U.S. patent application number 14/761895 was filed with the patent office on 2015-12-10 for methods for improving safety of blood-brain barrier transport.
The applicant listed for this patent is Genentech, Inc.. Invention is credited to Jessica Couch, Mark Dennis, Ryan Jefferson Watts, Joy Yu Zuchero.
Application Number | 20150353639 14/761895 |
Document ID | / |
Family ID | 48534514 |
Filed Date | 2015-12-10 |
United States Patent
Application |
20150353639 |
Kind Code |
A1 |
Watts; Ryan Jefferson ; et
al. |
December 10, 2015 |
METHODS FOR IMPROVING SAFETY OF BLOOD-BRAIN BARRIER TRANSPORT
Abstract
The present disclosure relates to compositions and methods for
improving the safety of blood-brain barrier receptor-mediated
blood-brain barrier transport.
Inventors: |
Watts; Ryan Jefferson;
(South San Francisco, CA) ; Yu Zuchero; Joy;
(South San Francisco, CA) ; Couch; Jessica; (South
San Francisco, CA) ; Dennis; Mark; (South San
Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
48534514 |
Appl. No.: |
14/761895 |
Filed: |
May 20, 2013 |
PCT Filed: |
May 20, 2013 |
PCT NO: |
PCT/US2013/041860 |
371 Date: |
July 17, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61763915 |
Feb 12, 2013 |
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61698495 |
Sep 7, 2012 |
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61649878 |
May 21, 2012 |
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Current U.S.
Class: |
424/134.1 ;
424/133.1; 424/136.1; 424/158.1; 424/178.1; 530/387.3;
530/391.7 |
Current CPC
Class: |
A61P 21/02 20180101;
A61P 35/00 20180101; A61P 25/28 20180101; C07K 16/44 20130101; C07K
2317/90 20130101; A61K 2039/505 20130101; C07K 2317/71 20130101;
A61K 2039/545 20130101; A61P 5/38 20180101; A61P 25/00 20180101;
A61K 39/39583 20130101; C07K 2319/30 20130101; C07K 2317/41
20130101; A61K 47/6879 20170801; A61P 43/00 20180101; A61P 19/08
20180101; C07K 16/40 20130101; C07K 16/2881 20130101; A61P 9/10
20180101; A61P 21/04 20180101; C07K 2317/24 20130101; C07K 2317/21
20130101; C07K 2317/31 20130101; A61P 21/00 20180101; A61P 25/16
20180101; A61P 25/14 20180101; A61P 9/00 20180101; C07K 2317/52
20130101; C07K 2317/94 20130101; A61K 35/18 20130101; A61K 47/6849
20170801; C07K 16/468 20130101; A61P 5/40 20180101; A61K 39/3955
20130101; C07K 2317/92 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/46 20060101 C07K016/46; A61K 39/395 20060101
A61K039/395; A61K 47/48 20060101 A61K047/48; C07K 16/44 20060101
C07K016/44; A61K 35/18 20060101 A61K035/18; C07K 16/40 20060101
C07K016/40 |
Claims
1. A method of transporting a compound across the blood-brain
barrier in a subject comprising exposing an antibody which binds
with low affinity to a blood-brain barrier receptor (BBB-R) coupled
to a compound to the blood-brain barrier such that the antibody
transports the compound coupled thereto across the blood-brain
barrier, wherein reduction of red blood cell levels in the subject
upon antibody administration to the subject is decreased or
eliminated.
2. A method of increasing exposure of the CNS of a subject to a
compound, wherein the compound is coupled to an antibody which
binds with low affinity to a BBB-R, thereby increasing the exposure
of the CNS to the compound, and wherein reduction of red blood cell
levels in the subject upon compound-coupled antibody administration
to the subject is decreased or eliminated.
3. A method of decreasing clearance of a compound administered to a
subject, wherein the compound is coupled to an antibody which binds
with low affinity to a BBB-R, such that the clearance of the
compound is decreased, and wherein reduction of red blood cell
levels in the subject upon compound-coupled antibody administration
to the subject is decreased or eliminated.
4. A method of increasing retention in the CNS of a compound
administered to a subject, wherein the compound is coupled to an
antibody which binds with low affinity to a BBB-R, such that the
retention in the CNS of the compound is increased, and wherein
reduction of red blood cell levels in the subject upon
compound-coupled antibody administration to the subject is
decreased or eliminated.
5. A method of optimizing the pharmcokinetics and/or
pharmacodynamics of a compound to be efficacious in the CNS in a
subject, wherein the compound is coupled to an antibody which binds
with low affinity to a BBB-R, and the antibody is selected such
that its affinity for the BBB-R after coupling to the compound
results in an amount of transport of the antibody conjugated to the
compound across the BBB that optimizes the pharmacokinetics and/or
pharmacodynamics of the compound in the CNS, wherein reduction of
red blood cell levels in the subject upon compound-coupled antibody
administration to the subject is decreased or eliminated.
6. A method of treating a neurological disorder in a mammal
comprising treating the mammal with an antibody that binds a BBB-R
and is coupled to a compound, wherein the antibody has been
selected to have a low affinity for the BBB-R and thereby improves
CNS uptake of the antibody and coupled compound, and wherein
reduction of red blood cell levels in the subject upon
compound-coupled antibody administration to the subject is
decreased or eliminated.
7. The method of any of claims 1-6, wherein the BBB-R is selected
from the group consisting of transferrin receptor (TfR), insulin
receptor, insulin-like growth factor receptor (IGF receptor), low
density lipoprotein receptor-related protein 8 (LRP8), low density
lipoprotein receptor-related protein 1 (LRP1), glucose transporter
1 (Glut1) and heparin-binding epidermal growth factor-like growth
factor (HB-EGF).
8. The method of claim 7, wherein the BBB-R is TfR.
9. The method of claim 8, wherein the red blood cells are immature
red blood cells.
10. The method of claim 9, wherein the immature red blood cells are
reticulocytes.
11. The method of claim 10, wherein reduction of reticulocyte
levels is accompanied by acute clinical symptoms.
12. The method of claim 11, wherein one or more properties of the
antibody have been modified to reduce the impact of the antibody on
reticulocyte levels and/or reduce the severity or presence of acute
clinical symptoms in the subject or mammal.
13. The method of claim 12, wherein the one or more properties are
selected from the effector function of the antibody Fc region, the
complement activation function of the antibody and the affinity of
the antibody for the BBB-R.
14. The method of claim 13, wherein the one or more properties are
selected from the effector function of the antibody Fc region and
the complement activation function of the antibody, and wherein the
effector function or complement activation function has been
reduced or eliminated relative to a wild-type antibody of the same
isotype.
15. The method of claim 14, wherein the effector function is
reduced or eliminated by a method selected from reduction of
glycosylation of the antibody, modification of the antibody isotype
to an isotype that naturally has reduced or eliminated effector
function, and modification of the Fc region.
16. The method of claim 15, wherein the glycosylation of the
antibody is reduced by a method selected from: production of the
antibody in an environment that does not permit wild-type
glycosylation; removal of carbohydrate groups already present on
the antibody; and modification of the antibody such that wild-type
glycosylation does not occur.
17. The method of claim 16, wherein the antibody is produced in a
non-mammalian cell production system, or where the antibody is
produced synthetically.
18. The method of claim 16, wherein the Fc region of the antibody
comprises a mutation at position 297 such that the wild-type
asparagine residue at that position is replaced with another amino
acid that interferes with glycosylation at that position.
19. The method of claim 15, wherein the effector function is
reduced or eliminated by at least one modification of the Fc
region.
20. The method of claim 19, wherein the effector function or
complement activation function is reduced or eliminated by deletion
of all or a portion of the Fc region, or by engineering the
antibody such that it does not include an Fc region or non-Fc
region competent for effector function or complement activation
function.
21. The method of claim 19, wherein the modification is selected
from: a point mutation of the Fc region to impair binding to one or
more Fc receptors selected from the following positions: 238, 239,
248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293,
294, 295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 335,
338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437,
438, and 439; a point mutation of the Fc region to impair binding
to C1q selected from the following positions: 270, 322, 329, and
321; eliminating some or all of the Fc region, and a point mutation
at position 132 of the CH1 domain.
22. The method of claim 11, wherein the dose amount and/or
frequency of administration is modulated to reduce the
concentration of antibody to which the red blood cells are
exposed.
23. The method of claim 11, wherein the antibody is modified to
comprise pH-sensitive binding to the BBB-R.
24. The method of claim 11, wherein a further compound is
administered in addition to the antibody.
25. The method of claim 24, wherein the further compound is
responsible for or contributes to the lack of reduction of
reticulocyte levels.
26. The method of claim 25, wherein the further compound protects
reticulocytes from antibody-related depletion or supports the
growth, development, or reestablishment of reticulocytes.
27. The method of claim 26, wherein the further compound is
selected from erythropoietin (EPO), an iron supplement, vitamin C,
folic acid, and vitamin B12.
28. The method of claim 26, wherein the further compound is red
blood cells or reticulocytes from the same or another subject.
29. The method of claim 13, wherein the affinity of the antibody
for the BBB-R is further decreased.
30. The method of any of claims 1-29, further comprising the step
of monitoring the subject for depletion of red blood cells.
31. The method of any of claims 1-30, wherein the compound is a
neurological disorder drug or an imaging agent.
32. The method of any of claims 2-30, wherein the increase or
decrease is measured relative to a wild-type antibody of the same
isotype not having lowered affinity for the BBB-R.
33. The method of any of claims 1-30, wherein the antibody does not
impair the binding of the BBB-R to one or more of its native
ligands.
34. The method of any of claims 1-30, wherein the blood-brain
barrier is in a mammal.
35. The method of claim 34, wherein the mammal has a neurological
disorder.
36. The method of claim 35, wherein the neurological disorder is
selected from the group consisting of Alzheimer's disease (AD),
stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS),
amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman's
syndrome, Liddle syndrome, Parkinson's disease, Pick's disease,
Paget's disease, cancer, and traumatic brain injury.
37. The method of claim 34 wherein the mammal is a human.
38. The method of any of claims 1-30, wherein the antibody has an
IC50 for the BBB-R from about 1 nM to about 100 .mu.M.
39. The method of claim 38, wherein the IC50 is from about 5 nM to
about 100 .mu.M.
40. The method of claim 38, wherein the IC50 is from about 50 nM to
about 100 .mu.M.
41. The method of claim 38, wherein the IC50 is from about 100 nM
to about 100 .mu.M.
42. The method of any of claims 1-30, wherein the antibody has an
affinity for the BBB-R from about 5 nM to about 50 .mu.M.
43. The method of any of claims 1-30, wherein the antibody coupled
to the compound has an affinity for the BBB-R from about 30 nM to
about 30 .mu.M.
44. The method of any of claims 1-30, wherein the antibody coupled
to the compound has an affinity for the BBB-R from about 30 nM to
about 1 .mu.M.
45. The method of any of claims 1-30, wherein the antibody coupled
to the compound has a dissociation half-life for the BBB-R from
about 30 seconds to about 5 minutes, or from about 30 seconds to
about 2 minutes.
46. The method of claim 8, wherein the antibody does not inhibit
the binding of TfR to transferrin.
47. The method of any of claims 1-30, wherein the antibody coupled
to the compound is administered at a therapeutic dose.
48. The method of claim 47, wherein the therapeutic dose is
BBB-R-saturating.
49. The method of any of claims 1-30, wherein the antibody is a
multispecific antibody and the compound optionally forms one
portion of the multispecific antibody.
50. The method of claim 49 wherein the multispecific antibody
comprises a first antigen binding site which binds the BBB-R and a
second antigen binding site which binds a brain antigen.
51. The method of claim 50, wherein the brain antigen is selected
from the group consisting of: beta-secretase 1 (BACE1), Abeta,
epidermal growth factor receptor (EGFR), human epidermal growth
factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4),
alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine
rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2,
gamma secretase, death receptor 6 (DR6), amyloid precursor protein
(APP), p75 neurotrophin receptor (p75NTR), and caspase 6.
52. The method of claim 51, wherein the multispecific antibody
binds both TfR and BACE1.
53. The method of claim 51, wherein the multispecific antibody
binds both TfR and Abeta.
54. A method of improving the safety in a subject of an antibody
that binds a BBB-R comprising modifying one or more properties of
the antibody such that administration of the antibody decreases or
eliminates reduction of red blood cell levels in the subject
observed upon administration of the unmodified antibody.
55. A method of making an antibody useful for transporting a
compound across the BBB with improved safety comprising selecting
an antibody specific for a blood-brain barrier receptor (BBB-R)
that has a desirably low affinity for the BBB-R, and modifying one
or more properties of the antibody such that administration of the
antibody decreases or eliminates reduction of red blood cell levels
in the subject observed upon administration of the unmodified
antibody.
56. The method of claim 54 or 55 wherein the BBB-R is selected from
the group consisting of transferrin receptor (TfR), insulin
receptor, insulin-like growth factor receptor (IGF receptor), low
density lipoprotein receptor-related protein 8 (LRP8), low density
lipoprotein receptor-related protein 1 (LRP1), glucose transporter
1 (Glut1) and heparin-binding epidermal growth factor-like growth
factor (HB-EGF).
57. The method of claim 56 wherein the BBB-R is transferrin
receptor (TfR).
58. The method of claim 57, wherein the red blood cells are
reticulocytes.
59. The method of claim 58, wherein reduction of reticulocyte
levels is accompanied by acute clinical symptoms.
60. The method of claim 59, wherein the one or more properties is
selected from the effector function of the antibody Fc region, the
complement activation function of the antibody and the affinity of
the antibody for the BBB-R.
61. The method of claim 60, wherein the one or more properties is
selected from the effector function of the antibody Fc region and
the complement activation function of the antibody, and wherein the
effector function or the complement activation function has been
reduced or eliminated relative to a wild-type antibody of the same
isotype.
62. The method of claim 61, wherein the effector function is
reduced or eliminated by a method selected from reduction of
glycosylation of the antibody, modification of the antibody isotype
to an isotype that naturally has reduced or eliminated effector
function, and modification of the Fc region.
63. The method of claim 62, wherein the glycosylation of the
antibody is reduced by a method selected from: production of the
antibody in an environment that does not permit wild-type
glycosylation; removal of carbohydrate groups already present on
the antibody; and modification of the antibody such that wild-type
glycosylation does not occur.
64. The method of claim 63, wherein the antibody is produced in a
non-mammalian cell production system, or where the antibody is
produced synthetically.
65. The method of claim 63, wherein the Fc region of the antibody
comprises a mutation at position 297 such that the wild-type
asparagine residue at that position is replaced with another amino
acid that interferes with glycosylation at that position
66. The method of claim 62, wherein the effector function or the
complement activation function is reduced or eliminated by at least
one modification of the Fc region or the non-Fc region of the
antibody.
67. The method of claim 66, wherein the effector function or
complement activation function is reduced or eliminated by deletion
of all or a portion of the Fc region, or by engineering the
antibody such that it does not include an Fc region competent for
effector function or complement activation function.
68. The method of claim 66, wherein the modification is selected
from: a point mutation of the Fc region to impair binding to one or
more Fc receptors selected from the following positions: 238, 239,
248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293,
294, 295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 338,
340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438,
and 439; a point mutation of the Fc region to impair binding to C1q
selected from the following positions: 270, 322, 329, and 321,
eliminating some or all of the Fc region, and a point mutation at
position 132 of the CH1 domain.
69. The method of claim 59, wherein the dose amount and/or
frequency of administration is modulated to reduce the
concentration of antibody to which the red blood cells are
exposed.
70. The method of claim 59, wherein the antibody is modified to
comprise pH-sensitive binding to the BBB-R.
71. The method of claim 60, wherein the affinity of the antibody
for the BBB-R is further decreased.
72. The method of claim 71, wherein the decrease is measured
relative to a wild-type antibody of the same isotype not having
lowered affinity for the BBB-R.
73. The method of claim 54 or 55, wherein the affinity for the
BBB-R is from about 1 nM to about 100 .mu.M.
74. The method of claim 73, wherein the antibody has an IC50 of
from about 1 nM to about 100 .mu.M.
75. The method of claim 73, wherein the IC50 is from about 5 nM to
about 100 .mu.M.
76. The method of claim 73, wherein the IC50 is from about 50 nM to
about 100 .mu.M.
77. The method of claim 73, wherein the IC50 is from about 100 nM
to about 100 .mu.M.
78. The method of any of claims 54-71, wherein the antibody has an
affinity for the BBB-R from about 5 nM to about 50 .mu.M.
79. The method of any of claims 54-71, wherein the antibody has a
dissociation half-life for the BBB-R from about 30 seconds to about
5 minutes, or from about 30 seconds to about 2 minutes.
80. The method of claim 54 or 55 wherein the antibody is selected
from a panel of antibodies based upon the affinity of the selected
antibody.
81. The method of claim 54 or 55, wherein the antibody is
engineered to have the affinity.
82. The method of claim 54 or 55 comprising coupling the antibody
with a therapeutic compound.
83. The method of claim 82 wherein the therapeutic compound is a
neurological disorder drug.
84. The method of claim 82, wherein the antibody coupled to the
compound has an affinity for the BBB-R from about 30 nM to about 30
.mu.M.
85. The method of claim 83, wherein the antibody is a multispecific
antibody and the compound optionally forms one portion of the
multispecific antibody.
86. The method of claim 54 or 55 wherein the antibody is a
multispecific antibody which comprises a first antigen binding site
which binds the BBB-R and a second antigen binding site which binds
a brain antigen.
87. The method of claim 86, wherein the brain antigen is selected
from the group consisting of: beta-secretase 1 (BACE1), Abeta,
epidermal growth factor receptor (EGFR), human epidermal growth
factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4),
alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine
rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2,
gamma secretase, death receptor 6 (DR6), amyloid precursor protein
(APP), p75 neurotrophin receptor (p75NTR), and caspase 6.
88. The method of claim 87, wherein the multispecific antibody
binds both TfR and BACE1.
89. The method of claim 87, wherein the multispecific antibody
binds both TfR and Abeta.
90. The method of any of claims 54-88, wherein the antibody does
not impair the binding of the BBB-R to one or more of its native
ligands.
91. The method of claim 90, wherein the antibody does not inhibit
the binding of TfR to transferrin.
92. An antibody which binds to a BBB-R, wherein the affinity of the
antibody for the BBB-R is from about 5 nM to about 50 .mu.M or the
dissociation half-life of the antibody for the BBB-R is from about
30 seconds to about 2 minutes, and wherein one or more properties
of the antibody have been modified to reduce at least one undesired
side effect on red blood cells.
93. The antibody of claim 92 wherein the BBB-R is selected from the
group consisting of transferrin receptor (TfR), insulin receptor,
insulin-like growth factor receptor (IGF receptor), low density
lipoprotein receptor-related protein 8 (LRP8), low density
lipoprotein receptor-related protein 1 (LRP1), glucose transporter
1 (Glut1) and heparin-binding epidermal growth factor-like growth
factor (HB-EGF).
94. The antibody of claim 93 wherein the BBB-R is transferrin
receptor (TfR).
95. The antibody of claim 94, wherein the red blood cells are
reticulocytes.
96. The antibody of claim 95, wherein the at least one undesired
side effect on red blood cells is selected from reduction of
reticulocyte levels and acute clinical symptoms.
97. The antibody of claim 96, wherein the one or more properties is
selected from the effector function of the antibody Fc region, the
complement activation function of the antibody and the affinity of
the antibody for the BBB-R.
98. The antibody of claim 97, wherein the one or more properties is
selected from the effector function of the antibody Fc region and
the complement activation function of the antibody, and wherein the
effector function or complement activation function has been
reduced or eliminated relative to a wild-type antibody of the same
isotype.
99. The antibody of claim 98, wherein the effector function is
reduced or eliminated by a method selected from reduction of
glycosylation of the antibody, modification of the antibody isotype
to an isotype that naturally has reduced or eliminated effector
function, and modification of the Fc region.
100. The antibody of claim 99, wherein the glycosylation of the
antibody is reduced by a method selected from: production of the
antibody in an environment that does not permit wild-type
glycosylation; removal of carbohydrate groups already present on
the antibody; and modification of the antibody such that wild-type
glycosylation does not occur.
101. The antibody of claim 100, wherein the antibody is produced in
a non-mammalian cell production system, or where the antibody is
produced synthetically.
102. The antibody of claim 100, wherein the Fc region of the
antibody comprises a mutation at position 297 such that the
wild-type asparagine residue at that position is replaced with
another amino acid that interferes with glycosylation at that
position.
103. The antibody of claim 99, wherein the effector function or
complement activation function is reduced or eliminated by at least
one modification of the Fc region or the non-Fc region.
104. The antibody of claim 103, wherein the effector function or
complement activation function is reduced or eliminated by deletion
of all or a portion of the Fc region, or by engineering the
antibody such that it does not include an Fc region competent for
effector function.
105. The antibody of claim 103, wherein the modification is
selected from: a point mutation of the Fc region to impair binding
to one or more Fc receptors selected from the following positions:
238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289,
292, 293, 294, 295, 296, 297, 298, 301, 303, 322, 324, 327, 329,
333, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435,
437, 438, and 439; a point mutation of the Fc region to impair
binding to C1q selected from the following positions: 270, 322,
329, and 321; eliminating some or all of the Fc region, and a point
mutation at position 132 of the CH1 domain.
106. The antibody of claim 97, wherein the antibody is modified to
comprise pH-sensitive binding to the BBB-R.
107. The antibody of claim 97, wherein the affinity of the antibody
for the BBB-R is further decreased.
108. The antibody of claim 107, wherein the decrease is measured
relative to a wild-type antibody of the same isotype not having
lowered affinity for the BBB-R.
109. The antibody of claim 92, wherein the affinity is measured as
an IC50.
110. The antibody of claim 92 wherein the antibody is selected from
a panel of antibodies based upon the affinity of the selected
antibody.
111. The antibody of claim 92 wherein the antibody is engineered to
have the affinity.
112. The antibody of claim 92, further coupled with a therapeutic
compound.
113. The antibody of claim 112 wherein the therapeutic compound is
a neurological disorder drug.
114. The antibody of claim 112, wherein the antibody coupled to the
compound has an affinity for the BBB-R from about 30 nM to about 30
.mu.M.
115. The antibody of claim 112, wherein the antibody is a
multispecific antibody and the compound optionally forms one
portion of the multispecific antibody.
116. The antibody of claim 92 wherein the antibody is a
multispecific antibody which comprises a first antigen binding site
which binds the BBB-R and a second antigen binding site which binds
a brain antigen.
117. The antibody of claim 116, wherein the brain antigen is
selected from the group consisting of: beta-secretase 1 (BACE1),
Abeta, epidermal growth factor receptor (EGFR), human epidermal
growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4),
alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine
rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2,
gamma secretase, death receptor 6 (DR6), amyloid precursor protein
(APP), p75 neurotrophin receptor (p75NTR), and caspase 6.
118. The antibody of claim 117, wherein the multispecific antibody
binds both TfR and BACE1.
119. The antibody of claim 117, wherein the multispecific antibody
binds both TfR and Abeta.
120. The antibody of any of claims 92-119, wherein the antibody
does not impair the binding of the BBB-R to one or more of its
native ligands.
121. The antibody of claim 120, wherein the antibody does not
inhibit the binding of TfR to transferrin.
122. Use of an antibody that binds with low affinity to a BBB-R and
that does not impact red blood cell levels for the manufacture of a
medicament for treating a neurological disorder.
123. Use of the antibody of any of claims 92-121 for the
manufacture of a medicament for treating a neurological
disorder.
124. An antibody that binds with low affinity to a BBB-R and which
does not impact red blood cell levels for use in treating a
neurological disorder.
125. An antibody of any of claims 92-121 for use in treating a
neurological disorder.
126. A method of treating a disease or disorder associated with or
caused by elevated red blood cell levels in a subject comprising
administering an anti-TfR antibody comprising at least partial
effector function to the subject.
127. The method of claim 126, wherein the administering step is at
a dose and/or dose frequency calibrated to minimize acute clinical
symptoms of the antibody administration.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions and methods
for improving the safety of blood-brain barrier receptor-mediated
blood-brain barrier transport.
BACKGROUND
[0002] Brain penetration of large molecule drugs is severely
limited by the largely impermeable blood-brain barrier (BBB). Among
the many strategies to overcome this obstacle is to utilize
transcytosis trafficking pathways of endogenous receptors expressed
at the brain capillary endothelium. Recombinant proteins such as
monoclonal antibodies have been designed against these receptors to
enable receptor-mediated delivery of large molecules to the brain.
