U.S. patent application number 12/253929 was filed with the patent office on 2009-06-18 for immunotherapy regimes dependent on apoe status.
This patent application is currently assigned to Wyeth. Invention is credited to Ronald Black, James Callaway, Lars Ekman, Davinder Gill, Keith M. Gregg, Michael Grundman, Jack Steven Jacobsen, Ivan Lieberburg, Lioudmila Tchistiakova, Angela Widom.
Application Number | 20090155256 12/253929 |
Document ID | / |
Family ID | 40568093 |
Filed Date | 2009-06-18 |
United States Patent
Application |
20090155256 |
Kind Code |
A1 |
Black; Ronald ; et
al. |
June 18, 2009 |
Immunotherapy Regimes Dependent On APOE Status
Abstract
The invention provides methods of immunotherapy of Alzheimer's
and similar diseases in which the regime administered to a patient
depends on the ApoE genotype of the patient.
Inventors: |
Black; Ronald; (Berwyn,
PA) ; Ekman; Lars; (La Jolla, CA) ;
Lieberburg; Ivan; (Berkeley, CA) ; Grundman;
Michael; (San Diego, CA) ; Callaway; James;
(San Diego, CA) ; Gregg; Keith M.; (Goodyear,
AZ) ; Jacobsen; Jack Steven; (Ramsey, NJ) ;
Gill; Davinder; (Andover, MA) ; Tchistiakova;
Lioudmila; (Andover, MA) ; Widom; Angela;
(Acton, MA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Wyeth
Madison
NJ
Elan Pharma International Limited
Athlone
|
Family ID: |
40568093 |
Appl. No.: |
12/253929 |
Filed: |
October 17, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61083827 |
Jul 25, 2008 |
|
|
|
60999423 |
Oct 17, 2007 |
|
|
|
Current U.S.
Class: |
424/133.1 ;
424/139.1; 530/387.1; 530/387.3; 536/23.1 |
Current CPC
Class: |
A61P 25/00 20180101;
C07K 16/18 20130101; C07K 16/2896 20130101; C07K 2317/56 20130101;
C07K 2317/24 20130101; A61P 25/28 20180101; C07K 2317/77 20130101;
A61P 9/00 20180101; C07K 2317/734 20130101; G06Q 99/00 20130101;
C07K 2317/732 20130101; C07K 2317/71 20130101; A61K 2039/505
20130101; A61P 7/10 20180101; C07K 2317/34 20130101 |
Class at
Publication: |
424/133.1 ;
424/139.1; 530/387.3; 536/23.1; 530/387.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/00 20060101 C07K016/00; C07H 21/00 20060101
C07H021/00; A61P 25/28 20060101 A61P025/28 |
Claims
1. A method of treating Alzheimer's disease, comprising
administering to a patient having zero ApoE4 alleles ("ApoE4
non-carrier patient") and Alzheimer's disease, an effective regime
of an antibody that specifically binds to a N-terminal epitope of
A.beta., wherein a dosage of the antibody within a range of about
0.15 mg/kg to about 2 mg/kg is administered by intravenous
infusion.
2. The method of claim 1, wherein antibody specifically binds to an
epitope within: residues 1-7 of A.beta.; residues 1-5 of A.beta.;
or residues 3-7 of A.beta..
3. (canceled)
4. The method of claim 2, wherein the dosage is administered every:
4 to 16 weeks; 10 to 14 weeks; or 13 weeks.
5. The method of claim 4, wherein the dosage is about 0.5 mg/kg to
about 1 mg/kg.
6. The method of claim 2, wherein the antibody is a humanized form
of a mouse 3D6 antibody (ATCC accession number PTA-5130), and
positions 234, 235 and 237 in the heavy chain constant region are
occupied by Ala, Ala and Ala respectively, wherein positions are
numbered by the EU numbering system.
7. The method of claim 5, wherein the antibody is bapineuzumab.
8. The method of claim 7, further comprising monitoring for
vasogenic edema.
9. The method of claim 8, comprising administering a corticosteroid
to the patient to treat vasogenic edema detected by the
monitoring.
10. A method of treating Alzheimer's disease, comprising
administering to an ApoE4 non-carrier patient an antibody that
specifically recognizes the N-terminal region of A.beta. in a
regime effective to maintain a mean serum concentration of the
antibody in the range of about 0.1 .mu.g/ml to about 60
.mu.g/ml.
11. (canceled)
12. The method of claim 10, wherein the maximum serum concentration
is within a range of about 4-28 .mu.g antibody/ml serum.
13-14. (canceled)
15. A method of treating Alzheimer's disease, comprising
administering to an ApoE4 non-carrier patient bapienuzumab in a
regime effective to achieve a mean plasma A.beta. concentration of
at least 450 pg/ml.
16. The method of claim 15, wherein the mean plasma A.beta.
concentration is in the range of about 600 pg/ml to about 3000
pg/ml.
17-18. (canceled)
19. A method of reducing cognitive decline in a patient having zero
ApoE4 alleles ("ApoE4 non-carrier patient"), comprising
administering to the patient bapineuzumab in a regime effective to
reduce the cognitive decline of the patient relative to a control
patient to whom the bapineuzumab is not administered, wherein a
dosage of the bapineuzumab within a range of about 0.15 mg/kg to
about 2 mg/kg is administered by intravenous infusion wherein: the
ApoE4 non-carrier patient and control patient have been diagnosed
with mild to moderate Alzheimer's disease; and the cognitive
decline is measured by ADAS-COG, NTB, MMSE or CDR-SB.
20-22. (canceled)
23. A method of reducing brain volume decline in a patient having
zero ApoE4 alleles ("ApoE4 non-carrier patient"), comprising
administering to the ApoE4 non-carrier patient bapineuzumab in a
regime effective to reduce the brain volume decline of the ApoE4
non-carrier patient relative to a control patient to whom the
bapineuzumab is not administered; wherein the ApoE4 non-carrier
patient and control patient have been diagnosed with mild to
moderate Alzheimer's disease; and, wherein a dosage of the antibody
within a range of about 0.15 mg/kg to about 2 mg/kg is administered
by intravenous infusion.
24-26. (canceled)
27. The method of claim 23, wherein the brain volume decline is
measured by MRI.
28. A method of treating Alzheimer's disease, comprising
subcutaneously administering to a patient having the disease and
one or two copies of an ApoE4 allele an effective regime of
bapineuzumab at a dose of 0.01-0.6 mg/kg and a frequency of between
weekly and monthly.
29. The method of claim 28 further comprising monitoring for
vasogenic edema.
30-33. (canceled)
34. A method of treating Alzheimer's disease, comprising
administering to a patient having the disease and one or two ApoE4
alleles an effective regime of bapineuzumab; and administering a
corticosteroid to the patient to treat vasogenic edema arising from
the administration of the bapineuzumab.
35. (canceled)
36. The method of claim 34, wherein the dose or frequency of
administration of the antibody is reduced or eliminated during the
vasogenic edema relative to the dose or frequency before the
vasogenic edema.
37. The method of claim 36, wherein the dose or frequency of
administration of the antibody is increased after resolution of the
vasogenic edema relative to the dose or frequency either before or
during the vasogenic edema.
38. A method of treating or effecting prophylaxis in a population
of patients of an amyloidogenic disease characterized by amyloid
deposits of A.beta. in the brain, comprising: administering
different regimes to different patients in the population depending
on which allelic forms of ApoE are present in the patients; wherein
at least one of the regimes comprises administering an antibody to
A.beta. to a patient.
39. The method of claim 38, wherein a first regime comprises
administering an antibody to A.beta. to a patient and a second
regime lacks an antibody to A.beta. or an agent that induces an
antibody to A.beta. and the first regime is administered to
patients having zero copies of an ApoE4 allele and the second
regime is administered to patients having one or two copies of an
ApoE4 allele.
40. The method of claim 38, wherein the different regimes comprise
first and second regimes each comprises administering an antibody
to A.beta.; and the second regime differs from the first regime in
at least one of (i)-(vi) below: (i) the dose of the antibody is
reduced; (ii) the frequency of administration of the antibody is
reduced; (iii) the capacity of the antibody to induce a clearing
response to amyloid deposits is reduced; (iv) the mean serum
concentration of the antibody is reduced; (v) the maximum serum
concentration of the antibody is reduced; (vi) the time of
initiation of treatment relative to disease progression is earlier;
whereby the first and second regimes are administered such that at
least one of (a), (b) and (c) occurs: (a) the second regime is
administered in patients having two copies of an ApoE4 allele and
the first regime in patients having zero copies of an ApoE4 allele;
(b) the second regime is administered in patients having one copy
of an ApoE4 allele and the first regime in patients having zero
copies of an ApoE4 allele; and/or (c) the second regime is
administered in patients having two copies of an ApoE4 allele and
the first regime is administered to patients having one copy of an
ApoE4 allele.
41. The method of claim 40, wherein a first regime comprises
administering a first antibody to A.beta. and the second regime
comprises administering a second antibody to A.beta. and the second
antibody has reduced binding to an Fc.gamma. receptor and/or C1q
relative to the first antibody, and the first antibody is
administered to patients having zero copies of an ApoE4 allele and
the second antibody is administered to patients having one or two
copies of an ApoE4 allele.
42. The method of claim 41, wherein the second antibody has one or
more mutations in the constant region that reduce binding to the
Fc.gamma. receptor and/or C1 q, the mutations not being present in
the first antibody.
43. The method of claim 42, wherein the one or more mutations
is/are at position(s) in a heavy chain constant region selected
from the group consisting of positions 234, 235, 236 and 237 (EU
numbering).
44. The method of claim 43, wherein the one or more mutations are
mutations at positions 234, 235 and 237.
45. The method of claim 44, wherein the one or more mutations are
L234A, L235A and G237A.
46. (canceled)
47. The method of any of claims 42-45, wherein the isotype of the
constant region is human IgG4.
48. The method of claim 41, wherein the first antibody is
bapineuzumab and the second antibody is an L234A, L235A, G237A
variant of bapineuzumab.
49-50. (canceled)
51. The method of claim 38 or, further comprising determining which
alleles of ApoE are present in the patient.
52-54. (canceled)
55. The method of claim 38, wherein the dose of the antibody and/or
the frequency of administration of the antibody and/or the capacity
of the antibody to induce a clearing response to amyloid deposits
is reduced in (a) patients having two ApoE4 alleles relative to
patients having one ApoE4 allele; and/or (b) patients having one
copy of an ApoE4 allele relative to patients having zero copies of
an ApoE4 allele, and/or (c) patients having two copies of an ApoE4
allele relative to patients having one copy of an ApoE4 allele.
56. (canceled)
57. The method of claim 38, wherein patients in the population
having one or two ApoE4 alleles are administered a dose of 0.15-1
mg/kg, and patients in the population having zero ApoE4 alleles are
administered a dose of 0.5-2 mg/kg of an antibody specifically
binding within residues 1-11 of A.beta..
58-59. (canceled)
60. The method of claim 38, wherein the patients in the population
having one or two ApoE4 alleles are administered an antibody with
reduced capacity to induce a clearing response to amyloid deposits
relative to bapineuzumab.
61. The method of claim 38, further comprising monitoring at least
some of the patients in the population for vasogenic edema.
62-63. (canceled)
64. The method of claim 38, wherein the antibody binds to an
epitope within residues 1-11 of A.beta..
65-68. (canceled)
69. The method of claim 38, wherein patients with one or two ApoE4
alleles are administered 1-3 doses of humanized 266 antibody
following by subsequent doses of bapineuzumab and patients with
zero ApoE4 alleles are administered the same total number of doses
but all with bapineuzumab.
70. The method of claim 38, wherein patients with one or two ApoE4
alleles are administered humanized 266 antibody and patients with
zero ApoE4 alleles are administered bapineuzumab.
71-74. (canceled)
75. A method of monitoring a population of patients undergoing
treatment or prophylaxis for Alzheimer's disease with an antibody
to A.beta., the method comprising: performing different monitoring
regimes in different patients in the population for vasogenic
edema, wherein the frequency of monitoring is greater for: (a)
patients having two copies of ApoE4 relative to patients having
zero copies of ApoE4; (b) patients having one copy of an ApoE4
allele relative to patients having zero copies of an ApoE4 allele;
and/or (c) patients having two copies of an ApoE4 allele relative
to patients having one copy of an ApoE4 allele.
76-84. (canceled)
85. A method of treating or effecting prophylaxis of a patient for
Alzheimer's disease, comprising administering to a patient with at
least one ApoE4 allele a humanized form of a mouse 3D6 antibody
(ATCC accession number PTA-5130), and positions 234, 235 and 237 in
the heavy chain constant region are occupied by Ala, Ala and Ala
respectively, wherein positions are numbered by the EU numbering
system, and monitoring the patient for vasogenic edema by MRI.
86-87. (canceled)
88. The method of claim 85, wherein the antibody is an L234A,
L235A, G237A variant of bapineuzumab comprising a humanized light
chain having an amino acid sequence comprising SEQ ID NO:48 and a
humanized heavy chain having an amino acid sequence comprising SEQ
ID NO:66 or 67.
89-96. (canceled)
97. A method of treating or effecting prophylaxis of Alzheimer's
disease in a patient, comprising administering an antibody to the
patient in a regime in which 0.15-2 mg/kg of antibody is
administered quarterly by intravenous administration, or at a dose
frequency and route of administration that generates an equivalent
average serum concentration or area under the curve, wherein the
antibody is an L234A, L235A, G237A variant of bapineuzumab
comprising a humanized light chain having an amino acid sequence
comprising SEQ ID NO:48 and a humanized heavy chain having an amino
acid sequence comprising SEQ ID NO:66 or 67.
98-105. (canceled)
106. A method of treating or effecting prophylaxis of Alzheimer's
disease in a population of patients, comprising administering an
antibody that specifically binds to an epitope within residues 1-11
of A.beta. to the patients, wherein the antibody is administered at
a dose of 0.15-1 mg/kg in patients of the population having one or
two ApoE4 alleles and a dose of 0.5-2.5 mg/kg in patients of the
population having zero ApoE4 alleles, and the mean dose is higher
in the patients having zero ApoE4 alleles.
107. The method of claim 106, wherein the antibody is a humanized
form of a mouse 3D6 antibody (ATCC accession number PTA-5130), and
positions 234, 235 and 237 in the heavy chain constant region are
occupied by Ala, Ala and Ala respectively, wherein positions are
numbered by the EU numbering system.
108. The method of claim 106, wherein the antibody is
bapineuzumab.
109. The method of claim 106, wherein the antibody is an L234A,
L235A, G237A variant of bapineuzumab comprising a humanized light
chain having an amino acid sequence comprising SEQ ID NO:48 and a
humanized heavy chain having an amino acid sequence comprising SEQ
ID NO:66 or 67.
110. The method of claim 106, wherein the dose is 0.5 mg/kg in
patients of the population having one or two ApoE4 alleles and 2
mg/kg in patients of the population having zero ApoE4 alleles.
111. A method of effecting prophylaxis of Alzheimer's disease in a
patient comprising administering an effective regime of an agent
that is an antibody to A.beta. or an agent that induces an antibody
to A.beta. on administration to a patient, wherein the patient has
at least one ApoE4 allele, wherein the patient has a mini-mental
test score of 27 or higher.
112-117. (canceled)
118. A method of treating or effecting prophylaxis of a disease
characterized by amyloid deposits of A.beta. in the brain in a
patient comprising administering a first regime and a second regime
each comprises administering an antibody to A.beta. to the patient;
monitoring the patient for vasogenic edema; maintaining the first
regime if vasogenic edema does not appear; and administering a
second regime to the patient if vasogenic edema does appear,
wherein the second regime differs relative to the first regime in
at least one of (i)-(v) below: (i) the dose of the antibody is
reduced; (ii) the frequency of administration of the antibody is
reduced; (iii) a different antibody with reduced capacity to bind
an Fc.gamma. receptor; (iv) a different antibody with reduced
capacity to bind C1q; (v) the antibody to A.beta. is not
administered; wherein the second regime is maintained at least for
the duration of the vasogenic edema.
119. (canceled)
120. The method of claim 118, wherein the first antibody is
bapineuzumab and the second antibody is an L234A, L235A, G237A
variant of bapineuzumab comprising a humanized light chain having
an amino acid sequence comprising SEQ ID NO:48 and a humanized
heavy chain having an amino acid sequence comprising SEQ ID NO:66
or 67.
121. A method of treating or effecting prophylaxis of Alzheimer's
disease in a patient population, comprising administering an
antibody that specifically binds to an epitope within residues 1-11
of A.beta. and has mutations in the constant region that reduce
binding to an Fc.gamma. receptor and/or C1q to the patient, wherein
the antibody is administered at the same dose and/or frequency to
each patient regardless of the number of ApoE4 alleles in the
patient.
122. The method of claim 121, wherein the antibody is an L234A,
L235A, and G237A variant of bapineuzumab comprising a humanized
light chain having an amino acid sequence comprising SEQ ID NO:48
and a humanized heavy chain having an amino acid sequence
comprising SEQ ID NO:66 or 67.
123. (canceled)
124. A method of treating or effecting prophylaxis of Alzheimer's
disease in a patient population, comprising administering an
antibody to A.beta. to some of the patients in the population,
wherein patients in the population having zero ApoE4 alleles
receive the antibody and patients in the population having one or
two ApoE4 alleles do not receive the antibody.
125-127. (canceled)
128. A method of treating or effecting prophylaxis of a disease
characterized by A.beta. deposits in the brain of patient
comprising administering an effective regime of a humanized
antibody to the patient; wherein the humanized antibody comprises a
mature light chain variable region sequence of SEQ ID NO:2 and a
mature heavy chain variable region sequence of SEQ ID NO:3, and a
human heavy chain constant of IgG1 isotype with L234A, L235A, and
G237A mutations, wherein position are numbered by the EU numbering
system.
129. The method of claim 128, wherein the patient has at least one
ApoE4 allele.
130. (canceled)
131. The method of claim 128, wherein the dose is 0.15-2 mg/kg.
132. The method of claim 128, further comprising monitoring the
patient by MRI for vasogenic edema.
133. (canceled)
134. A humanized form of a 10D5 antibody (ATCC accession number
PTA-5129) comprising a human heavy chain constant region with
L234A, L235A and G237A mutations, wherein positions are numbered by
the EU numbering system.
135. The humanized antibody of claim 134, comprising a light chain
variable region of SEQ ID NO:8 or SEQ ID NO: 73 and a heavy chain
variable region of SEQ ID NO:9 or SEQ ID NO:74.
136. (canceled)
137. A humanized form of a 12A11 antibody (ATCC accession number
PTA-7271) comprising a human heavy chain constant region with
L234A, L235A and G237A mutations, wherein positions are numbered by
the EU numbering system.
138. The humanized antibody of claim 137, comprising a light chain
variable region of SEQ ID NO:10 and a heavy chain variable region
of SEQ ID NO:11.
139. (canceled)
140. A humanized form of a 3D6 antibody (ATCC accession number
PTA-5130) comprising a human heavy chain constant region with
L234A, L235A and G237A mutations, wherein positions are numbered by
the EU numbering system.
141. (canceled)
142. The humanized antibody of claim 140, comprising a humanized
light chain having an amino acid sequence comprising SEQ ID NO:48
and a humanized heavy chain having an amino acid sequence
comprising SEQ ID NO:66 or 67.
143. (canceled)
144. An isolated nucleic acid having a sequence comprising SEQ ID
NO:68 provided that residues 1-57 encoding a signal sequence may or
may not be present.
145. An isolated humanized antibody comprising a mature light chain
variable region sequence of SEQ ID NO:2 and a mature heavy chain
variable region sequence of SEQ ID NO:3, and a human heavy chain
constant region of IgG isotype with L234A, L235A, and G237A
mutations, wherein positions are numbered by the EU numbering
system.
146. (canceled)
147. An isolated humanized form of a 12B4 antibody, wherein the
12B4 antibody is characterized by a light chain variable region
sequence of SEQ ID NO:31, and heavy chain variable region sequence
of SEQ ID NO:32, and a human heavy chain constant region of IgG
isotype with L234A, L235A, and G237A mutations, wherein positions
are numbered by the EU numbering system.
148. (canceled)
149. A humanized form of a 266 antibody (ATCC accession number
PTA-6123) comprising a human heavy chain constant region with
L234A, L235A and G237A mutations, wherein positions are numbered by
the EU numbering system.
150. The humanized antibody of claim 149, comprising a light chain
variable region of SEQ ID NO:33 and a heavy chain variable region
of SEQ ID NO:34.
151. (canceled)
152. An isolated antibody comprising a human heavy chain constant
region of isotype IgG1, wherein amino acids at positions 234, 235,
and 237 (EU numbering) are each alanine.
153. The antibody of claim 152, wherein no other amino acid from
positions 230-240 or 315-325 in the human heavy chain constant
region is occupied by an amino acid not naturally found at that
position in a human IgG1 constant region.
154. The antibody of claim 153, wherein no amino acid in the human
heavy chain constant region other than positions 234, 235 and 237
is occupied by an amino acid not naturally found at that position
in a human IgG1 constant region.
155. The antibody of claim 152, wherein the human heavy chain
constant region comprise CH1, hinge, CH2 and CH3 regions.
156. The antibody of claim 152, wherein the human heavy chain
constant region has an amino acid sequence comprising SEQ ID NO:66
or SEQ ID NO:67 or an allotype of either of these sequences.
157. (canceled)
158. The isolated antibody of claim 152 that is a fully human
antibody.
159. The isolated antibody of claim 152 that is a humanized
antibody.
160. (canceled)
161. A method of determining a regime for bapineuzumab
administration comprising providing instructions to a healthcare
professional that assists the healthcare professional determine a
regime of bapineuzumab to administer to a patient having zero
copies of an ApoE4 allele, wherein the regime is characterized by
administering 0.5-2 mg/kg of bapineuzumab quarterly by intravenous
administration, further comprising monitoring the patient for
vasogenic edema.
162-165. (canceled)
166. A method of determining a regime for bapineuzumab
administration comprising providing instructions to a healthcare
professional that assists the healthcare professional determine a
regime of bapineuzumab to administer to a patient having one or two
copies of an ApoE4 allele, wherein the instructions specify a
regime is characterized by administering bapineuzumab at a dose of
0.15-1 mg/kg quarterly by intravenous administration, or at a dose
frequency and route of administration and further comprising
monitoring the patient for vasogenic edema.
167-170. (canceled)
171. The method of claim 161 or claim 166, further comprises
providing bapineuzumab to a healthcare professional in combination
with the instructions.
172-177. (canceled)
178. A kit for bapineuzumab administration comprising instructions
to a healthcare professional that assist the healthcare
professional determine which regime of bapineuzumab to administer
to a patient having zero, one or two copies of an ApoE4 allele.
179-181. (canceled)
182. The kit of claim 178, wherein the instructions specify that
the regime is different for a patient having one or two copies of
an ApoE4 allele.
183-193. (canceled)
194. A method for improving the safety of bapineuzumab in patients
having one or two ApoE4 alleles, comprising advising the physician
to administer a lower dose of bapineuzumab to a patient having one
or two ApoE alleles relative to that of a patient having zero ApoE
alleles.
195. A method for improving the safety of bapineuzumab in patients
having one or two ApoE4 alleles, comprising advising the physician
to monitor the patient by MRI more frequently than a patient having
one or two ApoE alleles relative to that of a patient having zero
ApoE alleles.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] Provisional U.S. Application Nos. 60/999,423 and 61/083,827,
filed Oct. 17, 2007 and Jul. 25, 2008, respectively, are
incorporated by reference in their entirety for all purposes.
REFERENCE TO A SEQUENCE LISTING
[0002] The Substitute Sequence Listing written in file
Sequence_Listing_for.sub.--15270C-000420US.txt is 164,721 bytes and
was created on Oct. 14, 2008 for the instant application. The
information contained in this file is hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
I. General
[0003] Alzheimer's disease (AD) is a progressive disease resulting
in senile dementia. See generally Selkoe, TINS 16:403 (1993); Hardy
et al., WO 92/13069; Selkoe, J. Neuropathol. Exp. Neurol. 53:438
(1994); Duff et al., Nature 373:476 (1995); Games et al., Nature
373:523 (1995). Broadly speaking, the disease falls into two
categories: late onset, which occurs in old age (65+ years) and
early onset, which develops well before the senile period, i.e.,
between 35 and 60 years. In both types of disease, the pathology is
the same but the abnormalities tend to be more severe and
widespread in cases beginning at an earlier age. The disease is
characterized by at least two types of lesions in the brain,
neurofibrillary tangles and senile plaques. Neurofibrillary tangles
are intracellular deposits of microtubule associated tau protein
consisting of two filaments twisted about each other in pairs.
Senile plaques (i.e., amyloid plaques) are areas of disorganized
neuropile up to 150 .mu.m across with extracellular amyloid
deposits at the center which are visible by microscopic analysis of
sections of brain tissue. The accumulation of amyloid plaques
within the brain is also associated with Down's syndrome and other
cognitive disorders.
[0004] The principal constituent of the plaques is a peptide termed
A.beta. or .beta.-amyloid peptide. A.beta. peptide is a 4-kDa
internal fragment of 39-43 amino acids of a larger transmembrane
glycoprotein named amyloid precursor protein (APP). As a result of
proteolytic processing of APP by different secretase enzymes,
A.beta. is primarily found in both a short form, 40 amino acids in
length, and a long form, ranging from 42-43 amino acids in length.
Part of the hydrophobic transmembrane domain of APP is found at the
carboxy end of A.beta., and may account for the ability of A.beta.
to aggregate into plaques, particularly in the case of the long
form. Accumulation of amyloid plaques in the brain eventually leads
to neuronal cell death. The physical symptoms associated with this
type of neural deterioration characterize Alzheimer's disease.
[0005] Several mutations within the APP protein have been
correlated with the presence of Alzheimer's disease. See, e.g.,
Goate et al., Nature 349:704 (1991) (valine.sup.717 to isoleucine);
Chartier Harlan et al., Nature 353:844 (1991)) (valine.sup.717 to
glycine); Murrell et al., Science 254:97 (1991) (valine.sup.717 to
phenylalanine); Mullan et al., Nature Genet. 1:345 (1992) (a double
mutation changing lysine.sup.595-methionine.sup.596 to
asparagine.sup.595-leucine.sup.596). Such mutations are thought to
cause Alzheimer's disease by increased or altered processing of APP
to A.beta., particularly processing of APP to increased amounts of
the long form of A.beta. (i.e., A.beta.1-42 and A.beta.1-43).
Mutations in other genes, such as the presenilin genes, PS1 and
PS2, are thought indirectly to affect processing of APP to generate
increased amounts of long form A.beta. (see Hardy, TINS 20: 154
(1997)).
[0006] Apolipoprotein E (ApoE) encodes a cholesterol-processing
protein. The gene, which maps to 19q13.2, has three allelic
variants: ApoE4, ApoE3, and ApoE2. The frequency of the apoE4
version of the gene in the general population varies, but is always
less than 30% and frequently 8%-15%. ApoE3 is the most common form
and ApoE2 is the least common. Persons with one E4 allele usually
have about a two to three fold increased risk of developing
Alzheimer's disease. Persons with two E4 alleles (usually around 1%
of the population) have about a nine-fold increase in risk.
Nonetheless, even persons with two E4 alleles do not always get
Alzheimer's disease. At least one E4 allele is found in about 40%
of patients with late-onset Alzheimer's disease. Genetic screening
for E4 has not been routinely performed, because it has not been
known how to use this information for a therapeutic regime.
SUMMARY OF THE CLAIMED INVENTION
[0007] The invention provides a method of treating Alzheimer's
disease, comprising administering to a patient having zero ApoE4
alleles ("ApoE4 non-carrier patient") and Alzheimer's disease, an
effective regime of an antibody that specifically binds to an
N-terminal epitope of A.beta.. Optionally, the antibody
specifically binds to an epitope within residues 1-7 of A.beta., or
an epitope within residues 1-5 of A.beta., or an epitope within
residues 3-7 of A.beta.. Optionally, the dosage of the antibody
within a range of about 0.15 mg/kg to about 2 mg/kg is administered
by intravenous infusion. Optionally, the dosage is administered
every 4 to 16 weeks. Optionally, the dosage is administered every
10 to 14 weeks. Optionally, the dosage is administered every 13
weeks. Optionally, the dosage is about 0.5 mg/kg to about 1 mg/kg.
Optionally, the dosage is about 0.5 mg/kg to 2 mg/kg. Optionally,
the dosage is about 2 mg/kg. Optionally, the antibody is
bapineuzumab. Optionally, the method also involves monitoring for
vasogenic edema, and optionally administering a corticosteroid to
the patient to treat vasogenic edema detected by the
monitoring.
[0008] The invention also provides a method of reducing cognitive
decline in a patient having zero ApoE4 alleles ("ApoE4 non-carrier
patient"), comprising administering to the patient an antibody that
specifically binds to an N-terminal epitope of A.beta. in a regime
effective to reduce the cognitive decline of the patient relative
to a control patient to whom the antibody is not administered;
wherein: the ApoE4 non-carrier patient and control patient have
been diagnosed with mild to moderate Alzheimer's disease; and the
cognitive decline is measured by ADAS-COG, NTB, MMSE or CDR-SB.
Optionally, the antibody is administered by intravenous infusion at
a dosage within a range of about 0.15 mg/kg to about 2 mg/kg.
Optionally, the antibody is bapineuzumab. Optionally, the dosage is
about 0.5 mg/kg and the cognitive decline is measured by ADAS-COG.
Optionally, the dosage is about 2 mg/kg and the cognitive decline
is measured by ADAS-COG. Optionally, the cognitive decline is
measured by NTB. Optionally, the dosage is 0.5 mg/kg. Optionally,
the dosage is about 0.5 mg/kg and the cognitive decline is measured
by CDR. Optionally, the dosage is about 0.5 mg/kg and the cognitive
decline is measured by MMSE. Optionally, the dosage is about 2
mg/kg and the cognitive decline is measured by MMSE.
[0009] The invention also provides a method of reducing brain
volume decline in a patient having zero ApoE4 alleles ("ApoE4
non-carrier patient"), comprising administering to the ApoE4
non-carrier patient an antibody that specifically binds to an
N-terminal epitope of A.beta. in a regime effective to reduce the
brain volume decline of the ApoE4 non-carrier patient relative to a
control patient to whom the antibody is not administered; wherein
the ApoE4 non-carrier patient and control patient have been
diagnosed with mild to moderate Alzheimer's disease. Optionally,
the antibody is administered by intravenous infusion at a dosage
within a range of about 0.15 mg/kg to about 2 mg/kg. Optionally,
the antibody is bapineuzumab. Optionally, the dosage is about 0.5
mg/kg. Optionally, the dosage is about 2 mg/kg. Optionally, the
brain volume decline is measured by MRI.
[0010] The invention also provides a method of treating Alzheimer's
disease, comprising administering to an ApoE4 non-carrier patient
an antibody that specifically recognizes the N-terminal region of
A.beta. in a regime effective to maintain a mean serum
concentration of the antibody in the range of about 0.1 .mu.g/ml to
about 60 .mu.g/ml. Optionally, the range is about 0.4 .mu.g/ml to
about 20 .mu.g/ml. Optionally, the range is about 1 .mu.g/ml to
about 5 .mu.g/ml. Optionally, the maximum serum concentration of
the antibody in the patient less than about 28 .mu.g antibody/ml
serum. Optionally, the maximum serum concentration is within a
range of about 4-18 .mu.g antibody/ml serum. Optionally, the
antibody is bapineuzumab.
[0011] The invention also provides a method of treating Alzheimer's
disease, comprising administering to an ApoE4 non-carrier patient
an antibody that specifically recognizes the N-terminal region of
A.beta. in a regime effective to achieve a mean plasma A.beta.
concentration of at least 450 pg/ml. Optionally, the mean plasma
A.beta. concentration is in the range of about 600 pg/ml to about
3000 pg/ml. Optionally, the mean plasma A.beta. concentration is in
the range of about 700 pg/ml to about 2000 pg/ml. Optionally, the
mean plasma A.beta. concentration is in the range of about 700
pg/ml to about 2000 pg/ml. Optionally, the mean plasma A.beta.
concentration is in the range of about 800 pg/ml to about 1000
pg/ml.
[0012] The invention also provides a method of treating Alzheimer's
disease, comprising subcutaneously administering to a patient
having the disease and one or two copies of an ApoE4 allele an
effective regime of an antibody that binds to an N-terminal epitope
of A.beta.. Optionally, the method further comprises monitoring for
vasogenic edema. Optionally, the antibody is administered at a dose
of 0.01-0.6 mg/kg and a frequency of between weekly and monthly.
Optionally, the antibody is administered at a dose of 0.05-0.5
mg/kg. Optionally, the antibody is administered at a dose of
0.05-0.25 mg/kg. Optionally, the antibody is administered at a dose
of 0.015-0.2 mg/kg weekly to biweekly. Optionally, the antibody is
administered at a dose of 0.05-0.15 mg/kg weekly to biweekly.
Optionally, the antibody is administered at a dose of 0.05-0.07
mg/kg weekly. Optionally, the antibody is administered at a dose of
0.06 mg/kg weekly. Optionally, the antibody is administered at a
dose of 0.1 to 0.15 mg/kg biweekly. Optionally, the antibody is
administered at a dose of 0.1 to 0.3 mg/kg monthly. Optionally, the
antibody is administered at a dose of 0.2 mg/kg monthly.
Optionally, the antibody is administered at a dose of 1-40 mg and a
frequency of between weekly and monthly. Optionally, the antibody
is administered at a dose of 5-25 mg. Optionally, the antibody is
administered at a dose of 2.5-15 mg. Optionally, the antibody is
administered at a dose of 1-12 mg weekly to biweekly. Optionally,
the antibody is administered at a dose of 2.5-10 mg weekly to
biweekly. Optionally, the antibody is administered at a dose of
2.5-5 mg weekly. Optionally, the antibody is administered at a dose
of 4-5 mg weekly. Optionally, the antibody is administered at a
dose of 7-10 mg biweekly. Optionally, the method further comprises
monitoring for vasogenic edema.
[0013] The invention further comprises a method of treating
Alzheimer's disease, comprising administering to a patient having
the disease and one or two ApoE4 alleles an effective regime of an
antibody that binds to an N-terminal epitope of A.beta.;
administering a corticosteroid to the patient to treat vasogenic
edema arising from the administration of the antibody. Optionally,
the method further comprises monitoring the patient for vasogenic
edema. Optionally, the dose or frequency of administration of the
antibody is reduced or eliminated during the vasogenic edema
relative to the dose or frequency before the vasogenic edema.
Optionally, the dose or frequency of administration of the antibody
is increased after resolution of the vasogenic edema relative to
the dose or frequency either before or during the vasogenic
edema.
[0014] The invention further comprises a method of treating or
effecting prophylaxis in a population of patients of an
amyloidogenic disease characterized by amyloid deposits of A.beta.
in the brain, comprising: administering different regimes to
different patients in the population depending on which allelic
forms of ApoE are present in the patients; wherein at least one of
the regimes comprises administering an agent that is an antibody to
A.beta. or an agent that induces an antibody to A.beta. on
administration to a patient. Optionally, the different regimes each
comprise administering an agent that is an antibody to A.beta. or
an agent that induces an antibody to A.beta. on administration to a
patient; and the dose of the agent and/or the frequency of
administration of the agent and/or the capacity of the agent to
induce a clearing response to amyloid deposits and/or the mean
serum concentration of the agent or antibodies induced by the agent
and/or the maximum serum concentration of the agent or antibodies
induced by the agent is reduced and/or the time of initiation of
treatment relative to disease progression is earlier in (a)
patients having two copies of an ApoE4 allele relative to patients
having zero copies of an ApoE4 allele, and/or (b) patients having
one copy of an ApoE4 allele relative to patients having zero copies
of an ApoE4 allele, and/or (c) patients having two copies of an
ApoE4 allele relative to patients having one copy of an ApoE4
allele.
[0015] Optionally, a first regime comprises administering an agent
that is an antibody to A.beta. or an agent that induces an antibody
to A.beta. on administration to a patient and a second regime lacks
an antibody to A.beta. or an agent that induces an antibody to
A.beta. and the first regime is administered to patients having
zero copies of an ApoE4 allele and the second regime is
administered to patients having one or two copies of an ApoE4
allele. Optionally, a first regime comprises administering a first
antibody to A.beta. and the second regime comprises administering a
second antibody to A.beta. and the second antibody has reduced
binding to an Fc.gamma. receptor or C1q relative to the first
antibody, and the first antibody is administered to patients having
zero copies of an ApoE4 allele and the second antibody is
administered to patients having one or two copies of an ApoE4
allele. Optionally, the second antibody has one or more mutations
in the constant region that reduce binding to the Fc.gamma.
receptor and/or C1q, the mutations not being present in the first
antibody. Optionally, the one or more mutations is/are at
position(s) in a heavy chain constant region selected from the
group consisting of positions 234, 235, 236 and 237 (EU numbering).
Optionally, the one or more mutations are mutations at positions
234, 235 and 237. Optionally, the one or more mutations are L234A,
L235A and G237A. Optionally, the isotype of the constant region is
human IgG1. Optionally, the isotype of the constant region is human
IgG2 or IgG4. Optionally, the first antibody is bapineuzumab and
the second antibody is an L234A, L235A, G237A variant of
bapineuzumab. Optionally, a first regime comprises administering a
first antibody to A.beta. and a second regime comprises
administering a second antibody to A.beta., the first antibody
being of human IgG1 isotype and the second antibody of human IgG4
isotype, and the first antibody is administered to patients having
zero copies of an ApoE4 allele and the second antibody is
administered to patients having one or two copies of an ApoE4
allele.
[0016] In some methods, the disease is Alzheimer's disease. Some
methods further comprise determining which alleles of ApoE are
present in the patient.
[0017] Optionally, the different regimes differ in dose of the
agent administered. Optionally, the different regimes differ in
frequency of the agent administered. Optionally, the different
regimes differ in the type of agent administered.
[0018] Optionally, the dose of the agent and/or the frequency of
administration of the agent and/or the capacity of the agent to
induce a clearing response to amyloid deposits is reduced in (a)
patients having two ApoE4 alleles relative to patients having one
ApoE4 allele; and/or (b) patients having one copy of an ApoE4
allele relative to patients having zero copies of an ApoE4 allele,
and/or (c) patients having two copies of an ApoE4 allele relative
to patients having one copy of an ApoE4 allele. Optionally, the
dose of the agent and/or the frequency of administration of the
agent and/or the capacity of the agent to induce a clearing
response to amyloid deposits is reduced in patients having one or
two ApoE4 alleles relative to patients having zero ApoE4 alleles of
an ApoE4 allele. Optionally, patients in the population having one
or two ApoE4 alleles are administered a dose of 0.15-1 mg/kg, and
patients in the population having zero ApoE4 alleles are
administered a dose of 0.5-2 mg/kg of an antibody specifically
binding within residues 1-11 of A.beta.. Optionally, the patients
in the population having one or two ApoE4 alleles are administered
a lower dosage of agent than patients having zero ApoE4 alleles
until vasogenic edema has appeared and resolved, and the same
dosage of agent thereafter.
[0019] Optionally, the patients in the population having one or two
ApoE4 alleles are administered a lower frequency of the agent than
the patients having zero ApoE4 alleles until vasogenic edema has
appeared and resolved, and the same dosage of agent thereafter.
Optionally, the patients in the population having one or two ApoE4
alleles are administered an antibody with reduced capacity to
induce a clearing response to amyloid deposits relative to
bapineuzumab.
[0020] Optionally, the method further comprises monitoring at least
some of the patients in the population for vasogenic edema.
Optionally, the monitoring is performed by MRI. Optionally,
patients in the population with zero ApoE4 alleles are not
monitored by MRI. Optionally, the agent is an antibody binding to
an epitope within residues 1-11 of A.beta.. Optionally, the
antibody has human IgG1 isotype. Optionally, the antibody is
bapineuzumab. Optionally, the agent is an antibody having reduced
capacity to induce a clearing response to amyloid deposits relative
to bapineuzumab. Optionally, the antibody is an L234A, L235A, G237A
variant of bapineuzumab.
[0021] Optionally, wherein patients with one or two ApoE4 alleles
are administered 1-3 doses of humanized 266 antibody following by
subsequent doses of bapineuzumab and patients with zero ApoE4
alleles are administered the same total number of doses but all
with bapineuzumab. In some methods, the antibody is a humanized 266
antibody. Optionally, patients with one or two ApoE4 alleles are
administered humanized 266 and patients with zero ApoE4 alleles are
administered bapineuzumab.
