Modified fusion molecules for treatment of allergic disease

Modified fusion molecules for treatment of allergic disease

US 20060171942Al
(19) United States
(12) Patent Application Publication (10) Pub. No.: US 2006/0171942 A1
(43) Pub. Date:
Saxon et al.
(54)
MODIFIED FUSION MOLECULES FOR
Aug. 3, 2006
Publication Classi?cation
TREATMENT OF ALLERGIC DISEASE
(51)
(76) Inventors: Andrew Saxon, Santa Monica, CA
(US); Ke Zhang, Los Angeles, CA
(US); Daocheng Zhu, Los Angeles, CA
(Us)
(52)
Int. Cl.
A61K 39/395
00711 21/04
C12P 21/06
C07K 16/42
(2006.01)
(2006.01)
(2006.01)
(2006.01)
US. Cl. ................... ..424/133.1;435/69.1;435/328;
435/3201; 530/3872; 536/2353
(57)
Correspondence Address:
ABSTRACT
The present invention comprises a fusion molecule com
HELLER EHRMAN LLP
275 MIDDLEFIELD ROAD
prising a Fce fragment sequence including functionally
MENLO PARK, CA 94025-3506 (US)
of an lgE heavy chain (CHe2-CHe3 -CHe4 sequence) linked
active CH2, CH3 and CH4 domains of the constant region
at its C-terminus to the N-terminus of a second polypeptide
(21) Appl. No.:
11/050,113
including functionally active hinge, CH2 and CH3 domains
of the constant region of an lgGl heavy chain (Yhinge-CHYZ
Feb. 2, 2005
the fusion molecule and methods of treatment using the
fusion molecule.
CHy3 sequence), pharmaceutical compositions comprising
(22) Filed:
Patent Application Publication Aug. 3, 2006 Sheet 2 0f 19
US 2006/0171942 A1
Ala Ser 'I'hr Lys Gl'y Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
Ser ‘Th: Ser Gly' Gly Thr Ala Ala Len‘ Gly'icys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Le'u .Thr Ser
Gly Val 'His Thr' Phe Pro Ala Val 'Leu Gln Ser Ser Gly- Leu Tyr Ser
Leu Ser Ser' Val Val Thr Val‘ Pro Se: Ser Ser Leu Gly 'I'hr Gln Thr
' Tyr Ile 'Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys .Val _Glu Pro Lys Ser Cys Asp Lys Thr His 'I'hr Cys Pro Pro. Cys
Pro Ala Pro 'Gluleu Leu 6152 61y Pro Ser Val Phe Leu Phe Pro Pro ‘
Lys
Val
Tyr
Glu
H‘is'
Lys
Gin
Leu‘
Pro
Pro Lys
Val Val
Val’Asp
Gln Tyr
Gln Asn
Ala Len
Pro ‘Arg
Thr Lys
Ser Asp.
Asp
Asp
Gly
Asn
Trp
Thr
Val
Val
Ser
Met
Ala
Pro
Glln'
Ala
Leu
Ser
Glu
Thr
Met
His
Val
Tyr
Ile
Glu
His
Arg
Asn Glly Lys
Pro
Pro Ile
Glu
Gln Val
Asn
Val Ser
Ile
Val Glu
. Asn Tyr ‘Lys. 'J‘hr Thr Pro Pro
,Leu vTyr Ser Lys Leu v'I‘hr Val
Val Phe Ser Cys Ser Val Met
Ser'
Asp
Asn
Va'l
Glu
L15
Thr
Thr
Glu
Arg
Pro
Val
Val
Thr
Glu
Lys
Ser
Tyr Lys
Pro Glu
Val Lys
Thr Lys
Val Leu
Cys- Lys
Ser Lys
Pro Ser.