Strategies to maximize brain uptake while minimizing reverse
transcytosis back to the blood, and to also maximize the extent of
accumulation after therapeutic dosing have been addressed with the
finding that antibodies with low affinity to BBB receptors offer
the potential to substantially increase BBB transport and CNS
retention of associated therapeutic moieties/molecules relative to
typical high-affinity antibodies to such receptors (Atwal et al.,
Sci. Transl. Med. 3, 84ra43 (2011); Yu et al., Sci. Transl. Med. 25
May 2011: Vol. 3, Issue 84, p. 84ra44). However, the safety of
administration of such antibodies and conjugates has not been fully
explored.
SUMMARY
[0003] Monoclonal antibodies have vast therapeutic potential for
treatment of neurological or central nervous system (CNS) diseases,
but their passage into the brain is restricted by the blood-brain
barrier (BBB). Past studies have shown that a very small percentage
(approximately 0.1%) of an IgG circulating in the bloodstream
crosses through the BBB into the CNS (Felgenhauer, Klin. Wschr. 52:
1158-1164 (1974)), where the CNS concentration of the antibody may
be insufficient to permit a robust effect. It was previously found
that the percentage of the antibody that distributes into the CNS
could be improved by exploiting BBB receptors (ie, transferrin
receptor, insulin receptor, low density lipoprotein
receptor-related protein 8, glucose transporter 1 (Glut1) and the
like) (see, e.g., WO9502421). For example, the anti-BBB receptor
antibody can be made multispecific to target one or more desired
antigens in the CNS, or one or more heterologous molecules can be
coupled to the anti-BBB receptor antibody; in either case, the
anti-BBB receptor antibody can assist in delivering a therapeutic
molecule into the CNS across the BBB.
[0004] However, targeting a BBB receptor with a traditional
specific high-affinity antibody generally resulted in limited
increase in BBB transport. It was later found by Applicants that
the magnitude of antibody uptake into and distribution in the CNS
is inversely related to its binding affinity for the BBB receptor
amongst the anti-BBB antibodies studied. For example, a
low-affinity antibody to transferrin receptor (TfR) dosed at
therapeutic dose levels greatly improves BBB transport and CNS
retention of the anti-TfR antibody relative to a higher-affinity
anti-TfR antibody, and makes it possible to more readily attain
therapeutic concentrations in the CNS (Atwal et al., Sci. Transl.
Med. 3, 84ra43 (2011)). Proof of such BBB transport was achieved
using a bispecific antibody that binds both TfR and the amyloid
precursor protein (APP) cleavage enzyme, .beta.-secretase (BACE1).
A single systemic dose of the bispecific anti-TfR/BACE1 antibody
engineered using the methodology of the invention not only resulted
in significant antibody uptake in brain, but also dramatically
reduced levels of brain A.beta..sub.1-40 compared to monospecific
anti-BACE1 alone, suggesting that BBB penetrance affects the
potency of anti-BACE1. (Atwal et al., Sci. Transl. Med. 3, 84ra43
(2011); Yu et al., Sci. Transl. Med. 3, 84ra44 (2011)).
[0005] Those data and experiments highlighted several causative
mechanisms behind increasing uptake of an antibody into the CNS
using a lower-affinity antibody approach. First, high affinity
anti-BBB receptor (BBB-R) antibodies (e.g., anti-TfR.sup.A) limit
brain uptake by quickly saturating the BBB-R in the brain
vasculature, thus reducing the total amount of antibody taken up
into the brain and also restricting its distribution to the
vasculature. Strikingly, lowering affinity for the BBB-R improves
brain uptake and distribution, with a robust shift observed in
localization from the vasculature to neurons and associated
neuropil distributed within the CNS. Second, the lower affinity of
the antibody for the BBB-R is proposed to impair the ability of the
antibody to return to the vascular side of the BBB via the BBB-R
from the CNS side of the membrane because the overall affinity of
the antibody for the BBB-R is low and the local concentration of
the antibody on the CNS side of the BBB is non-saturating due to
the rapid dispersal of the antibody into the CNS compartment.
Third, in vivo, and as observed for the TfR system, antibodies with
less affinity for the BBB-R are not cleared from the system as
efficiently as those with greater affinity for the BBB-R, and thus
remain at higher circulating concentrations than their
higher-affinity counterparts. This is advantageous because the
circulating antibody levels of the lower-affinity antibody are
sustained at therapeutic levels for a longer period of time than
the higher-affinity antibody, which consequently improves uptake of
antibody in brain for a longer period of time. Furthermore, this
improvement in both plasma and brain exposure may reduce the
frequency of dosing in the clinic, which would have potential
benefit not only for patient compliance and convenience but also in
lessening any potential side effects or off-target effects of the
antibody and/or of a therapeutic compound coupled thereto.
[0006] The low-affinity BBB-R antibodies described in the
above-referenced work were selected/engineered to avoid
interference with the natural binding between transferrin and the
TfR, and thus to avoid potential iron transport-related side
effects. Nonetheless, upon administration of certain of these
antibodies in mice, some marked side effects were observed. The
mice displayed a primary response of robust depletion of
reticulocyte populations accompanied by rapid onset acute clinical
symptoms, as described in the Examples. Further in vitro studies
using a human erythroblast cell line and primary bone marrow cells
treated with anti-human TfR antibodies demonstrated that a robust
depletion of TfR-positive erythroid cells is also observable in
human cellular systems (see, e.g., Example 7). Though the mice
recovered from both the acute clinical symptoms and the decreased
reticulocyte levels in due course, avoiding or otherwise mitigating
this impact on reticulocytes is clearly desirable for an anti-TfR
antibody to be able to be used safely as a therapeutic
molecule.
[0007] Accordingly, the invention provides compositions and methods
that greatly reduce or eliminate the unwanted reduction in the
reticulocyte population upon anti-TfR administration while still
enabling the enhanced BBB transport, increased CNS distribution and
CNS retention provided by low-affinity anti-TfR antibodies
administered at therapeutic concentrations. The results described
herein show that the primary response to anti-TfR administration
(robust reticulocyte depletion and acute clinical signs) is driven
in large part by the antibody-dependent cell-mediated cytotoxicity
(ADCC) activity of the antibody, while the residual reticulocyte
depletion effect is mediated by the complement pathway. Several
general approaches to mitigate the observed effect of anti-TfR
antibodies on both the primary and residual reticulocyte depletion
are provided herein, and may be used singly or in combination.
[0008] In one approach, the effector function of the anti-BBB-R
antibody is reduced or eliminated in order to reduce or eliminate
ADCC activity. In another approach, the affinity of the anti-BBB-R
antibody for the BBB-R is further lessened such that interactions
of the antibody with the reticulocyte population are less
detrimental to that population. A third approach is directed to
reducing the amount of anti-BBB-R antibody that is present in the
plasma to reduce exposure of the reticulocyte population to
potentially detrimental concentrations of the antibody. A fourth
approach seeks to protect, stabilize and/or replenish reticulocyte
populations such that any potential depletion of the reticulocyte
population by administration of the anti-BBB-R antibody is avoided,
lessened, or mitigated.
[0009] Effector function reduction or elimination, as described
herein, may be accomplished by: (i) reduction or elimination of
wild-type mammalian glycosylation of the antibody, (for example, by
producing the antibody in an environment where such glycosylation
cannot occur, by mutating one or more carbohydrate attachment
points such that the antibody cannot be glycosylated, or by
chemically or enzymatically removing one or more carbohydrates from
the antibody after it has been glycosylated); (ii) by reduction or
elimination of the Fc receptor-binding capability of the anti-BBB-R
antibody (for example, by mutation of the Fc region, by deletion
within the Fc region or elimination of the Fc region); or (iii) by
utilization of an antibody isotype known to have minimal or no
effector function (ie., including but not limited to IgG4).
[0010] Decreasing antibody complement activation, as described
herein, may be accomplished by reduction or elimination of the C1q
binding capability of the anti-BBB-R antibody (for example, by
mutation of, deletion within or elimination of the Fc region, or by
modifying the non-Fc portion of the anti-BBB-R antibody), or by
otherwise suppressing activation or activity of the complement
system (for example, by co-administering one or more complement
pathway activation or complement pathway activity inhibitors).
[0011] When binding of anti-BBB-R antibody to BBB-R on
reticulocytes or other cell types triggers their depletion, as with
the anti-TfR antibodies exemplified herein, reduction of binding of
the antibodies to the BBB-R on the reticulocytes or other cell
types should in turn decrease the amount of reticulocyte or other
cell type depletion observed upon antibody administration. In fact,
this was demonstrated herein (see, e.g., FIG. 6B). The affinity of
the anti-BBB-R antibody for the BBB-R may be modified using any of
the methods described herein and as shown in the Examples.
[0012] Reducing the amount of anti-BBB-R antibody present in the
plasma in order to reduce exposure of the reticulocyte population
to potentially detrimental concentrations of the antibody may be
accomplished in several ways. One method is to simply decrease the
amount of the antibody that is dosed, potentially while also
increasing the frequency of the dosing, such that the maximal
concentration in the plasma is lowered but a sufficient serum level
is maintained for efficacy, while still below the threshold of the
cell-depleting side effect. Another method, which may be combined
with dosing modifications, is to select or engineer an anti-TfR
antibody that has pH-sensitive binding to TfR such that it binds to
cell surface TfR in the plasma at pH 7.4 with desirably low
affinity as described herein, but upon internalization into an
endosomal compartment, such binding to TfR is rapidly and
significantly reduced at the relatively lower pH of that
compartment (pH 5.5-6.0). Such dissociation may protect the
antibody from antigen-mediated clearance, or increase the amount of
antibody that is either delivered to the CNS or recycled back
across the BBB--in either case, the effective concentration of the
antibody is increased relative to an anti-TfR antibody that does
not comprise such pH sensitivity, without increasing the
administered dose of the antibody.
[0013] Protecting, stabilizing and/or replenishing reticulocyte
populations may be accomplished using pharmaceutical or physical
methods. In addition to the anti-BBB-R antibody, at least one
further therapeutic agent may be coadministered (simultaneously or
sequentially) that mitigates negative side effects of the antibody
on reticulocyte populations. Examples of such therapeutic agents
include, but are not limited to, erythropoietin (EPO), iron
supplements, vitamin C, folic acid, and vitamin B12. Physical
replacement of red blood cells (ie, reticulocytes) is also possible
by, for example, transfusion with similar cells, which may be from
another individual of similar blood type or may have been
previously extracted from the subject to whom the anti-BBB-R
antibody is administered.
[0014] One of ordinary skill in the art will appreciate that any
combination of the foregoing methods may be employed to engineer an
antibody (and/or dosage regimen for same) with the optimum balance
between (i) the desirably low affinity for the BBB-R that will
maximize transport of the antibody and any conjugated compounds
into the CNS; (ii) the affinity of the conjugated compound
(including as a nonlimiting example, a second or further
antigen-binding specificity in the anti-TfR antibody) for its CNS
antigen, since this is relevant to the amount of the compound that
needs to be present in the CNS to have a therapeutic effect; (iii)
the clearance rate of the anti-BBB-R antibody; and (iv) the impact
on reticulocyte populations.
[0015] It will also be appreciated that the reticulocyte-depleting
effect recognized herein of anti-TfR antibody administration may be
useful in the treatment of any disease or disorder where
overproliferation of reticulocytes is problematic. For example, in
congenital polycythemia or neoplastic polycythemia vera, raised red
blood cell counts due to hyperproliferation of, e.g.,
reticulocytes, results in thickening of blood and concomitant
physiological symptoms. Administration of an anti-TfR antibody of
the invention wherein at least partial effector function of the
antibody was preserved would permit selective removal of immature
reticulocyte populations without impacting normal transferrin
transport into the CNS. Dosing of such an antibody could be
modulated such that acute clinical symptoms could be minimized (ie,
by dosing at a very low dose or at widely-spaced intervals), as
well-understood in the art.
[0016] Anti-TfR/BACE1 and anti-TfR/Abeta are each promising and
novel therapeutic candidates for the treatment of Alzheimer's
disease. Furthermore, receptor mediated transport (RMT)-based
bispecific targeting technology opens the door for a wide range of
potential therapeutics for CNS diseases. The invention provides
methods of engineering BBB-penetrant therapeutics that greatly
improve transport across the BBB and CNS distribution of the
therapeutic without depletion of reticulocytes.
[0017] Accordingly, in a first embodiment, the invention provides a
method of transporting a compound across the blood-brain barrier in
a subject comprising exposing an antibody which binds with low
affinity to a blood-brain barrier receptor (BBB-R) coupled to a
compound to the blood-brain barrier such that the antibody
transports the compound coupled thereto across the blood-brain
barrier, wherein reduction of red blood cell levels in the subject
upon antibody administration to the subject is decreased or
eliminated. In one aspect, the BBB-R is selected from the group
consisting of transferrin receptor (TfR), insulin receptor,
insulin-like growth factor receptor (IGF receptor), low density
lipoprotein receptor-related protein 8 (LRP8), low density
lipoprotein receptor-related protein 1 (LRP1), glucose transporter
1 (Glut1) and heparin-binding epidermal growth factor-like growth
factor (HB-EGF). In another such aspect, the BBB-R is a human
BBB-R. In one such aspect, the BBB-R is TfR. In another such
aspect, the BBB-R is TfR, and the antibody does not inhibit TfR
activity. In another such aspect, the BBB-R is TfR and the antibody
does not inhibit the binding of TfR to transferrin.
[0018] In another aspect, the red blood cells are immature red
blood cells. In another such aspect, the immature red blood cells
are reticulocytes. In another aspect, reduction of reticulocyte
levels is accompanied by acute clinical symptoms. In another
aspect, the method further comprises the step of monitoring the
subject for depletion of red blood cells.
[0019] In another aspect, one or more properties of the antibody
have been modified to reduce the impact of the antibody on
reticulocyte levels and/or reduce the severity or presence of acute
clinical symptoms in the subject. In one such aspect, the affinity
of the antibody for the BBB-R is modified, i.e., decreased. In
another such aspect, the effector function of the antibody Fc
region is modified. In one such aspect, the effector function has
been reduced or eliminated relative to the effector function of a
wild-type antibody of the same isotype. In another such aspect, the
effector function is reduced or eliminated by reduction of
glycosylation of the antibody. In another such aspect, the
glycosylation of the antibody is reduced by production of the
antibody in an environment that does not permit wild-type
glycosylation. In one such aspect, the antibody is produced in a
non-mammalian cell production system. In another such aspect, the
antibody is produced synthetically. In another such aspect, the
glycosylation of the antibody is reduced by removal of carbohydrate
groups already present on the antibody. In another such aspect, the
glycosylation of the antibody is reduced by modification of the
antibody such that wild-type glycosylation does not occur. In
another such aspect, the Fc region of the antibody comprises a
mutation at position 297 such that the wild-type asparagine residue
at that position is replaced with another amino acid that
interferes with glycosylation at that position. In another aspect,
the effector function is reduced or eliminated by modification of
the antibody isotype to an isotype that naturally has reduced or
eliminated effector function.
[0020] In another aspect, the Fc region is modified to reduce or
eliminate effector function. In one such aspect, the effector
function is reduced or eliminated by at least one modification of
the Fc region. In one such aspect, the modification is a point
mutation of the Fc region to impair binding to one or more Fc
receptors selected from the following positions: 238, 239, 248,
249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294,
295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338,
340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438,
and 439. In another such aspect, the modification is elimination of
some or all of the Fc region. In another such aspect, the effector
function is reduced or eliminated by deletion of all or a portion
of the Fc region, or by engineering the antibody such that it does
not include an Fc region competent for effector function. In one
such aspect, the antibody is selected from a Fab or a single chain
antibody.
[0021] In another aspect, the Fc region and/or the non-Fc region of
the antibody is modified to reduce or eliminate activation of the
complement pathway by the antibody. In one such aspect, the
modification is a point mutation of the Fc region to impair binding
to C1q selected from the following positions: 270, 322, 329, and
321. In another such aspect, the modification is elimination of
some or all of the Fc region. In another such aspect,
complement-triggering function is reduced or eliminated by deletion
of all or a portion of the Fc region, or by engineering the
antibody such that it does not include an Fc region that engages
the complement pathway. In one such aspect, the antibody is
selected from a Fab or a single chain antibody. In another such
aspect, the non-Fc region of the antibody is modified to reduce or
eliminate activation of the complement pathway by the antibody. In
one such aspect, the modification is a point mutation of the CH1
region to impair binding to C3. In one such aspect, the point
mutation is at position 132 (see, e.g., Vidarte et al., (2001) J.
Biol. Chem. 276(41): 38217-38223).
[0022] In another aspect, the dose amount and/or frequency of
administration of the antibody is modulated to reduce the
concentration of the antibody to which the red blood cells are
exposed. In another aspect, the antibody is modified to comprise
pH-sensitive binding to the BBB-R.
[0023] In another aspect, a further compound is administered in
addition to the antibody and the coupled compound. In one such
aspect, the further compound is responsible for or contributes to
the lack of reduction of reticulocyte levels. In another such
aspect, the further compound inhibits or prevents the activation or
activity of the complement pathway (see, e.g., Mollnes and
Kirschfink (2006) Molec. Immunol. 43:107-121). In another such
aspect, the further compound protects reticulocytes from
antibody-related depletion. In another such aspect, the further
compound supports the growth, development, or reestablishment of
reticulocytes. In another aspect, the further compound is selected
from erythropoietin (EPO), an iron supplement, vitamin C, folic
acid and vitamin B12. In another aspect, the further compound is
red blood cells or reticulocytes from the same subject. In another
aspect, the further compound is red blood cells or reticulocytes
from another subject.
[0024] In another aspect, the compound is a neurological disorder
drug. In another aspect, the compound is an imaging agent. In
another aspect, the compound is labeled. In another aspect, the
antibody is labeled. In another aspect, the antibody does not
impair the binding of the BBB-R to one or more of its native
ligands. In another such aspect, the antibody specifically binds to
TfR in such a manner that it does not inhibit binding of the TfR to
transferrin. In another aspect, the BBB is in a mammal. In another
such aspect, the mammal is a human. In another such aspect, the
mammal has a neurological disorder. In another such aspect, the
neurological disorder is selected from the group consisting of
Alzheimer's disease (AD), stroke, dementia, muscular dystrophy
(MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS),
cystic fibrosis, Angelman's syndrome, Liddle syndrome, Parkinson's
disease, Pick's disease, Paget's disease, cancer, and traumatic
brain injury. In another aspect, the BBB is in a human.
[0025] In another aspect, the antibody has an IC50 for the BBB-R
from about 1 nM to about 100 .mu.M. In another such aspect, the
IC50 is from about 5 nM to about 100 .mu.M. In another such aspect,
the IC50 is from about 50 nM to about 100 .mu.M. In another such
aspect, the IC50 is from about 100 nM to about 100 .mu.M. In
another aspect, the antibody has an affinity for the BBB-R from
about 5 nM to about 50 .mu.M. In another aspect, the antibody has
an affinity for the BBB-R from about 30 nM to about 30 .mu.M. In
another such aspect, the antibody, when coupled to a compound, has
an affinity for the BBB-R from about 30 nM to about 1 .mu.M. In
another such aspect, the antibody, when coupled to a compound, has
an affinity for the BBB-R from about 50 nM to about 1 .mu.M. In
another such aspect, the compound-coupled antibody specifically
binds to TfR and has an affinity for TfR between those affinities
observed for the anti-TfR.sup.A/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has an
affinity for TfR between those affinities observed for the
anti-TfR.sup.D/BACE1 antibody and the anti-TfR.sup.E/BACE1
antibody. In another such aspect, the compound-coupled antibody
specifically binds to TfR and has an IC50 for TfR between those
IC50s observed for the anti-TfR.sup.A/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has an IC50
for TfR between those IC50s observed for the anti-TfR.sup.D/BACE1
antibody and the anti-TfR.sup.E/BACE1 antibody. In one aspect, the
affinity of the anti-BBB-R or anti-BBB-R/compound for the BBB-R is
measured using scatchard analysis. In another aspect, the affinity
of the anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured
using BIACORE analysis. In another aspect, the affinity of the
anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured using a
competition ELISA.
[0026] In another aspect, the dissociation half-life of the
antibody from the BBB-R to which it specifically binds is from
about 30 seconds to about 30 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 20
minutes. In another such aspect, the dissociation half-life is from
about 30 seconds to about 10 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 5 minutes.
In another such aspect, the dissociation half-life is from about 30
seconds to about 3 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 2 minutes.
In another such aspect, the dissociation half-life is about two
minutes. In another such aspect, the dissociation half-life is one
minute or less. In another such aspect, the compound-coupled
antibody specifically binds to TfR and has a dissociation half-life
for TfR between those dissociation half-lives observed for the
anti-TfR.sup.A/BACE1 antibody and the anti-TfR.sup.E/BACE1 antibody
from their respective binding to TfR. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has a
dissociation half-life for TfR between those dissociation
half-lives observed for the anti-TfR.sup.D/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody from their respective binding to TfR.
In another aspect, the dissociation half-life of the anti-BBB-R or
anti-BBB-R/compound for the BBB-R is measured using BIACORE
analysis. In another aspect, the dissociation half-life of the
anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured using a
competition binding assay, such as a competition ELISA. In another
aspect, the compound-coupled antibody is administered at a
therapeutic dose. In one such aspect, the therapeutic dose is a
dose that saturates the BBB-R to which the antibody specifically
binds. In another such aspect, the compound-coupled antibody is
administered at a dose and dose frequency that minimizes red blood
cell interaction with the compound-coupled antibody while still
facilitating compound delivery across the BBB into the CNS at
therapeutic levels.
[0027] In another aspect, the compound is covalently coupled to the
antibody. In one such aspect, the compound is joined to the
antibody by a linker. In one such aspect, the linker is cleavable.
In another such aspect, the linker is not cleavable. In another
such aspect, the compound is directly linked to the antibody. In
one such aspect, the antibody is a multispecific antibody and the
compound forms one portion of the multispecific antibody. In
another such aspect, the multispecific antibody comprises a first
antigen binding site which binds the BBB-R and a second antigen
binding site which binds a brain antigen. In another such aspect,
the brain antigen is selected from the group consisting of:
beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor
(EGFR), human epidermal growth factor receptor 2 (HER2), Tau,
apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion
protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin,
presenilin 1, presenilin 2, gamma secretase, death receptor 6
(DR6), amyloid precursor protein (APP), p75 neurotrophin receptor
(p75NTR), interleukin 6 receptor (IL6R), TNF receptor 1 (TNFR1),
interleukin 1 beta (IL1.beta.), and caspase 6. In another such
aspect, the multispecific antibody binds both TfR and BACE1. In
another such aspect, the multispecific antibody binds both TfR and
Abeta. In another such aspect, the multispecific antibody is
labeled. In another aspect, the compound is reversibly coupled to
the antibody such that the compound is released from the antibody
concurrent with or after BBB transport.
[0028] It will be appreciated that any of the foregoing aspects may
be applied singly or in combination with the foregoing
embodiment.
[0029] In another embodiment, the invention provides a method of
increasing exposure of the CNS of a subject to a compound, wherein
the compound is coupled to an antibody which binds with low
affinity to a BBB-R, thereby increasing the exposure of the CNS to
the compound, and wherein reduction of red blood cell levels in the
subject upon compound-coupled antibody administration to the
subject is decreased or eliminated. In one aspect, the BBB-R is
selected from the group consisting of transferrin receptor (TfR),
insulin receptor, insulin-like growth factor receptor (IGF
receptor), low density lipoprotein receptor-related protein 8
(LRP8), low density lipoprotein receptor-related protein 1 (LRP1),
glucose transporter 1 (Glut1) and heparin-binding epidermal growth
factor-like growth factor (HB-EGF). In another such aspect, the
BBB-R is a human BBB-R. In one such aspect, the BBB-R is TfR. In
another such aspect, the BBB-R is TfR, and the antibody does not
inhibit TfR activity. In another such aspect, the BBB-R is TfR and
the antibody does not inhibit the binding of TfR to
transferrin.
[0030] In another aspect, the red blood cells are immature red
blood cells. In another such aspect, the immature red blood cells
are reticulocytes. In another aspect, reduction of reticulocyte
levels is accompanied by acute clinical symptoms. In another
aspect, the method further comprises the step of monitoring the
subject for depletion of red blood cells.