[0022] The invention further provides a method of monitoring a
population of patients undergoing treatment or prophylaxis for a
disease characterized by amyloid deposits of A.beta. in the brain
with an agent that is an antibody to A.beta. or an agent that
induces an antibody to A.beta., the method comprising: performing
different monitoring regimes in different patients in the
population for vasogenic edema, wherein the frequency of monitoring
is greater for (a) patients having two copies of ApoE4 relative to
patients having zero copies of ApoE4 and/or (b) patients having one
copy of an ApoE4 allele relative to patients having zero copies of
an ApoE4 allele, and/or (c) patients having two copies of an ApoE4
allele relative to patients having one copy of an ApoE4 allele.
Optionally, the disease is Alzheimer's disease. Optionally, the
method further comprises determining which allelic forms of ApoE
are present in each patient in the population. Optionally, the
monitoring is by brain imaging. Optionally, the monitoring is by
MRI. Optionally, patients having one ApoE4 allele are monitored
more frequently than patients having zero ApoE4 alleles.
Optionally, patients having two ApoE4 alleles are monitored more
frequently than patients having one ApoE4 allele. Optionally,
patients having one ApoE4 allele are monitored more frequently than
patients having zero ApoE4 alleles. Optionally, patients having
zero ApoE4 alleles are not monitored by MRI for vasogenic
edema.
[0023] The invention further provides a method of treating or
effecting prophylaxis of a patient for a disease characterized by
amyloid deposits of A.beta. in the brain, comprising administering
to a patient with at least one ApoE4 allele an agent that is an
antibody to an epitope within residue 1-11 of A.beta. or an agent
that induces such an antibody to A.beta., and monitoring the
patient for vasogenic edema by MRI. Optionally, the agent is
bapineuzumab. Optionally, the agent is an L234A, L235A, G237A
variant of bapineuzumab.
[0024] The invention further provides a method of treating or
effecting prophylaxis of a disease characterized by amyloid
deposits of A.beta. in the brain in a patient having at least one
ApoE4 allele, comprising administering a first regime to the
patient before vasogenic edema appears, and a second regime after
vasogenic edema has resolved; wherein the first and second regimes
each comprise administering an agent that is an antibody to A.beta.
or an agent that induces an antibody to A.beta. on administration
to a patient; and the dose of the agent and/or the frequency of
administration of the agent and/or the capacity of the agent to
clear amyloid deposits is reduced in the first regime relative to
the second regime. Optionally, the disease is Alzheimer's disease.
Optionally, the patient has one or two ApoE4 alleles. Optionally,
the first and second regimes each comprises administering an
antibody that specifically binds to an epitope within residues 1-11
of A.beta. to the patient, and the antibody is administered at a
dose of 0.15-1 mg/kg before vasogenic edema appears and 0.5-2 mg/kg
after vasogenic edema has resolved. Optionally, the antibody is
bapineuzumab. Optionally, the antibody is a L234A, L235A, G237A
variant of bapineuzumab.
[0025] The invention further provides a method of treating or
effecting prophylaxis of Alzheimer's disease in a patient,
comprising administering to the patient an antibody that
specifically binds to an epitope within residues 1-11 of A.beta. to
a patient having one or two ApoE4 alleles, wherein the antibody is
administered in a regime in which 0.15-1 mg/kg of antibody is
administered quarterly by intravenous administration, or at a dose
frequency and route of administration that generates an equivalent
average serum concentration or area under the curve. Optionally,
the antibody is bapineuzumab. Optionally the dose is 0.5 mg/kg.
[0026] The invention further provides a method of treating or
effecting prophylaxis of Alzheimer's disease in a patient,
comprising administering to the patient an antibody that
specifically binds to an epitope within residues 1-11 of A.beta. to
a patient having zero ApoE4 alleles, wherein the dose of the
antibody is 0.5-2 mg/kg administered quarterly by intravenous
administration, or a dose frequency and route of administration
that generates an equivalent serum concentration or area under the
curve. Optionally, the antibody is an L234A, L235A, G237A variant
of bapineuzumab.
[0027] The invention further provides a method of treating or
effecting prophylaxis of Alzheimer's disease in a population of
patients, comprising administering an antibody that specifically
binds to an epitope within residues 1-11 of A.beta. to the
patients, wherein the antibody is administered at a dose of 0.15-1
mg/kg in patients of the population having one or two ApoE4 alleles
and a dose of 0.5-2.5 mg/kg in patients of the population having
zero ApoE4 alleles, and the mean dose is higher in the patients
having zero ApoE4 alleles. Optionally, the antibody is
bapineuzumab. Optionally, the antibody is an L234A, L235A, G237A
variant of bapineuzumab. Optionally, the dose is 0.5 mg/kg in
patients of the population having one or two ApoE4 alleles and 2
mg/kg in patients of the population having zero ApoE4 alleles.
[0028] The invention further provides a use of a measurement of
ApoE4 copy number is selecting from different regimes for treatment
or prophylaxis of a disease characterized by amyloid deposits in
the brain in the patient wherein the different regimes each
comprise administering an agent that is an antibody to A.beta. or
an agent that induces an antibody to A.beta. on administration to a
patient, and the dose of the agent and/or the frequency of
administration of the agent and/or the capacity of the agent to
induce a clearing response to amyloid deposits and/or the mean
serum concentration of the agent or antibodies induced by the agent
and/or the maximum serum concentration of the agent or antibodies
induced by the agent is reduced and/or the time of initiation of
treatment relative to disease progression is earlier in a regime
administered to (a) patients having two copies of an ApoE4 allele
relative to patients having zero copies of an ApoE4 allele, and/or
(b) patients having one copy of an ApoE4 allele relative to
patients having zero copies of an ApoE4 allele, and/or (c) patients
having two copies of an ApoE4 allele relative to patients having
one copy of an ApoE4.
[0029] The invention further provides a method of selecting a
regime for treatment or prophylaxis of a disease characterized by
amyloid deposits in the brain of a patient, the method comprising
determining the number of ApoE4 alleles present in a patient;
selecting from different regimes based on the number of ApoE4
alleles present, wherein the different regimes each comprise
administering an agent that is an antibody to A.beta. or an agent
that induces an antibody to A.beta. on administration to a patient,
and the dose of the agent and/or the frequency of administration of
the agent and/or the capacity of the agent to induce a clearing
response to amyloid deposits and/or the mean serum concentration of
the agent or antibodies induced by the agent and/or the maximum
serum concentration of the agent or antibodies induced by the agent
is reduced and/or the time of initiation of treatment relative to
disease progression is earlier in (a) patients having two copies of
an ApoE4 allele relative to patients having zero copies of an ApoE4
allele, and/or (b) patients having one copy of an ApoE4 allele
relative to patients having zero copies of an ApoE4 allele, and/or
(c) patients having two copies of an ApoE4 allele relative to
patients having one copy of an ApoE4.
[0030] The invention further provides a use of a measurement of
ApoE4 copy number in the manufacture of a medicament to treat
Alzheimer's disease, wherein the medicament comprises an antibody
to A.beta. or an agent that induces an antibody to A.beta..
[0031] The invention further provides a use of at least one agent
that is an antibody to A.beta. or an agent that induces an antibody
to A.beta. on administration to a patient in the manufacture of a
medicament for the treatment or prophylaxis of a disease
characterized by amyloid deposits in the brain of a patient by
different regimes depending on the number of ApoE4 alleles in the
patient, wherein the different regimes comprise administering an
agent to a patient and the dose of the agent and/or the frequency
of administration of the agent and/or the capacity of the agent to
induce a clearing response to amyloid deposits and/or the mean
serum concentration of the agent or antibodies induced by the agent
and/or the maximum serum concentration of the agent or antibodies
induced by the agent is reduced and/or the time of initiation of
treatment relative to disease progression is earlier in (a)
patients having two copies of an ApoE4 allele relative to patients
having zero copies of an ApoE4 allele, and/or (b) patients having
one copy of an ApoE4 allele relative to patients having zero copies
of an ApoE4 allele, and/or (c) patients having two copies of an
ApoE4 allele relative to patients having one copy of an ApoE4.
[0032] The invention further provides a method of treating or
effecting prophylaxis in a population of patients of an
amyloidogenic disease characterized by amyloid deposits of A.beta.
in the brain, comprising: administering different regimes to
different patients in the population depending on which allelic
forms of ApoE are present in the patients; wherein the different
regimes each comprise administering an agent that is an antibody to
A.beta. or an agent that induces an antibody to A.beta. on
administration to a patient; and the mean serum concentration of
the agent or antibodies induced by the agent and/or the maximum
concentration of the agent or antibodies induced by the agent is
reduced in patients having two copies of an ApoE4 allele relative
to patients having zero copies of an ApoE4 allele, and/or (b)
patients having one copy of an ApoE4 allele relative to patients
having zero copies of an ApoE4 allele, and/or (c) patients having
two copies of an ApoE4 allele relative to patients having one copy
of an ApoE4.
[0033] The invention further provides a method of treating or
effecting prophylaxis in a population of patients of an
amyloidogenic disease characterized by amyloid deposits of A.beta.
in the brain, comprising: determining the ApoE4 status of the
patient; administering different regimes to different patients in
the population depending on which allelic forms of ApoE are present
in the patients; wherein the different regimes each comprise
administering an agent that is an antibody to A.beta. or an agent
that induces an antibody to A.beta. on administration to a patient;
and the dose of the agent and/or the frequency of administration of
the agent and/or the capacity of the agent to induce a clearing
response to amyloid deposits and/or the mean serum concentration of
the agent or antibodies induced by the agent and/or the maximum
serum concentration of the agent or antibodies induced by the agent
is reduced and/or the time of initiation of treatment relative to
disease progression is earlier in (a) patients having two copies of
an ApoE4 allele relative to patients having zero copies of an ApoE4
allele, and/or (b) patients having one copy of an ApoE4 allele
relative to patients having zero copies of an ApoE4 allele, and/or
(c) patients having two copies of an ApoE4 allele relative to
patients having one copy of an ApoE4.
[0034] The invention further provides a humanized form of a 10D5
antibody comprising a human heavy chain constant region with L234A,
L235A and G237A mutations, wherein positions are numbered by the EU
numbering system. Optionally, the isotype is human IgG1, IgG2 or
IgG4, preferably IgG1. The 10D5 hybridoma was deposited with the
ATCC on Apr. 8, 2003 and assigned accession number PTA-5129. The
ATCC is located at 10801 University Blvd., Manassas, Va. 20110.
[0035] The invention further provides a humanized form of a 12A11
antibody comprising a humanized light chain variable region of SEQ
ID NO: 10 and a humanized heavy chain variable region of SEQ ID NO:
11 and a human heavy chain constant region with L234A, L235A and
G237A mutations, wherein positions are numbered by the EU numbering
system. Optionally, the isotype is human IgG1, IgG2 or IgG4,
preferably IgG1.
[0036] The invention further provides a humanized form of a 3D6
antibody comprising a human heavy chain constant region with L234A,
L235A and G237A mutations, wherein positions are numbered by the EU
numbering system. The 3D6 hybridoma was deposited with the ATCC on
Apr. 8, 2003 and assigned accession number PTA-5130. The ATCC is
located at 10801 University Blvd., Manassas, Va. 20110. Optionally,
the isotype is human IgG1, IgG2 or IgG4, preferably IgG1. The 3D6
hybridoma was deposited with the ATCC on Apr. 8, 2003.
[0037] The invention further provides an isolated humanized
antibody comprising a mature light chain variable region sequence
of SEQ ID NO: 2 and a mature heavy chain variable region sequence
of SEQ ID NO: 3, and a human heavy chain constant region of IgG
isotype with L234A, L235A, and G237A mutations, wherein positions
are numbered by the EU numbering system. Optionally, the isotype is
human IgG1 isotype.
[0038] The invention further provides an isolated humanized form of
a 12B4 antibody, wherein the 12B4 antibody is characterized by a
mature light chain variable region sequence of SEQ ID NO: 31 and a
mature heavy chain variable region sequence of SEQ ID NO: 32, and a
human heavy chain constant region of IgG isotype with L234A, L235A,
and G237A mutations, wherein positions are numbered by the EU
numbering system. Optionally, the isotype is human IgG1
isotype.
[0039] The invention further provides a method of treating or
effecting prophylaxis of a disease characterized by A.beta.
deposits in the brain of patient comprising administering an
effective regime of a humanized antibody to the patient; wherein
the humanized antibody comprises a mature light chain variable
region sequence of SEQ ID NO: 2 and a mature heavy chain variable
region sequence of SEQ ID NO: 3, and a human heavy chain constant
of IgG1 isotype with L234A, L235A, and G237A mutations, wherein
position are numbered by the EU numbering system. Optionally, the
patient has at least one ApoE4 allele. Optionally the dose is
0.15-1 mg/kg. Optionally, the dose is 0.15-2 mg/kg. Optionally, the
method further comprises monitoring the patient by MRI for
vasogenic edema. Optionally, the method is for treating a
population of the patients and the regime administered to different
patients in the population does not depend on the number of ApoE4
alleles present in a patient.
[0040] The invention further provides a method of effecting
prophylaxis of a disease characterized by deposits of A.beta.
deposits in the brain of a patient comprising administering an
effective regime of an agent that is an antibody to A.beta. or an
agent that induces an antibody to A.beta. on administration to a
patient, wherein the patient has at least one ApoE4 allele.
Optionally, the patient has two ApoE4 alleles. Optionally, the
patient is asymptomatic. Optionally, the patient has a mini-mental
test score of 27 or higher. Optionally, the patient has a
mini-mental test score of 20-26. Optionally, the patient is at
least sixty years of age. Optionally, the method further comprises
determining the number of ApoE4 alleles in the patient.
[0041] The invention further provides a method of treating or
effecting prophylaxis of a disease characterized by amyloid
deposits of A.beta. in the brain in a patient comprising
administering a first regime comprise administering an agent that
is an antibody to A.beta. or an agent that induces an antibody to
A.beta. to the patient; monitoring the patient for vasogenic edema;
maintaining the first regime if vasogenic edema does not appear;
and administering a second regime to the patient if vasogenic edema
does appear, wherein the second regime is a reduced dose of the
agent and/or a reduced frequency of the agent, and/or a different
agent with reduced capacity to bind an Fc.gamma. receptor and/or
C1q or is a lack of antibody to A.beta. or an agent that induces an
antibody to A.beta.; wherein the second regime is maintained at
least for the duration of the vasogenic edema. Optionally, the
agent in the first regime is an antibody that specifically binds to
an epitope within residues 1-11 of A.beta.. Optionally, the first
regime comprises administering a first antibody to A and the second
regime comprises administering a second antibody to A.beta. with
reduced capacity to find to an Fc.gamma. receptor and or C1q
relative to the first antibody. Optionally, the first antibody is
bapineuzumab and the second antibody is an L234A, L235A, G237A
variant of bapineuzumab.
[0042] The invention further provides a method of treating or
effecting prophylaxis of Alzheimer's disease in a patient
population, comprising administering an antibody that specifically
binds to an epitope within residues 1-11 of A.beta. and has
mutations in the constant region that reduce binding to an
Fc.gamma. receptor and or C1q to the patient, wherein the antibody
is administered at the same dose and/or frequency to each patient
regardless of the number of ApoE4 alleles in the patient.
Optionally, the antibody is an L234A, L235A, and G237A variant of
bapineuzumab. Optionally, the method further comprises a step of
monitoring the patient for vasogenic edema.
[0043] The invention further provides a method of treating or
effecting prophylaxis of Alzheimer's disease in a patient
population, comprising administering an agent that is an antibody
to A.beta. or which induces an antibody to A.beta. on
administration to some of the patients in the population, wherein
patients in the population having zero ApoE4 alleles receive the
agent and patients in the population having two ApoE4 alleles do
not receive the agent. Optionally, patients in the population
having one ApoE4 allele do not receive the agent. Optionally, the
antibody is administered by intravenous infusion at a dosage within
a range of about 0.15 mg/kg to about 2 mg/kg. Optionally, the
antibody is bapineuzumab. Optionally, the dosage is about 0.5
mg/kg. Optionally, the dosage is about 2 mg/kg. Optionally, the
brain volume decline is measured by MRI.
[0044] The invention further provides a method of determining a
regime for bapineuzumab administration, comprising providing
instructions to a healthcare professional that assists the
healthcare professional determine a regime of bapineuzumab to
administer to a patient having zero copies of an ApoE4 allele.
Optionally, the regime is characterized by administering
bapineuzumab at a dose of 0.5-2 mg/kg. Optionally, the regime is
characterized by administering 0.5-2 mg/kg of bapineuzumab
quarterly by intravenous administration, or at a dose frequency and
route of administration that generates an equivalent average serum
concentration or area under the curve. Optionally, the regime
further comprises monitoring the patient for vasogenic edema.
Optionally, the monitoring regime is different than the monitoring
regime for a patient having or two copies of an ApoE4 allele.
Optionally, the method further comprises the step of determining
the number of ApoE4 alleles present in a patient. Optionally, the
method further comprises providing bapineuzumab to a healthcare
professional. Optionally, the instructions and bapineuzumab are
provided in combination. Optionally, the regime further comprises
monitoring at the patient for vasogenic edema. Optionally, the
monitoring is performed by MRI. Optionally, the monitoring is by
brain imaging.
[0045] The invention further provides a method of determining a
regime for bapineuzumab administration comprising providing
instructions to a healthcare professional that assists the
healthcare professional determine a regime of bapineuzumab to
administer to a patient having one or two copies of an ApoE4
allele. Optionally, the regime is characterized by administering
bapineuzumab at a dose of 0.15-1 mg/kg. Optionally, the regime is
characterized by administering bapineuzumab at a dose of 0.15-1
mg/kg quarterly by intravenous administration, or at a dose
frequency and route of administration that generates an equivalent
average serum concentration or area under the curve. Optionally,
the determined regime comprises a first and a second regime,
wherein the first regime is administered to the patient before
vasogenic edema appears, and the second regime after vasogenic
edema has resolved; and wherein the first and second regimes each
comprise administering bapineuzumab; wherein the first regime
differs relative to the second regime in at least one of (i)-(ii)
below: (i) the dose of the bapineuzumab is reduced; (ii) the
frequency of administration of the bapineuzumab is reduced.
Optionally, the regime further comprises monitoring the patient for
vasogenic edema. Optionally, the monitoring regime is different
than the monitoring regime for a patient having or two copies of an
ApoE4 allele. Optionally, the method further comprises the step of
determining the number of ApoE4 alleles present in a patient.
Optionally, the method further comprises providing bapineuzumab to
a healthcare professional. Optionally, the instructions and
bapineuzumab are provided in combination. Optionally, the regime
further comprises monitoring at the patient for vasogenic edema.
Optionally, the monitoring is performed by MRI. Optionally, the
monitoring is by brain imaging. Optionally, the monitoring regime
is different than the monitoring regime for a patient having zero
copies of an ApoE4 allele. Optionally, the frequency of monitoring
is greater for: (a) patients having two copies of the ApoE4 allele
relative to patients having zero copies of an ApoE4 allele; (b)
patients having one copy of an ApoE4 allele relative to patients
having zero copies of an ApoE4 allele; and/or (c) patients having
two copies of an ApoE4 allele relative to patients having one copy
of an ApoE4 allele.
[0046] The invention further provides a kit for determining a
regime for bapineuzumab administration comprising instructions to a
healthcare professional that assist the healthcare professional
determine which regime of bapineuzumab to administer to a patient
having zero copies of an ApoE4 allele. Optionally, the instructions
specify a regime characterized by administering bapineuzumab at a
dose of 0.5-2 mg/kg. Optionally, the instructions specify
administering 0.5-2 mg/kg of bapineuzumab quarterly by intravenous
administration, or at a dose frequency and route of administration
that generates an equivalent average serum concentration or area
under the curve. Optionally, the instructions specify monitoring
the patient for vasogenic edema. Optionally, the instructions
specify that the monitoring regime is different that the monitoring
regime for a patient having one or two copies of an ApoE4 allele.
Optionally, the instructions specify that the determined regime
comprises a first and a second regime, wherein the first regime is
administered to the patient before vasogenic edema appears, and the
second regime after vasogenic edema has resolved; and wherein the
first and second regimes each comprise administering bapineuzumab;
wherein the first regime differs relative to the second regime in
at least one of (i)-(ii) below: (i) the dose of the bapineuzumab is
reduced; (ii) the frequency of administration of the bapineuzumab
is reduced. Optionally, the instructions specify determining the
number of ApoE4 alleles present in a patient. Optionally, the kit
further comprises bapineuzumab. Optionally, the instructions
specify monitoring at the patient for vasogenic edema. Optionally,
the instructions specify the monitoring is performed by MRI.
Optionally, the instructions specify the monitoring is by brain
imaging.
[0047] The invention further provides a kit for determining a
regime for bapineuzumab administration comprising instructions to a
healthcare professional that assist the healthcare professional
determine which regime of bapineuzumab to administer to a patient
having one or two copies of an ApoE4 allele. Optionally, the
instructions specify administering bapineuzumab at a dose of 0.15-1
mg/kg. Optionally, the instructions specify administering
bapineuzumab at a dose of 0.15-1 mg/kg quarterly by intravenous
administration, or at a dose frequency and route of administration
that generates an equivalent average serum concentration or area
under the curve. Optionally, the instructions specify that the
determined regime comprises a first and a second regime, wherein
the first regime is administered to the patient before vasogenic
edema appears, and the second regime after vasogenic edema has
resolved; and wherein the first and second regimes each comprise
administering bapineuzumab; wherein the first regime differs
relative to the second regime in at least one of (i)-(ii) below:
(i) the dose of the bapineuzumab is reduced; (ii) the frequency of
administration of the bapineuzumab is reduced. Optionally, the
instructions specify determining the number of ApoE4 alleles
present in a patient. Optionally, the kit further comprises
bapineuzumab. Optionally, the instructions specify monitoring at
the patient for vasogenic edema. Optionally, the instructions
specify the monitoring is performed by MRI. Optionally, the
instructions specify the monitoring is by brain imaging.
Optionally, the instructions specify the monitoring regime is
different that the monitoring regime for a patient having zero
copies of an ApoE4 allele. Optionally, the instructions specify
that the frequency of monitoring is greater for: (a) patients
having two copies of the ApoE4 allele relative to patients having
zero copies of an ApoE4 allele; (b) patients having one copy of an
ApoE4 allele relative to patients having zero copies of an ApoE4
allele; and/or (c) patients having two copies of an ApoE4 allele
relative to patients having one copy of an ApoE4 allele.
[0048] The invention further provides a method for improving the
safety of bapineuzumab in patients having one or two ApoE4 alleles,
comprising advising the physician to administer a lower dose of
bapineuzumab to a patient having one or two ApoE alleles relative
to that of a patient having zero ApoE alleles.
[0049] The invention further provides a method for improving the
safety of bapineuzumab in patients having one or two ApoE4 alleles,
comprising advising the physician to monitor the patient by MRI
more frequently than a patient having one or two ApoE alleles
relative to that of a patient having zero ApoE alleles.
[0050] The invention further provides an isolated antibody
comprising a human heavy chain constant region of isotype IgG1,
wherein amino acids at positions 234, 235, and 237 (EU numbering)
are each alanine. Optionally, no other amino acid from positions
230-240 or 315-325 in the human heavy chain constant region is
occupied by an amino acid not naturally found at that position in a
human IgG1 constant region. Optionally, no amino acid in the human
heavy chain constant region other than positions 234, 235 and 237
is occupied by an amino acid not naturally found at that position
in a human IgG1 constant region. Optionally, the human heavy chain
constant region comprise CH1, hinge, CH2 and CH3 regions.
Optionally, the human heavy chain constant region has an amino acid
sequence comprising SEQ ID NO:66 or SEQ ID NO:67 or an allotype of
either of these sequences. Optionally, the human heavy chain
constant region has an amino acid sequence comprising SEQ ID NO:66
or SEQ ID NO:67. Optionally, the antibody is a fully human
antibody. Optionally, the antibody is a humanized antibody.
Optionally, the antibody is chimeric antibody.
BRIEF DESCRIPTION OF THE FIGURES
[0051] FIG. 1 shows changes in ADAS-Cog, DAD, NTB and CDR-SB in
treated patients relative to placebo patients using a repeated
measures statistical model without assumption of linearity. Bars
above zero indicate improvement relative to placebo. MITT=modified
intent to treat.
[0052] FIG. 2 shows changes in ADAS-Cog, DAD, NTB and CDR-SB in
treated patients who completed the trials ("completers") relative
to placebo patients using a repeated measures statistical model
without assumption of linearity. Bars above zero indicate
improvement relative to placebo.
[0053] FIG. 3 shows changes in ADAS-Cog, DAD, NTB and CDR-SB in
ApoE4 carrier treated patients relative to placebo patients using a
repeated measures statistical model without assumption of
linearity. Bars above zero indicate improvement relative to
placebo.
[0054] FIG. 4 shows changes in ADAS-Cog, DAD, NTB and CDR-SB in
ApoE4 carrier treated patients who completed the trial relative to
placebo patients using a repeated measures statistical model
without assumption of linearity. Bars above zero indicate
improvement relative to placebo.
[0055] FIG. 5 shows changes in ADAS-Cog, DAD, NTB and CDR-SB in
ApoE4 non-carrier treated patients relative to placebo patients
using a repeated measures statistical model without assumption of
linearity. Bars above zero indicate improvement relative to
placebo.
[0056] FIG. 6 provides similar information to FIG. 5 except that
FIG. 6 shows changes based on the MMSE scale relative to
placebo.
[0057] FIG. 7 shows changes in ADAS-Cog, DAD, NTB and CDR-SB in
ApoE4 non-carrier treated patients who completed the trial relative
to placebo patients using a repeated measures statistical model
without assumption of linearity. Bars above zero indicate
improvement relative to placebo.
[0058] FIG. 8 shows similar information to FIG. 7 except that FIG.
8 shows changed based on the MMSE scale relative to placebo.
[0059] FIG. 9 shows changes in ADAS-cog, DAS, NTB and CDR-SB over
time in treated patients compared with placebos in an ApoE4
non-carrier population.
[0060] FIGS. 10, 11 and 12 show changes in BBSI in total population
(ApoE4 carriers and non-carriers), ApoE4 carriers and ApoE4
non-carriers respectively compared with placebo populations.
[0061] FIG. 13 shows CSF concentration of phospho-tau in treated
patients compared with placebo patients (without distinguishing
between ApoE4 genotypes).
[0062] FIG. 14 shows changes in serum concentration of bapineuzuab
in serum over time (left) and concentration of A.beta. in plasma
over time.
[0063] FIG. 15 shows an alignment of the CH2 domains of human IgG1
(SEQ ID NO: 95), IgG2 (SEQ ID NO: 96), and IgG4 (SEQ ID NO: 97)
with mouse IgG1 (SEQ ID NO: 98) and IgG2a (SEQ ID NO: 99).
[0064] FIG. 16 shows A.beta. plaque clearance by mouse microglia of
murine 3D6 IgG1 derivatives. MsIgG1 and MsIgG2a are murine
antibodies against irrelevant antigens. The 3D6 antibodies have the
variable region described herein. 3D6/Fc.gamma.R1 indicates the
single E233P mutant in the Fc binding region of the IgG1 constant
region. 3D6/C1q indicates the triple mutant in the C1q binding
region. See, e.g., Example 6 and Table 10.
[0065] FIGS. 17A & B show A.beta. plaque clearance by mouse
microglia of murine 3D6 IgG2a derivatives. IgG2a is a murine
antibody against an irrelevant antigen. The remaining antibodies
and conditions are described, e.g., in Example 6 and Table 10.
[0066] FIG. 18 shows A.beta. plaque clearance by mouse microglia of
humanized 3D6 derivatives (AAB). The antibodies and conditions are
described e.g., in Example 6 and Table 10.
[0067] FIG. 19 shows results of an in vitro assay measuring
engulfment of murine IgG-coated beads by mouse microglial cells.
Conditions are described in Example 6.
[0068] FIG. 20 shows a similar assay using the indicated humanized
antibodies. Conditions are described in Example 6.
[0069] FIG. 21 shows results of an ELISA assay measuring C1q
binding by the indicated humanized antibodies. See Example 7.
[0070] FIG. 22 shows the results of an antibody dependent
complement cytotoxicity assay using the indicated humanized
antibodies. Results are expressed as described in Example 7.
[0071] FIG. 23 shows results of an ELISA assay measuring C1q
binding by the indicated murine antibodies. See Example 8.
[0072] FIGS. 24-25 show the results of a contextual fear assay in
mice treated with the indicated humanized antibodies. Results are
compared between wild type and Tg2576 mice, as described in Example
9.
[0073] FIG. 26 shows the results of the ADCC activities of
anti-Lewis Y Ab02 antibodies. See Example 15.
[0074] FIG. 27 shows the results of the CDC (complement dependent
cytotoxicity) activities of anti-Lewis Y Ab02 antibodies. See
Example 15.
DEFINITIONS
[0075] The term "immunoglobulin" or "antibody" (used
interchangeably herein) refers to an antigen-binding protein having
a basic four-polypeptide chain structure consisting of two heavy
and two light chains, said chains being stabilized, for example, by
interchain disulfide bonds, which has the ability to specifically
bind antigen. Both heavy and light chains are folded into domains.
The term "domain" refers to a globular region of a heavy or light
chain polypeptide comprising peptide loops (e.g., comprising 3 to 4
peptide loops) stabilized, for example, by pleated sheet and/or
intrachain disulfide bond. Domains are further referred to herein
as "constant" or "variable", based on the relative lack of sequence
variation within the domains of various class members in the case
of a "constant" domain, or the significant variation within the
domains of various class members in the case of a "variable"
domain. "Constant" domains on the light chain are referred to
interchangeably as "light chain constant regions", "light chain
constant domains", "CL" regions or "CL" domains). "Constant"
domains on the heavy chain are referred to interchangeably as
"heavy chain constant regions", "heavy chain constant domains",
"CH" regions or "CH" domains). A heavy chain constant region is
also commonly understood to refer collectively to the domains
present in a full length constant region, which are CH1, hinge,
CH2, and CH3 domains in the case of antibodies of IgG isotype.
"Variable" domains on the light chain are referred to
interchangeably as "light chain variable regions", "light chain
variable domains", "VL" regions or "VL" domains). "Variable"
domains on the heavy chain are referred to interchangeably as
"heavy chain constant regions," "heavy chain constant domains,"
"CH" regions or "CH" domains).
[0076] The term "region" refers to a part or portion of an antibody
chain and includes constant or variable domains as defined herein,
as well as more discrete parts or portions of said domains. For
example, light chain variable domains or regions include
"complementarity determining regions" or "CDRs" interspersed among
"framework regions" or "FRs", as defined herein.
[0077] References to an antibody or immunoglobulin include intact
antibodies and binding fragments thereof. Typically, fragments
compete with the intact antibody from which they were derived for
specific binding to an antigen. Fragments include separate heavy
and light chains, Fab, Fab'F(ab').sub.2, Fabc, and Fv. Separate
chains include NANOBODIES.TM. (i.e., the isolated VH fragment of
the heavy chain of antibodies from camels or llamas, optionally
humanized). Isolated VH fragments can also be obtained from other
sources, such as human antibodies. Fragments are produced by
recombinant DNA techniques, or by enzymatic or chemical separation
of intact immunoglobulins. The term "antibody" also includes one or
more immunoglobulin chains that are chemically conjugated to, or
expressed as, fusion proteins with other proteins. The term
"antibody" also includes bispecific antibody. A bispecific or
bifunctional antibody is an artificial hybrid antibody having two
different heavy/light chain pairs and two different binding sites.
Bispecific antibodies can be produced by a variety of methods
including fusion of hybridomas or linking of Fab' fragments. (See,
e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321
(1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).)
[0078] "Specific binding" of an antibody means that the antibody
exhibits appreciable affinity for antigen or a preferred epitope
and, preferably, does not exhibit significant cross reactivity.
Appreciable or preferred binding includes binding with an affinity
of at least 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9 M.sup.-1, or
10.sup.10 M.sup.-1. Affinities greater than 10.sup.7 M.sup.-1,
preferably greater than 10.sup.8 M.sup.-1 are more preferred.
Values intermediate of those set forth herein are also intended to
be within the scope of the present invention and a preferred
binding affinity can be indicated as a range of affinities, for
example, 10.sup.6 to 10.sup.10 M.sup.-1, preferably 10.sup.7 to
10.sup.10 M.sup.-1, more preferably 10.sup.8 to 10.sup.10 M.sup.-1.
An antibody that "does not exhibit significant cross reactivity" is
one that will not appreciably bind to an undesirable entity (e.g.,
an undesirable proteinaceous entity). For example, an antibody that
specifically binds to A.beta. will appreciably bind A.beta. but
will not significantly react with non-A.beta. proteins or peptides
(e.g., non-A.beta. proteins or peptides included in plaques). An
antibody specific for a preferred epitope will, for example, not
significantly cross react with remote epitopes on the same protein
or peptide. Specific binding can be determined according to any
art-recognized means for determining such binding. Preferably,
specific binding is determined according to Scatchard analysis
and/or competitive binding assays.
[0079] The term "humanized immunoglobulin" or "humanized antibody"
refers to an immunoglobulin or antibody that includes at least one
humanized immunoglobulin or antibody chain (i.e., at least one
humanized light or heavy chain). The term "humanized immunoglobulin
chain" or "humanized antibody chain" (i.e., a "humanized
immunoglobulin light chain" or "humanized immunoglobulin heavy
chain") refers to an immunoglobulin or antibody chain (i.e., a
light or heavy chain, respectively) having a variable region that
includes a variable framework region (also known as variable region
framework) substantially from a human immunoglobulin or antibody
and complementarity determining regions (CDRs) (e.g., at least one
CDR, preferably two CDRs, more preferably three CDRs) substantially
from a non-human immunoglobulin or antibody (e.g., rodent, and
optionally, mouse), and further includes constant regions (e.g., at
least one constant region or portion thereof, in the case of a
light chain, and preferably three constant regions in the case of a
heavy chain). The term "humanized variable region" (e.g.,
"humanized light chain variable region" or "humanized heavy chain
variable region") refers to a variable region that includes a
variable framework region (also known as a variable region
framework) substantially from a human immunoglobulin or antibody
and complementarity determining regions (CDRs) substantially from a
non-human immunoglobulin or antibody.
[0080] The phrase "substantially from a human immunoglobulin or
antibody" or "substantially human" means that, when aligned to a
human immunoglobulin or antibody amino sequence for comparison
purposes, the region shares at least 80-90% (e.g., at least 90%),
preferably 90-95%, more preferably 95-99% identity (i.e., local
sequence identity) with the human framework or constant region
sequence, allowing, for example, for conservative substitutions,
consensus sequence substitutions, germline substitutions,
backmutations, and the like. The introduction of conservative
substitutions, consensus sequence substitutions, germline
substitutions, backmutations, and the like, is often referred to as
"optimization" of a humanized antibody or chain. The phrase
"substantially from a non-human immunoglobulin or antibody" or
"substantially non-human" means having an immunoglobulin or
antibody sequence at least 80-95%, preferably 90-95%, more
preferably, 96%, 97%, 98%, or 99% identical to that of a non-human
organism, e.g., a non-human mammal.
[0081] Accordingly, all regions or residues of a humanized
immunoglobulin or antibody, or of a humanized immunoglobulin or
antibody chain, except possibly the CDRs, are substantially
identical to the corresponding regions or residues of one or more
native human immunoglobulin sequences. The term "corresponding
region" or "corresponding residue" refers to a region or residue on
a second amino acid or nucleotide sequence which occupies the same
(i.e., equivalent) position as a region or residue on a first amino
acid or nucleotide sequence, when the first and second sequences
are optimally aligned for comparison purposes.
[0082] The terms "humanized immunoglobulin" or "humanized antibody"
are not intended to encompass chimeric immunoglobulins or
antibodies, as defined infra. Although humanized immunoglobulins or
antibodies are chimeric in their construction (i.e., comprise
regions from more than one species of protein), they include
additional features (i.e., variable regions comprising donor CDR
residues and acceptor framework residues) not found in chimeric
immunoglobulins or antibodies, as defined herein.
[0083] The term "chimeric immunoglobulin" or antibody refers to an
immunoglobulin or antibody whose variable regions derive from a
first species and whose constant regions derive from a second
species. Chimeric immunoglobulins or antibodies can be constructed,
for example by genetic engineering, from immunoglobulin gene
segments belonging to different species.
[0084] An "antigen" is an entity (e.g., a proteinaceous entity or
peptide) to which an antibody specifically binds.
[0085] The term "epitope" or "antigenic determinant" refers to a
site on an antigen to which an immunoglobulin or antibody (or
antigen binding fragment thereof) specifically binds. Epitopes can
be formed both from contiguous amino acids or noncontiguous amino
acids juxtaposed by tertiary folding of a protein. Epitopes formed
from contiguous amino acids are typically retained on exposure to
denaturing solvents whereas epitopes formed by tertiary folding are
typically lost on treatment with denaturing solvents. An epitope
typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14
or 15 amino acids in a unique spatial conformation. Methods of
determining spatial conformation of epitopes include, for example,
x-ray crystallography and 2-dimensional nuclear magnetic resonance.
See, e.g., Epitope Mapping Protocols in Methods in Molecular
Biology, Vol. 66, G. E. Morris, Ed. (1996).
[0086] Antibodies that recognize the same epitope can be identified
in a simple immunoassay showing the ability of one antibody to
block the binding of another antibody to a target antigen, i.e., a
competitive binding assay. Competitive binding is determined in an
assay in which the immunoglobulin under test inhibits specific
binding of a reference antibody to a common antigen, such as
A.beta.. Numerous types of competitive binding assays are known,
for example: solid phase direct or indirect radioimmunoassay (RIA),
solid phase direct or indirect enzyme immunoassay (EIA), sandwich
competition assay (see Stahli et al., Methods in Enzymology 9:242
(1983)); solid phase direct biotin-avidin EIA (see Kirkland et al.,
J. Immunol. 137:3614 (1986)); solid phase direct labelled assay,
solid phase direct labelled sandwich assay (see Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988));
solid phase direct label RIA using I-125 label (see Morel et al.,
Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA
(Cheung et al., Virology 176:546 (1990)); and direct labelled RIA
(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990). Typically,
such an assay involves the use of purified antigen bound to a solid
surface or cells bearing either of these, an unlabelled test
immunoglobulin and a labelled reference immunoglobulin. Competitive
inhibition is measured by determining the amount of label bound to
the solid surface or cells in the presence of the test
immunoglobulin. Usually the test immunoglobulin is present in
excess. Usually, when a competing antibody is present in excess, it
will inhibit specific binding of a reference antibody to a common
antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% or
more.
[0087] An epitope is also recognized by immunologic cells, for
example, B cells and/or T cells. Cellular recognition of an epitope
can be determined by in vitro assays that measure antigen-dependent
proliferation, as determined by .sup.3H-thymidine incorporation, by
cytokine secretion, by antibody secretion, or by antigen-dependent
killing (cytotoxic T lymphocyte assay).
[0088] Exemplary epitopes or antigenic determinants can be found
within the human amyloid precursor protein (APP), but are
preferably found within the A.beta. peptide of APP. Multiple
isoforms of APP exist, for example APP.sup.695, APP.sup.751 and
APP.sup.770. Amino acids within APP are assigned numbers according
to the sequence of the APP.sup.770 isoform (see e.g., GenBank
Accession No. P05067). The sequences of A.beta. peptides and their
relationship to the APP precursor are illustrated by FIG. 1 of
Hardy et al., TINS 20, 155-158 (1997). For example, A.beta.42 has
the sequence:
TABLE-US-00001 (SEQ ID NO: 1)
H.sub.2N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-
Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-
Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-
Val-Gly-Gly-Val-Val-Ile-Ala-OH.
[0089] Unless otherwise apparent from the context, reference to
A.beta. also includes natural allelic variations of the above
sequence, particularly those associated with hereditary disease,
such as the Arctic mutation, E693G, APP 770 numbering. A.beta.41,
A.beta.40 and A.beta.39 differ from A.beta.42 by the omission of
Ala, Ala-IIe, and Ala-IIe-Val respectively from the C-terminal end.
A.beta.43 differs from A.beta.42 by the presence of a threonine
residue at the C-terminus. Preferred epitopes or antigenic
determinants, as described herein, are located within the
N-terminus of the A.beta. peptide and include residues within amino
acids 1-11 of A.beta., preferably from residues 1-10, 1-3, 1-4,
1-5, 1-6, 1-7 or 3-7 of A.beta.42. Additional preferred epitopes or
antigenic determinants include residues 2-4, 5, 6, 7 or 8 of
A.beta., residues 3-5, 6, 7, 8 or 9 of A.beta., or residues 4-7, 8,
9 or 10 of A.beta.42. Other preferred epitopes occur within central
or C-terminal regions as described below.