Val‘ Lys
Gly- Gln
Val ,Thr
Ph‘e Asn
Pro 'Arg
Thr‘ Val
Val Ser Asn
Glu
‘J‘hr Ile
Ala
vTyr:
Leu Pro
Arg
Leu
Cys Leu
G-l-y
Trp
Ser Asn
Pro
Val Leu Asp‘ Ser Val Gly Ser
Asp Lys Ser Arg Trp Gln Gln'
His Glu Al‘a. Leu His Asn His
Gln Arg Ser Leo Ser Leu Ser Pro Gly Lys
'
Cys
Trp
Glu '
Leu
’
Lys
Asp
Phe
Glu
‘Val
Glu
Tyr
Asn
Phe Phe
Gly Asn
'I‘yr Gln
Patent Application Publication Aug. 3, 2006 Sheet 3 0f 19
US 2006/0171942 A1
Glu- Pro Lys 'Ser 'Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Gluv Leu Len Gly Gly Pro Se: Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr - Leu Met Ile Ser Arg_‘1'hr Pro Glu Val Thr'Cys val val
Val
Asp
"I‘yr
Asn
Leu
Arg
Lys
Asp
Gly
Asn
Trp
Pro
611:
Asn
Val
Val
Ser
Met
Ala
Pro
Gln
Ser His
6111 Val
Thr Tyr
Asn‘ Gly
-Pro\Ile
Gln Val
Val S‘er
Glu
His
Arg
Lys
Glu
Tyr
Leu
Asp'Pro
Asn Val
Val V'al
Glu 'I‘yr
Lys Thr
Thr Lei:
Thu-r Cys
Glu
Lys
Ser
Lys
Ile
Pro
Leu
Val
Thr
Val
Cys
Ser
Pro
Val
Lys
Lys'
Leu
-Lys~
Lys
Ser
Lys
Phe
Pro
Thr
Val
Ala
Arg
Gly
Asn :I‘rp Tyr
Arg' Glu G111
Val Leu 'His
Ser Asn Lys
Lys Val',Gln
AspGlu Leu
Phe Tyr Pro
Val
G111
Gln Ala
Pro
Thr
Se:
Asp Ile Ala Val Glu 'I‘rp Glu Ser'Asn Gly Gl'n Pro Glu Asn ' Asn 'Iyr
Ly's Thr' 'I‘hr ‘Pro Pro Val Len Asp Ser Val Gly Ser Phe Phe Leu Tyr
S_er Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln G'ly Asn Val Phe
Ser Cys' S‘e-r Val Met His Gin Ala’ Leu His Asn His Tyr Gln Gln Arg
.Ser vLou 'Ser Len Ser Pro Gly Ly;
'
'
Patent Application Publication Aug. 3, 2006 Sheet 5 0f 19
I
Ser
Ile
'I'yr
Gly
Tyr
Met
Asp
Thr
Pro
Phe
Thr
Ala
Phe
Asn
Gln
Ser
Pro
“Thr
Thr
Thr
Lys
~
'Ser
Asn
6111
Met
Ile
Cys
Thr
US 2006/0171942 A1
" “FIGURE 5
Pro Ser Val Phe Pro Leu Thr Arg Cys Cys Lys Asn
Ala Thr Ser Val Thr Leu Gly Cys Leu Ala Thr Gly
Pro
Thr
Ser'
Arg
Phe
Val
Leu
Leu
Val
Ser
Met
Pro
Leu
Ala
Val
Val-Thr
Ala Thr
Thr Val
His Thr
C'ys Ser
Trp
Thr
Ser
Pro
Arg
Asp
Leu
Gly
Ser
.Asp
Thr
Thr
Ala
Ser
Phe
Gly
Len
Trp'
Thr
Thr
Ser
Ser
Ala
Asp
Pro
Leu
Gly
Lys
".l‘rp
Pro
Asn
His
Gln .
