U.S. patent application number 15/566550 was filed with the patent office on 2018-03-29 for site-specific antibody-drug conjugates.
The applicant listed for this patent is ADC THERAPEUTICS S.A., Philip Wilson HOWARD, PATRICIUS HENDRIKUS CORNE VAN BERKEL. Invention is credited to PHILIP WILSON HOWARD, PATRICIUS HENDRIKUS CORNELIS VAN BERKEL.
Application Number | 20180086828 15/566550 |
Document ID | / |
Family ID | 53333832 |
Filed Date | 2018-03-29 |
United States Patent
Application |
20180086828 |
Kind Code |
A1 |
VAN BERKEL; PATRICIUS HENDRIKUS
CORNELIS ; et al. |
March 29, 2018 |
SITE-SPECIFIC ANTIBODY-DRUG CONJUGATES
Abstract
Site-specific antibody-drug conjugates are described, in
particular conjugates comprising pyrrolobenzodiazepines (PBDs)
having a labile protecting group in the form of a linker. The site
of conjugation, along with modification of the antiobody moiety,
allows for improved safety and efficacy of the ADC.
Inventors: |
VAN BERKEL; PATRICIUS HENDRIKUS
CORNELIS; (Lausanne, CH) ; HOWARD; PHILIP WILSON;
(Cambridge, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VAN BERKEL; PATRICIUS HENDRIKUS CORNE
HOWARD; Philip Wilson
ADC THERAPEUTICS S.A. |
Lausanne
Cambridge
EPALINGES |
|
CH
GB
CH |
|
|
Family ID: |
53333832 |
Appl. No.: |
15/566550 |
Filed: |
April 15, 2016 |
PCT Filed: |
April 15, 2016 |
PCT NO: |
PCT/EP2016/058379 |
371 Date: |
October 13, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/02 20180101;
C07D 487/04 20130101; C07K 2317/24 20130101; A61K 47/6867 20170801;
C07K 2317/53 20130101; Y02P 20/55 20151101; A61K 47/6803 20170801;
C07K 16/2803 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07D 487/04 20060101 C07D487/04; A61P 35/02 20060101
A61P035/02 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 15, 2015 |
GB |
1506402.5 |
Claims
1. A conjugate of formula L-(DL)p, where DL is of formula I or II:
##STR00153## wherein: L is an antibody (Ab) which binds CD22, and
which comprises an amino acid substitution of an interchain
cysteine residue by an amino acid that is not cysteine; when there
is a double bond present between C2' and C3', R.sup.12 is selected
from the group consisting of: (ia) C.sub.5-10 aryl group,
optionally substituted by one or more substituents selected from
the group comprising: halo, nitro, cyano, ether, carboxy, ester,
C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3
alkylene; (ib) C.sub.1-5 saturated aliphatic alkyl; (ic) C.sub.3-6
saturated cycloalkyl; (id) ##STR00154## wherein each of R.sup.21,
R.sup.22 and R.sup.23 are independently selected from H, C.sub.1-3
saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and
cyclopropyl, where the total number of carbon atoms in the R.sup.12
group is no more than 5; (ie) ##STR00155## wherein one of R.sup.25a
and R.sup.25b is H and the other is selected from: phenyl, which
phenyl is optionally substituted by a group selected from halo,
methyl, methoxy; pyridyl; and thiophenyl; and (if) ##STR00156##
where R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl;
C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which
phenyl is optionally substituted by a group selected from halo,
methyl, methoxy; pyridyl; and thiophenyl; when there is a single
bond present between C2' and C3', R.sup.12 is ##STR00157## where
R.sup.26a and R.sup.26b are independently selected from H, F,
C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and
alkenyl groups are optionally substituted by a group selected from
C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of
R.sup.26a and R.sup.26b is H, the other is selected from nitrile
and a C.sub.1-4 alkyl ester; R.sup.6 and R.sup.9 are independently
selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR', nitro,
Me.sub.3Sn and halo; where R and R' are independently selected from
optionally substituted C.sub.1-12 alkyl, C.sub.3-20 heterocyclyl
and C.sub.5-20 aryl groups; R' is selected from H, R, OH, OR, SH,
SR, NH.sub.2, NHR, NHRR', nitro, Me.sub.3Sn and halo; R'' is a
C.sub.3-12 alkylene group, which chain may be interrupted by one or
more heteroatoms, e.g. O, S, NR.sup.N2 (where R.sup.N2 is H or
C.sub.1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;
Y and Y' are selected from O, S, or NH; R.sup.6', R.sup.7',
R.sup.9' are selected from the same groups as R.sup.6, R.sup.7 and
R.sup.9 respectively; [Formula I] R.sup.L1' is a linker for
connection to the antibody (Ab); R.sup.11a is selected from OH,
OR.sup.A, where R.sup.A is C.sub.1-4 alkyl, and SO.sub.zM, where z
is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;
R.sup.20 and R.sup.21 either together form a double bond between
the nitrogen and carbon atoms to which they are bound or; R.sup.20
is selected from H and R.sup.C, where R.sup.C is a capping group;
R.sup.21 is selected from OH, OR.sup.A and SO.sub.zM; when there is
a double bond present between C2 and C3, R.sup.2 is selected from
the group consisting of: (ia) C.sub.5-10 aryl group, optionally
substituted by one or more substituents selected from the group
comprising: halo, nitro, cyano, ether, carboxy, ester, C.sub.1-7
alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene; (ib)
C.sub.1-5 saturated aliphatic alkyl; (ic) C.sub.3-6 saturated
cycloalkyl; (id) ##STR00158## wherein each of R.sup.11, R.sup.12
and R.sup.13 are independently selected from H, C.sub.1-3 saturated
alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where
the total number of carbon atoms in the R.sup.2 group is no more
than 5; (ie) ##STR00159## wherein one of R.sup.15a and R.sup.15b is
H and the other is selected from: phenyl, which phenyl is
optionally substituted by a group selected from halo, methyl,
methoxy; pyridyl; and thiophenyl; and (if) ##STR00160## where
R.sup.14 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3
alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is
optionally substituted by a group selected from halo, methyl,
methoxy; pyridyl; and thiophenyl; when there is a single bond
present between C2 and C3, R.sup.2 is ##STR00161## where R.sup.16a
and R.sup.16b are independently selected from H, F, C.sub.1-4
saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups
are optionally substituted by a group selected from C.sub.1-4 alkyl
amido and C.sub.1-4 alkyl ester; or, when one of R.sup.16a and
R.sup.16b is H, the other is selected from nitrile and a C.sub.1-4
alkyl ester; [Formula II] R.sup.22 is of formula IIIa, formula IIIb
or formula IIIc: (a) ##STR00162## where A is a C.sub.5-7 aryl
group, and either (i) Q.sup.1 is a single bond, and Q.sup.2 is
selected from a single bond and --Z--(CH.sub.2).sub.n--, where Z is
selected from a single bond, O, S and NH and n is from 1 to 3; or
(ii) Q.sup.1 is --CH.dbd.CH--, and Q.sup.2 is a single bond; (b)
##STR00163## where; R.sup.C1, R.sup.C2 and R.sup.C3 are
independently selected from H and unsubstituted C.sub.1-2 alkyl;
(c) ##STR00164## where Q is selected from O--R.sup.L2',
S--R.sup.L2' and NR.sup.N--R.sup.L2', and R.sup.N is selected from
H, methyl and ethyl X is selected from the group comprising:
O--R.sup.L2', S--R.sup.L2', CO.sub.2--R.sup.L2', CO--R.sup.L2',
NH--C(.dbd.O)--R.sup.L2', NHNH--R.sup.L2', CONHNH--R.sup.L2',
##STR00165## NR.sup.NR.sup.L2', wherein R.sup.N is selected from
the group comprising H and C.sub.1-4 alkyl; R.sup.L2' is a linker
for connection to the antibody (Ab); R.sup.10 and R.sup.11 either
together form a double bond between the nitrogen and carbon atoms
to which they are bound or; R.sup.10 is H and R.sup.11 is selected
from OH, OR.sup.A and SO.sub.zM; R.sup.30 and R.sup.31 either
together form a double bond between the nitrogen and carbon atoms
to which they are bound or; R.sup.30 is H and R.sup.31 is selected
from OH, OR.sup.A and SO.sub.zM; [Formula I and II] wherein the
conjugation of the drug moiety to the antibody is at an interchain
cysteine residue.
2. The conjugate according to claim 1, wherein the conjugate is
not: ##STR00166## ##STR00167##
3. The conjugate according to either claim 1 or claim 2, wherein
R.sup.7 is selected from H, OH and OR.
4. The conjugate according to claim 3, wherein R.sup.7 is a
C.sub.1-4 alkyloxy group.
5. The conjugate according to any one of claims 1 to 4, wherein Y
is O.
6. The conjugate according to any one of the preceding claims,
wherein R'' is C.sub.3-7 alkylene.
7. The conjugate according to any one of claims 1 to 6, wherein
R.sup.9 is H.
8. The conjugate according to any one of claims 1 to 7, wherein
R.sup.6 is selected from H and halo.
9. The conjugate according to any one of claims 1 to 8, wherein
there is a double bond between C2' and C3', and R.sup.12 is a
C.sub.5-7 aryl group.
10. The conjugate according to claim 9, wherein R.sup.12 is
phenyl.
11. The conjugate according to any one of claims 1 to 8, wherein
there is a double bond between C2' and C3', and R.sup.12 is a
C.sub.8-10 aryl group.
12. The conjugate according to any one of claims 9 to 11, wherein
R.sup.12 bears one to three substituent groups.
13. The conjugate according to any one of claims 9 to 12, wherein
the substituents are selected from methoxy, ethoxy, fluoro, chloro,
cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and
methyl-thiophenyl.
14. The conjugate according to any one of claims 1 to 8, wherein
there is a double bond between C2' and C3', and R.sup.12 is a
C.sub.1-5 saturated aliphatic alkyl group.
15. A compound according to claim 14, wherein R.sup.12 is methyl,
ethyl or propyl.
16. The conjugate according to any one of claims 1 to 8, wherein
there is a double bond between C2' and C3', and R.sup.12 is a
C.sub.3-6 saturated cycloalkyl group.
17. The conjugate according to claim 16, wherein R.sup.12 is
cyclopropyl.
18. The conjugate according to any one of claims 1 to 8, wherein
there is a double bond between C2' and C3', and R.sup.12 is a group
of formula: ##STR00168##
19. The conjugate according to claim 18, wherein the total number
of carbon atoms in the R.sup.12 group is no more than 4.
20. The conjugate according to claim 19, wherein the total number
of carbon atoms in the R.sup.12 group is no more than 3.
21. The conjugate according to any one of claims 18 to 20, wherein
one of R.sup.21, R.sup.22 and R.sup.23 is H, with the other two
groups being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3
alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
22. The conjugate according to any one of claims 18 to 20, wherein
two of R.sup.21, R.sup.22 and R.sup.23 are H, with the other group
being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3
alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
23. The conjugate according to any one of claims 1 to 8, wherein
there is a double bond between C2' and C3', and R.sup.12 is a group
of formula: ##STR00169##
24. The conjugate according to claim 23, wherein R.sup.12 is the
group: ##STR00170##
25. The conjugate according to any one of claims 1 to 8, wherein
there is a double bond between C2' and C3', and R.sup.12 is a group
of formula: ##STR00171##
26. The conjugate according to claim 25, wherein R.sup.24 is
selected from H, methyl, ethyl, ethenyl and ethynyl.
27. The conjugate according to claim 26, wherein R.sup.24 is
selected from H and methyl.
28. The conjugate according to any one of claims 1 to 8, wherein
there is a single bond between C2' and C3', R.sup.12 is
##STR00172## and R.sup.26a and R.sup.26b are both H.
29. The conjugate according to any one of claims 1 to 8, wherein
there is a single bond between C2' and C3', R.sup.12 is
##STR00173## and R.sup.26a and R.sup.26b are both methyl.
30. The conjugate according to any one of claims 1 to 8, wherein
there is a single bond between C2' and C3', R.sup.12 is
##STR00174## one of R.sup.26a and R.sup.26b is H, and the other is
selected from C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which
alkyl and alkenyl groups are optionally substituted. [Formula
I]
31. The conjugate according to any one of claims 1 to 30, wherein
there is a double bond between C2 and C3, and R.sup.2 is a
C.sub.5-7 aryl group.
32. The conjugate according to claim 31, wherein R.sup.2 is
phenyl.
33. The conjugate according to any one of claims 1 to 30, wherein
there is a double bond between C2 and C3, and R.sup.1 is a
C.sub.8-10 aryl group.
34. A compound according to any one of claims 31 to 33, wherein
R.sup.2 bears one to three substituent groups.
35. The conjugate according to any one of claims 31 to 34, wherein
the substituents are selected from methoxy, ethoxy, fluoro, chloro,
cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and
methyl-thiophenyl.
36. The conjugate according to any one of claims 1 to 30, wherein
there is a double bond between C2 and C3, and R.sup.2 is a
C.sub.1-5 saturated aliphatic alkyl group.
37. The conjugate according to claim 36, wherein R.sup.2 is methyl,
ethyl or propyl.
38. The conjugate according to any one of claims 1 to 30, wherein
there is a double bond between C2 and C3, and R.sup.2 is a
C.sub.3-6 saturated cycloalkyl group.
39. The conjugate according to claim 38, wherein R.sup.2 is
cyclopropyl.
40. The conjugate according to any one of claims 1 to 30, wherein
there is a double bond between C2 and C3, and R.sup.2 is a group of
formula: ##STR00175##
41. The conjugate according to claim 40, wherein the total number
of carbon atoms in the R.sup.2 group is no more than 4.
42. The conjugate according to claim 41, wherein the total number
of carbon atoms in the R.sup.2 group is no more than 3.
43. The conjugate according to any one of claims 40 to 42, wherein
one of R.sup.11, R.sup.12 and R.sup.13 is H, with the other two
groups being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3
alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
44. The conjugate according to any one of claims 40 to 42, wherein
two of R.sup.11, R.sup.12 and R.sup.13 are H, with the other group
being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3
alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
45. The conjugate according to any one of claims 1 to 30, wherein
there is a double bond between C2 and C3, and R.sup.2 is a group of
formula: ##STR00176##
46. The conjugate according to claim 45, wherein R.sup.2 is the
group: ##STR00177##
47. The conjugate according to any one of claims 1 to 30, wherein
there is a double bond between C2 and C3, and R.sup.2 is a group of
formula: ##STR00178##
48. The conjugate according to claim 47, wherein R.sup.14 is
selected from H, methyl, ethyl, ethenyl and ethynyl.
49. The conjugate according to claim 47, wherein R.sup.14 is
selected from H and methyl.
50. The conjugate according to any one of claims 1 to 30, wherein
there is a single bond between C2 and C3, R.sup.2 is ##STR00179##
and R.sup.16a and R.sup.16b are both H.
51. The conjugate according to any one of claims 1 to 30, wherein
there is a single bond between C2 and C3, R.sup.2 is ##STR00180##
and R.sup.16a and R.sup.16b are both methyl.
52. The conjugate according to any one of claims 1 to 30, wherein
there is a single bond between C2 and C3, R.sup.2 is ##STR00181##
one of R.sup.16a and R.sup.16b is H, and the other is selected from
C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and
alkenyl groups are optionally substituted.
53. The conjugate according to any one of claims 1 to 52, wherein
R.sup.11a is OH.
54. The conjugate according to any one of claims 1 to 53, wherein
R.sup.21 is OH.
55. The conjugate according to any one of claims 1 to 53, wherein
R.sup.21 is OMe.
56. The conjugate according to any one of claims 1 to 55, wherein
R.sup.20 is H.
57. The conjugate according to any one of claims 1 to 55, wherein
R.sup.20 is R.sup.C.
58. The conjugate according to claim 57, wherein R.sup.C is
selected from the group consisting of: Alloc, Fmoc, Boc, and
Troc.
59. The conjugate according to claim 57, wherein R.sup.C is
selected from the group consisting of: Teoc, Psec, Cbz and PNZ.
60. The conjugate according to claim 57, wherein R.sup.C is a
group: ##STR00182## where the asterisk indicates the point of
attachment to the N10 position, G.sup.2 is a terminating group,
L.sup.3 is a covalent bond or a cleavable linker L.sup.1, L.sup.2
is a covalent bond or together with OC(.dbd.O) forms a
self-immolative linker.
61. The conjugate according to claim 60, wherein G.sup.2 is Ac or
Moc or is selected from the group consisting of: Alloc, Fmoc, Boc,
Troc, Teoc, Psec, Cbz and PNZ.
62. The conjugate according to any one of claims 1 to 53, wherein
R.sup.20 and R.sup.21 together form a double bond between the
nitrogen and carbon atoms to which they are bound. [Formula II]
63. The conjugate according to any one of claims 1 to 30, wherein
R.sup.22 is of formula IIIa, and A is phenyl.
64. The conjugate according to any one of claims 1 to 30 and claim
63, wherein R.sup.22 is of formula IIa, and Q.sup.1 is a single
bond.
65. The conjugate according to claim 63, wherein Q.sup.2 is a
single bond.
66. The conjugate according to claim 63, wherein Q.sup.2 is
--Z--(CH.sub.2).sub.n--, Z is 0 or S and n is 1 or 2.
67. The conjugate according any one of claims 1 to 30 and claim 63,
wherein R.sup.22 is of formula IIIa, and Q.sup.1 is
--CH.dbd.CH--.
68. The conjugate according to any one of claims 1 to 30, wherein
R.sup.22 is of formula IIIb, and R.sup.C1, R.sup.C2 and R.sup.C3
are independently selected from H and methyl.
69. The conjugate according to claim 68, wherein R.sup.C1, R.sup.C2
and R.sup.C3 are all H.
70. The conjugate according to claim 68, wherein R.sup.C1, R.sup.C2
and R.sup.C3 are all methyl.
71. The conjugate according to any one of claims 1 to 30 and claims
63 to 70, wherein R.sup.22 is of formula IIIa or formula IIIb and X
is selected from O--R.sup.L2', S--R.sup.L2', CO.sub.2--R.sup.L2',
--N--C(.dbd.O)--R.sup.L2' and NH--R.sup.L2'.
72. The conjugate according to claim 71, wherein X is
NH--R.sup.L2'.
73. The conjugate according to any one of claims 1 to 30, wherein
R.sup.22 is of formula IIIc, and Q is NR.sup.N--R.sup.L2'.
74. The conjugate according to claim 73, wherein RN is H or
methyl.
75. The conjugate according to any one of claims 1 to 30, wherein
R.sup.22 is of formula IIIc, and Q is O--R.sup.L2' or
S--R.sup.L2'.
76. The conjugate according to any one of claims 1 to 30 and claims
63 to 75, wherein R.sup.11 is OH.
77. The conjugate according to any one of claims 1 to 30 and claims
63 to 75, wherein R.sup.11 is OMe.
78. The conjugate according to any one of claims 1 to 30 and claims
63 to 77, wherein R.sup.10 is H.
79. The conjugate according to any one of claims 1 to 30 and claims
63 to 75, wherein R.sup.10 and R.sup.11 together form a double bond
between the nitrogen and carbon atoms to which they are bound.
80. The conjugate according to any one of claims 1 to 30 and claims
63 to 79, wherein R.sup.31 is OH.
81. The conjugate according to any one of claims 1 to 30 and claims
63 to 79, wherein R.sup.31 is OMe.
82. The conjugate according to any one of claims 1 to 30 and claims
63 to 81, wherein R.sup.30 is H.
83. The conjugate according to any one of claims 1 to 30 and claims
63 to 79, wherein R.sup.30 and R.sup.31 together form a double bond
between the nitrogen and carbon atoms to which they are bound.
84. The conjugate according to any one of claims 1 to 83, wherein
R.sup.6', R.sup.7', R.sup.9', and Y' are the same as R.sup.6,
R.sup.7, R.sup.9, and Y.
85. The conjugate according to any one of claims 1 to 84 wherein,
wherein L-R.sup.L1' or L-R.sup.L2' is a group: ##STR00183## where
the asterisk indicates the point of attachment to the PBD, Ab is
the antibody, L.sup.1 is a cleavable linker, A is a connecting
group connecting L.sup.1 to the antibody, L.sup.2 is a covalent
bond or together with --OC(.dbd.O)-- forms a self-immolative
linker.
86. The conjugate of claim 85, wherein L.sup.1 is enzyme
cleavable.
87. The conjugate of claim 85 or claim 86, wherein L.sup.1
comprises a contiguous sequence of amino acids.
88. The conjugate of claim 87, wherein L.sup.1 comprises a
dipeptide and the group --X.sub.1--X.sub.2-- in dipeptide,
--NH--X.sub.1--X.sub.2--CO--, is selected from: -Phe-Lys-,
-Val-Ala-, -Val-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-,
-Ile-Cit-, -Phe-Arg-, -Trp-Cit-.
89. The conjugate according to claim 88, wherein the group
--X.sub.1--X.sub.2-- in dipeptide, --NH--X.sub.1--X.sub.2--CO--, is
selected from: -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-,
-Val-Cit-.
90. The conjugate according to claim 89, wherein the group
--X.sub.1--X.sub.2-- in dipeptide, --NH--X.sub.1--X.sub.2--CO--, is
-Phe-Lys-, -Val-Ala- or -Val-Cit-.
91. The conjugate according to any one of claims 88 to 90, wherein
the group X.sub.2--CO-- is connected to L.sup.2.
92. The conjugate according to any one of claims 88 to 91, wherein
the group NH--X.sub.1-- is connected to A.
93. The conjugate according to any one of claims 88 to 92, wherein
L.sup.2 together with OC(.dbd.O) forms a self-immolative
linker.
94. The conjugate according to claim 93, wherein C(.dbd.O)O and
L.sup.2 together form the group: ##STR00184## where the asterisk
indicates the point of attachment to the PBD, the wavy line
indicates the point of attachment to the linker L.sup.1, Y is NH,
O, C(.dbd.O)NH or C(.dbd.O)O, and n is 0 to 3.
95. The conjugate according to claim 94, wherein Y is NH.
96. The conjugate according to claim 94 or claim 95, wherein n is
0.
97. The conjugate according to claim 95, wherein L.sup.1 and
L.sup.2 together with --OC(.dbd.O)-- comprise a group selected
from: ##STR00185## where the asterisk indicates the point of
attachment to the PBD, and the wavy line indicates the point of
attachment to the remaining portion of the linker L.sup.1 or the
point of attachment to A.
98. The conjugate according to claim 97, wherein the wavy line
indicates the point of attachment to A.
99. The conjugate according to any one of claims 85 to 98, wherein
A is: ##STR00186## where the asterisk indicates the point of
attachment to L.sup.1, the wavy line indicates the point of
attachment to the antibody, and n is 0 to 6; or ##STR00187## where
the asterisk indicates the point of attachment to L.sup.1, the wavy
line indicates the point of attachment to the antibody, n is 0 or
1, and m is 0 to 30.
100. A conjugate according to claim 1 of formula ##STR00188##
##STR00189## ##STR00190##
101. The conjugate according to any one of claims 1 to 100 wherein
the antibody comprises: a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, or fragment thereof, wherein each of the
cysteines at positions 109 and 112 in SEQ ID NO: 110, if present,
is substituted by an amino acid that is not cysteine; a heavy chain
comprising the amino acid sequence of SEQ ID NO.120, or fragment
thereof, wherein each of the cysteines at positions 103, 106, and
109 in SEQ ID NO: 120, if present, is substituted by an amino acid
that is not cysteine; a heavy chain comprising the amino acid
sequence of SEQ ID NO.120, or fragment thereof, wherein each of the
cysteines at positions 14, 106, and 112 in SEQ ID NO: 120, if
present, is substituted by an amino acid that is not cysteine; a
heavy chain comprising the amino acid sequence of SEQ ID NO.130, or
fragment thereof, wherein each of the cysteines at positions 111,
114, 120, 126, 129, 135, 141, 144, 150, 156, and 159 in SEQ ID NO:
130, if present, is substituted by an amino acid that is not
cysteine; or a heavy chain comprising the amino acid sequence of
SEQ ID NO.140, or fragment thereof, wherein each of the cysteines
at positions 106 and 109 in SEQ ID NO: 140, if present, is
substituted by an amino acid that is not cysteine.
102. The conjugate according to claim 101 the cysteine at position
102 in SEQ ID NO: 120, if present, is also substituted by an amino
acid that is not cysteine.
103. The conjugate according to either one of claim 101 or 102
wherein the drug moiety is conjugated to the cysteine at position
103 of SEQ ID NO.110, the cysteine at position 14 of SEQ ID NO.120,
the cysteine at position 103 of SEQ ID NO.120, the cysteine at
position 14 of SEQ ID NO.130, or the cysteine at position 14 of SEQ
ID NO.140.
104. The conjugate according to any one of claims 101 to 103
wherein the antibody comprises: a light chain comprising the amino
acid sequence of SEQ ID NO. 150, or fragment thereof, wherein the
cysteine at position 105, if present, is substituted by an amino
acid that is not cysteine; or a light chain comprising the amino
acid sequence of SEQ ID NO. 160, or fragment thereof, wherein the
cysteine at position 102, if present, is substituted by an amino
acid that is not cysteine.
105. The conjugate according to any one of claims 1 to 100 wherein
the antibody comprises: a heavy chain comprising the amino acid
sequence of SEQ ID NO.113 and a light chain comprising the amino
acid sequence of SEQ ID NO.151, SEQ ID NO.152, SEQ ID NO.153, SEQ
ID NO.161, SEQ ID NO.162, or SEQ ID NO.163; optionally wherein the
drug moiety is conjugated to the cysteine at position 103 of SEQ ID
NO.113.
106. The conjugate according to any one of claims 1 to 100 wherein
the antibody comprises: a heavy chain comprising the amino acid
sequence of SEQ ID NO.114 and a light chain comprising the amino
acid sequence of SEQ ID NO.151, SEQ ID NO.152, SEQ ID NO.153, SEQ
ID NO.161, SEQ ID NO.162, or SEQ ID NO.163; optionally wherein the
drug moiety is conjugated to the cysteine at position 103 of SEQ ID
NO.114.
107. The conjugate according to any one of claims 1 to 100 wherein
the antibody comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.110 or fragment thereof, SEQ ID NO.120 or
fragment thereof, SEQ ID NO.130 or fragment thereof, or SEQ ID
NO.140 or fragment thereof.
108. The conjugate according to claim 107 wherein the drug moiety
is conjugated to the cysteine at position 103 of SEQ ID NO.110, the
cysteine at position 14 of SEQ ID NO.120, the cysteine at position
103 of SEQ ID NO.120, the cysteine at position 14 of SEQ ID NO.130,
or the cysteine at position 14 of SEQ ID NO.140.
109. The conjugate according to either one of claim 107 or 108
wherein the antibody comprises: a light chain comprising the amino
acid sequence of SEQ ID NO. 150, or fragment thereof, wherein the
cysteine at position 105, if present, is substituted by an amino
acid that is not cysteine; or a light chain comprising the amino
acid sequence of SEQ ID NO. 160, or fragment thereof, wherein the
cysteine at position 102, if present, is substituted by an amino
acid that is not cysteine.
110. The conjugate according to any one of claims 1 to 100 wherein
the antibody comprises: a heavy chain comprising the amino acid
sequence of SEQ ID NO.110 and light chain comprising the amino acid
sequence of SEQ ID NO.151, SEQ ID NO.152, SEQ ID NO.153, SEQ ID
NO.161, SEQ ID NO.162, or SEQ ID NO.163; optionally wherein the
drug moiety is conjugated to the cysteine at position 103 of SEQ ID
NO.110.
111. The conjugate according to any one of claims 1 to 100 wherein
the antibody comprises: a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, or fragment thereof, wherein the
cysteine at position 103 of SEQ ID NO.110, if present, is
substituted by an amino acid that is not cysteine; a heavy chain
comprising the amino acid sequence of SEQ ID NO.120, or fragment
thereof, wherein each of the cysteines at positions 14 and 103 of
SEQ ID NO.120, if present, is substituted by an amino acid that is
not cysteine; a heavy chain comprising the amino acid sequence of
SEQ ID NO.130, or fragment thereof, wherein the cysteine at
position 14 in SEQ ID NO: 130, if present, is substituted by an
amino acid that is not cysteine; or a heavy chain comprising the
amino acid sequence of SEQ ID NO.140, or fragment thereof, wherein
the cysteine at position 14 in SEQ ID NO: 140, if present, is
substituted by an amino acid that is not cysteine.
112. The conjugate according to claim 111 wherein the antibody
comprises a light chain comprising the amino acid sequence of SEQ
ID NO. 150 or SEQ ID NO. 160.
113. The conjugate according to any one of claims 1 to 100 wherein
the antibody comprises: a heavy chain comprising the amino acid
sequence of SEQ ID NO.111 and a light chain comprising the amino
acid sequence of SEQ ID NO.150 or SEQ ID NO.160.
114. The conjugate according to any one of claims 1 to 100 wherein
the antibody comprises: a heavy chain comprising the amino acid
sequence of SEQ ID NO.112 and a light chain comprising the amino
acid sequence of SEQ ID NO.150 or SEQ ID NO.160.
115. The conjugate according to any one of claims 112 to 114
wherein the drug moiety is conjugated to the cysteine at position
105 of SEQ ID NO.150, or the cysteine at position 102 of SEQ ID
NO.160.
116. The conjugate according to any one of claims 1 to 100 wherein
the antibody comprises: a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, or fragment thereof, wherein each of the
cysteines at positions 103, 109 and 112 in SEQ ID NO: 110, if
present, is substituted by an amino acid that is not cysteine; a
heavy chain comprising the amino acid sequence of SEQ ID NO.120, or
fragment thereof, wherein each of the cysteines at positions 14,
103, 106 and 109 in SEQ ID NO: 120, if present, is substituted by
an amino acid that is not cysteine; a heavy chain comprising the
amino acid sequence of SEQ ID NO.130, or fragment thereof, wherein
each of the cysteines at positions 14, 111, 114, 120, 126, 129,
135, 141, 144, 150, 156, and 159 in SEQ ID NO: 130, if present, is
substituted by an amino acid that is not cysteine; or a heavy chain
comprising the amino acid sequence of SEQ ID NO.140, or fragment
thereof, wherein each of the cysteines at positions 14, 106, and
109 in SEQ ID NO: 140, if present, is substituted by an amino acid
that is not cysteine.
117. The conjugate according to claim 116 wherein the antibody
comprises a light chain comprising the amino acid sequence of SEQ
ID NO. 150 or SEQ ID NO. 160.
118. The conjugate according to any one of claims 1 to 100 wherein
the antibody comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.115 and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160.
119. The conjugate according to any one of claims 1 to 100 wherein
the antibody comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.116 and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160.
120. The conjugate according to claim 117 wherein the drug moiety
is conjugated to the cysteine at position 105 of SEQ ID NO.150, the
cysteine at position 102 of SEQ ID NO.160.
121. The conjugate according to any one of claims 1 to 120 wherein
the antibody comprises a heavy chain having a substitution of the
amino acid at position 234 in the EU index set forth in Kabat
and/or a substitution of the residue at position 235 in the EU
index set forth in Kabat.
122. The conjugate according to claim 121 wherein the antibody
comprises a heavy chain having a substitution of the amino acid at
position 234 in the EU index set forth in Kabat and a substitution
of the residue at position 235 in the EU index set forth in
Kabat.
123. The conjugate according to to any one of claims 121 to 122
wherein the antibody comprises a heavy chain comprising the amino
acid sequence of SEQ ID NO.110, and wherein the leucine at position
117 and/or the leucine at position 118 is substituted by an amino
acid that is not leucine.
124. The conjugate according to claim 123 wherein the antibody
comprises a heavy chain comprising the amino acid sequence of SEQ
ID NO.110, and wherein the leucine at position 117 and the leucine
at position 118 are substituted by an amino acid that is not
leucine.
125. The conjugate according to any one of claims 121 to 122
wherein the antibody comprises a heavy chain comprising the amino
acid sequence of SEQ ID NO.130, and wherein the leucine at position
164 and/or the leucine at position 165 is substituted by an amino
acid that is not leucine.
126. The conjugate according to claim 125 wherein the antibody
comprises a heavy chain comprising the amino acid sequence of SEQ
ID NO.130, and wherein the leucine at position 164 and the leucine
at position 165 are substituted by an amino acid that is not
leucine.
127. The conjugate according to claim 121 wherein the antibody
comprises a heavy chain comprising the amino acid sequence of SEQ
ID NO.140, and wherein the leucine at position 115 is substituted
by an amino acid that is not leucine.
128. The conjugate according to any one of claims 1 to 127 wherein
the substituted amino acids are replaced by alanine, glycine,
valine, or isoleucine.
129. The conjugate according to claim 128 wherein the substituted
amino acids are replaced by alanine.
130. The conjugate according to any one of claims 1 to 129 wherein
the antibody comprises a VH domain having the amino acid sequence
of SEQ ID NO. 1.
131. The conjugate according to claim 130 wherein the antibody
further comprises a VL domain having the amino acid sequence of SEQ
ID NO. 2.
132. The conjugate according to any one of the preceding claims
wherein the antibody in an intact antibody.
133. The conjugate according to any one of the preceding claims
wherein the antibody is humanised, deimmunised or resurfaced.
134. The conjugate according to any one of the preceding claims
wherein the conjugate has a maximum tolerated dose in rat at least
2.0 mg/kg delivered as a single-dose.
135. The conjugate according to any one of the preceding claims
wherein the drug loading (p) of drugs (D) to antibody (Ab) is 2 or
4.
136. The conjugate according to any one of claims 1 to 135, for use
in therapy.
137. The conjugate according to any one of claims 1 to 135, for use
in the treatment of a proliferative disease in a subject.
138. The conjugate according to claim 137, wherein the disease is
cancer.
139. A pharmaceutical composition comprising the conjugate of any
one of claims 1 to 135 and a pharmaceutically acceptable diluent,
carrier or excipient.
140. The pharmaceutical composition of claim 139 further comprising
a therapeutically effective amount of a chemotherapeutic agent.
141. Use of a conjugate according to any one of claims 1 to 135 in
the preparation of a medicament for use in the treatment of a
proliferative disease in a subject.
142. A method of treating cancer comprising administering to a
patient the pharmaceutical composition of claim 139.
143. The method of claim 142 wherein the patient is administered a
chemotherapeutic agent, in combination with the conjugate.
Description
[0001] The present disclosure relates to site-specific
antibody-drug conjugates. Conjugates comprising
pyrrolobenzodiazepines (PBDs) having a labile protecting group in
the form of a linker to the antibody which binds CD22 are
described.
BACKGROUND
[0002] Antibody-Drug Conjugates
[0003] Antibody therapy has been established for the targeted
treatment of patients with cancer, immunological and angiogenic
disorders (Carter, P. (2006) Nature Reviews Immunology 6:343-357).
The use of antibody-drug conjugates (ADC), i.e. immunoconjugates,
for the local delivery of cytotoxic or cytostatic agents, i.e.
drugs to kill or inhibit tumor cells in the treatment of cancer,
targets delivery of the drug moiety to tumors, and intracellular
accumulation therein (Junutula, et al., 2008b Nature Biotech.,
26(8):925-932; Dornan et al (2009) Blood 114(13):2721-2729; U.S.
Pat. No. 7,521,541; U.S. Pat. No. 7,723,485; WO2009/052249;
McDonagh (2006) Protein Eng. Design & Sel. 19(7): 299-307;
Doronina et al (2006) Bioconj. Chem. 17:114-124; Erickson et al
(2006) Cancer Res. 66(8):1-8; Sanderson et al (2005) Clin. Cancer
Res. 11:843-852; Jeffrey et al (2005) J. Med. Chem. 48:1344-1358;
Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070).
[0004] The present inventors have developed particular
antibody-drug conjugates in which the antibody moiety is modified
so as to increase the safety and efficacy of the ADC.
[0005] Site-Specific Conjugation
[0006] In ADCs cytotoxic drugs have typically been conjugated to
the antibodies in a non-site-specific manner via lysine side chains
or by reducing interchain disulfide bonds present in the antibodies
to provide activated native cysteine sulfhydryl groups.
[0007] Site-specific conjugation of drug to antibody has also been
considered with a view to provide ADC populations with high
homogeneity and batch-to-batch consistency with respect to
drug-to-antibody ratio (DAR) and attachment site. Site-specific
attachment has typically been achieved by substituting a native
amino acid in the antibody with an amino acid such as cysteine, to
which a drug moiety can be conjugated (see Stimmel et al., JBC,
Vol. 275, No. 39, Issue of September 29, pp.
30445-30450--conjugation of an IgG S442C variant with
bromoacetyl-TMT); also Junutula et al., Nature Biotechnology, vol.
26, no. 8, pp. 925-932). Jujuntula et al. report that site-specific
ADCs in which drug moieties were attached to specific cysteine
residues engineered into the antibody seqeunce exhibited comparable
efficacy and reduced systemic toxicity compared to non-specifically
conjugated ADCs.
[0008] Other studies have investigated the biological
characteristics of ADCs comprising cytotoxic drug moieties
conjugated to antibodies at specific sites. For example,
WO2013/093809 discusses a number of engineered antibody constant
regions, a sub-set of which are exemplified as part of conjugates
to cytotoxic drugs such as monomethyl auristatin D (MMAD).
WO2011/005481 describes engineered antibody Fc regions for
site-specific conjugation, including exemplification of
biotin-PEG2-maleimide to a number of he engineered antibodies.
WO2006-065533 describes antibody Fc regions in which one or more of
the `native` interchain-disulphide-forming cysteines present in the
heavy and/or light chain is substituted with another amino acid, so
as to leave the complementary cysteine sulphydryl available for
conjugation to a drug moiety.
[0009] Strop et al., Chemistry & Biology 20, 161-167, Feb. 21,
2013 assessed the stability and pharakokinetics of a number of
site-specific ADCs which differed from each other only in the
location of the site used to conjugate the drug to the antibody.
The authors report that for the tested ADCs the conjugation site
influences the ADC stability and pharmacokinetics in a
species-dependent manner.
[0010] The present inventors have developed particular
antibody-drug conjugates in which the drug moiety is conjugated in
a site-specific manner.
SUMMARY
[0011] The present inventors have found that antibody-drug
conjugates where the Drug unit (D.sup.L) is conjugated to
particular interchain cysteine residues have unexpected and
advantageous properties. In particular, these newly developed ADCs
have advantageous manufacturing and pharmacological properties
which are described herein.
[0012] Accordingly, in a first aspect--in order to increase the
efficacy and efficiency of conjugation of Drug unit (D.sup.L) to
the desired interchain cysteine residue(s)--the antibody of the
conjugates described herein comprises one or more substitution of
an interchain cysteine residue by an amino acid that is not
cysteine.
[0013] The antibody of the conjugates described herein retains at
least one unsubstituted interchain cysteine residue for conjugation
of the drug moiety to the antibody. The number of retained
interchain cysteine residues in the antibody is greater than zero
but less than the total number of interchain cysteine residues in
the parent (native) antibody. Thus, in some embodiments, the
antibody has at least one, at least two, at least three, at least
four, at least five, at least six or at least seven interchain
cysteine residues. In typical embodiments, the antibody has an even
integral number of interchain cysteine residues (e.g., at least
two, four, six or eight). In some embodiments, the antibody has
less than eight interchain cysteine residues.
[0014] AbLJ
[0015] In some embodiments the antibody of the conjugates described
herein: (i) retain the unsubstituted hinge region interchain
cysteines, (ii) comprise light chains each having an amino acid
substitution of the interchain cysteine residue located in the
C.sub.L domain, and (iii) comprise heavy chains each retaining the
unsubstituted interchain cysteine located in the CH.sub.1 domain.
For example, In some embodiments the antibody of the conjugates
described herein: (i) retains unsubstituted HC226 and HC229
according to the EU index as set forth in Kabat, (ii) comprise
light chains each having an amino acid substitution of the
interchain cysteine residue .kappa.LC214 or .lamda.LC213 according
to the EU index as set forth in Kabat, and (iii) comprise heavy
chains each retaining the unsubstituted interchain cysteine HC220
according to the EU index as set forth in Kabat. Preferably the
drug moiety is conjugated to the unsubstituted interchain cysteine
located in the CH.sub.1 domain, for example to HC220 according to
the EU index as set forth in Kabat.
[0016] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0017] wherein
the cysteine at position 105 in SEQ ID NO: 150 or the cysteine at
position 102 in SEQ ID NO: 160, is substituted by an amino acid
that is not cysteine. Preferably the drug moiety is conjugated to
the cysteine at position 103 of SEQ ID NO.110.
[0018] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.120, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0019] wherein
the cysteine at position 105 in SEQ ID NO: 150 or the cysteine at
position 102 in SEQ ID NO: 160, is substituted by an amino acid
that is not cysteine. Preferably the drug moiety is conjugated to
the cysteine at position 14 of SEQ ID NO.120.
[0020] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
[0021] wherein the cysteine at position 105 in SEQ ID NO: 150 or
the cysteine at position 102 in SEQ ID NO: 160, is substituted by
an amino acid that is not cysteine. Preferably the drug moiety is
conjugated to the cysteine at position 14 of SEQ ID NO.130.
[0022] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0023] wherein
the cysteine at position 105 in SEQ ID NO: 150 or the cysteine at
position 102 in SEQ ID NO: 160, is substituted by an amino acid
that is not cysteine. Preferably the drug moiety is conjugated to
the cysteine at position 14 of SEQ ID NO.140.
[0024] AbHJ
[0025] In some embodiments the antibody of the conjugates described
herein: (i) retain the unsubstituted hinge region interchain
cysteines, (ii) comprise light chains each retaining the
unsubstituted interchain cysteine located in the C.sub.L domain,
and (iii) comprise heavy chains each having an amino acid
substitution of the interchain cysteine residue located in the
CH.sub.1 domain. For example, In some embodiments the antibody of
the conjugates described herein: (i) retains unsubstituted HC226
and HC229 according to the EU index as set forth in Kabat, (ii)
comprise light chains each retaining the unsubstituted interchain
cysteine .kappa.LC214 or .lamda.LC213 according to the EU index as
set forth in Kabat, and (iii) comprise heavy chains each having an
amino acid substitution of interchain cysteine HC220 according to
the EU index as set forth in Kabat. Preferably the drug moiety is
conjugated to the unsubstituted interchain cysteine located in the
C.sub.L domain, for example to .kappa.LC214 or .lamda.LC213
according to the EU index as set forth in Kabat.
[0026] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0027] wherein
the cysteine at position 103 in SEQ ID NO: 110 is substituted by an
amino acid that is not cysteine. Preferably the drug moiety is
conjugated to the cysteine at position 105 of SEQ ID NO.150, the
cysteine at position 102 of SEQ ID NO.160.
[0028] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.120, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0029] wherein
each of the cysteines at positions 14 and 103 in SEQ ID NO: 120 is
substituted by an amino acid that is not cysteine. Preferably the
drug moiety is conjugated to the cysteine at position 105 of SEQ ID
NO.150, the cysteine at position 102 of SEQ ID NO.160.
[0030] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0031] wherein
the cysteine at position 14 in SEQ ID NO: 130 is substituted by an
amino acid that is not cysteine. Preferably the drug moiety is
conjugated to the cysteine at position 105 of SEQ ID NO.150, the
cysteine at position 102 of SEQ ID NO.160.
[0032] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0033] wherein
the cysteine at position 14 in SEQ ID NO: 140 is substituted by an
amino acid that is not cysteine. Preferably the drug moiety is
conjugated to the cysteine at position 105 of SEQ ID NO.150, the
cysteine at position 102 of SEQ ID NO.160.
[0034] AbBJ
[0035] In some embodiments the antibody of the conjugates described
herein: (i) has an amino acid substitution of each of the hinge
region interchain cysteines, (ii) comprise light chains each having
an amino acid substitution of the interchain cysteine residue
located in the C.sub.L domain, and (iii) comprise heavy chains each
retaining the unsubstituted interchain cysteine located in the
CH.sub.1 domain. For example, in some embodiments the antibody of
the conjugates described herein: (i) has an amino acid substitution
of each of HC226 and HC229 according to the EU index as set forth
in Kabat, (ii) comprise light chains each having an amino acid
substitution of the interchain cysteine residue .kappa.LC214 or
.lamda.LC213 according to the EU index as set forth in Kabat, and
(iii) comprise heavy chains each retaining the unsubstituted
interchain cysteine HC220 according to the EU index as set forth in
Kabat. Preferably the drug moiety is conjugated to the
unsubstituted interchain cysteine located in the CH.sub.1 domain,
for example to HC220 according to the EU index as set forth in
Kabat.
[0036] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0037] wherein
each of the cysteines at positions 109 and 112 in SEQ ID NO: 110 is
substituted by an amino acid that is not cysteine; [0038] and
wherein the cysteine at position 105 in SEQ ID NO: 150 or the
cysteine at position 102 in SEQ ID NO: 160, is substituted by an
amino acid that is not cysteine. Preferably the drug moiety is
conjugated to the cysteine at position 103 of SEQ ID NO.110.
[0039] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.120, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0040] wherein
each of the cysteines at positions 103, 106, and 109 in SEQ ID NO:
120 is substituted by an amino acid that is not cysteine; [0041]
and wherein the cysteine at position 105 in SEQ ID NO: 150 or the
cysteine at position 102 in SEQ ID NO: 160, is substituted by an
amino acid that is not cysteine. In some embodiments, the cysteine
at position 102 in SEQ ID NO: 120 is also substituted by an amino
acid that is not cysteine. Preferably the drug moiety is conjugated
to the cysteine at position 14 of SEQ ID NO.120.
[0042] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.120, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0043] wherein
each of the cysteines at positions 14, 106, and 109 in SEQ ID NO:
120 is substituted by an amino acid that is not cysteine; [0044]
and wherein the cysteine at position 105 in SEQ ID NO: 150 or the
cysteine at position 102 in SEQ ID NO: 160, is substituted by an
amino acid that is not cysteine. In some embodiments, the cysteine
at position 102 in SEQ ID NO: 120 is also substituted by an amino
acid that is not cysteine. Preferably the drug moiety is conjugated
to the cysteine at position 103 of SEQ ID NO.120.
[0045] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0046] wherein
each of the cysteines at positions 111, 114, 120, 126, 129, 135,
141, 144, 150, 156, and 159 in SEQ ID NO: 130 is substituted by an
amino acid that is not cysteine; [0047] and wherein the cysteine at
position 105 in SEQ ID NO: 150 or the cysteine at position 102 in
SEQ ID NO: 160, is substituted by an amino acid that is not
cysteine. Preferably the drug moiety is conjugated to the cysteine
at position 14 of SEQ ID NO.130.
[0048] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0049] wherein
each of the cysteines at positions 106 and 109 in SEQ ID NO: 140 is
substituted by an amino acid that is not cysteine; [0050] and
wherein the cysteine at position 105 in SEQ ID NO: 150 or the
cysteine at position 102 in SEQ ID NO: 160, is substituted by an
amino acid that is not cysteine. Preferably the drug moiety is
conjugated to the cysteine at position 14 of SEQ ID NO.140.
[0051] AbDJ
[0052] In some embodiments the antibody of the conjugates described
herein: (i) has an amino acid substitution of each of the hinge
region interchain cysteines, (ii) comprises light chains each
retaining the unsubstituted interchain cysteine located in the
C.sub.L domain, and (iii) comprises heavy chains each having an
amino acid substitution of the interchain cysteine residue located
in the CH.sub.1 domain. For example, in some embodiments the
antibody of the conjugates described herein: (i) has an amino acid
substitution of each of HC226 and HC229 according to the EU index
as set forth in Kabat, (ii) comprises light chains each retaining
the unsubstituted interchain cysteine .kappa.LC214 or .lamda.LC213
according to the EU index as set forth in Kabat, and (iii)
comprises heavy chains each having an amino acid substitution of
interchain cysteine HC220 according to the EU index as set forth in
Kabat. Preferably the drug moiety is conjugated to the
unsubstituted interchain cysteine located in the C.sub.L domain,
for example to .kappa.LC214 or .lamda.LC213 according to the EU
index as set forth in Kabat.
[0053] In some embodiments, some embodiments, the antibody of the
conjugates described herein comprises a heavy chain comprising the
amino acid sequence of SEQ ID NO.110, and a light chain comprising
the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0054]
wherein each of the cysteines at positions 103, 109 and 112 in SEQ
ID NO: 110 is substituted by an amino acid that is not cysteine.
Preferably the drug moiety is conjugated to the cysteine at
position 105 of SEQ ID NO.150, the cysteine at position 102 of SEQ
ID NO.160.
[0055] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.120, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0056] wherein
each of the cysteines at positions 14, 103, 106 and 109 in SEQ ID
NO: 120 is substituted by an amino acid that is not cysteine.
Preferably the drug moiety is conjugated to the cysteine at
position 105 of SEQ ID NO.150, the cysteine at position 102 of SEQ
ID NO.160.
[0057] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0058] wherein
each of the cysteines at positions 14, 111, 114, 120, 126, 129,
135, 141, 144, 150, 156, and 159 in SEQ ID NO: 130 is substituted
by an amino acid that is not cysteine. Preferably the drug moiety
is conjugated to the cysteine at position 105 of SEQ ID NO.150, the
cysteine at position 102 of SEQ ID NO.160.
[0059] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0060] wherein
each of the cysteines at positions 14, 106, and 109 in SEQ ID NO:
140 is substituted by an amino acid that is not cysteine.
Preferably the drug moiety is conjugated to the cysteine at
position 105 of SEQ ID NO.150, the cysteine at position 102 of SEQ
ID NO.160.
[0061] The present inventors have further found that antibody-drug
conjugates wherein the antibody comprises specific mutations, or
combinations of mutations, in the heavy chain have unexpected and
advantageous properties. In particular, the present inventors have
identified antibody mutations in the heavy chain which reduce the
toxicity and increase the serum half-lives of the ADCs they are
incorporated into, as compared to otherwise identical ADCs
comprising antibodies which lack the specific mutations.
[0062] For example, in the IgG1 isotype the present inventors have
identified the Leucine residues at positions 234 and 235 in the EU
index set forth in Kabat (residues L117 and L118 in SEQ ID NO.110)
as residues which, when substituted by an amino acid that is not
leucine, allow for ADCs with advantageous properties.
[0063] Accordingly, in a second aspect the antibody of the
conjugates described herein comprises a heavy chain having a
substitution of the residue at position 234 in the EU index set
forth in Kabat and/or a substitution of the residue at position 235
in the EU index set forth in Kabat by any other amino acid (that
is, an amino acid that is not identical to that found in the
`wild-type` sequence). Preferably both the residues at position 234
and 235 in the EU index set forth in Kabat are substituted by any
other amino acid.
[0064] In some embodiments the antibody is an IgG1 isotype and the
leucine at position 234 in the EU index set forth in Kabat and/or
the leucine at position 235 in the EU index set forth in Kabat is
substituted by an amino acid that is not leucine. Preferably both
the leucines at position 234 and 235 in the EU index set forth in
Kabat are substituted by an amino acid that is not leucine, such as
alanine. One or both Leucines may be also substituted by other
amino acids which are not Leucine, such as Glycine, Valine, or
Isoleucine.
[0065] For example, in some embodiments the antibody of the
conjugates described herein comprises a heavy chain comprising the
amino acid sequence of SEQ ID NO.110, wherein the leucine at
position 117 and/or the leucine at position 118 is substituted by
an amino acid that is not leucine, such as alanine. Preferably both
the leucines at position 117 and 118 are substituted by an amino
acid that is not leucine, such as alanine. One or both Leucines may
be also substituted by other amino acids which are not Leucine,
such as Glycine, Valine, or Isoleucine.
[0066] In some embodiments the antibody is an IgG3 isotype and the
leucine at position 234 in the EU index set forth in Kabat and/or
the leucine at position 235 in the EU index set forth in Kabat is
substituted by an amino acid that is not leucine. Preferably both
the leucines at position 234 and 235 in the EU index set forth in
Kabat are substituted by an amino acid that is not leucine, such as
alanine. One or both Leucines may be also substituted by other
amino acids which are not Leucine, such as Glycine, Valine, or
Isoleucine.
[0067] For example, in some embodiments the antibody of the
conjugates described herein comprises a heavy chain comprising the
amino acid sequence of SEQ ID NO.130, wherein the leucine at
position 164 and/or the leucine at position 165 is substituted by
an amino acid that is not leucine, such as alanine. Preferably both
the leucines at position 164 and 165 are substituted by an amino
acid that is not leucine, such as alanine. One or both Leucines may
be also substituted by other amino acids which are not Leucine,
such as Glycine, Valine, or Isoleucine.
[0068] In some embodiments the antibody is an IgG4 isotype and the
leucine at position 235 in the EU index set forth in Kabat is
substituted by an amino acid that is not leucine, such as alanine.
The Leucine may be also substituted by other amino acids which are
not Leucine, such as Glycine, Valine, or Isoleucine.
[0069] For example, in some embodiments the antibody of the
conjugates described herein comprises a heavy chain comprising the
amino acid sequence of SEQ ID NO.140, wherein the leucine at
position 115 is substituted by an amino acid that is not leucine,
such as alanine. The Leucine may be also substituted by other amino
acids which are not Leucine, such as Glycine, Valine, or
Isoleucine.
[0070] The modifications described in the first aspect can be
advantageously combined in the same antibody with the modifications
described in the second aspect.
[0071] Accordingly, in a third aspect the antibody of the
conjugates described herein: [0072] (1) comprises one or more
substitution of an interchain cysteine residue by an amino acid
that is not cysteine and retains at least one unsubstituted
interchain cysteine residue for conjugation of the drug moiety to
the antibody; and [0073] (2) comprises a heavy chain having a
substitution of the residue at position 234 in the EU index set
forth in Kabat and/or a substitution of the residue at position 235
in the EU index set forth in Kabat by any other amino acid (that
is, an amino acid that is not identical to that found in the
`wild-type` sequence).
[0074] AbLJ(LALA)
[0075] In some embodiments the antibody of the conjugates described
herein: (i) retain the unsubstituted hinge region interchain
cysteines, (ii) comprise light chains each having an amino acid
substitution of the interchain cysteine residue located in the
C.sub.L domain, (iii) comprise heavy chains each retaining the
unsubstituted interchain cysteine located in the CH.sub.1 domain,
and (iv) comprise heavy chains each having an amino acid
substitution of the the residue at position 234 in the EU index set
forth in Kabat and/or a substitution of the residue at position 235
in the EU index set forth in Kabat.
[0076] For example, In some embodiments the antibody of the
conjugates described herein: (i) retains unsubstituted HC226 and
HC229 according to the EU index as set forth in Kabat, (ii)
comprise light chains each having an amino acid substitution of the
interchain cysteine residue .kappa.LC214 or .lamda.LC213 according
to the EU index as set forth in Kabat, (iii) comprise heavy chains
each retaining the unsubstituted interchain cysteine HC220
according to the EU index as set forth in Kabat, and (iv) comprise
heavy chains each having an amino acid substitution of the the
residue at position 234 in the EU index set forth in Kabat and/or a
substitution of the residue at position 235 in the EU index set
forth in Kabat by any other amino acid. Preferably both the
residues at position 234 and 235 in the EU index set forth in Kabat
are substituted. Preferably the drug moiety is conjugated to the
unsubstituted interchain cysteine located in the CH.sub.1 domain,
for example to HC220 according to the EU index as set forth in
Kabat.
[0077] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0078] wherein
the cysteine at position 105 in SEQ ID NO: 150 or the cysteine at
position 102 in SEQ ID NO: 160, is substituted by an amino acid
that is not cysteine; [0079] and wherein the leucine at position
117 in SEQ ID NO: 110 and/or the leucine at position 118 in SEQ ID
NO: 110 is substituted by an amino acid that is not leucine, such
as alanine. Preferably the drug moiety is conjugated to the
cysteine at position 103 of SEQ ID NO.110. Preferably both the
leucines at position 117 and 118 in SEQ ID NO: 110 are substituted
by an amino acid that is not leucine, such as alanine. One or both
Leucines may be also substituted by other amino acids which are not
Leucine, such as Glycine, Valine, or Isoleucine.
[0080] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0081] wherein
the cysteine at position 105 in SEQ ID NO: 150 or the cysteine at
position 102 in SEQ ID NO: 160, is substituted by an amino acid
that is not cysteine; [0082] and wherein the leucine at position
164 in SEQ ID NO: 130 and/or the leucine at position 165 in SEQ ID
NO: 130 is substituted by an amino acid that is not leucine, such
as alanine. Preferably the drug moiety is conjugated to the
cysteine at position 14 of SEQ ID NO.130. Preferably both the
leucines at position 164 and 165 in SEQ ID NO: 130 are substituted
by an amino acid that is not leucine, such as alanine. One or both
Leucines may be also substituted by other amino acids which are not
Leucine, such as Glycine, Valine, or Isoleucine.
[0083] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0084] wherein
the cysteine at position 105 in SEQ ID NO: 150 or the cysteine at
position 102 in SEQ ID NO: 160, is substituted by an amino acid
that is not cysteine; [0085] and wherein the leucine at position
115 in SEQ ID NO: 140 is substituted by an amino acid that is not
leucine, such as alanine. Preferably the drug moiety is conjugated
to the cysteine at position 14 of SEQ ID NO.140. The Leucine may be
also substituted by other amino acids which are not Leucine, such
as Glycine, Valine, or Isoleucine.
[0086] AbHJ(LALA)
[0087] In some embodiments the antibody of the conjugates described
herein: (i) retain the unsubstituted hinge region interchain
cysteines, (ii) comprise light chains each retaining the
unsubstituted interchain cysteine located in the C.sub.L domain,
(iii) comprise heavy chains each having an amino acid substitution
of the interchain cysteine residue located in the CH.sub.1 domain,
and (iv) comprise heavy chains each having an amino acid
substitution of the the residue at position 234 in the EU index set
forth in Kabat and/or a substitution of the residue at position 235
in the EU index set forth in Kabat.
[0088] For example, In some embodiments the antibody of the
conjugates described herein: (i) retains unsubstituted HC226 and
HC229 according to the EU index as set forth in Kabat, (ii)
comprise light chains each retaining the unsubstituted interchain
cysteine .kappa.LC214 or .lamda.LC213 according to the EU index as
set forth in Kabat, (iii) comprise heavy chains each having an
amino acid substitution of interchain cysteine HC220 according to
the EU index as set forth in Kabat, and (iv) comprise heavy chains
each having an amino acid substitution of the the residue at
position 234 in the EU index set forth in Kabat and/or a
substitution of the residue at position 235 in the EU index set
forth in Kabat by any other amino acid. Preferably both the
residues at position 234 and 235 in the EU index set forth in Kabat
are substituted. Preferably the drug moiety is conjugated to the
unsubstituted interchain cysteine located in the C.sub.L domain,
for example to .kappa.LC214 or .lamda.LC213 according to the EU
index as set forth in Kabat.
[0089] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0090] wherein
the cysteine at position 103 in SEQ ID NO: 110 is substituted by an
amino acid that is not cysteine; [0091] and wherein the leucine at
position 117 in SEQ ID NO: 110 and/or the leucine at position 118
in SEQ ID NO: 110 is substituted by an amino acid that is not
leucine, such as alanine. Preferably the drug moiety is conjugated
to the cysteine at position 105 of SEQ ID NO.150, the cysteine at
position 102 of SEQ ID NO.160. Preferably both the leucines at
position 117 and 118 in SEQ ID NO: 110 are substituted by an amino
acid that is not leucine, such as alanine. One or both Leucines may
be also substituted by other amino acids which are not Leucine,
such as Glycine, Valine, or Isoleucine.
[0092] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0093] wherein
the cysteine at position 14 in SEQ ID NO: 130 is substituted by an
amino acid that is not cysteine; [0094] and wherein the leucine at
position 164 in SEQ ID NO: 130 and/or the leucine at position 165
in SEQ ID NO: 130 is substituted by an amino acid that is not
leucine, such as alanine. Preferably the drug moiety is conjugated
to the cysteine at position 105 of SEQ ID NO.150, the cysteine at
position 102 of SEQ ID NO.160. Preferably both the leucines at
position 164 and 165 in SEQ ID NO: 130 are substituted by an amino
acid that is not leucine, such as alanine. One or both Leucines may
be also substituted by other amino acids which are not Leucine,
such as Glycine, Valine, or Isoleucine.
[0095] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0096] wherein
the cysteine at position 14 in SEQ ID NO: 140 is substituted by an
amino acid that is not cysteine; [0097] and wherein the leucine at
position 115 in SEQ ID NO: 140 is substituted by an amino acid that
is not leucine, such as alanine. Preferably the drug moiety is
conjugated to the cysteine at position 105 of SEQ ID NO.150, the
cysteine at position 102 of SEQ ID NO.160. The Leucine may be also
substituted by other amino acids which are not Leucine, such as
Glycine, Valine, or Isoleucine.
[0098] AbBJ(LALA)
[0099] In some embodiments the antibody of the conjugates described
herein: (i) has an amino acid substitution of each of the hinge
region interchain cysteines, (ii) comprise light chains each having
an amino acid substitution of the interchain cysteine residue
located in the C.sub.L domain, (iii) comprise heavy chains each
retaining the unsubstituted interchain cysteine located in the
CH.sub.1 domain, and (iv) comprise heavy chains each having an
amino acid substitution of the the residue at position 234 in the
EU index set forth in Kabat and/or a substitution of the residue at
position 235 in the EU index set forth in Kabat.
[0100] For example, in some embodiments the antibody of the
conjugates described herein: (i) has an amino acid substitution of
each of HC226 and HC229 according to the EU index as set forth in
Kabat, (ii) comprise light chains each having an amino acid
substitution of the interchain cysteine residue .kappa.LC214 or
.lamda.LC213 according to the EU index as set forth in Kabat, (iii)
comprise heavy chains each retaining the unsubstituted interchain
cysteine HC220 according to the EU index as set forth in Kabat, and
(iv) comprise heavy chains each having an amino acid substitution
of the the residue at position 234 in the EU index set forth in
Kabat and/or a substitution of the residue at position 235 in the
EU index set forth in Kabat by any other amino acid. Preferably
both the residues at position 234 and 235 in the EU index set forth
in Kabat are substituted. Preferably the drug moiety is conjugated
to the unsubstituted interchain cysteine located in the CH.sub.1
domain, for example to HC220 according to the EU index as set forth
in Kabat.
[0101] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0102] wherein
each of the cysteines at positions 109 and 112 in SEQ ID NO: 110 is
substituted by an amino acid that is not cysteine; [0103] and
wherein the cysteine at position 105 in SEQ ID NO: 150 or the
cysteine at position 102 in SEQ ID NO: 160, is substituted by an
amino acid that is not cysteine; [0104] and wherein the leucine at
position 117 in SEQ ID NO: 110 and/or the leucine at position 118
in SEQ ID NO: 110 is substituted by an amino acid that is not
leucine, such as alanine. Preferably the drug moiety is conjugated
to the cysteine at position 103 of SEQ ID NO.110. Preferably both
the leucines at position 117 and 118 in SEQ ID NO: 110 are
substituted by an amino acid that is not leucine, such as alanine.
One or both Leucines may be also substituted by other amino acids
which are not Leucine, such as Glycine, Valine, or Isoleucine.
[0105] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0106] wherein
each of the cysteines at positions 111, 114, 120, 126, 129, 135,
141, 144, 150, 156, and 159 in SEQ ID NO: 130 is substituted by an
amino acid that is not cysteine; [0107] and wherein the cysteine at
position 105 in SEQ ID NO: 150 or the cysteine at position 102 in
SEQ ID NO: 160, is substituted by an amino acid that is not
cysteine; [0108] and wherein the leucine at position 164 in SEQ ID
NO: 130 and/or the leucine at position 165 in SEQ ID NO: 130 is
substituted by an amino acid that is not leucine, such as alanine.
Preferably the drug moiety is conjugated to the cysteine at
position 14 of SEQ ID NO.130. Preferably both the leucines at
position 164 and 165 in SEQ ID NO: 130 are substituted by an amino
acid that is not leucine, such as alanine. One or both Leucines may
be also substituted by other amino acids which are not Leucine,
such as Glycine, Valine, or Isoleucine.
[0109] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0110] wherein
each of the cysteines at positions 106 and 109 in SEQ ID NO: 140 is
substituted by an amino acid that is not cysteine; [0111] and
wherein the cysteine at position 105 in SEQ ID NO: 150 or the
cysteine at position 102 in SEQ ID NO: 160, is substituted by an
amino acid that is not cysteine; [0112] and wherein the leucine at
position 115 in SEQ ID NO: 140 is substituted by an amino acid that
is not leucine, such as alanine. Preferably the drug moiety is
conjugated to the cysteine at position 14 of SEQ ID NO.140. The
Leucine may be also substituted by other amino acids which are not
Leucine, such as Glycine, Valine, or Isoleucine.
[0113] AbDJ(LALA)
[0114] In some embodiments the antibody of the conjugates described
herein: (i) has an amino acid substitution of each of the hinge
region interchain cysteines, (ii) comprises light chains each
retaining the unsubstituted interchain cysteine located in the
C.sub.L domain, (iii) comprises heavy chains each having an amino
acid substitution of the interchain cysteine residue located in the
CH.sub.1 domain, and (iv) comprise heavy chains each having an
amino acid substitution of the the residue at position 234 in the
EU index set forth in Kabat and/or a substitution of the residue at
position 235 in the EU index set forth in Kabat.
[0115] For example, in some embodiments the antibody of the
conjugates described herein: (i) has an amino acid substitution of
each of HC226 and HC229 according to the EU index as set forth in
Kabat, (ii) comprises light chains each retaining the unsubstituted
interchain cysteine .kappa.LC214 or .lamda.LC213 according to the
EU index as set forth in Kabat, (iii) comprises heavy chains each
having an amino acid substitution of interchain cysteine HC220
according to the EU index as set forth in Kabat, and (iv) comprise
heavy chains each having an amino acid substitution of the the
residue at position 234 in the EU index set forth in Kabat and/or a
substitution of the residue at position 235 in the EU index set
forth in Kabat by any other amino acid. Preferably both the
residues at position 234 and 235 in the EU index set forth in Kabat
are substituted. Preferably the drug moiety is conjugated to the
unsubstituted interchain cysteine located in the C.sub.L domain,
for example to .kappa.LC214 or .lamda.LC213 according to the EU
index as set forth in Kabat.
[0116] In some embodiments, some embodiments, the antibody of the
conjugates described herein comprises a heavy chain comprising the
amino acid sequence of SEQ ID NO.110, and a light chain comprising
the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0117]
wherein each of the cysteines at positions 103, 109 and 112 in SEQ
ID NO: 110 is substituted by an amino acid that is not cysteine;
[0118] and wherein the leucine at position 117 in SEQ ID NO: 110
and/or the leucine at position 118 in SEQ ID NO: 110 is substituted
by an amino acid that is not leucine, such as alanine. Preferably
the drug moiety is conjugated to the cysteine at position 105 of
SEQ ID NO.150, the cysteine at position 102 of SEQ ID NO.160.
Preferably both the leucines at position 117 and 118 in SEQ ID NO:
110 are substituted by an amino acid that is not leucine, such as
alanine. One or both Leucines may be also substituted by other
amino acids which are not Leucine, such as Glycine, Valine, or
Isoleucine.
[0119] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0120] wherein
each of the cysteines at positions 14, 111, 114, 120, 126, 129,
135, 141, 144, 150, 156, and 159 in SEQ ID NO: 130 is substituted
by an amino acid that is not cysteine; [0121] and wherein the
leucine at position 164 in SEQ ID NO: 130 and/or the leucine at
position 165 in SEQ ID NO: 130 is substituted by an amino acid that
is not leucine, such as alanine. Preferably the drug moiety is
conjugated to the cysteine at position 105 of SEQ ID NO.150, the
cysteine at position 102 of SEQ ID NO.160. Preferably both the
leucines at position 164 and 165 in SEQ ID NO: 130 are substituted
by an amino acid that is not leucine, such as alanine. One or both
Leucines may be also substituted by other amino acids which are not
Leucine, such as Glycine, Valine, or Isoleucine.
[0122] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0123] wherein
each of the cysteines at positions 14, 106, and 109 in SEQ ID NO:
140 is substituted by an amino acid that is not cysteine; [0124]
and wherein the leucine at position 115 in SEQ ID NO: 140 is
substituted by an amino acid that is not leucine, such as alanine.
Preferably the drug moiety is conjugated to the cysteine at
position 105 of SEQ ID NO.150, the cysteine at position 102 of SEQ
ID NO.160. The Leucine may be also substituted by other amino acids
which are not Leucine, such as Glycine, Valine, or Isoleucine.
BRIEF DESCRIPTION OF FIGURES
[0125] FIG. 1 Comparative systemic toxicitiy of site-specific ADCs,
as described in Example 7.
DETAILED DESCRIPTION
[0126] Described herein are conjugates comprising a
pyrrolobenzodiazepine (PBD) drug moiety with a labile C2 or N10
protecting group and an antibody which binds CD22, wherein the
antibody comprises an amino acid substitution of an interchain
cysteine residue by an amino acid that is not cysteine, and wherein
the drug moiety is conjugated to an interchain cysteine
residue.
[0127] Also described herein are conjugates comprising the
antibodies described herein conjugated to other (i.e. non-PBD)
functional moieties. Examples of a functional moiety include a drug
(PBD or non-PBD), a reporter, an organic moiety, and/or a binding
moiety.
[0128] Also contemplated are conjugates comprising an antibody
fragment as described herein, along with pharmaceutical
compositions comprising the conjugates. Example antibodies or
antibody fragment include scFv-Fc fusions and minibodies. Methods
of preparing the conjugates and using the conjugates are disclosed,
along with methods of using the conjugates to treat a number of
diseases.
[0129] Pyrrolobenzodiazepines
[0130] In some embodiments, the conjugates described herein
comprise a PBD drug moiety. Some pyrrolobenzodiazepines (PBDs) have
the ability to recognise and bond to specific sequences of DNA; the
preferred sequence is PuGPu. The first PBD antitumour antibiotic,
anthramycin, was discovered in 1965 (Leimgruber, et al., J. Am.
Chem. Soc., 87, 5793-5795 (1965); Leimgruber, et al., J. Am. Chem.
Soc., 87, 5791-5793 (1965)). Since then, a number of naturally
occurring PBDs have been reported, and over 10 synthetic routes
have been developed to a variety of analogues (Thurston, et al.,
Chem. Rev. 1994, 433-465 (1994); Antonow, D. and Thurston, D. E.,
Chem. Rev. 2011 111 (4), 2815-2864). Family members include
abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148
(1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206
(1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem.
Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758
(1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33,
665-667 (1980)), neothramycins A and B (Takeuchi, et al., J.
Antibiotics, 29, 93-96 (1976)), porothramycin (Tsunakawa, et al.,
J. Antibiotics, 41, 1366-1373 (1988)), prothracarcin (Shimizu, et
al, J. Antibiotics, 29, 2492-2503 (1982); Langley and Thurston, J.
Org. Chem., 52, 91-97 (1987)), sibanomicin (DC-102)(Hara, et al.,
J. Antibiotics, 41, 702-704 (1988); Itoh, et al., J. Antibiotics,
41, 1281-1284 (1988)), sibiromycin (Leber, et al., J. Am. Chem.
Soc., 110, 2992-2993 (1988)) and tomamycin (Arima, et al., J.
Antibiotics, 25, 437-444 (1972)). PBDs are of the general
structure:
##STR00001##
[0131] They differ in the number, type and position of
substituents, in both their aromatic A rings and pyrrolo C rings,
and in the degree of saturation of the C ring. In the B-ring there
is either an imine (N.dbd.C), a carbinolamine(NH--CH(OH)), or a
carbinolamine methyl ether (NH--CH(OMe)) at the N10-C11 position
which is the electrophilic centre responsible for alkylating DNA.
All of the known natural products have an (S)-configuration at the
chiral C11a position which provides them with a right-handed twist
when viewed from the C ring towards the A ring. This gives them the
appropriate three-dimensional shape for isohelicity with the minor
groove of B-form DNA, leading to a snug fit at the binding site
(Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11
(1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., 19,
230-237 (1986)). Their ability to form an adduct in the minor
groove, enables them to interfere with DNA processing, hence their
use as antitumour agents.
[0132] One pyrrolobenzodiazepine compound is described by Gregson
et al. (Chem. Commun. 1999, 797-798) as compound 1, and by Gregson
et al. (J. Med. Chem. 2001, 44, 1161-1174) as compound 4a. This
compound, also known as SG2000, is shown below:
##STR00002##
[0133] WO 2007/085930 describes the preparation of dimer PBD
compounds having linker groups for connection to a cell binding
agent, such as an antibody. The linker is present in the bridge
linking the monomer PBD units of the dimer.
[0134] WO 2011/130613 and WO 2011/130616 describe dimer PBD
compounds having linker groups for connection to a cell binding
agent, such as an antibody. The linker in these compounds is
attached to the PBD core via the C2 position, and are generally
cleaved by action of an enzyme on the linker group. In WO
2011/130598, the linker in these compounds is attached to one of
the available N10 positions on the PBD core, and are generally
cleaved by action of an enzyme on the linker group.
[0135] Conjugates Comprising PBD Drug Moieties
[0136] The present inventors have found that conjugates where the
Drug unit (D.sup.L) is conjugated to particular interchain cysteine
residues have unexpected and advantageous properties including
increased efficacy and stability, improved ease of manufacture, and
reduced systemic toxicity.
[0137] Accordingly, in one aspect the disclosure provides a
conjugate of formula L-(DL)p, where DL is of formula I or II::
##STR00003##
[0138] wherein:
[0139] L is an antibody (Ab) which binds CD22;
[0140] when there is a double bond present between C2' and C3',
R.sup.12 is selected from the group consisting of:
[0141] (ia) C.sub.5-10 aryl group, optionally substituted by one or
more substituents selected from the group comprising: halo, nitro,
cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7
heterocyclyl and bis-oxy-C.sub.1-3 alkylene;
[0142] (ib) C.sub.1-5 saturated aliphatic alkyl;
[0143] (ic) C.sub.3-6 saturated cycloalkyl;
[0144] (id)
##STR00004##
wherein each of R.sup.21, R.sup.22 and R.sup.23 are independently
selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl,
C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon
atoms in the R.sup.12 group is no more than 5;
[0145] (ie)
##STR00005##
wherein one of R.sup.25a and R.sup.25b is H and the other is
selected from: phenyl, which phenyl is optionally substituted by a
group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
and
[0146] (if)
##STR00006##
where R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl;
C.sub.2-3 alkenyl; C.sub.1-3 alkynyl; cyclopropyl; phenyl, which
phenyl is optionally substituted by a group selected from halo,
methyl, methoxy; pyridyl; and thiophenyl;
[0147] when there is a single bond present between C2' and C3',
[0148] R.sup.12 is
##STR00007##
where R.sup.26a and R.sup.26b are independently selected from H, F,
C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and
alkenyl groups are optionally substituted by a group selected from
C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of
R.sup.26a and R.sup.26b is H, the other is selected from nitrile
and a C.sub.1-4 alkyl ester;
[0149] R.sup.6 and R.sup.9 are independently selected from H, R,
OH, OR, SH, SR, NH.sub.2, NHR, NRR', nitro, Me.sub.3Sn and
halo;
[0150] where R and R' are independently selected from optionally
substituted C.sub.1-12 alkyl, C.sub.3-20 heterocyclyl and
C.sub.5-20 aryl groups;
[0151] R.sup.7 is selected from H, R, OH, OR, SH, SR, NH.sub.2,
NHR, NHRR', nitro, Me.sub.3Sn and halo;
[0152] R'' is a C.sub.3-12 alkylene group, which chain may be
interrupted by one or more heteroatoms, e.g. O, S, NR.sup.N2 (where
R.sup.N2 is H or C.sub.1-4 alkyl), and/or aromatic rings, e.g.
benzene or pyridine;
[0153] Y and Y' are selected from O, S, or NH;
[0154] R.sup.6', R.sup.7', R.sup.9' are selected from the same
groups as R.sup.6, R.sup.7 and R.sup.9 respectively;
[0155] [Formula I]
[0156] R.sup.L1' is a linker for connection to the antibody
(Ab);
[0157] R.sup.11a is selected from OH, OR.sup.A, where R.sup.A is
C.sub.1-4 alkyl, and SO.sub.zM, where z is 2 or 3 and M is a
monovalent pharmaceutically acceptable cation;
[0158] R.sup.20 and R.sup.21 either together form a double bond
between the nitrogen and carbon atoms to which they are bound
or;
[0159] R.sup.20 is selected from H and R.sup.C, where R.sup.C is a
capping group;
[0160] R.sup.21 is selected from OH, OR.sup.A and SO.sub.zM;
[0161] when there is a double bond present between C2 and C3,
R.sup.2 is selected from the group consisting of:
[0162] (ia) C.sub.5-10 aryl group, optionally substituted by one or
more substituents selected from the group comprising: halo, nitro,
cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7
heterocyclyl and bis-oxy-C.sub.1-3 alkylene;
[0163] (ib) C.sub.1-5 saturated aliphatic alkyl;
[0164] (ic) C.sub.3-6 saturated cycloalkyl;
[0165] (id)
##STR00008##
wherein each of R.sup.11, R.sup.12 and R.sup.13 are independently
selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl,
C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon
atoms in the R.sup.2 group is no more than 5;
[0166] (ie)
##STR00009##
wherein one of R.sup.15a and R.sup.15b is H and the other is
selected from: phenyl, which phenyl is optionally substituted by a
group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
and
[0167] (if)
##STR00010##
where R.sup.14 is selected from: H; C.sub.1-3 saturated alkyl;
C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which
phenyl is optionally substituted by a group selected from halo,
methyl, methoxy; pyridyl; and thiophenyl;
[0168] when there is a single bond present between C2 and C3,
[0169] R.sup.2 is
##STR00011##
where R.sup.16a and R.sup.16b are independently selected from H, F,
C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and
alkenyl groups are optionally substituted by a group selected from
C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of
R.sup.16a and R.sup.16b is H, the other is selected from nitrile
and a C.sub.1-4 alkyl ester;
[0170] [Formula II]
[0171] R.sup.22 is of formula IIIa, formula IIIb or formula
IIIc:
[0172] (a)
##STR00012##
[0173] where A is a C.sub.5-7 aryl group, and either
[0174] (i) Q.sup.1 is a single bond, and Q.sup.2 is selected from a
single bond and --Z--(CH.sub.2).sub.n--, where Z is selected from a
single bond, O, S and NH and n is from 1 to 3; or
[0175] (ii) Q.sup.1 is --CH.dbd.CH--, and Q.sup.2 is a single
bond;
[0176] (b)
##STR00013##
[0177] where;
[0178] R.sup.C1, R.sup.C2 and R.sup.C3 are independently selected
from H and unsubstituted C.sub.1-2 alkyl;
[0179] (c)
##STR00014##
[0180] where Q is selected from O--R.sup.L2', S--R.sup.L2' and
NR.sup.N--R.sup.L2', and R.sup.N is selected from H, methyl and
ethyl
[0181] X is selected from the group comprising: O--R.sup.L2',
S--R.sup.L2', CO.sub.2--R.sup.L2', CO--R.sup.L2',
NH--C(.dbd.O)--R.sup.L2', NHNH--R.sup.L2', CONHNH--R.sup.L2',
##STR00015##
NR.sup.NR.sup.L2', wherein R.sup.N is selected from the group
comprising H and C.sub.1-4 alkyl;
[0182] R.sup.L2' is a linker for connection to the antibody
(Ab);
[0183] R.sup.10 and R.sup.11 either together form a double bond
between the nitrogen and carbon atoms to which they are bound
or;
[0184] R.sup.10 is H and R.sup.11 is selected from OH, OR.sup.A and
SO.sub.zM;
[0185] R.sup.30 and R.sup.31 either together form a double bond
between the nitrogen and carbon atoms to which they are bound
or;
[0186] R.sup.30 is H and R.sup.31 is selected from OH, OR.sup.A and
SO.sub.zM. [Formula I and II] [0187] wherein: [0188] (1) the
antibody comprises an amino acid substitution of an interchain
cysteine residue by an amino acid that is not cysteine and the
conjugation of the drug moiety to the antibody is at an interchain
cysteine residue; and/or [0189] (2) the antibody comprises a heavy
chain having a substitution of the amino acid at position 234 in
the EU index set forth in Kabat and/or a substitution of the
residue at position 235 in the EU index set forth in Kabat.
[0190] In some embodiments, it may be preferred that the conjugate
is selected from a conjugate of formula ConjA, ConjB, ConjC, ConjD,
ConjE, ConjF, ConjG and ConjH:
##STR00016## ##STR00017## ##STR00018##
[0191] The link to the moiety shown is via a free S (active thiol)
of an interchain cysteine residue on the cell binding agent.
[0192] The subscript p in the formula I is an integer of from 1 to
20. Accordingly, the Conjugates comprise an antibody (Ab) as
defined herein covalently linked to at least one Drug unit by a
Linker unit. The Ligand unit, described more fully below, is a
targeting agent that binds to a target moiety. Accordingly, also
described herein are methods for the treatment of, for example,
various cancers and autoimmune disease. The drug loading is
represented by p, the number of drug molecules per antibody. Drug
loading may range from 1 to 20 Drug units (D.sup.L) per antibody.
For compositions, p represents the average drug loading of the
Conjugates in the composition, and p ranges from 1 to 20.
[0193] A second aspect of the disclosure provides a method of
making a conjugate according to the first aspect of the disclosure
comprising conjugating a compound of formula I.sup.L or
II.sup.L:
##STR00019##
[0194] to the antibody (Ab) as defined below, wherein:
[0195] R.sup.L1 is a linker suitable for conjugation to the
antibody (Ab);
[0196] R.sup.22L is of formula IIIa.sup.L, formula IIIb.sup.L or
formula IIIc.sup.L:
##STR00020##
[0197] where Q.sup.L is selected from O--R.sup.L2, S--R.sup.L2 and
NR.sup.N--R.sup.L2, and R.sup.N is sleected from H, methyl and
ethyl
[0198] X.sup.L is selected from the group comprising: O--R.sup.L2,
S--R.sup.L2, CO.sub.2--R.sup.L2, and CO--R.sup.L2, and
N.dbd.C.dbd.O--R.sup.L2, NHNH--R.sup.L2, and CONHNH--R.sup.L2,
##STR00021##
NR.sup.NR.sup.L, whereing R.sup.N is selected from the group
comprising H and C.sub.1-4 alkyl;
[0199] R.sup.L2 is a linker suitable for conjugation to the
antibody (Ab);
[0200] and all the remaining groups are as defined in the first
aspect.
[0201] Thus it may be preferred in the second aspect, that the
disclosure provides a method of making a conjugate selected from
the group consisting of ConjA, ConjB, ConjC, ConjD, ConjE, ConjF,
ConjG and ConjH comprising conjugating a compound which is selected
respectively from
##STR00022## ##STR00023## ##STR00024##
[0202] with an antibody as defined below.
[0203] Compounds A to E are disclosed in WO 2014/057073 and WO
2014/057074.
[0204] WO 2011/130613 discloses compound 51:
##STR00025##
[0205] WO 2013/041606 discloses Compound F (see compound 13e in WO
2013/041606). Compound F differs from compound 30 by only having a
(CH.sub.2).sub.3 tether between the PBD moieties, instead of a
(CH.sub.2).sub.5 tether, which reduces the lipophilicity of the
released PBD dimer. The linking group in compounds F and G is
attached to the C2-phenyl group in the para rather than meta
position.
[0206] Compound H has a cleavable protecting group on the second
imine group which avoids cross-reactions during its synthesis and
in the final product avoids the formation of carbinolamine and
carbinolamine methyl ethers. This protection also avoids the
presence of an reactive imine group in the molecule.
[0207] Compounds A, B, C, D, E, F, G and H have two sp.sup.2
centres in each C-ring, which may allow for stronger binding in the
minor groove of DNA, than for compounds with only one sp.sup.2
centre in each C-ring.
[0208] The drug linkers disclosed in WO 2010/043880, WO
2011/130613, WO 2011/130598, WO 2013/041606 and WO 2011/130616 may
be used in the present disclosure, and are incorporated herein by
reference. The drug linkers described herein may be synthesised as
described in these disclosures.
[0209] Delivery of PBD Compounds
[0210] The present disclosure is suitable for use in providing a
PBD compound to a preferred site in a subject. The conjugate may
allow the release of an active PBD compound that does not retain
any part of the linker. In such as case there is no stub present
that could affect the reactivity of the PBD compound.
[0211] ConjA would release the compound RelA:
##STR00026##
[0212] ConjB and ConjF would release the compound RelB:
##STR00027##
[0213] ConjC would release the compound RelC:
##STR00028##
[0214] ConjD would release the compound RelD:
##STR00029##
[0215] ConjE and ConjH would release the compound RelE:
##STR00030##
[0216] and ConjG would release the compound RelG:
##STR00031##
[0217] The speficied link between the PBD dimer and the antibody,
in the present disclosure is preferably stable extracellularly.
Before transport or delivery into a cell, the antibody-drug
conjugate (ADC) is preferably stable and remains intact, i.e. the
antibody remains linked to the drug moiety. The linkers are stable
outside the target cell and may be cleaved at some efficacious rate
inside the cell. An effective linker will: (i) maintain the
specific binding properties of the antibody; (ii) allow specific
intracellular delivery of the conjugate or drug moiety; (iii)
remain stable and intact, i.e. not cleaved, until the conjugate has
been delivered or transported to its targetted site; and (iv)
maintain a cytotoxic, cell-killing effect or a cytostatic effect of
the PBD drug moiety. Stability of the ADC may be measured by
standard analytical techniques such as in vitro cytotoxicity, mass
spectroscopy, HPLC, and the separation/analysis technique
LC/MS.
[0218] Delivery of the compounds of formulae RelA, RelB, RelC,
RelD, RelE or RelG is achieved at the desired activation site of
the conjugates of formulae ConjA, ConjB, ConjC, ConjD, ConjE,
ConhF, ConjG or ConjH by the action of an enzyme, such as
cathepsin, on the linking group, and in particular on the
valine-alanine dipeptide moiety.
[0219] The Antibody: Substitution of Interchain Cysteine
Residues
[0220] In a first aspect, the antibody of the conjugates described
herein comprise an amino acid substitution of an interchain
cysteine residue by an amino acid that is not cysteine.
[0221] Interchain Cysteine Residues
[0222] Naturally occurring antibodies generally include two larger
heavy chains and two smaller light chains. In the case of native
full-length antibodies, these chains join together to form a
"Y-shaped" protein. Heavy chains and light chains include cysteine
amino acids that can be joined to one another via disulphide
linkages. Heavy chains are joined to one another in an antibody by
disulphide linkages between cysteine amino acids in each chain.
Light chains are joined to heavy chains also by disulphide linkages
between cysteine amino acids in the chains. Such disulphide
linkages generally are formed between thiol side chain moieties of
the free cysteine amino acids. The cysteine amino acids which
typically take part in these interchain disulphide linkages in
naturally occurring antibodies are described herein as "interchain
cysteine residues" or "interchain cysteines". For example, three
particular cysteine amino acids in each IgG1 isotype heavy chain
(`HC`-220, 226, and 229 in the EU index set forth in Kabat) and one
particular cysteine in each light chain (`LC`-.kappa.(kappa)214 or
.lamda.(lambda)213) are "interchain cysteines" as they generally
participate in disulphide linkages between the antibody chains.
[0223] The interchain cysteine residues are located in the CL
domain of the light chain, the CH.sub.1 domain of the heavy chain,
and in the hinge region. The number of interchain cysteine residues
in an antibody depends on the antibody isotype.