[0090] An N-terminal epitope of A.beta. means an epitope with
residues 1-11. An epitope within a C-terminal region means an
epitope within residues 29-43, and an epitope within a central
regions means an epitope with residues 12-28
[0091] "Soluble" or "dissociated" A.beta. refers to non-aggregating
or disaggregated A.beta. polypeptide.
[0092] "Insoluble" A.beta. refers to aggregating A.beta.
polypeptide, for example, A.beta. held together by noncovalent
bonds. A.beta. (e.g., A.beta.42) is believed to aggregate, at least
in part, due to the presence of hydrophobic residues at the
C-terminus of the peptide (part of the transmembrane domain of
APP). One method to prepare soluble A.beta. is to dissolve
lyophilized peptide in neat DMSO with sonication. The resulting
solution is centrifuged to remove any insoluble particulates.
[0093] The term "Fc region" refers to a C-terminal region of an IgG
antibody, in particular, the C-terminal region of the heavy
chain(s) of said IgG antibody. Although the boundaries of the Fc
region of an IgG heavy chain can vary slightly, a Fc region is
typically defined as spanning from about amino acid residue Cys226
to the carboxyl-terminus of an IgG heavy chain(s).
[0094] The term "effector function" refers to an activity that
resides in the Fc region of an antibody (e.g., an IgG antibody) and
includes, for example, the ability of the antibody to bind effector
molecules such as complement and/or Fc receptors, which can control
several immune functions of the antibody such as effector cell
activity, lysis, complement-mediated activity, antibody clearance,
and antibody half-life. Effector function can also be influenced by
mutations in the hinge region.
[0095] The term "effector molecule" refers to a molecule that is
capable of binding to the Fc region of an antibody (e.g., an IgG
antibody) including a complement protein or a Fc receptor.
[0096] The term "effector cell" refers to a cell capable of binding
to the Fc portion of an antibody (e.g., an IgG antibody) typically
via an Fc receptor expressed on the surface of the effector cell
including, but not limited to, lymphocytes, e.g., antigen
presenting cells and T cells.
[0097] The term "Kabat numbering" unless otherwise stated, is
defined as the numbering of the residues as in Kabat et al.
(Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991)), incorporated herein by reference.
[0098] The term "Fc receptor" or "FcR" refers to a receptor that
binds to the Fc region of an antibody. Typical Fc receptors which
bind to an Fc region of an antibody (e.g., an IgG antibody)
include, but are not limited to, receptors of the Fc.gamma.RI,
Fc.gamma.RII, and Fc.gamma.RIII subclasses, including allelic
variants and alternatively spliced forms of these receptors. Fc
receptors are reviewed in Ravetch and Kinet, Annu. Rev. Immunol
9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de
Haas et al., J. Lab. Clin. Med. 126:330-41 (1995).
[0099] The term "adjuvant" refers to a compound that when
administered in conjunction with an antigen augments and/or
redirects the immune response to the antigen, but when administered
alone does not generate an immune response to the antigen.
Adjuvants can augment an immune response by several mechanisms
including lymphocyte recruitment, stimulation of B and/or T cells,
and stimulation of macrophages.
[0100] The area under the curve (AUC) is the area under the curve
in a plot of concentration of drug in plasma against time. In an
individual patient, the area under the curve represents the area
under the curve based on that patient. In a population of patients,
the area under the curve represents the mean area under the curve
for a comparable time interval of different patients in the
population.
[0101] The mean serum concentration in an individual patient
represents the mean concentration of an antibody (or induced
antibodies for an active agent) over a period of time. The mean
serum concentration in a population of patients represents the mean
of the mean serum concentrations of the individual patients over
comparable periods of time.
[0102] The maximum serum concentration in an individual patient
represents the maximum concentration of an antibody (or induced
antibodies for an active agent) during a course of treatment. The
maximum serum concentration in a population of individuals
represents the mean of maximum concentrations of the antibody or
induces antibodies between individuals in the population.
[0103] For brevity, the term "ApoE4 carrier" is sometimes used to
refer to patients have in one or two ApoE4 alleles and "ApoE4
noncarrier", ApoE4 non-carrier" or "non-ApoE4 carrier" to refer to
patients having zero ApoE4 alleles.
DETAILED DESCRIPTION OF THE INVENTION
I. General
[0104] The invention provides methods of immunotherapy of
Alzheimer's and similar diseases in which the regime administered
to a patient depends on the ApoE genotype of the patient. The
methods are based in part on (1) the observation that certain
immunotherapy regimes lead to higher instances in the appearance of
vasogenic edema (VE) in patients having an ApoE4 allele (E4) than
in patients lacking an E4 allele, and more frequently still in
patients having two E4 alleles, and/or (2) the initial observation
of differential efficacy in ApoE4 carrier patients compared to
ApoE4 non-carrier patients or patients receiving at least six doses
compared to patients receiving less than six doses. The results
also show that frequency of cases of vasogenic edema increases with
dose frequency and amount.
[0105] Although practice of the invention is not dependent on an
understanding of mechanism, it is hypothesized that the association
of the vasogenic edema with an ApoE4 genotype may stem from a
greater deposition of A.beta. deposits and hence induction of a
greater clearing response when antibodies bind to the deposits.
Clearing of amyloid deposits may lead to vasogenic edema by any or
all of several mechanisms. Removal of amyloid from blood vessel
walls (vascular amyloid) may cause leakiness of blood vessels; more
amyloid in perivascular space may cause slower drainage of
interstitial fluid, and/or net increased flow of amyloid from
intravascular compartment to brain parenchyma may lead to osmotic
gradients. Although vasogenic edema effect is usually asymptomatic
and reversible and does not preclude further treatment, it is
desirable nevertheless to adjust the therapeutic regime to reduce
the risk of vasogenic edema occurring.
[0106] The invention thus provides methods in which the
immunotherapy regime is varied, for example to adjust the
phagocytic response, depending on the ApoE status of the patient.
Although the phagocytic response is useful in clearing amyloid
deposits, the response, can optionally be controlled to avoid
vasogenic edema. In general, patients having two E4 alleles, who
are most susceptible to the vasogenic edema are administered either
a lower dose or a lower frequency of the same agent as patients
with zero E4 alleles, or are administered a different agent that is
less prone to induce a phagocytic response or receive the agent
through an alternate mode of administration, such as, for example,
subcutaneous administration. Patients with one E4 allele can be
treated the same as either patients with zero or two E4 alleles or
a treatment can be customized for them in which the dose and/or
frequency of administration is intermediate between that
administered to patients with zero or two ApoE4 alleles.
II. APOE
[0107] Human ApoE has the UniProtKB/Swiss-Prot entry accession
number P02649. The E2, E3, and E4 variants are described in
Genomics 3:373-379 (1988), J. Biol. Chem. 259:5495-5499 (1984); and
Proc. Natl. Acad. Sci. U.S.A. 82:3445-3449 (1985). Association of
the E4 form with late onset Alzheimer's disease has been reported
by e.g., Corder, Science 261, 921-3 (1993); Farrer, JAMA, 278,
1349-56 (1997); and Saunders, Neurology 43, 1467-72 (1993). The
allelic forms present in any individual can be determined by many
conventional techniques, such as direct sequencing, use of
GeneChip.RTM. arrays or the like, allele-specific probes,
single-base extension methods, allelic specific extension. Allelic
forms can also be determined at the protein level by ELISA using
antibodies specific for different allelic expression products. Kits
for genetic and immunological analysis are commercially available
(e.g., Innogenetics, Inc.; Graceful Earth, Inc.). Determination of
allelic forms are usually made in vitro, that is, on samples
removed and never returned to a patient.
III. Different Strategies for Treating or Monitoring Depending on
ApoE
A. Different Treatment Regimes
[0108] Some immunotherapy regimes for immunotherapy of Alzheimer's
and other diseases have been associated with vasogenic edema (VE)
in the brain of some patients. Generally, the incidence of VE is
greater in ApoE4 carriers than in ApoeE4 non-carriers and in
patients receiving higher doses of certain agents in certain
immunotherapy regimes. VE has been observed on magnetic resonance
imaging (MRI) as high signal intensities on the fluid-attenuated
inversion recovery (FLAIR) sequence involving cerebral
abnormalities and gyral swelling. VE generally is observed after
the first or second administration of the immunotherapeutic agent,
although it has been observed after the third or fourth
administration. Most patients with VE discovered on MRI are
asymptomatic. VE is heterogeneous on presentation, and MRI findings
in a particular patient may vary over time. The gyral swelling and
to some extent, the larger magnetic resonance (MR) changes seen on
FLAIR differentiate VE from the commonly observed white matter
changes seen on FLAIR in both normal elderly and Alzheimer's
disease patients (Hentschel et al., 2005; de Leeuw et al.
2001).
[0109] Vasogenic edema (VE) is characterized by an increase in
extracellular fluid volume due to increased permeability of brain
capillary endothelial cells to macromolecular serum proteins (e.g.,
albumin). VE may be the result of increased brain capillary
permeability. Clinical symptoms observed in patients with VE, when
existent, are varied and to date have been largely mild in nature.
Of the cases of VE observed on regularly scheduled MRI, the
majority of patients are asymptomatic. Clinical observations
associated with the symptomatic cases of VE have included altered
mental states (e.g., increased confusion, lethargy, disorientation,
and hallucinations), vomiting, headache, gait difficulties, visual
disturbances, fatigue, irritability, ataxia, decreased appetite,
and diarrhea.
[0110] As summarized above, the invention provides different
treatment regimes depending on whether a patient has zero, one or
two E4 alleles. Thus, in a population of treated individuals, those
having zero E4 alleles can be treated differently from those having
two alleles. Those having one E4 allele can be treated differently
(in an intermediate fashion) to those with either zero or two E4
alleles or can be grouped with individuals having zero or two the
E4 allele in any of the regimes that follow. It follows that
individuals having one E4 allele can be treated differently than
individuals with zero alleles and/or that individual with two ApoE4
alleles can be treated differently than individuals with one ApoE4
allele. Ongoing experience with some immunotherapeutic agents
suggests that VE is more likely to occur at doses greater than 5
mg/kg (see PCT/US07/09499).
[0111] In some methods, ApoE4 status is the only genetic marker
determining different treatment regimes in different patients. In
other methods, differential treatment regimes can be based on ApoE4
in combination with other genetic markers associated with
Alzheimer's disease susceptibility or resistance.
[0112] A population of treated individuals optionally has
sufficient total number of patients and sufficient numbers of
subpopulations with different numbers of ApoE4 alleles that an
association between different treatment regimes and different ApoE4
alleles can be seen relative to a random assignment of the
different regimes with a statistical confidence of at least 95%.
For example, the treated population can consist of at least 100,
500 or 1000 individuals of who 10-70% and more typically 30-50%
have at least one an ApoE4 allele. A treated population can also
(i.e., optionally) be recognized as the total population treated
with a particular drug produced by a particular manufacturer.
[0113] In some methods, as discussed in greater detail below,
individuals having zero ApoE4 alleles are administered an agent in
a regime designed to achieve efficacy as assessed from one or more
clinical endpoints, such as, for example, cognitive measures (e.g.,
ADAS-cog, NTB, DAD, MMSE, CDR-SB, NPI), biomarkers (e.g., CSF tau),
and brain volume (e.g., BBSI, VBSI), as well as other parameters,
such as, for example desirable safety, pharmacokinetics and
pharmacodynamics. In some methods, one or two E4 alleles are
administered a reduced dose and/or frequency of the same agent as
individuals with zero E4 alleles. A goal of such method is to
deliver a reduced mean serum concentration of the agent over a
period of time (reduced area under the curve) and/or to reduce the
maximum peak concentration. This can be accomplished for example,
by reducing the dose and administering at the same frequency, or
reducing the frequency and administering at the same dose or
administering at reduced dose and frequency. If the dose is reduced
but the frequency kept constant, the dose is usually reduced
between 10-90%, often about 30-75 or 40-60%. If the frequency is
reduced, but the dose kept constant, then the frequency is
typically reduced between two and five fold. Sometimes, the
frequency is reduced by simply omitting an occasional dose or two
consecutive doses from the regime administered to patients with
zero ApoE4 alleles. Such doses can for example be omitted during
the period a patient is experiencing vasogenic edema.
[0114] In other methods, individual having one or two E4 alleles
are administered a reduce dose of the agent at an increased
frequency relative to individuals having zero E4 alleles. For,
example, the dose can be halved and the frequency doubled. In such
methods, the total drug delivered to the two subpopulations over
time (i.e., area under the curve) can be the same within
experimental error, but the maximum plasma concentration is lower
in individuals having two E4 alleles. For example, in patients
having one or two E4 alleles the maximum serum concentration of
antibody is preferably below 14 .mu.g/ml and for patients having
zero alleles, the maximum serum concentration of antibody is
preferably below 28 .mu.g/ml.
[0115] In other methods, treatment is administered at different
stages relative to disease progression depending on ApoE4 status.
In such methods, treatment is administered earlier in patients
having two ApoE4 alleles relative to patients having zero ApoE4
alleles or in patients having one ApoE4 allele relative to patients
having zero ApoE4 alleles and/or in patients having two ApoE4
alleles relative to patients having one ApoE4 allele. Disease
progression can be measured by e.g., the MMSE scale on which a
score of 27 to 20 is considered normal, and 20-26 considered mild
Alzheimer's. Thus, for example, the mean MMSE score of non-ApoE4
carriers on commencement of treatment can be higher than that of
ApoE4 carriers (patients with one or two ApoE4 alleles).
Optionally, treatment of ApoE4 carriers can be begun
prophylactically before clinical symptoms are evident. Such
patients can be identified by screening populations for ApoE4
status. Treatment can be commenced on detecting such status or
subsequently when the patient reaches a certain age (e.g., 55, 60
or 65 years) when there is a high risk of Alzheimer's developing.
Although understanding of mechanism is not required for practice of
such methods, it is believed that early treatment of ApoE4 carriers
may be beneficial because the ApoE4 allele reduces capacity to
repair neuronal damage, and/or because deposition of A.beta. is
greater in such patients.
[0116] In some methods, treatment is administered by a different
route in patients having zero ApoE4 alleles and patients having one
ApoE4 allele and/or patients having two ApoE4 alleles. For example,
treatment can be administered intravenously in patients having zero
ApoE4 alleles and subcutaneously in patients having one or two
alleles. The dosage is typically greater and/or frequency of
administration less in such non-ApoE4 carrier patients relative to
ApoE4 carrier patients.
[0117] In some methods, a positive response to treatment (i.e.,
inhibition of cognitive decline or inhibition of decline in brain
volume) takes longer to develop in ApoE4 carriers than
non-carriers. The greater time may reflect reduced capacity for
neuronal repair and/or greater amyloid burden in such patients;
and/or use of a less potent treatment regime. In such methods,
treatment can be administered for at least one year and optionally
at least 2, 3 or 4 years before ceasing treatment for lack of
effect. In some methods, treatment is administered for at least six
quarterly administrations.
[0118] As noted, agents are sometimes provided with a label
contraindicating use in ApoE4 carriers. Such agents can be used in
methods of treatment in which only non-ApoE4 carriers receive an
agent of the invention (i.e., an antibody that binds to A.beta. or
an agent that induces such an antibody). In such methods ApoE4
carriers do not receive an antibody that binds to A.beta. or an
agent that induces such an antibody but can receive other
treatments such memantine.
[0119] Methods in which dose and/or frequency of administration are
reduced depending on ApoE4 are most useful for agents that initiate
a clearing response against amyloid deposits. In general, such
agents are antibodies binding to an epitope within A.beta. 1-11,
and which have an Fc region, or fragments of A.beta. that induce
such antibodies (i.e., contain an epitope within A.beta. 1-11).
Antibodies binding to epitopes within central or C-terminal regions
of A.beta. usually bind predominantly to soluble forms of A.beta.
rather than amyloid deposits, and thus initiate little, if any
clearing response against amyloid deposits, particularly dense or
vascular deposits.
[0120] Examples of suitable dosages ranges and frequencies for
administration are provided below. Different dosages and/or
frequencies of administration for patients with different E4 status
can be selected from within such ranges of dose and frequency. For
example, patients with one or two E4 alleles can be administered a
dose of 0.1 to 1 mg/kg antibody by intravenous infusion every
thirteen weeks, and patients with zero E4 alleles can be
administered a dose of 1 to 2 mg/kg every thirteen weeks.
Optionally, patients with two E4 alleles are administered a dose of
0.15 to 0.5 mg/kg, patients with one E4 allele are administered a
dose of 0.15 to 1 mg/kg (e.g., 0.5 to 1 mg/kg) and patients with
zero E4 alleles are administered a dose of 0.15-2 mg/kg (e.g., 1-2
mg/kg) every thirteen weeks. In a preferred regime, patients with
one or two E4 alleles are administered a dose of 0.5 mg/kg of an
antibody binding to an epitope within residues 1-11 of A.beta.
(e.g., bapineuzumab) and patients with zero E4 alleles a dose of 2
mg/kg. The doses are administered intravenously at quarterly
intervals until vasogenic edema appears (if it does). After
vasogenic edema appears, the next dose is missed and thereafter,
patients return to the quarterly dosing schedule at a lower dose of
0.15 mg/kg. If vasogenic edema appears again treatment can be
terminated. Patients with zero E4 alleles are administered a dose
of 0.5-2 mg/kg, with individually patients with zero E4 alleles
optionally receiving doses of 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg and
2.0 mg/kg.
[0121] As another example, patients with two E4 alleles are given a
first dose of 0.5 mg/kg, and subsequent doses of 1 mg/kg.
Alternatively, patients with two E4 alleles are given a first dose
of 0.5 mg/kg, second and third doses of 1 mg/kg and subsequent
doses of 2.0 mg/kg.
[0122] As another example, patients with zero E4 alleles can be
administered a dose of 0.015-0.2 mg/kg antibody subcutaneously once
per week and patients with two E4 alleles can be administered the
same dose every two weeks. Equivalent regimes to any of the above
can be devised by varying either the amount or frequency or route
of administration to deliver the same area under the curve (i.e.,
mean dose integrated with time) of antibody to the serum.
[0123] In some methods, patients with one or two E4 alleles are
administered agent to achieve a lower mean serum concentration of
antibody over time than patients with zero E4 alleles. The lower
mean serum concentration is maintained over a period of at least
one or threes month, and usually three months to one year, or
indefinitely. The mean serum concentration of all such patients is
preferably within the range 2-7 .mu.g antibody/ml serum with that
for patients with one or two E4 alleles being lower than that for
patients with zero E4 alleles. For example patients with zero E4
alleles can be administered to achieve a mean serum concentration
of antibody within a range of 4.5-7 .mu.g antibody/ml and patients
with one or two E4 alleles can be administered agent to achieve a
mean serum concentration in the range of 2-4.5 .mu.g
antibody/ml.
[0124] In such methods, individuals within any subpopulation
defined by presence of two, one or zero E4 alleles are usually
administered the same regime. However, the regime can also be
customized for individuals within a subpopulation. In this case,
the mean dose and/or frequency and/or average serum concentration
and/or maximum concentration of agent or antibodies induced by the
agent in a subpopulation of individuals with two E4 alleles is
lower than that of individuals having zero E4 alleles.
[0125] In some methods, a different agent is administered to
individuals with two E4 alleles than individuals with zero E4
alleles. The different agents usually differ in their capacity to
induce a clearing response against amyloid deposits (i.e.,
preexisting deposits). Such a capacity can be tested, for example,
in an ex vivo clearing assay as described by U.S. Pat. No.
6,750,324. In brief, an antibody and microglial cells are incubated
with an amyloid deposit from a diseased Alzheimer's patient or
transgenic mouse model, and the clearing reaction is monitored
using a labelled antibody to A.beta.. Clearing capacity of active
agents can be similarly tested using sera induced by the active
agent as a source of antibody for the assay. Clearing capacity of
both passive and active agents can also be evaluated in a
transgenic mouse model as also described U.S. Pat. No. 6,750,324 or
in a human patient by MRI monitoring. Optionally, the clearing
response is measured in an assay that distinguishes between compact
and diffuse amyloid deposits. Differences in clearing capacity of
some antibodies are more evident or only evident when the
comparison is made with respect to clearing capacity of compact
amyloid deposits. Optionally, the clearing response is evaluated
from a reduction in clearing of vascular amyloid of a mutated
antibody relative to an isotype matched otherwise-identical
antibody. Vascular amyloid clearing can be assessed by a
statistical significant difference between populations of animal
models or human patients treated with a mutated antibody and an
otherwise-identical isotype-matched antibody without the
mutations.
[0126] Additionally or alternatively to assays measuring a clearing
response, some antibodies suitable for use in the methods of the
invention can be recognized by reduced binding to C1q and/or to
Fc.gamma. receptor(s). Capacity to bind C1q and/or an Fc.gamma.
receptor can be reduced by mutations near the hinge region of a
heavy chain as discussed in more detail below. Reduced capacity can
be determined, for example, by comparing a mutated antibody with an
isotype matched otherwise identical antibody lacking the
mutation(s) present in the mutated antibody (i.e., having residues
from a wild type human constant region (e.g., bapineuzumab vs.
AAB-003), or by comparing otherwise identical antibodies having
different isotypes (e.g., human IgG1 versus human IgG4).
[0127] Some antibodies having reduced capacity to bind C1q and/or
Fc.gamma. receptor(s) reduce micro-hemorrhaging relative to isotype
matched controls but retain at least some activity in inhibiting
cognitive decline and/or clearing amyloid deposits. In some
antibodies, reduced amyloid clearing capacity is mainly associated
with reduced clearing capacity of vascular amyloid and/or compact
amyloid deposits and not with diffuse amyloid deposits. Such
antibodies offer a potentially improved efficacy:side-effects
profile, particularly for use in ApoE4 carriers.
[0128] Antibodies having reduced binding to C1q and/or an Fc.gamma.
receptor can be used in differential methods of treatment as
described above. For example, an antibody with reduced binding to
C1q and/or and Fc.gamma. receptor can be administered to patients
having one or two ApoE4 alleles and an otherwise identical antibody
without the mutation(s) to patients with zero ApoE4 alleles.
Alternatively, an antibody with reduced binding to C1q and/or an
Fc.gamma. receptor can be administered to patients irrespective of
the number of ApoE4 alleles.
[0129] Antibodies with constant regions mutated to reduce C1q
and/or Fc.gamma. receptor binding are sometimes administered at
higher dosages than otherwise identical antibodies without the
mutation. For some such antibodies, the dosage can be adjusted
upward to achieve an equivalent therapeutic effect with reduced
side effects.
[0130] Clearing capacity is affected both by the epitope
specificity of an antibody (or antibodies induced by a fragment for
active administration) and on the presence of, and type of effector
function of the antibody, in particular by the capacity of the Fc
region if present to bind to Fc.gamma. receptors. Although clearing
amyloid deposits is one useful mechanism of action, agents that
lack the capacity to clear deposits can be useful by other
mechanisms, such as binding to soluble A.beta. and/or soluble
oligomeric forms of A.beta.. Such binding may reduce toxicity of
such species and/or inhibit their aggregating to form deposits
among other possible mechanisms.
[0131] Agents with a propensity to induce such a clearing response
include antibodies binding to an epitope within residues 1-11 and
particularly 1-7 of A.beta., particularly such antibodies having a
human IgG1 isotype, which interacts most strongly with Fc.gamma.
receptors. Fragments of A.beta. that contain epitopes within
residues 1-11 and particularly 1-7 are similarly effective in
inducing a clearing response. Optionally, agents which initiate a
clearing response, can be provided with a label contraindicating
use to patients with one or two ApoE4 alleles. Agents with less or
no propensity to induce a clearing response include antibodies to
A.beta. that have isotypes other than human IgG1, antibodies that
lack an Fc region (e.g., Fab fragments, Fv fragments, or
Nanobodies), or antibodies with Fc regions mutated by genetic
engineering to reduce interactions with Fc.gamma. receptors. Such
agents also include antibodies that specifically bind to an epitope
within a region of A.beta. other than residues 1-11, (i.e., to a
mid-epitope or C-terminal epitope, as described above) and
antibodies that specifically bind to soluble or oligomeric forms of
A.beta. without binding to amyloid deposits. Such agents also
include fragments of A.beta. that lack epitopes within residues
1-11 of A.beta.. In such methods, individuals having two E4 alleles
are administered an agent with a lower tendency to induce a
phagocytic clearing response than individuals having zero alleles.
For example, individuals having zero E4 alleles can be administered
an antibody binding to an epitope within residues 1-11 of A.beta.
and having human IgG1 isotype and individuals having two E4 alleles
can be administered the same antibody except that the antibody is a
Fab fragment or has an isotype other than human IgG1 or has an
engineered Fc region to reduce binding to Fc.gamma. receptors. The
agent administered to individuals having two E4 alleles can also be
an antibody to a mid or C-terminal epitope of A.beta. or a fragment
of A.beta. from a mid or C-terminal region (i.e., lacking an
epitope from within A.beta. 1-11).
[0132] In some methods, patients with two E4 alleles are
administered an antibody having an epitope within a mid or
C-terminal regions for one or more initial doses and an antibody
having an epitope within an N-terminal region for subsequent doses.
Such an antibody can be a humanized 266 antibody, a humanized 2H6
antibody, a deglycosylated humanized 2H6 antibody or RN1219. Such
an antibody can also be a humanized antibody that specifically
binds to an epitope within A028-40 or A.beta.33-40. The initial
doses preferably consist of 1, 2 or 3 doses. Patients having zero
alleles can be administered an antibody having an epitope within an
N-terminal region.
[0133] The different regimes administered to different patients
depending on their E4 status can be maintained indefinitely.
However, such is not usually necessary. It has been found that the
vasogenic edema side effect associated with the E4 allele usually
occurs by the third dose, if at all. Thus, once patients have
received about 2-3 doses of treatment, patients having one or two
ApoE4 alleles who have not developed vasogenic edema probably will
not develop it, and can thereafter, if desired, be treated by the
same regime as patients having zero E4 alleles. Likewise patients
with one or two ApoE4 alleles who do develop vasogenic edema
notwithstanding the present differential treatment regime usually
resolve this condition and can thereafter, if desired, be treated
in similar fashion to patients having zero E4 alleles. Optionally,
the dose is titrated up after recovering from vasogenic edema to
that used for non-carriers.
[0134] Vasogenic edema typically resolves of its own accord.
However, resolution can be facilitated if desired by administration
of a corticosteroid.
[0135] Agents can be packaged with labels indicating differential
treatment procedures dependent on ApoE4 status consistent with any
of the above regimes or combinations thereof.
B. Different Monitoring Regimes
[0136] Alternatively or additionally, the invention provides
different monitoring regimes for patients depending on their E4
status. Vasogenic edema is an increase in brain volume from leakage
of plasma into the interstitial space. Once extravasated, fluid is
retained outside the vasculature, mostly in the white matter of the
brain. Vasogenic edema can be monitored by brain imaging
particularly by MRI, Positron Emission Tomography (PET Imaging) or
Fluid Attenuated Inversion Recovery (FLAIR) sequence imaging (See
Pediatric Neurology, 20(3):241-243; AJNR, 26:825-830; NEJM,
334(8):494-500; Pediatr Nephrol, 18:1161-1166; Internal Medicine
Journal, 35:83-90; JNNP, 68:790-79 1; AJNR, 23:1038-1048; Pak J Med
Sci, 21(2):149-154 and, AJNR, 21:1199-1209). Vasogenic edema
presents with a high signal intensity in white matter. The
vasogenic edema observed is often asymptomatic but can also be
accompanied by headache, nausea, vomiting, confusion, seizures,
visual abnormalities, altered mental functioning, ataxia, frontal
symptoms, parietal symptoms, stupor, and focal neurological
signs.
[0137] According to the present methods, patients with two E4
alleles can be subjected to brain imaging more frequently than
patients having zero E4 alleles. For example, patients with two
copies of E4 can be imaged before beginning treatment and quarterly
thereafter, whereas patients with zero E4 alleles can be imaged
before beginning treatment and annually or biannually thereafter.
Alternatively, brain imaging can be omitted altogether in patients
having zero E4 alleles. Patients having one E4 allele can be imaged
with intermediate frequency between patients having zero and two E4
alleles, or can be grouped with patients having either zero or two
E4 alleles. It follows that patients with one E4 allele can be
monitored differently (e.g., more frequently) than patients with
zero E4 alleles and patients with two E4 alleles can be monitored
differently (e.g., more frequently) than patients with one E4
allele.
[0138] In patients developing vasogenic edema, monitoring can be
continued during the vasogenic edema and for about a year after
symptoms resolve. Thereafter, assuming no neurologic findings,
monitoring can optionally be performed six monthly or annually.
[0139] Agents can be packaged with labels indicating differential
monitoring procedures dependent on ApoE4 status consistent with any
of the above regimes or combinations thereof.
C. Universal Treatment or Monitoring Regimes
[0140] Although ApoE4 carriers and non-carriers can have different
responses to treatment as discussed above, and some treatment
regimes that are safe and effective in ApoE4 carriers are also safe
and effective, although not necessarily optimal, in non-ApoE4
carriers and can be used in both types of patients without regard
to ApoE status of the patients. In some such regimes, the agent is
an antibody that binds to an N-terminal epitope of A.beta. having
mutation(s) in its constant region that reduce binding to an
Fc.gamma. receptor and/or C1q. AAB-003 is an example of such an
antibody. In other regimes, the dose and/or frequency and/or the
maximal serum concentration and/or mean serum concentration of an
administered or induced antibody are constrained within limits as
described in PCT/US2007/009499 and further summarized below to
reduce the risk of vasogenic edema.
IV. Agents
A. Antibodies
[0141] A variety of antibodies to A.beta. have been described in
the patent and scientific literature for use in immunotherapy of
Alzheimer's disease, some of which are in clinical trials (see,
e.g., U.S. Pat. No. 6,750,324). Such antibodies can specifically
bind to an N-terminal epitope, a mid (i.e., central)-epitope or a
C-terminal epitope as defined above. Some antibodies are N-terminal
specific (i.e., such antibodies specifically bind to the N-terminus
of A.beta. without binding to APP). As noted above antibodies
binding to epitopes within residues 1-10, 1-3, 1-4, 1-5, 1-6, 1-7
or 3-7 of A.beta.42 or within residues 2-4, 5, 6, 7 or 8 of
A.beta., or within residues 3-5, 6, 7, 8 or 9 of A.beta., or within
residues 4-7, 8, 9 or 10 of A.beta.42 can be used. Some antibodies
are C-terminal specific (i.e., specifically bind to a C-terminus of
A.beta. without binding to APP) Antibodies can be polyclonal or
monoclonal. Polyclonal sera typically contain mixed populations of
antibodies specifically binding to several epitopes along the
length of APP. However, polyclonal sera can be specific to a
particular segment of A.beta. such as A.beta. 1-11) without
specifically binding to other segments of A.beta.. Preferred
antibodies are chimeric, humanized (including veneered antibodies)
(see Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989)
and WO 90/07861, U.S. Pat. No. 5,693,762, U.S. Pat. No. 5,693,761,
U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,530,101 and Winter, U.S.
Pat. No. 5,225,539), or human (Lonberg et al., WO 93/12227 (1993);
U.S. Pat. No. 5,877,397, U.S. Pat. No. 5,874,299, U.S. Pat. No.
5,814,318, U.S. Pat. No. 5,789,650, U.S. Pat. No. 5,770,429, U.S.
Pat. No. 5,661,016, U.S. Pat. No. 5,633,425, U.S. Pat. No.
5,625,126, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,545,806, Nature
148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996),
Kucherlapati, WO 91/10741 (1991)) EP1481008, Bleck, Bioprocessing
Journal 1 (September/October 2005), US 2004132066, US 2005008625,
WO 04/072266, WO 05/065348, WO 05/069970, and WO 06/055778.
[0142] 3D6 antibody, 10D5 and variants thereof are examples of
antibodies that can be used. Both are described in US 20030165496,
US 20040087777, WO 02/46237, and WO 04/080419, WO 02/088306 and WO
02/088307. 10D5 antibodies are also described in US 20050142131.
Additional 3D6 antibodies are described in US 20060198851 and
PCT/US05/45614. 3D6 is a monoclonal antibody (mAb) that
specifically binds to an N-terminal epitope located in the human
.beta.-amyloid peptide, specifically, residues 1-5. By comparison,
10D5 is a mAb that specifically binds to an N-terminal epitope
located in the human .beta.-amyloid peptide, specifically, residues
3-6. A cell line producing the 3D6 monoclonal antibody (RB96
3D6.32.2.4) was deposited with the American Type Culture Collection
(ATCC), Manassas, Va. 20108, USA on Apr. 8, 2003 under the terms of
the Budapest Treaty and assigned accession number PTA-5130. A cell
line producing the 10D5 monoclonal antibody (RB44 10D5.19.21) was
deposited with the ATCC on Apr. 8, 2003 under the terms of the
Budapest Treaty and assigned accession number PTA-5129.
[0143] Bapineuzumab (International Non-Proprietary Name designated
by the World Health Organization) means a humanized 3D6 antibody
comprising a light chain having a mature variable region having the
amino acid sequence designated SEQ ID NO: 2 and a heavy chain
having a mature variable region having the amino acid sequence
designated SEQ ID NO: 3. (The heavy and light chain constant
regions of the antibody designated bapineuzumab by WHO are human
IgG1 and human kappa respectively.) A humanized light chain
including variable and constant regions is designated SEQ ID NO: 48
below, and a humanized heavy chain including variable and constant
regions is designated SEQ ID NO: 66 or 67 (SEQ ID NO: 66 having an
additional C-terminal lysine relative to SEQ ID NO: 67).
[0144] Humanized 3D6 Light Chain Variable Region
TABLE-US-00002 (SEQ ID NO: 2) Asp Val Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser
Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln
Lys Pro Gly Gln Ser Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp
Gln Gly Thr His Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys
[0145] Humanized 3D6 Heavy Chain Variable Region
TABLE-US-00003 (SEQ ID NO: 3) Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Asn Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr
Ser Asp Asn Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser
[0146] A second version of humanized 3D6 antibody comprising a
light chain having a mature variable region having the amino acid
sequence designated SEQ ID NO: 4 and a heavy chain having a mature
variable region having the amino acid sequence designated SEQ ID
NO: 5 is shown below.
[0147] Humanized 3D6 Light Chain Variable Region
TABLE-US-00004 (SEQ ID NO: 4) Tyr Val Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser
Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln
Lys Pro Gly Gln Ser Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp
Gln Gly Thr His Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys
[0148] Humanized 3D6 Heavy Chain Variable Region
TABLE-US-00005 (SEQ ID NO: 5) Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Asn Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr
Ser Asp Asn Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr
Tyr Cys Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser
[0149] A third version of humanized 3D6 antibody comprising a light
chain having the amino acid sequence designated SEQ ID NO: 6 and a
heavy chain having the amino acid sequence designated SEQ ID NO: 7
is described in US 2005/0090648 A1 published on Apr. 28, 2005
issued as U.S. Pat. No. 7,318,923, which is incorporated herein by
reference for all purposes.
[0150] Humanized 3D6 Light Chain
TABLE-US-00006 (SEQ ID NO: 6) Asp Val Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser
Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln
Arg Pro Gly Gln Ser Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp
Gln Gly Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys
[0151] Humanized 3D6 Heavy Chain
TABLE-US-00007 (SEQ ID NO: 7) Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly
Phe Thr Phe Ser Asn Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr
Ser Asp Asn Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Gln Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys.
[0152] A version of humanized 10D5 antibody comprising a light
chain having a mature variable region having the amino acid
sequence designated SEQ ID NO: 8 and a heavy chain having a mature
variable region having the amino acid sequence designated SEQ ID
NO: 9 is shown below.
[0153] Humanized 10D5 Light Chain Variable Region
TABLE-US-00008 (SEQ ID NO: 8) Asp Val Leu Met Thr Gln Thr Pro Leu
Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser
Gln Asn Ile Ile His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile Lys Lys Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe
Gln Gly Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
Glu
[0154] Humanized 10D5 Heavy Chain Variable Region
TABLE-US-00009 (SEQ ID NO: 9) Gln Ala Thr Leu Lys Glu Ser Gly Pro
Gly Ile Leu Gln Ser Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly
Phe Ser Leu Ser Thr Ser Gly Met Gly Val Ser Trp Ile Arg Gln Pro Ser
Gly Lys Gly Leu Glu Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg
Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Arg
Lys Gln Val Phe Leu Lys Ile Thr Ser Val Asp Pro Ala Asp Thr Ala Thr
Tyr Tyr Cys Val Arg Arg Pro Ile Thr Pro Val Leu Val Asp Ala Met Asp
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
[0155] 12A11 or a chimeric or humanized or nanobody form thereof is
a preferred antibody. The 12A11 antibody or a variant thereof, is
described in US 20050118651, US 20060198851, WO 04/108895, and WO
06/066089, all of which are incorporated by reference in their
entirety herein for all purposes.
[0156] 12A11 is a mAb that specifically binds to an N-terminal
epitope located in the human .beta.-amyloid peptide, specifically,
residues 3-7. A cell line producing the 12A11 monoclonal antibody
was deposited at the ATCC (American Type Culture Collection, 10801
University Boulevard, Manassas, Va. 20110-2209) on Dec. 12, 2005
and assigned ATCC accession number PTA-7271.
[0157] A preferred version of the humanized 12A11 antibody is
version 1 comprising a light chain having the amino acid sequence
designated SEQ ID NO: 10 and a heavy chain having the amino acid
sequence designated SEQ ID NO: 11. Version 1 of humanized 12A11 is
described in US 20050118651 A1 published on Jun. 2, 2005, which is
incorporated herein by reference for all purposes.
[0158] Humanized 12A11 Light Chain
TABLE-US-00010 (SEQ ID NO: 10) Asp Val Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln
Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe
Gln Ser Ser His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys
[0159] Humanized 12A11 Heavy Chain Variable Region (version 1)
TABLE-US-00011 (SEQ ID NO: 11) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys
Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser
[0160] A second version of the humanized 12A11 antibody comprising
a light chain having the amino acid sequence designated SEQ ID NO:
10 and a heavy chain having the amino acid sequence designated SEQ
ID NO: 12 (version 2) is described in US 20050118651 A1 published
on Jun. 2, 2005, which is incorporated herein by reference for all
purposes.
[0161] Humanized 12A11 Heavy Chain Variable Region (version 2)
TABLE-US-00012 (SEQ ID NO: 12) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Thr Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser
[0162] A third version of the humanized 12A11 antibody comprising a
light chain having the amino acid sequence designated SEQ ID NO: 10
and a heavy chain having the amino acid sequence designated SEQ ID
NO: 13 (version 2.1) is described in US 20050118651 A1 published on
Jun. 2, 2005, which is incorporated herein by reference for all
purposes.
[0163] Humanized 12A11Heavy Chain Variable Region (version 2.1)
TABLE-US-00013 (SEQ ID NO: 13) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Thr Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser
[0164] A fourth version of the humanized 12A11 antibody comprising
a light chain having the amino acid sequence designated SEQ ID NO:
10 and a heavy chain having the amino acid sequence designated SEQ
ID NO: 14 (version 3) is described in WO 02/088306 published on
Jun. 2, 2005, which is incorporated herein by reference for all
purposes.
[0165] Humanized 12A11 Heavy Chain Variable Region (version 3)
TABLE-US-00014 (SEQ ID NO: 14) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Thr Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Tin Val Thr Val Ser Ser
[0166] A fifth version of the humanized 12A11 antibody comprising a
light chain having the amino acid sequence designated SEQ ID NO: 10
and a heavy chain having the amino acid sequence designated SEQ ID
NO: 15 (version 4.1) is described in US 20050118651 A1 published on
Jun. 2, 2005, which is incorporated herein by reference for all
purposes.
Humanized 12A11 Heavy Chain Variable Region (version 4.1)
TABLE-US-00015 (SEQ ID NO: 15) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Thr Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys
Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser
[0167] A sixth version of the humanized 12A11 antibody comprising a
light chain having the amino acid sequence designated SEQ ID NO: 10
and a heavy chain having the amino acid sequence designated SEQ ID
NO: 16 (version 4.2) is described in US 20050118651 A1 published on
Jun. 2, 2005, which is incorporated herein by reference for all
purposes.
[0168] Humanized 12A11 Heavy Chain Variable Region (version
4.2)
TABLE-US-00016 (SEQ ID NO: 16) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys
Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser
[0169] An seventh version of the humanized 12A11 antibody
comprising a light chain having the amino acid sequence designated
SEQ ID NO: 10 and a heavy chain having the amino acid sequence
designated SEQ ID NO: 17 (version 4.3) is described in US
20050118651 A1 published on Jun. 2, 2005, which is incorporated
herein by reference for all purposes.
