ES2573662T3 - Utilización de mimotopos de epítopos de alfa-sinucleína para el tratamiento de enfermedades de cuerpos de Lewy - Google Patents

Utilización de mimotopos de epítopos de alfa-sinucleína para el tratamiento de enfermedades de cuerpos de Lewy Download PDF

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ES2573662T3
ES2573662T3 ES10747562.6T ES10747562T ES2573662T3 ES 2573662 T3 ES2573662 T3 ES 2573662T3 ES 10747562 T ES10747562 T ES 10747562T ES 2573662 T3 ES2573662 T3 ES 2573662T3
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Markus Mandler
Harald Weninger
Radmila Santic
Christian Lahsnig
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    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

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Abstract

Composición que comprende por lo menos un péptido o polipéptido que comprende la secuencia de aminoácidos siguiente: (X1)nX2X3X4X5GX6P(X7)m (Fórmula I), en la que: X1 es cualquier residuo aminoácido, X2 es un residuo aminoácido seleccionado de entre el grupo que consiste en lisina (K), arginina (R), alanina (A) e histidina (H), X3 es un residuo aminoácido seleccionado de entre el grupo que consiste en asparagina (N), glutamina (Q), serina (S), glicina (G) y alanina (A), preferentemente asparagina (N), serina (S), glicina (G) y alanina (A), X4 es un residuo aminoácido seleccionado de entre el grupo que consiste en ácido glutámico (E), ácido aspártico (D) y alanina (A), X5 es un residuo aminoácido seleccionado de entre el grupo que consiste en ácido glutámico (E) y ácido aspártico (D), X6 es un residuo aminoácido seleccionado de entre el grupo que consiste en alanina (A) y tirosina (Y), X7 es cualquier residuo aminoácido, n y m, independientemente, son 0 o un número entero superior a 0, y en la que la secuencia de aminoácidos según la fórmula I no es idéntica al, o no comprende el fragmento polipeptídico 7-mero de la alfa-sinucleína que presenta la secuencia de aminoácidos KNEEGAP, presentando dicho por lo menos un péptido o polipéptido una capacidad de unión a un anticuerpo que es específico para un epítopo de alfa-sinucleína que comprende la secuencia de aminoácidos KNEEGAP para la utilización en la prevención y/o el tratamiento de las sinucleinopatías.

