid having a length of 1 nucleotide. In another embodiment of the spacer of t is made by neucleotides the spacer, In one of the embodiments neucleotides the spacer is a D-spacer. In another embodiment neucleotides the spacer is a linker. In one embodiments the oligonucleotide has the formula 5' XP1SP2T 3', where X is a domain of TLR activation, P1is a palindrome, S is a spacer, R2is a palindrome, and T represents a 3'-tail length of 0-100 nucleotides. In one of the embodiments X is a TCG, TTCG or TTTCG. In another embodiment T is the length of 5-50 nucleotides. In yet another embodiment of the T has a length of 5-10 nucleotides. In one of embodiments S is a nucleic acid having a length of from 1 to 50 nucleotides. In another embodiment S is a nucleic acid having a length of 1 nucleotide. In another embodiment S is neucleotides the spacer. In one of the embodiments neucleotides the spacer is a D-spacer. In another embodiment neucleotides the spacer is a linker. In another embodiment the oligonucleotide is not antimuslim the oligonucleotide or a ribozyme. In one of the embodiments of the P1is A - and T-rich, In another embodiment of the P1includes at least 4 I. In another embodiment of the P2is a perfect palindrome. In another embodiment R2is G-C-Bo is etym. In yet another embodiment of the P2represents CGGCGCX1GCGCCG, where X1represents T or nothing.

In one of the embodiments of the oligonucleotide includes at least one phosphorothioate communication. In another embodiment all vnutrikletochnye connection of the oligonucleotide are phosphorothioate links. In another embodiment, the oligonucleotide includes at least one connection, such phosphodiester bond. In another embodiment of the communication, such fosfomifira is fosfodiesterazu communication. In another embodiment of the lipophilic group is conjugated to the oligonucleotide. In one of the embodiments of the lipophilic group is a cholesterol.

In one embodiments the TLR agonist-9 for use in the present invention is a CpG oligonucleotide R-class with the 5'-domain of TLR activation and at least two complementarity-containing regions: the 5'- and 3'complementarity-containing parts, where each complementarity-containing section has a length of at least 8 nucleotides and connected to each other directly or via a spacer, where the oligonucleotide includes at least one pyrimidine-purine (YpR) dinucleotide and where at least one of the complementarity-containing sites is not a perfect palindrome. In one of the embodiments of the oligonucleotide vkluchaet least one demetilirovanny CpG dinucleotide. In another embodiment the domain of TLR activation is a TCG, TTCG, TTTCG, TYpR, TTYpR, TTTYpR, UCG, UUCG, UUUCG, TTT or TTTT. In another embodiment the domain of TLR activation is within 5'-complementarity-containing area. In another embodiment the domain of TLR activation is directly 5' relative to the 5'-complementarity-containing area. In another embodiment the 3'complementarity-containing section has a length of at least 10 nucleotides In yet another embodiment the 5'complementarity-containing section has a length of at least 10 nucleotides. In one embodiments the 3'complementarity-containing section includes demetilirovanny CpG dinucleotide. In another embodiment the 3'complementarity-containing unit includes two neetilirovannyj dinucleotide CpG. In another embodiment the 5'complementarity-containing section includes demetilirovanny CpG dinucleotide. In another embodiment the 5'complementarity-containing unit includes two neetilirovannyj dinucleotide CpG. In another embodiment of the complementarity-containing parts include at least one nucleotide analogue. In another embodiment of the complementarity-containing areas form intermolecular duplex. In one of the embodiments of the intramolecular duplex includes at least one pair of bases are not Watson-Cricova type. In another voploscheni the base pair are not Watson-Cricova type represents G-T, G-A, G-G or C-A. In one of the embodiments of the complementarity-containing areas form intermolecular duplexes. In another embodiment, at least one intermolecular duplexes includes at least one pair of bases are not Watson-Cricova type. In another embodiment of a base pair are not Watson-Cricova type represents G-T, G - A, G-G or C-A. In yet another embodiment of the complementarity-containing areas contain incorrect mating. In yet another embodiment of the complementarity-containing plots contain two incorrect mating. In another embodiment of the complementarity-containing areas contain intermediate nucleotide. In another embodiment of the complementarity-containing plots contain two intermediate nucleotide.

In one of the embodiments of the 5'- and 3'-complementarity-containing areas are of importance to the stability of the duplex is at least 25. In another embodiment the 5'- and 3'complementarity-containing areas are of importance to the stability of the duplex is at least 30. In another embodiment the 5'- and 3'complementarity-containing areas are of importance to the stability of the duplex is at least 35. In another embodiment of the complementarity-containing areas are of importance to the stability of the duplex of at least 40. In another embodiment of the complementarity-containing parts have a value of stability on the Plex at least 45. In another embodiment of the complementarity-containing areas are of importance to the stability of the duplex is at least 50. In another embodiment of the complementarity-containing areas are of importance to the stability of the duplex is at least 55. In another embodiment of the complementarity-containing areas are of importance to the stability of the duplex is at least 60. In another embodiment of the complementarity-containing areas are of importance to the stability of the duplex is at least 65.

In another embodiment two complementarity-containing area is connected directly. In another embodiment two palindromic plot are connected by 3'-3'-connection. In another embodiment two complementarity-containing area overlap by one nucleotide. In another embodiment two complementarity-containing area overlap by two nucleotides. In another embodiment two complementarity-containing area do not overlap. In another embodiment two complementarity-containing area is connected via a spacer. In another embodiment, the spacer is a nucleic acid having a length of 1-50 nucleotides. In another embodiment, the spacer is a nucleic acid having a length of 1 nucleotide. In one embodiments the spacer is neucleotides the spacer. In another embodiment of ninuki tiny the spacer is a D-spacer. In yet another embodiment neucleotides the spacer is a linker.

In one of the embodiments of the oligonucleotide R-class has the formula 5' XNSPT 3', where X is a domain of TLR activation, N represents an imperfect palindrome, P is a palindrome, S is a spacer, and T represents a 3'-tail length of 0-100 nucleotides. In another embodiment X is a TCG, TTCG, or TTTCG. In another embodiment T is the length of 5-50 nucleotides. In another embodiment of the T has a length of 5-10 nucleotides. In another embodiment S is a nucleic acid having a length of 1-50 nucleotides. In another embodiment S is a nucleic acid having a length of 1 nucleotide. In another embodiment S is neucleotides the spacer. In another embodiment neucleotides the spacer is a D-spacer. In another embodiment neucleotides the spacer is a linker. In another embodiment the oligonucleotide is not antimuslim the oligonucleotide or a ribozyme. In another embodiment N is A - and T-rich. In another embodiment N includes at least 4 T. In another embodiment R is a perfect palindrome. In another embodiment R is G-C-rich. In another embodiment R represents CGGCGCX1GCGCCG, where X1is a T or anything. Another is lewinii oligonucleotide includes at least one phosphorothioate communication. In another embodiment all mezhnukleotidnyh connection of the oligonucleotide are phosphorothioate communication. In another embodiment, the oligonucleotide includes at least one connection, such phosphodiester bond. In another embodiment of the communication, such fosfomifira is fosfodiesterazu communication. In another embodiment of the lipophilic group is conjugated to the oligonucleotide. In one of the embodiments of the lipophilic group is a cholesterol.

In one of the embodiments of CpG-oligonucleotides "P class" according to the invention have the following sequence of nucleic acid; 5' TCGTCGACGATCGGCGCGCGCCG 3' (SEQ ID NO:607).

In these sequences, all communication can be phosphorothioate communication. In another embodiment, one or more ties can be phosphodieterase, preferably between "C" and "G" CpG motif, giving semi CpG oligonucleotide. In any of these sequences ethyl-uridine or halogen may be substituted for a 5' T; examples of the halogen substitutions include, but are not limited to, substitution of the bromine-uridine or iodine-uridine.

A non-limiting example of the oligonucleotides P-class includes:

5' T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G 3'

where * refers to phosphorothioates communication, and _ refers to a phosphodiester bond.

In one of the embodiments of all mezhnukleotidnyh communication CpG oligonucleotides described in donnezac, are phosphodiesterase bonds ("flexible" oligonucleotides, as described in PCT application WO 2007/026190). In another embodiment of CpG-oligonucleotides according to the invention make resistant to degradation (e.g., stabilize). "Stabilized oligonucleotide" refers to an oligonucleotide, which is relatively resistant to degradation in vivo (for example by Exo - or endonucleases). Stabilization of nucleic acids can be achieved by modifications of the skeleton. Oligonucleotides having phosphorothioate communication, provide maximum activity and protect the oligonucleotide from degradation of intracellular Exo - and endonucleases.

Immunostimulatory oligonucleotides may have a Chimera skeleton, which represents the combination fosfolipidnyh and phosphorothioate links. For the purposes of the present invention chimeric skeleton refers to the partially stabilized the skeleton, where at least one mezhnukleotidnyh communication is fosfomifira or similar fosfomifira and where at least one other mezhnukleotidnyh relationship is a stable mezhnukleotidnyh link, where at least one fosfomifira or similar fosfomifira connection and at least one stable relationship is different. When fosfomifira communication is mostly within the CpG motif, such is olekuly called "semi-flexible", as described in PCT application WO 2007/026190.

Other modified oligonucleotides include combinations fosfolipidnyh, phosphorothioate, methylphosphonate, methylphosphonothioate, phosphorodithioate and/or para-ethoxy.

Because boranophosphate communication, as reported, are stable compared to phosphodieterase relationships within the chimeric nature of the skeleton, boranophosphate communication can be classified either as such fosfomifira or stable, depending on the context. For example, chimeric skeleton according to the present invention in some embodiments may include at least one fosfodiesterazu (fosfodiesterazu or similar fosfomifira) linkage and at least one boranophosphate (stable) relationship. In other embodiments chimeric skeleton according to the present invention may include boranophosphate (phosphodieterase or similar fosfomifira) and phosphorothioate (stable) connection. "Stable mezhnukleotidnyh communication" means mezhnukleotidnyh communication, which is relatively resistant to degradation in vivo (for example by Exo - or endonucleases) compared to fosfomifira mezhnukleotidnyh communication. Preferred stabilized mezhnukleotidnyh networks include, without limitation, phosphorothioates, phosphoro itiative, methylphosphonate and methylphosphonothioate. Other stable mezhnukleotidnyh networks include, without limitation, peptide, alkyl, dephospho and other communications, as described above.

Modified skeletons, such as phosphorothioate, can be synthesized using automated methods using either phosphoramidate or H-phosphonate chemistry. Aryl - and alkylphosphonate can be obtained, for example, as described in U.S. patent No. 4469863; and alkylphosphocholine (in which the charged oxygen group is alkylated, as described in U.S. patent No. 5023243 and European patent No. 092574) can be obtained by using an automated solid-phase synthesis using commercially available reagents. Methods of obtaining other modifications and substitutions DNA skeleton have been described. Uhlmann E et al. (1990) Chem Rev 90:544; J Goodchild (1990) Bioconjugate Chem 1:165. Methods of obtaining chimeric oligonucleotides are also known. For example, in patents issued in Uhlmann and others described such methods.

Mixed skeleton-modified one can be synthesized as described in PCT application WO 2007/026190.

The oligonucleotides according to the invention may also include other modifications. These include non-ionic analogs of DNA, such as alkyl - and aryl-phosphates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), FOC is dietery and alkylphosphocholine. in which the charged oxygen group is alkylated. Nucleic acids that contain a diol, such as tetraethylene glycol or hexamethyleneimine, either on one or on both ends, as was also shown to be substantially resistant to nuclease degradation.

The size of the CpG-oligonucleotide (i.e., the number of nucleotide residues along the length of the oligonucleotide) can also contribute to stimulating the activity of the oligonucleotide. To facilitate entry into cells, CpG-oligonucleotide according to the invention preferably has a minimum length of 6 nucleotide residues. Oligonucleotides of any size greater than 6 nucleotides (even many so on, ad in length) is able to induce an immune response, if present in sufficient immunostimulatory motifs, because of larger oligonucleotides are degraded inside the cells. In some embodiments CpG oligonucleotides have a length of from 6 to 100 nucleotides, preferably a length of from 8 to 30 nucleotides. In an important embodiment of the nucleic acids and oligonucleotides of the invention are not expressing plasmids or vectors.

In one of the embodiments of the CpG-oligonucleotide described in this application contains replacement or modification, such as at the bases and/or sugars, as described in paragraphs 134-147 in WO 2007/026190.

In one of the embodiments of the CpG oligonucleotide for us is oasea the invention are chemically modified. Examples of chemical modifications are known to the expert and are described, for example, in UhSmanri E. et al. (1990), Chem. Rev. 90:543, S. Agrawal, Ed., Humana Press, Totowa, USA 199.3; Crooke, S. T. et al. (1996) Annu. Rev. Pharmacol. Toxicol. 36:107-129; and J. Hunziker et al., (1995), Mod. Synth. Methods 7:331-417. Oligonucleotide according to the invention can have one or more modifications, where each modification is localized in specific fosfomifira magnoliopsida the bridge and/or in specific β-D-ribose link and/or specific natural position of the nucleoside base compared to the oligonucleotide with the same sequence which is composed of natural DNA or RNA.

In some embodiments of the invention CpG-containing nucleic acid can be simply mixed with immunogenic carriers according to methods known to experts in the art (see, for example, WO 03/024480).

In a particular embodiment of the present invention, any vaccine described in this application contains from 20 μg to 20 mg CpG oligonucleotide, preferably from 0.1 mg to 10 mg CpG oligonucleotide, preferably from 0.2 mg to 5 mg CpG oligonucleotide, preferably from 0.3 mg to 3 mg CpG oligonucleotide, more preferably from 0.4 to 2 mg CpG oligonucleotide, more preferably from 0.5 to 1.5 mg CpG oligonucleotide. In the preferred embodiment, any vaccine described in this application contains from about 0.5 to 1 mg CpG-about what gonucleotide.

Preferred adjuvants for use in the present invention are alum, QS21, CpG one, alum in combination with one CpG, Iscomatrix and Iscomatrix in combination with one CpG.