[0170] Humanized 12A11 Heavy Chain Variable Region (version
4.3)
TABLE-US-00017 (SEQ ID NO: 17) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys
Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser
[0171] A eighth version of the humanized 12A11 antibody comprising
a light chain having the amino acid sequence designated SEQ ID NO:
10 and a heavy chain having the amino acid sequence designated SEQ
ID NO: 18 (version 4.4) is described in US 20050118651 A1 published
on Jun. 2, 2005, which is incorporated herein by reference for all
purposes.
[0172] Humanized 12A11 Heavy Chain Variable Region (version
4.4)
TABLE-US-00018 (SEQ ID NO: 18) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser
[0173] A ninth version of the humanized 12A11 antibody comprising a
light chain having the amino acid sequence designated SEQ ID NO: 10
and a heavy chain having the amino acid sequence designated SEQ ID
NO: 19 (version 5.1) is described in US 20050118651 A1 published on
Jun. 2, 2005, which is incorporated herein by reference for all
purposes.
[0174] Humanized 12A11 Heavy Chain Variable Region (version
5.1)
TABLE-US-00019 (SEQ ID NO: 19) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Thr Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys
Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser
[0175] A tenth version of the humanized 12A11 antibody comprising a
light chain having the amino acid sequence designated SEQ ID NO: 10
and a heavy chain having the amino acid sequence designated SEQ ID
NO: 20 (version 5.2) is described in US 20050118651 A1 published on
Jun. 2, 2005, which is incorporated herein by reference for all
purposes.
[0176] Humanized 12A11 Heavy Chain Variable Region (version
5.2)
TABLE-US-00020 (SEQ ID NO: 20) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Thr Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys
Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser
[0177] An eleventh version of the humanized 12A11 antibody
comprising a light chain having the amino acid sequence designated
SEQ ID NO: 10 and a heavy chain having the amino acid sequence
designated SEQ ID NO: 21 (version 5.3) is described in US
20050118651 A1 published on Jun. 2, 2005, which is incorporated
herein by reference for all purposes.
[0178] Humanized 12A11 Heavy Chain Variable Region (version
5.3)
TABLE-US-00021 (SEQ ID NO: 21) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Thr Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser Val
[0179] A twelfth version of the humanized 12A11 antibody comprising
a light chain having the amino acid sequence designated SEQ ID NO:
10 and a heavy chain having the amino acid sequence designated SEQ
ID NO: 22 (version 5.4) is described in US 20050118651 A1 published
on Jun. 2, 2005, which is incorporated herein by reference for all
purposes.
[0180] Humanized 12A11 Heavy Chain Variable Region (version
5.4)
TABLE-US-00022 (SEQ ID NO: 22) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys
Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser Val
[0181] A thirteenth version of the humanized 12A11 antibody
comprising a light chain having the amino acid sequence designated
SEQ ID NO: 10 and a heavy chain having the amino acid sequence
designated SEQ ID NO: 23 (version 5.5) is described in US
20050118651 A1 published on Jun. 2, 2005, which is incorporated
herein by reference for all purposes.
[0182] Humanized 12A11 Heavy Chain Variable Region (version
5.5)
TABLE-US-00023 (SEQ ID NO: 23) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser
[0183] A fourteenth version of the humanized 12A11 antibody
comprising a light chain having the amino acid sequence designated
SEQ ID NO: 10 and a heavy chain having the amino acid sequence
designated SEQ ID NO: 24 (version 5.6) is described in US
20050118651 A1 published on Jun. 2, 2005, which is incorporated
herein by reference for all purposes.
[0184] Humanized 12A11 Heavy Chain Variable Region (version
5.6)
TABLE-US-00024 (SEQ ID NO: 24) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser
[0185] A fifteenth version of the humanized 12A11 antibody
comprising a light chain having the amino acid sequence designated
SEQ ID NO: 10 and a heavy chain having the amino acid sequence
designated SEQ ID NO: 25 (version 6.1) is described in US
20050118651 A1 published on Jun. 2, 2005, which is incorporated
herein by reference for all purposes.
[0186] Humanized 12A11 Heavy Chain Variable Region (version
6.1)
TABLE-US-00025 (SEQ ID NO: 25) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Thr Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys
Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser
[0187] A sixteenth version of the humanized 12A11 antibody
comprising a light chain having the amino acid sequence designated
SEQ ID NO: 10 and a heavy chain having the amino acid sequence
designated SEQ ID NO: 26 (version 6.2) is described in US
20050118651 A1 published on Jun. 2, 2005, which is incorporated
herein by reference for all purposes.
[0188] Humanized 12A11 Heavy Chain Variable Region (version
6.2)
TABLE-US-00026 (SEQ ID NO: 26) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Thr Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser
[0189] A seventeenth version of the humanized 12A11 antibody
comprising a light chain having the amino acid sequence designated
SEQ ID NO: 10 and a heavy chain having the amino acid sequence
designated SEQ ID NO: 27 (version 6.3) is described in US
20050118651 A1 published on Jun. 2, 2005, which is incorporated
herein by reference for all purposes.
[0190] Humanized 12A11 Heavy Chain Variable Region (version
6.3)
TABLE-US-00027 (SEQ ID NO: 27) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Thr Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser
[0191] A eighteenth version of the humanized 12A11 antibody
comprising a light chain having the amino acid sequence designated
SEQ ID NO: 10 and a heavy chain having the amino acid sequence
designated SEQ ID NO: 28 (version 6.4) is described in US
20050118651 A1 published on Jun. 2, 2005, which is incorporated
herein by reference for all purposes.
[0192] Humanized 12A11 Heavy Chain Variable Region (version
6.4)
TABLE-US-00028 (SEQ ID NO: 28) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser
[0193] A nineteenth version of the humanized 12A11 antibody
comprising a light chain having the amino acid sequence designated
SEQ ID NO: 10 and a heavy chain having the amino acid sequence
designated SEQ ID NO: 29 (version 7) is described in US 20050118651
A1 published on Jun. 2, 2005, which is incorporated herein by
reference for all purposes.
[0194] Humanized 12A11 Heavy Chain Variable Region (version 7)
TABLE-US-00029 (SEQ ID NO: 29) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Thr Leu Ser Thr Ser Gly Met Ser Val Gly Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys
Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser
[0195] A twentieth version of the humanized 12A11 antibody
comprising a light chain having the amino acid sequence designated
SEQ ID NO: 10 and a heavy chain having the amino acid sequence
designated SEQ ID NO: 30 (version 8) is described in US 20050118651
A1 published on Jun. 2, 2005, which is incorporated herein by
reference for all purposes.
[0196] Humanized 12A11 Heavy Chain Variable Region (version 8)
TABLE-US-00030 (SEQ ID NO: 30) Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly
Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys
Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser
[0197] Other exemplary antibodies include 12B4 antibody or variant
thereof, as described in US 20040082762A1 and WO 03/077858. 12B4 is
a mAb that specifically binds to an N-terminal epitope located in
the human .beta.-amyloid peptide, specifically, residues 3-7. The
light (SEQ ID NO: 31) and heavy chain (SEQ ID NO: 32) of 12B4 have
the following variable regions (not including signal
sequences).
TABLE-US-00031 (Seq ID NO: 31) Asp Val Leu Met Thr Gln Thr Pro Leu
Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser
Gln Asn Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe
Gln Gly Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
Lys (SEQ ID NO: 32) Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu
Gln Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu
Ser Thr Asn Gly Met Gly Val Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly
Leu Glu Trp Leu Ala His Ile Tyr Trp Asp Glu Asp Lys Arg Tyr Asn Pro
Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val
Phe Leu Lys Ile Thr Asn Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr Cys
Ala Arg Arg Arg Ile Ile Tyr Asp Val Glu Asp Tyr Phe Asp Tyr Trp Gly
Gln Gly Thr Thr Leu Thr Val Ser Ser
[0198] Other exemplary antibodies are 6C6 antibody, or a variant
thereof, as described in a US 20060165682 and WO 06/06604. 6C6 is a
mAb that specifically binds to an N-terminal epitope located in the
human .beta.-amyloid peptide, specifically, residues 3-7. A cell
line producing the antibody 6C6 was deposited on Nov. 1, 2005, with
the ATCC under the terms of the Budapest Treaty and assigned
accession number PTA-7200.
[0199] Other exemplary antibodies are 2H3 antibody and variants
thereof as described in US 20060257396. 2H3 is a mAb that
specifically binds to an N-terminal epitope located in the human
.beta.-amyloid peptide, specifically, residues 2-7. A cell line
producing the antibody 2H3 was deposited on Dec. 13, 2005, with the
ATCC under the terms of the Budapest Treaty and assigned accession
number PTA-7267.
[0200] Other exemplary antibodies include 3A3 and variants thereof
as described in US 20060257396. 3A3 is a mAb that specifically
binds to an N-terminal epitope located in the human .beta.-amyloid
peptide, specifically, residues 3-7. A cell line producing the
antibody 3A3 was deposited on Dec. 13, 2005, with the ATCC under
the terms of the Budapest Treaty and assigned accession number
PTA-7269.
[0201] Other exemplary antibodies are 2B1, 1C2 or 9G8. Cell lines
producing the antibodies 2B1, 1C2 and 9G8 were deposited on Nov. 1,
2005, with the ATCC under the terms of the Budapest Treaty and were
assigned accession numbers PTA-7202, PTA-7199 and PTA-7201,
respectively.
[0202] Another exemplary antibody is a humanized 266 antibody or
variant thereof. The 266 antibody binds to an epitope between
residues 13-28 of A.beta.. A cell line producing the antibody 266
antibody was deposited on Jul. 20, 2004 with the ATCC under the
terms of the Budapest Treaty and assigned accession numer PTA-6123.
Humanized forms of the 266 antibody are described in US
20040265308, US 20040241164, WO 03/016467, and U.S. Pat. No.
7,195,761. The light (SEQ ID NO: 33) and heavy chain (SEQ ID NO:
34) of the 266 antibody have the following variable region
sequences (not including signal sequences).
TABLE-US-00032 (SEQ ID NO: 33) Asp Xaa Val Met Thr Gln Xaa Pro Leu
Ser Leu Pro Val Xaa Xaa Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu Xaa Tyr Ser Asp Gly Asn Ala Tyr Leu His Trp Phe Leu Gln
Lys Pro Gly Gln Ser Pro Xaa Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Xaa Gly Val Tyr Tyr Cys Ser
Gln Ser Thr His Val Pro Trp Thr Phe Gly Xaa Gly Thr Xaa Xaa Gln Ile
Lys Arg
wherein: Xaa at position 2 is Val or Ile; Xaa at position 7 is Ser
or Thr; Xaa at position 14 is Thr or Ser; Xaa at position 15 is Leu
or Pro; Xaa at position 30 is Ile or Val; Xaa at position 50 is
Arg, Gln, or Lys; Xaa at position 88 is Val or Leu; Xaa at position
105 is Gln or Gly; Xaa at position 108 is Lys or Arg; and Xaa at
position 109 is Val or Leu; and
TABLE-US-00033 (SEQ ID NO: 34) Xaa Val Gln Leu Val Glu Xaa Gly Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Arg Tyr Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Xaa Leu Val Ala Gln Ile Asn Ser Val Gly Asn Ser Thr Tyr Tyr
Pro Asp Xaa Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Xaa Xaa Asn
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Xaa Asp Thr Ala Val Tyr
Tyr Cys Ala Ser Gly Asp Tyr Trp Gly Gln Gly Thr Xaa Val Thr Val Ser
Ser
wherein: Xaa at position 1 is Glu or Gln; Xaa at position 7 is Ser
or Leu; Xaa at position 46 is Glu, Val, Asp, or Ser; Xaa at
position 63 is Thr or Ser; Xaa at position 75 is Ala, Ser, Val or
Thr; Xaa at position 76 is Lys or Arg; Xaa at position 89 is Glu or
Asp; and Xaa at position 107 is Leu or Thr.
[0203] An exemplary humanized 266 antibody comprises the following
light chain (SEQ ID NO: 35) and heavy chain (SEQ ID NO: 36)
sequences (not including signal sequences).
TABLE-US-00034 (SEQ ID NO: 35) Asp Val Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu Ile Tyr Ser Asp Gly Asn Ala Tyr Leu His Trp Phe Leu Gln
Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser
Gln Ser Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys (SEQ ID NO: 36) Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Arg Tyr Ser Met Ser Trp Val Ary Gln Ala Pro
Gly Lys Gly Leu Glu Leu Val Ala Gln Ile Asn Ser Val Gly Asn Ser Thr
Tyr Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys Ala Ser Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Va Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Ary Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
[0204] The antibody can also be 15C11 or a humanized form thereof
(see US 20060165682), which specifically binds to an epitope within
A.beta.15-24.
[0205] The antibody can also be a humanized form of 20C2 or a
variant thereof. Such antibodies are described, e.g., in US
2007081998. The core linear epitope for 20C2 corresponds to amino
acid residues 3-8 of A.beta.1-42, with a conformational epitope
that is dependent upon elements from within residues 17-42 of
A.beta.. The light (SEQ ID NO: 37) and heavy chain (SEQ ID NO: 38)
of humanized 20C2 antibody (version 1) have the following variable
region sequences (not including signal sequences).
TABLE-US-00035 (SEQ ID NO: 37) Asp Val Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Ile Leu His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln
Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe
Gln Gly Ser Leu Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys (SEQ ID NO: 38) Gln Val Thr Leu Lys Glu Ser Gly Pro Ala Leu Val
Lys Pro Thr Gln Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu
Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala
Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Ser Tyr Asn Pro
Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val
Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys
Ala Arg Arg Gln Leu Gly Leu Arg Ser Ile Asp Ala Met Asp Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser
[0206] An additional humanized 20C2 antibody (version 2) comprises
the light chain variable region sequence of SEQ ID NO: 37 and the
heavy chain variable region sequence of SEQ ID NO: 39 (not
including signal sequence).
TABLE-US-00036 (SEQ ID NO: 39) Gln Val Thr Leu Lys Glu Ser Gly Pro
Ala Leu Val Lys Pro Thr Gln Thr Leu Thr Leu Thr Cys Thr Leu Ser Gly
Phe Ser Leu Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro
Gly Lys Ala Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Ser
Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys
Asn Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr
Tyr Tyr Cys Ala Arg Arg Gln Leu Gly Leu Arg Ser Ile Asp Ala Met Asp
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser
[0207] Another antibody that can be used according to the invention
is C705 or a variant thereof, which binds an epitope comprising
amino acids 7-12 of the A.beta. peptide, as described in WO
05/028511. The C705 antibody comprises the light chain variable
region sequence of SEQ ID NO: 40 and heavy chain variable region of
SEQ ID NO: 41, signal sequence underlined.
TABLE-US-00037 (SEQ ID NO: 40) Met Lys Leu Pro Val Arg Leu Leu Val
Leu Met Phe Trp Ile Pro Gly Ser Ser Ser Asp Val Met Met Thr Gln Thr
Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr
Met Gln Lys Pro Gly Gln Ser Pro Met Leu Leu Ile Tyr Lys Val Ser Asn
Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Lys Ile Ser Ser Val Glu Ala Glu Asp Leu Gly Val Phe Tyr
Cys Phe Gln Gly Ser Arg Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
Glu Leu Lys Arg (SEQ ID NO: 41) Met Asp Arg Leu Thr Ser Ser Phe Leu
Leu Leu Ile Val Pro Ala Tyr Val Leu Ser Gln Val Thr Leu Lys Glu Ser
Gly Pro Gly Ile Leu Gln Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe
Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Gly Val Ser Trp Ile Arg Gln
Pro Ser Gly Lys Gly Leu Glu Trp Leu Ala His Ile Tyr Trp Asp Asp Asp
Lys Arg Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr
Ser Arg Asn Gln Val Phe Leu Lys Ile Thr Ser Val Asp Thr Thr Asp Thr
Ala Thr Tyr Tyr Cys Thr Arg Ser Ser Gly Ser Ile Val Ile Ala Thr Gly
Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
[0208] Another antibody that can be used according to the invention
is C706 or a variant thereof, which binds to an epitope comprising
amino acids 6-11 of the A.beta. peptide, as described in WO
05/028511. The C706 antibody comprises the light chain variable
region sequence of SEQ ID NO: 42, and the heavy chain variable
region sequence of SEQ ID NO: 43. Signal sequences are
underlined.
TABLE-US-00038 (SEQ ID NO: 42) Met Asp Phe Gln Val Gln Ile Phe Ser
Phe Leu Leu Ile Ser Ala Ser Val Ile Ile Ser Arg Gly Gln Ile Val Leu
Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met
Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys
Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Ser Ser Arg Leu Ala Ser
Gly Val Pro Ser Arg Phe Ser Gly Gly Gly Ser Gly Thr Ser Tyr Ser Pro
Thr Ile Ser Asn Met Glu Ala Glu Asp Ala Ala Thr Tyr Phe Cys Gln Asn
Trp Arg Ser Ser Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
(SEQ ID NO: 43) Met Glu Trp Thr Trp Val Phe Leu Phe Leu Leu Ser Val
Thr Ala Gly Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Met Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr
Phe Ser Thr Ser Trp Ile Glu Trp Ile Lys Gln Arg Pro Gly His Gly Leu
Glu Trp Ile Gly Glu Val Leu Pro Gly Ser Gly Lys Ser Asn His Asn Ala
Asn Phe Lys Gly Arg Ala Thr Phe Thr Ala Asp Thr Ala Ser Asn Thr Ala
Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
Ala Arg Glu Gly Ser Asn Asn Asn Ala Leu Ala Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ala
[0209] Other antibodies that can be used according to the invention
include humanized 2286 antibodies and variants thereof. These
antibodies recognize an epitope comprising amino acids 28-40 of the
A.beta. peptide, as described in US 20070160616. A humanized 2286
antibody (version 1) comprises the light chain variable region of
SEQ ID NO: 44 and the heavy chain variable region of SEQ ID NO: 45
(not including signal sequences).
TABLE-US-00039 (SEQ ID NO: 44)
DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAPKLLIYY
TSSLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYRKLPYTFGG GTKVEIKR (SEQ ID
NO: 45) EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMNWVRQAPGKGLEWVSE
INPDSSTINYTPSLKDRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARQM
GYWGQGTTLTVSS
[0210] Another version of humanized 2286 comprises the light chain
variable region of SEQ ID NO: 44 and the heavy chain variable
region of SEQ ID NO: 46 (not including signal sequences).
TABLE-US-00040 (SEQ ID NO: 46)
QVQLQESGPGLVKPSETLSLTCTVSGFDFSRYWMNWIRQPPGKGLEWIGE
INPDSSTINYTPSLKDRVTISKDTSKNQFSLKLSSVTAADTAVYYCARQM
GYWGQGTLVTVSS
[0211] Additional antibodies that can be used according to the
invention are a fourth version of humanized 3D6 and a second
version of humanized 10D5, as disclosed in U.S. Pat. Nos. 7,318,923
and 7,320,790, respectively. These antibodies bind to the
N-terminus of the A.beta. peptide, as explained above. The
humanized 3D6 (version 4) comprises the light chain variable region
sequence of SEQ ID NO: 71 and the heavy chain variable region
sequence of SEQ ID NO: 72.
TABLE-US-00041 (SEQ ID NO: 71) Asp Val Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Leu GlyGln Pro Ala Ser Ile Ser Cys Lys Ser Ser
Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln
Arg Pro Gly Gln Ser Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp
Gln Gly Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Arg (SEQ ID NO: 72) Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr
Phe Ser Asn Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser Asp
Asn Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser
[0212] The humanized 10D5 antibody (version 2) comprises the light
chain variable region sequence of SEQ ID NO: 73 and the heavy chain
variable region sequence of SEQ ID NO: 74.
TABLE-US-00042 (SEQ ID NO: 73) Asp Val Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Asn Ile Ile His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln
Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe
Gln Gly Ser His Val Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Arg (SEQ ID NO: 74) Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu
Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser
Leu Ser Thr Ser Gly Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys
Ala Leu Glu Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn
Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln
Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr
Cys Val Arg Arg Pro Ile Thr Pro Val Leu Val Asp Ala Met Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser
[0213] Another exemplary antibody is humanized 2E7, as disclosed in
WO 07/113,172. The 2E7 antibody binds residues 1-12 of A.beta.
peptide, but not 2-13, or longer variants of the peptide. Humanized
2E7 antibody (version 1) comprises light chain variable region
sequence of SEQ ID NO: 75 and heavy chain variable region sequence
of SEQ ID NO: 76.
TABLE-US-00043 (SEQ ID NO: 75)
DIVMTQSPLSLPVTPGEPASISCRVSQSLLHSNGYTYLHWYLQKPGQSPQ
LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQTRHVP YTFGGGTKVEIK
(SEQ ID NO: 76) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDNGMAWVRQAPGKGLEWVSF
ISNLAYSIDYADTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVSGT
WFAYWGQGTLVTVSS
[0214] A second version of humanized 2E7 antibody comprises the
light chain variable region of SEQ ID NO: 75 and the heavy chain
variable region sequence of SEQ ID NO: 77 (see, e.g., WO
07/113,172).
TABLE-US-00044 (SEQ ID NO: 77)
EVQLVESGGGLVQPGGSLRLSCAVSGFTFSDNGMAWVRQAPGKGLEWVSF
ISNLAYSIDYADTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVSGT
WFAYWGQGTLVTVSS
[0215] Humanized 2E7 antibody (version 3) comprises the light chain
variable region sequence of SEQ ID NO: 75 and the heavy chain
variable region sequence of SEQ ID NO: 78 (see, e.g., WO
07/113,172).
TABLE-US-00045 (SEQ ID NO: 78)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDNGMAWVRQAPGKGLEWISF
ISNLAYSIDYADTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVSGT
WFAYWGQGTLVTVSS
[0216] An additional antibody that can be used according to the
invention includes humanized 9TL antibody (ATCC accession numbers
PTA-6124 and PTA-6125), as described in WO 06/036291. The heavy and
light chain variable regions, without signal sequences, are shown
as SEQ ID NO: 79 and SEQ ID NO: 80, respectively.
TABLE-US-00046 (SEQ ID NO: 79)
QVQLVQSGAEVKKPGASVKVSCKASGYYTEAYYIHWVRQAPGQGLEWMGR
IDPATGNTKYAPRLQDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASLY SLPVYWGQGTTVTVSS
(SEQ ID NO: 80) DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSDAKTYLNWFQQRPGQSPR
RLIYQISRLDPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGTHYP
VLFGQGTRLEIKRT
[0217] Humanized versions of the 6G antibody can also be used
according to the invention. The heavy and light chain variable
regions, without signal sequences, are shown as SEQ ID NOs:81 and
82, respectively.
TABLE-US-00047 (SEQ ID NO: 81)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYAIHWVRQAPGQGLEWMGF
TSPYSGVSNYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARFD
NYDRGYVRDYWGQGTLV (SEQ ID NO: 82)
DIVMTQSPDSLAVSLGERATINCRASESVDNDRISFLNWYQQKPGQPPKL
LIYAATKQGTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEFPW
SFGGGTKVEIKRTV
[0218] Additional antibodies that can be used according to the
invention are humanized versions of the 2.1 antibody, as described
in WO 06/081171. These antibodies rely on the CDRs of the murine
2.1 antibody and substitute residues from the human VKII A19/JK4
light chain variable framework region. The heavy chain variable
framework region used for substitution is roughly based on VH 2-70.
An exemplary humanized 2.1 antibody comprises the heavy and light
chain variable regions, without signal sequences, shown as SEQ ID
NOs: 83 and 84, respectively.
TABLE-US-00048 (SEQ ID NO: 83)
QVTLKESGPALVKPTQTLTLTCTFSGFSLRTSGMGVGWIRQPPGKALEWL
AHIWWDDDKSYNPSLKSQLTISKDTSKNQVVLTMTNMDPVDTATYYCARR
NYYYDDYFAYWGQGTLVTVSS (SEQ ID NO: 84)
DVLMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYLQRPGQSPK
LLIYKVSNRFSGVPDRESGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVP LTFGAGTKLEIK
[0219] Other antibodies that can be used according to the invention
include CW1181 and CW1185 antibodies. These antibodies specifically
bind to two regions of the A.beta. peptide, as described in WO
03/070760 and US 20050196399. The first region comprises AEFRHDSGY
(SEQ ID NO: 85) or a fragment thereof (e.g., AEFRHD (SEQ ID NO:
86), or EFRHDSG (SEQ ID NO: 87), EFRHD (SEQ ID NO: 88)) and second
region comprises the amino acid sequence YEVHHQKLVFFAEDVG (SEQ ID
NO: 89) or a fragment thereof (e.g., VFFA (SEQ ID NO: 90), or
QKLFFAEDV (SEQ ID NO: 91)).
[0220] An additional antibody that can be used according to the
invention is the monoclonal NAB61 antibody. NAB61 binds
A.beta.1-11, but does not bind to full length APP or C99, as
disclosed in WO 07/062,088. Similarly, the monoclonal 82E1 antibody
can be used according to the invention. 82E1 binds the N-terminus
of the A.beta. peptide, but not full length APP, as disclosed in US
20080025988.
[0221] Other antibodies of the invention are anti-ADDL antibodies.
Such antibodies have been generated and selected for the ability to
bind ADDLs specifically, without binding to A.beta. monomer or
amyloid fibrils. See e.g., WO 04/031400.
[0222] Other antibodies that can be used include (i) the catalytic
antibody ABP 102 (Abzyme, from Abiogen Pharma); (ii) ACI-01 Ab7 C2
(AC Immune Genentech); (iii) AZD-3102 (AstraZeneca/Dyax); (iv) IVIg
(Gammagard S/D Immune Globulin Intravenous (Human), from Baxter
Bioscience); (v) BAN 2401 (BioArctic Neuroscience AB/Eisai Co.
Ltd.; (vi) R1450 (Hoffman-La Roche/MorphoSys); (vii) LY2062430 (Eli
Lilly); (viii) h3D6 (Eli Lilly); (ix) ACU-5A5 (.alpha. ADDL mAb
from Merck/Acumen); .alpha.-amyloidspheroid (ASPD) antibody
(Mitsubishi Pharma Corp.); (xi) the antibody derived from PBMCs of
an AN1792 patient (Neurimmune Therapeutics AG); (xii) BC05
(Takeda); (xiii) the CEN701-CEN706 antibodies (Centocor/Johnson
& Johnson); and (xiv) PF-04360365 (also called RN-1219 (h2286),
from Pfizer/Rinat Neurosciences). Each of these antibodies can be
used according to any of the methods of the invention.
[0223] The ABP 102 antibody cleaves aggregated A.beta. as
described, e.g., in U.S. Pat. No. 6,387,674 and WO 99/06536. The
ACI-01 Ab7 C2 antibody binds the A.beta. peptide between residues
10-20 and is described in US 20070166311. The IVIg Gammagard SD
Immune Globulin antibody is described, e.g., on the Baxter
Bioscience website at Baxter.com. The BAN 2401 antibody is a
humanized antibody that binds A.beta. protofibrils, and is
described, e.g., in WO 05/123775. The human R-1450 HuCAL antibody
has a dual 266/3D6 epitope. The humanized LY2062430 antibody (IgG)
binds the A.beta. peptide between residues 16-23, and is described,
e.g., in U.S. Pat. No. 7,195,761. The humanized h3D6 antibody binds
the A.beta. peptide at residues 1-5, and is described, e.g., in
U.S. Pat. No. 7,318,923. The BC05 antibody binds a C terminal
A.beta. epitope, as described by Asami-Odaka et al. (2005)
Neurodegenerative Diseases 2:36-43. The CEN701-CEN706 antibodies
are described, e.g., in WO 05/028511. The humanized PF-04360365
antibody binds the A.beta. peptide between residues 28-40 and is
described, e.g., in WO 04/032868.
[0224] Any of the antibodies or antibody fragments described herein
can be designed or prepared using standard methods, as disclosed,
e.g., in US 20040038304, US 20070020685, US 200601660184, US
20060134098, US 20050255552, US 20050130266, US 2004025363, US
20040038317, US 20030157579, and U.S. Pat. No. 7,335,478.
[0225] Any of the antibodies described above can be produced with
different isotypes or mutant isotypes to control the extent of
binding to different Fc.gamma. receptors. Antibodies lacking an Fc
region (e.g., Fab fragments) lack binding to Fc.gamma. receptors.
Selection of isotype also affects binding to Fc.gamma. receptors.
The respective affinities of various human IgG isotypes for the
three Fc.gamma. receptors, Fc.gamma.RI, Fc.gamma.RII, and
Fc.gamma.RIII, have been determined. (See Ravetch & Kinet,
Annu. Rev. Immunol. 9, 457 (1991)). Fc.gamma.RI is a high affinity
receptor that binds to IgGs in monomeric form, and the latter two
are low affinity receptors that bind IgGs only in multimeric form.
In general, both IgG1 and IgG3 have significant binding activity to
all three receptors, IgG4 to Fc.gamma.RI, and IgG2 to only one type
of Fc.gamma.RII called IIa.sub.LR (see Parren et al., J. Immunol.
148, 695 (1992). Therefore, human isotype IgG1 is usually selected
for stronger binding to Fc.gamma. receptors is desired, and IgG2 is
usually selected for weaker binding.
[0226] Mutations on, adjacent, or close to sites in the hinge link
region (e.g., replacing residues 234, 235, 236 and/or 237 with
another residue) in all of the isotypes reduce affinity for
Fc.gamma. receptors, particularly Fc.gamma.RI receptor (see, e.g.,
U.S. Pat. No. 6,624,821). Optionally, positions 234, 236 and/or 237
are substituted with alanine and position 235 with glutamine. (See,
e.g., U.S. Pat. No. 5,624,821.) Position 236 is missing in the
human IgG2 isotype. Exemplary segments of amino acids for positions
234, 235 and 237 for human IgG2 are Ala Alai Gly, Val Ala Ala, Ala
Ala Ala, Val Glu Ala, and Ala Glu Ala. A preferred combination of
mutants is L234A, L235A, and G237A for human isotype IgG1. A
particular preferred antibody is bapineuzumab having human isotype
IgG and these three mutations of the Fc region. Other substitutions
that decrease binding to Fc.gamma. receptors are an E233P mutation
(particularly in mouse IgG1) and D265A (particularly in mouse
IgG2a). Other examples of mutations and combinations of mutations
reducing Fc and/or C1q binding are described in the Examples
(E318A/K320A/R322A (particularly in mouse IgG1),
L235A/E318A/K320A/K322A (particularly in mouse IgG2a). Similarly,
residue 241 (Ser) in human IgG4 can be replaced, e.g., with proline
to disrupt Fc binding.
[0227] Additional mutations can be made to the constant region to
modulate effector activity. For example, mutations can be made to
the IgG2a constant region at A330S, P331S, or both. For IgG4,
mutations can be made at E233P, F234V and L235A, with G236 deleted,
or any combination thereof. IgG4 can also have one or both of the
following mutations S228P and L235E. The use of disrupted constant
region sequences to modulate effector function is further
described, e.g., in WO 06/118,959 and WO 06/036291.
[0228] Additional mutations can be made to the constant region of
human IgG to modulate effector activity (see, e.g., WO 06/03291).
These include the following substitutions: (i) A327G, A330S, P331S;
(ii) E233P, L234V, L235A, G236 deleted; (iii) E233P, L234V, L235A;
(iv) E233P, L234V, L235A, G236 deleted, A327G, A330S, P331S; and
(v) E233P, L234V, L235A, A327G, A330S, P331S to human IgG1.
[0229] The affinity of an antibody for the FcR can be altered by
mutating certain residues of the heavy chain constant region. For
example, disruption of the glycosylation site of human IgG1 can
reduce FcR binding, and thus effector function, of the antibody
(see, e.g., WO 06/036291). The tripeptide sequences NXS, NXT, and
NXC, where X is any amino acid other than proline, are the
enzymatic recognition sites for glycosylation of the N residue.
Disruption of any of the tripeptide amino acids, particularly in
the CH2 region of IgG, will prevent glycosylation at that site. For
example, mutation of N297 of human IgG1 prevents glycosylation and
reduces FcR binding to the antibody.
[0230] The sequences of several exemplary humanized 3D6 antibodies
and their components parts are shown below. Human constant regions
show allotypic variation and isoallallotypic variation between
different individuals, that is, the constant regions can differ in
different individuals at one or more polymorphic positions.
Isoallotypes differ from allotypes in that sera recognizing an
isoallotype binds to a non-polymorphic region of a one or more
other isotypes. The allotype of the IgG1 constant region shown
below is 3D6 (AAB-001) is G1mz which has Glu at position 356 and
Met at position 358. The allotype of the kappa constant region
shown below is Km3, which has an Ala at position 153 and a Val at
position 191. A different allotye Km(1) has Val and Leu at
positions 153 and 191 respectively. Allotypic variants are reviewed
by J Immunogen 3: 357-362 (1976) and Loghem,. Monogr Allergy 19:
40-51 (1986). Other allotypic and isoallotypic variants of the
illustrated constant regions are included. Also included are
constant regions having any permutation of residues occupying
polymorphic positions in natural allotypes. Examples of other heavy
chain IgG1 allotypes include: G1m(f), G1m(a) and G1m(x). G1m(f)
differs from G1m(z) in that it has an Arg instead of a Lys at
position 214. G1m(a) has amino acids Arg, Asp, Glu, Leu at
positions 355-358.
[0231] Humanized 3D6 Full Length Light Chain (signal sequence
underlined) (bapineuzumab and AAB-003)
TABLE-US-00049 (SEQ ID NO: 47)
MDMRVPAQLLGLLMLWVSGSSGDVVMTQSPLSLPVTPGEPASISCKSSQS
LLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTL
KISRVEAEDVGVYYCWQGTHFPRTFGQGTKVEIKRTVAAPSVFIFPPSDE
QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
[0232] Humanized 3D6 Full Length Light Chain, Not Including Signal
Sequence (bapineuzumab and AAB-003)
TABLE-US-00050 (SEQ ID NO: 48)
DVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQ
RLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFP
RTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC
[0233] DNA encoding humanized 3D6 Light Chain Coding Sequence
(signal sequence underlined) (bapineuzumab and AAB-003)
TABLE-US-00051 (SEQ ID NO: 49)
ATGGACATGCGCGTGCCCGCCCAGCTGCTGGGCCTGCTGATGCTGTGGGT
GTCCGGCTCCTCCGGCGACGTGGTGATGACCCAGTCCCCCCTGTCCCTGC
CCGTGACCCCCGGCGAGCCCGCCTCCATCTCCTGCAAGTCCTCCCAGTCC
CTGCTGGACTCCGACGGCAAGACCTACCTGAACTGGCTGCTGCAGAAGCC
CGGCCAGTCCCCCCAGCGCCTGATCTACCTGGTGTCCAAGCTGGACTCCG
GCGTGCCCGACCGCTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTG
AAGATCTCCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCTGGCA
GGGCACCCACTTCCCCCGCACCTTCGGCCAGGGCACCAAGGTGGAGATCA
AGCGTACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAG
CAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTA
TCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGG
GTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTAC
AGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAA
AGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAA
AGAGCTTCAACAGGGGAGAGTGTTAG
[0234] Human Heavy Chain Constant Region, IgG1 Isotype,
L234A/G237A
TABLE-US-00052 (SEQ ID NO: 50)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0235] The C-terminal K residue can be absent, as indicated
below.
TABLE-US-00053 (SEQ ID NO: 51)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG.
[0236] Humanized 3D6 Full Length Heavy Chain (IgG1 Isotype,
L234A/G237A) including signal sequence (underlined)
TABLE-US-00054 (SEQ ID NO: 52)
MEFGLSWLFLVAILKGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSN
YGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFTISRDNSKNTLYL
QMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC
PAPEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPGK
[0237] The C-terminal K residue can be absent, as indicated
below.
TABLE-US-00055 (SEQ ID NO: 53)
MEFGLSWLFLVAWKGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSNY
GMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFTISRDNSKNTLYLQ
MNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP
APEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPG.
[0238] Humanized 3D6 Full Length Heavy Chain Not Including Signal
Sequence (IgG1 Isotype, L234A/G237A)
TABLE-US-00056 (SEQ ID NO: 54)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVAS
IRSGGGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYD
HYSGSSDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEALGAPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0239] The C-terminal K residue can be absent, as indicated
below.
TABLE-US-00057 (SEQ ID NO: 55)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVAS
IRSGGGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYD
HYSGSSDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEALGAPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.
[0240] Human Heavy Chain Constant Region, IgG4 Isotype, S241P
(Kabat numbering); S228P (EU numbering)
TABLE-US-00058 (SEQ ID NO: 56)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYEPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES
KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED
PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG
NVFSCSVMHEALHNHYTQKSLSLSLGK
[0241] The C-terminal K residue can be absent, as indicated
below.
TABLE-US-00059 (SEQ ID NO: 57)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES
KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED
PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG
NVFSCSVMHEALHNHYTQKSLSLSLG
[0242] Humanized 3D6 Full Length Heavy Chain (IgG4 Isotype, S241P),
Including Signal Sequence (underlined)
TABLE-US-00060 (SEQ ID NO: 58)
MEFGLSWLFLVAILKGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSN
YGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFTISRDNSKNTLYL
QMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSSASTKGPSVFPLA
PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP
EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL
HNHYTQKSLSLSLGK
[0243] The C-terminal K residue can be absent, as indicated
below.
TABLE-US-00061 (SEQ ID NO: 59)
MEFGLSWLFLVAILKGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSN
YGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRETISRDNSKNTLYL
QMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSSASTKGPSVFPLA
PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP
EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL
HNHYTQKSLSLSLG.
[0244] Humanized 3D6 Heavy Chain, Not Including Signal Sequence
(IgG4 Isotype, S241P)
TABLE-US-00062 (SEQ ID NO: 60)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVAS
IRSGGGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYD
HYSGSSDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY
TCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKGLPSSLEKTISKAKGQPREPQVYTL
PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
[0245] The C-terminal K residue can be absent, as indicated
below.
TABLE-US-00063 (SEQ ID NO: 61)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVAS
IRSGGGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYD
HYSGSSDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY
TCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL
PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.
[0246] Human Heavy Chain Constant Region, IgG1 Isotype (AAB-003),
L234A/L235A/G237A
TABLE-US-00064 (SEQ ID NO: 62)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTRNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0247] The C-terminal K residue can be absent, as indicated
below.
TABLE-US-00065 (SEQ ID NO: 63)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPWKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMLIEALHNHYTQKSLSLSPG.
[0248] Humanized 3D6 Full Length Heavy Chain Including Signal
Sequence (IgG1 isotype, L234A/L235A/G237A): AAB-003
TABLE-US-00066 (SEQ ID NO: 64)
MEFGLSWLFLVAILKGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSN
YGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRiFTISRDNSKNTLY
LQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSSASTKGPSVFPL
APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP
CPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK
[0249] The C-terminal K residue can be absent, as indicated
below.
TABLE-US-00067 (SEQ ID NO: 65)
MEFGLSWLFLVAILKGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSN
YGMSWVRQAPGKGLEWVASTRSGGGRTYYSDNVKGRFTISRDNSKNTLYL
QMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC
PAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPG.
[0250] Humanized 3D6 Heavy Chain, Not Including Signal Sequence
(IgG1 isotype, L234A/L235A/G237A): AAB-003
TABLE-US-00068 (SEQ ID NO: 66)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVAS
IRSGGGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYD
HYSGSSDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0251] The C-terminal K residue can be absent, as indicated
below.
TABLE-US-00069 (SEQ ID NO: 67)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVAS
IRSGGGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYD
HYSGSSDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.
[0252] DNA encoding humanized 3D6 Heavy Chain Coding Region
including Signal Sequence (underlined) (IgG1 isotype,
L234A/L235A/G237A): AAB-003
TABLE-US-00070 (SEQ ID NO: 68)
ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGT
CCAGTGTGAGGTGCAGCTGCTGGAGTCCGGCGGCGGCCTGGTGCAGCCCG
GCGGCTCCCTGCGCCTGTCCTGCGCCGCCTCCGGCTTCACCTTCTCCAAC
TACGGCATGTCCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGT
GGCCTCCATCCGCTCCGGCGGCGGCCGCACCTACTACTCCGACAACGTGA
AGGGCCGCTTCACCATCTCCCGCGACAACTCCAAGAACACCCTGTACCTG
CAGATGAACTCCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGTGCG
CTACGACCACTACTCCGGCTCCTCCGACTACTGGGGCCAGGGCACCCTGG
TGACCGTGTCCTCCGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCA
CCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGT
CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCC
TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCA
GACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACA
AGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGC
CCAGCACCTGAAGCCGCTGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGG
TGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG
GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT
GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA
GCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC
ACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTG
GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGG TAAATGA
[0253] Full-length heavy chain of bapineuzumab, not including
signal sequence, IgG1 isotype, no Fc mutations
TABLE-US-00071 (SEQ ID NO: 69)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVAS
IRSGGGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYD
HYSGSSDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0254] The C-terminal K residue can be absent, as indicated
below.