[0224] Nature of Substitutions
[0225] As noted above, the antibody of the conjugates described
herein comprise an amino acid substitution of an interchain
cysteine residue by an amino acid that is not cysteine. The amino
acid substituted for an interchain cysteine typically does not
include a thiol moiety, and often is a valine, serine, threonine,
alanine, glycine, leucine, isoleucine, other naturally occurring
amino acid, or non-naturally occurring amino acid. In some
preferred embodiments, the amino acid substitution is a valine for
the interchain cysteine residue.
[0226] In some embodiments, one or more or all interchain cysteines
are `substituted` for no amino acid; that is, the one or more or
all interchain cysteines is deleted and not replaced by another
amino acid. Accordingly, in some embodiments the phrase ". . . a
light chain comprising the amino acid sequence of SEQ ID NO. XXX
wherein the cysteine at position YYY in SEQ ID NO: XXX, is
substituted by an amino acid that is not cysteine." Has the same
meaning as ". . . a light chain comprising the amino acid sequence
of SEQ ID NO. XXX wherein the cysteine at position YYY in SEQ ID
NO: XXX, is deleted."
[0227] For example, SEQ ID NO.153 as disclosed herein is an example
of "a light chain comprising the amino acid sequence of SEQ ID NO.
150 wherein the cysteine at position 105 in SEQ ID NO: 150, is
substituted by an amino acid that is not cysteine" wherein the
cysteine is substituted for no amino acid i.e. deleted.
[0228] In embodiments comprising "a light chain comprising the
amino acid sequence of SEQ ID NO. 160 wherein the cysteine at
position 102 in SEQ ID NO: 160, is deleted" the serine at positon
103 is also preferably deleted. See, for example, SEQ ID NO:
163.
[0229] Even when not explicitly stated, the terms "substituted" and
"a substitution" as used herein in reference to amino acids is used
to mean the replacement of an amino acid residue with a
different--that is, non-identical--amino acid residue (or with no
amino acid residue--that is, a deletion--as explained above). Thus,
an amino acid residue nominally `replacement` by an identical
reisdue--for example replacing a cysteine residue with a cysteine
residue--is not considered "substituted" or "a substitution".
[0230] As used herein, "substitution of a leucine by an amino acid
which is not leucine" means the replacement of the specified with
any non-leucine amino acid. This can be--for example--Asp, Glu,
Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, Cys, Gly, Ala, Val, Ile,
Phe, Trp, Pro, or Met, but is preferably Gly, Ala, Val, or Ile, and
most preferably Ala,
[0231] The statement in this "Nature of substitutions" section are
applicable to all three aspects of the disclosure described
herein.
[0232] Retention of Unsubstituted Interchain Cysteines
[0233] The antibody of the conjugates described herein retains at
least one unsubstituted interchain cysteine residue for conjugation
of the drug moiety to the antibody. The number of retained
interchain cysteine residues in the antibody is greater than zero
but less than the total number of interchain cysteine residues in
the parent (native) antibody. Thus, in some embodiments, the
antibody has at least one, at least two, at least three, at least
four, at least five, at least six or at least seven interchain
cysteine residues. In typical embodiments, the antibody has an even
integral number of interchain cysteine residues (e.g., at least
two, four, six or eight). In some embodiments, the antibody has
less than eight interchain cysteine residues.
[0234] In some embodiments, the antibody of the conjugates
described herein retains the unsubstituted hinge region interchain
cysteines. For example, in some embodiments the antibody retains
unsubstituted HC226 and HC229 according to the EU index as set
forth in Kabat.
[0235] In some embodiments, the antibody of the conjugates
described herein has an amino acid substitution of each of the
hinge region interchain cysteines. For example, in some embodiments
the antibody has an amino acid substitution of each of HC226 and
HC229 according to the EU index as set forth in Kabat.
[0236] In some embodiments, the antibody of the conjugates
described herein retains at least one unsubstituted hinge region
interchain cysteine. For example, in some embodiments the antibody
retains an unsubstituted HC226 according to the EU index as set
forth in Kabat. In some embodiments the antibody retains an
unsubstituted HC229 according to the EU index as set forth in
Kabat. In some embodiments each heavy chain retains exactly one
(i.e. not more than one) unsubstituted hinge region interchain
cysteine.
[0237] In some embodiments, the antibody of the conjugates
described herein has the amino acid substitution of valine for each
of the hinge region interchain cysteines. For example, in some
embodiments the antibody has the amino acid substitution of valine
each of HC226 and HC229 according to the EU index as set forth in
Kabat
[0238] Embodiments Defined Using the EU Index of Kabat
[0239] In some embodiments, the antibody of the conjugates
described herein comprise: (i) a light chain having an amino acid
substitution of the interchain cysteine residue located in the
C.sub.L domain, and (ii) a heavy chain retaining the unsubstituted
interchain cysteine located in the CH.sub.1 domain. For example, in
some embodiments, the antibody of the conjugates described herein
comprise: (i) a light chain having an amino acid substitution of
the interchain cysteine residue .kappa.LC214 or .lamda.LC213
according to the EU index as set forth in Kabat, and (ii) a heavy
chain retaining the unsubstituted interchain cysteine HC220
according to the EU index as set forth in Kabat. Preferably the
drug moiety is conjugated to the unsubstituted interchain cysteine
located in the CH.sub.1 domain, for example to HC220 according to
the EU index as set forth in Kabat.
[0240] In some embodiments, the antibody of the conjugates
described herein comprise: (i) light chains each having an amino
acid substitution of the interchain cysteine residue located in the
C.sub.L domain, and (ii) heavy chains each retaining the
unsubstituted interchain cysteine located in the CH.sub.1 domain.
For example, in some embodiments, the antibody of the conjugates
described herein comprise: (i) light chains each having an amino
acid substitution of the interchain cysteine residue .kappa.LC214
or .lamda.LC213 according to the EU index as set forth in Kabat,
and (ii) heavy chains each retaining the unsubstituted interchain
cysteine HC220 according to the EU index as set forth in Kabat.
Preferably the drug moiety is conjugated to the unsubstituted
interchain cysteine located in the CH.sub.1 domain, for example to
HC220 according to the EU index as set forth in Kabat.
[0241] In some embodiments, the antibody of the conjugates
described herein comprise: (i) a light chain retaining the
unsubstituted interchain cysteine located in the C.sub.L domain,
and (ii) a heavy chain having an amino acid substitution of the
interchain cysteine residue located in the CH.sub.1 domain. For
example, in some embodiments, the antibody of the conjugates
described herein comprise: (i) a light chain retaining the
unsubstituted interchain cysteine .kappa.LC214 or .lamda.LC213
according to the EU index as set forth in Kabat, and (ii) a heavy
chain having an amino acid substitution of the interchain cysteine
residue HC220 according to the EU index as set forth in Kabat. In
some embodiments the drug moiety is conjugated to the unsubstituted
interchain cysteine located in the C.sub.L domain, for example to
.kappa.LC214 or .lamda.LC213 according to the EU index as set forth
in Kabat.
[0242] In some embodiments, the antibody of the conjugates
described herein comprise: (i) light chains each retaining the
unsubstituted interchain cysteine located in the C.sub.L domain,
and (ii) heavy chains each having an amino acid substitution of the
interchain cysteine residue located in the CH.sub.1 domain. For
example, in some embodiments, the antibody of the conjugates
described herein comprise: (i) light chains each retaining the
unsubstituted interchain cysteine .kappa.LC214 or .lamda.LC213
according to the EU index as set forth in Kabat, and (ii) heavy
chains each having an amino acid substitution of the interchain
cysteine residue HC220 according to the EU index as set forth in
Kabat. In some embodiments the drug moiety is conjugated to the
unsubstituted interchain cysteine located in the C.sub.L domain,
for example to .kappa.LC214 or .lamda.LC213 according to the EU
index as set forth in Kabat.
[0243] AbLJ
[0244] In some embodiments the antibody of the conjugates described
herein: (i) retain the unsubstituted hinge region interchain
cysteines, (ii) comprise a light chain having an amino acid
substitution of the interchain cysteine residue located in the
C.sub.L domain, and (iii) comprise a heavy chain retaining the
unsubstituted interchain cysteine located in the CH.sub.1 domain.
For example, In some embodiments the antibody of the conjugates
described herein: (i) retains unsubstituted HC226 and HC229
according to the EU index as set forth in Kabat, (ii) comprise a
light chain having an amino acid substitution of the interchain
cysteine residue .kappa.LC214 or .lamda.LC213 according to the EU
index as set forth in Kabat, and (iii) comprise a heavy chain
retaining the unsubstituted interchain cysteine HC220 according to
the EU index as set forth in Kabat. Preferably the drug moiety is
conjugated to the unsubstituted interchain cysteine located in the
CH.sub.1 domain, for example to HC220 according to the EU index as
set forth in Kabat.
[0245] In some embodiments the antibody of the conjugates described
herein: (i) retain the unsubstituted hinge region interchain
cysteines, (ii) comprise light chains each having an amino acid
substitution of the interchain cysteine residue located in the
C.sub.L domain, and (iii) comprise heavy chains each retaining the
unsubstituted interchain cysteine located in the CH.sub.1 domain.
For example, In some embodiments the antibody of the conjugates
described herein: (i) retains unsubstituted HC226 and HC229
according to the EU index as set forth in Kabat, (ii) comprise
light chains each having an amino acid substitution of the
interchain cysteine residue .kappa.LC214 or .lamda.LC213 according
to the EU index as set forth in Kabat, and (iii) comprise heavy
chains each retaining the unsubstituted interchain cysteine HC220
according to the EU index as set forth in Kabat. Preferably the
drug moiety is conjugated to the unsubstituted interchain cysteine
located in the CH.sub.1 domain, for example to HC220 according to
the EU index as set forth in Kabat.
[0246] AbHJ
[0247] In some embodiments the antibody of the conjugates described
herein: (i) retain the unsubstituted hinge region interchain
cysteines, (ii) comprise a light chain retaining the unsubstituted
interchain cysteine located in the C.sub.L domain, and (iii)
comprise a heavy chain having an amino acid substitution of the
interchain cysteine residue located in the CH.sub.1 domain. For
example, In some embodiments the antibody of the conjugates
described herein: (i) retains unsubstituted HC226 and HC229
according to the EU index as set forth in Kabat, (ii) comprise a
light chain retaining the unsubstituted interchain cysteine
.kappa.LC214 or .lamda.LC213 according to the EU index as set forth
in Kabat, and (iii) comprise a heavy chain having an amino acid
substitution of interchain cysteine HC220 according to the EU index
as set forth in Kabat. Preferably the drug moiety is conjugated to
the unsubstituted interchain cysteine located in the C.sub.L
domain, for example to .kappa.LC214 or .lamda.LC213 according to
the EU index as set forth in Kabat.
[0248] In some embodiments the antibody of the conjugates described
herein: (i) retain the unsubstituted hinge region interchain
cysteines, (ii) comprise light chains each retaining the
unsubstituted interchain cysteine located in the C.sub.L domain,
and (iii) comprise heavy chains each having an amino acid
substitution of the interchain cysteine residue located in the
CH.sub.1 domain. For example, In some embodiments the antibody of
the conjugates described herein: (i) retains unsubstituted HC226
and HC229 according to the EU index as set forth in Kabat, (ii)
comprise light chains each retaining the unsubstituted interchain
cysteine .kappa.LC214 or .lamda.LC213 according to the EU index as
set forth in Kabat, and (iii) comprise heavy chains each having an
amino acid substitution of interchain cysteine HC220 according to
the EU index as set forth in Kabat. Preferably the drug moiety is
conjugated to the unsubstituted interchain cysteine located in the
C.sub.L domain, for example to .kappa.LC214 or .lamda.LC213
according to the EU index as set forth in Kabat.
[0249] AbBJ
[0250] In some embodiments the antibody of the conjugates described
herein: (i) has an amino acid substitution of each of the hinge
region interchain cysteines, (ii) comprise a light chain having an
amino acid substitution of the interchain cysteine residue located
in the C.sub.L domain, and (iii) comprise a heavy chain retaining
the unsubstituted interchain cysteine located in the CH.sub.1
domain. For example, in some embodiments the antibody of the
conjugates described herein: (i) has an amino acid substitution of
each of HC226 and HC229 according to the EU index as set forth in
Kabat, (ii) comprise a light chain having an amino acid
substitution of the interchain cysteine residue .kappa.LC214 or
.lamda.LC213 according to the EU index as set forth in Kabat, and
(iii) comprise a heavy chain retaining the unsubstituted interchain
cysteine HC220 according to the EU index as set forth in Kabat.
Preferably the drug moiety is conjugated to the unsubstituted
interchain cysteine located in the CH.sub.1 domain, for example to
HC220 according to the EU index as set forth in Kabat.
[0251] In some embodiments the antibody of the conjugates described
herein: (i) has an amino acid substitution of each of the hinge
region interchain cysteines, (ii) comprise light chains each having
an amino acid substitution of the interchain cysteine residue
located in the C.sub.L domain, and (iii) comprise heavy chains each
retaining the unsubstituted interchain cysteine located in the
CH.sub.1 domain. For example, in some embodiments the antibody of
the conjugates described herein: (i) has an amino acid substitution
of each of HC226 and HC229 according to the EU index as set forth
in Kabat, (ii) comprise light chains each having an amino acid
substitution of the interchain cysteine residue .kappa.LC214 or
.lamda.LC213 according to the EU index as set forth in Kabat, and
(iii) comprise heavy chains each retaining the unsubstituted
interchain cysteine HC220 according to the EU index as set forth in
Kabat. Preferably the drug moiety is conjugated to the
unsubstituted interchain cysteine located in the CH.sub.1 domain,
for example to HC220 according to the EU index as set forth in
Kabat.
[0252] In some embodiments the antibody of the conjugates described
herein: (i) has the amino acid substitution of valine for each of
the hinge region interchain cysteines, (ii) comprises a light chain
having an amino acid substitution of the interchain cysteine
residue located in the C.sub.L domain, and (iii) comprises a heavy
chain retaining the unsubstituted interchain cysteine located in
the CH.sub.1 domain. For example, in some embodiments the antibody
of the conjugates described herein: (i) has the amino acid
substitution of valine for each of HC226 and HC229 according to the
EU index as set forth in Kabat, (ii) comprises a light chain having
an amino acid substitution of the interchain cysteine residue
.kappa.LC214 or .lamda.LC213 according to the EU index as set forth
in Kabat, and (iii) comprises a heavy chain retaining the
unsubstituted interchain cysteine HC220 according to the EU index
as set forth in Kabat. Preferably the drug moiety is conjugated to
the unsubstituted interchain cysteine located in the CH.sub.1
domain, for example to HC220 according to the EU index as set forth
in Kabat.
[0253] In some embodiments the antibody of the conjugates described
herein: (i) has the amino acid substitution of valine for each of
the hinge region interchain cysteines, (ii) comprises light chains
each having an amino acid substitution of the interchain cysteine
residue located in the C.sub.L domain, and (iii) comprises heavy
chains each retaining the unsubstituted interchain cysteine located
in the CH.sub.1 domain. For example, in some embodiments the
antibody of the conjugates described herein: (i) has the amino acid
substitution of valine for each of HC226 and HC229 according to the
EU index as set forth in Kabat, (ii) comprises light chains each
having an amino acid substitution of the interchain cysteine
residue .kappa.LC214 or .lamda.LC213 according to the EU index as
set forth in Kabat, and (iii) comprises heavy chains each retaining
the unsubstituted interchain cysteine HC220 according to the EU
index as set forth in Kabat. Preferably the drug moiety is
conjugated to the unsubstituted interchain cysteine located in the
CH.sub.1 domain, for example to HC220 according to the EU index as
set forth in Kabat.
[0254] AbDJ
[0255] In some embodiments the antibody of the conjugates described
herein: (i) has the amino acid substitution of valine for each of
the hinge region interchain cysteines, (ii) comprises a light chain
retaining the unsubstituted interchain cysteine located in the
C.sub.L domain, and (iii) comprises a heavy chain having an amino
acid substitution of the interchain cysteine residue located in the
CH.sub.1 domain. For example, in some embodiments the antibody of
the conjugates described herein: (i) has an amino acid substitution
of each of HC226 and HC229 according to the EU index as set forth
in Kabat, (ii) comprises a light chain retaining the unsubstituted
interchain cysteine .kappa.LC214 or .lamda.LC213 according to the
EU index as set forth in Kabat, and (iii) comprises a heavy chain
having an amino acid substitution of interchain cysteine HC220
according to the EU index as set forth in Kabat. Preferably the
drug moiety is conjugated to the unsubstituted interchain cysteine
located in the C.sub.L domain, for example to .kappa.LC214 or
.lamda.LC213 according to the EU index as set forth in Kabat.
[0256] In some embodiments the antibody of the conjugates described
herein: (i) has an amino acid substitution of each of the hinge
region interchain cysteines, (ii) comprises light chains each
retaining the unsubstituted interchain cysteine located in the
C.sub.L domain, and (iii) comprises heavy chains each having an
amino acid substitution of the interchain cysteine residue located
in the CH.sub.1 domain. For example, in some embodiments the
antibody of the conjugates described herein: (i) has an amino acid
substitution of each of HC226 and HC229 according to the EU index
as set forth in Kabat, (ii) comprises light chains each retaining
the unsubstituted interchain cysteine .kappa.LC214 or .lamda.LC213
according to the EU index as set forth in Kabat, and (iii)
comprises heavy chains each having an amino acid substitution of
interchain cysteine HC220 according to the EU index as set forth in
Kabat. Preferably the drug moiety is conjugated to the
unsubstituted interchain cysteine located in the C.sub.L domain,
for example to .kappa.LC214 or .lamda.LC213 according to the EU
index as set forth in Kabat.
[0257] In some embodiments the antibody of the conjugates described
herein: (i) has an amino acid substitution of each of the hinge
region interchain cysteines, (ii) comprises a light chain retaining
the unsubstituted interchain cysteine located in the C.sub.L
domain, and (iii) comprises a heavy chain having an amino acid
substitution of the interchain cysteine residue located in the
CH.sub.1 domain. For example, in some embodiments the antibody of
the conjugates described herein: (i) has the amino acid
substitution of valine for each of HC226 and HC229 according to the
EU index as set forth in Kabat, (ii) comprises a light chain
retaining the unsubstituted interchain cysteine .kappa.LC214 or
.lamda.LC213 according to the EU index as set forth in Kabat, and
(iii) comprises a heavy chain having an amino acid substitution of
interchain cysteine HC220 according to the EU index as set forth in
Kabat. Preferably the drug moiety is conjugated to the
unsubstituted interchain cysteine located in the C.sub.L domain,
for example to .kappa.LC214 or .lamda.LC213 according to the EU
index as set forth in Kabat.
[0258] In some embodiments the antibody of the conjugates described
herein: (i) has the amino acid substitution of valine for each of
the hinge region interchain cysteines, (ii) comprises light chains
each retaining the unsubstituted interchain cysteine located in the
C.sub.L domain, and (iii) comprises heavy chains each having an
amino acid substitution of the interchain cysteine residue located
in the CH.sub.1 domain. For example, in some embodiments the
antibody of the conjugates described herein: (i) has the amino acid
substitution of valine for each of HC226 and HC229 according to the
EU index as set forth in Kabat, (ii) comprises light chains each
retaining the unsubstituted interchain cysteine .kappa.LC214 or
.lamda.LC213 according to the EU index as set forth in Kabat, and
(iii) comprises heavy chains each having an amino acid substitution
of interchain cysteine HC220 according to the EU index as set forth
in Kabat. Preferably the drug moiety is conjugated to the
unsubstituted interchain cysteine located in the C.sub.L domain,
for example to .kappa.LC214 or .lamda.LC213 according to the EU
index as set forth in Kabat.
[0259] Corrspondence Between the Kabat System and the Disclosed
Sequences
[0260] The following Table 1 illustrates positions of interchain
cysteines in the heavy chain constant region and light chain
constant region of particular antibody isotypes according to the EU
index as set forth in Kabat and with reference to the sequences
disclosed herein. Each of the interchain cysteine positions present
in an antibody or antibody fragment may be substituted with an
amino acid that is not a cysteine.
TABLE-US-00001 TABLE 1 Antibody Kabat EU/SEQ Isotype ID NO Position
of Cysteine HC Kabat EU position 131 220 n/a n/a 226 229 IgG1
Corresponding n/a 103 n/a n/a 109 112 position in SEQ ID NO: 110
IgG2 Corresponding 14 103 n/a n/a 106 109 position in SEQ ID NO:
120 IgG3 Corresponding 14 n/a n/a n/a 111 114 position in SEQ ID
NO: 130 IgG4 Corresponding 14 n/a n/a n/a 106 109 position in SEQ
ID NO: 140 LC .kappa. Kabat EU position 214 Corresponding 105
position in SEQ ID NO: 150 .lamda. Kabat EU position 213
Corresponding 102 position in SEQ ID NO: 160
[0261] Heavy Chain and Light Chain Embodiments Defined Using
Disclosed Sequences
[0262] AbLJ Heavy Chain
[0263] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.110 or fragment thereof, SEQ ID NO.120 or
fragment thereof, SEQ ID NO.130 or fragment thereof, or SEQ ID
NO.140 or fragment thereof. Preferably the drug moiety is
conjugated to the cysteine at position 103 of SEQ ID NO.110, the
cysteine at position 14 of SEQ ID NO.120, the cysteine at position
14 of SEQ ID NO.130, or the cysteine at position 14 of SEQ ID
NO.140.
[0264] AbHJ Heavy Chain
[0265] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, or fragment thereof, wherein the
cysteine at position 103 of SEQ ID NO.110, if present, is
substituted by an amino acid that is not cysteine. For example, SEQ
ID NO. 111 discloses a heavy chain comprising the amino acid
sequence of SEQ ID NO.110 wherein the cysteine at position 103 of
SEQ ID NO.110 is substituted by a serine residue. SEQ ID NO. 112
discloses a heavy chain comprising the amino acid sequence of SEQ
ID NO.110 wherein the cysteine at position 103 of SEQ ID NO.110 is
substituted by a valine residue.
[0266] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.120, or fragment thereof, wherein the
cysteine at positions 14 of SEQ ID NO.120, if present, is
substituted by an amino acid that is not cysteine.
[0267] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, or fragment thereof, wherein the
cysteine at position 14 in SEQ ID NO: 130, if present, is
substituted by an amino acid that is not cysteine.
[0268] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, or fragment thereof, wherein the
cysteine at position 14 in SEQ ID NO: 140, if present, is
substituted by an amino acid that is not cysteine.
[0269] AbBJ Heavy Chain
[0270] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, or fragment thereof, wherein each of the
cysteines at positions 109 and 112 in SEQ ID NO: 110, if present,
is substituted by an amino acid that is not cysteine. For example,
SEQ ID NO: 113 dislcoses a heavy chain comprising the amino acid
sequence of SEQ ID NO.110 wherein each of the cysteines at
positions 109 and 112 in SEQ ID NO: 110 is substituted by a serine
residue. SEQ ID NO: 114 dislcoses a heavy chain comprising the
amino acid sequence of SEQ ID NO.110 wherein each of the cysteines
at positions 109 and 112 in SEQ ID NO: 110 is substituted by a
valine residue. Preferably the drug moiety is conjugated to the
cysteine at position 103 of SEQ ID NO.110. In some embodiments, the
cysteine at position 109 in SEQ ID NO: 110, if present, is
substituted by an amino acid that is not cysteine, and the cysteine
at position 112 in SEQ ID NO: 110, if present, is unsubstituted. In
some embodiments, the cysteine at position 112 in SEQ ID NO: 110,
if present, is substituted by an amino acid that is not cysteine,
and the cysteine at position 109 in SEQ ID NO: 110, if present, is
unsubstituted.
[0271] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.120, or fragment thereof, wherein each of the
cysteines at positions 103, 106, and 109 in SEQ ID NO: 120, if
present, is substituted by an amino acid that is not cysteine. In
some embodiments, the cysteine at position 102 in SEQ ID NO: 120,
if present, is also substituted by an amino acid that is not
cysteine. In some embodiments, all but one of the cysteines at
positions 103, 106, 109, and 102 in SEQ ID NO: 120, if present, are
substituted by an amino acid that is not cysteine. For example, in
some embodiments, the cysteine at position 103, 106, 109, or 102 in
SEQ ID NO: 120, if present, is unsubstituted. Preferably the drug
moiety is conjugated to the cysteine at position 14 of SEQ ID
NO.120.
[0272] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, or fragment thereof, wherein each of the
cysteines at positions 111, 114, 120, 126, 129, 135, 141, 144, 150,
156, and 159 in SEQ ID NO: 130, if present, is substituted by an
amino acid that is not cysteine. In some embodiments, all but one
of the cysteines at positions 111, 114, 120, 126, 129, 135, 141,
144, 150, 156, and 159 in SEQ ID NO: 130, if present, are
substituted by an amino acid that is not cysteine. For example, in
some embodiments, the cysteine at position 111, 114, 120, 126, 129,
135, 141, 144, 150, 156, or 159 in SEQ ID NO: 130, if present, is
unsubstituted. Preferably the drug moiety is conjugated to the
cysteine at position 14 of SEQ ID NO.130.
[0273] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, or fragment thereof, wherein each of the
cysteines at positions 106 and 109 in SEQ ID NO: 140, if present,
is substituted by an amino acid that is not cysteine. In some
embodiments, the cysteine at position 106 in SEQ ID NO: 140, if
present, is substituted by an amino acid that is not cysteine, and
the cysteine at position 109 in SEQ ID NO: 140, if present, is
unsubstituted. In some embodiments, the cysteine at position 109 in
SEQ ID NO: 140, if present, is substituted by an amino acid that is
not cysteine, and the cysteine at position 106 in SEQ ID NO: 140,
if present, is unsubstituted. Preferably the drug moiety is
conjugated to the cysteine at position 14 of SEQ ID NO.140.
[0274] AbDJ Heavy Chain
[0275] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, or fragment thereof, wherein each of the
cysteines at positions 103, 109 and 112 in SEQ ID NO: 110, if
present, is substituted by an amino acid that is not cysteine. For
example, SEQ ID NO: 115 discloses a heavy chain comprising the
amino acid sequence of SEQ ID NO.110 wherein each of the cysteines
at positions 103, 109 and 112 in SEQ ID NO: 110 is substituted by a
serine residue. SEQ ID NO: 116 discloses a heavy chain comprising
the amino acid sequence of SEQ ID NO.110 wherein each of the
cysteines at positions 103, 109 and 112 in SEQ ID NO: 110 is
substituted by a valine residue. In some embodiments, the cysteine
at position 109 in SEQ ID NO: 110, if present, is substituted by an
amino acid that is not cysteine, and the cysteine at position 112
in SEQ ID NO: 110, if present, is unsubstituted. In some
embodiments, the cysteine at position 112 in SEQ ID NO: 110, if
present, is substituted by an amino acid that is not cysteine, and
the cysteine at position 109 in SEQ ID NO: 110, if present, is
unsubstituted.
[0276] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.120, or fragment thereof, wherein each of the
cysteines at positions 14, 103, 106 and 109 in SEQ ID NO: 120, if
present, is substituted by an amino acid that is not cysteine. In
some embodiments, all but one of the cysteines at positions 103,
106, 109, and 102 in SEQ ID NO: 120, if present, are substituted by
an amino acid that is not cysteine. For example, in some
embodiments, the cysteine at position 103, 106, 109, or 102 in SEQ
ID NO: 120, if present, is unsubstituted.
[0277] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, or fragment thereof, wherein each of the
cysteines at positions 14, 111, 114, 120, 126, 129, 135, 141, 144,
150, 156, and 159 in SEQ ID NO: 130, if present, is substituted by
an amino acid that is not cysteine. In some embodiments, all but
one of the cysteines at positions 111, 114, 120, 126, 129, 135,
141, 144, 150, 156, and 159 in SEQ ID NO: 130, if present, are
substituted by an amino acid that is not cysteine. For example, in
some embodiments, the cysteine at position 111, 114, 120, 126, 129,
135, 141, 144, 150, 156, or 159 in SEQ ID NO: 130, if present, is
unsubstituted.
[0278] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, or fragment thereof, wherein each of the
cysteines at positions 14, 106, and 109 in SEQ ID NO: 140, if
present, is substituted by an amino acid that is not cysteine. In
some embodiments, the cysteine at position 106 in SEQ ID NO: 140,
if present, is substituted by an amino acid that is not cysteine,
and the cysteine at position 109 in SEQ ID NO: 140, if present, is
unsubstituted. In some embodiments, the cysteine at position 109 in
SEQ ID NO: 140, if present, is substituted by an amino acid that is
not cysteine, and the cysteine at position 106 in SEQ ID NO: 140,
if present, is unsubstituted.
[0279] Light Chains
[0280] In some embodiments, the antibody of the conjugates
described herein comprises a light chain comprising the amino acid
sequence of SEQ ID NO. 150, or fragment thereof, or SEQ ID NO. 160
or fragment thereof. Preferably the drug moiety is conjugated to
the cysteine at position 105 of SEQ ID NO.150, the cysteine at
position 102 of SEQ ID NO.160.
[0281] In some embodiments, the antibody of the conjugates
described herein comprises a light chain comprising the amino acid
sequence of SEQ ID NO. 150, or fragment thereof, wherein the
cysteine at position 105, if present, is substituted by an amino
acid that is not cysteine. For example, SEQ ID NO. 151 discloses a
light chain comprising the amino acid sequence of SEQ ID NO. 150
wherein the cysteine at position 105 is substituted by a serine
residue. SEQ ID NO. 152 discloses a light chain comprising the
amino acid sequence of SEQ ID NO. 150 wherein the cysteine at
position 105 is substituted by a valine residue. SEQ ID NO. 153
discloses a light chain having the amino acid sequence of SEQ ID
NO. 150, wherein the cysteine at position 105 has been deleted.
[0282] In some embodiments, the antibody of the conjugates
described herein comprises a light chain comprising the amino acid
sequence of SEQ ID NO. 160, or fragment thereof, wherein the
cysteine at position 102, if present, is substituted by an amino
acid that is not cysteine. For example, SEQ ID NO. 161 discloses a
light chain comprising the amino acid sequence of SEQ ID NO. 160
wherein the cysteine at position 102 is substituted by a serine
residue. SEQ ID NO. 162 discloses a light chain comprising the
amino acid sequence of SEQ ID NO. 160 wherein the cysteine at
position 102 is substituted by a valine residue. SEQ ID NO. 163
discloses a light chain having the amino acid sequence of SEQ ID
NO. 160, wherein the cysteine at position 102 and the serine at
position 103 have been deleted.
[0283] Immunoglobulin Embodiments Defined Using Disclosed Sequences
AbLJ IgG1
[0284] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0285] wherein
the cysteine at position 105 in SEQ ID NO: 150 or the cysteine at
position 102 in SEQ ID NO: 160, is substituted by an amino acid
that is not cysteine. Preferably the drug moiety is conjugated to
the cysteine at position 103 of SEQ ID NO.110.
[0286] AbLJ IgG2
[0287] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.120, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0288] wherein
the cysteine at position 105 in SEQ ID NO: 150 or the cysteine at
position 102 in SEQ ID NO: 160, is substituted by an amino acid
that is not cysteine. Preferably the drug moiety is conjugated to
the cysteine at position 14 of SEQ ID NO.120.
[0289] AbLJ IgG3
[0290] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0291] wherein
the cysteine at position 105 in SEQ ID NO: 150 or the cysteine at
position 102 in SEQ ID NO: 160, is substituted by an amino acid
that is not cysteine. Preferably the drug moiety is conjugated to
the cysteine at position 14 of SEQ ID NO.130.
[0292] AbLJ IgG4
[0293] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0294] wherein
the cysteine at position 105 in SEQ ID NO: 150 or the cysteine at
position 102 in SEQ ID NO: 160, is substituted by an amino acid
that is not cysteine. Preferably the drug moiety is conjugated to
the cysteine at position 14 of SEQ ID NO.140.
[0295] AbHJ IgG1
[0296] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0297] wherein
the cysteine at position 103 in SEQ ID NO: 110 is substituted by an
amino acid that is not cysteine. Preferably the drug moiety is
conjugated to the cysteine at position 105 of SEQ ID NO.150, the
cysteine at position 102 of SEQ ID NO.160.
[0298] AbHJ IgG2
[0299] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.120, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0300] wherein
each of the cysteines at positions 14 and 103 in SEQ ID NO: 120 is
substituted by an amino acid that is not cysteine. Preferably the
drug moiety is conjugated to the cysteine at position 105 of SEQ ID
NO.150, the cysteine at position 102 of SEQ ID NO.160.
[0301] AbHJ IgG3
[0302] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0303] wherein
the cysteine at position 14 in SEQ ID NO: 130 is substituted by an
amino acid that is not cysteine. Preferably the drug moiety is
conjugated to the cysteine at position 105 of SEQ ID NO.150, the
cysteine at position 102 of SEQ ID NO.160.
[0304] AbHJ IgG4
[0305] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0306] wherein
the cysteine at position 14 in SEQ ID NO: 140 is substituted by an
amino acid that is not cysteine. Preferably the drug moiety is
conjugated to the cysteine at position 105 of SEQ ID NO.150, the
cysteine at position 102 of SEQ ID NO.160.
[0307] AbBJ IgG1
[0308] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0309] wherein
each of the cysteines at positions 109 and 112 in SEQ ID NO: 110 is
substituted by an amino acid that is not cysteine; [0310] and
wherein the cysteine at position 105 in SEQ ID NO: 150 or the
cysteine at position 102 in SEQ ID NO: 160, is substituted by an
amino acid that is not cysteine.
[0311] Preferably the drug moiety is conjugated to the cysteine at
position 103 of SEQ ID NO.110.
[0312] In some embodiments the cysteines at positions 109 and 112
in SEQ ID NO: 110 are substituted for valine. In some embodiments
the cysteine at position 105 in SEQ ID NO: 150 or the cysteine at
position 102 in SEQ ID NO: 160 is substituted by serine.
[0313] AbBJ IgG2A
[0314] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.120, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0315] wherein
each of the cysteines at positions 103, 106, and 109 in SEQ ID NO:
120 is substituted by an amino acid that is not cysteine; [0316]
and wherein the cysteine at position 105 in SEQ ID NO: 150 or the
cysteine at position 102 in SEQ ID NO: 160, is substituted by an
amino acid that is not cysteine.
[0317] In some embodiments, the cysteine at position 102 in SEQ ID
NO: 120 is also substituted by an amino acid that is not
cysteine.
[0318] Preferably the drug moiety is conjugated to the cysteine at
position 14 of SEQ ID NO.120.
[0319] In some embodiments the cysteines at positions 103, 106, and
109 in SEQ ID NO: 120 are substituted for valine. In some
embodiments the cysteine at position 105 in SEQ ID NO: 150 or the
cysteine at position 102 in SEQ ID NO: 160, is substituted by
serine.
[0320] AbBJ IgG2B
[0321] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.120, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0322] wherein
each of the cysteines at positions 14, 106, and 109 in SEQ ID NO:
120 is substituted by an amino acid that is not cysteine; [0323]
and wherein the cysteine at position 105 in SEQ ID NO: 150 or the
cysteine at position 102 in SEQ ID NO: 160, is substituted by an
amino acid that is not cysteine.
[0324] In some embodiments, the cysteine at position 102 in SEQ ID
NO: 120 is also substituted by an amino acid that is not
cysteine.
[0325] Preferably the drug moiety is conjugated to the cysteine at
position 103 of SEQ ID NO.120.
[0326] In some embodiments the cysteines at positions 14, 106, and
109 in SEQ ID NO: 120 are substituted for valine. In some
embodiments the cysteine at position 105 in SEQ ID NO: 150 or the
cysteine at position 102 in SEQ ID NO: 160, is substituted by
serine.
[0327] AbBJ IgG3
[0328] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0329] wherein
each of the cysteines at positions 111, 114, 120, 126, 129, 135,
141, 144, 150, 156, and 159 in SEQ ID NO: 130 is substituted by an
amino acid that is not cysteine; [0330] and wherein the cysteine at
position 105 in SEQ ID NO: 150 or the cysteine at position 102 in
SEQ ID NO: 160, is substituted by an amino acid that is not
cysteine.
[0331] Preferably the drug moiety is conjugated to the cysteine at
position 14 of SEQ ID NO.130.
[0332] In some embodiments each of the cysteines at positions 111,
114, 120, 126, 129, 135, 141, 144, 150, 156, and 159 in SEQ ID NO:
130 for valine.
[0333] In some embodiments the cysteine at position 105 in SEQ ID
NO: 150 or the cysteine at position 102 in SEQ ID NO: 160, is
substituted by serine.
[0334] AbBJ IgG4
[0335] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0336] wherein
each of the cysteines at positions 106 and 109 in SEQ ID NO: 140 is
substituted by an amino acid that is not cysteine; [0337] and
wherein the cysteine at position 105 in SEQ ID NO: 150 or the
cysteine at position 102 in SEQ ID NO: 160, is substituted by an
amino acid that is not cysteine.
[0338] Preferably the drug moiety is conjugated to the cysteine at
position 14 of SEQ ID NO.140.
[0339] Preferably the drug moiety is conjugated to the cysteine at
position 14 of SEQ ID NO.140.
[0340] In some embodiments each of the cysteines at positions 106
and 109 in SEQ ID NO: 140 are substituted for valine. In some
embodiments the cysteine at position 105 in SEQ ID NO: 150 or the
cysteine at position 102 in SEQ ID NO: 160, is substituted by
serine.
[0341] AbDJ IgG1
[0342] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0343] wherein
each of the cysteines at positions 103, 109 and 112 in SEQ ID NO:
110 is substituted by an amino acid that is not cysteine.
[0344] Preferably the drug moiety is conjugated to the cysteine at
position 105 of SEQ ID NO.150, the cysteine at position 102 of SEQ
ID NO.160.
[0345] AbDJ IgG2
[0346] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.120, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0347] wherein
each of the cysteines at positions 14, 103, 106 and 109 in SEQ ID
NO: 120 is substituted by an amino acid that is not cysteine.
[0348] Preferably the drug moiety is conjugated to the cysteine at
position 105 of SEQ ID NO.150, the cysteine at position 102 of SEQ
ID NO.160.
[0349] AbDJ IgG3
[0350] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0351] wherein
each of the cysteines at positions 14, 111, 114, 120, 126, 129,
135, 141, 144, 150, 156, and 159 in SEQ ID NO: 130 is substituted
by an amino acid that is not cysteine.
[0352] Preferably the drug moiety is conjugated to the cysteine at
position 105 of SEQ ID NO.150, the cysteine at position 102 of SEQ
ID NO.160.
[0353] AbDJ IgG4
[0354] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0355] wherein
each of the cysteines at positions 14, 106, and 109 in SEQ ID NO:
140 is substituted by an amino acid that is not cysteine.
[0356] Preferably the drug moiety is conjugated to the cysteine at
position 105 of SEQ ID NO.150, the cysteine at position 102 of SEQ
ID NO.160.
[0357] The Antibody: Substitution of Kabat EU Residues 234 and/or
235
[0358] In a second aspect, the antibody of the conjugates described
herein comprises a heavy chain having a substitution of the residue
at position 234 in the EU index set forth in Kabat and/or a
substitution of the residue at position 235 in the EU index set
forth in Kabat. It has been unexpectedly found that ADCs in which
the antibody bears one, or preferably both, of these substitutions
have improved tolerability and increased serum half-lives as
compared to otherwise identical ADCs comprising antibodies which
lack the specific mutations.
[0359] Substitution at Kabat EU 234/235
[0360] Hezareh, M. et al., Journal of Virology, Vol. 75, No. 24,
pp. 12161-12168 (2001) discloses an IgG1 antibody mutant comprising
a heavy chain in which the leucine residue at Kabat EU 234 and the
leucine residue at Kabat EU 235 are both substituted for alanine;
the antibody is described in that reference as "IgG1 b12 (L234A,
L235A)". Hazareh et al. does not disclose the IgG1 b12 (L234A,
L235A) as part of an ADC.
[0361] Hazareh et al. report that introduction of the L234A/L235A
double mutation resulted in complete loss of antibody binding by
the Fc(gamma)R and C1q proteins, with consequent abolition of both
antibody-dependent cellular cytotoxicity (ADCC) and
complement-dependent cytotoxicity (CDC).
[0362] Wines, B. D., et al., Journal of Immmunology, Vol. 164, pp.
5313-5318 (2000) shares authors with Hazareh et al. and also
describes an L234A/L235A double mutant. There the authors report
that the L234A/L235A double mutant slightly reduces (<25%)
antibody binding to the FcRn receptor. The FcRn receptor is known
to have an important role in antibody recycling, with increased
antibody/FcRn affinity reported to extend antibody half-life in
vivo and improve anti-tumour activity (see Zalevsky, J., Nature
Biotechnology 28, 157-159 (2010) [doi:10.1038/nbt.1601]). However,
in view of the size of the decrease in FcRn affinity, the authors
of Hazareh et al. conclude that the L234A/L235A double mutation is
not expected to significantly reduce the antibody's serum
half-life.
[0363] Contrary to the expectation following from the above
disclosures, it has been found that the ADCs disclosed herein which
comprise a heavy chain having substitutions of the residues at
positions 234 and 235 in the EU index set forth in Kabat actually
have increased serum half-lives as compared to otherwise identical
ADCs comprising antibodies which lack the mutations. Furthermore,
the ADCs comprising a heavy chain having substitutions of the
residues at positions 234 and 235 in the EU index set forth also
exhibit improved tolerability / reduced toxicity as compared to
otherwise identical ADCs comprising antibodies which lack the
mutations.
[0364] Embodiments Defined Using the EU Index of Kabat
[0365] Accordingly, in a second aspect the antibody of the
conjugates described herein comprises a heavy chain having a
substitution of the residue at position 234 in the EU index set
forth in Kabat and/or a substitution of the residue at position 235
in the EU index set forth in Kabat. Preferably both the residues at
position 234 and 235 in the EU index set forth in Kabat are
substituted by any other amino acid.
[0366] In some embodiments the antibody is an IgG1 isotype and the
leucine at position 234 in the EU index set forth in Kabat and/or
the leucine at position 235 in the EU index set forth in Kabat is
substituted by an amino acid that is not leucine.
[0367] In some embodiments the antibody is an IgG3 isotype and the
leucine at position 234 in the EU index set forth in Kabat and/or
the leucine at position 235 in the EU index set forth in Kabat is
substituted by an amino acid that is not leucine.
[0368] In some embodiments the antibody is an IgG4 isotype and the
leucine at position 235 in the EU index set forth in Kabat is
substituted by an amino acid that is not leucine, such as
alanine.
[0369] Corrspondence Between the Kabat System and the Disclosed
Sequences.
[0370] The following Table 2 illustrates positions of corresponding
residues in the heavy chain constant region of particular antibody
isotypes according to the EU index as set forth in Kabat and with
reference to the sequences disclosed herein.
TABLE-US-00002 TABLE 2 Antibody Isotype Kabat EU/SEQ ID NO Position
of Residue HC Kabat EU position 234 235 IgG1 Corresponding position
in 117 118 SEQ ID NO: 110 IgG2 Corresponding position in -- -- SEQ
ID NO: 120 IgG3 Corresponding position in 164 165 SEQ ID NO: 130
IgG4 Corresponding position in -- 115 SEQ ID NO: 140
[0371] Immunoglobulin Embodiments Defined Using Disclosed
Sequences
[0372] In some embodiments the antibody of the conjugates described
herein comprises a heavy chain comprising the amino acid sequence
of SEQ ID NO.110, wherein the leucine at position 117 and/or the
leucine at position 118 is substituted by an amino acid that is not
leucine, such as alanine. Preferably both the leucines at position
117 and 118 are substituted by an amino acid that is not leucine,
such as alanine.
[0373] In some embodiments the antibody of the conjugates described
herein comprises a heavy chain comprising the amino acid sequence
of SEQ ID NO.130, wherein the leucine at position 164 and/or the
leucine at position 165 is substituted by an amino acid that is not
leucine, such as alanine. Preferably both the leucines at position
164 and 165 are substituted by an amino acid that is not leucine,
such as alanine.
[0374] In some embodiments the antibody of the conjugates described
herein comprises a heavy chain comprising the amino acid sequence
of SEQ ID NO.140, wherein the leucine at position 115 is
substituted by an amino acid that is not leucine, such as
alanine.
[0375] The Antibody: Substitution of Interchain Cysteine Residues
Combined with Substitution of Kabat EU Residues 234 and/or 235
[0376] The modifications described in the first aspect can be
advantageously combined in the same antibody with the modifications
described in the second aspect. Accordingly, in a third aspect the
antibody of the conjugates described herein: [0377] (1) comprises
one or more substitution of an interchain cysteine residue by an
amino acid that is not cysteine and retains at least one
unsubstituted interchain cysteine residue for conjugation of the
drug moiety to the antibody; and [0378] (2) comprises a heavy chain
having a substitution of the residue at position 234 in the EU
index set forth in Kabat and/or a substitution of the residue at
position 235 in the EU index set forth in Kabat by any other amino
acid (that is, an amino acid that is not identical to that found in
the `wild-type` sequence).
[0379] Embodiments Defined Using the Kabat EU Numbering
[0380] AbLJ(LALA)
[0381] In some embodiments the antibody of the conjugates described
herein: (i) retain the unsubstituted hinge region interchain
cysteines, (ii) comprise light chains each having an amino acid
substitution of the interchain cysteine residue located in the
C.sub.L domain, (iii) comprise heavy chains each retaining the
unsubstituted interchain cysteine located in the CH.sub.1 domain,
and (iv) comprise heavy chains each having an amino acid
substitution of the the residue at position 234 in the EU index set
forth in Kabat and/or a substitution of the residue at position 235
in the EU index set forth in Kabat.
[0382] For example, In some embodiments the antibody of the
conjugates described herein: (i) retains unsubstituted HC226 and
HC229 according to the EU index as set forth in Kabat, (ii)
comprise light chains each having an amino acid substitution of the
interchain cysteine residue .kappa.LC214 or .lamda.LC213 according
to the EU index as set forth in Kabat, (iii) comprise heavy chains
each retaining the unsubstituted interchain cysteine HC220
according to the EU index as set forth in Kabat, and (iv) comprise
heavy chains each having an amino acid substitution of the the
residue at position 234 in the EU index set forth in Kabat and/or a
substitution of the residue at position 235 in the EU index set
forth in Kabat by any other amino acid. Preferably both the
residues at position 234 and 235 in the EU index set forth in Kabat
are substituted. Preferably the drug moiety is conjugated to the
unsubstituted interchain cysteine located in the CH.sub.1 domain,
for example to HC220 according to the EU index as set forth in
Kabat.
[0383] AbHJ(LALA)
[0384] In some embodiments the antibody of the conjugates described
herein: (i) retain the unsubstituted hinge region interchain
cysteines, (ii) comprise light chains each retaining the
unsubstituted interchain cysteine located in the C.sub.L domain,
(iii) comprise heavy chains each having an amino acid substitution
of the interchain cysteine residue located in the CH.sub.1 domain,
and (iv) comprise heavy chains each having an amino acid
substitution of the the residue at position 234 in the EU index set
forth in Kabat and/or a substitution of the residue at position 235
in the EU index set forth in Kabat.
[0385] For example, In some embodiments the antibody of the
conjugates described herein: (i) retains unsubstituted HC226 and
HC229 according to the EU index as set forth in Kabat, (ii)
comprise light chains each retaining the unsubstituted interchain
cysteine .kappa.LC214 or .lamda.LC213 according to the EU index as
set forth in Kabat, (iii) comprise heavy chains each having an
amino acid substitution of interchain cysteine HC220 according to
the EU index as set forth in Kabat, and (iv) comprise heavy chains
each having an amino acid substitution of the the residue at
position 234 in the EU index set forth in Kabat and/or a
substitution of the residue at position 235 in the EU index set
forth in Kabat by any other amino acid. Preferably both the
residues at position 234 and 235 in the EU index set forth in Kabat
are substituted. Preferably the drug moiety is conjugated to the
unsubstituted interchain cysteine located in the C.sub.L domain,
for example to .kappa.LC214 or .lamda.LC213 according to the EU
index as set forth in Kabat.
[0386] AbBJ(LALA)
[0387] In some embodiments the antibody of the conjugates described
herein: (i) has an amino acid substitution of each of the hinge
region interchain cysteines, (ii) comprise light chains each having
an amino acid substitution of the interchain cysteine residue
located in the C.sub.L domain, (iii) comprise heavy chains each
retaining the unsubstituted interchain cysteine located in the
CH.sub.1 domain, and (iv) comprise heavy chains each having an
amino acid substitution of the the residue at position 234 in the
EU index set forth in Kabat and/or a substitution of the residue at
position 235 in the EU index set forth in Kabat.
[0388] For example, in some embodiments the antibody of the
conjugates described herein: (i) has an amino acid substitution of
each of HC226 and HC229 according to the EU index as set forth in
Kabat, (ii) comprise light chains each having an amino acid
substitution of the interchain cysteine residue .kappa.LC214 or
.lamda.LC213 according to the EU index as set forth in Kabat, (iii)
comprise heavy chains each retaining the unsubstituted interchain
cysteine HC220 according to the EU index as set forth in Kabat, and
(iv) comprise heavy chains each having an amino acid substitution
of the the residue at position 234 in the EU index set forth in
Kabat and/or a substitution of the residue at position 235 in the
EU index set forth in Kabat by any other amino acid. Preferably
both the residues at position 234 and 235 in the EU index set forth
in Kabat are substituted. Preferably the drug moiety is conjugated
to the unsubstituted interchain cysteine located in the CH.sub.1
domain, for example to HC220 according to the EU index as set forth
in Kabat.
[0389] AbDJ(LALA)
[0390] In some embodiments the antibody of the conjugates described
herein: (i) has an amino acid substitution of each of the hinge
region interchain cysteines, (ii) comprises light chains each
retaining the unsubstituted interchain cysteine located in the
C.sub.L domain, (iii) comprises heavy chains each having an amino
acid substitution of the interchain cysteine residue located in the
CH.sub.1 domain, and (iv) comprise heavy chains each having an
amino acid substitution of the the residue at position 234 in the
EU index set forth in Kabat and/or a substitution of the residue at
position 235 in the EU index set forth in Kabat.
[0391] For example, in some embodiments the antibody of the
conjugates described herein: (i) has an amino acid substitution of
each of HC226 and HC229 according to the EU index as set forth in
Kabat, (ii) comprises light chains each retaining the unsubstituted
interchain cysteine .kappa.LC214 or .lamda.LC213 according to the
EU index as set forth in Kabat, (iii) comprises heavy chains each
having an amino acid substitution of interchain cysteine HC220
according to the EU index as set forth in Kabat, and (iv) comprise
heavy chains each having an amino acid substitution of the the
residue at position 234 in the EU index set forth in Kabat and/or a
substitution of the residue at position 235 in the EU index set
forth in Kabat by any other amino acid. Preferably both the
residues at position 234 and 235 in the EU index set forth in Kabat
are substituted. Preferably the drug moiety is conjugated to the
unsubstituted interchain cysteine located in the C.sub.L domain,
for example to .kappa.LC214 or .lamda.LC213 according to the EU
index as set forth in Kabat.
[0392] Embodiments Defined Using Disclosed Sequences
[0393] AbLJ(LALA)
[0394] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0395] wherein
the cysteine at position 105 in SEQ ID NO: 150 or the cysteine at
position 102 in SEQ ID NO: 160, is substituted by an amino acid
that is not cysteine; [0396] and wherein the leucine at position
117 and/or the leucine at position 118 is substituted by an amino
acid that is not leucine, such as alanine. Preferably the drug
moiety is conjugated to the cysteine at position 103 of SEQ ID
NO.110. Preferably both the leucines at position 117 and 118 are
substituted by an amino acid that is not leucine, such as
alanine.
[0397] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0398] wherein
the cysteine at position 105 in SEQ ID NO: 150 or the cysteine at
position 102 in SEQ ID NO: 160, is substituted by an amino acid
that is not cysteine; [0399] and wherein the leucine at position
164 and/or the leucine at position 165 is substituted by an amino
acid that is not leucine, such as alanine. Preferably the drug
moiety is conjugated to the cysteine at position 14 of SEQ ID
NO.130. Preferably both the leucines at position 164 and 165 are
substituted by an amino acid that is not leucine, such as
alanine.
[0400] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0401] wherein
the cysteine at position 105 in SEQ ID NO: 150 or the cysteine at
position 102 in SEQ ID NO: 160, is substituted by an amino acid
that is not cysteine; [0402] and wherein the leucine at position
115 is substituted by an amino acid that is not leucine, such as
alanine. Preferably the drug moiety is conjugated to the cysteine
at position 14 of SEQ ID NO.140.
[0403] AbHJ(LALA)
[0404] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0405] wherein
the cysteine at position 103 in SEQ ID NO: 110 is substituted by an
amino acid that is not cysteine; [0406] and wherein the leucine at
position 117 and/or the leucine at position 118 is substituted by
an amino acid that is not leucine, such as alanine. Preferably the
drug moiety is conjugated to the cysteine at position 105 of SEQ ID
NO.150, the cysteine at position 102 of SEQ ID NO.160. Preferably
both the leucines at position 117 and 118 are substituted by an
amino acid that is not leucine, such as alanine.
[0407] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0408] wherein
the cysteine at position 14 in SEQ ID NO: 130 is substituted by an
amino acid that is not cysteine; [0409] and wherein the leucine at
position 164 and/or the leucine at position 165 is substituted by
an amino acid that is not leucine, such as alanine. Preferably the
drug moiety is conjugated to the cysteine at position 105 of SEQ ID
NO.150, the cysteine at position 102 of SEQ ID NO.160. Preferably
both the leucines at position 164 and 165 are substituted by an
amino acid that is not leucine, such as alanine.
[0410] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0411] wherein
the cysteine at position 14 in SEQ ID NO: 140 is substituted by an
amino acid that is not cysteine; [0412] and wherein the leucine at
position 115 is substituted by an amino acid that is not leucine,
such as alanine. Preferably the drug moiety is conjugated to the
cysteine at position 105 of SEQ ID NO.150, the cysteine at position
102 of SEQ ID NO.160.
[0413] AbBJ(LALA)
[0414] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0415] wherein
each of the cysteines at positions 109 and 112 in SEQ ID NO: 110 is
substituted by an amino acid that is not cysteine; [0416] and
wherein the cysteine at position 105 in SEQ ID NO: 150 or the
cysteine at position 102 in SEQ ID NO: 160, is substituted by an
amino acid that is not cysteine; [0417] and wherein the leucine at
position 117 and/or the leucine at position 118 is substituted by
an amino acid that is not leucine, such as alanine. Preferably the
drug moiety is conjugated to the cysteine at position 103 of SEQ ID
NO.110. Preferably both the leucines at position 117 and 118 are
substituted by an amino acid that is not leucine, such as
alanine.
[0418] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0419] wherein
each of the cysteines at positions 111, 114, 120, 126, 129, 135,
141, 144, 150, 156, and 159 in SEQ ID NO: 130 is substituted by an
amino acid that is not cysteine; [0420] and wherein the cysteine at
position 105 in SEQ ID NO: 150 or the cysteine at position 102 in
SEQ ID NO: 160, is substituted by an amino acid that is not
cysteine; [0421] and wherein the leucine at position 164 and/or the
leucine at position 165 is substituted by an amino acid that is not
leucine, such as alanine. Preferably the drug moiety is conjugated
to the cysteine at position 14 of SEQ ID NO.130. Preferably both
the leucines at position 164 and 165 are substituted by an amino
acid that is not leucine, such as alanine.
[0422] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0423] wherein
each of the cysteines at positions 106 and 109 in SEQ ID NO: 140 is
substituted by an amino acid that is not cysteine; [0424] and
wherein the cysteine at position 105 in SEQ ID NO: 150 or the
cysteine at position 102 in SEQ ID NO: 160, is substituted by an
amino acid that is not cysteine; [0425] and wherein the leucine at
position 115 is substituted by an amino acid that is not leucine,
such as alanine. Preferably the drug moiety is conjugated to the
cysteine at position 14 of SEQ ID NO.140.
[0426] AbDJ(LALA)
[0427] In some embodiments, some embodiments, the antibody of the
conjugates described herein comprises a heavy chain comprising the
amino acid sequence of SEQ ID NO.110, and a light chain comprising
the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0428]
wherein each of the cysteines at positions 103, 109 and 112 in SEQ
ID NO: 110 is substituted by an amino acid that is not cysteine;
[0429] and wherein the leucine at position 117 and/or the leucine
at position 118 is substituted by an amino acid that is not
leucine, such as alanine. Preferably the drug moiety is conjugated
to the cysteine at position 105 of SEQ ID NO.150, the cysteine at
position 102 of SEQ ID NO.160. Preferably both the leucines at
position 117 and 118 are substituted by an amino acid that is not
leucine, such as alanine.
[0430] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.130, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0431] wherein
each of the cysteines at positions 14, 111, 114, 120, 126, 129,
135, 141, 144, 150, 156, and 159 in SEQ ID NO: 130 is substituted
by an amino acid that is not cysteine; [0432] and wherein the
leucine at position 164 and/or the leucine at position 165 is
substituted by an amino acid that is not leucine, such as alanine.
Preferably the drug moiety is conjugated to the cysteine at
position 105 of SEQ ID NO.150, the cysteine at position 102 of SEQ
ID NO.160. Preferably both the leucines at position 164 and 165 are
substituted by an amino acid that is not leucine, such as
alanine.
[0433] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, and a light chain comprising the amino
acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160; [0434] wherein
each of the cysteines at positions 14, 106, and 109 in SEQ ID NO:
140 is substituted by an amino acid that is not cysteine; [0435]
and wherein the leucine at position 115 is substituted by an amino
acid that is not leucine, such as alanine. Preferably the drug
moiety is conjugated to the cysteine at position 105 of SEQ ID
NO.150, the cysteine at position 102 of SEQ ID NO.160.
[0436] Conjugate/Antibody Properties
[0437] Maximum Tolerated Dose (MTD)
[0438] The conjugates described herein have been found to be
well-tolerated in in vivo disease models, allowing for reduced
side-effects in subjects receiving the conjugates. Accordingly, in
some embodiments the conjugates described herein have a higher MTD
than an otherwise identical conjugate where the drug moieties are
to the antibody at non-site specifically. MTD is typically tested
in animals such as mouse (for example, Mus musculus), rat (for
example, Rattus norvegicus), or monkey (for example, Macaca
fascicularis). In some embodiments, the conjugates described herein
have an MTD in rat of at least 1 mg/kg delivered as a single-dose,
for example at least 1.2 mg/kg, at least 1.4 mg/kg, at least 1.6
mg/kg, at least 1.8 mg/kg, at least 2.0 mg/kg, at least 2.2 mg/kg,
at least 2.4 mg/kg, at least 2.6 mg/kg, at least 2.8 mg/kg, at
least 3.0 mg/kg, at least 4.0 mg/kg, or at least 5.0 mg/kg
delivered as a single-dose.
[0439] Therapeutic Index
[0440] In some embodiments the site-specific conjugates described
herein have an improved therapeutic index as compared to an
otherwise identical non site-specific conjugate. In some
embodiments the therapeutic index for a site specific conjugate
descried herein is at least 2% higher than an otherwise identical
non site-specific conjugate. That is, if the non site-specific
conjugate has a therapeutic index of 100:1, the site specific
conjugate has a therapeutic index of at least 102:1. In some
embodiments the therapeutic index for a site specific conjugate
descried herein is at least 5% higher than an otherwise identical
non site-specific conjugate, for example at least 5% higher, at
least 7% higher, at least 10% higher, at least 12% higher, at least
15% higher, at least 20% higher, at least 25% higher, at least 30%
higher, at least 40% higher, at least 50% higher, at least 70%
higher, at least 100% higher, at least 150% higher, or at least
200% higher than an otherwise identical non site-specific
conjugate.
[0441] Systemic Toxicity
[0442] Strop et al., Chemistry & Biology 20, 161-167, Feb. 21,
2013 reported that the conjugation site of the drug moiety on the
antibody can influence the stability and pharmacokinetics of an
ADC.
[0443] The relative systemic toxicity of a site-specific ADC newly
described herein was compared to that of a known type of
site-specific ADC--see Example 7 and FIG. 1. The site-specific ADC
newly described herein was not observed to induce significant
systemic toxicity, in contrast to the known site-specific ADC.
[0444] Antibody Affinity
[0445] In some embodiments, the site-specific conjugate has the
same affinity for the cognate antigen as compared to an otherwise
identical non site-specific conjugate. In some embodiments, the
site-specific conjugate has a greater affinity for the cognate
antigen as compared to an otherwise identical non site-specific
conjugate. In some embodiments the site-specific conjugate binds
the cognate antigen with a dissociation constant (Kd) of at least
10.sup.--6 M, such as at least 5.times.10.sup.-7 M, at least
10.sup.-7 M, at least 5.times.10.sup.-8 M, at least 10.sup.-9 M,
such as at least 5.times.10.sup.-10 M, at least 10.sup.-10 M, at
least 5.times.10.sup.-11 M, at least 10.sup.-11 M, at least
5.times.10.sup.-12 M, at least 10.sup.-12 M, at least
5.times.10.sup.-13 M, at least 10.sup.-13 M, at least
5.times.10.sup.-14 M, at least 10.sup.-14 M, at least
5.times.10.sup.-15 M, or at least 10.sup.-15 M. In one embodiment
the site-specific conjugate competitively inhibits the in vivo
and/or in vitro binding to the cognate antigen of an otherwise
identical non site-specific conjugate.
[0446] As used herein, "binds [antigen X]" is used to mean the
antibody binds [antigen X] with a higher affinity than a
non-specific partner such as Bovine Serum Albumin (BSA, Genbank
accession no. CAA76847, version no. CAA76847.1 GI:3336842, record
update date: Jan. 7, 2011 02:30 PM). In some embodiments the
antibody binds [antigen X] with an association constant (Ka) at
least 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000,
10.sup.4, 10.sup.5 or 10.sup.6-fold higher than the antibody's
association constant for BSA, when measured at physiological
conditions. The antibodies of the disclosure can typically bind
[antigen X] with a high affinity. For example, in some embodiments
the antibody can bind [antigen X] with a KD equal to or less than
about 10.sup.-6 M, such as 1.times.10.sup.-6, 10.sup.-7, 10.sup.-8,
10.sup.-9, 10.sup.-10, 10.sup.-11, 10.sup.-12, 10.sup.-13 or
10.sup.-14 M.
[0447] Effective Dose
[0448] In some embodiments the site-specific conjugate has an EC50
of less than 35 ng/ml, such as less than 30 ng/ml, less than 25
ng/ml, less than 20 ng/ml, or less than 15 ng/ml. In some
embodiments the EC50 of the site-specific conjugate is no higher
than an otherwise identical non site-specific conjugate. In some
embodiments the EC50 of the site-specific conjugate is at least 2
ng/ml lower than an otherwise identical non site-specific
conjugate, for example at least 5 ng/ml lower, at least 10 ng/ml
lower, at least 15 ng/ml lower, at least 20 ng/ml lower, at least
25 ng/ml lower, or at least 30 ng/ml lower.
[0449] Ease of Manufacture
[0450] Embodiments of the site-specific ADCs newly described herein
allow for simplification of the ADC manufacture procedure.
[0451] For example, in a cysteine engineered IgG version such as
those described in Junutula et al., Nature Biotechnology, vol. 26,
no. 8, pp. 925-932, additional cysteines are engineered into the
IgG1 to allow for site-specific conjugation on the engineered
cysteines. When such cysteine engineered IgG are recombinantly
expressed in mammalian cells, the engineered cysteines are
typically capped with other sulphydryl containing molecules such as
GSH, cysteine etc. In order to release the engineered cysteines for
conjugation, the molecule must be reduced. This typically will also
reduce the interchain disulphide bond between the heavy and light
chains, as well as those in the hinge region. This reduction of
native interchain cysteines is undesireable, since drug conjugation
can also occur on these native cysteines. Thus, the antibody
molecule must be re-oxidized to re-establish these native
interchain disulphide bonds before the cysteines engineered into
the antibody can be conjugated to the drug.
[0452] Incontrast, the present disclosure specifically contemplates
embodiemnts where the antibody comprises only two interchain
cycteins suitable for conjugation (for example, one on each heavy
chain) with the other interchain cycteine residues present in a
native antibody having been substituted for an amino acid which is
not cysteine. This format allows the complex-reduction-reoxidation
procedure described above to be dispensed with. Instead a straight
forward reduction-conjugation procedure can be followed. This is
possible because the site-specific antibody formats described
herein typically do not contain interchain cysteines that are not
ultimately intended to be conjugated to drug moiteies. For example,
in preferred embodiments the site-specific antibody contains only
two interchain cycteins suitable for conjugation (for example, one
on each heavy chain). It is therefore not necessary to reoxidize
the antibody molecule after the initial reduction step. Instead the
molecule is reduced with a reducatant such as TCEP which reduces
the (two) remaining interchain cysteines (with the other interchain
cysteines having been substituted for amino acids which are not
cysteine). The reduced cysteine sulphhydryl moiteis can then be
conjugated to the drug-linker.
[0453] In the preferred embodiments where there are only two
intercahin cysteines, it is not possible to generate IgG species
with DAR 3 or higher. This can be advantageous, since higher DAR
species can contribute to ADC toxicity--see Jununtula et al.,
(Nature Biotech 26_925-932 (2008)).
[0454] The newly described site-specific ADCs also avoid other
potential manufacturing problems. For example, during the analysis
of cysteine engineered IgGs secreted by stably transfected Chinese
Hamster Ovary (CHO) cells, the existence of Triple Light Chain
antibodies (3LC) has been observed; the 3LC species appears to be
the product of a disulfide bond formed between an extra light chain
and an additional cysteine engineered into an IgG (Gomez et al.,
Biotechnol. Bioeng. 105(4)_748-60 (2010); Gomez et al., Biotechnol.
Prog. 26(5)_1438-1445 (2010)). The newly described site-specific
ADCs do not have inseted cysteines in the light chain, so have no
potential to form contamination 3LC species.
[0455] Terminal Half-Life
[0456] In some embodiments, conjugates in which the antibody
comprises a heavy chain having a substitution of the residue at
position 234 in the EU index set forth in Kabat and/or a
substitution of the residue at position 235 in the EU index set
forth in Kabat have improved terminal half-life as compared to
another otherwise identical conjugate lacking the 234/235
substitution(s). The terminal-half life may be measured as
described herein in Example 6. Accordingly, in some embodiments
conjugates in which the antibody comprises a heavy chain having a
substitution of the residue at position 234 in the EU index set
forth in Kabat and/or a substitution of the residue at position 235
in the EU index set forth in Kabat have a half-life which is at
least 110% of the half-life of an otherwise identical conjugate
lacking the 234/235 substitution(s); for example at least 115% of
the half-life, at least 120% of the half-life, at least 125% of the
half-life, at least 130% of the half-life, at least 135% of the
half-life, at least 140% of the half-life, at least 145% of the
half-life, at least 150% of the half-life, at least 160% of the
half-life, at least 170% of the half-life, at least 180% of the
half-life, at least 190% of the half-life, or at least 200% of the
half-life of an otherwise identical conjugate lacking the 234/235
substitution(s).
[0457] Antigen Binding
[0458] The antibody of the conjugates described herein is an
antibody (Ab) which binds CD22. That is, the conjugates described
herein are conjugates comprising antibodies which specifically bind
to CD22.
[0459] In some embodiments, CD22 polypeptide corresponds to Genbank
accession no. BAB15489, version no. BAB15489.1 GI:10439338, record
update date: Sep. 11, 2006 11:24 PM. In one embodiment, the nucleic
acid encoding CD22 polypeptide corresponds to Genbank accession no
AK026467, version no. AK026467.1 GI:10439337, record update date:
Sep. 11, 2006 11:24 PM.
[0460] In one aspect the antibody is an antibody that binds to
CD22, the antibody comprising a VH domain having the sequence
according to SEQ ID NO. 1.
[0461] The antibody may further comprise a VL domain. In some
embodiments the antibody further comprises a VL domain having the
sequence according to SEQ ID NO. 2.
[0462] In some embodiments the antibody comprises a VH domain
paired with a VL domain, the VH and VL domains having the sequences
of SEQ ID NO. 1 paired with SEQ ID NO. 2.
[0463] The VH and VL domain(s) may pair so as to form an antibody
antigen binding site that binds CD22.
[0464] In some embodiments the antibody is an intact antibody
comprising a VH domain paired with a VL domain, the VH and VL
domains having sequences of SEQ ID NO. 1 paired with SEQ ID NO.
2.
[0465] In aspect the antibody is an antibody as described herein
which has been modified (or further modified) as described below.
In some embodiments the antibody is a humanised, deimmunised or
resurfaced version of an antibody disclosed herein.
Some Embodiments
[0466] Listed below are some specifically contemplated
embodiments.
[0467] Substitution of Interchain Cysteine Residues
[0468] AbLJ-CD22 IgG1
[0469] An antibody of the conjugates described herein comprising a
heavy chain comprising the amino acid sequence of SEQ ID NO.110, a
light chain comprising the amino acid sequence of SEQ ID NO. 150 or
SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a
VL domain having the sequence SEQ ID NO. 2; [0470] wherein the
cysteine at position 105 in SEQ ID NO: 150 or the cysteine at
position 102 in SEQ ID NO: 160, is substituted by an amino acid
that is not cysteine. Preferably the drug moiety is conjugated to
the cysteine at position 103 of SEQ ID NO.110.
[0471] An antibody of the conjugates described herein comprising:
[0472] a heavy chain comprising the amino acid sequence of SEQ ID
NO.110; [0473] a light chain comprising the amino acid sequence of
SEQ ID NO.151, SEQ ID NO.152, SEQ ID NO.153, SEQ ID NO.161, SEQ ID
NO.162, or SEQ ID NO.163;a VH domain having the sequence SEQ ID NO.
1; and [0474] a VL domain having the sequence SEQ ID NO. 2.
[0475] An antibody of the conjugates described herein comprising:
[0476] a heavy chain comprising the amino acid sequence of SEQ ID
NO.110; [0477] a light chain comprising the amino acid sequence of
SEQ ID NO.151; [0478] a VH domain having the sequence SEQ ID NO. 1;
and [0479] a VL domain having the sequence SEQ ID NO. 2.
[0480] An antibody of the conjugates described herein comprising:
[0481] a heavy chain comprising the amino acid sequence of SEQ ID
NO.110; [0482] a light chain comprising the amino acid sequence of
SEQ ID NO.152; [0483] a VH domain having the sequence SEQ ID NO. 1;
and [0484] a VL domain having the sequence SEQ ID NO. 2.
[0485] An antibody of the conjugates described herein comprising:
[0486] a heavy chain comprising the amino acid sequence of SEQ ID
NO.110; [0487] a light chain comprising the amino acid sequence of
SEQ ID NO.153; [0488] a VH domain having the sequence SEQ ID NO. 1;
and [0489] a VL domain having the sequence SEQ ID NO. 2.
[0490] An antibody of the conjugates described herein comprising:
[0491] a heavy chain comprising the amino acid sequence of SEQ ID
NO.110; [0492] a light chain comprising the amino acid sequence of
SEQ ID NO.161; [0493] a VH domain having the sequence SEQ ID NO. 1;
and [0494] a VL domain having the sequence SEQ ID NO. 2.
[0495] An antibody of the conjugates described herein comprising:
[0496] a heavy chain comprising the amino acid sequence of SEQ ID
NO.110; [0497] a light chain comprising the amino acid sequence of
SEQ ID NO.162; [0498] a VH domain having the sequence SEQ ID NO. 1;
and [0499] a VL domain having the sequence SEQ ID NO. 2.
[0500] An antibody of the conjugates described herein comprising:
[0501] a heavy chain comprising the amino acid sequence of SEQ ID
NO.110; [0502] a light chain comprising the amino acid sequence of
SEQ ID NO.163; [0503] a VH domain having the sequence SEQ ID NO. 1;
and [0504] a VL domain having the sequence SEQ ID NO. 2.
[0505] AbHJ-CD22 IgG1
[0506] An antibody of the conjugates described herein comprising a
heavy chain comprising the amino acid sequence of SEQ ID NO.110, a
light chain comprising the amino acid sequence of SEQ ID NO. 150 or
SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a
VL domain having the sequence SEQ ID NO. 2; [0507] wherein the
cysteine at position 103 in SEQ ID NO: 110 is substituted by an
amino acid that is not cysteine. Preferably the drug moiety is
conjugated to the cysteine at position 105 of SEQ ID NO.150, the
cysteine at position 102 of SEQ ID NO.160.
[0508] An antibody of the conjugates described herein comprising:
[0509] a heavy chain comprising the amino acid sequence of SEQ ID
NO.111; [0510] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0511] a VH domain having the
sequence SEQ ID NO. 1; and [0512] a VL domain having the sequence
SEQ ID NO. 2.
[0513] An antibody of the conjugates described herein comprising:
[0514] a heavy chain comprising the amino acid sequence of SEQ ID
NO.112; [0515] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0516] a VH domain having the
sequence SEQ ID NO. 1; and [0517] a VL domain having the sequence
SEQ ID NO. 2.
[0518] AbBJ-CD22 IgG1
[0519] An antibody of the conjugates described herein comprising a
heavy chain comprising the amino acid sequence of SEQ ID NO.110, a
light chain comprising the amino acid sequence of SEQ ID NO. 150 or
SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a
VL domain having the sequence SEQ ID NO. 2; [0520] wherein each of
the cysteines at positions 109 and 112 in SEQ ID NO: 110 is
substituted by an amino acid that is not cysteine; [0521] and
wherein the cysteine at position 105 in SEQ ID NO: 150 or the
cysteine at position 102 in SEQ ID NO: 160, is substituted by an
amino acid that is not cysteine. Preferably the drug moiety is
conjugated to the cysteine at position 103 of SEQ ID NO.110.
Preferably the cysteines at positions 109 and 112 in SEQ ID NO: 110
are substituted by valine.
[0522] An antibody of the conjugates described herein comprising:
[0523] a heavy chain comprising the amino acid sequence of SEQ ID
NO.113; [0524] a light chain comprising the amino acid sequence of
SEQ ID NO.151, SEQ ID NO.152, SEQ ID NO.153, SEQ ID NO.161, SEQ ID
NO.162, or SEQ ID NO.163; [0525] a VH domain having the sequence
SEQ ID NO. 1; and [0526] a VL domain having the sequence SEQ ID NO.
2.
[0527] An antibody of the conjugates described herein comprising:
[0528] a heavy chain comprising the amino acid sequence of SEQ ID
NO.113; [0529] a light chain comprising the amino acid sequence of
SEQ ID NO.151; [0530] a VH domain having the sequence SEQ ID NO. 1;
and [0531] a VL domain having the sequence SEQ ID NO. 2.
[0532] An antibody of the conjugates described herein comprising:
[0533] a heavy chain comprising the amino acid sequence of SEQ ID
NO.113; [0534] a light chain comprising the amino acid sequence of
SEQ ID NO.152; [0535] a VH domain having the sequence SEQ ID NO. 1;
and [0536] a VL domain having the sequence SEQ ID NO. 2.
[0537] An antibody of the conjugates described herein comprising:
[0538] a heavy chain comprising the amino acid sequence of SEQ ID
NO.113; [0539] a light chain comprising the amino acid sequence of
SEQ ID NO.153; [0540] a VH domain having the sequence SEQ ID NO. 1;
and [0541] a VL domain having the sequence SEQ ID NO. 2.
[0542] An antibody of the conjugates described herein comprising:
[0543] a heavy chain comprising the amino acid sequence of SEQ ID
NO.113; [0544] a light chain comprising the amino acid sequence of
SEQ ID NO.161; [0545] a VH domain having the sequence SEQ ID NO. 1;
and [0546] a VL domain having the sequence SEQ ID NO. 2.
[0547] An antibody of the conjugates described herein comprising:
[0548] a heavy chain comprising the amino acid sequence of SEQ ID
NO.113; [0549] a light chain comprising the amino acid sequence of
SEQ ID NO.162; [0550] a VH domain having the sequence SEQ ID NO. 1;
and [0551] a VL domain having the sequence SEQ ID NO. 2.
[0552] An antibody of the conjugates described herein comprising:
[0553] a heavy chain comprising the amino acid sequence of SEQ ID
NO.113; [0554] a light chain comprising the amino acid sequence of
SEQ ID NO.163; [0555] a VH domain having the sequence SEQ ID NO. 1;
and [0556] a VL domain having the sequence SEQ ID NO. 2.
[0557] An antibody of the conjugates described herein comprising:
[0558] a heavy chain comprising the amino acid sequence of SEQ ID
NO.114; [0559] a light chain comprising the amino acid sequence of
SEQ ID NO.151, SEQ ID NO.152, SEQ ID NO.153, SEQ ID NO.161, SEQ ID
NO.162, or SEQ ID NO.163; [0560] a VH domain having the sequence
SEQ ID NO. 1; and [0561] a VL domain having the sequence SEQ ID NO.
2.
[0562] An antibody of the conjugates described herein comprising:
[0563] a heavy chain comprising the amino acid sequence of SEQ ID
NO.114; [0564] a light chain comprising the amino acid sequence of
SEQ ID NO.151; [0565] a VH domain having the sequence SEQ ID NO. 1;
and [0566] a VL domain having the sequence SEQ ID NO. 2.
[0567] An antibody of the conjugates described herein comprising:
[0568] a heavy chain comprising the amino acid sequence of SEQ ID
NO.114; [0569] a light chain comprising the amino acid sequence of
SEQ ID NO.152; [0570] a VH domain having the sequence SEQ ID NO. 1;
and [0571] a VL domain having the sequence SEQ ID NO. 2.
[0572] An antibody of the conjugates described herein comprising:
[0573] a heavy chain comprising the amino acid sequence of SEQ ID
NO.114; [0574] a light chain comprising the amino acid sequence of
SEQ ID NO.153; [0575] a VH domain having the sequence SEQ ID NO. 1;
and [0576] a VL domain having the sequence SEQ ID NO. 2.
[0577] An antibody of the conjugates described herein comprising:
[0578] a heavy chain comprising the amino acid sequence of SEQ ID
NO.114; [0579] a light chain comprising the amino acid sequence of
SEQ ID NO.161; [0580] a VH domain having the sequence SEQ ID NO. 1;
and [0581] a VL domain having the sequence SEQ ID NO. 2.
[0582] An antibody of the conjugates described herein comprising:
[0583] a heavy chain comprising the amino acid sequence of SEQ ID
NO.114; [0584] a light chain comprising the amino acid sequence of
SEQ ID NO.162; [0585] a VH domain having the sequence SEQ ID NO. 1;
and [0586] a VL domain having the sequence SEQ ID NO. 2.
[0587] An antibody of the conjugates described herein comprising:
[0588] a heavy chain comprising the amino acid sequence of SEQ ID
NO.114; [0589] a light chain comprising the amino acid sequence of
SEQ ID NO.163; [0590] a VH domain having the sequence SEQ ID NO. 1;
and [0591] a VL domain having the sequence SEQ ID NO. 2.
[0592] An antibody of the conjugates described herein comprising a
heavy chain comprising the amino acid sequence of SEQ ID NO.110, a
light chain comprising the amino acid sequence of SEQ ID NO. 150 or
SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a
VL domain having the sequence SEQ ID NO. 2; [0593] wherein the
cysteine at positions 109 in SEQ ID NO: 110 is substituted by an
amino acid that is not cysteine and the cysteine at positions 112
in SEQ ID NO: 110 is unsubstituted; [0594] and wherein the cysteine
at position 105 in SEQ ID NO: 150 or the cysteine at position 102
in SEQ ID NO: 160, is substituted by an amino acid that is not
cysteine. Preferably the drug moieties are conjugated to the
cysteines at positions 103 and 112 of SEQ ID NO.110. Preferably the
cysteine at position 109 in SEQ ID NO: 110 is substituted by
valine.
[0595] An antibody of the conjugates described herein comprising a
heavy chain comprising the amino acid sequence of SEQ ID NO.110, a
light chain comprising the amino acid sequence of SEQ ID NO. 150 or
SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a
VL domain having the sequence SEQ ID NO. 2; [0596] wherein the
cysteine at positions 112 in SEQ ID NO: 110 is substituted by an
amino acid that is not cysteine and the cysteine at positions 109
in SEQ ID NO: 110 is unsubstituted; [0597] and wherein the cysteine
at position 105 in SEQ ID NO: 150 or the cysteine at position 102
in SEQ ID NO: 160, is substituted by an amino acid that is not
cysteine. Preferably the drug moieties are conjugated to the
cysteines at positions 103 and 109 of SEQ ID NO.110. Preferably the
cysteine at position 112 in SEQ ID NO: 110 is substituted by
valine.
[0598] AbDJ-CD22 IgG1
[0599] An antibody of the conjugates described herein comprising a
heavy chain comprising the amino acid sequence of SEQ ID NO.110, a
light chain comprising the amino acid sequence of SEQ ID NO. 150 or
SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a
VL domain having the sequence SEQ ID NO. 2; [0600] wherein each of
the cysteines at positions 103, 109 and 112 in SEQ ID NO: 110 is
substituted by an amino acid that is not cysteine. Preferably the
drug moiety is conjugated to the cysteine at position 105 of SEQ ID
NO.150, or the cysteine at position 102 of SEQ ID NO.160.
Preferably the cysteines at positions 109 and 112 in SEQ ID NO: 110
are substituted by valine.
[0601] An antibody of the conjugates described herein comprising:
[0602] a heavy chain comprising the amino acid sequence of SEQ ID
NO.115; [0603] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0604] a VH domain having the
sequence SEQ ID NO. 1; and [0605] a VL domain having the sequence
SEQ ID NO. 2.
[0606] An antibody of the conjugates described herein comprising:
[0607] a heavy chain comprising the amino acid sequence of SEQ ID
NO.116; [0608] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0609] a VH domain having the
sequence SEQ ID NO. 1; and [0610] a VL domain having the sequence
SEQ ID NO. 2.
[0611] An antibody of of the conjugates described herein comprising
a heavy chain comprising the amino acid sequence of SEQ ID NO.110,
a light chain comprising the amino acid sequence of SEQ ID NO. 150
or SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1,
and a VL domain having the sequence SEQ ID NO. 2; [0612] wherein
each of the cysteines at positions 109 and 112 in SEQ ID NO: 110
are substituted by an amino acid that is not cysteine and the
cysteine at positions 103 in SEQ ID NO: 110 is unsubstituted.
Preferably the drug moieties are conjugated to: (i) the cysteine at
position 105 of SEQ ID NO.150, or the cysteine at position 102 of
SEQ ID NO.160; and (ii) the cysteine at position 103 of SEQ ID
NO.110. Preferably the cysteines at positions 109 and 112 in SEQ ID
NO: 110 are substituted by valine.
[0613] An antibody of of the conjugates described herein comprising
a heavy chain comprising the amino acid sequence of SEQ ID NO.110,
a light chain comprising the amino acid sequence of SEQ ID NO. 150
or SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1,
and a VL domain having the sequence SEQ ID NO. 2; [0614] wherein
each of the cysteines at positions 103 and 112 in SEQ ID NO: 110
are substituted by an amino acid that is not cysteine and the
cysteine at position 109 in SEQ ID NO: 110 is unsubstituted.
Preferably the drug moieties are conjugated to: (i) the cysteine at
position 105 of SEQ ID NO.150, or the cysteine at position 102 of
SEQ ID NO.160; and (ii) the cysteine at position 109 of SEQ ID
NO.110. Preferably the cysteine at position 112 in SEQ ID NO: 110
is substituted by valine.
[0615] An antibody of of the conjugates described herein comprising
a heavy chain comprising the amino acid sequence of SEQ ID NO.110,
a light chain comprising the amino acid sequence of SEQ ID NO. 150
or SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1,
and a VL domain having the sequence SEQ ID NO. 2; [0616] wherein
each of the cysteines at positions 103 and 109 in SEQ ID NO: 110
are substituted by an amino acid that is not cysteine and the
cysteine at position 112 in SEQ ID NO: 110 is unsubstituted.
Preferably the drug moieties are conjugated to: (i) the cysteine at
position 105 of SEQ ID NO.150, or the cysteine at position 102 of
SEQ ID NO.160; and (ii) the cysteine at position 112 of SEQ ID
NO.110. Preferably the cysteine at position 109 in SEQ ID NO: 110
is substituted by valine.
[0617] Substitution of Kabat EU Residues 234 and/or 235
[0618] An antibody of of the conjugates described herein comprising
a heavy chain comprising the amino acid sequence of SEQ ID NO.110,
a light chain, a VH domain having the sequence SEQ ID NO. 1, and a
VL domain having the sequence SEQ ID NO. 2; [0619] wherein the
leucine at position 117 of SEQ ID NO.110 and/or the leucine at
position 118 of SEQ ID NO.110 is substituted by an amino acid that
is not leucine.
[0620] An antibody of the conjugates described herein comprising:
[0621] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1101; [0622] a light chain; [0623] a VH domain having the
sequence SEQ ID NO. 1; and [0624] a VL domain having the sequence
SEQ ID NO. 2.
[0625] An antibody of the conjugates described herein comprising:
[0626] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1102; [0627] a light chain; [0628] a VH domain having the
sequence SEQ ID NO. 1; and [0629] a VL domain having the sequence
SEQ ID NO. 2.
[0630] An antibody of the conjugates described herein comprising:
[0631] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1103; [0632] a light chain; [0633] a VH domain having the
sequence SEQ ID NO. 1; and [0634] a VL domain having the sequence
SEQ ID NO. 2.
[0635] An antibody of the conjugates described herein comprising:
[0636] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1104; [0637] a light chain; [0638] a VH domain having the
sequence SEQ ID NO. 1; and [0639] a VL domain having the sequence
SEQ ID NO. 2.
[0640] An antibody of the conjugates described herein comprising:
[0641] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1105; [0642] a light chain; [0643] a VH domain having the
sequence SEQ ID NO. 1; and [0644] a VL domain having the sequence
SEQ ID NO. 2.
[0645] An antibody of the conjugates described herein comprising:
[0646] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1106; [0647] a light chain; [0648] a VH domain having the
sequence SEQ ID NO. 1; and [0649] a VL domain having the sequence
SEQ ID NO. 2.
[0650] An antibody of of the conjugates described herein comprising
a heavy chain comprising the amino acid sequence of SEQ ID NO.130,
a light chain, a VH domain having the sequence SEQ ID NO. 1, and a
VL domain having the sequence SEQ ID NO. 2; [0651] wherein the
leucine at position 164 of SEQ ID NO.130 and/or the leucine at
position 165 of SEQ ID NO.130 is substituted by an amino acid that
is not leucine.
[0652] An antibody of the conjugates described herein comprising:
[0653] a heavy chain comprising the amino acid sequence of SEQ ID
NO.131; [0654] a light chain; [0655] a VH domain having the
sequence SEQ ID NO. 1; and [0656] a VL domain having the sequence
SEQ ID NO. 2.
[0657] An antibody of the conjugates described herein comprising:
[0658] a heavy chain comprising the amino acid sequence of SEQ ID
NO.132; [0659] a light chain; [0660] a VH domain having the
sequence SEQ ID NO. 1; and [0661] a VL domain having the sequence
SEQ ID NO. 2.
[0662] An antibody of the conjugates described herein comprising:
[0663] a heavy chain comprising the amino acid sequence of SEQ ID
NO.133; [0664] a light chain; [0665] a VH domain having the
sequence SEQ ID NO. 1; and [0666] a VL domain having the sequence
SEQ ID NO. 2.