The pharmaceutical compositions according to the invention

The invention also suggested that pharmaceutical compositions containing the antigenic PCSK9 peptide of the invention or immunogenic composition, the drug together with one or more pharmaceutically acceptable(and) excipient(s) and possibly combined with one or more adjuvants (as adjuvant, as described above). The term "excipient" is used in this application to describe any ingredient other than the active ingredient, i.e., the antigenic PCSK9 peptide of the invention, ultimately associated with immunogenic carrier and possibly combined with one or more adjuvants. The choice of excipient(s) will essentially depend on factors such as the particular mode of administration, the effect of excipient on solubility and stability, and the nature of the dosage form. As used in this application, a "pharmaceutically acceptable excipient" includes any and all solvents, dispersion medium, coatings, antibacterial and antifungal agents, isotonic and slow absorption agents, etc. that are physiologically compatible. Some examples of the pharmacist who Cesky acceptable excipients are water, saline, phosphate buffer, dextrose, glycerol, ethanol, etc., and combinations thereof. In many cases it is preferable to include isotonic agents, for example, sugar, polyalcohol, such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are moisturizing agents or small amounts of auxiliary substances such as moisturizing or emulsifying agents, preservatives or buffers, which enhance the shelf life and efficacy of the active ingredient.

The pharmaceutical compositions of the present invention and methods for their preparation will be obvious to experts in the given field of technology. Such compositions and methods for their preparation can be found, for example, in Remington''s Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). The pharmaceutical composition preferably is prepared in accordance with the conditions of GMP (Good Manufacturing Practice, good manufacturing practices).

The pharmaceutical composition according to the invention can be obtained, packaging or sell no, as a single dose or multiple single doses. Used in this application "single dose" represents the number of pharmaceutical composition containing a specified amount of the active ingredient. The amount of active ingredient what is the usual dose of the active ingredient, which is administered to a subject, or a convenient fraction of such a dosage such as, for example, half or a third of such a dosage.

Any method of administration of peptides or proteins, adopted in this area, can accordingly be used for peptides or proteins according to the invention.

The pharmaceutical compositions according to the invention is generally suitable for parenteral administration. Used in this application "parenteral pharmaceutical composition includes any route of administration characterized by physical gap in the tissue of the subject, and the introduction of the pharmaceutical composition through the breach in the tissue, thus usually resulting in immediate introduction into the blood stream, into muscle, or into an internal organ. Parenteral administration thus includes, but is not limited to, the introduction of a pharmaceutical composition by injection of the composition by applying the composition through a surgical incision, by application of the composition through dynarnical non-surgical wound, and the like. In particular, it is assumed that the parenteral administration includes, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, be, intracranial, intra-articular injection or details who Ziya; and how dialysis and infusion of the kidneys. Preferred embodiments include intravenous, subcutaneous, intradermal and intramuscular route, even more preferred embodiments are intramuscular or subcutaneous route.

Drugs pharmaceutical compositions suitable for parenteral administration, generally contain the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such preparations can be prepared, packaged, or sold in a form suitable for bolus injection or continuous infusion. Injectable preparations can be prepared, packaged, or sold in a standard dosage form such as capsules or mnogorazovye containers containing a preservative. Preparations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous carriers, pastes, etc., These preparations may additionally contain one or more additional ingredients including, but not limited to, suspendida, stabilizing or dispersing agents. In one of the embodiments of the preparation for parenteral administration the active ingredient offered in dry (i.e. powder or granular) form for the appropriate recovery Napo is the diameter (for example, sterile pyrogen-free water) prior to parenteral introduction of the recovered composition. Parenteral preparations include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but for some applications they may be more preferably prepared in the form of a sterile non-aqueous solution or as a dried form to be used in combination with a suitable excipient such as sterile pyrogen-free water. Typical forms for parenteral administration include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or solutions of dextrose. Such dosage forms can be suitably buffered, if desired. Other parenterally administered drugs that are useful include preparations that contain the active ingredient in microcrystalline form, in the form of microparticles or in a liposomal preparation. Preparations for parenteral administration can be prepared for immediate and/or modified release. Composition with modified release include deferred, delayed, pulsed, controlled, targeted and programmed release.

For example, in one aspect sterile injectable races the thieves can be prepared by incorporating the anti-PCSKQ peptide, preferably associated with immunogenic carrier, possibly in combination with one or more adjuvants, in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above. if required, followed by sterilization by filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile filler, which contains the basic dispersion medium and other necessary ingredients from above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation is vacuum drying and freeze-drying, which give a powder of the active ingredient plus any additional desired ingredient from its solution, pre-sterilized by filtration. The proper fluidity of the solution can be provided, for example, through the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and the use of surfactants. Prolonged absorption of injectable compositions can be carried out, for example, by incorporating in the composition an agent that slows the absorption of, for example, monostearate salts and gelatin.

Typical, non-limiting pharmaceutical composition p. the invention is a product in the form of a sterile aqueous solution, having a pH that ranges from about 5.0 to 6.5, and containing from about 0.1 mg/ml to about 20 mg/ml peptide according to the invention, from about 1 millimolar to about 100 millimolar his-tag buffer, from about 0.01 mg/ml to about 10 mg/ml Polysorbate 80, from about 100 millimolar to about 400 millimolar of trehalose and about of 0.01 millimolar to about 1.0 millimolar dihydrate disodium if EDTA.

Antigenic PCSK9 peptides according to the invention can also be entered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture or in the form of fractions or mixed components, for example, mixed with a suitable pharmaceutically acceptable excipient) from a dry powder inhaler, such as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably atomizer using electrohydrodynamics to create a fine dust) or a nebulizer with the use or without the use of a suitable propellant, or in the form of nose drops.

The pressurized container, pump, spray, atomizer, or nebulizer, typically contains a solution or suspension of the antibodies according to the invention, containing, for example, a suitable agent for dispersion, solubilization, or sustained release of active agent, a propellant(s) in ka is este solvent.

Before use in a dry powder drug or suspension, the drug is usually crushed to a size suitable for delivery by inhalation (typically less than 5 microns). This can be achieved using any suitable grinding method such as grinding in a spiral jet mill, grinding in a jet mill, fluidized bed processing supercritical fluid with the formation of the nanoparticles, homogenization at high pressure or spray drying.

Capsules, blister packaging and cartridges for use in an inhaler or insufflator can be prepared for maintenance powder mixture of compounds according to the invention, a suitable powder base and modifier efficiency.

The preparation of a suitable solution for use in an atomizer using electrohydrodynamics to obtain a fine powder may contain a suitable dose of the antigenic PCSK9 peptide of the invention to activate, and the volume of activation can vary, for example from 1 μl to 100 μl.

Suitable flavoring agents such as menthol and levomenthol, or sweeteners, such as saccharin and saccharin sodium, can be added to these preparations according to the invention, intended for inhaled/intranasal.

Preparations for inhalation/intranasal may be the prigotovleny for immediate and/or modified release. Preparations for the modified release include deferred, delayed, pulsed, controlled, targeted and programmed release.

In the case of dry powder inhalers and aerosols, the unit dosage is determined by the valve, which delivers a measured quantity. Unit according to the invention are typically arranged to introduce metered dose or "zilch" antibodies according to the invention. Total daily dose usually can be entered as a single dose or more often as divided doses throughout the day.

Pharmaceutical composition containing antigenic PCSK9 peptide may also be prepared for oral administration. Oral administration may include ingestion, so that the connection enters the gastrointestinal tract, and/or transbukkalno, lingual or sublingual introduction, with which the connection enters the bloodstream directly from the mouth.

Preparations suitable for oral administration include solid, semi-solid and liquid systems, such as tablets, soft or hard capsules containing multi - or nano-particles, liquids or powders; lozenges (including liquid-filled); chewing gum; gels; quickly dispersible dosage forms; film, ovule; sprays; and transbukkalno/mucoadhesive the patch.

Liquid preparations include suspensions, solutions, syrups and elixirs. Such drugs can be used as fillers in soft or hard capsules (made, for example, gelatin or hydroxypropylmethylcellulose) and usually contain a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspendida agents. Liquid preparations can also be obtained by recovering a solid form, for example from sachet.

The composition of the invention can be used for the treatment, mitigation or prevention of PCSK9-mediated disorders or symptoms in a subject at risk of developing or suffering from such a disorder or symptoms by stimulating the immune response in the specified entity using immunotherapy. Immunotherapy can include the primary immunization, and then further, for example, one, two, three or more repeated immunizations.

"Immunologically effective amount" antigenic PCSK9 peptide of the invention or composition is an amount that is delivered to the subject is a mammal or in a single dose or as part of a series, which is effective for the induction of an immune response is against PCSK9 in the specified subject. This number varies depending on the health and physical condition of the individual, which are treated, the taxonomic group of individual who are treated, the immune system of the individual to synthesize antibodies, the preparation of vaccines and other relevant factors. Assume that the number is relatively broad range that can be determined by routine tests.

"Pharmaceutically effective dose" or "therapeutically effective dose" is a dose that is required to treat, prevent or alleviate one or more of the PCSK9-related disorder or symptoms in the subject. Pharmaceutically effective dose depends, in part, on the particular compound that is administered, the severity of symptoms, the sensitivity of the subject to side effects, type of disease, the composition, the route of administration, the type of mammal, which is being treated, the physical characteristics of the specific question of a mammal, such as health and physical condition, concomitant treatment, the ability of the immune system of an individual to synthesize antibodies, the degree of desired protection, and other factors that are known to experts in the field of medicine. For preventive purposes, the amount of peptide in each dose is chosen in the form of a number, which is induces an immunoprotective response without significant, adverse side effects in typical vaccines. After the initial vaccination, subjects may receive one or more repeated immunization with adequate intervals.

It should be understood that the specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound, the age, body weight, General health, sex, diet, time of administration, route of administration, and rate of excretion, combination of drugs and the severity of the particular disease being treated.

For example, the antigenic PCSK9 peptide or pharmaceutical composition according to the invention it is possible to introduce the subject in a dose of from about 0.1 µg to about 5 mg, e.g., from about 0.1 μg to about 5 μg, from about 5 μg to about 10 μg, from about 10 μg to about 25 μg, from about 25 μg to about 50 μg, from about 50 μg to about 100 μg, from about 100 μg to about 500 μg, from about 500 μg to about 1 mg, from about 1 mg to about 2 mg, with optimal re-immunization, which hide those, for example, after 1 week, 2 weeks, 3 weeks, 4 weeks, two months, three months, 6 months and/or year.

In some embodiments injected with a single dose of the antigenic PCSK9 peptide or pharmaceutical composition according to the invention. In other embodiments administered multiple doses of antigenic PCSK9 peptide or pharmaceutical is th compositions according to the invention. The frequency of injection may vary depending on any of a variety of factors, such as severity of symptoms, extent desired immunoassay, whether the composition for prophylactic or therapeutic purposes, and so on, for Example, in some embodiments the antigenic PCSK9 peptide or pharmaceutical composition according to the invention is administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week five times per week six times per week, in a day (qod), daily (qd), twice a day (qid), or three times daily (tid). When using the compositions according to the invention for preventive purposes, they usually impose both initial and repeated doses. Assume that repeated doses are sufficiently separated by adequate periods of time, or preferably be given annually or at such a time, when the levels of circulating antibodies fall below the desired level. Repeated doses may consist of antigenic PCSK9 peptide in the absence of initial immunogenic molecules of the carrier. Such booster designs can include alternative immunogenic carrier or may not contain any media. Such booster compositions can be prepared either with adjuvant or without it.

About aiternate introduction antigenic PCSK9 peptide according to the invention, for example, the period of time during which impose antigenic PCSK9 peptide can vary depending on any of a variety of factors, for example, the reaction of the patient, and so on, for Example, the antigenic PCSK9 peptide can be entered within a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years or more.

In the present description also provides various methods of treatment, which include the introduction of antigenic PCSK9 peptide according to the invention. Consider methods of treatment include methods of inducing an immune response in an individual against his own PCSK9 and methods of prevention, mitigation or treatment of PCSK9-related disorder or symptom in the individual.

In one aspect of the present invention, a method for treatment, prevention or relief of PCSK9-related disorder or symptom in a subject, comprising introducing a therapeutically effective amount of the antigenic peptide PCSK according to the invention or immunogenic or pharmaceutical composition to the specified entity.

In another aspect of the present invention, a method of inducing an immune response against PCSK9 for a subject, comprising introducing a therapeutically or immunogen effective amount of the antigenic PCSK9 peptide of the invention or immunogenic or pharmaceutical composition to the specified entity.

PCSK9-associated disease or PCSK9-mediated disease is a disease in which inhibition of PCSK9 activity or inhibition of the interaction of PCSK9 with the LDL receptor may be useful.

"Treating", "treat" and "treatment" refer to the way of relief or cancellation of a biological disorder and/or at least one of the accompanying symptoms. As used in this application, "facilitate" a disease, disorder or condition means reducing the severity and/or frequency of symptoms of the disease, disorder or condition. In addition, references in this application to "treatment" include references to curative, palliative and prophylactic treatment. The specified subject is preferably a human, and can be male or female of any age.

Other aspects of the invention relate to the antigenic PCSK9 peptide according to the invention or immunogenic compositions or pharmaceutical compositions for the application of the Oia as a medicinal product, preferably for the treatment, mitigation or prevention of PCSK9-related disorder.

In another aspect of the present invention proposed the use of antigenic PCSK9 peptide of the invention or immunogenic compositions or pharmaceutical compositions in the manufacture of medicines, preferably for the treatment of PCSK9-Svyaznoi disorders.

In particular, the invention relates to the antigenic PCSK9 peptide of the invention or immunogenic or pharmaceutical composition for use as a medicine, preferably for the treatment, mitigation or prevention of diseases associated with high cholesterol levels.

In another aspect of the present invention proposed the use of antigenic PCSK9 peptide of the invention or immunogenic compositions or pharmaceutical compositions in the manufacture of a medicinal product, it is preferable to reduce the level of LDL-cholesterol in blood of a subject in need thereof.

In some aspects, uses or methods according to the invention specified PCSK9-related disorder selected from the group consisting of high cholesterol, a condition associated with elevated LDL-cholesterol, for example, lipid disorders (e.g., hyperlipidemia, hyperlipidemia, type 1, type 2, type 3, type 4 or ti is 5, secondary hypertriglyceridemia, hypercholesterolemia, familial hypercholesterolemia, xanthomatosis, deficit holesterinesterzy), atherosclerotic conditions (e.g., atherosclerosis), coronary artery disease and cardiovascular diseases.

In another aspect of the present invention proposed the use of antigenic PCSK9 peptide of the invention or immunogenic compositions or pharmaceutical compositions in the manufacture of a medicinal product for the treatment or relief of diseases in which activation of the LDL receptor or inhibition of the interaction between PCSK9 and LDL receptor are useful.

In another aspect of the present invention proposed the use of antigenic PCSK9 peptide of the invention or immunogenic compositions or pharmaceutical compositions in the manufacture of a medicine for the treatment of Alzheimer's disease.

In other aspects of the uses or methods according to the invention the specified subject is a mammal, preferably human.