TABLE-US-00072 (SEQ ID NO: 70)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVAS
IRSGGGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYD
HYSGSSDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
[0255] In some antibodies, positions 234, 235, and 237 of a human
IgG heavy chain constant region can be AAA respectively, LLA
respectively, LAG respectively, ALG respectively, AAG respectively,
ALA respectively, or LAA respectively. As shown above, AAB-003 is
an L234A, L235A, and G237A variant of bapineuzumab (i.e., having
identical amino acid sequences to bapineuzumab except for the
L234A, L235A, and G237A mutations, alanine (A) being the variant
amino acid). Like bapineuzumab, AAB-003 has a full-length human
kappa light chain constant region and a full-length human IgG1
heavy chain constant region (in either bapineuzumab or AAB-003, a
C-terminal lysine residue is sometimes cleaved intracellularly and
is sometimes missing from the final product).
[0256] Although the three mutations in AAB-003 are close to the
hinge region rather than the complement binding region, AAB-003 has
reduced binding to both Fc.gamma. receptors and to C1q, relative to
bapineuzumab. Thus, the AAB-003 antibody has reduced capacity to
induce both phagocytosis and the complement cascade. Furthermore,
AAB-003 displays less binding to human Fc.gamma.RII than an
otherwise identical antibody with fewer than the three mutations
present in AAB-003 (e.g., one with substitutions at residues 234
and 237), indicating that all three mutations in the AAB-003 Fc
region contribute to reducing effector function. Mutation of the
heavy chain constant region to reduce interaction with Fc.gamma.
receptor(s) and or C1q can reduce microhemorrhaging in a mouse
model without eliminating useful activities. Microhemorraghing in
mice is one factor that may contribute to vasogenic edema occurring
in humans. Antibodies bearing such mutations retain the ability to
inhibit cognitive decline as well as ability to clear amyloid
deposits.
[0257] Similarly heavy chain constant region mutants can also be
combined with the variable region sequences described above, e.g.,
for humanized 12A11 and 12B4 antibodies. The following table shows
exemplary combinations of heavy chain variable regions and heavy
chain constant regions with mutation(s) for antibodies described
above. The heavy chains shown in the table for a particular
antibody e.g., 12A11, can be paired with any of the light chain
variable regions described above for that antibody linked to a
light chain constant region (e.g., a human kappa light chain
constant region as follows:
TABLE-US-00073 (SEQ ID NO: 85)
RTVAAPSVEWPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
or an allotype or isoallotype thereof.
TABLE-US-00074 TABLE 1 Correlation of Full Length Heavy Chain SEQ
ID NOS with Respective Variable and Constant Region SEQ ID NOS
Heavy Chain Heavy Chain Antibody Variable region Constant region
10D5 (version 1) 9 50 9 51 9 56 9 57 9 62 9 63 12B4 32 50 32 51 32
56 32 57 32 62 32 63 12A11 (version 1) 11 50 11 51 11 56 11 57 11
62 11 63 12A11 (version 2) 12 50 12 51 12 56 12 57 12 62 12 63
12A11 (version 2.1) 13 50 13 51 13 56 13 57 13 62 13 63 12A11
(version 3) 14 50 14 51 14 56 14 57 14 62 14 63 12A11 (version 4.1)
15 50 15 51 15 56 15 57 15 62 15 63 12A11 (version 4.2) 16 50 16 51
16 56 16 57 16 62 16 63 12A11 (version 4.3) 17 50 17 51 17 56 17 57
17 62 17 63 12A11 (version 4.4) 18 50 18 51 18 56 18 57 18 62 18 63
12A11 (version 5.1) 19 50 19 51 19 56 19 57 19 62 19 63 12A11
(version 5.2) 20 50 20 51 20 56 20 57 20 62 20 63 12A11 (version
5.3) 21 50 21 51 21 56 21 57 21 62 21 63 12A11 (version 5.4) 22 50
22 51 22 56 22 57 22 62 22 63 12A11 (version 5.5) 23 50 23 51 23 56
23 57 23 62 23 63 12A11 (version 5.6) 24 50 24 51 24 56 24 57 24 62
24 63 12A11 (version 6.1) 25 50 25 51 25 56 25 57 25 62 25 63 12A11
(version 6.2) 26 50 26 51 26 56 26 57 26 62 26 63 12A11 (version
6.3) 27 50 27 51 27 56 27 57 27 62 27 63 12A11 (version 6.4) 28 50
28 51 28 56 28 57 28 62 28 63 12A11 (version 7) 29 50 29 51 29 56
29 57 29 62 29 63 12A11 (version 8) 30 50 30 51 30 56 30 57 30 62
30 63 12B4 32 50 32 51 32 56 32 57 32 62 32 63 266 34 50 34 51 34
56 34 57 34 62 34 63 20C2 (version 1) 38 50 38 51 38 56 38 57 38 62
38 63 20C2 (version 2) 39 50 39 51 39 56 39 57 39 62 39 63 C705 41
50 41 51 41 56 41 57 41 62 41 63 C706 43 50 43 51 43 56 43 57 43 62
43 63 2286 (version 1) 45 50 45 51 45 56 45 57 45 62 45 63 2286
(version 2) 46 50 46 51 46 56 46 57 46 62 46 63 3D6 (version 4) 72
50 72 51 72 56 72 57 72 62 72 63 10D6 (version 2) 74 50 74 51 74 56
74 57 74 62 74 63 2E7 (version 1) 76 50 76 51 76 56 76 57 76 62 76
63 2E7 (version 2) 77 50 77 51 77 56 77 57 77 62 77 63 2E7 (version
3) 78 50 78 51 78 56 78 57 78 62 78 63 9TL 79 50 79 51 79 56 79 57
79 62 79 63 6G 81 50 81 51 81 56 81 57 81 62 81 63 2.1 82 50 82 51
82 56 82 57 82 62 82 63
[0258] Amino acids in the constant region are numbered by alignment
with the human antibody EU (see Cunningham et al., J. Biol. Chem.,
9, 3161 (1970)). That is, the heavy and light chains of an antibody
are aligned with the heavy and light chains of EU to maximize amino
acid sequence identity and each amino acid in the antibody is
assigned the same number as the corresponding amino acid in EU. The
EU numbering system is conventional (see generally, Kabat et al.,
Sequences of Protein of Immunological Interest, NIH Publication No.
91-3242, US Department of Health and Human Services (1991)).
[0259] The affinity of an antibody for complement component C1q can
be altered by mutating at least one of the amino acid residues 318,
320, and 322 of the heavy chain to a residue having a different
side chain. Other suitable alterations for altering, e.g., reducing
or abolishing, specific C1q-binding to an antibody include changing
any one of residues 318 (Glu), 320 (Lys) and 322 (Lys), to Ala. C1q
binding activity can be abolished by replacing any one of the three
specified residues with a residue having an inappropriate
functionality on its side chain. It is not necessary to replace the
ionic residues only with Ala to abolish C1q binding. It is also
possible to use other alkyl-substituted non-ionic residues, such as
Gly, Ile, Leu, or Val, or such aromatic non-polar residues as Phe,
Tyr, Trp and Pro in place of any one of the three residues in order
to abolish C1q binding. In addition, it is also be possible to use
such polar non-ionic residues as Ser, Thr, Cys, and Met in place of
residues 320 and 322, but not 318, to abolish C1q binding activity.
Replacement of the 318 (Glu) residue by a polar residue may modify
but not abolish C1q binding activity. Replacing residue 297 (Asn)
with Ala results in removal of lytic activity while only slightly
reducing (about three fold weaker) affinity for C1q. This
alteration destroys the glycosylation site and the presence of
carbohydrate that is required for complement activation. Any other
substitution at this site also destroys the glycosylation site.
[0260] Additional mutations that can affect Clq binding to the
constant region of human IgG1 include those described, e.g., in WO
06/036291. In this case, at least one of the following
substitutions can be made to reduce C1q binding: D270A, K322A,
P329A, and P311S. Each of these mutations, including those at
residues 297, 318, and 320 can be made individually or in
combination.
[0261] Antibodies with heavy chain constant region mutations that
reduce binding to Fc.gamma. receptor(s) and/or C1q can be used in
any of the methods of the invention. Preferably, such antibodies
have reduced binding relative to an otherwise identical antibody
lacking the mutation of at least 50% to at least one Fc.gamma.
receptor and/or to C1q.
B. A.beta. Fragments
[0262] Numerous fragments of A.beta. have been now been described
in the scientific and patent literature as agents for active
immunotherapy (see, e.g., U.S. Pat. No. 6,750,324, US 20040213800;
US 20070134762). In general, fragments including an epitope within
residues 1-11 of A.beta. induce antibodies that bind Fc.gamma.
receptors and induce a clearing response against amyloid deposits,
whereas fragments lacking an epitope within residues 1-11 of
A.beta. induce antibodies that bind preferentially or exclusively
to soluble forms of A.beta. rather than plaques and induces little
if any clearing response against amyloid deposits.
[0263] Preferred fragment for inducing antibodies that bind to
amyloid deposits and induce a clearing response are N-terminal
fragments beginning at residues 1-3 of A.beta. and ending at
residues 7-11 of A.beta.. Exemplary N-terminal fragments include
A.beta.1-5, 1-6, 1-7, 1-10, 3-7, 1-3, and 1-4 with 1-7 being
particularly preferred. A class of exemplary fragments includes
fragments beginning at a residue between 1-3 (inclusive) and ending
at a residue between 7-11 (inclusive).
[0264] Preferred fragments for inducing antibodies to soluble
A.beta., which induce little, if any, clearing response against
amyloid deposits include A.beta.15-21, A.beta. 16-22, A.beta.17-23,
A.beta.18-24, A.beta.19-25, A.beta.015-22, A.beta.16-23,
A.beta.17-24, A.beta.18-25, A.beta.15-23, A.beta.16-24,
A.beta.17-25, A.beta.18-26, A.beta.15-24, A.beta.16-25, and
A.beta.15-25. A.beta.16-23 is particularly preferred meaning s a
fragment including residues 16-23 of A.beta. and lacking other
residues of A.beta.. Also preferred are C-terminal fragments of
A.beta.42 or 43 of 5-10 and preferably 7-10 contiguous amino acids.
Analogous C-terminal fragments of A.beta.40, or 39 can also be
used. These fragments can generate an antibody response that
includes end-specific antibodies. Fragments preferably lack T-cell
epitopes that would induce T-cells against A.beta.. Generally,
T-cell epitopes are greater than 10 contiguous amino acids.
Therefore, preferred fragments of A.beta. are of size 5-10 or
preferably 7-10 contiguous amino acids; i.e., sufficient length to
generate an antibody response without generating a T-cell response.
Absence of T-cell epitopes is preferred because these epitopes are
not needed for immunogenic activity of fragments, and may cause an
undesired inflammatory response in a subset of patients.
[0265] Agents to induce antibodies to A.beta. that can be used in
the methods of the invention also include (i) ACI-24 (AC Immune);
(ii) Affitopes AD02 and AD02 (Affiris GmbH); (iii) Arctic
Immunotherapeutic KLVFFAGDV (SEQ ID NO: 92) (BioArctic
Neuroscience/Eisai); (iv) A.beta.1-15-K-K-A.beta.1-15 (Brigham
& Women's Hospital); (v) .beta.-Vax.TM. and Recall-Vax.TM.
(Intellect Neurosciences); (vi) K6-A.beta.1-30 (Intellect
Neurosciences/NYU); (vii) V-950 (Merck); (viii) CAD106
(Novartis/Cytos); (ix) A.beta. DCtag.TM. nanoparticle adjuvant
(Prana Biotechnology/PRIMABioMed); (x) PX106 (also
2A.beta.1-11-PADRE, from Pharmexa/Lundbeck); (xi) A.beta.4-10
conjugated to a T cell epitope (U. Toronto); and (xii) p3102 and
p3075 (United Biomedical).
[0266] ACI-24 is an A.beta.1-15 liposome construct with
A.beta.1-15-K-K-16C palmitic acid inserted into a liposomal
bilayer. These compounds are described in US 2004/0242845, WO
05/081872, US 2007/0281006, and US 2006/0073158. Affitopes AD01 and
AD02 are mimotopes from the N-terminus of A.beta., as described in
WO 06/005707. The Arctic Immunotherapeutic is derived from
A.beta.22 of E692G, as described in US 20020162129 and US
20070248606. A.beta.1-15-K-K-A.beta.1-15 represents two linked
N-terminal A.beta. fragments, as described in WO 05/012330 and WO
02/0123553. .beta.-Vax.TM., Recall-Vax.TM. and K6-A.beta.1-30 are
A.beta. fragments linked to a T cell epitope, as described in WO
01/42306. V-950 is an 8-mer A.beta. peptide linked to a multivalent
linear peptide with at least one spacer and a multivalent branched
multiple antigen peptide, as described in WO 06/121656. CAD 106 is
a Q.beta. carrier (an RNA VLP) linked to an N-terminal A.beta.
peptide, as described in WO 04/016282. The A.beta. DCtag.TM.
nanoparticle adjuvant is described, e.g., in WO 02/00245. PX106 is
a A.beta.1-11 peptide linked to a T cell epitope called a "pan DR
epitope peptide (PADRE)," as described in U.S. Pat. No. 7,135,181.
p3102 and p3075 are A.beta.1-14 peptides linked by a spacer to a T
cell epitope (e.g., measles epitope), as described in US
20030068325 US 20040247612, U.S. Pat. No. 6,906,169, and WO
02/096350.
[0267] Fragments are usually natural A.beta. peptides but can
include unnatural amino acids or modifications of N or C terminal
amino acids at a one, two, five, ten or even all positions. For
example, the natural aspartic acid residue at position 1 and/or 7
of A.beta. can be replaced with iso-aspartic acid. Examples of
unnatural amino acids are D, alpha, alpha-disubstituted amino
acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline,
.gamma.-carboxyglutamate, epsilon-N,N,N-trimethyllysine,
epsilon-N-acetyllysine, O-phosphoserine, N-acetylserine,
N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,
omega-N-methylarginine, .beta.-alanine, ornithine, norleucine,
norvaline, hydroxproline, thyroxine, .gamma.-amino butyric acid,
homoserine, citrulline, and isoaspartic acid. Some therapeutic
agents of the invention are all-D peptides, e.g., all-D A.beta. or
all-D A.beta. fragment, and all-D peptide analogs. Fragments can be
screened for prophylactic or therapeutic efficacy in transgenic
animal models in comparison with untreated or placebo controls.
[0268] Fragments are typically conjugated to carrier molecules,
which provide a T-cell epitope, and thus promote an immune response
against the fragment conjugated to the carrier. A single agent can
be linked to a single carrier, multiple copies of an agent can be
linked to multiple copies of a carrier, which are in turn linked to
each other, multiple copies of an agent can be linked to a single
copy of a carrier, or a single copy of an agent can be linked to
multiple copies of a carrier, or different carriers. Suitable
carriers include serum albumins, keyhole limpet hemocyanin,
immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid,
or a toxoid from other pathogenic bacteria, such as diphtheria
(e.g., CRM197), E. coli, cholera, or H. pylori, or an attenuated
toxin derivative. T cell epitopes are also suitable carrier
molecules. Some conjugates can be formed by linking agents of the
invention to an immunostimulatory polymer molecule (e.g.,
tripalmitoyl-S-glycerine cysteine (Pam.sub.3Cys), mannan (a mannose
polymer), or glucan (a .beta.1.fwdarw.2 polymer)), cytokines (e.g.,
IL-1, IL-1 alpha and .beta. peptides, IL-2, .gamma.-INF, IL-10,
GM-CSF), and chemokines (e.g., MIP1-.alpha. and .beta., and
RANTES). Immunogenic agents can also be linked to peptides that
enhance transport across tissues, as described in O'Mahony, WO
97/17613 and WO 97/17614. Immunogens may be linked to the carries
with or with out spacers amino acids (e.g., gly-gly).
[0269] Additional carriers include virus-like particles. Virus-like
particles (VLPs), also called pseudovirions or virus-derived
particles, represent subunit structures composed of multiple copies
of a viral capsid and/or envelope protein capable of self assembly
into VLPs of defined spherical symmetry in vivo. (Powilleit, et
al., (2007) PLoS ONE 2(5):e415.) These particles have been found to
be useful as antigen delivery systems. VLPs can be produced and
readily purified in large quantities and due to their particulate
nature and high molecular weights. VLPs induce an immune response
without additional application of an adjuvant. (Ulrich et al.,
(1996) Intervirology 39:126-132.) Exemplary chimeric particles
useful as VLP antigen delivery systems include those based on
hepatitis B virus, human immunodeficiency virus (HIV), yeast
retrotransposon Ty, yeast totivirus L-A, parvovirus, influenza
virus, Norwalk virus, rotavirus, adeno-associated virus, bluetongue
virus, hepatitis A virus, human papillomavirus, measles virus,
polyoma virus and RNA phage virus, as well as those based on
various retroviruses and lentiviruses. For review, see Lechner, et
al. (2002) Intervirology 45:212-217.
[0270] The core protein of hepatitis B virus (HBcAg) is a common
VLP used for carrying foreign antigens (see Koletzki et al., (1997)
J Gen Vir 78:2049-2053). Briefly, HBcAg can be used as a core to
construct VLPs that present extended foreign protein segments. The
method employs a construct having a linker sequence between the a
C-terminally truncated HBcAg and a foreign protein sequence that
contains a stop codon. Truncated HBcAg/foreign protein chimera is
expressed utilizing a read through mechanism based on the opal
TGA-Trp mutation for expression in an E. coli suppressor strain.
The method described by Koletzki et al. allows for incorporation of
long foreign protein sequences into VLPs, allowing for a greater
variety of antigens to be carried by the VLP.
[0271] The HIV virus Gag protein can be used as an antigen carrier
system (see Griffiths et al., (1993) J. Virol. 67(6):3191-3198).
Griffiths utilized the V3 loop of HIV, which is the principle
neutralizing determinant of the HIV envelope. The Gag:V3 fusion
proteins assembled in vivo into hybrid Gag particles, designated
virus-derived particles (VDPs). The VDPs induce both humoral and
cellular responses. As the V3 loop contains a CTL epitope,
immunization with Gag:V3 induces a CTL response to the V3 protein
portion of the VLP.
[0272] A hybrid HIV:Ty VLP can also be used (see Adams et al.,
(1987) Nature 329(3):68-70). The HIV:Ty VLP employs the p1 protein
of the yeast transposon Ty. The first 381 amino acids of p1 are
sufficient for VLP formation. The HIV:Ty fusion proteins are
capable of assembling into VLPs in vivo, as well as inducing an
immune response to the HIV antigen carried by the VLP. VLPs using
the Ty p1 protein can also contain p1 fused to the whole of an
alpha2-interferon, the product of the bovine papilloma virus E1 and
E2 genes, and a portion of an influenza hemagglutinin. Each of
these Ty fusions formed VLPs and were capable of inducing
production of antisera to the non-Ty VLP component.
[0273] VLPs can also be designed from variants of the yeast
totivirus L-A (see Powilleit et al. (2007) PLOS One 2(5):e415). The
Pol gene of the L-A virus can be replaced with an appropriate
antigen to induce a specific immune response, demonstrating that
yeast VLPs are effective antigen carriers.
[0274] Recombinant, nonreplicative parvovirus-like particles can
also be used as antigen carriers. (Sedlik, et al. (1997) PNAS
94:7503-7508.) These particles allow the carried antigens into the
cytosol so they enter the class I-restricted immunological pathway,
thus stimulating cytotoxic T-lymphocyte (CTL) mediated responses.
Sedlik specifically used PPV:VLP, which contained the VP2 capsid
protein of the parvovirus and residues 118-132 from the lymphocytic
choriomeningitis virus (LCMV) was inserted into the VP2 capsid
protein. The PPV:VLP containing LCMV was capable of inducing an
immune response to LCMV and elicited immunological protection
against lethal viral doses in pre-immunized mice.
[0275] VLPs can also comprise replication incompetent influenza
that lack the influenza NS2 gene, the gene essential for viral
replication. (Watanabe, et al. (1996) J Virol. 76(2):767-773.)
These VLPs infect mammalian cells and allow expression of foreign
proteins.
[0276] Norwalk virus (NV)-based VLPs can also be used as vehicles
for immunogen delivery. (Ball, et al. (1999) Gastroenterology
117:40-48.) The NV genome has three open reading frames (ORFs 1-3).
Recombinant baculovirus expression of ORFs 2 and 3 allows for
spontaneous assembly of high yields of recombinant Norwalk virus
(rNV) VLPs.
[0277] Some conjugates can be formed by linking agents of the
invention to at least one T cell epitope. Some T cell epitopes are
promiscuous whereas other T cell epitopes are universal.
Promiscuous T cell epitopes are capable of enhancing the induction
of T cell immunity in a wide variety of subjects displaying various
HLA types. In contrast to promiscuous T cell epitopes, universal T
cell epitopes are capable of enhancing the induction of T cell
immunity in a large percentage, e.g., at least 75%, of subjects
displaying various HLA molecules encoded by different HLA-DR
alleles.
[0278] A large number of naturally occurring T-cell epitopes exist,
such as, tetanus toxoid (e.g., the P2 and P30 epitopes), Hepatitis
B surface antigen, pertussis, toxoid, measles virus F protein,
Chlamydia trachomatis major outer membrane protein, diphtheria
toxoid, Plasmodium falciparum circumsporozoite T, Plasmodium
falciparum CS antigen, Schistosoma mansoni triose phosphate
isomerase, Escherichia coli TraT, and Influenza virus hemagglutinin
(HA). The immunogenic peptides of the invention can also be
conjugated to the T-cell epitopes described in Sinigaglia F. et
al., Nature, 336:778-780 (1988); Chicz R. M. et al., J. Exp. Med.,
178:27-47 (1993); Hammer J. et al., Cell 74:197-203 (1993); Falk K.
et al., Immunogenetics, 39:230-242 (1994); WO 98/23635; and,
Southwood S. et al. J. Immunology, 160:3363-3373 (1998).
[0279] Carriers also include virus-like particles (see US
20040141984).
[0280] Fragments are often administered with pharmaceutically
acceptable adjuvants. The adjuvant increases the titer of induced
antibodies and/or the binding affinity of induced antibodies
relative to the situation if the peptide were used alone. A variety
of adjuvants can be used in combination with an immunogenic
fragment of A.beta., to elicit an immune response. Preferred
adjuvants augment the intrinsic response to an immunogen without
causing conformational changes in the immunogen that affect the
qualitative form of the response. Preferred adjuvants include
aluminum hydroxide and aluminum phosphate, 3 De-O-acylated
monophosphoryl lipid A (MPL.TM.) (see GB 2220211 (RIBI ImmunoChem
Research Inc., Hamilton, Mont., now part of Corixa). Stimulon.TM.
QS-21 is a triterpene glycoside or saponin isolated from the bark
of the Quillaja Saponaria Molina tree found in South America (see
Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach
(eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Pat. No.
5,057,540), (Aquila BioPharmaceuticals, Framingham, Mass.; now
Antigenics, Inc., New York, N.Y.). Other adjuvants are oil in water
emulsions (such as squalene or peanut oil), optionally in
combination with immune stimulants, such as monophosphoryl lipid A
(see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)), pluronic
polymers, and killed mycobacteria. Another adjuvant is CpG (WO
98/40100). Adjuvants can be administered as a component of a
therapeutic composition with an active agent or can be administered
separately, before, concurrently with, or after administration of
the therapeutic agent.
[0281] A preferred class of adjuvants is aluminum salts (alum),
such as alum hydroxide, alum phosphate, alum sulfate. Such
adjuvants can be used with or without other specific
immunostimulating agents such as MPL or 3-DMP, QS-21, polymeric or
monomeric amino acids such as polyglutamic acid or polylysine.
Another class of adjuvants is oil-in-water emulsion formulations.
Such adjuvants can be used with or without other specific
immunostimulating agents such as muramyl peptides (e.g.,
N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'dipalmitoyl-sn-
-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE),
N-acetylglucsaminyl-N-acetylmuramyl-L-A1-D-isoglu-L-Ala-dipalmitoxy
propylamide (DTP-DPP) Theramide.TM.), or other bacterial cell wall
components. Oil-in-water emulsions include (a) MF59 (WO 90/14837),
containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally
containing various amounts of MTP-PE) formulated into submicron
particles using a microfluidizer such as Model 110Y microfluidizer
(Microfluidics, Newton Mass.), (b) SAP, containing 10% Squalene,
0.4% Tween 80, 5% pluronic-blocked polymer L 121, and thr-MDP,
either microfluidized into a submicron emulsion or vortexed to
generate a larger particle size emulsion, and (c) Ribi.TM. adjuvant
system (RAS), (Ribi ImmunoChem, Hamilton, Mont.) containing 2%
squalene, 0.2% Tween 80, and one or more bacterial cell wall
components from the group consisting of monophosphorylipid A (MPL),
trehalose dimycolate (TDM), and cell wall skeleton (CWS),
preferably MPL+CWS (Detox.TM.).
[0282] Another class of preferred adjuvants is saponin adjuvants,
such as Stimulon.TM. (QS-21, Aquila, Framingham, Mass.) or
particles generated therefrom such as ISCOMs (immunostimulating
complexes) and ISCOMATRIX. Other adjuvants include RC-529, GM-CSF
and Complete Freund's Adjuvant (CFA) and Incomplete Freund's
Adjuvant (IFA). Other adjuvants include cytokines, such as
interleukins (e.g., IL-1 .alpha. and .beta. peptides, IL-2, IL-4,
IL-6, IL-12, IL13, and IL-15), macrophage colony stimulating factor
(M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF),
tumor necrosis factor (TNF), chemokines, such as MIP1.alpha. and
.beta. and RANTES. Another class of adjuvants is glycolipid
analogues including N-glycosylamides, N-glycosylureas and
N-glycosylcarbamates, each of which is substituted in the sugar
residue by an amino acid, as immuno-modulators or adjuvants (see
U.S. Pat. No. 4,855,283). Heat shock proteins, e.g., HSP70 and
HSP90, may also be used as adjuvants.
[0283] An adjuvant can be administered with an immunogen as a
single composition, or can be administered before, concurrent with
or after administration of the immunogen. Immunogen and adjuvant
can be packaged and supplied in the same vial or can be packaged in
separate vials and mixed before use. Immunogen and adjuvant are
typically packaged with a label indicating the intended therapeutic
application. If immunogen and adjuvant are packaged separately, the
packaging typically includes instructions for mixing before use.
The choice of an adjuvant and/or carrier depends on the stability
of the immunogenic formulation containing the adjuvant, the route
of administration, the dosing schedule, the efficacy of the
adjuvant for the species being vaccinated, and, in humans, a
pharmaceutically acceptable adjuvant is one that has been approved
or is approvable for human administration by pertinent regulatory
bodies. For example, Complete Freund's adjuvant is not suitable for
human administration. Alum, MPL and QS-21 are preferred.
Optionally, two or more different adjuvants can be used
simultaneously. Preferred combinations include alum with MPL, alum
with QS-21, MPL with QS-21, MPL or RC-529 with GM-CSF, and alum,
QS-21 and MPL together. Also, Incomplete Freund's adjuvant can be
used (Chang et al., Advanced Drug Delivery Reviews 32, 173-186
(1998)), optionally in combination with any of alum, QS-21, and MPL
and all combinations thereof.
V. Patients Amenable to Treatment
[0284] The present regimes are useful for treatment of any disease
characterized by amyloid deposits of A.beta. in the brain. As well
as Alzheimer's disease, such diseases include Down's syndrome,
Parkinson's disease, mild-cognitive impairment, and vascular
amyloid disease. Patients amenable to treatment include individuals
at risk of disease but not showing symptoms, as well as patients
presently showing symptoms. In the case of Alzheimer's disease,
virtually anyone is at risk of suffering from Alzheimer's disease
if he or she lives long enough. Therefore, the present methods can
be administered prophylactically to the general population without
the need for any assessment of the risk of the subject patient. The
present methods can also be useful for individuals who have a known
genetic risk of Alzheimer's disease. Such individuals include those
having relatives who have experienced this disease, and those whose
risk is determined by analysis of genetic or biochemical markers.
Genetic markers of risk toward Alzheimer's disease include
mutations in the APP gene, particularly mutations at position 717
and positions 670 and 671 referred to as the Hardy and Swedish
mutations respectively (see Hardy, supra). Other markers of risk
are mutations in the presenilin genes, PS1 and PS2, and ApoE4,
family history of A.beta., hypercholesterolemia or atherosclerosis.
Individuals presently suffering from Alzheimer's disease can be
recognized from characteristic dementia, as well as the presence of
risk factors described above. In addition, a number of diagnostic
tests are available for identifying individuals who have AD. These
include measurement of CSF tau and A.beta.42 levels. Elevated tau
and decreased A.beta.42 levels signify the presence of A.beta..
Individuals suffering from Alzheimer's disease can also be
diagnosed by ADRDA criteria as discussed in the Examples
section.
[0285] In asymptomatic patients, treatment can begin at any age
(e.g., 10, 20, 30). Usually, however, it is not necessary to begin
treatment until a patient reaches 40, 50, 60 or 70 years of age.
Treatment typically entails multiple dosages over a period of time.
Treatment can be monitored by assaying antibody levels over time.
If the response falls, a booster dosage is indicated. In the case
of potential Down's syndrome patients, treatment can begin
antenatally by administering therapeutic agent to the mother or
shortly after birth.
[0286] Patients amenable to treatment include patients 50 to 87
years of age, patients suffering from mild to moderate Alzheimer's
disease, patients having an MMSE score of 14-26, patients having a
diagnosis of probable Alzheimer's disease based on Neurological and
Communicative Disorders and Stroke-Alzheimer's disease Related
Disorders (NINCDS-ADRDA) criteria, and/or patients having an Rosen
Modified Hachinski Ischemic score less than or equal to 4. Patients
with MRI an scan consistent with the diagnosis of Alzheimer's
disease, i.e., that there are no other abnormalities present on the
MRI that could be attributed to other diseases, e.g. stroke,
traumatic brain injury, arachnoid cysts, tumors, etc are also
amendable to treatment.
VI. Treatment Regimes
[0287] In prophylactic applications, agents or pharmaceutical
compositions or medicaments containing the same are administered to
a patient susceptible to, or otherwise at risk of, Alzheimer's
disease in an amount sufficient to eliminate or reduce the risk,
lessen the severity, or delay the outset of the disease, including
biochemical, histologic and/or behavioral symptoms of the disease,
its complications and intermediate pathological phenotypes
presenting during development of the disease. In therapeutic
applications, compositions or medicaments are administered to a
patient suspected of, or already suffering from such a disease in
an amount sufficient to cure, or at least partially arrest, the
symptoms of the disease (biochemical, histologic and/or
behavioral), including its complications and intermediate
pathological phenotypes in development of the disease.
[0288] Effective doses of the compositions of the present
invention, for the treatment of the above described conditions vary
depending upon many different factors, including means of
administration, target site, physiological state of the patient,
whether the patient is human or an animal, other medications
administered, and whether treatment is prophylactic or
therapeutic.
[0289] Optionally, antibodies are administered to achieve a mean
serum concentration of administered antibody of 0.1-60, 0.4-20, or
1-15 .mu.g/ml in a patient. These ranges bracket the demonstrated
effective concentrations in mice and humans allowing some margin
for error in measurement and individual patient variation. The
serum concentration can be determined by actual measurement or
predicted from standard pharmacokinetics (e.g., WinNonline Version
4.0.1 (Pharsight Corporation, Cary, USA)) based on the amount of
antibody administered, frequency of administration, route of
administration and antibody half-life.
[0290] The mean antibody concentration in the serum is optionally
within a range of 1-10, 1-5 or 2-4 .mu.g/ml. It is also optional to
maintain a maximum serum concentration of the antibody in the
patient less than about 28 .mu.g antibody/ml serum for maximizing
therapeutic benefit relative to the occurrence of possible side
effects, particularly vascular edema. A preferred maximum serum
concentration is within a range of about 4-28 .mu.g antibody/ml
serum. The combination of maximum serum less than about 28 .mu.g
antibody/mil serum and an mean serum concentration of the antibody
in the patient is below about 7 .mu.g antibody/ml serum is
particularly beneficial. Optionally, the mean concentration is
within a range of about 2-7 .mu.g antibody/ml serum.
[0291] The concentration of A.beta. in plasma following antibody
administration changes roughly in parallel with changes of antibody
serum concentration. In other words, plasma concentration of
A.beta. is highest after a dose of antibody and then declines as
the concentration of antibody declines between doses. The dose and
regime of antibody administration can be varied to obtain a desired
level of A.beta. in plasma. In such methods, the mean plasma
concentration of antibody can be at least 450 pg/ml or for example,
within a range of 600-30000 pg/ml or 700-2000 pg/ml or 800-1000
pg/ml.
[0292] The preferred dosage ranges for antibodies are from about
0.01 to 5 mg/kg, and more usually 0.1 to 3 mg/kg or 0.15-2 mg/kg or
0.15-1.5 mg/kg, of the host body weight. Subjects can be
administered such doses daily, on alternative days, weekly,
biweekly, monthly, quarterly, or according to any other schedule
determined by empirical analysis. An exemplary treatment entails
administration in multiple dosages over a prolonged period, for
example, of at least six months. Additional exemplary treatment
regimes entail administration once per every two weeks or once a
month or once every 3 to 6 months.
[0293] For intravenous administration, doses of 0.1 mg/kg to 2
mg/kg, and preferably 0.5 mg/kg or 1.5 mg/kg administered
intravenously quarterly are suitable. Preferred doses of antibody
for monthly intravenous administration occur in the range of
0.1-1.0 mg/kg antibody or preferably 0.5-1.0 mg/kg antibody.
[0294] For more frequent dosing, e.g., from weekly to monthly
dosing, subcutaneous administration is preferred. Subcutaneous
dosing is easier to administer and can reduce maximum serum
concentrations relative to intravenous dosing. The doses used for
subcutaneous dosing are usually in the range of 0.01 to 0.6 mg/kg
or 0.01-0.35 mg/kg, preferably, 0.05-0.25 mg/kg. For weekly or
biweekly dosing, the dose is preferably in the range of 0.015-0.2
mg/kg, or 0.05-0.15 mg/kg. For weekly dosing, the dose is
preferably 0.05 to 0.07 mg/kg, e.g., about 0.06 mg/kg. For biweekly
dosing, the dose is preferably 0.1 to 0.15 mg/kg. For monthly
dosing, the dose is preferably 0.1 to 0.3 mg/kg or about 0.2 mg/kg.
Monthly dosing includes dosing by the calendar month or lunar month
(i.e., every four weeks). Here as elsewhere in the application,
dosages expressed in mg/kg can be converted to absolute mass
dosages by multiplying by the mass of a typical patient (e.g., 70
or 75 kg) typically rounding to a whole number. Other regimes are
described by e.g., PCT/US2007/009499. The dosage and frequency can
be varied within these guidelines based on the ApoE status of the
patient as discussed above.
[0295] The amount of an agent for active administration varies from
1-500 .mu.g per patient and more usually from 5-100 .mu.g per
injection for human administration. Exemplary dosages per injection
are 3, 10, 30, or 90 .mu.g for each human injection. The mass of
immunogen also depends on the mass ratio of immunogenic epitope
within the immunogen to the mass of immunogen as a whole.
Typically, 10.sup.-3 to 10.sup.-5 micromoles of immunogenic epitope
are used for each immunization of immunogen. The timing of
injections can vary significantly from once a day, to once a year,
to once a decade. On any given day that a dosage of immunogen is
given, the dosage is greater than 1 .mu.g/patient and usually
greater than 10 .mu.g/patient if adjuvant is also administered, and
greater than 10 .mu.g/patient and usually greater than 100
.mu.g/patient in the absence of adjuvant. A typical regimen
consists of an immunization followed by booster injections at time
intervals, such as 6 week intervals. Another regimen consists of an
immunization followed by booster injections 1, 2 and 12 months
later. Another regimen entails an injection every two months for
life Alternatively, booster injections can be on an irregular basis
as indicated by monitoring of immune response. The dosage and
frequency can be varied such that antibodies induced by an active
agent have mean serum concentrations within a range of 0.1-60,
0.4-20, or 1-15 or 2-7 .mu.g/ml as in passive administration. The
dosage and frequency can be varied within these guidelines based on
the ApoE status of the patient as discussed above.
VII. Exemplary Regimes Depending On Carrier Status
[0296] The invention provides methods of treating non-carrier
patients having Alzheimer's disease (e.g., mild or moderate) in
which an effective regime of an antibody that specifically binds to
an N-terminal epitope of A.beta. is administered to such a patient.
The antibody can for example bind to an epitope within residues
1-11, 1-7, 1-5, or 3-7 of A.beta.. Optionally, the antibody is
bapineuzumab. The dosage of the antibody can be within a range of
about 0.15 mg/kg to 2 mg/kg administered by intravenous infusion.
Optionally, the dosage is about 0.5 mg/kg to about 1 mg/kg The
dosage can be administered for example every 8-16 weeks, every 1-14
weeks or every 13 weeks.
[0297] The invention also provides methods of reducing cognitive
decline in a non-carrier patient having been diagnosed with mild or
moderate Alzheimer's disease. The method entails administering an
effective regime of an antibody that specifically binds to an
N-terminal epitope of A.beta. to such a patient. The antibody can
for example bind to an epitope within residues 1-11, 1-7, 1-5, or
3-7 of A.beta.. Optionally, the antibody is bapineuzumab. The
dosage of the antibody can be within a range of about 0.15 mg/kg to
2 mg/kg administered by intravenous infusion. Optionally, the
dosage is about 0.5 mg/kg to about 1 mg/kg The dosage can be
administered for example every 8-16 weeks, every 1-14 weeks or
every 13 weeks. Cognitive decline can be measured by comparing the
patient being treated with the cognitive decline in a population of
control patients also of non-carrier status and having mild or
moderate Alzheimer's disease (e.g., a control population in a
clinical trial). Cognitive ability can be measured by scales such
as ADAS-COG, NTB, MMSE or CDR-SB. The rate of change in such a
scale (points over time) in a patient can be compared with the mean
decline in a population of control patients as described above.
[0298] The invention also provides methods of reducing brain volume
decline in a non-carrier patient having been diagnosed with mild or
moderate Alzheimer's disease. The method entails administering an
effective regime of an antibody that specifically binds to an
N-terminal epitope of A.beta. to such a patient. The antibody can
for example bind to an epitope within residues 1-11, 1-7, 1-5, or
3-7 of A.beta.. Optionally, the antibody is bapineuzumab. The
dosage of the antibody can be within a range of about 0.15 mg/kg to
2 mg/kg administered by intravenous infusion. Optionally, the
dosage is about 0.5 mg/kg to about 1 mg/kg The dosage can be
administered for example every 8-16 weeks, every 1-14 weeks or
every 13 weeks. Brain volume can be measured by MRI. Change in
brain volume in a patient can be compared with the mean decline in
brain volume in a population of control patients also of
non-carrier status and having mild or moderate Alzheimer's disease
(e.g., a control population in a clinical trial).
[0299] The invention also provides methods of treating non-carrier
patients having Alzheimer's disease (e.g., mild or moderate) in
which a regime of an antibody that specifically binds to an
N-terminal epitope of A.beta. is administered to such a patient.
The regime is effective to maintain a mean serum concentration of
the antibody in the range of about 0.1 .mu.g/ml to about 60
.mu.g/ml, optionally 0.4-20 or 1-5 .mu.g/ml. Additionally or
alternatively, the regime is administered to maintain a mean plasma
concentration of A.beta. of 600-3000 pg/ml, 700-2000 pg/ml or
800-100 pg/ml. Optionally, the antibody in such methods is
bapineuzumab.
[0300] The invention also provides methods of treating a patient
who is an ApoE4 carrier and has Alzheimer's disease in which the
antibody administered has a constant region mutation that reduces
binding to C1q and/or and Fc.gamma. receptor(s). Optionally, the
antibody is an antibody that binds to an epitope within an
N-terminal region of A.beta.. Optionally, the antibody is AAB-003.
Optionally, the patients are monitored, e.g., quarterly, by MRI for
vasogenic edema. If vasogenic edema-develops the frequency or dose
can be reduced or eliminated. Vasogenic edema can optionally be
treated with a corticosteroid. After resolution of vasogenic edema,
administration of treatment can be resumed. Optionally, the dose is
increased over time.
