Humanized Antibodies That Recognize Alpha-Synuclein

59 Pages·2015·3.69 MB·English

US 20150056187Al
(19) United States
(12) Patent Application Publication (10) Pub. No.: US 2015/0056187 A1
Saldanha et al. (43) Pub. Date: Feb. 26, 2015
(54) HUMANIZED ANTIBODIES THAT (60) Provisional application No. 61/591,835, ?led on Jan.
RECOGNIZE ALPHA-SYNUCLEIN 27, 2012, provisional application No. 61/711,207,
?led on Oct. 8, 2012.
(71) Applicant: NEOTOPE BIOSCIENCES
LIMITED, Dublin (IE) Publication Classi?cation
(72) Inventors: Jose Saldanha, En?eld (GB); Tarlochan (51) Int. Cl.
S. Nijjar, Orinda, CA (US) C07K 16/18 (2006.01)
(52) US. Cl.
(73) Assignee; NEOTOPE BIOSCIENCES CPC ........... .. C07K 16/18 (2013.01); C07K 2317/56
LIMITED, Dublin (IE) (2013.01); C07K2317/76 (2013.01); C07K
2317/565 (2013.01); C07K2317/567 (2013.01);
(21) APPL NO; 14/340 355 C07K 231 7/71 (2013.01); C07K 2317/24
’ (2013.01)
(22) Filed: Jul. 24’ 2014 USPC ................................... .. 424/1331; 424/1721
(57) ABSTRACT
Related U's' Apphcatlon Data The present application discloses humanized 1H7 antibodies.
(63) Continuation of application No. 13/750,983, ?led on The antibodies bind to human alpha synuclein and can be
Jan. 25, 2013, now Pat. No. 8,790,644. used for treatment and diagnosis of Lewy body disease.
Patent Application Publication Feb. 26, 2015 Sheet 1 0f 8 US 2015/0056187 A1
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Numbering Numbering
mlH7VH
Kabat Kabat Kabat
Patent Application Publication Feb. 26, 2015 Sheet 2 0f 8 US 2015/0056187 A1
0300100000
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Numbering Numbering Numbering
BAC02037
mlH7VH m1H7VH m1H7VH
Kabat Kabat Kabat
Patent Application Publication Feb. 26, 2015 Sheet 3 0f 8 US 2015/0056187 A1
Patent Application Publication Feb. 26, 2015 Sheet 4 0f 8 US 2015/0056187 A1
Patent Application Publication Feb. 26, 2015 Sheet 5 0f 8 US 2015/0056187 A1
murine 1H7 antiboby
game: Fc=4-3 Ligand: 1H7 mouse Sample: Synuclein Temp: 25°C
40 -
3350 " l/ ' \
Response
2250- -- ' >
15
\ _~ \\
10- ' // “\m\_ "T" ‘ M
5" / <
0...
'550 b 50 160 150 260 250 $0 5
Time
FIG. 3A
chimeric 1H7 antiboby
game: Fc=2-1 Ligand: 1H7 chi Sample: Synuclein Temp: 25°C
25-~
20:
Response
15-
W
5...
0..
35100 $0 0 5'0 160 150 260 250 360 350 460 s
Time
FIG. 3B
humanized 1H7 an’cibob?al
game: Fc=2-1 Ligand: 1 7 h313 Sample: Synuclein Temp: 25°C
40-
30 -
10-
0..
10
20
—30-
40 -
50—
-100 ~50 0 5'0 160 1:50 260 s
Time
FIG. 3C
Patent Application Publication Feb. 26, 2015 Sheet 6 0f 8 US 2015/0056187 A1
kon(1lMs) kdis(1ls)
Patent Application Publication Feb. 26, 2015 Sheet 7 0f 8 US 2015/0056187 A1
Correct Quadrant
Time
FIG. 5A
Patent Application Publication Feb. 26, 2015 Sheet 8 0f 8 US 2015/0056187 A1
Beam D - Speed
10cmlseca
FIG. 5A FIG. 65
US 2015/0056187 A1 Feb. 26, 2015
HUMANIZED ANTIBODIES THAT Kabat CDRs of SEQ ID NO:44, and being at least 90%
RECOGNIZE ALPHA-SYNUCLEIN identical to SEQ ID NO:44, and a light chain comprising the
three Kabat CDRs of SEQ ID NO:45, and being at least 90%
CROSS-REFERENCE TO RELATED identical to SEQ ID NO:45. In some antibodies, the mature
APPLICATION heavy chain variable region is at least 95%, 96%, 97%, 98%,
[0001] This is a continuation application of US. applica or 99% identical to SEQ ID NO:44 and mature light chain
tion Ser. No. 13/750,983; ?led Jan. 25, 2013, Which claims variable region is at least 95%, 96%, 97%, 98%, or 99%
priority to US. Provisional Patent Application No. 61/591, identical to SEQ ID NO:45. In some antibodies, position L46
835, ?led Jan. 27, 2012 and US. Provisional Patent Applica (Kabat numbering) can be occupied by F; position L49 (Ka
tion No. 61/711,207, ?led Oct. 8, 2012, Which are incorpo bat numbering) can be occupied by C; position L83 (Kabat
rated by reference in their entirety for all purposes. numbering) can be occupied by A; position H1 1 (Kabat num
bering) can be occupied by L; position H28 (Kabat number
BACKGROUND ing) can be occupied by S; position H38 (Kabat numbering)
can be occupied by K; position H48 (Kabat numbering) can
[0002] Synucleinopathies, including Lewy body diseases
be occupied by I; position H67 (Kabat numbering) can be
(LBDs) are characterized by degeneration of the dopaminer
occupied byA; position H69 (Kabat numbering) can be occu
gic system, motor alterations, cognitive impairment, and for
pied by L; position H71 (Kabat numbering) can be occupied
mation of Lewy bodies (LBs) and/or Lewy neurites. (McK
by A; and/or position H91 (Kabat numbering) can be occu
eith et al., Neurology (1996) 47: 1 1 13-24). Synucleinopathies
pied by F. In some of such antibodies, position H97 (Kabat
include Parkinson’s disease (including idiopathic Parkin
numbering) can be occupied by S. In some of such antibodies
son’s disease), Diffuse Lewy Body Disease (DLBD) also
the amino acid sequence of the mature heavy chain variable