In other aspects of the uses or methods according to the invention the specified subject is suffering from the specified PSCK9-associated disorders. Alternatively, if the specified subject has a risk of occurrence of the specified PCSK9-related disorder, for example, due to the presence of one or the more risk factors (for example, hypertension, Smoking, diabetes, obesity, or hyperhomocysteinemia).

Antigenic PCSK9 peptide of the invention or immunogenic composition or a pharmaceutical composition useful for subjects who cannot tolerate therapy with another agent that reduces the level of cholesterol, or for subjects in whom therapy with another agent that reduces the level of cholesterol leads to inadequate results (for example, subjects who experience a lack of reduction in LDL-c when latinboy therapy). Antigenic PCSK9 peptide of the invention described in this application, you can enter the subject with elevated LDL-cholesterol.

Preferably, a subject with elevated cholesterol is a person with a total cholesterol levels in plasma of 200 mg/DL or higher. Preferably, a subject with elevated cholesterol is a subject-a person with an LDL-cholesterol of 160 mg/DL or higher.

Total cholesterol levels in plasma and LDL-cholesterol was measured using standard methods in blood samples obtained after the subsequent starvation. Protocols for measuring levels of total cholesterol in plasma and LDL-cholesterol are well known to the person skilled in the technical field.

In one of the embodiments of the antigenic PCSK9 peptide or immunogenic composition or a pharmaceutical to the impositio administered together with another agent, two agents can be entered sequentially in any order or simultaneously. In some embodiments the antigenic PCSK9 peptide or immunogenic composition or pharmaceutical composition is administered to a subject, who is also receiving treatment with a second agent (e.g., a second agent that lowers cholesterol). Agents that lower cholesterol include statins, substances that increase the secretion of bile acids, Niacin, derivatives fibroeva acid and long-chain alpha, omega-dicarboxylic acids. Statins inhibit cholesterol synthesis by blocking HMG-COA (3-hydroxy-3-methylglutaryl-coenzyme A), the key enzyme in the biosynthesis of cholesterol. Examples of statins include lovastatin, pravastatin, atorvastatin, tseriwastatina, fluvastatin and simvastatin. Substances that increase the secretion of bile acids, interrupt the recirculation of bile acids from the intestine to the liver. Examples of these agents include cholestyramine and colestipol hydrochloride. Examples fibroevoy acid derivatives are clofibrate, and gemfibrozil. Long-chain alpha, omega-dicarboxylic acids are described, for example, Bisgaier et al., 1998, J. Lipid Res. 39:17-30; WO 98/30530; U.S. patent No. 4689344; WO 99/00116; U.S. patent No. 5756344; U.S. patent No. 3773946; U.S. patent No. 4689344; U.S. patent No. 4689344; U.S. patent No. 4689344; and U.S. patent No. 3930024); ethers (see, for example, p is the awning U.S. No. 4711896; U.S. patent No. 5756544; U.S. patent No. 6506799). Phosphate dolichol (U.S. patent No. 4613593) and derivatives of solidilin (oxazolidinedione) (U.S. patent No. 4287200) can also be used to reduce cholesterol levels. The mode of combination therapy may be additive, or it can lead to synergistic results (for example, to a greater reduction in cholesterol than expected for the combined use of the two agents). In some embodiments, combination therapy with antigenic PCSK9 peptide or immunogenic composition or pharmaceutical composition and a statin lead to synergistic results (for example, a synergistic reduction of cholesterol). In some subjects this may allow to reduce the dosage of statin to achieve the desired cholesterol level.

EXAMPLES

The following examples are designed to provide professionals in this field a full description of the invention and the description of how to make and use the present invention, and are not intended to limit the scope of what the inventors consider their invention, and the following experiments are not considered unique. Attempts have been made to ensure the accuracy in terms of numbers (e.g., amounts, temperature, etc), but you should consider some experiments the material error and bias. Unless indicated otherwise, parts are parts by weight, molecular weight is an average molecular weight, temperature is expressed in degrees Celsius, and the pressure corresponds to the atmospheric or close to it. Can be used standard abbreviations, e.g., p. O., pair(s) of the grounds; and so on, O., a thousand(and) base pairs; PL, picoliter(s); s or sec, second(s); min, minute(s); h, hour(s); and.K. the amino acid(s); N., nucleotide(s); V/m, intramuscular(but); in b/W, intraperitoneal(but); p/C, subcutaneous(but); and the like.

Example 1 - Selecting antigenic PCSK9 peptides in the domain of PCSK9-EGF-A surface interaction of LDL receptor

The structure of the human PCSK9 binding to the EGF domain-A receptor LDL was determined and reported (Kwon et al., PNAS 105, 1820-1825, 2008). This structural information (PDB: 3BPS) used together with information about the structure of the free PCSK9, PDB: 2P4E (Cunningham et al., Nature Structural &Molecular Biology, 14; 413-419, 2007) to construct the following peptides, which will correspond to areas of importance for the interaction of PCSK9-LDL receptor (see Fig 1).

Peptide 1. ASSDCSTCFV (SEQ ID NO:19)

Peptide 2. GTRFHRQASK (SEQ ID NO:63)

Peptide 3. IQSDHREIEGRV (SEQ ID NO:109)

Peptide 4. SGRDAGVAKGA (SEQ ID NO:153)

Peptide 5. SIPWNLERITP (SEQ ID NO:184)

Since the peptides 1-4 represent loops in the structure of PCSK9, the corresponding sequence (SEQ ID NO:19, 63, 109 and 153) made and added Cys, Cys-Gly or Lys-linkers, providing binding through both ends with the VLP carrier to provide conformational mimetic natural loop structures (from VR_9.1 to VR_9.4 in Table 1). In addition to this also made cyklinowanie options peptides 2-4 (from VR_9.6 to VR_9.9 in Table 1), which provided a Cys residue for binding with VLPs. Peptide 1 did with Lys-Gly-Gly N-terminal linker to link so that two Cys residue were free for disulfide bond, they form the native structure of PCSK9. Peptide 5 is N-end of the Mature protestirovanny forms of human PCSK9 and communicates via added to the end of a cysteine residue to the N-end was free for the detection of antibodies (VR 9.5 Table 1). The following table (table 1) described 9 peptides obtained for evaluation of candidate vaccines.

Table 1
Peptide sequence
PeptideSequenceSEQ ID NO:
VR_9.1KGGASSDCSTCFV313
VR_9.2CGGTRFHRQASKC 314
VR_9.3CGIQSDHREIEGRVC315
VR_9.4CSGRDAGVAKGAC316
VR_9.5SIPWNLERITPC317
VR_9.6ASK-Cys(H)-GDGTRFHRQ318
VR_9.7AG-Cys(H)-GTRFHRQ319
VR_9.8GRV-Cys(H)-IQSDHREIE320
VR_9.9AGVAKGAG-Cys(H)-SGRD321
Underlining indicates cysteine residues used for conjugation and double underline indicates GC or KGG-linker.

Example 2 - Getting the Peptide-the VLP conjugates to assess candidates for the role of vaccines

Peptides were synthesized using standard Fmoc Protocol on amide resin CLEAR. The reaction of a combination of amino acids was performed using a 5-fold excess of Fmoc-protected amino acids activated with 1 equiv. HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea what I hexaphosphate) in the presence of HOBt (hydroxybenzotriazole) and NMM (N-methylmorpholine). Removal of the Fmoc protecting group was achieved using a mixture of 20% piperidine/DMF. The peptide bound to the resin, then tsalala and groups protecting the side chains were removed simultaneously with reagent D (TFA/H20/DODT: 89/3/8). The peptide was obtained with a free N-end and emitirovannykh To-end. The crude peptide was purified to homogeneity by HPLC using a column VEINS 130 C18 and gradient water/acetonitrile in the presence of 0.1% TFA (triperoxonane acid). The purified peptide was subjected to vacuum drying using lyophilizate. The peptide was analyzed using mass spectrometry (LC-MS) and received satisfactory information.

Qβ the VLP used in this study were obtained using bacterial fermentation in E. coli strain BL21 (DE3), containing plasmid RET encoding a Monomeric protein of 14 kDa: MAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQP SRNRKNYKVQVKIQNPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY (Genbank ID: M99039). Fermentation induce at OD600 of 0.8 using IPTG (isopropylthioxanthone) and leave overnight in terrific broth (TB) with kanamycin. The VLP, which organizes itself in the cell host, then was purified from the cell sediment from fermentation using the method described in patent application EP 1736538 with the following differences: after the destruction of cells in the clarified homogenate was treated with ammonium sulfate at 50% saturation and the cell precipitate was isolated centrifuging what W. Then the precipitate was dissolved in HEPES buffer and dialyzed against HEPES buffer before carrying out the first stage column in the published method. After stages ion-exchange column and hydroxyapatite column material was purified using additional stages of the anion-exchange column and subjected to sterile filtration by the end no material the VLP, which were analyzed by gel-chromatography, SDS page-ordinator (polyacrylamide gel electrophoresis with sodium dodecyl sulfate) and electron microscopy with acceptable results.

Conjugation of peptides through cysteine residues:

Qβ the VLP activated using either N-gamma-maleimido-butyrylcholinesterase of ester (GMBS) or more long Succinimidyl-6-[β-maleimidopropionamide]hexanoate-linking reagent. The procedure of application of the two reagents was similar: solid reagent was dissolved in dimethyl sulfoxide (DMSO) and added to a solution of the VLP with at least a 10-fold molar excess. Reaction activation was left not less than 90 minutes and the solution then was absoluely using desalting columns NAP-25 in phosphate-buffered saline, Dulbecco (DPBS) with 5 mm EDTA or phosphate-buffered saline, Dulbecco (DPBS), which was modified by adding solid NaCl (14.6 g/l). If desired, a solution of proteins is slightly concentrated using 10 kDa of microconcentrators-centrifuges before the next reaction conjugation.

Before the reaction of conjugating the peptides were dissolved in an aliquot of DPBS pH 7.4 with 5 mm EDTA as an additive. The concentration of peptide in solution was 10 mg/ml Dissolved peptide was added to the aliquot TSER-immobilized reducing agent (Pierce Chemical), which was washed in DPBS containing 5 mm EDTA. An aliquot of the peptides was incubated with stirring in the presence of TSAR the gel for approximately 1 hour, after which an aliquot was centrifuged in microcentrifuge and the solid precipitate was discarded. The supernatant, containing the recovered peptide was added directly to activated the VLP, which has been prepared previously, One of the alternatives to this procedure, however, is the addition of solid peptide directly to the sample activated Q(3 the VLP. Both methods work equally well for obtaining the peptide-the VLP conjugates.

The reaction between VLPs and the recovered peptides were left to proceed for at least thirty minutes with careful mixing. At the end of the reaction period, each sample was absoluely in PBS, Dulbecco (DPBS) using desalting columns NAP-10 or NAP-25 (GE Healthcare). Desalted conjugated peptides were analyzed for protein content using analysis by Bradford (Kumasi brilliant blue, Pierce Chemical Co) or protein analysis with ICA (bicinchoninic acid) (Pierc Chemical Co), and through PAG-ordinator and gel chromatography. Conjugated products were subjected to sterilization by filtration using a 0.22 μm filter and stored at 2-8°C before use. Special attention was paid to the samples during storage to prevent freezing or exposed to extreme temperatures.

Conjugation of peptide PCSK9 with CRM197 were performed using BAANS (N-hydroxysuccinimidyl ester bromoxynil acid, Sigma B8271). First CRM197 activated by interaction with BAANS in 0.1 M sodium carbonate pH 8.3 with a molar ratio of 100 in a cold room for 90 minutes. The reaction mixture was passed through demineralization Zeba column and collecting the eluate. The PCSK9 peptide at 10 mg/ml were incubated with an equal volume of immobilized TSAR reducing gel (Thermo Scientific) at room temperature for 1 hour and collected after centrifugation through a 0.2 μm filter. Activated CRM197 then mixed with the processed peptide in a cold room during the night, with subsequent active dialysis in PBS buffer. The conjugate was recovered, concentrated and sterilized by passing through 0.22 μm filter. The protein concentration was determined using analysis of Kumasi blue (Thermo Scientific).

Conjugation of peptides through amines

For the conjugation of peptides to Qβ through amine residues, for instance the peptide VR_9.1, performed the following procedure. Qβ first was derivateservlet by adding solid succinic anhydride with at least a 10-fold molar excess relative to the VLP monomer. Succinic anhydride was left to dissolve and the derivatization reaction was left to proceed for at least 30 minutes. After that, the sample was absoluely using desalting columns NAP-25 in phosphate-buffered saline, Dulbecco (DPBS) with 5 mm EDTA. Then the following reagents were added in this order at no less than a 10-fold molar excess relative to the VLP monomer: Solid peptide, N-hydroxysultaine and, finally, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. After adding the reagents in the above procedure the sample was incubated at room temperature and the reaction was left to proceed for at least 30 minutes, after which the VLP-peptide conjugate was subjected to desalting using desalting columns NAP-25 in phosphate-buffered saline, Dulbecco (DPBS).

The degree of conjugation for the VLP-peptide samples was measured using SDS page-ordinator, and the increase in molecular weight was observed in all samples, which is consistent with the addition of peptide to the VLP-protein monomer. In addition, samples were tested by HPLC gel-chromatographic analysis (using column Tosoh PWXL5000 HPLC) and found that they sod is rat collected the VLP when compared with unconjugated samples of the VLP. Signs of the VLP-peptide conjugation are summarized in Table 2.