[0031] In another aspect, one or more properties of the antibody
have been modified to reduce the impact of the antibody on
reticulocyte levels and/or reduce the severity or presence of acute
clinical symptoms in the subject. In one such aspect, the affinity
of the antibody for the BBB-R is modified, i.e., decreased. In
another such aspect, the effector function of the antibody Fc
region is modified. In one such aspect, the effector function has
been reduced or eliminated relative to the effector function of a
wild-type antibody of the same isotype. In another such aspect, the
effector function is reduced or eliminated by reduction of
glycosylation of the antibody. In another such aspect, the
glycosylation of the antibody is reduced by production of the
antibody in an environment that does not permit wild-type
glycosylation. In one such aspect, the antibody is produced in a
non-mammalian cell production system. In another such aspect, the
antibody is produced synthetically. In another such aspect, the
glycosylation of the antibody is reduced by removal of carbohydrate
groups already present on the antibody. In another such aspect, the
glycosylation of the antibody is reduced by modification of the
antibody such that wild-type glycosylation does not occur. In
another such aspect, the Fc region of the antibody comprises a
mutation at position 297 such that the wild-type asparagine residue
at that position is replaced with another amino acid that
interferes with glycosylation at that position. In another aspect,
the effector function is reduced or eliminated by modification of
the antibody isotype to an isotype that naturally has reduced or
eliminated effector function.
[0032] In another aspect, the Fc region is modified to reduce or
eliminate effector function. In one such aspect, the effector
function is reduced or eliminated by at least one modification of
the Fc region. In one such aspect, the modification is a point
mutation of the Fc region to impair binding to one or more Fc
receptors selected from the following positions: 238, 239, 248,
249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294,
295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338,
340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438,
and 439. In another such aspect, the modification is elimination of
some or all of the Fc region. In another such aspect, the effector
function is reduced or eliminated by deletion of all or a portion
of the Fc region, or by engineering the antibody such that it does
not include an Fc region competent for effector function. In one
such aspect, the antibody is selected from a Fab or a single chain
antibody.
[0033] In another aspect, the Fc region and/or the non-Fc region of
the antibody is modified to reduce or eliminate activation of the
complement pathway by the antibody. In one such aspect, the
modification is a point mutation of the Fc region to impair binding
to C1q selected from the following positions: 270, 322, 329, and
321. In another such aspect, the modification is elimination of
some or all of the Fc region. In another such aspect,
complement-triggering function is reduced or eliminated by deletion
of all or a portion of the Fc region, or by engineering the
antibody such that it does not include an Fc region that engages
the complement pathway. In one such aspect, the antibody is
selected from a Fab or a single chain antibody. In another such
aspect, the non-Fc region of the antibody is modified to reduce or
eliminate activation of the complement pathway by the antibody. In
one such aspect, the modification is a point mutation of the CH1
region to impair binding to C3. In one such aspect, the point
mutation is at position 132 (see, e.g., Vidarte et al., (2001) J.
Biol. Chem. 276(41): 38217-38223).
[0034] In another aspect, the dose amount and/or frequency of
administration of the antibody is modulated to reduce the
concentration of the antibody to which the red blood cells are
exposed. In another aspect, the antibody is modified to comprise
pH-sensitive binding to the BBB-R.
[0035] In another aspect, a further compound is administered in
addition to the antibody and the coupled compound. In one such
aspect, the further compound is responsible for or contributes to
the lack of reduction of reticulocyte levels. In another such
aspect, the further compound inhibits or prevents the activation or
activity of the complement pathway (see, e.g., Mollnes and
Kirschfink (2006) Molec. Immunol. 43:107-121). In another such
aspect, the further compound protects reticulocytes from
antibody-related depletion. In another such aspect, the further
compound supports the growth, development, or reestablishment of
reticulocytes. In another aspect, the further compound is selected
from erythropoietin (EPO), an iron supplement, vitamin C, folic
acid and vitamin B12. In another aspect, the further compound is
red blood cells or reticulocytes from the same subject. In another
aspect, the further compound is red blood cells or reticulocytes
from another subject.
[0036] In another aspect, the compound is a neurological disorder
drug. In another aspect, the compound is an imaging agent. In
another aspect, the compound is labeled. In another aspect, the
antibody is labeled. In another aspect, the antibody does not
impair the binding of the BBB-R to one or more of its native
ligands. In another such aspect, the antibody specifically binds to
TfR in such a manner that it does not inhibit binding of the TfR to
transferrin. In another aspect, the antibody-coupled compound is
administered to a mammal. In another such aspect, the mammal is a
human. In another such aspect, the mammal has a neurological
disorder. In another such aspect, the neurological disorder is
selected from the group consisting of Alzheimer's disease (AD),
stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS),
amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman's
syndrome, Liddle syndrome, Parkinson's disease, Pick's disease,
Paget's disease, cancer, and traumatic brain injury.
[0037] In another aspect, the increase in CNS exposure to the
compound is measured relative to the CNS exposure of a compound
coupled with a typical antibody not having lowered affinity for the
BBB-R. In another aspect, the increase in CNS exposure to the
compound is measured as a ratio of the amount of the compound found
in the CNS relative to the amount found in the serum after
administration. In another such aspect, the increase in CNS
exposure results in a ratio of greater than 0.1%. In another
aspect, the increase in CNS exposure to the compound is measured
relative to the CNS exposure of a compound in the absence of a
coupled antibody. In another aspect, the increase in CNS exposure
to the compound is measured by imaging. In another aspect, the
increase in CNS exposure to the compound is measured by an indirect
readout such as a modification of one or more physiological
symptoms.
[0038] In another aspect, the antibody has an IC50 for the BBB-R
from about 1 nM to about 100 .mu.M. In another such aspect, the
IC50 is from about 5 nM to about 100 .mu.M. In another such aspect,
the IC50 is from about 50 nM to about 100 .mu.M. In another such
aspect, the IC50 is from about 100 nM to about 100 .mu.M. In
another aspect, the antibody has an affinity for the BBB-R from
about 5 nM to about 50 .mu.M. In another aspect, the antibody has
an affinity for the BBB-R from about 30 nM to about 30 .mu.M. In
another such aspect, the antibody, when coupled to a compound, has
an affinity for the BBB-R from about 30 nM to about 1 .mu.M. In
another such aspect, the antibody, when coupled to a compound, has
an affinity for the BBB-R from about 50 nM to about 1 .mu.M. In
another such aspect, the compound-coupled antibody specifically
binds to TfR and has an affinity for TfR between those affinities
observed for the anti-TfR.sup.A/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has an
affinity for TfR between those affinities observed for the
anti-TfR.sup.D/BACE1 antibody and the anti-TfR.sup.E/BACE1
antibody. In another such aspect, the compound-coupled antibody
specifically binds to TfR and has an IC50 for TfR between those
IC50s observed for the anti-TfR.sup.A/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has an IC50
for TfR between those IC50s observed for the anti-TfR.sup.D/BACE1
antibody and the anti-TfR.sup.E/BACE1 antibody. In one aspect, the
affinity of the anti-BBB-R or anti-BBB-R/compound for the BBB-R is
measured using scatchard analysis. In another aspect, the affinity
of the anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured
using BIACORE analysis. In another aspect, the affinity of the
anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured using a
competition ELISA.
[0039] In another aspect, the dissociation half-life of the
antibody from the BBB-R to which it specifically binds is from
about 30 seconds to about 30 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 20
minutes. In another such aspect, the dissociation half-life is from
about 30 seconds to about 10 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 5 minutes.
In another such aspect, the dissociation half-life is from about 30
seconds to about 3 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 2 minutes.
In another such aspect, the dissociation half-life is about two
minutes. In another such aspect, the dissociation half-life is one
minute or less. In another such aspect, the compound-coupled
antibody specifically binds to TfR and has a dissociation half-life
for TfR between those dissociation half-lives observed for the
anti-TfR.sup.A/BACE1 antibody and the anti-TfR.sup.E/BACE1 antibody
from their respective binding to TfR. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has a
dissociation half-life for TfR between those dissociation
half-lives observed for the anti-TfR.sup.D/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody from their respective binding to TfR.
In another aspect, the dissociation half-life of the anti-BBB-R or
anti-BBB-R/compound for the BBB-R is measured using BIACORE
analysis. In another aspect, the dissociation half-life of the
anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured using a
competition binding assay, such as a competition ELISA.
[0040] In another aspect, the compound-coupled antibody is
administered at a therapeutic dose. In one such aspect, the
therapeutic dose is a dose that saturates the BBB-R to which the
antibody specifically binds. In another such aspect, the
compound-coupled antibody is administered at a dose and dose
frequency that minimizes red blood cell interaction with the
compound-coupled antibody while still facilitating compound
delivery across the BBB into the CNS at therapeutic levels.
[0041] In another aspect, the compound is covalently coupled to the
antibody. In one such aspect, the compound is joined to the
antibody by a linker. In one such aspect, the linker is cleavable.
In another such aspect, the linker is not cleavable. In another
such aspect, the compound is directly linked to the antibody. In
one such aspect, the antibody is a multispecific antibody and the
compound forms one portion of the multispecific antibody. In
another such aspect, the multispecific antibody comprises a first
antigen binding site which binds the BBB-R and a second antigen
binding site which binds a brain antigen. In another such aspect,
the brain antigen is selected from the group consisting of:
beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor
(EGFR), human epidermal growth factor receptor 2 (HER2), Tau,
apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion
protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin,
presenilin 1, presenilin 2, gamma secretase, death receptor 6
(DR6), amyloid precursor protein (APP), p75 neurotrophin receptor
(p75NTR), interleukin 6 receptor (IL6R), TNF receptor 1 (TNFR1),
interleukin 1 beta (IL1.beta.), and caspase 6. In another such
aspect, the multispecific antibody binds both TfR and BACE1. In
another such aspect, the multispecific antibody binds both TfR and
Abeta. In another such aspect, the multispecific antibody is
labeled. In another aspect, the compound is reversibly coupled to
the antibody such that the compound is released from the antibody
concurrent with or after BBB transport.
[0042] It will be appreciated that any of the foregoing aspects may
be applied singly or in combination with the foregoing
embodiment.
[0043] In another embodiment, the invention provides a method of
decreasing clearance of a compound administered to a subject,
wherein the compound is coupled to an antibody which binds with low
affinity to a BBB-R, such that the clearance of the compound is
decreased, and wherein reduction of red blood cell levels in the
subject upon compound-coupled antibody administration to the
subject is decreased or eliminated. In one aspect, the BBB-R is
selected from the group consisting of transferrin receptor (TfR),
insulin receptor, insulin-like growth factor receptor (IGF
receptor), low density lipoprotein receptor-related protein 8
(LRP8), low density lipoprotein receptor-related protein 1 (LRP1),
glucose transporter 1 (Glut1) and heparin-binding epidermal growth
factor-like growth factor (HB-EGF). In another such aspect, the
BBB-R is a human BBB-R. In one such aspect, the BBB-R is TfR. In
another such aspect, the BBB-R is TfR, and the antibody does not
inhibit TfR activity. In another such aspect, the BBB-R is TfR and
the antibody does not inhibit the binding of TfR to
transferrin.
[0044] In another aspect, the red blood cells are immature red
blood cells. In another such aspect, the immature red blood cells
are reticulocytes. In another aspect, reduction of reticulocyte
levels is accompanied by acute clinical symptoms. In another
aspect, the method further comprises the step of monitoring the
subject for depletion of red blood cells.
[0045] In another aspect, one or more properties of the antibody
have been modified to reduce the impact of the antibody on
reticulocyte levels and/or reduce the severity or presence of acute
clinical symptoms in the subject. In one such aspect, the affinity
of the antibody for the BBB-R is modified, i.e., decreased. In
another such aspect, the effector function of the antibody Fc
region is modified. In one such aspect, the effector function has
been reduced or eliminated relative to the effector function of a
wild-type antibody of the same isotype. In another such aspect, the
effector function is reduced or eliminated by reduction of
glycosylation of the antibody. In another such aspect, the
glycosylation of the antibody is reduced by production of the
antibody in an environment that does not permit wild-type
glycosylation. In one such aspect, the antibody is produced in a
non-mammalian cell production system. In another such aspect, the
antibody is produced synthetically. In another such aspect, the
glycosylation of the antibody is reduced by removal of carbohydrate
groups already present on the antibody. In another such aspect, the
glycosylation of the antibody is reduced by modification of the
antibody such that wild-type glycosylation does not occur. In
another such aspect, the Fc region of the antibody comprises a
mutation at position 297 such that the wild-type asparagine residue
at that position is replaced with another amino acid that
interferes with glycosylation at that position. In another aspect,
the effector function is reduced or eliminated by modification of
the antibody isotype to an isotype that naturally has reduced or
eliminated effector function.
[0046] In another aspect, the Fc region is modified to reduce or
eliminate effector function. In one such aspect, the effector
function is reduced or eliminated by at least one modification of
the Fc region. In one such aspect, the modification is a point
mutation of the Fc region to impair binding to one or more Fc
receptors selected from the following positions: 238, 239, 248,
249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294,
295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338,
340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438,
and 439. In another such aspect, the modification is elimination of
some or all of the Fc region. In another such aspect, the effector
function is reduced or eliminated by deletion of all or a portion
of the Fc region, or by engineering the antibody such that it does
not include an Fc region competent for effector function. In one
such aspect, the antibody is selected from a Fab or a single chain
antibody.
[0047] In another aspect, the Fc region and/or the non-Fc region of
the antibody is modified to reduce or eliminate activation of the
complement pathway by the antibody. In one such aspect, the
modification is a point mutation of the Fc region to impair binding
to C1q selected from the following positions: 270, 322, 329, and
321. In another such aspect, the modification is elimination of
some or all of the Fc region. In another such aspect,
complement-triggering function is reduced or eliminated by deletion
of all or a portion of the Fc region, or by engineering the
antibody such that it does not include an Fc region that engages
the complement pathway. In one such aspect, the antibody is
selected from a Fab or a single chain antibody. In another such
aspect, the non-Fc region of the antibody is modified to reduce or
eliminate activation of the complement pathway by the antibody. In
one such aspect, the modification is a point mutation of the CH1
region to impair binding to C3. In one such aspect, the point
mutation is at position 132 (see, e.g., Vidarte et al., (2001) J.
Biol. Chem. 276(41): 38217-38223).
[0048] In another aspect, the dose amount and/or frequency of
administration of the antibody is modulated to reduce the
concentration of the antibody to which the red blood cells are
exposed. In another aspect, the antibody is modified to comprise
pH-sensitive binding to the BBB-R.
[0049] In another aspect, a further compound is administered in
addition to the antibody and the coupled compound. In one such
aspect, the further compound is responsible for or contributes to
the lack of reduction of reticulocyte levels. In another such
aspect, the further compound inhibits or prevents the activation or
activity of the complement pathway (see, e.g., Mollnes and
Kirschfink (2006) Molec. Immunol. 43:107-121). In another such
aspect, the further compound protects reticulocytes from
antibody-related depletion. In another such aspect, the further
compound supports the growth, development, or reestablishment of
reticulocytes. In another aspect, the further compound is selected
from erythropoietin (EPO), an iron supplement, vitamin C, folic
acid and vitamin B12. In another aspect, the further compound is
red blood cells or reticulocytes from the same subject. In another
aspect, the further compound is red blood cells or reticulocytes
from another subject.
[0050] In another aspect, the compound is a neurological disorder
drug. In another aspect, the compound is an imaging agent. In
another aspect, the compound is labeled. In another aspect, the
antibody is labeled. In another aspect, the antibody does not
impair the binding of the BBB-R to one or more of its native
ligands. In another such aspect, the antibody specifically binds to
TfR in such a manner that it does not inhibit binding of the TfR to
transferrin. In another aspect, the subject is a mammal. In another
such aspect, the mammal is a human. In another such aspect, the
mammal has a neurological disorder. In another such aspect, the
neurological disorder is selected from the group consisting of
Alzheimer's disease (AD), stroke, dementia, muscular dystrophy
(MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS),
cystic fibrosis, Angelman's syndrome, Liddle syndrome, Parkinson's
disease, Pick's disease, Paget's disease, cancer, and traumatic
brain injury.
[0051] In another aspect, the decrease in clearance of the compound
is measured relative to the clearance of a compound coupled with a
typical antibody not having lowered affinity for the BBB-R. In
another aspect, the decrease in clearance of the compound is
measured relative to the clearance of the compound in the absence
of a coupled antibody.
[0052] In another aspect, the antibody has an IC50 for the BBB-R
from about 1 nM to about 100 .mu.M. In another such aspect, the
IC50 is from about 5 nM to about 100 .mu.M. In another such aspect,
the IC50 is from about 50 nM to about 100 .mu.M. In another such
aspect, the IC50 is from about 100 nM to about 100 .mu.M. In
another aspect, the antibody has an affinity for the BBB-R from
about 5 nM to about 50 .mu.M. In another aspect, the antibody has
an affinity for the BBB-R from about 30 nM to about 30 .mu.M. In
another such aspect, the antibody, when coupled to a compound, has
an affinity for the BBB-R from about 30 nM to about 1 .mu.M. In
another such aspect, the antibody, when coupled to a compound, has
an affinity for the BBB-R from about 50 nM to about 1 .mu.M. In
another such aspect, the compound-coupled antibody specifically
binds to TfR and has an affinity for TfR between those affinities
observed for the anti-TfR.sup.A/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has an
affinity for TfR between those affinities observed for the
anti-TfR.sup.D/BACE1 antibody and the anti-TfR.sup.E/BACE1
antibody. In another such aspect, the compound-coupled antibody
specifically binds to TfR and has an IC50 for TfR between those
IC50s observed for the anti-TfR.sup.A/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has an IC50
for TfR between those IC50s observed for the anti-TfR.sup.D/BACE1
antibody and the anti-TfR.sup.E/BACE1 antibody. In one aspect, the
affinity of the anti-BBB-R or anti-BBB-R/compound for the BBB-R is
measured using scatchard analysis. In another aspect, the affinity
of the anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured
using BIACORE analysis. In another aspect, the affinity of the
anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured using a
competition ELISA.
[0053] In another aspect, the dissociation half-life of the
antibody from the BBB-R to which it specifically binds is from
about 30 seconds to about 30 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 20
minutes. In another such aspect, the dissociation half-life is from
about 30 seconds to about 10 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 5 minutes.
In another such aspect, the dissociation half-life is from about 30
seconds to about 3 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 2 minutes.
In another such aspect, the dissociation half-life is about two
minutes. In another such aspect, the dissociation half-life is one
minute or less. In another such aspect, the compound-coupled
antibody specifically binds to TfR and has a dissociation half-life
for TfR between those dissociation half-lives observed for the
anti-TfR.sup.A/BACE1 antibody and the anti-TfR.sup.E/BACE1 antibody
from their respective binding to TfR. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has a
dissociation half-life for TfR between those dissociation
half-lives observed for the anti-TfR.sup.D/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody from their respective binding to TfR.
In another aspect, the dissociation half-life of the anti-BBB-R or
anti-BBB-R/compound for the BBB-R is measured using BIACORE
analysis. In another aspect, the dissociation half-life of the
anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured using a
competition binding assay, such as a competition ELISA.
[0054] In another aspect, the compound-coupled antibody is
administered at a therapeutic dose. In one such aspect, the
therapeutic dose is a dose that saturates the BBB-R to which the
antibody specifically binds. In another such aspect, the
compound-coupled antibody is administered at a dose and dose
frequency that minimizes red blood cell interaction with the
compound-coupled antibody while still facilitating compound
delivery across the BBB into the CNS at therapeutic levels.
[0055] In another aspect, the compound is covalently coupled to the
antibody. In one such aspect, the compound is joined to the
antibody by a linker. In one such aspect, the linker is cleavable.
In another such aspect, the linker is not cleavable. In another
such aspect, the compound is directly linked to the antibody. In
one such aspect, the antibody is a multispecific antibody and the
compound forms one portion of the multispecific antibody. In
another such aspect, the multispecific antibody comprises a first
antigen binding site which binds the BBB-R and a second antigen
binding site which binds a brain antigen. In another such aspect,
the brain antigen is selected from the group consisting of:
beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor
(EGFR), human epidermal growth factor receptor 2 (HER2), Tau,
apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion
protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin,
presenilin 1, presenilin 2, gamma secretase, death receptor 6
(DR6), amyloid precursor protein (APP), p75 neurotrophin receptor
(p75NTR), interleukin 6 receptor (IL6R), TNF receptor 1 (TNFR1),
interleukin 1 beta (IL1.beta.), and caspase 6. In another such
aspect, the multispecific antibody binds both TfR and BACE1. In
another such aspect, the multispecific antibody binds both TfR and
Abeta. In another such aspect, the multispecific antibody is
labeled. In another aspect, the compound is reversibly coupled to
the antibody such that the compound is released from the antibody
concurrent with or after BBB transport.
[0056] It will be appreciated that any of the foregoing aspects may
be applied singly or in combination with the foregoing
embodiment.
[0057] A method of increasing retention in the CNS of a compound
administered to a subject, wherein the compound is coupled to an
antibody which binds with low affinity to a BBB-R, such that the
retention in the CNS of the compound is increased, and wherein
reduction of red blood cell levels in the subject upon
compound-coupled antibody administration to the subject is
decreased or eliminated. In one aspect, the BBB-R is selected from
the group consisting of transferrin receptor (TfR), insulin
receptor, insulin-like growth factor receptor (IGF receptor), low
density lipoprotein receptor-related protein 8 (LRP8), low density
lipoprotein receptor-related protein 1 (LRP1), glucose transporter
1 (Glut1) and heparin-binding epidermal growth factor-like growth
factor (HB-EGF). In another such aspect, the BBB-R is a human
BBB-R. In one such aspect, the BBB-R is TfR. In another such
aspect, the BBB-R is TfR, and the antibody does not inhibit TfR
activity. In another such aspect, the BBB-R is TfR and the antibody
does not inhibit the binding of TfR to transferrin.
[0058] In another aspect, the red blood cells are immature red
blood cells. In another such aspect, the immature red blood cells
are reticulocytes. In another aspect, reduction of reticulocyte
levels is accompanied by acute clinical symptoms. In another
aspect, the method further comprises the step of monitoring the
subject for depletion of red blood cells.
[0059] In another aspect, one or more properties of the antibody
have been modified to reduce the impact of the antibody on
reticulocyte levels and/or reduce the severity or presence of acute
clinical symptoms in the subject. In one such aspect, the affinity
of the antibody for the BBB-R is modified, i.e., decreased. In
another such aspect, the effector function of the antibody Fc
region is modified. In one such aspect, the effector function has
been reduced or eliminated relative to the effector function of a
wild-type antibody of the same isotype. In another such aspect, the
effector function is reduced or eliminated by reduction of
glycosylation of the antibody. In another such aspect, the
glycosylation of the antibody is reduced by production of the
antibody in an environment that does not permit wild-type
glycosylation. In one such aspect, the antibody is produced in a
non-mammalian cell production system. In another such aspect, the
antibody is produced synthetically. In another such aspect, the
glycosylation of the antibody is reduced by removal of carbohydrate
groups already present on the antibody. In another such aspect, the
glycosylation of the antibody is reduced by modification of the
antibody such that wild-type glycosylation does not occur. In
another such aspect, the Fc region of the antibody comprises a
mutation at position 297 such that the wild-type asparagine residue
at that position is replaced with another amino acid that
interferes with glycosylation at that position. In another aspect,
the effector function is reduced or eliminated by modification of
the antibody isotype to an isotype that naturally has reduced or
eliminated effector function.
[0060] In another aspect, the Fc region is modified to reduce or
eliminate effector function. In one such aspect, the effector
function is reduced or eliminated by at least one modification of
the Fc region. In one such aspect, the modification is a point
mutation of the Fc region to impair binding to one or more Fc
receptors selected from the following positions: 238, 239, 248,
249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294,
295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338,
340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438,
and 439. In another such aspect, the modification is elimination of
some or all of the Fc region. In another such aspect, the effector
function is reduced or eliminated by deletion of all or a portion
of the Fc region, or by engineering the antibody such that it does
not include an Fc region competent for effector function. In one
such aspect, the antibody is selected from a Fab or a single chain
antibody.
[0061] In another aspect, the Fc region and/or the non-Fc region of
the antibody is modified to reduce or eliminate activation of the
complement pathway by the antibody. In one such aspect, the
modification is a point mutation of the Fc region to impair binding
to C1q selected from the following positions: 270, 322, 329, and
321. In another such aspect, the modification is elimination of
some or all of the Fc region. In another such aspect,
complement-triggering function is reduced or eliminated by deletion
of all or a portion of the Fc region, or by engineering the
antibody such that it does not include an Fc region that engages
the complement pathway. In one such aspect, the antibody is
selected from a Fab or a single chain antibody. In another such
aspect, the non-Fc region of the antibody is modified to reduce or
eliminate activation of the complement pathway by the antibody. In
one such aspect, the modification is a point mutation of the CH1
region to impair binding to C3. In one such aspect, the point
mutation is at position 132 (see, e.g., Vidarte et al., (2001) J.
Biol. Chem. 276(41): 38217-38223).