[0667] An antibody of the conjugates described herein comprising:
[0668] a heavy chain comprising the amino acid sequence of SEQ ID
NO.134; [0669] a light chain; [0670] a VH domain having the
sequence SEQ ID NO. 1; and [0671] a VL domain having the sequence
SEQ ID NO. 2.
[0672] An antibody of the conjugates described herein comprising:
[0673] a heavy chain comprising the amino acid sequence of SEQ ID
NO.135; [0674] a light chain; [0675] a VH domain having the
sequence SEQ ID NO. 1; and [0676] a VL domain having the sequence
SEQ ID NO. 2.
[0677] An antibody of the conjugates described herein comprising:
[0678] a heavy chain comprising the amino acid sequence of SEQ ID
NO.136; [0679] a light chain; [0680] a VH domain having the
sequence SEQ ID NO. 1; and [0681] a VL domain having the sequence
SEQ ID NO. 2.
[0682] An antibody of of the conjugates described herein comprising
a heavy chain comprising the amino acid sequence of SEQ ID NO.140,
a light chain, a VH domain having the sequence SEQ ID NO. 1, and a
VL domain having the sequence SEQ ID NO. 2; [0683] wherein the
leucine at position 115 of SEQ ID NO.140 is substituted by an amino
acid that is not leucine.
[0684] An antibody of the conjugates described herein comprising:
[0685] a heavy chain comprising the amino acid sequence of SEQ ID
NO.141; [0686] a light chain; [0687] a VH domain having the
sequence SEQ ID NO. 1; and [0688] a VL domain having the sequence
SEQ ID NO. 2.
[0689] An antibody of the conjugates described herein comprising:
[0690] a heavy chain comprising the amino acid sequence of SEQ ID
NO.142; [0691] a light chain; [0692] a VH domain having the
sequence SEQ ID NO. 1; and [0693] a VL domain having the sequence
SEQ ID NO. 2.
[0694] An antibody of the conjugates described herein comprising:
[0695] a heavy chain comprising the amino acid sequence of SEQ ID
NO.143; [0696] a light chain; [0697] a VH domain having the
sequence SEQ ID NO. 1; and [0698] a VL domain having the sequence
SEQ ID NO. 2.
[0699] An antibody of the conjugates described herein comprising:
[0700] a heavy chain comprising the amino acid sequence of SEQ ID
NO.144; [0701] a light chain; [0702] a VH domain having the
sequence SEQ ID NO. 1; and [0703] a VL domain having the sequence
SEQ ID NO. 2.
[0704] Substitution of Interchain Cysteine Residues Combined with
Substitution of Kabat EU Residues 234 and/or 235
[0705] AbLJ(LALA) IgG1
[0706] An antibody of the conjugates described herein comprising a
heavy chain comprising the amino acid sequence of SEQ ID NO.110, a
light chain comprising the amino acid sequence of SEQ ID NO. 150 or
SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a
VL domain having the sequence SEQ ID NO. 2; [0707] wherein the
cysteine at position 105 in SEQ ID NO: 150 or the cysteine at
position 102 in SEQ ID NO: 160, is substituted by an amino acid
that is not cysteine; [0708] and wherein the leucine at position
117 of SEQ ID NO.110 and/or the leucine at position 118 of SEQ ID
NO.110 is substituted by an amino acid that is not leucine.
Preferably the drug moiety is conjugated to the cysteine at
position 103 of SEQ ID NO.110.
[0709] An antibody of the conjugates described herein comprising:
[0710] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1101, SEQ ID NO.1102, SEQ ID NO.1103, SEQ ID NO.1104, SEQ ID
NO.1105, SEQ ID NO.1106; [0711] a light chain comprising the amino
acid sequence of SEQ ID NO.151, SEQ ID NO.152, SEQ ID NO.153, SEQ
ID NO.161, SEQ ID NO.162, or SEQ ID NO.163; [0712] a VH domain
having the sequence SEQ ID NO. 1; and [0713] a VL domain having the
sequence SEQ ID NO. 2.
[0714] An antibody of the conjugates described herein comprising:
[0715] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1103; [0716] a light chain comprising the amino acid sequence of
SEQ ID NO.151; [0717] a VH domain having the sequence SEQ ID NO. 1;
and [0718] a VL domain having the sequence SEQ ID NO. 2.
[0719] An antibody of the conjugates described herein comprising:
[0720] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1103; [0721] a light chain comprising the amino acid sequence of
SEQ ID NO.152; [0722] a VH domain having the sequence SEQ ID NO. 1;
and [0723] a VL domain having the sequence SEQ ID NO. 2.
[0724] An antibody of the conjugates described herein comprising:
[0725] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1103; [0726] a light chain comprising the amino acid sequence of
SEQ ID NO.153; [0727] a VH domain having the sequence SEQ ID NO. 1;
and [0728] a VL domain having the sequence SEQ ID NO. 2.
[0729] An antibody of the conjugates described herein comprising:
[0730] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1103; [0731] a light chain comprising the amino acid sequence of
SEQ ID NO.161; [0732] a VH domain having the sequence SEQ ID NO. 1;
and [0733] a VL domain having the sequence SEQ ID NO. 2.
[0734] An antibody of the conjugates described herein comprising:
[0735] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1103; [0736] a light chain comprising the amino acid sequence of
SEQ ID NO.162; [0737] a VH domain having the sequence SEQ ID NO. 1;
and [0738] a VL domain having the sequence SEQ ID NO. 2.
[0739] An antibody of the conjugates described herein comprising:
[0740] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1103; [0741] a light chain comprising the amino acid sequence of
SEQ ID NO.163; [0742] a VH domain having the sequence SEQ ID NO. 1;
and [0743] a VL domain having the sequence SEQ ID NO. 2.
[0744] AbHJ(LALA) IgG1
[0745] An antibody of the conjugates described herein comprising a
heavy chain comprising the amino acid sequence of SEQ ID NO.110, a
light chain comprising the amino acid sequence of SEQ ID NO. 150 or
SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a
VL domain having the sequence SEQ ID NO. 2; [0746] wherein the
cysteine at position 103 in SEQ ID NO: 110 is substituted by an
amino acid that is not cysteine; [0747] and wherein the leucine at
position 117 of SEQ ID NO.110 and/or the leucine at position 118 of
SEQ ID NO.110 is substituted by an amino acid that is not leucine.
Preferably the drug moiety is conjugated to the cysteine at
position 105 of SEQ ID NO.150, the cysteine at position 102 of SEQ
ID NO.160.
[0748] An antibody of the conjugates described herein comprising:
[0749] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1111; [0750] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0751] a VH domain having the
sequence SEQ ID NO. 1; and [0752] a VL domain having the sequence
SEQ ID NO. 2.
[0753] An antibody of the conjugates described herein comprising:
[0754] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1112; [0755] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0756] a VH domain having the
sequence SEQ ID NO. 1; and [0757] a VL domain having the sequence
SEQ ID NO. 2.
[0758] An antibody of the conjugates described herein comprising:
[0759] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1113; [0760] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0761] a VH domain having the
sequence SEQ ID NO. 1; and [0762] a VL domain having the sequence
SEQ ID NO. 2.
[0763] An antibody of the conjugates described herein comprising:
[0764] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1114; [0765] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0766] a VH domain having the
sequence SEQ ID NO. 1; and [0767] a VL domain having the sequence
SEQ ID NO. 2.
[0768] An antibody of the conjugates described herein comprising:
[0769] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1115; [0770] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0771] a VH domain having the
sequence SEQ ID NO. 1; and [0772] a VL domain having the sequence
SEQ ID NO. 2.
[0773] An antibody of the conjugates described herein comprising:
[0774] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1116; [0775] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0776] a VH domain having the
sequence SEQ ID NO. 1; and [0777] a VL domain having the sequence
SEQ ID NO. 2.
[0778] An antibody of the conjugates described herein comprising:
[0779] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1121; [0780] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0781] a VH domain having the
sequence SEQ ID NO. 1; and [0782] a VL domain having the sequence
SEQ ID NO. 2.
[0783] An antibody of the conjugates described herein comprising:
[0784] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1122; [0785] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0786] a VH domain having the
sequence SEQ ID NO. 1; and [0787] a VL domain having the sequence
SEQ ID NO. 2.
[0788] An antibody of the conjugates described herein comprising:
[0789] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1123; [0790] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0791] a VH domain having the
sequence SEQ ID NO. 1; and [0792] a VL domain having the sequence
SEQ ID NO. 2.
[0793] An antibody of the conjugates described herein comprising:
[0794] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1124; [0795] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0796] a VH domain having the
sequence SEQ ID NO. 1; and [0797] a VL domain having the sequence
SEQ ID NO. 2.
[0798] An antibody of the conjugates described herein comprising:
[0799] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1125; [0800] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0801] a VH domain having the
sequence SEQ ID NO. 1; and [0802] a VL domain having the sequence
SEQ ID NO. 2.
[0803] An antibody of the conjugates described herein comprising:
[0804] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1126; [0805] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0806] a VH domain having the
sequence SEQ ID NO. 1; and [0807] a VL domain having the sequence
SEQ ID NO. 2.
[0808] AbBJ(LALA) IgG1
[0809] An antibody of the conjugates described herein comprising a
heavy chain comprising the amino acid sequence of SEQ ID NO.110, a
light chain comprising the amino acid sequence of SEQ ID NO. 150 or
SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a
VL domain having the sequence SEQ ID NO. 2; [0810] wherein each of
the cysteines at positions 109 and 112 in SEQ ID NO: 110 is
substituted by an amino acid that is not cysteine; [0811] and
wherein the cysteine at position 105 in SEQ ID NO: 150 or the
cysteine at position 102 in SEQ ID NO: 160, is substituted by an
amino acid that is not cysteine; [0812] and wherein the leucine at
position 117 of SEQ ID NO.110 and/or the leucine at position 118 of
SEQ ID NO.110 is substituted by an amino acid that is not leucine.
Preferably the drug moiety is conjugated to the cysteine at
position 103 of SEQ ID NO.110. Preferably the cysteines at
positions 109 and 112 in SEQ ID NO: 110 are substituted by
valine.
[0813] An antibody of the conjugates described herein comprising:
[0814] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1131, SEQ ID NO.1132, SEQ ID NO.1133, SEQ ID NO.1134, SEQ ID
NO.1135, SEQ ID NO.1136; [0815] a light chain comprising the amino
acid sequence of SEQ ID NO.151, SEQ ID NO.152, SEQ ID NO.153, SEQ
ID NO.161, SEQ ID NO.162, or SEQ ID NO.163; [0816] a VH domain
having the sequence SEQ ID NO. 1; and [0817] a VL domain having the
sequence SEQ ID NO. 2.
[0818] An antibody of the conjugates described herein comprising:
[0819] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1133; [0820] a light chain comprising the amino acid sequence of
SEQ ID NO.151; [0821] a VH domain having the sequence SEQ ID NO. 1;
and [0822] a VL domain having the sequence SEQ ID NO. 2.
[0823] An antibody of the conjugates described herein comprising:
[0824] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1133; [0825] a light chain comprising the amino acid sequence of
SEQ ID NO.152; [0826] a VH domain having the sequence SEQ ID NO. 1;
and [0827] a VL domain having the sequence SEQ ID NO. 2.
[0828] An antibody of the conjugates described herein comprising:
[0829] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1133; [0830] a light chain comprising the amino acid sequence of
SEQ ID NO.153; [0831] a VH domain having the sequence SEQ ID NO. 1;
and [0832] a VL domain having the sequence SEQ ID NO. 2.
[0833] An antibody of the conjugates described herein comprising:
[0834] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1133; [0835] a light chain comprising the amino acid sequence of
SEQ ID NO.161; [0836] a VH domain having the sequence SEQ ID NO. 1;
and [0837] a VL domain having the sequence SEQ ID NO. 2.
[0838] An antibody of the conjugates described herein comprising:
[0839] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1133; [0840] a light chain comprising the amino acid sequence of
SEQ ID NO.162; [0841] a VH domain having the sequence SEQ ID NO. 1;
and [0842] a VL domain having the sequence SEQ ID NO. 2.
[0843] An antibody of the conjugates described herein comprising:
[0844] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1133; [0845] a light chain comprising the amino acid sequence of
SEQ ID NO.163; [0846] a VH domain having the sequence SEQ ID NO. 1;
and [0847] a VL domain having the sequence SEQ ID NO. 2.
[0848] An antibody of the conjugates described herein comprising:
[0849] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1141, SEQ ID NO.1142, SEQ ID NO.1143, SEQ ID NO.1144, SEQ ID
NO.1145, SEQ ID NO.1146; [0850] a light chain comprising the amino
acid sequence of SEQ ID NO.151, SEQ ID NO.152, SEQ ID NO.153, SEQ
ID NO.161, SEQ ID NO.162, or SEQ ID NO.163; [0851] a VH domain
having the sequence SEQ ID NO. 1; and [0852] a VL domain having the
sequence SEQ ID NO. 2.
[0853] An antibody of the conjugates described herein comprising:
[0854] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1143; [0855] a light chain comprising the amino acid sequence of
SEQ ID NO.151; [0856] a VH domain having the sequence SEQ ID NO. 1;
and [0857] a VL domain having the sequence SEQ ID NO. 2.
[0858] An antibody of the conjugates described herein comprising:
[0859] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1143; [0860] a light chain comprising the amino acid sequence of
SEQ ID NO.152; [0861] a VH domain having the sequence SEQ ID NO. 1;
and [0862] a VL domain having the sequence SEQ ID NO. 2.
[0863] An antibody of the conjugates described herein comprising:
[0864] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1143; [0865] a light chain comprising the amino acid sequence of
SEQ ID NO.153; [0866] a VH domain having the sequence SEQ ID NO. 1;
and [0867] a VL domain having the sequence SEQ ID NO. 2.
[0868] An antibody of the conjugates described herein comprising:
[0869] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1143; [0870] a light chain comprising the amino acid sequence of
SEQ ID NO.161; [0871] a VH domain having the sequence SEQ ID NO. 1;
and [0872] a VL domain having the sequence SEQ ID NO. 2.
[0873] An antibody of the conjugates described herein comprising:
[0874] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1143; [0875] a light chain comprising the amino acid sequence of
SEQ ID NO.162; [0876] a VH domain having the sequence SEQ ID NO. 1;
and [0877] a VL domain having the sequence SEQ ID NO. 2.
[0878] An antibody of the conjugates described herein comprising:
[0879] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1143; [0880] a light chain comprising the amino acid sequence of
SEQ ID NO.163; [0881] a VH domain having the sequence SEQ ID NO. 1;
and [0882] a VL domain having the sequence SEQ ID NO. 2.
[0883] AbDJ591 IgG1
[0884] An antibody of the conjugates described herein comprising a
heavy chain comprising the amino acid sequence of SEQ ID NO.110, a
light chain comprising the amino acid sequence of SEQ ID NO. 150 or
SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a
VL domain having the sequence SEQ ID NO. 2; [0885] wherein each of
the cysteines at positions 103, 109 and 112 in SEQ ID NO: 110 is
substituted by an amino acid that is not cysteine; [0886] and
wherein the leucine at position 117 of SEQ ID NO.110 and/or the
leucine at position 118 of SEQ ID NO.110 is substituted by an amino
acid that is not leucine. Preferably the drug moiety is conjugated
to the cysteine at position 105 of SEQ ID NO.150, or the cysteine
at position 102 of SEQ ID NO.160. Preferably the cysteines at
positions 109 and 112 in SEQ ID NO: 110 are substituted by
valine.
[0887] An antibody of the conjugates described herein comprising:
[0888] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1151; [0889] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0890] a VH domain having the
sequence SEQ ID NO. 1; and [0891] a VL domain having the sequence
SEQ ID NO. 2.
[0892] An antibody of the conjugates described herein comprising:
[0893] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1152; [0894] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0895] a VH domain having the
sequence SEQ ID NO. 1; and [0896] a VL domain having the sequence
SEQ ID NO. 2.
[0897] An antibody of the conjugates described herein comprising:
[0898] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1153; [0899] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0900] a VH domain having the
sequence SEQ ID NO. 1; and [0901] a VL domain having the sequence
SEQ ID NO. 2.
[0902] An antibody of the conjugates described herein comprising:
[0903] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1154; [0904] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0905] a VH domain having the
sequence SEQ ID NO. 1; and [0906] a VL domain having the sequence
SEQ ID NO. 2.
[0907] An antibody of the conjugates described herein comprising:
[0908] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1155; [0909] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0910] a VH domain having the
sequence SEQ ID NO. 1; and [0911] a VL domain having the sequence
SEQ ID NO. 2.
[0912] An antibody of the conjugates described herein comprising:
[0913] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1156; [0914] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0915] a VH domain having the
sequence SEQ ID NO. 1; and [0916] a VL domain having the sequence
SEQ ID NO. 2.
[0917] An antibody of the conjugates described herein comprising:
[0918] a heavy chain comprising the amino acid sequence of SEQ ID
NO. 116; [0919] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0920] a VH domain having the
sequence SEQ ID NO. 1; and [0921] a VL domain having the sequence
SEQ ID NO. 2.
[0922] An antibody of the conjugates described herein comprising:
[0923] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1161; [0924] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0925] a VH domain having the
sequence SEQ ID NO. 1; and [0926] a VL domain having the sequence
SEQ ID NO. 2.
[0927] An antibody of the conjugates described herein comprising:
[0928] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1162; [0929] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0930] a VH domain having the
sequence SEQ ID NO. 1; and [0931] a VL domain having the sequence
SEQ ID NO. 2.
[0932] An antibody of the conjugates described herein comprising:
[0933] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1163; [0934] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0935] a VH domain having the
sequence SEQ ID NO. 1; and [0936] a VL domain having the sequence
SEQ ID NO. 2.
[0937] An antibody of the conjugates described herein comprising:
[0938] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1164; [0939] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0940] a VH domain having the
sequence SEQ ID NO. 1; and [0941] a VL domain having the sequence
SEQ ID NO. 2.
[0942] An antibody of the conjugates described herein comprising:
[0943] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1165; [0944] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0945] a VH domain having the
sequence SEQ ID NO. 1; and [0946] a VL domain having the sequence
SEQ ID NO. 2.
[0947] An antibody of the conjugates described herein comprising:
[0948] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1166; [0949] a light chain comprising the amino acid sequence of
SEQ ID NO.150 or SEQ ID NO.160; [0950] a VH domain having the
sequence SEQ ID NO. 1; and [0951] a VL domain having the sequence
SEQ ID NO. 2.
[0952] Definitions
[0953] Numbering of Amino Acid Positions in Immunoglobulin (Ig)
Molecules
[0954] The numbering of the amino acids used herein is according to
the numbering system of the EU index as set forth in Kabat et al.
(1991, NIH Publication 91-3242, National Technical Information
Service, Springfield, Va., hereinafter "Kabat"). The "EU index as
set forth in Kabat" refers to the residue numbering of the human
IgG 1 EU antibody as described in Kabat et al. supra.
[0955] In the case of substitutions in, for example, IgG2, IgG3,
and IgG4 (or of IgA1, IgA2, IgD, IgE, IgM etc.) the skilled person
can readily use sequence alignment programs such as NCBI BLAST.RTM.
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) to align the sequences
with IgG1 to determine which residues of the desired isoform
correspond to the Kabat positions described herein.
[0956] Antibody
[0957] The term "antibody" as used encompasses any molecule
comprising an antibody antigen-binding site (as, for example,
formed by a paired VH domain and a VL domain). Thus, for example,
the term "antibody" encompasses monoclonal antibodies (including
intact monoclonal antibodies), polyclonal antibodies, multispecific
antibodies formed from at least two different epitope binding
fragments (e.g., bispecific antibodies), human antibodies,
humanized antibodies, camelised antibodies, chimeric antibodies,
single-chain antibodies (such as scFv fusions with CH3), antibody
fragments that exhibit the desired biological activity (e.g. the
antigen binding portion; for exampleminibodies), and anti-idiotypic
(anti-Id) antibodies, intrabodies, and epitope-binding fragments of
any of the above, so long as they exhibit the desired biological
activity, for example, the ability to bind the cognate antigen.
Antibodies may be murine, human, humanized, chimeric, or derived
from other species. In one embodiment the antibody is a
single-chain Fv antibody fused to a CH3 domain (scFv-CH3). In one
embodiment the antibody is a single-chain Fv antibody fused to a Fc
region (scFv-Fc). In one embodiment the antibody is a minibody.
[0958] An antibody is a protein generated by the immune system that
is capable of recognizing and binding to a specific antigen.
(Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno
Biology, 5th Ed., Garland Publishing, New York). A target antigen
generally has numerous binding sites, also called epitopes,
recognized by CDRs on multiple antibodies. Each antibody that
specifically binds to a different epitope has a different
structure. Thus, one antigen may have more than one corresponding
antibody. An antibody includes an intact immunoglobulin molecule or
an immunologically active portion of a intact immunoglobulin
molecule, i.e., a molecule that contains an antigen binding site
that immunospecifically binds an antigen of a target of interest or
part thereof, such targets including but not limited to, cancer
cell or cells that produce autoimmune antibodies associated with an
autoimmune disease.
[0959] In particular, antibodies include immunoglobulin molecules
and immunologically active fragments of immunoglobulin molecules,
i.e., molecules that contain at least one antigen binding site. The
antibody can be of any isotype (e.g. IgG, IgE, IgM, IgD, and IgA),
class (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass, or
allotype (e.g. human G1m1, G1m2, G1m3, non-G1m1 [that, is any
allotype other than G1m1], G1m17, G2m23, G3m21, G3m28, G3m11, G3m5,
G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1,
A2m2, Km1, Km2 and Km3) of antibody molecule. The immunoglobulins
can be derived from any species, including human, murine, or rabbit
origin.
[0960] An "intact antibody" herein is one comprising VL and VH
domains, as well as a light chain constant domain (CL) and heavy
chain constant domains, CH1, CH2 and CH3. The constant domains may
be native sequence constant domains (e.g. human native sequence
constant domains) or amino acid sequence variant thereof. The
intact antibody may have one or more "effector functions" which
refer to those biological activities attributable to the Fc region
(a native sequence Fc region or amino acid sequence variant Fc
region) of an antibody. Examples of antibody effector functions
include C1q binding; complement dependent cytotoxicity; Fc receptor
binding; antibody-dependent cell-mediated cytotoxicity (ADCC);
phagocytosis; and down regulation of cell surface receptors such as
B cell receptor and BCR.
[0961] Antibody Heavy Chain Constant Region, or a Portion
Thereof
[0962] The terms "antibody heavy chain constant region", "Fc
region", "Fc domain" and "Fc", as used herein refer to the portion
of an antibody molecule that correlates to a crystallizable
fragment obtained by papain digestion of an IgG molecule.
[0963] As used herein, the terms "Fc region", "Fc domain" and "Fc"
relate to the constant region of an antibody excluding the first
constant region immunoglobulin domain and further relates to
portions of that region. Thus, Fc refers to the last two constant
region immunoglobulin domains of IgA, IgD, and IgG, and the last
three constant region immunoglobulin domains of IgE and IgM, and
the flexible hinge N-terminal to these domains, or portions
thereof. For IgA and IgM, Fc may include the J chain.
[0964] For IgG, Fc comprises immunoglobulin domains Cy2 and Cy3 (C
gamma 2 and C gamma 3) and the hinge between Cy1 (C gamma 1) and
Cy2 (C gamma 2). Although the boundaries of the Fc region may vary,
the human IgG heavy chain Fc region is usually defined to comprise
residues C226 or P230 to its carboxyl-terminus, as numbered
according to the numbering system of the EU index as set forth in
Kabat et al. supra. Typically, the Fc domain comprises from about
amino acid residue 236 to about 447 of the human IgG1 constant
domain.
[0965] Fc polypeptide may refer to this region in isolation, or
this region in the context of an antibody, or an antigen-binding
portion thereof, or Fc fusion protein.
[0966] The "intact heavy chain constant region" comprises the Fc
region and further comprises the CH1 domain and hinge as well as
the CH2 and CH3 (and, optionally, CH4 of IgA and IgE) domains of
the IgG heavy chain.
[0967] "Hinge region" as used herein, is generally defined as
stretching from Glu216 to Pro230 of human IgG1 (Burton, 1985,
Malec. Immunol. 22: 161-206), and refers to the portion of an IgG
molecule comprising the C-terminal portion of the CH1 domain and
the N-terminal portion of the CH2 domain. Exemplary hinge regions
for human IgG1, IgG2, IgG2 and IgG4 and mouse IgG1 and IgG2A are
provided in U.S. Pat. No. 6,165,476, at the Table shown at column
4, line 54 to column 5, line 15, and also illustrated, for example,
in Janeway et al., 1999, Immunology: The Immune System in Health
and Disease, 4th ed. (Elsevier Science Ltd.); Bloom et al., 1997,
Protein Science 6:407-415; Humphreys et al., 1997, J. Immunol.
Methods 209:193-202. Hinge regions of other IgG isotypes may be
aligned with the IgG 1 sequence by placing the first and last
cysteine residues forming inter-heavy chain S--S bonds in the same
positions.
[0968] The "lower hinge region" of an Fc region is normally defined
as the stretch of residues immediately C-terminal to the hinge
region, i.e. residues 233 to 239 of the Fe region The term "IgG
hinge-Fc region" or "hinge-Fc fragment" as used herein refers to a
hinge region (approximately residues 216-230) and an Fc region
(residues 231-44 7) C-terminal thereto.
[0969] The term "fragment" is used herein to describe a portion of
sequence that is shorter than the full-length sequence disclosed
herein. Preferably antibodies comprising "fragments" as disclosed
herein retain the ability to bind the target antigen, most
preferably with a specific binding activity of about 70% or more
compared to of an otherwise identical antibody comprising the
full-length sequence disclosed herein (for example, about 10% or
more, 50% or more, 75% or more, 80% or more, 85% or more, 90% or
more, 95% or more of the binding activity). In certain embodiments,
the specific binding activity is in vitro. The specific binding
activity sometimes is quantified by an in vitro homogeneous assay
or an in vitro heterogeneous assay. In some embodiments the
specific binding activity is in vivo, and sometimes, the specific
binding activity is determined in situ. In some embodiments a
"fragment" is at least 50 amino acids long, such as at least 75, at
least 100, at least 150, at least 200, at least 250, or at least
300 amino acids long.
[0970] Sequence Modifications
[0971] The sequences of the antibody heavy chain variable regions
and/or the light chain variable regions disclosed herein may be
modified by substitution, insertion or deletion. Amino acid
sequences that are substantially the same as the sequences
described herein include sequences comprising conservative amino
acid substitutions, as well as amino acid deletions and/or
insertions. A conservative amino acid substitution refers to the
replacement of a first amino acid by a second amino acid that has
chemical and/or physical properties (e.g., charge, structure,
polarity, hydrophobicity/hydrophilicity) that are similar to those
of the first amino acid. Preferred conservative substitutions are
those wherein one amino acid is substituted for another within the
groups of amino acids indicated herein below:
[0972] Amino acids having polar side chains (Asp, Glu, Lys, Arg,
His, Asn, Gin, Ser, Thr, Tyr, and Cys)
[0973] Amino acids having non-polar side chains (Gly, Ala, Val,
Leu, Ile, Phe, Trp, Pro, and Met)
[0974] Amino acids having aliphatic side chains (Gly, Ala Val, Leu,
Ile)
[0975] Amino acids having cyclic side chains (Phe, Tyr, Trp, His,
Pro)
[0976] Amino acids having aromatic side chains (Phe, Tyr, Trp)
[0977] Amino acids having acidic side chains (Asp, Glu)
[0978] Amino acids having basic side chains (Lys, Arg, His)
[0979] Amino acids having amide side chains (Asn, Gln)
[0980] Amino acids having hydroxy side chains (Ser, Thr)
[0981] Amino acids having sulphur-containing side chains (Cys,
Met),
[0982] Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser,
Thr)
[0983] Hydrophilic, acidic amino acids (Gin, Asn, Glu, Asp),
and
[0984] Hydrophobic amino acids (Leu, Ile, Val)
[0985] Particular preferred conservative amino acids substitution
groups are: Val-Leu-Ile, Phe-Tyr, Lys-Arg, Ala-Val, and
Asn-Gln.
[0986] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain having an amino acid
sequence with 80% or more amino acid sequence identity (for
example, about 85% or more, 86% or more, 87% or more, 88% or more,
89% or more, 90% or more, 91% or more, 92% or more, 93% or more,
94% or more, 95% or more, 96% or more, 97% or more, 98% or more,
99% or more sequence identity) to a heavy chain described herein.
In some embodiments, the antibody of the conjugates described
herein comprises a light chain having an amino acid sequence with
80% or more amino acid sequence identity (for example, about 85% or
more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or
more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or
more, 96% or more, 97% or more, 98% or more, 99% or more sequence
identity) to a light chain described herein.
[0987] In some embodiments, the antibody of the conjugates
described herein comprises a heavy chain having an amino acid
sequence identical to the amino acid sequence of a heavy chain
described herein, except that it includes 1, 2, 3, 4, 5, 6, 7, 8, 9
or 10 amino acid modifications (e.g., substitutions, insertions
and/or deletions) relative to the amino acid sequence of the heavy
chain described herein. In some embodiments, the antibody of the
conjugates described herein comprises a light chain having an amino
acid sequence identical to the amino acid sequence of a light chain
described herein, except that it includes 1, 2, 3, 4, 5, 6, 7, 8, 9
or 10 amino acid modifications (e.g., substitutions, insertions
and/or deletions) relative to the amino acid sequence of the light
chain described herein.
[0988] Reduction of Immunogenicity
[0989] The antibodies disclosed herein may be modified. For
example, to make them less immunogenic to a human subject. This may
be achieved using any of a number of techniques familiar to the
person skilled in the art. Some of these techniques are described
in more detail below.
[0990] Humanisation
[0991] Techniques to reduce the in vivo immunogenicity of a
non-human antibody or antibody fragment include those termed
"humanisation".
[0992] A "humanized antibody" refers to a polypeptide comprising at
least a portion of a modified variable region of a human antibody
wherein a portion of the variable region, preferably a portion
substantially less than the intact human variable domain, has been
substituted by the corresponding sequence from a non-human species
and wherein the modified variable region is linked to at least
another part of another protein, preferably the constant region of
a human antibody. The expression "humanized antibodies" includes
human antibodies in which one or more complementarity determining
region ("CDR") amino acid residues and/or one or more framework
region ("FW" or "FR") amino acid residues are substituted by amino
acid residues from analogous sites in rodent or other non-human
antibodies. The expression "humanized antibody" also includes an
immunoglobulin amino acid sequence variant or fragment thereof that
comprises an FR having substantially the amino acid sequence of a
human immunoglobulin and a CDR having substantially the amino acid
sequence of a non-human immunoglobulin.
[0993] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. Or, looked at another way, a humanized
antibody is a human antibody that also contains selected sequences
from non-human (e.g. murine) antibodies in place of the human
sequences. A humanized antibody can include conservative amino acid
substitutions or non-natural residues from the same or different
species that do not significantly alter its binding and/or biologic
activity. Such antibodies are chimeric antibodies that contain
minimal sequence derived from non-human immunoglobulins.
[0994] There are a range of humanisation techniques, including `CDR
grafting`, `guided selection`, `deimmunization`, `resurfacing`
(also known as `veneering`), `composite antibodies`, `Human String
Content Optimisation` and framework shuffling.
[0995] CDR Grafting
[0996] In this technique, the humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a
complementary-determining region (CDR) of the recipient antibody
are replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat, camel, bovine, goat, or rabbit having
the desired properties (in effect, the non-human CDRs are `grafted`
onto the human framework). In some instances, framework region (FR)
residues of the human immunoglobulin are replaced by corresponding
non-human residues (this may happen when, for example, a particular
FR residue has significant effect on antigen binding).
[0997] Furthermore, humanized antibodies can comprise residues that
are found neither in the recipient antibody nor in the imported CDR
or framework sequences. These modifications are made to further
refine and maximize antibody performance. Thus, in general, a
humanized antibody will comprise all of at least one, and in one
aspect two, variable domains, in which all or all of the
hypervariable loops correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin sequence. The humanized antibody
optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), or that of a human
immunoglobulin.
[0998] Guided Selection
[0999] The method consists of combining the V.sub.H or V.sub.L
domain of a given non-human antibody specific for a particular
epitope with a human V.sub.H or V.sub.L library and specific human
V domains are selected against the antigen of interest. This
selected human VH is then combined with a VL library to generate a
completely human VH.times.VL combination. The method is described
in Nature Biotechnology (N.Y.) 12, (1994) 899-903.
[1000] Composite Antibodies
[1001] In this method, two or more segments of amino acid sequence
from a human antibody are combined within the final antibody
molecule. They are constructed by combining multiple human VH and
VL sequence segments in combinations which limit or avoid human T
cell epitopes in the final composite antibody V regions. Where
required, T cell epitopes are limited or avoided by, exchanging V
region segments contributing to or encoding a T cell epitope with
alternative segments which avoid T cell epitopes. This method is
described in US 2008/0206239 A1.
[1002] Deimmunization
[1003] This method involves the removal of human (or other second
species) T-cell epitopes from the V regions of the therapeutic
antibody (or other molecule). The therapeutic antibodies V-region
sequence is analysed for the presence of MHC class II- binding
motifs by, for example, comparison with databases of MHC-binding
motifs (such as the "motifs" database hosted at www.wehi.edu.au).
Alternatively, MHC class II- binding motifs may be identified using
computational threading methods such as those devised by Altuvia et
al. (J. Mol. Biol. 249 244-250 (1995)); in these methods,
consecutive overlapping peptides from the V-region sequences are
testing for their binding energies to MHC class II proteins. This
data can then be combined with information on other sequence
features which relate to successfully presented peptides, such as
amphipathicity, Rothbard motifs, and cleavage sites for cathepsin B
and other processing enzymes.
[1004] Once potential second species (e.g. human) T-cell epitopes
have been identified, they are eliminated by the alteration of one
or more amino acids. The modified amino acids are usually within
the T-cell epitope itself, but may also be adjacent to the epitope
in terms of the primary or secondary structure of the protein (and
therefore, may not be adjacent in the primary structure). Most
typically, the alteration is by way of substitution but, in some
circumstances amino acid addition or deletion will be more
appropriate.
[1005] All alterations can be accomplished by recombinant DNA
technology, so that the final molecule may be prepared by
expression from a recombinant host using well established methods
such as Site Directed Mutagenesis. However, the use of protein
chemistry or any other means of molecular alteration is also
possible.
[1006] Resurfacing
[1007] This method involves: [1008] (a) determining the
conformational structure of the variable region of the non-human
(e.g. rodent) antibody (or fragment thereof) by constructing a
three-dimensional model of the non-human antibody variable region;
[1009] (b) generating sequence alignments using relative
accessibility distributions from x-ray crystallographic structures
of a sufficient number of non-human and human antibody variable
region heavy and light chains to give a set of heavy and light
chain framework positions wherein the alignment positions are
identical in 98% of the sufficient number of non-human antibody
heavy and light chains; [1010] (c) defining for the non-human
antibody to be humanized, a set of heavy and light chain surface
exposed amino acid residues using the set of framework positions
generated in step (b); [1011] (d) identifying from human antibody
amino acid sequences a set of heavy and light chain surface exposed
amino acid residues that is most closely identical to the set of
surface exposed amino acid residues defined in step (c), wherein
the heavy and light chain from the human antibody are or are not
naturally paired; [1012] (e) substituting, in the amino acid
sequence of the non-human antibody to be humanized, the set of
heavy and light chain surface exposed amino acid residues defined
in step (c) with the set of heavy and light chain surface exposed
amino acid residues identified in step (d); [1013] (f) constructing
a three-dimensional model of the variable region of the non-human
antibody resulting from the substituting specified in step (e);
[1014] (g) identifying, by comparing the three-dimensional models
constructed in steps (a) and (f), any amino acid residues from the
sets identified in steps (c) or (d), that are within 5 Angstroms of
any atom of any residue of the complementarity determining regions
of the non-human antibodt to be humanized; and (h1) changing any
residues identified in step (g) from the human to the original
non-human amino acid residue to thereby define a non-human antibody
humanizing set of surface exposed amino acid residues; with the
proviso that step (a) need not be conducted first, but must be
conducted prior to step (g).
[1015] Superhumanization
[1016] The method compares the non-human sequence with the
functional human germline gene repertoire. Those human genes
encoding canonical structures identical or closely related to the
non-human sequences are selected. Those selected human genes with
highest homology within the CDRs are chosen as FR donors. Finally,
the non-human CDRs are grafted onto these human FRs. This method is
described in patent WO 2005/079479 A2.
[1017] Human String Content Optimization
[1018] This method compares the non-human (e.g. mouse) sequence
with the repertoire of human germline genes and the differences are
scored as Human String Content (HSC) that quantifies a sequence at
the level of potential MHC/T-cell epitopes. The target sequence is
then humanized by maximizing its HSC rather than using a global
identity measure to generate multiple diverse humanized variants
(described in Molecular Immunology, 44, (2007) 1986-1998).
[1019] Framework Shuffling
[1020] The CDRs of the non-human antibody are fused in-frame to
cDNA pools encompassing all known heavy and light chain human
germline gene frameworks. Humanised antibodies are then selected by
e.g. panning of the phage displayed antibody library. This is
described in Methods 36, 43-60 (2005).
[1021] Epitope Binding Domain
[1022] As used herein, the term epitope binding domain refers to a
domain which is able to specifically recognize and bind an
antigenic epitope. The classic example of an epitope binding domain
would be an antibody paratope comprising a V.sub.H domain and a
V.sub.L domain forming an antigen binding site.
[1023] The sequences of the antibody heavy chain variable regions
and/or the light chain variable regions disclosed herein may be
modified by, for example, insertions, substitutions and/or
deletions to the extent that the epitope binding domain maintains
the ability to bind to the cognate antigen. The skilled person can
ascertain the maintenance of this activity by performing the
functional assays described herein, or known in the art.
Accordingly, in some embodiments the heavy chain variable region
comprises no more than 20 insertions, substitutions and/or
deletions, such as no more than 15, no more than 10, no more than
9, no more than 8, no more than 7, no more than 6, no more than 5,
no more than 4, no more than 3, no more than 2, or no more than 1
insertion, substitution and/or deletion. In some embodiments the
light chain variable region comprises no more than 20 insertions,
substitutions and/or deletions, such as no more than 15, no more
than 10, no more than 9, no more than 8, no more than 7, no more
than 6, no more than 5, no more than 4, no more than 3, no more
than 2, or no more than 1 insertion, substitution and/or deletion.
In some embodiments the antibodies of the disclosure include
comprising VH and VL domains with amino acid sequences that are
identical to the sequences described herein.
[1024] Therapeutic Index
[1025] As used herein, the term "therapeutic index is used as a
comparison of the amount of a therapeutic agent that causes the
therapeutic effect to the amount that causes death (in animal
studies) or toxicity (in human studies).
Therapeutic index=LD.sub.50/ED.sub.50 (animal studies), or
=TD50/ED50 (human studies),
[1026] where LD=lethal dose for 50% of the population, TD=toxic
dose for 50% of the population, and ED=minimum effective dose for
50% of the population. The levels of "effective" and "toxic" doses
can be readily determined by a medical practitioner or person
skilled in the art. When comparing the therapeutic indexes of the
site-specific and non-site-specific conjugates, the levels of
"effective" and "toxic" are determined in an identical manner
[1027] Otherwise Identical
[1028] The term "otherwise identical non site-specific conjugate"
as used herein refers to a conjugate which is identical to the
defined or claimed site-specific conjugate in all respects apart
from the position(s) at which the Drug units (D.sup.L) are
conjugated to antibody heavy chain constant region, or a portion
thereof. Specifically, in the defined or claimed site-specific
conjugate Drug units (D.sup.L) are uniformly and consistently
conjugated to the specified residue(s), whereas in an otherwise
identical non site-specific the degree and position of conjugation
of Drug unit (D.sup.L) to the antibody is variable from batch to
batch.
[1029] For example, in one embodiment of a site specific
antibody-drug conjugate of the disclosure there are two Drug units
(D.sup.L), one conjugated to each of the position 442 residues
(kabat numbering) of the two antibody heavy chain constant regions,
or a portions thereof. The `otherwise identical non site-specific
conjugate` for this example would be an antibody with identical
amino acid sequence and polypeptide structure, also with two
conjugated Drug unit (D.sup.L); however, the Drug unist (D.sup.L)
would not uniformly and consistently conjugated to each 442
position, but rather conjugated to a selection of different
positions the precise combination of which varies from conjugate to
conjugate within a population (for example, conjugation may be via
lysine side chains or by reduced interchain disulfide bonds).
[1030] As described herein, properties such as affinity,
therapeutic index and stability are bulk properties measured at a
population level, as opposed to being measured at a molecular
level. Thus, the comparisons made herein between the properties of
a site-specific conjugate and an "otherwise identical non
site-specific conjugate" are comparisons of properties exhibited by
populations of those molecules.
[1031] Functional Moieties
[1032] The humanised antibody of the disclosure may be conjugated
to a functional moiety. Examples of functional moieties include an
amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a
nucleotide, an oligonucleotide, a nucleic acid, a drug, a hormone,
a lipid, a lipid assembly, a synthetic polymer, a polymeric
microparticle, a biological cell, a virus, a reporter (such as a
fluorophore, a chromophore, or a dye), a toxin, a hapten, an
enzyme, a binding member (such as an antibody, or an antibody
fragment), a radioisotope, solid matrixes, semisolid matrixes and
combinations thereof, or an organic moiety.
[1033] Examples of a drug include a cytotoxic agent, a
chemotherapeutic agent, a peptide, a peptidomimetic, a protein
scaffold, DNA, RNA, siRNA, microRNA, and a peptidonucleic acid. In
preferred embodiments the functional moiety is a PBD drug moiety.
In other embodiments the humanised antibody is conjugated to a
therapeutic agent or drug moiety that modifies a given biological
response. Therapeutic agents or drug moieties are not to be
construed as limited to classical chemical therapeutic agents. For
example, the drug moiety may be a protein or polypeptide possessing
a desired biological activity. Such proteins may include, for
example, a toxin such as abrin, ricin A, pseudomonas exotoxin,
cholera toxin, or diphtheria toxin; a protein such as tumor
necrosis factor, .alpha.-interferon, .beta.-interferon, nerve
growth factor, platelet derived growth factor, tissue plasminogen
activator, an apoptotic agent, e.g., TNF-.alpha., TNF-.beta., AIM I
(see, International Publication No. WO 97/33899), AIM II (see,
International Publication No. WO 97/34911), Fas Ligand (Takahashi
et al., 1994, J Immunol., 6: 1567), and VEGf (see, International
Publication No. WO 99/23105), a thrombotic agent or an
anti-angiogenic agent, e.g., angiostatin or endostatin; or, a
biological response modifier such as, for example, a lymphokine
(e.g., interleukin-1 ("IL-I"), interleukin-2 ("IL-2"),
interleukin-4 ("IL-4"), interleukin-6 ("IL-6"), interleukin-7
("IL-7"), interleukin-9 ("IL-9"), interleukin-15 ("IL-15"),
interleukin-12 ("IL-12"), granulocyte macrophage colony stimulating
factor ("GMCSF"), and granulocyte colony stimulating factor
("G-CSF")), or a growth factor (e.g.,growth hormone ("GH")).
[1034] Examples of a reporter include a fluorophore, a chromophore,
a radionuclide, and an enzyme. Such antibody-reporter conjugates
can be useful for monitoring or prognosing the development or
progression of a disorder (such as, but not limited to cancer) as
part of a clinical testing procedure, such as determining the
efficacy of a particular therapy. Such diagnosis and detection can
accomplished by fusing or conjugating the antibody to detectable
substances including, but not limited to various enzymes, such as
but not limited to horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, or acetylcholinesterase; prosthetic groups,
such as but not limited to streptavidin/biotin and avidin/biotin;
fluorescent materials, such as but not limited to, umbelliferone,
fluorescein, fluorescein isothiocynate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; luminescent materials, such as but not limited to,
bioluminescent materials, such as but not limited to, luciferase,
luciferin, and aequorin; radioactive materials, such as but not
limited to, bismuth (.sup.213Bi), carbon (.sup.14C), chromium
(.sup.51Cr), cobalt (.sup.57Co), fluorine (.sup.18F), gadolinium
(.sup.153Gd, .sup.159Gd), gallium (.sup.68Ga, .sup.67Ga), germanium
(.sup.68Ge), holmium (166Ho) indium (115.sub.In, 113In, .sub.112In,
.sup.111In), iodine (131I, .sup.125I, .sup.123I, .sup.121I),
lanthanium (.sup.140La), lutetium (.sup.177Lu), manganese
(.sup.54Mn), molybdenum (.sup.99Mo), palladium (.sup.103Pd),
phosphorous (.sup.32P), praseodymium (.sup.142Pr), promethium
(.sup.149Pm), rhenium (186Re, .sup.188Re), rhodium (.sup.105Rh),
ruthemium (.sup.97Ru), samarium (.sup.153Sm), scandium (.sup.47Sc),
selenium (.sup.75Se), strontium (.sup.85Sr), sulfur (3.sup.5S),
technetium (.sup.99Tc), thallium (.sup.201Ti), tin (.sup.113Sn,
.sup.117Sn), tritium (.sup.3H), xenon (.sup.133Xe), ytterbium
(.sup.169Yb, .sup.175Yb), yttrium (.sup.90Y), zinc (.sup.65Zn);
positron emitting metals using various positron emission
tomographies, and nonradioactive paramagnetic metal ions.
[1035] Examples of a binding member include an antibody or antibody
fragment, and biotin and/or streptavidin.
[1036] A toxin, cytotoxin or cytotoxic agent includes any agent
that is detrimental to cells. Examples of toxins include
radioisotopes such as .sup.131I, a ribosome inactivating protein
such as pseudomonas exotoxin (PE38 fragment), plant or bacterial
toxins such as ricin, the a-chain of ricin, saporin, pokeweed
antiviral protein, diphtheria toxin, or Pseudomonas exotoxin A
(Kreitman and Pastan (1998) Adv. Drug Delivery Rev. 31:53.). Other
toxins and cytotoxins include, e.g., a cytostatic or cytocidal
agent, or a radioactive metal ion, e.g., alpha-emitters. Examples
include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide,
emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide
and analogs or homo logs thereof, antimetabolites (e.g.,
methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-fluorouracil decarbazine), alkylating agents (e.g.,
mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BCNU)
and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,
streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II)
(DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine). Chemical toxins can also be taken from the group
chosen from duocarmycin (U.S. Pat. Nos. 5,703,080; 4,923,990),
methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C,
vindesine, mitomycin C, cisplatinum, etoposide, bleomycin and
5-fluorouracil. Examples of chemotherapeutic agents also include
Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside
(Ara-C), Cyclophosphamide, Thiotepa, Taxotere (docetaxel),
Busulfan, Cytoxin, Taxol, Methotrexate, In one embodiment, the
cytotoxic agent is chosen from an enediyne, a lexitropsin, a
duocarmycin, a taxane, a puromycin, a dolastatin, a maytansinoid,
and a vinca alkaloid. In other embodiments, the cytotoxic agent is
paclitaxel, docetaxel, CC-I 065, SN-3 8, topotecan,
morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin,
dolastatin-10, echinomycin, combretastatin, calicheamicin,
maytansine, DM-I, an auristatin or other dolastatin derivatives,
such as auristatin E or auristatin F, AEB, AEVB, AEFP, MMAE
(monomethylauristatin E), MMAF (monomethylauristatin F),
eleutherobin or netropsin. In certain embodiments, the cytoxic
agent is Maytansine or Maytansinoids, and derivatives thereof,
wherein an antibodies (full length or fragments) of the disclosure
are conjugated to one or more maytansinoid molecules. Maytansinoids
are mitototic inhibitors which act by inhibiting tubulin
polymerization. In other embodimetns the toxin is a small molecule
or protein toxins, such as, but not limited to abrin, brucine,
cicutoxin, diphtheria toxin, batrachotoxin, botulism toxin, shiga
toxin, endotoxin, Pseudomonas exotoxin, Pseudomonas endotoxin,
tetanus toxin, pertussis toxin, anthrax toxin, cholera toxin,
falcarinol, fumonisin B1, fumonisin B2, aflatoxin, maurotoxin,
agitoxin, charybdotoxin, margatoxin, slotoxin, scyllatoxin,
hefutoxin, calciseptine, taicatoxin, calcicludine, geldanamycin,
gelonin, lotaustralin, ocratoxin A, patulin, ricin, strychnine,
trichothecene, zearlenone, and tetradotoxin. Enzymatically active
toxins and fragments thereof which can be used include diphtheria A
chain, non-binding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
Momordica charantia inhibitor, curcin, crotin, Sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin and the tricothecenes.
[1037] The humanized antibody may be modified by conjugation to an
organic moiety. Such modification can produce an antibody or
antigen-binding fragment with improved pharmacokinetic properties
(e.g., increased in vivo serum half-life). The organic moiety can
be a linear or branched hydrophilic polymeric group, fatty acid
group, or fatty acid ester group. In particular embodiments, the
hydrophilic polymeric group can have a molecular weight of about
800 to about 120,000 Daltons and can be a polyalkane glycol (e.g.,
polyethylene glycol (PEG), polypropylene glycol (PPG)),
carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone,
and the fatty acid or fatty acid ester group can comprise from
about eight to about forty carbon atoms. In certain embodiments,
the cytotoxic or cytostatic agent is a dolastatin. In more specific
embodiments, the dolastatin is of the auristatin class. In a
specific embodiment of the disclosure, the cytotoxic or cytostatic
agent is MMAE. In another specific embodiment of the disclosure,
the cytotoxic or cytostatic agent is AEFP. In another specific
embodiment of the disclosure, the cytotoxic or cytostatic agent is
MMAF.
[1038] The humanized antibody and antigen-binding fragments can
comprise one or more organic moieties that are covalently bonded,
directly or indirectly, to the antibody. Each organic moiety that
is bonded to an antibody or antigen-binding fragment described
herein can independently be a hydrophilic polymeric group, a fatty
acid group or a fatty acid ester group. As used herein, the term
"fatty acid" encompasses mono-carboxylic acids and di-carboxylic
acids. A "hydrophilic polymeric group," as the term is used herein,
refers to an organic polymer that is more soluble in water than in
octane. For example, polylysine is more soluble in water than in
octane. Thus, an antibody modified by the covalent attachment of
polylysine is encompassed by the present disclosure. Hydrophilic
polymers suitable for modifying antibodies described herein can be
linear or branched and include, for example, polyalkane glycols
(e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the
like), carbohydrates (e.g., dextran, cellulose, oligosaccharides,
polysaccharides and the like), polymers of hydrophilic amino acids
(e.g., polylysine, polyarginine, polyaspartate and the like),
polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide
and the like) and polyvinyl pyrolidone. Preferably, the hydrophilic
polymer that modifies the antibody described herein has a molecular
weight of about 800 to about 150,000 Daltons as a separate
molecular entity. For example PEG5000 and PEG20,000, wherein the
numerical component of the name is the average molecular weight of
the polymer in Daltons, can be used. The hydrophilic polymeric
group can be substituted with one to about six alkyl, fatty acid or
fatty acid ester groups. Hydrophilic polymers that are substituted
with a fatty acid or fatty acid ester group can be prepared by
employing suitable methods. For example, a polymer comprising an
amine group can be coupled to a carboxylate of the fatty acid or
fatty acid ester, and an activated carboxylate (e.g., activated
with N,N-carbonyl diimidazole) on a fatty acid or fatty acid ester
can be coupled to a hydroxyl group on a polymer.
[1039] Fatty acids and fatty acid esters suitable for modifying
antibodies described herein can be saturated or can contain one or
more units of unsaturation. Fatty acids that are suitable for
modifying antibodies described herein include, for example,
n-dodecanoate (C12, laurate), n-tetradecanoate (C14, myristate),
n-octadecanoate (C18, stearate), n-eicosanoate (C20, arachidate),
n-docosanoate (C22, behenate), n-triacontanoate (C30),
n-tetracontanoate (C40), cis-.delta. 9-octadecanoate (C18, oleate),
all cis-.delta. 5,8,11,14-eicosatetraenoate (C20, arachidonate),
octanedioic acid, tetradecanedioic acid, octadecanedioic acid,
docosanedioic acid, and similar faty acids. Suitable fatty acid
esters include mono-esters of dicarboxylic acids that comprise a
linear or branched lower alkyl group. The lower alkyl group can
comprise from one to about twelve, preferably one to about six,
carbon atoms.
[1040] The above conjugates can be prepared using suitable methods,
such as by reaction with one or more modifying agents: a "modifying
agent" as the term is used herein, refers to a suitable organic
group (e.g., hydrophilic polymer, a fatty acid, a fatty acid ester)
that comprises an activating group; aAn "activating group" is a
chemical moiety or functional group that can, under appropriate
conditions, react with a second chemical group thereby forming a
covalent bond between the modifying agent and the second chemical
group.
[1041] For example, amine-reactive activating groups include
electrophilic groups such as tosylate, mesylate, halo (chloro,
bromo, fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the
like. Activating groups that can react with thiols include, for
example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfides,
5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An
aldehyde functional group can be coupled to amine- or
hydrazide-containing molecules, and an azide group can react with a
trivalent phosphorous group to form phosphoramidate or
phosphorimide linkages. Suitable methods to introduce activating
groups into molecules are known in the art (see for example,
Hernanson, G. T., Bioconjugate Techniques, Academic Press: San
Diego, Calif. (1996)). An activating group can be bonded directly
to the organic group (e.g., hydrophilic polymer, fatty acid, fatty
acid ester), or through a linker moiety, for example a divalent
C1-C12 group wherein one or more carbon atoms can be replaced by a
heteroatom such as oxygen, nitrogen or sulfur. Suitable linker
moieties include, for example, tetraethylene glycol, --(CH2)3-,
--NH--(CH2)6-NH--, --(CH2)2-NH-- and
--CH2-O--CH2-CH2-O--CH2-CH2-O--CH--NH--. Modifying agents that
comprise a linker moiety can be produced, for example, by reacting
a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine,
mono-Boc-diaminohexane) with a fatty acid in the presence of
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form an
amide bond between the free amine and the fatty acid carboxylate.
The Boc protecting group can be removed from the product by
treatment with trifluoroacetic acid (TFA) to expose a primary amine
that can be coupled to another carboxylate as described, or can be
reacted with maleic anhydride and the resulting product cyclized to
produce an activated maleimido derivative of the fatty acid. (See,
for example, Thompson, et al., WO 92/16221 the entire teachings of
which are incorporated herein by reference.)
[1042] The above conjugates can be produced by reacting a human
antibody or antigen-binding fragment with a modifying agent. For
example, the organic moieties can be bonded to the antibody in a
non-site-specific manner by employing an amine-reactive modifying
agent, for example, an NHS ester of PEG. Modified human antibodies
or antigen-binding fragments can also be prepared by reducing
disulfide bonds (e.g., inter-chain disulfide bonds) of an antibody
or antigen-binding fragment. The reduced antibody or
antigen-binding fragment can then be reacted with a thiol-reactive
modifying agent to produce the modified antibody described herein.
Modified human antibodies and antigen-binding fragments comprising
an organic moiety that is bonded to specific sites of an antibody
described herein can be prepared using suitable methods, such as
reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153
(1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994);
Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al.,
Bioorg. Chem., 24(1): 59-68 (1996); Capellas et al., Biotechnol.
Bioeng., 56(4):456-463 (1997)), and the methods described in
Hermanson, G. T., Bioconjugate Techniques, Academic Press: San
Diego, Calif. (1996).
[1043] Pharmaceutically Acceptable Cations
[1044] Examples of pharmaceutically acceptable monovalent and
divalent cations are discussed in Berge, et al., J. Pharm. Sci.,
66, 1-19 (1977), which is incorporated herein by reference.
[1045] The pharmaceutically acceptable cation may be inorganic or
organic.
[1046] Examples of pharmaceutically acceptable monovalent inorganic
cations include, but are not limited to, alkali metal ions such as
Na.sup.+ and K.sup.+. Examples of pharmaceutically acceptable
divalent inorganic cations include, but are not limited to,
alkaline earth cations such as Ca.sup.2+ and Mg.sup.2+. Examples of
pharmaceutically acceptable organic cations include, but are not
limited to, ammonium ion (i.e. NH.sub.4.sup.+) and substituted
ammonium ions (e.g. NH.sub.3R.sup.+, NH.sub.2R.sub.2.sup.+,
NHR.sub.3.sup.+, NR.sub.4.sup.+). Examples of some suitable
substituted ammonium ions are those derived from: ethylamine,
diethylamine, dicyclohexylamine, triethylamine, butylamine,
ethylenediamine, ethanolamine, diethanolamine, piperazine,
benzylamine, phenylbenzylamine, choline, meglumine, and
tromethamine, as well as amino acids, such as lysine and arginine.
An example of a common quaternary ammonium ion is
N(CH.sub.3).sub.4.sup.+.
[1047] Substituents
[1048] The phrase "optionally substituted" as used herein, pertains
to a parent group which may be unsubstituted or which may be
substituted.
[1049] Unless otherwise specified, the term "substituted" as used
herein, pertains to a parent group which bears one or more
substituents. The term "substituent" is used herein in the
conventional sense and refers to a chemical moiety which is
covalently attached to, or if appropriate, fused to, a parent
group. A wide variety of substituents are well known, and methods
for their formation and introduction into a variety of parent
groups are also well known.
[1050] Examples of substituents are described in more detail
below.
[1051] C.sub.1-12 alkyl: The term "C.sub.1-12 alkyl" as used
herein, pertains to a monovalent moiety obtained by removing a
hydrogen atom from a carbon atom of a hydrocarbon compound having
from 1 to 12 carbon atoms, which may be aliphatic or alicyclic, and
which may be saturated or unsaturated (e.g. partially unsaturated,
fully unsaturated). The term "C.sub.1-4 alkyl" as used herein,
pertains to a monovalent moiety obtained by removing a hydrogen
atom from a carbon atom of a hydrocarbon compound having from 1 to
4 carbon atoms, which may be aliphatic or alicyclic, and which may
be saturated or unsaturated (e.g. partially unsaturated, fully
unsaturated). Thus, the term "alkyl" includes the sub-classes
alkenyl, alkynyl, cycloalkyl, etc., discussed below.
[1052] Examples of saturated alkyl groups include, but are not
limited to, methyl (C.sub.l), ethyl (C.sub.2), propyl (C.sub.3),
butyl (C.sub.4), pentyl (C.sub.5), hexyl (C.sub.6) and heptyl
(C.sub.7).
[1053] Examples of saturated linear alkyl groups include, but are
not limited to, methyl (C.sub.1), ethyl (C.sub.2), n-propyl
(C.sub.3), n-butyl (C.sub.4), n-pentyl (amyl) (C.sub.5), n-hexyl
(C.sub.6) and n-heptyl (C.sub.7).
[1054] Examples of saturated branched alkyl groups include
iso-propyl (C.sub.3), iso-butyl (C.sub.4), sec-butyl (C.sub.4),
tert-butyl (C.sub.4), iso-pentyl (C.sub.5), and neo-pentyl
(C.sub.5).
[1055] C.sub.2-12 Alkenyl: The term "C.sub.2-12 alkenyl" as used
herein, pertains to an alkyl group having one or more carbon-carbon
double bonds.
[1056] Examples of unsaturated alkenyl groups include, but are not
limited to, ethenyl (vinyl, --CH.dbd.CH.sub.2), 1-propenyl
(--CH.dbd.CH--CH.sub.3), 2-propenyl (allyl, --CH--CH.dbd.CH.sub.2),
isopropenyl (1-methylvinyl, --C(CH.sub.3).dbd.CH.sub.2), butenyl
(C.sub.4), pentenyl (C.sub.5), and hexenyl (C.sub.6).
[1057] C.sub.2-12 alkynyl: The term "C.sub.2-12 alkynyl" as used
herein, pertains to an alkyl group having one or more carbon-carbon
triple bonds.
[1058] Examples of unsaturated alkynyl groups include, but are not
limited to, ethynyl (--C.ident.CH) and 2-propynyl (propargyl,
--CH.sub.2--C.ident.CH).
[1059] C.sub.3-12 cycloalkyl: The term "C.sub.3-12 cycloalkyl" as
used herein, pertains to an alkyl group which is also a cyclyl
group; that is, a monovalent moiety obtained by removing a hydrogen
atom from an alicyclic ring atom of a cyclic hydrocarbon
(carbocyclic) compound, which moiety has from 3 to 7 carbon atoms,
including from 3 to 7 ring atoms.
[1060] Examples of cycloalkyl groups include, but are not limited
to, those derived from: [1061] saturated monocyclic hydrocarbon
compounds:
[1062] cyclopropane (C.sub.3), cyclobutane (C.sub.4), cyclopentane
(C.sub.5), cyclohexane (C.sub.6), cycloheptane (C.sub.7),
methylcyclopropane (C.sub.4), dimethylcyclopropane (C.sub.5),
methylcyclobutane (C.sub.5), dimethylcyclobutane (C.sub.6),
methylcyclopentane (C.sub.6), dimethylcyclopentane (C.sub.7) and
methylcyclohexane (C.sub.7);
[1063] unsaturated monocyclic hydrocarbon compounds: [1064]
cyclopropene (C.sub.3), cyclobutene (C.sub.4), cyclopentene
(C.sub.5), cyclohexene (C.sub.6), methylcyclopropene (C.sub.4),
dimethylcyclopropene (C.sub.5), methylcyclobutene (C.sub.5),
dimethylcyclobutene (C.sub.6), methylcyclopentene (C.sub.6),
dimethylcyclopentene (C.sub.7) and methylcyclohexene (C.sub.7);
and
[1065] saturated polycyclic hydrocarbon compounds: [1066] norcarane
(C.sub.7), norpinane (C.sub.7), norbornane (C.sub.7).
[1067] C.sub.3-20 heterocyclyl: The term "C.sub.3-20 heterocyclyl"
as used herein, pertains to a monovalent moiety obtained by
removing a hydrogen atom from a ring atom of a heterocyclic
compound, which moiety has from 3 to 20 ring atoms, of which from 1
to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7
ring atoms, of which from 1 to 4 are ring heteroatoms.
[1068] In this context, the prefixes (e.g. C.sub.3-20, C.sub.3-7,
C.sub.5-6, etc.) denote the number of ring atoms, or range of
number of ring atoms, whether carbon atoms or heteroatoms. For
example, the term "C.sub.5-6heterocyclyl", as used herein, pertains
to a heterocyclyl group having 5 or 6 ring atoms.
[1069] Examples of monocyclic heterocyclyl groups include, but are
not limited to, those derived from:
[1070] N.sub.1: aziridine (C.sub.3), azetidine (C.sub.4),
pyrrolidine (tetrahydropyrrole) (C.sub.5), pyrroline (e.g.,
3-pyrroline, 2,5-dihydropyrrole) (C.sub.5), 2H-pyrrole or
3H-pyrrole (isopyrrole, isoazole) (C.sub.5), piperidine (C.sub.6),
dihydropyridine (C.sub.6), tetrahydropyridine (C.sub.6), azepine
(C.sub.7);
[1071] O.sub.1: oxirane (C.sub.3), oxetane (C.sub.4), oxolane
(tetrahydrofuran) (C.sub.5), oxole (dihydrofuran) (C.sub.5), oxane
(tetrahydropyran) (C.sub.6), dihydropyran (C.sub.6), pyran
(C.sub.6), oxepin (C.sub.7);
[1072] S.sub.1: thiirane (C.sub.3), thietane (C.sub.4), thiolane
(tetrahydrothiophene) (C.sub.5), thiane (tetrahydrothiopyran)
(C.sub.6), thiepane (C.sub.7);
[1073] O.sub.2: dioxolane (C.sub.5), dioxane (C.sub.6), and
dioxepane (C.sub.7);
[1074] O.sub.3: trioxane (C.sub.6);
[1075] N.sub.2: imidazolidine (C.sub.5), pyrazolidine (diazolidine)
(C.sub.5), imidazoline (C.sub.5), pyrazoline (dihydropyrazole)
(C.sub.5), piperazine (C.sub.6);
[1076] N.sub.1O.sub.1: tetrahydrooxazole (C.sub.5), dihydrooxazole
(C.sub.5), tetrahydroisoxazole (C.sub.5), dihydroisoxazole
(C.sub.5), morpholine (C.sub.6), tetrahydrooxazine (C.sub.6),
dihydrooxazine (C.sub.6), oxazine (C.sub.6);
[1077] N.sub.1S.sub.1: thiazoline (C.sub.5), thiazolidine
(C.sub.5), thiomorpholine (C.sub.6);
[1078] N.sub.2O.sub.1: oxadiazine (C.sub.6);
[1079] O.sub.1S.sub.1: oxathiole (C.sub.5) and oxathiane (thioxane)
(C.sub.6); and,
[1080] N.sub.1O.sub.1S.sub.1: oxathiazine (C.sub.6).
[1081] Examples of substituted monocyclic heterocyclyl groups
include those derived from saccharides, in cyclic form, for
example, furanoses (C.sub.5), such as arabinofuranose,
lyxofuranose, ribofuranose, and xylofuranse, and pyranoses
(C.sub.6), such as allopyranose, altropyranose, glucopyranose,
mannopyranose, gulopyranose, idopyranose, galactopyranose, and
talopyranose.
[1082] C.sub.5-20 aryl: The term "C.sub.5-20 aryl", as used herein,
pertains to a monovalent moiety obtained by removing a hydrogen
atom from an aromatic ring atom of an aromatic compound, which
moiety has from 3 to 20 ring atoms. The term "C.sub.5-7 aryl", as
used herein, pertains to a monovalent moiety obtained by removing a
hydrogen atom from an aromatic ring atom of an aromatic compound,
which moiety has from 5 to 7 ring atoms and the term "C.sub.5-10
aryl", as used herein, pertains to a monovalent moiety obtained by
removing a hydrogen atom from an aromatic ring atom of an aromatic
compound, which moiety has from 5 to 10 ring atoms. Preferably,
each ring has from 5 to 7 ring atoms.
[1083] In this context, the prefixes (e.g. C.sub.3-20, C.sub.5-7,
C.sub.5-6, C.sub.5-10, etc.) denote the number of ring atoms, or
range of number of ring atoms, whether carbon atoms or heteroatoms.
For example, the term "C.sub.5-6 aryl" as used herein, pertains to
an aryl group having 5 or 6 ring atoms.
[1084] The ring atoms may be all carbon atoms, as in "carboaryl
groups".
[1085] Examples of carboaryl groups include, but are not limited
to, those derived from benzene (i.e. phenyl) (C.sub.6), naphthalene
(C.sub.10), azulene (C.sub.10), anthracene (C.sub.14), phenanthrene
(C.sub.14), naphthacene (C.sub.18), and pyrene (C.sub.16).
[1086] Examples of aryl groups which comprise fused rings, at least
one of which is an aromatic ring, include, but are not limited to,
groups derived from indane (e.g. 2,3-dihydro-1H-indene) (C.sub.9),
indene (C.sub.9), isoindene (C.sub.9), tetraline
(1,2,3,4-tetrahydronaphthalene (C.sub.10), acenaphthene (C.sub.12),
fluorene (C.sub.13), phenalene (C.sub.13), acephenanthrene
(C.sub.15), and aceanthrene (C.sub.16).
[1087] Alternatively, the ring atoms may include one or more
heteroatoms, as in "heteroaryl groups". Examples of monocyclic
heteroaryl groups include, but are not limited to, those derived
from:
[1088] N.sub.1: pyrrole (azole) (C.sub.5), pyridine (azine)
(C.sub.6);
[1089] O.sub.1: furan (oxole) (C.sub.5);
[1090] S.sub.1: thiophene (thiole) (C.sub.5);
[1091] N.sub.1O.sub.1: oxazole (C.sub.5), isoxazole (C.sub.5),
isoxazine (C.sub.6);
[1092] N.sub.2O.sub.1: oxadiazole (furazan) (C.sub.5);
[1093] N.sub.3O.sub.1: oxatriazole (C.sub.5);
[1094] N.sub.1S.sub.1: thiazole (C.sub.5), isothiazole
(C.sub.5);
[1095] N.sub.2: imidazole (1,3-diazole) (C.sub.5), pyrazole
(1,2-diazole) (C.sub.5), pyridazine (1,2-diazine) (C.sub.6),
pyrimidine (1,3-diazine) (C.sub.6) (e.g., cytosine, thymine,
uracil), pyrazine (1,4-diazine) (C.sub.6);
[1096] N.sub.3: triazole (C.sub.5), triazine (C.sub.6); and,
[1097] N.sub.4: tetrazole (C.sub.5).
[1098] Examples of heteroaryl which comprise fused rings, include,
but are not limited to: [1099] C9 (with 2 fused rings) derived from
benzofuran (O.sub.1), isobenzofuran (O.sub.1), indole (N.sub.1),
isoindole (N.sub.1), indolizine (N.sub.1), indoline (N.sub.1),
isoindoline (N.sub.1), purine (N.sub.4) (e.g., adenine, guanine),
benzimidazole (N.sub.2), indazole (N.sub.2), benzoxazole
(N.sub.1O.sub.1), benzisoxazole (N.sub.1O.sub.1), benzodioxole
(O.sub.2), benzofurazan (N.sub.2O.sub.1), benzotriazole (N.sub.3),
benzothiofuran (S.sub.1), benzothiazole benzothiadiazole
(N.sub.2S);
[1100] C.sub.10 (with 2 fused rings) derived from chromene
(O.sub.1), isochromene (O.sub.1), chroman (O.sub.1), isochroman
(O.sub.1), benzodioxan (O.sub.2), quinoline (N.sub.1), isoquinoline
(N.sub.1), quinolizine (N.sub.1), benzoxazine (N.sub.1O.sub.1),
benzodiazine (N.sub.2), pyridopyridine (N.sub.2), quinoxaline
(N.sub.2), quinazoline (N.sub.2), cinnoline (N.sub.2), phthalazine
(N.sub.2), naphthyridine (N.sub.2), pteridine (N.sub.4);
[1101] C.sub.11 (with 2 fused rings) derived from benzodiazepine
(N.sub.2);
[1102] C.sub.13 (with 3 fused rings) derived from carbazole
(N.sub.1), dibenzofuran (O.sub.1), dibenzothiophene (S.sub.1),
carboline (N.sub.2), perimidine (N.sub.2), pyridoindole (N.sub.2);
and,
[1103] C.sub.14 (with 3 fused rings) derived from acridine
(N.sub.1), xanthene (O.sub.1), thioxanthene (S.sub.1), oxanthrene
(O.sub.2), phenoxathiin (O.sub.1S.sub.1), phenazine (N.sub.2),
phenoxazine (N.sub.1O.sub.1), phenothiazine (N.sub.1S.sub.1),
thianthrene (S.sub.2), phenanthridine (N.sub.1), phenanthroline
(N.sub.2), phenazine (N.sub.2).
[1104] The above groups, whether alone or part of another
substituent, may themselves optionally be substituted with one or
more groups selected from themselves and the additional
substituents listed below.
[1105] Halo: --F, --Cl, --Br, and --I.
[1106] Hydroxy: --OH.
[1107] Ether: --OR, wherein R is an ether substituent, for example,
a C.sub.1-7 alkyl group (also referred to as a C.sub.1-7 alkoxy
group, discussed below), a C.sub.3-20 heterocyclyl group (also
referred to as a C.sub.3-20 heterocyclyloxy group), or a C.sub.5-20
aryl group (also referred to as a C.sub.5-20 aryloxy group),
preferably a C.sub.1-7alkyl group.
[1108] Alkoxy: --OR, wherein R is an alkyl group, for example, a
C.sub.1-7 alkyl group. Examples of C.sub.1-7 alkoxy groups include,
but are not limited to, --OMe (methoxy), --OEt (ethoxy), --O(nPr)
(n-propoxy), --O(iPr) (isopropoxy), --O(nBu) (n-butoxy), --O(sBu)
(sec-butoxy), --O(iBu) (isobutoxy), and --O(tBu) (tert-butoxy).
[1109] Acetal: --CH(OR.sup.1)(OR.sup.2), wherein R.sup.1 and
R.sup.2 are independently acetal substituents, for example, a
C.sub.1-7 alkyl group, a C.sub.3-20 heterocyclyl group, or a
C.sub.5-20 aryl group, preferably a C.sub.1-7 alkyl group, or, in
the case of a "cyclic" acetal group, R.sup.1 and R.sup.2, taken
together with the two oxygen atoms to which they are attached, and
the carbon atoms to which they are attached, form a heterocyclic
ring having from 4 to 8 ring atoms. Examples of acetal groups
include, but are not limited to, --CH(OMe).sub.2, --CH(OEt).sub.2,
and --CH(OMe)(OEt).
[1110] Hemiacetal: --CH(OH)(OR.sup.1), wherein R.sup.1 is a
hemiacetal substituent, for example, a C.sub.1-7 alkyl group, a
C.sub.3-20 heterocyclyl group, or a C.sub.5-20 aryl group,
preferably a C.sub.1-7 alkyl group. Examples of hemiacetal groups
include, but are not limited to, --CH(OH)(OMe) and
--CH(OH)(OEt).
[1111] Ketal: --CR(OR.sup.1)(OR.sup.2), where R.sup.1 and R.sup.2
are as defined for acetals, and R is a ketal substituent other than
hydrogen, for example, a C.sub.1-7 alkyl group, a C.sub.3-20
heterocyclyl group, or a C.sub.5-20 aryl group, preferably a
C.sub.1-7 alkyl group. Examples ketal groups include, but are not
limited to, --C(Me)(OMe).sub.2, --C(Me)(OEt).sub.2,
--C(Me)(OMe)(OEt), --C(Et)(OMe).sub.2, --C(Et)(OEt).sub.2, and
--C(Et)(OMe)(OEt).
[1112] Hemiketal: --CR(OH)(OR.sup.1), where R.sup.1 is as defined
for hemiacetals, and R is a hemiketal substituent other than
hydrogen, for example, a C.sub.1-7 alkyl group, a C.sub.3-20
heterocyclyl group, or a C.sub.5-20 aryl group, preferably a
C.sub.1-7 alkyl group. Examples of hemiacetal groups include, but
are not limited to, --C(Me)(OH)(OMe), --C(Et)(OH)(OMe),
--C(Me)(OH)(OEt), and --C(Et)(OH)(OEt).
[1113] Oxo (keto, -one): .dbd.O.
[1114] Thione (thioketone): .dbd.S.
[1115] Imino (imine): .dbd.NR, wherein R is an imino substituent,
for example, hydrogen, C.sub.1-7 alkyl group, a C.sub.3-20
heterocyclyl group, or a C.sub.5-20 aryl group, preferably hydrogen
or a C.sub.1-7 alkyl group. Examples of ester groups include, but
are not limited to, .dbd.NH, .dbd.NMe, .dbd.NEt, and .dbd.NPh.
[1116] Formyl (carbaldehyde, carboxaldehyde): --C(.dbd.O)H.
[1117] Acyl (keto): --C(.dbd.O)R, wherein R is an acyl substituent,
for example, a C.sub.1-7 alkyl group (also referred to as C.sub.1-7
alkylacyl or C.sub.1-7 alkanoyl), a C.sub.3-20 heterocyclyl group
(also referred to as C.sub.3-20 heterocyclylacyl), or a C.sub.5-20
aryl group (also referred to as C.sub.5-20 arylacyl), preferably a
C.sub.1-7 alkyl group. Examples of acyl groups include, but are not
limited to, --C(.dbd.O)CH.sub.3 (acetyl),
--C(.dbd.O)CH.sub.2CH.sub.3 (propionyl),
--C(.dbd.O)C(CH.sub.3).sub.3 (t-butyryl), and --C(.dbd.O)Ph
(benzoyl, phenone).
[1118] Carboxy (carboxylic acid): --C(.dbd.O)OH.
[1119] Thiocarboxy (thiocarboxylic acid): --C(.dbd.S)SH.
[1120] Thiolocarboxy (thiolocarboxylic acid): --C(.dbd.O)SH.
[1121] Thionocarboxy (thionocarboxylic acid): --C(.dbd.S)OH.
[1122] Imidic acid: --C(.dbd.NH)OH.
[1123] Hydroxamic acid: --C(.dbd.NOH)OH.
[1124] Ester (carboxylate, carboxylic acid ester, oxycarbonyl):
--C(.dbd.O)OR, wherein R is an ester substituent, for example, a
C.sub.1-7 alkyl group, a C.sub.3-20 heterocyclyl group, or a
C.sub.5-20 aryl group, preferably a C.sub.1-7 alkyl group. Examples
of ester groups include, but are not limited to,
--C(.dbd.O)OCH.sub.3, --C(.dbd.O)OCH.sub.2CH.sub.3,
--C(.dbd.O)OC(CH.sub.3).sub.3, and --C(.dbd.O)OPh.
[1125] Acyloxy (reverse ester): --OC(.dbd.O)R, wherein R is an
acyloxy substituent, for example, a C.sub.1-7 alkyl group, a
C.sub.3-20 heterocyclyl group, or a C.sub.5-20 aryl group,
preferably a C.sub.1-7 alkyl group. Examples of acyloxy groups
include, but are not limited to, --OC(.dbd.O)CH.sub.3 (acetoxy),
--OC(.dbd.O)CH.sub.2CH.sub.3, --OC(.dbd.O)C(CH.sub.3).sub.3,
--OC(.dbd.O)Ph, and --OC(.dbd.O)CH.sub.2Ph.
[1126] Oxycarboyloxy: --OC(.dbd.O)OR, wherein R is an ester
substituent, for example, a C.sub.1-7 alkyl group, a C.sub.3-20
heterocyclyl group, or a C.sub.5-20 aryl group, preferably a
C.sub.1-7 alkyl group. Examples of ester groups include, but are
not limited to, --OC(.dbd.O)OCH.sub.3,
--OC(.dbd.O)0CH.sub.2CH.sub.3, --OC(.dbd.O)OC(CH.sub.3).sub.3, and
--OC(.dbd.O)OPh.
[1127] Amino: --NR.sup.1R.sup.2, wherein R.sup.1 and R.sup.2 are
independently amino substituents, for example, hydrogen, a
C.sub.1-7 alkyl group (also referred to as C.sub.1-7 alkylamino or
di-C.sub.1-7alkylamino), a C.sub.3-20 heterocyclyl group, or a
C.sub.5-20 aryl group, preferably H or a C.sub.1-7 alkyl group, or,
in the case of a "cyclic" amino group, R.sup.1 and R.sup.2, taken
together with the nitrogen atom to which they are attached, form a
heterocyclic ring having from 4 to 8 ring atoms. Amino groups may
be primary (--NH.sub.2), secondary (--NHR.sup.1), or tertiary
(--NHR.sup.1R.sup.2), and in cationic form, may be quaternary
(--.sup.+NR.sup.1R.sup.2R.sup.3). Examples of amino groups include,
but are not limited to, --NH.sub.2, --NHCH.sub.3,
--NHC(CH.sub.3).sub.2, --N(CH.sub.3).sub.2,
--N(CH.sub.2CH.sub.3).sub.2, and --NHPh. Examples of cyclic amino
groups include, but are not limited to, aziridino, azetidino,
pyrrolidino, piperidino, piperazino, morpholino, and
thiomorpholino.
[1128] Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide):
--C(.dbd.O)NR.sup.1R.sup.2, wherein R.sup.1 and R.sup.2 are
independently amino substituents, as defined for amino groups.
Examples of amido groups include, but are not limited to,
--C(.dbd.O)NH.sub.2, --C(.dbd.O)NHCH.sub.3,
--C(.dbd.O)N(CH.sub.3).sub.2, --C(.dbd.O)NHCH.sub.2CH.sub.3, and
--C(.dbd.O)N(CH.sub.2CH.sub.3).sub.2, as well as amido groups in
which R.sup.1 and R.sup.2, together with the nitrogen atom to which
they are attached, form a heterocyclic structure as in, for
example, piperidinocarbonyl, morpholinocarbonyl,
thiomorpholinocarbonyl, and piperazinocarbonyl.
[1129] Thioamido (thiocarbamyl): --C(.dbd.S)NR.sup.1R.sup.2,
wherein R.sup.1 and R.sup.2 are independently amino substituents,
as defined for amino groups. Examples of amido groups include, but
are not limited to, --C(.dbd.S)NH.sub.2, --C(.dbd.S)NHCH.sub.3,
--C(.dbd.S)N(CH.sub.3).sub.2, and
--C(.dbd.S)NHCH.sub.2CH.sub.3.
[1130] Acylamido (acylamino): --NR.sup.1C(.dbd.O)R.sup.2, wherein
R.sup.1 is an amide substituent, for example, hydrogen, a C.sub.1-7
alkyl group, a C.sub.3-20 heterocyclyl group, or a C.sub.5-20 aryl
group, preferably hydrogen or a C.sub.1-7 alkyl group, and R.sup.2
is an acyl substituent, for example, a C.sub.1-7 alkyl group, a
C.sub.3-20 heterocyclyl group, or a C.sub.5-20aryl group,
preferably hydrogen or a C.sub.1-7 alkyl group. Examples of
acylamide groups include, but are not limited to,
--NHC(.dbd.O)CH.sub.3 , --NHC(.dbd.O)CH.sub.2CH.sub.3, and
--NHC(.dbd.O)Ph. R.sup.1 and R.sup.2 may together form a cyclic
structure, as in, for example, succinimidyl, maleimidyl, and
phthalimidyl:
##STR00032##
[1131] Aminocarbonyloxy: --OC(.dbd.O)NR.sup.1R.sup.2, wherein
R.sup.1 and R.sup.2 are independently amino substituents, as
defined for amino groups. Examples of aminocarbonyloxy groups
include, but are not limited to, --OC(.dbd.O)NH.sub.2,
--OC(.dbd.O)NHMe, --OC(.dbd.O)NMe.sub.2, and
--OC(.dbd.O)NEt.sub.2.
[1132] Ureido: --N(R.sup.1)CONR.sup.2R.sup.3 wherein R.sup.2 and
R.sup.3 are independently amino substituents, as defined for amino
groups, and R.sup.1 is a ureido substituent, for example, hydrogen,
a C.sub.1-7 alkyl group, a C.sub.3-20 heterocyclyl group, or a
C.sub.5-20 aryl group, preferably hydrogen or a C.sub.1-7 alkyl
group. Examples of ureido groups include, but are not limited to,
--NHCONH.sub.2, --NHCONHMe, --NHCONHEt, --NHCONMe.sub.2,
--NHCONEt.sub.2, --NMeCONH.sub.2, --NMeCONHMe, --NMeCONHEt,
--NMeCONMe.sub.2, and --NMeCONEt.sub.2.
[1133] Guanidino: --NH--C(.dbd.NH)NH.sub.2.
[1134] Tetrazolyl: a five membered aromatic ring having four
nitrogen atoms and one carbon atom,
##STR00033##
[1135] Imino: .dbd.NR, wherein R is an imino substituent, for
example, for example, hydrogen, a C.sub.1-7 alkyl group, a
C.sub.3-20 heterocyclyl group, or a C.sub.5-20 aryl group,
preferably H or a C.sub.1-7alkyl group. Examples of imino groups
include, but are not limited to, .dbd.NH, .dbd.NMe, and
.dbd.NEt.
[1136] Amidine (amidino): --C(.dbd.NR)NR.sub.2, wherein each R is
an amidine substituent, for example, hydrogen, a C.sub.1-7alkyl
group, a C.sub.3-20 heterocyclyl group, or a C.sub.5-20 aryl group,
preferably H or a C.sub.1-7alkyl group. Examples of amidine groups
include, but are not limited to, --C(.dbd.NH)NH.sub.2,
--C(.dbd.NH)NMe.sub.2, and --C(.dbd.NMe)NMe.sub.2.
[1137] Nitro: --NO.sub.2.
[1138] Nitroso: --NO.
[1139] Azido: --N.sub.3.
[1140] Cyano (nitrile, carbonitrile): --CN.
[1141] Isocyano: --NC.
[1142] Cyanato: --OCN.
[1143] Isocyanato: --NCO.
[1144] Thiocyano (thiocyanato): --SCN.
[1145] Isothiocyano (isothiocyanato): --NCS.
[1146] Sulfhydryl (thiol, mercapto): --SH.
[1147] Thioether (sulfide): --SR, wherein R is a thioether
substituent, for example, a C.sub.1-7 alkyl group (also referred to
as a C.sub.1-7 alkylthio group), a C.sub.3-20 heterocyclyl group,
or a C.sub.5-20 aryl group, preferably a C.sub.1-7 alkyl group.
Examples of C.sub.1-7 alkylthio groups include, but are not limited
to, --SCH.sub.3 and --SCH.sub.2CH.sub.3.
[1148] Disulfide: --SS--R, wherein R is a disulfide substituent,
for example, a C.sub.1-7 alkyl group, a C.sub.3-20 heterocyclyl
group, or a C.sub.5-20 aryl group, preferably a C.sub.1-7 alkyl
group (also referred to herein as C.sub.1-7 alkyl disulfide).
Examples of C.sub.1-7 alkyl disulfide groups include, but are not
limited to, --SSCH.sub.3 and --SSCH.sub.2CH.sub.3.
[1149] Sulfine (sulfinyl, sulfoxide): --S(.dbd.O)R, wherein R is a
sulfine substituent, for example, a C.sub.1-7 alkyl group, a
C.sub.3-20 heterocyclyl group, or a C.sub.5-20 aryl group,
preferably a C.sub.1-7 alkyl group. Examples of sulfine groups
include, but are not limited to, --S(.dbd.O)CH.sub.3 and
--S(.dbd.O)CH.sub.2CH.sub.3.
[1150] Sulfone (sulfonyl): --S(.dbd.O).sub.2R, wherein R is a
sulfone substituent, for example, a C.sub.1-7 alkyl group, a
C.sub.3-20 heterocyclyl group, or a C.sub.5-20 aryl group,
preferably a C.sub.1-7 alkyl group, including, for example, a
fluorinated or perfluorinated C.sub.1-7 alkyl group. Examples of
sulfone groups include, but are not limited to,
--S(.dbd.O).sub.2CH.sub.3 (methanesulfonyl, mesyl),
--S(.dbd.O).sub.2CF.sub.3 (triflyl),
--S(.dbd.O).sub.2CH.sub.2CH.sub.3 (esyl),
--S(.dbd.O).sub.2C.sub.4F.sub.9 (nonaflyl),
--S(.dbd.O).sub.2CH.sub.2CF.sub.3 (tresyl),
--S(.dbd.O).sub.2CH.sub.2CH.sub.2NH.sub.2 (tauryl),
--S(.dbd.O).sub.2Ph (phenylsulfonyl, besyl), 4-methylphenylsulfonyl
(tosyl), 4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl
(brosyl), 4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl),
and 5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).
[1151] Sulfinic acid (sulfino): --S(.dbd.O)OH, --SO.sub.2H.
[1152] Sulfonic acid (sulfo): --S(.dbd.O).sub.2OH, --SO.sub.3H.
[1153] Sulfinate (sulfinic acid ester): --S(.dbd.O)OR; wherein R is
a sulfinate substituent, for example, a C.sub.1-7 alkyl group, a
C.sub.3-20 heterocyclyl group, or a C.sub.5-20 aryl group,
preferably a C.sub.1-7 alkyl group. Examples of sulfinate groups
include, but are not limited to, --S(.dbd.O)OCH.sub.3
(methoxysulfinyl; methyl sulfinate) and
--S(.dbd.O)OCH.sub.2CH.sub.3 (ethoxysulfinyl; ethyl sulfinate).