Table 2
The VLP-Peptide conjugates
PeptideAdding activated the VLPThe amount of time to relax. peptidePercent of entering the VLPApproximate substitution (µg peptide in MrVLP monomer)
VR_9.14 mg5 mg40-60%25 mcg
VR_9.23 mg5 mg40-60%125 mcg

VR_9.33 mg5 mg40-60%125 mcg
VR_9.43 mg5 mg40-60%125 mcg
VR_9.53 mg 5 mg40-60%50 mcg
VR_9.63 mg5 mg40-60%125 mcg
VR_9.73 mg5 mg40-60%125 mcg
VR_9.83 mg5 mg40-60%125 mcg
VR_9.93 mg5 mg40-60%125 mcg
VR_9.103 mg5 mg40-60%125 mcg
VR_9.113 mg2 mg40-60%125 mcg
VR_9.123 mg2 mg40-60%125 mcg
VR_9.133 mg40-60%125 mcg
VR_9.143 mg2 mg40-60%125 mcg
VR_9.153 mg2 mg40-60%125 mcg
VR_9.163 mg2 mg40-60%125 mcg
VR_9.1710 mg9.5 mg95%82 mcg
VR_9.1810 mg8,7 mg90%74 mcg
VR_9.1910 mg7,6 mg80%58 mcg
VR_9.2010 mg8,7 mg95%86 mcg
VR_9.2110 mg 8.8 mg95%92 mcg
VR_9.2210 mg6.6 mg85%62 mcg
VR_9.2310 mg10.0 mg90%85 mcg
VR_9.2410 mg10.5 mg75%64 mcg
VR_9.2510 mg5.1 mg40%1 mcg
VR_9.2610 mg10.0 mg60%123 mcg
VR_9.2710 mg9.6 mg60%136 mcg
VR_9.2810 mg9,4 mg65%153 mcg
VR_9.2910 mg 4,2 mg75%19 mcg
VR_9.3010 mg4.4 mg63%15 mcg
VR_9.3110 mg4.4 mg70%13 mcg
VR_9.3210 mg4.4 mg63%15 mcg
VR_9.3310 mg7.4 mg40%18 mcg
VR_9.3410 mg7,3 mg50%23 mcg
VR_9.357.5 mg4.3 mg61%16 mcg
* As determined using SDS page-ordinator and densitometric calculations

Example 3: the Immunogenicity of the peptide PCSK9

This study aimed at assessing how effective peptides, conjugatively is with the VLP Qbeta (as detailed above in Example 2) in the induction or antibody-based test response, which may contact the human and mouse PCSK9. Female Balb/c mice (6-8 weeks) were injected by intramuscular (volume of 50 ál in each muscle Tibialis anterior) at day 0, 21 and 42 of the VLP-peptide conjugates prepared with alum with CpG formula 5' TCGTCGTTnTCGGTGCTTTT 3'. One group of control mice were immunized by the VLP associated with the control (non-PCSK9) peptide, following the same Protocol, and the second control group were left unimmunized. An autopsy was performed on the 49th day. At necropsy, 400-600 ál of blood was collected from euthanized mice by heart puncture using an anticoagulant. The blood was centrifuged to separate plasma, which was stored frozen until testing.

IgG or antibody-based test answers on a full-size recombinant human PCSK9 protein was measured using a colorimetric ELISA method. Serial dilution was obtained from serum samples and tested in the analysis.

Method 1 ELISA human PCSK9: on 384-well plates to analyze binding (Coming International, cat. No. 3700) was applied 25 µl/well concentrate human PCSK9 protein, diluted to 1 μg/ml with 0.01 M PBS pH 7.4 and incubated on a rocking chair with K. so for 3 hours. After washing × 2 PBS pH 7.4, the plates were blocked with 80 μl/well of 0.01 M PBS/1% BSA, incubated at K. I. within 1 hour before the final premiani the m × 3 0.01 M PBS pH 7.4/0.05% of Tween 20. On the following day prepared 8 point ½ log serial dilution of each sample, starting with a dilution of 1:25 (diluent PBS/1%BCA), 25 μl/well, serial dilution was transferred in duplicate to a tablet covered with human PCSK9, then incubated with shaking at K. so for 1.5 hours. After washing × 3 0.01 M PBS pH 7.4/0.05% of Tween 20, 25 µl/well total the detecting IgG antibodies (rabbit anti-mu IgG-Fc, cat. No. a-130A Bethyl Laboratories) at a dilution of 1:6000) was added to 0.01 M PBS pH 7.4/1% BSA, then incubated with shaking at K. T. for 1 hour. After washing × 5 0.01 M PBS pH 7.4/0.05% of Tween 20, was added 25 μl/well of a set of goat anti-rabbit conjugate with horseradish peroxidase Bio-Rad (Bio-Rad, cat. No. 172-1019) 1:3000 0.01 M PBS pH 7.4/0.05% of Tween 20 pH 7.4, then incubated with shaking at K. T. for 1 hour. After washing × 4 0.01 M PBS pH 7.4/0.05% of Tween 20 and then only × 1 0.01 M PBS pH 7.4 was added 25 μl/well of mouse substrate Typer HRP (Bio-Rad, cat. No. 172-1064) and the plates were incubated at K. I. within 30 minutes. Added 25 μl/well of 2% oxalic acid and then read the absorbance at 405 nm.

Method 1 ELISA murine PCSK9: 96-hole plates to ELISA Thermo Immunolon 4HBX put 100 μl of 1 μg/ml recombinant murine PCSK9 in PBS over night at 4°C. After washing the plates were blocked with 300 ml PBS/0.5% BSA (Sigma A7030) for 1 hour, washed 4 × with 300 μl PBS/0.01% of Tween-20 was added 100 μl of serial is x the dilution of plasma samples (in PBS/0.5% BSA). After incubation with gentle rocking for 1 hour at room temperature tablets were washed 4x with 300 ál PBS/0.01% of Tween-20 and to each well was added 100 μl of a dilution of 1:5000 goat antimisting IgG-HRP (horseradish peroxidase; Pierce 31430). The plates were incubated at room temperature with gentle rocking for 45 minutes and then washed × 7 300 μl PBS/0.01% of Tween-20. Then add 100 ál of TMB substrate (Sigma T-4444), the colorimetric reaction was stopped after 4 minutes by adding 2 N. sulfuric acid and read the absorbance at 450 nm.

Data analysis: titration Curves constructed for each test sample (sample dilution of anti-takeover provisions). The titer of the sample (subsequently converted into a reciprocal titer) is then used as the serum dilution that reaches the cut-off values of optical density (O. D.) of 1.0 or 0.5.

Measurement of cholesterol in plasma/serum

Cholesterol in samples of plasma and serum were measured using the test kit WAKO Cholesterol E (cat. No. 439-17501) according to the manufacturer's instructions. Cultivation of cholesterol standard or test samples of plasma/serum (volume 4 ml) was added to the wells of the 96-hole tablet and added 196 ál received cholesterol reagent. Tablet incubated for 5 minutes at 37°C and the absorption of the developed color in accordance with their 30 minute read at 600 nm.

Measurement of the interaction between PCSK9 and the extracellular domain of the LDL receptor

Interrupt link LDLR and PCSK9 using mouse plasma

determined by TR-FRET analysis (analysis of resolution in time resonance energy transfer fluorescence) using a fluorophore-labeled extracellular domain of LDLR (LDLR-ECD) and a full-sized protein PCSK9 wild type. LDLR-ECD (R&D system, cat No. 2148-LD/CF)) were labeled with europium (GE heaithcare, cat. No. RA) according to the manufacturer's instructions (at a molar ratio of Eu:LDL.R 6:1). PCSK9 was biotinilated using Biotin-XX-SSE (Pierce, cat. No. 21237) at a molar ratio of bnoTHH:PCSK9 8. Analysis of TR-FRET was performed with 5 nm LDLR-Eu+3, 30 nm PCSK9-Biotin and 50 nm Alexa FSuor 647, conjugated with streptavidin (SA647, Invitrogen, cat. No. S21374) in 20 μl of buffer for analysis (20 mm Hepes, pH 7.0, 150 mm NaCl2, 0.1 mm CaCl2and 0.05% (wt./about.) BSA) in 384-well black plates (Corning, cat. No. 3676). Serial dilution of mouse plasma pre-incubated with PCSK9-Biotin in K. so for 30 minutes in a humidified chamber, followed by mixing with LDLR and streptavidin^A. After an additional 60-minute incubation at K. so in a humid atmosphere in the dark tablets read on a tablet reader Perkin Elmer Victor2, using a time delay of 50 μs and a box of 400 μs. Data are presented as the ratio of signal (665 nm/615 nm)×1000.

Results: as shown in Fig.2, all the peptides used is administered as immunogens, were able to induce or antibody-based test responses to the intact full-size human PCSK9 protein, some induced higher responses than others. In all cases, these responses cross react with murine PCSK9, as shown in Fig 3. In Fig.4 shows that immunization with peptide VR_9.5 also resulted in lower levels of cholesterol in the plasma, and Fig.5 and 6 shows that VR_9.5 and VR_9.6 induced serum or antibody-based test answers that could inhibit the interaction between PCSK9 and LDL receptor by analysis of resonance energy transfer fluorescence (FRET).

Example 4 - Design of peptides corresponding to portions other than the receptor domain EGF-A. present in PDB: 3BPS:

The LDL receptor is a multidomain protein, the extracellular domains which consist of N-terminal ligand-binding domain, three EGF-like repeats (EGF-A is one) β-propeller domain and O-linked sugar domain" (Kwon et al., PNAS 105, 1820-1825, 2008). PDB file 3GCX describes in detail the structure of PCSK9 in complex with a soluble form only domain EGF-A receptor LDL, therefore, the inventors hypothesized that there may be more implicit interaction between PCSK9 and LDL receptor, which cannot be derived from direct analysis of the molecules represented in the PDB: 3GCX. Examples of the quiet sections are described in detail in Fig.7, and were specifically identified two sequences in PCSK9, which could act as an additional alleged receptor interaction surfaces (NAQDQPVTLGTL and INEAWFPEDQRVL - see Fig.8).

SIPWNLERIIP (SEQ ID NO:332) (mouse)

NAQDQPVTLGTL (SEQ ID NO:227)

INEAWFPEDQRVL (SEQ ID NO:263)

INMAWFPEDQQVL (SEQ ID NO:360) (mouse)

When using mouse PCSK9 sequences found in public databases, were identified murine homologues (as shown in Table 3). The inventors have also suggested that part of the amino acid sequence contained in the peptide VR_9.5 (described in Example 1), can also interact with parts of the LDL receptor, is not visible in the PDB: 3BPS (Fig.8). This concept was investigated by specifying the sequence VR_9.5 by changing the amino acid linker and the orientation of the conjugation. The inventors have also identified a peptide corresponding to mouse homologue VR_9.5 (peptide VR_9.10) Received a number of peptide sequences of the above described approach was modified by adding amino acids to ensure chemical conjugation, and was evaluated as candidates for the role of the vaccine peptides described in Table 3).

Table 3
Peptide sequence
PeptideSequenceSEQ ID NO:
VR_9.10SIPWNLERIIPCMouse322
VR_9.11CGGSIPWNLERIIPMouse323
VR_9.12SIPWNLERIIPGGCMouse324
VR_9.13CGGNAQDQPVTLGTLMouse and man325
VR_9.14NAQDQPVTLGTLGGCMouse and man326
VR_9.15CGGINMAWFPEDQQVLMouse327
VR_9.16INMAWFPEDQQVLGGCMouse328
Underlined residues indicate amino acids that were added for conjugation purposes.

Example 5

Peptides VR_9.10-VR_9.16 (and VR_9.5 for comparison) conjugatively with Qβ the VLP as described in example 2, and used to immunize mice or antibody-based test and evaluation of responses to PCSK9, as described in example 3, with the VLP unconjugated used as a control immunogen. As shown in Fig.9 and 10, all peptides induced antibodies that can recognize the intact full-size mouse PCSK9 in ELISA analysis.

Example 6

On the basis of observation of the reduction of cholesterol induced by immunization with peptide VR_9.5, representing the N-end of the Mature protestirovanny forms of human PCSK9 (SEQ ID NO:184), the inventors hypothesized that, since the split prodomain immature PCSK9, as it is known, remains associated with the Mature protein PCSK9, areas of this predomina (SEQ ID NO:329) are also possible targets of antibodies to reduce cholesterol levels.

MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQ (SEQ ID NO:329)

Specific, non-limiting examples of peptides of interest found within the C-terminal sequence produmannoho plot and exposed on the surface of sequences and loops, including areas containing known people have genetic mutations loss of function and acquisition functions:

VDYIEEDSSVFAQ (SEQ ID NO:308)

RCAKDPWRLPGT (SEQ ID NO:309)

AQAARRGYLTKIL (SEQ ID NO:310)

GDYEELVLALRSEEDG (SEQ ID NO:311)

FLVKMSGDLLELALKLP (SEQ ID NO:312)

The above peptides and their shortening synthesize added at the N - or C-end-linkers (e.g., CGG or GGC) or in the form of cicletanine or otherwise conformationally constrained molecules and is associated with Qβ VLPs as described in example 2, and used for immunization of mice or antibody-based test and evaluation of responses to PCSK9, as described in example 3.

Example 7

Peptides VR_9.5 and 9, 10 conjugatively with Qβ the VLP or CRM197, as described in example 2, and used for immunization of mice (BALB/c or C57BL/6) using TiterMax Gold, alum or alum-CpG adjuvant, a Peptide derived from hepatitis b virus (amino acids 28-39 (IPQSLDSWWTSL)) conjugatively with Qβ the VLP or CRM197 and used as a control immunogen.

The method used ELISA differed slightly from the method described in example 3: Method ELISA was performed as follows for ELISA human and mouse PCSK9 (method 2): 384-hole tablets with high binding (Greiner bio-one 781061) was applied 25 µl/well concentrate human or murine PCSK9 protein, diluted to 1 μg/ml 1×PBS pH 7.4, and incubated at 4°C over night. On the following day the plates were blocked using 25 μl/well 1×PBS/0,05% Tween-20/1% BSA, incubated on a rocking chair with K. so within 1 hour. 10 point ½ log serial dilution of each of the first sample was prepared, starting with a dilution of 1:50 or 1:500 (diluent 1×PBS/0,05% Tween-20), 25 μl/well, serial dilution was transferred in duplicate to a tablet coated with human or murine PCSK9, then incubated with shaking at K. T. for 1 hour. After washing × 3 1×PBS pH 7.4/0.05% of Tween-20 was added 25 μl/well total the detecting IgG antibodies (goat antimachine IgG, HRP, Invitrogen M30107) at a dilution of 1:3000 with 1×PBS pH 7.4/0.05% of Tween-20, then incubated with shaking at K. T. for 1 hour. After washing × 5 1×PBS pH 7.4/0.05% of Tween 20, was added 25 μl/well of a set of Bio-Rad TMB EIA Buffer Substrate (Bio-Rad, cat. No. 172-1067) and the plates were incubated for 15 minutes. Was added to 12.5 μl/well 1 N. sulfuric acid and then read the absorbance at 450 nm.

Results: Peptides VR_9.5 and 9.10 conjugated to Qβ the VLP or CRM197, were able to induce or antibody-based test responses to the intact full-sized human and mouse PCSK9 protein (see Fig.11 and 12). In Fig.13 and 14, and Table 4 shows that the peptides VR_9.5 and 9.10 conjugated with different media, and in the presence of various adjuvants lead to lower levels of serum cholesterol.