[0062] In another aspect, the dose amount and/or frequency of
administration of the antibody is modulated to reduce the
concentration of the antibody to which the red blood cells are
exposed. In another aspect, the antibody is modified to comprise
pH-sensitive binding to the BBB-R.
[0063] In another aspect, a further compound is administered in
addition to the antibody and the coupled compound. In one such
aspect, the further compound is responsible for or contributes to
the lack of reduction of reticulocyte levels. In another such
aspect, the further compound inhibits or prevents the activation or
activity of the complement pathway (see, e.g., Mollnes and
Kirschfink (2006) Molec. Immunol. 43:107-121). In another such
aspect, the further compound protects reticulocytes from
antibody-related depletion. In another such aspect, the further
compound supports the growth, development, or reestablishment of
reticulocytes. In another aspect, the further compound is selected
from erythropoietin (EPO), an iron supplement, vitamin C, folic
acid and vitamin B12. In another aspect, the further compound is
red blood cells or reticulocytes from the same subject. In another
aspect, the further compound is red blood cells or reticulocytes
from another subject.
[0064] In another aspect, the compound is a neurological disorder
drug. In another aspect, the compound is an imaging agent. In
another aspect, the compound is labeled. In another aspect, the
antibody is labeled. In another aspect, the antibody does not
impair the binding of the BBB-R to one or more of its native
ligands. In another such aspect, the antibody specifically binds to
TfR in such a manner that it does not inhibit binding of the TfR to
transferrin. In another aspect, the compound is administered to a
mammal. In another such aspect, the mammal is a human. In another
such aspect, the mammal has a neurological disorder. In another
such aspect, the neurological disorder is selected from the group
consisting of Alzheimer's disease (AD), stroke, dementia, muscular
dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral
sclerosis (ALS), cystic fibrosis, Angelman's syndrome, Liddle
syndrome, Parkinson's disease, Pick's disease, Paget's disease,
cancer, and traumatic brain injury.
[0065] In another aspect, the increase in CNS retention of the
compound is measured relative to the CNS retention of a compound
coupled with a typical antibody not having lowered affinity for the
BBB-R. In another aspect, the increase in CNS retention of the
compound is measured as a ratio of the amount of the compound found
in the CNS relative to the amount found in the serum at one or more
time points after administration. In another such aspect, the
increase in CNS retention results in a ratio of greater than 0.1%
at one or more time points after administration. In another aspect,
the increase in CNS retention of the compound is measured relative
to the CNS retention of a compound in the absence of a coupled
antibody. In another aspect, the increase in CNS retention of the
compound is measured by imaging. In another aspect, the increase in
CNS retention of the compound is measured by an indirect readout
such as a modification of one or more physiological symptoms.
[0066] In another aspect, the antibody has an IC50 for the BBB-R
from about 1 nM to about 100 .mu.M. In another such aspect, the
IC50 is from about 5 nM to about 100 .mu.M. In another such aspect,
the IC50 is from about 50 nM to about 100 .mu.M. In another such
aspect, the IC50 is from about 100 nM to about 100 .mu.M. In
another aspect, the antibody has an affinity for the BBB-R from
about 5 nM to about 50 .mu.M. In another aspect, the antibody has
an affinity for the BBB-R from about 30 nM to about 30 .mu.M. In
another such aspect, the antibody, when coupled to a compound, has
an affinity for the BBB-R from about 30 nM to about 1 .mu.M. In
another such aspect, the antibody, when coupled to a compound, has
an affinity for the BBB-R from about 50 nM to about 1 .mu.M. In
another such aspect, the compound-coupled antibody specifically
binds to TfR and has an affinity for TfR between those affinities
observed for the anti-TfR.sup.A/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has an
affinity for TfR between those affinities observed for the
anti-TfR.sup.D/BACE1 antibody and the anti-TfR.sup.E/BACE1
antibody. In another such aspect, the compound-coupled antibody
specifically binds to TfR and has an IC50 for TfR between those
IC50s observed for the anti-TfR.sup.A/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has an IC50
for TfR between those IC50s observed for the anti-TfR.sup.D/BACE1
antibody and the anti-TfR.sup.E/BACE1 antibody. In one aspect, the
affinity of the anti-BBB-R or anti-BBB-R/compound for the BBB-R is
measured using scatchard analysis. In another aspect, the affinity
of the anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured
using BIACORE analysis. In another aspect, the affinity of the
anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured using a
competition ELISA.
[0067] In another aspect, the dissociation half-life of the
antibody from the BBB-R to which it specifically binds is from
about 30 seconds to about 30 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 20
minutes. In another such aspect, the dissociation half-life is from
about 30 seconds to about 10 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 5 minutes.
In another such aspect, the dissociation half-life is from about 30
seconds to about 3 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 2 minutes.
In another such aspect, the dissociation half-life is about two
minutes. In another such aspect, the dissociation half-life is one
minute or less. In another such aspect, the compound-coupled
antibody specifically binds to TfR and has a dissociation half-life
for TfR between those dissociation half-lives observed for the
anti-TfR.sup.A/BACE1 antibody and the anti-TfR.sup.E/BACE1 antibody
from their respective binding to TfR. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has a
dissociation half-life for TfR between those dissociation
half-lives observed for the anti-TfR.sup.D/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody from their respective binding to TfR.
In another aspect, the dissociation half-life of the anti-BBB-R or
anti-BBB-R/compound for the BBB-R is measured using BIACORE
analysis. In another aspect, the dissociation half-life of the
anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured using a
competition binding assay, such as a competition ELISA.
[0068] In another aspect, the compound-coupled antibody is
administered at a therapeutic dose. In one such aspect, the
therapeutic dose is a dose that saturates the BBB-R to which the
antibody specifically binds. In another such aspect, the
compound-coupled antibody is administered at a dose and dose
frequency that minimizes red blood cell interaction with the
compound-coupled antibody while still facilitating compound
delivery across the BBB into the CNS at therapeutic levels.
[0069] In another aspect, the compound is covalently coupled to the
antibody. In one such aspect, the compound is joined to the
antibody by a linker. In one such aspect, the linker is cleavable.
In another such aspect, the linker is not cleavable. In another
such aspect, the compound is directly linked to the antibody. In
one such aspect, the antibody is a multispecific antibody and the
compound forms one portion of the multispecific antibody. In
another such aspect, the multispecific antibody comprises a first
antigen binding site which binds the BBB-R and a second antigen
binding site which binds a brain antigen. In another such aspect,
the brain antigen is selected from the group consisting of:
beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor
(EGFR), human epidermal growth factor receptor 2 (HER2), Tau,
apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion
protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin,
presenilin 1, presenilin 2, gamma secretase, death receptor 6
(DR6), amyloid precursor protein (APP), p75 neurotrophin receptor
(p75NTR), interleukin 6 receptor (IL6R), TNF receptor 1 (TNFR1),
interleukin 1 beta (IL1.beta.), and caspase 6. In another such
aspect, the multispecific antibody binds both TfR and BACE1. In
another such aspect, the multispecific antibody binds both TfR and
Abeta. In another such aspect, the multispecific antibody is
labeled. In another aspect, the compound is reversibly coupled to
the antibody such that the compound is released from the antibody
concurrent with or after BBB transport.
[0070] It will be appreciated that any of the foregoing aspects may
be applied singly or in combination with the foregoing
embodiment.
[0071] In another embodiment, the invention provides a method of
optimizing the pharmcokinetics and/or pharmacodynamics of a
compound to be efficacious in the CNS of a subject, wherein the
compound is coupled to an antibody which binds with low affinity to
a BBB-R, and the antibody is selected such that its affinity for
the BBB-R after coupling to the compound results in an amount of
transport of the antibody conjugated to the compound across the BBB
that optimizes the pharmacokinetics and/or pharmacodynamics of the
compound in the CNS, wherein reduction of red blood cell levels in
the subject upon compound-coupled antibody administration to the
subject is decreased or eliminated. In one aspect, the BBB-R is
selected from the group consisting of transferrin receptor (TfR),
insulin receptor, insulin-like growth factor receptor (IGF
receptor), low density lipoprotein receptor-related protein 8
(LRP8), low density lipoprotein receptor-related protein 1 (LRP1),
glucose transporter 1 (Glut1) and heparin-binding epidermal growth
factor-like growth factor (HB-EGF). In another such aspect, the
BBB-R is a human BBB-R. In one such aspect, the BBB-R is TfR. In
another such aspect, the BBB-R is TfR, and the antibody does not
inhibit TfR activity. In another such aspect, the BBB-R is TfR and
the antibody does not inhibit the binding of TfR to
transferrin.
[0072] In another aspect, the red blood cells are immature red
blood cells. In another such aspect, the immature red blood cells
are reticulocytes. In another aspect, reduction of reticulocyte
levels is accompanied by acute clinical symptoms. In another
aspect, the method further comprises the step of monitoring the
subject for depletion of red blood cells.
[0073] In another aspect, one or more properties of the antibody
have been modified to reduce the impact of the antibody on
reticulocyte levels and/or reduce the severity or presence of acute
clinical symptoms in the subject. In one such aspect, the affinity
of the antibody for the BBB-R is modified, i.e., decreased. In
another such aspect, the effector function of the antibody Fc
region is modified. In one such aspect, the effector function has
been reduced or eliminated relative to the effector function of a
wild-type antibody of the same isotype. In another such aspect, the
effector function is reduced or eliminated by reduction of
glycosylation of the antibody. In another such aspect, the
glycosylation of the antibody is reduced by production of the
antibody in an environment that does not permit wild-type
glycosylation. In one such aspect, the antibody is produced in a
non-mammalian cell production system. In another such aspect, the
antibody is produced synthetically. In another such aspect, the
glycosylation of the antibody is reduced by removal of carbohydrate
groups already present on the antibody. In another such aspect, the
glycosylation of the antibody is reduced by modification of the
antibody such that wild-type glycosylation does not occur. In
another such aspect, the Fc region of the antibody comprises a
mutation at position 297 such that the wild-type asparagine residue
at that position is replaced with another amino acid that
interferes with glycosylation at that position. In another aspect,
the effector function is reduced or eliminated by modification of
the antibody isotype to an isotype that naturally has reduced or
eliminated effector function.
[0074] In another aspect, the Fc region is modified to reduce or
eliminate effector function. In one such aspect, the effector
function is reduced or eliminated by at least one modification of
the Fc region. In one such aspect, the modification is a point
mutation of the Fc region to impair binding to one or more Fc
receptors selected from the following positions: 238, 239, 248,
249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294,
295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338,
340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438,
and 439. In another such aspect, the modification is elimination of
some or all of the Fc region. In another such aspect, the effector
function is reduced or eliminated by deletion of all or a portion
of the Fc region, or by engineering the antibody such that it does
not include an Fc region competent for effector function. In one
such aspect, the antibody is selected from a Fab or a single chain
antibody.
[0075] In another aspect, the Fc region and/or the non-Fc region of
the antibody is modified to reduce or eliminate activation of the
complement pathway by the antibody. In one such aspect, the
modification is a point mutation of the Fc region to impair binding
to C1q selected from the following positions: 270, 322, 329, and
321. In another such aspect, the modification is elimination of
some or all of the Fc region. In another such aspect,
complement-triggering function is reduced or eliminated by deletion
of all or a portion of the Fc region, or by engineering the
antibody such that it does not include an Fc region that engages
the complement pathway. In one such aspect, the antibody is
selected from a Fab or a single chain antibody. In another such
aspect, the non-Fc region of the antibody is modified to reduce or
eliminate activation of the complement pathway by the antibody. In
one such aspect, the modification is a point mutation of the CH1
region to impair binding to C3. In one such aspect, the point
mutation is at position 132 (see, e.g., Vidarte et al., (2001) J.
Biol. Chem. 276(41): 38217-38223).
[0076] In another aspect, the dose amount and/or frequency of
administration of the antibody is modulated to reduce the
concentration of the antibody to which the red blood cells are
exposed. In another aspect, the antibody is modified to comprise
pH-sensitive binding to the BBB-R.
[0077] In another aspect, a further compound is administered in
addition to the antibody and the coupled compound. In one such
aspect, the further compound is responsible for or contributes to
the lack of reduction of reticulocyte levels. In another such
aspect, the further compound inhibits or prevents the activation or
activity of the complement pathway (see, e.g., Mollnes and
Kirschfink (2006) Molec. Immunol. 43:107-121). In another such
aspect, the further compound protects reticulocytes from
antibody-related depletion. In another such aspect, the further
compound supports the growth, development, or reestablishment of
reticulocytes. In another aspect, the further compound is selected
from erythropoietin (EPO), an iron supplement, vitamin C, folic
acid and vitamin B12. In another aspect, the further compound is
red blood cells or reticulocytes from the same subject. In another
aspect, the further compound is red blood cells or reticulocytes
from another subject.
[0078] In another aspect, the compound is a neurological disorder
drug. In another aspect, the compound is an imaging agent. In
another aspect, the compound is labeled. In another aspect, the
antibody is labeled. In another aspect, the antibody does not
impair the binding of the BBB-R to one or more of its native
ligands. In another such aspect, the antibody specifically binds to
TfR in such a manner that it does not inhibit binding of the TfR to
transferrin. In another aspect, the BBB is in a mammal. In another
such aspect, the mammal is a human. In another such aspect, the
mammal has a neurological disorder. In another such aspect, the
neurological disorder is selected from the group consisting of
Alzheimer's disease (AD), stroke, dementia, muscular dystrophy
(MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS),
cystic fibrosis, Angelman's syndrome, Liddle syndrome, Parkinson's
disease, Pick's disease, Paget's disease, cancer, and traumatic
brain injury. In another aspect, the BBB is in a human.
[0079] In one aspect, the optimizing may include the generation of
a series of antibody-compound complexes in which each antibody has
a different affinity for the BBB-R, and assessing the
pharmacokinetics and/or pharmacodynamics of each in the CNS. In
another aspect, optimizing may be relative to a known standard,
such as, but not limited to, the pharmacokinetics and/or
pharmacodynamics of the compound when directly introduced into the
CNS or when introduced to the subject in the absence of a coupled
anti-BBB-R antibody.
[0080] In another aspect, the antibody has an IC50 for the BBB-R
from about 1 nM to about 100 .mu.M. In another such aspect, the
IC50 is from about 5 nM to about 100 .mu.M. In another such aspect,
the IC50 is from about 50 nM to about 100 .mu.M. In another such
aspect, the IC50 is from about 100 nM to about 100 .mu.M. In
another aspect, the antibody has an affinity for the BBB-R from
about 5 nM to about 50 .mu.M. In another aspect, the antibody has
an affinity for the BBB-R from about 30 nM to about 30 .mu.M. In
another such aspect, the antibody, when coupled to a compound, has
an affinity for the BBB-R from about 30 nM to about 1 .mu.M. In
another such aspect, the antibody, when coupled to a compound, has
an affinity for the BBB-R from about 50 nM to about 1 .mu.M. In
another such aspect, the compound-coupled antibody specifically
binds to TfR and has an affinity for TfR between those affinities
observed for the anti-TfR.sup.A/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has an
affinity for TfR between those affinities observed for the
anti-TfR.sup.D/BACE1 antibody and the anti-TfR.sup.E/BACE1
antibody. In another such aspect, the compound-coupled antibody
specifically binds to TfR and has an IC50 for TfR between those
IC50s observed for the anti-TfR.sup.A/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has an IC50
for TfR between those IC50s observed for the anti-TfR.sup.D/BACE1
antibody and the anti-TfR.sup.E/BACE1 antibody. In one aspect, the
affinity of the anti-BBB-R or anti-BBB-R/compound for the BBB-R is
measured using scatchard analysis. In another aspect, the affinity
of the anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured
using BIACORE analysis. In another aspect, the affinity of the
anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured using a
competition ELISA.
[0081] In another aspect, the dissociation half-life of the
antibody from the BBB-R to which it specifically binds is from
about 30 seconds to about 30 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 20
minutes. In another such aspect, the dissociation half-life is from
about 30 seconds to about 10 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 5 minutes.
In another such aspect, the dissociation half-life is from about 30
seconds to about 3 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 2 minutes.
In another such aspect, the dissociation half-life is about two
minutes. In another such aspect, the dissociation half-life is one
minute or less. In another such aspect, the compound-coupled
antibody specifically binds to TfR and has a dissociation half-life
for TfR between those dissociation half-lives observed for the
anti-TfR.sup.A/BACE1 antibody and the anti-TfR.sup.E/BACE1 antibody
from their respective binding to TfR. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has a
dissociation half-life for TfR between those dissociation
half-lives observed for the anti-TfR.sup.D/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody from their respective binding to TfR.
In another aspect, the dissociation half-life of the anti-BBB-R or
anti-BBB-R/compound for the BBB-R is measured using BIACORE
analysis. In another aspect, the dissociation half-life of the
anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured using a
competition binding assay, such as a competition ELISA.
[0082] In another aspect, the compound-coupled antibody is
administered at a therapeutic dose. In one such aspect, the
therapeutic dose is a dose that saturates the BBB-R to which the
antibody specifically binds. In another such aspect, the
compound-coupled antibody is administered at a dose and dose
frequency that minimizes red blood cell interaction with the
compound-coupled antibody while still facilitating compound
delivery across the BBB into the CNS at therapeutic levels.
[0083] In another aspect, the compound is covalently coupled to the
antibody. In one such aspect, the compound is joined to the
antibody by a linker. In one such aspect, the linker is cleavable.
In another such aspect, the linker is not cleavable. In another
such aspect, the compound is directly linked to the antibody. In
one such aspect, the antibody is a multispecific antibody and the
compound forms one portion of the multispecific antibody. In
another such aspect, the multispecific antibody comprises a first
antigen binding site which binds the BBB-R and a second antigen
binding site which binds a brain antigen. In another such aspect,
the brain antigen is selected from the group consisting of:
beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor
(EGFR), human epidermal growth factor receptor 2 (HER2), Tau,
apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion
protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin,
presenilin 1, presenilin 2, gamma secretase, death receptor 6
(DR6), amyloid precursor protein (APP), p75 neurotrophin receptor
(p75NTR), interleukin 6 receptor (IL6R), TNF receptor 1 (TNFR1),
interleukin 1 beta (IL1.beta.), and caspase 6. In another such
aspect, the multispecific antibody binds both TfR and BACE1. In
another such aspect, the multispecific antibody binds both TfR and
Abeta. In another such aspect, the multispecific antibody is
labeled. In another aspect, the compound is reversibly coupled to
the antibody such that the compound is released from the antibody
concurrent with or after BBB transport.
[0084] It will be appreciated that any of the foregoing aspects may
be applied singly or in combination with the foregoing
embodiment.
[0085] In another embodiment the invention provides a method of
treating a neurological disorder in a mammal comprising treating
the mammal with an antibody that binds a BBB-R and is coupled to a
compound, wherein the antibody has been selected to have a low
affinity for the BBB-R and thereby improves CNS uptake of the
antibody and coupled compound, and wherein reduction of red blood
cell levels in the subject upon compound-coupled antibody
administration to the subject is decreased or eliminated. In one
aspect, the BBB-R is selected from the group consisting of
transferrin receptor (TfR), insulin receptor, insulin-like growth
factor receptor (IGF receptor), low density lipoprotein
receptor-related protein 8 (LRP8), low density lipoprotein
receptor-related protein 1 (LRP1), glucose transporter 1 (Glut1)
and heparin-binding epidermal growth factor-like growth factor
(HB-EGF). In another such aspect, the BBB-R is a human BBB-R. In
one such aspect, the BBB-R is TfR. In another such aspect, the
BBB-R is TfR, and the antibody does not inhibit TfR activity. In
another such aspect, the BBB-R is TfR and the antibody does not
inhibit the binding of TfR to transferrin.
[0086] In another aspect, the red blood cells are immature red
blood cells. In another such aspect, the immature red blood cells
are reticulocytes. In another aspect, reduction of reticulocyte
levels is accompanied by acute clinical symptoms. In another
aspect, the method further comprises the step of monitoring the
subject for depletion of red blood cells.
[0087] In another aspect, one or more properties of the antibody
have been modified to reduce the impact of the antibody on
reticulocyte levels and/or reduce the severity or presence of acute
clinical symptoms in the subject. In one such aspect, the affinity
of the antibody for the BBB-R is modified, i.e., decreased. In
another such aspect, the effector function of the antibody Fc
region is modified. In one such aspect, the effector function has
been reduced or eliminated relative to the effector function of a
wild-type antibody of the same isotype. In another such aspect, the
effector function is reduced or eliminated by reduction of
glycosylation of the antibody. In another such aspect, the
glycosylation of the antibody is reduced by production of the
antibody in an environment that does not permit wild-type
glycosylation. In one such aspect, the antibody is produced in a
non-mammalian cell production system. In another such aspect, the
antibody is produced synthetically. In another such aspect, the
glycosylation of the antibody is reduced by removal of carbohydrate
groups already present on the antibody. In another such aspect, the
glycosylation of the antibody is reduced by modification of the
antibody such that wild-type glycosylation does not occur. In
another such aspect, the Fc region of the antibody comprises a
mutation at position 297 such that the wild-type asparagine residue
at that position is replaced with another amino acid that
interferes with glycosylation at that position. In another aspect,
the effector function is reduced or eliminated by modification of
the antibody isotype to an isotype that naturally has reduced or
eliminated effector function.
[0088] In another aspect, the Fc region is modified to reduce or
eliminate effector function. In one such aspect, the effector
function is reduced or eliminated by at least one modification of
the Fc region. In one such aspect, the modification is a point
mutation of the Fc region to impair binding to one or more Fc
receptors selected from the following positions: 238, 239, 248,
249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294,
295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338,
340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438,
and 439. In another such aspect, the modification is elimination of
some or all of the Fc region. In another such aspect, the effector
function is reduced or eliminated by deletion of all or a portion
of the Fc region, or by engineering the antibody such that it does
not include an Fc region competent for effector function. In one
such aspect, the antibody is selected from a Fab or a single chain
antibody.
[0089] In another aspect, the dose amount and/or frequency of
administration of the antibody is modulated to reduce the
concentration of the antibody to which the red blood cells are
exposed. In another aspect, the antibody is modified to comprise
pH-sensitive binding to the BBB-R.
[0090] In another aspect, a further compound is administered in
addition to the antibody and the coupled compound. In one such
aspect, the further compound is responsible for or contributes to
the lack of reduction of reticulocyte levels. In another such
aspect, the further compound inhibits or prevents the activation or
activity of the complement pathway (see, e.g., Mollnes and
Kirschfink (2006) Molec. Immunol. 43:107-121). In another such
aspect, the further compound protects reticulocytes from
antibody-related depletion. In another such aspect, the further
compound supports the growth, development, or reestablishment of
reticulocytes. In another aspect, the further compound is selected
from erythropoietin (EPO), an iron supplement, vitamin C, folic
acid and vitamin B12. In another aspect, the further compound is
red blood cells or reticulocytes from the same subject. In another
aspect, the further compound is red blood cells or reticulocytes
from another subject.
[0091] In another aspect, the compound is a neurological disorder
drug. In another aspect, the compound is an imaging agent. In
another aspect, the compound is labeled. In another aspect, the
antibody is labeled. In another aspect, the antibody does not
impair the binding of the BBB-R to one or more of its native
ligands. In another such aspect, the antibody specifically binds to
TfR in such a manner that it does not inhibit binding of the TfR to
transferrin. In one aspect, the mammal is a human. In another such
aspect, the mammal has a neurological disorder. In another such
aspect, the neurological disorder is selected from the group
consisting of Alzheimer's disease (AD), stroke, dementia, muscular
dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral
sclerosis (ALS), cystic fibrosis, Angelman's syndrome, Liddle
syndrome, Parkinson's disease, Pick's disease, Paget's disease,
cancer, and traumatic brain injury.
[0092] In one aspect, the treating results in lessening or
elimination of disorder symptoms. In another aspect, the treating
results in amelioration of the neurological disorder.