[1154] Sulfonate (sulfonic acid ester): --S(.dbd.O).sub.2OR,
wherein R is a sulfonate substituent, for example, a C.sub.1-7
alkyl group, a C.sub.3-20 heterocyclyl group, or a C.sub.5-20 aryl
group, preferably a C.sub.1-7 alkyl group. Examples of sulfonate
groups include, but are not limited to, --S(.dbd.O).sub.2OCH.sub.3
(methoxysulfonyl; methyl sulfonate) and
--S(.dbd.O).sub.2OCH.sub.2CH.sub.3 (ethoxysulfonyl; ethyl
sulfonate).
[1155] Sulfinyloxy: --OS(.dbd.O)R, wherein R is a sulfinyloxy
substituent, for example, a C.sub.1-7 alkyl group, a C.sub.3-20
heterocyclyl group, or a C.sub.5-20 aryl group, preferably a
C.sub.1-7 alkyl group. Examples of sulfinyloxy groups include, but
are not limited to, --OS(.dbd.O)CH.sub.3 and
--OS(.dbd.O)CH.sub.2CH.sub.3.
[1156] Sulfonyloxy: --OS(.dbd.O).sub.2R, wherein R is a sulfonyloxy
substituent, for example, a C.sub.1-7 alkyl group, a C.sub.3-20
heterocyclyl group, or a C.sub.5-20 aryl group, preferably a
C.sub.1-7 alkyl group. Examples of sulfonyloxy groups include, but
are not limited to, --OS(.dbd.O).sub.2CH.sub.3 (mesylate) and
--OS(.dbd.O).sub.2CH.sub.2CH.sub.3 (esylate).
[1157] Sulfate: --OS(.dbd.O).sub.2OR; wherein R is a sulfate
substituent, for example, a C.sub.1-7 alkyl group, a C.sub.3-20
heterocyclyl group, or a C.sub.5-20 aryl group, preferably a
C.sub.1-7 alkyl group. Examples of sulfate groups include, but are
not limited to, --OS(.dbd.O).sub.2OCH.sub.3 and
--SO(.dbd.O).sub.2OCH.sub.2CH.sub.3.
[1158] Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide):
--S(.dbd.O)NR.sup.1R.sup.2, wherein R.sup.1 and R.sup.2 are
independently amino substituents, as defined for amino groups.
Examples of sulfamyl groups include, but are not limited to,
--S(.dbd.O)NH.sub.2, --S(.dbd.O)NH(CH.sub.3),
--S(.dbd.O)N(CH.sub.3).sub.2, --S(.dbd.O)NH(CH.sub.2CH.sub.3),
--S(.dbd.O)N(CH.sub.2CH.sub.3).sub.2, and --S(.dbd.O)NHPh.
[1159] Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide):
--S(.dbd.O).sub.2NR.sup.1R.sup.2, wherein R.sup.1 and R.sup.2 are
independently amino substituents, as defined for amino groups.
Examples of sulfonamido groups include, but are not limited to,
--S(.dbd.O).sub.2NH.sub.2, --S(.dbd.O).sub.2NH(CH.sub.3),
--S(.dbd.O).sub.2N(CH.sub.3).sub.2,
--S(.dbd.O).sub.2NH(CH.sub.2CH.sub.3),
--S(.dbd.O).sub.2N(CH.sub.2CH.sub.3).sub.2, and
--S(.dbd.O).sub.2NHPh.
[1160] Sulfamino: --NR.sup.1S(.dbd.O).sub.2OH, wherein R.sup.1 is
an amino substituent, as defined for amino groups. Examples of
sulfamino groups include, but are not limited to,
--NHS(.dbd.O).sub.2OH and --N(CH.sub.3)S(.dbd.O).sub.2OH.
[1161] Sulfonamino: --NR.sup.1S(.dbd.O).sub.2R, wherein R.sup.1 is
an amino substituent, as defined for amino groups, and R is a
sulfonamino substituent, for example, a C.sub.1-7 alkyl group, a
C.sub.3-20 heterocyclyl group, or a C.sub.5-20 aryl group,
preferably a C.sub.1-7 alkyl group. Examples of sulfonamino groups
include, but are not limited to, --NHS(.dbd.O).sub.2CH.sub.3 and
--N(CH.sub.3)S(.dbd.O).sub.2C.sub.6H.sub.5.
[1162] Sulfinamino: --NR.sup.1S(.dbd.O)R, wherein R.sup.1 is an
amino substituent, as defined for amino groups, and R is a
sulfinamino substituent, for example, a C.sub.1-7 alkyl group, a
C.sub.3-20 heterocyclyl group, or a C.sub.5-20 aryl group,
preferably a C.sub.1-7 alkyl group. Examples of sulfinamino groups
include, but are not limited to, --NHS(.dbd.O)CH.sub.3 and
--N(CH.sub.3)S(.dbd.O)C.sub.6H.sub.5.
[1163] Phosphino (phosphine): --PR.sub.2, wherein R is a phosphino
substituent, for example, --H, a C.sub.1-7 alkyl group, a
C.sub.3-20 heterocyclyl group, or a C.sub.5-20 aryl group,
preferably --H, a C.sub.1-7 alkyl group, or a C.sub.5-20 aryl
group. Examples of phosphino groups include, but are not limited
to, --PH.sub.2, --P(CH.sub.3).sub.2, --P(CH.sub.2CH.sub.3).sub.2,
--P(t-Bu).sub.2, and --P(Ph).sub.2.
[1164] Phospho: --P(.dbd.O).sub.2.
[1165] Phosphinyl (phosphine oxide): --P(.dbd.O)R.sub.2, wherein R
is a phosphinyl substituent, for example, a C.sub.1-7 alkyl group,
a C.sub.3-20 heterocyclyl group, or a C.sub.5-20 aryl group,
preferably a C.sub.1-7 alkyl group or a C.sub.5-20 aryl group.
Examples of phosphinyl groups include, but are not limited to,
--P(.dbd.O)(CH.sub.3).sub.2, --P(.dbd.O)(CH.sub.2CH.sub.3).sub.2,
--P(.dbd.O)(t-Bu).sub.2, and --P(.dbd.O)(Ph).sub.2.
[1166] Phosphonic acid (phosphono): --P(.dbd.O)(OH).sub.2.
[1167] Phosphonate (phosphono ester): --P(.dbd.O)(OR).sub.2, where
R is a phosphonate substituent, for example, --H, a C.sub.1-7 alkyl
group, a C.sub.3-20 heterocyclyl group, or a C.sub.5-20 aryl group,
preferably --H, a C.sub.1-7 alkyl group, or a C.sub.5-20 aryl
group. Examples of phosphonate groups include, but are not limited
to, --P(.dbd.O)(OCH.sub.3).sub.2,
--P(.dbd.O)(OCH.sub.2CH.sub.3).sub.2, --P(.dbd.O)(O-t-Bu).sub.2,
and --P(.dbd.O)(OPh).sub.2.
[1168] Phosphoric acid (phosphonooxy): --OP(.dbd.O)(OH).sub.2.
[1169] Phosphate (phosphonooxy ester): --OP(.dbd.O)(OR).sub.2,
where R is a phosphate substituent, for example, --H, a C.sub.1-7
alkyl group, a C.sub.3- 20 heterocyclyl group, or a C.sub.5-20 aryl
group, preferably --H, a C.sub.1-7 alkyl group, or a C.sub.5-20
aryl group. Examples of phosphate groups include, but are not
limited to, --OP(.dbd.O)(OCH.sub.3).sub.2,
--OP(.dbd.O)(OCH.sub.2CH.sub.3).sub.2, --OP(.dbd.O)(O-t-Bu).sub.2,
and --OP(.dbd.O)(OPh).sub.2.
[1170] Phosphorous acid: --OP(OH).sub.2.
[1171] Phosphite: --OP(OR).sub.2, where R is a phosphite
substituent, for example, --H, a C.sub.1-7 alkyl group, a C.sub.3-
20 heterocyclyl group, or a C.sub.5-20 aryl group, preferably --H,
a C.sub.1-7 alkyl group, or a C.sub.5-20 aryl group. Examples of
phosphite groups include, but are not limited to,
--OP(OCH.sub.3).sub.2, --OP(OCH.sub.2CH.sub.3).sub.2,
--OP(O-t-Bu).sub.2, and --OP(OPh).sub.2.
[1172] Phosphoramidite: --OP(OR.sup.1)--NR.sup.2.sub.2, where
R.sup.1 and R.sup.2 are phosphoramidite substituents, for example,
--H, a (optionally substituted) C.sub.1-7 alkyl group, a C.sub.3-20
heterocyclyl group, or a C.sub.5-20 aryl group, preferably --H, a
C.sub.1-7 alkyl group, or a C.sub.5-20 aryl group. Examples of
phosphoramidite groups include, but are not limited to,
--OP(OCH.sub.2CH.sub.3)--N(CH.sub.3).sub.2,
--OP(OCH.sub.2CH.sub.3)--N(i-Pr).sub.2, and
--OP(OCH.sub.2CH.sub.2CN)--N(i-Pr).sub.2.
[1173] Phosphoramidate: --OP(.dbd.O)(OR.sup.1)--NR.sup.2.sub.2,
where R.sup.1 and R.sup.2 are phosphoramidate substituents, for
example, --H, a (optionally substituted) C.sub.1-7 alkyl group, a
C.sub.3-20 heterocyclyl group, or a C.sub.5-20 aryl group,
preferably --H, a C.sub.1-7 alkyl group, or a C.sub.5-20 aryl
group. Examples of phosphoramidate groups include, but are not
limited to, --OP(.dbd.O)(OCH.sub.2CH.sub.3)--N(CH.sub.3).sub.2,
--OP(.dbd.O)(OCH.sub.2CH.sub.3)--N(i-Pr).sub.2, and
--OP(.dbd.O)(OCH.sub.2CH.sub.2CN)--N(i-Pr).sub.2.
[1174] Alkylene
[1175] C.sub.3-12 alkylene: The term "C.sub.3-12 alkylene", as used
herein, pertains to a bidentate moiety obtained by removing two
hydrogen atoms, either both from the same carbon atom, or one from
each of two different carbon atoms, of a hydrocarbon compound
having from 3 to 12 carbon atoms (unless otherwise specified),
which may be aliphatic or alicyclic, and which may be saturated,
partially unsaturated, or fully unsaturated. Thus, the term
"alkylene" includes the sub-classes alkenylene, alkynylene,
cycloalkylene, etc., discussed below.
[1176] Examples of linear saturated C.sub.3-12 alkylene groups
include, but are not limited to, --(CH.sub.2).sub.n-- where n is an
integer from 3 to 12, for example, --CH.sub.2CH.sub.2CH.sub.2--
(propylene), --CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- (butylene),
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- (pentylene) and
--CH.sub.2CH.sub.2CH.sub.2CH--.sub.2CH.sub.2CH.sub.2CH.sub.2--
(heptylene).
[1177] Examples of branched saturated C.sub.3-12 alkylene groups
include, but are not limited to, --CH(CH.sub.3)CH.sub.2--,
--CH(CH.sub.3)CH.sub.2CH.sub.2--,
--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH(CH.sub.3)CH.sub.2--,
--CH.sub.2CH(CH.sub.3)CH.sub.2CH.sub.2--, --CH(CH.sub.2CH.sub.3)--,
--CH(CH.sub.2CH.sub.3)CH.sub.2--, and
--CH.sub.2CH(CH.sub.2CH.sub.3)CH.sub.2--.
[1178] Examples of linear partially unsaturated C.sub.3-12 alkylene
groups (C.sub.3-12 alkenylene, and alkynylene groups) include, but
are not limited to, --CH.dbd.CH--CH.sub.2--,
--CH.sub.2--CH.dbd.CH.sub.2--, --CH.dbd.CH--CH.sub.2--CH.sub.2--,
--CH.dbd.CH--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.dbd.CH--CH.dbd.CH--, --CH.dbd.CH--CH.dbd.CH--OH.sub.2--,
--CH.dbd.CH--CH.dbd.CH--CH.sub.2--CH.sub.2--,
--CH.dbd.CH--CH.sub.2--CH.dbd.CH--,
--CH.dbd.CH--CH.sub.2--CH.sub.2--CH.dbd.CH--, and
--CH.sub.2--C.ident.C--CH.sub.2--.
[1179] Examples of branched partially unsaturated C.sub.3-12
alkylene groups (C.sub.3-12 alkenylene and alkynylene groups)
include, but are not limited to, --C(CH.sub.3).dbd.CH--,
--C(CH.sub.3).dbd.CH--CH.sub.2--, --CH.dbd.CH--CH(CH.sub.3)-- and
--C.dbd.C--CH(CH.sub.3)--.
[1180] Examples of alicyclic saturated C.sub.3-12 alkylene groups
(C.sub.3-12 cycloalkylenes) include, but are not limited to,
cyclopentylene (e.g. cyclopent-1,3-ylene), and cyclohexylene (e.g.
cyclohex-1,4-ylene).
[1181] Examples of alicyclic partially unsaturated C.sub.3-12
alkylene groups (C.sub.3-12 cycloalkylenes) include, but are not
limited to, cyclopentenylene (e.g. 4-cyclopenten-1,3-ylene),
cyclohexenylene (e.g. 2-cyclohexen-1,4-ylene;
3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-1,4-ylene).
[1182] Carbamate nitrogen protecting group: the term "carbamate
nitrogen protecting group" pertains to a moiety which masks the
nitrogen in the imine bond, and these are well known in the art.
These groups have the following structure:
##STR00034##
[1183] wherein R'.sup.10 is R as defined above. A large number of
suitable groups are described on pages 503 to 549 of Greene, T. W.
and Wuts, G. M., Protective Groups in Organic Synthesis, 3.sup.rd
Edition, John Wiley & Sons, Inc., 1999, which is incorporated
herein by reference.
[1184] Hemi-aminal nitrogen protecting group: the term "hemi-aminal
nitrogen protecting group" pertains to a group having the following
structure:
##STR00035##
[1185] wherein R'.sup.10 is R as defined above. A large number of
suitable groups are described on pages 633 to 647 as amide
protecting groups of Greene, T. W. and Wuts, G. M., Protective
Groups in Organic Synthesis, 3.sup.rd Edition, John Wiley &
Sons, Inc., 1999, which is incorporated herein by reference.
[1186] The groups Carbamate nitrogen protecting group and
Hemi-aminal nitrogen protecting group may be jointly termed a
"nitrogen protecting group for synthesis".
[1187] Conjugates
[1188] The present disclosure provides a conjugate comprising a PBD
compound connected to the antibody via a Linker Unit.
[1189] In one embodiment, the conjugate comprises the antibody
connected to a spacer connecting group, the spacer connected to a
trigger, the trigger connected to a self-immolative linker, and the
self-immolative linker connected to the N10 position of the PBD
compound. Such a conjugate is illustrated below:
[1190] where Ab is the antibody as defined above and PBD is a
pyrrolobenzodiazepine compound (D), as described herein. The
illustration shows the portions that correspond to R.sup.L', A,
L.sup.1 and L.sup.2 in certain embodiments of the disclosure.
R.sup.L' may be either R.sup.L1' or R.sup.L2'. D is D.sup.L with
R.sup.L1' or R.sup.L2' removed.
[1191] The present disclosure is suitable for use in providing a
PBD compound to a preferred site in a subject. In the preferred
embodiments, the conjugate allows the release of an active PBD
compound that does not retain any part of the linker. There is no
stub present that could affect the reactivity of the PBD
compound.
[1192] The linker attaches the antibody to the PBD drug moiety D
through covalent bond(s). The linker is a bifunctional or
multifunctional moiety which can be used to link one or more drug
moiety (D) and an antibody unit (Ab) to form antibody-drug
conjugates (ADC). The linker (R.sup.L') may be stable outside a
cell, i.e. extracellular, or it may be cleavable by enzymatic
activity, hydrolysis, or other metabolic conditions. Antibody-drug
conjugates (ADC) can be conveniently prepared using a linker having
reactive functionality for binding to the drug moiety and to the
antibody. A cysteine thiol, or an amine, e.g. N-terminus or amino
acid side chain such as lysine, of the antibody (Ab) can form a
bond with a functional group of a linker or spacer reagent, PBD
drug moiety (D) or drug-linker reagent (D.sup.L, D-R.sup.L), where
R.sup.L can be R.sup.L1 or R.sup.L2.
[1193] The linkers of the ADC preferably prevent aggregation of ADC
molecules and keep the ADC freely soluble in aqueous media and in a
monomeric state.
[1194] The linkers of the ADC are preferably stable
extracellularly. Before transport or delivery into a cell, the
antibody-drug conjugate (ADC) is preferably stable and remains
intact, i.e. the antibody remains linked to the drug moiety. The
linkers are stable outside the target cell and may be cleaved at
some efficacious rate inside the cell. An effective linker will:
(i) maintain the specific binding properties of the antibody; (ii)
allow intracellular delivery of the conjugate or drug moiety; (iii)
remain stable and intact, i.e. not cleaved, until the conjugate has
been delivered or transported to its targetted site; and (iv)
maintain a cytotoxic, cell-killing effect or a cytostatic effect of
the PBD drug moiety. Stability of the ADC may be measured by
standard analytical techniques such as mass spectroscopy, HPLC, and
the separation/analysis technique LC/MS.
[1195] Covalent attachment of the antibody and the drug moiety
requires the linker to have two reactive functional groups, i.e.
bivalency in a reactive sense. Bivalent linker reagents which are
useful to attach two or more functional or biologically active
moieties, such as peptides, nucleic acids, drugs, toxins,
antibodies, haptens, and reporter groups are known, and methods
have been described their resulting conjugates (Hermanson, G. T.
(1996) Bioconjugate Techniques; Academic Press: New York, p
234-242).
[1196] In another embodiment, the linker may be substituted with
groups which modulate aggregation, solubility or reactivity. For
example, a sulfonate substituent may increase water solubility of
the reagent and facilitate the coupling reaction of the linker
reagent with the antibody or the drug moiety, or facilitate the
coupling reaction of Ab-L with D.sup.L, or D.sup.L-L with Ab,
depending on the synthetic route employed to prepare the ADC.
[1197] In one embodiment, L-R.sup.L' is a group:
##STR00036##
[1198] where the asterisk indicates the point of attachment to the
Drug Unit (D), Ab is the antibody (L), L.sup.1 is a linker, A is a
connecting group connecting L.sup.1 to the antibody, L.sup.2 is a
covalent bond or together with --OC(.dbd.O)-- forms a
self-immolative linker, and L.sup.1 or L.sup.2 is a cleavable
linker.
[1199] L.sup.1 is preferably the cleavable linker, and may be
referred to as a trigger for activation of the linker for
cleavage.
[1200] The nature of L.sup.1 and L.sup.2, where present, can vary
widely. These groups are chosen on the basis of their cleavage
characteristics, which may be dictated by the conditions at the
site to which the conjugate is delivered. Those linkers that are
cleaved by the action of enzymes are preferred, although linkers
that are cleavable by changes in pH (e.g. acid or base labile),
temperature or upon irradiation (e.g. photolabile) may also be
used. Linkers that are cleavable under reducing or oxidising
conditions may also find use in the present disclosure.
[1201] L.sup.1 may comprise a contiguous sequence of amino acids.
The amino acid sequence may be the target substrate for enzymatic
cleavage, thereby allowing release of L-R.sup.L' from the N10
position.
[1202] In one embodiment, L.sup.1 is cleavable by the action of an
enzyme. In one embodiment, the enzyme is an esterase or a
peptidase.
[1203] In one embodiment, L.sup.2 is present and together with
--C(.dbd.O)O-- forms a self-immolative linker. In one embodiment,
L.sup.2 is a substrate for enzymatic activity, thereby allowing
release of L-R.sup.L' from the N10 position.
[1204] In one embodiment, where L.sup.1 is cleavable by the action
of an enzyme and L.sup.2 is present, the enzyme cleaves the bond
between L.sup.1 and L.sup.2.
[1205] L.sup.1 and L.sup.2, where present, may be connected by a
bond selected from: [1206] --C(.dbd.O)NH--, [1207] --C(.dbd.O)O--,
[1208] --NHC(.dbd.O)--, [1209] OC(.dbd.O)--, [1210]
--OC(.dbd.O)O--, [1211] --NHC(.dbd.O)O--, [1212] --OC(.dbd.O)NH--,
and [1213] --NHC(.dbd.O)NH--.
[1214] An amino group of L.sup.1 that connects to L.sup.2 may be
the N-terminus of an amino acid or may be derived from an amino
group of an amino acid side chain, for example a lysine amino acid
side chain.
[1215] A carboxyl group of L.sup.1 that connects to L.sup.2 may be
the C-terminus of an amino acid or may be derived from a carboxyl
group of an amino acid side chain, for example a glutamic acid
amino acid side chain.
[1216] A hydroxyl group of L.sup.1 that connects to L.sup.2 may be
derived from a hydroxyl group of an amino acid side chain, for
example a serine amino acid side chain.
[1217] The term "amino acid side chain" includes those groups found
in: (i) naturally occurring amino acids such as alanine, arginine,
asparagine, aspartic acid, cysteine, glutamine, glutamic acid,
glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine,
and valine; (ii) minor amino acids such as ornithine and
citrulline; (iii) unnatural amino acids, beta-amino acids,
synthetic analogs and derivatives of naturally occurring amino
acids; and (iv) all enantiomers, diastereomers, isomerically
enriched, isotopically labelled (e.g. .sup.2H, .sup.3H, .sup.14C,
.sup.15N), protected forms, and racemic mixtures thereof.
[1218] In one embodiment, --C(.dbd.O)O-- and L.sup.2 together form
the group:
##STR00037## [1219] where the asterisk indicates the point of
attachment to the N10 position, the wavy line indicates the point
of attachment to the linker L.sup.1, Y is --N(H)--, --O--,
--C(.dbd.O)N(H)-- or --C(.dbd.O)O--, and n is 0 to 3. The phenylene
ring is optionally substituted with one, two or three substituents
as described herein. In one embodiment, the phenylene group is
optionally substituted with halo, NO.sub.2, R or OR.
[1220] In one embodiment, Y is NH.
[1221] In one embodiment, n is 0 or 1. Preferably, n is 0.
[1222] Where Y is NH and n is 0, the self-immolative linker may be
referred to as a p-aminobenzylcarbonyl linker (PABC).
[1223] The self-immolative linker will allow for release of the
protected compound when a remote site is activated, proceeding
along the lines shown below (for n=0):
##STR00038## [1224] where L* is the activated form of the remaining
portion of the linker. These groups have the advantage of
separating the site of activation from the compound being
protected. As described above, the phenylene group may be
optionally substituted.
[1225] In one embodiment described herein, the group L* is a linker
L.sup.1 as described herein, which may include a dipeptide
group.
[1226] In another embodiment, --C(.dbd.O)O-- and L.sup.2 together
form a group selected from:
##STR00039## [1227] where the asterisk, the wavy line, Y, and n are
as defined above. Each phenylene ring is optionally substituted
with one, two or three substituents as described herein. In one
embodiment, the phenylene ring having the Y substituent is
optionally substituted and the phenylene ring not having the Y
substituent is unsubstituted. In one embodiment, the phenylene ring
having the Y substituent is unsubstituted and the phenylene ring
not having the Y substituent is optionally substituted.
[1228] In another embodiment, --C(.dbd.O)O-- and L.sup.2 together
form a group selected from:
##STR00040## [1229] where the asterisk, the wavy line, Y, and n are
as defined above, E is O, S or NR, D is N, CH, or CR, and F is N,
CH, or CR.
[1230] In one embodiment, D is N.
[1231] In one embodiment, D is CH.
[1232] In one embodiment, E is O or S.
[1233] In one embodiment, F is CH.
[1234] In a preferred embodiment, the linker is a cathepsin labile
linker.
[1235] In one embodiment, L.sup.1 comprises a dipeptide The
dipeptide may be represented as --NH--X.sub.1--X.sub.2--CO--, where
--NH-- and --CO-- represent the N- and C-terminals of the amino
acid groups X.sub.1 and X.sub.2 respectively. The amino acids in
the dipeptide may be any combination of natural amino acids. Where
the linker is a cathepsin labile linker, the dipeptide may be the
site of action for cathepsin-mediated cleavage.
[1236] Additionally, for those amino acids groups having carboxyl
or amino side chain functionality, for example Glu and Lys
respectively, CO and NH may represent that side chain
functionality.
[1237] In one embodiment, the group --X.sub.1--X.sub.2-- in
dipeptide, --NH--X.sub.1--X.sub.2--CO--, is selected from: [1238]
-Phe-Lys-, [1239] -Val-Ala-, [1240] -Val-Lys-, [1241] -Ala-Lys-,
[1242] -Phe-Cit-, [1243] -Leu-Cit-, [1244] Ile-Cit-, [1245]
-Phe-Arg-, [1246] -Trp-Cit-,
[1247] where Cit is citrulline.
[1248] Preferably, the group --X.sub.1--X.sub.2-- in dipeptide,
--NH--X.sub.1--X.sub.2--CO--, is selected from: [1249] -Phe-Lys-,
[1250] -Val-Ala-, [1251] -Val-Lys-, [1252] -Ala-Lys-, [1253]
-Val-Cit-.
[1254] Most preferably, the group --X.sub.1--X.sub.2-- in
dipeptide, --NH--X.sub.1--X.sub.2--CO--, is -Phe-Lys- or
-Val-Ala-.
[1255] Other dipeptide combinations may be used, including those
described by Dubowchik et al., Bioconjugate Chemistry, 2002,
13,855-869, which is incorporated herein by reference.
[1256] In one embodiment, the amino acid side chain is derivatised,
where appropriate. For example, an amino group or carboxy group of
an amino acid side chain may be derivatised.
[1257] In one embodiment, an amino group NH.sub.2 of a side chain
amino acid, such as lysine, is a derivatised form selected from the
group consisting of NHR and NRR'.
[1258] In one embodiment, a carboxy group COOH of a side chain
amino acid, such as aspartic acid, is a derivatised form selected
from the group consisting of COOR, CONH.sub.2, CONHR and
CONRR'.
[1259] In one embodiment, the amino acid side chain is chemically
protected, where appropriate. The side chain protecting group may
be a group as discussed below in relation to the group R.sup.L. The
present inventors have established that protected amino acid
sequences are cleavable by enzymes. For example, it has been
established that a dipeptide sequence comprising a Boc side
chain-protected Lys residue is cleavable by cathepsin.
[1260] Protecting groups for the side chains of amino acids are
well known in the art and are described in the Novabiochem Catalog.
Additional protecting group strategies are set out in Protective
Groups in Organic Synthesis, Greene and Wuts.
[1261] Possible side chain protecting groups are shown below for
those amino acids having reactive side chain functionality: [1262]
Arg: Z, Mtr, Tos; [1263] Asn: Trt, Xan; [1264] Asp: Bzl, t-Bu;
[1265] Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt; [1266] Glu: Bzl, t-Bu;
[1267] Gln: Trt, Xan; [1268] His: Boc, Dnp, Tos, Trt; [1269] Lys:
Boc, Z-Cl, Fmoc, Z, Alloc; [1270] Ser: Bzl, TBDMS, TBDPS; [1271]
Thr: Bz; [1272] Trp: Boc; [1273] Tyr: BzL, Z, Z--Br.
[1274] In one embodiment, the side chain protection is selected to
be orthogonal to a group provided as, or as part of, a capping
group, where present. Thus, the removal of the side chain
protecting group does not remove the capping group, or any
protecting group functionality that is part of the capping
group.
[1275] In other embodiments of the disclosure, the amino acids
selected are those having no reactive side chain functionality. For
example, the amino acids may be selected from: Ala, Gly, Ile, Leu,
Met, Phe, Pro, and Val.
[1276] In one embodiment, the dipeptide is used in combination with
a self-immolative linker. The self-immolative linker may be
connected to --X.sub.2--.
[1277] Where a self-immolative linker is present, --X.sub.2-- is
connected directly to the self-immolative linker. Preferably the
group --X.sub.2--CO-- is connected to Y, where Y is NH, thereby
forming the group --X.sub.2--CO--NH--.
[1278] --NH--X.sub.1-- is connected directly to A. A may comprise
the functionality --CO-- thereby to form an amide link with
--X.sub.1--.
[1279] In one embodiment, L.sup.1 and L.sup.2 together with
--OC(.dbd.O)-- comprise the group NH--X.sub.1--X.sub.2--CO--PABC--.
The PABC group is connected directly to the N10 position.
Preferably, the self-immolative linker and the dipeptide together
form the group --NH-Phe-Lys-CO--NH--PABC--, which is illustrated
below:
##STR00041## [1280] where the asterisk indicates the point of
attachment to the N10 position, and the wavy line indicates the
point of attachment to the remaining portion of the linker L.sup.1
or the point of attachment to A. Preferably, the wavy line
indicates the point of attachment to A. The side chain of the Lys
amino acid may be protected, for example, with Boc, Fmoc, or Alloc,
as described above.
[1281] Alternatively, the self-immolative linker and the dipeptide
together form the group --NH-Val-Ala-CO--NH--PABC--, which is
illustrated below:
##STR00042## [1282] where the asterisk and the wavy line are as
defined above.
[1283] Alternatively, the self-immolative linker and the dipeptide
together form the group --NH-Val-Cit-CO--NH--PABC--, which is
illustrated below:
##STR00043## [1284] where the asterisk and the wavy line are as
defined above.
[1285] In one embodiment, A is a covalent bond. Thus, L.sup.1 and
the antibody are directly connected. For example, where L.sup.1
comprises a contiguous amino acid sequence, the N-terminus of the
sequence may connect directly to the antibody.
[1286] Thus, where A is a covalent bond, the connection between the
antibody and L.sup.1 may be selected from: [1287] --C(.dbd.O)NH--,
[1288] --C(.dbd.O)O--, [1289] --NHC(.dbd.O)--, [1290]
--OC(.dbd.O)--, [1291] --OC(.dbd.O)O--, [1292] --NHC(.dbd.O)O--,
[1293] --OC(.dbd.O)NH--, [1294] --NHC(.dbd.O)NH--, [1295]
--C(.dbd.O)NHC(.dbd.O)--, [1296] --S--, [1297] --S--S--, [1298]
---CH.sub.2C(.dbd.O)--, and [1299] .dbd.N--NH--.
[1300] An amino group of L.sup.1 that connects to the antibody may
be the N-terminus of an amino acid or may be derived from an amino
group of an amino acid side chain, for example a lysine amino acid
side chain.
[1301] An carboxyl group of L.sup.1 that connects to the antibody
may be the C-terminus of an amino acid or may be derived from a
carboxyl group of an amino acid side chain, for example a glutamic
acid amino acid side chain.
[1302] A hydroxyl group of L.sup.1 that connects to the antibody
may be derived from a hydroxyl group of an amino acid side chain,
for example a serine amino acid side chain.
[1303] A thiol group of L.sup.1 that connects to the antibody may
be derived from a thiol group of an amino acid side chain, for
example a serine amino acid side chain.
[1304] The comments above in relation to the amino, carboxyl,
hydroxyl and thiol groups of L.sup.1 also apply to the
antibody.
[1305] In one embodiment, L.sup.2 together with --OC(.dbd.O)--
represents:
##STR00044## [1306] where the asterisk indicates the point of
attachment to the N10 position, the wavy line indicates the point
of attachment to L.sup.1, n is 0 to 3, Y is a covalent bond or a
functional group, and E is an activatable group, for example by
enzymatic action or light, thereby to generate a self-immolative
unit. The phenylene ring is optionally further substituted with
one, two or three substituents as described herein. In one
embodiment, the phenylene group is optionally further substituted
with halo, NO.sub.2, R or OR. Preferably n is 0 or 1, most
preferably 0.
[1307] E is selected such that the group is susceptible to
activation, e.g. by light or by the action of an enzyme. E may be
--NO.sub.2 or glucoronic acid. The former may be susceptible to the
action of a nitroreductase, the latter to the action of a
.beta.-glucoronidase.
[1308] In this embodiment, the self-immolative linker will allow
for release of the protected compound when E is activated,
proceeding along the lines shown below (for n=0):
##STR00045## [1309] where the asterisk indicates the point of
attachment to the N10 position, E* is the activated form of E, and
Y is as described above. These groups have the advantage of
separating the site of activation from the compound being
protected. As described above, the phenylene group may be
optionally further substituted.
[1310] The group Y may be a covalent bond to L.sup.1.
[1311] The group Y may be a functional group selected from: [1312]
--C(.dbd.O)-- [1313] --NH-- [1314] --O-- [1315] --C(.dbd.O)NH--,
[1316] --C(.dbd.O)O--, [1317] --NHC(.dbd.O)--, [1318]
--OC(.dbd.O)--, [1319] --OC(.dbd.O)O--, [1320] --NHC(.dbd.O)O--,
[1321] --OC(.dbd.O)NH--, [1322] --NHC(.dbd.O)NH--, [1323]
--NHC(.dbd.O)NH, [1324] --C(.dbd.O)NHC(.dbd.O)--, and [1325]
--S--.
[1326] Where L.sup.1 is a dipeptide, it is preferred that Y is
--NH-- or --C(.dbd.O)--, thereby to form an amide bond between
L.sup.1 and Y. In this embodiment, the dipeptide sequence need not
be a substrate for an enzymatic activity.
[1327] In another embodiment, A is a spacer group. Thus, L.sup.1
and the antibody are indirectly connected.
[1328] L.sup.1 and A may be connected by a bond selected from:
[1329] --C(.dbd.O)NH--, [1330] --C(.dbd.O)O--, [1331]
--NHC(.dbd.O)--, [1332] --OC(.dbd.O)--, [1333] --OC(.dbd.O)O--,
[1334] --NHC(.dbd.O)O--, [1335] --OC(.dbd.O)NH--, and [1336]
--NHC(.dbd.O)NH--.
[1337] In one embodiment, the group A is:
##STR00046## [1338] where the asterisk indicates the point of
attachment to L.sup.1, the wavy line indicates the point of
attachment to the antibody, and n is 0 to 6. In one embodiment, n
is 5.
[1339] In one embodiment, the group A is:
##STR00047## [1340] where the asterisk indicates the point of
attachment to L.sup.1, the wavy line indicates the point of
attachment to the antibody, and n is 0 to 6. In one embodiment, n
is 5.
[1341] In one embodiment, the group A is:
##STR00048## [1342] where the asterisk indicates the point of
attachment to L.sup.1, the wavy line indicates the point of
attachment to the antibody, n is 0 or 1, and m is 0 to 30. In a
preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4
to 8, and most preferably 4 or 8. In another embodiment, m is 10 to
30, and preferably 20 to 30. Alternatively, m is 0 to 50. In this
embodiment, m is preferably 10-40 and n is 1.
[1343] In one embodiment, the group A is:
##STR00049## [1344] where the asterisk indicates the point of
attachment to L.sup.1, the wavy line indicates the point of
attachment to the antibody, n is 0 or 1, and m is 0 to 30. In a
preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4
to 8, and most preferably 4 or 8. In another embodiment, m is 10 to
30, and preferably 20 to 30. Alternatively, m is 0 to 50. In this
embodiment, m is preferably 10-40 and n is 1.
[1345] In one embodiment, the connection between the antibody and A
is through a thiol residue of the antibody and a maleimide group of
A.
[1346] In one embodiment, the connection between the antibody and A
is:
##STR00050## [1347] where the asterisk indicates the point of
attachment to the remaining portion of A and the wavy line
indicates the point of attachment to the remaining portion of the
antibody. In this embodiment, the S atom is typically derived from
the antibody.
[1348] In each of the embodiments above, an alternative
functionality may be used in place of the maleimide-derived group
shown below:
##STR00051## [1349] where the wavy line indicates the point of
attachment to the antibody as before, and the asterisk indicates
the bond to the remaining portion of the A group.
[1350] In one embodiment, the maleimide-derived group is replaced
with the group:
##STR00052## [1351] where the wavy line indicates point of
attachment to the antibody, and the asterisk indicates the bond to
the remaining portion of the A group.
[1352] In one embodiment, the maleimide-derived group is replaced
with a group, which optionally together with the antibody, is
selected from: [1353] --C(.dbd.O)NH--, [1354] --C(.dbd.O)O--,
[1355] --NHC(.dbd.O)--, [1356] --OC(.dbd.O)--, [1357]
--OC(.dbd.O)O--, [1358] --NHC(.dbd.O)O--, [1359] --OC(.dbd.O)NH--,
[1360] --NHC(.dbd.O)NH--, [1361] --NHC(.dbd.O)NH, [1362]
--C(.dbd.O)NHC(.dbd.O)--, [1363] --S--, [1364] --S--S--, [1365]
--CH.sub.2C(.dbd.O)-- [1366] --C(.dbd.O)CH.sub.2--, [1367]
.dbd.N--NH--, and [1368] --NH--N.dbd..
[1369] In one embodiment, the maleimide-derived group is replaced
with a group, which optionally together with the antibody, is
selected from:
##STR00053## [1370] where the wavy line indicates either the point
of attachment to the antibody or the bond to the remaining portion
of the A group, and the asterisk indicates the other of the point
of attachment to the antibody or the bond to the remaining portion
of the A group.
[1371] Other groups suitable for connecting L.sup.1 to the antibody
are described in WO 2005/082023.
[1372] In one embodiment, the Connecting Group A is present, the
Trigger L1 is present and Self-Immolative Linker L.sup.2 is absent.
Thus, L.sup.1 and the Drug unit are directly connected via a bond.
Equivalently in this embodiment, L.sup.2 is a bond. This may be
particularly relevant when D.sup.L is of Formula II.
[1373] L.sup.1 and D may be connected by a bond selected from:
[1374] --C(.dbd.O)N<, [1375] --C(.dbd.O)O--, [1376]
--NHC(.dbd.O)--, [1377] --OC(.dbd.O)--, [1378] --OC(.dbd.O)O--,
[1379] --NHC(.dbd.O)O--, [1380] --OC(.dbd.O)N<, and [1381]
--NHC(.dbd.O)N<,
[1382] where N< or O- are part of D.
[1383] In one embodiment, L.sup.1 and D are preferably connected by
a bond selected from: [1384] --C(.dbd.O)N<, and [1385]
--NHC(.dbd.O)--.
[1386] In one embodiment, L.sup.1 comprises a dipeptide and one end
of the dipeptide is linked to D. As described above, the amino
acids in the dipeptide may be any combination of natural amino
acids and non-natural amino acids. In some embodiments, the
dipeptide comprises natural amino acids. Where the linker is a
cathepsin labile linker, the dipeptide is the site of action for
cathepsin-mediated cleavage. The dipeptide then is a recognition
site for cathepsin.
[1387] In one embodiment, the group --X.sub.1--X.sub.2-- in
dipeptide, --NH--X.sub.1--X.sub.2--CO--, is selected from: [1388]
-Phe-Lys-, [1389] -Val-Ala-, [1390] -Val-Lys-, [1391] -Ala-Lys-,
[1392] -Val-Cit-, [1393] -Phe-Cit-, [1394] -Leu-Cit-, [1395]
-Ile-Cit-, [1396] -Phe-Arg-, and [1397] -Trp-Cit-;
[1398] where Cit is citrulline. In such a dipeptide, --NH-- is the
amino group of X.sub.1, and CO is the carbonyl group of
X.sub.2.
[1399] Preferably, the group --X.sub.1--X.sub.2-- in dipeptide,
--NH--X.sub.1--X.sub.2--CO--, is selected from: [1400] -Phe-Lys-,
[1401] -Val-Ala-, [1402] -Val-Lys-, [1403] -Ala-Lys-, and [1404]
-Val-Cit-.
[1405] Most preferably, the group --X.sub.1--X.sub.2-- in
dipeptide, --NH--X.sub.1--X.sub.2--CO--, is -Phe-Lys- or
-Val-Ala-.
[1406] Other dipeptide combinations of interest include: [1407]
-Gly-Gly-, [1408] -Pro-Pro-, and [1409] -Val-Glu-.
[1410] Other dipeptide combinations may be used, including those
described above.
[1411] In one embodiment, L.sup.1- D is:
##STR00054## [1412] where --NH--X.sub.1--X.sub.2--CO is the
dipeptide, --N< is part of the Drug unit, the asterisk indicates
the points of attachment to the remainder of the Drug unit, and the
wavy line indicates the point of attachment to the remaining
portion of L.sup.1 or the point of attachment to A. Preferably, the
wavy line indicates the point of attachment to A.
[1413] In one embodiment, the dipeptide is valine-alanine and
L.sup.1-D is:
##STR00055## [1414] where the asterisks, --N< and the wavy line
are as defined above.
[1415] In one embodiment, the dipeptide is phenylalnine-lysine and
L.sup.1-D is:
##STR00056## [1416] where the asterisks, --N< and the wavy line
are as defined above.
[1417] In one embodiment, the dipeptide is valine-citrulline.
[1418] In one embodiment, the groups A-L.sup.1 are:
##STR00057## [1419] where the asterisk indicates the point of
attachment to L.sup.2 or D, the wavy line indicates the point of
attachment to the Ligand unit, and n is 0 to 6. In one embodiment,
n is 5.
[1420] In one embodiment, the groups A-L.sup.1 are:
##STR00058## [1421] where the asterisk indicates the point of
attachment to L.sup.2 or D, the wavy line indicates the point of
attachment to the Ligand unit, and n is 0 to 6. In one embodiment,
n is 5.
[1422] In one embodiment, the groups A-L.sup.1 are:
##STR00059## [1423] where the asterisk indicates the point of
attachment to L.sup.2 or D, the wavy line indicates the point of
attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a
preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4
to 8, most preferably 4 or 8.
[1424] In one embodiment, the groups A-L.sup.1 are:
##STR00060## [1425] where the asterisk indicates the point of
attachment to L.sup.2 or D, the wavy line indicates the point of
attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a
preferred embodiment, n is 1 and m is 0 to 10, 1 to 7, preferably 3
to 7, most preferably 3 or 7.
[1426] In one embodiment, the groups A-L.sup.1 are:
##STR00061## [1427] where the asterisk indicates the point of
attachment to L.sup.2 or D, the wavy line indicates the point of
attachment to the Ligand unit, and n is 0 to 6. In one embodiment,
n is 5.
[1428] In one embodiment, the groups A-L.sup.1 are:
##STR00062## [1429] where the asterisk indicates the point of
attachment to L.sup.2 or D, the wavy line indicates the point of
attachment to the Ligand unit, and n is 0 to 6. In one embodiment,
n is 5.
[1430] In one embodiment, the groups A-L.sup.1 are:
##STR00063## [1431] where the asterisk indicates the point of
attachment to L.sup.2 or D, the wavy line indicates the point of
attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a
preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4
to 8, most preferably 4 or 8.
[1432] In one embodiment, the groups A-L.sup.1 is:
##STR00064## [1433] where the asterisk indicates the point of
attachment to L.sup.2 or D, the wavy line indicates the point of
attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a
preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4
to 8, most preferably 4 or 8.
[1434] In one embodiment, the groups A-L.sup.1 are:
##STR00065## [1435] where the asterisk indicates the point of
attachment to L.sup.2 or D, S is a sulfur group of the Ligand unit,
the wavy line indicates the point of attachment to the rest of the
Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
[1436] In one embodiment, the group A-L.sup.1 are:
##STR00066## [1437] where the asterisk indicates the point of
attachment to L.sup.2 or D, S is a sulfur group of the Ligand unit,
the wavy line indicates the point of attachment to the remainder of
the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
[1438] In one embodiment, the groups A.sup.1-L.sup.1 are:
##STR00067## [1439] where the asterisk indicates the point of
attachment to L.sup.2 or D, S is a sulfur group of the Ligand unit,
the wavy line indicates the point of attachment to the remainder of
the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred
embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8,
most preferably 4 or 8.
[1440] In one embodiment, the groups A.sup.1-L.sup.1 are:
##STR00068## [1441] where the asterisk indicates the point of
attachment to L.sup.2 or D, the wavy line indicates the point of
attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a
preferred embodiment, n is 1 and m is 0 to 10, 1 to 7, preferably 4
to 8, most preferably 4 or 8.
[1442] In one embodiment, the groups A.sup.1-L.sup.1 are:
##STR00069## [1443] where the asterisk indicates the point of
attachment to L.sup.2 or D, the wavy line indicates the point of
attachment to the remainder of the Ligand unit, and n is 0 to 6. In
one embodiment, n is 5.
[1444] In one embodiment, the groups A.sup.1-L.sup.1 are:
##STR00070## [1445] where the asterisk indicates the point of
attachment to L.sup.2 or D, the wavy line indicates the point of
attachment to the remainder of the Ligand unit, and n is 0 to 6. In
one embodiment, n is 5.
[1446] In one embodiment, the groups A.sup.1-L.sup.1 are:
##STR00071## [1447] where the asterisk indicates the point of
attachment to L.sup.2 or D, the wavy line indicates the point of
attachment to the remainder of the Ligand unit, n is 0 or 1, and m
is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1
to 8, preferably 4 to 8, most preferably 4 or 8.
[1448] In one embodiment, the groups A.sup.1-L.sup.1 are:
##STR00072## [1449] where the asterisk indicates the point of
attachment to L.sup.2 or D, the wavy line indicates the point of
attachment to the remainder of the Ligand unit, n is 0 or 1, and m
is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1
to 8, preferably 4 to 8, most preferably 4 or 8.
[1450] The group R.sup.L' is derivable from the group R.sup.L. The
group R.sup.L may be converted to a group R.sup.L' by connection of
an antibody to a functional group of R.sup.L. Other steps may be
taken to convert R.sup.L to R.sup.L'. These steps may include the
removal of protecting groups, where present, or the installation of
an appropriate functional group.
[1451] R.sup.L
[1452] Linkers can include protease-cleavable peptidic moieties
comprising one or more amino acid units. Peptide linker reagents
may be prepared by solid phase or liquid phase synthesis methods
(E. Schroder and K. Lubke, The Peptides, volume 1, pp 76-136 (1965)
Academic Press) that are well known in the field of peptide
chemistry, including t-BOC chemistry (Geiser et al "Automation of
solid-phase peptide synthesis" in Macromolecular Sequencing and
Synthesis, Alan R. Liss, Inc., 1988, pp. 199-218) and Fmoc/HBTU
chemistry (Fields, G. and Noble, R. (1990) "Solid phase peptide
synthesis utilizing 9-fluoroenylmethoxycarbonyl amino acids", Int.
J. Peptide Protein Res. 35:161-214), on an automated synthesizer
such as the Rainin Symphony Peptide Synthesizer (Protein
Technologies, Inc., Tucson, Ariz.), or Model 433 (Applied
Biosystems, Foster City, Calif.).
[1453] Exemplary amino acid linkers include a dipeptide, a
tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides
include: valine-citrulline (vc or val-cit), alanine-phenylalanine
(af or ala-phe). Exemplary tripeptides include:
glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine
(gly-gly-gly). Amino acid residues which comprise an amino acid
linker component include those occurring naturally, as well as
minor amino acids and non-naturally occurring amino acid analogs,
such as citrulline. Amino acid linker components can be designed
and optimized in their selectivity for enzymatic cleavage by a
particular enzymes, for example, a tumor-associated protease,
cathepsin B, C and D, or a plasmin protease.
[1454] Amino acid side chains include those occurring naturally, as
well as minor amino acids and non-naturally occurring amino acid
analogs, such as citrulline. Amino acid side chains include
hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,
p-hydroxybenzyl, --CH.sub.2OH, --CH(OH)CH.sub.3,
--CH.sub.2CH.sub.2SCH.sub.3, --CH.sub.2CONH.sub.2, --CH.sub.2COOH,
--CH.sub.2CH.sub.2CONH.sub.2, --CH.sub.2CH.sub.2COOH,
--(CH.sub.2).sub.3NHC(.dbd.NH)NH.sub.2, --(CH.sub.2).sub.3NH.sub.2,
--(CH.sub.2).sub.3NHCOCH.sub.3, --(CH.sub.2).sub.3NHCHO,
--(CH.sub.2).sub.4NHC(.dbd.NH)NH.sub.2, --(CH.sub.2).sub.4NH.sub.2,
--(CH.sub.2).sub.4NHCOCH.sub.3, --(CH.sub.2).sub.4NHCHO,
--(CH.sub.2).sub.3NHCONH.sub.2, --(CH.sub.2).sub.4NHCONH.sub.2,
--CH.sub.2CH.sub.2CH(OH)CH.sub.2NH.sub.2, 2-pyridylmethyl-,
3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl, as well as
the following structures:
##STR00073##
[1455] When the amino acid side chains include other than hydrogen
(glycine), the carbon atom to which the amino acid side chain is
attached is chiral. Each carbon atom to which the amino acid side
chain is attached is independently in the (S) or (R) configuration,
or a racemic mixture. Drug-linker reagents may thus be
enantiomerically pure, racemic, or diastereomeric.
[1456] In exemplary embodiments, amino acid side chains are
selected from those of natural and non-natural amino acids,
including alanine, 2-amino-2-cyclohexylacetic acid,
2-amino-2-phenylacetic acid, arginine, asparagine, aspartic acid,
cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,
leucine, lysine, methionine, norleucine, phenylalanine, proline,
serine, threonine, tryptophan, tyrosine, valine,
.gamma.-aminobutyric acid, .alpha.,.alpha.-dimethyl
.gamma.-aminobutyric acid, .beta.,.beta.-dimethyl
.gamma.-aminobutyric acid, ornithine, and citrulline (Cit).
[1457] An exemplary valine-citrulline (val-cit or vc) dipeptide
linker reagent useful for constructing a linker-PBD drug moiety
intermediate for conjugation to an antibody, having a
para-aminobenzylcarbamoyl (PAB) self-immolative spacer has the
structure:
##STR00074##
[1458] where Q is C.sub.1-C.sub.8 alkyl, --O--(C.sub.1--C.sub.8
alkyl), -halogen, --NO.sub.2 or --CN; and m is an integer ranging
from 0-4.
[1459] An exemplary phe-lys(Mtr) dipeptide linker reagent having a
p-aminobenzyl group can be prepared according to Dubowchik, et al.
(1997) Tetrahedron Letters, 38:5257-60, and has the structure:
##STR00075##
[1460] where Mtr is mono-4-methoxytrityl, Q is C.sub.1-C.sub.8
alkyl, --O--(C.sub.1-C.sub.8 alkyl), -halogen, --NO.sub.2 or --CN;
and m is an integer ranging from 0-4.
[1461] The "self-immolative linker" PAB
(para-aminobenzyloxycarbonyl), attaches the drug moiety to the
antibody in the antibody drug conjugate (Carl et al (1981) J. Med.
Chem. 24:479-480; Chakravarty et al (1983) J. Med. Chem.
26:638-644; U.S. Pat. No. 6,214,345; US20030130189; US20030096743;
U.S. Pat. No. 6,759,509; US20040052793; U.S. Pat. No. 6,218,519;
U.S. Pat. No. 6,835,807; U.S. Pat. No. 6,268,488; US20040018194;
WO98/13059; US20040052793; U.S. Pat. No. 6,677,435; U.S. Pat. No.
5,621,002; US20040121940; WO2004/032828). Other examples of
self-immolative spacers besides PAB include, but are not limited
to: (i) aromatic compounds that are electronically similar to the
PAB group such as 2-aminoimidazol-5-methanol derivatives (Hay et
al. (1999) Bioorg. Med. Chem. Lett. 9:2237), thiazoles (U.S. Pat.
No. 7,375,078), multiple, elongated PAB units (de Groot et al
(2001) J. Org. Chem. 66:8815-8830); and ortho or
para-aminobenzylacetals; and (ii) homologated styryl PAB analogs
(U.S. Pat. No. 7,223,837). Spacers can be used that undergo
cyclization upon amide bond hydrolysis, such as substituted and
unsubstituted 4-aminobutyric acid amides (Rodrigues et al (1995)
Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1]
and bicyclo[2.2.2] ring systems (Storm et al (1972) J. Amer. Chem.
Soc. 94:5815) and 2-aminophenylpropionic acid amides (Amsberry, et
al (1990) J. Org. Chem. 55:5867). Elimination of amine-containing
drugs that are substituted at glycine (Kingsbury et al (1984) J.
Med. Chem. 27:1447) are also examples of self-immolative spacers
useful in ADC.
[1462] In one embodiment, a valine-citrulline dipeptide PAB analog
reagent has a 2,6 dimethyl phenyl group and has the structure:
##STR00076##
[1463] Linker reagents useful for the antibody drug conjugates of
the disclosure include, but are not limited to: BMPEO, BMPS, EMCS,
GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH,
sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,
sulfo-SMCC, and sulfo-SMPB, and SVSB
(succinimidyl-(4-vinylsulfone)benzoate), and bis-maleimide
reagents: DTME, BMB, BMDB, BMH, BMOE,
1,8-bis-maleimidodiethyleneglycol (BM(PEO).sub.2), and
1,11-bis-maleimidotriethyleneglycol (BM(PEO).sub.3), which are
commercially available from Pierce Biotechnology, Inc.,
ThermoScientific, Rockford, Ill., and other reagent suppliers.
Bis-maleimide reagents allow the attachment of a free thiol group
of a cysteine residue of an antibody to a thiol-containing drug
moiety, label, or linker intermediate, in a sequential or
concurrent fashion. Other functional groups besides maleimide,
which are reactive with a thiol group of an antibody, PBD drug
moiety, or linker intermediate include iodoacetamide,
bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide,
isocyanate, and isothiocyanate.
##STR00077##
[1464] Other embodiments of linker reagents are:
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP),
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP, Carlsson et al
(1978) Biochem. J. 173:723-737), succinimidyl-4-(N-maleimidomethyl)
cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional
derivatives of imidoesters (such as dimethyl adipimidate HCl),
active esters (such as disuccinimidyl suberate), aldehydes (such as
glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)
hexanediamine), bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). Useful linker reagents can
also be obtained via other commercial sources, such as Molecular
Biosciences Inc. (Boulder, Colo.), or synthesized in accordance
with procedures described in Toki et al (2002) J. Org. Chem.
67:1866-1872; U.S. Pat. No. 6,214,345; WO 02/088172; US 2003130189;
US2003096743; WO 03/026577; WO 03/043583; and WO 04/032828.
[1465] The Linker may be a dendritic type linker for covalent
attachment of more than one drug moiety through a branching,
multifunctional linker moiety to an antibody (US 2006/116422; US
2005/271615; de Groot et al (2003) Angew. Chem. Int. Ed.
42:4490-4494; Amir et al (2003) Angew. Chem. Int. Ed. 42:4494-4499;
Shamis et al (2004) J. Am. Chem. Soc. 126:1726-1731; Sun et al
(2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215;
Sun et al (2003) Bioorganic & Medicinal Chemistry 11:1761-1768;
King et al (2002) Tetrahedron Letters 43:1987-1990). Dendritic
linkers can increase the molar ratio of drug to antibody, i.e.
loading, which is related to the potency of the ADC. Thus, where an
antibody bears only one reactive cysteine thiol group, a multitude
of drug moieties may be attached through a dendritic or branched
linker.
[1466] One exemplary embodiment of a dendritic type linker has the
structure:
##STR00078##
[1467] where the asterisk indicate the point of attachment to the
N10 position of a PBD moiety.
[1468] R.sup.C, Capping Group
[1469] The conjugate of the first aspect of the disclosure may have
a capping group R.sup.C at the N10 position.
[1470] The group R.sup.C is removable from the N10 position of the
PBD moiety to leave an N10-C11 imine bond, a carbinolamine, a
substituted carbinolamine, where QR.sup.11 is OSO.sub.3M, a
bisulfite adduct, a thiocarbinolamine, a substituted
thiocarbinolamine, or a substituted carbinalamine.
[1471] In one embodiment, R.sup.C, may be a protecting group that
is removable to leave an N10-C11 imine bond, a carbinolamine, a
substituted cabinolamine, or, where QR.sup.11 is OSO.sub.3M, a
bisulfite adduct. In one embodiment, R.sup.C is a protecting group
that is removable to leave an N10-C11 imine bond.
[1472] The group R.sup.C is intended to be removable under the same
conditions as those required for the removal of the group R.sup.10,
for example to yield an N10-C11 imine bond, a carbinolamine and so
on. The capping group acts as a protecting group for the intended
functionality at the N10 position. The capping group is intended
not to be reactive towards an antibody. For example, R.sup.C is not
the same as R.sup.L.
[1473] Compounds having a capping group may be used as
intermediates in the synthesis of dimers having an imine monomer.
Alternatively, compounds having a capping group may be used as
conjugates, where the capping group is removed at the target
location to yield an imine, a carbinolamine, a substituted
cabinolamine and so on. Thus, in this embodiment, the capping group
may be referred to as a therapeutically removable nitrogen
protecting group, as defined in the inventors' earlier application
WO 00/12507.
[1474] In one embodiment, the group R.sup.C is removable under the
conditions that cleave the linker R.sup.L of the group R.sup.10.
Thus, in one embodiment, the capping group is cleavable by the
action of an enzyme.
[1475] In an alternative embodiment, the capping group is removable
prior to the connection of the linker R.sup.L to the antibody. In
this embodiment, the capping group is removable under conditions
that do not cleave the linker R.sup.L.
[1476] Where a compound includes a functional group G.sup.1 to form
a connection to the antibody, the capping group is removable prior
to the addition or unmasking of G.sup.1.
[1477] The capping group may be used as part of a protecting group
strategy to ensure that only one of the monomer units in a dimer is
connected to an antibody.
[1478] The capping group may be used as a mask for a N10-C11 imine
bond. The capping group may be removed at such time as the imine
functionality is required in the compound. The capping group is
also a mask for a carbinolamine, a substituted cabinolamine, and a
bisulfite adduct, as described above.
[1479] R.sup.C may be an N10 protecting group, such as those groups
described in the inventors' earlier application, WO 00/12507. In
one embodiment, R.sup.C is a therapeutically removable nitrogen
protecting group, as defined in the inventors' earlier application,
WO 00/12507.
[1480] In one embodiment, R.sup.C is a carbamate protecting
group.
[1481] In one embodiment, the carbamate protecting group is
selected from: [1482] Alloc, Fmoc, Boc, Troc, Teoc, Psec, Cbz and
PNZ.
[1483] Optionally, the carbamate protecting group is further
selected from Moc.
[1484] In one embodiment, R.sup.C is a linker group R.sup.L lacking
the functional group for connection to the antibody.
[1485] This application is particularly concerned with those
R.sup.C groups which are carbamates.
[1486] In one embodiment, R.sup.C is a group:
##STR00079## [1487] where the asterisk indicates the point of
attachment to the N10 position, G.sup.2 is a terminating group,
L.sup.3 is a covalent bond or a cleavable linker L.sup.1, L.sup.2
is a covalent bond or together with OC(.dbd.O) forms a
self-immolative linker.
[1488] Where L.sup.3 and L.sup.2 are both covalent bonds, G.sup.2
and OC(.dbd.O) together form a carbamate protecting group as
defined above.
[1489] L.sup.1 is as defined above in relation to R.sup.10.
[1490] L.sup.2 is as defined above in relation to R.sup.10.
[1491] Various terminating groups are described below, including
those based on well known protecting groups.
[1492] In one embodiment L.sup.3 is a cleavable linker L.sup.1, and
L.sup.2, together with OC(.dbd.O), forms a self-immolative linker.
In this embodiment, G.sup.2 is Ac (acetyl) or Moc, or a carbamate
protecting group selected from: [1493] Alloc, Fmoc, Boc, Troc,
Teoc, Psec, Cbz and PNZ.
[1494] Optionally, the carbamate protecting group is further
selected from Moc.
[1495] In another embodiment, G.sup.2 is an acyl group
--C(.dbd.O)G.sup.3, where G.sup.3 is selected from alkyl (including
cycloalkyl, alkenyl and alkynyl), heteroalkyl, heterocyclyl and
aryl (including heteroaryl and carboaryl). These groups may be
optionally substituted. The acyl group together with an amino group
of L.sup.3 or L.sup.2, where appropriate, may form an amide bond.
The acyl group together with a hydroxy group of L.sup.3 or L.sup.2,
where appropriate, may form an ester bond.
[1496] In one embodiment, G.sup.3 is heteroalkyl. The heteroalkyl
group may comprise polyethylene glycol. The heteroalkyl group may
have a heteroatom, such as O or N, adjacent to the acyl group,
thereby forming a carbamate or carbonate group, where appropriate,
with a heteroatom present in the group L.sup.3 or L.sup.2, where
appropriate.
[1497] In one embodiment, G.sup.3 is selected from NH.sub.2, NHR
and NRR'. Preferably, G.sup.3 is NRR'.
[1498] In one embodiment G.sup.2 is the group:
##STR00080## [1499] where the asterisk indicates the point of
attachment to L.sup.3, n is 0 to 6 and G.sup.4 is selected from OH,
OR, SH, SR, COOR, CONH.sub.2, CONHR, CONRR', NH.sub.2, NHR, NRR',
NO.sub.2, and halo. The groups OH, SH, NH.sub.2 and NHR are
protected. In one embodiment, n is 1 to 6, and preferably n is 5.
In one embodiment, G.sup.4 is OR, SR, COOR, CONH.sub.2, CONHR,
CONRR', and NRR'. In one embodiment, G.sup.4 is OR, SR, and NRR'.
Preferably G.sup.4 is selected from OR and NRR', most preferably
G.sup.4 is OR. Most preferably G.sup.4 is OMe.
[1500] In one embodiment, the group G.sup.2 is:
##STR00081## [1501] where the asterisk indicates the point of
attachment to L.sup.3, and n and G.sup.4 are as defined above.
[1502] In one embodiment, the group G.sup.2 is:
##STR00082## [1503] where the asterisk indicates the point of
attachment to L.sup.3, n is 0 or 1, m is 0 to 50, and G.sup.4 is
selected from OH, OR, SH, SR, COOR, CONH.sub.2, CONHR, CONRR',
NH.sub.2, NHR, NRR', NO.sub.2, and halo. In a preferred embodiment,
n is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most
preferably 4 or 8. In another embodiment, n is 1 and m is 10 to 50,
preferably 20 to 40. The groups OH, SH, NH.sub.2 and NHR are
protected. In one embodiment, G.sup.4 is OR, SR, COOR, CONH.sub.2,
CONHR, CONRR', and NRR'. In one embodiment, G.sup.4 is OR, SR, and
NRR'. Preferably G.sup.4 is selected from OR and NRR', most
preferably G.sup.4 is OR. Preferably G.sup.4 is OMe.
[1504] In one embodiment, the group G.sup.2 is:
##STR00083## [1505] where the asterisk indicates the point of
attachment to L.sup.3, and n, m and G.sup.4 are as defined
above.
[1506] In one embodiment, the group G.sup.2 is:
##STR00084##
[1507] where n is 1-20, m is 0-6, and G.sup.4 is selected from OH,
OR, SH, SR, COOR, CONH.sub.2, CONHR, CONRR', NH.sub.2, NHR, NRR',
NO.sub.2, and halo. In one embodiment, n is 1-10. In another
embodiment, n is 10 to 50, preferably 20 to 40. In one embodiment,
n is 1. In one embodiment, m is 1. The groups OH, SH, NH.sub.2 and
NHR are protected. In one embodiment, G.sup.4 is OR, SR, COOR,
CONH.sub.2, CONHR, CONRR', and NRR'. In one embodiment, G.sup.4 is
OR, SR, and NRR'. Preferably G.sup.4 is selected from OR and NRR',
most preferably G.sup.4 is OR. Preferably G.sup.4 is OMe.
[1508] In one embodiment, the group G.sup.2 is:
##STR00085## [1509] where the asterisk indicates the point of
attachment to L.sup.3, and n, m and G.sup.4 are as defined
above.
[1510] In each of the embodiments above G.sup.4 may be OH, SH,
NH.sub.2 and NHR. These groups are preferably protected.
[1511] In one embodiment, OH is protected with Bzl, TBDMS, or
TBDPS.
[1512] In one embodiment, SH is protected with Acm, Bzl, Bzl-OMe,
Bzl-Me, or Trt.
[1513] In one embodiment, NH.sub.2 or NHR are protected with Boc,
Moc, Z-Cl, Fmoc, Z, or Alloc.
[1514] In one embodiment, the group G.sup.2 is present in
combination with a group L.sup.3, which group is a dipeptide.
[1515] The capping group is not intended for connection to the
antibody. Thus, the other monomer present in the dimer serves as
the point of connection to the antibody via a linker.
[1516] Accordingly, it is preferred that the functionality present
in the capping group is not available for reaction with an
antibody. Thus, reactive functional groups such as OH, SH,
NH.sub.2, COOH are preferably avoided. However, such functionality
may be present in the capping group if protected, as described
above.
Embodiments
[1517] Embodiments of the present disclosure include ConjA wherein
the antibody is as defined above.
[1518] Embodiments of the present disclosure include ConjB wherein
the antibody is as defined above.
[1519] Embodiments of the present disclosure include ConjC wherein
the antibody is as defined above.
[1520] Embodiments of the present disclosure include ConjD wherein
the antibody is as defined above.
[1521] Embodiments of the present disclosure include ConjE wherein
the antibody is as defined above.
[1522] Embodiments of the present disclosure include ConjF wherein
the antibody is as defined above.
[1523] Embodiments of the present disclosure include ConjG wherein
the antibody is as defined above.
[1524] Embodiments of the present disclosure include ConjH wherein
the antibody is as defined above.
[1525] Drug Loading
[1526] The drug loading is the average number of PBD drugs per
antibody, e.g. antibody. Where the compounds of the disclosure are
bound to native cysteines, drug loading may range from 1 to 8 drugs
(D.sup.L) per antibody, i.e. where 1, 2, 3, 4, 5, 6, 7, and 8 drug
moieties are covalently attached to the antibody. Compositions of
conjgates include collections of antibodies, conjugated with a
range of drugs, from 1 to 8. Where the compounds of the disclosure
are bound to lysines, drug loading may range from 1 to 80 drugs
(D.sup.L) per antibody, although an upper limit of 40, 20, 10 or 8
may be preferred. Compositions of conjugates include collections of
antibodies, conjugated with a range of drugs, from 1 to 80, 1 to
40, 1 to 20, 1 to 10 or 1 to 8.
[1527] The average number of drugs per antibody in preparations of
ADC from conjugation reactions may be characterized by conventional
means such as UV, reverse phase HPLC, HIC, mass spectroscopy, ELISA
assay, and electrophoresis. The quantitative distribution of ADC in
terms of p may also be determined. By ELISA, the averaged value of
p in a particular preparation of ADC may be determined (Hamblen et
al (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al (2005)
Clin. Cancer Res. 11:843-852). However, the distribution of p
(drug) values is not discernible by the antibody-antigen binding
and detection limitation of ELISA. Also, ELISA assay for detection
of antibody-drug conjugates does not determine where the drug
moieties are attached to the antibody, such as the heavy chain or
light chain fragments, or the particular amino acid residues. In
some instances, separation, purification, and characterization of
homogeneous ADC where p is a certain value from ADC with other drug
loadings may be achieved by means such as reverse phase HPLC or
electrophoresis. Such techniques are also applicable to other types
of conjugates.
[1528] For some antibody-drug conjugates, p may be limited by the
number of attachment sites on the antibody. For example, an
antibody may have only one or several cysteine thiol groups, or may
have only one or several sufficiently reactive thiol groups through
which a linker may be attached. Higher drug loading, e.g. p>5,
may cause aggregation, insolubility, toxicity, or loss of cellular
permeability of certain antibody-drug conjugates.
[1529] Typically, fewer than the theoretical maximum of drug
moieties are conjugated to an antibody during a conjugation
reaction. An antibody may contain, for example, many lysine
residues that do not react with the drug-linker intermediate (D-L)
or linker reagent. Only the most reactive lysine groups may react
with an amine-reactive linker reagent. Also, only the most reactive
cysteine thiol groups may react with a thiol-reactive linker
reagent. Generally, antibodies do not contain many, if any, free
and reactive cysteine thiol groups which may be linked to a drug
moiety. Most cysteine thiol residues in the antibodies of the
compounds exist as disulfide bridges and must be reduced with a
reducing agent such as dithiothreitol (DTT) or TCEP, under partial
or total reducing conditions. The loading (drug/antibody ratio) of
an ADC may be controlled in several different manners, including:
(i) limiting the molar excess of drug-linker intermediate (D-L) or
linker reagent relative to antibody, (ii) limiting the conjugation
reaction time or temperature, and (iii) partial or limiting
reductive conditions for cysteine thiol modification.
[1530] Certain antibodies have reducible interchain disulfides,
i.e. cysteine bridges. Antibodies may be made reactive for
conjugation with linker reagents by treatment with a reducing agent
such as DTT (dithiothreitol). Each cysteine bridge will thus form,
theoretically, two reactive thiol nucleophiles. Additional
nucleophilic groups can be introduced into antibodies through the
reaction of lysines with 2-iminothiolane (Traut's reagent)
resulting in conversion of an amine into a thiol. Reactive thiol
groups may be introduced into the antibody (or fragment thereof) by
engineering one, two, three, four, or more cysteine residues (e.g.,
preparing mutant antibodies comprising one or more non-native
cysteine amino acid residues). U.S. Pat. No. 7,521,541 teaches
engineering antibodies by introduction of reactive cysteine amino
acids.
[1531] Cysteine amino acids may be engineered at reactive sites in
an antibody and which do not form intrachain or intermolecular
disulfide linkages (Junutula, et al., 2008b Nature Biotech.,
26(8):925-932; Dornan et al (2009) Blood 114(13):2721-2729; U.S.
Pat. No. 7,521,541; U.S. Pat. No. 7,723,485; WO2009/052249). The
engineered cysteine thiols may react with linker reagents or the
drug-linker reagents of the present disclosure which have
thiol-reactive, electrophilic groups such as maleimide or
alpha-halo amides to form ADC with cysteine engineered antibodies
and the PBD drug moieties. The location of the drug moiety can thus
be designed, controlled, and known. The drug loading can be
controlled since the engineered cysteine thiol groups typically
react with thiol-reactive linker reagents or drug-linker reagents
in high yield. Engineering an IgG antibody to introduce a cysteine
amino acid by substitution at a single site on the heavy or light
chain gives two new cysteines on the symmetrical antibody. A drug
loading near 2 can be achieved with near homogeneity of the
conjugation product ADC.
[1532] Alternatively, site-specific conjugation can be achieved by
engineering antibodies to contain unnatural amino acids in their
heavy and/or light chains as described by Axup et al. ((2012), Proc
Natl Acad Sci USA. 109(40):16101-16116). The unnatural amino acids
provide the additional advantage that orthogonal chemistry can be
designed to attach the linker reagent and drug.
[1533] Where more than one nucleophilic or electrophilic group of
the antibody reacts with a drug-linker intermediate, or linker
reagent followed by drug moiety reagent, then the resulting product
is a mixture of ADC compounds with a distribution of drug moieties
attached to an antibody, e.g. 1, 2, 3, etc. Liquid chromatography
methods such as polymeric reverse phase (PLRP) and hydrophobic
interaction (HIC) may separate compounds in the mixture by drug
loading value. Preparations of ADC with a single drug loading value
(p) may be isolated, however, these single loading value ADCs may
still be heterogeneous mixtures because the drug moieties may be
attached, via the linker, at different sites on the antibody.
[1534] Thus the antibody-drug conjugate compositions of the
disclosure include mixtures of antibody-drug conjugate compounds
where the antibody has one or more PBD drug moieties and where the
drug moieties may be attached to the antibody at various amino acid
residues.
[1535] In one embodiment, the average number of dimer
pyrrolobenzodiazepine groups per antibody is in the range 1 to 20.
In some embodiments the range is selected from 1 to 8, 2 to 8, 2 to
6, 2 to 4, and 4 to 8.
[1536] In some embodiments, there is one dimer
pyrrolobenzodiazepine group per antibody.
[1537] Includes Other Forms
[1538] Unless otherwise specified, included in the above are the
well known ionic, salt, solvate, and protected forms of these
substituents. For example, a reference to carboxylic acid (--COOH)
also includes the anionic (carboxylate) form (--COO.sup.-), a salt
or solvate thereof, as well as conventional protected forms.
Similarly, a reference to an amino group includes the protonated
form (--N.sup.+HR.sup.1R.sup.2), a salt or solvate of the amino
group, for example, a hydrochloride salt, as well as conventional
protected forms of an amino group. Similarly, a reference to a
hydroxyl group also includes the anionic form (--O.sup.-), a salt
or solvate thereof, as well as conventional protected forms.
[1539] Salts
[1540] It may be convenient or desirable to prepare, purify, and/or
handle a corresponding salt of the active compound, for example, a
pharmaceutically-acceptable salt. Examples of pharmaceutically
acceptable salts are discussed in Berge, et al., J. Pharm. Sci.,
66, 1-19 (1977).
[1541] For example, if the compound is anionic, or has a functional
group which may be anionic (e.g. --COOH may be --COO.sup.-), then a
salt may be formed with a suitable cation. Examples of suitable
inorganic cations include, but are not limited to, alkali metal
ions such as Na.sup.+ and K.sup.+, alkaline earth cations such as
Ca.sup.2+ and Mg.sup.2+, and other cations such as Al.sup.+3.
Examples of suitable organic cations include, but are not limited
to, ammonium ion (i.e. NH.sub.4.sup.+) and substituted ammonium
ions (e.g. NH.sub.3R.sup.+, NH.sub.2R.sub.2.sup.+, NHR.sub.3.sup.+,
NR.sub.4.sup.+). Examples of some suitable substituted ammonium
ions are those derived from: ethylamine, diethylamine,
dicyclohexylamine, triethylamine, butylamine, ethylenediamine,
ethanolamine, diethanolamine, piperazine, benzylamine,
phenylbenzylamine, choline, meglumine, and tromethamine, as well as
amino acids, such as lysine and arginine. An example of a common
quaternary ammonium ion is N(CH.sub.3).sub.4.sup.+.
[1542] If the compound is cationic, or has a functional group which
may be cationic (e.g. --NH.sub.2 may be --NH.sub.3.sup.+), then a
salt may be formed with a suitable anion. Examples of suitable
inorganic anions include, but are not limited to, those derived
from the following inorganic acids: hydrochloric, hydrobromic,
hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and
phosphorous.
[1543] Examples of suitable organic anions include, but are not
limited to, those derived from the following organic acids:
2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic,
camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic,
ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic,
glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic,
lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic,
oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic,
phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic,
sulfanilic, tartaric, toluenesulfonic, trifluoroacetic acid and
valeric. Examples of suitable polymeric organic anions include, but
are not limited to, those derived from the following polymeric
acids: tannic acid, carboxymethyl cellulose.
[1544] Solvates
[1545] It may be convenient or desirable to prepare, purify, and/or
handle a corresponding solvate of the active compound. The term
"solvate" is used herein in the conventional sense to refer to a
complex of solute (e.g. active compound, salt of active compound)
and solvent. If the solvent is water, the solvate may be
conveniently referred to as a hydrate, for example, a mono-hydrate,
a di-hydrate, a tri-hydrate, etc.
[1546] The disclosure includes compounds where a solvent adds
across the imine bond of the PBD moiety, which is illustrated below
where the solvent is water or an alcohol (R.sup.AOH, where R.sup.A
is C.sub.1-4 alkyl):
##STR00086##
[1547] These forms can be called the carbinolamine and
carbinolamine ether forms of the PBD (as described in the section
relating to R.sup.10 above). The balance of these equilibria depend
on the conditions in which the compounds are found, as well as the
nature of the moiety itself.
[1548] These particular compounds may be isolated in solid form,
for example, by lyophilisation.
[1549] Isomers
[1550] Certain compounds of the disclosure may exist in one or more
particular geometric, optical, enantiomeric, diasteriomeric,
epimeric, atropic, stereoisomeric, tautomeric, conformational, or
anomeric forms, including but not limited to, cis- and trans-forms;
E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-,
and meso-forms; D- and L-forms; d- and I-forms; (+) and (-) forms;
keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal-
and anticlinal-forms; .alpha.- and .beta.-forms; axial and
equatorial forms; boat-, chair-, twist-, envelope-, and
halfchair-forms; and combinations thereof, hereinafter collectively
referred to as "isomers" (or "isomeric forms").
[1551] The term "chiral" refers to molecules which have the
property of non-superimposability of the mirror image partner,
while the term "achiral" refers to molecules which are
superimposable on their mirror image partner.
[1552] The term "stereoisomers" refers to compounds which have
identical chemical constitution, but differ with regard to the
arrangement of the atoms or groups in space.
[1553] "Diastereomer" refers to a stereoisomer with two or more
centers of chirality and whose molecules are not mirror images of
one another. Diastereomers have different physical properties, e.g.
melting points, boiling points, spectral properties, and
reactivities. Mixtures of diastereomers may separate under high
resolution analytical procedures such as electrophoresis and
chromatography.
[1554] "Enantiomers" refer to two stereoisomers of a compound which
are non-superimposable mirror images of one another.
[1555] Stereochemical definitions and conventions used herein
generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of
Chemical Terms (1984) McGraw-Hill Book Company, New York; and
Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds",
John Wiley & Sons, Inc., New York, 1994. The compounds of the
disclosure may contain asymmetric or chiral centers, and therefore
exist in different stereoisomeric forms. It is intended that all
stereoisomeric forms of the compounds of the disclosure, including
but not limited to, diastereomers, enantiomers and atropisomers, as
well as mixtures thereof such as racemic mixtures, form part of the
present disclosure. Many organic compounds exist in optically
active forms, i.e., they have the ability to rotate the plane of
plane-polarized light. In describing an optically active compound,
the prefixes D and L, or R and S, are used to denote the absolute
configuration of the molecule about its chiral center(s). The
prefixes d and I or (+) and (-) are employed to designate the sign
of rotation of plane-polarized light by the compound, with (-) or I
meaning that the compound is levorotatory. A compound prefixed with
(+) or d is dextrorotatory. For a given chemical structure, these
stereoisomers are identical except that they are mirror images of
one another. A specific stereoisomer may also be referred to as an
enantiomer, and a mixture of such isomers is often called an
enantiomeric mixture. A 50:50 mixture of enantiomers is referred to
as a racemic mixture or a racemate, which may occur where there has
been no stereoselection or stereospecificity in a chemical reaction
or process. The terms "racemic mixture" and "racemate" refer to an
equimolar mixture of two enantiomeric species, devoid of optical
activity.
[1556] Note that, except as discussed below for tautomeric forms,
specifically excluded from the term "isomers", as used herein, are
structural (or constitutional) isomers (i.e. isomers which differ
in the connections between atoms rather than merely by the position
of atoms in space). For example, a reference to a methoxy group,
--OCH.sub.3, is not to be construed as a reference to its
structural isomer, a hydroxymethyl group, --CH.sub.2OH. Similarly,
a reference to ortho-chlorophenyl is not to be construed as a
reference to its structural isomer, meta-chlorophenyl. However, a
reference to a class of structures may well include structurally
isomeric forms falling within that class (e.g. C.sub.1-7 alkyl
includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-,
and tert-butyl; methoxyphenyl includes ortho-, meta-, and
para-methoxyphenyl).
[1557] The above exclusion does not pertain to tautomeric forms,
for example, keto-, enol-, and enolate-forms, as in, for example,
the following tautomeric pairs: keto/enol (illustrated below),
imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime,
thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.
##STR00087##
[1558] The term "tautomer" or "tautomeric form" refers to
structural isomers of different energies which are interconvertible
via a low energy barrier. For example, proton tautomers (also known
as prototropic tautomers) include interconversions via migration of
a proton, such as keto-enol and imine-enamine isomerizations.
Valence tautomers include interconversions by reorganization of
some of the bonding electrons.
[1559] Note that specifically included in the term "isomer" are
compounds with one or more isotopic substitutions. For example, H
may be in any isotopic form, including .sup.1H, .sup.2H (D), and
.sup.3H (T); C may be in any isotopic form, including .sup.12C,
.sup.13C, and .sup.14C; O may be in any isotopic form, including
.sup.16O and .sup.18O; and the like.
[1560] Examples of isotopes that can be incorporated into compounds
of the disclosure include isotopes of hydrogen, carbon, nitrogen,
oxygen, phosphorous, fluorine, and chlorine, such as, but not
limited to .sup.2H (deuterium, D), .sup.3H (tritium), 11C, 13C,
14C, .sup.15N, .sup.18F, .sup.31P, .sup.32P, .sup.35S, .sup.36Cl,
and .sup.125I. Various isotopically labeled compounds of the
present disclosure, for example those into which radioactive
isotopes such as 3H, 13C, and 14C are incorporated. Such
isotopically labelled compounds may be useful in metabolic studies,
reaction kinetic studies, detection or imaging techniques, such as
positron emission tomography (PET) or single-photon emission
computed tomography (SPECT) including drug or substrate tissue
distribution assays, or in radioactive treatment of patients.
Deuterium labelled or substituted therapeutic compounds of the
disclosure may have improved DMPK (drug metabolism and
pharmacokinetics) properties, relating to distribution, metabolism,
and excretion (ADME). Substitution with heavier isotopes such as
deuterium may afford certain therapeutic advantages resulting from
greater metabolic stability, for example increased in vivo
half-life or reduced dosage requirements. An 18F labeled compound
may be useful for PET or SPECT studies. Isotopically labeled
compounds of this disclosure and prodrugs thereof can generally be
prepared by carrying out the procedures disclosed in the schemes or
in the examples and preparations described below by substituting a
readily available isotopically labeled reagent for a
non-isotopically labeled reagent. Further, substitution with
heavier isotopes, particularly deuterium (i.e., 2H or D) may afford
certain therapeutic advantages resulting from greater metabolic
stability, for example increased in vivo half-life or reduced
dosage requirements or an improvement in therapeutic index. It is
understood that deuterium in this context is regarded as a
substituent. The concentration of such a heavier isotope,
specifically deuterium, may be defined by an isotopic enrichment
factor. In the compounds of this disclosure any atom not
specifically designated as a particular isotope is meant to
represent any stable isotope of that atom.
[1561] Unless otherwise specified, a reference to a particular
compound includes all such isomeric forms, including (wholly or
partially) racemic and other mixtures thereof. Methods for the
preparation (e.g. asymmetric synthesis) and separation (e.g.
fractional crystallisation and chromatographic means) of such
isomeric forms are either known in the art or are readily obtained
by adapting the methods taught herein, or known methods, in a known
manner.
[1562] Biological Activity
[1563] In Vitro Cell Proliferation Assays
[1564] Generally, the cytotoxic or cytostatic activity of an
antibody-drug conjugate (ADC) is measured by: exposing mammalian
cells having receptor proteins to the antibody of the ADC in a cell
culture medium; culturing the cells for a period from about 6 hours
to about 5 to 7 days; and measuring cell viability. Cell-based in
vitro assays are used to measure viability (proliferation),
cytotoxicity, and induction of apoptosis (caspase activation) of an
ADC of the disclosure.
[1565] The in vitro potency of antibody-drug conjugates can be
measured by a cell proliferation assay. The CellTiter-Glo.RTM.
Luminescent Cell Viability Assay is a commercially available
(Promega Corp., Madison, Wis.), homogeneous assay method based on
the recombinant expression of Coleoptera luciferase (U.S. Pat. Nos.
5,583,024; 5,674,713 and 5,700,670). This cell proliferation assay
determines the number of viable cells in culture based on
quantitation of the ATP present, an indicator of metabolically
active cells (Crouch et al (1993) J. lmmunol. Meth. 160:81-88; U.S.
Pat. No. 6,602,677). The CellTiter-Glo.RTM. Assay is conducted in
96 well format, making it amenable to automated high-throughput
screening (HTS) (Cree et al (1995) AntiCancer Drugs 6:398-404). The
homogeneous assay procedure involves adding the single reagent
(CellTiter-Glo.RTM. Reagent) directly to cells cultured in
serum-supplemented medium. Cell washing, removal of medium and
multiple pipetting steps are not required. The system detects as
few as 15 cells/well in a 384-well format in 10 minutes after
adding reagent and mixing. The cells may be treated continuously
with ADC, or they may be treated and separated from ADC. Generally,
cells treated briefly, i.e. 3 hours, showed the same potency
effects as continuously treated cells.
[1566] The homogeneous "add-mix-measure" format results in cell
lysis and generation of a luminescent signal proportional to the
amount of ATP present. The amount of ATP is directly proportional
to the number of cells present in culture. The CellTiter-Glo.RTM.
Assay generates a "glow-type" luminescent signal, produced by the
luciferase reaction, which has a half-life generally greater than
five hours, depending on cell type and medium used. Viable cells
are reflected in relative luminescence units (RLU). The substrate,
Beetle Luciferin, is oxidatively decarboxylated by recombinant
firefly luciferase with concomitant conversion of ATP to AMP and
generation of photons.
[1567] The in vitro potency of antibody-drug conjugates can also be
measured by a cytotoxicity assay. Cultured adherent cells are
washed with PBS, detached with trypsin, diluted in complete medium,
containing 10% FCS, centrifuged, re-suspended in fresh medium and
counted with a haemocytometer. Suspension cultures are counted
directly. Monodisperse cell suspensions suitable for counting may
require agitation of the suspension by repeated aspiration to break
up cell clumps.
[1568] The cell suspension is diluted to the desired seeding
density and dispensed (100 .mu.l per well) into black 96 well
plates. Plates of adherent cell lines are incubated overnight to
allow adherence. Suspension cell cultures can be used on the day of
seeding.
[1569] A stock solution (1 ml) of ADC (20 .mu.pg/ml) is made in the
appropriate cell culture medium. Serial 10-fold dilutions of stock
ADC are made in 15 ml centrifuge tubes by serially transferring 100
.mu.l to 900 .mu.l of cell culture medium.
[1570] Four replicate wells of each ADC dilution (100 .mu.l) are
dispensed in 96-well black plates, previously plated with cell
suspension (100 .mu.l), resulting in a final volume of 200 .mu.l.
Control wells receive cell culture medium (100 .mu.l).
[1571] If the doubling time of the cell line is greater than 30
hours, ADC incubation is for 5 days, otherwise a four day
incubation is done.
[1572] At the end of the incubation period, cell viability is
assessed with the Alamar blue assay.
[1573] AlamarBlue (Invitrogen) is dispensed over the whole plate
(20 .mu.l per well) and incubated for 4 hours. Alamar blue
fluorescence is measured at excitation 570 nm, emission 585 nm on
the Varioskan flash plate reader. Percentage cell survival is
calculated from the mean fluorescence in the ADC treated wells
compared to the mean fluorescence in the control wells.
[1574] Use
[1575] The conjugates of the disclosure may be used to provide a
PBD compound at a target location.
[1576] The target location is preferably a proliferative cell
population, such as a population of proliferative cancer cells.
Other targets locations include a quiescent cell population, such
as a population of quiescent cancer cells, or a population of
cancer stem cells The antibody is an antibody for an antigen
present on a proliferative cell population.
[1577] In one embodiment the antigen is absent or present at a
reduced level in a non-proliferative cell population compared to
the amount of antigen present in the proliferative cell population,
for example a tumour cell population.
[1578] At the target location the linker may be cleaved so as to
release a compound RelA, RelB, RelC, RelD, RelE or RelG. Thus, the
conjugate may be used to selectively provide a compound RelA, RelB,
RelC, RelD, RelE or RelG to the target location.
[1579] The linker may be cleaved by an enzyme present at the target
location.
[1580] The target location may be in vitro, in vivo or ex vivo.