Example 8

Additional peptides (SEQ ID NO:312, 420, 421, 422, 423, 425, 426, 427, 428, 445, 482, 525 and 563) were selected from predomina and C-terminal site of the catalytic domain of PCSK9 for surface exposure and Association with Muta is the second acquisition function or loss function, identified in humans. Received a number of peptide sequences of the above described approach was modified by adding amino acids to ensure chemical conjugation and evaluated as candidates for the role of vaccines (table 5, peptides 9.23-9.35).

Table 5
Peptide sequence
PeptideSequenceSEQ ID NO:
VR_9.5SIPWNLERITPCPeople317
VR_9.10SIPWNLERIIPCMouse322
VR_9.17SIPWNLERIGGCHuman and mouse401
VR_9.18SIPWNLERGGCHuman and mouse402
VR_9.19SIPWNLEGGCHuman and mouse 403
VR_9.20CGGSGRDAGVAKGAPeople404
VR_9.21CGGSGRDAGVAKGTMouse405
VR_9.22RDAGVAKGGCPeople406
VR_9.23CSRHLAQASQELQPeople407
VR_9.24CRSRPSAKASWVQMouse408
VR_9.25CGGDYEELVLALRPeople409
VR_9.26CGGDYEELMLALPMouse410
VR_9.27LVLALRSEEDGGCPeople411
VR_9.28LMLALPSQEDGGCMouse412

VR_9.29AKDPWRLPGGCPeople413
VR_9.30SKEAWRLPGGCMouse414
VR_9.31CGGAARRGYLTKPeople415
VR_9.32CGGAARRGYVIKMouse416
VR_9.33FLVKMSGDU-ELALKLPGGCPeople417
VR_9.34FLVKMSSDLLGLALKLPGGCMouse418
VR_9.35CGGEEDSSVFAQPeople419
Underlined residues indicate amino acids that were added for conjugation purposes.

Example 9

Peptides VR_9.17-VR_9.35 (and VR_9.5) conjugatively with Qβ the VLP as described in example 2, and used to immunize mice or antibody-based test and evaluation of responses to PCSK9, as described in the example 3. Peptide derived from hepatitis b virus (amino acids 28-39 (IPQSLDSWWTSL)) conjugatively with Qβ the VLP and used as a control immunogen. Method no: 2 was used for ELISA.

Result: As shown in Fig.15 and 16, the majority of peptides conjugated to Qβ the VLP, were able to induce or antibody-based test responses to the intact full-PCSK9. Or antibody-based test, the answer is not found for peptides 9.31 and 9.32. In some cases (peptides 9.23, 9.24, 9.27, 9.28, 9.29, 9.30, 9.33 and 9.34) or antibody-based test responses were species-specific. Immunization with peptides 9.5, 9.18 and 9.29 also resulted in lowering serum cholesterol, whereas immunization with peptides 9.17, 9.19, 9.30, 9.31 and 9.32 led to a decline in cholesterol levels (table 6, Fig.17).

VR_9.19The VLPAlum+CpG871,71,7P>0,05P>0,05
VR_9.20The VLPAlum+CpG7to 85.24,0F>0,05 P>0,05
VR_9.21The VLPAlum+CpG877,12,0P>0,05P>0,05
VR_9.22The VLPAlum+CpG884,52,6P>0,05P>0,05
VR_9.23The VLPAlum+CpG879,01,8P>0,05P>0,05
VR_9.24The VLPAlum+CpG876,92,1P>0,05P>0,05
VR_9.25The VLPAlum+CpG8 77,32,5P>0,05P>0,05
VR_9.26The VLPAlum+CpG879,22,2P>0,05P>0,05
VR_9.27The VLPAlum+CpG871,83,4P>0,05P>0,05
VR_9.28The VLPAlum+CpG876,62,2P>0,05P>0,05
VR_9.29The VLPAlum+CpG867,01,6P<0,05P>0,05
VR_9.30LP Alum+CpG870,42,3P>0,05P>0,05
VR_9.31The VLPAlum+CpG871,01,7P>0,05P>0,05
VR_9.32The VLPAlum+CpG872,81,0P>0,05P>0,05
VR_9.33The VLPAlum+CpG875,52,2P>0,05P>0,05
VR_9.34The VLPAlum+CpG876,02,3P>0,05P>0,05
VR_9.35The VLPAlum+CpG874,52,2P>0,05P>0,05
Statistics(a): univariate analysis of variance ANOVA for statistical comparison with using the criterion of Bonferroni (post-hoc), showing p values of the test groups compared with the untreated group.
Statistics(b): univariate analysis of variance ANOVA for statistical comparison using the criterion of Bonferroni (post-hoc), showing p values of the test groups in comparison with the control peptide group.

The sequence listing:

SEQ ID NO:1IGASSDCSTCFVSSEQ ID NO:53DGTRFHRQASKCDS
SEQ ID NO:2IGASSDCSTCFVSEQ ID NO:54DGTRFHRQASKCD
SEQ ID NO:3IGASSDCSTCFSEQ ID NO:55DGTRFHRQASKC
SEQ ID NO:4IGASSDCSTCSEQ ID NO:56DGTRFHRQASK
SEQ ID NO:5IGASSDCSTSEQ ID NO:57DGTRFHRQAS
SEQ ID NO:6IGASSDCSSEQ ID NO:58DGTRFHRQA
SEQ ID NO:7IGASSDCSEQ ID NO:59DGTRFHRQ
SEQ ID NO:8IGASSDSEQ ID NO:60GTRFHRQASKCDS
SEQ ID NO:9IGASSSEQ ID NO:61GTRFHRQASKCD
SEQ ID NO:10GASSDCSTCFVSSEQ ID NO:62GTRFHRQASKC
SEQ ID NO:11GASSDCSTCFVSEQ ID NO:63GTRFHRQASK
SEQ ID NO:12GASSDCSTCFSEQ ID NO:64GTRFHRQAS

SEQ ID NO:13 GASSDCSTCSEQ ID NO:65GTRFKRQA
SEQ ID NO:14GASSDCSTSEQ ID NO:66GTRFHRQ
SEQ ID NO:15GASSDCSSEQ ID NO:67TRFHRQASKCDS
SEQ ID NO:16GAS S DCSEQ ID NO:68TRFHRQASKCD
SEQ ID NO:17GASSDSEQ ID NO:69TRFHRQASKC
SEQ ID NO:18ASSDCSTCFVSSEQ ID NO:70TRFHRQASK
SEQ ID NO:19ASSDCSTCFVSEQ ID NO:71TRFHRQAS
SEQ ID NO:20ASSUCSTCFSEQ ID NO:72TRFHRQA
SEQ ID NO:21ASSDCSTCSEQ ID NO:73TRFHRQ
SEQ ID NO:22ASSDCSTSEQ ID NO:74RFHRQASKCDS
SEQ ID NO:23ASSDCSSEQ ID NO:75RFHRQASKCD
SEQ ID NO:24ASS DCSEQ ID NO:76RFHRQASKC
SEQ ID NO:25SSDCSTCFVSSEQ ID NO:77RFHRQASK
SEQ ID NO:26SSDCSTCFVSEQ ID NO:78RFHRQAS
SEQ ID NO:27SSDCSTCFSEQ ID NO:79RFHRQA
SEQ ID NO:28SSDCSTCSEQ ID NO:80RFHRQ
SEQ ID NO:29SSDCSTSEQ ID NO:81FHRQASKCDS
SEQ ID NO:30SSDCSSEQ ID NO:82FHRQASKCD
SEQ ID NO:31SDCSTCFVSSEQ ID NO:83FHRQASKC
SEQ ID NO:32SDCSTCFV SEQ ID NO:84FHRQASK
SEQ ID NO:33SDCSTCFSEQ ID NO:85FHRQAS
SEQ ID NO:34SDCSTCSEQ ID NO:86FHRQA
SEQ ID NO:35SDCSTSEQ ID NO:87HRQASKCDS
SEQ ID NO:36DCSTCFVSSEQ ID NO:88HRQASKCD
SEQ ID NO:37DCSTCFVSEQ ID NO:89HRQASKC
SEQ ID NO:38DCSTCFSEQ ID NO:90HRQASK
SEQ ID NO:39DCSTCSEQ ID NO:91HRQAS
SEQ ID NO:40CSTCFVSSEQ ID NO:92RQASKCDS
SEQ ID NO:41CSTCFVSEQ ID NO:93RQASKCD
SEQ ID NO:42 CSTCFSEQ ID NO:94RQASKC
SEQ ID NO:43STCFVSSEQ ID NO:95RQASK
SEQ ID NO:44STCFVSEQ ID NO:96QASKCDS
SEQ ID NO:45TCFVSSEQ ID NO:97QASKCD
SEQ ID NO:46EDGTRFHRQASKCDSSEQ ID NO:98QASKC
SEQ ID NO:47EDGTRFHRQASKCDSEQ ID NO:99ASKCDS
SEQ ID NO:48EDGTRFHRQASKCSEQ ID NO:100ASKCD
SEQ ID NO:49EDGTRFHRQASKSEQ ID NO:101SKCDS
SEQ ID NO:50EDGTRFHRQASSEQ ID NO:102SIQSDHREIEGRVM

SEQ ID NO:51EDGTRFHRQA SEQ ID NO:103SIQSDHREIEGRV
SEQ ID NO:52EDGTRFHRQSEQ ID NO:104STQSDHREIEGR
SEQ ID NO:105SIOSDHREIEGSEQ ID NO:156SGRDAGVA
SEQ ID NO:106SIQSDHREIESEQ ID NO:157GRDAGVAKGAS
SEQ ID NO:107SIQSDHREISEQ ID NO:158GRDAGVAKGA
SEQ ID NO:108IQSDHREIEGRVMSEQ ID NO:159GRDAGVAKG
SEQ ID NO:109IQSDHREIEGRVSEQ ID NO:160GRDAGVAK
SEQ ID NO:110IQSDHREIEGRSEQ ID NO:161GRDAGVA
SEQ ID NO:111IQSDHREIEGSEQ ID NO:162RDAGVAKGAS
SEQ ID NO:112IQSDHREIESEQ ID NO:163RDAGVAKGA
SEQ ID NO:113IQSDHREISEQ ID NO:164RDAGVAKG
SEQ ID NO:114QSDHREIEGRVMSEQ ID NO:165RDAGVAK
SEQ ID NO:115QSDHREIEGRVSEQ ID NO:166RDAGVA
SEQ ID NO:116QSDHREIEGRSEQ ID NO:167DAGVAKGAS
SEQ ID NO:117QSDHREIEGSEQ ID NO:168DAGVAKGA
SEQ ID NO:118QSDHREIESEQ ID NO:169DAGVAKG
SEQ ID NO:119QSDHREISEQ ID NO:170DAGVAK
SEQ ID NO:120SDHREIEGRVMSEQ ID NO:171DAGVA
SEQ ID NO:121SDHREIEGRV SEQ ID NO:172AGVAKGAS
SEQ ID NO:122SDHREIEGRSEQ ID NO:173AGVAKGA
SEQ ID NO:123SDHREIEGSEQ ID NO:174AGVAKG
SEQ ID NO:124SDHREIESEQ ID NO:175AGVAK
SEQ ID NO:125SDHREISEQ ID NO:176GVAKGAS
SEQ ID NO:126DHREIEGRVMSEQ ID NO:177GVAKGA
SEQ ID NO:127DHRETEGRVSEQ ID NO:178GVAKG
SEQ ID NO:128DHREIEGRSEQ ID NO:179VAKGAS
SEQ ID NO:129DHREIEGSEQ ID NO:180VAKGA
SEQ ID NO:130DHREIESEQ ID NO:181AKGAS
SEQ ID NO:131/td> DHREISEQ ID NO:182SIPWNLERITPPR
SEQ ID NO:132HREIEGRVMSEQ ID NO:183SIPWNLERITPP
SEQ ID NO:133HREIEGRVSEQ ID NO:184SIPWNLERITP
SEQ ID NO:134HREIEGRSEQ ID NO:185SIPWNLERIT
SEQ ID NO:135HREIEGSEQ ID NO:186SIPWNLERI
SEQ ID NO:136HREIESEQ ID NO:187SIPWNLER
SEQ ID NO:137RETEGRVMSEQ ID NO:188SIPWNLE
SEQ ID NO:138REIEGRVSEQ ID NO:189SIPWNL
SEQ ID NO:139REIEGRSEQ ID NO:190SIPWN

SEQ ID NO:140 REIEGSEQ ID NO:191IPWNLERITPPR
SEQ ID NO:141EIEGRVMSEQ ID NO:192IPWNLERITPP
SEQ ID NO:142EIEGRVSEQ ID NO:193IPWNLERITP
SEQ ID NO:143EIEGRSEQ ID NO:194IPWNLERIT
SEQ ID NO:144IEGRVMSEQ ID NO:195IPWNLERI
SEQ ID NO:145IEGRVSEQ ID NO:196IPWNLER
SEQ ID NO:146EGRVMSEQ ID NO:197IPWNLE
SEQ ID NO:147VSGRDAGVAKGASSEQ ID NO:198IPWNL
SEQ ID NO:148VSGRDAGVAKGASEQ ID NO:199PWNLERITPPR
SEQ ID NO:149VSGRDAGVAKGSEQ ID NO:200WNLERITPP
SEQ ID NO:150VSGRDAGVAKSEQ ID NO:201PWNLERITP
SEQ ID NO:151VSGRDAGVASEQ ID NO:202PWNLERIT
SEQ ID NO:152SGRDAGVAKGASSEQ ID NO:203PWNLERI
SEQ ID NO:153SGRDAGVAKGASEQ ID NO:204PWNLER
SEQ ID NO:154SGRDAGVAKGSEQ ID NO:205PWNLE
SEQ ID NO:155SGRDAGVAKSEQ ID NO:206WNLERITPPR
SEQ ID NO:207WNLERITPPSEQ ID NO:258PVTLGT
SEQ ID NO:208WNLERITPSEQ ID NO:259PVTLG
SEQ ID NO:209WNLERIT SEQ ID NO:260VTLGTL
SEQ ID NO:210WNLERISEQ ID NO:261VTLGT
SEQ ID NO:211WNLERSEQ ID NO:262TLGTL
SEQ ID NO:212NLERITPPRSEQ ID NO:263INEAWFPEDQRVL
SEQ ID NO:213NLERITPPSEQ ID NO:264INEAWFPEDQRV
SEQ ID NO:214NLERITPSEQ ID NO:265INEAWFPEDQR
SEQ ID NO:215NLERITSEQ ID NO:266INEAWFPEDQ
SEQ ID NO:216HELPERSSEQ ID NO:267INEAWFPED
SEQ ID NO:217LERITPPRSEQ ID NO:268INEAWFPE
SEQ ID NO:218LERITPPSEQ ID NO:269INEAWFP
SEQ ID NO:219LERITPSEQ ID NO:270INEAWF
SEQ ID NO:220LERITSEQ ID NO:271INEAW
SEQ ID NO:221ERITPPRSEQ ID NO:272NEAWFPEDQRVL
SEQ ID NO:222ERITPPSEQ ID NO:273NEAWFPEDQRV
SEQ ID NO:223ERITPSEQ ID NO:274NEAWFPEDQR
SEQ ID NO:224RITPPRSEQ ID NO:275NEAWFPEDQ
SEQ ID NO:225RITPPSEQ ID NO:276NEAWFPED
SEQ ID NO:226ITPPRSEQ ID NO:277NEAWFPE
SEQ ID NO:227NAQDQPVTLGTLSEQ ID NO:278NEAWFP