Description

imagen1
imagen2
imagen3
imagen4
imagen5
imagen6
imagen7
imagen8
sinucleína humana recombinante partiendo del suero 2, respectivamente. En general, se analizaron los sueros mediante la reacción contra péptidos acoplados con albúmina de suero bovino (ASB) y proteínas de longitud completa recombinantes que se inmovilizaron sobre placas de ELIAS. Se determinaron los títulos utilizando anticuerpos específicos anti-IgG de ratón. Para ejemplos de inmunogenicidad contra el péptido inyectado y la alfa
5 sinucleína, ver las Tablas 5 y 6.
2. Resultados
2.1. Identificación de un mAb específico de alfa-sinucleína: la figura 2 ilustra la caracterización del anticuerpo 10 monoclonal específico de la alfa-sinucleína 12-9-9 (IgG1, kappa) derivado de la fusión Affiris 6.
2.2. Cribado para mimotopos específicos de alfa-sinucleína:
2.2.1. Expresión fágica PhD 7, PhD 12 y PhD C7C y cribado mutacional. 15
2.2.1.1. Cribado con anticuerpo monoclonal dirigido contra LGKNEEGAPQ
Mediante el cribado de las bibliotecas de expresión fágica PhD 7, PhD 12 y PhD C7C y el desplazamiento selectivo de aminoácidos individuales, se identificó un total de 60 secuencias (ver la Tabla 1, ID18-77).
20 La Tabla 1 muestra ejemplos de todos los péptidos utilizados.
SEC ID
nº de péptido secuencia
1
p4446 Alfa-sinucleína (figura 1; P37840)
2
p4447 Beta-sinucleína; Q16143
3
p4453 LGKNEEGAPQC
4
p4454 MGKGEEGYPQC
5
p5397 GKNEEGAPQC
6
p5398 KNEEGAPQC
7
p5399 NEEGAPQC
8
p5400 EEGAPQC
9
p5401 EGAPQC
10
p5402 LGKNEEGAPC
11
p5403 LGKNEEGAC
12
p5404 LGKNEEGC
13
p5405 LGKNEEC
14
p5406 LGKNEC
15
p5435 CKNEEGAP
16
p5436 KNEEGAPC
17
p1253 DAEFRHDSGY-C
18
p5437 ANEEGAPC
19
p5438 KAEEGAPC
20
p5439 KNAEGAPC
21
p5440 KNEAGAPC
22
p5441 KNEEAAPC
23
p5442 KNEEGAAC
24
p5443 RNEEGAPC
25
p5444 HNEEGAPC
26
p5445 KNDEGAPC
27
p5446 KNEDGAPC
28
p5447 KQEEGAPC
29
p5448 KSEEGAPC
30
p5449 KNDDGAPC
31
p5461 KPSFKNEC
32
p5462 QPSFAMEC
33
p5463 SPSFKQEC
34
p5464 TPSWKGEC
35
p5465 DPSFALEC
36
p5466 LPSFRLEC
37
p5467 EPNSRMDC
38
p5468 QPSSKLDC
39
p5469 HIHQSKFFDAPPC
40
p5547 QASFAMEC
10
41
p5548 TASWKGEC
42
p5549 QASSKLDC
43
p5550 QPAFAMEC
44
p5551 TPAWKGEC
45
p5552 QPASKLDC
46
p5553 QPSFAMAC
47
p5554 TPSWKGAC
48
p5555 QPSSKLAC
49
p5556 APSWKGEC
50
p5557 TPSAKGEC
51
p5558 TPSWAGEC
52
p5559. TPSWKAEC
53
p5560 CTPSWKGE
54
p5587 RNDEGAPC
55
p5588 RNEDGAPC
56
p5589 RQEEGAPC
57
p5590 RSEEGAPC
58
p5591 ANDEGAPC
59
p5592 ANEDGAPC
60
p5593 HSEEGAPC
61
p5594 ASEEGAPC
62
p5595 HNEDGAPC
63
p5596 HNDEGAPC
64
p5597 RNAEGAPC
65
p5598 HNAEGAPC
66
p5599 KSAEGAPC
67
p5600 KSDEGAPC
68
p5601 KSEDGAPC
69
p5602 RQDEGAPC
70
p5603 RQEDGAPC
71
p5604 HSAEGAPC
72
p5605 RSAEGAPC
73
p5606 RSDEGAPC
74
p5607 RSEDGAPC
75
p5608 HSDEGAPC
76
p5609 HSEDGAPC
77
p5610 RQDDGAPC
La Tabla 2 muestra ejemplos de péptidos y de la capacidad de unión de los mismos en comparación con el epítopo original.
número interno
secuencia unión observación
p5435
CKNEEGAP 2 original
p5436
KNEEGAPC 2 original
p5437
ANEEGAPC 2 mimotopo
p5438
KAEEGAPC 1 mimotopo
p5439
KNAEGAPC 2 mimotopo
p5443
RNEEGAPC 2 mimotopo