[1581] The antibody-drug conjugate (ADC) compounds of the
disclosure include those with utility for anticancer activity. In
particular, the compounds include an antibody conjugated, i.e.
covalently attached by a linker, to a PBD drug moiety, i.e. toxin.
When the drug is not conjugated to an antibody, the PBD drug has a
cytotoxic effect. The biological activity of the PBD drug moiety is
thus modulated by conjugation to an antibody. The antibody-drug
conjugates (ADC) of the disclosure selectively deliver an effective
dose of a cytotoxic agent to tumor tissue whereby greater
selectivity, i.e. a lower efficacious dose, may be achieved.
[1582] Thus, in one aspect, the present disclosure provides a
conjugate compound as described herein for use in therapy.
[1583] In a further aspect there is also provides a conjugate
compound as described herein for use in the treatment of a
proliferative disease. A second aspect of the present disclosure
provides the use of a conjugate compound in the manufacture of a
medicament for treating a proliferative disease.
[1584] One of ordinary skill in the art is readily able to
determine whether or not a candidate conjugate treats a
proliferative condition for any particular cell type. For example,
assays which may conveniently be used to assess the activity
offered by a particular compound are described in the examples
below.
[1585] The term "proliferative disease" pertains to an unwanted or
uncontrolled cellular proliferation of excessive or abnormal cells
which is undesired, such as, neoplastic or hyperplastic growth,
whether in vitro or in vivo.
[1586] Examples of proliferative conditions include, but are not
limited to, benign, pre-malignant, and malignant cellular
proliferation, including but not limited to, neoplasms and tumours
(e.g. histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung
cancer, small cell lung cancer, gastrointestinal cancer, bowel
cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate
cancer, testicular cancer, liver cancer, kidney cancer, bladder
cancer, pancreas cancer, brain cancer, sarcoma, osteosarcoma,
Kaposi's sarcoma, melanoma), lymphomas, leukemias, psoriasis, bone
diseases, fibroproliferative disorders (e.g. of connective
tissues), and atherosclerosis. Cancers of particular interest
include, but are not limited to prostate cancers, leukemias and
ovarian cancers.
[1587] Any type of cell may be treated, including but not limited
to, lung, gastrointestinal (including, e.g. bowel, colon), breast
(mammary), ovarian, prostate, liver (hepatic), kidney (renal),
bladder, pancreas, brain, and skin.
[1588] Disorders of particular interest include, but are not
limited to, non-Hodgkin Lymphoma including diffuse large B-cell
lymphoma (DLBCL), follicular lymphoma, (FL), Mantle Cell lymphoma
(MCL), chronic lymphatic lymphoma (CLL) nd leukemias such as Hairy
cell leukemia (HCL), Hairy cell leukemia variant (HCL-v) and Acute
Lymphoblastic Leukaemia (ALL).
[1589] It is contemplated that the antibody-drug conjugates (ADC)
of the present disclosure may be used to treat various diseases or
disorders, e.g. characterized by the overexpression of a tumor
antigen. Exemplary conditions or hyperproliferative disorders
include benign or malignant tumors; leukemia, haematological, and
lymphoid malignancies. Others include neuronal, glial, astrocytal,
hypothalamic, glandular, macrophagal, epithelial, stromal,
blastocoelic, inflammatory, angiogenic and immunologic, including
autoimmune, disorders.
[1590] Generally, the disease or disorder to be treated is a
hyperproliferative disease such as cancer. Examples of cancer to be
treated herein include, but are not limited to, carcinoma,
lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
More particular examples of such cancers include squamous cell
cancer (e.g. epithelial squamous cell cancer), lung cancer
including small-cell lung cancer, non-small cell lung cancer,
adenocarcinoma of the lung and squamous carcinoma of the lung,
cancer of the peritoneum, hepatocellular cancer, gastric or stomach
cancer including gastrointestinal cancer, pancreatic cancer,
glioblastoma, cervical cancer, ovarian cancer, liver cancer,
bladder cancer, hepatoma, breast cancer, colon cancer, rectal
cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney or renal cancer, prostate cancer,
vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma,
penile carcinoma, as well as head and neck cancer.
[1591] Autoimmune diseases for which the ADC compounds may be used
in treatment include rheumatologic disorders (such as, for example,
rheumatoid arthritis, Sjogren's syndrome, scleroderma, lupus such
as SLE and lupus nephritis, polymyositis/dermatomyositis,
cryoglobulinemia, anti-phospholipid antibody syndrome, and
psoriatic arthritis), osteoarthritis, autoimmune gastrointestinal
and liver disorders (such as, for example, inflammatory bowel
diseases (e.g. ulcerative colitis and Crohn's disease), autoimmune
gastritis and pernicious anemia, autoimmune hepatitis, primary
biliary cirrhosis, primary sclerosing cholangitis, and celiac
disease), vasculitis (such as, for example, ANCA-associated
vasculitis, including Churg-Strauss vasculitis, Wegener's
granulomatosis, and polyarteriitis), autoimmune neurological
disorders (such as, for example, multiple sclerosis, opsoclonus
myoclonus syndrome, myasthenia gravis, neuromyelitis optica,
Parkinson's disease, Alzheimer's disease, and autoimmune
polyneuropathies), renal disorders (such as, for example,
glomerulonephritis, Goodpasture's syndrome, and Berger's disease),
autoimmune dermatologic disorders (such as, for example, psoriasis,
urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and
cutaneous lupus erythematosus), hematologic disorders (such as, for
example, thrombocytopenic purpura, thrombotic thrombocytopenic
purpura, post-transfusion purpura, and autoimmune hemolytic
anemia), atherosclerosis, uveitis, autoimmune hearing diseases
(such as, for example, inner ear disease and hearing loss),
Behcet's disease, Raynaud's syndrome, organ transplant, and
autoimmune endocrine disorders (such as, for example,
diabetic-related autoimmune diseases such as insulin-dependent
diabetes mellitus (IDDM), Addison's disease, and autoimmune thyroid
disease (e.g. Graves' disease and thyroiditis)). More preferred
such diseases include, for example, rheumatoid arthritis,
ulcerative colitis, ANCA-associated vasculitis, lupus, multiple
sclerosis, Sjogren's syndrome, Graves' disease, IDDM, pernicious
anemia, thyroiditis, and glomerulonephritis.
[1592] Methods of Treatment
[1593] The conjugates of the present disclosure may be used in a
method of therapy. Also provided is a method of treatment,
comprising administering to a subject in need of treatment a
therapeutically-effective amount of a conjugate compound of the
disclosure. The term "therapeutically effective amount" is an
amount sufficient to show benefit to a patient. Such benefit may be
at least amelioration of at least one symptom. The actual amount
administered, and rate and time-course of administration, will
depend on the nature and severity of what is being treated.
Prescription of treatment, e.g. decisions on dosage, is within the
responsibility of general practitioners and other medical
doctors.
[1594] A compound of the disclosure may be administered alone or in
combination with other treatments, either simultaneously or
sequentially dependent upon the condition to be treated. Examples
of treatments and therapies include, but are not limited to,
chemotherapy (the administration of active agents, including, e.g.
drugs, such as chemotherapeutics); surgery; and radiation
therapy.
[1595] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer, regardless of mechanism of action. Classes
of chemotherapeutic agents include, but are not limited to:
alkylating agents, antimetabolites, spindle poison plant alkaloids,
cytotoxic/antitumor antibiotics, topoisomerase inhibitors,
antibodies, photosensitizers, and kinase inhibitors.
Chemotherapeutic agents include compounds used in "targeted
therapy" and conventional chemotherapy.
[1596] Examples of chemotherapeutic agents include: erlotinib
(TARCEVA.RTM., Genentech/OSI Pharm.), docetaxel (TAXOTERE.RTM.,
Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No.
51-21-8), gemcitabine (GEMZAR.RTM., Lilly), PD-0325901 (CAS No.
391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum(II),
CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel
(TAXOL.RTM., Bristol-Myers Squibb Oncology, Princeton, N.J.),
trastuzumab (HERCEPTIN.RTM., Genentech), temozolomide
(4-methyl-5-oxo- 2,3,4,6,8-pentazabicyclo [4.3.0]
nona-2,7,9-triene- 9-carboxamide, CAS No. 85622-93-1, TEMODAR.RTM.,
TEMODAL.RTM., Schering Plough), tamoxifen
((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine,
NOLVADEX.RTM., ISTUBAL.RTM., VALODEX.RTM.), and doxorubicin
(ADRIAMYCIN.RTM.), Akti-1/2, HPPD, and rapamycin. More examples of
chemotherapeutic agents include: oxaliplatin (ELOXATIN.RTM.,
Sanofi), bortezomib (VELCADE.RTM., Millennium Pharm.), sutent
(SUNITINIB.RTM., SU11248, Pfizer), letrozole (FEMARA.RTM.,
Novartis), imatinib mesylate (GLEEVEC.RTM., Novartis), XL-518 (Mek
inhibitor, Exelixis, WO 2007/044515), ARRY-886 (Mek inhibitor,
AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (P13K inhibitor,
Semafore Pharmaceuticals), BEZ-235 (P13K inhibitor, Novartis),
XL-147 (P13K inhibitor, Exelixis), PTK787/ZK 222584 (Novartis),
fulvestrant (FASLODEX.RTM., AstraZeneca), leucovorin (folinic
acid), rapamycin (sirolimus, RAPAMUNE.RTM., Wyeth), lapatinib
(TYKERB.RTM., GSK572016, Glaxo Smith Kline), lonafarnib
(SARASAR.TM., SCH 66336, Schering Plough), sorafenib (NEXAVAR.RTM.,
BAY43-9006, Bayer Labs), gefitinib (IRESSA.RTM., AstraZeneca),
irinotecan (CAMPTOSAR.RTM., CPT-11, Pfizer), tipifarnib
(ZARNESTRA.TM., Johnson & Johnson), ABRAXANE.TM.
(Cremophor-free), albumin-engineered nanoparticle formulations of
paclitaxel (American Pharmaceutical Partners, Schaumberg, II),
vandetanib (rINN, ZD6474, ZACTIMA.RTM., AstraZeneca),
chloranmbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus
(TORISEL.RTM., Wyeth), pazopanib (GlaxoSmithKline), canfosfamide
(TELCYTA.RTM., Telik), thiotepa and cyclosphosphamide
(CYTOXAN.RTM., NEOSAR.RTM.); alkyl sulfonates such as busulfan,
improsulfan and piposulfan; aziridines such as benzodopa,
carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and
trimethylomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analog
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogs);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogs, KW-2189
and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, chlorophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosoureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics (e.g. calicheamicin, calicheamicin
gamma1l, calicheamicin omegal1 (Angew Chem. Intl. Ed. Engl. (1994)
33:183-186); dynemicin, dynemicin A; bisphosphonates, such as
clodronate; an esperamicin; as well as neocarzinostatin chromophore
and related chromoprotein enediyne antibiotic chromophores),
aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,
cactinomycin, carabicin, carminomycin, carzinophilin,
chromomycinis, dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as
denopterin, methotrexate, pteropterin, trimetrexate; purine analogs
such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;
pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine,
carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine,
floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals
such as aminoglutethimide, mitotane, trilostane; folic acid
replenisher such as frolinic acid; aceglatone; aldophosphamide
glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elfornithine; elliptinium acetate; an epothilone; etoglucid;
gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids
such as maytansine and ansamitocins; mitoguazone; mitoxantrone;
mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;
losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine;
PSK.RTM. polysaccharide complex (JHS Natural Products, Eugene,
Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic
acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes
(especially T-2 toxin, verracurin A, roridin A and anguidine);
urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin and carboplatin;
vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;
vincristine; vinorelbine (NAVELBINE.RTM.); novantrone; teniposide;
edatrexate; daunomycin; aminopterin; capecitabine (XELODA.RTM.,
Roche); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;
difluoromethylornithine (DMFO); retinoids such as retinoic acid;
and pharmaceutically acceptable salts, acids and derivatives of any
of the above.
[1597] Also included in the definition of "chemotherapeutic agent"
are: (i) anti-hormonal agents that act to regulate or inhibit
hormone action on tumors such as anti-estrogens and selective
estrogen receptor modulators (SERMs), including, for example,
tamoxifen (including NOLVADEX.RTM.; tamoxifen citrate), raloxifene,
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and FARESTON.RTM. (toremifine citrate); (ii) aromatase
inhibitors that inhibit the enzyme aromatase, which regulates
estrogen production in the adrenal glands, such as, for example,
4(5)-imidazoles, aminoglutethimide, MEGASE.RTM. (megestrol
acetate), AROMASIN.RTM. (exemestane; Pfizer), formestanie,
fadrozole, RIVISOR.RTM. (vorozole), FEMARA.RTM. (letrozole;
Novartis), and ARIMIDEX.RTM. (anastrozole; AstraZeneca); (iii)
anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide, and goserelin; as well as troxacitabine (a
1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase
inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipid
kinase inhibitors; (vi) antisense oligonucleotides, particularly
those which inhibit expression of genes in signaling pathways
implicated in aberrant cell proliferation, for example, PKC-alpha,
Raf and H-Ras, such as oblimersen (GENASENSE.RTM., Genta Inc.);
(vii) ribozymes such as VEGF expression inhibitors (e.g.,
ANGIOZYME.RTM.); (viii) vaccines such as gene therapy vaccines, for
example, ALLOVECTIN.RTM., LEUVECTIN.RTM., and VAXID.RTM.;
PROLEUKIN.RTM. rIL-2; topoisomerase 1 inhibitors such as
LURTOTECAN.RTM.; ABARELIX.RTM. rmRH; (ix) anti-angiogenic agents
such as bevacizumab (AVASTIN.RTM., Genentech); and pharmaceutically
acceptable salts, acids and derivatives of any of the above.
[1598] Also included in the definition of "chemotherapeutic agent"
are therapeutic antibodies such as alemtuzumab (Campath),
bevacizumab (AVASTIN.RTM., Genentech); cetuximab (ERBITUX.RTM.,
lmclone); panitumumab (VECTIBIX.RTM., Amgen), rituximab
(RITUXAN.RTM., Genentech/Biogen Idec), ofatumumab (ARZERRA.RTM.,
GSK), pertuzumab (PERJETA.TM., OMNITARG.TM., 2C4, Genentech),
trastuzumab (HERCEPTIN.RTM., Genentech), tositumomab (Bexxar,
Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin
(MYLOTARGO, Wyeth).
[1599] Humanized monoclonal antibodies with therapeutic potential
as chemotherapeutic agents in combination with the conjugates of
the disclosure include: alemtuzumab, apolizumab, aselizumab,
atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine,
cantuzumab mertansine, cedelizumab, certolizumab pegol,
cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab,
epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab
ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab,
lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab,
natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab,
omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab,
pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab,
reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab,
siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab,
talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab,
tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab,
and visilizumab.
[1600] Pharmaceutical compositions according to the present
disclosure, and for use in accordance with the present disclosure,
may comprise, in addition to the active ingredient, i.e. a
conjugate compound, a pharmaceutically acceptable excipient,
carrier, buffer, stabiliser or other materials well known to those
skilled in the art. Such materials should be non-toxic and should
not interfere with the efficacy of the active ingredient. The
precise nature of the carrier or other material will depend on the
route of administration, which may be oral, or by injection, e.g.
cutaneous, subcutaneous, or intravenous.
[1601] Pharmaceutical compositions for oral administration may be
in tablet, capsule, powder or liquid form. A tablet may comprise a
solid carrier or an adjuvant. Liquid pharmaceutical compositions
generally comprise a liquid carrier such as water, petroleum,
animal or vegetable oils, mineral oil or synthetic oil.
Physiological saline solution, dextrose or other saccharide
solution or glycols such as ethylene glycol, propylene glycol or
polyethylene glycol may be included. A capsule may comprise a solid
carrier such a gelatin.
[1602] For intravenous, cutaneous or subcutaneous injection, or
injection at the site of affliction, the active ingredient will be
in the form of a parenterally acceptable aqueous solution which is
pyrogen-free and has suitable pH, isotonicity and stability. Those
of relevant skill in the art are well able to prepare suitable
solutions using, for example, isotonic vehicles such as Sodium
Chloride Injection, Ringer's Injection, Lactated Ringer's
Injection. Preservatives, stabilisers, buffers, antioxidants and/or
other additives may be included, as required.
[1603] Formulations
[1604] While it is possible for the conjugate compound to be used
(e.g., administered) alone, it is often preferable to present it as
a composition or formulation.
[1605] In one embodiment, the composition is a pharmaceutical
composition (e.g., formulation, preparation, medicament) comprising
a conjugate compound, as described herein, and a pharmaceutically
acceptable carrier, diluent, or excipient.
[1606] In one embodiment, the composition is a pharmaceutical
composition comprising at least one conjugate compound, as
described herein, together with one or more other pharmaceutically
acceptable ingredients well known to those skilled in the art,
including, but not limited to, pharmaceutically acceptable
carriers, diluents, excipients, adjuvants, fillers, buffers,
preservatives, anti-oxidants, lubricants, stabilisers,
solubilisers, surfactants (e.g., wetting agents), masking agents,
colouring agents, flavouring agents, and sweetening agents.
[1607] In one embodiment, the composition further comprises other
active agents, for example, other therapeutic or prophylactic
agents.
[1608] Suitable carriers, diluents, excipients, etc. can be found
in standard pharmaceutical texts. See, for example, Handbook of
Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash),
2001 (Synapse Information Resources, Inc., Endicott, New York,
USA), Remington's Pharmaceutical Sciences, 20th edition, pub.
Lippincott, Williams & Wilkins, 2000; and Handbook of
Pharmaceutical Excipients, 2nd edition, 1994.
[1609] Another aspect of the present disclosure pertains to methods
of making a pharmaceutical composition comprising admixing at least
one [.sup.11C]-radiolabelled conjugate or conjugate-like compound,
as defined herein, together with one or more other pharmaceutically
acceptable ingredients well known to those skilled in the art,
e.g., carriers, diluents, excipients, etc. If formulated as
discrete units (e.g., tablets, etc.), each unit contains a
predetermined amount (dosage) of the active compound.
[1610] The term "pharmaceutically acceptable," as used herein,
pertains to compounds, ingredients, materials, compositions, dosage
forms, etc., which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of the subject in
question (e.g., human) without excessive toxicity, irritation,
allergic response, or other problem or complication, commensurate
with a reasonable benefit/risk ratio. Each carrier, diluent,
excipient, etc. must also be "acceptable" in the sense of being
compatible with the other ingredients of the formulation.
[1611] The formulations may be prepared by any methods well known
in the art of pharmacy. Such methods include the step of bringing
into association the active compound with a carrier which
constitutes one or more accessory ingredients. In general, the
formulations are prepared by uniformly and intimately bringing into
association the active compound with carriers (e.g., liquid
carriers, finely divided solid carrier, etc.), and then shaping the
product, if necessary.
[1612] The formulation may be prepared to provide for rapid or slow
release; immediate, delayed, timed, or sustained release; or a
combination thereof.
[1613] Formulations suitable for parenteral administration (e.g.,
by injection), include aqueous or non-aqueous, isotonic,
pyrogen-free, sterile liquids (e.g., solutions, suspensions), in
which the active ingredient is dissolved, suspended, or otherwise
provided (e.g., in a liposome or other microparticulate). Such
liquids may additional contain other pharmaceutically acceptable
ingredients, such as anti-oxidants, buffers, preservatives,
stabilisers, bacteriostats, suspending agents, thickening agents,
and solutes which render the formulation isotonic with the blood
(or other relevant bodily fluid) of the intended recipient.
Examples of excipients include, for example, water, alcohols,
polyols, glycerol, vegetable oils, and the like. Examples of
suitable isotonic carriers for use in such formulations include
Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's
Injection. Typically, the concentration of the active ingredient in
the liquid is from about 1 ng/ml to about 10 .mu.g/ml, for example
from about 10 ng/ml to about 1 .mu.g/ml. The formulations may be
presented in unit-dose or multi-dose sealed containers, for
example, ampoules and vials, and may be stored in a freeze-dried
(lyophilised) condition requiring only the addition of the sterile
liquid carrier, for example water for injections, immediately prior
to use. Extemporaneous injection solutions and suspensions may be
prepared from sterile powders, granules, and tablets.
[1614] Dosage
[1615] It will be appreciated by one of skill in the art that
appropriate dosages of the conjugate compound, and compositions
comprising the conjugate compound, can vary from patient to
patient. Determining the optimal dosage will generally involve the
balancing of the level of therapeutic benefit against any risk or
deleterious side effects. The selected dosage level will depend on
a variety of factors including, but not limited to, the activity of
the particular compound, the route of administration, the time of
administration, the rate of excretion of the compound, the duration
of the treatment, other drugs, compounds, and/or materials used in
combination, the severity of the condition, and the species, sex,
age, weight, condition, general health, and prior medical history
of the patient. The amount of compound and route of administration
will ultimately be at the discretion of the physician,
veterinarian, or clinician, although generally the dosage will be
selected to achieve local concentrations at the site of action
which achieve the desired effect without causing substantial
harmful or deleterious side-effects.
[1616] Administration can be effected in one dose, continuously or
intermittently (e.g., in divided doses at appropriate intervals)
throughout the course of treatment. Methods of determining the most
effective means and dosage of administration are well known to
those of skill in the art and will vary with the formulation used
for therapy, the purpose of the therapy, the target cell(s) being
treated, and the subject being treated. Single or multiple
administrations can be carried out with the dose level and pattern
being selected by the treating physician, veterinarian, or
clinician.
[1617] In general, a suitable dose of the active compound is in the
range of about 100 ng to about 25 mg (more typically about 1 .mu.g
to about 10 mg) per kilogram body weight of the subject per day.
Where the active compound is a salt, an ester, an amide, a prodrug,
or the like, the amount administered is calculated on the basis of
the parent compound and so the actual weight to be used is
increased proportionately.
[1618] In one embodiment, the active compound is administered to a
human patient according to the following dosage regime: about 100
mg, 3 times daily.
[1619] In one embodiment, the active compound is administered to a
human patient according to the following dosage regime: about 150
mg, 2 times daily.
[1620] In one embodiment, the active compound is administered to a
human patient according to the following dosage regime: about 200
mg, 2 times daily.
[1621] However in one embodiment, the conjugate compound is
administered to a human patient according to the following dosage
regime: about 50 or about 75 mg, 3 or 4 times daily.
[1622] In one embodiment, the conjugate compound is administered to
a human patient according to the following dosage regime: about 100
or about 125 mg, 2 times daily.
[1623] The dosage amounts described above may apply to the
conjugate (including the PBD moiety and the linker to the antibody)
or to the effective amount of PBD compound provided, for example
the amount of compound that is releasable after cleavage of the
linker.
[1624] For the prevention or treatment of disease, the appropriate
dosage of an ADC of the disclosure will depend on the type of
disease to be treated, as defined above, the severity and course of
the disease, whether the molecule is administered for preventive or
therapeutic purposes, previous therapy, the patient's clinical
history and response to the antibody, and the discretion of the
attending physician. The molecule is suitably administered to the
patient at one time or over a series of treatments. Depending on
the type and severity of the disease, about 1 .mu.g/kg to 15 mg/kg
(e.g. 0.1-20 mg/kg) of molecule is an initial candidate dosage for
administration to the patient, whether, for example, by one or more
separate administrations, or by continuous infusion. A typical
daily dosage might range from about 1 .mu.g/kg to 100 mg/kg or
more, depending on the factors mentioned above. An exemplary dosage
of ADC to be administered to a patient is in the range of about 0.1
to about 10 mg/kg of patient weight. For repeated administrations
over several days or longer, depending on the condition, the
treatment is sustained until a desired suppression of disease
symptoms occurs. An exemplary dosing regimen comprises a course of
administering an initial loading dose of about 4 mg/kg, followed by
additional doses every week, two weeks, or three weeks of an ADC.
Other dosage regimens may be useful. The progress of this therapy
is easily monitored by conventional techniques and assays.
[1625] Treatment
[1626] The term "treatment," as used herein in the context of
treating a condition, pertains generally to treatment and therapy,
whether of a human or an animal (e.g., in veterinary applications),
in which some desired therapeutic effect is achieved, for example,
the inhibition of the progress of the condition, and includes a
reduction in the rate of progress, a halt in the rate of progress,
regression of the condition, amelioration of the condition, and
cure of the condition. Treatment as a prophylactic measure (i.e.,
prophylaxis, prevention) is also included.
[1627] The term "therapeutically-effective amount," as used herein,
pertains to that amount of an active compound, or a material,
composition or dosage from comprising an active compound, which is
effective for producing some desired therapeutic effect,
commensurate with a reasonable benefit/risk ratio, when
administered in accordance with a desired treatment regimen.
[1628] Similarly, the term "prophylactically-effective amount," as
used herein, pertains to that amount of an active compound, or a
material, composition or dosage from comprising an active compound,
which is effective for producing some desired prophylactic effect,
commensurate with a reasonable benefit/risk ratio, when
administered in accordance with a desired treatment regimen.
[1629] Preparation of Drug Conjugates
[1630] Antibody drug conjugates may be prepared by several routes,
employing organic chemistry reactions, conditions, and reagents
known to those skilled in the art, including reaction of a
nucleophilic group of an antibody with a drug-linker reagent. This
method may be employed to prepare the antibody-drug conjugates of
the disclosure.
[1631] Nucleophilic groups on antibodies include, but are not
limited to side chain thiol groups, e.g. cysteine. Thiol groups are
nucleophilic and capable of reacting to form covalent bonds with
electrophilic groups on linker moieties such as those of the
present disclosure. Certain antibodies have reducible interchain
disulfides, i.e. cysteine bridges. Antibodies may be made reactive
for conjugation with linker reagents by treatment with a reducing
agent such as DTT (Cleland's reagent, dithiothreitol) or TCEP
(tris(2-carboxyethyl)phosphine hydrochloride; Getz et al (1999)
Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.).
Each cysteine disulfide bridge will thus form, theoretically, two
reactive thiol nucleophiles. Additional nucleophilic groups can be
introduced into antibodies through the reaction of lysines with
2-iminothiolane (Traut's reagent) resulting in conversion of an
amine into a thiol.
[1632] The Subject/Patient
[1633] The subject/patient may be an animal, mammal, a placental
mammal, a marsupial (e.g., kangaroo, wombat), a monotreme (e.g.,
duckbilled platypus), a rodent (e.g., a guinea pig, a hamster, a
rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a
rabbit), avian (e.g., a bird), canine (e.g., a dog), feline (e.g.,
a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g.,
a sheep), bovine (e.g., a cow), a primate, simian (e.g., a monkey
or ape), a monkey (e.g., marmoset, baboon), an ape (e.g., gorilla,
chimpanzee, orangutang, gibbon), or a human.
[1634] Furthermore, the subject/patient may be any of its forms of
development, for example, a foetus. In one preferred embodiment,
the subject/patient is a human.
[1635] Further Preferences
[1636] The following preferences may apply to all aspects of the
disclosure as described above, or may relate to a single aspect.
The preferences may be combined together in any combination.
[1637] In some embodiments, R.sup.6', R.sup.7', R.sup.9', and Y'
are preferably the same as R.sup.6, R.sup.7, R.sup.9, and Y
respectively.
[1638] Dimer Link
[1639] Y and Y' are preferably O.
[1640] R'' is preferably a C.sub.3-7 alkylene group with no
substituents. More preferably R'' is a C.sub.3, C.sub.5 or C.sub.7
alkylene. Most preferably, R'' is a C.sub.3 or C.sub.5
alkylene.
[1641] R.sup.6 to R.sup.9
[1642] R.sup.9 is preferably H.
[1643] R.sup.6 is preferably selected from H, OH, OR, SH, NH.sub.2,
nitro and halo, and is more preferably H or halo, and most
preferably is H.
[1644] R.sup.7 is preferably selected from H, OH, OR, SH, SR,
NH.sub.2, NHR, NRR', and halo, and more preferably independently
selected from H, OH and OR, where R is preferably selected from
optionally substituted C.sub.1-7 alkyl, C.sub.3-10 heterocyclyl and
C.sub.5-10 aryl groups. R may be more preferably a C.sub.1-4 alkyl
group, which may or may not be substituted. A substituent of
interest is a C.sub.5-6 aryl group (e.g. phenyl). Particularly
preferred substituents at the 7- positions are OMe and OCH.sub.2Ph.
Other substituents of particular interest are dimethylamino (i.e.
--NMe.sub.2); --(OC.sub.2H.sub.4).sub.qOMe, where q is from 0 to 2;
nitrogen-containing C.sub.6 heterocyclyls, including morpholino,
piperidinyl and N-methyl-piperazinyl.
[1645] These preferences apply to R.sup.9', R.sup.6' and R.sup.7'
respectively.
[1646] .sub.R12
[1647] When there is a double bond present between C2' and C3',
R.sup.12 is selected from:
[1648] (a) C.sub.5-10 aryl group, optionally substituted by one or
more substituents selected from the group comprising: halo, nitro,
cyano, ether, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and
bis-oxy-C.sub.1-3 alkylene;
[1649] (b) C.sub.1-5 saturated aliphatic alkyl;
[1650] (c) C.sub.3-6 saturated cycloalkyl;
[1651] (d)
##STR00088##
wherein each of R.sup.21, R.sup.22 and R.sup.23 are independently
selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl,
C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon
atoms in the R.sup.12 group is no more than 5;
[1652] (e)
##STR00089##
wherein one of R.sup.25a and R.sup.25b is H and the other is
selected from: phenyl, which phenyl is optionally substituted by a
group selected from halo methyl, methoxy; pyridyl; and thiophenyl;
and
[1653] (f)
##STR00090##
where R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl;
C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which
phenyl is optionally substituted by a group selected from halo
methyl, methoxy; pyridyl; and thiophenyl.
[1654] When R.sup.12 is a C.sub.5-10 aryl group, it may be a
C.sub.5-7 aryl group. A C.sub.5-7 aryl group may be a phenyl group
or a C.sub.5-7 heteroaryl group, for example furanyl, thiophenyl
and pyridyl. In some embodiments, R.sup.12 is preferably phenyl. In
other embodiments, R.sup.12 is preferably thiophenyl, for example,
thiophen-2-yl and thiophen-3-yl.
[1655] When R.sup.12 is a C.sub.5-10 aryl group, it may be a
C.sub.8-10 aryl, for example a quinolinyl or isoquinolinyl group.
The quinolinyl or isoquinolinyl group may be bound to the PBD core
through any available ring position. For example, the quinolinyl
may be quinolin-2-yl, quinolin-3-yl, quinolin-4y1, quinolin-5-yl,
quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these
quinolin-3-yl and quinolin-6-yl may be preferred. The isoquinolinyl
may be isoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4y1,
isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and
isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl
may be preferred.
[1656] When R.sup.12 is a C.sub.5-10 aryl group, it may bear any
number of substituent groups. It preferably bears from 1 to 3
substituent groups, with 1 and 2 being more preferred, and singly
substituted groups being most preferred. The substituents may be
any position.
[1657] Where R.sup.12 is C.sub.5-7 aryl group, a single substituent
is preferably on a ring atom that is not adjacent the bond to the
remainder of the compound, i.e. it is preferably 13 or y to the
bond to the remainder of the compound. Therefore, where the
C.sub.5-7 aryl group is phenyl, the substituent is preferably in
the meta- or para- positions, and more preferably is in the
para-position.
[1658] Where R.sup.12 is a C.sub.8-10 aryl group, for example
quinolinyl or isoquinolinyl, it may bear any number of substituents
at any position of the quinoline or isoquinoline rings. In some
embodiments, it bears one, two or three substituents, and these may
be on either the proximal and distal rings or both (if more than
one substituent).
[1659] R.sup.12 Substituents, When R.sup.12 is a C.sub.5-10 Aryl
Group
[1660] If a substituent on R.sup.12 when R.sup.12 is a C.sub.5-10
aryl group is halo, it is preferably F or Cl, more preferably
Cl.
[1661] If a substituent on R.sup.12 when R.sup.12 is a C.sub.5-10
aryl group is ether, it may in some embodiments be an alkoxy group,
for example, a C.sub.1-7 alkoxy group (e.g. methoxy, ethoxy) or it
may in some embodiments be a C.sub.5-7 aryloxy group (e.g phenoxy,
pyridyloxy, furanyloxy). The alkoxy group may itself be further
substituted, for example by an amino group (e.g.
dimethylamino).
[1662] If a substituent on R.sup.12 when R.sup.12 is a C.sub.5-10
aryl group is C.sub.1-7 alkyl, it may preferably be a C.sub.1-4
alkyl group (e.g. methyl, ethyl, propryl, butyl).
[1663] If a substituent on R.sup.12 when R.sup.12 is a C.sub.5-10
aryl group is C.sub.3-7 heterocyclyl, it may in some embodiments be
C.sub.6 nitrogen containing heterocyclyl group, e.g. morpholino,
thiomorpholino, piperidinyl, piperazinyl. These groups may be bound
to the rest of the PBD moiety via the nitrogen atom. These groups
may be further substituted, for example, by C.sub.1-4 alkyl groups.
If the C.sub.6 nitrogen containing heterocyclyl group is
piperazinyl, the said further substituent may be on the second
nitrogen ring atom.
[1664] If a substituent on R.sup.12 when R.sup.12 is a C.sub.5-10
aryl group is bis-oxy-C.sub.1-3 alkylene, this is preferably
bis-oxy-methylene or bis-oxy-ethylene.
[1665] If a substituent on R.sup.12 when R.sup.12 is a C.sub.5-10
aryl group is ester, this is preferably methyl ester or ethyl
ester.
[1666] Particularly preferred substituents when R.sup.12 is a
C.sub.5-10 aryl group include methoxy, ethoxy, fluoro, chloro,
cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and
methyl-thiophenyl. Other particularly preferred substituent for
R.sup.12 are dimethylaminopropyloxy and carboxy.
[1667] Particularly preferred substituted R.sup.12 groups when
R.sup.12 is a C.sub.5-10 aryl group include, but are not limited
to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl,
3-ethoxy-phenyl, 4-fluoro-phenyl, 4-chloro-phenyl,
3,4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4-cyanophenyl,
4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl
and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and
naphthyl. Another possible substituted R.sup.12 group is
4-nitrophenyl. R.sup.12 groups of particular interest include
4-(4-methylpiperazin-1-yl)phenyl and
3,4-bisoxymethylene-phenyl.
[1668] When R.sup.12 is Cis saturated aliphatic alkyl, it may be
methyl, ethyl, propyl, butyl or pentyl. In some embodiments, it may
be methyl, ethyl or propyl (n-pentyl or isopropyl). In some of
these embodiments, it may be methyl. In other embodiments, it may
be butyl or pentyl, which may be linear or branched.
[1669] When R.sup.12 is C.sub.3-6 saturated cycloalkyl, it may be
cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some
embodiments, it may be cyclopropyl.
[1670] When R.sup.12 is
##STR00091##
each of R.sup.21, R.sup.22 and R.sup.23 are independently selected
from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3
alkynyl and cyclopropyl, where the total number of carbon atoms in
the R.sup.12 group is no more than 5. In some embodiments, the
total number of carbon atoms in the R.sup.12 group is no more than
4 or no more than 3.
[1671] In some embodiments, one of R.sup.21, R.sup.22 and R.sup.23
is H, with the other two groups being selected from H, C.sub.1-3
saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and
cyclopropyl.
[1672] In other embodiments, two of R.sup.21, R.sup.22 and R.sup.23
are H, with the other group being selected from H, C.sub.1-3
saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and
cyclopropyl.
[1673] In some embodiments, the groups that are not H are selected
from methyl and ethyl. In some of these embodiments, the groups
that re not H are methyl.
[1674] In some embodiments, R.sup.21 is H.
[1675] In some embodiments, R.sup.22 is H.
[1676] In some embodiments, R.sup.23 is H.
[1677] In some embodiments, R.sup.21 and R.sup.22 are H.
[1678] In some embodiments, R.sup.21 and R.sup.23 are H.
[1679] In some embodiments, R.sup.22 and R.sup.23 are H.
[1680] An R.sup.12 group of particular interest is:
##STR00092##
[1681] When R.sup.12 is
##STR00093##
one of R.sup.25a and R.sup.25b is H and the other is selected from:
phenyl, which phenyl is optionally substituted by a group selected
from halo, methyl, methoxy; pyridyl; and thiophenyl. In some
embodiments, the group which is not H is optionally substituted
phenyl. If the phenyl optional substituent is halo, it is
preferably fluoro. In some embodiment, the phenyl group is
unsubstituted.
[1682] When R.sup.12 is
##STR00094##
R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3
alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is
optionally substituted by a group selected from halo methyl,
methoxy; pyridyl; and thiophenyl. If the phenyl optional
substituent is halo, it is preferably fluoro. In some embodiment,
the phenyl group is unsubstituted.
[1683] In some embodiments, R.sup.24 is selected from H, methyl,
ethyl, ethenyl and ethynyl. In some of these embodiments, R.sup.24
is selected from H and methyl.
[1684] When there is a single bond present between C2' and C3',
[1685] R.sup.12 is
##STR00095##
where R.sup.26a and R.sup.26b are independently selected from H, F,
C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and
alkenyl groups are optionally substituted by a group selected from
C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of
R.sup.26a and R.sup.26b is H, the other is selected from nitrile
and a C.sub.1-4 alkyl ester.
[1686] In some embodiments, it is preferred that R.sup.26a and
R.sup.26b are both H.
[1687] In other embodiments, it is preferred that R.sup.26a and
R.sup.26b are both methyl.
[1688] In further embodiments, it is preferred that one of
R.sup.26a and R.sup.26b is H, and the other is selected from
C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and
alkenyl groups are optionally substituted. In these further
embodiment, it may be further preferred that the group which is not
H is selected from methyl and ethyl.
[1689] R.sup.2
[1690] The above preferences for R.sup.12 apply equally to
R.sup.2.
[1691] R.sup.22
[1692] In some embodiments, R.sup.22 is of formula Ila.
[1693] A in R.sup.22 when it is of formula Ila may be phenyl group
or a C.sub.5-7 heteroaryl group, for example furanyl, thiophenyl
and pyridyl. In some embodiments, A is preferably phenyl.
[1694] Q.sup.2-X may be on any of the available ring atoms of the
C.sub.5-7 aryl group, but is preferably on a ring atom that is not
adjacent the bond to the remainder of the compound, i.e. it is
preferably .beta. or .gamma. to the bond to the remainder of the
compound. Therefore, where the C.sub.5-7 aryl group (A) is phenyl,
the substituent (Q.sup.2-X) is preferably in the meta- or
para-positions, and more preferably is in the para-position.
[1695] In some embodiments, Q.sup.1 is a single bond. In these
embodiments, Q.sup.2 is selected from a single bond and
--Z--(CH.sub.2).sub.n--, where Z is selected from a single bond, O,
S and NH and is from 1 to 3. In some of these embodiments, Q.sup.2
is a single bond. In other embodiments, Q.sup.2 is
--Z--(CH.sub.2).sub.n--. In these embodiments, Z may be O or S and
n may be 1 or n may be 2. In other of these embodiments, Z may be a
single bond and n may be 1.
[1696] In other embodiments, Q.sup.1 is --CH.dbd.CH--.
[1697] In other embodiments, R.sup.22 is of formula Ilb. In these
embodiments, R.sup.C1, R.sup.C2 and R.sup.C3 are independently
selected from H and unsubstituted C.sub.1-2 alkyl. In some
preferred embodiments, R.sup.C1, R.sup.C2 and R.sup.C3 are all H.
In other embodiments, R.sup.C1, R.sup.C2 and R.sup.C3 are all
methyl. In certain embodiments, R.sup.C1, R.sup.C2 and R.sup.C3 are
independently selected from H and methyl.
[1698] X is a group selected from the list comprising:
O--R.sup.L2', S--R.sup.L2', CO.sub.2--R.sup.L2', CO--R.sup.L2',
NH--C(.dbd.O)--R.sup.L2', NHNH--R.sup.L2', CONHNH--R.sup.L2',
##STR00096##
NR.sup.NR.sup.L2', wherein R.sup.N is selected from the group
comprising H and C.sub.1-4 alkyl. X may preferably be: OH, SH,
CO.sub.2H, --N.dbd.C.dbd.O or NHR.sup.N, and may more preferably
be: O--R.sup.L2', S--R.sup.L2', CO.sub.2--R.sup.L2',
--NH--C(.dbd.O)--R.sup.L2' or NH--R.sup.L2'. Particularly preferred
groups include: O--R.sup.L2', S--R.sup.L2' and NH--R.sup.L2', with
NH--R.sup.L2' being the most preferred group.
[1699] In some embodiments R.sup.22 is of formula Ilc. In these
embodiments, it is preferred that Q is NR.sup.N--R.sup.L2'. In
other embodiments, Q is O--R.sup.L2'. In further embodiments, Q is
S--R.sup.L2'. R.sup.N is preferably selected from H and methyl. In
some embodiment, R.sup.N is H. In other embodiments, R.sup.N is
methyl.
[1700] In some embodiments, R.sup.22 may be -A-CH.sub.2--X and
-A-X. In these embodiments, X may be O--R.sup.L2', S--R.sup.L2',
CO.sub.2--R.sup.L2', CO--R.sup.L2' and NH--R.sup.L2'. In
particularly preferred embodiments, X may be NH--R.sup.L2'.
[1701] R.sup.10, R.sup.11
[1702] In some embodiments, R.sup.10 and R.sup.11 together form a
double bond between the nitrogen and carbon atoms to which they are
bound.
[1703] In some embodiments, R.sup.11 is OH.
[1704] In some embodiments, R.sup.11 is OMe.
[1705] In some embodiments, R.sup.11 is SO.sub.zM, where z is 2 or
3 and M is a monovalent pharmaceutically acceptable cation.
[1706] R.sup.11a
[1707] In some embodiments, R.sup.11a is OH.
[1708] In some embodiments, R.sup.11a is OMe.
[1709] In some embodiments, R.sup.11a is SO.sub.zM, where z is 2 or
3 and M is a monovalent pharmaceutically acceptable cation.
[1710] R.sup.20, R.sup.21
[1711] In some embodiments, R.sup.20 and R.sup.21 together form a
double bond between the nitrogen and carbon atoms to which they are
bound.
[1712] In some embodiments R.sup.20 is H.
[1713] In some embodiments, R.sup.20 is R.sup.C.
[1714] In some embodiments, R.sup.21 is OH.
[1715] In some embodiments, R.sup.21 is OMe.
[1716] In some embodiments, R.sup.21 is SO.sub.zM, where z is 2 or
3 and M is a monovalent pharmaceutically acceptable cation.
[1717] R.sup.30 , R.sup.31
[1718] In some embodiments, R.sup.30 and R.sup.31 together form a
double bond between the nitrogen and carbon atoms to which they are
bound.
[1719] In some embodiments, R.sup.31 is OH.
[1720] In some embodiments, R.sup.31 is OMe.
[1721] In some embodiments, R.sup.31 is SO.sub.zM, where z is 2 or
3 and M is a monovalent pharmaceutically acceptable cation.
[1722] M and z
[1723] It is preferred that M is a monovalent pharmaceutically
acceptable cation, and is more preferably Na.sup.+.
[1724] z is preferably 3.
[1725] Preferred conjugates of the first aspect of the present
disclosure may have a D.sup.L of formula Ia:
##STR00097##
[1726] where
[1727] R.sup.L1', R.sup.20 and R.sup.21 are as defined above;
[1728] n is 1 or 3;
[1729] R.sup.1a is methyl or phenyl; and
[1730] R.sup.2a is selected from:
##STR00098##
[1731] Preferred conjugates of the first aspect of the present
disclosure may have a D.sup.L of formula Ib:
##STR00099##
[1732] where
[1733] R.sup.L1', R.sup.20 and R.sup.21 are as defined above;
[1734] n is 1 or 3; and
[1735] R.sup.1a is methyl or phenyl.
[1736] Preferred conjugates of the first aspect of the present
disclosure may have a D.sup.L of formula Ic:
##STR00100##
[1737] where R.sup.L2', R.sup.10, R.sup.11, R.sup.30 and R.sup.31
are as defined above
[1738] n is 1 or 3;
[1739] R.sup.12a is selected from:
##STR00101##
[1740] the amino group is at either the meta or para positions of
the phenyl group.
[1741] Preferred conjugates of the first aspect of the present
disclosure may have a D.sup.L of formula Id:
##STR00102##
[1742] where R.sup.L2', R.sup.10, R.sup.11, R.sup.30 and R.sup.31
are as defined above;
[1743] n is 1 or 3;
[1744] R.sup.1a is methyl or phenyl;
[1745] R.sup.12a is selected from:
##STR00103##
[1746] Preferred conjugates of the first aspect of the present
disclosure may have a D.sup.L of formula Ie:
##STR00104##
[1747] where R.sup.L2', R.sup.10, R.sup.11, R.sup.30 and R.sup.31
are as defined above;
[1748] n is 1 or 3;
[1749] R.sup.1a is methyl or phenyl;
[1750] R.sup.12a is selected from:
##STR00105##
EXAMPLES
[1751] General Experimental Methods
[1752] Optical rotations were measured on an ADP 220 polarimeter
(Bellingham Stanley Ltd.) and concentrations (c) are given in g/100
mL. Melting points were measured using a digital melting point
apparatus (Electrothermal). IR spectra were recorded on a
Perkin-Elmer Spectrum 1000 FT IR Spectrometer. .sup.1H and .sup.13C
NMR spectra were acquired at 300 K using a Bruker Avance NMR
spectrometer at 400 and 100 MHz, respectively. Chemical shifts are
reported relative to TMS (.delta.=0.0 ppm), and signals are
designated as s (singlet), d (doublet), t (triplet), dt (double
triplet), dd (doublet of doublets), ddd (double doublet of
doublets) or m (multiplet), with coupling constants given in Hertz
(Hz). Mass spectroscopy (MS) data were collected using a Waters
Micromass ZQ instrument coupled to a Waters 2695 HPLC with a Waters
2996 PDA. Waters Micromass ZQ parameters used were: Capillary (kV),
3.38; Cone (V), 35; Extractor (V), 3.0; Source temperature
(.degree. C.), 100; Desolvation Temperature (.degree. C.), 200;
Cone flow rate (L/h), 50; De-solvation flow rate (L/h), 250.
High-resolution mass spectroscopy (HRMS) data were recorded on a
Waters Micromass QTOF Global in positive W-mode using metal-coated
borosilicate glass tips to introduce the samples into the
instrument. Thin Layer Chromatography (TLC) was performed on silica
gel aluminium plates (Merck 60, F.sub.254), and flash
chromatography utilised silica gel (Merck 60, 230-400 mesh ASTM).
Except for the HOBt (NovaBiochem) and solid-supported reagents
(Argonaut), all other chemicals and solvents were purchased from
Sigma-Aldrich and were used as supplied without further
purification. Anhydrous solvents were prepared by distillation
under a dry nitrogen atmosphere in the presence of an appropriate
drying agent, and were stored over 4 .ANG. molecular sieves or
sodium wire. Petroleum ether refers to the fraction boiling at
40-60.degree. C.
[1753] General LC/MS Conditions:
[1754] The HPLC (Waters Alliance 2695) was run using a mobile phase
of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid
0.1%). Gradient: initial composition 5% B held over 1.0 min, then
increase from 5% B to 95% B over a 3 min period. The composition
was held for 0.1 min at 95% B, then returned to 5% B in 0.03
minutes and hold there for 0.87 min. Total gradient run time equals
5 minutes.
[1755] Flow rate 3.0 mL/min, 400 .mu.L was split via a zero dead
volume tee piece which passes into the mass spectrometer.
Wavelength detection range: 220 to 400 nm. Function type: diode
array (535 scans). Column: Phenomenex Onyx Monolithic C18
50.times.4.60 mm.
[1756] The reverse phase flash purification conditions were as
follows: The Flash purification system (Varian 971-Fp) was run
using a mobile phase of water (A) and acetonitrile (B). Gradient:
initial composition 5% B over 20 C.V. (Column Volume) then 5% B to
70% B within 60 C.V. The composition was held for 15 C.V. at 95% B,
and then returned to 5% B in 5 C.V. and held at 5% B for 10 C.V.
Total gradient run time equals 120 C.V. Flow rate 6.0 mL/min.
Wavelength detection range: 254 nm. Column: Agilent AX1372-1
SF10-5.5gC8.
[1757] Preparative HPLC: Reverse-phase ultra-high-performance
liquid chromatography (UPLC) was carried out on Phenomenex Gemini
NX 5p C-18 columns of the following dimensions: 150.times.4.6 mm
for analysis, and 150.times.21.20 mm for preparative work. All UPLC
experiments were performed with gradient conditions. Eluents used
were solvent A (H.sub.2O with 0.1% Formic acid) and solvent B
(CH.sub.3CN with 0.1% Formic acid). Flow rates used were 1.0 ml/min
for analytical, and 20.0 ml/min for preparative HPLC. Detection was
at 254 and 280 nm.
Example 1
Formation of Conjugates
[1758] Conjugation of AbHJ, AbDJ, AbBJ
[1759] Antibodies AbHJ, AbDJ, AbBJ were prepared for reduction in a
buffer containing 1 mM EDTA in PBS pH 7.4 at an antibody
concentration of 1-10 mg/mL. TCEP reductant was added to the batch
as a 50-fold molar excess with respect to the antibody and the
reduction mixture was heated at +37.degree. C. for 3 hours in an
incubator with slow orbital shaking. After confirming by RP-HPLC
that reduction was complete, the antibody was cooled down to room
temperature and buffer exchanged into PBS buffer containing 1 mM
EDTA to remove excess TCEP. Reduced antibody was reoxidised by the
addition of 50 mM dehydroascorbic acid (DHAA) as a 50 fold molar
excess with respect to antibody, and the reoxidation mixture is
allowed to proceed for a total of 2 hours with HPLC monitoring,
then sterile filtered to remove DHAA. Conjugation was initiated by
the addition of 10 mM drug linker stock diluted into DMSO (to a
final 10% v/v concentration) and 10 fold excess relative to the
antibody. The conjugation reaction was incubated for 16 hours at
room temperature. Post conjugation the reaction was quenched with a
10 fold molar excess of N-acetyl cysteine and incubated for an
additional 30 mins. The final product was exchanged into
formulation buffer (30 mM Histidine, 200 mM sorbitol, 0.02%
Tween-20) and analysed by SEC, HIC, RP-HPLC.
[1760] Conjugation of AbLJ
[1761] Initial attempts to conjugate AbLJ directly or following
complete reduction/re-oxidation results in a complete lack of
conjugation confirming that the unpaired heavy chain Cys were
disulphide bridged together and would re-oxidise at the same rate
as the heavy-heavy disulphide bonds. A site specific reduction
process based on literature precedent (mAbs 1:6, 563-571;
November/December, 2009) was attempted both in solution and on a
resin. Both approaches were successful but the solid phase approach
had certain practical advantages: [1762] Avoided the need for
process optimization to increase protein concentration during
reduction--to maintain concentration during subsequent steps [1763]
Results in concentration not dilution of the reduced antibody
[1764] Ensures excellent toxin linker removal which can require
multiple passes down G25 or TFF for a solution based process
[1765] It is expected that many resins will be capable of
supporting this process with the requirement for the resin being:
[1766] Ability to capture the educed antibody from the reduction
process [1767] Lack of affinity/binding of Cys [1768] No blocking
of the target free thiol
[1769] An example of a resin likely to work for this is Protein
A.
[1770] Solid Phase
[1771] AbLJ (25.5 mg, 5.1 mg/mL in PBS) was conjugated with
Compound E in a multi-step process. In the first step the AbLJ
antibody was buffer exchanged into 20mM HEPES pH 8.0 via G25 column
chromatography (NAP25, GE Healthcare) and diluted to 1 mg/mL.
Cysteine was then added to 5 mM final concentration from a freshly
prepared stock of 500 mM in deionised water. The site specific
reduction process was allowed to proceed for 90 minutes at
37.degree. C. The reduced AbLJ was then captured on a 2 mL column
of protein L mimetic resin to achieve fast and complete removal of
the reductant (FabSorbent F1 P HF, Prometic biosciences Ltd). The
column was immediately washed with 20 column volumes of phosphate
buffered saline (PBS) and then with PBS containing 5% v/v of
dimethylacetamide (DMA). The resin was suspended in 10 mL of PBS,
5% v/v DMA containing Compound E at 5 fold molar excess over
antibody and allowed to conjugate for 60 minutes at room
temperature. The column was then washed with 20 column volumes of
PBS containing 5% v/v of dimethylacetamide (DMA) and then 20 column
volumes of phosphate buffered saline (PBS). The purified conjugate
was then eluted from the resin with 0.1 M Glycine pH 3.0 and
immediately buffer exchanged into 30 mM Histidine, 200 mM sorbitol
pH 6 via G25 column chromatography (HiTrap G25, GE Healthcare).
Polysorbate 20 was then added to 0.01% w/v from a freshly prepared
stock of 1% w/v Polysorbate 20 in deionised water. The formulated
conjugate was then subjected to a sterilizing grade filtration via
a 0.22 pm, polyethersulfone membrane (Steriflip, EMD
Millipore).
[1772] The AbLJ-ConjE ADC was analysed by hydrophobic interaction
chromatography (HIC) to determine the amount of DAR2 relative to
unwanted DAR<2 and DAR>2 species. The percentage of on-target
heavy-chain conjugation was determined by RP-HPLC and monomer
content be size exclusion chromatography.
[1773] Solution Phase
[1774] AbLJ (25.5 mg, 5.1 mg/mL in PBS) was conjugated with
Compound E in a multi-step process. In the first step the AbLJ
antibody was buffer exchanged into 20 mM HEPES pH 8.0 via G25
column chromatography (NAP25, GE Healthcare) and diluted to 1
mg/mL. Cysteine was then added to 5 mM final concentration from a
freshly prepared stock of 500 mM in deionised water. The site
specific reduction process was allowed to proceed for 90 minutes at
37.degree. C. The reduced AbLJ was then buffer exchanged into PBS,
5% v/v DMA via G25 column chromatography (NAP25, GE Healthcare) and
Compound E added at a 5 fold molar excess over antibody and allowed
to conjugate for 60 minutes at room temperature. The conjugate was
then buffer exchanged into 30 mM Histidine, 200 mM sorbitol pH 6
via G25 column chromatography (HiTrap G25, GE Healthcare).
Polysorbate 20 was then added to 0.01% w/v from a freshly prepared
stock of 1% w/v Polysorbate 20 in deionised water. The formulated
conjugate was then subjected to a sterilizing grade filtration via
a 0.22 .mu.m, polyethersulfone membrane (Steriflip, EMD
Millipore).
[1775] The AbLJ-ConjE ADC was analysed by hydrophobic interaction
chromatography (HIC) to determine the amount of DAR2 relative to
unwanted DAR<2 and DAR>2 species. The percentage of on-target
heavy-chain conjugation was determined by RP-HPLC and monomer
content be size exclusion chromatography.
[1776] Conjugation #2 of AbLJ
[1777] AbLJ-ConjE
[1778] 4 mL (approx. 5 mg/mL) AbLJ in PBS is buffer exchanged into
20 mM Tris/Cl, 1 M Lysine, 5 mM EDTA pH 8.0 using a G25 fine
desalting column (GE Healthcare HiPrep 26/10).
[1779] The antibody was diluted to 1 mg/mL (approx. 20 mL volume)
based on UV absorbance and reduction initiated by the addition of
N-acetyl cysteine (500 mM NAC in water, Sigma A7250) to 5 mM final
concentration. The reduction process was allowed to proceed for 75
minutes. The reduction process is stopped by removal of the NAC by
binding the reduced protein in batch mode to a protein A mimetic
resin.
[1780] 2 mL Fabsorbent.TM. F1P HF (Prometics Biosciences) was
pre-equilibrated with phosphate buffered saline, filtered to remove
the PBS and then suspended in the reduced antibody solution and
mixed gently on a roller for 15 minutes. The resin is washed 5
times with 10 mL of 20 mM Tris/Cl, 5 mM EDTA. The washed resin was
then suspended in 10 mL volumes of 20 mM Tris/Cl, 5 mM EDTA, 5% v/v
Dimethylacetamide (DMA). Compoud E was added to 5 equivalents
relative to total antibody from a 10 mM stock solution in DMA. This
conjugation reaction was mixed gently on a roller for 60 minutes.
The resin bound conjugate was then washed sequentially with
3.times.10mL of PBS/5% v/v DMA followed by 3.times.10 mL of
PBS.
[1781] The conjugate was released from the resin by suspending the
resin in 10 mL of 0.1 M Glycine pH 3.0 for 5 minutes and the
conjugate containing supernatant collected by filtering off the
resin The elution process was repeated and the two elution
fractions combined and immediately formulated by buffer exchange
into 30 M Histidine/Cl, 200 mM sorbitol pH 6.0 using a G25 fine
desalting column (GE Healthcare PD10 or HiPrep 26/10). Polysorbate
20 was then added to 0.02% w/v from a 10% w/v stock solution in
water.
[1782] The final formulated conjugate was 0.2um filtered
(Steriflip-GP PES filtration unit, Merck Millipore).
[1783] Site-specific conjugation to the heavy chain and average DAR
are determined by RP-HPLC (PLRP) and monomer content by size
exclusion chromatography as described above. The final conjugate
haad an average DAR of 1.8 and a monomer/HMW content of 95.2 and
1.6% respectively.
[1784] Conjugation of AbLJ(LALA)
[1785] AbLJ(LALA)-ConjE
[1786] The AbLJ(LALA) antibody was conjugated to Compound E exactly
as described above for Conjugation #2 of AbLJ.
[1787] The final conjugate had an average DAR of 1.8 and a
monomer/HMW content of 95% and 1.8% respectively.
[1788] DAR Determination
[1789] Antibody or ADC (ca. 35 .mu.g in 35 .mu.L) was reduced by
addition of 10 .mu.L borate buffer (100 mM, pH 8.4) and 5 .mu.L DTT
(0.5 M in water), and heated at 37.degree. C. for 15 minutes. The
sample was diluted with 1 volume of acetonitrile:water:formic acid
(49%:49%:2% v/v), and injected onto a Widepore 3.6.mu. XB-C18
150.times.2.1 mm (P/N 00F-4482-AN) column (Phenomenex Aeris) at
80.degree. C., in a UPLC system (Shimadzu Nexera) with a flow rate
of 1 ml/min equilibrated in 75% Buffer A (Water, Trifluoroacetic
acid (0.1% v/v) (TFA), 25% buffer B (Acetonitrile:water:TFA
90%:10%:0.1% v/v). Bound material was eluted using a gradient from
25% to 55% buffer B in 10 min. Peaks of UV absorption at 214 nm
were integrated. The following peaks were identified for each ADC
or antibody: native antibody light chain (L0), native antibody
heavy chain (HO), and each of these chains with added drug-linkers
(labelled L1 for light chain with one drug and H1, H2, H3 for heavy
chain with 1, 2 or 3 attached drug-linkers). The UV chromatogram at
330 nm was used for identification of fragments containing
drug-linkers (i.e., L1, H1, H2, H3).
[1790] A PBD/protein molar ratio was calculated for both light
chains and heavy chains:
Drug Protein ratio on light chain = % Area at 214 nm for L 1 % Area
at 214 nm for L 0 and L 1 ##EQU00001## Drug Protein ratio on heavy
chain = n = 0 3 n .times. ( % area at 214 for Hn ) n = 0 3 % area
at 214 for Hn ##EQU00001.2##
[1791] Final DAR is calculated as:
DAR - 2 .times. ( Drug Protein ratio on light chain + Drug Protein
ratio on heavy chain ) ##EQU00002##
[1792] DAR measurement is carried out at 214 nm because it
minimises interference from drug-linker absorbance.
TABLE-US-00003 AbHJ- AbDJ- AbBJ- AbLJ- Test ConjE ConjE ConjE ConjE
Visual Clear, Clear, Clear, 0.63 colourless, colourless,
colourless, particulate particulate particulate free free free C
(by A280/330 0.77* 1.0* Nd* Nd nm)in mg/ml* C (SEC 214 nm) 0.88*
Nd* 1.18* 1.8 in mg/mL* DAR by HIC 1.5 1.9 1.7 1.8 DAR by PLRP 1.5
1.9 1.8 100% SEC (% 99.4% 98.1% 95.6% Nd monomer) Free drug-
<LOD <LOD <LOD Nd linker DMA DMA DMA DMA 0.63 not used not
used not used *Two concentration methods were used: SEC (214 nm) vs
known concentration reference sample or A280/A330 as described in
patent. When data was available concentration was recalculated
using this formula.
Example 2
In Vitro Cytotoxicity of Conjugates
[1793] Cytotoxicity Assay
[1794] The concentration and viability of cultures of suspended
cells (at up to 1.times.10.sup.6/ml) were determined by mixing 1:1
with Trypan blue and counting clear (live)/blue (dead) cells with a
haemocytometer. The cell suspension was diluted to the required
seeding density (generally 10.sup.5/ml) and dispensed into 96-well
flat bottomed plates. For Alamar blue assay, 100 .mu.l/well was
dispensed in black-well plates. For MTS assay, 50 .mu.l/well was
dispensed in clear-well plates. A stock solution (1 ml) of ADC (20
.mu.g/ml) was made by dilution of filter-sterile ADC into cell
culture medium. A set of 8.times.10-fold dilutions of stock ADC
were made in a 24 well plate by serial transfer of 100 .mu.l onto
900 .mu.l of cell culture medium. Each ADC dilution (100 pl/well
for Alamar blue, 50 .mu.l/well for MTS) was dispensed into 4
replicate wells of the 96-well plate, containing cell suspension.
Control wells received the same volume of culture medium only.
After incubation for 4 days, cell viability was measured by either
Alamar blue or MTS assay.
[1795] AlamarBlue.RTM. (Invitrogen, catalogue number DAL1025) was
dispensed (20 .mu.l per well) into each well and incubated for 4
hours at 37.degree. C. in the CO.sub.2-gassed incubator. Well
fluorescence was measured at excitation 570 nm, emission 585 nm.
Cell survival (%) was calculated from the ratio of mean
fluorescence in the 4 ADC-treated wells compared to the mean
fluorescence in the 4 control wells (100%).
[1796] MTS (Promega, catalogue number G5421) was dispensed (20
.mu.l per well) into each well and incubated for 4 hours at
37.degree. C. in the CO.sub.2-gassed incubator. Absorbance was
measured at 490 nm. Cell survival (%) was calculated from the mean
absorbance in the 4 ADC-treated wells compared to the mean
absorbance in the 4 control wells (100%). Dose response curves were
generated from the mean data of 3 replicate experiments and the
EC.sub.50 was determined by fitting data to a sigmoidal
dose-response curve with variable slope using Prism (GraphPad, San
Diego, Calif.).
[1797] Results
[1798] In order to produce site-specific versions of the ADCs,
engineered versions of the AbJ antibody was conjugated the PBD
warhead linker ConjE. The engineered AbJ antibodies were
transiently produced in CHO cells. The in vitro cytotoxic efficacy
of the site-specific ADCs were compared to wild-type AbJ-ADC
conjugate (AbJ-ConjE).
[1799] AbJ.fwdarw. [1800] An antibody comprising: [1801] a heavy
chain comprising the amino acid sequence of SEQ ID NO.110; [1802] a
light chain comprising the amino acid sequence of SEQ ID NO.150;
[1803] a VH domain; and [1804] a VL domain.
[1805] AbJ-ConjE.fwdarw.AbJ stochastically conjugated to Compound
E
[1806] AbHJ-ConjE.fwdarw. [1807] An antibody comprising: [1808] a
heavy chain comprising the amino acid sequence of SEQ ID NO.111;
[1809] a light chain comprising the amino acid sequence of SEQ ID
NO.150; [1810] a VH domain; and [1811] a VL domain; [1812]
conjugated to Compound E at C105 of SEQ ID NO.150.
[1813] AbDJ-ConjE.fwdarw. [1814] An antibody comprising: [1815] a
heavy chain comprising the amino acid sequence of SEQ ID NO.115;
[1816] a light chain comprising the amino acid sequence of SEQ ID
NO.150; [1817] a VH domain; and [1818] a VL domain; [1819]
conjugated to Compound E at C105 of SEQ ID NO.150.
[1820] AbBJ-ConjE.fwdarw. [1821] An antibody comprising: [1822] a
heavy chain comprising the amino acid sequence of SEQ ID NO.113;
[1823] a light chain comprising the amino acid sequence of SEQ ID
NO.151; [1824] a VH domain; and [1825] a VL domain; [1826]
conjugated to Compound Eat C103 of SEQ ID NO.113.
[1827] AbLJ-ConjE.fwdarw. [1828] An antibody comprising: [1829] a
heavy chain comprising the amino acid sequence of SEQ ID NO.110;
[1830] a light chain comprising the amino acid sequence of SEQ ID
NO.151; [1831] a VH domain; and [1832] a VL domain; [1833]
conjugated to Compound Eat C103 of SEQ ID NO.110.
TABLE-US-00004 [1833] Binding EC50 Cytotoxicity IC50 ADC candidate
(ng/ml) (ng/ml) AbJ 59 -- AbJ-ConjE 44 56 AbHJ-ConjE 55 18
AbDJ-ConjE 44 12 AbBJ-ConjE 49 23
[1834] No significant differences were reported in the EC50 values
when the site-specific AbJ conjugates were compared to the
corresponding wild-type conjugates.
Example 3
In Vivo Efficacy of Site-Specific and Non-Site Specific
Conjugates
[1835] 8 to 12 weeks old male CB.17 SCID mice were implanted with
1.times.10.sup.7 tumor cells in 50% Matrigel s.c. in flank. On Day
1 of the study, mice bearing established xenografts (average size
of 100-150 mm.sup.3) were sorted into treatment groups (n=10), and
dosing was initiated at either 0.33 mg/kg or 1.0 mg/kg. Tumors were
measured twice per week until the study was ended.
[1836] Results
[1837] The various ADCs were tested in the xenograft model. At 0.3
mg/kg qd.times.1, AbHJ-ConjE and AbBJ-ConjE were equally
efficacious providing tumor stasis for 30 days. AbDJ-ConjE was
slightly more efficacious providing tumor stasis for up to 35 days.
At 1.0 mg/kg qd.times.1, AbBJ-ConjE, AbHJ-ConjE and AbDJ-ConjE
provided tumor stasis for 55, 70 and >95 days.
Example 4
Plasma/Serum Sstability of Site-Specific and Non-Site Specific
Conjugates
[1838] Stochastically conjugated ADCs (AbJ) and site-specifically
conjugated ADCs ADCs were spiked in cyno or human plasma or PBS at
a concentration of 60 ug/ml and incubated at 37.degree. C. for 24
h, one and three weeks.
[1839] After one week samples were harvested and in vitro
cytoxicity of the ADCs was determined. ADC instability would result
in a loss of potenty on the cells due to release of warhead from
the ADC.
[1840] Gl.sub.50 data were generated by least squares fitting
OD.sub.490 data derived from the CellTiter 96.RTM. AQueous One
Solution Cell Proliferation Assay (MTS) to a sigmoidal, 4PL X is
log(concentration) algorithm using Graph Pad Prism v6.03. Cells
were cultured for 6 days with the ADC-plasma mix, before MTS assay
as described in the application.
TABLE-US-00005 GI.sub.50 (ng/ml) in cells Days at 37.degree. C.
before storage at -80.degree. C. Unfrozen until assay control 0 1 7
21 Human plasma stability AbJ-ConjE 16.8 65.0 95.9 62.4 480.9
AbBJ-ConjE 12.8 22.5 18.1 48.0 287.1 AbHJ-ConjE 11.3 9.0 10.7 39.5
234.8 AbDJ-ConjE 7.1 7.2 7.6 20.2 258.2 Cynomolgus monkey plasma
stability AbJ-ConjE 16.8 26.2 32.1 74.4 111.8 AbBJ-ConjE 12.8 14.0
19.6 56.7 74.4 AbHJ-ConjE 11.3 9.8 13.3 24.3 44.4 AbDJ-ConjE 7.1
7.6 8.7 13.0 48.2
[1841] AbBJ-ConjE, AbDJ-ConjE and AbHJ-ConjE showed improved
stability when compared to the stochastic conjugate AbJ-ConjE in
human and cynomolgos plasma upon 1, 7 or 21 days incubation at
37.degree. C.
Example 5
Tolerability of Different Site-Specific Conjugates
[1842] The effect of the mutation of the residues at Kabat EU
positions 234 and 235 on the tolerability of the ADCs to rats was
investigated.
[1843] Single dose studies were performed in male sprague-dawley
rats, with necropsy on day 21 following dosing. Bodyweights and
food consumption were monitored frequently with in-life sampling
for clinical pathology (blood on days 8 and 21) and repeated
sampling for pharmacokinetics. At necropsy, macroscopic
observations were taken with selected organs weighed and retained
for possible histopathology.
[1844] Results
[1845] AbLJ-ConjE.fwdarw. [1846] An antibody comprising: [1847] a
heavy chain comprising the amino acid sequence of SEQ ID NO.1103;
[1848] a light chain comprising the amino acid sequence of SEQ ID
NO.151; [1849] a VH domain; and [1850] a VL domain; [1851]
conjugated to Compound Eat C103 of SEQ ID NO.1103.
[1852] AbLJ(LALA)-ConjE.fwdarw. [1853] An antibody comprising:
[1854] a heavy chain comprising the amino acid sequence of SEQ ID
NO.1103; [1855] a light chain comprising the amino acid sequence of
SEQ ID NO.151; [1856] a VH domain; and [1857] a VL domain; [1858]
conjugated to Compound Eat C103 of SEQ ID NO.1103.
[1859] The VH and VL domains present in the AbLJ-ConjE conjugate
were identical to those present in the AbLJ(LALA)-ConjE
conjugate.
TABLE-US-00006 Rat toxicology study AbLJ-ConjE AbLJ(LALA)-ConjE
observations.sup.1 (2 mg/kg) (2 mg/kg) Clinical observations
Moderate raised hair/ Mild raised hair/ hunched posture & pale
hunched posture extremities Bodyweight gain.sup.2 -78% -45%
Haematology.sup.3 Reticulocytes -93% -56% Platelets -72% -60%
Neutrophils -98% -97% Anemia Minimal Minimal Organ weights.sup.4
Liver -23% -12% Lung +16% +16% Thymus -81% -73% Spleen -41% -33%
Kidney -27% -17% Testis -23% -19% .sup.121 day study, single dose
on day 1 (male SD rats) .sup.2associated with reduced food intake
.sup.3nadir on day 8, trending towards recovery by day 21
.sup.4absolute organ weights
[1860] The results indicate that mutation of the residues at Kabat
EU positions 234 and 235 substantially improves ADC
tolerability.
Example 6
Pharmacokinetics of Different Site-Specific Conjugates
[1861] The effect of the mutation of the residues at Kabat EU
positions 234 and 235 on the pharmacokinetics was investigated.
AbLJ-ConjE and AbLJ(LALA)-ConjE as described above in Example 5
were used.
[1862] Rats were dosed with 2 mg/kg of ADC and serum samples were
taken frequently until day 20. A a fit-for-purpose ELISA was
developed for measuring conjugated antibody. Calibration curve, QCs
and study samples were diluted in a low adhesion plate and added to
a plate coated with a mouse monoclonal antibody directed against
anti-SG3249 . After incubation and washing, the plate was incubated
with a mouse monoclonal antibody to human Fc-HRP conjugated.
[1863] As substrate, 3,3',5,5'-Tetramethylbenzidine (TMB) was used,
the reaction stopped with 1 M HCl and the plate read at 450 nm
absorbance at a Versamax plate reader. The Lower Limit Of
Quantification (LLOQ) was 750 ng/ml in rat serum. All samples were
measured using the PBD-ADC specific assay and the measured terminal
half-lifes (mean of three animals) for AbLJ(LALA)-ConjE and
AbLJ-ConjE were calculated using Phoenix 64 WinNonlin 6.4
(Pharsight) software.
[1864] Results
TABLE-US-00007 Terminal Half life ADC (h) AbLJ(LALA)-ConjE 306.3
AbLJ-ConjE 200.1
[1865] The results indicate that mutation of the residues at Kabat
EU positions 234 and 235 substantially improves ADC terminal
half-life.
Example 7
Reduced Systemic Toxicity
[1866] AbCJ specific for human antigen X, was engineered to contain
a cysteine instead of a serine at position 442 (designated as
AbCJX) and conjugated to drug-linkers ConjH and ConjE.
[1867] The toxicity of AbCJX-ConjH and AbCJX-ConjE in cynomolgos
monkey was compared to that of AbBJX-ConjE (the AbBJ-ConjE antibody
described above in Example 2, specific for human antigen X).
[1868] The study used three cynomolgus monkeys per group (males or
females), the monkeys being approximately 3 years old (4 kg) at
dosing. All animals were dosed once on day 1, with data presented
up to day 22 for surviving animals.
[1869] Results
[1870] Due to adverse clinical signs, including bleeding associated
with marked platelet depletion, animals were either found dead or
euthanised early with AbCJX-ConjH (by day 13) and with AbCJX-ConjE
(by day 16); see FIG. 1. AbBJX-ConjE did not induce significant
platelet depletion and monkeys received a second dose at day
21.
Example 8
##STR00106## ##STR00107##
[1871] (a)
(S)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-(4-methylpiperazin-1
-yl)phenyl)-5,11
-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-py-
rrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5,11-dioxo-10-((2-(trimet-
hylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1
H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl trifluoromethanesulfonate
(82)
[1872] Pd(PPh.sub.3).sub.4 (20.6 mg, 0.018 mmol) was added to a
stirred mixture of the bis-enol triflate 12 (500 mg, 0.44
mmol)(Compound 8a in WO 2010/043880), N-methyl piperazine boronic
ester (100 mg, 0.4 mmol), Na.sub.2CO.sub.3 (218 mg, 2.05 mmol),
MeOH (2.5 mL), toluene (5 mL) and water (2.5 mL). The reaction
mixture was allowed to stir at 30.degree. C. under a nitrogen
atmosphere for 24 hours after which time all the boronic ester has
consumed. The reaction mixture was then evaporated to dryness
before the residue was taken up in EtOAc (100 mL) and washed with
H.sub.2O (2.times.50 mL), brine (50 mL), dried (MgSO.sub.4),
filtered and evaporated under reduced pressure to provide the crude
product. Purification by flash chromatography (gradient elution:
80:20 v/v Hexane/EtOAc to 60:40 v/v Hexane/EtOAc) afforded product
82 as a yellowish foam (122.6 mg, 25%).
[1873] LC/MS 3.15 min (ES+) m/z (relative intensity) 1144
([M.times.H].sup.+, 20%).
(b) (9H-fluoren-9-yl)methyl
((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-(4-methylpipe-
razin-1-yl)phenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,1-
1,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5,1-
1-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-py-
rrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)--
3-methyl-1-oxobutan-2-yl)carbamate (83)
[1874] PBD-triflate 82 (359 mg, 0.314 mmol), boronic pinacol ester
20 (250 mg, 0.408 mmol) (Compound 20 in WO 2014/057073) and
triethylamine (0.35 mL, 2.51 mmol) were dissolved in a mixture of
toluene/MeOH/H.sub.2O, 2:1:1 (3 mL). The microwave vessel was
purged and filled with argon three times before
tetrakis(triphenylphosphine)palladium(0) (21.7 mg, 0.018 mmol) was
added and the reaction mixture placed in the microwave at
80.degree. C. for 10 minutes. Subsequently, CH.sub.2Cl.sub.2 (100
mL) was added and the organics were washed with water (2.times.50
mL) and brine (50 mL) before being dried with MgSO.sub.4, filtered
and the volatiles removed by rotary evaporation under reduced
pressure. The crude product was purified by silica gel
chromatography column (CHCl.sub.3/MeOH, 100% to 9:1) to afford pure
83 (200 mg, 43% yield).
[1875] LC/MS 3.27 min (ES+) m/z (relative intensity) 1478
([M+H].sup.+, 100%).
(c) (9H-fluoren-9-yl)methyl
((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-(4-methylpipe-
razin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-
-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-
-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carba-
mate (84)
[1876] A solution of Super-Hydride.RTM. (0.34 mL, 1M in THF) was
added dropwise to a solution of SEM-dilactam 83 (200 mg, 0.135
mmol) in THF (5 mL) at -78.degree. C. under an argon atmosphere.
The addition was completed over 5 minutes in order to maintain the
internal temperature of the reaction mixture constant. After 20
minutes, an aliquot was quenched with water for LC/MS analysis,
which revealed that the reaction was complete. Water (20 mL) was
added to the reaction mixture and the cold bath was removed. The
organic layer was extracted with EtOAc (3.times.30 mL) and the
combined organics were washed with brine (50 mL), dried with
MgSO.sub.4, filtered and the solvent removed by rotary evaporation
under reduced pressure. The crude product was dissolved in MeOH (6
mL), CH.sub.2Cl.sub.2 (3 mL), water (1 mL) and enough silica gel to
form a thick stirring suspension. After 5 days, the suspension was
filtered through a sintered funnel and washed with
CH.sub.2Cl.sub.2/MeOH (9:1) (100 mL) until the elution of the
product was complete. The organic layer was washed with brine
(2.times.50 mL), dried with MgSO.sub.4, filtered and the solvent
removed by rotary evaporation under reduced pressure. Purification
by silica gel column chromatography (100% CHCl.sub.3 to 96%
CHCl.sub.3/4% MeOH) afforded the product 84 as a yellow solid (100
mg, 63%). LC/MS 2.67 min (ES+) m/z (relative intensity) 1186
([M+H].sup.+, 5%).
(d)
(S)-2-amino-N-((S)-1-((4-((R)-7-methoxy-8-(3-(((R)-7-methoxy-2-(4-(4-m-
ethylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benz-
odiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benz-
odiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide
(85)
[1877] Excess piperidine was added (0.1 mL, 1 mmol) to a solution
of PBD 84 (36.4 mg, 0.03 mmol) in DMF (0.9 mL). The mixture was
allowed to stir at room temperature for 20 min, at which point the
reaction had gone to completion (as monitored by LC/MS). The
reaction mixture was diluted with CH.sub.2Cl.sub.2 (50 mL) and the
organic phase was washed with H.sub.2O (3.times.50 mL) until
complete piperidine removal. The organic phase was dried over
MgSO.sub.4, filtered and excess solvent removed by rotary
evaporation under reduced pressure to afford crude product 85 which
was used as such in the next step. LC/MS 2.20 min (ES+) m/z
(relative intensity) 964 ([M+H].sup.+, 5%).
(e)
1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-((2S)-1-(((2-
S)-1-((4-(7-methoxy-8-(3-((7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)--
5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propox-
y)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)-
amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21-
,24-octaoxaheptacosan-27-amide (86)
[1878] EDCI hydrochloride (8 mg, 0.042 mmol) was added to a
suspension of Maleimide-PEG.sub.8-acid (25 mg, 0.042 mmol) in dry
CH.sub.2Cl.sub.2 (4 mL) under argon atmosphere. PBD 85 (42 mg,
crude) was added straight away and stirring was maintained until
the reaction was complete (3 hours). The reaction was diluted with
CH.sub.2Cl.sub.2 and the organic phase was washed with H.sub.2O and
brine before being dried over MgSO.sub.4, filtered and excess
solvent removed by rotary evaporation under reduced pressure by
rotary evaporation under reduced pressure. The product was purified
by careful silica gel chromatography (slow elution starting with
100% CHCl.sub.3 up to 9:1 CHCl.sub.3/MeOH) followed by reverse
phase HPLC to remove unreacted maleimide-PEG.sub.8-acid. The
product 86 was isolated in 10% over two steps (6.6 mg). LC/MS 1.16
min (ES+) m/z (relative intensity) 770.20 ([M+2H].sup.+, 40%).
Example 9
Alternative Synthesis of Compound 83
##STR00108##
[1879] (9H-fluoren-9-yl)methyl
((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-(4-methylpipe-
razin-1-yl)phenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,1-
1,11a-tetrahydro-1 H-pyrrolo[2,1
-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5,11-dioxo-10-((2-(trimethylsilyl-
)ethoxy)methyl)-5,10,11,11a-tetrahydro-1 H-pyrrolo[2,1
-c][,4]benzodiazepin-2-yl)phenyl)amino)-1
-oxopropan-2-yl)amino)-3-methyl-1 -oxobutan-2-yl)carbamate (83)
[1880] PBD-triflate 21 (469 mg, 0.323 mmol)(Compound 21 in WO
2014/057073), boronic pinacol ester (146.5 mg, 0.484 mmol) and
Na.sub.2CO.sub.3 (157 mg, 1.48 mmol) were dissolved in a mixture of
toluene/MeOH/H.sub.2O, 2:1:1 (10 mL). The reaction flask was purged
with argon three times before
tetrakis(triphenylphosphine)palladium(0) (7.41 mg, 0.0064 mmol) was
added and the reaction mixture heated to 30.degree. C. overnight.
The solvents were removed under reduced pressure and the residue
was taken up in H.sub.2O (50 mL) and extracted with EtOAc
(3.times.50 mL). The combined organics were washed with brine (100
mL), dried with MgSO.sub.4, filtered and the volatiles removed by
rotary evaporation under reduced pressure. The crude product was
purified by silica gel column chromatography (CHCl.sub.3 100% to
CHCl.sub.3/MeOH 95%:5%) to afford pure 83 in 33% yield (885 mg).
LC/MS 3.27 min (ES+) m/z (relative intensity) 1478 ([M+H].sup.+,
100%).
Example 10
##STR00109## ##STR00110##
[1881] (a)
(S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1
-yl)phenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a--
tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-5,1-
1-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,
10,11,11a-tetrahydro-1 H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl
trifluoromethanesulfonate (88)
[1882] Pd(PPh.sub.3).sub.4 (30 mg, 26 pmol) was added to a stirred
mixture of the bis-enol triflate 87 (1 g, 0.87 mmol)(Compound 8b in
WO 2010/043880), 4-(4-methylpiperazin-1-yl)phenylboronic acid,
pinacol ester (264 mg, 0.87 mmol), Na.sub.2CO.sub.3 (138 mg, 1.30
mmol), EtOH (5 mL), toluene (10 mL) and water (5 mL). The reaction
mixture was allowed to stir under a nitrogen atmosphere overnight
at room temperature after which time the complete consumption of
starting material was observed by TLC (EtOAc) and LC/MS (1.52 min
(ES+) m/z (relative intensity) 1171.40 ([M +H].sup.+, 100)). The
reaction mixture was diluted with EtOAc (400 mL) and washed with
H.sub.2O (2.times.300 mL), brine (200 mL), dried (MgSO.sub.4),
filtered and evaporated under reduced pressure to provide the crude
product. Purification by flash chromatography (gradient elution:
100:0 v/v EtOAc/MeOH to 85:15 v/v EtOAc/MeOH) afforded the
asymmetrical triflate 88 (285 mg, 28%). .sup.1H NMR (400 MHz,
CDCl3) .delta. 7.39 (s, 1H), 7.37-7.29 (m, 4H), 7.23 (d, J=2.8 Hz,
2H), 7.14 (t, J=2.0 Hz, 1H), 6.89 (d, J=9.0 Hz, 2H), 5.54 (d,
J=10.0 Hz, 2H), 4.71 (dd, J=10.0, 2.6 Hz, 2H), 4.62 (td, J =10.7,
3.5 Hz, 2H), 4.13-4.01 (m, 4H), 3.97-3.87 (m, 8H), 3.85-3.75 (m,
2H), 3.74-3.63 (m, 2H), 3.31-3.22 (m, 4H), 3.14 (tdd, J=16.2, 10.8,
2.2 Hz, 2H), 2.73-2.56 (m, 4H), 2.38 (d, J=2.4 Hz, 3H), 2.02-1.92
(m, 4H), 1.73 (dd, J=9.4, 6.0 Hz, 2H), 1.04-0.90 (m, 4H),
0.05--0.00 (m, 18H). MS (ES.sup.+) m/z (relative intensity) 1171.40
([M+H].sup.+, 100).
(b) (9H-fluoren-9-yl)methyl
((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-(4-methylpip-
erazin-1-yl)phenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,-
11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)o-
xy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydr-
o-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2--
yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (89)
[1883] Pd(PPh.sub.3).sub.4 (8 mg, 7 pmol) was added to a stirred
mixture of the asymmetrical triflate 88 (269 mg, 0.23 mmol),
Fmoc-Val-Ala-4-aminophenylboronic acid, pinacol ester 20 (210 mg,
0.34 mmol), Na.sub.2CO.sub.3 (36.5 mg, 0.34 mmol), EtOH (5 mL),
toluene (10 mL), THF (1 mL), and water (5 mL). The reaction mixture
was allowed to stir under a nitrogen atmosphere at 35.degree. C.
for 2 hours after which time the complete consumption of starting
material was observed by TLC (80:20 v/v EtOAc/MeOH) and LC/MS (1.68
min (ES+) m/z (relative intensity) 1508.10 ([M+H].sup.+, 100)). The
reaction mixture was diluted with EtOAc (100 mL) and washed with
H.sub.2O (1.times.100 mL), brine (200 mL), dried (MgSO.sub.4),
filtered and evaporated under reduced pressure to provide the crude
product. Purification by flash chromatography (gradient elution:
100:0 v/v EtOAc/MeOH to 80:20 v/v EtOAc/MeOH) afforded the SEM
protected dimer 89 (240 mg, 69%). .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.42 (s, 1H), 7.76 (d, J=7.5 Hz, 2H), 7.63-7.49 (m, 4H),
7.45-7.28 (m, 9H), 7.25 (d, J=2.9 Hz, 1H), 6.87 (t, J=14.0 Hz, 2H),
6.41 (s, 1H), 5.63-5.49 (m, 2H), 5.25 (s, 1H), 4.71 (d, J=10.1 Hz,
2H), 4.68-4.57 (m, 2H), 4.49 (d, J=6.7 Hz, 2H), 4.20 (s, 1H),
4.16-4.02 (m, 4H), 4.00-3.87 (m, 7H), 3.86-3.61 (m, 7H), 3.30-3.21
(m, 4H), 3.19-3.05 (m, 2H), 2.69-2.54 (m, 4H), 2.37 (s, 3H),
2.04-1.92 (m, 4H), 1.91-1.79 (m, 4H), 1.72 (s, 2H), 1.46 (d, J=6.9
Hz, 3H), 1.04-0.82 (m, 8H), 0.04- -0.02 (m, 18H). MS (ES.sup.+) m/z
(relative intensity) 1508.10 ([M +H].sup.+, 100).