SEQ ID NO:228 NAQDQPVTLGTSEQ ID NO:279NEAWF
SEQ ID NO:229NAQDQPVTLGSEQ ID NO:280EAWFPEDQRVL
SEQ ID NO:230NAQDQPVTLSEQ ID NO:281EAWFPEDQRV
SEQ ID NO:231NAQDQPVTSEQ ID NO:282EAWFPEDQR
SEQ ID NO:232NAQDQPVSEQ ID NO:283EAWFPEDQ
SEQ ID NO:233NAQDQPSEQ ID NO:284EAWFPED
SEQ ID NO:234NAQDQSEQ ID NO:285EAWFPE
SEQ ID NO:235AQDQPVTLGTLSEQ ID NO:286EAWFP
SEQ ID NO:236AQDQPVTLGTSEQ ID NO:287AWFPEDQRVL
SEQ ID NO:237AQDQPVTLGSEQ ID NO:288SEQ ID NO:238AQDQPVTLSEQ ID NO:289AWFPEDQR
SEQ ID NO:239AQDQPVTSEQ ID NO:290AWFPEDQ
SEQ ID NO:240AQDQPVSEQ ID NO:291AWFPED
SEQ ID NO:241AQDQPSEQ ID NO:292AWFPE
SEQ ID NO:242QDQPVTLGTLSEQ ID NO:293WFPEDQRVL
SEQ ID NO:243QDQPVTLGTSEQ ID NO:294WFPEDQRV
SEQ ID NO:244QDQPVTLGSEQ ID NO:295WFPEDQR
SEQ ID NO:245QDQPVTLSEQ ID NO:296WFPEDQ
SEQ ID NO:246QDQPVTSEQ ID NO:297WFPED
SEQ ID NO:247QDQPV SEQ ID NO:298FPEDQRVL
SEQ ID NO:248DQPVTLGTLSEQ ID NO:299FPEDQRV
SEQ ID NO:249DQPVTLGTSEQ ID NO:300FPEDQR
SEQ ID NO:250DQPVTLGSEQ ID NO:301FPEDQ
SEQ ID NO:251DQPVTLSEQ ID NO:302PEDQRVL
SEQ ID NO:252DQPVTSEQ ID NO:303PEDQRV
SEQ ID NO:253QPVTLGTLSEQ ID NO:304PEDQR
SEQ ID NO:254QPVTLGTSEQ ID NO:305EDQRVL
SEQ ID NO:255QPVTLGSEQ ID NO:306EDQRV
SEQ ID NO:256QPVTLSEQ ID NO:307DQRVL
SEQ ID NO:257PVTLGTLSEQ ID NO:308VDYIEEDSSVFAQ
SEQ ID NO:309RCAKDPWRLPGTSEQ ID NO:319AGCGTRFHRQ
SEQ ID NO:310AQAARRGYLTKILSEQ ID NO:320GRVCIQSDHREIE
SEQ ID NO:311GDYEELVLALRSEEDGSEQ ID NO:321AGVAKGAGCSGRD
SEQ ID NO:312FLVKMSGDLLELALKLPSEQ ID NO:322SIPWNLERIIPC
SEQ ID NO:313KGGASSDCSTCFVSEQ ID NO:323CGGSIPWNLERIIP
SEQ ID NO:314CGGTRFHRQASKCSEQ ID NO:324SIPWNLERIIPGGC
SEQ ID NO:315CGIQSDHREIEGRVCSEQ ID NO:325CGGNAQDQPVTLGTL

SEQ ID NO:316CSGRDAGVAKGACSEQ ID NO:326NAQDQPVTLGTGGC
SEQ ID NO:317SIPWNLERITPCSEQ ID NO:327CGGINMAWFPEDQQVL
SEQ ID NO:318ASKCGDGTRFHRQSEQ ID NO:328NMAWFPEDQQVLGGC
SEQ ID NO:329MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQ
SEQ ID NO:330SIPWNLERIIPAWSEQ ID NO:369NMAWFPEDQQVL
SEQ ID NO:331SIPWNLERIIPASEQ ID NO:370NMAWFPEDQQV
SEQ ID NO:332SIPWNLERIIPSEQ ID NO:371NMAWFPEDQQ
SEQ ID NO:333SIPWNLERIISEQ ID NO:372NMAWFPEDQ
SEQ ID NO:334IPWNLERIIPAWSEQ ID NO:373NMAWFPED
SEQ ID NO:335IPWNLERIIPAWSEQ ID NO:374NMAWFPE
SEQ ID NO:336IPWNLERTIPSEQ ID NO:375NMAWFP
SEQ ID NO:337IPWNLERIISEQ ID NO:376NMAWF
SEQ ID NO:338PWNLERIIPAWSEQ ID NO:377MAWFPEDQQVL
SEQ ID NO:339PWNLERII PASEQ ID NO:378MAWFPEDQQV
SEQ ID NO:340PWNLERIIPSEQ ID NO:379MAWFPEDQQ
SEQ ID NO:341PWNLERIISEQ ID NO:380MAWFPEDQ
SEQ ID NO:342WNLERIIPAWSEQ ID NO:381MAWFPED
SEQ ID NO:343WNLERIIPASEQ ID NO:382MAWFPE
SEQ ID NO:344WNLERIIPSEQ ID NO:383MAWFP
SEQ ID NO:345WNLERII SEQ ID NO:384AWFPEDQQVL
SEQ ID NO:346NLERIIPAWSEQ ID NO:385AWFPEDQQV
SEQ ID NO:347NLERIIPASEQ ID NO:386AWFPEDQQ
SEQ ID NO:348NLERIIPSEQ ID NO:387WFPEDQQVL
SEQ ID NO:349NLERIISEQ ID NO:388WFPEDQQV
SEQ ID NO:350LERIIPAWSEQ ID NO:389WFPEDQQ
SEQ ID NO:351LERTIPASEQ ID NO:390FPEDQQVL
SEQ ID NO:352LERITPSEQ ID NO:391FPEDQQV
SEQ ID NO:353LERIISEQ ID NO:392FPEDQQ
SEQ ID NO:354ERIIPAWSEQ ID NO:393PEDQQVL
SQ ID NO:355 ERIIPASEQ ID NO:394PEDQQV
SEQ ID NO:356ERIIPSEQ ID NO:395PEDQQ
SEQ ID NO:357RIIPAWSEQ ID NO:396EDQQVL
SEQ ID NO:358RIIPASEQ ID NO:397EDQQV
SEQ ID NO:359IIPAWSEQ ID NO:398DQQVL
SEQ ID NO:360INMAWFPEDQQVL
SEQ ID NO:361INMAWFPEDQQV
SEQ ID NO:362INMAWFPEDQQ

td align="center"> CAKDPW
SEQ ID NO:363INMAWFPEDQ
SEQ ID NO:364INMAWFPED
SEQ ID NO:365INMAWFPE
SEQ ID NO:366INMAWFP
SEQ ID NO:367INMAWF
SEQ ID NO:368INMAW
SEQ ID NO:399MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYWVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQSIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSTQSDHREIEGRVMVTDFENVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGASMRSLRVLNCQGKGTVSGTLIGLEFIRKSQLVQPVGPLVVLLPLAGGYSRVLNAACQRLARAGVVLVTAAGNFRDDACLYSPASAPEVITVGATNAQDQPVTLGTLGTNFGRCVDLFAPGELIIGASSDCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAKEPEDQRVLTPKLVAALPPSTHCAGWCLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGFAMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHKEVEDLGTHKPPVLRPRGQPNQCVGHREASJHASCCHAPGLECKVKEHGIFAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTCSTSEGAVTAVAICCRSRHLAQASQELQ
SEQ ID NO:400MGTHCSAWLRWPLLFLLPPLLLLLLLLCPTGAGAODEUGDYEELMLALPSUEDGLADEAAHVATATFRRCGKEAWPLPGTYIVVLMEETQRLQIEQTAHRLQTRAARRGYVIKVLHIFYDLFPGFLVKMSSDLLGLALKLPHVEYIEEDSFVFAQSIPWNLERIIPAWHQTEEDRSPDGSSQVEVYLLDTSIQGAHREIERVTITPFNSVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGTSLHSLRVLNCQGKGTVSGTLIGLEFIRKSQLIQPSGPLVVLLPLAGGYSRILNAACRHLARTGVVLVAAAGNFRDDACLYSPASAPEVITVGATNAQDQPVTLGTLGTNFGRCVDLFAPGKDIIGASSDCSTCFMSQSGTSQAAAHVAGIVARMLSREPTLTLAELRQRLIHFSTKDVTNMAWFPEDQQVITPNLVATLPFSTHETGGQLLCRTVWSAHSGPTRTATATARCAPEEELLSCSSFSRSGPRRGDWIEAIGGQQVCKALNAFGGEGVYAVARCCLVPRANCSTHNTPAARAGLETHVHCHQKDHVLTGCSFHWEVEDLSVRRQPALRSRRQPGQCVGHQAASVYASCCHAPGLFCKIKEHGTSGPSEQVTVACEAGWTLTGCNVLPGASLTLGAYSVDNLCVARVHDTARADRTSGEATVAAAICCRSRPSAKASWVQ
SEQ ID NO:401SIPWNLERTGGCSEQ ID NO:434RCAKDP
SEQ ID NO:402SIPWNLERGGCSEQ ID NO:435RCAKD
SEQ ID NO:403SIPWNLEGGCSEQ ID NO:436CAKDPWRLPGT
SEQ ID NO:404CGGSGRDAGVAKGASEQ ID NO:437CAKDPWRLPG
SEQ ID NO:405CGGSGRDAGVAKGTSEQ ID NO:438CAKDPWRLP
SEQ ID NO:406RDAGVAKGGCSEQ ID NO:439CAKDPWRL
SEQ ID NO:407CSRHLAQASQELQSEQ ID NO:440CAKDPWR
SEQ ID NO:408CRSRPSAKASWVQSEQ ID NO:441
SEQ ID NO:409CGGDYEELVLALRSEQ ID NO:442CAKDP

PWRLPGT QAARRGY
SEQ ID NO:410CGGDYEELMLALPSEQ ID NO:443AKDPWRLPGT
SEQ ID NO:411LVLALRSEEDGGCSEQ ID NO:444AKDPWRLPG
SEQ ID NO:412LMLALPSQEDGGCSEQ ID NO:445AKDPWRLP
SEQ ID NO:413AKDPWRLPGGCSEQ ID NO:446AKDPWRL
SEQ ID NO:414SKEAWRLPGGCSEQ ID NO:447AKDPWR
SEQ ID NO:415CGGAARRGYLTKSEQ ID NO:448AKDPW
SEQ ID NO:416CGGAARRGYVIKSEQ ID NO:449KDPWRLPGT
SEQ ID NO:417FLVKMSGDLLELALKLPGGCSEQ ID NO:450KDPWRLPG
SEQ ID NO:418FLVKMSSDLLGLALKLPGGCSEQ ID NO:451KDPWRLP
SEQ ID NO:419CGGEEDSSVFAQSEQ ID NO:452KDPWRL
SEQ ID NO:420SRHLAQASQELQSEQ ID NO:453KDPWR
SEQ ID NO:421DYEELVLALRSEQ ID NO:454DPWRLPGT
SEQ ID NO:422LVLALRSEEDGSEQ ID NO:455DPWRLFG
SEQ ID NO:423EEDSSVFAQSEQ ID NO:456DPWRLP
SEQ ID NO:424SGRDAGVAKGTSEQ ID NO:457DPWRL
SEQ ID NO:425RSRPSAKASWVQSEQ ID NO:458
SEQ ID NO:426GDYEELMLALPSEQ ID NO:459PWRLPG
SEQ ID NO:427LMLALPSQEDSEQ ID NO:460PKRLP
SEQ ID NO:428FLVKMSSDLLGLALKLPSEQ ID NO:461WRLPGT
SEQ ID NO:429RCAKDFWRLPGSEQ ID NO:462WRLPG
SEQ ID NO:430RCAKDPWRLPSEQ ID NO:464AQAARRGYLTKT
SEQ ID NO:431RCAKDPWRLSEQ ID NO:465AQAARRGYLTK
SEQ ID NO:432RCAKDPWRSEQ ID NO:466AQAARRGYLT
SEQ ID NO:433RCAKDPWSEQ ID NO:467AQAARRGYL
SEQ ID NO:468AQAARRGY SEQ ID NO:519CSKEAWRL
SEQ ID NO:469AQAARRGSEQ ID NO:520CSKEAWR
SEQ ID NO:470AQAARRSEQ ID NO:521CSKEAW
SEQ ID NO:471AQAARSEQ ID NO:522CSKEA
SEQ ID NO:472QAARRGYLTKILSEQ ID NO:523SKEAWRLPGT
SEQ ID NO:473QAARRGYLTKISEQ ID NO:524SKEAWRLPG
SEQ ID NO:474QAARRGYLTKSEQ ID NO:525SKEAWRLP
SEQ ID NO:475QAARRGYLTSEQ ID NO:526SKEAWRL
SEQ ID NO:476QAARRGYLSEQ ID NO:527SKEAWR
SEQ ID NO:477SEQ ID NO:528SKEAW
SEQ ID NO:478QAARRGSEQ ID NO:529KEAWRLPGT
SEQ ID NO:479QAARRSEQ ID NO:530KEAWRLPG
SEQ ID NO:480AARRGYLTKILSEQ ID NO:531KEAWRLP