Val -‘
Thr
Val Lys ‘Ile Leu Gln Ser Ser'Cys Asp Gly Gly Gly His Phe' ‘Pro Pro
Thr Ile Gln 'Leu Leu Cys Leu Val 'Ser Gly Tyr Thr Pro Gly Thr Ile
Asn ‘Ile Thr Trp Len Glu Asp Gly Gln Val Met Asp Val Asp Leu Ser
Thr Al'a
Leu Thr
Gln Val
Ala AspPro Phe
Val' Asp
Ala Ser
Arg Asn
_' Asp'Trp
Leu. Pro
Ala Ala
Arg ‘Asp
Asp Ile
Arg His
val Phe
Glu Phe
Ser Thr
Leu Ser
Thr '_I‘yr
Ser Asn
Asp Leu
Leu Ala
Gly' Lys
Gly Thr
Ile Glu
Arg Ala
Pro Glu
Lys Arg
Ser'Val
Ser Thr
Ser Arg
Ile Cys
Thr
Gln
Gln
Pro
Phe
Pro
Pro
Leu
Gly
Leu
Val
Thr
Gln
Thr
Leu
Arg
Gln Glu Gly Glu
Lys His 'J‘rp Leu
Gly His Thr Phe
Arg Gl'y Val Ser
lle Arg Lys Ser
Ser Lys Gly Thr
Val Asn His Ser
Thr ‘Val Thr Ser
Glu Thr Tyr Gln
Met Argv Ser Thr
Tyr Ala Phe' Ala
Leu Ala ,Cys Leu
Trp Leu ‘His Asn
v(Sln Pro Arg Lys
611.: Val "I‘hr' Arg'
Ala Val ‘His Glu
Leu Ala Ser 'I'hr Gln 'Ser Glu
Ser Asp Arg Thr Tyr Thr, Cys
Glu
Ala
Pro
Val
Thr
Thr
Cys
Thr
Thr
Ile
Glu
Thr
‘Ala
_-Ala
Asp
Ty:
Thr
Asn
Arg
Leu'
Arg
Ser
Leu
Ile
Leu
Lys
Pro
Val
Thr Lys 'Lys
Ser Arg Pro
Thr Cys Leu
Thr -Trp Ser
G11: G11: Lys
Val Gly. Thr
Thr His Pro
Cys
Se:
val
Arg
Gln
Arg
His
Lys
Pro
Gln
Val
Lys
Thr‘
Glu
Asn
Gln
Gly
Ser
Trp
Phe
Len
Ser
Arg
Ser
Glu
Ala
Phe
Gly
Pro
Met
Pro
Gly
Pro
Gly
Pro
Asp
Phe.
Glu Trp Glu Gln Lys Asp
Ala Ser Pro Ser 6111 Thr
Val Gln Arg Ala Val Ser Val As‘n Pro Gly Lys
'
‘
Patent Application Publication Aug. 3, 2006 Sheet 6 0f 19
US 2006/0171942 A1
FIGURE6.
Phe Thr Pro Pro Thr Val Lys Ile‘ Leu Gln Ser Ser Cys Asp Gly Gly
Glyv His Phe Pro Pro Thr Ile Gln Leu Leu Cys L‘eu Val. Ser Gly Tyr
Thr' Pro Gly_'Thr Ile Asn Ile Thr _Trp Leu Glu Asp Gly_ Gln Val Met
. Asp Val Asp Leu Ser Thr Ala Ser Thr 'Thr Gln 611: Gig Gl'u .Leu Ala
so: Thr Gl'n Ser Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser Asp
Arg
Ser
Leu
Ile
Leu
Lys
Th-r
Thr
Ser
Thr
'Thr
Gl-u
'I'yr
Lys
Arg
'Cys
Thr
Lys
Pro
.L'eu
Cys
Cys
Ser
Val
Trp Ser Arg
Glu Lys Gln'
Gln
Ala
Pro
Val
Val
Asp
Phe
Asp
Ala Ser
Arg Asn
Thr
Ser
Asp
Len