p5444
HNEEGAPC 2 mimotopo
p5445
KNDEGAPC 2 mimotopo
p5446
KNEDGAPC 2 mimotopo
p5447
KQEEGAPC 2 mimotopo
p5448
KSEEGAPC 2 mimotopo
p5449
KNDDGAPC 2 mimotopo
p5398
KNEEGAPQC 2 original
p5402
LGKNEEGAPC 2 original
p5397
GKNEEGAPQC 2 original
p4454
MGKGEEGYPQC 2 original
p4453
LGKNEEGAPQC 2 original
p5461
KPSFKNEC 2 mimotopo
p5462
QPSFAMEC 2 mimotopo
p5463
SPSFKQEC 2 mimotopo
p5464
TPSWKGEC 2 mimotopo
11
p5465
DPSFALEC 2 mimotopo
p5466
LPSFRLEC 1 mimotopo
p5468
QPSSKLDC 2 mimotopo
p5547
QASFAMEC 1 mimotopo
p5548
TASWKGEC 2 mimotopo
p5549
QASSKLDC 1 mimotopo
p5551
TPAWKGEC 1 mimotopo
p5556
APSWKGEC 2 mimotopo
p5558
TPSWAGEC 2 mimotopo
p5560
CTPSWKGE 2 mimotopo
p5587
--RNDEGAPC 2 mimotopo
p5588
--RNEDGAPC 2 mimotopo
p5589
--RQEEGAPC 2 mimotopo
p5590
--RSEEGAPC 2 mimotopo
p5591
--ANDEGAPC 2 mimotopo
p5593
--HSEEGAPC 1 mimotopo
p5594
--ASEEGAPC 1 mimotopo
p5596
--HNDEGAPC 2 mimotopo
p5597
--RNAEGAPC 2 mimotopo
p5598
--HNAEGAPC 1 mimotopo
p5599
--KSAEGAPC 2 mimotopo
p5600
--KSDEGAPC 2 mimotopo
p5601
--KSEDGAPC 2 mimotopo
p5602
--RQDEGAPC 2 mimotopo
p5603
--RQEDGAPC 2 mimotopo
p5604
--HSAEGAPC 1 mimotopo
p5605
--RSAEGAPC 2 mimotopo
p5606
--RSDEGAPC 2 mimotopo
p5607
--RSEDGAPC 2 mimotopo
p5608
--HSDEGAPC 1 mimotopo
p5610
--RQDDGAPC 2 mimotopo
p4446
2 α-Syn
Tabla 2: ejemplos de epítopos y mimotopos de alfa-sinucleína ligantes del anticuerpo monoclonal 12-9-9.
La capacidad de unión se codificó según el código siguiente: 5
0: ninguna unión detectable en el ELISA a 12-9-9
1: unión débil: unión del mimotopo más débil que la de la secuencia original mínima p5436
2: unión fuerte: unión del mimotopo similar a la de la secuencia original mínima p5436
10 2.3. Caracterización in vitro de los mimotopos identificados en un cribado (expresión fágica y cribado de péptidos) con un anticuerpo monoclonal dirigido contra la alfa-sinucleína:
Las figuras 2 y 3 muestran ejemplos representativos de los ensayos de unión e inhibición utilizados para caracterizar los mimotopos in vitro. Los datos obtenidos se resumen en las Tablas 2 y 3, respectivamente. 15 Tabla 3: ensayo de inhibición
denominación
secuencia competencia observación
p5435
CKNEEGAP 1 original
p5436
KNEEGAPC 2 original
p5439
KNAEGAPC 1 mimotopo
p5443
RNEEGAPC 2 mimotopo
p5445
KNDEGAPC 2 mimotopo
p5446
KNEDGAPC 1 mimotopo
p5448
KSEEGAPC 1 mimotopo
p5449
KNDDGAPC 1 mimotopo
p5398
KNEEGAPQC 2 original
p5402
LGKNEEGAPC 2 original
p5397
GKNEEGAPQC 2 original
12
imagen9
imagen10

Claims (1)

  1. imagen1
    imagen2
ES10747562.6T 2009-08-21 2010-08-20 Utilización de mimotopos de epítopos de alfa-sinucleína para el tratamiento de enfermedades de cuerpos de Lewy Active ES2573662T3 (es)

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AT0132409A AT508638B1 (de) 2009-08-21 2009-08-21 Verwendung von peptiden und polypeptiden zur behandlung und/oder prävention von synukleinopathien
PCT/AT2010/000303 WO2011020133A1 (en) 2009-08-21 2010-08-20 Use of mimotopes of alpha-synuclein epitopes for treating lewy body diseases

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