[0093] In another aspect, the antibody has an IC50 for the BBB-R
from about 1 nM to about 100 .mu.M. In another such aspect, the
IC50 is from about 5 nM to about 100 .mu.M. In another such aspect,
the IC50 is from about 50 nM to about 100 .mu.M. In another such
aspect, the IC50 is from about 100 nM to about 100 .mu.M. In
another aspect, the antibody has an affinity for the BBB-R from
about 5 nM to about 50 .mu.M. In another aspect, the antibody has
an affinity for the BBB-R from about 30 nM to about 30 .mu.M. In
another such aspect, the antibody, when coupled to a compound, has
an affinity for the BBB-R from about 30 nM to about 1 .mu.M. In
another such aspect, the antibody, when coupled to a compound, has
an affinity for the BBB-R from about 50 nM to about 1 .mu.M. In
another such aspect, the compound-coupled antibody specifically
binds to TfR and has an affinity for TfR between those affinities
observed for the anti-TfR.sup.A/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has an
affinity for TfR between those affinities observed for the
anti-TfR.sup.D/BACE1 antibody and the anti-TfR.sup.E/BACE1
antibody. In another such aspect, the compound-coupled antibody
specifically binds to TfR and has an IC50 for TfR between those
IC50s observed for the anti-TfR.sup.A/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has an IC50
for TfR between those IC50s observed for the anti-TfR.sup.D/BACE1
antibody and the anti-TfR.sup.E/BACE1 antibody. In one aspect, the
affinity of the anti-BBB-R or anti-BBB-R/compound for the BBB-R is
measured using scatchard analysis. In another aspect, the affinity
of the anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured
using BIACORE analysis. In another aspect, the affinity of the
anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured using a
competition ELISA.
[0094] In another aspect, the dissociation half-life of the
antibody from the BBB-R to which it specifically binds is from
about 30 seconds to about 30 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 20
minutes. In another such aspect, the dissociation half-life is from
about 30 seconds to about 10 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 5 minutes.
In another such aspect, the dissociation half-life is from about 30
seconds to about 3 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 2 minutes.
In another such aspect, the dissociation half-life is about two
minutes. In another such aspect, the dissociation half-life is one
minute or less. In another such aspect, the compound-coupled
antibody specifically binds to TfR and has a dissociation half-life
for TfR between those dissociation half-lives observed for the
anti-TfR.sup.A/BACE1 antibody and the anti-TfR.sup.E/BACE1 antibody
from their respective binding to TfR. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has a
dissociation half-life for TfR between those dissociation
half-lives observed for the anti-TfR.sup.D/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody from their respective binding to TfR.
In another aspect, the dissociation half-life of the anti-BBB-R or
anti-BBB-R/compound for the BBB-R is measured using BIACORE
analysis. In another aspect, the dissociation half-life of the
anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured using a
competition binding assay, such as a competition ELISA.
[0095] In another aspect, the compound-coupled antibody is
administered at a therapeutic dose. In one such aspect, the
therapeutic dose is a dose that saturates the BBB-R to which the
antibody specifically binds. In another such aspect, the
compound-coupled antibody is administered at a dose and dose
frequency that minimizes red blood cell interaction with the
compound-coupled antibody while still facilitating compound
delivery across the BBB into the CNS at therapeutic levels.
[0096] In another aspect, the compound is covalently coupled to the
antibody. In one such aspect, the compound is joined to the
antibody by a linker. In one such aspect, the linker is cleavable.
In another such aspect, the linker is not cleavable. In another
such aspect, the compound is directly linked to the antibody. In
one such aspect, the antibody is a multispecific antibody and the
compound forms one portion of the multispecific antibody. In
another such aspect, the multispecific antibody comprises a first
antigen binding site which binds the BBB-R and a second antigen
binding site which binds a brain antigen. In another such aspect,
the brain antigen is selected from the group consisting of:
beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor
(EGFR), human epidermal growth factor receptor 2 (HER2), Tau,
apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion
protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin,
presenilin 1, presenilin 2, gamma secretase, death receptor 6
(DR6), amyloid precursor protein (APP), p75 neurotrophin receptor
(p75NTR), interleukin 6 receptor (IL6R), TNF receptor 1 (TNFR1),
interleukin 1 beta (IL1.beta.), and caspase 6. In another such
aspect, the multispecific antibody binds both TfR and BACE1. In
another such aspect, the multispecific antibody binds both TfR and
Abeta. In another such aspect, the multispecific antibody is
labeled. In another aspect, the compound is reversibly coupled to
the antibody such that the compound is released from the antibody
concurrent with or after BBB transport.
[0097] It will be appreciated that any of the foregoing aspects may
be applied singly or in combination with the foregoing
embodiment.
[0098] In another embodiment, the invention provides a method of
improving the safety in a subject of an antibody that binds a BBB-R
comprising modifying one or more properties of the antibody such
that administration of the antibody decreases or eliminates
reduction of red blood cell levels in the subject observed upon
administration of the unmodified antibody. In one aspect, the BBB-R
is selected from the group consisting of transferrin receptor
(TfR), insulin receptor, insulin-like growth factor receptor (IGF
receptor), low density lipoprotein receptor-related protein 8
(LRP8), low density lipoprotein receptor-related protein 1 (LRP1),
glucose transporter 1 (Glut1) and heparin-binding epidermal growth
factor-like growth factor (HB-EGF). In another such aspect, the
BBB-R is a human BBB-R. In one such aspect, the BBB-R is TfR. In
another such aspect, the BBB-R is TfR, and the antibody does not
inhibit TfR activity. In another such aspect, the BBB-R is TfR and
the antibody does not inhibit the binding of TfR to
transferrin.
[0099] In another aspect, the red blood cells are immature red
blood cells. In another such aspect, the immature red blood cells
are reticulocytes. In another aspect, reduction of reticulocyte
levels is accompanied by acute clinical symptoms.
[0100] In another aspect, one or more properties of the antibody
have been modified to reduce the impact of the antibody on
reticulocyte levels and/or reduce the severity or presence of acute
clinical symptoms in the subject. In one such aspect, the affinity
of the antibody for the BBB-R is modified, i.e., decreased. In
another such aspect, the modification of the affinity of the
antibody is measured relative to a wild-type antibody of the same
isotype not having modified (i.e., decreased) affinity for the
BBB-R. In another such aspect, the effector function of the
antibody Fc region is modified. In one such aspect, the effector
function has been reduced or eliminated relative to the effector
function of a wild-type antibody of the same isotype. In another
such aspect, the effector function is reduced or eliminated by
reduction of glycosylation of the antibody. In another such aspect,
the glycosylation of the antibody is reduced by production of the
antibody in an environment that does not permit wild-type
glycosylation. In one such aspect, the antibody is produced in a
non-mammalian cell production system. In another such aspect, the
antibody is produced synthetically. In another such aspect, the
glycosylation of the antibody is reduced by removal of carbohydrate
groups already present on the antibody. In another such aspect, the
glycosylation of the antibody is reduced by modification of the
antibody such that wild-type glycosylation does not occur. In
another such aspect, the Fc region of the antibody comprises a
mutation at position 297 such that the wild-type asparagine residue
at that position is replaced with another amino acid that
interferes with glycosylation at that position. In another aspect,
the effector function is reduced or eliminated by modification of
the antibody isotype to an isotype that naturally has reduced or
eliminated effector function.
[0101] In another aspect, the Fc region is modified to reduce or
eliminate effector function. In one such aspect, the effector
function is reduced or eliminated by at least one modification of
the Fc region. In one such aspect, the modification is a point
mutation of the Fc region to impair binding to one or more Fc
receptors selected from the following positions: 238, 239, 248,
249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294,
295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338,
340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438,
and 439. In another such aspect, the modification is elimination of
some or all of the Fc region. In another such aspect, the effector
function is reduced or eliminated by deletion of all or a portion
of the Fc region, or by engineering the antibody such that it does
not include an Fc region competent for effector function. In one
such aspect, the antibody is selected from a Fab or a single chain
antibody.
[0102] In another aspect, the Fc region and/or the non-Fc region of
the antibody is modified to reduce or eliminate activation of the
complement pathway by the antibody. In one such aspect, the
modification is a point mutation of the Fc region to impair binding
to C1q selected from the following positions: 270, 322, 329, and
321. In another such aspect, the modification is elimination of
some or all of the Fc region. In another such aspect,
complement-triggering function is reduced or eliminated by deletion
of all or a portion of the Fc region, or by engineering the
antibody such that it does not include an Fc region that engages
the complement pathway. In one such aspect, the antibody is
selected from a Fab or a single chain antibody. In another such
aspect, the non-Fc region of the antibody is modified to reduce or
eliminate activation of the complement pathway by the antibody. In
one such aspect, the modification is a point mutation of the CH1
region to impair binding to C3. In one such aspect, the point
mutation is at position 132 (see, e.g., Vidarte et al., (2001) J.
Biol. Chem. 276(41): 38217-38223).
[0103] In another aspect, the dose amount and/or frequency of
administration of the antibody is modulated to reduce the
concentration of the antibody to which the red blood cells are
exposed. In another aspect, the antibody is modified to comprise
pH-sensitive binding to the BBB-R.
[0104] In another aspect, the antibody is coupled with a
therapeutic compound. In another such aspect, the compound is a
neurological disorder drug. In another aspect, the compound is an
imaging agent. In another aspect, the compound is labeled. In
another aspect, the antibody is labeled. In another aspect, the
antibody does not impair the binding of the BBB-R to one or more of
its native ligands. In another such aspect, the antibody
specifically binds to TfR in such a manner that it does not inhibit
binding of the TfR to transferrin. In another aspect, the BBB is in
a mammal. In another such aspect, the mammal is a human. In another
such aspect, the mammal has a neurological disorder. In another
such aspect, the neurological disorder is selected from the group
consisting of Alzheimer's disease (AD), stroke, dementia, muscular
dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral
sclerosis (ALS), cystic fibrosis, Angelman's syndrome, Liddle
syndrome, Parkinson's disease, Pick's disease, Paget's disease,
cancer, and traumatic brain injury. In another aspect, the BBB is
in a human.
[0105] In another aspect, the antibody has an IC50 for the BBB-R
from about 1 nM to about 100 .mu.M. In another such aspect, the
IC50 is from about 5 nM to about 100 .mu.M. In another such aspect,
the IC50 is from about 50 nM to about 100 .mu.M. In another such
aspect, the IC50 is from about 100 nM to about 100 .mu.M. In
another aspect, the antibody has an affinity for the BBB-R from
about 5 nM to about 50 .mu.M. In another aspect, the antibody has
an affinity for the BBB-R from about 30 nM to about 30 .mu.M. In
another such aspect, the antibody, when coupled to a compound, has
an affinity for the BBB-R from about 30 nM to about 1 .mu.M. In
another such aspect, the antibody, when coupled to a compound, has
an affinity for the BBB-R from about 50 nM to about 1 .mu.M. In
another such aspect, the compound-coupled antibody specifically
binds to TfR and has an affinity for TfR between those affinities
observed for the anti-TfR.sup.A/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has an
affinity for TfR between those affinities observed for the
anti-TfR.sup.D/BACE1 antibody and the anti-TfR.sup.E/BACE1
antibody. In another such aspect, the compound-coupled antibody
specifically binds to TfR and has an IC50 for TfR between those
IC50s observed for the anti-TfR.sup.A/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has an IC50
for TfR between those IC50s observed for the anti-TfR.sup.D/BACE1
antibody and the anti-TfR.sup.E/BACE1 antibody. In one aspect, the
affinity of the anti-BBB-R or anti-BBB-R/compound for the BBB-R is
measured using scatchard analysis. In another aspect, the affinity
of the anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured
using BIACORE analysis. In another aspect, the affinity of the
anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured using a
competition ELISA.
[0106] In another aspect, the dissociation half-life of the
antibody from the BBB-R to which it specifically binds is from
about 30 seconds to about 30 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 20
minutes. In another such aspect, the dissociation half-life is from
about 30 seconds to about 10 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 5 minutes.
In another such aspect, the dissociation half-life is from about 30
seconds to about 3 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 2 minutes.
In another such aspect, the dissociation half-life is about two
minutes. In another such aspect, the dissociation half-life is one
minute or less. In another such aspect, the compound-coupled
antibody specifically binds to TfR and has a dissociation half-life
for TfR between those dissociation half-lives observed for the
anti-TfR.sup.A/BACE1 antibody and the anti-TfR.sup.E/BACE1 antibody
from their respective binding to TfR. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has a
dissociation half-life for TfR between those dissociation
half-lives observed for the anti-TfR.sup.D/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody from their respective binding to TfR.
In another aspect, the dissociation half-life of the anti-BBB-R or
anti-BBB-R/compound for the BBB-R is measured using BIACORE
analysis. In another aspect, the dissociation half-life of the
anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured using a
competition binding assay, such as a competition ELISA.
[0107] In another aspect, the antibody is selected from a panel of
antibodies based upon the affinity of the selected antibody. In
another aspect, the antibody is engineered to have the desired
affinity. In one such aspect, the antibody is generated using any
art-known protein engineering methodology including, but not
limited to, phage display, yeast display, random mutagenesis, and
site-directed mutagenesis.
[0108] In another aspect, the compound-coupled antibody is
administered at a therapeutic dose. In one such aspect, the
therapeutic dose is a dose that saturates the BBB-R to which the
antibody specifically binds. In another such aspect, the
compound-coupled antibody is administered at a dose and dose
frequency that minimizes red blood cell interaction with the
compound-coupled antibody while still facilitating compound
delivery across the BBB into the CNS at therapeutic levels.
[0109] In another aspect, the compound is covalently coupled to the
antibody. In one such aspect, the compound is joined to the
antibody by a linker. In one such aspect, the linker is cleavable.
In another such aspect, the linker is not cleavable. In another
such aspect, the compound is directly linked to the antibody. In
one such aspect, the antibody is a multispecific antibody and the
compound forms one portion of the multispecific antibody. In
another such aspect, the multispecific antibody comprises a first
antigen binding site which binds the BBB-R and a second antigen
binding site which binds a brain antigen. In another such aspect,
the brain antigen is selected from the group consisting of:
beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor
(EGFR), human epidermal growth factor receptor 2 (HER2), Tau,
apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion
protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin,
presenilin 1, presenilin 2, gamma secretase, death receptor 6
(DR6), amyloid precursor protein (APP), p75 neurotrophin receptor
(p75NTR), interleukin 6 receptor (IL6R), TNF receptor 1 (TNFR1),
interleukin 1 beta (IL1.beta.), and caspase 6. In another such
aspect, the multispecific antibody binds both TfR and BACE1. In
another such aspect, the multispecific antibody binds both TfR and
Abeta. In another such aspect, the multispecific antibody is
labeled. In another aspect, the compound is reversibly coupled to
the antibody such that the compound is released from the antibody
concurrent with or after BBB transport.
[0110] It will be appreciated that any of the foregoing aspects may
be applied singly or in combination with the foregoing
embodiment.
[0111] In another embodiment, the invention provides a method of
making an antibody useful for transporting a compound across the
BBB with improved safety comprising selecting an antibody specific
for a blood-brain barrier receptor (BBB-R) that has a desirably low
affinity for the BBB-R, and modifying one or more properties of the
antibody such that administration of the antibody decreases or
eliminates reduction of red blood cell levels in the subject
observed upon administration of an unmodified antibody. In one
aspect, the BBB-R is selected from the group consisting of
transferrin receptor (TfR), insulin receptor, insulin-like growth
factor receptor (IGF receptor), low density lipoprotein
receptor-related protein 8 (LRP8), low density lipoprotein
receptor-related protein 1 (LRP1), glucose transporter 1 (Glut1)
and heparin-binding epidermal growth factor-like growth factor
(HB-EGF). In another such aspect, the BBB-R is a human BBB-R. In
one such aspect, the BBB-R is TfR. In another such aspect, the
BBB-R is TfR, and the antibody does not inhibit TfR activity. In
another such aspect, the BBB-R is TfR and the antibody does not
inhibit the binding of TfR to transferrin.
[0112] In another aspect, the red blood cells are immature red
blood cells. In another such aspect, the immature red blood cells
are reticulocytes. In another aspect, reduction of reticulocyte
levels is accompanied by acute clinical symptoms.
[0113] In another aspect, one or more properties of the antibody
have been modified to reduce the impact of the antibody on
reticulocyte levels and/or reduce the severity or presence of acute
clinical symptoms in the subject. In one such aspect, the affinity
of the antibody for the BBB-R is modified, i.e., decreased. In
another such aspect, the modification of the affinity of the
antibody is measured relative to a wild-type antibody of the same
isotype not having modified (i.e., decreased) affinity for the
BBB-R. In another such aspect, the effector function of the
antibody Fc region is modified. In one such aspect, the effector
function has been reduced or eliminated relative to the effector
function of a wild-type antibody of the same isotype. In another
such aspect, the effector function is reduced or eliminated by
reduction of glycosylation of the antibody. In another such aspect,
the glycosylation of the antibody is reduced by production of the
antibody in an environment that does not permit wild-type
glycosylation. In one such aspect, the antibody is produced in a
non-mammalian cell production system. In another such aspect, the
antibody is produced synthetically. In another such aspect, the
glycosylation of the antibody is reduced by removal of carbohydrate
groups already present on the antibody. In another such aspect, the
glycosylation of the antibody is reduced by modification of the
antibody such that wild-type glycosylation does not occur. In
another such aspect, the Fc region of the antibody comprises a
mutation at position 297 such that the wild-type asparagine residue
at that position is replaced with another amino acid that
interferes with glycosylation at that position. In another aspect,
the effector function is reduced or eliminated by modification of
the antibody isotype to an isotype that naturally has reduced or
eliminated effector function.
[0114] In another aspect, the Fc region is modified to reduce or
eliminate effector function. In one such aspect, the effector
function is reduced or eliminated by at least one modification of
the Fc region. In one such aspect, the modification is a point
mutation of the Fc region to impair binding to one or more Fc
receptors selected from the following positions: 238, 239, 248,
249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294,
295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338,
340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438,
and 439. In another such aspect, the modification is elimination of
some or all of the Fc region. In another such aspect, the effector
function is reduced or eliminated by deletion of all or a portion
of the Fc region, or by engineering the antibody such that it does
not include an Fc region competent for effector function. In one
such aspect, the antibody is selected from a Fab or a single chain
antibody.
[0115] In another aspect, the Fc region and/or the non-Fc region of
the antibody is modified to reduce or eliminate activation of the
complement pathway by the antibody. In one such aspect, the
modification is a point mutation of the Fc region to impair binding
to C1q selected from the following positions: 270, 322, 329, and
321. In another such aspect, the modification is elimination of
some or all of the Fc region. In another such aspect,
complement-triggering function is reduced or eliminated by deletion
of all or a portion of the Fc region, or by engineering the
antibody such that it does not include an Fc region that engages
the complement pathway. In one such aspect, the antibody is
selected from a Fab or a single chain antibody. In another such
aspect, the non-Fc region of the antibody is modified to reduce or
eliminate activation of the complement pathway by the antibody. In
one such aspect, the modification is a point mutation of the CH1
region to impair binding to C3. In one such aspect, the point
mutation is at position 132 (see, e.g., Vidarte et al., (2001) J.
Biol. Chem. 276(41): 38217-38223).
[0116] In another aspect, the dose amount and/or frequency of
administration of the antibody is modulated to reduce the
concentration of the antibody to which the red blood cells are
exposed. In another aspect, the antibody is modified to comprise
pH-sensitive binding to the BBB-R.
[0117] In another aspect, the antibody is coupled with a
therapeutic compound. In another such aspect, the compound is a
neurological disorder drug. In another aspect, the compound is an
imaging agent. In another aspect, the compound is labeled. In
another aspect, the antibody is labeled. In another aspect, the
antibody does not impair the binding of the BBB-R to one or more of
its native ligands. In another such aspect, the antibody
specifically binds to TfR in such a manner that it does not inhibit
binding of the TfR to transferrin. In another aspect, the BBB is in
a mammal. In another such aspect, the mammal is a human. In another
such aspect, the mammal has a neurological disorder. In another
such aspect, the neurological disorder is selected from the group
consisting of Alzheimer's disease (AD), stroke, dementia, muscular
dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral
sclerosis (ALS), cystic fibrosis, Angelman's syndrome, Liddle
syndrome, Parkinson's disease, Pick's disease, Paget's disease,
cancer, and traumatic brain injury. In another aspect, the BBB is
in a human.
[0118] In another aspect, the antibody has an IC50 for the BBB-R
from about 1 nM to about 100 .mu.M. In another such aspect, the
IC50 is from about 5 nM to about 100 .mu.M. In another such aspect,
the IC50 is from about 50 nM to about 100 .mu.M. In another such
aspect, the IC50 is from about 100 nM to about 100 .mu.M. In
another aspect, the antibody has an affinity for the BBB-R from
about 5 nM to about 50 .mu.M. In another aspect, the antibody has
an affinity for the BBB-R from about 30 nM to about 30 .mu.M. In
another such aspect, the antibody, when coupled to a compound, has
an affinity for the BBB-R from about 30 nM to about 1 .mu.M. In
another such aspect, the antibody, when coupled to a compound, has
an affinity for the BBB-R from about 50 nM to about 1 .mu.M. In
another such aspect, the compound-coupled antibody specifically
binds to TfR and has an affinity for TfR between those affinities
observed for the anti-TfR.sup.A/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has an
affinity for TfR between those affinities observed for the
anti-TfR.sup.D/BACE1 antibody and the anti-TfR.sup.E/BACE1
antibody. In another such aspect, the compound-coupled antibody
specifically binds to TfR and has an IC50 for TfR between those
IC50s observed for the anti-TfR.sup.A/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has an IC50
for TfR between those IC50s observed for the anti-TfR.sup.D/BACE1
antibody and the anti-TfR.sup.E/BACE1 antibody. In one aspect, the
affinity of the anti-BBB-R or anti-BBB-R/compound for the BBB-R is
measured using scatchard analysis. In another aspect, the affinity
of the anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured
using BIACORE analysis. In another aspect, the affinity of the
anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured using a
competition ELISA.
[0119] In another aspect, the dissociation half-life of the
antibody from the BBB-R to which it specifically binds is from
about 30 seconds to about 30 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 20
minutes. In another such aspect, the dissociation half-life is from
about 30 seconds to about 10 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 5 minutes.
In another such aspect, the dissociation half-life is from about 30
seconds to about 3 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 2 minutes.
In another such aspect, the dissociation half-life is about two
minutes. In another such aspect, the dissociation half-life is one
minute or less. In another such aspect, the compound-coupled
antibody specifically binds to TfR and has a dissociation half-life
for TfR between those dissociation half-lives observed for the
anti-TfR.sup.A/BACE1 antibody and the anti-TfR.sup.E/BACE1 antibody
from their respective binding to TfR. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has a
dissociation half-life for TfR between those dissociation
half-lives observed for the anti-TfR.sup.D/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody from their respective binding to TfR.
In another aspect, the dissociation half-life of the anti-BBB-R or
anti-BBB-R/compound for the BBB-R is measured using BIACORE
analysis. In another aspect, the dissociation half-life of the
anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured using a
competition binding assay, such as a competition ELISA.
[0120] In another aspect, the antibody is selected from a panel of
antibodies based upon the affinity of the selected antibody. In
another aspect, the antibody is engineered to have the desired
affinity. In one such aspect, the antibody is generated using any
art-known protein engineering methodology including, but not
limited to, phage display, yeast display, random mutagenesis, and
site-directed mutagenesis.
[0121] In another aspect, the compound-coupled antibody is
administered at a therapeutic dose. In one such aspect, the
therapeutic dose is a dose that saturates the BBB-R to which the
antibody specifically binds. In another such aspect, the
compound-coupled antibody is administered at a dose and dose
frequency that minimizes red blood cell interaction with the
compound-coupled antibody while still facilitating compound
delivery across the BBB into the CNS at therapeutic levels.
[0122] In another aspect, the compound is covalently coupled to the
antibody. In one such aspect, the compound is joined to the
antibody by a linker. In one such aspect, the linker is cleavable.
In another such aspect, the linker is not cleavable. In another
such aspect, the compound is directly linked to the antibody. In
one such aspect, the antibody is a multispecific antibody and the
compound forms one portion of the multispecific antibody. In
another such aspect, the multispecific antibody comprises a first
antigen binding site which binds the BBB-R and a second antigen
binding site which binds a brain antigen. In another such aspect,
the brain antigen is selected from the group consisting of:
beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor
(EGFR), human epidermal growth factor receptor 2 (HER2), Tau,
apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion
protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin,
presenilin 1, presenilin 2, gamma secretase, death receptor 6
(DR6), amyloid precursor protein (APP), p75 neurotrophin receptor
(p75NTR), interleukin 6 receptor (IL6R), TNF receptor 1 (TNFR1),
interleukin 1 beta (IL1.beta.), and caspase 6. In another such
aspect, the multispecific antibody binds both TfR and BACE1. In
another such aspect, the multispecific antibody binds both TfR and
Abeta. In another such aspect, the multispecific antibody is
labeled. In another aspect, the compound is reversibly coupled to
the antibody such that the compound is released from the antibody
concurrent with or after BBB transport.
[0123] It will be appreciated that any of the foregoing aspects may
be applied singly or in combination with the foregoing
embodiment.