.6151
Gly
Tyr
Asn
Leu
Ala
Gln
Pro
Phe
Pro
Lys Pro
Thr Leu
Gly
Arg
‘Ile
Se:
His
Gly
Arg
Lys
Val Asn
'I'hr Val
Thr
Val
Lys
Gly
Phe w Glu
S'er Ala
Ser. Pro
Thr val
Asp_
Tyr _
Thr
Asn '
315' Ser. Thr Arg
Thr Seerw Thr Leu
Pro Val Gly Thr Arg Asp Trp Ile Glu Gly Glu Thr Tyr Gln Cys Arg
Val Thr His Pro Hi's Leu Pro Arg Ala ~Leu Met Arg Ser Th: Th: Lys
Thr Ser Gly Pro Arg Ala Ala Pro Glu Val 'Iyr Ala Phe Ala Th: Pro
Glu T'rp‘vPro Gly Ser Arg As'p Lys Arg Thr L_eu Ala, Cys Leu Ile 6111
A511 Phe Met Pr‘o Glu Asp Ile Ser val Gln Trp Leu His Asn Glu Val
.Gln Len Pro ‘Asp Alaw Arg His Ser Thr Thr Gln Pro Arg Lys Thr ‘Lys
I Gly Ser Gly Phe Phe \fal Phe Ser Ar-g Leu Glu Val T-hr Arg Ala Glu I
- TrpGlu Gln Lys Asp Glu Phe Ile Cys'Arg Ala Val His Glu Ala Al‘a
'S_er Pro Ser Gln Thr Val Gln Arg Ala Val Ser Val Asn Pro Gly Lys
Patent Application Publication Aug. 3, 2006 Sheet 12 0f 19
US 2006/0171942 A1
FIGURE 9B: GE2 S mutant DNA and Protein Sequence
1 0 a 1 GACAGCACCAAGAAGTGTGCAGATTCCAACCCGAGAGGGGTGAGCGCCTACCTAAGCCGG
361DSTKKCADSNPRGVSAYLSR
1141 CCCAGCCCGTTCGACCTGTTCATCCGCAAGTCGCCCACGATCACCTGTCTGGTGGTGGAC
381
P
S
P
F
D
L
F
I
R
K
S
P
T
I
T
C
L
V
V
D
1201 CTGGCACCCAGCAAGGGGACCGTGAACCTGACCTGGTCCCGGGCCAGTGGGAAGCCTGTG
401
L
A
P
S
K
G
T
V
N
L
T
W
S
R
A
S
G
K
P
V
1261 AACCACTCCACCAGAAAGGAGGAGAAGCAGCGCAATGGCACGTTAACCGTCACGTCCACC
421
N
H
S
T
R
K
E
E
‘K
Q
R
N
G
T
L
T
V
T
S
T
1321 CTGCCGGTGGGCACCCGAGACTGGATCGAGGGGGAGACCTACCAGTGCAGGGTGACCCAC
441
1381
461
L
P
V
G
T
R
D
W
I
E
G
E
T
Y
Q
C
R
V
T
H
CCCCACCTGCCCAGGGCCCTCATGCGGTCCACGACCAAGACCAGCGGCCCGCGTGCTGCC
P
H
L
P
R
A
L
M
R
S
T
T
K
T-
S
G
P
R
A
A
1441 CCGGAAGTCTATGCGTTTGCGACGCCGGAGTGGCCGGGGAGCCGGGACAAGCGCACCC(TC
481 0 P
E
V
Y
A
F
A
T
P
E
W
P
G
S
R
D
K
R
T
L
1501 GCCTGCCTGATCCAGAACTTCATGCCTGAGGACATCTCGGTGCAGTGGCTGCACAACGAG
501
A
C
L
I
Q
N
F
M
P
E
D
I
S
V
Q
W
L
H
N
E
1561 GTGCAGCTCCCGGACGCCCGGCACAGCACGACGCAG-CCCCGCAAGACCAAGGGCTCCGGC
521
V
Q
L
P
D
A
R
H
S
T
T
Q
P
R
K
T
K
G
S
G