(c) (9H-fluoren-9-yl)methyl
((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-(4-methylpip-
erazin-1 -yl)phenyl)-5-oxo-5,11
a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-5-o-
xo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-
-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate
(90)
[1884] Super hydride (0.358 mL, 0.358 mmol, 1.0 M in THF) was added
dropwise to a stirred solution of the SEM-tetralactam 89 (216 mg,
0.143 mmol) in anhydrous THF (10 mL) at -78.degree. C. The reaction
mixture was allowed to stir for 3 hours after which time the
complete conversion of starting material directly was observed by
LC/MS (1.37 min (ES+) m/z (relative intensity) 608.15
(([M+2H].sup.2+)/2, 100)). The reaction mixture was carefully
diluted with H.sub.2O (100 mL) and extracted with DCM (100 mL). The
organic layers was washed with brine (100 mL), dried over
MgSO.sub.4, filtered and evaporated under reduced pressure to
provide the intermediate SEM-carbinolamine. The white solids were
immediately dissolved in MeOH (100 mL), DCM (10mL) and H.sub.2O (20
mL) and treated with flash silica gel (50 g). The thick suspension
was allowed to stir at room temperature for 4 days after which time
the formation of a significant quantity of desired product was
observed by TLC (90:10 v/v CHCl.sub.3/MeOH). The reaction mixture
was filtered through a porosity 3 sinter funnel and the pad rinsed
slowly and thoroughly with 90:10 v/v CHCl.sub.3/MeOH until no
further product eluted (checked by TLC). The filtrate was washed
with brine (100 mL), dried (MgSO.sub.4), filtered and evaporated in
vacuo, followed by high vacuum drying, to provide the crude
product. Purification by flash chromatography (gradient elution:
HPLC grade 98:2 v/v CHCl.sub.3/MeOH to 88:12 v/v CHCl.sub.3/MeOH)
gave 90 as a mixture of carbinolamine ethers and imine (80 mg,
46%). .sup.1H NMR (400 MHz, CDCl3) .delta. 8.52 (s, 1H), 7.87 (d,
J=3.9 Hz, 2H), 7.75 (d, J=7.5 Hz, 2H), 7.66-7.26 (m, 12H), 6.90 (d,
J=8.8 Hz, 2H), 6.81 (s, 1H), 6.64 (d, J=6.0 Hz, 1H), 5.37 (d, J=5.7
Hz, 1H), 4.74-4.58 (m, 2H), 4.54-4.31 (m, 4H), 4.26-3.98 (m, 6H),
3.94 (s, 2H), 3.86 (dd, J=13.6, 6.6 Hz, 1H), 3.63-3.48 (m, 2H),
3.37 (dd, J=16.5, 5.6 Hz, 2H), 3.31-3.17 (m, 4H), 2.66-2.51 (m,
4H), 2.36 (s, 3H), 2.16 (d, J=5.1 Hz, 1H), 2.06-1.88 (m, 4H),
1.78-1.55 (m, 6H), 1.46 (d, J=6.8 Hz, 3H), 0.94 (d, J=6.8 Hz, 6H).
MS (ES.sup.+) m/z (relative intensity) 608.15 (([M +2H].sup.2+)/2,
100).
(d)
1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-((S)-1-(((S)-
-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)p-
henyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy-
)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2--
yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,1-
2,15,18,21,24-octaoxaheptacosan-27-amide (91)
[1885] Piperidine (0.2 mL) was added to a solution of 90 (77 mg,
63.4 pmol) in DMF (1 mL). The reaction mixture was allowed to stir
for 20 minutes. The reaction mixture was carefully diluted with DCM
(50 mL) and washed with water (50 mL). The organic layers was
washed with brine (100 mL), dried over MgSO.sub.4, filtered and
evaporated under reduced pressure to provide the unprotected valine
intermediate. The crude residue was immediately redissolved in
chloroform (5 mL). Mal(Peg).sub.8-acid (56 mg, 95 .mu.mol) and EDCI
(18 mg, 95 .mu.mol) were added, followed by methanol (0.1 mL). The
reaction was allowed to stir for 3 hours at room temperature at
which point completion was observed by TLC and LC/MS (1.19 min
(ES+) m/z (relative intensity) 784.25 (([M+2H].sup.2+)/2, 100)).
The reaction mixture was diluted with chloroform (50 mL), washed
with water (100 mL), dried (MgSO.sub.4), filtered and evaporated in
vacuo, followed by high vacuum drying, to provide the crude
product. Purification by flash chromatography (gradient elution:
HPLC grade 96:4 v/v CHCl.sub.3/MeOH to 90:10 v/v CHCl.sub.3/MeOH)
gave 91 as a yellow solid (43 mg, 43%). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 8.73 (s, 1H), 7.88 (dd, J=7.6, 3.9 Hz, 2H),
7.75 (d, J=8.6 Hz, 2H), 7.52 (d, J=2.0 Hz, 2H), 7.44 (s, 1H),
7.40-7.28 (m, 4H), 6.91 (d, J=8.8 Hz, 2H), 6.81 (s, 2H), 6.69 (s,
2H), 6.48 (s, 1 H), 4.72-4.63 (m, 1H), 4.46-4.34 (m, 2H), 4.25-4.03
(m, 6H), 3.95 (s, 4H), 3.84 (dd, J=17.2, 10.1 Hz, 4H), 3.72-3.46
(m, 30H), 3.44-3.32 (m, 4H), 3.30-3.20 (m, 4H), 2.75-2.63 (m, 1H),
2.59 (s, 4H), 2.55-2.43 (m, 3H), 2.37 (s, 3H), 2.29 (dd, J=12.7,
6.7 Hz, 1 H), 2.03-1.89 (m, 4H), 1.72 (d, J=22.7 Hz, 8H), 1.46 (d,
J=7.2 Hz, 3H), 1.01 (dd, J=11.5, 6.9 Hz, 6H). MS (ES.sup.+) m/z
(relative intensity) 784.25 (([M+2H].sup.2+)/2, 100).
Example 11
(i)
(S)-((pentane-1,5-diylbis(oxy))bis(2-amino-5-methoxy-4,1-phenylene))bi-
s((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyr-
rol-1-yl)methanone) (98)
##STR00111##
[1886] (a)
(S,R)-((pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phen-
ylene))bis(((2S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxypyrr-
olidin-1-yl)methanone) (94)
[1887] Anhydrous DMF (approx. 0.5 mL) was added dropwise to a
stirred suspension of
4,4'-(pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitrobenzoic acid)
(92) (36.64 g, 74.0 mmol) and oxalyl chloride (18.79 mL, 0.222 mol,
3.0 eq.) in anhydrous DCM (450 mL) until vigorous effervescence
occurred and the reaction mixture was left to stir overnight. The
reaction mixture was evaporated to dryness, and triturated with
diethyl ether. The resulting yellow precipitate was filtered from
solution, washed with diethyl ether (100 mL) and immediately added
to a solution of (3R,5S)-5-((tert-butyldimethylsilyloxy)methyl)
pyrrolidin-3-ol (93) (39.40 g, 0.170 mol, 2.3 eq.) and anhydrous
triethylamine (82.63 mL, 0.592 mol, 8 eq.) in anhydrous DCM (400
mL) at -40.degree. C. The reaction mixture was allowed to slowly
warm to room temperature (over 2.5 hours) after which, LCMS
analysis indicated complete reaction. DCM (250 mL) was added and
the mixture was transferred into a separating funnel. The organic
layer was washed successively with 0.1M HCl (2.times.800 mL),
saturated NaHCO.sub.3 (500 mL) and brine (300 mL). After drying
over MgSO.sub.4 and filtration, evaporation of the solvent left the
product as a yellow foam (62.8 g, 92%). LC/MS: RT 1.96 min; MS
(ES+) m/z (relative intensity)921.45 ([M+H].sup.+, 100).
(b)
(5S,5'S)-1,1'-(4,4'-(pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitroben-
zoyl))bis(5-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidin-3-one)
(95)
[1888] Trichloroisocyanuric acid (21.86 g, 94.07 mmol, 1.4 eq) was
added in one portion to a solution of diol 94 (61.90 g, 67.20 mmol)
and TEMPO (2.10 g, 13.44 mmol, 0.2 eq) in anhydrous DCM (500 mL)
under an atmosphere of argon at 0.degree. C. The reaction mixture
was stirred at 0.degree. C. for 20 minutes after which, LCMS
analysis of the reaction mixture showed complete reaction. The
reaction mixture was diluted with DCM (400 mL) and washed with
saturated sodium bicarbonate (500 mL), 0.2 M sodium thiosulfate
solution (600 mL), brine (400 mL) and dried (MgSO.sub.4).
Evaporation of the solvent gave the crude product. Flash
chromatography [gradient elution 80% n-hexane/20% ethyl acetate to
100% ethyl acetate] gave pure 95 as yellow solid (49.30 g, 80%).
LC/MS: RT 2.03 min; MS (ES+) m/z (relative intensity) 917.55
([M+H].sup.+, 100).
(c)
(5S,5'S)-1,1'-(4,4'-(pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitroben-
zoyl))bis(5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrole--
3,1-diyl) bis(trifluoromethanesulfonate), (96)
[1889] Triflic anhydride (24.19 mL, 0.144 mol, 6.0 eq) was added
dropwise to a vigorously stirred solution of bis-ketone 95 (21.98
g, 23.96 mmol) in anhydrous DCM (400 mL) containing 2,6-lutidine
(22.33 mL, 0.192 mol, 8.0 eq) at -40.degree. C. The reaction
mixture was stirred at -40.degree. C. for 30 min after which, LCMS
analysis indicated complete reaction. Reaction mixture was rapidly
diluted with DCM (500 mL) and washed with ice-cold water (600 mL),
ice-cold saturated sodium bicarbonate (400 mL) and brine (500 mL),
dried over MgSO.sub.4, filtered and evaporated to leave a crude
brown oil. Flash chromatography [gradient elution 80% n-hexane/20%
ethyl acetate to 66% n-hexane/33% ethyl acetate] gave pure 96 as a
brown foam (16.40 g, 58%). LC/MS: RT 2.28 min; MS (ES+) m/z
(relative intensity) no data.
(d)
(5)-((pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bi-
s((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyr-
rol-1-yl)methanone) (97)
[1890] Triflate 96 (5.06 g, 4.29 mmol), methyl boronic acid (1.80
g, 30.00 mmol, 7 eq) and triphenylarsine (1.05 g, 3.43 mmol, 0.8
eq) were dissolved in anhydrous dioxane and stirred under argon. Pd
(II) bisbenzonitrile chloride was then added and the reaction
mixture heated rapidly to 80.degree. C. for 20 min. Reaction
mixture cooled, filtered through Celite (washed through with ethyl
acetate), filtrate washed with water (500 mL), brine (500 mL),
dried over MgSO.sub.4, filtered and evaporated. Flash
chromatography [gradient elution 50% n-hexane/50% ethyl acetate]
gave pure 97 as a brown foam (4.31 g, 59%). LC/MS: RT 2.23 min; MS
(ES+) m/z (relative intensity) 913.50 ([M+H].sup.+, 100).
(e)
(S)-((pentane-1,5-diylbis(oxy))bis(2-amino-5-methoxy-4,1-phenylene))bi-
s((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyr-
rol-1-yl)methanone) (98)
[1891] Zinc dust (26.48 g, 0.405 mol, 36.0 eq) was added in one
portion to a solution of bis-nitro compound 97 (10.26 g, 11.24
mmol) in 5% formic acid/methanol (200 mL) keeping the temperature
between 25-30.degree. C. with the aid of a cold water bath. The
reaction was stirred at 30.degree. C. for 20 minutes after which,
LCMS showed complete reaction. The reaction mixture was filtered
through Celite to remove the excess zinc, which was washed with
ethyl acetate (600 mL). The organic fractions were washed with
water (500 mL), saturated sodium bicarbonate (500 mL) and brine
(400 mL), dried over MgSO.sub.4 and evaporated. Flash
chromatography [gradient elution 100% chloroform to 99%
chloroform/1% methanol] gave pure 98 as an orange foam (6.22 g,
65%). LC/MS: RT 2.20 min; MS (ES+) m/z (relative intensity) 853.50
([M+H].sup.+, 100).
(ii)
4-((R)-2-((R)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propan-
amido)benzyl
4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetrad-
ecanamido)benzyl
((S)-(pentane-1,5-diylbis(oxy))bis(2-((S)-2-(hydroxymethyl)-4-methyl-2,3--
dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5,1-phenylene))dicarbamate
(103)
##STR00112## ##STR00113##
[1892] (a) Allyl
(5-((5-(5-amino-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl--
2,3-dihydro-1H-pyrrole-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(-
((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1
H-pyrrole-1-carbonyl)-4-methoxyphenyl)carbamate (99)
[1893] Pyridine (1.156 mL, 14.30 mmol, 1.5 eq) was added to a
solution of the bis-aniline 98 (8.14 g, 9.54 mmol) in anhydrous DCM
(350 mL) at -78.degree. C. under an atmosphere of argon. After 5
minutes, allyl chloroformate (0.911 mL, 8.58 mmol, 0.9 eq) was
added and the reaction mixture allowed to warm to room temperature.
The reaction mixture was diluted with DCM (250 mL), washed with
saturated CuSO.sub.4 solution (400 mL), saturated sodium
bicarbonate (400 mL) and brine (400 mL), dried over MgSO.sub.4.
Flash chromatography [gradient elution 66% n-hexane/33% ethyl
acetate to 33% n-hexane/66% ethyl acetate] gave pure 99 as an
orange foam (3.88 g, 43%). LC/MS: RT 2.27 min; MS (ES+) m/z
(relative intensity) 937.55 ([M+H].sup.+, 100).
(b) Allyl
4-((10S,13S)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-di-
azatetradecanamido)benzyl
((S)-(pentane-1,5-diylbis(oxy))bis(2-((S)-2-(((tert-butyldimethylsilyl)ox-
y)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5,1-phenyl-
ene))dicarbamate (100)
[1894] Triethylamine (0.854 mL, 6.14 mmol, 2.2 eq) was added to a
stirred solution of the aniline 99 (2.62 g, 2.79 mmol) and
triphosgene (0.30 g, 1.00 mmol, 0.36 eq) in anhydrous THF (50 mL)
under argon 0.degree. C. The reaction mixture was stirred at room
temperature for 5 minutes. LCMS analysis of an aliquot quenched
with methanol, showed formation of the isocyanate. A solution of
mPEG.sub.2-Val-Ala-PAB-OH (1.54 g, 3.63 mmol, 1.3 eq) and
triethylamine (0.583 mL, 4.19 mmol, 1.5 eq) in dry THF (50 mL) was
added in one portion and the resulting mixture was stirred
overnight at 40.degree. C. The solvent of the reaction mixture was
evaporated leaving a crude product. Flash chromatography [gradient
elution 100% chloroform to 98% chloroform/2% methanol] gave pure
100 as a light orange solid (2.38 g, 62%). LC/MS: RT 2.29 min; MS
(ES+) m/z (relative intensity) no data.
(c)
4-((10S,13S)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatet-
radecanamido)benzyl
(5-((5-(5-amino-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl--
2,3-dihydro-1H-pyrrole-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(-
((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-ca-
rbonyl)-4-methoxyphenyl)carbamate (101)
[1895] Tetrakis(triphenylphosphine)palladium (39 mg, 0.034 mmol,
0.02 eq) was added to a stirred solution of 100 (2.35 g, 1.69 mmol)
and pyrrolidine (0.35 mL, 4.24 mmol, 2.5 eq) in anhydrous DCM (25
mL) under argon at room temperature. Reaction mixture allowed to
stir for 45 min then diluted with DCM (100 mL), washed with
saturated ammonium chloride solution (100mL), brine (100mL), dried
over MgSO.sub.4, filtered and evaporated. Flash chromatography
[gradient elution 100% chloroform to 95% chloroform/5% methanol]
gave pure 101 as a yellow solid (1.81 g, 82%). LC/MS: RT 2.21 min;
MS (ES+) m/z (relative intensity) 1303.65 ([M+H].sup.+, 100).
(d)
4-((R)-2-((R)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propana-
mido)benzyl
4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetrad-
ecanamido)benzyl
((S)-(pentane-1,5-diylbis(oxy))bis(2-((S)-2-(((tert-butyldimethylsilyl)ox-
y)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5,1-phenyl-
ene))dicarbamate (102)
[1896] Triethylamine (0.419 mL, 3.01 mmol, 2.2 eq) was added to a
stirred solution of the aniline 101 (1.78 g, 1.37 mmol) and
triphosgene (0.15 g, 0.49 mmol, 0.36 eq) in anhydrous THF (50 mL)
under argon 0.degree. C. The reaction mixture was stirred at room
temperature for 5 min. LCMS analysis of an aliquot quenched with
methanol, showed formation of the isocyanate. A solution of
Alloc-Val-Ala-PAB-OH (0.67 g, 1.78 mmol, 1.3 eq) and triethylamine
(0.29 mL, 2.05 mmol, 1.5 eq) in dry THF (45 mL) was added in one
portion and the resulting mixture was stirred overnight at
40.degree. C. The solvent of the reaction mixture was evaporated
leaving a crude product. Flash chromatography [gradient elution
100% ethyl acetate to 97% ethyl acetate/3% methanol] gave pure 102
as a pale yellow solid (1.33 g, 57%).
[1897] LC/MS: RT 2.21 min; MS (ES+) m/z (relative intensity) no
data.
(e)
4-((R)-2-((R)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propana-
mido)benzyl
4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetrad-
ecanamido)benzyl
((S)-(pentane-1,5-diylbis(oxy))bis(2-((S)-2-(hydroxymethyl)-4-methyl-2,3--
dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5,1-phenylene))dicarbamate
(103)
[1898] Tetra-n-butylammonium fluoride (1 M, 1.52 mL, 1.52 mmol, 2.0
eq) was added to a solution of the TBS protected compound 102 (1.30
g, 0.76 mmol) in anhydrous THF (15 mL). The reaction mixture was
stirred at room temperature for 4 hours. The reaction mixture was
diluted with chloroform (100 mL) and washed sequentially with water
(40 mL) and brine (40 mL). The organic phase was dried over
MgSO.sub.4 and evaporated to leave a yellow solid. Flash
chromatography [gradient elution 95% ethyl acetate/5% methanol to
90% ethyl acetate/10% methanol] gave pure 103 as a pale yellow
solid (1.00 g, 89%). LC/MS: RT 1.60 min; MS (ES+) m/z (relative
intensity) 1478.45 (100).
(iii)
(11S,11aS)-4-((2R,5R)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-is-
opropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-tria-
zaheptatriacontanamido)benzyl
11-hydroxy-8-((5-(((11S,11aS)-11-hydroxy-10-(((4-((10R,13R)-10-isopropyl--
13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamido)benzyl)oxy)carbon-
yl)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]-
benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-
-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (106)
##STR00114##
[1899] (a)
(11S,11aS)-4-((R)-2-((R)-2-(((allyloxy)carbonyl)amino)-3-methyl-
butanamido)propanamido)benzyl
11-hydroxy-8-((5-(((11S,11aS)-11-hydroxy-10-(((4-((10R,13R)-10-isopropyl--
13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamido)benzyl)oxy)carbon-
yl)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]-
benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-
-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (104)
[1900] Dess-Martin periodinane (0.59 g, 1.38 mmol, 2.1 eq) was
added to a stirred solution of 103 (0.97 g, 0.66 mmol) in anhydrous
DCM under argon at room temperature. The reaction mixture was
allowed to stir for 4 hours. Reaction mixture diluted with DCM (100
mL), washed with saturated sodium bicarbonate solution (3.times.100
mL), water (100 mL), brine (100 mL), dried over MgSO.sub.4,
filtered and evaporated. Flash chromatography [gradient elution
100% chloroform to 95% chloroform/5% methanol] gave pure 104 as a
pale yellow solid (0.88 g, 90%). LC/MS: RT 1.57 min; MS (ES+) m/z
(relative intensity) 1473.35 (100).
(b) (11 S,11
aS)-4-((R)-2-((R)-2-amino-3-methylbutanamido)propanamido)benzyl
11-hydroxy-8-((5-(((11S,11
aS)-11-hydroxy-10-(((4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-d-
ioxa-9,12-diazatetradecanamido)benzyl)oxy)carbonyl)-7-methoxy-2-methyl-5-o-
xo-5,10,11,11 a-tetrahydro-1 H-pyrrolo[2,1
-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11
a-dihydro-1 H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate
(105)
[1901] Tetrakis(triphenylphosphine)palladium (5 mg, 0.004 mmol,
0.06 eq) was added to a solution of 104 (105 mg, 0.071 mmol) and
pyrrolidine (7 .mu.L, 0.086 mmol, 1.2 eq) in anhydrous DCM (5 mL).
The reaction mixture was stirred 15 minutes then diluted with
chloroform (50 mL) and washed sequentially with saturated aqueous
ammonium chloride (30 mL) and brine (30mL). The organic phase was
dried over magnesium sulphate, filtered and evaporated. Flash
chromatography [gradient elution 100% chloroform to 90%
chloroform/10% methanol] gave pure 105 as a pale yellow solid (54
mg, 55%). LC/MS: RT 1.21 min; MS (ES+) m/z (relative intensity)
1389.50 (100).
(c)
(11S,11aS)-4-((2R,5R)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isop-
ropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triaza-
heptatriacontanamido)benzyl
11-hydroxy-8-((5-(((11S,11aS)-11-hydroxy-10-(((4-((10R,13R)-10-isopropyl--
13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamido)benzyl)oxy)carbon-
yl)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]-
benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-
-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (106)
[1902] N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (28 mg, 0.146
mmol, 1 eq) was added to a solution of 105 (203 mg, 0.146 mmol) and
maleimide-PEG.sub.8 acid (87 mg, 0.146 mmol) in chloroform (5 mL).
The reaction was stirred for 1.5 h then diluted with chloroform (50
mL), washed with water (50 mL), brine (30 mL), dried over magnesium
sulphate, filtered and evaporated. Flash chromatography [gradient
elution 100% DCM to 90% DCM/10% methanol] gave 106 as a pale yellow
solid (205 mg, 72%). LC/MS: RT 5.75 min; MS (ES+) m/z (relative
intensity) 982.90 (100), 1963.70 (5).
Example 12
Activity of Released Compounds
[1903] K562 Assay
[1904] K562 human chronic myeloid leukaemia cells were maintained
in RPM1 1640 medium supplemented with 10% fetal calf serum and 2 mM
glutamine at 37.degree. C. in a humidified atmosphere containing 5%
CO.sub.2 and were incubated with a specified dose of drug for 1
hour or 96 hours at 37.degree. C. in the dark. The incubation was
terminated by centrifugation (5 min, 300 g) and the cells were
washed once with drug-free medium. Following the appropriate drug
treatment, the cells were transferred to 96-well microtiter plates
(10.sup.4 cells per well, 8 wells per sample). Plates were then
kept in the dark at 37.degree. C. in a humidified atmosphere
containing 5% CO.sub.2. The assay is based on the ability of viable
cells to reduce a yellow soluble tetrazolium salt,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
(MTT, Aldrich-Sigma), to an insoluble purple formazan precipitate.
Following incubation of the plates for 4 days (to allow control
cells to increase in number by approximately 10 fold), 20 .mu.L of
MTT solution (5 mg/mL in phosphate-buffered saline) was added to
each well and the plates further incubated for 5 h. The plates were
then centrifuged for 5 min at 300 g and the bulk of the medium
pipetted from the cell pellet leaving 10-20 .mu.L per well. DMSO
(200 .mu.L) was added to each well and the samples agitated to
ensure complete mixing. The optical density was then read at a
wavelength of 550 nm on a Titertek Multiscan ELISA plate reader,
and a dose-response curve was constructed. For each curve, an
IC.sub.50 value was read as the dose required to reduce the final
optical density to 50% of the control value.
ABBREVIATIONS
[1905] Ac acetyl [1906] Acm acetamidomethyl [1907] Alloc
allyloxycarbonyl [1908] Boc di-tert-butyl dicarbonate [1909] t-Bu
tert-butyl [1910] Bzl benzyl, where Bzl-OMe is methoxybenzyl and
Bzl-Me is methylbenzene [1911] Cbz or Z benzyloxy-carbonyl, where
Z-Cl and Z-Br are chloro- and bromobenzyloxy carbonyl respectively
[1912] DMF N,N-dimethylformamide [1913] Dnp dinitrophenyl [1914]
DTT dithiothreitol [1915] Fmoc 9H-fluoren-9-ylmethoxycarbonyl
[1916] imp N-10 imine protecting group:
3-(2-methoxyethoxy)propanoate-Val-Ala-PAB [1917] MC--OSu
maleimidocaproyl-O--N-succinimide [1918] Moc methoxycarbonyl [1919]
MP maleimidopropanamide [1920] Mtr
4-methoxy-2,3,6-trimethtylbenzenesulfonyl [1921] PAB
para-aminobenzyloxycarbonyl [1922] PEG ethyleneoxy [1923] PNZ
p-nitrobenzyl carbamate [1924] Psec
2-(phenylsulfonyl)ethoxycarbonyl [1925] TBDMS
tert-butyldimethylsilyl [1926] TBDPS tert-butyldiphenylsilyl [1927]
Teoc 2-(trimethylsilyl)ethoxycarbonyl [1928] Tos tosyl [1929] Troc
2,2,2-trichlorethoxycarbonyl chloride [1930] Trt trityl [1931] Xan
xanthyl
Statements of Invention
[1932] 1. A conjugate of formula L-(DL)p, where DL is of formula I
or II:
##STR00115##
[1933] wherein:
[1934] L is an antibody (Ab) which binds CD22;
[1935] when there is a double bond present between C2' and C3',
R.sup.12 is selected from the group consisting of:
[1936] (ia) C.sub.5-10 aryl group, optionally substituted by one or
more substituents selected from the group comprising: halo, nitro,
cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7
heterocyclyl and bis-oxy-C.sub.1-3 alkylene;
[1937] (ib) C.sub.1-5 saturated aliphatic alkyl;
[1938] (ic) C.sub.3-6 saturated cycloalkyl;
[1939] (id)
##STR00116##
wherein each of R.sup.21, R.sup.22 and R.sup.23 are independently
selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl,
C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon
atoms in the R.sup.12 group is no more than 5;
[1940] (ie)
##STR00117##
wherein one of R.sup.25a a and R.sup.25b is H and the other is
selected from: phenyl, which phenyl is optionally substituted by a
group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
and
[1941] (if)
##STR00118##
where R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl;
C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which
phenyl is optionally substituted by a group selected from halo,
methyl, methoxy; pyridyl; and thiophenyl;
[1942] when there is a single bond present between C2' and C3',
[1943] R.sup.12 is
##STR00119##
where R.sup.26a and R.sup.26b are independently selected from H, F,
C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and
alkenyl groups are optionally substituted by a group selected from
C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of
R.sup.26a and R.sup.26b is H, the other is selected from nitrile
and a C.sub.1-4 alkyl ester;
[1944] R.sup.6 and R.sup.9 are independently selected from H, R,
OH, OR, SH, SR, NH.sub.2, NHR, NRR', nitro, Me.sub.3Sn and
halo;
[1945] where R and R' are independently selected from optionally
substituted C.sub.1-12 alkyl, C.sub.3-20 heterocyclyl and
C.sub.5-20 aryl groups;
[1946] R.sup.7 is selected from H, R, OH, OR, SH, SR, NH.sub.2,
NHR, NHRR', nitro, Me.sub.3Sn and halo;
[1947] R'' is a C.sub.3-12 alkylene group, which chain may be
interrupted by one or more heteroatoms, e.g. O, S, NR.sup.N2 (where
R.sup.N2 is H or C.sub.1-4 alkyl), and/or aromatic rings, e.g.
benzene or pyridine;
[1948] Y and Y' are selected from O, S, or NH;
[1949] R.sup.6', R.sup.7', R.sup.9' are selected from the same
groups as R.sup.6, R.sup.7 and R.sup.9 respectively;
[1950] [Formula I]
[1951] R.sup.L1' is a linker for connection to the antibody
(Ab);
[1952] R.sup.11a is selected from OH, OR.sup.A, where R.sup.A is
C.sub.1-4 alkyl, and SO.sub.zM, where z is 2 or 3 and M is a
monovalent pharmaceutically acceptable cation;
[1953] R.sup.20 and R.sup.21 either together form a double bond
between the nitrogen and carbon atoms to which they are bound
or;
[1954] R.sup.20 is selected from H and R.sup.C, where R.sup.C is a
capping group;
[1955] R.sup.21 is selected from OH, OR.sup.A and SO.sub.zM;
[1956] when there is a double bond present between C2 and C3,
R.sup.2 is selected from the group consisting of:
[1957] (ia) C.sub.5-10 aryl group, optionally substituted by one or
more substituents selected from the group comprising: halo, nitro,
cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7
heterocyclyl and bis-oxy-C.sub.1-3 alkylene;
[1958] (ib) C.sub.1-5 saturated aliphatic alkyl;
[1959] (ic) C.sub.3-6 saturated cycloalkyl;
[1960] (id)
##STR00120##
wherein each of R.sup.11, R.sup.12 and R.sup.13 are independently
selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl,
C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon
atoms in the R.sup.2 group is no more than 5;
[1961] (ie)
##STR00121##
wherein one of R.sup.15a and R.sup.15b is H and the other is
selected from: phenyl, which phenyl is optionally substituted by a
group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
and
[1962] (if)
##STR00122##
where R.sup.14 is selected from: H; C.sub.1-3 saturated alkyl;
C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which
phenyl is optionally substituted by a group selected from halo,
methyl, methoxy; pyridyl; and thiophenyl;
[1963] when there is a single bond present between C2 and C3,
[1964] R.sup.2 is
##STR00123##
where R.sup.16a and R.sup.16b are independently selected from H, F,
C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and
alkenyl groups are optionally substituted by a group selected from
C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of
R.sup.16a and R.sup.16b is H, the other is selected from nitrile
and a C.sub.1-4 alkyl ester;
[1965] [Formula II]
[1966] R.sup.22 is of formula IIIa, formula IIIb or formula
IIIc:
[1967] (a)
##STR00124##
where A is a C.sub.5-7 aryl group, and either
[1968] (i) Q.sup.1 is a single bond, and Q.sup.2 is selected from a
single bond and --Z--(CH.sub.2).sub.n--, where Z is selected from a
single bond, O, S and NH and n is from 1 to 3; or
[1969] (ii) Q.sup.1 is --CH.dbd.CH--, and Q.sup.2 is a single
bond;
[1970] (b)
##STR00125##
[1971] where;
[1972] R.sup.C1, R.sup.C2 and R.sup.C3 are independently selected
from H and unsubstituted C.sub.1-2 alkyl;
[1973] (c)
##STR00126##
[1974] where Q is selected from O--R.sup.L2', S--R.sup.L2' and
NR.sup.N--R.sup.L2', and R.sup.N is selected from H, methyl and
ethyl
[1975] X is selected from the group comprising: O--R.sup.L2',
S--R.sup.L2', CO.sub.2--R.sup.L2', CO--R.sup.L2',
NH--C(.dbd.O)--R.sup.L2', NHNH--R.sup.L2', CONHNH--R.sup.L2',
##STR00127##
NR.sup.NR.sup.L2', wherein R.sup.N is selected from the group
comprising H and C.sub.1-4 alkyl;
[1976] R.sup.L2' is a linker for connection to the antibody
(Ab);
[1977] R.sup.10 and R.sup.11 either together form a double bond
between the nitrogen and carbon atoms to which they are bound
or;
[1978] R.sup.10 is H and R.sup.11 is selected from OH, OR.sup.A and
SO.sub.zM;
[1979] R.sup.30 and R.sup.31 either together form a double bond
between the nitrogen and carbon atoms to which they are bound
or;
[1980] R.sup.30 is H and R.sup.31 is selected from OH, OR.sup.A and
SO.sub.zM.
[1981] 2. The conjugate according to statement 1, wherein the
conjugate is not:
##STR00128## ##STR00129##
[1982] 3. The conjugate according to either statement 1 or
statement 2, wherein R.sup.7 is selected from H, OH and OR.
[1983] 4. The conjugate according to statement 3, wherein R.sup.7
is a C.sub.1-4 alkyloxy group.
[1984] 5. The conjugate according to any one of statements 1 to 4,
wherein Y is O.
[1985] 6. The conjugate according to any one of the preceding
statements, wherein R'' is C.sub.3-7 alkylene.
[1986] 7. The conjugate according to any one of statements 1 to 6,
wherein R.sup.9 is H.
[1987] 8. The conjugate according to any one of statements 1 to 7,
wherein R.sup.6 is selected from H and halo.
[1988] 9. The conjugate according to any one of statements 1 to 8,
wherein there is a double bond between C2' and C3', and R.sup.12 is
a C.sub.5-7 aryl group.
[1989] 10. The conjugate according to statement 9, wherein R.sup.12
is phenyl.
[1990] 11. The conjugate according to any one of statements 1 to 8,
wherein there is a double bond between C2' and C3', and R.sup.12 is
a C.sub.8-10 aryl group.
[1991] 12. The conjugate according to any one of statements 9 to
11, wherein R.sup.12 bears one to three substituent groups.
[1992] 13. The conjugate according to any one of statements 9 to
12, wherein the substituents are selected from methoxy, ethoxy,
fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl,
morpholino and methyl-thiophenyl.
[1993] 14. The conjugate according to any one of statements 1 to 8,
wherein there is a double bond between C2' and C3', and R.sup.12 is
a C.sub.1-5 saturated aliphatic alkyl group.
[1994] 15. A compound according to statement 14, wherein R.sup.12
is methyl, ethyl or propyl.
[1995] 16. The conjugate according to any one of statements 1 to 8,
wherein there is a double bond between C2' and C3', and R.sup.12 is
a C.sub.3-6 saturated cycloalkyl group.
[1996] 17. The conjugate according to statement 16, wherein
R.sup.12 is cyclopropyl.
[1997] 18. The conjugate according to any one of statements 1 to 8,
wherein there is a double bond between C2' and C3', and R.sup.12 is
a group of formula:
##STR00130##
[1998] 19. The conjugate according to statement 18, wherein the
total number of carbon atoms in the R.sup.12 group is no more than
4.
[1999] 20. The conjugate according to statement 19, wherein the
total number of carbon atoms in the R.sup.12 group is no more than
3.
[2000] 21. The conjugate according to any one of statements 18 to
20, wherein one of R.sup.21, R.sup.22 and R.sup.23 is H, with the
other two groups being selected from H, C.sub.1-3 saturated alkyl,
C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
[2001] 22. The conjugate according to any one of statements 18 to
20, wherein two of R21, R22 and R.sup.23 are H, with the other
group being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3
alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
[2002] 23. The conjugate according to any one of statements 1 to 8,
wherein there is a double bond between C2' and C3', and R.sup.12 is
a group of formula:
##STR00131##
[2003] 24. The conjugate according to statement 23, wherein
R.sup.12 is the group:
##STR00132##
[2004] 25. The conjugate according to any one of statements 1 to 8,
wherein there is a double bond between C2' and C3', and R.sup.12 is
a group of formula:
##STR00133##
[2005] 26. The conjugate according to statement 25, wherein
R.sup.24 is selected from H, methyl, ethyl, ethenyl and
ethynyl.
[2006] 27. The conjugate according to statement 26, wherein
R.sup.24 is selected from H and methyl.
[2007] 28. The conjugate according to any one of statements 1 to 8,
wherein there is a single bond between C2' and C3', R.sup.12 is
##STR00134##
and R.sup.26a and R.sup.26b are both H.
[2008] 29. The conjugate according to any one of statements 1 to 8,
wherein there is a single bond between C2' and C3', R.sup.12 is
##STR00135##
and R.sup.26a and R.sup.26b are both methyl.
[2009] 30. The conjugate according to any one of statements 1 to 8,
wherein there is a single bond between C2' and C3', R.sup.12 is
##STR00136##
one of R.sup.26a and R.sup.26b is H, and the other is selected from
C.sub.1-.sub.4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and
alkenyl groups are optionally substituted.
[2010] [Formula I]
[2011] 31. The conjugate according to any one of statements 1 to
30, wherein there is a double bond between C2 and C3, and R.sup.2
is a C.sub.5-7 aryl group.
[2012] 32. The conjugate according to statement 31, wherein R.sup.2
is phenyl.
[2013] 33. The conjugate according to any one of statements 1 to
30, wherein there is a double bond between C2 and C3, and R.sup.1
is a C.sub.8-10 aryl group.
[2014] 34. A compound according to any one of statements 31 to 33,
wherein R.sup.2 bears one to three substituent groups.
[2015] 35. The conjugate according to any one of statements 31 to
34, wherein the substituents are selected from methoxy, ethoxy,
fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl,
morpholino and methyl-thiophenyl.
[2016] 36. The conjugate according to any one of statements 1 to
30, wherein there is a double bond between C2 and C3, and R.sup.2
is a C.sub.1-5 saturated aliphatic alkyl group.
[2017] 37. The conjugate according to statement 36, wherein R.sup.2
is methyl, ethyl or propyl.
[2018] 38. The conjugate according to any one of statements 1 to
30, wherein there is a double bond between C2 and C3, and R.sup.2
is a C.sub.3-6 saturated cycloalkyl group.
[2019] 39. The conjugate according to statement 38, wherein R.sup.2
is cyclopropyl.
[2020] 40. The conjugate according to any one of statements 1 to
30, wherein there is a double bond between C2 and C3, and R.sup.2
is a group of formula:
##STR00137##
[2021] 41. The conjugate according to statement 40, wherein the
total number of carbon atoms in the R.sup.2 group is no more than
4.
[2022] 42. The conjugate according to statement 41, wherein the
total number of carbon atoms in the R.sup.2 group is no more than
3.
[2023] 43. The conjugate according to any one of statements 40 to
42, wherein one of R.sup.11, R.sup.12 and R.sup.13 is H, with the
other two groups being selected from H, C.sub.1-3 saturated alkyl,
C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
[2024] 44. The conjugate according to any one of statements 40 to
42, wherein two of R.sub.11, R.sub.12 and R.sup.13 are H, with the
other group being selected from H, C.sub.1-3 saturated alkyl,
C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
[2025] 45. The conjugate according to any one of statements 1 to
30, wherein there is a double bond between C2 and C3, and R.sup.2
is a group of formula:
##STR00138##
[2026] 46. The conjugate according to statement 45, wherein R.sup.2
is the group:
##STR00139##
[2027] 47. The conjugate according to any one of statements 1 to
30, wherein there is a double bond between C2 and C3, and R.sup.2
is a group of formula:
##STR00140##
[2028] 48. The conjugate according to statement 47, wherein
R.sup.14 is selected from H, methyl, ethyl, ethenyl and
ethynyl.
[2029] 49. The conjugate according to statement 47, wherein
R.sup.14 is selected from H and methyl.
[2030] 50. The conjugate according to any one of statements 1 to
30, wherein there is a single bond between C2 and C3, R.sup.2
is
##STR00141##
and R.sup.16a and R.sup.16b are both H.
[2031] 51. The conjugate according to any one of statements 1 to
30, wherein there is a single bond between C2 and C3, R.sup.2
is
##STR00142##
and R.sup.16a and R.sup.16b are both methyl.
[2032] 52. The conjugate according to any one of statements 1 to
30, wherein there is a single bond between C2 and C3, R.sup.2
is
##STR00143##
one of R.sup.16a and R.sup.16b is H, and the other is selected from
C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and
alkenyl groups are optionally substituted.
[2033] 53. The conjugate according to any one of statements 1 to
52, wherein R.sup.11a is OH.
[2034] 54. The conjugate according to any one of statements 1 to
53, wherein R.sup.21 is OH.
[2035] 55. The conjugate according to any one of statements 1 to
53, wherein R.sup.21 is OMe.
[2036] 56. The conjugate according to any one of statements 1 to
55, wherein R.sup.2.degree. is H.
[2037] 57. The conjugate according to any one of statements 1 to
55, wherein R.sup.2.degree. is Re.
[2038] 58. The conjugate according to statement 57, wherein R.sup.C
is selected from the group consisting of: Alloc, Fmoc, Boc, and
Troc.
[2039] 59. The conjugate according to statement 57, wherein R.sup.C
is selected from the group consisting of: Teoc, Psec, Cbz and
PNZ.
[2040] 60. The conjugate according to statement 57, wherein R.sup.C
is a group:
##STR00144## [2041] where the asterisk indicates the point of
attachment to the N10 position, G.sup.2 is a terminating group,
L.sup.3 is a covalent bond or a cleavable linker L.sup.1, L.sup.2
is a covalent bond or together with OC(.dbd.O) forms a
self-immolative linker.
[2042] 61. The conjugate according to statement 60, wherein G.sup.2
is Ac or Moc or is selected from the group consisting of: Alloc,
Fmoc, Boc, Troc, Teoc, Psec, Cbz and PNZ.
[2043] 62. The conjugate according to any one of statements 1 to
53, wherein R.sup.20 and R.sup.21 together form a double bond
between the nitrogen and carbon atoms to which they are bound.
[2044] [Formula II]
[2045] 63. The conjugate according to any one of statements 1 to
30, wherein R.sup.22 is of formula IIIa, and A is phenyl.
[2046] 64. The conjugate according to any one of statements 1 to 30
and statement 63, wherein R.sup.22 is of formula IIa, and Q.sup.1
is a single bond.
[2047] 65. The conjugate according to statement 63, wherein Q.sup.2
is a single bond.
[2048] 66. The conjugate according to statement 63, wherein Q.sup.2
is --Z--(CH.sub.2).sub.n--, Z is O or S and n is 1 or 2.
[2049] 67. The conjugate according any one of statements 1 to 30
and statement 63, wherein R.sup.22 is of formula IIIa, and Q.sup.1
is --CH.dbd.CH--.
[2050] 68. The conjugate according to any one of statements 1 to
30, wherein R.sup.22 is of formula IIIb,
[2051] and R.sup.C1, R.sup.C2 and R.sup.C3 are independently
selected from H and methyl.
[2052] 69. The conjugate according to statement 68, wherein
R.sup.C1, R.sup.C2 and R.sup.C3 are all H.
[2053] 70. The conjugate according to statement 68, wherein
R.sup.C1, R.sup.C2 and R.sup.C3 are all
[2054] 71. The conjugate according to any one of statements 1 to 30
and statements 63 to 70, wherein R.sup.22 is of formula IIIa or
formula IIIb and X is selected from O--R.sup.L2', S--R.sup.L2',
CO.sub.2--R.sup.L2', --N--C(.dbd.O)--R.sup.L2' and
NH--R.sup.L2'.
[2055] 72. The conjugate according to statement 71, wherein X is
NH--R.sup.L2'.
[2056] 73. The conjugate according to any one of statements 1 to
30, wherein R.sup.22 is of formula IIIc, and Q is
NR.sup.N--R.sup.L2'.
[2057] 74. The conjugate according to statement 73, wherein RN is H
or methyl.
[2058] 75. The conjugate according to any one of statements 1 to
30, wherein R.sup.22 is of formula IIIc, and Q is O--R.sup.L2' or
S--R.sup.L2'.
[2059] 76. The conjugate according to any one of statements 1 to 30
and statements 63 to 75, wherein R.sup.11 is OH.
[2060] 77. The conjugate according to any one of statements 1 to 30
and statements 63 to 75, wherein R.sup.11 is OMe.
[2061] 78. The conjugate according to any one of statements 1 to 30
and statements 63 to 77, wherein R.sup.10 is H.
[2062] 79. The conjugate according to any one of statements 1 to 30
and statements 63 to 75, wherein R.sup.10 and R.sup.11 together
form a double bond between the nitrogen and carbon atoms to which
they are bound.
[2063] 80. The conjugate according to any one of statements 1 to 30
and statements 63 to 79, wherein R.sup.31 is OH.
[2064] 81. The conjugate according to any one of statements 1 to 30
and statements 63 to 79, wherein R.sup.31 is OMe.
[2065] 82. The conjugate according to any one of statements 1 to 30
and statements 63 to 81, wherein R.sup.30 is H.
[2066] 83. The conjugate according to any one of statements 1 to 30
and statements 63 to 79, wherein R.sup.30 and R.sup.31 together
form a double bond between the nitrogen and carbon atoms to which
they are bound.
[2067] 84. The conjugate according to any one of statements 1 to
83, wherein R.sup.6', R.sup.7', R.sup.9', and Y' are the same as
R.sup.6, R.sup.7, R.sup.9, and Y.
[2068] 85. The conjugate according to any one of statements 1 to 84
wherein, wherein L-R.sup.L1' or L-R.sup.L2' is a group:
##STR00145## [2069] where the asterisk indicates the point of
attachment to the PBD, Ab is the antibody, L.sup.1 is a cleavable
linker, A is a connecting group connecting L.sup.1 to the antibody,
L.sup.2 is a covalent bond or together with --OC(.dbd.O)-- forms a
self-immolative linker.
[2070] 86. The conjugate of statement 85, wherein L.sup.1 is enzyme
cleavable.
[2071] 87. The conjugate of statement 85 or statement 86, wherein
L.sup.1 comprises a contiguous sequence of amino acids.
[2072] 88. The conjugate of statement 87, wherein L.sup.1 comprises
a dipeptide and the group --X.sub.1--X.sub.2-- in dipeptide,
--NH--X.sub.1--X.sub.2--CO--, is selected from: [2073] -Phe-Lys-,
[2074] -Val-Ala-, [2075] -Val-Lys-, [2076] -Ala-Lys-, [2077]
-Val-Cit-, [2078] -Phe-Cit-, [2079] -Leu-Cit-, [2080] -Ile-Cit-,
[2081] -Phe-Arg-, [2082] -Trp-Cit-.
[2083] 89. The conjugate according to statement 88, wherein the
group --X.sub.1--X.sub.2-- in dipeptide,
--NH--X.sub.1--X.sub.2--CO--, is selected from: [2084] -Phe-Lys-,
[2085] -Val-Ala-, [2086] -Val-Lys-, [2087] -Ala-Lys-, [2088]
-Val-Cit-.
[2089] 90. The conjugate according to statement 89, wherein the
group --X.sub.1--X.sub.2-- in dipeptide,
--NH--X.sub.1--X.sub.2--CO--, is -Phe-Lys-, -Val-Ala- or
-Val-Cit-.
[2090] 91. The conjugate according to any one of statements 88 to
90, wherein the group X.sub.2--CO-- is connected to L.sup.2.
[2091] 92. The conjugate according to any one of statements 88 to
91, wherein the group NH--X.sub.1-- is connected to A.
[2092] 93. The conjugate according to any one of statements 88 to
92, wherein L.sup.2 together with OC(.dbd.O) forms a
self-immolative linker.
[2093] 94. The conjugate according to statement 93, wherein
C(.dbd.O)O and L.sup.2 together form the group:
##STR00146## [2094] where the asterisk indicates the point of
attachment to the PBD, the wavy line indicates the point of
attachment to the linker L.sup.1, Y is NH, O, C(.dbd.O)NH or
C(.dbd.O)O, and n is 0 to 3.
[2095] 95. The conjugate according to statement 94, wherein Y is
NH.
[2096] 96. The conjugate according to statement 94 or statement 95,
wherein n is 0.
[2097] 97. The conjugate according to statement 95, wherein L.sup.1
and L.sup.2 together with --OC(.dbd.O)-- comprise a group selected
from:
##STR00147## [2098] where the asterisk indicates the point of
attachment to the PBD, and the wavy line indicates the point of
attachment to the remaining portion of the linker L.sup.1 or the
point of attachment to A.
[2099] 98. The conjugate according to statement 97, wherein the
wavy line indicates the point of attachment to A.
[2100] 99. The conjugate according to any one of statements 85 to
98, wherein A is:
##STR00148## [2101] where the asterisk indicates the point of
attachment to L.sup.1, the wavy line indicates the point of
attachment to the antibody, and n is 0 to 6; or
[2101] ##STR00149## [2102] where the asterisk indicates the point
of attachment to L.sup.1, the wavy line indicates the point of
attachment to the antibody, n is 0 or 1, and m is 0 to 30.
[2103] 100. A conjugate according to statement 1 of formula
##STR00150## ##STR00151## ##STR00152##
[2104] 101. The conjugate according to any one of statements 1 to
100 wherein the antibody comprises an amino acid substitution of an
interchain cysteine residue by an amino acid that is not cysteine
and the conjugation of the drug moiety to the antibody is at an
interchain cysteine residue.
[2105] 102. The conjugate according to statement 101 wherein the
antibody comprises a heavy chain comprising the amino acid sequence
of SEQ ID NO.110 or fragment thereof, SEQ ID NO.120 or fragment
thereof, SEQ ID NO.130 or fragment thereof, or SEQ ID NO.140 or
fragment thereof.
[2106] 103. The conjugate according to statement 102 wherein the
drug moiety is conjugated to the cysteine at position 103 of SEQ ID
NO.110, the cysteine at position 14 of SEQ ID NO.120, the cysteine
at position 103 of SEQ ID NO.120, the cysteine at position 14 of
SEQ ID NO.130, or the cysteine at position 14 of SEQ ID NO.140.
[2107] 104. The conjugate according to either one of statements 102
or 103 wherein the antibody comprises: [2108] a light chain
comprising the amino acid sequence of SEQ ID NO. 150, or fragment
thereof, wherein the cysteine at position 105, if present, is
substituted by an amino acid that is not cysteine; or [2109] a
light chain comprising the amino acid sequence of SEQ ID NO. 160,
or fragment thereof, wherein the cysteine at position 102, if
present, is substituted by an amino acid that is not cysteine.
[2110] 105. The conjugate according to statement 101 wherein the
antibody comprises: [2111] a heavy chain comprising the amino acid
sequence of SEQ ID NO.110 and light chain comprising the amino acid
sequence of SEQ ID NO.151, SEQ ID NO.152, SEQ ID NO.153, SEQ ID
NO.161, SEQ ID NO.162, or SEQ ID NO.163; [2112] optionally wherein
the drug moiety is conjugated to the cysteine at position 103 of
SEQ ID NO.110.
[2113] 106. The conjugate according to statement 101 wherein the
antibody comprises: [2114] a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, or fragment thereof, wherein the
cysteine at position 103 of SEQ ID NO.110, if present, is
substituted by an amino acid that is not cysteine; [2115] a heavy
chain comprising the amino acid sequence of SEQ ID NO.120, or
fragment thereof, wherein each of the cysteines at positions 14 and
103 of SEQ ID NO.120, if present, is substituted by an amino acid
that is not cysteine; [2116] a heavy chain comprising the amino
acid sequence of SEQ ID NO.130, or fragment thereof, wherein the
cysteine at position 14 in SEQ ID NO: 130, if present, is
substituted by an amino acid that is not cysteine; or [2117] a
heavy chain comprising the amino acid sequence of SEQ ID NO.140, or
fragment thereof, wherein the cysteine at position 14 in SEQ ID NO:
140, if present, is substituted by an amino acid that is not
cysteine.
[2118] 107. The conjugate according to statement 106 wherein the
antibody comprises a light chain comprising the amino acid sequence
of SEQ ID NO. 150 or SEQ ID NO. 160.
[2119] 108. The conjugate according to statement 101 wherein the
antibody comprises: [2120] a heavy chain comprising the amino acid
sequence of SEQ ID NO.111 and a light chain comprising the amino
acid sequence of SEQ ID NO.150 or SEQ ID NO.160.
[2121] 109. The conjugate according to statement 101 wherein the
antibody comprises: [2122] a heavy chain comprising the amino acid
sequence of SEQ ID NO.112 and a light chain comprising the amino
acid sequence of SEQ ID NO.150 or SEQ ID NO.160.
[2123] 110. The conjugate according to any one of statements 107 to
109 wherein the drug moiety is conjugated to the cysteine at
position 105 of SEQ ID NO.150, or the cysteine at position 102 of
SEQ ID NO.160.
[2124] 111. The conjugate according to statement 101 wherein the
antibody comprises: [2125] a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, or fragment thereof, wherein each of the
cysteines at positions 109 and 112 in SEQ ID NO: 110, if present,
is substituted by an amino acid that is not cysteine; [2126] a
heavy chain comprising the amino acid sequence of SEQ ID NO.120, or
fragment thereof, wherein each of the cysteines at positions 103,
106, and 109 in SEQ ID NO: 120, if present, is substituted by an
amino acid that is not cysteine; [2127] a heavy chain comprising
the amino acid sequence of SEQ ID NO.120, or fragment thereof,
wherein each of the cysteines at positions 14, 106, and 112 in SEQ
ID NO: 120, if present, is substituted by an amino acid that is not
cysteine; [2128] a heavy chain comprising the amino acid sequence
of SEQ ID NO.130, or fragment thereof, wherein each of the
cysteines at positions 111, 114, 120, 126, 129, 135, 141, 144, 150,
156, and 159 in SEQ ID NO: 130, if present, is substituted by an
amino acid that is not cysteine; or [2129] a heavy chain comprising
the amino acid sequence of SEQ ID NO.140, or fragment thereof,
wherein each of the cysteines at positions 106 and 109 in SEQ ID
NO: 140, if present, is substituted by an amino acid that is not
cysteine.
[2130] 112. The conjugate according to statement 111 the cysteine
at position 102 in SEQ ID NO: 120, if present, is also substituted
by an amino acid that is not cysteine.
[2131] 113. The conjugate according to either one of statements 111
or 112 wherein the drug moiety is conjugated to the cysteine at
position 103 of SEQ ID NO.110, the cysteine at position 14 of SEQ
ID NO.120, the cysteine at position 103 of SEQ ID NO.120, the
cysteine at position 14 of SEQ ID NO.130, or the cysteine at
position 14 of SEQ ID NO.140.
[2132] 114. The conjugate according to any one of statements 111 to
113 wherein the antibody comprises: [2133] a light chain comprising
the amino acid sequence of SEQ ID NO. 150, or fragment thereof,
wherein the cysteine at position 105, if present, is substituted by
an amino acid that is not cysteine; or [2134] a light chain
comprising the amino acid sequence of SEQ ID NO. 160, or fragment
thereof, wherein the cysteine at position 102, if present, is
substituted by an amino acid that is not cysteine.
[2135] 115. The conjugate according to statement 101 wherein the
antibody comprises: [2136] a heavy chain comprising the amino acid
sequence of SEQ ID NO.113 and a light chain comprising the amino
acid sequence of SEQ ID NO.151, SEQ ID NO.152, SEQ ID NO.153, SEQ
ID NO.161, SEQ ID NO.162, or SEQ ID NO.163; [2137] optionally
wherein the drug moiety is conjugated to the cysteine at position
103 of SEQ ID NO.113.
[2138] 116. The conjugate according to statement 101 wherein the
antibody comprises: [2139] a heavy chain comprising the amino acid
sequence of SEQ ID NO.114 and a light chain comprising the amino
acid sequence of SEQ ID NO.151, SEQ ID NO.152, SEQ ID NO.153, SEQ
ID NO.161, SEQ ID NO.162, or SEQ ID NO.163; [2140] optionally
wherein the drug moiety is conjugated to the cysteine at position
103 of SEQ ID NO.114.
[2141] 117. The conjugate according to statement 101 wherein the
antibody comprises: [2142] a heavy chain comprising the amino acid
sequence of SEQ ID NO.110, or fragment thereof, wherein each of the
cysteines at positions 103, 109 and 112 in SEQ ID NO: 110, if
present, is substituted by an amino acid that is not cysteine;
[2143] a heavy chain comprising the amino acid sequence of SEQ ID
NO.120, or fragment thereof, wherein each of the cysteines at
positions 14, 103, 106 and 109 in SEQ ID NO: 120, if present, is
substituted by an amino acid that is not cysteine; [2144] a heavy
chain comprising the amino acid sequence of SEQ ID NO.130, or
fragment thereof, wherein each of the cysteines at positions 14,
111, 114, 120, 126, 129, 135, 141, 144, 150, 156, and 159 in SEQ ID
NO: 130, if present, is substituted by an amino acid that is not
cysteine; or [2145] a heavy chain comprising the amino acid
sequence of SEQ ID NO.140, or fragment thereof, wherein each of the
cysteines at positions 14, 106, and 109 in SEQ ID NO: 140, if
present, is substituted by an amino acid that is not cysteine.
[2146] 118. The conjugate according to statement 117 wherein the
antibody comprises a light chain comprising the amino acid sequence
of SEQ ID NO. 150 or SEQ ID NO. 160.
[2147] 119. The conjugate according to statement 101 wherein the
antibody comprises a heavy chain comprising the amino acid sequence
of SEQ ID NO.115 and a light chain comprising the amino acid
sequence of SEQ ID NO. 150 or SEQ ID NO. 160.
[2148] 120. The conjugate according to statement 101 wherein the
antibody comprises a heavy chain comprising the amino acid sequence
of SEQ ID NO.116 and a light chain comprising the amino acid
sequence of SEQ ID NO. 150 or SEQ ID NO. 160.
[2149] 121. The conjugate according to statement 118 wherein the
drug moiety is conjugated to the cysteine at position 105 of SEQ ID
NO.150, the cysteine at position 102 of SEQ ID NO.160
[2150] 122. The conjugate according to any one of statements 1 to
100 wherein the antibody comprises a heavy chain having a
substitution of the amino acid at position 234 in the EU index set
forth in Kabat and/or a substitution of the residue at position 235
in the EU index set forth in Kabat.
[2151] 123. The conjugate according to statement 122 wherein the
antibody comprises a heavy chain having a substitution of the amino
acid at position 234 in the EU index set forth in Kabat and a
substitution of the residue at position 235 in the EU index set
forth in Kabat.
[2152] 124. The conjugate according to statement 122 wherein the
antibody comprises a heavy chain comprising the amino acid sequence
of SEQ ID NO.110, and wherein the leucine at position 117 and/or
the leucine at position 118 is substituted by an amino acid that is
not leucine.
[2153] 125. The conjugate according to statement 124 wherein the
antibody comprises a heavy chain comprising the amino acid sequence
of SEQ ID NO.110, and wherein the leucine at position 117 and the
leucine at position 118 are substituted by an amino acid that is
not leucine.
[2154] 126. The conjugate according to statement 122 wherein the
antibody comprises a heavy chain comprising the amino acid sequence
of SEQ ID NO.130, and wherein the leucine at position 164 and/or
the leucine at position 165 is substituted by an amino acid that is
not leucine.
[2155] 127. The conjugate according to statement 126 wherein the
antibody comprises a heavy chain comprising the amino acid sequence
of SEQ ID NO.130, and wherein the leucine at position 164 and the
leucine at position 165 are substituted by an amino acid that is
not leucine.
[2156] 128. The conjugate according to statement 122 wherein the
antibody comprises a heavy chain comprising the amino acid sequence
of SEQ ID NO.140, and wherein the leucine at position 115 is
substituted by an amino acid that is not leucine.
[2157] 129. The conjugate according to any one of statements 102 to
121 wherein: [2158] the leucine at position 117 in SEQ ID NO: 110
and/or the leucine at position 118 in SEQ ID NO: 110 is substituted
by an amino acid that is not leucine; [2159] the leucine at
position 164 in SEQ ID NO: 130 and/or the leucine at position 165
in SEQ ID NO: 130 is substituted by an amino acid that is not
leucine; or [2160] the leucine at position 115 in SEQ ID NO: 140 is
substituted by an amino acid that is not leucine.
[2161] 130. The conjugate according to statement 129 wherein:
[2162] the leucine at position 117 in SEQ ID NO: 110 and the
leucine at position 118 in SEQ ID NO: 110 are substituted by an
amino acid that is not leucine; or [2163] the leucine at position
164 in SEQ ID NO: 130 and the leucine at position 165 in SEQ ID NO:
130 are substituted by an amino acid that is not leucine.
[2164] 131. The conjugate according to any one of statements 122 to
130 wherein the substituted amino acids are replaced by alanine,
glycine, valine, or isoleucine.
[2165] 132. The conjugate according to any one of statements 122 to
131 wherein the substituted amino acids are replaced by
alanine.
[2166] 133. The conjugate according to any one of statements 1 to
132 wherein the antibody comprises a VH domain having the amino
acid sequence of SEQ ID NO. 1.
[2167] 134. The conjugate according to statement 133 wherein the
antibody further comprises a VL domain having the amino acid
sequence of SEQ ID NO. 2.
[2168] 135. The conjugate according to any one of the preceding
statements wherein the antibody in an intact antibody.
[2169] 136. The conjugate according to any one of the preceding
statements wherein the antibody is humanised, deimmunised or
resurfaced.
[2170] 137. The conjugate according to any one of the preceding
statements wherein the conjugate has a maximum tolerated dose in
rat at least 2.0 mg/kg delivered as a single-dose.
[2171] 138. The conjugate according to any one of the preceding
statements wherein the drug loading (p) of drugs (D) to antibody
(Ab) is 2 or 4.
[2172] 139. The conjugate according to any one of statements 1 to
138, for use in therapy.
[2173] 140. The conjugate according to any one of statements 1 to
138, for use in the treatment of a proliferative disease in a
subject.
[2174] 141. The conjugate according to statement 140, wherein the
disease is cancer.
[2175] 142. A pharmaceutical composition comprising the conjugate
of any one of statements 1 to 138 and a pharmaceutically acceptable
diluent, carrier or excipient.
[2176] 143. The pharmaceutical composition of statement 142 further
comprising a therapeutically effective amount of a chemotherapeutic
agent.
[2177] 144. Use of a conjugate according to any one of statements 1
to 138 in the preparation of a medicament for use in the treatment
of a proliferative disease in a subject.
[2178] 145. A method of treating cancer comprising administering to
a patient the pharmaceutical composition of statement 142.
[2179] 146. The method of statement 145 wherein the patient is
administered a chemotherapeutic agent, in combination with the
conjugate.
TABLE-US-00008 SEQUENCES (Epratuzumab VH): SEQ ID NO. 1
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGYINPRNDYTE
YNQNFKDKATITADESTNTAYMELSSLRSEDTAFYFCARRDITTFYWGQG (Epratuzumab
VL): SEQ ID NO. 2
DIQLTQSPSSLSASVGDRVTMSCKSSQSVLYSANHKNYLAWYQQKPGKAPKLLIYWASTRE
SGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYLSSWTFGQG CD22 (IgG1 HC
constant region) SEQ ID NO. 110
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, L117A) SEQ ID
NO. 1101
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEALGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, L118A) SEQ ID
NO. 1102
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, L117A &
L118A) SEQ ID NO. 1103
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, L117G &
L118G) SEQ ID NO. 1104
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEGGGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, L117V &
L118V) SEQ ID NO. 1105
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEVVGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, L117I &
L118I) SEQ ID NO. 1106
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEIIGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, HJ C.fwdarw.S)
SEQ ID NO. 111
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, HJ C.fwdarw.S,
L117A) SEQ ID NO. 1111
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPEALGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, HJ C.fwdarw.S,
L118A) SEQ ID NO. 1112
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, HJ C.fwdarw.S,
L117A & L118A) SEQ ID NO. 1113
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, HJ C.fwdarw.S,
L117G & L118G) SEQ ID NO. 1114
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPEGGGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, HJ C.fwdarw.S,
L117V & L118V) SEQ ID NO. 1115
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPEVVGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, HJ C.fwdarw.S,
L117I & L118I) SEQ ID NO. 1116
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPEIIGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, HJ C.fwdarw.V)
SEQ ID NO. 112
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSVDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, HJ C.fwdarw.V,
L117A) SEQ ID NO. 1121
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSVDKTHTCPPCPAPEALGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, HJ C.fwdarw.V,
L118A) SEQ ID NO. 1122
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSVDKTHTCPPCPAPELAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, HJ C.fwdarw.V,
L117A & L118A) SEQ ID NO. 1123
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSVDKTHTCPPCPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, HJ C.fwdarw.V,
L117G & L118G) SEQ ID NO. 1124
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSVDKTHTCPPCPAPEGGGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, HJ C.fwdarw.V,
L117V & L118V) SEQ ID NO. 1125
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSVDKTHTCPPCPAPEVVGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, HJ C.fwdarw.V,
L117I & L118I) SEQ ID NO. 1126
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSVDKTHTCPPCPAPEIIGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, BJ C.fwdarw.S)
SEQ ID NO. 113
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTSPPSPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, BJ C.fwdarw.S,
L117A) SEQ ID NO. 1131
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTSPPSPAPEALGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, BJ C.fwdarw.S,
L118A) SEQ ID NO. 1132
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTSPPSPAPELAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, BJ C.fwdarw.S,
L117A & L118A) SEQ ID NO. 1133
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTSPPSPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, BJ C.fwdarw.S,
L117G & L118G) SEQ ID NO. 1134
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTSPPSPAPEGGGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, BJ C.fwdarw.S,
L117V & L118V) SEQ ID NO. 1135
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTSPPSPAPEVVGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, BJ C.fwdarw.S,
L117I & L118I) SEQ ID NO. 1136
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTSPPSPAPEIIGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, BJ C.fwdarw.V)
SEQ ID NO. 114
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTVPPVPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, BJ C.fwdarw.V,
L117A) SEQ ID NO. 1141
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTVPPVPAPEALGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, BJ C.fwdarw.V,
L118A) SEQ ID NO. 1142
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTVPPVPAPELAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, BJ C.fwdarw.V,
L117A & L118A) SEQ ID NO. 1143
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTVPPVPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, BJ C.fwdarw.V,
L117G & L118G) SEQ ID NO. 1144
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTVPPVPAPEGGGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, BJ C.fwdarw.V,
L117V & L118V) SEQ ID NO. 1145
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTVPPVPAPEVVGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, BJ C.fwdarw.V,
L117I & L118I) SEQ ID NO. 1146
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTVPPVPAPEIIGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, DJ C.fwdarw.S)
SEQ ID NO. 115
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTSPPSPAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, DJ C.fwdarw.S,
L117A) SEQ ID NO. 1151
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTSPPSPAPEALGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, DJ C.fwdarw.S,
L118A) SEQ ID NO. 1152
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTSPPSPAPELAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, DJ C.fwdarw.S,
L117A & L118A) SEQ ID NO. 1153
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTSPPSPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, DJ C.fwdarw.S,
L117G & L118G) SEQ ID NO. 1154
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTSPPSPAPEGGGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, DJ C.fwdarw.S,
L117V & L118V) SEQ ID NO. 1155
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTSPPSPAPEVVGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, DJ C.fwdarw.S,
L117I & L118I) SEQ ID NO. 1156
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTSPPSPAPEIIGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, DJ C.fwdarw.V)
SEQ ID NO. 116
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSVDKTHTVPPVPAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, DJ C.fwdarw.V,
L117A) SEQ ID NO. 1161
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSVDKTHTVPPVPAPEALGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, DJ C.fwdarw.V,
L118A) SEQ ID NO. 1162
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSVDKTHTVPPVPAPELAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, DJ C.fwdarw.V,
L117A & L118A) SEQ ID NO. 1163
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSVDKTHTVPPVPAPEAAGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, DJ C.fwdarw.V,
L117G & L118G) SEQ ID NO. 1164
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSVDKTHTVPPVPAPEGGGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, DJ C.fwdarw.V,
L117V & L118V) SEQ ID NO. 1165
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSVDKTHTVPPVPAPEVVGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 HC constant region, DJ C.fwdarw.V,
L117I & L118I) SEQ ID NO. 1166
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSVDKTHTVPPVPAPEIIGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK (IgG2 HC constant region) SEQ ID NO. 120
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVV
SVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGK (IgG3 HC constant region) SEQ ID NO. 130
ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCD
TPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDIAVEWESSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEAL
HNHFTQKSLSLSPGK (IgG3 HC constant region, L164A) SEQ ID NO. 131
ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCD
TPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPEALGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDIAVEWESSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEAL
HNHFTQKSLSLSPGK (IgG3 HC constant region, L165A) SEQ ID NO. 132
ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCD
TPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELAGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDIAVEWESSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEAL
HNHFTQKSLSLSPGK (IgG3 HC constant region, L164A & L165A) SEQ ID
NO. 133
ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCD
TPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPEAAGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDIAVEWESSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEAL
HNHFTQKSLSLSPGK (IgG3 HC constant region, L164G & L165G) SEQ ID
NO. 134
ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCD
TPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPEGGGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDIAVEWESSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEAL
HNHFTQKSLSLSPGK (IgG3 HC constant region, L164V & L165V) SEQ ID
NO. 135
ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCD
TPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPEVVGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDIAVEWESSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEAL
HNHFTQKSLSLSPGK (IgG3 HC constant region, L164I & L165I) SEQ ID
NO. 136
ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCD
TPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPEIIGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALH
NHFTQKSLSLSPGK (IgG4 HC constant region) SEQ ID NO. 140
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF
SCSVMHEALHNHYTQKSLSLSLGK (IgG4 HC constant region, L115A) SEQ ID
NO. 141
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFAGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF
SCSVMHEALHNHYTQKSLSLSLGK (IgG4 HC constant region, L115G) SEQ ID
NO. 142
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFGGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF
SCSVMHEALHNHYTQKSLSLSLGK (IgG4 HC constant region, L115V) SEQ ID
NO. 143
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFVGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF
SCSVMHEALHNHYTQKSLSLSLGK (IgG4 HC constant region, L115I) SEQ ID
NO. 144
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFIGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS
CSVMHEALHNHYTQKSLSLSLGK (.kappa.LC constant region) SEQ ID NO. 150
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (.kappa.LC constant
region, C105S) SEQ ID NO. 151
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES (.kappa.LC constant
region, C105V)) SEQ ID NO. 152
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEV (.kappa.LC constant
region, C105del)) SEQ ID NO. 153
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE (.lamda.LC constant
region) SEQ ID NO. 160
KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN
NKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (.lamda.LC constant
region, C102S) SEQ ID NO. 161
KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN
NKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTESS (.lamda.LC constant
region, C102V) SEQ ID NO. 162
KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN
NKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEVS (.lamda.LC constant
region, C102 & S103del) SEQ ID NO. 163
KAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN
NKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTE
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 1166 <210> SEQ ID NO 1 <211> LENGTH: 109
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Epratuzumab VH <400> SEQUENCE: 1 Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Leu His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly
Tyr Ile Asn Pro Arg Asn Asp Tyr Thr Glu Tyr Asn Gln Asn Phe 50 55
60 Lys Asp Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr
65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr
Phe Cys 85 90 95 Ala Arg Arg Asp Ile Thr Thr Phe Tyr Trp Gly Gln
Gly 100 105 <210> SEQ ID NO 2 <211> LENGTH: 106
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Epratuzumab VL <400> SEQUENCE: 2 Asp Ile Gln Leu Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Met
Ser Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ala Asn His
Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala
Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55
60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr
65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys
His Gln 85 90 95 Tyr Leu Ser Ser Trp Thr Phe Gly Gln Gly 100 105
<210> SEQ ID NO 3 <400> SEQUENCE: 3 000 <210> SEQ
ID NO 4 <400> SEQUENCE: 4 000 <210> SEQ ID NO 5
<400> SEQUENCE: 5 000 <210> SEQ ID NO 6 <400>
SEQUENCE: 6 000 <210> SEQ ID NO 7 <400> SEQUENCE: 7 000
<210> SEQ ID NO 8 <400> SEQUENCE: 8 000 <210> SEQ
ID NO 9 <400> SEQUENCE: 9 000 <210> SEQ ID NO 10
<400> SEQUENCE: 10 000 <210> SEQ ID NO 11 <400>
SEQUENCE: 11 000 <210> SEQ ID NO 12 <400> SEQUENCE: 12
000 <210> SEQ ID NO 13 <400> SEQUENCE: 13 000
<210> SEQ ID NO 14 <400> SEQUENCE: 14 000 <210>
SEQ ID NO 15 <400> SEQUENCE: 15 000 <210> SEQ ID NO 16
<400> SEQUENCE: 16 000 <210> SEQ ID NO 17 <400>
SEQUENCE: 17 000 <210> SEQ ID NO 18 <400> SEQUENCE: 18
000 <210> SEQ ID NO 19 <400> SEQUENCE: 19 000
<210> SEQ ID NO 20 <400> SEQUENCE: 20 000 <210>
SEQ ID NO 21 <400> SEQUENCE: 21 000 <210> SEQ ID NO 22
<400> SEQUENCE: 22 000 <210> SEQ ID NO 23 <400>
SEQUENCE: 23 000 <210> SEQ ID NO 24 <400> SEQUENCE: 24
000 <210> SEQ ID NO 25 <400> SEQUENCE: 25 000
<210> SEQ ID NO 26 <400> SEQUENCE: 26 000 <210>
SEQ ID NO 27 <400> SEQUENCE: 27 000 <210> SEQ ID NO 28
<400> SEQUENCE: 28 000 <210> SEQ ID NO 29 <400>
SEQUENCE: 29 000 <210> SEQ ID NO 30 <400> SEQUENCE: 30
000 <210> SEQ ID NO 31 <400> SEQUENCE: 31 000
<210> SEQ ID NO 32 <400> SEQUENCE: 32 000 <210>
SEQ ID NO 33 <400> SEQUENCE: 33 000 <210> SEQ ID NO 34
<400> SEQUENCE: 34 000 <210> SEQ ID NO 35 <400>
SEQUENCE: 35 000 <210> SEQ ID NO 36 <400> SEQUENCE: 36
000 <210> SEQ ID NO 37 <400> SEQUENCE: 37 000
<210> SEQ ID NO 38 <400> SEQUENCE: 38 000 <210>
SEQ ID NO 39 <400> SEQUENCE: 39 000 <210> SEQ ID NO 40
<400> SEQUENCE: 40 000 <210> SEQ ID NO 41 <400>
SEQUENCE: 41 000 <210> SEQ ID NO 42 <400> SEQUENCE: 42
000 <210> SEQ ID NO 43 <400> SEQUENCE: 43 000
<210> SEQ ID NO 44 <400> SEQUENCE: 44 000 <210>
SEQ ID NO 45 <400> SEQUENCE: 45 000 <210> SEQ ID NO 46
<400> SEQUENCE: 46 000 <210> SEQ ID NO 47 <400>
SEQUENCE: 47 000 <210> SEQ ID NO 48 <400> SEQUENCE: 48
000 <210> SEQ ID NO 49 <400> SEQUENCE: 49 000
<210> SEQ ID NO 50 <400> SEQUENCE: 50 000 <210>
SEQ ID NO 51 <400> SEQUENCE: 51 000 <210> SEQ ID NO 52
<400> SEQUENCE: 52 000 <210> SEQ ID NO 53 <400>
SEQUENCE: 53 000 <210> SEQ ID NO 54 <400> SEQUENCE: 54
000 <210> SEQ ID NO 55 <400> SEQUENCE: 55 000
<210> SEQ ID NO 56 <400> SEQUENCE: 56 000 <210>
SEQ ID NO 57 <400> SEQUENCE: 57 000 <210> SEQ ID NO 58
<400> SEQUENCE: 58 000 <210> SEQ ID NO 59 <400>
SEQUENCE: 59 000 <210> SEQ ID NO 60 <400> SEQUENCE: 60
000 <210> SEQ ID NO 61 <400> SEQUENCE: 61 000
<210> SEQ ID NO 62 <400> SEQUENCE: 62 000 <210>
SEQ ID NO 63 <400> SEQUENCE: 63 000 <210> SEQ ID NO 64
<400> SEQUENCE: 64 000 <210> SEQ ID NO 65 <400>
SEQUENCE: 65 000 <210> SEQ ID NO 66 <400> SEQUENCE: 66
000 <210> SEQ ID NO 67 <400> SEQUENCE: 67 000
<210> SEQ ID NO 68 <400> SEQUENCE: 68 000 <210>
SEQ ID NO 69 <400> SEQUENCE: 69 000 <210> SEQ ID NO 70
<400> SEQUENCE: 70 000 <210> SEQ ID NO 71 <400>
SEQUENCE: 71 000 <210> SEQ ID NO 72 <400> SEQUENCE: 72
000 <210> SEQ ID NO 73 <400> SEQUENCE: 73 000
<210> SEQ ID NO 74 <400> SEQUENCE: 74 000 <210>
SEQ ID NO 75 <400> SEQUENCE: 75 000 <210> SEQ ID NO 76
<400> SEQUENCE: 76 000 <210> SEQ ID NO 77 <400>
SEQUENCE: 77 000 <210> SEQ ID NO 78 <400> SEQUENCE: 78
000 <210> SEQ ID NO 79 <400> SEQUENCE: 79 000
<210> SEQ ID NO 80 <400> SEQUENCE: 80 000 <210>
SEQ ID NO 81 <400> SEQUENCE: 81 000 <210> SEQ ID NO 82
<400> SEQUENCE: 82 000 <210> SEQ ID NO 83 <400>
SEQUENCE: 83 000 <210> SEQ ID NO 84 <400> SEQUENCE: 84
000 <210> SEQ ID NO 85 <400> SEQUENCE: 85 000
<210> SEQ ID NO 86 <400> SEQUENCE: 86 000 <210>
SEQ ID NO 87 <400> SEQUENCE: 87 000 <210> SEQ ID NO 88
<400> SEQUENCE: 88 000 <210> SEQ ID NO 89 <400>
SEQUENCE: 89 000 <210> SEQ ID NO 90 <400> SEQUENCE: 90
000 <210> SEQ ID NO 91 <400> SEQUENCE: 91 000