[0124] In another embodiment, the invention provides an antibody
which binds to a blood-brain barrier receptor (BBB-R), wherein the
affinity of the antibody for the BBB-R is from about 5 nM to about
50 .mu.M, and wherein one or more properties of the antibody have
been modified to reduce at least one undesired side effect on red
blood cells. In one aspect, the BBB-R is selected from the group
consisting of transferrin receptor (TfR), insulin receptor,
insulin-like growth factor receptor (IGF receptor), low density
lipoprotein receptor-related protein 8 (LRP8), low density
lipoprotein receptor-related protein 1 (LRP1), glucose transporter
1 (Glut1) and heparin-binding epidermal growth factor-like growth
factor (HB-EGF). In another such aspect, the BBB-R is a human
BBB-R. In one such aspect, the BBB-R is TfR. In another such
aspect, the BBB-R is TfR, and the antibody does not inhibit TfR
activity. In another such aspect, the BBB-R is TfR and the antibody
does not inhibit the binding of TfR to transferrin.
[0125] In another aspect, the red blood cells are immature red
blood cells. In another such aspect, the immature red blood cells
are reticulocytes. In another aspect, reduction of reticulocyte
levels is accompanied by acute clinical symptoms.
[0126] In another aspect, one or more properties of the antibody
have been modified to reduce the impact of the antibody on
reticulocyte levels and/or reduce the severity or presence of acute
clinical symptoms in the subject. In one such aspect, the affinity
of the antibody for the BBB-R is modified, i.e., decreased. In
another such aspect, the modification of the affinity of the
antibody is measured relative to a wild-type antibody of the same
isotype not having modified (i.e., decreased) affinity for the
BBB-R. In another such aspect, the effector function of the
antibody Fc region is modified. In one such aspect, the effector
function has been reduced or eliminated relative to the effector
function of a wild-type antibody of the same isotype. In another
such aspect, the effector function is reduced or eliminated by
reduction of glycosylation of the antibody. In another such aspect,
the glycosylation of the antibody is reduced by production of the
antibody in an environment that does not permit wild-type
glycosylation. In one such aspect, the antibody is produced in a
non-mammalian cell production system. In another such aspect, the
antibody is produced synthetically. In another such aspect, the
glycosylation of the antibody is reduced by removal of carbohydrate
groups already present on the antibody. In another such aspect, the
glycosylation of the antibody is reduced by modification of the
antibody such that wild-type glycosylation does not occur. In
another such aspect, the Fc region of the antibody comprises a
mutation at position 297 such that the wild-type asparagine residue
at that position is replaced with another amino acid that
interferes with glycosylation at that position. In another aspect,
the effector function is reduced or eliminated by modification of
the antibody isotype to an isotype that naturally has reduced or
eliminated effector function.
[0127] In another aspect, the Fc region is modified to reduce or
eliminate effector function. In one such aspect, the effector
function is reduced or eliminated by at least one modification of
the Fc region. In one such aspect, the modification is a point
mutation of the Fc region to impair binding to one or more Fc
receptors selected from the following positions: 238, 239, 248,
249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294,
295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338,
340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438,
and 439. In another such aspect, the modification is elimination of
some or all of the Fc region. In another such aspect, the effector
function is reduced or eliminated by deletion of all or a portion
of the Fc region, or by engineering the antibody such that it does
not include an Fc region competent for effector function. In one
such aspect, the antibody is selected from a Fab or a single chain
antibody.
[0128] In another aspect, the Fc region and/or the non-Fc region of
the antibody is modified to reduce or eliminate activation of the
complement pathway by the antibody. In one such aspect, the
modification is a point mutation of the Fc region to impair binding
to C1q selected from the following positions: 270, 322, 329, and
321. In another such aspect, the modification is elimination of
some or all of the Fc region. In another such aspect,
complement-triggering function is reduced or eliminated by deletion
of all or a portion of the Fc region, or by engineering the
antibody such that it does not include an Fc region that engages
the complement pathway. In one such aspect, the antibody is
selected from a Fab or a single chain antibody. In another such
aspect, the non-Fc region of the antibody is modified to reduce or
eliminate activation of the complement pathway by the antibody. In
one such aspect, the modification is a point mutation of the CH1
region to impair binding to C3. In one such aspect, the point
mutation is at position 132 (see, e.g., Vidarte et al., (2001) J.
Biol. Chem. 276(41): 38217-38223).
[0129] In another aspect, the antibody is coupled with a
therapeutic compound. In another such aspect, the compound is a
neurological disorder drug. In another aspect, the compound is an
imaging agent. In another aspect, the compound is labeled. In
another aspect, the antibody is labeled. In another aspect, the
antibody does not impair the binding of the BBB-R to one or more of
its native ligands. In another such aspect, the antibody
specifically binds to TfR in such a manner that it does not inhibit
binding of the TfR to transferrin. In another aspect, the BBB is in
a mammal. In another such aspect, the mammal is a human. In another
such aspect, the mammal has a neurological disorder. In another
such aspect, the neurological disorder is selected from the group
consisting of Alzheimer's disease (AD), stroke, dementia, muscular
dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral
sclerosis (ALS), cystic fibrosis, Angelman's syndrome, Liddle
syndrome, Parkinson's disease, Pick's disease, Paget's disease,
cancer, and traumatic brain injury. In another aspect, the BBB is
in a human.
[0130] In another aspect, the antibody has an IC50 for the BBB-R
from about 30 nM to about 30 .mu.M. In another such aspect, the
antibody, when coupled to a compound, has an affinity for the BBB-R
from about 30 nM to about 1 .mu.M. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has an
affinity for TfR between those affinities observed for the
anti-TfR.sup.A/BACE1 antibody and the anti-TfR.sup.E/BACE1
antibody. In another such aspect, the compound-coupled antibody
specifically binds to TfR and has an affinity for TfR between those
affinities observed for the anti-TfR.sup.D/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has an IC50
for TfR between those IC50s observed for the anti-TfR.sup.A/BACE1
antibody and the anti-TfR.sup.E/BACE1 antibody. In another such
aspect, the compound-coupled antibody specifically binds to TfR and
has an IC50 for TfR between those IC50s observed for the
anti-TfR.sup.D/BACE1 antibody and the anti-TfR.sup.E/BACE1
antibody. In one aspect, the affinity of the anti-BBB-R or
anti-BBB-R/compound for the BBB-R is measured using scatchard
analysis. In another aspect, the affinity of the anti-BBB-R or
anti-BBB-R/compound for the BBB-R is measured using BIACORE
analysis. In another aspect, the affinity of the anti-BBB-R or
anti-BBB-R/compound for the BBB-R is measured using a competition
ELISA.
[0131] In another aspect, the dissociation half-life of the
antibody from the BBB-R to which it specifically binds is from
about 30 seconds to about 30 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 20
minutes. In another such aspect, the dissociation half-life is from
about 30 seconds to about 10 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 5 minutes.
In another such aspect, the dissociation half-life is from about 30
seconds to about 3 minutes. In another such aspect, the
dissociation half-life is from about 30 seconds to about 2 minutes.
In another such aspect, the dissociation half-life is about two
minutes. In another such aspect, the dissociation half-life is one
minute or less. In another such aspect, the compound-coupled
antibody specifically binds to TfR and has a dissociation half-life
for TfR between those dissociation half-lives observed for the
anti-TfR.sup.A/BACE1 antibody and the anti-TfR.sup.E/BACE1 antibody
from their respective binding to TfR. In another such aspect, the
compound-coupled antibody specifically binds to TfR and has a
dissociation half-life for TfR between those dissociation
half-lives observed for the anti-TfR.sup.D/BACE1 antibody and the
anti-TfR.sup.E/BACE1 antibody from their respective binding to TfR.
In another aspect, the dissociation half-life of the anti-BBB-R or
anti-BBB-R/compound for the BBB-R is measured using BIACORE
analysis. In another aspect, the dissociation half-life of the
anti-BBB-R or anti-BBB-R/compound for the BBB-R is measured using a
competition binding assay, such as a competition ELISA.
[0132] In another aspect, the antibody is selected from a panel of
antibodies based upon the affinity of the selected antibody. In
another aspect, the antibody is engineered to have the desired
affinity. In one such aspect, the antibody is generated using any
art-known protein engineering methodology including, but not
limited to, phage display, yeast display, random mutagenesis, and
site-directed mutagenesis.
[0133] In another aspect, a compound is covalently coupled to the
antibody. In one such aspect, the compound is joined to the
antibody by a linker. In one such aspect, the linker is cleavable.
In another such aspect, the linker is not cleavable. In another
such aspect, the compound is directly linked to the antibody. In
one such aspect, the antibody is a multispecific antibody and the
compound forms one portion of the multispecific antibody. In
another such aspect, the multispecific antibody comprises a first
antigen binding site which binds the BBB-R and a second antigen
binding site which binds a brain antigen. In another such aspect,
the brain antigen is selected from the group consisting of:
beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor
(EGFR), human epidermal growth factor receptor 2 (HER2), Tau,
apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion
protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin,
presenilin 1, presenilin 2, gamma secretase, death receptor 6
(DR6), amyloid precursor protein (APP), p75 neurotrophin receptor
(p75NTR), interleukin 6 receptor (IL6R), TNF receptor 1 (TNFR1),
interleukin 1 beta (IL1.beta.), and caspase 6. In another such
aspect, the multispecific antibody binds both TfR and BACE1. In
another such aspect, the multispecific antibody binds both TfR and
Abeta. In another such aspect, the multispecific antibody is
labeled. In another aspect, the compound is reversibly coupled to
the antibody such that the compound is released from the antibody
concurrent with or after BBB transport.
[0134] It will be appreciated that any of the foregoing aspects may
be applied singly or in combination with the foregoing
embodiment.
[0135] In another embodiment, the invention provides the use of an
antibody that binds with low affinity to a BBB-R and that does not
reduce red blood cell levels for the manufacture of a medicament
for treating a neurological disorder. Any of the foregoing
described low-affinity anti-BBB-R antibodies or any of the
low-affinity anti-BBB-R antibodies described elsewhere herein may
be used in the method.
[0136] In another embodiment, the invention provides an antibody
that binds with low affinity to a BBB-R and that does not reduce
red blood cell levels for use in treating a neurological disorder.
Any of the foregoing described low-affinity anti-BBB-R antibodies
or any of the low-affinity anti-BBB-R antibodies described
elsewhere herein may be used in the method.
[0137] In another embodiment, the invention provides a method of
transporting a therapeutic compound, such as a neurological
disorder drug, across the blood-brain barrier comprising exposing
the anti-BBB-R antibody coupled with a neurological disorder drug
to the blood-brain barrier such that the antibody transports the
neurological disorder drug coupled thereto across the blood-brain
barrier, wherein the antibody does not reduce red blood cell
levels.
[0138] The invention additionally provides a method of treating a
neurological disorder in a mammal comprising treating the mammal
with a multispecific antibody that binds both a blood-brain barrier
receptor (BBB-R) and a brain antigen, wherein the anti-BBB-R
antibody has been selected to have a low affinity for the BBB-R and
thereby improves brain uptake of the anti-brain antigen antibody,
and wherein administration of the antibody does not decrease red
blood cell levels.
[0139] The invention additionally provides a method of treating a
disease or disorder associated with or caused by elevated red blood
cell levels in a subject comprising administering an anti-TfR
antibody comprising at least partial effector function to the
subject. In one aspect, the administering step is at a dose and/or
dose frequency calibrated to minimize acute clinical symptoms of
the antibody administration.
[0140] It will be understood that any of the foregoing methods and
compositions of the invention may be combined with one another
and/or with the further aspects of the invention described in the
specification herein.
BRIEF DESCRIPTION OF THE FIGURES
[0141] FIGS. 1A-1E depict the results of experiments assessing the
affinities of anti-transferrin receptor ("TfR") and
anti-TfR/beta-secretase ("BACE1") variants for TfR, as well as
concentrations of the antibody and A.beta..sub.1-40 after
administration in mice, as described in Example 1. The competitive
ELISA assay results in FIG. 1A show that anti-TfR/BACE1 variants
and anti-TfR variants have distinct affinities for TfR. FIGS. 1B
and 1D show, respectively, mean serum and brain antibody
concentrations in wild-type mice after a single 50 mg/kg
intravenous injection of control IgG, anti-BACE1, or an
anti-TfR/BACE1 variant (n=6 per group). FIGS. 1C and 1E show,
respectively, plasma and brain concentrations of A.beta..sub.1-40
in these same treated mice, as a marker of the activity of the
injected antibody.
[0142] FIG. 2A is a schematic depiction of red blood cell (RBC)
maturation in the bone marrow, showing progression from the
pro-erythroblast (Pro-EB), to basophilic erythroblast (Baso-EB), to
polychromatic erythroblast (Poly-EB), to orthochromatic
erythroblast (Ortho-EB) and finally to the reticulocyte.
Reticulocytes are released from the bone marrow to the circulation
where they mature to RBCs. During the later stages of maturation in
the bone marrow, erythroid precursors synthesize the
iron-containing protein hemoglobin, which requires a concomitant
increase in TfR expression. Transferrin receptors are shed with the
cessation of hemoglobin synthesis and cell proliferation as cells
mature through the reticulocyte stage, such that mature RBCs do not
express TfR. The relative number of TfR present at each cell stage
of RBC maturation is indicated in the graph at the top of the
figure, based on data from Iacpetta et al., Biochim. Biophys. Acta
687: 204-210 (1982). FIGS. 2B and 2C depict the results of
experiments assessing the impact of anti-TfR and anti-TfR/BACE1
administration on reticulocytes in mice, as described in Example
2A. FIG. 2B depicts the results of experiments testing the impact
of intravenously administered anti-TfR.sup.D, anti-TfR.sup.D/BACE1
or control IgG on the percent of the immature reticulocyte fraction
from whole blood of wild-type mice at 1 hour post-dose (n=6 per
group). FIG. 2C depicts the results of experiments testing the
impact of intravenously administered anti-TfR.sup.A/BACE1,
anti-TfR.sup.D/BACE1 or control IgG on total reticulocyte counts in
whole blood of wild-type mice at 24 hours or 7 days post-dose (n=6
per group). All data are shown as mean.+-.SEM. FIGS. 2D and 2E
depict mean brain Abeta.sub.1-40 concentrations in wild-type mice
after a single 50 mg/kg intravenous injection of control IgG, or 5
mg/kg, 25 mg/kg or 50 mg/kg injections of anti-TfR.sup.D/BACE1
(FIG. 2D) or anti-TfR.sup.A/BACE1 (FIG. 2E) (n=6 per group). FIGS.
2F-2H depict the results of experiments assessing the
pharmacokinetics of anti-TfR.sup.A/BACE1 and anti-TfR.sup.D/BACE1
in comparison with control at 5 mg/kg, 25 mg/kg or 50 mg/kg dose
levels. FIG. 2F provides measurements of brain antibody
concentration at the indicated time points. FIG. 2G provides
measurements of plasma antibody concentration at the indicated time
points. FIG. 2H provides measurements of plasma Abeta levels at the
indicated time points.
[0143] FIGS. 3A-3E depict the results of experiments assessing the
impact of elimination of effector function (FIGS. 3A-3C) or
elimination of complement function (FIGS. 3D and 3E) on
reticulocyte depletion by various anti-TfR antibodies, as described
in Example 2B. Total reticulocyte counts in whole blood are shown
from wild-type mice (FIGS. 3A and 3C), Fc.gamma..sup.-/-
(B6.129P2-Fcer1gtm1Rav N12) mice (FIG. 3B), or C3.sup.-/- mice
(FIG. 3D) 24 hours after intravenous injection of antibody at the
indicated dose, as compared to control IgG (n=6 per group). FIG. 3E
depicts the results of experiments assessing the effect of
impairment of the complement system on the previously observed
depletion of reticulocytes by anti-TfR. Wild-type or C3 knockout
mice were intravenously administered 50 mg/kg of a control IgG or
an anti-TfR.sup.D/control IgG mixture (n=6 per group).
[0144] FIGS. 4A and 4B depict the results of in vitro experiments
assessing the induction of antibody-dependent cell-mediated
cytotoxicity (ADCC) (FIG. 4A) or complement-dependent cytotoxicity
(CDC) (FIG. 4B) by anti-TfR.sup.A, anti-TfR.sup.A/BACE1, or control
IgG in mouse erythroleukemic blasts at a range of antibody
concentrations, as described in Example 2B.
[0145] FIGS. 5A-5C depict the results of experiments assessing
whether elimination of Fc binding or BACE1 binding impacts
reticulocyte depletion by monospecific or bispecific anti-TfR
antibodies, as described in Example 2C. Total reticulocyte counts
are shown for wild-type mice (n=6 per group) 24 hours after
intravenous injection of the indicated F(ab).sub.2 or control IgG
(FIGS. 5A and 5B) or bispecific antibody (FIG. 5C).
[0146] FIGS. 6A-6C depict the results of experiments assessing the
impact of reducing affinity to TfR on reticulocyte depletion and
brain TfR expression, as described in Example 3. FIGS. 6A and 6B
depict total reticulocyte counts in wild-type mice 24 hours after
intravenous injection of the indicated anti-TfR/BACE1 variant
antibody, compared to control IgG. FIG. 6C shows quantification of
brain TfR expression level by Western blot from whole mouse brain
lysates 4 days after an intravenous injection of control IgG,
anti-TfR.sup.A/BACE1, or anti-TfR.sup.D/BACE1 at the indicated dose
(n=3 per group). Quantification of TfR expression was normalized to
actin and the data are shown as mean.+-.SEM.
[0147] FIGS. 7A-7C depict the results of experiments assessing
whether TfR antibody treatment affected blood-brain barrier
permeability, as described in Example 4. Wild-type mice were
intravenously administered 50 mg/kg of control IgG or 25 mg/kg of
each of the co-injected antibody combinations. Mean antibody uptake
in brain 24 hours after intravenous injection was measured using a
generic human-Fc ELISA (FIG. 7A) or a BACE1-ectodomain ELISA (FIG.
7B). FIG. 7C shows a quantification of A.beta..sub.1-40
concentrations in mouse brain after intravenous injection of
control IgG or co-injection of antibodies (n=6 per group).
[0148] FIGS. 8A-8F depict the results of experiments assessing the
impact of multiple doses of anti-TfR.sup.D/BACE1 on reticulocyte
levels in treated mice, as described in Example 5. Wild-type mice
were intravenously dosed once weekly with 25 mg/kg of control IgG
or anti-TfR.sup.D/BACE1. FIGS. 8A and 8B, respectively, depict
observed plasma and brain antibody concentrations at 24 hours, 4
days and 7 days following two or four doses of antibody. It should
be noted that the Y-axis scale in FIG. 8A is in .mu.M while the
Y-axis scale in FIG. 8B is in nM. The corresponding average
A.beta..sub.1-40 concentrations in plasma (FIG. 8C) and brain (FIG.
8D) were also measured. FIG. 8E shows the total reticulocyte count
in mice 24 hours after the second and fourth dose, and 7 days after
the fourth dose of control IgG or anti-TfR.sup.D/BACE1. FIG. 8F
shows a graph depicting the results of a quantification of brain
TfR expression level by Western blot from whole mouse brain lysates
after 4 weekly doses of control IgG or anti-TfR.sup.D/BACE1.
Quantification of TfR expression was normalized to actin and data
are shown as mean.+-.SEM.
[0149] FIGS. 9A-9B and 10A-10D depict the results of experiments
assessing the impact of an effectorless anti-TfR/BACE1 antibody on
erythrocyte subpopulations in blood and bone marrow in mice.
Distinct populations of Ter119-positive erythrocyte lineage in both
(FIG. 9A) blood and (FIG. 9B) bone marrow are distinguished by
their TfR expression and cell size (as determined by forward
scatter profile) using flow cytometry (Paniga et al., PLoS One 6, 9
(2011)). Ter119-positive cell subsets in bone marrow were defined
as EryA=large, TfR-positive early basophilic erythroblasts,
EryB=small, TfR-positive polychromatic erythroblasts, and
EryC=TfR-negative mature erythrocytes. FIGS. 9C and 9D show a
time-course of the total Ter119-positive erythroid population
(reticulocytes and red blood cells; 9C) and TfR-positive
reticulocytes (9D) in blood after dosing with anti-TfR.sup.D/BACE1
compared to control IgG (n=6/group). FIGS. 10A to 10D provide
graphs of the quantification of distinct erythrocyte subpopulations
(EryA, EryB, EryC) in bone marrow following anti-TfR.sup.D/BACE1 or
control IgG dosing (n=6/group).
[0150] FIGS. 11A-11B and 12A-12D depict the results of experiments
analyzing the impact of affinity and effector function of an
anti-TfR/BACE1 antibody on erythrocyte populations in blood and
bone marrow in mice. FIGS. 11A-11B show the quantification of total
Ter119-positive erythrocyte populations (FIG. 11A) and TfR-positive
reticulocyte populations (FIG. 11B) in blood following effectorless
anti-TfR.sup.A/BACE1 (Fc-) and anti-TfR.sup.D/BACE1 (Fc-), full
effector function anti-TfR.sup.D/BACE1 (Fc+), or control IgG dosing
(n=6/group). FIGS. 12A-12D provide the quantification of distinct
erythrocyte subpopulations (total Ter119-positive erythrocyte
lineage in FIG. 12A; EryA in FIG. 12B; EryB in FIG. 12C; and EryC
in FIG. 12D) in bone marrow following dosing of effectorless
anti-TfR.sup.A/BACE1 (Fc-) and anti-TfR.sup.D/BACE1 (Fc-), full
effector function anti-TfR.sup.D/BACE1 (Fc+), or control IgG dosing
(n=6/group).
[0151] FIGS. 13A-B and FIGS. 14A-B depict the results of
experiments assessing the impact of effector function status on
ADCC activity of anti-human TfR ("anti-hTFR") antibodies in a human
erythroblast cell line or primary human bone marrow mononuclear
cells, as described in Example 7.
[0152] FIGS. 15A-B depict the light and heavy chain amino acid
sequences of anti-BACE1 clone YW412.8 obtained from a naive sort of
the natural diversity phage display library and affinity-matured
forms of YW412.8. FIG. 15A depicts the variable light (VL) sequence
alignments (SEQ ID NOs. 1-6). FIG. 13B depicts the variable heavy
(VH) sequence alignments (SEQ ID Nos. 7-8). In both figures, the
HVR sequences for each clone are indicated by the boxed regions,
with the first box indicating HVR-L1 (FIG. 15A) or HVR-H1 (FIG.
15B), the second box indicating HVR-L2 (FIG. 15A) or HVR-H2 (FIG.
15B), and the third box indicating HVR-L3 (FIG. 15A) or HVR-H3
(FIG. 15B).
[0153] FIGS. 16A-B depict the light and heavy chain amino acid
sequences of anti-BACE1 antibody clone Fab 12 obtained from a naive
sort of a synthetic diversity phage display library and
affinity-matured forms of Fab 12. FIG. 16A depicts the light chain
sequence alignments (SEQ ID NOs. 9-12). FIG. 16B depicts the heavy
chain sequence alignments (SEQ ID NO. 13). In both figures, the HVR
sequences for each clone are indicated by the boxed regions, with
the first box indicating HVR-L1 (FIG. 16A) or HVR-H1 (FIG. 16B),
the second box indicating HVR-L2 (FIG. 16A) or HVR-H2 (FIG. 16B),
and the third box indicating HVR-L3 (FIG. 16A) or HVR-H3 (FIG.
16B).
[0154] FIGS. 17A-B depict the heavy chain (FIG. 17A; SEQ ID NO. 14)
and light chain (FIG. 17B; SEQ ID NO. 15) of an exemplary
anti-Abeta antibody.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
I. Definitions
[0155] The "blood-brain barrier" or "BBB" refers to the
physiological barrier between the peripheral circulation and the
brain and spinal cord (i.e., the CNS) which is formed by tight
junctions within the brain capillary endothelial plasma membranes,
creating a tight barrier that restricts the transport of molecules
into the brain, even very small molecules such as urea (60
Daltons). The blood-brain barrier within the brain, the
blood-spinal cord barrier within the spinal cord, and the
blood-retinal barrier within the retina are contiguous capillary
barriers within the CNS, and are herein collectively referred to a
the blood-brain barrier or BBB. The BBB also encompasses the
blood-CSF barrier (choroid plexus) where the barrier is comprised
of ependymal cells rather than capillary endothelial cells.
[0156] The "central nervous system" or "CNS" refers to the complex
of nerve tissues that control bodily function, and includes the
brain and spinal cord.