[0301] The invention also provides methods of treating a patient
diagnosed with probable Alzheimer's disease, irrespective of ApoE4
status. In such methods, an effective regime of an antibody that
specifically binds to an N-terminal region of A.beta. is
administered. The antibody has a constant region mutation that
reduces binding to C1q and/or and Fc.gamma. receptor relative to an
otherwise identical antibody without the mutation. Optionally, the
antibody is an antibody that binds to an epitope within an
N-terminal region of A.beta.. Optionally, the antibody is AAB-003.
Optionally, the patients are monitored, e.g., quarterly, by MRI for
vasogenic edema. If vasogenic edema develops the frequency or dose
can be reduced or eliminated. Vasogenic edema can optionally be
treated with a corticosteroid. After resolution of vasogenic edema,
administration of treatment can be resumed. Optionally, the dose is
increased over time after resolution of vasogenic edema.
[0302] The invention provides methods of treating an ApoE carrier
patient with Alzheimer disease comprising subcutaneously
administering to a patient having the disease an antibody that
specifically binds to an N-terminal epitope of A.beta.. Optionally,
the antibody is administered at a dose of 0.01-0.6 mg/kg and a
frequency of between weekly and monthly. Optionally, the antibody
is administered at a dose of 0.05-0.5 mg/kg. Optionally, the
antibody is administered at a dose of 0.05-0.25 mg/kg. Optionally,
the antibody is administered at a dose of 0.015-0.2 mg/kg weekly to
biweekly. Optionally, the antibody is administered at a dose of
0.05-0.15 mg/kg weekly to biweekly. Optionally, the antibody is
administered at a dose of 0.05-0.07 mg/kg weekly. Optionally, the
antibody is administered at a dose of 0.06 mg/kg weekly.
Optionally, the antibody is administered at a dose of 0.1 to 0.15
mg/kg biweekly. Optionally, the antibody is administered at a dose
of 0.1 to 0.3 mg/kg monthly. Optionally, the antibody is
administered at a dose of 0.2 mg/kg monthly.
[0303] The invention also provides methods of treating an ApoE4
carrier patient having Alzheimer disease comprising subcutaneously
administering to a patient having the disease an antibody that
specifically binds to an N-terminal fragment of A.beta., wherein
the antibody is administered at a dose of 1-40 mg and a frequency
of between weekly and monthly. Optionally, the antibody is
administered at a dose of 5-25 mg. Optionally, the antibody is
administered at a dose of 2.5-15 mg. Optionally, the antibody is
administered at a dose of 1-12 mg weekly to biweekly. Optionally,
the antibody is administered at a dose of 2.5-10 mg weekly to
biweekly. Optionally, the antibody is administered at a dose of
2.5-5 mg weekly. Optionally, the antibody is administered at a dose
of 4-5 mg weekly. Optionally, the antibody is administered at a
dose of 7-10 mg biweekly.
VIII. Pharmaceutical Compositions
[0304] Agents of the invention are often administered as
pharmaceutical compositions comprising an active therapeutic agent,
i.e., and a variety of other pharmaceutically acceptable
components. See Remington's Pharmaceutical Science (15th ed., Mack
Publishing Company, Easton, Pa. (1980)). The preferred form depends
on the intended mode of administration and therapeutic application.
The compositions can also include, depending on the formulation
desired, pharmaceutically-acceptable, non-toxic carriers or
diluents, which are defined as vehicles commonly used to formulate
pharmaceutical compositions for animal or human administration. The
diluent is selected so as not to affect the biological activity of
the combination. Examples of such diluents are distilled water,
physiological phosphate-buffered saline, Ringer's solutions,
dextrose solution, and Hank's solution. In addition, the
pharmaceutical composition or formulation may also include other
carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic
stabilizers and the like.
[0305] Pharmaceutical compositions can also include large, slowly
metabolized macromolecules such as proteins, polysaccharides such
as chitosan, polylactic acids, polyglycolic acids and copolymers
(such as latex functionalized Sepharose.TM., agarose, cellulose,
and the like), polymeric amino acids, amino acid copolymers, and
lipid aggregates (such as oil droplets or liposomes). Additionally,
these carriers can function as immunostimulating agents (i.e.,
adjuvants).
[0306] Agents are typically administered parenterally. Antibodies
are usually administered intravenously or subcutaneously. Agents
for inducing an active immune response are usually administered
subcutaneously or intramuscularly. For parenteral administration,
agents of the invention can be administered as injectable dosages
of a solution or suspension of the substance in a physiologically
acceptable diluent with a pharmaceutical carrier that can be a
sterile liquid such as water oils, saline, glycerol, or ethanol.
Additionally, auxiliary substances, such as wetting or emulsifying
agents, surfactants, pH buffering substances and the like can be
present in compositions. Other components of pharmaceutical
compositions are those of petroleum, animal, vegetable, or
synthetic origin, for example, peanut oil, soybean oil, and mineral
oil. In general, glycols such as propylene glycol or polyethylene
glycol are preferred liquid carriers, particularly for injectable
solutions. Antibodies can be administered in the form of a depot
injection or implant preparation, which can be formulated in such a
manner as to permit a sustained release of the active
ingredient.
[0307] Some preferred formulations are described in US 20060193850.
A preferred formulation has a pH of about 5.5 to about 6.5,
comprises i. at least one A.beta. antibody at a concentration of
about 1 mg/ml to about 30 mg/ml; ii. mannitol at a concentration of
about 4% w/v or NaCl at a concentration of about 150 mM; iii. about
5 mM to about 10 mM histidine or succinate; and iv. 10 mM
methionine. Optionally, the formulation also includes polysorbate
80 at a concentration of about 0.001% w/v to about 0.01% w/v.
Optionally, the formulation has a pH of about 6.0 to about 6.5 and
comprises about 10 mg/ml A.beta. antibody, about 10 mM histidine
and about 4% w/v mannitol and about 0.005% w/v polysorbate 80
Optionally, the formulation has a pH of about 6.0 to about 6.2 and
comprises about 20 mg/ml A.beta. antibody, about 10 mM histidine,
about 4% w/v mannitol and about 0.005% w/v polysorbate 80.
Optionally, the formulation has a pH of about 6.0 to about 6.2 and
comprises about 30 mg/ml A.beta. antibody, about 10 mM histidine,
about 4% w/v mannitol and about 0.005% w/v polysorbate 80.
[0308] Typically, compositions are prepared as injectables, either
as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid vehicles prior to injection
can also be prepared. The preparation also can be emulsified or
encapsulated in liposomes or micro particles such as polylactide,
polyglycolide, or copolymer for enhanced adjuvant effect, as
discussed above (see Langer, Science 249: 1527 (1990) and Hanes,
Advanced Drug Delivery Reviews 28:97 (1997)). The agents of this
invention can be administered in the form of a depot injection or
implant preparation, which can be formulated in such a manner as to
permit a sustained or pulsatile release of the active
ingredient.
[0309] Additional formulations suitable for other modes of
administration include oral, intranasal, and pulmonary
formulations, suppositories, and transdermal applications. For
suppositories, binders and carriers include, for example,
polyalkylene glycols or triglycerides; such suppositories can be
formed from mixtures containing the active ingredient in the range
of 0.5% to 10%, preferably 1%-2%. Oral formulations include
excipients, such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, and
magnesium carbonate. These compositions take the form of solutions,
suspensions, tablets, pills, capsules, sustained release
formulations or powders and contain 10%-95% of active ingredient,
preferably 25%-70%.
IX. Kits and Labels
[0310] The invention provides kits containing an antibody binding
to an N-terminal epitope of A.beta.. The antibody is typically
provided in lyophilized or solution form in a vial, optionally in a
single-dose form. The antibody in the vial is typically sterile and
manufactured under GMP conditions. The kits can also include
diluents, syringes, needles, intravenous or subcutaneous drips and
the like. The kits typically contain instructions (e.g., a package
insert or label) for use. In some kits, the instructions specify
whether the antibody is to be provided to ApoE4 carriers or
non-carriers or can be provided to both. The instructions can also
specify that the antibody is not to be provided to ApoE4 carriers.
In some kits, the instructions can provide information or sources
for ApoE testing.
[0311] In some kits, the instructions specify results that can be
achieved by administering the antibody. The results can include an
inhibition of cognitive decline. The instructions can also include
a measure of cognitive decline in a control patient (typically a
mean value from a population of such patients) for purposes of
comparison. Cognitive decline can be measured, by for example,
ADAS-COG, NTB, MMSE or CDR-SB Likewise, the instructions can refer
to inhibition of decrease in brain volume or inhibition of
ventricular volume. The instructions can also include a measure of
decrease in brain volume or inhibition of ventricular volume in a
control patient (typically a mean value from a population of such
patients for purposes of comparison).
[0312] In some kits, the instructions specify potential side
effects including vasogenic edema. The instructions can also
specify a monitoring regime, such as performing MRI at quarterly,
six monthly or annual intervals. The instructions can specify
different monitoring regimes for ApoE4 non-carriers and carriers as
discussed above. The instructions can also specify altered dosing
schedules on occurrence and/or resolution of vasogenic edema and
treatment measures for vasogenic edema, such as
corticosteroids.
[0313] The kits can also include instructions for patients for whom
treatment is contraindicated such as prior brain injury, CVA, brain
tumor, multiple lacunes, venothrombotic disease, anticoagulation
(heparin/coumadin) or atrial fibrillation. The kits can also
provide instructions for route (e.g., subcutaneous), dosage amount
or frequency of dosing.
X. Antibodies with Mutated IgG1 Constant Region
[0314] The invention provides a human IgG1 constant region, in
which amino acids at positions 234, 235, and 237 (EU numbering) are
each alanine, and isolated antibodies or fusion proteins containing
such a constant region. Such antibodies include human antibodies,
humanized antibodies and chimeric antibodies as described above.
Examples of such antibodies include antibodies to A.beta.,
antibodies to the Lewis Y antigen and the 5T4 tumor antigen, such
as described in the Examples. Fusion proteins include the
extracellular domains of receptors (e.g., TNF-alpha receptor)
linked to a constant region. Methods for fusing or conjugating
polypeptides to the constant regions of antibodies are described
by, e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046,
5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095,
5,112,946; EP 0 307 434; EP 0 367 166; EP 0 394 827).
[0315] Antibodies or fusion proteins incorporating these mutations
can offer advantages of the IgG1 isotype including pharmacokinetics
and ease of manufacture, but also have reduced or eliminated
effector function relative to an otherwise identical antibody
lacking these mutations. Effector function is typically impaired in
binding to one or more Fc gamma receptors, binding to C1Q,
antibody-dependent cellular cytotoxicity and/or antibody-dependent
complement activity. In some antibodies, all of these activities
are reduced or eliminated. An activity is considered eliminated if
there is no detectable difference beyond experimental error in that
activity between an antibody having the above three mutations and
an otherwise identical control antibody without the mutations.
[0316] Typically, a mutated constant region includes CH1, hinge,
CH2 and CH3 domains. However, the CH1 domain is sometimes replaced
particularly in fusion proteins with a synthetic linker. Some
constant regions contain a full-length IgG1 constant region with
the possible exception of a C-terminal lysine residue. Exemplary
sequences of a mutated constant region are provided by SEQ ID NOS:
62 and 63. These sequences differ in the 62 contains a C-terminal
lysine not present in 63.
[0317] The sequences 62 and 63 represent the G1mz allotype of human
IgG1. Other examples of allotypes have been provided above.
Allotypes are natural polymorphic variations in the human IgG1
constant region that differ between different individuals at the
polymorphic position. The G1mz allotype has Glu at position 356 and
Met at position 358.
[0318] Other allotypic variants of SEQ ID NOS. 62 and 63 are
included. Also included are human IgG1 constant regions having
alanine residues at positions 234, 235 and 237 any permutation of
residues occupying polymorphic positions in natural allotypes.
[0319] Mutated IgG1 constant regions having alanine at positions
234, 235 and 237 can have additional mutations present relative to
a natural human IgG1 constant region. As an example in which
additional mutations can be present, alanine mutations at positions
234, 235 and 237 can be combined with mutations at positions 428
and/or 250 as described in U.S. Pat. No. 7,365,168. Mutations at
positions 428 and 250 can result in increased half life. Additional
mutations that can be combined with mutations at positions 234, 235
and 237 have been described in Section IV A in connection with
antibodies that bind A.beta.. Some such constant regions have no
additional mutations present. Some such constant regions have no
additional mutations present in and around regions of the IgG1
constant region affecting Fc gamma receptor and/or complement
binding (e.g., residues 230-240 and 325-325 by EU numbering). The
omission of a C-terminal lysine residue by intracellular processing
is not considered to be a mutation. Likewise, naturally occurring
amino acids occupying polymorphic sites differing between allotypes
are considered natural rather than mutant amino acids.
XI. Experimental Models, Assays and Diagnostics
A. Animal Models
[0320] Such models include, for example, mice bearing a 717 (APP770
numbering) mutation of APP described by Games et al., supra, and
mice bearing a 670/671 (APP770 numbering) Swedish mutation of APP
such as described by McConlogue et al., U.S. Pat. No. 5,612,486 and
Hsiao et al., Science, 274, 99 (1996); Staufenbiel et al., Proc.
Natl. Acad. Sci. USA, 94:13287-13292 (1997); Sturchler-Pierrat et
al., Proc. Natl. Acad. Sci. USA, 94:13287-13292 (1997); Borchelt et
al., Neuron, 19:939-945 (1997)); Richards et al., J. Neurosci.
23:8989-9003, 2003; Cheng, Nat. Med. 10(11): 1190-2, 2004 Hwang et
al., Exp Neurol. 2004 Mar. Mutations of APP suitable for inclusion
in transgenic animals include conversion of the wild-type Val717
(APP770 numbering) codon to a codon for Ile, Phe, Gly, Tyr, Leu,
Ala, Pro, Trp, Met, Ser, Thr, Asn, or Gln. A preferred substitution
for Val717 is Phe. Another suitable mutation is the arctic mutation
E693G (APP 770 numbering). The PSAPP mouse, which has both amyloid
precursor protein and presenilin transgenes, is described by
Takeuchi et al., American Journal of Pathology. 2000; 157:331-339.
A triple transgenic mouse having amyloid precursor protein,
presenilin and tau transgenes is described by LaFerla, (2003),
Neuron 39, 409-421. Another useful transgenic mouse has both APP
and TGF-.beta. transgenes. Protein encoding sequences in transgenes
are in operable linkage with one or more suitable regulatory
elements for neural expression. Such elements include the PDGF,
prion protein and Thy-1 promoters. Another useful transgenic mouse
has an APP transgene with both a Swedish and 717 mutation. Another
useful transgenic mouse has an APP transgene with an arctic
mutation (E693G).
B. Assays to Detect Amyloid Related Pathologies
[0321] Contextual fear conditioning assays. Contextual fear
conditioning (CFC) is a common form of learning that is
exceptionally reliable and rapidly acquired in most animals, for
example, mammals. Test animals learn to fear a previously neutral
stimulus and/or environment because of its association with an
aversive experience. (see, e.g., Fanselow, Anim. Learn. Behav.
18:264-270 (1990); Wehner et al., Nature Genet. 17:331-334. (1997);
Caldarone et al., Nature Genet. 17:335-337 (1997)).
[0322] Contextual fear conditioning is especially useful for
determining cognitive function or dysfunction, e.g., as a result of
disease or a disorder, such as a neurodegenerative disease or
disorder, an A.beta.-related disease or disorder, an amyloidogenic
disease or disorder, the presence of an unfavorable genetic
alteration effecting cognitive function (e.g., genetic mutation,
gene disruption, or undesired genotype), and/or the efficacy of an
agent, e.g., an A.beta. conjugate agent, on cognitive ability.
Accordingly, the CFC assay provides a method for independently
testing and/or validating the therapeutic effect of agents for
preventing or treating a cognitive disease or disorder, and in
particular, a disease or disorder affecting one or more regions of
the brains, e.g., the hippocampus, subiculum, cingulated cortex,
prefrontal cortex, perirhinal cortex, sensory cortex, and medial
temporal lobe (see US 2008145373).
C. Phagocytosis Assays to Determine Antibody Effector Function
[0323] Antibodies can be screened for clearing an amyloid deposit
in an ex vivo assay. A tissue sample from a brain of a patient with
Alzheimer's disease or an animal model having characteristic
Alzheimer's pathology is contacted with phagocytic cells bearing an
Fc.gamma. receptor, such as microglial cells, and the antibody
under test in a medium in vitro. The phagocytic cells can be a
primary culture or a cell line, such as BV-2, C8-B4, or THP-1. A
series of measurements is made of the amount of amyloid deposit in
the reaction mixture, starting from a baseline value before the
reaction has proceeded, and one or more test values during the
reaction. The antigen can be detected by staining, for example,
with a fluorescently labelled antibody to A.beta. or other
component of amyloid plaques. A reduction relative to baseline
during the reaction of the amyloid deposits indicates that the
antibody under test has clearing activity.
[0324] Generally, isotype controls are added to ensure that the
appropriate Fc-Fc.gamma. receptor interaction is being observed.
Additional controls include use of non-specific antibodies,
and/antibodies with a known affinity for the F.gamma.c receptors on
the phagocytic cells. Such assays can be carried out with human or
non-human tissues and phagocytic cells, and human, non-human, or
humanized antibodies.
[0325] A variation on the ex vivo phagocytosis assay eliminates the
need for an A.beta.-containing tissue, although still allowing
detection of the interaction between a particular antibody and
Fc.gamma. receptors. In this case, the assay relies on a solid
matrix which is coated with antibody. The solid matrix is generally
in a form that can be engulfed by a phagocytic cell, e.g., a bead
or particle on the order of nanometers to several microns in size.
The solid matrix can be conjugated to a detectable moiety, e.g., a
fluorophore, so that the particle can be traced. Kits and materials
for phagocytosis assays of this sort are commercially available,
e.g., from Beckman Coulter (Fullerton, Calif.) and Molecular Probes
(Eugene, Oreg.). An example of such an assay is provided in the
Examples section.
D. Complement Binding Assays
[0326] Antibody effector function can also be determined by
detecting the ability of an antibody to interact with complement,
in particular, the C1q polypeptide (see, e.g., Mansouri et al.
(1999) Infect. Immun. 67:1461). In the case of A.beta.-specific
antibody, a solid matrix (e.g., a multiwell plate) can be coated
with A.beta., and exposed to antibody, and, in turn, exposed to
labelled C1q. Alternatively, C1q can be attached to the matrix, and
labelled antibody added. Alternatively, the antibody can be
attached to the matrix and exposed to C1q, followed by detection of
C1q. Such in vitro binding assays are common in the art and are
amenable to modification and optimization as necessary.
E. Diagnostic Methods
[0327] Cognitive function assessment tools. A number of tools exist
to quantify the cognition and mental function of dementia patients.
These include the NTB, DAD, ADAS, MMSE, CDR-SOB, NINCDS-ADRDA
criteria, and the RMHI (Rosen Modified Hachinski Ischemic) score.
These tools are generally known in the art.
[0328] The NTB (Neuropsychological Test Battery) is composed of
nine well-accepted tests of memory and executive function. The test
battery is acceptable in the most recent EMEA guidance. Patients
are generally assessed in the following memory tests periodically:
Weschsler Memory Scale Visual Paired Associates; Weschsler Memory
Scale Verbal Paired Associates; and Rey Auditory Verbal Learning
Test. The Executive function tests include: Wechsler Memory Scale
Digit Span; Controlled Word Association Test; and Category Naming
Test. This test is sensitive to change in mild A.beta. patients and
clinical effects of amyloid lowering agents.
[0329] The DAD (Disability Assessment for Dementia) test was
developed and validated to measure the functional disability of
patients with Alzheimer's disease (Gelinas et al. (1999) Am J Occup
Ther 53:471-81.) Caregivers answer questions about the patients'
ability to perform both instrumental and basic activities of daily
living that had been attempted in the preceding two weeks. The
proportion of DAD activities successfully completed out of those
attempted is then determined and reported as a percentage.
[0330] The ADAS-Cog refers to the cognitive portion of the
Alzheimer's Disease Assessment Scale (see Rosen, et al. (1984) Am J
Psychiatry 141:1356-64.) The test consists of eleven tasks that
measure disturbances in memory, language, praxis, attention and
other cognitive abilities.
[0331] The NINCDS-ADRDA (Neurological and Communicative Disorders
and Stroke-Alzheimer's disease Related Disorders Assessment)
measures eight criteria affected in Alzheimer's: memory, language,
perceptual skills, attention, constructive abilities, orientation,
problem solving, and functional abilities (McKhann et al. (1984)
Neurology 34: 939-44)
[0332] The MMSE (Mini Mental State Exam), CDR-SOB (Clinical
Dementia Rating--Sum of Boxes, and RMHI (Rosen Modified Hachinki
Ischemic) score are also known in the art (see, e.g., Folstein et
al. (1975) J Psych Res 12: 189-198; Morris (1993) Neurology 43:
2412-2414; and Rosen et al. (1980) Ann Neurol. 17:486-488).
[0333] Biomarkers. Biomarkers for Alzheimer's symptomology in
humans can be measured using MRI volumetrics, blood and CSF protein
levels, and PET (positron emission topography). For example,
biomarkers to support antibody-A.beta. engagement include A.beta.40
and A.beta.42 in the CSF and plasma, and amyloid plaque imaging,
e.g., by PET. Biomarkers pointing to disease modification include
brain morphology (MRI), CSF tau and phosphotau levels, and again,
amyloid plaque imaging.
XII. Examples
Example 1
Phase 1 Trial
[0334] 111 patients with a diagnosis of probable Alzheimer's
disease (mild to moderate) were administered the humanized antibody
bapineuzumab at doses ranging from 0.15 to 2.0 mg/kg in a multiple
ascending dose study (MAD). Antibody was administered by
intravenous infusion every thirteen weeks until the dosing regime
is complete. Patients were also classified for ApoE4 status. Table
2 shows that eleven patients in the study experienced vasogenic
edema detected by MRI. Table 2 also shows symptoms experienced in
some of these patients; in other patients the vasogenic edema was
asymptomatic. Table 3 shows the risk of vasogenic edema stratified
by genotype irrespective of dose. The risk is only 2% in patients
lacking an E4 allele but is 35% in patients with two E4 alleles.
Table 4 shows the risk of vasogenic edema in only the highest dose
group (2 mg/kg). The risk of vasogenic edema for patients with two
E4 alleles is 60% and that for patients with one allele is 35%.
[0335] Table 5 shows the risk of vasogenic edema at different
dosages. The risk of vasogenic edema is very low for all genotypes
for doses between 0.15-0.5 mg/ml but starts to become significant
for patients with two E4 alleles at a dose of 1 mg/kg and for
patients with one E4 allele at 2 mg/kg. These data indicate that
the risk of vasogenic edema is dependent on both ApoE genotype and
dose and patients.
TABLE-US-00075 TABLE 2 Study Dose (mg/kg) Dose # E4 status Symptoms
SAD 5 1 ND -- SAD 5 1 ND -- SAD 5 1 ND dizziness, confusion MAD
0.15 2 4/4 abn gait, confusion MAD 1 1 4/4 visual MAD 1 1 4/4 --
MAD 1 2 3/4 -- MAD 2 1 4/4 -- MAD 2 1 3/4 -- MAD 2 1 4/4 confusion
MAD 2 1 3/4 -- MAD 2 1 3/4 HA, lethargy, confusion MAD 2 2 3/4 --
PET 2 1 3/4 -- MAD 2 3 4/4 --
TABLE-US-00076 TABLE 3 ApoE.sub.4 VE cases VE % genotype genotype/
% cases/patients of patients (alleles) total VE cases of VE cases
exposed exposed 2 6/11 55% 6/17 35% 1 4/11 36% 4/52 8% 0 1/11 9%
1/42 2%
TABLE-US-00077 TABLE 4 ApoE.sub.4 VE cases VE % genotype genotype/
% cases/patients of patients (alleles) total VE cases of VE cases
exposed exposed 2 3/7 43% 3/5 60% 1 3/7 43% 3/9 33% 0 1/7 14% 1/14
7%
TABLE-US-00078 TABLE 5 Number of patients (number developing
vasogenic edema) ApoE4 copy # 0.15 mg/kg 0.5 mg/kg 1.0 mg/kg 2.0
mg/kg 0 13 (0) 11 (0) 9 (0) 14 (1) 1 15 (0) 14 (0) 14 (1) 9 (3) 2 3
(1) 4 (0) 5 (2) 5 (3)
Example 2
Phase 2 Trial
[0336] A randomized double-blind placebo-controlled multiple
ascending dose study was conducted on a population of 234 patients
randomized from an initial population of 317 screened patients.
Patients were assessed for ApoE4 carrier status, but carriers
(homozygous and heterozygous) and non-carriers received the same
treatment. Inclusion criteria were: probable A.beta. diagnosis;
aged 50-85 years; MMSE score 16-26; Rosen Modified Hachinski
Ischemic score .ltoreq.4; Living at home or in a community dwelling
with a capable caregiver; MRI consistent with diagnosis of A.beta.;
MRI scan of sufficient quality for volumetric analysis; stable
doses of medication for treatment of non-excluded conditions;
stable doses of AchEIs and/or memantine for 120 days prior to
screen. The main exclusion criteria were: current manifestation of
a major psychiatric disorder (e.g., major depressive disorder);
current systemic illness likely to result in deterioration of the
patient's condition; history or evidence of a clinically important
auto-immune disease or disorder of the immune system; history of
any of the following: clinically evident stroke, clinically
important carotid or vertebro-basilar stenosis/plaque, seizures,
cancer within the last 5 years, alcohol/drug dependence within last
2 years, myocardial infarction within the last 2 years, a
significant neurologic disease (other than A.beta.) that might
affect cognition. Kits of the invention and their accompanying
labels or package inserts can provide exclusions for patients
meeting any of the above exclusion criteria and any subcombinations
thereof.
[0337] Four dose levels were employed (0.15, 0.5, 1.0 and 2.0
mg/kg) together with a placebo. 124 patients received bapineuzumab
and 110 received a placebo. Of those patients, 122 and 107,
respectively, were analyzed for efficacy. Bapineuzumab was supplied
as a sterile aqueous solution in 5 ml vials containing: 100 mg of
bapineuzumab (20 mg/mL), 10 mM histidine, 10 mM methionine, 4%
mannitol, 0.005% polysorbate-80 (vegetable-derived), pH of 6.0. The
placebo was supplied in matching vials containing the same
constituents except for bapineuzumab. The study medication was
diluted in normal saline and administered as a 100 ml intravenous
(IV) infusion over .about.1 hour
[0338] The treatment period was for 18 months with 6 intravenous
infusions at 13 week intervals. Safety follow-up visits, including
MRI scans occurred 6 weeks following each dose. Following the
treatment period patients were either monitored with a 1 year
safety follow up for continued treatment in open label extension.
The primary objective of the trial was to evaluate the safety and
tolerability of bapineuzumab in patients with mild to moderate
Alzheimer's disease. The primary endpoints for the study were
(Alzheimer Disease Assessment Scale-Cognitive Subscale (ADAS-Cog),
Disability Assessment Scale for Dementia (DAD) together with safety
and tolerability). The ADAS-Cog 12 contains an additional test
involving delayed recall of a ten item word list relative to the
ADAS-Cog 11. The secondary objective of the study was to evaluate
the efficacy of bapineuzumab in patients with mild to moderate
Alzhiemer's disease. Other end points were neuropsychological test
battery (NTB), neuropsychiatric inventory (NPI), clinical dementia
rating sum of boxes (CDR-SB), MRI brain volumetrics, and CSF
measures.
[0339] A summary of the total population, the populations broken
down by dosage group and populations broken down by carrier status
is provided is the following tables.
TABLE-US-00079 TABLE 6 Demographics and Patient Characteristics All
Placebo All Bapineuzumab N = 107 N = 122 Age 67.9 70.1 Gender (% F)
59.8 50.0 Ethnicity (% Caucasian) 95.3 96.7 Years Since Onset 3.7
3.5 ApoE4 (% carrier) 69.8 60.5 Screening MMSE 20.7 20.9 %
Cholinesterase or 96.3 95.1 Memantine Use
TABLE-US-00080 TABLE 7 % Con # of patients Avg Avg Disease Disease
Severity APOE Alz Wk Bapineuzumab MMSE Age Duration Mild Moderate
Carrier Meds Baseline 78 0.15 mg/kg 20 70 4 29% 71% 64% 100% 31 24
Placebo 20 64 4 33% 65% 46% 96% 26 17 0.5 mg/kg 21 71 4 48% 51% 58%
91% 33 17 Placebo 21 69 4 43% 57% 86% 93% 28 21 1.0 mg/kg 21 69 3
43% 55% 69% 97% 29 25 Placebo 21 69 4 36% 69% 75% 93% 26 21 2.0
mg/kg 2 70 3 63% 34% 53% 90% 29 17 Placebo 21 69 3 56% 44% 70% 100%
27 22 All 21 70 4 46% 53% 61% 95% 122 83 Bapineuzumab All Placebo
21 68 4 42% 59% 69% 96% 107 81
TABLE-US-00081 TABLE 8 Carrier Non-carrier Placebo Bapineuzumab
Placebo Bapineuzumab N = 74 N = 72 N = 32 N = 47 Age 68.6 71.2 66.1
69.1 Gender (% F) 59.5 48.6 62.5 51.1 Ethnicity 97.3 97.2 90.6 95.7
(% Caucasian) Years Since Onset 3.8 3.7 3.5 3.0 Screening MMSE 21.0
20.6 19.8 21.4 % Cholinesterase or 95.9 98.6 96.9 89.4 Memantine
Use
[0340] Comparison of the various dosage cohorts with placebo using
a linear model of cognitive decline on ADAS-COG and DAD scales did
not achieve statistical significance for any of the dosage cohorts
or the combined dosage cohorts population.
[0341] The data were reanalyzed using a statistical model not
assuming linear decline (a) based on all of the patients in whom
efficacy was determined and (b) based only on patients who had
received all six dosages ("completers") and not including patients
who had dropped out for various reasons. The non-linear model is
believed to be more accurate because the cognitive abilities do not
necessarily decline linearly with time.
[0342] The results using the non-linear decline model for all of
the patients in whom efficacy was determined (ApoE4 carriers and
non-carriers combined) are shown in FIG. 1. MITT (modified intent
to treat) analysis was done using the repeated measures model
without assumption of linearity. Bars above the X-axis represent a
favorable result (i.e., inhibited decline) relative to placebo.
Although statistical significance was not obtained, a trend was
observed for the combined dosage cohorts using the ADAS-cog and NTB
scales (0.1.gtoreq.p.gtoreq.0.05).
[0343] The results for the completer populations (ApoE4 carriers
and non-carriers combined) are shown in FIG. 2. Completers were
defined as patients who received all 6 infusions and an efficacy
assessment at week 78. Bars above the axis indicate improvement
relative to placebo. Statistical significance was obtained for the
combined dosage cohorts for ADAS-cog and DAD measurements and a
positive trend (0.1.gtoreq.p.gtoreq.0.05) was found for NTB
measurement.
[0344] Separate analyses were performed for ApoE4 carriers and
non-carriers using the non-linear model and (a) all treated
patients in whom efficacy was determined and (b) completers.
[0345] FIG. 3 shows the results for all ApoE4 carrier patients in
which efficacy was measured. Statistical significance was not found
for any of the cognitive scales. Again, MITT analysis used repeated
measures model without assumption of linearity. FIG. 4 shows the
analysis for ApoE4 carrier completers, as defined above. Again,
statistical significance was not found by any of the scales
(ADAS-cog, DAD, NTB, and CDR-SB). However, favorable directional
changes (bars above the axis) were found particularly for the
ADAS-cog and DAD measurements.
[0346] FIGS. 5 and 6 show the results for all ApoE4 non-carrier
patients in whom efficacy was measured. Statistical significance
was obtained for ADAS-cog, NTB, CDR-SB and MMSE measurements for
the combined dosage cohorts. Bars above the axis indicate
improvement relative to placebo. FIG. 9 shows time course analysis
of these parameters (ADAS-cog, upper left, DAD, upper right, NTB,
lower left, CDR-SB, lower right). The decline in cognitive
performance for treated patients was less than that of placebo at
all time points on the ADAS-cog, NTB and CDR-SB scales. FIGS. 7 and
8 show the analysis for ApoE4 non-carrier completers, as defined
above. Statistical significance was again obtained for ADAS-cog,
NTB, CDR-SB and MMSE measurements. Again, bars above the axis
indicate improvement relative to placebo.
[0347] MRI was performed up to seven times per patient during the
study six weeks after each infusion. Changes in the brain were
assessed by brain volume, ventricular volume, brain boundary shift
integral and ventricular boundary shift integral. The boundary
shift integral (BSI) as a measure of cerebral volume changes
derived from registered repeat three-dimensional magnetic resonance
scans. The BSI determines the total volume through which the
boundaries of a given cerebral structure have moved and, hence, the
volume change, directly from voxel intensities. The ventricular
shift integral is a similar measurement of ventricular space
changes. Both of these parameters increase as Alzheimer's disease
progresses. Thus, inhibition of the increase in these parameters
relative to placebo shows a positive (i.e., desired) effect of
treatment.
[0348] In the total treated population (carriers and non-carriers)
no significant differences were found for changes in brain volume
measured by brain boundary shift integral or ventricular volume
measured by ventricular boundary shift integral over 78 weeks
compared with the placebo population.
[0349] In the treated non-ApoE4 carrier population brain volume
decline was significantly lower than the non-ApoE4 placebo
population (mean -10.7 cc; 95% CI: -18.0 to -3.4; p=0.004). The
increase in ventricular volume compared to placebo was also reduced
but the change did not reach statistical significance. There was no
significant change in brain volume compared with the ApoE4 placebo
population. However, the ventricular volume increased significantly
compared to placebo (mean 2.5 cc; 95% CI: 0.1 to 5. 1;
p=0.037).
[0350] The changes of BBSI in the total population, ApoE4 carrier
population and ApoE4 non-carrier population are shown in FIGS.
10-12. FIG. 12 (ApoE4 non-carriers) shows a statistically
significant separation between the lines for treated patients and
placebo. The change in brain volume was reduced in the treated
population relative to placebo at all measured time points. FIG. 10
(combined ApoE4 carriers and non-carriers) shows separation of the
lines for treated and placebo patients but the results did not
reach statistical significance. FIG. 11 (ApoE4 carriers) shows the
lines for treated and placebo patients are virtually superimposed.
Analysis used repeated measures model with time as categorical,
adjusting for APOE4 carrier status. Baseline was whole brain volume
and MMSE stratum.
[0351] A trend was observed for reduction in CSF phospho-tau in the
bapineuzumab treated patient population relative to the placebo
treated population at 52 weeks into the trials (FIG. 13).
Phospho-tau is a biomarker associated with Alzheimer's disease. No
significant differences were found between CSF levels of tau and
A.beta.42 between all treated patients and controls. The figure is
based on ANCOVA analysis, adjusted for baseline value. One outlier
was excluded in the 0.15 mg/kg placebo dose cohort.
[0352] Treatment was generally safe and well tolerated. Vasogenic
edema (VE) occurred only in bapineuzumab treated patients. VE
occurred with greater frequency in ApoE4 carriers (10) than
non-carriers (2) and at greater frequency with increasing dose,
there being 8, 3, 0 and 1 episodes at doses of 2.0, 1.0, 0.5 and
0.15 mg/kg respectively. All VE episodes occurred after the first
or second dose. Most episodes of VE were detected only by MRI and
had no detected clinical symptoms. The VE episodes resolved over
weeks to months. In one patient, the VE was treated with steroids.
Excluding VE, and excluding the 0.15 mg/kg cohort (which contained
patients with more advanced disease than other cohorts), serious
adverse events were similar between treated and placebo groups.
Adverse events were generally mild to moderate, transient,
considered unrelated to study drug, occurred in relatively small
proportion of patients and did not appear to be dose-related.
[0353] Serum concentration of bapineuzumab and plasma concentration
of A.beta. were measured in treated patients over time for the
different dosage cohorts as shown in FIG. 14. The Cmax for serum
bapineuzumab ranged from about 3.5-50 .mu.g/ml in the different
dosage cohorts from 0.15 mg/kg to 2.0 mg/kg. The profile of mean
plasma concentration of A.beta. mirrored that of mean serum
bapineuzumab with the concentration of plasma A.beta. rising on
dosing with bapineuzumab and declining as the concentration of
bapineuzumab declined. The concentration of plasma A.beta. ranged
from about 500-3000 pg/ml. The variation of plasma concentration of
A.beta. between different dosage cohorts showed less variation than
the variation between doses. For example, increasing the dose from
0.15 mg/kg to 2 mg/kg increases plasma A.beta. by about a factor of
2.
[0354] The PK parameters after the first infusion of bapineuzumab
are summarized in Table 9 below.
TABLE-US-00082 TABLE 9 Dose Cmax Cavg Cmin Tmax AUCinf CL/F Vz/F
T1/2 (mg/kg) (.mu.g/mL) (.mu.g/mL) (.mu.g/mL) (days) (.mu.g h/mL)
(mL/hr/kg) (mL/kg) (days) 0.15 4.6 0.7 0.1.dagger-dbl. 0.1 1794
0.09 76.2 26.7 0.5* 17.7 3.0 1.1.dagger-dbl. 0.4 7165 0.07 63.7
26.4 1.0 28.0 5.5 1.8.dagger-dbl. 0.1 13499 0.08 75.4 28.4 2.0 56.3
9.5* 1.7.dagger-dbl. 0.1 21802* 0.09* 65.8* 20.5* N = 6 unless
otherwise specified; *n = 5 .dagger-dbl.trough values of 2nd
infusion; all values below limit of quantification for trough of
1st infusion
Abbreviations: Cavg--Average concentration over 13 weeks;
Cmin--Minimum concentration ("trough"); Tmax--Time of maximum
concentration; AUC inf--Area under Concentration vs. time curve
extrapolated to infinity; CLss/F--ratio of the extravascular
clearance at steady state (CLss) and extent of bioavailability (F);
Vz/F--ratio of apparent volume of distribution at steady state (Vz)
and F; t 1/2--elimination (or terminal) half-life in days.
Conclusions
[0355] 1. The trial provides evidence that ApoE4 carriers and
non-carriers react differently to immunotherapy.
[0356] 2. The trial provides evidence that vasogenic edema occurs
more frequently in ApoE4 carriers and at higher dosages.
[0357] 3. The trial provides statistically significant evidence of
efficacy in non-ApoE4 carriers and in patients receiving at least 6
doses of bapineuzumab (ApoE4 carriers and non-carriers).
[0358] 4. The trial provides evidence of trends or favorable
directional changes in a total population (ApoE4 carriers and
non-carriers) and ApoE4-carrier population by some measures.
Statistical significance might be shown with larger populations.
Alternative treatment regimes in these patients such as discussed
above are likely to improve efficacy as discussed above.
[0359] 5. The trial provides evidence that the treatment is
generally safe and well tolerated.
Example 3
Clinical Study of Subcutaneous Administration of Bapineuzumab in
Alzheimer's Patients
[0360] Subcutaneous injections are generally easier to administer,
which can be a consideration for patients with impaired mental
function and coordination, or caregivers administering to an
uncooperative patient. It is also easier to do at home, which is
less upsetting to the patient, as well as less expensive. Finally,
subcutaneous administration usually results in a lower peak
concentration of the composition (Cmax) in the patient's system
than intravenous. The reduced peak can reduce the likelihood of
vasogenic edema.
[0361] For these reasons, a clinical study was designed for
subcutaneous administration of bapineuzumab. The primary endpoints
for the initial study are safety and bioavailability. Once these
are established for subcutaneous administration, the cognitive
tests described above will be administered to determine
efficacy.
[0362] Under the initial regime, bapineuzumab is administered
subcutaneously to patients every 13 weeks for 24 months, for a
total of 9 doses. All patients receive a dose of 0.5 mg/kg.
Patients are screened and periodically monitored as described in
the above examples, e.g., for blood levels of the antibody, heart
function, and vasogenic edema.
Example 4
Design of Specific Mouse and Human Antibodies
[0363] Variants of humanized and mouse 3D6 antibodies differing in
isotype and or constant region mutations were constructed to test
effects of reducing effector function on amyloid deposit clearing,
cognitive function and microhemorrhaging. Mice treated with
antibodies to A.beta. proteins often exhibit signs of
microhemorrhage in cerebral vessels, which is one factor that my be
related to the vasogenic edema observed in human patients
undergoing similar treatment.