<210> SEQ ID NO 92 <400> SEQUENCE: 92 000 <210>
SEQ ID NO 93 <400> SEQUENCE: 93 000 <210> SEQ ID NO 94
<400> SEQUENCE: 94 000 <210> SEQ ID NO 95 <400>
SEQUENCE: 95 000 <210> SEQ ID NO 96 <400> SEQUENCE: 96
000 <210> SEQ ID NO 97 <400> SEQUENCE: 97 000
<210> SEQ ID NO 98 <400> SEQUENCE: 98 000 <210>
SEQ ID NO 99 <400> SEQUENCE: 99 000 <210> SEQ ID NO 100
<400> SEQUENCE: 100 000 <210> SEQ ID NO 101 <400>
SEQUENCE: 101 000 <210> SEQ ID NO 102 <400> SEQUENCE:
102 000 <210> SEQ ID NO 103 <400> SEQUENCE: 103 000
<210> SEQ ID NO 104 <400> SEQUENCE: 104 000 <210>
SEQ ID NO 105 <400> SEQUENCE: 105 000 <210> SEQ ID NO
106 <400> SEQUENCE: 106 000 <210> SEQ ID NO 107
<400> SEQUENCE: 107 000 <210> SEQ ID NO 108 <400>
SEQUENCE: 108 000 <210> SEQ ID NO 109 <400> SEQUENCE:
109 000 <210> SEQ ID NO 110 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG1
HC constant region <400> SEQUENCE: 110 Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
<210> SEQ ID NO 111 <211> LENGTH: 330 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
HJ C->S <400> SEQUENCE: 111 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 112 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
HJ C->V <400> SEQUENCE: 112 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Val Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 113 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
BJ C->S <400> SEQUENCE: 113 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Ser Pro Pro Ser 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 114 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
BJ C->V <400> SEQUENCE: 114 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Val Pro Pro Val 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 115 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
DJ C->S <400> SEQUENCE: 115 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr
Ser Pro Pro Ser 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 116 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
DJ C->V <400> SEQUENCE: 116 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Val Asp Lys Thr His Thr
Val Pro Pro Val 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 117 <400> SEQUENCE: 117 000 <210> SEQ ID NO
118 <400> SEQUENCE: 118 000 <210> SEQ ID NO 119
<400> SEQUENCE: 119 000 <210> SEQ ID NO 120 <211>
LENGTH: 326 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic IgG2 HC constant region <400> SEQUENCE: 120 Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn
Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys
Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val
Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150
155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His
Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys
Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275
280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210>
SEQ ID NO 121 <400> SEQUENCE: 121 000 <210> SEQ ID NO
122 <400> SEQUENCE: 122 000 <210> SEQ ID NO 123
<400> SEQUENCE: 123 000 <210> SEQ ID NO 124 <400>
SEQUENCE: 124 000 <210> SEQ ID NO 125 <400> SEQUENCE:
125 000 <210> SEQ ID NO 126 <400> SEQUENCE: 126 000
<210> SEQ ID NO 127 <400> SEQUENCE: 127 000 <210>
SEQ ID NO 128 <400> SEQUENCE: 128 000 <210> SEQ ID NO
129 <400> SEQUENCE: 129 000 <210> SEQ ID NO 130
<211> LENGTH: 377 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG3 HC constant region <400>
SEQUENCE: 130 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105
110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg
115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro
Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys
Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser
His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 195 200 205 Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 225 230
235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp
Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Phe Thr Gln 355
360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 <210> SEQ
ID NO 131 <211> LENGTH: 377 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic IgG3 HC constant region, L164A
<400> SEQUENCE: 131 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys
Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro
Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro
Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro
Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Ala Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val
Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 195 200 205
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210
215 220 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala
Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330
335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile
340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Phe
Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375
<210> SEQ ID NO 132 <211> LENGTH: 377 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG3 HC constant region,
L165A <400> SEQUENCE: 132 Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70
75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His
Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro
Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr
Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp
Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190
Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 195
200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu 210 215 220 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp
Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315
320 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370
375 <210> SEQ ID NO 133 <211> LENGTH: 377 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG3 HC constant
region, L164A & L165A <400> SEQUENCE: 133 Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly
Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser
Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys
Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro
Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165
170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290
295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn
Asn 305 310 315 320 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser
Pro Gly Lys 370 375 <210> SEQ ID NO 134 <211> LENGTH:
377 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG3
HC constant region, L164G & L165G <400> SEQUENCE: 134 Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10
15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr
Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu
Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140
Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145
150 155 160 Ala Pro Glu Gly Gly Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265
270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln
Pro Glu Asn Asn 305 310 315 320 Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Phe Thr Gln 355 360 365 Lys Ser Leu
Ser Leu Ser Pro Gly Lys 370 375 <210> SEQ ID NO 135
<211> LENGTH: 377 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG3 HC constant region, L164V & L165V
<400> SEQUENCE: 135 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys
Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro
Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro
Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro
Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Val Val Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val
Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 195 200 205
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210
215 220 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala
Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330
335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile
340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Phe
Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375
<210> SEQ ID NO 136 <211> LENGTH: 377 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG3 HC constant region,
L164I & L165I <400> SEQUENCE: 136 Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50
55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr
Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp
Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys
Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser
Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro
Glu Ile Ile Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180
185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300
Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305
310 315 320 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly
Lys 370 375 <210> SEQ ID NO 137 <400> SEQUENCE: 137 000
<210> SEQ ID NO 138 <400> SEQUENCE: 138 000 <210>
SEQ ID NO 139 <400> SEQUENCE: 139 000 <210> SEQ ID NO
140 <211> LENGTH: 327 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic IgG4 HC constant region <400>
SEQUENCE: 140 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys
Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105
110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230
235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp
Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser
Leu Gly Lys 325 <210> SEQ ID NO 141 <211> LENGTH: 327
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG4
HC constant region, L115A <400> SEQUENCE: 141 Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr
Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro
Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Ala Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165
170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290
295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> SEQ
ID NO 142 <211> LENGTH: 327 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic IgG4 HC constant region, L115G
<400> SEQUENCE: 142 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala
Pro 100 105 110 Glu Phe Gly Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210
215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu
Ser Leu Ser Leu Gly Lys 325 <210> SEQ ID NO 143 <211>
LENGTH: 327 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic IgG4 HC constant region, L115V <400> SEQUENCE: 143
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5
10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys
Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Val
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135
140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260
265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325
<210> SEQ ID NO 144 <211> LENGTH: 327 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG4 HC constant region,
L115I <400> SEQUENCE: 144 Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70
75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys
Pro Ala Pro 100 105 110 Glu Phe Ile Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195
200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315
320 Leu Ser Leu Ser Leu Gly Lys 325 <210> SEQ ID NO 145
<400> SEQUENCE: 145 000 <210> SEQ ID NO 146 <400>
SEQUENCE: 146 000 <210> SEQ ID NO 147 <400> SEQUENCE:
147 000 <210> SEQ ID NO 148 <400> SEQUENCE: 148 000
<210> SEQ ID NO 149 <400> SEQUENCE: 149 000 <210>
SEQ ID NO 150 <211> LENGTH: 105 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: kappa LC constant region <400>
SEQUENCE: 150 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu 1 5 10 15 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro 20 25 30 Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly 35 40 45 Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr 50 55 60 Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 65 70 75 80 Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 85 90 95 Thr
Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> SEQ ID NO 151
<211> LENGTH: 105 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: kappa LC constant region, C105S <400> SEQUENCE:
151 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
1 5 10 15 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro 20 25 30 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly 35 40 45 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr 50 55 60 Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His 65 70 75 80 Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val 85 90 95 Thr Lys Ser Phe
Asn Arg Gly Glu Ser 100 105 <210> SEQ ID NO 152 <211>
LENGTH: 105 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: kappa
LC constant region, C105V <400> SEQUENCE: 152 Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 1 5 10 15 Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 20 25 30
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 35
40 45 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr 50 55 60 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His 65 70 75 80 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val 85 90 95 Thr Lys Ser Phe Asn Arg Gly Glu Val
100 105 <210> SEQ ID NO 153 <211> LENGTH: 104
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: kappa LC
constant region, C105del <400> SEQUENCE: 153 Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 1 5 10 15 Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 20 25 30
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 35
40 45 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr 50 55 60 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His 65 70 75 80 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val 85 90 95 Thr Lys Ser Phe Asn Arg Gly Glu 100
<210> SEQ ID NO 154 <400> SEQUENCE: 154 000 <210>
SEQ ID NO 155 <400> SEQUENCE: 155 000 <210> SEQ ID NO
156 <400> SEQUENCE: 156 000 <210> SEQ ID NO 157
<400> SEQUENCE: 157 000 <210> SEQ ID NO 158 <400>
SEQUENCE: 158 000 <210> SEQ ID NO 159 <400> SEQUENCE:
159 000 <210> SEQ ID NO 160 <211> LENGTH: 103
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: lambda LC
constant region <400> SEQUENCE: 160 Lys Ala Ala Pro Ser Val
Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 1 5 10 15 Gln Ala Asn Lys
Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 20 25 30 Gly Ala
Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 35 40 45
Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 50
55 60 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
Arg 65 70 75 80 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
Glu Lys Thr 85 90 95 Val Ala Pro Thr Glu Cys Ser 100 <210>
SEQ ID NO 161 <211> LENGTH: 103 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: lambda LC constant region, C102S
<400> SEQUENCE: 161 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro
Pro Ser Ser Glu Glu Leu 1 5 10 15 Gln Ala Asn Lys Ala Thr Leu Val
Cys Leu Ile Ser Asp Phe Tyr Pro 20 25 30 Gly Ala Val Thr Val Ala
Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 35 40 45 Gly Val Glu Thr
Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 50 55 60 Ala Ser
Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 65 70 75 80
Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 85
90 95 Val Ala Pro Thr Glu Ser Ser 100 <210> SEQ ID NO 162
<211> LENGTH: 103 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: lambda LC constant region, C102V <400> SEQUENCE:
162 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu
1 5 10 15 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
Tyr Pro 20 25 30 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser
Pro Val Lys Ala 35 40 45 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln
Ser Asn Asn Lys Tyr Ala 50 55 60 Ala Ser Ser Tyr Leu Ser Leu Thr
Pro Glu Gln Trp Lys Ser His Arg 65 70 75 80 Ser Tyr Ser Cys Gln Val
Thr His Glu Gly Ser Thr Val Glu Lys Thr 85 90 95 Val Ala Pro Thr
Glu Val Ser 100 <210> SEQ ID NO 163 <211> LENGTH: 101
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: lambda LC
constant region, C102&S103del <400> SEQUENCE: 163 Lys Ala
Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 1 5 10 15
Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 20
25 30 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys
Ala 35 40 45 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
Lys Tyr Ala 50 55 60 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln
Trp Lys Ser His Arg 65 70 75 80 Ser Tyr Ser Cys Gln Val Thr His Glu
Gly Ser Thr Val Glu Lys Thr 85 90 95 Val Ala Pro Thr Glu 100
<210> SEQ ID NO 164 <400> SEQUENCE: 164 000 <210>
SEQ ID NO 165 <400> SEQUENCE: 165 000 <210> SEQ ID NO
166 <400> SEQUENCE: 166 000 <210> SEQ ID NO 167
<400> SEQUENCE: 167 000 <210> SEQ ID NO 168 <400>
SEQUENCE: 168 000 <210> SEQ ID NO 169 <400> SEQUENCE:
169 000 <210> SEQ ID NO 170 <400> SEQUENCE: 170 000
<210> SEQ ID NO 171 <400> SEQUENCE: 171 000 <210>
SEQ ID NO 172 <400> SEQUENCE: 172 000 <210> SEQ ID NO
173 <400> SEQUENCE: 173 000 <210> SEQ ID NO 174
<400> SEQUENCE: 174 000 <210> SEQ ID NO 175 <400>
SEQUENCE: 175 000 <210> SEQ ID NO 176 <400> SEQUENCE:
176 000 <210> SEQ ID NO 177 <400> SEQUENCE: 177 000
<210> SEQ ID NO 178 <400> SEQUENCE: 178 000 <210>
SEQ ID NO 179 <400> SEQUENCE: 179 000 <210> SEQ ID NO
180 <400> SEQUENCE: 180 000 <210> SEQ ID NO 181
<400> SEQUENCE: 181 000 <210> SEQ ID NO 182 <400>
SEQUENCE: 182 000 <210> SEQ ID NO 183 <400> SEQUENCE:
183 000 <210> SEQ ID NO 184 <400> SEQUENCE: 184 000
<210> SEQ ID NO 185 <400> SEQUENCE: 185 000 <210>
SEQ ID NO 186 <400> SEQUENCE: 186 000 <210> SEQ ID NO
187 <400> SEQUENCE: 187 000 <210> SEQ ID NO 188
<400> SEQUENCE: 188 000 <210> SEQ ID NO 189 <400>
SEQUENCE: 189 000 <210> SEQ ID NO 190 <400> SEQUENCE:
190 000 <210> SEQ ID NO 191 <400> SEQUENCE: 191 000
<210> SEQ ID NO 192 <400> SEQUENCE: 192 000 <210>
SEQ ID NO 193 <400> SEQUENCE: 193 000 <210> SEQ ID NO
194 <400> SEQUENCE: 194 000 <210> SEQ ID NO 195
<400> SEQUENCE: 195 000 <210> SEQ ID NO 196 <400>
SEQUENCE: 196 000 <210> SEQ ID NO 197 <400> SEQUENCE:
197 000 <210> SEQ ID NO 198 <400> SEQUENCE: 198 000
<210> SEQ ID NO 199 <400> SEQUENCE: 199 000 <210>
SEQ ID NO 200 <400> SEQUENCE: 200 000 <210> SEQ ID NO
201 <400> SEQUENCE: 201 000 <210> SEQ ID NO 202
<400> SEQUENCE: 202 000 <210> SEQ ID NO 203 <400>
SEQUENCE: 203 000 <210> SEQ ID NO 204 <400> SEQUENCE:
204 000 <210> SEQ ID NO 205 <400> SEQUENCE: 205 000
<210> SEQ ID NO 206 <400> SEQUENCE: 206 000 <210>
SEQ ID NO 207 <400> SEQUENCE: 207 000 <210> SEQ ID NO
208 <400> SEQUENCE: 208 000 <210> SEQ ID NO 209
<400> SEQUENCE: 209 000 <210> SEQ ID NO 210 <400>
SEQUENCE: 210 000 <210> SEQ ID NO 211 <400> SEQUENCE:
211 000 <210> SEQ ID NO 212 <400> SEQUENCE: 212 000
<210> SEQ ID NO 213 <400> SEQUENCE: 213 000 <210>
SEQ ID NO 214 <400> SEQUENCE: 214 000 <210> SEQ ID NO
215 <400> SEQUENCE: 215 000 <210> SEQ ID NO 216
<400> SEQUENCE: 216 000 <210> SEQ ID NO 217 <400>
SEQUENCE: 217 000 <210> SEQ ID NO 218 <400> SEQUENCE:
218 000 <210> SEQ ID NO 219 <400> SEQUENCE: 219 000
<210> SEQ ID NO 220 <400> SEQUENCE: 220 000 <210>
SEQ ID NO 221 <400> SEQUENCE: 221 000 <210> SEQ ID NO
222 <400> SEQUENCE: 222 000 <210> SEQ ID NO 223
<400> SEQUENCE: 223 000 <210> SEQ ID NO 224 <400>
SEQUENCE: 224 000 <210> SEQ ID NO 225 <400> SEQUENCE:
225 000 <210> SEQ ID NO 226 <400> SEQUENCE: 226 000
<210> SEQ ID NO 227 <400> SEQUENCE: 227 000 <210>
SEQ ID NO 228 <400> SEQUENCE: 228 000 <210> SEQ ID NO
229 <400> SEQUENCE: 229 000 <210> SEQ ID NO 230
<400> SEQUENCE: 230 000 <210> SEQ ID NO 231 <400>
SEQUENCE: 231 000 <210> SEQ ID NO 232 <400> SEQUENCE:
232 000 <210> SEQ ID NO 233 <400> SEQUENCE: 233 000
<210> SEQ ID NO 234 <400> SEQUENCE: 234 000 <210>
SEQ ID NO 235 <400> SEQUENCE: 235 000 <210> SEQ ID NO
236 <400> SEQUENCE: 236 000 <210> SEQ ID NO 237
<400> SEQUENCE: 237 000 <210> SEQ ID NO 238 <400>
SEQUENCE: 238 000 <210> SEQ ID NO 239 <400> SEQUENCE:
239 000 <210> SEQ ID NO 240 <400> SEQUENCE: 240 000
<210> SEQ ID NO 241 <400> SEQUENCE: 241 000 <210>
SEQ ID NO 242 <400> SEQUENCE: 242 000 <210> SEQ ID NO
243 <400> SEQUENCE: 243 000 <210> SEQ ID NO 244
<400> SEQUENCE: 244 000 <210> SEQ ID NO 245 <400>
SEQUENCE: 245 000 <210> SEQ ID NO 246 <400> SEQUENCE:
246 000 <210> SEQ ID NO 247 <400> SEQUENCE: 247 000
<210> SEQ ID NO 248 <400> SEQUENCE: 248 000 <210>
SEQ ID NO 249 <400> SEQUENCE: 249 000 <210> SEQ ID NO
250 <400> SEQUENCE: 250 000 <210> SEQ ID NO 251
<400> SEQUENCE: 251 000 <210> SEQ ID NO 252 <400>
SEQUENCE: 252 000 <210> SEQ ID NO 253 <400> SEQUENCE:
253 000 <210> SEQ ID NO 254 <400> SEQUENCE: 254 000
<210> SEQ ID NO 255 <400> SEQUENCE: 255 000 <210>
SEQ ID NO 256 <400> SEQUENCE: 256 000 <210> SEQ ID NO
257 <400> SEQUENCE: 257 000 <210> SEQ ID NO 258
<400> SEQUENCE: 258 000 <210> SEQ ID NO 259 <400>
SEQUENCE: 259 000 <210> SEQ ID NO 260 <400> SEQUENCE:
260 000 <210> SEQ ID NO 261 <400> SEQUENCE: 261 000
<210> SEQ ID NO 262 <400> SEQUENCE: 262 000 <210>
SEQ ID NO 263 <400> SEQUENCE: 263 000 <210> SEQ ID NO
264 <400> SEQUENCE: 264 000 <210> SEQ ID NO 265
<400> SEQUENCE: 265 000 <210> SEQ ID NO 266 <400>
SEQUENCE: 266 000 <210> SEQ ID NO 267 <400> SEQUENCE:
267 000 <210> SEQ ID NO 268 <400> SEQUENCE: 268 000
<210> SEQ ID NO 269 <400> SEQUENCE: 269 000 <210>
SEQ ID NO 270 <400> SEQUENCE: 270 000 <210> SEQ ID NO
271 <400> SEQUENCE: 271 000 <210> SEQ ID NO 272
<400> SEQUENCE: 272 000 <210> SEQ ID NO 273 <400>
SEQUENCE: 273 000 <210> SEQ ID NO 274 <400> SEQUENCE:
274 000 <210> SEQ ID NO 275 <400> SEQUENCE: 275 000
<210> SEQ ID NO 276 <400> SEQUENCE: 276 000 <210>
SEQ ID NO 277 <400> SEQUENCE: 277 000 <210> SEQ ID NO
278 <400> SEQUENCE: 278 000 <210> SEQ ID NO 279
<400> SEQUENCE: 279 000 <210> SEQ ID NO 280 <400>
SEQUENCE: 280 000 <210> SEQ ID NO 281 <400> SEQUENCE:
281 000 <210> SEQ ID NO 282 <400> SEQUENCE: 282 000
<210> SEQ ID NO 283 <400> SEQUENCE: 283 000 <210>
SEQ ID NO 284 <400> SEQUENCE: 284 000 <210> SEQ ID NO
285 <400> SEQUENCE: 285 000 <210> SEQ ID NO 286
<400> SEQUENCE: 286 000 <210> SEQ ID NO 287 <400>
SEQUENCE: 287 000 <210> SEQ ID NO 288 <400> SEQUENCE:
288 000 <210> SEQ ID NO 289 <400> SEQUENCE: 289 000
<210> SEQ ID NO 290 <400> SEQUENCE: 290 000 <210>
SEQ ID NO 291 <400> SEQUENCE: 291 000 <210> SEQ ID NO
292 <400> SEQUENCE: 292 000 <210> SEQ ID NO 293
<400> SEQUENCE: 293 000 <210> SEQ ID NO 294 <400>
SEQUENCE: 294 000 <210> SEQ ID NO 295 <400> SEQUENCE:
295 000 <210> SEQ ID NO 296 <400> SEQUENCE: 296 000
<210> SEQ ID NO 297 <400> SEQUENCE: 297 000 <210>
SEQ ID NO 298 <400> SEQUENCE: 298 000 <210> SEQ ID NO
299 <400> SEQUENCE: 299 000 <210> SEQ ID NO 300
<400> SEQUENCE: 300 000 <210> SEQ ID NO 301 <400>
SEQUENCE: 301 000 <210> SEQ ID NO 302 <400> SEQUENCE:
302 000 <210> SEQ ID NO 303 <400> SEQUENCE: 303 000
<210> SEQ ID NO 304 <400> SEQUENCE: 304 000 <210>
SEQ ID NO 305 <400> SEQUENCE: 305 000 <210> SEQ ID NO
306 <400> SEQUENCE: 306 000 <210> SEQ ID NO 307
<400> SEQUENCE: 307 000 <210> SEQ ID NO 308 <400>
SEQUENCE: 308 000 <210> SEQ ID NO 309 <400> SEQUENCE:
309 000 <210> SEQ ID NO 310 <400> SEQUENCE: 310 000
<210> SEQ ID NO 311 <400> SEQUENCE: 311 000 <210>
SEQ ID NO 312 <400> SEQUENCE: 312 000 <210> SEQ ID NO
313 <400> SEQUENCE: 313 000 <210> SEQ ID NO 314
<400> SEQUENCE: 314 000 <210> SEQ ID NO 315 <400>
SEQUENCE: 315 000 <210> SEQ ID NO 316 <400> SEQUENCE:
316 000 <210> SEQ ID NO 317 <400> SEQUENCE: 317 000
<210> SEQ ID NO 318 <400> SEQUENCE: 318 000 <210>
SEQ ID NO 319 <400> SEQUENCE: 319 000 <210> SEQ ID NO
320 <400> SEQUENCE: 320 000 <210> SEQ ID NO 321
<400> SEQUENCE: 321 000 <210> SEQ ID NO 322 <400>
SEQUENCE: 322 000 <210> SEQ ID NO 323 <400> SEQUENCE:
323 000 <210> SEQ ID NO 324 <400> SEQUENCE: 324 000
<210> SEQ ID NO 325 <400> SEQUENCE: 325 000 <210>
SEQ ID NO 326 <400> SEQUENCE: 326 000 <210> SEQ ID NO
327 <400> SEQUENCE: 327 000 <210> SEQ ID NO 328
<400> SEQUENCE: 328 000 <210> SEQ ID NO 329 <400>
SEQUENCE: 329 000 <210> SEQ ID NO 330 <400> SEQUENCE:
330 000 <210> SEQ ID NO 331 <400> SEQUENCE: 331 000
<210> SEQ ID NO 332 <400> SEQUENCE: 332 000 <210>
SEQ ID NO 333 <400> SEQUENCE: 333 000 <210> SEQ ID NO
334 <400> SEQUENCE: 334 000 <210> SEQ ID NO 335
<400> SEQUENCE: 335 000 <210> SEQ ID NO 336 <400>
SEQUENCE: 336 000 <210> SEQ ID NO 337 <400> SEQUENCE:
337 000 <210> SEQ ID NO 338 <400> SEQUENCE: 338 000
<210> SEQ ID NO 339 <400> SEQUENCE: 339 000 <210>
SEQ ID NO 340 <400> SEQUENCE: 340 000 <210> SEQ ID NO
341 <400> SEQUENCE: 341 000 <210> SEQ ID NO 342
<400> SEQUENCE: 342 000 <210> SEQ ID NO 343 <400>
SEQUENCE: 343 000 <210> SEQ ID NO 344 <400> SEQUENCE:
344 000 <210> SEQ ID NO 345 <400> SEQUENCE: 345 000
<210> SEQ ID NO 346 <400> SEQUENCE: 346 000 <210>
SEQ ID NO 347 <400> SEQUENCE: 347 000 <210> SEQ ID NO
348 <400> SEQUENCE: 348 000 <210> SEQ ID NO 349
<400> SEQUENCE: 349 000 <210> SEQ ID NO 350 <400>
SEQUENCE: 350 000 <210> SEQ ID NO 351 <400> SEQUENCE:
351 000 <210> SEQ ID NO 352 <400> SEQUENCE: 352 000
<210> SEQ ID NO 353 <400> SEQUENCE: 353 000 <210>
SEQ ID NO 354 <400> SEQUENCE: 354 000 <210> SEQ ID NO
355 <400> SEQUENCE: 355 000 <210> SEQ ID NO 356
<400> SEQUENCE: 356 000 <210> SEQ ID NO 357 <400>
SEQUENCE: 357 000 <210> SEQ ID NO 358 <400> SEQUENCE:
358 000 <210> SEQ ID NO 359 <400> SEQUENCE: 359 000
<210> SEQ ID NO 360 <400> SEQUENCE: 360 000 <210>
SEQ ID NO 361 <400> SEQUENCE: 361 000 <210> SEQ ID NO
362 <400> SEQUENCE: 362 000 <210> SEQ ID NO 363
<400> SEQUENCE: 363 000 <210> SEQ ID NO 364 <400>
SEQUENCE: 364 000 <210> SEQ ID NO 365 <400> SEQUENCE:
365 000 <210> SEQ ID NO 366 <400> SEQUENCE: 366 000
<210> SEQ ID NO 367 <400> SEQUENCE: 367 000 <210>
SEQ ID NO 368 <400> SEQUENCE: 368 000 <210> SEQ ID NO
369 <400> SEQUENCE: 369 000 <210> SEQ ID NO 370
<400> SEQUENCE: 370 000 <210> SEQ ID NO 371 <400>
SEQUENCE: 371 000 <210> SEQ ID NO 372 <400> SEQUENCE:
372 000 <210> SEQ ID NO 373 <400> SEQUENCE: 373 000
<210> SEQ ID NO 374 <400> SEQUENCE: 374 000 <210>
SEQ ID NO 375 <400> SEQUENCE: 375 000 <210> SEQ ID NO
376 <400> SEQUENCE: 376 000 <210> SEQ ID NO 377
<400> SEQUENCE: 377 000 <210> SEQ ID NO 378 <400>
SEQUENCE: 378 000 <210> SEQ ID NO 379 <400> SEQUENCE:
379 000 <210> SEQ ID NO 380 <400> SEQUENCE: 380 000
<210> SEQ ID NO 381 <400> SEQUENCE: 381 000 <210>
SEQ ID NO 382 <400> SEQUENCE: 382 000 <210> SEQ ID NO
383 <400> SEQUENCE: 383 000 <210> SEQ ID NO 384
<400> SEQUENCE: 384 000 <210> SEQ ID NO 385 <400>
SEQUENCE: 385 000 <210> SEQ ID NO 386 <400> SEQUENCE:
386 000 <210> SEQ ID NO 387 <400> SEQUENCE: 387 000
<210> SEQ ID NO 388 <400> SEQUENCE: 388 000 <210>
SEQ ID NO 389 <400> SEQUENCE: 389 000 <210> SEQ ID NO
390 <400> SEQUENCE: 390 000 <210> SEQ ID NO 391
<400> SEQUENCE: 391 000 <210> SEQ ID NO 392 <400>
SEQUENCE: 392 000 <210> SEQ ID NO 393 <400> SEQUENCE:
393 000 <210> SEQ ID NO 394 <400> SEQUENCE: 394 000
<210> SEQ ID NO 395 <400> SEQUENCE: 395 000 <210>
SEQ ID NO 396 <400> SEQUENCE: 396 000 <210> SEQ ID NO
397 <400> SEQUENCE: 397 000 <210> SEQ ID NO 398
<400> SEQUENCE: 398 000 <210> SEQ ID NO 399 <400>
SEQUENCE: 399 000 <210> SEQ ID NO 400 <400> SEQUENCE:
400 000 <210> SEQ ID NO 401 <400> SEQUENCE: 401 000
<210> SEQ ID NO 402 <400> SEQUENCE: 402 000 <210>
SEQ ID NO 403 <400> SEQUENCE: 403 000 <210> SEQ ID NO
404 <400> SEQUENCE: 404 000 <210> SEQ ID NO 405
<400> SEQUENCE: 405 000 <210> SEQ ID NO 406 <400>
SEQUENCE: 406 000 <210> SEQ ID NO 407 <400> SEQUENCE:
407 000 <210> SEQ ID NO 408 <400> SEQUENCE: 408 000
<210> SEQ ID NO 409 <400> SEQUENCE: 409 000 <210>
SEQ ID NO 410 <400> SEQUENCE: 410 000 <210> SEQ ID NO
411 <400> SEQUENCE: 411 000 <210> SEQ ID NO 412
<400> SEQUENCE: 412 000 <210> SEQ ID NO 413 <400>
SEQUENCE: 413 000 <210> SEQ ID NO 414 <400> SEQUENCE:
414 000 <210> SEQ ID NO 415 <400> SEQUENCE: 415 000
<210> SEQ ID NO 416 <400> SEQUENCE: 416 000 <210>
SEQ ID NO 417 <400> SEQUENCE: 417 000 <210> SEQ ID NO
418 <400> SEQUENCE: 418 000 <210> SEQ ID NO 419
<400> SEQUENCE: 419 000 <210> SEQ ID NO 420 <400>
SEQUENCE: 420 000 <210> SEQ ID NO 421 <400> SEQUENCE:
421 000 <210> SEQ ID NO 422 <400> SEQUENCE: 422 000
<210> SEQ ID NO 423 <400> SEQUENCE: 423 000 <210>
SEQ ID NO 424 <400> SEQUENCE: 424 000 <210> SEQ ID NO
425 <400> SEQUENCE: 425 000 <210> SEQ ID NO 426
<400> SEQUENCE: 426 000 <210> SEQ ID NO 427 <400>
SEQUENCE: 427 000 <210> SEQ ID NO 428 <400> SEQUENCE:
428 000 <210> SEQ ID NO 429 <400> SEQUENCE: 429 000
<210> SEQ ID NO 430 <400> SEQUENCE: 430 000 <210>
SEQ ID NO 431 <400> SEQUENCE: 431 000 <210> SEQ ID NO
432 <400> SEQUENCE: 432 000 <210> SEQ ID NO 433
<400> SEQUENCE: 433 000 <210> SEQ ID NO 434 <400>
SEQUENCE: 434 000 <210> SEQ ID NO 435 <400> SEQUENCE:
435 000 <210> SEQ ID NO 436 <400> SEQUENCE: 436 000
<210> SEQ ID NO 437 <400> SEQUENCE: 437 000 <210>
SEQ ID NO 438 <400> SEQUENCE: 438 000 <210> SEQ ID NO
439 <400> SEQUENCE: 439 000 <210> SEQ ID NO 440
<400> SEQUENCE: 440 000 <210> SEQ ID NO 441 <400>
SEQUENCE: 441 000 <210> SEQ ID NO 442 <400> SEQUENCE:
442 000 <210> SEQ ID NO 443 <400> SEQUENCE: 443 000
<210> SEQ ID NO 444 <400> SEQUENCE: 444 000 <210>
SEQ ID NO 445 <400> SEQUENCE: 445 000 <210> SEQ ID NO
446 <400> SEQUENCE: 446 000 <210> SEQ ID NO 447
<400> SEQUENCE: 447 000 <210> SEQ ID NO 448 <400>
SEQUENCE: 448 000 <210> SEQ ID NO 449 <400> SEQUENCE:
449 000 <210> SEQ ID NO 450 <400> SEQUENCE: 450 000
<210> SEQ ID NO 451 <400> SEQUENCE: 451 000 <210>
SEQ ID NO 452 <400> SEQUENCE: 452 000 <210> SEQ ID NO
453 <400> SEQUENCE: 453 000 <210> SEQ ID NO 454
<400> SEQUENCE: 454 000 <210> SEQ ID NO 455 <400>
SEQUENCE: 455 000 <210> SEQ ID NO 456 <400> SEQUENCE:
456 000 <210> SEQ ID NO 457 <400> SEQUENCE: 457 000
<210> SEQ ID NO 458 <400> SEQUENCE: 458 000 <210>
SEQ ID NO 459 <400> SEQUENCE: 459 000 <210> SEQ ID NO
460 <400> SEQUENCE: 460 000 <210> SEQ ID NO 461
<400> SEQUENCE: 461 000 <210> SEQ ID NO 462 <400>
SEQUENCE: 462 000 <210> SEQ ID NO 463 <400> SEQUENCE:
463 000 <210> SEQ ID NO 464 <400> SEQUENCE: 464 000
<210> SEQ ID NO 465 <400> SEQUENCE: 465 000 <210>
SEQ ID NO 466 <400> SEQUENCE: 466 000 <210> SEQ ID NO
467 <400> SEQUENCE: 467 000 <210> SEQ ID NO 468
<400> SEQUENCE: 468 000 <210> SEQ ID NO 469 <400>
SEQUENCE: 469 000 <210> SEQ ID NO 470 <400> SEQUENCE:
470 000 <210> SEQ ID NO 471 <400> SEQUENCE: 471 000
<210> SEQ ID NO 472 <400> SEQUENCE: 472 000 <210>
SEQ ID NO 473 <400> SEQUENCE: 473 000 <210> SEQ ID NO
474 <400> SEQUENCE: 474 000 <210> SEQ ID NO 475
<400> SEQUENCE: 475 000 <210> SEQ ID NO 476 <400>
SEQUENCE: 476 000 <210> SEQ ID NO 477 <400> SEQUENCE:
477 000 <210> SEQ ID NO 478 <400> SEQUENCE: 478 000
<210> SEQ ID NO 479 <400> SEQUENCE: 479 000 <210>
SEQ ID NO 480 <400> SEQUENCE: 480 000 <210> SEQ ID NO
481 <400> SEQUENCE: 481 000 <210> SEQ ID NO 482
<400> SEQUENCE: 482 000 <210> SEQ ID NO 483 <400>
SEQUENCE: 483 000 <210> SEQ ID NO 484 <400> SEQUENCE:
484 000 <210> SEQ ID NO 485 <400> SEQUENCE: 485 000
<210> SEQ ID NO 486 <400> SEQUENCE: 486 000 <210>
SEQ ID NO 487 <400> SEQUENCE: 487 000 <210> SEQ ID NO
488 <400> SEQUENCE: 488 000 <210> SEQ ID NO 489
<400> SEQUENCE: 489 000 <210> SEQ ID NO 490 <400>
SEQUENCE: 490 000 <210> SEQ ID NO 491 <400> SEQUENCE:
491 000 <210> SEQ ID NO 492 <400> SEQUENCE: 492 000
<210> SEQ ID NO 493 <400> SEQUENCE: 493 000 <210>
SEQ ID NO 494 <400> SEQUENCE: 494 000 <210> SEQ ID NO
495 <400> SEQUENCE: 495 000 <210> SEQ ID NO 496
<400> SEQUENCE: 496 000 <210> SEQ ID NO 497 <400>
SEQUENCE: 497 000 <210> SEQ ID NO 498 <400> SEQUENCE:
498 000 <210> SEQ ID NO 499 <400> SEQUENCE: 499 000
<210> SEQ ID NO 500 <400> SEQUENCE: 500 000 <210>
SEQ ID NO 501 <400> SEQUENCE: 501 000 <210> SEQ ID NO
502 <400> SEQUENCE: 502 000 <210> SEQ ID NO 503
<400> SEQUENCE: 503 000 <210> SEQ ID NO 504 <400>
SEQUENCE: 504 000 <210> SEQ ID NO 505 <400> SEQUENCE:
505 000 <210> SEQ ID NO 506 <400> SEQUENCE: 506 000
<210> SEQ ID NO 507 <400> SEQUENCE: 507 000 <210>
SEQ ID NO 508 <400> SEQUENCE: 508 000 <210> SEQ ID NO
509 <400> SEQUENCE: 509 000 <210> SEQ ID NO 510
<400> SEQUENCE: 510 000 <210> SEQ ID NO 511 <400>
SEQUENCE: 511 000 <210> SEQ ID NO 512 <400> SEQUENCE:
512 000 <210> SEQ ID NO 513 <400> SEQUENCE: 513 000
<210> SEQ ID NO 514 <400> SEQUENCE: 514 000 <210>
SEQ ID NO 515 <400> SEQUENCE: 515 000 <210> SEQ ID NO
516 <400> SEQUENCE: 516 000 <210> SEQ ID NO 517
<400> SEQUENCE: 517 000 <210> SEQ ID NO 518 <400>
SEQUENCE: 518 000 <210> SEQ ID NO 519 <400> SEQUENCE:
519 000 <210> SEQ ID NO 520 <400> SEQUENCE: 520 000
<210> SEQ ID NO 521 <400> SEQUENCE: 521 000 <210>
SEQ ID NO 522 <400> SEQUENCE: 522 000 <210> SEQ ID NO
523 <400> SEQUENCE: 523 000 <210> SEQ ID NO 524
<400> SEQUENCE: 524 000 <210> SEQ ID NO 525 <400>
SEQUENCE: 525 000 <210> SEQ ID NO 526 <400> SEQUENCE:
526 000 <210> SEQ ID NO 527 <400> SEQUENCE: 527 000
<210> SEQ ID NO 528 <400> SEQUENCE: 528 000 <210>
SEQ ID NO 529 <400> SEQUENCE: 529 000 <210> SEQ ID NO
530 <400> SEQUENCE: 530 000 <210> SEQ ID NO 531
<400> SEQUENCE: 531 000 <210> SEQ ID NO 532 <400>
SEQUENCE: 532 000 <210> SEQ ID NO 533 <400> SEQUENCE:
533 000 <210> SEQ ID NO 534 <400> SEQUENCE: 534 000
<210> SEQ ID NO 535 <400> SEQUENCE: 535 000 <210>
SEQ ID NO 536 <400> SEQUENCE: 536 000 <210> SEQ ID NO
537 <400> SEQUENCE: 537 000 <210> SEQ ID NO 538
<400> SEQUENCE: 538 000 <210> SEQ ID NO 539 <400>
SEQUENCE: 539 000 <210> SEQ ID NO 540 <400> SEQUENCE:
540 000 <210> SEQ ID NO 541 <400> SEQUENCE: 541 000
<210> SEQ ID NO 542 <400> SEQUENCE: 542 000 <210>
SEQ ID NO 543 <400> SEQUENCE: 543 000 <210> SEQ ID NO
544 <400> SEQUENCE: 544 000 <210> SEQ ID NO 545
<400> SEQUENCE: 545 000 <210> SEQ ID NO 546 <400>
SEQUENCE: 546 000 <210> SEQ ID NO 547 <400> SEQUENCE:
547 000 <210> SEQ ID NO 548 <400> SEQUENCE: 548 000
<210> SEQ ID NO 549 <400> SEQUENCE: 549 000 <210>
SEQ ID NO 550 <400> SEQUENCE: 550 000 <210> SEQ ID NO
551 <400> SEQUENCE: 551 000 <210> SEQ ID NO 552
<400> SEQUENCE: 552 000 <210> SEQ ID NO 553 <400>
SEQUENCE: 553 000 <210> SEQ ID NO 554 <400> SEQUENCE:
554 000 <210> SEQ ID NO 555 <400> SEQUENCE: 555 000
<210> SEQ ID NO 556 <400> SEQUENCE: 556 000 <210>
SEQ ID NO 557 <400> SEQUENCE: 557 000 <210> SEQ ID NO
558 <400> SEQUENCE: 558 000 <210> SEQ ID NO 559
<400> SEQUENCE: 559 000 <210> SEQ ID NO 560 <400>
SEQUENCE: 560 000 <210> SEQ ID NO 561 <400> SEQUENCE:
561 000 <210> SEQ ID NO 562 <400> SEQUENCE: 562 000
<210> SEQ ID NO 563 <400> SEQUENCE: 563 000 <210>
SEQ ID NO 564 <400> SEQUENCE: 564 000 <210> SEQ ID NO
565 <400> SEQUENCE: 565 000 <210> SEQ ID NO 566
<400> SEQUENCE: 566 000 <210> SEQ ID NO 567 <400>
SEQUENCE: 567 000 <210> SEQ ID NO 568 <400> SEQUENCE:
568 000 <210> SEQ ID NO 569 <400> SEQUENCE: 569 000
<210> SEQ ID NO 570 <400> SEQUENCE: 570 000 <210>
SEQ ID NO 571 <400> SEQUENCE: 571 000 <210> SEQ ID NO
572 <400> SEQUENCE: 572 000 <210> SEQ ID NO 573
<400> SEQUENCE: 573 000 <210> SEQ ID NO 574 <400>
SEQUENCE: 574 000 <210> SEQ ID NO 575 <400> SEQUENCE:
575 000 <210> SEQ ID NO 576 <400> SEQUENCE: 576 000
<210> SEQ ID NO 577 <400> SEQUENCE: 577 000 <210>
SEQ ID NO 578 <400> SEQUENCE: 578 000 <210> SEQ ID NO
579 <400> SEQUENCE: 579 000 <210> SEQ ID NO 580
<400> SEQUENCE: 580 000 <210> SEQ ID NO 581 <400>
SEQUENCE: 581 000 <210> SEQ ID NO 582 <400> SEQUENCE:
582 000 <210> SEQ ID NO 583 <400> SEQUENCE: 583 000
<210> SEQ ID NO 584 <400> SEQUENCE: 584 000 <210>
SEQ ID NO 585 <400> SEQUENCE: 585 000 <210> SEQ ID NO
586 <400> SEQUENCE: 586 000 <210> SEQ ID NO 587
<400> SEQUENCE: 587 000 <210> SEQ ID NO 588 <400>
SEQUENCE: 588 000 <210> SEQ ID NO 589 <400> SEQUENCE:
589 000 <210> SEQ ID NO 590 <400> SEQUENCE: 590 000
<210> SEQ ID NO 591 <400> SEQUENCE: 591 000 <210>
SEQ ID NO 592 <400> SEQUENCE: 592 000 <210> SEQ ID NO
593 <400> SEQUENCE: 593 000 <210> SEQ ID NO 594
<400> SEQUENCE: 594 000 <210> SEQ ID NO 595 <400>
SEQUENCE: 595 000 <210> SEQ ID NO 596 <400> SEQUENCE:
596 000 <210> SEQ ID NO 597 <400> SEQUENCE: 597 000
<210> SEQ ID NO 598 <400> SEQUENCE: 598 000 <210>
SEQ ID NO 599 <400> SEQUENCE: 599 000 <210> SEQ ID NO
600 <400> SEQUENCE: 600 000 <210> SEQ ID NO 601
<400> SEQUENCE: 601 000 <210> SEQ ID NO 602 <400>
SEQUENCE: 602 000 <210> SEQ ID NO 603 <400> SEQUENCE:
603 000 <210> SEQ ID NO 604 <400> SEQUENCE: 604 000
<210> SEQ ID NO 605 <400> SEQUENCE: 605 000 <210>
SEQ ID NO 606 <400> SEQUENCE: 606 000 <210> SEQ ID NO
607 <400> SEQUENCE: 607 000 <210> SEQ ID NO 608
<400> SEQUENCE: 608 000 <210> SEQ ID NO 609 <400>
SEQUENCE: 609 000 <210> SEQ ID NO 610 <400> SEQUENCE:
610 000 <210> SEQ ID NO 611 <400> SEQUENCE: 611 000
<210> SEQ ID NO 612 <400> SEQUENCE: 612 000 <210>
SEQ ID NO 613 <400> SEQUENCE: 613 000 <210> SEQ ID NO
614 <400> SEQUENCE: 614 000 <210> SEQ ID NO 615
<400> SEQUENCE: 615 000 <210> SEQ ID NO 616 <400>
SEQUENCE: 616 000 <210> SEQ ID NO 617 <400> SEQUENCE:
617 000 <210> SEQ ID NO 618 <400> SEQUENCE: 618 000
<210> SEQ ID NO 619 <400> SEQUENCE: 619 000 <210>
SEQ ID NO 620 <400> SEQUENCE: 620 000 <210> SEQ ID NO
621 <400> SEQUENCE: 621 000 <210> SEQ ID NO 622
<400> SEQUENCE: 622 000 <210> SEQ ID NO 623 <400>
SEQUENCE: 623 000 <210> SEQ ID NO 624 <400> SEQUENCE:
624 000 <210> SEQ ID NO 625 <400> SEQUENCE: 625 000
<210> SEQ ID NO 626 <400> SEQUENCE: 626 000 <210>
SEQ ID NO 627 <400> SEQUENCE: 627 000 <210> SEQ ID NO
628 <400> SEQUENCE: 628 000 <210> SEQ ID NO 629
<400> SEQUENCE: 629 000 <210> SEQ ID NO 630 <400>
SEQUENCE: 630 000 <210> SEQ ID NO 631 <400> SEQUENCE:
631 000 <210> SEQ ID NO 632 <400> SEQUENCE: 632 000
<210> SEQ ID NO 633 <400> SEQUENCE: 633 000 <210>
SEQ ID NO 634 <400> SEQUENCE: 634 000 <210> SEQ ID NO
635 <400> SEQUENCE: 635 000 <210> SEQ ID NO 636
<400> SEQUENCE: 636 000 <210> SEQ ID NO 637 <400>
SEQUENCE: 637 000 <210> SEQ ID NO 638 <400> SEQUENCE:
638 000 <210> SEQ ID NO 639 <400> SEQUENCE: 639 000
<210> SEQ ID NO 640 <400> SEQUENCE: 640 000 <210>
SEQ ID NO 641 <400> SEQUENCE: 641 000 <210> SEQ ID NO
642 <400> SEQUENCE: 642 000 <210> SEQ ID NO 643
<400> SEQUENCE: 643 000 <210> SEQ ID NO 644 <400>
SEQUENCE: 644 000 <210> SEQ ID NO 645 <400> SEQUENCE:
645 000 <210> SEQ ID NO 646 <400> SEQUENCE: 646 000
<210> SEQ ID NO 647 <400> SEQUENCE: 647 000 <210>
SEQ ID NO 648 <400> SEQUENCE: 648 000 <210> SEQ ID NO
649 <400> SEQUENCE: 649 000 <210> SEQ ID NO 650
<400> SEQUENCE: 650 000 <210> SEQ ID NO 651 <400>
SEQUENCE: 651 000 <210> SEQ ID NO 652 <400> SEQUENCE:
652 000 <210> SEQ ID NO 653 <400> SEQUENCE: 653 000
<210> SEQ ID NO 654 <400> SEQUENCE: 654 000 <210>
SEQ ID NO 655 <400> SEQUENCE: 655 000 <210> SEQ ID NO
656 <400> SEQUENCE: 656 000 <210> SEQ ID NO 657
<400> SEQUENCE: 657 000 <210> SEQ ID NO 658 <400>
SEQUENCE: 658 000 <210> SEQ ID NO 659 <400> SEQUENCE:
659 000 <210> SEQ ID NO 660 <400> SEQUENCE: 660 000
<210> SEQ ID NO 661 <400> SEQUENCE: 661 000 <210>
SEQ ID NO 662 <400> SEQUENCE: 662 000 <210> SEQ ID NO
663 <400> SEQUENCE: 663 000 <210> SEQ ID NO 664
<400> SEQUENCE: 664 000 <210> SEQ ID NO 665 <400>
SEQUENCE: 665 000 <210> SEQ ID NO 666 <400> SEQUENCE:
666 000 <210> SEQ ID NO 667 <400> SEQUENCE: 667 000
<210> SEQ ID NO 668 <400> SEQUENCE: 668 000 <210>
SEQ ID NO 669 <400> SEQUENCE: 669 000 <210> SEQ ID NO
670 <400> SEQUENCE: 670 000 <210> SEQ ID NO 671
<400> SEQUENCE: 671 000 <210> SEQ ID NO 672 <400>
SEQUENCE: 672 000 <210> SEQ ID NO 673 <400> SEQUENCE:
673 000 <210> SEQ ID NO 674 <400> SEQUENCE: 674 000
<210> SEQ ID NO 675 <400> SEQUENCE: 675 000 <210>
SEQ ID NO 676 <400> SEQUENCE: 676 000 <210> SEQ ID NO
677 <400> SEQUENCE: 677 000 <210> SEQ ID NO 678
<400> SEQUENCE: 678 000 <210> SEQ ID NO 679 <400>
SEQUENCE: 679 000 <210> SEQ ID NO 680 <400> SEQUENCE:
680 000 <210> SEQ ID NO 681 <400> SEQUENCE: 681 000
<210> SEQ ID NO 682 <400> SEQUENCE: 682 000 <210>
SEQ ID NO 683 <400> SEQUENCE: 683 000 <210> SEQ ID NO
684 <400> SEQUENCE: 684 000 <210> SEQ ID NO 685
<400> SEQUENCE: 685 000 <210> SEQ ID NO 686 <400>
SEQUENCE: 686 000 <210> SEQ ID NO 687 <400> SEQUENCE:
687 000 <210> SEQ ID NO 688 <400> SEQUENCE: 688 000
<210> SEQ ID NO 689 <400> SEQUENCE: 689 000 <210>
SEQ ID NO 690 <400> SEQUENCE: 690 000 <210> SEQ ID NO
691 <400> SEQUENCE: 691 000 <210> SEQ ID NO 692
<400> SEQUENCE: 692 000 <210> SEQ ID NO 693 <400>
SEQUENCE: 693 000 <210> SEQ ID NO 694 <400> SEQUENCE:
694 000 <210> SEQ ID NO 695 <400> SEQUENCE: 695 000
<210> SEQ ID NO 696 <400> SEQUENCE: 696 000 <210>
SEQ ID NO 697 <400> SEQUENCE: 697 000 <210> SEQ ID NO
698 <400> SEQUENCE: 698 000 <210> SEQ ID NO 699
<400> SEQUENCE: 699 000 <210> SEQ ID NO 700 <400>
SEQUENCE: 700 000 <210> SEQ ID NO 701 <400> SEQUENCE:
701 000 <210> SEQ ID NO 702 <400> SEQUENCE: 702 000
<210> SEQ ID NO 703 <400> SEQUENCE: 703 000 <210>
SEQ ID NO 704 <400> SEQUENCE: 704 000 <210> SEQ ID NO
705 <400> SEQUENCE: 705 000 <210> SEQ ID NO 706
<400> SEQUENCE: 706 000 <210> SEQ ID NO 707 <400>
SEQUENCE: 707 000 <210> SEQ ID NO 708 <400> SEQUENCE:
708 000 <210> SEQ ID NO 709 <400> SEQUENCE: 709 000
<210> SEQ ID NO 710 <400> SEQUENCE: 710 000 <210>
SEQ ID NO 711 <400> SEQUENCE: 711 000 <210> SEQ ID NO
712 <400> SEQUENCE: 712 000 <210> SEQ ID NO 713
<400> SEQUENCE: 713 000 <210> SEQ ID NO 714 <400>
SEQUENCE: 714 000 <210> SEQ ID NO 715 <400> SEQUENCE:
715 000 <210> SEQ ID NO 716 <400> SEQUENCE: 716 000
<210> SEQ ID NO 717 <400> SEQUENCE: 717 000 <210>
SEQ ID NO 718 <400> SEQUENCE: 718 000 <210> SEQ ID NO
719 <400> SEQUENCE: 719 000 <210> SEQ ID NO 720
<400> SEQUENCE: 720 000 <210> SEQ ID NO 721 <400>
SEQUENCE: 721 000 <210> SEQ ID NO 722 <400> SEQUENCE:
722 000 <210> SEQ ID NO 723 <400> SEQUENCE: 723 000
<210> SEQ ID NO 724 <400> SEQUENCE: 724 000 <210>
SEQ ID NO 725 <400> SEQUENCE: 725 000 <210> SEQ ID NO
726 <400> SEQUENCE: 726 000 <210> SEQ ID NO 727
<400> SEQUENCE: 727 000 <210> SEQ ID NO 728 <400>
SEQUENCE: 728 000 <210> SEQ ID NO 729 <400> SEQUENCE:
729 000 <210> SEQ ID NO 730 <400> SEQUENCE: 730 000
<210> SEQ ID NO 731 <400> SEQUENCE: 731 000 <210>
SEQ ID NO 732 <400> SEQUENCE: 732 000 <210> SEQ ID NO
733 <400> SEQUENCE: 733 000 <210> SEQ ID NO 734
<400> SEQUENCE: 734 000 <210> SEQ ID NO 735 <400>
SEQUENCE: 735 000 <210> SEQ ID NO 736 <400> SEQUENCE:
736 000 <210> SEQ ID NO 737 <400> SEQUENCE: 737 000
<210> SEQ ID NO 738 <400> SEQUENCE: 738 000 <210>
SEQ ID NO 739 <400> SEQUENCE: 739 000 <210> SEQ ID NO
740 <400> SEQUENCE: 740 000 <210> SEQ ID NO 741
<400> SEQUENCE: 741 000 <210> SEQ ID NO 742 <400>
SEQUENCE: 742 000 <210> SEQ ID NO 743 <400> SEQUENCE:
743 000 <210> SEQ ID NO 744 <400> SEQUENCE: 744 000
<210> SEQ ID NO 745 <400> SEQUENCE: 745 000 <210>
SEQ ID NO 746 <400> SEQUENCE: 746 000 <210> SEQ ID NO
747 <400> SEQUENCE: 747 000 <210> SEQ ID NO 748
<400> SEQUENCE: 748 000 <210> SEQ ID NO 749 <400>
SEQUENCE: 749 000 <210> SEQ ID NO 750 <400> SEQUENCE:
750 000 <210> SEQ ID NO 751 <400> SEQUENCE: 751 000
<210> SEQ ID NO 752 <400> SEQUENCE: 752 000 <210>
SEQ ID NO 753 <400> SEQUENCE: 753 000 <210> SEQ ID NO
754 <400> SEQUENCE: 754 000 <210> SEQ ID NO 755
<400> SEQUENCE: 755 000 <210> SEQ ID NO 756 <400>
SEQUENCE: 756 000 <210> SEQ ID NO 757 <400> SEQUENCE:
757 000 <210> SEQ ID NO 758 <400> SEQUENCE: 758 000
<210> SEQ ID NO 759 <400> SEQUENCE: 759 000 <210>
SEQ ID NO 760 <400> SEQUENCE: 760 000 <210> SEQ ID NO
761 <400> SEQUENCE: 761 000 <210> SEQ ID NO 762
<400> SEQUENCE: 762 000 <210> SEQ ID NO 763 <400>
SEQUENCE: 763 000 <210> SEQ ID NO 764 <400> SEQUENCE:
764 000 <210> SEQ ID NO 765 <400> SEQUENCE: 765 000
<210> SEQ ID NO 766 <400> SEQUENCE: 766 000 <210>
SEQ ID NO 767 <400> SEQUENCE: 767 000 <210> SEQ ID NO
768 <400> SEQUENCE: 768 000 <210> SEQ ID NO 769
<400> SEQUENCE: 769 000 <210> SEQ ID NO 770 <400>
SEQUENCE: 770 000 <210> SEQ ID NO 771 <400> SEQUENCE:
771 000 <210> SEQ ID NO 772 <400> SEQUENCE: 772 000
<210> SEQ ID NO 773 <400> SEQUENCE: 773 000 <210>
SEQ ID NO 774 <400> SEQUENCE: 774 000 <210> SEQ ID NO
775 <400> SEQUENCE: 775 000 <210> SEQ ID NO 776
<400> SEQUENCE: 776 000 <210> SEQ ID NO 777 <400>
SEQUENCE: 777 000 <210> SEQ ID NO 778 <400> SEQUENCE:
778 000 <210> SEQ ID NO 779 <400> SEQUENCE: 779 000
<210> SEQ ID NO 780 <400> SEQUENCE: 780 000 <210>
SEQ ID NO 781 <400> SEQUENCE: 781 000 <210> SEQ ID NO
782 <400> SEQUENCE: 782 000 <210> SEQ ID NO 783
<400> SEQUENCE: 783 000 <210> SEQ ID NO 784 <400>
SEQUENCE: 784 000 <210> SEQ ID NO 785 <400> SEQUENCE:
785 000 <210> SEQ ID NO 786 <400> SEQUENCE: 786 000
<210> SEQ ID NO 787 <400> SEQUENCE: 787 000 <210>
SEQ ID NO 788 <400> SEQUENCE: 788 000 <210> SEQ ID NO
789 <400> SEQUENCE: 789 000 <210> SEQ ID NO 790
<400> SEQUENCE: 790 000 <210> SEQ ID NO 791 <400>
SEQUENCE: 791 000 <210> SEQ ID NO 792 <400> SEQUENCE:
792 000 <210> SEQ ID NO 793 <400> SEQUENCE: 793 000
<210> SEQ ID NO 794 <400> SEQUENCE: 794 000 <210>
SEQ ID NO 795 <400> SEQUENCE: 795 000 <210> SEQ ID NO
796 <400> SEQUENCE: 796 000 <210> SEQ ID NO 797
<400> SEQUENCE: 797 000 <210> SEQ ID NO 798 <400>
SEQUENCE: 798 000 <210> SEQ ID NO 799 <400> SEQUENCE:
799 000 <210> SEQ ID NO 800 <400> SEQUENCE: 800 000
<210> SEQ ID NO 801 <400> SEQUENCE: 801 000 <210>
SEQ ID NO 802 <400> SEQUENCE: 802 000 <210> SEQ ID NO
803 <400> SEQUENCE: 803 000 <210> SEQ ID NO 804
<400> SEQUENCE: 804 000 <210> SEQ ID NO 805 <400>
SEQUENCE: 805 000 <210> SEQ ID NO 806 <400> SEQUENCE:
806 000 <210> SEQ ID NO 807 <400> SEQUENCE: 807 000
<210> SEQ ID NO 808 <400> SEQUENCE: 808 000 <210>
SEQ ID NO 809 <400> SEQUENCE: 809 000 <210> SEQ ID NO
810 <400> SEQUENCE: 810 000 <210> SEQ ID NO 811
<400> SEQUENCE: 811 000 <210> SEQ ID NO 812 <400>
SEQUENCE: 812 000 <210> SEQ ID NO 813 <400> SEQUENCE:
813 000 <210> SEQ ID NO 814 <400> SEQUENCE: 814 000
<210> SEQ ID NO 815 <400> SEQUENCE: 815 000 <210>
SEQ ID NO 816 <400> SEQUENCE: 816 000 <210> SEQ ID NO
817 <400> SEQUENCE: 817 000 <210> SEQ ID NO 818
<400> SEQUENCE: 818 000 <210> SEQ ID NO 819 <400>
SEQUENCE: 819 000 <210> SEQ ID NO 820 <400> SEQUENCE:
820 000 <210> SEQ ID NO 821 <400> SEQUENCE: 821 000
<210> SEQ ID NO 822 <400> SEQUENCE: 822 000 <210>
SEQ ID NO 823 <400> SEQUENCE: 823 000 <210> SEQ ID NO
824 <400> SEQUENCE: 824 000 <210> SEQ ID NO 825
<400> SEQUENCE: 825 000 <210> SEQ ID NO 826 <400>
SEQUENCE: 826 000 <210> SEQ ID NO 827 <400> SEQUENCE:
827 000 <210> SEQ ID NO 828 <400> SEQUENCE: 828 000
<210> SEQ ID NO 829 <400> SEQUENCE: 829 000 <210>
SEQ ID NO 830 <400> SEQUENCE: 830 000 <210> SEQ ID NO
831 <400> SEQUENCE: 831 000 <210> SEQ ID NO 832
<400> SEQUENCE: 832 000 <210> SEQ ID NO 833 <400>
SEQUENCE: 833 000 <210> SEQ ID NO 834 <400> SEQUENCE:
834 000 <210> SEQ ID NO 835 <400> SEQUENCE: 835 000
<210> SEQ ID NO 836 <400> SEQUENCE: 836 000 <210>
SEQ ID NO 837 <400> SEQUENCE: 837 000 <210> SEQ ID NO
838 <400> SEQUENCE: 838 000 <210> SEQ ID NO 839
<400> SEQUENCE: 839 000 <210> SEQ ID NO 840 <400>
SEQUENCE: 840 000 <210> SEQ ID NO 841 <400> SEQUENCE:
841 000 <210> SEQ ID NO 842 <400> SEQUENCE: 842 000
<210> SEQ ID NO 843 <400> SEQUENCE: 843 000 <210>
SEQ ID NO 844 <400> SEQUENCE: 844 000 <210> SEQ ID NO
845 <400> SEQUENCE: 845 000 <210> SEQ ID NO 846
<400> SEQUENCE: 846 000 <210> SEQ ID NO 847 <400>
SEQUENCE: 847 000 <210> SEQ ID NO 848 <400> SEQUENCE:
848 000 <210> SEQ ID NO 849 <400> SEQUENCE: 849 000
<210> SEQ ID NO 850 <400> SEQUENCE: 850 000 <210>
SEQ ID NO 851 <400> SEQUENCE: 851 000 <210> SEQ ID NO
852 <400> SEQUENCE: 852 000 <210> SEQ ID NO 853
<400> SEQUENCE: 853 000 <210> SEQ ID NO 854 <400>
SEQUENCE: 854 000 <210> SEQ ID NO 855 <400> SEQUENCE:
855 000 <210> SEQ ID NO 856 <400> SEQUENCE: 856 000
<210> SEQ ID NO 857 <400> SEQUENCE: 857 000 <210>
SEQ ID NO 858 <400> SEQUENCE: 858 000 <210> SEQ ID NO
859 <400> SEQUENCE: 859 000 <210> SEQ ID NO 860
<400> SEQUENCE: 860 000 <210> SEQ ID NO 861 <400>
SEQUENCE: 861 000 <210> SEQ ID NO 862 <400> SEQUENCE:
862 000 <210> SEQ ID NO 863 <400> SEQUENCE: 863 000
<210> SEQ ID NO 864 <400> SEQUENCE: 864 000 <210>
SEQ ID NO 865 <400> SEQUENCE: 865 000 <210> SEQ ID NO
866 <400> SEQUENCE: 866 000 <210> SEQ ID NO 867
<400> SEQUENCE: 867 000 <210> SEQ ID NO 868 <400>
SEQUENCE: 868 000 <210> SEQ ID NO 869 <400> SEQUENCE:
869 000 <210> SEQ ID NO 870 <400> SEQUENCE: 870 000
<210> SEQ ID NO 871 <400> SEQUENCE: 871 000 <210>
SEQ ID NO 872 <400> SEQUENCE: 872 000 <210> SEQ ID NO
873 <400> SEQUENCE: 873 000 <210> SEQ ID NO 874
<400> SEQUENCE: 874 000 <210> SEQ ID NO 875 <400>
SEQUENCE: 875 000 <210> SEQ ID NO 876 <400> SEQUENCE:
876 000 <210> SEQ ID NO 877 <400> SEQUENCE: 877 000
<210> SEQ ID NO 878 <400> SEQUENCE: 878 000 <210>
SEQ ID NO 879 <400> SEQUENCE: 879 000 <210> SEQ ID NO
880 <400> SEQUENCE: 880 000 <210> SEQ ID NO 881
<400> SEQUENCE: 881 000 <210> SEQ ID NO 882 <400>
SEQUENCE: 882 000 <210> SEQ ID NO 883 <400> SEQUENCE:
883 000 <210> SEQ ID NO 884 <400> SEQUENCE: 884 000
<210> SEQ ID NO 885 <400> SEQUENCE: 885 000 <210>
SEQ ID NO 886 <400> SEQUENCE: 886 000 <210> SEQ ID NO
887 <400> SEQUENCE: 887 000 <210> SEQ ID NO 888
<400> SEQUENCE: 888 000 <210> SEQ ID NO 889 <400>
SEQUENCE: 889 000 <210> SEQ ID NO 890 <400> SEQUENCE:
890 000 <210> SEQ ID NO 891 <400> SEQUENCE: 891 000
<210> SEQ ID NO 892 <400> SEQUENCE: 892 000 <210>
SEQ ID NO 893 <400> SEQUENCE: 893 000 <210> SEQ ID NO
894 <400> SEQUENCE: 894 000 <210> SEQ ID NO 895
<400> SEQUENCE: 895 000 <210> SEQ ID NO 896 <400>
SEQUENCE: 896 000 <210> SEQ ID NO 897 <400> SEQUENCE:
897 000 <210> SEQ ID NO 898 <400> SEQUENCE: 898 000
<210> SEQ ID NO 899 <400> SEQUENCE: 899 000 <210>
SEQ ID NO 900 <400> SEQUENCE: 900 000 <210> SEQ ID NO
901 <400> SEQUENCE: 901 000 <210> SEQ ID NO 902
<400> SEQUENCE: 902 000 <210> SEQ ID NO 903 <400>
SEQUENCE: 903 000 <210> SEQ ID NO 904 <400> SEQUENCE:
904 000 <210> SEQ ID NO 905 <400> SEQUENCE: 905 000
<210> SEQ ID NO 906 <400> SEQUENCE: 906 000 <210>
SEQ ID NO 907 <400> SEQUENCE: 907 000 <210> SEQ ID NO
908 <400> SEQUENCE: 908 000 <210> SEQ ID NO 909
<400> SEQUENCE: 909 000 <210> SEQ ID NO 910 <400>
SEQUENCE: 910 000 <210> SEQ ID NO 911 <400> SEQUENCE:
911 000 <210> SEQ ID NO 912 <400> SEQUENCE: 912 000
<210> SEQ ID NO 913 <400> SEQUENCE: 913 000 <210>
SEQ ID NO 914 <400> SEQUENCE: 914 000 <210> SEQ ID NO
915 <400> SEQUENCE: 915 000 <210> SEQ ID NO 916
<400> SEQUENCE: 916 000 <210> SEQ ID NO 917 <400>
SEQUENCE: 917 000 <210> SEQ ID NO 918 <400> SEQUENCE:
918 000 <210> SEQ ID NO 919 <400> SEQUENCE: 919 000
<210> SEQ ID NO 920 <400> SEQUENCE: 920 000 <210>
SEQ ID NO 921 <400> SEQUENCE: 921 000 <210> SEQ ID NO
922 <400> SEQUENCE: 922 000 <210> SEQ ID NO 923
<400> SEQUENCE: 923 000 <210> SEQ ID NO 924 <400>
SEQUENCE: 924 000 <210> SEQ ID NO 925 <400> SEQUENCE:
925 000 <210> SEQ ID NO 926 <400> SEQUENCE: 926 000
<210> SEQ ID NO 927 <400> SEQUENCE: 927 000 <210>
SEQ ID NO 928 <400> SEQUENCE: 928 000 <210> SEQ ID NO
929 <400> SEQUENCE: 929 000 <210> SEQ ID NO 930
<400> SEQUENCE: 930 000 <210> SEQ ID NO 931 <400>
SEQUENCE: 931 000 <210> SEQ ID NO 932 <400> SEQUENCE:
932 000 <210> SEQ ID NO 933 <400> SEQUENCE: 933 000
<210> SEQ ID NO 934 <400> SEQUENCE: 934 000 <210>
SEQ ID NO 935 <400> SEQUENCE: 935 000 <210> SEQ ID NO
936 <400> SEQUENCE: 936 000 <210> SEQ ID NO 937
<400> SEQUENCE: 937 000 <210> SEQ ID NO 938 <400>
SEQUENCE: 938 000 <210> SEQ ID NO 939 <400> SEQUENCE:
939 000 <210> SEQ ID NO 940 <400> SEQUENCE: 940 000
<210> SEQ ID NO 941 <400> SEQUENCE: 941 000 <210>
SEQ ID NO 942 <400> SEQUENCE: 942 000 <210> SEQ ID NO
943 <400> SEQUENCE: 943 000 <210> SEQ ID NO 944
<400> SEQUENCE: 944 000 <210> SEQ ID NO 945 <400>
SEQUENCE: 945 000 <210> SEQ ID NO 946 <400> SEQUENCE:
946 000 <210> SEQ ID NO 947 <400> SEQUENCE: 947 000
<210> SEQ ID NO 948 <400> SEQUENCE: 948 000 <210>
SEQ ID NO 949 <400> SEQUENCE: 949 000 <210> SEQ ID NO
950 <400> SEQUENCE: 950 000 <210> SEQ ID NO 951
<400> SEQUENCE: 951 000 <210> SEQ ID NO 952 <400>
SEQUENCE: 952 000 <210> SEQ ID NO 953 <400> SEQUENCE:
953 000 <210> SEQ ID NO 954 <400> SEQUENCE: 954 000
<210> SEQ ID NO 955 <400> SEQUENCE: 955 000 <210>
SEQ ID NO 956 <400> SEQUENCE: 956 000 <210> SEQ ID NO
957 <400> SEQUENCE: 957 000 <210> SEQ ID NO 958
<400> SEQUENCE: 958 000 <210> SEQ ID NO 959 <400>
SEQUENCE: 959 000 <210> SEQ ID NO 960 <400> SEQUENCE:
960 000 <210> SEQ ID NO 961 <400> SEQUENCE: 961 000
<210> SEQ ID NO 962 <400> SEQUENCE: 962 000 <210>
SEQ ID NO 963 <400> SEQUENCE: 963 000 <210> SEQ ID NO
964 <400> SEQUENCE: 964 000 <210> SEQ ID NO 965
<400> SEQUENCE: 965 000 <210> SEQ ID NO 966 <400>
SEQUENCE: 966 000 <210> SEQ ID NO 967 <400> SEQUENCE:
967 000 <210> SEQ ID NO 968 <400> SEQUENCE: 968 000
<210> SEQ ID NO 969 <400> SEQUENCE: 969 000 <210>
SEQ ID NO 970 <400> SEQUENCE: 970 000 <210> SEQ ID NO
971 <400> SEQUENCE: 971 000 <210> SEQ ID NO 972
<400> SEQUENCE: 972 000 <210> SEQ ID NO 973 <400>
SEQUENCE: 973 000 <210> SEQ ID NO 974 <400> SEQUENCE:
974 000 <210> SEQ ID NO 975 <400> SEQUENCE: 975 000
<210> SEQ ID NO 976 <400> SEQUENCE: 976 000 <210>
SEQ ID NO 977 <400> SEQUENCE: 977 000 <210> SEQ ID NO
978 <400> SEQUENCE: 978 000 <210> SEQ ID NO 979
<400> SEQUENCE: 979 000 <210> SEQ ID NO 980 <400>
SEQUENCE: 980 000 <210> SEQ ID NO 981 <400> SEQUENCE:
981 000 <210> SEQ ID NO 982 <400> SEQUENCE: 982 000
<210> SEQ ID NO 983 <400> SEQUENCE: 983 000 <210>
SEQ ID NO 984 <400> SEQUENCE: 984 000 <210> SEQ ID NO
985 <400> SEQUENCE: 985 000 <210> SEQ ID NO 986
<400> SEQUENCE: 986 000 <210> SEQ ID NO 987 <400>
SEQUENCE: 987 000 <210> SEQ ID NO 988 <400> SEQUENCE:
988 000 <210> SEQ ID NO 989 <400> SEQUENCE: 989 000
<210> SEQ ID NO 990 <400> SEQUENCE: 990 000 <210>
SEQ ID NO 991 <400> SEQUENCE: 991 000 <210> SEQ ID NO
992 <400> SEQUENCE: 992 000 <210> SEQ ID NO 993
<400> SEQUENCE: 993 000 <210> SEQ ID NO 994 <400>
SEQUENCE: 994 000 <210> SEQ ID NO 995 <400> SEQUENCE:
995 000 <210> SEQ ID NO 996 <400> SEQUENCE: 996 000
<210> SEQ ID NO 997 <400> SEQUENCE: 997 000 <210>
SEQ ID NO 998 <400> SEQUENCE: 998 000 <210> SEQ ID NO
999 <400> SEQUENCE: 999 000 <210> SEQ ID NO 1000
<400> SEQUENCE: 1000 000 <210> SEQ ID NO 1001
<400> SEQUENCE: 1001 000 <210> SEQ ID NO 1002
<400> SEQUENCE: 1002 000 <210> SEQ ID NO 1003
<400> SEQUENCE: 1003 000 <210> SEQ ID NO 1004
<400> SEQUENCE: 1004 000 <210> SEQ ID NO 1005
<400> SEQUENCE: 1005 000 <210> SEQ ID NO 1006
<400> SEQUENCE: 1006 000 <210> SEQ ID NO 1007
<400> SEQUENCE: 1007 000 <210> SEQ ID NO 1008
<400> SEQUENCE: 1008 000 <210> SEQ ID NO 1009
<400> SEQUENCE: 1009 000 <210> SEQ ID NO 1010
<400> SEQUENCE: 1010 000 <210> SEQ ID NO 1011
<400> SEQUENCE: 1011 000 <210> SEQ ID NO 1012
<400> SEQUENCE: 1012 000 <210> SEQ ID NO 1013
<400> SEQUENCE: 1013 000 <210> SEQ ID NO 1014
<400> SEQUENCE: 1014 000 <210> SEQ ID NO 1015
<400> SEQUENCE: 1015 000 <210> SEQ ID NO 1016
<400> SEQUENCE: 1016 000 <210> SEQ ID NO 1017
<400> SEQUENCE: 1017 000 <210> SEQ ID NO 1018
<400> SEQUENCE: 1018 000 <210> SEQ ID NO 1019
<400> SEQUENCE: 1019 000 <210> SEQ ID NO 1020
<400> SEQUENCE: 1020 000 <210> SEQ ID NO 1021
<400> SEQUENCE: 1021 000 <210> SEQ ID NO 1022
<400> SEQUENCE: 1022 000 <210> SEQ ID NO 1023
<400> SEQUENCE: 1023 000 <210> SEQ ID NO 1024
<400> SEQUENCE: 1024 000 <210> SEQ ID NO 1025
<400> SEQUENCE: 1025 000 <210> SEQ ID NO 1026
<400> SEQUENCE: 1026 000 <210> SEQ ID NO 1027
<400> SEQUENCE: 1027 000 <210> SEQ ID NO 1028
<400> SEQUENCE: 1028 000 <210> SEQ ID NO 1029
<400> SEQUENCE: 1029 000 <210> SEQ ID NO 1030
<400> SEQUENCE: 1030 000 <210> SEQ ID NO 1031
<400> SEQUENCE: 1031 000 <210> SEQ ID NO 1032
<400> SEQUENCE: 1032 000 <210> SEQ ID NO 1033
<400> SEQUENCE: 1033 000 <210> SEQ ID NO 1034
<400> SEQUENCE: 1034 000 <210> SEQ ID NO 1035
<400> SEQUENCE: 1035 000 <210> SEQ ID NO 1036
<400> SEQUENCE: 1036 000 <210> SEQ ID NO 1037
<400> SEQUENCE: 1037 000 <210> SEQ ID NO 1038
<400> SEQUENCE: 1038 000 <210> SEQ ID NO 1039
<400> SEQUENCE: 1039 000 <210> SEQ ID NO 1040
<400> SEQUENCE: 1040 000 <210> SEQ ID NO 1041
<400> SEQUENCE: 1041 000 <210> SEQ ID NO 1042
<400> SEQUENCE: 1042 000 <210> SEQ ID NO 1043
<400> SEQUENCE: 1043 000 <210> SEQ ID NO 1044
<400> SEQUENCE: 1044 000 <210> SEQ ID NO 1045
<400> SEQUENCE: 1045 000 <210> SEQ ID NO 1046
<400> SEQUENCE: 1046 000 <210> SEQ ID NO 1047
<400> SEQUENCE: 1047 000 <210> SEQ ID NO 1048
<400> SEQUENCE: 1048 000 <210> SEQ ID NO 1049
<400> SEQUENCE: 1049 000 <210> SEQ ID NO 1050
<400> SEQUENCE: 1050 000 <210> SEQ ID NO 1051
<400> SEQUENCE: 1051 000 <210> SEQ ID NO 1052
<400> SEQUENCE: 1052 000 <210> SEQ ID NO 1053
<400> SEQUENCE: 1053 000 <210> SEQ ID NO 1054
<400> SEQUENCE: 1054 000 <210> SEQ ID NO 1055
<400> SEQUENCE: 1055 000 <210> SEQ ID NO 1056
<400> SEQUENCE: 1056 000 <210> SEQ ID NO 1057
<400> SEQUENCE: 1057 000 <210> SEQ ID NO 1058
<400> SEQUENCE: 1058 000 <210> SEQ ID NO 1059
<400> SEQUENCE: 1059 000 <210> SEQ ID NO 1060
<400> SEQUENCE: 1060 000 <210> SEQ ID NO 1061
<400> SEQUENCE: 1061 000 <210> SEQ ID NO 1062
<400> SEQUENCE: 1062 000 <210> SEQ ID NO 1063
<400> SEQUENCE: 1063 000 <210> SEQ ID NO 1064
<400> SEQUENCE: 1064 000 <210> SEQ ID NO 1065
<400> SEQUENCE: 1065 000 <210> SEQ ID NO 1066
<400> SEQUENCE: 1066 000 <210> SEQ ID NO 1067
<400> SEQUENCE: 1067 000 <210> SEQ ID NO 1068
<400> SEQUENCE: 1068 000 <210> SEQ ID NO 1069
<400> SEQUENCE: 1069 000 <210> SEQ ID NO 1070
<400> SEQUENCE: 1070 000 <210> SEQ ID NO 1071
<400> SEQUENCE: 1071 000 <210> SEQ ID NO 1072
<400> SEQUENCE: 1072 000 <210> SEQ ID NO 1073
<400> SEQUENCE: 1073 000 <210> SEQ ID NO 1074
<400> SEQUENCE: 1074 000 <210> SEQ ID NO 1075
<400> SEQUENCE: 1075 000 <210> SEQ ID NO 1076
<400> SEQUENCE: 1076 000 <210> SEQ ID NO 1077
<400> SEQUENCE: 1077 000 <210> SEQ ID NO 1078
<400> SEQUENCE: 1078 000 <210> SEQ ID NO 1079
<400> SEQUENCE: 1079 000 <210> SEQ ID NO 1080
<400> SEQUENCE: 1080 000 <210> SEQ ID NO 1081
<400> SEQUENCE: 1081 000 <210> SEQ ID NO 1082
<400> SEQUENCE: 1082 000 <210> SEQ ID NO 1083
<400> SEQUENCE: 1083 000 <210> SEQ ID NO 1084
<400> SEQUENCE: 1084 000 <210> SEQ ID NO 1085
<400> SEQUENCE: 1085 000 <210> SEQ ID NO 1086
<400> SEQUENCE: 1086 000 <210> SEQ ID NO 1087
<400> SEQUENCE: 1087 000 <210> SEQ ID NO 1088
<400> SEQUENCE: 1088 000 <210> SEQ ID NO 1089
<400> SEQUENCE: 1089 000 <210> SEQ ID NO 1090
<400> SEQUENCE: 1090 000 <210> SEQ ID NO 1091
<400> SEQUENCE: 1091 000 <210> SEQ ID NO 1092
<400> SEQUENCE: 1092 000 <210> SEQ ID NO 1093
<400> SEQUENCE: 1093 000 <210> SEQ ID NO 1094
<400> SEQUENCE: 1094 000 <210> SEQ ID NO 1095
<400> SEQUENCE: 1095 000 <210> SEQ ID NO 1096
<400> SEQUENCE: 1096 000 <210> SEQ ID NO 1097
<400> SEQUENCE: 1097 000 <210> SEQ ID NO 1098
<400> SEQUENCE: 1098 000 <210> SEQ ID NO 1099
<400> SEQUENCE: 1099 000 <210> SEQ ID NO 1100
<400> SEQUENCE: 1100 000 <210> SEQ ID NO 1101
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG1 HC constant region, L117A <400>
SEQUENCE: 1101 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105
110 Pro Ala Pro Glu Ala Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230
235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO 1102
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG1 HC constant region, L118A <400>
SEQUENCE: 1102 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105
110 Pro Ala Pro Glu Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230
235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO 1103
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG1 HC constant region, L117A & L118A
<400> SEQUENCE: 1103 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210
215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO
1104 <211> LENGTH: 330 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic IgG1 HC constant region, L117G &
L118G <400> SEQUENCE: 1104 Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65
70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Gly Gly Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 1105 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
L117V & L118V <400> SEQUENCE: 1105 Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Val Val Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
<210> SEQ ID NO 1106 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, L117I & L118I <400> SEQUENCE: 1106 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ile Ile
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330 <210> SEQ ID NO 1107 <400> SEQUENCE: 1107 000
<210> SEQ ID NO 1108 <400> SEQUENCE: 1108 000
<210> SEQ ID NO 1109 <400> SEQUENCE: 1109 000
<210> SEQ ID NO 1110 <400> SEQUENCE: 1110 000
<210> SEQ ID NO 1111 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, HJ C->S, L117A <400> SEQUENCE: 1111 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Ser Asp
Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330 <210> SEQ ID NO 1112 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, HJ C->S, L118A <400> SEQUENCE: 1112 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Ser Asp
Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Ala
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330 <210> SEQ ID NO 1113 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, HJ C->S, L117A & L118A <400> SEQUENCE: 1113
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325 330 <210> SEQ ID NO 1114 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG1
HC constant region, HJ C->S, L117G & L118G <400>
SEQUENCE: 1114 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys 100 105
110 Pro Ala Pro Glu Gly Gly Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230
235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO 1115
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG1 HC constant region, HJ C->S, L117V
& L118V <400> SEQUENCE: 1115 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Val Val Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 1116 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
HJ C->S, L117I & L118I <400> SEQUENCE: 1116 Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Ser
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ile
Ile Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150
155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275
280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330 <210> SEQ ID NO 1117 <400> SEQUENCE: 1117 000
<210> SEQ ID NO 1118 <400> SEQUENCE: 1118 000
<210> SEQ ID NO 1119 <400> SEQUENCE: 1119 000
<210> SEQ ID NO 1120 <400> SEQUENCE: 1120 000
<210> SEQ ID NO 1121 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, HJ C->V, L117A <400> SEQUENCE: 1121 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Val Asp
Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330 <210> SEQ ID NO 1122 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, HJ C->V, L118A <400> SEQUENCE: 1122 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Val Asp
Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Ala
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330 <210> SEQ ID NO 1123 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, HJ C->V, L117A & L118A <400> SEQUENCE: 1123
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys
Ser Val Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325 330 <210> SEQ ID NO 1124 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG1
HC constant region, HJ C->V, L117G & L118G <400>
SEQUENCE: 1124 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Pro Lys Ser Val Asp Lys Thr His Thr Cys Pro Pro Cys 100 105
110 Pro Ala Pro Glu Gly Gly Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230
235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO 1125
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG1 HC constant region, HJ C->V, L117V
& L118V <400> SEQUENCE: 1125 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Val Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Val Val Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 1126 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
HJ C->V, L117I & L118I <400> SEQUENCE: 1126 Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Val
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ile
Ile Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150
155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275
280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330 <210> SEQ ID NO 1127 <400> SEQUENCE: 1127 000
<210> SEQ ID NO 1128 <400> SEQUENCE: 1128 000
<210> SEQ ID NO 1129 <400> SEQUENCE: 1129 000
<210> SEQ ID NO 1130 <400> SEQUENCE: 1130 000
<210> SEQ ID NO 1131 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, BJ C->S, L117A <400> SEQUENCE: 1131 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Ser Pro Pro Ser 100 105 110 Pro Ala Pro Glu Ala Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330 <210> SEQ ID NO 1132 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, BJ C->S, L118A <400> SEQUENCE: 1132 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Ser Pro Pro Ser 100 105 110 Pro Ala Pro Glu Leu Ala
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330 <210> SEQ ID NO 1133 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, BJ C->S, L117A & L118A <400> SEQUENCE: 1133
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ser 100 105 110 Pro Ala Pro
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325 330 <210> SEQ ID NO 1134 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG1
HC constant region, BJ C->S, L117G & L118G <400>
SEQUENCE: 1134 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ser 100 105
110 Pro Ala Pro Glu Gly Gly Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230
235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO 1135
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG1 HC constant region, BJ C->S, L117V
& L118V <400> SEQUENCE: 1135 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Ser Pro Pro Ser 100 105 110 Pro Ala Pro Glu Val Val Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 1136 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
BJ C->S, L117I & L118I <400> SEQUENCE: 1136 Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Ser Pro Pro Ser 100 105 110 Pro Ala Pro Glu Ile
Ile Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150
155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275
280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330 <210> SEQ ID NO 1137 <400> SEQUENCE: 1137 000
<210> SEQ ID NO 1138 <400> SEQUENCE: 1138 000
<210> SEQ ID NO 1139 <400> SEQUENCE: 1139 000
<210> SEQ ID NO 1140 <400> SEQUENCE: 1140 000
<210> SEQ ID NO 1141 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, BJ C->V, L117A <400> SEQUENCE: 1141 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Val Pro Pro Val 100 105 110 Pro Ala Pro Glu Ala Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330 <210> SEQ ID NO 1142 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, BJ C->V, L118A <400> SEQUENCE: 1142 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Val Pro Pro Val 100 105 110 Pro Ala Pro Glu Leu Ala
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330 <210> SEQ ID NO 1143 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, BJ C->V, L117A & L118A <400> SEQUENCE: 1143
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Val Pro Pro Val 100 105 110 Pro Ala Pro
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325 330 <210> SEQ ID NO 1144 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG1
HC constant region, BJ C->V, L117G & L118G <400>
SEQUENCE: 1144 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Val Pro Pro Val 100 105
110 Pro Ala Pro Glu Gly Gly Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230
235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO 1145
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG1 HC constant region, BJ C->V, L117V
& L118V <400> SEQUENCE: 1145 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Val Pro Pro Val 100 105 110 Pro Ala Pro Glu Val Val Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 1146 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
BJ C->V, L117I & L118I <400> SEQUENCE: 1146 Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Val Pro Pro Val 100 105 110 Pro Ala Pro Glu Ile
Ile Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150
155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275
280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330 <210> SEQ ID NO 1147 <400> SEQUENCE: 1147 000
<210> SEQ ID NO 1148 <400> SEQUENCE: 1148 000
<210> SEQ ID NO 1149 <400> SEQUENCE: 1149 000
<210> SEQ ID NO 1150 <400> SEQUENCE: 1150 000
<210> SEQ ID NO 1151 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, DJ C->S, L117A <400> SEQUENCE: 1151 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Ser Asp
Lys Thr His Thr Ser Pro Pro Ser 100 105 110 Pro Ala Pro Glu Ala Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330 <210> SEQ ID NO 1152 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, DJ C->S, L118A <400> SEQUENCE: 1152 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Ser Asp
Lys Thr His Thr Ser Pro Pro Ser 100 105 110 Pro Ala Pro Glu Leu Ala
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330 <210> SEQ ID NO 1153 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, DJ C->S, L117A & L118A <400> SEQUENCE: 1153
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys
Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser 100 105 110 Pro Ala Pro
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325 330 <210> SEQ ID NO 1154 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG1
HC constant region, DJ C->S, L117G & L118G <400>
SEQUENCE: 1154 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser 100 105
110 Pro Ala Pro Glu Gly Gly Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230
235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO 1155
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG1 HC constant region, DJ C->S, L117V
& L118V <400> SEQUENCE: 1155 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr
Ser Pro Pro Ser 100 105 110 Pro Ala Pro Glu Val Val Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 1156 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
DJ C->S, L117I & L118I <400> SEQUENCE: 1156 Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Ser
Asp Lys Thr His Thr Ser Pro Pro Ser 100 105 110 Pro Ala Pro Glu Ile
Ile Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150
155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275
280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330 <210> SEQ ID NO 1157 <400> SEQUENCE: 1157 000
<210> SEQ ID NO 1158 <400> SEQUENCE: 1158 000
<210> SEQ ID NO 1159 <400> SEQUENCE: 1159 000
<210> SEQ ID NO 1160 <400> SEQUENCE: 1160 000
<210> SEQ ID NO 1161 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, DJ C->V, L117A <400> SEQUENCE: 1161 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Val Asp
Lys Thr His Thr Val Pro Pro Val 100 105 110 Pro Ala Pro Glu Ala Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330 <210> SEQ ID NO 1162 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, DJ C->V, L118A <400> SEQUENCE: 1162 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Val Asp
Lys Thr His Thr Val Pro Pro Val 100 105 110 Pro Ala Pro Glu Leu Ala
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330 <210> SEQ ID NO 1163 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, DJ C->V, L117A & L118A <400> SEQUENCE: 1163
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys
Ser Val Asp Lys Thr His Thr Val Pro Pro Val 100 105 110 Pro Ala Pro
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325 330 <210> SEQ ID NO 1164 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG1
HC constant region, DJ C->V, L117G & L118G <400>
SEQUENCE: 1164 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Pro Lys Ser Val Asp Lys Thr His Thr Val Pro Pro Val 100 105
110 Pro Ala Pro Glu Gly Gly Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230
235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO 1165
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG1 HC constant region, DJ C->V, L117V
& L118V <400> SEQUENCE: 1165 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Val Asp Lys Thr His Thr
Val Pro Pro Val 100 105 110 Pro Ala Pro Glu Val Val Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 1166 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
DJ C->V, L117I & L118I <400> SEQUENCE: 1166 Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Val
Asp Lys Thr His Thr Val Pro Pro Val 100 105 110 Pro Ala Pro Glu Ile
Ile Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150
155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275
280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 1166
<210> SEQ ID NO 1 <211> LENGTH: 109 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Epratuzumab VH <400>
SEQUENCE: 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30 Trp Leu His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Arg Asn Asp
Tyr Thr Glu Tyr Asn Gln Asn Phe 50 55 60 Lys Asp Lys Ala Thr Ile
Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Phe Cys 85 90 95 Ala Arg
Arg Asp Ile Thr Thr Phe Tyr Trp Gly Gln Gly 100 105 <210> SEQ
ID NO 2 <211> LENGTH: 106 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Epratuzumab VL <400> SEQUENCE: 2
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Leu Tyr
Ser 20 25 30 Ala Asn His Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr
Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Phe Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu
Asp Ile Ala Thr Tyr Tyr Cys His Gln 85 90 95 Tyr Leu Ser Ser Trp
Thr Phe Gly Gln Gly 100 105 <210> SEQ ID NO 3 <400>
SEQUENCE: 3 000 <210> SEQ ID NO 4 <400> SEQUENCE: 4 000
<210> SEQ ID NO 5 <400> SEQUENCE: 5 000 <210> SEQ
ID NO 6 <400> SEQUENCE: 6 000 <210> SEQ ID NO 7
<400> SEQUENCE: 7 000 <210> SEQ ID NO 8 <400>
SEQUENCE: 8 000 <210> SEQ ID NO 9 <400> SEQUENCE: 9 000
<210> SEQ ID NO 10 <400> SEQUENCE: 10 000 <210>
SEQ ID NO 11 <400> SEQUENCE: 11 000 <210> SEQ ID NO 12
<400> SEQUENCE: 12 000 <210> SEQ ID NO 13 <400>
SEQUENCE: 13 000 <210> SEQ ID NO 14 <400> SEQUENCE: 14
000 <210> SEQ ID NO 15 <400> SEQUENCE: 15 000
<210> SEQ ID NO 16 <400> SEQUENCE: 16 000 <210>
SEQ ID NO 17 <400> SEQUENCE: 17 000 <210> SEQ ID NO 18
<400> SEQUENCE: 18 000 <210> SEQ ID NO 19 <400>
SEQUENCE: 19 000 <210> SEQ ID NO 20 <400> SEQUENCE: 20
000 <210> SEQ ID NO 21 <400> SEQUENCE: 21 000
<210> SEQ ID NO 22 <400> SEQUENCE: 22 000 <210>
SEQ ID NO 23 <400> SEQUENCE: 23 000 <210> SEQ ID NO 24
<400> SEQUENCE: 24 000 <210> SEQ ID NO 25 <400>
SEQUENCE: 25 000 <210> SEQ ID NO 26 <400> SEQUENCE: 26
000 <210> SEQ ID NO 27 <400> SEQUENCE: 27 000
<210> SEQ ID NO 28 <400> SEQUENCE: 28 000
<210> SEQ ID NO 29 <400> SEQUENCE: 29 000 <210>
SEQ ID NO 30 <400> SEQUENCE: 30 000 <210> SEQ ID NO 31
<400> SEQUENCE: 31 000 <210> SEQ ID NO 32 <400>
SEQUENCE: 32 000 <210> SEQ ID NO 33 <400> SEQUENCE: 33
000 <210> SEQ ID NO 34 <400> SEQUENCE: 34 000
<210> SEQ ID NO 35 <400> SEQUENCE: 35 000 <210>
SEQ ID NO 36 <400> SEQUENCE: 36 000 <210> SEQ ID NO 37
<400> SEQUENCE: 37 000 <210> SEQ ID NO 38 <400>
SEQUENCE: 38 000 <210> SEQ ID NO 39 <400> SEQUENCE: 39
000 <210> SEQ ID NO 40 <400> SEQUENCE: 40 000
<210> SEQ ID NO 41 <400> SEQUENCE: 41 000 <210>
SEQ ID NO 42 <400> SEQUENCE: 42 000 <210> SEQ ID NO 43
<400> SEQUENCE: 43 000 <210> SEQ ID NO 44 <400>
SEQUENCE: 44 000 <210> SEQ ID NO 45 <400> SEQUENCE: 45
000 <210> SEQ ID NO 46 <400> SEQUENCE: 46 000
<210> SEQ ID NO 47 <400> SEQUENCE: 47 000 <210>
SEQ ID NO 48 <400> SEQUENCE: 48 000 <210> SEQ ID NO 49
<400> SEQUENCE: 49 000 <210> SEQ ID NO 50 <400>
SEQUENCE: 50 000 <210> SEQ ID NO 51 <400> SEQUENCE: 51
000 <210> SEQ ID NO 52 <400> SEQUENCE: 52 000
<210> SEQ ID NO 53 <400> SEQUENCE: 53 000 <210>
SEQ ID NO 54 <400> SEQUENCE: 54 000 <210> SEQ ID NO 55