[0157] A "blood-brain barrier receptor" (abbreviated "BBB-R"
herein) is a transmembrane receptor protein expressed on brain
endothelial cells which is capable of transporting molecules across
the blood-brain barrier. Examples of BBB-R include, but are not
limited to: transferrin receptor (TfR), insulin receptor,
insulin-like growth factor receptor (IGF-R), low density
lipoprotein receptors including without limitation low density
lipoprotein receptor-related protein 1 (LRP1) and low density
lipoprotein receptor-related protein 8 (LRP8), glucose transporter
1 (Glut1) and heparin-binding epidermal growth factor-like growth
factor (HB-EGF). An exemplary BBB-R herein is transferrin receptor
(TfR).
[0158] The "transferrin receptor" ("TfR") is a transmembrane
glycoprotein (with a molecular weight of about 180,000) composed of
two disulphide-bonded sub-units (each of apparent molecular weight
of about 90,000) involved in iron uptake in vertebrates. In one
embodiment, the TfR herein is human TfR comprising the amino acid
sequence as set forth in Schneider et al. Nature 311: 675-678
(1984), for example.
[0159] A "neurological disorder" as used herein refers to a disease
or disorder which affects the CNS and/or which has an etiology in
the CNS. Exemplary CNS diseases or disorders include, but are not
limited to, neuropathy, amyloidosis, cancer, an ocular disease or
disorder, viral or microbial infection, inflammation, ischemia,
neurodegenerative disease, seizure, behavioral disorders, and a
lysosomal storage disease. For the purposes of this application,
the CNS will be understood to include the eye, which is normally
sequestered from the rest of the body by the blood-retina barrier.
Specific examples of neurological disorders include, but are not
limited to, neurodegenerative diseases (including, but not limited
to, Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger
syndrome, olivopontocerebellar atrophy, Parkinson's disease,
multiple system atrophy, striatonigral degeneration, tauopathies
(including, but not limited to, Alzheimer disease and supranuclear
palsy), prion diseases (including, but not limited to, bovine
spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome,
kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting
disease, and fatal familial insomnia), bulbar palsy, motor neuron
disease, and nervous system heterodegenerative disorders
(including, but not limited to, Canavan disease, Huntington's
disease, neuronal ceroid-lipofuscinosis, Alexander's disease,
Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome,
Halervorden-Spatz syndrome, lafora disease, Rett syndrome,
hepatolenticular degeneration, Lesch-Nyhan syndrome, and
Unverricht-Lundborg syndrome), dementia (including, but not limited
to, Pick's disease, and spinocerebellar ataxia), cancer (e.g. of
the CNS, including brain metastases resulting from cancer elsewhere
in the body).
[0160] A "neurological disorder drug" is a drug or therapeutic
agent that treats one or more neurological disorder(s).
Neurological disorder drugs of the invention include, but are not
limited to, antibodies, peptides, proteins, natural ligands of one
or more CNS target(s), modified versions of natural ligands of one
or more CNS target(s), aptamers, inhibitory nucleic acids (i.e.,
small inhibitory RNAs (siRNA) and short hairpin RNAs (shRNA)),
ribozymes, and small molecules, or active fragments of any of the
foregoing. Exemplary neurological disorder drugs of the invention
are described herein and include, but are not limited to:
antibodies, aptamers, proteins, peptides, inhibitory nucleic acids
and small molecules and active fragments of any of the foregoing
that either are themselves or specifically recognize and/or act
upon (i.e., inhibit, activate, or detect) a CNS antigen or target
molecule such as, but not limited to, amyloid precursor protein or
portions thereof, amyloid beta, beta-secretase, gamma-secretase,
tau, alpha-synuclein, parkin, huntingtin, DR6, presenilin, ApoE,
glioma or other CNS cancer markers, and neurotrophins Non-limiting
examples of neurological disorder drugs and the disorders they may
be used to treat are provided in the following Table 1:
TABLE-US-00001 TABLE 1 Non-limiting examples of neurological
disorder drugs and the corresponding disorders they may be used to
treat Drug Neurological disorder Anti-BACE1 Antibody Alzheimer's,
acute and chronic brain injury, stroke Anti-Abeta Antibody
Alzheimer's disease Anti-Tau Antibody Alzheimer's disease,
tauopathies Neurotrophin Stroke, acute brain injury, spinal cord
injury Brain-derived neurotrophic factor (BDNF), Chronic brain
injury (Neurogenesis) Fibroblast growth factor 2 (FGF-2)
Anti-Epidermal Growth Factor Receptor Brain cancer (EGFR)-antibody
Glial cell-line derived neural factor Parkinson's disease (GDNF)
Brain-derived neurotrophic factor (BDNF) Amyotrophic lateral
sclerosis, depression Lysosomal enzyme Lysosomal storage disorders
of the brain Ciliary neurotrophic factor (CNTF) Amyotrophic lateral
sclerosis Neuregulin-1 Schizophrenia Anti-HER2 antibody (e.g.
trastuzumab, Brain metastasis from HER2-positive pertuzumab, etc.)
cancer Anti-VEGF antibody (e.g., bevacizumab) Recurrent or newly
diagnosed glioblastoma, recurrent malignant glioma, brain
metastasis
[0161] An "imaging agent" is a compound that has one or more
properties that permit its presence and/or location to be detected
directly or indirectly. Examples of such imaging agents include
proteins and small molecule compounds incorporating a labeled
moiety that permits detection.
[0162] A "CNS antigen" or "brain antigen" is an antigen expressed
in the CNS, including the brain, which can be targeted with an
antibody or small molecule. Examples of such antigens include,
without limitation: beta-secretase 1 (BACE1), amyloid beta (Abeta),
epidermal growth factor receptor (EGFR), human epidermal growth
factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4),
alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine
rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2,
gamma secretase, death receptor 6 (DR6), amyloid precursor protein
(APP), p75 neurotrophin receptor (p75NTR), interleukin 6 receptor
(IL6R), TNF receptor 1 (TNFR1), interleukin 1 beta (IL1.beta.), and
caspase 6. In one embodiment, the antigen is BACE1.
[0163] The term "BACE1," as used herein, refers to any native
beta-secretase 1 (also called .beta.-site amyloid precursor protein
cleaving enzyme 1, membrane-associated aspartic protease 2,
memapsin 2, aspartyl protease 2 or Asp2) from any vertebrate
source, including mammals such as primates (e.g. humans) and
rodents (e.g., mice and rats), unless otherwise indicated. The term
encompasses "full-length," unprocessed BACE1 as well as any form of
BACE1 which results from processing in the cell. The term also
encompasses naturally occurring variants of BACE1, e.g., splice
variants or allelic variants. The amino acid sequence of an
exemplary BACE1 polypeptide is the sequence for human BACE1,
isoform A as reported in Vassar et al., Science 286:735-741 (1999),
which is incorporated herein by reference in its entirety. Several
other isoforms of human BACE1 exist including isoforms B, C and D.
See UniProtKB/Swiss-Prot Entry P56817, which is incorporated herein
by reference in its entirety.
[0164] The terms "anti-beta-secretase antibody", "anti-BACE1
antibody", "an antibody that binds to beta-secretase" and "an
antibody that binds to BACE1" refer to an antibody that is capable
of binding BACE1 with sufficient affinity such that the antibody is
useful as a diagnostic and/or therapeutic agent in targeting BACE1.
In one embodiment, the extent of binding of an anti-BACE1 antibody
to an unrelated, non-BACE1 protein is less than about 10% of the
binding of the antibody to BACE1 as measured, e.g., by a
radioimmunoassay (RIA). In certain embodiments, an antibody that
binds to BACE1 has a dissociation constant (Kd) of .ltoreq.1 .mu.M,
.ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM,
.ltoreq.0.01 nM, or .ltoreq.0.001 nM (e.g. 10.sup.-8M or less, e.g.
from 10.sup.-8M to 10.sup.-13 M, e.g., from 10.sup.-9 M to
10.sup.-13 M). In certain embodiments, an anti-BACE1 antibody binds
to an epitope of BACE1 that is conserved among BACE1 from different
species and isoforms. In one embodiment, an antibody is provided
that binds to the epitope on BACE1 bound by anti-BACE1 antibody
YW412.8.31. In other embodiments, an antibody is provided that
binds to an exosite within BACE1 located in the catalytic domain of
BACE1. In one embodiment an antibody is provided that competes with
the peptides identified in Kornacker et al., Biochem.
44:11567-11573 (2005), which is incorporated herein by reference in
its entirety, (i.e., Peptides 1, 2, 3, 1-11, 1-10, 1-9, 1-8, 1-7,
1-6, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, 4, 5,
6, 5-10, 5-9, scrambled, Y5A, P6A, Y7A, F8A, I9A, P10A and L11A)
for binding to BACE1. Exemplary BACE1 antibody sequences are
depicted in FIG. 15A-B and FIG. 16A-B. One exemplary antibody
herein comprises the variable domains of the antibody YW412.8.31
(e.g. as in FIGS. 15A-B).
[0165] A "native sequence" protein herein refers to a protein
comprising the amino acid sequence of a protein found in nature,
including naturally occurring variants of the protein. The term as
used herein includes the protein as isolated from a natural source
thereof or as recombinantly produced.
[0166] The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies, polyclonal antibodies,
multispecific antibodies (e.g. bispecific antibodies) formed from
at least two intact antibodies, and antibody fragments so long as
they exhibit the desired biological activity.
[0167] "Antibody fragments" herein comprise a portion of an intact
antibody which retains the ability to bind antigen. Examples of
antibody fragments are well known in the art (see, e.g., Nelson,
MAbs (2010) 2(1): 77-83) and include but are not limited to Fab,
Fab', F(ab').sub.2, and Fv fragments; diabodies; linear antibodies;
single-chain antibody molecules including but not limited to
single-chain variable fragments (scFv), fusions of light and/or
heavy-chain antigen-binding domains with or without a linker (and
optionally in tandem); and monospecific or multispecific
antigen-binding molecules formed from antibody fragments
(including, but not limited to multispecific antibodies constructed
from multiple variable domains which lack Fc regions).
[0168] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variants that may arise during production of the
monoclonal antibody, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations that
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. In addition to their
specificity, the monoclonal antibodies are advantageous in that
they are uncontaminated by other immunoglobulins. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (see, e.g., U.S.
Pat. No. 4,816,567). The "monoclonal antibodies" may also be
isolated from phage antibody libraries using the techniques
described in Clackson et al., Nature, 352:624-628 (1991) and Marks
et al., J. Mol. Biol., 222:581-597 (1991), for example. Specific
examples of monoclonal antibodies herein include chimeric
antibodies, humanized antibodies, and human antibodies, including
antigen-binding fragments thereof.
[0169] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (U.S. Pat. No. 4,816,567; Morrison et
al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric
antibodies of interest herein include "primatized" antibodies
comprising variable domain antigen-binding sequences derived from a
non-human primate (e.g. Old World Monkey, such as baboon, rhesus or
cynomolgus monkey) and human constant region sequences (U.S. Pat.
No. 5,693,780).
[0170] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable regions correspond to those
of a non-human immunoglobulin and all or substantially all of the
FRs are those of a human immunoglobulin sequence, except for FR
substitution(s) as noted above. The humanized antibody optionally
also will comprise at least a portion of an immunoglobulin constant
region, typically that of a human immunoglobulin. For further
details, see Jones et al., Nature 321:522-525 (1986); Riechmann et
al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.
2:593-596 (1992).
[0171] A "human antibody" herein is one comprising an amino acid
sequence structure that corresponds with the amino acid sequence
structure of an antibody obtainable from a human B-cell, and
includes antigen-binding fragments of human antibodies. Such
antibodies can be identified or made by a variety of techniques,
including, but not limited to: production by transgenic animals
(e.g., mice) that are capable, upon immunization, of producing
human antibodies in the absence of endogenous immunoglobulin
production (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci.
USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);
Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Pat.
Nos. 5,591,669, 5,589,369 and 5,545,807)); selection from phage
display libraries expressing human antibodies or human antibody
fragments (see, for example, McCafferty et al., Nature 348:552-553
(1990); Johnson et al., Current Opinion in Structural Biology
3:564-571 (1993); Clackson et al., Nature, 352:624-628 (1991);
Marks et al., J. Mol. Biol. 222:581-597 (1991); Griffith et al.,
EMBO J. 12:725-734 (1993); U.S. Pat. Nos. 5,565,332 and 5,573,905);
generation via in vitro activated B cells (see U.S. Pat. Nos.
5,567,610 and 5,229,275); and isolation from human
antibody-producing hybridomas.
[0172] A "multispecific antibody" herein is an antibody having
binding specificities for at least two different epitopes.
Exemplary multispecific antibodies may bind both a BBB-R and a
brain antigen. Multispecific antibodies can be prepared as
full-length antibodies or antibody fragments (e.g. F(ab').sub.2
bispecific antibodies). Engineered antibodies with two, three or
more (e.g. four) functional antigen binding sites are also
contemplated (see, e.g., US Appln No. US 2002/0004587 A1, Miller et
al.). Multispecific antibodies can be prepared as full length
antibodies or as antibody fragments.
[0173] Antibodies herein include "amino acid sequence variants"
with altered antigen-binding or biological activity. Examples of
such amino acid alterations include antibodies with enhanced
affinity for antigen (e.g. "affinity matured" antibodies), and
antibodies with altered Fc region, if present, e.g. with altered
(increased or diminished) antibody dependent cellular cytotoxicity
(ADCC) and/or complement dependent cytotoxicity (CDC) (see, for
example, WO 00/42072, Presta, L. and WO 99/51642, Iduosogie et
al.); and/or increased or diminished serum half-life (see, for
example, WO00/42072, Presta, L.).
[0174] An "affinity modified variant" has one or more substituted
hypervariable region or framework residues of a parent antibody
(e.g. of a parent chimeric, humanized, or human antibody) that
alter (increase or reduce) affinity. A convenient way for
generating such substitutional variants uses phage display.
Briefly, several hypervariable region sites (e.g. 6-7 sites) are
mutated to generate all possible amino substitutions at each site.
The antibody variants thus generated are displayed in a monovalent
fashion from filamentous phage particles as fusions to the gene III
product of M13 packaged within each particle. The phage-displayed
variants are then screened for their biological activity (e.g.
binding affinity). In order to identify candidate hypervariable
region sites for modification, alanine scanning mutagenesis can be
performed to identify hypervariable region residues contributing
significantly to antigen binding. Alternatively, or additionally,
it may be beneficial to analyze a crystal structure of the
antigen-antibody complex to identify contact points between the
antibody and its target. Such contact residues and neighboring
residues are candidates for substitution according to the
techniques elaborated herein. Once such variants are generated, the
panel of variants is subjected to screening and antibodies with
altered affinity may be selected for further development.
[0175] A "pH-sensitive antibody variant" is an antibody variant
which has a different binding binding affinity for a target antigen
at a first pH than it does for that target antigen at a different
pH. As a nonlimiting example, an anti-TfR antibody of the invention
may be selected for or engineered to have pH-sensitive binding to
TfR such that it binds with desirably low affinity (as described
herein) to cell surface TfR in the plasma at pH 7.4, but upon
internalization into an endosomal compartment, rapidly dissociates
from TfR at the relatively lower pH (pH 5.5-6.0); such dissociation
may protect the antibody from antigen-mediated clearance, and
increase the amount of antibody that is either delivered to the CNS
or recycled back across the BBB--in either case, the effective
concentration of the antibody is increased relative to an anti-TfR
antibody that does not comprise such pH sensitivity (see, e.g.,
Chaparro-Riggers et al. J. Biol. Chem. 287(14): 11090-11097; Igawa
et al., Nature Biotechnol. 28(11): 1203-1208). The desired
combination of affinities at the serum pH and the endosomal
compartment pH can be readily determined for a particular BBB-R and
conjugated compound by one of ordinary skill in the art.
[0176] The antibody herein may be conjugated with a "heterologous
molecule" for example to increase half-life or stability or
otherwise improve the antibody. For example, the antibody may be
linked to one of a variety of non-proteinaceous polymers, e.g.,
polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes,
or copolymers of polyethylene glycol and polypropylene glycol.
Antibody fragments, such as Fab', linked to one or more PEG
molecules are an exemplary embodiment of the invention. In another
example, the heterologous molecule is a therapeutic compound or a
visualization agent (ie., a detectable label), and the antibody is
being used to transport such heterologous molecule across the BBB.
Examples of heterologous molecules include, but are not limited to,
a chemical compound, a peptide, a polymer, a lipid, a nucleic acid,
and a protein.
[0177] The antibody herein may be a "glycosylation variant" such
that any carbohydrate attached to the Fc region, if present, is
altered, either modified in presence/absence, or modified in type.
For example, antibodies with a mature carbohydrate structure that
lacks fucose attached to an Fc region of the antibody are described
in US Pat Appl No US 2003/0157108 (Presta, L.). See also US
2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with a
bisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached
to an Fc region of the antibody are referenced in WO 2003/011878,
Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al.
Antibodies with at least one galactose residue in the
oligosaccharide attached to an Fc region of the antibody are
reported in WO 1997/30087, Patel et al. See, also, WO 1998/58964
(Raju, S.) and WO 1999/22764 (Raju, S.) concerning antibodies with
altered carbohydrate attached to the Fc region thereof. See also US
2005/0123546 (Umana et al.) describing antibodies with modified
glycosylation. Mutation of the consensus glycosylation sequence in
the Fc region (Asn-X-Ser/Thr at positions 297-299, where X cannot
be proline), for example by mutating the Asn of this sequence to
any other amino acid, by placing a Pro at position 298, or by
modifying position 299 to any amino acid other than Ser or Thr
should abrogate glycosylation at that position (see, e.g., Fares
Al-Ejeh et al., Clin. Cancer Res. (2007) 13:5519s-5527s; Imperiali
and Shannon, Biochemistry (1991) 30(18): 4374-4380; Katsuri,
Biochem J. (1997) 323(Pt 2): 415-419; Shakin-Eshleman et al., J.
Biol. Chem. (1996) 271: 6363-6366).
[0178] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody that are responsible for
antigen binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR" (e.g.
residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain
variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the
heavy chain variable domain; Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues
from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2)
and 91-96 (L3) in the light chain variable domain and 26-32 (H1),
53-55 (H2) and 96-101 (H3) in the heavy chain variable domain;
Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework" or
"FR" residues are those variable domain residues other than the
hypervariable region residues as herein defined.
[0179] A "full length antibody" is one which comprises an
antigen-binding variable region as well as a light chain constant
domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The
constant domains may be native sequence constant domains (e.g.
human native sequence constant domains) or amino acid sequence
variants thereof.
[0180] A "naked antibody" is an antibody (as herein defined) that
is not conjugated to a heterologous molecule, such as a cytotoxic
moiety, polymer, or radiolabel.
[0181] Antibody "effector functions" refer to those biological
activities of an antibody that result in activation of the immune
system other than activation of the complement pathway. Such
activities are largely found in the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody.
Examples of antibody effector functions include, for example, Fc
receptor binding and antibody-dependent cell-mediated cytotoxicity
(ADCC). In one embodiment, the antibody herein essentially lacks
effector function. In another embodiment, the antibody herein
retains minimal effector function. Methods of modifying or
eliminating effector function are well-known in the art and
include, but are not limited to, eliminating all or a portion of
the Fc region responsible for the effector function (ie, using an
antibody or antibody fragment in a format lacking all or a portion
of the Fc region such as, but not limited to, a Fab fragment, a
single-chain antibody, and the like as described herein and as
known in the art; modifying the Fc region at one or more amino acid
positions to eliminate effector function (Fc binding-impacting:
positions 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272,
278, 289, 292, 293, 294, 295, 296, 297, 298, 301, 303, 322, 324,
327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416,
419, 434, 435, 437, 438, and 439; and modifying the glycosylation
of the antibody (including, but not limited to, producing the
antibody in an environment that does not permit wild-type mammalian
glycosylation, removing one or more carbohydrate groups from an
already-glycosylated antibody, and modifying the antibody at one or
more amino acid positions to eliminate the ability of the antibody
to be glycosylated at those positions (including, but not limited
to N297G and N297A).
[0182] Antibody "complement activation" functions, or properties of
an antibody that enable or trigger "activation of the complement
pathway" are used interchangeably, and refer to those biological
activities of an antibody that engage or stimulate the complement
pathway of the immune system in a subject. Such activities include,
e.g., C1q binding and complement dependent cytotoxicity (CDC), and
may be mediated by both the Fc portion and the non-Fc portion of
the antibody. Methods of modifying or eliminating complement
activation function are well-known in the art and include, but are
not limited to, eliminating all or a portion of the Fc region
responsible for complement activation (ie., using an antibody or
antibody fragment in a format lacking all or a portion of the Fc
region such as, but not limited to, a Fab fragment, a single-chain
antibody, and the like as described herein and as known in the art,
or modifying the Fc region at one or more amino acid positions to
eliminate or lessen interactions with complement components or the
ability to activate complement components, such as positions 270,
322, 329 and 321, known to be involved in C1q binding), and
modifying or eliminating a portion of the non-Fc region responsible
for complement activation (ie, modifying the CH1 region at position
132 (see, e.g., Vidarte et al., (2001) J. Biol. Chem. 276(41):
38217-38223)).
[0183] Depending on the amino acid sequence of the constant domain
of their heavy chains, full length antibodies can be assigned to
different "classes". There are five major classes of full length
antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may
be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2,
IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that
correspond to the different classes of antibodies are called alpha,
delta, epsilon, gamma, and mu, respectively. The subunit structures
and three-dimensional configurations of different classes of
immunoglobulins are well known.
[0184] The term "recombinant antibody", as used herein, refers to
an antibody (e.g. a chimeric, humanized, or human antibody or
antigen-binding fragment thereof) that is expressed by a
recombinant host cell comprising nucleic acid encoding the
antibody. Examples of "host cells" for producing recombinant
antibodies include: (1) mammalian cells, for example, Chinese
Hamster Ovary (CHO), COS, myeloma cells (including Y0 and NS0
cells), baby hamster kidney (BHK), Hela and Vero cells; (2) insect
cells, for example, sf9, sf21 and Tn5; (3) plant cells, for example
plants belonging to the genus Nicotiana (e.g. Nicotiana tabacum);
(4) yeast cells, for example, those belonging to the genus
Saccharomyces (e.g. Saccharomyces cerevisiae) or the genus
Aspergillus (e.g. Aspergillus niger); (5) bacterial cells, for
example Escherichia coli cells or Bacillus subtilis cells, etc.
[0185] As used herein, "specifically binding" or "binds
specifically to" refers to an antibody selectively or
preferentially binding to an antigen. The binding affinity is
generally determined using a standard assay, such as Scatchard
analysis, or surface plasmon resonance technique (e.g. using
BIACORE.RTM.).
[0186] An "antibody that binds to the same epitope" as a reference
antibody refers to an antibody that blocks binding of the reference
antibody to its antigen in a competition assay by 50% or more, and
conversely, the reference antibody blocks binding of the antibody
to its antigen in a competition assay by 50% or more. In one
embodiment, an anti-BACE1 antibody binds to the BACE1 epitope bound
by YW412.8.31.
[0187] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents a cellular function and/or
causes cell death or destruction. Cytotoxic agents include, but are
not limited to, radioactive isotopes (e.g., At.sup.211, I.sup.131,
I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive isotopes of Lu);
chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin,
vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,
melphalan, mitomycin C, chlorambucil, daunorubicin or other
intercalating agents); growth inhibitory agents; enzymes and
fragments thereof such as nucleolytic enzymes; antibiotics; toxins
such as small molecule toxins or enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof.
[0188] An "effective amount" of an agent, e.g., a pharmaceutical
formulation, refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic or
prophylactic result.
[0189] The term "Fc region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain that contains at least a
portion of the constant region. The term includes native sequence
Fc regions and variant Fc regions. In one embodiment, a human IgG
heavy chain Fc region extends from Cys226, or from Pro230, to the
carboxyl-terminus of the heavy chain. However, the C-terminal
lysine (Lys447) of the Fc region may or may not be present. Unless
otherwise specified herein, numbering of amino acid residues in the
Fc region or constant region is according to the EU numbering
system, also called the EU index, as described in Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.,
1991.
[0190] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0191] An "immunoconjugate" is an antibody conjugated to one or
more heterologous molecule(s), including but not limited to a label
or cytotoxic agent. Optionally such conjugation is via a
linker.
[0192] A "linker" as used herein is a structure that covalently or
non-covalently connects the anti-BBB-R antibody to heterologous
molecule. In certain embodiments, a linker is a peptide. In other
embodiments, a linker is a chemical linker.
[0193] A "label" is a marker coupled with the antibody herein and
used for detection or imaging. Examples of such labels include:
radiolabel, a fluorophore, a chromophore, or an affinity tag. In
one embodiment, the label is a radiolabel used for medical imaging,
for example tc99m or I123, or a spin label for nuclear magnetic
resonance (NMR) imaging (also known as magnetic resonance imaging,
mri), such as iodine-123 again, iodine-131, indium-111,
fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,
manganese, iron, etc.