known as Dementia With Lewy Bodies (DLB), Lewy body
region is SEQ ID NO:44 and the amino acid sequence of the
variant of Alzheimer’s disease (LBV), Combined Athe
mature light chain variable region is SEQ ID NO:45 except
imer’s and Parkinson disease, pure autonomic failure and
multiple system atrophy (MSA; e.g., Olivopontocerebellar provided that position L46 (Kabat numbering) can be occu
Atrophy, Striatonigral Degeneration and Shy-Drager Syn pied by L or F and/or position L49 (Kabat numbering) can be
occupied by Y or C and/or position L83 (Kabat numbering)
drome). Several nonmotor signs and symptoms are thought to
can be occupied by F or A, and/or position H11 (Kabat num
be harbingers for synucleinopathies in the prodromal phase of
bering) can be occupied by V or L, and/or position H28
the diseases (i.e., the presymptomatic, subclinical, preclini
(Kabat numbering) can be occupied by T or S, and/or position
cal, or premotor period). Such early signs include, for
H38 (Kabat numbering) can be occupied by R or K, and/or
example, REM sleep behavior disorder (RBD), loss of smell
position H48 (Kabat numbering) can be occupied by M or I,
and constipation (Mahowald et al., Neurology (2010) 75:488
and/ or position H67 (Kabat numbering) can be occupied by V
489). Lewy body diseases continue to be a common cause for
orA, and/ or position H69 (Kabat numbering) can be occupied
movement disorders and cognitive deterioration in the aging
by M or L, and/or position H71 (Kabat numbering) can be
population (Galasko et al., Arch. Neurol. (1994) 51 :888-95).
occupied by T or A, and/ or position H91 (Kabat numbering)
[0003] Alpha-synuclein is part of a large family of proteins
can be occupied byY or F, and/or H97 (Kabat numbering) can
including beta- and gamma-synuclein and synoretin. Alpha
be occupied by Cor S. In some of such antibodies, position
synuclein is expressed in the normal state associated With
H71 (Kabat numbering) is occupied by A. In some of such
synapses and is believed to play a role in neural plasticity,
antibodies, position H67 (Kabat numbering) is occupied by
learning and memory. Several studies have implicated alpha
A, position H71 (Kabat numbering) is occupied byA. In some
synuclein With a central role in PD pathogenesis. The protein
of such antibodies, position L46 (Kabat numbering) is occu
can aggregate to form insoluble ?brils in pathological condi
pied by F. In some of such antibodies, position L46 (Kabat
tions. For example, synuclein accumulates in LBs (Spillantini
numbering) is occupied by F, position L49 (Kabat number
et al., Nature (1997) 388:839-40; Takeda et al., J. Pathol.
ing) is occupied by C. In some of such antibodies, position
(1998) 152:367-72; Wakabayashi et al., Neurosci. Lett.
L46 (Kabat numbering) is occupied by F, position L49 (Kabat
(1997) 239:45-8). Mutations in the alpha-synuclein gene co
numbering) is occupied by Y. In some of such antibodies,
segregate With rare familial forms of parkinsonism (Kruger et
position H67 (Kabat numbering) is occupied by A, position
al., Nature Gen. (1998) 18: 106-8; Polymeropoulos, et al.,
H71 (Kabat numbering) is occupied by A, L46 (Kabat num
Science (1997) 276:2045-7). Over expression of alpha
bering) is occupied by F, and position L49 (Kabat numbering)
synuclein in transgenic mice (Masliah et al., Science (2000)
is occupied by C. In some of such antibodies, position H11
287: 1265-9) and Drosophila (Feany et al., Nature (2000)
(Kabat numbering) is occupied by L and position H38 (Kabat
404:394-8) mimics several pathological aspects of Lewy
numbering) is occupied by K. In some of such antibodies,
body disease. In addition, it has been suggested that soluble
position H11 (Kabat numbering) is occupied by V and posi
oligomers of synuclein may be neurotoxic (Conway et al.,
tion H38 (Kabat numbering) is occupied by R. In some of
Proc. Natl. Acad. Sci. USA (2000) 97:571-576; Volles et al.,
such antibodies, position H28 (Kabat numbering) is occupied
J. Biochemistry (2003) 42:7871-7878). The accumulation of
a p a-synuc e1nW1t srmr armorp o og1ca an neuro og1ca position H69 (Kabat numbering) is occupied by L, position
alterations in species and animal models as diverse as
H91 (Kabat numbering) is occupied by F. In some of such
humans, mice, and ?ies suggests that this molecule contrib
antibodies, position H28 (Kabat numbering) is occupied by T,
utes to the development of Lewy body disease.
position H48 (Kabat numbering) is occupied by M, position
H69 (Kabat numbering) is occupied by M, position H91
SUMMARY OF THE CLAIMED INVENTION
(Kabat numbering) is occupied by Y In some of such anti
[0004] The invention provides antibodies comprising a bodies, position L83 (Kabat numbering) is occupied by A. In
mature heavy chain variable region comprising the three some of such antibodies, position L83 (Kabat numbering) is