<400> SEQUENCE: 55 000 <210> SEQ ID NO 56 <400>
SEQUENCE: 56 000 <210> SEQ ID NO 57 <400> SEQUENCE: 57
000 <210> SEQ ID NO 58 <400> SEQUENCE: 58 000
<210> SEQ ID NO 59 <400> SEQUENCE: 59 000 <210>
SEQ ID NO 60 <400> SEQUENCE: 60 000 <210> SEQ ID NO 61
<400> SEQUENCE: 61 000 <210> SEQ ID NO 62 <400>
SEQUENCE: 62 000 <210> SEQ ID NO 63 <400> SEQUENCE: 63
000 <210> SEQ ID NO 64 <400> SEQUENCE: 64 000
<210> SEQ ID NO 65 <400> SEQUENCE: 65 000 <210>
SEQ ID NO 66 <400> SEQUENCE: 66 000 <210> SEQ ID NO 67
<400> SEQUENCE: 67 000 <210> SEQ ID NO 68 <400>
SEQUENCE: 68 000 <210> SEQ ID NO 69 <400> SEQUENCE: 69
000 <210> SEQ ID NO 70 <400> SEQUENCE: 70 000
<210> SEQ ID NO 71 <400> SEQUENCE: 71 000 <210>
SEQ ID NO 72 <400> SEQUENCE: 72 000 <210> SEQ ID NO 73
<400> SEQUENCE: 73 000 <210> SEQ ID NO 74 <400>
SEQUENCE: 74 000 <210> SEQ ID NO 75 <400> SEQUENCE: 75
000 <210> SEQ ID NO 76 <400> SEQUENCE: 76 000
<210> SEQ ID NO 77 <400> SEQUENCE: 77 000 <210>
SEQ ID NO 78 <400> SEQUENCE: 78 000 <210> SEQ ID NO 79
<400> SEQUENCE: 79 000 <210> SEQ ID NO 80 <400>
SEQUENCE: 80 000 <210> SEQ ID NO 81 <400> SEQUENCE: 81
000 <210> SEQ ID NO 82 <400> SEQUENCE: 82 000
<210> SEQ ID NO 83 <400> SEQUENCE: 83 000 <210>
SEQ ID NO 84 <400> SEQUENCE: 84 000 <210> SEQ ID NO 85
<400> SEQUENCE: 85 000 <210> SEQ ID NO 86 <400>
SEQUENCE: 86 000 <210> SEQ ID NO 87 <400> SEQUENCE: 87
000 <210> SEQ ID NO 88 <400> SEQUENCE: 88 000
<210> SEQ ID NO 89 <400> SEQUENCE: 89 000 <210>
SEQ ID NO 90 <400> SEQUENCE: 90 000 <210> SEQ ID NO 91
<400> SEQUENCE: 91 000 <210> SEQ ID NO 92 <400>
SEQUENCE: 92 000 <210> SEQ ID NO 93 <400> SEQUENCE: 93
000 <210> SEQ ID NO 94 <400> SEQUENCE: 94 000
<210> SEQ ID NO 95 <400> SEQUENCE: 95 000 <210>
SEQ ID NO 96 <400> SEQUENCE: 96 000 <210> SEQ ID NO 97
<400> SEQUENCE: 97 000 <210> SEQ ID NO 98 <400>
SEQUENCE: 98 000 <210> SEQ ID NO 99 <400> SEQUENCE: 99
000 <210> SEQ ID NO 100 <400> SEQUENCE: 100 000
<210> SEQ ID NO 101 <400> SEQUENCE: 101 000 <210>
SEQ ID NO 102 <400> SEQUENCE: 102 000 <210> SEQ ID NO
103 <400> SEQUENCE: 103 000 <210> SEQ ID NO 104
<400> SEQUENCE: 104 000 <210> SEQ ID NO 105 <400>
SEQUENCE: 105 000 <210> SEQ ID NO 106 <400> SEQUENCE:
106 000 <210> SEQ ID NO 107 <400> SEQUENCE: 107 000
<210> SEQ ID NO 108 <400> SEQUENCE: 108 000 <210>
SEQ ID NO 109 <400> SEQUENCE: 109 000 <210> SEQ ID NO
110 <211> LENGTH: 330 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic IgG1 HC constant region <400>
SEQUENCE: 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105
110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230
235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO 111
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG1 HC constant region, HJ C->S
<400> SEQUENCE: 111 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Arg Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210
215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO
112 <211> LENGTH: 330 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic IgG1 HC constant region, HJ C->V
<400> SEQUENCE: 112 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Arg Val Glu Pro Lys Ser Val Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp 145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165
170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290
295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
<210> SEQ ID NO 113 <211> LENGTH: 330 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
BJ C->S <400> SEQUENCE: 113 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Ser Pro Pro Ser 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 114 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
BJ C->V <400> SEQUENCE: 114 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Val Pro Pro Val 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 115 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
DJ C->S <400> SEQUENCE: 115 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr
Ser Pro Pro Ser 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
<210> SEQ ID NO 116 <211> LENGTH: 330 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
DJ C->V <400> SEQUENCE: 116 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Val Asp Lys Thr His Thr
Val Pro Pro Val 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 117 <400> SEQUENCE: 117 000 <210> SEQ ID NO
118 <400> SEQUENCE: 118 000 <210> SEQ ID NO 119
<400> SEQUENCE: 119 000 <210> SEQ ID NO 120 <211>
LENGTH: 326 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic IgG2 HC constant region <400> SEQUENCE: 120 Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn
Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys
Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val
Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150
155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His
Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys
Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275
280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210>
SEQ ID NO 121 <400> SEQUENCE: 121 000 <210> SEQ ID NO
122 <400> SEQUENCE: 122 000 <210> SEQ ID NO 123
<400> SEQUENCE: 123 000 <210> SEQ ID NO 124 <400>
SEQUENCE: 124 000 <210> SEQ ID NO 125 <400> SEQUENCE:
125 000 <210> SEQ ID NO 126 <400> SEQUENCE: 126 000
<210> SEQ ID NO 127 <400> SEQUENCE: 127 000 <210>
SEQ ID NO 128 <400> SEQUENCE: 128 000 <210> SEQ ID NO
129 <400> SEQUENCE: 129 000 <210> SEQ ID NO 130
<211> LENGTH: 377 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG3 HC constant region <400>
SEQUENCE: 130 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly
Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser
Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys
Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro
Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165
170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290
295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn
Asn 305 310 315 320 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser
Pro Gly Lys 370 375 <210> SEQ ID NO 131 <211> LENGTH:
377 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG3
HC constant region, L164A <400> SEQUENCE: 131 Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly
Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser
Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys
Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro
Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160
Ala Pro Glu Ala Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165
170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290
295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn
Asn 305 310 315 320 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser
Pro Gly Lys 370 375 <210> SEQ ID NO 132 <211> LENGTH:
377 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG3
HC constant region, L165A <400> SEQUENCE: 132 Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly
Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser
Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys
Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro
Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160
Ala Pro Glu Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165
170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290
295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn
Asn 305 310 315 320 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser
Pro Gly Lys 370 375 <210> SEQ ID NO 133 <211> LENGTH:
377 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG3
HC constant region, L164A & L165A <400> SEQUENCE: 133 Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10
15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50
55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr
Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp
Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys
Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser
Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180
185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300
Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305
310 315 320 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly
Lys 370 375 <210> SEQ ID NO 134 <211> LENGTH: 377
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG3
HC constant region, L164G & L165G <400> SEQUENCE: 134 Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10
15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr
Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu
Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140
Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145
150 155 160 Ala Pro Glu Gly Gly Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265
270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln
Pro Glu Asn Asn 305 310 315 320 Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Phe Thr Gln 355 360 365 Lys Ser Leu
Ser Leu Ser Pro Gly Lys 370 375 <210> SEQ ID NO 135
<211> LENGTH: 377 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG3 HC constant region, L164V & L165V
<400> SEQUENCE: 135 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys
Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro
Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro
Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro
Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Val Val Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val
Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 195 200 205
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210
215 220 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala
Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330
335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile
340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Phe
Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375
<210> SEQ ID NO 136 <211> LENGTH: 377 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG3 HC constant region,
L164I & L165I <400> SEQUENCE: 136 Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50
55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65
70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr
His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr
Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp
Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys
Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu
Ile Ile Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185
190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser
Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310
315 320 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys
370 375 <210> SEQ ID NO 137 <400> SEQUENCE: 137 000
<210> SEQ ID NO 138 <400> SEQUENCE: 138 000 <210>
SEQ ID NO 139 <400> SEQUENCE: 139 000 <210> SEQ ID NO
140 <211> LENGTH: 327 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic IgG4 HC constant region <400>
SEQUENCE: 140 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys
Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105
110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230
235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp
Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser
Leu Gly Lys 325 <210> SEQ ID NO 141 <211> LENGTH: 327
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG4
HC constant region, L115A <400> SEQUENCE: 141 Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr
Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro
Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Ala Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165
170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290
295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> SEQ
ID NO 142 <211> LENGTH: 327 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic IgG4 HC constant region, L115G
<400> SEQUENCE: 142 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser
Cys Pro Ala Pro 100 105 110 Glu Phe Gly Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185
190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310
315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> SEQ ID NO 143
<211> LENGTH: 327 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG4 HC constant region, L115V <400>
SEQUENCE: 143 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys
Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105
110 Glu Phe Val Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230
235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp
Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser
Leu Gly Lys 325 <210> SEQ ID NO 144 <211> LENGTH: 327
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG4
HC constant region, L115I <400> SEQUENCE: 144 Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr
Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro
Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Ile Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165
170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290
295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> SEQ
ID NO 145 <400> SEQUENCE: 145 000 <210> SEQ ID NO 146
<400> SEQUENCE: 146 000 <210> SEQ ID NO 147 <400>
SEQUENCE: 147 000 <210> SEQ ID NO 148 <400> SEQUENCE:
148 000 <210> SEQ ID NO 149 <400> SEQUENCE: 149 000
<210> SEQ ID NO 150 <211> LENGTH: 105 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: kappa LC constant region <400>
SEQUENCE: 150 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu 1 5 10 15 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro 20 25 30 Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly 35 40 45 Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr
50 55 60 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His 65 70 75 80 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val 85 90 95 Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
105 <210> SEQ ID NO 151 <211> LENGTH: 105 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: kappa LC constant region,
C105S <400> SEQUENCE: 151 Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu 1 5 10 15 Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro 20 25 30 Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 35 40 45 Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 50 55 60 Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 65 70
75 80 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val 85 90 95 Thr Lys Ser Phe Asn Arg Gly Glu Ser 100 105
<210> SEQ ID NO 152 <211> LENGTH: 105 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: kappa LC constant region, C105V
<400> SEQUENCE: 152 Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu 1 5 10 15 Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro 20 25 30 Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 35 40 45 Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 50 55 60 Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 65 70 75 80
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 85
90 95 Thr Lys Ser Phe Asn Arg Gly Glu Val 100 105 <210> SEQ
ID NO 153 <211> LENGTH: 104 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: kappa LC constant region, C105del <400>
SEQUENCE: 153 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu 1 5 10 15 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro 20 25 30 Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly 35 40 45 Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr 50 55 60 Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 65 70 75 80 Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 85 90 95 Thr
Lys Ser Phe Asn Arg Gly Glu 100 <210> SEQ ID NO 154
<400> SEQUENCE: 154 000 <210> SEQ ID NO 155 <400>
SEQUENCE: 155 000 <210> SEQ ID NO 156 <400> SEQUENCE:
156 000 <210> SEQ ID NO 157 <400> SEQUENCE: 157 000
<210> SEQ ID NO 158 <400> SEQUENCE: 158 000 <210>
SEQ ID NO 159 <400> SEQUENCE: 159 000 <210> SEQ ID NO
160 <211> LENGTH: 103 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: lambda LC constant region <400> SEQUENCE:
160 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu
1 5 10 15 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
Tyr Pro 20 25 30 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser
Pro Val Lys Ala 35 40 45 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln
Ser Asn Asn Lys Tyr Ala 50 55 60 Ala Ser Ser Tyr Leu Ser Leu Thr
Pro Glu Gln Trp Lys Ser His Arg 65 70 75 80 Ser Tyr Ser Cys Gln Val
Thr His Glu Gly Ser Thr Val Glu Lys Thr 85 90 95 Val Ala Pro Thr
Glu Cys Ser 100 <210> SEQ ID NO 161 <211> LENGTH: 103
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: lambda LC
constant region, C102S <400> SEQUENCE: 161 Lys Ala Ala Pro
Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 1 5 10 15 Gln Ala
Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 20 25 30
Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 35
40 45 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr
Ala 50 55 60 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
Ser His Arg 65 70 75 80 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser
Thr Val Glu Lys Thr 85 90 95 Val Ala Pro Thr Glu Ser Ser 100
<210> SEQ ID NO 162 <211> LENGTH: 103 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: lambda LC constant region, C102V
<400> SEQUENCE: 162 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro
Pro Ser Ser Glu Glu Leu 1 5 10 15 Gln Ala Asn Lys Ala Thr Leu Val
Cys Leu Ile Ser Asp Phe Tyr Pro 20 25 30 Gly Ala Val Thr Val Ala
Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 35 40 45 Gly Val Glu Thr
Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 50 55 60 Ala Ser
Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 65 70 75 80
Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 85
90 95 Val Ala Pro Thr Glu Val Ser 100 <210> SEQ ID NO 163
<211> LENGTH: 101 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: lambda LC constant region, C102&S103del
<400> SEQUENCE: 163 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro
Pro Ser Ser Glu Glu Leu 1 5 10 15
Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 20
25 30 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys
Ala 35 40 45 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
Lys Tyr Ala 50 55 60 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln
Trp Lys Ser His Arg 65 70 75 80 Ser Tyr Ser Cys Gln Val Thr His Glu
Gly Ser Thr Val Glu Lys Thr 85 90 95 Val Ala Pro Thr Glu 100
<210> SEQ ID NO 164 <400> SEQUENCE: 164 000 <210>
SEQ ID NO 165 <400> SEQUENCE: 165 000 <210> SEQ ID NO
166 <400> SEQUENCE: 166 000 <210> SEQ ID NO 167
<400> SEQUENCE: 167 000 <210> SEQ ID NO 168 <400>
SEQUENCE: 168 000 <210> SEQ ID NO 169 <400> SEQUENCE:
169 000 <210> SEQ ID NO 170 <400> SEQUENCE: 170 000
<210> SEQ ID NO 171 <400> SEQUENCE: 171 000 <210>
SEQ ID NO 172 <400> SEQUENCE: 172 000 <210> SEQ ID NO
173 <400> SEQUENCE: 173 000 <210> SEQ ID NO 174
<400> SEQUENCE: 174 000 <210> SEQ ID NO 175 <400>
SEQUENCE: 175 000 <210> SEQ ID NO 176 <400> SEQUENCE:
176 000 <210> SEQ ID NO 177 <400> SEQUENCE: 177 000
<210> SEQ ID NO 178 <400> SEQUENCE: 178 000 <210>
SEQ ID NO 179 <400> SEQUENCE: 179 000 <210> SEQ ID NO
180 <400> SEQUENCE: 180 000 <210> SEQ ID NO 181
<400> SEQUENCE: 181 000 <210> SEQ ID NO 182 <400>
SEQUENCE: 182 000 <210> SEQ ID NO 183 <400> SEQUENCE:
183 000 <210> SEQ ID NO 184 <400> SEQUENCE: 184 000
<210> SEQ ID NO 185 <400> SEQUENCE: 185 000 <210>
SEQ ID NO 186 <400> SEQUENCE: 186 000 <210> SEQ ID NO
187 <400> SEQUENCE: 187 000 <210> SEQ ID NO 188
<400> SEQUENCE: 188 000 <210> SEQ ID NO 189 <400>
SEQUENCE: 189 000 <210> SEQ ID NO 190 <400> SEQUENCE:
190 000 <210> SEQ ID NO 191 <400> SEQUENCE: 191 000
<210> SEQ ID NO 192 <400> SEQUENCE: 192 000 <210>
SEQ ID NO 193 <400> SEQUENCE: 193 000 <210> SEQ ID NO
194 <400> SEQUENCE: 194 000 <210> SEQ ID NO 195
<400> SEQUENCE: 195 000 <210> SEQ ID NO 196 <400>
SEQUENCE: 196 000
<210> SEQ ID NO 197 <400> SEQUENCE: 197 000 <210>
SEQ ID NO 198 <400> SEQUENCE: 198 000 <210> SEQ ID NO
199 <400> SEQUENCE: 199 000 <210> SEQ ID NO 200
<400> SEQUENCE: 200 000 <210> SEQ ID NO 201 <400>
SEQUENCE: 201 000 <210> SEQ ID NO 202 <400> SEQUENCE:
202 000 <210> SEQ ID NO 203 <400> SEQUENCE: 203 000
<210> SEQ ID NO 204 <400> SEQUENCE: 204 000 <210>
SEQ ID NO 205 <400> SEQUENCE: 205 000 <210> SEQ ID NO
206 <400> SEQUENCE: 206 000 <210> SEQ ID NO 207
<400> SEQUENCE: 207 000 <210> SEQ ID NO 208 <400>
SEQUENCE: 208 000 <210> SEQ ID NO 209 <400> SEQUENCE:
209 000 <210> SEQ ID NO 210 <400> SEQUENCE: 210 000
<210> SEQ ID NO 211 <400> SEQUENCE: 211 000 <210>
SEQ ID NO 212 <400> SEQUENCE: 212 000 <210> SEQ ID NO
213 <400> SEQUENCE: 213 000 <210> SEQ ID NO 214
<400> SEQUENCE: 214 000 <210> SEQ ID NO 215 <400>
SEQUENCE: 215 000 <210> SEQ ID NO 216 <400> SEQUENCE:
216 000 <210> SEQ ID NO 217 <400> SEQUENCE: 217 000
<210> SEQ ID NO 218 <400> SEQUENCE: 218 000 <210>
SEQ ID NO 219 <400> SEQUENCE: 219 000 <210> SEQ ID NO
220 <400> SEQUENCE: 220 000 <210> SEQ ID NO 221
<400> SEQUENCE: 221 000 <210> SEQ ID NO 222 <400>
SEQUENCE: 222 000 <210> SEQ ID NO 223 <400> SEQUENCE:
223 000 <210> SEQ ID NO 224 <400> SEQUENCE: 224 000
<210> SEQ ID NO 225 <400> SEQUENCE: 225 000 <210>
SEQ ID NO 226 <400> SEQUENCE: 226 000 <210> SEQ ID NO
227 <400> SEQUENCE: 227 000 <210> SEQ ID NO 228
<400> SEQUENCE: 228 000 <210> SEQ ID NO 229 <400>
SEQUENCE: 229 000 <210> SEQ ID NO 230 <400> SEQUENCE:
230 000 <210> SEQ ID NO 231 <400> SEQUENCE: 231 000
<210> SEQ ID NO 232 <400> SEQUENCE: 232 000
<210> SEQ ID NO 233 <400> SEQUENCE: 233 000 <210>
SEQ ID NO 234 <400> SEQUENCE: 234 000 <210> SEQ ID NO
235 <400> SEQUENCE: 235 000 <210> SEQ ID NO 236
<400> SEQUENCE: 236 000 <210> SEQ ID NO 237 <400>
SEQUENCE: 237 000 <210> SEQ ID NO 238 <400> SEQUENCE:
238 000 <210> SEQ ID NO 239 <400> SEQUENCE: 239 000
<210> SEQ ID NO 240 <400> SEQUENCE: 240 000 <210>
SEQ ID NO 241 <400> SEQUENCE: 241 000 <210> SEQ ID NO
242 <400> SEQUENCE: 242 000 <210> SEQ ID NO 243
<400> SEQUENCE: 243 000 <210> SEQ ID NO 244 <400>
SEQUENCE: 244 000 <210> SEQ ID NO 245 <400> SEQUENCE:
245 000 <210> SEQ ID NO 246 <400> SEQUENCE: 246 000
<210> SEQ ID NO 247 <400> SEQUENCE: 247 000 <210>
SEQ ID NO 248 <400> SEQUENCE: 248 000 <210> SEQ ID NO
249 <400> SEQUENCE: 249 000 <210> SEQ ID NO 250
<400> SEQUENCE: 250 000 <210> SEQ ID NO 251 <400>
SEQUENCE: 251 000 <210> SEQ ID NO 252 <400> SEQUENCE:
252 000 <210> SEQ ID NO 253 <400> SEQUENCE: 253 000
<210> SEQ ID NO 254 <400> SEQUENCE: 254 000 <210>
SEQ ID NO 255 <400> SEQUENCE: 255 000 <210> SEQ ID NO
256 <400> SEQUENCE: 256 000 <210> SEQ ID NO 257
<400> SEQUENCE: 257 000 <210> SEQ ID NO 258 <400>
SEQUENCE: 258 000 <210> SEQ ID NO 259 <400> SEQUENCE:
259 000 <210> SEQ ID NO 260 <400> SEQUENCE: 260 000
<210> SEQ ID NO 261 <400> SEQUENCE: 261 000 <210>
SEQ ID NO 262 <400> SEQUENCE: 262 000 <210> SEQ ID NO
263 <400> SEQUENCE: 263 000 <210> SEQ ID NO 264
<400> SEQUENCE: 264 000 <210> SEQ ID NO 265 <400>
SEQUENCE: 265 000 <210> SEQ ID NO 266 <400> SEQUENCE:
266 000 <210> SEQ ID NO 267 <400> SEQUENCE: 267 000
<210> SEQ ID NO 268 <400> SEQUENCE: 268 000
<210> SEQ ID NO 269 <400> SEQUENCE: 269 000 <210>
SEQ ID NO 270 <400> SEQUENCE: 270 000 <210> SEQ ID NO
271 <400> SEQUENCE: 271 000 <210> SEQ ID NO 272
<400> SEQUENCE: 272 000 <210> SEQ ID NO 273 <400>
SEQUENCE: 273 000 <210> SEQ ID NO 274 <400> SEQUENCE:
274 000 <210> SEQ ID NO 275 <400> SEQUENCE: 275 000
<210> SEQ ID NO 276 <400> SEQUENCE: 276 000 <210>
SEQ ID NO 277 <400> SEQUENCE: 277 000 <210> SEQ ID NO
278 <400> SEQUENCE: 278 000 <210> SEQ ID NO 279
<400> SEQUENCE: 279 000 <210> SEQ ID NO 280 <400>
SEQUENCE: 280 000 <210> SEQ ID NO 281 <400> SEQUENCE:
281 000 <210> SEQ ID NO 282 <400> SEQUENCE: 282 000
<210> SEQ ID NO 283 <400> SEQUENCE: 283 000 <210>
SEQ ID NO 284 <400> SEQUENCE: 284 000 <210> SEQ ID NO
285 <400> SEQUENCE: 285 000 <210> SEQ ID NO 286
<400> SEQUENCE: 286 000 <210> SEQ ID NO 287 <400>
SEQUENCE: 287 000 <210> SEQ ID NO 288 <400> SEQUENCE:
288 000 <210> SEQ ID NO 289 <400> SEQUENCE: 289 000
<210> SEQ ID NO 290 <400> SEQUENCE: 290 000 <210>
SEQ ID NO 291 <400> SEQUENCE: 291 000 <210> SEQ ID NO
292 <400> SEQUENCE: 292 000 <210> SEQ ID NO 293
<400> SEQUENCE: 293 000 <210> SEQ ID NO 294 <400>
SEQUENCE: 294 000 <210> SEQ ID NO 295 <400> SEQUENCE:
295 000 <210> SEQ ID NO 296 <400> SEQUENCE: 296 000
<210> SEQ ID NO 297 <400> SEQUENCE: 297 000 <210>
SEQ ID NO 298 <400> SEQUENCE: 298 000 <210> SEQ ID NO
299 <400> SEQUENCE: 299 000 <210> SEQ ID NO 300
<400> SEQUENCE: 300 000 <210> SEQ ID NO 301 <400>
SEQUENCE: 301 000 <210> SEQ ID NO 302 <400> SEQUENCE:
302 000 <210> SEQ ID NO 303 <400> SEQUENCE: 303 000
<210> SEQ ID NO 304 <400> SEQUENCE: 304
000 <210> SEQ ID NO 305 <400> SEQUENCE: 305 000
<210> SEQ ID NO 306 <400> SEQUENCE: 306 000 <210>
SEQ ID NO 307 <400> SEQUENCE: 307 000 <210> SEQ ID NO
308 <400> SEQUENCE: 308 000 <210> SEQ ID NO 309
<400> SEQUENCE: 309 000 <210> SEQ ID NO 310 <400>
SEQUENCE: 310 000 <210> SEQ ID NO 311 <400> SEQUENCE:
311 000 <210> SEQ ID NO 312 <400> SEQUENCE: 312 000
<210> SEQ ID NO 313 <400> SEQUENCE: 313 000 <210>
SEQ ID NO 314 <400> SEQUENCE: 314 000 <210> SEQ ID NO
315 <400> SEQUENCE: 315 000 <210> SEQ ID NO 316
<400> SEQUENCE: 316 000 <210> SEQ ID NO 317 <400>
SEQUENCE: 317 000 <210> SEQ ID NO 318 <400> SEQUENCE:
318 000 <210> SEQ ID NO 319 <400> SEQUENCE: 319 000
<210> SEQ ID NO 320 <400> SEQUENCE: 320 000 <210>
SEQ ID NO 321 <400> SEQUENCE: 321 000 <210> SEQ ID NO
322 <400> SEQUENCE: 322 000 <210> SEQ ID NO 323
<400> SEQUENCE: 323 000 <210> SEQ ID NO 324 <400>
SEQUENCE: 324 000 <210> SEQ ID NO 325 <400> SEQUENCE:
325 000 <210> SEQ ID NO 326 <400> SEQUENCE: 326 000
<210> SEQ ID NO 327 <400> SEQUENCE: 327 000 <210>
SEQ ID NO 328 <400> SEQUENCE: 328 000 <210> SEQ ID NO
329 <400> SEQUENCE: 329 000 <210> SEQ ID NO 330
<400> SEQUENCE: 330 000 <210> SEQ ID NO 331 <400>
SEQUENCE: 331 000 <210> SEQ ID NO 332 <400> SEQUENCE:
332 000 <210> SEQ ID NO 333 <400> SEQUENCE: 333 000
<210> SEQ ID NO 334 <400> SEQUENCE: 334 000 <210>
SEQ ID NO 335 <400> SEQUENCE: 335 000 <210> SEQ ID NO
336 <400> SEQUENCE: 336 000 <210> SEQ ID NO 337
<400> SEQUENCE: 337 000 <210> SEQ ID NO 338 <400>
SEQUENCE: 338 000 <210> SEQ ID NO 339 <400> SEQUENCE:
339 000 <210> SEQ ID NO 340 <400> SEQUENCE: 340
000 <210> SEQ ID NO 341 <400> SEQUENCE: 341 000
<210> SEQ ID NO 342 <400> SEQUENCE: 342 000 <210>
SEQ ID NO 343 <400> SEQUENCE: 343 000 <210> SEQ ID NO
344 <400> SEQUENCE: 344 000 <210> SEQ ID NO 345
<400> SEQUENCE: 345 000 <210> SEQ ID NO 346 <400>
SEQUENCE: 346 000 <210> SEQ ID NO 347 <400> SEQUENCE:
347 000 <210> SEQ ID NO 348 <400> SEQUENCE: 348 000
<210> SEQ ID NO 349 <400> SEQUENCE: 349 000 <210>
SEQ ID NO 350 <400> SEQUENCE: 350 000 <210> SEQ ID NO
351 <400> SEQUENCE: 351 000 <210> SEQ ID NO 352
<400> SEQUENCE: 352 000 <210> SEQ ID NO 353 <400>
SEQUENCE: 353 000 <210> SEQ ID NO 354 <400> SEQUENCE:
354 000 <210> SEQ ID NO 355 <400> SEQUENCE: 355 000
<210> SEQ ID NO 356 <400> SEQUENCE: 356 000 <210>
SEQ ID NO 357 <400> SEQUENCE: 357 000 <210> SEQ ID NO
358 <400> SEQUENCE: 358 000 <210> SEQ ID NO 359
<400> SEQUENCE: 359 000 <210> SEQ ID NO 360 <400>
SEQUENCE: 360 000 <210> SEQ ID NO 361 <400> SEQUENCE:
361 000 <210> SEQ ID NO 362 <400> SEQUENCE: 362 000
<210> SEQ ID NO 363 <400> SEQUENCE: 363 000 <210>
SEQ ID NO 364 <400> SEQUENCE: 364 000 <210> SEQ ID NO
365 <400> SEQUENCE: 365 000 <210> SEQ ID NO 366
<400> SEQUENCE: 366 000 <210> SEQ ID NO 367 <400>
SEQUENCE: 367 000 <210> SEQ ID NO 368 <400> SEQUENCE:
368 000 <210> SEQ ID NO 369 <400> SEQUENCE: 369 000
<210> SEQ ID NO 370 <400> SEQUENCE: 370 000 <210>
SEQ ID NO 371 <400> SEQUENCE: 371 000 <210> SEQ ID NO
372 <400> SEQUENCE: 372 000 <210> SEQ ID NO 373
<400> SEQUENCE: 373 000 <210> SEQ ID NO 374 <400>
SEQUENCE: 374 000 <210> SEQ ID NO 375 <400> SEQUENCE:
375 000 <210> SEQ ID NO 376
<400> SEQUENCE: 376 000 <210> SEQ ID NO 377 <400>
SEQUENCE: 377 000 <210> SEQ ID NO 378 <400> SEQUENCE:
378 000 <210> SEQ ID NO 379 <400> SEQUENCE: 379 000
<210> SEQ ID NO 380 <400> SEQUENCE: 380 000 <210>
SEQ ID NO 381 <400> SEQUENCE: 381 000 <210> SEQ ID NO
382 <400> SEQUENCE: 382 000 <210> SEQ ID NO 383
<400> SEQUENCE: 383 000 <210> SEQ ID NO 384 <400>
SEQUENCE: 384 000 <210> SEQ ID NO 385 <400> SEQUENCE:
385 000 <210> SEQ ID NO 386 <400> SEQUENCE: 386 000
<210> SEQ ID NO 387 <400> SEQUENCE: 387 000 <210>
SEQ ID NO 388 <400> SEQUENCE: 388 000 <210> SEQ ID NO
389 <400> SEQUENCE: 389 000 <210> SEQ ID NO 390
<400> SEQUENCE: 390 000 <210> SEQ ID NO 391 <400>
SEQUENCE: 391 000 <210> SEQ ID NO 392 <400> SEQUENCE:
392 000 <210> SEQ ID NO 393 <400> SEQUENCE: 393 000
<210> SEQ ID NO 394 <400> SEQUENCE: 394 000 <210>
SEQ ID NO 395 <400> SEQUENCE: 395 000 <210> SEQ ID NO
396 <400> SEQUENCE: 396 000 <210> SEQ ID NO 397
<400> SEQUENCE: 397 000 <210> SEQ ID NO 398 <400>
SEQUENCE: 398 000 <210> SEQ ID NO 399 <400> SEQUENCE:
399 000 <210> SEQ ID NO 400 <400> SEQUENCE: 400 000
<210> SEQ ID NO 401 <400> SEQUENCE: 401 000 <210>
SEQ ID NO 402 <400> SEQUENCE: 402 000 <210> SEQ ID NO
403 <400> SEQUENCE: 403 000 <210> SEQ ID NO 404
<400> SEQUENCE: 404 000 <210> SEQ ID NO 405 <400>
SEQUENCE: 405 000 <210> SEQ ID NO 406 <400> SEQUENCE:
406 000 <210> SEQ ID NO 407 <400> SEQUENCE: 407 000
<210> SEQ ID NO 408 <400> SEQUENCE: 408 000 <210>
SEQ ID NO 409 <400> SEQUENCE: 409 000 <210> SEQ ID NO
410 <400> SEQUENCE: 410 000 <210> SEQ ID NO 411
<400> SEQUENCE: 411 000 <210> SEQ ID NO 412
<400> SEQUENCE: 412 000 <210> SEQ ID NO 413 <400>
SEQUENCE: 413 000 <210> SEQ ID NO 414 <400> SEQUENCE:
414 000 <210> SEQ ID NO 415 <400> SEQUENCE: 415 000
<210> SEQ ID NO 416 <400> SEQUENCE: 416 000 <210>
SEQ ID NO 417 <400> SEQUENCE: 417 000 <210> SEQ ID NO
418 <400> SEQUENCE: 418 000 <210> SEQ ID NO 419
<400> SEQUENCE: 419 000 <210> SEQ ID NO 420 <400>
SEQUENCE: 420 000 <210> SEQ ID NO 421 <400> SEQUENCE:
421 000 <210> SEQ ID NO 422 <400> SEQUENCE: 422 000
<210> SEQ ID NO 423 <400> SEQUENCE: 423 000 <210>
SEQ ID NO 424 <400> SEQUENCE: 424 000 <210> SEQ ID NO
425 <400> SEQUENCE: 425 000 <210> SEQ ID NO 426
<400> SEQUENCE: 426 000 <210> SEQ ID NO 427 <400>
SEQUENCE: 427 000 <210> SEQ ID NO 428 <400> SEQUENCE:
428 000 <210> SEQ ID NO 429 <400> SEQUENCE: 429 000
<210> SEQ ID NO 430 <400> SEQUENCE: 430 000 <210>
SEQ ID NO 431 <400> SEQUENCE: 431 000 <210> SEQ ID NO
432 <400> SEQUENCE: 432 000 <210> SEQ ID NO 433
<400> SEQUENCE: 433 000 <210> SEQ ID NO 434 <400>
SEQUENCE: 434 000 <210> SEQ ID NO 435 <400> SEQUENCE:
435 000 <210> SEQ ID NO 436 <400> SEQUENCE: 436 000
<210> SEQ ID NO 437 <400> SEQUENCE: 437 000 <210>
SEQ ID NO 438 <400> SEQUENCE: 438 000 <210> SEQ ID NO
439 <400> SEQUENCE: 439 000 <210> SEQ ID NO 440
<400> SEQUENCE: 440 000 <210> SEQ ID NO 441 <400>
SEQUENCE: 441 000 <210> SEQ ID NO 442 <400> SEQUENCE:
442 000 <210> SEQ ID NO 443 <400> SEQUENCE: 443 000
<210> SEQ ID NO 444 <400> SEQUENCE: 444 000 <210>
SEQ ID NO 445 <400> SEQUENCE: 445 000 <210> SEQ ID NO
446 <400> SEQUENCE: 446 000 <210> SEQ ID NO 447
<400> SEQUENCE: 447 000
<210> SEQ ID NO 448 <400> SEQUENCE: 448 000 <210>
SEQ ID NO 449 <400> SEQUENCE: 449 000 <210> SEQ ID NO
450 <400> SEQUENCE: 450 000 <210> SEQ ID NO 451
<400> SEQUENCE: 451 000 <210> SEQ ID NO 452 <400>
SEQUENCE: 452 000 <210> SEQ ID NO 453 <400> SEQUENCE:
453 000 <210> SEQ ID NO 454 <400> SEQUENCE: 454 000
<210> SEQ ID NO 455 <400> SEQUENCE: 455 000 <210>
SEQ ID NO 456 <400> SEQUENCE: 456 000 <210> SEQ ID NO
457 <400> SEQUENCE: 457 000 <210> SEQ ID NO 458
<400> SEQUENCE: 458 000 <210> SEQ ID NO 459 <400>
SEQUENCE: 459 000 <210> SEQ ID NO 460 <400> SEQUENCE:
460 000 <210> SEQ ID NO 461 <400> SEQUENCE: 461 000
<210> SEQ ID NO 462 <400> SEQUENCE: 462 000 <210>
SEQ ID NO 463 <400> SEQUENCE: 463 000 <210> SEQ ID NO
464 <400> SEQUENCE: 464 000 <210> SEQ ID NO 465
<400> SEQUENCE: 465 000 <210> SEQ ID NO 466 <400>
SEQUENCE: 466 000 <210> SEQ ID NO 467 <400> SEQUENCE:
467 000 <210> SEQ ID NO 468 <400> SEQUENCE: 468 000
<210> SEQ ID NO 469 <400> SEQUENCE: 469 000 <210>
SEQ ID NO 470 <400> SEQUENCE: 470 000 <210> SEQ ID NO
471 <400> SEQUENCE: 471 000 <210> SEQ ID NO 472
<400> SEQUENCE: 472 000 <210> SEQ ID NO 473 <400>
SEQUENCE: 473 000 <210> SEQ ID NO 474 <400> SEQUENCE:
474 000 <210> SEQ ID NO 475 <400> SEQUENCE: 475 000
<210> SEQ ID NO 476 <400> SEQUENCE: 476 000 <210>
SEQ ID NO 477 <400> SEQUENCE: 477 000 <210> SEQ ID NO
478 <400> SEQUENCE: 478 000 <210> SEQ ID NO 479
<400> SEQUENCE: 479 000 <210> SEQ ID NO 480 <400>
SEQUENCE: 480 000 <210> SEQ ID NO 481 <400> SEQUENCE:
481 000 <210> SEQ ID NO 482 <400> SEQUENCE: 482 000
<210> SEQ ID NO 483 <400> SEQUENCE: 483 000
<210> SEQ ID NO 484 <400> SEQUENCE: 484 000 <210>
SEQ ID NO 485 <400> SEQUENCE: 485 000 <210> SEQ ID NO
486 <400> SEQUENCE: 486 000 <210> SEQ ID NO 487
<400> SEQUENCE: 487 000 <210> SEQ ID NO 488 <400>
SEQUENCE: 488 000 <210> SEQ ID NO 489 <400> SEQUENCE:
489 000 <210> SEQ ID NO 490 <400> SEQUENCE: 490 000
<210> SEQ ID NO 491 <400> SEQUENCE: 491 000 <210>
SEQ ID NO 492 <400> SEQUENCE: 492 000 <210> SEQ ID NO
493 <400> SEQUENCE: 493 000 <210> SEQ ID NO 494
<400> SEQUENCE: 494 000 <210> SEQ ID NO 495 <400>
SEQUENCE: 495 000 <210> SEQ ID NO 496 <400> SEQUENCE:
496 000 <210> SEQ ID NO 497 <400> SEQUENCE: 497 000
<210> SEQ ID NO 498 <400> SEQUENCE: 498 000 <210>
SEQ ID NO 499 <400> SEQUENCE: 499 000 <210> SEQ ID NO
500 <400> SEQUENCE: 500 000 <210> SEQ ID NO 501
<400> SEQUENCE: 501 000 <210> SEQ ID NO 502 <400>
SEQUENCE: 502 000 <210> SEQ ID NO 503 <400> SEQUENCE:
503 000 <210> SEQ ID NO 504 <400> SEQUENCE: 504 000
<210> SEQ ID NO 505 <400> SEQUENCE: 505 000 <210>
SEQ ID NO 506 <400> SEQUENCE: 506 000 <210> SEQ ID NO
507 <400> SEQUENCE: 507 000 <210> SEQ ID NO 508
<400> SEQUENCE: 508 000 <210> SEQ ID NO 509 <400>
SEQUENCE: 509 000 <210> SEQ ID NO 510 <400> SEQUENCE:
510 000 <210> SEQ ID NO 511 <400> SEQUENCE: 511 000
<210> SEQ ID NO 512 <400> SEQUENCE: 512 000 <210>
SEQ ID NO 513 <400> SEQUENCE: 513 000 <210> SEQ ID NO
514 <400> SEQUENCE: 514 000 <210> SEQ ID NO 515
<400> SEQUENCE: 515 000 <210> SEQ ID NO 516 <400>
SEQUENCE: 516 000 <210> SEQ ID NO 517 <400> SEQUENCE:
517 000 <210> SEQ ID NO 518 <400> SEQUENCE: 518 000
<210> SEQ ID NO 519 <400> SEQUENCE: 519 000
<210> SEQ ID NO 520 <400> SEQUENCE: 520 000 <210>
SEQ ID NO 521 <400> SEQUENCE: 521 000 <210> SEQ ID NO
522 <400> SEQUENCE: 522 000 <210> SEQ ID NO 523
<400> SEQUENCE: 523 000 <210> SEQ ID NO 524 <400>
SEQUENCE: 524 000 <210> SEQ ID NO 525 <400> SEQUENCE:
525 000 <210> SEQ ID NO 526 <400> SEQUENCE: 526 000
<210> SEQ ID NO 527 <400> SEQUENCE: 527 000 <210>
SEQ ID NO 528 <400> SEQUENCE: 528 000 <210> SEQ ID NO
529 <400> SEQUENCE: 529 000 <210> SEQ ID NO 530
<400> SEQUENCE: 530 000 <210> SEQ ID NO 531 <400>
SEQUENCE: 531 000 <210> SEQ ID NO 532 <400> SEQUENCE:
532 000 <210> SEQ ID NO 533 <400> SEQUENCE: 533 000
<210> SEQ ID NO 534 <400> SEQUENCE: 534 000 <210>
SEQ ID NO 535 <400> SEQUENCE: 535 000 <210> SEQ ID NO
536 <400> SEQUENCE: 536 000 <210> SEQ ID NO 537
<400> SEQUENCE: 537 000 <210> SEQ ID NO 538 <400>
SEQUENCE: 538 000 <210> SEQ ID NO 539 <400> SEQUENCE:
539 000 <210> SEQ ID NO 540 <400> SEQUENCE: 540 000
<210> SEQ ID NO 541 <400> SEQUENCE: 541 000 <210>
SEQ ID NO 542 <400> SEQUENCE: 542 000 <210> SEQ ID NO
543 <400> SEQUENCE: 543 000 <210> SEQ ID NO 544
<400> SEQUENCE: 544 000 <210> SEQ ID NO 545 <400>
SEQUENCE: 545 000 <210> SEQ ID NO 546 <400> SEQUENCE:
546 000 <210> SEQ ID NO 547 <400> SEQUENCE: 547 000
<210> SEQ ID NO 548 <400> SEQUENCE: 548 000 <210>
SEQ ID NO 549 <400> SEQUENCE: 549 000 <210> SEQ ID NO
550 <400> SEQUENCE: 550 000 <210> SEQ ID NO 551
<400> SEQUENCE: 551 000 <210> SEQ ID NO 552 <400>
SEQUENCE: 552 000 <210> SEQ ID NO 553 <400> SEQUENCE:
553 000 <210> SEQ ID NO 554 <400> SEQUENCE: 554 000
<210> SEQ ID NO 555 <400> SEQUENCE: 555
000 <210> SEQ ID NO 556 <400> SEQUENCE: 556 000
<210> SEQ ID NO 557 <400> SEQUENCE: 557 000 <210>
SEQ ID NO 558 <400> SEQUENCE: 558 000 <210> SEQ ID NO
559 <400> SEQUENCE: 559 000 <210> SEQ ID NO 560
<400> SEQUENCE: 560 000 <210> SEQ ID NO 561 <400>
SEQUENCE: 561 000 <210> SEQ ID NO 562 <400> SEQUENCE:
562 000 <210> SEQ ID NO 563 <400> SEQUENCE: 563 000
<210> SEQ ID NO 564 <400> SEQUENCE: 564 000 <210>
SEQ ID NO 565 <400> SEQUENCE: 565 000 <210> SEQ ID NO
566 <400> SEQUENCE: 566 000 <210> SEQ ID NO 567
<400> SEQUENCE: 567 000 <210> SEQ ID NO 568 <400>
SEQUENCE: 568 000 <210> SEQ ID NO 569 <400> SEQUENCE:
569 000 <210> SEQ ID NO 570 <400> SEQUENCE: 570 000
<210> SEQ ID NO 571 <400> SEQUENCE: 571 000 <210>
SEQ ID NO 572 <400> SEQUENCE: 572 000 <210> SEQ ID NO
573 <400> SEQUENCE: 573 000 <210> SEQ ID NO 574
<400> SEQUENCE: 574 000 <210> SEQ ID NO 575 <400>
SEQUENCE: 575 000 <210> SEQ ID NO 576 <400> SEQUENCE:
576 000 <210> SEQ ID NO 577 <400> SEQUENCE: 577 000
<210> SEQ ID NO 578 <400> SEQUENCE: 578 000 <210>
SEQ ID NO 579 <400> SEQUENCE: 579 000 <210> SEQ ID NO
580 <400> SEQUENCE: 580 000 <210> SEQ ID NO 581
<400> SEQUENCE: 581 000 <210> SEQ ID NO 582 <400>
SEQUENCE: 582 000 <210> SEQ ID NO 583 <400> SEQUENCE:
583 000 <210> SEQ ID NO 584 <400> SEQUENCE: 584 000
<210> SEQ ID NO 585 <400> SEQUENCE: 585 000 <210>
SEQ ID NO 586 <400> SEQUENCE: 586 000 <210> SEQ ID NO
587 <400> SEQUENCE: 587 000 <210> SEQ ID NO 588
<400> SEQUENCE: 588 000 <210> SEQ ID NO 589 <400>
SEQUENCE: 589 000 <210> SEQ ID NO 590 <400> SEQUENCE:
590 000 <210> SEQ ID NO 591 <400> SEQUENCE: 591
000 <210> SEQ ID NO 592 <400> SEQUENCE: 592 000
<210> SEQ ID NO 593 <400> SEQUENCE: 593 000 <210>
SEQ ID NO 594 <400> SEQUENCE: 594 000 <210> SEQ ID NO
595 <400> SEQUENCE: 595 000 <210> SEQ ID NO 596
<400> SEQUENCE: 596 000 <210> SEQ ID NO 597 <400>
SEQUENCE: 597 000 <210> SEQ ID NO 598 <400> SEQUENCE:
598 000 <210> SEQ ID NO 599 <400> SEQUENCE: 599 000
<210> SEQ ID NO 600 <400> SEQUENCE: 600 000 <210>
SEQ ID NO 601 <400> SEQUENCE: 601 000 <210> SEQ ID NO
602 <400> SEQUENCE: 602 000 <210> SEQ ID NO 603
<400> SEQUENCE: 603 000 <210> SEQ ID NO 604 <400>
SEQUENCE: 604 000 <210> SEQ ID NO 605 <400> SEQUENCE:
605 000 <210> SEQ ID NO 606 <400> SEQUENCE: 606 000
<210> SEQ ID NO 607 <400> SEQUENCE: 607 000 <210>
SEQ ID NO 608 <400> SEQUENCE: 608 000 <210> SEQ ID NO
609 <400> SEQUENCE: 609 000 <210> SEQ ID NO 610
<400> SEQUENCE: 610 000 <210> SEQ ID NO 611 <400>
SEQUENCE: 611 000 <210> SEQ ID NO 612 <400> SEQUENCE:
612 000 <210> SEQ ID NO 613 <400> SEQUENCE: 613 000
<210> SEQ ID NO 614 <400> SEQUENCE: 614 000 <210>
SEQ ID NO 615 <400> SEQUENCE: 615 000 <210> SEQ ID NO
616 <400> SEQUENCE: 616 000 <210> SEQ ID NO 617
<400> SEQUENCE: 617 000 <210> SEQ ID NO 618 <400>
SEQUENCE: 618 000 <210> SEQ ID NO 619 <400> SEQUENCE:
619 000 <210> SEQ ID NO 620 <400> SEQUENCE: 620 000
<210> SEQ ID NO 621 <400> SEQUENCE: 621 000 <210>
SEQ ID NO 622 <400> SEQUENCE: 622 000 <210> SEQ ID NO
623 <400> SEQUENCE: 623 000 <210> SEQ ID NO 624
<400> SEQUENCE: 624 000 <210> SEQ ID NO 625 <400>
SEQUENCE: 625 000 <210> SEQ ID NO 626 <400> SEQUENCE:
626 000 <210> SEQ ID NO 627
<400> SEQUENCE: 627 000 <210> SEQ ID NO 628 <400>
SEQUENCE: 628 000 <210> SEQ ID NO 629 <400> SEQUENCE:
629 000 <210> SEQ ID NO 630 <400> SEQUENCE: 630 000
<210> SEQ ID NO 631 <400> SEQUENCE: 631 000 <210>
SEQ ID NO 632 <400> SEQUENCE: 632 000 <210> SEQ ID NO
633 <400> SEQUENCE: 633 000 <210> SEQ ID NO 634
<400> SEQUENCE: 634 000 <210> SEQ ID NO 635 <400>
SEQUENCE: 635 000 <210> SEQ ID NO 636 <400> SEQUENCE:
636 000 <210> SEQ ID NO 637 <400> SEQUENCE: 637 000
<210> SEQ ID NO 638 <400> SEQUENCE: 638 000 <210>
SEQ ID NO 639 <400> SEQUENCE: 639 000 <210> SEQ ID NO
640 <400> SEQUENCE: 640 000 <210> SEQ ID NO 641
<400> SEQUENCE: 641 000 <210> SEQ ID NO 642 <400>
SEQUENCE: 642 000 <210> SEQ ID NO 643 <400> SEQUENCE:
643 000 <210> SEQ ID NO 644 <400> SEQUENCE: 644 000
<210> SEQ ID NO 645 <400> SEQUENCE: 645 000 <210>
SEQ ID NO 646 <400> SEQUENCE: 646 000 <210> SEQ ID NO
647 <400> SEQUENCE: 647 000 <210> SEQ ID NO 648
<400> SEQUENCE: 648 000 <210> SEQ ID NO 649 <400>
SEQUENCE: 649 000 <210> SEQ ID NO 650 <400> SEQUENCE:
650 000 <210> SEQ ID NO 651 <400> SEQUENCE: 651 000
<210> SEQ ID NO 652 <400> SEQUENCE: 652 000 <210>
SEQ ID NO 653 <400> SEQUENCE: 653 000 <210> SEQ ID NO
654 <400> SEQUENCE: 654 000 <210> SEQ ID NO 655
<400> SEQUENCE: 655 000 <210> SEQ ID NO 656 <400>
SEQUENCE: 656 000 <210> SEQ ID NO 657 <400> SEQUENCE:
657 000 <210> SEQ ID NO 658 <400> SEQUENCE: 658 000
<210> SEQ ID NO 659 <400> SEQUENCE: 659 000 <210>
SEQ ID NO 660 <400> SEQUENCE: 660 000 <210> SEQ ID NO
661 <400> SEQUENCE: 661 000 <210> SEQ ID NO 662
<400> SEQUENCE: 662 000 <210> SEQ ID NO 663
<400> SEQUENCE: 663 000 <210> SEQ ID NO 664 <400>
SEQUENCE: 664 000 <210> SEQ ID NO 665 <400> SEQUENCE:
665 000 <210> SEQ ID NO 666 <400> SEQUENCE: 666 000
<210> SEQ ID NO 667 <400> SEQUENCE: 667 000 <210>
SEQ ID NO 668 <400> SEQUENCE: 668 000 <210> SEQ ID NO
669 <400> SEQUENCE: 669 000 <210> SEQ ID NO 670
<400> SEQUENCE: 670 000 <210> SEQ ID NO 671 <400>
SEQUENCE: 671 000 <210> SEQ ID NO 672 <400> SEQUENCE:
672 000 <210> SEQ ID NO 673 <400> SEQUENCE: 673 000
<210> SEQ ID NO 674 <400> SEQUENCE: 674 000 <210>
SEQ ID NO 675 <400> SEQUENCE: 675 000 <210> SEQ ID NO
676 <400> SEQUENCE: 676 000 <210> SEQ ID NO 677
<400> SEQUENCE: 677 000 <210> SEQ ID NO 678 <400>
SEQUENCE: 678 000 <210> SEQ ID NO 679 <400> SEQUENCE:
679 000 <210> SEQ ID NO 680 <400> SEQUENCE: 680 000
<210> SEQ ID NO 681 <400> SEQUENCE: 681 000 <210>
SEQ ID NO 682 <400> SEQUENCE: 682 000 <210> SEQ ID NO
683 <400> SEQUENCE: 683 000 <210> SEQ ID NO 684
<400> SEQUENCE: 684 000 <210> SEQ ID NO 685 <400>
SEQUENCE: 685 000 <210> SEQ ID NO 686 <400> SEQUENCE:
686 000 <210> SEQ ID NO 687 <400> SEQUENCE: 687 000
<210> SEQ ID NO 688 <400> SEQUENCE: 688 000 <210>
SEQ ID NO 689 <400> SEQUENCE: 689 000 <210> SEQ ID NO
690 <400> SEQUENCE: 690 000 <210> SEQ ID NO 691
<400> SEQUENCE: 691 000 <210> SEQ ID NO 692 <400>
SEQUENCE: 692 000 <210> SEQ ID NO 693 <400> SEQUENCE:
693 000 <210> SEQ ID NO 694 <400> SEQUENCE: 694 000
<210> SEQ ID NO 695 <400> SEQUENCE: 695 000 <210>
SEQ ID NO 696 <400> SEQUENCE: 696 000 <210> SEQ ID NO
697 <400> SEQUENCE: 697 000 <210> SEQ ID NO 698
<400> SEQUENCE: 698 000
<210> SEQ ID NO 699 <400> SEQUENCE: 699 000 <210>
SEQ ID NO 700 <400> SEQUENCE: 700 000 <210> SEQ ID NO
701 <400> SEQUENCE: 701 000 <210> SEQ ID NO 702
<400> SEQUENCE: 702 000 <210> SEQ ID NO 703 <400>
SEQUENCE: 703 000 <210> SEQ ID NO 704 <400> SEQUENCE:
704 000 <210> SEQ ID NO 705 <400> SEQUENCE: 705 000
<210> SEQ ID NO 706 <400> SEQUENCE: 706 000 <210>
SEQ ID NO 707 <400> SEQUENCE: 707 000 <210> SEQ ID NO
708 <400> SEQUENCE: 708 000 <210> SEQ ID NO 709
<400> SEQUENCE: 709 000 <210> SEQ ID NO 710 <400>
SEQUENCE: 710 000 <210> SEQ ID NO 711 <400> SEQUENCE:
711 000 <210> SEQ ID NO 712 <400> SEQUENCE: 712 000
<210> SEQ ID NO 713 <400> SEQUENCE: 713 000 <210>
SEQ ID NO 714 <400> SEQUENCE: 714 000 <210> SEQ ID NO
715 <400> SEQUENCE: 715 000 <210> SEQ ID NO 716
<400> SEQUENCE: 716 000 <210> SEQ ID NO 717 <400>
SEQUENCE: 717 000 <210> SEQ ID NO 718 <400> SEQUENCE:
718 000 <210> SEQ ID NO 719 <400> SEQUENCE: 719 000
<210> SEQ ID NO 720 <400> SEQUENCE: 720 000 <210>
SEQ ID NO 721 <400> SEQUENCE: 721 000 <210> SEQ ID NO
722 <400> SEQUENCE: 722 000 <210> SEQ ID NO 723
<400> SEQUENCE: 723 000 <210> SEQ ID NO 724 <400>
SEQUENCE: 724 000 <210> SEQ ID NO 725 <400> SEQUENCE:
725 000 <210> SEQ ID NO 726 <400> SEQUENCE: 726 000
<210> SEQ ID NO 727 <400> SEQUENCE: 727 000 <210>
SEQ ID NO 728 <400> SEQUENCE: 728 000 <210> SEQ ID NO
729 <400> SEQUENCE: 729 000 <210> SEQ ID NO 730
<400> SEQUENCE: 730 000 <210> SEQ ID NO 731 <400>
SEQUENCE: 731 000 <210> SEQ ID NO 732 <400> SEQUENCE:
732 000 <210> SEQ ID NO 733 <400> SEQUENCE: 733 000
<210> SEQ ID NO 734 <400> SEQUENCE: 734 000
<210> SEQ ID NO 735 <400> SEQUENCE: 735 000 <210>
SEQ ID NO 736 <400> SEQUENCE: 736 000 <210> SEQ ID NO
737 <400> SEQUENCE: 737 000 <210> SEQ ID NO 738
<400> SEQUENCE: 738 000 <210> SEQ ID NO 739 <400>
SEQUENCE: 739 000 <210> SEQ ID NO 740 <400> SEQUENCE:
740 000 <210> SEQ ID NO 741 <400> SEQUENCE: 741 000
<210> SEQ ID NO 742 <400> SEQUENCE: 742 000 <210>
SEQ ID NO 743 <400> SEQUENCE: 743 000 <210> SEQ ID NO
744 <400> SEQUENCE: 744 000 <210> SEQ ID NO 745
<400> SEQUENCE: 745 000 <210> SEQ ID NO 746 <400>
SEQUENCE: 746 000 <210> SEQ ID NO 747 <400> SEQUENCE:
747 000 <210> SEQ ID NO 748 <400> SEQUENCE: 748 000
<210> SEQ ID NO 749 <400> SEQUENCE: 749 000 <210>
SEQ ID NO 750 <400> SEQUENCE: 750 000 <210> SEQ ID NO
751 <400> SEQUENCE: 751 000 <210> SEQ ID NO 752
<400> SEQUENCE: 752 000 <210> SEQ ID NO 753 <400>
SEQUENCE: 753 000 <210> SEQ ID NO 754 <400> SEQUENCE:
754 000 <210> SEQ ID NO 755 <400> SEQUENCE: 755 000
<210> SEQ ID NO 756 <400> SEQUENCE: 756 000 <210>
SEQ ID NO 757 <400> SEQUENCE: 757 000 <210> SEQ ID NO
758 <400> SEQUENCE: 758 000 <210> SEQ ID NO 759
<400> SEQUENCE: 759 000 <210> SEQ ID NO 760 <400>
SEQUENCE: 760 000 <210> SEQ ID NO 761 <400> SEQUENCE:
761 000 <210> SEQ ID NO 762 <400> SEQUENCE: 762 000
<210> SEQ ID NO 763 <400> SEQUENCE: 763 000 <210>
SEQ ID NO 764 <400> SEQUENCE: 764 000 <210> SEQ ID NO
765 <400> SEQUENCE: 765 000 <210> SEQ ID NO 766
<400> SEQUENCE: 766 000 <210> SEQ ID NO 767 <400>
SEQUENCE: 767 000 <210> SEQ ID NO 768 <400> SEQUENCE:
768 000 <210> SEQ ID NO 769 <400> SEQUENCE: 769 000
<210> SEQ ID NO 770 <400> SEQUENCE: 770 000
<210> SEQ ID NO 771 <400> SEQUENCE: 771 000 <210>
SEQ ID NO 772 <400> SEQUENCE: 772 000 <210> SEQ ID NO
773 <400> SEQUENCE: 773 000 <210> SEQ ID NO 774
<400> SEQUENCE: 774 000 <210> SEQ ID NO 775 <400>
SEQUENCE: 775 000 <210> SEQ ID NO 776 <400> SEQUENCE:
776 000 <210> SEQ ID NO 777 <400> SEQUENCE: 777 000
<210> SEQ ID NO 778 <400> SEQUENCE: 778 000 <210>
SEQ ID NO 779 <400> SEQUENCE: 779 000 <210> SEQ ID NO
780 <400> SEQUENCE: 780 000 <210> SEQ ID NO 781
<400> SEQUENCE: 781 000 <210> SEQ ID NO 782 <400>
SEQUENCE: 782 000 <210> SEQ ID NO 783 <400> SEQUENCE:
783 000 <210> SEQ ID NO 784 <400> SEQUENCE: 784 000
<210> SEQ ID NO 785 <400> SEQUENCE: 785 000 <210>
SEQ ID NO 786 <400> SEQUENCE: 786 000 <210> SEQ ID NO
787 <400> SEQUENCE: 787 000 <210> SEQ ID NO 788
<400> SEQUENCE: 788 000 <210> SEQ ID NO 789 <400>
SEQUENCE: 789 000 <210> SEQ ID NO 790 <400> SEQUENCE:
790 000 <210> SEQ ID NO 791 <400> SEQUENCE: 791 000
<210> SEQ ID NO 792 <400> SEQUENCE: 792 000 <210>
SEQ ID NO 793 <400> SEQUENCE: 793 000 <210> SEQ ID NO
794 <400> SEQUENCE: 794 000 <210> SEQ ID NO 795
<400> SEQUENCE: 795 000 <210> SEQ ID NO 796 <400>
SEQUENCE: 796 000 <210> SEQ ID NO 797 <400> SEQUENCE:
797 000 <210> SEQ ID NO 798 <400> SEQUENCE: 798 000
<210> SEQ ID NO 799 <400> SEQUENCE: 799 000 <210>
SEQ ID NO 800 <400> SEQUENCE: 800 000 <210> SEQ ID NO
801 <400> SEQUENCE: 801 000 <210> SEQ ID NO 802
<400> SEQUENCE: 802 000 <210> SEQ ID NO 803 <400>
SEQUENCE: 803 000 <210> SEQ ID NO 804 <400> SEQUENCE:
804 000 <210> SEQ ID NO 805 <400> SEQUENCE: 805 000
<210> SEQ ID NO 806 <400> SEQUENCE: 806
000 <210> SEQ ID NO 807 <400> SEQUENCE: 807 000
<210> SEQ ID NO 808 <400> SEQUENCE: 808 000 <210>
SEQ ID NO 809 <400> SEQUENCE: 809 000 <210> SEQ ID NO
810 <400> SEQUENCE: 810 000 <210> SEQ ID NO 811
<400> SEQUENCE: 811 000 <210> SEQ ID NO 812 <400>
SEQUENCE: 812 000 <210> SEQ ID NO 813 <400> SEQUENCE:
813 000 <210> SEQ ID NO 814 <400> SEQUENCE: 814 000
<210> SEQ ID NO 815 <400> SEQUENCE: 815 000 <210>
SEQ ID NO 816 <400> SEQUENCE: 816 000 <210> SEQ ID NO
817 <400> SEQUENCE: 817 000 <210> SEQ ID NO 818
<400> SEQUENCE: 818 000 <210> SEQ ID NO 819 <400>
SEQUENCE: 819 000 <210> SEQ ID NO 820 <400> SEQUENCE:
820 000 <210> SEQ ID NO 821 <400> SEQUENCE: 821 000
<210> SEQ ID NO 822 <400> SEQUENCE: 822 000 <210>
SEQ ID NO 823 <400> SEQUENCE: 823 000 <210> SEQ ID NO
824 <400> SEQUENCE: 824 000 <210> SEQ ID NO 825
<400> SEQUENCE: 825 000 <210> SEQ ID NO 826 <400>
SEQUENCE: 826 000 <210> SEQ ID NO 827 <400> SEQUENCE:
827 000 <210> SEQ ID NO 828 <400> SEQUENCE: 828 000
<210> SEQ ID NO 829 <400> SEQUENCE: 829 000 <210>
SEQ ID NO 830 <400> SEQUENCE: 830 000 <210> SEQ ID NO
831 <400> SEQUENCE: 831 000 <210> SEQ ID NO 832
<400> SEQUENCE: 832 000 <210> SEQ ID NO 833 <400>
SEQUENCE: 833 000 <210> SEQ ID NO 834 <400> SEQUENCE:
834 000 <210> SEQ ID NO 835 <400> SEQUENCE: 835 000
<210> SEQ ID NO 836 <400> SEQUENCE: 836 000 <210>
SEQ ID NO 837 <400> SEQUENCE: 837 000 <210> SEQ ID NO
838 <400> SEQUENCE: 838 000 <210> SEQ ID NO 839
<400> SEQUENCE: 839 000 <210> SEQ ID NO 840 <400>
SEQUENCE: 840 000 <210> SEQ ID NO 841 <400> SEQUENCE:
841 000 <210> SEQ ID NO 842 <400> SEQUENCE: 842
000 <210> SEQ ID NO 843 <400> SEQUENCE: 843 000
<210> SEQ ID NO 844 <400> SEQUENCE: 844 000 <210>
SEQ ID NO 845 <400> SEQUENCE: 845 000 <210> SEQ ID NO
846 <400> SEQUENCE: 846 000 <210> SEQ ID NO 847
<400> SEQUENCE: 847 000 <210> SEQ ID NO 848 <400>
SEQUENCE: 848 000 <210> SEQ ID NO 849 <400> SEQUENCE:
849 000 <210> SEQ ID NO 850 <400> SEQUENCE: 850 000
<210> SEQ ID NO 851 <400> SEQUENCE: 851 000 <210>
SEQ ID NO 852 <400> SEQUENCE: 852 000 <210> SEQ ID NO
853 <400> SEQUENCE: 853 000 <210> SEQ ID NO 854
<400> SEQUENCE: 854 000 <210> SEQ ID NO 855 <400>
SEQUENCE: 855 000 <210> SEQ ID NO 856 <400> SEQUENCE:
856 000 <210> SEQ ID NO 857 <400> SEQUENCE: 857 000
<210> SEQ ID NO 858 <400> SEQUENCE: 858 000 <210>
SEQ ID NO 859 <400> SEQUENCE: 859 000 <210> SEQ ID NO
860 <400> SEQUENCE: 860 000 <210> SEQ ID NO 861
<400> SEQUENCE: 861 000 <210> SEQ ID NO 862 <400>
SEQUENCE: 862 000 <210> SEQ ID NO 863 <400> SEQUENCE:
863 000 <210> SEQ ID NO 864 <400> SEQUENCE: 864 000
<210> SEQ ID NO 865 <400> SEQUENCE: 865 000 <210>
SEQ ID NO 866 <400> SEQUENCE: 866 000 <210> SEQ ID NO
867 <400> SEQUENCE: 867 000 <210> SEQ ID NO 868
<400> SEQUENCE: 868 000 <210> SEQ ID NO 869 <400>
SEQUENCE: 869 000 <210> SEQ ID NO 870 <400> SEQUENCE:
870 000 <210> SEQ ID NO 871 <400> SEQUENCE: 871 000
<210> SEQ ID NO 872 <400> SEQUENCE: 872 000 <210>
SEQ ID NO 873 <400> SEQUENCE: 873 000 <210> SEQ ID NO
874 <400> SEQUENCE: 874 000 <210> SEQ ID NO 875
<400> SEQUENCE: 875 000 <210> SEQ ID NO 876 <400>
SEQUENCE: 876 000 <210> SEQ ID NO 877 <400> SEQUENCE:
877 000 <210> SEQ ID NO 878
<400> SEQUENCE: 878 000 <210> SEQ ID NO 879 <400>
SEQUENCE: 879 000 <210> SEQ ID NO 880 <400> SEQUENCE:
880 000 <210> SEQ ID NO 881 <400> SEQUENCE: 881 000
<210> SEQ ID NO 882 <400> SEQUENCE: 882 000 <210>
SEQ ID NO 883 <400> SEQUENCE: 883 000 <210> SEQ ID NO
884 <400> SEQUENCE: 884 000 <210> SEQ ID NO 885
<400> SEQUENCE: 885 000 <210> SEQ ID NO 886 <400>
SEQUENCE: 886 000 <210> SEQ ID NO 887 <400> SEQUENCE:
887 000 <210> SEQ ID NO 888 <400> SEQUENCE: 888 000
<210> SEQ ID NO 889 <400> SEQUENCE: 889 000 <210>
SEQ ID NO 890 <400> SEQUENCE: 890 000 <210> SEQ ID NO
891 <400> SEQUENCE: 891 000 <210> SEQ ID NO 892
<400> SEQUENCE: 892 000 <210> SEQ ID NO 893 <400>
SEQUENCE: 893 000 <210> SEQ ID NO 894 <400> SEQUENCE:
894 000 <210> SEQ ID NO 895 <400> SEQUENCE: 895 000
<210> SEQ ID NO 896 <400> SEQUENCE: 896 000 <210>
SEQ ID NO 897 <400> SEQUENCE: 897 000 <210> SEQ ID NO
898 <400> SEQUENCE: 898 000 <210> SEQ ID NO 899
<400> SEQUENCE: 899 000 <210> SEQ ID NO 900 <400>
SEQUENCE: 900 000 <210> SEQ ID NO 901 <400> SEQUENCE:
901 000 <210> SEQ ID NO 902 <400> SEQUENCE: 902 000
<210> SEQ ID NO 903 <400> SEQUENCE: 903 000 <210>
SEQ ID NO 904 <400> SEQUENCE: 904 000 <210> SEQ ID NO
905 <400> SEQUENCE: 905 000 <210> SEQ ID NO 906
<400> SEQUENCE: 906 000 <210> SEQ ID NO 907 <400>
SEQUENCE: 907 000 <210> SEQ ID NO 908 <400> SEQUENCE:
908 000 <210> SEQ ID NO 909 <400> SEQUENCE: 909 000
<210> SEQ ID NO 910 <400> SEQUENCE: 910 000 <210>
SEQ ID NO 911 <400> SEQUENCE: 911 000 <210> SEQ ID NO
912 <400> SEQUENCE: 912 000 <210> SEQ ID NO 913
<400> SEQUENCE: 913 000 <210> SEQ ID NO 914
<400> SEQUENCE: 914 000 <210> SEQ ID NO 915 <400>
SEQUENCE: 915 000 <210> SEQ ID NO 916 <400> SEQUENCE:
916 000 <210> SEQ ID NO 917 <400> SEQUENCE: 917 000
<210> SEQ ID NO 918 <400> SEQUENCE: 918 000 <210>
SEQ ID NO 919 <400> SEQUENCE: 919 000 <210> SEQ ID NO
920 <400> SEQUENCE: 920 000 <210> SEQ ID NO 921
<400> SEQUENCE: 921 000 <210> SEQ ID NO 922 <400>
SEQUENCE: 922 000 <210> SEQ ID NO 923 <400> SEQUENCE:
923 000 <210> SEQ ID NO 924 <400> SEQUENCE: 924 000
<210> SEQ ID NO 925 <400> SEQUENCE: 925 000 <210>
SEQ ID NO 926 <400> SEQUENCE: 926 000 <210> SEQ ID NO
927 <400> SEQUENCE: 927 000 <210> SEQ ID NO 928
<400> SEQUENCE: 928 000 <210> SEQ ID NO 929 <400>
SEQUENCE: 929 000 <210> SEQ ID NO 930 <400> SEQUENCE:
930 000 <210> SEQ ID NO 931 <400> SEQUENCE: 931 000
<210> SEQ ID NO 932 <400> SEQUENCE: 932 000 <210>
SEQ ID NO 933 <400> SEQUENCE: 933 000 <210> SEQ ID NO
934 <400> SEQUENCE: 934 000 <210> SEQ ID NO 935
<400> SEQUENCE: 935 000 <210> SEQ ID NO 936 <400>
SEQUENCE: 936 000 <210> SEQ ID NO 937 <400> SEQUENCE:
937 000 <210> SEQ ID NO 938 <400> SEQUENCE: 938 000
<210> SEQ ID NO 939 <400> SEQUENCE: 939 000 <210>
SEQ ID NO 940 <400> SEQUENCE: 940 000 <210> SEQ ID NO
941 <400> SEQUENCE: 941 000 <210> SEQ ID NO 942
<400> SEQUENCE: 942 000 <210> SEQ ID NO 943 <400>
SEQUENCE: 943 000 <210> SEQ ID NO 944 <400> SEQUENCE:
944 000 <210> SEQ ID NO 945 <400> SEQUENCE: 945 000
<210> SEQ ID NO 946 <400> SEQUENCE: 946 000 <210>
SEQ ID NO 947 <400> SEQUENCE: 947 000 <210> SEQ ID NO
948 <400> SEQUENCE: 948 000 <210> SEQ ID NO 949
<400> SEQUENCE: 949 000
<210> SEQ ID NO 950 <400> SEQUENCE: 950 000 <210>
SEQ ID NO 951 <400> SEQUENCE: 951 000 <210> SEQ ID NO
952 <400> SEQUENCE: 952 000 <210> SEQ ID NO 953
<400> SEQUENCE: 953 000 <210> SEQ ID NO 954 <400>
SEQUENCE: 954 000 <210> SEQ ID NO 955 <400> SEQUENCE:
955 000 <210> SEQ ID NO 956 <400> SEQUENCE: 956 000
<210> SEQ ID NO 957 <400> SEQUENCE: 957 000 <210>
SEQ ID NO 958 <400> SEQUENCE: 958 000 <210> SEQ ID NO
959 <400> SEQUENCE: 959 000 <210> SEQ ID NO 960
<400> SEQUENCE: 960 000 <210> SEQ ID NO 961 <400>
SEQUENCE: 961 000 <210> SEQ ID NO 962 <400> SEQUENCE:
962 000 <210> SEQ ID NO 963 <400> SEQUENCE: 963 000
<210> SEQ ID NO 964 <400> SEQUENCE: 964 000 <210>
SEQ ID NO 965 <400> SEQUENCE: 965 000 <210> SEQ ID NO
966 <400> SEQUENCE: 966 000 <210> SEQ ID NO 967
<400> SEQUENCE: 967 000 <210> SEQ ID NO 968 <400>
SEQUENCE: 968 000 <210> SEQ ID NO 969 <400> SEQUENCE:
969 000 <210> SEQ ID NO 970 <400> SEQUENCE: 970 000
<210> SEQ ID NO 971 <400> SEQUENCE: 971 000 <210>
SEQ ID NO 972 <400> SEQUENCE: 972 000 <210> SEQ ID NO
973 <400> SEQUENCE: 973 000 <210> SEQ ID NO 974
<400> SEQUENCE: 974 000 <210> SEQ ID NO 975 <400>
SEQUENCE: 975 000 <210> SEQ ID NO 976 <400> SEQUENCE:
976 000 <210> SEQ ID NO 977 <400> SEQUENCE: 977 000
<210> SEQ ID NO 978 <400> SEQUENCE: 978 000 <210>
SEQ ID NO 979 <400> SEQUENCE: 979 000 <210> SEQ ID NO
980 <400> SEQUENCE: 980 000 <210> SEQ ID NO 981
<400> SEQUENCE: 981 000 <210> SEQ ID NO 982 <400>
SEQUENCE: 982 000 <210> SEQ ID NO 983 <400> SEQUENCE:
983 000 <210> SEQ ID NO 984 <400> SEQUENCE: 984 000
<210> SEQ ID NO 985 <400> SEQUENCE: 985 000
<210> SEQ ID NO 986 <400> SEQUENCE: 986 000 <210>
SEQ ID NO 987 <400> SEQUENCE: 987 000 <210> SEQ ID NO
988 <400> SEQUENCE: 988 000 <210> SEQ ID NO 989
<400> SEQUENCE: 989 000 <210> SEQ ID NO 990 <400>
SEQUENCE: 990 000 <210> SEQ ID NO 991 <400> SEQUENCE:
991 000 <210> SEQ ID NO 992 <400> SEQUENCE: 992 000
<210> SEQ ID NO 993 <400> SEQUENCE: 993 000 <210>
SEQ ID NO 994 <400> SEQUENCE: 994 000 <210> SEQ ID NO
995 <400> SEQUENCE: 995 000 <210> SEQ ID NO 996
<400> SEQUENCE: 996 000 <210> SEQ ID NO 997 <400>
SEQUENCE: 997 000 <210> SEQ ID NO 998 <400> SEQUENCE:
998 000 <210> SEQ ID NO 999 <400> SEQUENCE: 999 000
<210> SEQ ID NO 1000 <400> SEQUENCE: 1000 000
<210> SEQ ID NO 1001 <400> SEQUENCE: 1001 000
<210> SEQ ID NO 1002 <400> SEQUENCE: 1002 000
<210> SEQ ID NO 1003 <400> SEQUENCE: 1003 000
<210> SEQ ID NO 1004 <400> SEQUENCE: 1004 000
<210> SEQ ID NO 1005 <400> SEQUENCE: 1005 000
<210> SEQ ID NO 1006 <400> SEQUENCE: 1006 000
<210> SEQ ID NO 1007 <400> SEQUENCE: 1007 000
<210> SEQ ID NO 1008 <400> SEQUENCE: 1008 000
<210> SEQ ID NO 1009 <400> SEQUENCE: 1009 000
<210> SEQ ID NO 1010 <400> SEQUENCE: 1010 000
<210> SEQ ID NO 1011 <400> SEQUENCE: 1011 000
<210> SEQ ID NO 1012 <400> SEQUENCE: 1012 000
<210> SEQ ID NO 1013 <400> SEQUENCE: 1013 000
<210> SEQ ID NO 1014 <400> SEQUENCE: 1014 000
<210> SEQ ID NO 1015 <400> SEQUENCE: 1015 000
<210> SEQ ID NO 1016 <400> SEQUENCE: 1016 000
<210> SEQ ID NO 1017 <400> SEQUENCE: 1017 000
<210> SEQ ID NO 1018 <400> SEQUENCE: 1018 000
<210> SEQ ID NO 1019 <400> SEQUENCE: 1019 000
<210> SEQ ID NO 1020 <400> SEQUENCE: 1020 000
<210> SEQ ID NO 1021 <400> SEQUENCE: 1021 000
<210> SEQ ID NO 1022 <400> SEQUENCE: 1022 000
<210> SEQ ID NO 1023 <400> SEQUENCE: 1023 000
<210> SEQ ID NO 1024 <400> SEQUENCE: 1024 000
<210> SEQ ID NO 1025 <400> SEQUENCE: 1025 000
<210> SEQ ID NO 1026 <400> SEQUENCE: 1026 000
<210> SEQ ID NO 1027 <400> SEQUENCE: 1027 000
<210> SEQ ID NO 1028 <400> SEQUENCE: 1028 000
<210> SEQ ID NO 1029 <400> SEQUENCE: 1029 000
<210> SEQ ID NO 1030 <400> SEQUENCE: 1030 000
<210> SEQ ID NO 1031 <400> SEQUENCE: 1031 000
<210> SEQ ID NO 1032 <400> SEQUENCE: 1032 000
<210> SEQ ID NO 1033 <400> SEQUENCE: 1033 000
<210> SEQ ID NO 1034 <400> SEQUENCE: 1034 000
<210> SEQ ID NO 1035 <400> SEQUENCE: 1035 000
<210> SEQ ID NO 1036 <400> SEQUENCE: 1036 000
<210> SEQ ID NO 1037 <400> SEQUENCE: 1037 000
<210> SEQ ID NO 1038 <400> SEQUENCE: 1038 000
<210> SEQ ID NO 1039 <400> SEQUENCE: 1039 000
<210> SEQ ID NO 1040 <400> SEQUENCE: 1040 000
<210> SEQ ID NO 1041 <400> SEQUENCE: 1041 000
<210> SEQ ID NO 1042 <400> SEQUENCE: 1042 000
<210> SEQ ID NO 1043 <400> SEQUENCE: 1043 000
<210> SEQ ID NO 1044 <400> SEQUENCE: 1044 000
<210> SEQ ID NO 1045 <400> SEQUENCE: 1045 000
<210> SEQ ID NO 1046 <400> SEQUENCE: 1046 000
<210> SEQ ID NO 1047 <400> SEQUENCE: 1047 000
<210> SEQ ID NO 1048 <400> SEQUENCE: 1048 000
<210> SEQ ID NO 1049 <400> SEQUENCE: 1049 000
<210> SEQ ID NO 1050 <400> SEQUENCE: 1050 000
<210> SEQ ID NO 1051 <400> SEQUENCE: 1051 000
<210> SEQ ID NO 1052 <400> SEQUENCE: 1052 000
<210> SEQ ID NO 1053 <400> SEQUENCE: 1053 000
<210> SEQ ID NO 1054 <400> SEQUENCE: 1054 000
<210> SEQ ID NO 1055 <400> SEQUENCE: 1055 000
<210> SEQ ID NO 1056 <400> SEQUENCE: 1056 000
<210> SEQ ID NO 1057 <400> SEQUENCE: 1057
000 <210> SEQ ID NO 1058 <400> SEQUENCE: 1058 000
<210> SEQ ID NO 1059 <400> SEQUENCE: 1059 000
<210> SEQ ID NO 1060 <400> SEQUENCE: 1060 000
<210> SEQ ID NO 1061 <400> SEQUENCE: 1061 000
<210> SEQ ID NO 1062 <400> SEQUENCE: 1062 000
<210> SEQ ID NO 1063 <400> SEQUENCE: 1063 000
<210> SEQ ID NO 1064 <400> SEQUENCE: 1064 000
<210> SEQ ID NO 1065 <400> SEQUENCE: 1065 000
<210> SEQ ID NO 1066 <400> SEQUENCE: 1066 000
<210> SEQ ID NO 1067 <400> SEQUENCE: 1067 000
<210> SEQ ID NO 1068 <400> SEQUENCE: 1068 000
<210> SEQ ID NO 1069 <400> SEQUENCE: 1069 000
<210> SEQ ID NO 1070 <400> SEQUENCE: 1070 000
<210> SEQ ID NO 1071 <400> SEQUENCE: 1071 000
<210> SEQ ID NO 1072 <400> SEQUENCE: 1072 000
<210> SEQ ID NO 1073 <400> SEQUENCE: 1073 000
<210> SEQ ID NO 1074 <400> SEQUENCE: 1074 000
<210> SEQ ID NO 1075 <400> SEQUENCE: 1075 000
<210> SEQ ID NO 1076 <400> SEQUENCE: 1076 000
<210> SEQ ID NO 1077 <400> SEQUENCE: 1077 000
<210> SEQ ID NO 1078 <400> SEQUENCE: 1078 000
<210> SEQ ID NO 1079 <400> SEQUENCE: 1079 000
<210> SEQ ID NO 1080 <400> SEQUENCE: 1080 000
<210> SEQ ID NO 1081 <400> SEQUENCE: 1081 000
<210> SEQ ID NO 1082 <400> SEQUENCE: 1082 000
<210> SEQ ID NO 1083 <400> SEQUENCE: 1083 000
<210> SEQ ID NO 1084 <400> SEQUENCE: 1084 000
<210> SEQ ID NO 1085 <400> SEQUENCE: 1085 000
<210> SEQ ID NO 1086 <400> SEQUENCE: 1086 000
<210> SEQ ID NO 1087 <400> SEQUENCE: 1087 000
<210> SEQ ID NO 1088 <400> SEQUENCE: 1088 000
<210> SEQ ID NO 1089 <400> SEQUENCE: 1089 000
<210> SEQ ID NO 1090 <400> SEQUENCE: 1090 000
<210> SEQ ID NO 1091 <400> SEQUENCE: 1091 000
<210> SEQ ID NO 1092 <400> SEQUENCE: 1092 000
<210> SEQ ID NO 1093 <400> SEQUENCE: 1093
000 <210> SEQ ID NO 1094 <400> SEQUENCE: 1094 000
<210> SEQ ID NO 1095 <400> SEQUENCE: 1095 000
<210> SEQ ID NO 1096 <400> SEQUENCE: 1096 000
<210> SEQ ID NO 1097 <400> SEQUENCE: 1097 000
<210> SEQ ID NO 1098 <400> SEQUENCE: 1098 000
<210> SEQ ID NO 1099 <400> SEQUENCE: 1099 000
<210> SEQ ID NO 1100 <400> SEQUENCE: 1100 000
<210> SEQ ID NO 1101 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, L117A <400> SEQUENCE: 1101 Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50
55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180
185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305
310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
<210> SEQ ID NO 1102 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, L118A <400> SEQUENCE: 1102 Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50
55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Ala Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180
185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305
310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
<210> SEQ ID NO 1103 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, L117A & L118A <400> SEQUENCE: 1103 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225
230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO 1104
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG1 HC constant region, L117G & L118G
<400> SEQUENCE: 1104 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110 Pro Ala Pro Glu Gly Gly Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210
215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO
1105 <211> LENGTH: 330 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic IgG1 HC constant region, L117V &
L118V <400> SEQUENCE: 1105 Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65
70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Val Val Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 1106 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
L117I & L118I <400> SEQUENCE: 1106 Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ile Ile Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
<210> SEQ ID NO 1107 <400> SEQUENCE: 1107 000
<210> SEQ ID NO 1108 <400> SEQUENCE: 1108 000
<210> SEQ ID NO 1109 <400> SEQUENCE: 1109 000
<210> SEQ ID NO 1110 <400> SEQUENCE: 1110 000
<210> SEQ ID NO 1111 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, HJ C->S, L117A <400> SEQUENCE: 1111 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Ser Asp
Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330 <210> SEQ ID NO 1112 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, HJ C->S, L118A <400> SEQUENCE: 1112 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Ser Asp
Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Ala
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330 <210> SEQ ID NO 1113 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, HJ C->S, L117A & L118A <400> SEQUENCE: 1113
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325 330 <210> SEQ ID NO 1114 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
HJ C->S, L117G & L118G <400> SEQUENCE: 1114 Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Ser
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Gly
Gly Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150
155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275
280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330 <210> SEQ ID NO 1115 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG1
HC constant region, HJ C->S, L117V & L118V <400>
SEQUENCE: 1115 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys 100 105
110 Pro Ala Pro Glu Val Val Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230
235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO 1116
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG1 HC constant region, HJ C->S, L117I
& L118I <400> SEQUENCE: 1116 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ile Ile Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 1117 <400> SEQUENCE: 1117 000 <210> SEQ ID NO
1118 <400> SEQUENCE: 1118 000 <210> SEQ ID NO 1119
<400> SEQUENCE: 1119 000 <210> SEQ ID NO 1120
<400> SEQUENCE: 1120 000 <210> SEQ ID NO 1121
<211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
HJ C->V, L117A <400> SEQUENCE: 1121 Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Val Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
<210> SEQ ID NO 1122 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, HJ C->V, L118A <400> SEQUENCE: 1122 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Val Asp
Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Ala
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330 <210> SEQ ID NO 1123 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, HJ C->V, L117A & L118A <400> SEQUENCE: 1123
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys
Ser Val Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325 330 <210> SEQ ID NO 1124 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG1
HC constant region, HJ C->V, L117G & L118G <400>
SEQUENCE: 1124 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95
Arg Val Glu Pro Lys Ser Val Asp Lys Thr His Thr Cys Pro Pro Cys 100
105 110 Pro Ala Pro Glu Gly Gly Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225
230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO 1125
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG1 HC constant region, HJ C->V, L117V
& L118V <400> SEQUENCE: 1125 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Val Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Val Val Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 1126 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
HJ C->V, L117I & L118I <400> SEQUENCE: 1126 Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Val
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ile
Ile Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150
155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275
280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330 <210> SEQ ID NO 1127 <400> SEQUENCE: 1127 000
<210> SEQ ID NO 1128 <400> SEQUENCE: 1128 000
<210> SEQ ID NO 1129 <400> SEQUENCE: 1129 000
<210> SEQ ID NO 1130 <400> SEQUENCE: 1130 000
<210> SEQ ID NO 1131 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, BJ C->S, L117A <400> SEQUENCE: 1131 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys
85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro
Pro Ser 100 105 110 Pro Ala Pro Glu Ala Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID
NO 1132 <211> LENGTH: 330 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic IgG1 HC constant region, BJ C->S,
L118A <400> SEQUENCE: 1132 Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65
70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Ser Pro Pro Ser 100 105 110 Pro Ala Pro Glu Leu Ala Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 1133 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
BJ C->S, L117A & L118A <400> SEQUENCE: 1133 Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Ser Pro Pro Ser 100 105 110 Pro Ala Pro Glu Ala
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150
155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275
280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330 <210> SEQ ID NO 1134 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG1
HC constant region, BJ C->S, L117G & L118G <400>
SEQUENCE: 1134 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ser 100 105
110 Pro Ala Pro Glu Gly Gly Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225
230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO 1135
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG1 HC constant region, BJ C->S, L117V
& L118V <400> SEQUENCE: 1135 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Ser Pro Pro Ser 100 105 110 Pro Ala Pro Glu Val Val Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 1136 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
BJ C->S, L117I & L118I <400> SEQUENCE: 1136 Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Ser Pro Pro Ser 100 105 110 Pro Ala Pro Glu Ile
Ile Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150
155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275
280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330 <210> SEQ ID NO 1137 <400> SEQUENCE: 1137 000
<210> SEQ ID NO 1138 <400> SEQUENCE: 1138 000
<210> SEQ ID NO 1139 <400> SEQUENCE: 1139 000
<210> SEQ ID NO 1140 <400> SEQUENCE: 1140 000
<210> SEQ ID NO 1141 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, BJ C->V, L117A <400> SEQUENCE: 1141 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Val Pro Pro Val 100 105 110 Pro Ala Pro Glu Ala Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155
160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325 330 <210> SEQ ID NO 1142 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG1
HC constant region, BJ C->V, L118A <400> SEQUENCE: 1142
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Val Pro Pro Val 100 105 110 Pro Ala Pro
Glu Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325 330 <210> SEQ ID NO 1143 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG1
HC constant region, BJ C->V, L117A & L118A <400>
SEQUENCE: 1143 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Val Pro Pro Val 100 105
110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230
235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO 1144
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG1 HC constant region, BJ C->V, L117G
& L118G <400> SEQUENCE: 1144 Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Val Pro Pro Val 100 105 110 Pro Ala Pro Glu Gly Gly Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>
SEQ ID NO 1145 <211> LENGTH: 330 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic IgG1 HC constant region,
BJ C->V, L117V & L118V <400> SEQUENCE: 1145
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Val Pro Pro Val 100 105 110 Pro Ala Pro
Glu Val Val Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325 330 <210> SEQ ID NO 1146 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG1
HC constant region, BJ C->V, L117I & L118I <400>
SEQUENCE: 1146 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Val Pro Pro Val 100 105
110 Pro Ala Pro Glu Ile Ile Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230
235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO 1147
<400> SEQUENCE: 1147 000 <210> SEQ ID NO 1148
<400> SEQUENCE: 1148 000 <210> SEQ ID NO 1149
<400> SEQUENCE: 1149 000 <210> SEQ ID NO 1150
<400> SEQUENCE: 1150 000 <210> SEQ ID NO 1151
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG1 HC constant region, DJ C->S, L117A
<400> SEQUENCE: 1151 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Arg Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro
Ser 100 105 110 Pro Ala Pro Glu Ala Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210
215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO
1152 <211> LENGTH: 330 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic IgG1 HC constant region, DJ C->S,
L118A
<400> SEQUENCE: 1152 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Arg Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro
Ser 100 105 110 Pro Ala Pro Glu Leu Ala Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210
215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO
1153 <211> LENGTH: 330 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic IgG1 HC constant region, DJ C->S,
L117A & L118A <400> SEQUENCE: 1153 Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Ser Asp Lys Thr
His Thr Ser Pro Pro Ser 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
<210> SEQ ID NO 1154 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, DJ C->S, L117G & L118G <400> SEQUENCE: 1154
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys
Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser 100 105 110 Pro Ala Pro
Glu Gly Gly Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325 330 <210> SEQ ID NO 1155 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG1
HC constant region, DJ C->S, L117V & L118V <400>
SEQUENCE: 1155 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser 100 105
110 Pro Ala Pro Glu Val Val Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130
135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250
255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 325 330 <210> SEQ ID NO 1156 <211> LENGTH:
330 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG1
HC constant region, DJ C->S, L117I & L118I <400>
SEQUENCE: 1156 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser 100 105
110 Pro Ala Pro Glu Ile Ile Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230
235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO 1157
<400> SEQUENCE: 1157 000 <210> SEQ ID NO 1158
<400> SEQUENCE: 1158 000 <210> SEQ ID NO 1159
<400> SEQUENCE: 1159 000 <210> SEQ ID NO 1160
<400> SEQUENCE: 1160 000 <210> SEQ ID NO 1161
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic IgG1 HC constant region, DJ C->V, L117A
<400> SEQUENCE: 1161 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Arg Val Glu Pro Lys Ser Val Asp Lys Thr His Thr Val Pro Pro
Val 100 105 110 Pro Ala Pro Glu Ala Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210
215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO
1162 <211> LENGTH: 330 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic IgG1 HC constant region, DJ C->V,
L118A <400> SEQUENCE: 1162 Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65
70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Val Asp Lys Thr His Thr
Val Pro Pro Val 100 105 110
Pro Ala Pro Glu Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115
120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235
240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO 1163 <211>
LENGTH: 330 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic IgG1 HC constant region, DJ C->V, L117A & L118A
<400> SEQUENCE: 1163 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Arg Val Glu Pro Lys Ser Val Asp Lys Thr His Thr Val Pro Pro
Val 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210
215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO
1164 <211> LENGTH: 330 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic IgG1 HC constant region, DJ C->V,
L117G & L118G <400> SEQUENCE: 1164 Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Val Asp Lys Thr
His Thr Val Pro Pro Val 100 105 110 Pro Ala Pro Glu Gly Gly Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
<210> SEQ ID NO 1165 <211> LENGTH: 330 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic IgG1 HC constant
region, DJ C->V, L117V & L118V <400> SEQUENCE: 1165
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys
Ser Val Asp Lys Thr His Thr Val Pro Pro Val 100 105 110 Pro Ala Pro
Glu Val Val Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250
255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325 330 <210> SEQ ID NO 1166 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic IgG1
HC constant region, DJ C->V, L117I & L118I <400>
SEQUENCE: 1166 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg
Val Glu Pro Lys Ser Val Asp Lys Thr His Thr Val Pro Pro Val 100 105
110 Pro Ala Pro Glu Ile Ile Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230
235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 330
* * * * *
References