1621 TTCTTCGTCTTCAGCCGTCTAGAGGTGACCAGGGCCGAATGGGAGCAGAAAGATGAGTTC
541,
F
F
V
F
S
R
L
E
V
T
R
A
E
W
E
Q
K
D
E
F
1681 ATCTGCCGTG'CAGTCCATGAGGCAGCTAGCCCCTCACAGACCGTCCAGCGAGCGGTGTCT
561
I
C
R
A
V
H
1741 GTAAATCCCGGTAAATGA
581
V
N
P
G
K
*
E
A
A
S
P
S
Q
T
V
Q
R
A
V
S
1 14 o
380
1200
400
1260
420
1320
440
1380
460
I 1440
480
1500
500
1560
520
1620
540
1680
560
1740
580
1758
586
Patent Application Publication Aug. 3, 2006 Sheet 14 0f 19
US 2006/0171942 A1
FIGURE 10B: GEZ H mutant DNA and Protein Sequence
1081 GACAGCACCAAGAAGTGTGCAGATTCCAACCCGAGAGGGGTGAGCGCCTACCTAAGCCGG
361
D
S
T
K
K
C
A
D
S
N
P
R
G
V
S
A
Y
L
S
R
1141 CCCAGCCCGTTCGACCTGTTCATCCGCAAGTCGCCCACGATCACCTGTCTGGTGGTGGAC
381
P
S
P
F
D
L
F
I
R
K
S
P
T
I
T
C
L
V
V
D
1201 CTGGCACCCAGCAAGGGGACCGTGAACCTGACCTGGTCCCGGGCCAGTGGGAAGCCTGTG
401
L
A
P
S
K
G
T
V
N
L
T
W
S
R
A
S
G
K
P
V
1261 AACCACTCCACCAGAAAGGAGGAGAAGCAGCGCAATGGCACGTTAACCGTCACGTCCACC
421
N
H
S
T
R
K
E
E
K
Q
R ' N
G
T
L
T
V
T
S
T
1321 CTGCCGGTGGGCACCCGAGACTGGATCGAGGGGGAGACCTACCAGTGCAGGGTGACCCAC
441
L
P
V
G
T
R
D
W
I
E
G
E
T
Y
Q
C
R
V
T
H
1381 CCCCACCTGCCCAGGGCCCTCATGCGGTCCACGACCAAGACCAGCGGCCCGCGTGCTGCC
461
P
H
L
P
R
A
L
M
R
S
T
T
K
T
S
G
P
R
A
A
1441 CCGGAAGTCTATGCGTTTGCGACGCCGGAGTGGCCGGGGAGCCGGGACAAGCGCACCCTC
481
P
E
V
Y
A
F
A
T
P
E
W
P
G
S
R
D
K
R
T
L
1501 GCCTGCCTGATCCAGAACTTCATGCCTGAGGACATCTCGGTGCAGTGGCTGCACAACGAG
501
A
C
L
I
Q
N
F
M
P
E
D
I
S
V
Q
W
L
H
N
E
1561 GTGCAGCTCCCGGACGCCCGGCACAGCACGACGCAGCCCCGCAAGACCAAGGGCTCCGGC
521
V
Q
L
P
D
A
R
H
S
T
T
Q
P
R
K
T
K
G
S
G
1621 TTCTTCGTCT'I‘CAGCCGTCTAGAGGTGACCAGGGCCGAATGGGAGCAGAAAGATGAGTTC
541
F
F
V
F
S
R
L
E
V
T
R
A
E
W
E
Q
K
D
E
F
1681 ATCTGCCGTGCAGTCCATGAGGCAGCTAGCCCCTCACAGACCGTCCAGCGAGCGGTGTCT
561
I
C
R
A
V
H
1741 GTAAATCCCGGTAAATGA
581
V N
P
G
K
*
E
A
A
S
P
S
Q
T
V
Q
R
A
V
S
1140
380
1200
400
1260
420
1320
I 440
1380
460
1440
480
1500
500
1560
520
1620
540
1680
560
1740
580
1758
586
Patent Application Publication Aug. 3, 2006 Sheet 15 0f 19
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