SEQ ID NO:481AARRGYLTKISEQ ID NO:532KEAWRL
SEQ ID NO:482AARRGYLTKSEQ ID NO:533KEAWR
SEQ ID NO:483AARRGYLTSEQ ID NO:534EAWRLPGT
SEQ ID NO:484AARRGYLSEQ ID NO:535EAWRLPG
SEQ ID NO:485AARRGYSEQ ID NO:536EAWRLP
SEQ IDNO:486 AARRGSEQ !D NO:537EAWRL
SEQ ID NO:487ARRGYLTKILSEQ ID NO:538AWRLPGT
SEQ ID NO:488ARRGYLTKISEQ ID NO:539AWRLPG
SEQ ID NO:489ARRGYLTKSEQ ID NO:540AWRLP
SEQ ID NO:490ARRGYLTSEQ ID NO:541WRLPGT
SEQ ID NO:491ARRGYLSEQ ID NO:542WRLPG
SEQ ID NO:492ARRGYSEQ ID NO:543RLPGT
SEQ ID NO:493RRGYLTKILSEQ ID NO:544TRAARRGYVIKVL
SEQ ID NO:494RRGYLTKISEQ ID NO:545TRAARRGYVIKV
SEQ ID NO:495RRGYLTKSEQ ID NO:546 TRAARRGYVIK
SEQ ID NO:496RRGYLTSEQ ID NO:547TRAARRGYVI
SEQ ID NO:497RRGYLSEQ ID NO:548TRAARRGYV
SEQ ID NO:498RGYLTKTLSEQ ID NO:549TRAARRGY
SEQ ID NO:499RGYLTKISEQ ID NO:550TRAARRG
SEQ ID NO:500RGYLTKSEQ ID NO:551TRAARR
SEQ ID NO:501RGYLTSEQ ID NO:552TRAAR
SEQ ID NO:502GYLTKILSEQ ID NO:553RAARRGYVIKVL
SEQ ID NO:503GYLTKISEQ ID NO:554RAARRGYVIKV
SEQ ID NO:504GYLTKSEQ ID NO:555RAARRGYVIK
SEQ ID NO:505YLTKIL SEQ ID NO:556RAARRGYVI
SEQ ID NO:506YLTKISEQ ID NO:557RAARRCYV
SEQ ID NO:507LTKILSEQ ID NO:558PAARRGY
SEQ ID NO:508RCSKEAWRLPGTSEQ ID NO:559RAARRG
SEQ ID NO:509RCSKEAWRLPGSEQ ID NO:560RAARR
SEQ ID NO:510RCSKEAWRLPSEQ ID NO:561AARRGYVIKVL
SEQ ID NO:511RCSKEAWRLSEQ ID NO:562AARRGYVI KV
SEQ ID NO:512RCSKEAWRSEQ ID NO:563AAPRGYVIK
SEQ ID NO:513RCSKEAWSEQ ID NO:564AARRGYVI
SEQ ID NO:514RCSKEASEQ ID NO:565AARRGYV
SEQ ID NO:515RCSKESEQ ID NO:566AARRGY
SEQ ID NO:516CSKEAWRLPGTSEQ ID NO:567AARRG
SEQ ID NO:517CSKEAWRLPGSEQ ID NO:568ARRGYVTKVL
SEQ ID NO:518CSKEAWRLPSEQ ID NO:569ARRGYVIKV

SEQ ID NO 570ARRGYVIK
SEQ ID NO:571ARRGYVI
SEQ ID NO:572ARRGYV
SEQ ID NO:573ARRGY
SEQ ID NO:574RRGYVIKVL
SEQ ID NO:575RRGYVIKV
SEQ ID NO:576RRGYVIK
SEQ ID NO:577RRGYVI
SEQ ID NO:578RRGYV
SEQ ID NO:579RGYIKVL
SEQ ID NO:580RGYVIKV
SEQ ID NO:581RGYVIK
SEQ ID NO:582RGYVI
SEQ ID NO:583GYVTKVL
SEQ ID NO:584GYVIKV
SEQ ID NO:585GYVIK
SEQ ID NO:586YVIKVL
SEQ ID NO:587YVIKV
SEQ ID NO:588VIKVL
SEQ ID NO:589TCGTCGTTTTTCGGTGCTTTT
SEQ ID NO:590TCGTCGTTTTTCGGTCGTTTT
SEQ ID NO:591TCGTCGTTTTGTCGTTTTGTCGTT
SEQ ID NO:592TCGTCGTTTCGTCGTTTTGTCGTT
SEQ ID NO:593TCGTCGTTTTGTCGTTTTTTTCGA
SEQ ID NO:594TCGCGTCGTTCGGCGCGCGCCG
SEQ ID NO:595TCGTCGACGTTCGGCGCGCGCCG
SEQ ID NO:596TCGGACGTTCGGCGCGCGCCG
SEQ D NO:597 TCGGACGTTCGGCGCGCCG
SEQ ID NO:598TCGCGTCGTTCGGCGCGCCG
SEQ ID NO:599TCGACGTTCGGCGCGCGCCG
SEQ ID NO:600TCGACGTTCGGCGCGCCG
SEQ ID NO:601TCGCGTCGTTCGGCGCCG
SEQ ID NO:602TCGCGACGTTCGGCGCGCGCCG
SEQ ID NO:603TCGTCGTTTTCGGCGCGCGCCG
SEQ ID NO:604TCGTCGTTTTCGGCGGCCGCCG
SEQ ID NO:605TCGTCGTTTTACGGCGCCGTGCCG
SEQ ID NO:606TCGTCGTTTTCGGCGCGCGCCGT
SEQ ID NO:607TCGTCGACGATCGGCGCGCGCCG

1. The immunogen to induce an immune response against PCSK9 protein containing antigenic PCSK9 peptide selected from the group consisting of SEQ ID NO: 182, 183, 184, 185, 186, 187, 188, 317, 401, 402 and 403, where the specified antigenic PCSK9 peptide anywhereman with immunogenic carrier selected from CRM197 or Qbeta virus-like particles (the VLP).

2. The immunogen to induce an immune response against PCSK9 protein containing antigenic PCSK9 peptide selected from the group consisting of SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 314, 318 and 319, where specified antigenic PCSK9 peptide anywhereman with immunogenic carrier selected from CRM197 or Qbeta the VLP.

3. Immunogen under item 1, where the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO: 182-188.

4. Immunogen under item 2, where the antigenic PCSK9 peptide is selected from the group consisting of SEQ ID NO: 46-73.

5. Immunogen under item 3 or 4, where the specified antigenic PCSK9 peptide further comprises any of the following:
at its C-end of the linker having the formula (G)nC (G)nSC or (G)nK, where n is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10,
at its N end of the linker having the formula C(G)n, CS(G)n or K(G)n, where n is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, or
at its C-end of the linker having the formula (G)nC (G)nSC or (G)nK, where n is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and at its N-end linker having the formula C(G)n, CS(G)n or K(G)n, where n is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

6. Immunogen under item 3 or 4, where the specified antigenic PCSK9 peptide further comprises any of the following:
at its C-end of the linker having the formula (G)nC (G)nSC or (G)nK, where n is an integer selected from the group consisting of 0, 1 or 2,
at its N end of the linker with four the Ulu C(G)n, CS(G)n or K(G)n, where n is an integer selected from the group consisting of 0, 1, or 2, or
at its C-end of the linker having the formula (G)nC (G)nSC or (G)nK, where n is an integer selected from the group consisting of 0, 1 or 2, and at its N-end linker having the formula C(G)n, CS(G)n or K(G)n, where n is an integer selected from the group consisting of 0, 1 or 2.

7. Immunogen under item 3 or 4, where the specified antigenic PCSK9 peptide further comprises a cysteine at its C-end.

8. Immunogen under item 3 or 4, where the specified antigenic PCSK9 peptide further comprises a group of CG or cysteine at its N end.

9. Immunogen under item 3 or 4, where the specified antigenic PCSK9 peptide further comprises CGG at its N end.

10. Immunogen under item 3 or 4, where the specified antigenic PCSK9 peptide further comprises GGC at its C-end.

11. Immunogen under item 1 or 2, where the specified antigenic PCSK9 peptide cycletour and contains cysteine, (G)nC - or(G)n-fragment, where n is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

12. Immunogen under item 1 or 2, where the specified antigenic PCSK9 peptide cycletour and contains cysteine, GC, or CG-fragment.

13. Immunogen under item 1 or 2, where the specified antigenic PCSK9 peptide further comprises KG or KGG at its N end.

14. Immunogen under item 1 or 2, where the antigenic PCSK9 peptide of conform is largely limited.

15. The composition for inducing an immune response against PCSK9 protein containing at least two of the immunogen in immunologically effective amounts, where the first immunogen is an immunogen under item 1.

16. The composition for inducing an immune response against PCSK9 protein containing at least two of the immunogen in immunologically effective amounts, where the first immunogen is an immunogen under item 2.

17. The composition for inducing an immune response against PCSK9 protein containing at least two of the immunogen in immunologically effective amounts, where the first immunogen is an immunogen under item 1, and where the second immunogen is an immunogen under item 2.

18. The composition according to p. 17, where the antigenic PCSK9 peptide in the first immunogen selected from the group consisting of SEQ ID NO: 182, 183, 184, 185, 186, 187 and 188, and where the antigenic PCSK9 peptide of the second immunogen selected from the group consisting of SEQ ID NO: 67, 68, 69, 70, 71, 72 and 73.

19. Immunogenic composition for inducing an immune response against PCSK9 protein containing composition of immunogens in immunologically effective amounts under item 17, further containing at least one adjuvant.

20. Immunogenic composition according to p. 19, where the specified adjuvant selected from alum, CpG one (oligodeoxynucleotide), QS21 and Iscomatrix.

21. Immunogenic composicao p. 19, where specified adjuvant selected from QS21, alum in combination with one CpG or Iscomatrix in combination with one CpG.

22. Immunogenic composition according to p. 19, where the specified adjuvant selected from alum in combination with one CpG.

23. Immunogenic composition according to p. 19, where one CpG chosen from:
5' TCGTCGI I I I ICGGTGCTTTT 3', or
5' TCGTCG I I I I ICGGTCGTTTT 3', or
5' TCGTCGTTTTGTCGTTTTGTCGTT 3'.

24. The pharmaceutical composition intended for the prevention, cure or relief of PCSK9-related disorder, comprising:
in a therapeutically effective amount of the immunogen under item 1 or 2, the composition under item 17 or immunogenic composition according to p. 19; and
pharmaceutically acceptable excipient.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pyrazolopyridine derivatives of formula (I) , a based pharmaceutical composition, and using them for treating and/or preventing disorders or conditions related to nictonamide adenine dinucleotide phosphatoxidase (NADPH-oxidase), as well as to a method for preparing them and an intermediate of formula (VIII) . In general formula (I), G1 is specified in H; and optionally substituted heteroaryl-C1-C6-alkyl; G2 is specified in H; optionally substituted C1-C6-alkyl; optionally substituted C2-C6-alkenyl; optionally substituted C2-C6-alkinyl; optionally substituted aryl; optionally substituted C1-C6-alkylaryl; optionally substituted aryl-C1-C6-alkyl; optionally substituted heteroaryl; optionally substituted C1-C6-alkylheteroaryl; optionally substituted heteroaryl-C1-C6-alkyl; optionally substituted C2-C6-alkenylaryl; optionally substituted aryl-C2-C6-alkenyl; optionally substituted C2-C6-alkenylheteroaryl; optionally substituted heteroaryl-C2-C6-alkenyl; optionally substituted C3-C8-cycloalkyl; optionally substituted C3-C8-heterocycloalkyl; optionally substituted C1-C6-alkyl-C3-C8-cycloalkyl; optionally substituted C3-C8-cycloalkyl-C1-C6-alkyl; optionally substituted C1-C6-alkyl-C3-C8-heterocycloalkyl and optionally substituted C3-C8-heterocycloalkyl-C1-C6-alkyl; G3 is specified in H; optionally substituted amino; optionally substituted aminoalkyl; optionally substituted aminocarbonyl; optionally substituted alkoxy, optionally substituted alkoxy-C1-C6-alkyl; optionally substituted carbonyl; optionally substituted C1-C6-alkyl; optionally substituted C2-C6-alkenyl; optionally substituted C2-C6-alkinyl; optionally substituted aryl; optionally substituted aryl-C1-C6-alkyl; optionally substituted C1-C6-alkylaryl: optionally substituted heteroaryl; optionally substituted C1-C6-alkylheteroaryl; optionally substituted heteroaryl-C1-C6-alkyl; optionally substituted C2-C6-alkenylaryl; optionally substituted aryl-C2-C6-alkenyl; optionally substituted C2-C6-alkenylheteroaryl; optionally substituted heteroaryl-C2-C6-alkenyl; optionally substituted C3-C8-cycloalkyl; optionally substituted C3-C8-heterocycloalkyl; optionally substituted C1-C6-alkyl-C3-C8-cycloalkyl; optionally substituted C3-C8-cycloalkyl-C1-C6-alkyl; optionally substituted C1-C6-alkyl-C3-C8-hterocycloalkyl and optionally substituted C3-C8-heterocycloalkyl-C1-C6-alkyl; G4 is specified in -NR2-C(O)-R1 and -(CHR3)m-(CH2)n-R4, G5 represents H.

EFFECT: preparing the pharmaceutical composition for treating and/or preventing the disorders and conditions related to nictonamide adenine dinucleotide phosphatoxidase.

16 cl, 3 tbl, 87 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, namely to endocrinology, and can be used for treating non-alcoholic liver disease accompanying type 2 diabetes mellitus. The declared preparation Mexicor provides reducing manifestations of cytolysis and cholestasis, decreasing the steatosis index, enables improving metabolic lipid and glycaemic values and reducing insulin resistance. Mexicor is applied in a daily therapeutically effective dose of 100 mg 4 times a day for at least 16 weeks.

EFFECT: high pharmacological activity of Mexicor has been shown by achieving the pronounced and stable elimination of fatty liver disease that enables reducing the length of treatment with no side effects.

2 cl, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: pharmaceutical composition contains aleglitezar or its sodium salt in a dose of 0.01 to 0.9 mg.

EFFECT: pharmaceutical composition of aleglitezar is used for treating or preventing type II diabetes mellitus or cardiovascular diseases.

32 cl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology. Characterised is application of therapeutically efficient quantity of peptide GGF2, which contains domain, similar to epidermal growth factor (EGF-like), in treatment or prevention of heart failure in mammal by injection of said peptide every 48 hours in dose, which constitutes from approximately 0.001 mg/kg to approximately 10 mg/kg.

EFFECT: invention improves therapeutic effect with introduction of neuroregulin with minimisation of any potential side effects.