[0364] An alignment of the CH2 domains of human IgG1, IgG2, and
IgG4 with mouse IgG1 and IgG2a are shown in FIG. 15. The alignment
highlights the residues responsible for FcR and C1q binding. The
C1q binding motif is conserved across species and isotypes. The FcR
binding motif is conserved in human IgG1, IgG4, and murine
IgG2a.
[0365] The following table discloses the particular modifications
made to the CH2 region of the heavy chain. The amino acid numbering
is by the EU system. The format is wildtype residue, position,
mutant residue.
TABLE-US-00083 TABLE 10 3D6 Derivative Antibodies 3D6 Derivative
Antibody Isotype (species) Mutated Residues Bapineuzumab Control
IgG1 (human) -- AAB-001 Humanized 3D6 2m IgG1 (human) L234A/G237A
(Fc.gamma.R) (EU numbering) Humanized 3D6 3m IgG1 (human)
L234A/L235A/G237A (Fc.gamma.R) AAB-003 (EU numbering) Humanized 3D6
1m IgG4 (human) S241P (hinge region) (Kabat numbering) 3D6 Control
IgG1 (mouse) -- 3D6 1m (Fc.gamma.R) IgG1 (mouse) E233P 3D6 3m (C1q)
IgG1 (mouse) E318A/K320A/R322A 3D6 4m (Clq) IgG1 (mouse)
E318A/K320A/R322A/E233P 3D6 Control IgG2a (mouse) -- 3D6 1m
(Fc.gamma.R) IgG2a (mouse) D265A 3D6 4m (Fc.gamma.R, C1q) IgG2a
(mouse) L235A/E318A/K320A/K322A
[0366] The epitope-binding regions of 3D6 derivative antibodies are
the same, and the kinetics of A.beta. binding are comparable. Table
11 discloses the kinetics of the Fc receptor binding to the 3D6
derivative antibodies listed in Table 10. These values were
generated as follows.
[0367] For the humanized 3D6 derivative antibodies, the following
assay conditions were used. A Biacore 3000 and CM5 chip coated with
penta-His (SEQ ID NO: 93) antibody (Qiagen, Cat # 34660) was used
in combination with His-tagged domains of human Fc.gamma.RI,
Fc.gamma.RII, and Fc.gamma.RII (R&D Systems, Cat # 1257-Fc,
1330-CD, 1597-Fc). Each receptor was separately captured in one
flow cell of the sensor chip by the penta-His (SEQ ID NO: 93)
antibody. A solution of the antibody to be tested was injected to
enable measurements of association and dissociation rates to the
captured receptor. After measurements were completed, the receptors
and experimental antibodies were removed by injection of buffer at
pH2.5. The flow cell was then ready for the next cycle. Each cycle
was carried out in duplicate, and the same conditions (e.g.,
concentrations, flow rates, and timing) were used for each
sample.
[0368] As indicated by the values in Table 11, bapineuzumab
(unmodified Fc region) bound to all of the human Fc.gamma.R
receptors with relatively high affinity. KD for Fc.gamma.RI was in
the nm range, while KD for Fc.gamma.RII and III were in the .mu.m
range. For the latter two, the sensorgrams showed typical fast-on,
fast-off kinetics. IgG4 isotype had similar binding to Fc.gamma.RI,
but did not bind Fc.gamma.RIII, as expected. The two IgG1
derivatives, Hu 3D6 2 m and 3 m, did not show detectable binding to
either Fc.gamma.RI or Fc.gamma.RIII.
[0369] For the mouse 3D6 derivative antibodies, similar methods
were used to determine binding to mouse Fc.gamma.RI, II, and III.
Fc.gamma.RI and III are activating receptors, while Fc.gamma.RII is
generally considered to be inhibitory. The antibodies tested were
3D6 IgG2a, 3D6 IgG1, and the IgG1 mutants, 3D61 m, 3 m and 4 m.
Results are expressed as a relative percentage of 3D6 IgG2a
binding. As shown in Table 11, 3D6 IgG2a was the only antibody with
detectable Fc.gamma.RI binding ability. 3D6 IgG1 and the 3D6 3 m
IgG1 had similar Fc.gamma.RII and III binding profiles.
TABLE-US-00084 TABLE 11 Fc Receptor Binding Ability of 3D6
Antibodies Relative Binding Capability* (%) Human 3D6 Derivative
Human Fc.gamma.RI** Human Fc.gamma.RII** Fc.gamma.RIII**
Bapineuzumab Control 100 100 100 Humanized 3D6 1m 85-95 40-50 0
Humanized 3D6 2m 0 40-50 0 Humanized 3D6 3m 0 8-12 0 AAB-003 Mouse
Mouse Fc.gamma.RI** Mouse Fc.gamma.RII** Fc.gamma.RIII** 3D6
Control IgG2a 100*** 100 100 3D6 Control IgG1 0 180 70 3D6 1m IgG1
0 15 10 3D6 3m IgG1 0 180 70 3D6 4m IgG1 0 25 15 *Defined as the
amount of binding in (RU) relative to that of IgG2a control at the
steady state **The mFc.gamma.RI and mFc.gamma.RIII are activating
receptors, mFc.gamma.RII is an inhibitory receptor. Another potent
activating receptor, mFc.gamma.RIV, is not commercially available.
***A steady-state binding was not reached. Kinetic fitting led to
an estimate of K.sub.D in the nanomolar range.
[0370] The above results show that that the Hu 3D6 3 m (AAB-003)
antibody has the most reduced Fc gamma receptor binding of the
three tested. Of those tested, the 3D6 .mu.m IgG1 mouse mutant
antibody was the most similar to AAB-003, in that its Fc.gamma.R
binding was reduced to near 10% of normal.
Example 5
Mouse Studies of 3D6 Derivative Antibodies
Study Design
[0371] One-year old PDAPP mice were exposed to a 6 month treatment
paradigm with control or the 3D6 derivative antibodies described in
Table 10. The negative control was a mouse IgG2a antibody to an
irrelevant, non-amyloid epitope. The mice were injected IP with 3
mg/kg of the indicated antibody each week.
[0372] Serum antibody concentrations were tested over the course of
the study by ELISA. Levels were comparable in all groups. After six
months, the mice were sacrificed and perfused. Brain sections and
tissues were prepared according to known methods (Johnson-Wood et
al. (1997) Proc. Natl. Acad. Sci., USA 94:1550-55).
[0373] Amyloid burden was measured in the cortex and hippocampus of
transgenic mice. Results in Table 12A and 12B are indicated as
percentage reduction of area with amyloid (p values indicate
significant difference compared to IgG2a control antibody).
TABLE-US-00085 TABLE 12A Cortical Amyloid Burden (% reduction)
Control 3D6 Control 3D6 Control 3D6 1m IgG1 3D6 3m IgG1 IgG2a IgG2a
IgG1 (Fc.gamma.R) (C1q) Median % 6.25076 0.757259 1.24205 2.06056
1.50084 Area Range 0.069-17.073 0-9.646 0-17.799 0-24.531 0-17.069
% Change -- 88 80 67 76 Control p < 0.0001 p < 0.0001 p <
0.003 p < 0.0001 IgG2a % Change -- -- -- 165.9 120.8 3D6 IgG1
Number 32 34 36 36 34
TABLE-US-00086 TABLE 12B Hippocampal Amyloid Burden (% reduction)
Control 3D6 Control 3D6 Control 3D6 1m IgG1 3D6 3m IgG1 IgG2a IgG2a
IgG1 (Fc.gamma.R) (C1q) Median % 20.36 8.462 12.29 12.18 8.435 Area
Range 4.707-35.79 1.467-17.59 0.2449-18.61 0-26.99 0.8445-18.61 %
Change -- 58 40 40 59 Control -- p < 0.0001 p < 0.0001 p <
0.0001 p < 0.0001 IgG2a % Change -- -- -- 0.895 31.4 3D6 IgG1
number 34 34 37 37 34
[0374] The above results indicate that all of the 3D6 antibodies
(IgG2a, IgG1 and mutants) significantly reduced amyloid burden
relative to negative controls. Differences between the tested
antibodies were not statistically significant.
[0375] The effect of the 3D6 derivative antibodies was then tested
on vascular amyloid ratings. Table 13 shows the number of mice with
the indicated vascular amyloid rating and the percentage of animals
with a rating of 4 or greater (p values indicate significant
difference compared to 3D6 IgG2a antibody).
TABLE-US-00087 TABLE 13 % of Mice Having Vascular Amyloid Moderate
Percentage with None-little (0-3) (4+) moderate rating Control
IgG2a 11 24 69 p < 0.0001 3D6 Control IgG2a 27 7 21 -- 3D6
Control IgG1 12 25 68 p < 0.0001 3D6 1m (Fc.gamma.R) IgG1 15 21
58 p < 0.0016 3D6 3m (C1q) IgG1 20 17 46 <0.0434
[0376] The above data show that the positive control 3D6 IgG2a
significantly reduced vascular amyloid relative to the irrelevant
IgG2a antibody. The reduction with 3D6 IgG2a was also statistically
significant relative to that with 3D6 IgG1, 3D61 m IgG1 and 3D6 3 m
IgG1. Differences between 3D6 IgG1, 3D61 m IgG1 and 3D6 3 m IgG1
and control IgG2a were not statistically significant.
[0377] To determine whether the 3D6 antibody derivatives cause
microhemorrhage in mice, hemosiderin levels, a marker for
microhemorrhage, were examined in brain sections of mice treated
with 3 mg/kg antibody. Staining was carried out with 2% potassium
ferrocyanide in 2% hydrochloric acid, followed by a counterstain in
a 1% neutral red solution. Table 14 indicates the percentage and
absolute number of mice with the indicated level of hemosiderin
staining. The results demonstrate that 3D6 .mu.m IgG1 (Fc.gamma.R)
and 3D6 3 m IgG1 (C1q), which are shown above to be effective in
clearing amyloid plaques, reduce microhemorrhage levels relative to
3D6 IgG2a. Differences between 3D6 IgG1, 3D6 .mu.m IgG1 and 3D6 3 m
IgG1 did not reach statistical significance, although the
difference between 3D61m IgG1 and 3D6 IgG1 showed a trend. (p
values indicate significant difference compared to 3D6 IgG2a
antibody).
TABLE-US-00088 TABLE 14 Microhemorrhage level: 0 1 2 3 Control
IgG2a 68% (23) 32% (11) 0% (0) 0% (0) p < 0.0001 3D6 Control
IgG2a 9% (3) 42% (14) 27% (9) 21% (7) -- 3D6 Control IgG1 38% (14)
46% (17) 3% (1) 13% (5) p < 0.0023 3D6 1m IgG1 51% (19) 49% (18)
0% (0) 0% (0) (Fc.gamma.R) p < 0.0001 3D6 3m IgG1 (C1q) 53% (19)
42% (15) 0% (0) 5% (2) p < 0.0001
Example 6
Phagocytosis Assays
Materials and Methods
[0378] Ex vivo plaque phagocytosis assays: Frozen brain sections
from PDAPP mice were pre-incubated with 3D6 IgG1 and the effector
function mutants described in Table 10 (3D61 m (Fc.gamma.RI) and
3D6 3 m (C1q), both mouse IgG1 isotype). 3D6 IgG2a was used as a
positive control and irrelevant IgG1 and IgG2a antibodies were used
as isotype controls. Sections were treated with 0.3 or 3 .mu.g/ml
antibody for 30 minutes prior to addition of mouse microglia, at 5%
CO.sub.2 at 37 C. The co-cultures were extracted the next day.
Remaining A.beta. was measured by ELISA (266 antibody for capture,
and 3D6-B for reporter) to assess A.beta. clearance.
[0379] Phagocytosis of murine IgG2a derivatives was tested. These
experiments included: 3D6 IgG2a (positive control); non-specific
IgG2a (negative control); 3D6 .mu.m (Fc.gamma.R1, IgG2a isotype);
and 3D6 4 m (Fc.gamma.R1/C1q) antibodies. Conditions were similar
to those described above.
[0380] Non-plaque phagocytosis was additionally determined for
humanized 3D6 (Hu 3D6 IgG1) and the effector mutants described in
Table 10 (Hu 3D6 2 m IgG1, Hu 3D6 3 m IgG1, and Hu 3D61 m IgG4).
The negative control was an irrelevant human IgG1 antibody. Assay
and detection conditions were otherwise the same.
[0381] In vitro assays: For the mouse antibody assays of
fluorescently conjugated bead phagocytosis, 10 .mu.M FluoroSphere
particles (5.times.10.sup.6) were opsonized with 1 mg/ml of mouse
F(ab'2), 3D6 IgG2a, 3D6 IgG1, or the 3D6 Fc.gamma.R mutant for 2
hrs at RT with rotation. Following 2 hrs, beads were washed with 1
ml of PBS 3 times to remove unbound IgG. Opsonized particles were
added (1:10) to mouse microglia for the murine 3D6 Ig2a (3D62a)
experiments. Beads were incubated with the cells for 90 min at 37
C. Unbound particles were then washed away with PBS. Cells were
stained with DiffQuick for 30 sec for each stain and phagocytosis
was visualized by light microscopy. Controls for this assay were
un-opsonized beads (unlabelled) (to detect non-specific engulfment)
and pre-treatment with human Fc-fragments (3D62a+FC)(to block
Fc.gamma.R1).
[0382] For humanized antibody assays, conditions and detection were
the same. However, the antibodies were: no antibody (unlabelled;
negative control), irrelevant human IgG1 (Human IgG1; positive
control), Hu 3D6 IgG1, Hu 3D6 2 m IgG1, Hu 3D6 3 m IgG1, and Hu 3D6
.mu.m IgG4. The phagocytic cells were human THP-1 cells
(differentiated with PMA).
Results
[0383] Ex vivo plague phagocytosis assays: The murine 3D6 IgG1
antibody and its effector mutants (3D61 m (Fc.gamma.R1) and 3D6 3 m
(C1q)) were assayed to assess their ability to facilitate amyloid
clearance (see FIG. 16). The 3D6 IgG2a antibody stimulated more
robust clearance than 3D6 IgG1, 3D61 m (Fc.gamma.R1) and 3D6 3 m
(C1q). Stimulation of phagocytosis by 3D6 IgG1, 3D6 .mu.m
(Fc.gamma.R1) and 3D6 3 m (C1q) was greater than the negative
control. Mutations to the Fc domain of 3D6 IgG1 do not appear to
significantly dampen its ability to stimulate clearance in the ex
vivo clearance assay.
[0384] For the IgG2a 3D6 derivatives, the mutants stimulated
clearance equivalent to wild-type 3D6 IgG2a and to a greater degree
relative to an irrelevant IgG2 isotype matched control (see FIGS.
17A & B). Thus, neither of the mutants completely inhibited
A.beta. phagocytosis.
[0385] In the humanized antibody assays, mutations to the effector
region of the Hu 3D6 IgG1 retained significant clearing activity
relative to the negative control. Hu 3D6 IgG1 stimulated clearance
in the ex vivo A.beta. plaque clearance assay, and the effector
region mutants had moderately impaired function. Hu 3D6 IgG4
induced phagocytosis to the same extent as Hu 3D6 IgG1, and
mutation to the IgG4 hinge region of 3D6 did not appear to change
its effector function (see FIG. 18).
[0386] In vitro bead phagocytosis assays: To determine if the ex
vivo results were specific for A.beta. clearance and whether the Fc
mutation in the 3D6 IgG1 altered its effector function,
non-specific Fc-mediated bead phagocytosis assays were performed.
In the mouse antibody bead phagocytosis assay, the 3D6 IgG2a
isotype antibody mediated more efficient phagocytosis than 3D6 IgG1
(see FIG. 19). The Fc mutation in 3D6 IgG1 did not significantly
diminish the ability to stimulate phagocytosis, as compared to the
positive control 3D6 IgG2a, indicating that the Fc mutation in 3D6
IgG1 was moderately effective in reducing phagocytosis.
[0387] In the humanized antibody assay, the effect of the Fc
mutation seen in the ex vivo plaque phagocytosis assay was verified
on Fc-mediated bead phagocytosis. Again, the mutations in the Fc
portion of humanized 3D6 diminished its ability to mediate
phagocytosis of fluorescent beads and there was no significant
difference between the 2 m and 3 m mutants. Again, the
theoretically ineffective IgG4 isotype mediated removal to the same
extent as the IgG1 isotype (see FIG. 20). Mutation to the IgG4
hinge region of 3D6 does not appear to change its effector
function.
Example 7
C1q Binding Ability of Humanized 3D6 Derivatives
[0388] The humanized 3D6 derivatives were tested for ability to
bind C1q and induce a complement response. A standard C1q dilution
series protocol was followed, as described below. Similar protocols
are described, e.g., in Idusogie et al. (2000) J. Immunol. 164:
4178-4184.
[0389] Purified A.beta. was coated on to ELISA plates and exposed
to one of the following humanized 3D6 antibodies at the
concentrations indicated in FIG. 21: Hu 3D6 2 m (IgG1), Hu 3D6 3 m
(IgG1), Hu 3D6 .mu.m (IgG4), and unmodified Hu 3D6 (IgG1). The
ELISA plates were washed and then blocked with 0.02% Casein
solution in PBS for 3 to 24 hours with slow agitation. The blocking
solution was removed with another step of washing.
[0390] Next, purified human C1q (191391, MP Biomedicals) was added
to the ELISA plates, with 2 ug C1q/ml assay buffer starting the
2.times. dilution series. C1q was allowed to bind for 2 hours with
agitation. Following another wash step, 100 .mu.l/well anti-C1q
antibody (Rb anti human C1q FITC conjugated cat# F010 DBS
(dbiosys.com)) used at 1:200 was added for 1 hour with agitation.
Results were compared to a blank with no anti-C1q antibody.
[0391] As shown in FIG. 21, the humanized 3D6 derivative antibodies
did not significantly interact with C1q. This is in contrast to
bapineuzumab, which does not have mutations in the Fc region.
[0392] The derivative antibodies were tested for ability to induce
complement-mediated lysis of HEK 293 cells expressing A.beta. on
the surface. A standard .sup.51Cr release assay was used, as
described in Phillips et al. (2000)Cancer Res. 60:6977-84; Aprile
et al. (1981) Clin. Exp. Immunol. 46:565-76.
[0393] The target cells were HEK293 cells (ATCC, CRL-1573) that
expressed a fusion protein with the A.beta. epitope detected by 3D6
(DAEFR (SEQ ID NO: 94)) on the surface. The A.beta.-containing
sequence was inserted into the pDisplay vector (Invitrogen). The
pDisplay vector was altered to remove the HA tag and instead start
with the A.beta.-containing peptide after leader sequence. A stable
pool of HEK 293 was moved forward to the ADCC assay.
[0394] For labeling, 10.sup.7 cells were suspended in 2 ml RPMI 10%
FCS and added 250uCi of .sup.51Cr (NEN catalog #NEZ-030;
sodium.sup.51chromate in saline). Cells were incubated for 1 hour
at 37 C with occasional agitation. At the end of the incubation, 10
ml RPMI with 10% FCS was added. Cells were spun down so the
supernatant could be removed, and resuspended in 10 ml RPMI
containing 10% FCS. Cells were again incubated, at room temperature
for 1.5 hours with occasional agitation, to allow excess .sup.51Cr
to bleed from the cells. Target cells were washed 3 times with 10
ml RPMI, and a final time in 10 ml RPMI containing 10% FCS. Cells
were resuspended in RPMI with 10% FCS to a concentration of
10.sup.6 cells/ml.
[0395] Effector cells were collected from human blood. Briefly,
blood was diluted 1:1 with PBS and layered over Ficoll (Sigma
Histopaque 1077). The column was spun for 20 min, 1200.times.g,
with no brake at 20 C. Cells at the interface were collected;
washed once with 2-3 volumes PBS, and twice with RPMI containing
10% FCS. NK enrichment is detected with antibodies to CD3 and
CD56.
[0396] Effector cells and target cells were added to 96 well plates
at a ratio of 25:1 (effector:target) in a total volume of 200
.mu.l. The following control samples were included: Spontaneous
lysis (containing target cells with no effectors) and Total lysis
(leave wells empty) was included. The cells were incubated for 5
hours at 37 C. Just before harvest, 100 .mu.l 0.1% Triton X-100 was
added to the Total lysis sample to release .sup.51Cr. The reactions
were harvested onto filter units with a Skatron harvester
(Molecular Devices) and total .sup.51Cr was detected.
[0397] To calculate % lysis, the average cpm and standard deviation
was determined for each sample. The % Maximum .sup.51Cr Release is
determined with the following formula:
(Experimental-Spontaneous).times.100(Total-Spontaneous)
[0398] Consistent with the results of the C1q binding assay, the
humanized 3D6 effector function mutant derivative antibodies were
not effective at inducing complement lysis of the
A.beta.-expressing HEK 293 cells (see FIG. 22).
Example 8
ELISA Assay Measuring C1q Binding Ability of Murine 3D6
Derivatives
Materials and Methods
[0399] A 96-well fluorescent plate was coated with 1, 3, or 6
.mu.g/ml of various antibodies in 100 .mu.l well coating buffer
overnight at 4 C. After coating, plates were washed and blocked
with 200 .mu.l Casein Elisa Block for 1 hr at RT. Plates were
washed and 100 .mu.l of 2 .mu.g/ml human C1q in diluent buffer was
added for 2 hrs at RT. After 2 hrs, plates were washed and
FITC-labelled rabbit anti-C1q (1:1000) was added for 1 hr. Plates
were washed twice and read at 494/517 on the fluorescent plate
reader in PBS. The following mouse antibody samples were tested:
IgG2a, IgG2b, 3D6 IgG2a, IgG1, 3D6 IgG1, and the 3D6 IgG1 C1q
mutant.
Results
[0400] The highest level of C1q binding was observed for IgG2a and
3D6 IgG2a (see FIG. 23). C1q binding to IgG1 and 3D6 IgG1 was
significantly lower than IgG2a. The mutation in 3D6 IgG1 C1q
binding domain suppressed this binding further.
Example 9
Contextual Fear Conditioning (CFC) Assay
[0401] Tg2576 transgenic mice and wild-type littermate controls
were individually housed for at least 2 weeks prior to any testing
and allowed ad libitum access to food and water. CFC occurred in
operant chambers (Med Associates, Inc.) constructed from aluminum
sidewalls and PLEXIGLAS ceiling, door and rear wall. Each chamber
was equipped with a floor through which a foot shock could be
administered. In addition, each chamber had 2 stimulus lights, one
house light and a solenoid. Lighting, the footshock (US) and the
solenoid (CS) were all controlled by a PC running MED-PC software.
Chambers were located in a sound isolated room in the presence of
red light.
[0402] Mice (n=8-12/genotype/treatment) were trained and tested on
two consecutive days. The Training Phase consisted of placing mice
in the operant chambers, illuminating both the stimulus and house
lights and allowing them to explore for 2 minutes. At the end of
the two minutes, a footshock (US; 1.5 mAmp) was administered for 2
seconds. This procedure was repeated and 30 seconds after the
second foot shock the mice were removed from the chambers and
returned to their home cages.
[0403] Twenty hours after training, animals were returned to the
chambers in which they had previously been trained. Freezing
behavior, in the same environment in which they had received the
shock ("Context"), was then recorded using time sampling in 10
seconds bins for 5 minutes (30 sample points). Freezing was defined
as the lack of movement except that required for respiration. At
the end of the 5 minute Context test mice were returned to their
home cages.
[0404] Approximately 20-week old wild-type mice and Tg2576
transgenic mice were administered a single dose of treatment
antibody by intraperitoneal injection at 24 hours prior to the
training phase of the CFC. Treatment antibodies were: (i)
non-specific IgG1 antibody; (ii) Hu 3D6 3 m (Fc.gamma.R) (also
called AAB-003); and (iii) bapineuzumab (also called AAB-001).
[0405] FIG. 24 demonstrates the results. Control-treated wild type
mice showed about 40% freeze, while in comparison, control-treated
transgenic mice exhibited a severe deficit in contextual memory.
When administered at 30 mg/kg, the Hu 3D6 3 m antibody restored
cognitive function to wild type levels. Furthermore, the effector
function mutant had the same effect on contextual memory as the
parent antibody, bapineuzumab.
[0406] The effect of the Hu 3D6 3 m antibody on contextual memory
was observed over time. FIG. 25 illustrates that treatment with 30
mg/kg Hu 3D6 3 m antibody provided wild type levels of cognition at
least 5 days post-administration.
[0407] In summary, the above examples show that Hu 3D6 3 m results
in similar cognition improvements as bapineuzumab. This is despite
the fact that the derivative antibody does not significantly bind
to Fc receptors or C1q, or induce phagocytosis or ADCC
activity.
Example 10
Mouse Studies with 3D6 4 m (Fc.gamma.R/C1q) IgG2a and Hu 3D6 3 m
IgG1 (AAB-003)
Study Design
[0408] One-year old PDAPP mice are exposed to a 6 month treatment
paradigm with control; 3D6 4 m (Fc.gamma.R/C1q) IgG2a; or Hu 3D6 3
m IgG1 (see Table 10). Negative controls include a mouse IgG2a
antibody and a human IgG1 antibody to an irrelevant, non-amyloid
epitope. Positive controls include 3D6 IgG2a and Hu 3D6 IgG1. The
mice are split into dosage cohorts and injected IP at weekly
intervals with 3, 30, or 300 mg/kg of the indicated antibody.
Experimental conditions are as described in Example 5.
[0409] After 6 months, the mice are sacrificed and brain tissue
harvested as described above. Tissues are examined for cortical and
hippocampal Ab and amyloid burden, vascular amyloid, and
microhemorrhage.
Example 11
Cynomolgus Monkey Studies with Hu 3D6 3 m IgG1 (AAB-003)
Study Design
[0410] Cynomolgus monkeys are treated with Hu 3D6 3 m IgG1
(AAB-003). The negative control includes a human IgG1 antibody to
an irrelevant, non-amyloid epitope. The positive control include Hu
3D6 IgG1 (Bapineuzumab). Monkeys are split into dosage cohorts
receiving either 15, 50, or 150 mg/kg of the indicated antibody.
Each cohort is further split into IV and SC administration
groups.
[0411] Monkeys are injected weekly for 13 weeks, with a 2 month
observation period. At the end of the study, the monkeys are
sacrificed and brain tissue harvested. Tissues are examined for
cortical and hippocampal A.beta. and amyloid burden, vascular
amyloid, and microhemorrhage.
Example 12
Single Ascending Dose (SAD) Study in Humans of Hu 3D6 3 m (AAB-003)
Antibody
[0412] Mild to moderate Alzheimer's patients, including ApoE4
carriers and non-carriers, are divided into cohorts for intravenous
(IV) or subcutaneous (SC) injection with AAB-003 antibody. The
cohorts are given a single dose with a 12 month follow up, and
monitored throughout by an independent safety monitoring
committee.
[0413] The goal of the study is to increase the exposure equivalent
to at least 5 mg/kg of intravenous Bapineuzumab (unless signs of
vasogenic edema are observed). At this dose of Bapineuzumab, VE was
observed in 3 of 10 patients.
[0414] The SC cohorts include at least two subcutaneous dosage
levels. These patients are be observed for bioavailability of the
antibody and linearity thereof.
[0415] All patients are screened (e.g., for ApoE status) and
monitored as described in Example 1. For all cohorts, safety
monitoring includes MRI monitoring. MRI results are compared to
those from the Bapineuzumab study described in the above examples.
Efficacy is measured by cognitive metrics (e.g., NTB, DAD,
ADAS-Cog,); plasma A.beta. levels; CSF levels of amyloid, tau, and
phosphotau; and amyloid imaging.
[0416] Certain biomarkers are tracked in each patient during the
study. Biomarkers to support A.beta. binding by the antibody
include A.beta.40 and A.beta.42 in the CSF and plasma, and amyloid
plaque imaging, e.g., by PET. Biomarkers pointing to disease
modification include MRI, CSF tau and phosphotau levels, and again,
amyloid plaque imaging.
Example 13
Pharmacokinetic Profiles of Hu 3D6 3 m (AAB-003) in Tg2576 and Wild
Type Mice
[0417] Tg2576 transgenic mice and wild type controls were dosed
with AAB-003 subcutaneously (SC) or intraperitoneally (IP) to
determine bioavailability of the antibody. The profile was typical
for therapeutic antibody.
[0418] AAB-003 was eliminated slowly, with a T.sub.1/2 of 66-160
hours. There was low volume distribution (71-96) and good exposure
(as measured by AUC).
[0419] Some differences between the wild type and transgenic mice
were apparent. For example, wild type mice had higher AUC and
T.sub.1/2. The transgenic mice had slightly higher levels of
anti-AAB-003 antibodies.
Example 14
Pharmacokinetic Profiles of Hu 3D6 3 m (AAB-003) in Cynomolgus
Monkeys
[0420] 10 mg/kg Hu 3D6 3 m or bapineuzumab were administered
intravenously (IV) to cynomolgus monkeys (3 animals/antibody
treatment) to compare the pharmacokinetic profiles and determine
whether the effector function mutation had any effect. The results
were comparable between the two antibodies, and typical for
therapeutic antibodies in general. There was low clearance
(0.16.+-.0.06 ml/hr/kg), small volume of distribution (.about.62
ml/kg), and long elimination half-life (309.+-.226 hours). One of
the three animals tested positive for antibodies against
AAB-003.
[0421] The same antibody doses were administered subcutaneously
(SC). Bioavailability was good, approximating 69%, and the
half-life ranged from 21-445 hours. Two of the three animals tested
positive for antibodies against AAB-003.
Example 15
Effect of Fc Mutations on the Effector Function of an Anti-Lewis Y
Antibody
[0422] To determine the effect of mutations in the low hinge region
of human IgG1 on the effector function of antibodies with different
antigen specificity, we designed antibodies to the Lewis Y (LeY)
antigen. LeY is a type 2 blood group related difucosylated
oligosaccharide that is mainly expressed in epithelial cancers,
including breast, pancreas, colon, ovary, gastric, and lung. LeY
does not appear to be expressed on tumors of neuroectodermal or
mesodermal origin.
[0423] The anti-LeY Ab02 antibody was generated with one of three
heavy chain constant regions: (i) wild type human IgG1; (ii) wild
type human IgG4; and (iii) human IgG1 with two effector region
mutations, L234A and G237A (see SEQ ID NOs:50 and 51). IgG4 has
been shown to have reduced effector function in other systems.
[0424] For the ADCC (antibody-dependent complement cytotoxicity)
assay, LeY-overexpressing N87 human gastric adenocarcinoma cells
were used as target cells, and freshly isolated human PBMC were
used as effector cells. Effector and target cells were plated at a
ratio of 50:1 in 96 well plates. Antibody was applied at varying
concentrations (0.1, 1 and 10 .mu.g/ml) in triplicate with medium,
effector and target cell controls, and antibody controls. The ADCC
activities of anti-Lewis Y Ab02 versions are presented in FIG.
26.
[0425] For the CDC (complement dependent cytotoxicity) assay, LeY
positive tumor cells (A431 LeY) were plated in 96 well plates with
varying amount of antibody (0.1, 1 and 10 .mu.g/ml). Diluted human
complement (1:100), was added to each well. Tests were done in
triplicate at a final volume of 100 .mu.l/ml with medium, cells
alone, and antibody and complement controls. After 4 hours
incubation at 37 C, plates were removed and equilibrated to 22
C.
[0426] An equal volume of CytoTox-One.TM. was added to each well,
and incubated for 10 minutes at 22 C. As a positive control, 2
.mu.l of lysis buffer per well (in triplicate) was added to
generate a maximum LDH (lactate dehydrogenase) release in control
wells. The enzymatic reaction was stopped by adding 50 .mu.l of
stop solution. The resulting fluorescence was recorded with an
excitation wavelength of 560 nm and an emission wavelength of 590
nm. The % of complement-related cell lysis was calculated as % of
total LDH release (FIG. 27).
[0427] In spite of the L234A and G237A mutations in IgG1, the
mutant antibody fully retained its capacity to mediate both ADCC
and CDC against Lewis Y expressing tumor cells, as compared to wild
type IgG1.
Example 16
Effect of Fc Mutations on the Effector Function of Anti-5T4
Antibody
[0428] To investigate further the effect of Fc mutations in human
IgG1 on the effector function of antibodies with different antigen
specificity, we designed antibodies to the oncofetal protein 5T4.
5T4 is a tumor-associated protein displayed on the cell membrane of
various carcinomas, and is a promising target for anti-tumor
vaccine development and for antibody directed therapies.
[0429] The anti-5T4 antibody was generated with different
combinations of mutations in the heavy chain constant region. The
heavy chains used were: (i) wild type human IgG1; (ii) wild type
human IgG4; (iii) human IgG1, L234A and L235A; (iv) human IgG1,
L234A and G237A; (v) human IgG1, L235A and G237A; and (vi) human
IgG1 with three effector region mutations, L234A, L235A, and G237A
(see SEQ ID NOs:62 and 63).
[0430] Human breast carcinoma cell line MDAMB435, stably
transfected with 5T4 antigen, was used for the ADCC and CDC assays.
The ADCC assay of anti-5T4 antibodies was as described in Example
15, using freshly isolated human PBMC as effector cells at an
effector:target cell ratio 50:1. MDAMB435-Neo transfected cells
were used as a negative control. The results of ADCC activity
(maximum specific cytotoxicity at the antibody concentration 10
ug/ml) are summarized in Table 15.
TABLE-US-00089 TABLE 15 ADCC activity of anti-5T4 antibodies
against 5T4 positive and negative human breast carcinoma cell line
MDAMB435 MDAMB345-5T4 MDAMB-Neo Antibody % specific cytotoxicity %
specific cytotoxicity 5T4-IgG1wt 81 3 5T4-IgG1 78 2 L234A/G237A
5T4-IgG1 15 2 L234A/L235A 5T4-IgG1 27 2 L235A/G237A 5T4-IgG1 2 2
L234A/L235A/G237A 5T4-IgG1 5 3 N297A 5T4-IgG4 2 2
[0431] To evaluate an effect of Fc mutations on the complement
induced cytotoxicity, human breast carcinoma MDAMB435-5T4 cells
were incubated with diluted human complement as described in the
Example 15. The results of CDC assays are presented in the Table
16.
TABLE-US-00090 TABLE 16 CDC activity of anti-5T4 antibodies against
5T4 positive and negative human breast carcinoma cell line MDAMB435
MDAMB345-5T4 MDAMB-Neo Antibody % specific cytotoxicity % specific
cytotoxicity 5T4-IgG1wt 90 2 5T4-IgG1 72 2 L234A/G237A 5T4-IgG1 5 2
L3234A/L235A 5T4-IgG1 19 2 L235A/G237A 5T4-IgG1 1 1
L234A/L235A/G237A 5T4-IgG1 1 1 N297A 5T4-IgG4 1 1
[0432] The introduction of two mutations in the low hinge region of
human IgG1 in any of the combinations tried (L234A/L235;
L234A/G237A; L235A/G237A) only partially reduced ADCC and CDC
activity with L235A/G237A showing the higher residual effecter
function capabilities. However, anti-5T4 antibody with three
mutations in the IgG1 low hinge region (L234A/L235A/G237A)
demonstrated completely abolished ADCC and CDC activities.
Conclusions
[0433] The Examples provide a number of comparisons of Fc region
mutant antibodies with different antigen specificities. Example 6
describes an ADCC assay using A.beta.-specific antibodies with IgG1
Fc mutations at either L234A and G237A (double mutant), or L234,
L235A, and G237A (triple mutant). Both the double and triple
mutants had significantly reduced function (see FIG. 22). Example
15 describes ADCC and CDC assays using LeY-specific antibodies with
IgG1 mutations at L234A and G237A. In this case, the mutant
antibody retained effector function (see FIGS. 26 and 27). Finally,
Example 16 compares IgG1 Fc mutants of 5T4-specific antibodies.
Each of the double mutants (L234A/L235; L234A/G237A; L235A/G237A)
retained more effector activity than the triple mutant
(L234A/L235A/G237A) (see Tables 15 and 16). The effector activity
of the L234A/L235 double mutant, however, was reduced to nearly the
same level as that of the triple mutant.
[0434] The above results demonstrate that the effect of the
hinge-region mutations can depend on a number of factors, including
target antigen density on the cell surface. However, the data
indicate that disruptions at all three positions are necessary to
eliminate effector activity.
[0435] The above examples are illustrative only and do not define
the invention; other variants will be readily apparent to those of
ordinary skill in the art. The scope of the invention is
encompassed by the claims of any patent(s) issuing herefrom. The
scope of the invention should, therefore, be determined not with
reference to the above description, but instead should be
determined with reference to the issued claims along with their
full scope of equivalents. All publications, references, accession
numbers, and patent documents cited in this application are
incorporated by reference in their entirety for all purposes to the
same extent as if each individual publication or patent document
were so individually denoted.
Sequence CWU 1
1
99142PRTHomo sapiens 1Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu
Val His His Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser
Asn Lys Gly Ala Ile Ile20 25 30Gly Leu Met Val Gly Gly Val Val Ile
Ala35 402112PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 2Asp Val Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Lys
Ser Ser Gln Ser Leu Leu Asp Ser20 25 30Asp Gly Lys Thr Tyr Leu Asn
Trp Leu Leu Gln Lys Pro Gly Gln Ser35 40 45Pro Gln Arg Leu Ile Tyr
Leu Val Ser Lys Leu Asp Ser Gly Val Pro50 55 60Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val
Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly85 90 95Thr His
Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys100 105
1103119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 3Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Asn Tyr20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val35 40 45Ala Ser Ile Arg Ser Gly Gly Gly
Arg Thr Tyr Tyr Ser Asp Asn Val50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Val Arg Tyr Asp
His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly100 105 110Thr Leu
Val Thr Val Ser Ser1154112PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 4Tyr Val Val Met Thr Gln
Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile
Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser20 25 30Asp Gly Lys Thr
Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser35 40 45Pro Gln Arg
Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro50 55 60Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly85
90 95Thr His Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys100 105 1105119PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 5Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Tyr20 25 30Gly Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val35 40 45Ala Ser Ile Arg Ser Gly
Gly Gly Arg Thr Tyr Tyr Ser Asp Asn Val50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys85 90 95Val Arg
Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly100 105
110Thr Leu Val Thr Val Ser Ser1156219PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
6Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5
10 15Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp
Ser20 25 30Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly
Gln Ser35 40 45Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser
Gly Val Pro50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Trp Gln Gly85 90 95Thr His Phe Pro Arg Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys100 105 110Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu115 120 125Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe130 135 140Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155
160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys210 2157449PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 7Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Gly
Ser Gly Phe Thr Phe Ser Asn Tyr20 25 30Gly Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val35 40 45Ala Ser Ile Arg Ser Gly
Gly Gly Arg Thr Tyr Tyr Ser Asp Asn Val50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Val Arg
Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly100 105
110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu165 170 175Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser180 185 190Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro195 200 205Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys210 215
220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp260 265 270Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn275 280 285Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val290 295 300Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys325 330
335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr340 345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys405 410 415Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu420 425 430Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly435 440
445Lys8112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 8Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu
Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser
Gln Asn Ile Ile His Ser20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Tyr
Leu Gln Lys Pro Gly Gln Ser35 40 45Pro Lys Leu Leu Ile Tyr Lys Val
Ser Asn Arg Phe Ser Gly Val Pro50 55 60Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Lys Lys Val Glu Ala
Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly85 90 95Ser His Val Pro
Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Glu100 105
1109123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 9Gln Ala Thr Leu Lys Glu Ser Gly Pro Gly Ile
Leu Gln Ser Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly
Phe Ser Leu Ser Thr Ser20 25 30Gly Met Gly Val Ser Trp Ile Arg Gln
Pro Ser Gly Lys Gly Leu Glu35 40 45Trp Leu Ala His Ile Tyr Trp Asp
Asp Asp Lys Arg Tyr Asn Pro Ser50 55 60Leu Lys Ser Arg Leu Thr Ile
Ser Lys Asp Thr Ser Arg Lys Gln Val65 70 75 80Phe Leu Lys Ile Thr
Ser Val Asp Pro Ala Asp Thr Ala Thr Tyr Tyr85 90 95Cys Val Arg Arg
Pro Ile Thr Pro Val Leu Val Asp Ala Met Asp Tyr100 105 110Trp Gly
Gln Gly Thr Ser Val Thr Val Ser Ser115 12010112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
10Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1
5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His
Ser20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly
Gln Ser35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
Gly Val Pro50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Phe Gln Ser85 90 95Ser His Val Pro Leu Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys100 105 11011120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
11Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly Phe Ser Leu Ser Thr
Ser20 25 30Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly
Leu Glu35 40 45Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr
Asn Pro Ser50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser
Lys Asn Thr Val65 70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr85 90 95Cys Ala Arg Arg Thr Thr Thr Ala Asp
Tyr Phe Ala Tyr Trp Gly Gln100 105 110Gly Thr Thr Val Thr Val Ser
Ser115 12012120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 12Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Phe Ser Gly Phe Thr Leu Ser Thr Ser20 25 30Gly Met Ser Val Gly Trp
Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu35 40 45Trp Val Ala His Ile
Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser50 55 60Leu Lys Ser Arg
Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Leu65 70 75 80Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr85 90 95Cys
Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly Gln100 105
110Gly Thr Thr Val Thr Val Ser Ser115 12013120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
13Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly Phe Thr Leu Ser Thr
Ser20 25 30Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly
Leu Glu35 40 45Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr
Asn Pro Ser50 55 60Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser
Lys Asn Thr Leu65 70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr85 90 95Cys Ala Arg Arg Thr Thr Thr Ala Asp
Tyr Phe Ala Tyr Trp Gly Gln100 105 110Gly Thr Thr Val Thr Val Ser
Ser115 12014120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 14Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Phe Ser Gly Phe Thr Leu Ser Thr Ser20 25 30Gly Met Ser Val Gly Trp
Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu35 40 45Trp Val Ala His Ile
Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser50 55 60Leu Lys Ser Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu65 70 75 80Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr85 90 95Cys
Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly Gln100 105
110Gly Thr Thr Val Thr Val Ser Ser115 12015120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
15Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly Phe Thr Leu Ser Thr
Ser20 25 30Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly
Leu Glu35 40 45Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr
Asn Pro Ser50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser
Lys Asn Thr Val65 70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr85 90 95Cys Ala Arg Arg Thr Thr Thr Ala Asp
Tyr Phe Ala Tyr Trp Gly Gln100 105 110Gly Thr Thr Val Thr Val Ser
Ser115 12016120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 16Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Phe Ser Gly Phe Ser Leu Ser Thr Ser20 25 30Gly Met Ser Val Gly Trp
Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu35 40 45Trp Val Ala His Ile
Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser50 55 60Leu Lys Ser Arg
Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val65 70 75 80Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr85 90 95Cys
Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly Gln100 105
110Gly Thr Thr Val Thr Val Ser Ser115 12017120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
17Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly Phe Ser Leu Ser Thr
Ser20 25 30Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly
Leu Glu35 40 45Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr
Asn Pro Ser50 55 60Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Thr Ser
Lys Asn Thr Val65 70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr85 90 95Cys Ala Arg Arg Thr Thr Thr Ala Asp
Tyr Phe Ala Tyr Trp Gly Gln100 105 110Gly Thr Thr Val Thr Val Ser
Ser115 12018120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 18Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Phe Ser Gly Phe Ser Leu Ser Thr Ser20 25 30Gly Met Ser Val Gly Trp
Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu35 40 45Trp Leu Ala His Ile
Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser50 55 60Leu Lys Ser Arg
Leu Thr Ile Ser Lys Asp Thr Ser
Lys Asn Thr Leu65 70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr85 90 95Cys Ala Arg Arg Thr Thr Thr Ala Asp
Tyr Phe Ala Tyr Trp Gly Gln100 105 110Gly Thr Thr Val Thr Val Ser
Ser115 12019120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 19Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Phe Ser Gly Phe Thr Leu Ser Thr Ser20 25 30Gly Met Ser Val Gly Trp
Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu35 40 45Trp Val Ala His Ile
Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser50 55 60Leu Lys Ser Arg
Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val65 70 75 80Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr85 90 95Cys
Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly Gln100 105
110Gly Thr Thr Val Thr Val Ser Ser115 12020120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
20Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly Phe Thr Leu Ser Thr
Ser20 25 30Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly
Leu Glu35 40 45Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr
Asn Pro Ser50 55 60Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Thr Ser
Lys Asn Thr Val65 70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr85 90 95Cys Ala Arg Arg Thr Thr Thr Ala Asp
Tyr Phe Ala Tyr Trp Gly Gln100 105 110Gly Thr Thr Val Thr Val Ser
Ser115 12021121PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 21Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Phe Ser Gly Phe Thr Leu Ser Thr Ser20 25 30Gly Met Ser Val Gly Trp
Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu35 40 45Trp Leu Ala His Ile
Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser50 55 60Leu Lys Ser Arg
Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Leu65 70 75 80Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr85 90 95Cys
Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly Gln100 105
110Gly Thr Thr Val Thr Val Ser Ser Val115 12022121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
22Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly Phe Ser Leu Ser Thr
Ser20 25 30Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly
Leu Glu35 40 45Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr
Asn Pro Ser50 55 60Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Thr Ser
Lys Asn Thr Val65 70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr85 90 95Cys Ala Arg Arg Thr Thr Thr Ala Asp
Tyr Phe Ala Tyr Trp Gly Gln100 105 110Gly Thr Thr Val Thr Val Ser
Ser Val115 12023120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 23Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Phe Ser Gly Phe Ser Leu Ser Thr Ser20 25 30Gly Met Ser Val Gly Trp
Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu35 40 45Trp Val Ala His Ile
Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser50 55 60Leu Lys Ser Arg
Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Leu65 70 75 80Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr85 90 95Cys
Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly Gln100 105
110Gly Thr Thr Val Thr Val Ser Ser115 12024120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
24Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly Phe Ser Leu Ser Thr
Ser20 25 30Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly
Leu Glu35 40 45Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr
Asn Pro Ser50 55 60Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Thr Ser
Lys Asn Thr Leu65 70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr85 90 95Cys Ala Arg Arg Thr Thr Thr Ala Asp
Tyr Phe Ala Tyr Trp Gly Gln100 105 110Gly Thr Thr Val Thr Val Ser
Ser115 12025120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 25Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Phe Ser Gly Phe Thr Leu Ser Thr Ser20 25 30Gly Met Ser Val Gly Trp
Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu35 40 45Trp Val Ala His Ile
Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser50 55 60Leu Lys Ser Arg
Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val65 70 75 80Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr85 90 95Cys
Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly Gln100 105
110Gly Thr Thr Val Thr Val Ser Ser115 12026120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
26Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly Phe Thr Leu Ser Thr
Ser20 25 30Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly
Leu Glu35 40 45Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr
Asn Pro Ser50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser
Lys Asn Thr Leu65 70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr85 90 95Cys Ala Arg Arg Thr Thr Thr Ala Asp
Tyr Phe Ala Tyr Trp Gly Gln100 105 110Gly Thr Thr Val Thr Val Ser
Ser115 12027120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 27Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Phe Ser Gly Phe Thr Leu Ser Thr Ser20 25 30Gly Met Ser Val Gly Trp
Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu35 40 45Trp Leu Ala His Ile
Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser50 55 60Leu Lys Ser Arg
Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Leu65 70 75 80Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr85 90 95Cys
Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly Gln100 105
110Gly Thr Thr Val Thr Val Ser Ser115 12028120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
28Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly Phe Ser Leu Ser Thr
Ser20 25 30Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly
Leu Glu35 40 45Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr
Asn Pro Ser50 55 60Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Thr Ser
Lys Asn Thr Leu65 70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr85 90 95Cys Ala Arg Arg Thr Thr Thr Ala Asp
Tyr Phe Ala Tyr Trp Gly Gln100 105 110Gly Thr Thr Val Thr Val Ser
Ser115 12029120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 29Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Phe Ser Gly Phe Thr Leu Ser Thr Ser20 25 30Gly Met Ser Val Gly Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu35 40 45Trp Leu Ala His Ile
Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser50 55 60Leu Lys Ser Arg
Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val65 70 75 80Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr85 90 95Cys
Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly Gln100 105
110Gly Thr Thr Val Thr Val Ser Ser115 12030120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
30Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly Phe Ser Leu Ser Thr
Ser20 25 30Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly
Leu Glu35 40 45Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr
Asn Pro Ser50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser
Lys Asn Thr Val65 70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr85 90 95Cys Ala Arg Arg Thr Thr Thr Ala Asp
Tyr Phe Ala Tyr Trp Gly Gln100 105 110Gly Thr Thr Val Thr Val Ser
Ser115 12031112PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 31Asp Val Leu Met Thr Gln Thr Pro
Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys
Arg Ser Ser Gln Asn Ile Val His Ser20 25 30Asn Gly Asn Thr Tyr Leu
Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser35 40 45Pro Lys Leu Leu Ile
Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro50 55 60Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg
Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly85 90 95Ser
His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys100 105
11032123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 32Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile
Leu Gln Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly
Phe Ser Leu Ser Thr Asn20 25 30Gly Met Gly Val Ser Trp Ile Arg Gln
Pro Ser Gly Lys Gly Leu Glu35 40 45Trp Leu Ala His Ile Tyr Trp Asp
Glu Asp Lys Arg Tyr Asn Pro Ser50 55 60Leu Lys Ser Arg Leu Thr Ile
Ser Lys Asp Thr Ser Asn Asn Gln Val65 70 75 80Phe Leu Lys Ile Thr
Asn Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr85 90 95Cys Ala Arg Arg
Arg Ile Ile Tyr Asp Val Glu Asp Tyr Phe Asp Tyr100 105 110Trp Gly
Gln Gly Thr Thr Leu Thr Val Ser Ser115 12033113PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
33Asp Xaa Val Met Thr Gln Xaa Pro Leu Ser Leu Pro Val Xaa Xaa Gly1
5 10 15Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Xaa Tyr
Ser20 25 30Asp Gly Asn Ala Tyr Leu His Trp Phe Leu Gln Lys Pro Gly
Gln Ser35 40 45Pro Xaa Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
Gly Val Pro50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Xaa Gly Val
Tyr Tyr Cys Ser Gln Ser85 90 95Thr His Val Pro Trp Thr Phe Gly Xaa
Gly Thr Xaa Xaa Glu Ile Lys100 105 110Arg34112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
34Xaa Val Gln Leu Val Glu Xaa Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg
Tyr20 25 30Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Xaa
Leu Val35 40 45Ala Gln Ile Asn Ser Val Gly Asn Ser Thr Tyr Tyr Pro
Asp Xaa Val50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Xaa Xaa
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Xaa Asp
Thr Ala Val Tyr Tyr Cys85 90 95Ala Ser Gly Asp Tyr Trp Gly Gln Gly
Thr Xaa Val Thr Val Ser Ser100 105 11035219PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
35Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1
5 10 15Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Ile Tyr
Ser20 25 30Asp Gly Asn Ala Tyr Leu His Trp Phe Leu Gln Lys Pro Gly
Gln Ser35 40 45Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
Gly Val Pro50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Ser Gln Ser85 90 95Thr His Val Pro Trp Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys100 105 110Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu115 120 125Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe130 135 140Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155
160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys210 21536447PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 36Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Arg Tyr20 25 30Ser Met Ser Trp Val Ala
Arg Tyr Gln Ala Pro Gly Lys Gly Leu Glu35 40 45Leu Val Ala Gln Ile
Asn Ser Val Gly Asn Ser Thr Tyr Tyr Pro Asp50 55 60Thr Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr65 70 75 80Leu Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr85 90 95Tyr
Cys Ala Ser Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val100 105
110Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser115 120 125Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys130 135 140Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu145 150 155 160Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu165 170 175Tyr Ser Leu Ser Ser Val
Ala Val Thr Val Pro Ser Ser Ser Leu Gly180 185 190Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys195 200 205Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys210 215
220Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu225 230
235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu245 250 255Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys275 280 285Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu290 295 300Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys325 330 335Ala Lys
Gly Gln Pro Ala Arg Tyr Glu Pro Gln Val Tyr Thr Leu Pro340 345
350Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg405 410 415Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu420 425 430His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys435 440
44537112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 37Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Ile Leu His Ser20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Tyr
Leu Gln Lys Pro Gly Gln Ser35 40 45Pro Gln Leu Leu Ile Tyr Lys Val
Ser Asn Arg Phe Ser Gly Val Pro50 55 60Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala
Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly85 90 95Ser Leu Val Pro
Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys100 105
11038123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 38Gln Val Thr Leu Lys Glu Ser Gly Pro Ala Leu
Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly
Phe Ser Leu Ser Thr Ser20 25 30Gly Met Gly Val Gly Trp Ile Arg Gln
Pro Pro Gly Lys Ala Leu Glu35 40 45Trp Leu Ala His Ile Trp Trp Asp
Asp Asp Lys Ser Tyr Asn Pro Ser50 55 60Leu Lys Ser Arg Leu Thr Ile
Ser Lys Asp Thr Ser Lys Asn Gln Val65 70 75 80Val Leu Thr Met Thr
Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr85 90 95Cys Ala Arg Arg
Gln Leu Gly Leu Arg Ser Ile Asp Ala Met Asp Tyr100 105 110Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser115 12039122PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
39Gln Val Thr Leu Lys Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1
5 10 15Thr Leu Thr Leu Thr Cys Thr Leu Ser Gly Phe Ser Leu Ser Thr
Ser20 25 30Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala
Leu Glu35 40 45Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Ser Tyr
Asn Pro Ser50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser
Lys Asn Gln Val65 70 75 80Val Leu Thr Met Thr Asn Met Asp Pro Val
Asp Thr Ala Thr Tyr Tyr85 90 95Cys Ala Arg Arg Gln Leu Gly Leu Arg
Ser Ile Asp Ala Met Asp Tyr100 105 110Trp Gly Gln Gly Thr Thr Val
Thr Val Ser115 12040132PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 40Met Lys Leu Pro Val Arg
Leu Leu Val Leu Met Phe Trp Ile Pro Gly1 5 10 15Ser Ser Ser Asp Val
Met Met Thr Gln Thr Pro Leu Ser Leu Pro Val20 25 30Ser Leu Gly Asp
Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu35 40 45Val His Ser
Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Met Gln Lys Pro50 55 60Gly Gln
Ser Pro Met Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser65 70 75
80Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr85
90 95Leu Lys Ile Ser Ser Val Glu Ala Glu Asp Leu Gly Val Phe Tyr
Cys100 105 110Phe Gln Gly Ser Arg Val Pro Leu Thr Phe Gly Ala Gly
Thr Lys Leu115 120 125Glu Leu Lys Arg13041142PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
41Met Asp Arg Leu Thr Ser Ser Phe Leu Leu Leu Ile Val Pro Ala Tyr1
5 10 15Val Leu Ser Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu
Gln20 25 30Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe
Ser Leu35 40 45Ser Thr Ser Gly Met Gly Val Ser Trp Ile Arg Gln Pro
Ser Gly Lys50 55 60Gly Leu Glu Trp Leu Ala His Ile Tyr Trp Asp Asp
Asp Lys Arg Tyr65 70 75 80Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile
Ser Lys Asp Thr Ser Arg85 90 95Asn Gln Val Phe Leu Lys Ile Thr Ser
Val Asp Thr Thr Asp Thr Ala100 105 110Thr Tyr Tyr Cys Thr Arg Ser
Ser Gly Ser Ile Val Ile Ala Thr Gly115 120 125Phe Ala Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ala130 135 14042128PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
42Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1
5 10 15Val Ile Ile Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala
Ile20 25 30Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser
Ala Ser35 40 45Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser
Gly Thr Ser50 55 60Pro Lys Arg Trp Ile Tyr Asp Ser Ser Arg Leu Ala
Ser Gly Val Pro65 70 75 80Ser Arg Phe Ser Gly Gly Gly Ser Gly Thr
Ser Tyr Ser Pro Thr Ile85 90 95Ser Asn Met Glu Ala Glu Asp Ala Ala
Thr Tyr Phe Cys Gln Asn Trp100 105 110Arg Ser Ser Pro Thr Phe Gly
Ala Gly Thr Lys Leu Glu Leu Lys Arg115 120 12543138PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
43Met Glu Trp Thr Trp Val Phe Leu Phe Leu Leu Ser Val Thr Ala Gly1
5 10 15Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Met
Lys20 25 30Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr
Thr Phe35 40 45Ser Thr Ser Trp Ile Glu Trp Ile Lys Gln Arg Pro Gly
His Gly Leu50 55 60Glu Trp Ile Gly Glu Val Leu Pro Gly Ser Gly Lys
Ser Asn His Asn65 70 75 80Ala Asn Phe Lys Gly Arg Ala Thr Phe Thr
Ala Asp Thr Ala Ser Asn85 90 95Thr Ala Tyr Met Gln Leu Ser Ser Leu
Thr Ser Glu Asp Ser Ala Val100 105 110Tyr Tyr Cys Ala Arg Glu Gly
Ser Asn Asn Asn Ala Leu Ala Tyr Trp115 120 125Gly Gln Gly Thr Leu
Val Thr Val Ser Ala130 13544108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 44Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile35 40 45Tyr Tyr Thr
Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Arg Lys Leu Pro Tyr85
90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg100
10545113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 45Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Asp Phe Ser Arg Tyr20 25 30Trp Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val35 40 45Ser Glu Ile Asn Pro Asp Ser Ser
Thr Ile Asn Tyr Thr Pro Ser Leu50 55 60Lys Asp Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Arg Gln Met
Gly Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser100 105
110Ser46113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 46Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
Phe Asp Phe Ser Arg Tyr20 25 30Trp Met Asn Trp Ile Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Ile35 40 45Gly Glu Ile Asn Pro Asp Ser Ser
Thr Ile Asn Tyr Thr Pro Ser Leu50 55 60Lys Asp Arg Val Thr Ile Ser
Lys Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Arg Gln Met
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser100 105
110Ser47241PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 47Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Met Leu Trp1 5 10 15Val Ser Gly Ser Ser Gly Asp Val Val Met
Thr Gln Ser Pro Leu Ser20 25 30Leu Pro Val Thr Pro Gly Glu Pro Ala
Ser Ile Ser Cys Lys Ser Ser35 40 45Gln Ser Leu Leu Asp Ser Asp Gly
Lys Thr Tyr Leu Asn Trp Leu Leu50 55 60Gln Lys Pro Gly Gln Ser Pro
Gln Arg Leu Ile Tyr Leu Val Ser Lys65 70 75 80Leu Asp Ser Gly Val
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr85 90 95Asp Phe Thr Leu
Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val100 105 110Tyr Tyr
Cys Trp Gln Gly Thr His Phe Pro Arg Thr Phe Gly Gln Gly115 120
125Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe
Ile130 135 140Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala
Ser Val Val145 150 155 160Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys165 170 175Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu180 185 190Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu195 200 205Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr210 215 220His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu225 230 235
240Cys48219PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 48Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser
Gln Ser Leu Leu Asp Ser20 25 30Asp Gly Lys Thr Tyr Leu Asn Trp Leu
Leu Gln Lys Pro Gly Gln Ser35 40 45Pro Gln Arg Leu Ile Tyr Leu Val
Ser Lys Leu Asp Ser Gly Val Pro50 55 60Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala
Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly85 90 95Thr His Phe Pro
Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys100 105 110Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu115 120
125Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe130 135 140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu180 185 190Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser195 200 205Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys210 21549726DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
49atggacatgc gcgtgcccgc ccagctgctg ggcctgctga tgctgtgggt gtccggctcc
60tccggcgacg tggtgatgac ccagtccccc ctgtccctgc ccgtgacccc cggcgagccc
120gcctccatct cctgcaagtc ctcccagtcc ctgctggact ccgacggcaa
gacctacctg 180aactggctgc tgcagaagcc cggccagtcc ccccagcgcc
tgatctacct ggtgtccaag 240ctggactccg gcgtgcccga ccgcttctcc
ggctccggct ccggcaccga cttcaccctg 300aagatctccc gcgtggaggc
cgaggacgtg ggcgtgtact actgctggca gggcacccac 360ttcccccgca
ccttcggcca gggcaccaag gtggagatca agcgtactgt ggctgcacca
420tctgtcttca tcttcccgcc atctgatgag cagttgaaat ctggaactgc
ctctgttgtg 480tgcctgctga ataacttcta tcccagagag gccaaagtac
agtggaaggt ggataacgcc 540ctccaatcgg gtaactccca ggagagtgtc
acagagcagg acagcaagga cagcacctac 600agcctcagca gcaccctgac
gctgagcaaa gcagactacg agaaacacaa agtctacgcc 660tgcgaagtca
cccatcaggg cctgagctcg cccgtcacaa agagcttcaa caggggagag 720tgttag
72650330PRTHomo sapiens 50Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90 95Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys100 105 110Pro
Ala Pro Glu Ala Leu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu180 185 190His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200 205Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly210 215 220Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235
240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn290 295 300Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys325 33051329PRTHomo sapiens 51Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40
45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50
55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys100
105 110Pro Ala Pro Glu Ala Leu Gly Ala Pro Ser Val Phe Leu Phe Pro
Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200 205Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn290 295 300Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln
Lys Ser Leu Ser Leu Ser Pro Gly32552468PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
52Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly1
5 10 15Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe35 40 45Ser Asn Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu50 55 60Glu Trp Val Ala Ser Ile Arg Ser Gly Gly Gly Arg
Thr Tyr Tyr Ser65 70 75 80Asp Asn Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn85 90 95Thr Leu Tyr Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val100 105 110Tyr Tyr Cys Val Arg Tyr Asp
His Tyr Ser Gly Ser Ser Asp Tyr Trp115 120 125Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro130 135 140Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr145 150 155
160Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr165 170 175Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro180 185 190Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr195 200 205Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn210 215 220His Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser225 230 235 240Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Leu245 250 255Gly Ala
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu260 265
270Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser275 280 285His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu290 295 300Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr305 310 315 320Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn325 330 335Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro340 345 350Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln355 360 365Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val370 375
380Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val385 390 395 400Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro405 410 415Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr420 425 430Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val435 440 445Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu450 455 460Ser Pro Gly
Lys46553467PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 53Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val
Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe35 40 45Ser Asn Tyr Gly Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu50 55 60Glu Trp Val Ala Ser Ile Arg
Ser Gly Gly Gly Arg Thr Tyr Tyr Ser65 70 75 80Asp Asn Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn85 90 95Thr Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val100 105 110Tyr Tyr
Cys Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp115 120
125Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro130 135 140Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr145 150 155 160Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr165 170 175Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro180 185 190Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr195 200 205Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn210 215 220His Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser225 230 235
240Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Leu245 250 255Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu260 265 270Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser275 280 285His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu290 295 300Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr305 310 315 320Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn325 330 335Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro340 345
350Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln355 360 365Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val370 375 380Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val385 390 395 400Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro405 410 415Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr420 425 430Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val435 440 445Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu450 455
460Ser Pro Gly46554449PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 54Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr20 25 30Gly Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val35 40 45Ala Ser Ile
Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser Asp Asn Val50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85
90 95Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln
Gly100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu165 170 175Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser180 185 190Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro195 200
205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Leu
Gly Ala Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser245 250 255Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp260 265 270Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn275 280 285Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val290 295 300Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315
320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr340 345 350Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu370 375 380Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys405 410 415Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu420 425
430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly435 440 445Lys55448PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 55Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr20 25 30Gly Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val35 40 45Ala Ser Ile
Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser Asp Asn Val50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85
90 95Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln
Gly100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu165 170 175Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser180 185 190Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro195 200
205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Leu
Gly Ala Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser245 250 255Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp260 265 270Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn275 280 285Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val290 295 300Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315
320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr340 345 350Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu370 375 380Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys405 410 415Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu420 425
430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly435 440 44556327PRTHomo sapiens 56Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr
Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90
95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
Pro100 105 110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe165 170 175Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp180 185 190Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu195 200
205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg210 215 220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys225 230 235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp245 250 255Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys260 265 270Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser275 280 285Arg Leu Thr Val
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser290 295 300Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315
320Leu Ser Leu Ser Leu Gly Lys32557326PRTHomo sapiens 57Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr
Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40
45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50
55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys
Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys
Val Asp Lys85 90 95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro
Cys Pro Ala Pro100 105 110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys115 120 125Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val130 135 140Asp Val Ser Gln Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe165 170 175Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp180 185
190Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu195 200 205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg210 215 220Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp245 250 255Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys260 265
270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser290 295 300Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu
Gly32558465PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 58Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val
Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe35 40 45Ser Asn Tyr Gly Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu50 55 60Glu Trp Val Ala Ser Ile Arg
Ser Gly Gly Gly Arg Thr Tyr Tyr Ser65 70 75 80Asp Asn Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn85 90 95Thr Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val100 105 110Tyr Tyr
Cys Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp115 120
125Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro130 135 140Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
Glu Ser Thr145 150 155 160Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr165 170 175Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro180 185 190Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr195 200 205Val Pro Ser Ser
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp210 215 220His Lys
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr225 230 235
240Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
Pro245 250 255Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser260 265 270Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser Gln Glu Asp275 280 285Pro Glu Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn290 295 300Ala Lys Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Tyr Arg Val305 310 315 320Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu325 330 335Tyr Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys340 345
350Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr355 360 365Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr370 375 380Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu385 390 395 400Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu405 410 415Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys420 425 430Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu435 440 445Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly450 455
460Lys46559464PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 59Met Glu Phe Gly Leu Ser Trp Leu
Phe Leu Val Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln20 25 30Pro Gly Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe35 40 45Ser Asn Tyr Gly Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu50 55 60Glu Trp Val Ala
Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser65 70 75 80Asp Asn
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn85 90 95Thr
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val100 105
110Tyr Tyr Cys Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr
Trp115 120 125Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro130 135 140Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser
Thr Ser Glu Ser Thr145 150 155 160Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr165 170 175Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro180 185 190Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr195 200 205Val Pro
Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp210 215
220His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys
Tyr225 230 235 240Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
Leu Gly Gly Pro245 250 255Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser260 265 270Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Glu Asp275 280 285Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn290 295 300Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val305 310 315 320Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu325 330
335Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
Lys340 345 350Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr355 360 365Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr370 375 380Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu385 390 395 400Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu405 410 415Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys420 425 430Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu435 440
445Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
Gly450 455 46060446PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 60Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asn Tyr20 25 30Gly Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val35 40 45Ala Ser Ile Arg Ser
Gly Gly Gly Arg Thr Tyr Tyr Ser Asp Asn Val50 55 60Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Val
Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly100 105
110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe115 120 125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
Ala Ala Leu130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu165 170 175Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser180 185 190Ser Ser Leu Gly
Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro195 200 205Ser Asn
Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro210 215
220Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val
Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser
Gln Glu Asp Pro Glu Val260 265 270Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr275 280 285Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Tyr Arg Val Val Ser Val290 295 300Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser325 330
335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro340 345 350Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu Tyr
Ser Arg Leu Thr Val Asp Lys Ser Arg Trp405 410 415Gln Glu Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His420 425 430Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys435 440
44561445PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 61Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Asn Tyr20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val35 40 45Ala Ser Ile Arg Ser Gly Gly Gly
Arg Thr Tyr Tyr Ser Asp Asn Val50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Val Arg Tyr Asp
His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly100 105 110Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe115 120
125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala
Leu130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser180 185 190Ser Ser Leu Gly Thr Lys
Thr Tyr Thr Cys Asn Val Asp His Lys Pro195 200 205Ser Asn Thr Lys
Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro210 215 220Cys Pro
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe225 230 235
240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
Pro Glu Val260 265 270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr275 280 285Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser Val290 295 300Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser325 330 335Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro340 345
350Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr Val Asp Lys Ser Arg Trp405 410 415Gln Glu Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His420 425 430Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Leu Gly435 440 44562330PRTHomo sapiens
62Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1
5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys100 105 110Pro Ala Pro Glu Ala Ala Gly
Ala Pro Ser Val Phe Leu Phe Pro Pro115 120 125Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys130 135 140Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155
160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly210 215 220Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr245 250 255Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn260 265
270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys325 33063329PRTHomo sapiens 63Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90
95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys100 105 110Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu
Phe Pro Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys130 135 140Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr245 250 255Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn260 265 270Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe275 280 285Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn290 295 300Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly32564468PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
64Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val
Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe35 40 45Ser Asn Tyr Gly Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu50 55 60Glu Trp Val Ala Ser Ile Arg
Ser Gly Gly Gly Arg Thr Tyr Tyr Ser65 70 75 80Asp Asn Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn85 90 95Thr Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val100 105 110Tyr Tyr
Cys Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp115 120
125Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro130 135 140Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr145 150 155 160Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr165 170 175Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro180 185 190Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr195 200 205Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn210 215 220His Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser225 230 235
240Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala245 250 255Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu260 265 270Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser275 280 285His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu290 295 300Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr305 310 315 320Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn325 330 335Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro340 345
350Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln355 360 365Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val370 375 380Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val385 390 395 400Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro405 410 415Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr420 425 430Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val435 440 445Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu450 455
460Ser Pro Gly Lys46565467PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 65Met Glu Phe Gly Leu Ser
Trp Leu Phe Leu Val Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln20 25 30Pro Gly Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe35 40 45Ser Asn Tyr
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu50 55 60Glu Trp
Val Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser65 70 75
80Asp Asn Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn85
90 95Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val100 105 110Tyr Tyr Cys Val Arg Tyr Asp His Tyr Ser Gly Ser Ser
Asp Tyr Trp115 120 125Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro130 135 140Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr145 150 155 160Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr165 170 175Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro180 185 190Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr195 200
205Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn210 215 220His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser225 230 235 240Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Ala Ala245 250 255Gly Ala Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu260 265 270Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser275 280 285His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu290 295 300Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr305 310 315
320Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn325 330 335Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro340 345 350Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln355 360 365Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val370 375 380Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val385 390 395 400Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro405 410 415Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr420 425
430Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val435 440 445Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu450 455 460Ser Pro Gly46566449PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
66Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn
Tyr20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val35 40 45Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser
Asp Asn Val50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys85 90 95Val Arg Tyr Asp His Tyr Ser Gly Ser
Ser Asp Tyr Trp Gly Gln Gly100 105 110Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe115 120 125Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu130 135 140Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys210 215 220Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Ala Ala Gly Ala Pro225 230 235 240Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser245 250 255Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp260 265
270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys325 330 335Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr340 345 350Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr355 360 365Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu370 375
380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu420 425 430Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly435 440 445Lys67448PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
67Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn
Tyr20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val35 40 45Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser
Asp Asn Val50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys85 90 95Val Arg Tyr Asp His Tyr Ser Gly Ser
Ser Asp Tyr Trp Gly Gln Gly100 105 110Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe115 120 125Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu130 135 140Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys210 215 220Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Ala Ala Gly Ala Pro225 230 235 240Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser245 250 255Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp260 265
270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys325 330 335Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr340 345 350Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr355 360 365Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu370 375
380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu420 425 430Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly435 440 445681407DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
68atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgtgag
60gtgcagctgc tggagtccgg cggcggcctg gtgcagcccg gcggctccct gcgcctgtcc
120tgcgccgcct ccggcttcac cttctccaac tacggcatgt cctgggtgcg
ccaggccccc 180ggcaagggcc tggagtgggt ggcctccatc cgctccggcg
gcggccgcac ctactactcc 240gacaacgtga agggccgctt caccatctcc
cgcgacaact ccaagaacac cctgtacctg 300cagatgaact ccctgcgcgc
cgaggacacc gccgtgtact actgcgtgcg ctacgaccac 360tactccggct
cctccgacta ctggggccag ggcaccctgg tgaccgtgtc ctccgcgtcg
420accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc
tgggggcaca 480gcggccctgg gctgcctggt caaggactac ttccccgaac
cggtgacggt gtcgtggaac 540tcaggcgccc tgaccagcgg cgtgcacacc
ttcccggctg tcctacagtc ctcaggactc 600tactccctca gcagcgtggt
gaccgtgccc tccagcagct tgggcaccca gacctacatc 660tgcaacgtga
atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct
720tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aagccgctgg
ggcaccgtca 780gtcttcctct tccccccaaa acccaaggac accctcatga
tctcccggac ccctgaggtc 840acatgcgtgg tggtggacgt gagccacgaa
gaccctgagg tcaagttcaa ctggtacgtg 900gacggcgtgg aggtgcataa
tgccaagaca aagccgcggg aggagcagta caacagcacg 960taccgtgtgg
tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac
1020aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat
ctccaaagcc 1080aaagggcagc cccgagaacc acaggtgtac accctgcccc
catcccggga ggagatgacc 1140aagaaccagg tcagcctgac ctgcctggtc
aaaggcttct atcccagcga catcgccgtg 1200gagtgggaga gcaatgggca
gccggagaac aactacaaga ccacgcctcc cgtgctggac 1260tccgacggct
ccttcttcct ctatagcaag ctcaccgtgg acaagagcag gtggcagcag
1320gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta
cacgcagaag 1380agcctctccc tgtccccggg taaatga 140769449PRTHomo
sapiens 69Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Asn Tyr20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val35 40 45Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr
Tyr Ser Asp Asn Val50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Val Arg Tyr Asp His Tyr Ser
Gly Ser Ser Asp Tyr Trp Gly Gln Gly100 105 110Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe115 120 125Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu130 135 140Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150
155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys210 215 220Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser245 250 255Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp260 265
270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys325 330 335Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr340 345 350Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr355 360 365Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu370 375
380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu420 425 430Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly435 440 445Lys70448PRTHomo sapiens
70Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn
Tyr20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val35 40 45Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser
Asp Asn Val50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys85 90 95Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly
Gln Gly100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu130 135 140Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu165 170 175Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser180 185 190Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro195 200
205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser245 250 255Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp260 265 270Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn275 280 285Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val290 295 300Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315
320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr340 345 350Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu370 375 380Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys405 410 415Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu420 425
430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly435 440 44571113PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 71Asp Val Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys
Lys Ser Ser Gln Ser Leu Leu Asp Ser20 25 30Asp Gly Lys Thr Tyr Leu
Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser35 40 45Pro Arg Arg Leu Ile
Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro50 55 60Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly85 90 95Thr
His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys100 105
110Arg72119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 72Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly
Phe Thr Phe Ser Asn Tyr20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val35 40 45Ala Ser Ile Arg Ser Gly Gly Gly
Arg Thr Tyr Tyr Ser Asp Asn Val50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Glu Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Val Arg Tyr Asp
His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly100 105 110Thr Leu
Val Thr Val Ser Ser11573113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 73Asp Val Val Met Thr Gln
Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Gln Pro Ala Ser Ile
Ser Cys Arg Ser Ser Gln Asn Ile Ile His Ser20 25 30Asn Gly Asn Thr
Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser35 40 45Pro Arg Leu
Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro50 55 60Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly85
90 95Ser His Val Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys100 105 110Arg74123PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 74Gln Val Thr Leu Lys Glu
Ser Gly Pro Val Leu Val Lys Pro Thr Glu1 5 10 15Thr Leu Thr Leu Thr
Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser20 25 30Gly Met Gly Val
Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu35 40 45Trp Leu Ala
His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser50 55 60Leu Lys
Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val65 70 75
80Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr85
90 95Cys Val Arg Arg Pro Ile Thr Pro Val Leu Val Asp Ala Met Asp
Tyr100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser115
12075112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 75Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Val Ser
Gln Ser Leu Leu His Ser20 25 30Asn Gly Tyr Thr Tyr Leu His Trp Tyr
Leu Gln Lys Pro Gly Gln Ser35 40 45Pro Gln Leu Leu Ile Tyr Lys Val
Ser Asn Arg Phe Ser Gly Val Pro50 55 60Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala
Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Thr85 90 95Arg His Val Pro
Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys100 105
11076115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 76Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Asp Asn20 25 30Gly Met Ala Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val35 40 45Ser Phe Ile Ser Asn Leu Ala Tyr
Ser Ile Asp Tyr Ala Asp Thr Val50 55 60Thr Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Val Ser Gly Thr
Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr100 105 110Val Ser
Ser11577115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 77Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Val Ser Gly
Phe Thr Phe Ser Asp Asn20 25 30Gly Met Ala Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val35 40 45Ser Phe Ile Ser Asn Leu Ala Tyr
Ser Ile Asp Tyr Ala Asp Thr Val50 55 60Thr Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Val Ser Gly Thr
Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr100 105 110Val Ser
Ser11578115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 78Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Asp Asn20 25 30Gly Met Ala Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Ile35 40 45Ser Phe Ile Ser Asn Leu Ala Tyr
Ser Ile Asp Tyr Ala Asp Thr Val50 55 60Thr Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Val Ser Gly Thr
Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr100 105 110Val Ser
Ser11579116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 79Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Tyr Thr Glu Ala Tyr20 25 30Tyr Ile His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met35 40 45Gly Arg Ile Asp Pro Ala Thr Gly
Asn Thr Lys Tyr Ala Pro Arg Leu50 55 60Gln Asp Arg Val Thr Met Thr
Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Ser Leu Tyr
Ser Leu Pro Val Tyr Trp Gly Gln Gly Thr Thr Val100 105 110Thr Val
Ser Ser11580114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 80Asp Val Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys
Lys Ser Ser Gln Ser Leu Leu Tyr Ser20 25 30Asp Ala Lys Thr Tyr Leu
Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser35 40 45Pro Arg Arg Leu Ile
Tyr Gln Ile Ser Arg Leu Asp Pro Gly Val Pro50 55 60Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Gly85 90 95Thr
His Tyr Pro Val Leu Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys100 105
110Arg Thr81117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 81Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Thr Tyr20 25 30Ala Ile His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45Gly Phe Thr Ser Pro
Tyr Ser Gly Val Ser Asn Tyr Asn Gln Lys Phe50 55 60Lys Gly Arg Val
Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala
Arg Phe Asp Asn Tyr Asp Arg Gly Tyr Val Arg Asp Tyr Trp Gly100 105
110Gln Gly Thr Leu Val11582114PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 82Asp Ile Val Met Thr Gln
Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile
Asn Cys Arg Ala Ser Glu Ser Val Asp Asn Asp20 25 30Arg Ile Ser Phe
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro35 40 45Lys Leu Leu
Ile Tyr Ala Ala Thr Lys Gln Gly Thr Gly Val Pro Asp50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75
80Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Lys85
90 95Glu Phe Pro Trp Ser Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg100 105 110Thr Val83121PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 83Gln Val Thr Leu Lys Glu
Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr
Cys Thr Phe Ser Gly Phe Ser Leu Arg Thr Ser20 25 30Gly Met Gly Val
Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu35 40 45Trp Leu Ala
His Ile Trp Trp Asp Asp Asp Lys Ser Tyr Asn Pro Ser50 55 60Leu Lys
Ser Gln Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val65 70 75
80Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr85
90 95Cys Ala Arg Arg Asn Tyr Tyr Tyr Asp Asp Tyr Phe Ala Tyr Trp
Gly100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser115
12084112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 84Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Leu Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Ile Val His Ser20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Tyr
Leu Gln Arg Pro Gly Gln Ser35 40 45Pro Lys Leu Leu Ile Tyr Lys Val
Ser Asn Arg Phe Ser Gly Val Pro50 55 60Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala
Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly85 90 95Ser His Val Pro
Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys100 105
110859PRTHomo sapiens 85Ala Glu Phe Arg His Asp Ser Gly Tyr1
5866PRTHomo sapiens 86Ala Glu Phe Arg His Asp1 5877PRTHomo sapiens
87Glu Phe Arg His Asp Ser Gly1 5885PRTHomo sapiens 88Glu Phe Arg
His Asp1 58916PRTHomo sapiens 89Tyr Glu Val His His Gln Lys Leu Val
Phe Phe Ala Glu Asp Val Gly1 5 10 15904PRTHomo sapiens 90Val Phe
Phe Ala1919PRTHomo sapiens 91Gln Lys Leu Phe Phe Ala Glu Asp Val1
5929PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 92Lys Leu Val Phe Phe Ala Gly Asp Val1
5935PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 5xHis tag 93His His His His His1 5945PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
94Asp Ala Glu Phe Arg1 595110PRTHomo sapiens 95Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val20 25 30Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr35 40 45Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu50 55 60Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65 70 75
80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys85
90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys100 105
11096109PRTHomo sapiens 96Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro1 5 10 15Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val20 25 30Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val35 40 45Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln50 55 60Phe Asn Ser Thr Phe Arg
Val Val Ser Val Leu Thr Val Val His Gln65 70 75 80Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly85 90 95Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys100 10597110PRTHomo sapiens
97Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1
5 10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val20 25 30Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
Trp Tyr35 40 45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu50 55 60Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His65 70 75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys85 90 95Gly Leu Pro Ser Ser Ile Glu Lys Thr
Ile Ser Lys Ala Lys100 105 11098107PRTMus sp. 98Val Pro Glu Val Ser
Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp1
5 10 15Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val
Asp20 25 30Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val
Asp Asp35 40 45Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu
Gln Phe Asn50 55 60Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met
His Gln Asp Trp65 70 75 80Leu Asn Gly Lys Glu Phe Lys Cys Arg Val
Asn Ser Ala Ala Phe Pro85 90 95Ala Pro Ile Glu Lys Thr Ile Ser Lys
Thr Lys100 10599110PRTMus sp. 99Ala Pro Asn Leu Leu Gly Gly Pro Ser
Val Phe Ile Phe Pro Pro Lys1 5 10 15Ile Lys Asp Val Leu Met Ile Ser
Leu Ser Pro Ile Val Thr Cys Val20 25 30Val Val Asp Val Ser Glu Asp
Asp Pro Asp Val Gln Ile Ser Trp Phe35 40 45Val Asn Asn Val Glu Val
His Thr Ala Gln Thr Gln Thr His Arg Glu50 55 60Asp Tyr Asn Ser Thr
Leu Arg Val Val Ser Ala Leu Pro Ile Gln His65 70 75 80Gln Asp Trp
Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys85 90 95Asp Leu
Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys100 105 110
* * * * *