14 cl, 15 dwg, 11 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to phenol derivatives of formula (1), wherein R1 represents C1-C6 alkyl group, C1-C6 alkynyl group, C1-C6 halogen alkyl group, C1-C6 alkyl sulphanyl group or a halogen atom, R2 represents a cyano group or a halogen atom, R3 represents a hydrogen atom, and X represents -S(=O)2. Besides, the invention refers to a drug preparation containing a compound of formula (I) as an active ingredient.

EFFECT: phenol derivatives of formula (1) characterised by the high urine concentration of the permanent compound, and possess the uricosuric action.

11 cl, 1 dwg, 2 tbl, 42 ex

FIELD: biotechnologies.

SUBSTANCE: invention relates to compositions containing vasoactive intestinal peptide (VIP) or its fragments, and to application of such compositions in treatment of aorta fibrosis.

EFFECT: group of inventions is effective in treatment and prophylaxis of aorta fibrosis.

10 cl, 4 dwg, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to pharmaceutics and represents a pharmaceutical composition for parenteral administration containing sub-micron particles of dosocahexaenoic acid ester dispersed in a water phase with the use of mixture of at least two surfactants specified in a) at least one fatty acid polyoxyethylene ester and b) at least one phospholipide derivative, as well as a method for preparing the above pharmaceutical composition.

EFFECT: invention provides higher pharmacological activity.

14 cl, 3 dwg, 3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: what is presented is a fused protein that is a Notch1 antagonist, which consists of a human Fc region fused with the EGF-like repeat 1-13 of Notch1 or the EGF-like repeat 1-24 of Notch1. Fc-portion is localised on a carboxy-terminal portion of the EGF-repeat. There are described a pharmaceutical composition for the protein-based Notch signal transmission inhibition and using it for preparing the pharmaceutical composition for treating an individual suffering from: tumour; ovarian cancer; metabolic disorder; vascular proliferative retinopathy. What is presented is using the fused protein for producing the pharmaceutical composition for inhibition: angiogenesis in the individual; physiological lymphangiogenesis or pathological lymphangiogenesis in the individual; tumour deposits in the individual.

EFFECT: using the invention provides the proteins expressed in a supernatant at a level by several times more than the fused protein containing the EGF-like repeats 1-36 of Notch1; they penetrate into the tumour better, maintain a ligand-binding ability with the fused protein containing the repeats 1-24, binds to DLL4 and JAG1, whereas the fused protein containing the repeats 1-13 only binds to DLL4, but not to JAG1 that can find application in therapy of various diseases related to the Notch1 activity.

18 cl, 124 dwg, 10 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of general formula (1), which possess an affinity to the µ-opiod receptor and the ORL1-receptor. The invention also relates to the application of the said compounds for obtaining medications, which can be used in treatment of fear, stress and associated with stress syndromes, depressions, epilepsy, Alzheimer's disease, senile dementia, general cognitive dysfunctions, learning and memory disorders (as nootropic), withdrawal syndromes, alcohol and/or drug abuse and/or abuse of medications and/or alcohol, narcotic and medication addiction, etc. In general formula (1) (1) Y1, Y1', Y2, Y2', Y3, Y3', Y4 and Y4' in each case stand for -H; Q stands for -R0, -C(=O)-R0, -C(=O)OR0, -C(=O)NHR0, -C(=O)N(R0)2 or-C(=NH)-R0; R0 in each case stands for -C1-8-aliphate, -C3-12-cycloaliphate, -aryl, -heteroaryl, -C1-8-aliphate-C3-12-cycloaliphate, -C1-8-aliphate-aryl, -C1-8-aliphate-heteroaryl, -C3-8-cycloaliphate-C1-8-aliphate, -C3-8-cycloaliphate-aryl or -C3-8-cycloaliphate-heteroaryl; R1 and R2 independently on each other stand for -C-1-8-aliphate; R3 stands for -C1-8-aliphate, -aryl, -heteroaryl or -C1-8-aliphate-C3-12-cycloaliphate; n stands for 0; X stands for -NRA-;RA stands for -C1-8-aliphate; RB stands for -C1-8-aliphate; on condition that R1, R2, RA and RB simultaneously do not stand for the non-substituted-C1-8-aliphate.

EFFECT: increased efficiency of the application of the compounds.

9 cl, 11 tbl, 164 ex

FIELD: chemistry.

SUBSTANCE: invention relates to bi- and polycyclic substituted isoquinoline and isoquinolinones of formula (I), or to its stereoisomeric and/or tautomeric forms and/or to its pharmaceutically acceptable salts, where R1 represents OH; R3, R4, R5 and R8 represents H; R7 represents halogen or (C1-C6) alkyl; R6 represents one (C1-C4)alkylene, bound to a cycloalkyl ring, in which (C1-C4)alkylene forms the second bond with the other carbon atom of the cycloalkyl ring with the formation of a bicyclic ring system, where in the bicyclic ring system one carbon atom is substituted with a group, independently selected from O, S or SO2; and if m and s equal 2 or m equals 3 and s equals 1, R6 represents a group CH2-CH-(CH2)2, which via one group CH2 is bound to the cycloalkyl ring, and two other CH2 groups are bound to different carbon atoms of the cycloalkyl ring, and if m equals 3 and s equals 3, R6 represents two methylene groups, bound to different carbon atoms of the cycloalkyl ring, where the methylene groups or group CH2-CH-(CH2)2 are bound to the carbon atoms of the cycloalkyl ring and form an adamantane system of formula (XX) , where L can be bound to any secondary or tertiary carbon atom, or R6 together with R11 and an N atom form (C5) heterocycloalkyl, bound with the cycloalkyl residue in the form of a spirocyclic ring system, where the bicyclic ring system, or the adamantane system, or a ring system, containing (C5) heterocycloalkyl, represent non-substituted or optionally substituted with substituent R9; R9 represents (C1-C6)alkyl, (C2-C6)alkenyl, (C6)aryl or cyclopropyl R11 and R12 independently on each other represent H or (C1-C6)alkylene-(C6)aryl; n equals 0 or 1; m equals 2 or 3; s equals 1, 2 or 3; L represents O; its stereoisomeric and/or tautomeric forms and/or its pharmaceutically acceptable salts. The invention also relates to the application of a formula (I) compound.

EFFECT: novel bi- and polycyclic isoquinoline and isoquinolinone derivatives, useful as inhibitors of Rho-kinase, are obtained.

22 cl, 22 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to obstetrics and gynaecology, and can be used for treating and managing the pregnant women suffering drug-induced hepatitis. Establishing a diagnosis of drug-induced hepatitis requires oral administration of the herbal hepatoprotectors Carsil, or Hepabene, or the vitamin-free essential phospholipids Eslidin per os or Essentiale forte N intravenously, per os in standard doses with a cessation of any other drug-induced therapy, in the 1st trimester of pregnancy in case of observing a symptom-free increase of transaminase activity within 3 normal values and no counter-indications. In the 2nd trimester, a serum transaminase level up to 5 normal values requires prescribing ademetionine 400 mg intravenously N 7-10, then per os 1,600 mg/day. The therapeutic course makes 1 month. Ademetionine 800 mg intravenously N 10 + ursodeoxycholic acid 12 mg/kg/day are prescribed if observing the daily serum transaminase level of 5 to 10 normal values. Prescribing ademetionine 800 mg/day intravenously + ursodeoxycholic acid 12 mg/kg/day + prednisolone 90 mg/day is used for the daily serum transaminase level of more than 10 normal values. In the 3rd trimester, the daily serum transaminase level of below 5 normal values requires administering ademetionine 800 mg/day intravenously N10 + ursodeoxycholic acid 13 mg/kg/day. The transaminase level of 5 to 10 normal values requires prescribing ademetionine 800 mg/day intravenously + ursodeoxycholic acid 13 mg/kg/day + prednisolone 90 mg/day intravenously. A progressive increase of the daily transaminase level of above 10 normal values with underlying therapy with ademetionine 800 mg/day intravenously, ursodeoxycholic acid 13 mg/kg/day, prednisolone 120 mg/day intravenously with 3 days expected, a decrease of the daily transaminase level or a stabilization thereof are accompanied by continuing the therapy with the progression follow-up, including hepatic cytolysis values - an increase of the hepatic cytolysis values; an increase thereof is accompanied by prescribing the adequate therapy. The postpartum therapy is continued completely until liver function tests are normalised.

EFFECT: method enables preventing the pregnancy complications ensured by the timely diagnosis of drug-induced hepatitis in the pregnant women, the adequate drug-induced therapy differentiated by gestational age and a degree of manifestation of hepatic cytolysis and aiming at reducing a hepatic pathological process.

6 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to therapy and infectious diseases, and can be used for preventing and treating bronchial asthma, complicating respiratory viral infections and other respiratory inflammatory diseases. That is ensured by a combined inhibition of p-gene of the respiratory syncytial virus (further - RSV) and anti-inflammatory murine il-5 gene; p-gene of the RSV required for the cell replication is inhibited using a created molecule of small interfering RNA (siRNA) - SEQ ID NO 1 (siP1), while anti-inflammatory murine il-5 gene is inhibited using a created molecule of siRNA - SEQ ID NO 15 (siIL4-408). They are used to develop preparations containing therapeutically effective amounts thereof to be used intranasally.

EFFECT: method enables relieving signs of the allergic inflammation and a severity of RSV-infection by inhibiting anti-inflammatory murine il-5 gene and inhibiting the RSV virus replication.

2 cl, 17 dwg, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to peptide derivatives of general formula

,

their stereoisomers, mixtures and/or pharmaceutically acceptable salts, to methods for preparing them, to pharmaceutical compositions containing them, to using them for treating, preventing and/or diagnosing conditions, disorders and/or pathologies involving sstr1, sstr2, sstr3, sstr4 and/or sstr5 somatostatin receptor expression.

EFFECT: preparing the compositions for preventing and/or diagnosing the conditions, disorders and/or pathologies involving sstr1, sstr2, sstr3, sstr4 and/or sstr5 somatostatin receptor expression.

14 cl, 5 tbl, 33 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine and concerns methods of treating growth hormone or insulin-like growth factor 1 deficiency in a patient involving administering an immunogenic amount of a vaccine containing a chimeric somatostatin-14 based polypeptide bound to inactivated chloramphenicol acetyltransferase (CAT), and an adjuvant; the vaccine for treating the patient having growth hormone or insulin-like growth factor 1 deficiency; a method of treating obesity in the patient involving administering the immunogenic amount of the vaccine.

EFFECT: group of inventions provide the immunogenicity for somatostatin and higher release of endogenously produced growth hormone and/or insulin-like growth factor 1.

22 cl, 8 ex, 6 dwg, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: there are presented: a composition for treating cancer containing (a) a conjugate containing (i) a polypeptide vector containing an amino acid sequence min. 70% identical to AngioPep-2 (SEQ ID NO: 97); and (ii) paclitaxel or a paclitaxel analogue conjugated with the above polypeptide; (b) an optional tonicity agent; (c) a buffer agent; (d) an excipient; (e) a polyoxyethylene ester of fatty acid; and (f) 0.01-8% DMSO (versions), a method for preparing (versions), a method of treating cancer, a container comprising the above composition, and a kit comprising the container with the above composition.

EFFECT: invention differs by the fact that the declared conjugates (eg ANG 1005) may pass through the hematoencephalic barrier, be characterised by high absorption by the targeted cells, manifest higher stability or be removed from the cell in a lesser degree, while the based compositions may be dissolved in an aqueous solution; they differ by stability and the minimal decomposition at temperatures within the range of 40°C and 50°C.

53 cl, 3 dwg, 28 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to application of peptide, which has sequence originating from amino acid sequence of protein SNAP-25, for treatment of pain and/or inflammation.

EFFECT: obtaining novel composition.

9 cl, 1 dwg, 1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: group concerns a prolonged delivery of a compstatin analogue, as well as an optional additional active agent when released from a microscopic gel-like inclusion formed if a liquid composition containing the compstatin analogue is introduced into an extravascular space, such as a vitreous chamber of eye in a mammalian body. The invention also refers to a method of treating an individual suffering age-related macular degeneration (ARMD).

EFFECT: improving the compstatin delivery system for the complement system inhibition required for treating ARMD.

58 cl, 4 ex, 6 tbl, 5 dwg

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine and pharmaceutics, and concerns a method of treating pleural mesothelioma by administering a conjugate containing a targeting peptide containing a NGR motif, and cytokine representing TNF specified in a group consisting of TNFα, TNFβ, and also refers to a pharmaceutical formulation containing the above conjugate dissolved in suitable buffers.

EFFECT: group of inventions provides the therapeutic effectiveness, relative safety and low toxicity profile for the patients.

9 cl, 2 ex, 2 tbl

FIELD: biotechnologies.

SUBSTANCE: invention proposes compounds of labyrinthpeptins A1, A2, or A3 of formula (I) , where {A}, {B}, {C}, R1-R6, m and n have the values specified in the formula of the invention. Compounds are obtained at fermentation of Actinomadura namibiensis DSM 6313 strain under acceptable conditions in cultural environment till one or more compounds of formula (I) are formed. The invention proposes the deoxyribonucleic acid (DNA) coding preprolabyrinthpeptin A2, and the deoxyribonucleic acid (DNA) coding preprolabyrinthpeptin A1, as well as preprolabyrinthpeptins A1 and A2, and prolabyrinthpeptins A1 and A2. Labyrinthpeptins of formula (I) are used for therapy of infections caused by gram-positive bacteria, virus infections and/or neuropathic pain caused by inflammation.

EFFECT: improving therapy efficiency.

24 cl, 4 tbl, 20 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to dermatology and dentistry, and can be applied for treatment of lichen ruber planus of oral cavity mucosa. For this purpose at the background of traditional therapy applied is combination of two medications immunomax and gepon. Immunomax is introduced intramuscularly in dose 200 U, one time per day on 1, 2, 3, 8, 9 and 10-th days of treatment. Gepon is applied by irrigation of affected areas of oral cavity mucosa with 0.04% solution, daily during the entire treatment course.

EFFECT: method makes it possible to achieve high efficiency of treatment and contributes both to correction of immune disorders and local manifestations of disease on oral cavity mucosa.

1 ex, 3 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to pharmaceutical industry, particularly to compositions for treating and/or preventing obesity. The composition contains the peptide compound Pro-Val-Asn-Phe-Lys-Phe-Leu-Ser-His in water containing a salt solution in the physiologically acceptable concentration. A therapeutic agent containing the above composition can be presented in the form of spray/drops applicable for nasal, subglossal or oral administration. A dosage form of the nasal or subglossal spray/drops, as well as oral film provides ease of administration and makes it applicable by the patient in need of treating and/or preventing obesity.

EFFECT: compositions and agents are effective for treating obesity.

45 cl, 4 tbl, 2 ex

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