[0194] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g., cows, sheep,
cats, dogs, and horses), primates (e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and
rats). In certain embodiments, the individual or subject is a
human.
[0195] An "isolated" antibody is one which has been separated from
a component of its natural environment. In some embodiments, an
antibody is purified to greater than 95% or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE,
isoelectric focusing (IEF), capillary electrophoresis) or
chromatographic (e.g., ion exchange or reverse phase HPLC) methods.
For review of methods for assessment of antibody purity, see, e.g.,
Flatman et al., J. Chromatogr. B 848:79-87 (2007).
[0196] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0197] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0198] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical formulation, other than an active
ingredient, which is nontoxic to a subject., A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0199] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of the
individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, antibodies of
the invention are used to delay development of a disease or to slow
the progression of a disease.
II. Compositions and Methods
[0200] A. Production of Anti-BBB-R Antibodies and Conjugates
Thereof
[0201] The methods and articles of manufacture of the present
invention use, or incorporate, an antibody that binds to a BBB-R.
The BBB-R antigen to be used for production of, or screening for,
antibodies may be, e.g., a soluble form of or a portion thereof
(e.g. the extracellular domain) of the BBB-R containing the desired
epitope. Alternatively, or additionally, cells expressing BBB-R at
their cell surface can be used to generate, or screen for,
antibodies. Other forms and presentations of BBB-R useful for
generating antibodies will be apparent to those skilled in the art.
Examples of BBB-Rs herein include transferrin receptor (TfR),
insulin receptor, insulin-like growth factor receptor (IGF-R), low
density lipoprotein receptor-related protein 1 (LRP1) and LRP8 etc,
glucose transporter 1 (Glut1) and heparin-binding epidermal growth
factor-like growth factor (HB-EGF).
[0202] According to the present invention, a "low affinity"
anti-BBB-R (e.g. anti-TfR) antibody is selected based on the data
herein demonstrating that such antibodies display improved CNS (for
example, brain) uptake. In order to identify such low affinity
antibodies, various assays for measuring antibody affinity are
available including, without limitation: Scatchard assay and
surface plasmon resonance technique (e.g. using BIACORE.RTM.).
According to one embodiment of the invention, the antibody has an
affinity for the BBB-R antigen (e.g. for TfR) from about 5 nM, or
from about 20 nM, or from about 100 nM, to about 50 .mu.M, or to
about 30 .mu.M, or to about 10 .mu.M, or to about 1 .mu.M, or to
about 500 nM. Thus, the affinity may be in the range from about 5
nM to about 50 .mu.M, or in the range from about 20 nM to about 30
.mu.M, or in the range from about 30 nM to about 30 .mu.M, or in
the range from about 50 nM to about 1 .mu.M, or in the range from
about 100 nM to about 500 nM, e.g. as measured by Scatchard
analysis or BIACORE.RTM.. In another embodiment of the invention,
the antibody has a dissociation half-life from the BBB-R antigen
(e.g. for TfR) of less than 1 minute, less than 2 minutes, less
than 3 minutes, less than four minutes, less than 5 minutes, or
less than 10 minutes to about 20 minutes, or to about 30 minutes,
as measured by competition binding analysis or BIACORE.RTM..
[0203] Thus, the invention provides a method of making an antibody
useful for transporting a neurological disorder drug across the
blood-brain barrier comprising selecting an antibody from a panel
of antibodies against a blood-brain barrier receptor (BBB-R)
because it has an affinity for the BBB-R which is in the range from
about 5 nM, or from about 20 nM, or from about 100 nM, to about 50
.mu.M, or to about 30 .mu.M, or to about 10 .mu.M, or to about 1
.mu.M, or to about 500 mM. Thus, the affinity may be in the range
from about 5 nM to about 50 .mu.M, or in the range from about 20 nM
to about 30 .mu.M, or in the range from about 30 nM to about 30
.mu.M, or in the range from about 50 nM to about 1 .mu.M, or in the
range from about 100 nM to about 500 nM, e.g. as measured by
Scatchard analysis or BIACORE.RTM.. As will be understood by one of
ordinary skill in the art, conjugating a heterologous
molecule/compound to an antibody will often decrease the affinity
of the antibody for its target due, e.g., to steric hindrance or
even to elimination of one binding arm if the antibody is made
multispecific with one or more arms binding to a different antigen
than the antibody's original target. In one embodiment, a low
affinity antibody of the invention specific for TfR conjugated to
BACE1 had a Kd for TfR as measured by BIACORE of about 30 nM. In
another embodiment, a low affinity antibody of the invention
specific for TfR conjugated to BACE1 had a Kd for TfR as measured
by BIACORE of about 600 nM. In another embodiment, a low affinity
antibody of the invention specific for TfR conjugated to BACE1 had
a Kd for TfR as measured by BIACORE of about 20 .mu.M. In another
embodiment, a low affinity antibody of the invention specific for
TfR conjugated to BACE1 had a Kd for TfR as measured by BIACORE of
about 30 .mu.M.
[0204] One exemplary assay for evaluating antibody affinity is by
Scatchard analysis. For example, the anti-BBB-R antibody of
interest can be iodinated using the lactoperoxidase method (Bennett
and Horuk, Methods in Enzymology 288 pg. 134-148 (1997)). A
radiolabeled anti-BBB-R antibody is purified from free .sup.125I-Na
by gel filtration using a NAP-5 column and its specific activity
measured. Competition reaction mixtures of 50 .mu.L containing a
fixed concentration of iodinated antibody and decreasing
concentrations of serially diluted unlabeled antibody are placed
into 96-well plates. Cells transiently expressing BBB-R are
cultured in growth media, consisting of Dulbecco's modified eagle's
medium (DMEM) (Genentech) supplemented with 10% FBS, 2 mM
L-glutamine and 1.times.penicillin-streptomycin at 37.degree. C. in
5% CO.sub.2. Cells are detached from the dishes using Sigma Cell
Dissociation Solution and washed with binding buffer (DMEM with 1%
bovine serum albumin, 50 mM HEPES, pH 7.2, and 0.2% sodium azide).
The washed cells are added at an approximate density of 200,000
cells in 0.2 mL of binding buffer to the 96-well plates containing
the 50-.mu.L competition reaction mixtures. The final concentration
of the unlabeled antibody in the competition reaction with cells is
varied, starting at 1000 nM and then decreasing by 1:2 fold
dilution for 10 concentrations and including a zero-added,
buffer-only sample. Competition reactions with cells for each
concentration of unlabeled antibody are assayed in triplicate.
Competition reactions with cells are incubated for 2 hours at room
temperature. After the 2-hour incubation, the competition reactions
are transferred to a filter plate and washed four times with
binding buffer to separate free from bound iodinated antibody. The
filters are counted by gamma counter and the binding data are
evaluated using the fitting algorithm of Munson and Rodbard (1980)
to determine the binding affinity of the antibody.
[0205] An exemplary scatchard analysis using the compositions of
the invention may be performed as follows. Anti-TFR.sup.A was
iodinated using the lactoperoxidase method (Bennett and Horuk,
Methods in Enzymology 288 pg. 134-148 (1997)). Radiolabeled
anti-TFR.sup.A was purified from free .sup.125I-Na by gel
filtration using a NAP-5 column; purified anti-TFR.sup.A had a
specific activity of 19.82 .mu.Ci/.mu.g. Competition reaction
mixtures of 50 .mu.L containing a fixed concentration of iodinated
antibody and decreasing concentrations of serially diluted
unlabeled antibody were placed into 96-well plates. The 293 cells
transiently expressing murine TfR were cultured in growth media,
consisting of Dulbecco's modified eagle's medium (DMEM) (Genentech)
supplemented with 10% FBS, 2 mM L-glutamine and
1.times.penicillin-streptomycin at 37.degree. C. in 5% CO.sub.2.
Cells were detached from the dishes using Sigma Cell Dissociation
Solution and washed with binding buffer (DMEM with 1% bovine serum
albumin, 50 mM HEPES, pH 7.2, and 0.2% sodium azide). The washed
cells were added at an approximate density of 200,000 cells in 0.2
mL of binding buffer to the 96-well plates containing the 50-.mu.L
competition reaction mixtures. The final concentration of the
iodinated antibody in each competition reaction with cells was 100
pM (134,000 cpm per 0.25 mL). The final concentration of the
unlabeled antibody in the competition reaction with cells varied,
starting at 1000 nM and then decreasing by 1:2 fold dilution for 10
concentrations and including a zero-added, buffer-only sample.
Competition reactions with cells for each concentration of
unlabeled antibody were assayed in triplicate. Competition
reactions with cells were incubated for 2 hours at room
temperature. After the 2-hour incubation, the competition reactions
were transferred to a Millipore Multiscreen filter plate and washed
four times with binding buffer to separate free from bound
iodinated antibody. The filters were counted on a Wallac Wizard
1470 gamma counter (PerkinElmer Life and Analytical Sciences;
Waltham, Mass.). The binding data were evaluated using New Ligand
software (Genentech), which uses the fitting algorithm of Munson
and Rodbard (1980) to determine the binding affinity of the
antibody.
[0206] An exemplary BIACORE.RTM. analysis using the compositions of
the invention may be performed as follows. Kd was measured using
surface plasmon resonance assays using a BIACORE.RTM.-2000
(BIAcore, Inc., Piscataway, N.J.) at 25.degree. C. using anti-human
Fc kit (BiAcore Inc., Piscataway, N.J.). Briefly, carboxymethylated
dextran biosensor chips (CM5, BIACORE, Inc.) were activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Anti-human Fc antibody was diluted with 10 mM sodium
acetate, pH 4.0, to 50 .mu.g/ml before injection at a flow rate of
5 .mu.l/minute to achieve approximately 10000 response units (RU)
of coupled protein. Following the injection of antibody, 1 M
ethanolamine was injected to block unreacted groups. For kinetics
measurements, monospecific or multispecific anti-TfR antibody
variants were injected in HBS-P to reach about 220 RU, then
two-fold serial dilutions of MuTfR-His (0.61 nM to 157 nM) were
injected in HBS-P at 25.degree. C. at a flow rate of approximately
30 .mu.l/min. Association rates (kon) and dissociation rates (koff)
were calculated using a simple one-to-one Langmuir binding model
(BIACORE.RTM. Evaluation Software version 3.2) by simultaneously
fitting the association and dissociation sensorgrams. The
equilibrium dissociation constant (Kd) was calculated as the ratio
koff/kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881
(1999)
[0207] According to another embodiment, Kd is measured using
surface plasmon resonance assays with a BIACORE.RTM.-2000 device
(BIAcore, Inc., Piscataway, N.J.) at 25.degree. C. using anti-human
Fc kit (BiAcore Inc., Piscataway, N.J.). Briefly, carboxymethylated
dextran biosensor chips (CM5, BIACORE, Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Anti-human Fc antibody is diluted with 10 mM sodium
acetate, pH 4.0, to 50 .mu.g/ml before injection at a flow rate of
5 .mu.l/minute to achieve approximately 10000 response units (RU)
of coupled protein. Following the injection of antibody, 1 M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, anti-BBB-R antibody variants are injected in HBS-P to
reach about 220 RU, then two-fold serial dilutions of BBB-R-His
(0.61 nM to 157 nM) are injected in HBS-P at 25.degree. C. at a
flow rate of approximately 30 .mu.l/min. Association rates (kon)
and dissociation rates (koff) are calculated using a simple
one-to-one Langmuir binding model (BIACORE.RTM. Evaluation Software
version 3.2) by simultaneously fitting the association and
dissociation sensorgrams. The equilibrium dissociation constant
(Kd) is calculated as the ratio koff/kon. See, e.g., Chen et al.,
J. Mol. Biol. 293:865-881 (1999).
[0208] A surrogate measurement for the affinity of one or more
antibodies for the BBB-R is its half maximal inhibitory
concentration (IC50), a measure of how much of the antibody is
needed to inhibit the binding of a known BBB-R ligand to the BBB-R
by 50%. Several methods of determining the IC50 for a given
compound are art-known; a common approach is to perform a
competition binding assay, such as that described herein in the
examples, i.e. with regard to FIG. 1A. In general, a high IC50
indicates that more of the antibody is required to inhibit binding
of the known ligand, and thus that the antibody's affinity for that
ligand is relatively low. Conversely, a low IC50 indicates that
less of the antibody is required to inhibit binding of the known
ligand, and thus that the antibody's affinity for that ligand is
relatively high.
[0209] An exemplary competitive ELISA assay to measure IC50 is one
in which increasing concentrations of anti-TfR or anti-TfR/brain
antigen (i.e., anti-TfR/BACE1, anti-TfR/Abeta and the like) variant
antibodies are used to compete against biotinylated TfR.sup.A for
binding to TfR. The anti-TfR competition ELISA was performed in
Maxisorp plates (Neptune, N.J.) coated with 2.5 .mu.g/ml of
purified murine TfR extracellular domain in PBS at 4.degree. C.
overnight. Plates were washed with PBS/0.05% Tween 20 and blocked
using Superblock blocking buffer in PBS (Thermo Scientific, Hudson,
N.H.). A titration of each individual anti-TfR or anti-TfR/brain
antigen (i.e., anti-TfR/BACE1 or anti-TfR/Abeta) (1:3 serial
dilution) was combined with biotinylated anti-TfR.sup.A (0.5 nM
final concentration) and added to the plate for 1 hour at room
temperature. Plates were washed with PBS/0.05% Tween 20, and
HRP-streptavidin (Southern Biotech, Birmingham) was added to the
plate and incubated for 1 hour at room temperature. Plates were
washed with PBS/0.05% Tween 20, and biotinylated anti-TfR.sup.A
bound to the plate was detected using TMB substrate (BioFX
Laboratories, Owings Mills).
[0210] In one embodiment, the low affinity anti-BBB-R antibody
herein is coupled with a label and/or neurological disorder drug or
imaging agent in order to more efficiently transport the label
and/or drug or imaging agent across the BBB. Such coupling can be
achieved by chemical cross-linkers or by generating fusion
proteins, etc.
[0211] Covalent conjugation can either be direct or via a linker.
In certain embodiments, direct conjugation is by construction of a
protein fusion (i.e., by genetic fusion of the two genes encoding
the BBB-R antibody and the neurological disorder drug and
expression as a single protein). In certain embodiments, direct
conjugation is by formation of a covalent bond between a reactive
group on one of the two portions of the anti-BBB-R antibody and a
corresponding group or acceptor on the neurological drug. In
certain embodiments, direct conjugation is by modification (i.e.,
genetic modification) of one of the two molecules to be conjugated
to include a reactive group (as nonlimiting examples, a sulfhydryl
group or a carboxyl group) that forms a covalent attachment to the
other molecule to be conjugated under appropriate conditions. As
one nonlimiting example, a molecule (i.e., an amino acid) with a
desired reactive group (i.e., a cysteine residue) may be introduced
into, e.g., the anti-BBB-R antibody and a disulfide bond formed
with the neurological drug. Methods for covalent conjugation of
nucleic acids to proteins are also known in the art (i.e.,
photocrosslinking, see, e.g., Zatsepin et al. Russ. Chem. Rev. 74:
77-95 (2005)) Non-covalent conjugation can be by any nonconvalent
attachment means, including hydrophobic bonds, ionic bonds,
electrostatic interactions, and the like, as will be readily
understood by one of ordinary skill in the art. Conjugation may
also be performed using a variety of linkers. For example, an
anti-BBB-R antibody and a neurological drug may be conjugated using
a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Peptide linkers, comprised of from one to twenty amino acids joined
by peptide bonds, may also be used. In certain such embodiments,
the amino acids are selected from the twenty naturally-occurring
amino acids. In certain other such embodiments, one or more of the
amino acids are selected from glycine, alanine, proline,
asparagine, glutamine and lysine. The linker may be a "cleavable
linker" facilitating release of the neurological drug upon delivery
to the brain. For example, an acid-labile linker,
peptidase-sensitive linker, photolabile linker, dimethyl linker or
disulfide-containing linker (Chari et al., Cancer Res. 52:127-131
(1992); U.S. Pat. No. 5,208,020) may be used.
[0212] The invention herein expressly contemplates, but is not
limited to, conjugates prepared with cross-linker reagents
including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC,
MBS, MPBH, SBAP, SIA, SLAB, SMCC, SMPB, SMPH, sulfo-EMCS,
sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and
sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which
are commercially available (e.g., from Pierce Biotechnology, Inc.,
Rockford, Ill., U.S.A).
[0213] For a neuropathy disorder, a neurological drug may be
selected that is an analgesic including, but not limited to, a
narcotic/opioid analgesic (i.e., morphine, fentanyl, hydrocodone,
meperidine, methadone, oxymorphone, pentazocine, propoxyphene,
tramadol, codeine and oxycodone), a nonsteroidal anti-inflammatory
drug (NSAID) (i.e., ibuprofen, naproxen, diclofenac, diflunisal,
etodolac, fenoprofen, flurbiprofen, indomethacin, ketorolac,
mefenamic acid, meloxicam, nabumetone, oxaprozin, piroxicam,
sulindac, and tolmetin), a corticosteroid (i.e., cortisone,
prednisone, prednisolone, dexamethasone, methylprednisolone and
triamcinolone), an anti-migraine agent (i.e., sumatriptin,
almotriptan, frovatriptan, sumatriptan, rizatriptan, eletriptan,
zolmitriptan, dihydroergotamine, eletriptan and ergotamine),
acetaminophen, a salicylate (i.e., aspirin, choline salicylate,
magnesium salicylate, diflunisal, and salsalate), a anti-convulsant
(i.e., carbamazepine, clonazepam, gabapentin, lamotrigine,
pregabalin, tiagabine, and topiramate), an anaesthetic (i.e.,
isoflurane, trichloroethylene, halothane, sevoflurane, benzocaine,
chloroprocaine, cocaine, cyclomethycaine, dimethocaine,
propoxycaine, procaine, novocaine, proparacaine, tetracaine,
articaine, bupivacaine, carticaine, cinchocaine, etidocaine,
levobupivacaine, lidocaine, mepivacaine, piperocaine, prilocaine,
ropivacaine, trimecaine, saxitoxin and tetrodotoxin), and a
cox-2-inhibitor (i.e., celecoxib, rofecoxib, and valdecoxib). For a
neuropathy disorder with vertigo involvement, a neurological drug
may be selected that is an anti-vertigo agent including, but not
limited to, meclizine, diphenhydramine, promethazine and diazepam.
For a neuropathy disorder with nausea involvement, a neurological
drug may be selected that is an anti-nausea agent including, but
not limited to, promethazine, chlorpromazine, prochlorperazine,
trimethobenzamide, and metoclopramide. For a neurodegenerative
disease, a neurological drug may be selected that is a growth
hormone or neurotrophic factor; examples include but are not
limited to brain-derived neurotrophic factor (BDNF), nerve growth
factor (NGF), neurotrophin-4/5, fibroblast growth factor (FGF)-2
and other FGFs, neurotrophin (NT)-3, erythropoietin (EPO),
hepatocyte growth factor (HGF), epidermal growth factor (EGF),
transforming growth factor (TGF)-alpha, TGF-beta, vascular
endothelial growth factor (VEGF), interleukin-1 receptor antagonist
(IL-1ra), ciliary neurotrophic factor (CNTF), glial-derived
neurotrophic factor (GDNF), neurturin, platelet-derived growth
factor (PDGF), heregulin, neuregulin, artemin, persephin,
interleukins, glial cell line derived neurotrophic factor (GFR),
granulocyte-colony stimulating factor (CSF),
granulocyte-macrophage-CSF, netrins, cardiotrophin-1, hedgehogs,
leukemia inhibitory factor (LIF), midkine, pleiotrophin, bone
morphogenetic proteins (BMPs), netrins, saposins, semaphorins, and
stem cell factor (SCF).
[0214] For cancer, a neurological drug may be selected that is a
chemotherapeutic agent. Examples of chemotherapeutic agents include
alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphor-amide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,
MARINOL.RTM.); beta-lapachone; lapachol; colchicines; betulinic
acid; a camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e. g., calicheamicin, especially
calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew,
Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including
dynemicin A; an esperamicin; as well as neocarzinostatin
chromophore and related chromoprotein enediyne antibiotic
chromophores), aclacinomysins, actinomycin, authramycin, azaserine,
bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin,
chromomycinis, dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM. doxorubicin (including
morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin
C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elfornithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;
procarbazine; PSK.RTM. polysaccharide complex (JHS Natural
Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine
(ELDISINE.RTM., FILDESIN.RTM.); dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside
("Ara-C"); thiotepa; taxoids, e.g., TAXOL.RTM. paclitaxel
(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE.TM.
Cremophor-free, albumin-engineered nanoparticle formulation of
paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),
and TAXOTERE.RTM. doxetaxel (Rhone-Poulenc Rorer, Antony, France);
chloranbucil; gemcitabine (GEMZAR.RTM.); 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine (VELBAN.RTM.); platinum; etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN.RTM.);
oxaliplatin; leucovovin; vinorelbine (NAVELBINE.RTM.); novantrone;
edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase
inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such
as retinoic acid; capecitabine (XELODA.RTM.); pharmaceutically
acceptable salts, acids or derivatives of any of the above; as well
as combinations of two or more of the above such as CHOP, an
abbreviation for a combined therapy of cyclophosphamide,
doxorubicin, vincristine, and prednisolone, and FOLFOX, an
abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovovin.
[0215] Also included in this definition of chemotherapeutic agents
are anti-hormonal agents that act to regulate, reduce, block, or
inhibit the effects of hormones that can promote the growth of
cancer, and are often in the form of systemic, or whole-body
treatment. They may be hormones themselves. Examples include
anti-estrogens and selective estrogen receptor modulators (SERMs),
including, for example, tamoxifen (including NOLVADEX.RTM.
tamoxifen), EVISTA.RTM. raloxifene, droloxifene,
4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone,
and FARESTON.RTM. toremifene; anti-progesterones; estrogen receptor
down-regulators (ERDs); agents that function to suppress or shut
down the ovaries, for example, leutinizing hormone-releasing
hormone (LHRH) agonists such as LUPRON.RTM. and ELIGARD.RTM.
leuprolide acetate, goserelin acetate, buserelin acetate and
tripterelin; other anti-androgens such as flutamide, nilutamide and
bicalutamide; and aromatase inhibitors that inhibit the enzyme
aromatase, which regulates estrogen production in the adrenal
glands, such as, for example, 4(5)-imidazoles, aminoglutethimide,
MEGASE.RTM. megestrol acetate, AROMASIN.RTM. exemestane,
formestanie, fadrozole, RIVISOR.RTM. vorozole, FEMARA.RTM.
letrozole, and ARIMIDEX.RTM. anastrozole. In addition, such
definition of chemotherapeutic agents includes bisphosphonates such
as clodronate (for example, BONEFOS.RTM. or OSTAC.RTM.),
DIDROCAL.RTM. etidronate, NE-58095, ZOMETA.RTM. zoledronic
acid/zoledronate, FOSAMAX.RTM. alendronate, AREDIA.RTM.
pamidronate, SKELID.RTM. tiludronate, or ACTONEL.RTM. risedronate;
as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine
analog); antisense oligonucleotides, particularly those that
inhibit expression of genes in signaling pathways implicated in
aberrant cell proliferation, such as, for example, PKC-alpha, Raf,
H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such
as THERATOPE.RTM. vaccine and gene therapy vaccines, for example,
ALLOVECTIN.RTM. vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM.
vaccine; LURTOTECAN.RTM. topoisomerase 1 inhibitor; ABARELIX.RTM.
rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase
small-molecule inhibitor also known as GW572016); and
pharmaceutically acceptable salts, acids or derivatives of any of
the above.
[0216] Another group of compounds that may be selected as
neurological drugs for cancer treatment or prevention are
anti-cancer immunoglobulins (including, but not limited to,
trastuzumab, pertuzumab, bevacizumab, alemtuxumab, cetuximab,
gemtuzumab ozogamicin, ibritumomab tiuxetan, panitumumab and
rituximab). In some instances, antibodies in conjunction with a
toxic label or conjugate may be used to target and kill desired
cells (i.e., cancer cells), including, but not limited to,
tositumomab with a .sup.131I radiolabel, or trastuzumab
emtansine.
[0217] For an ocular disease or disorder, a neurological drug may
be selected that is an anti-angiogenic ophthalmic agent (i.e.,
bevacizumab, ranibizumab and pegaptanib), an ophthalmic glaucoma
agent (i.e., carbachol, epinephrine, demecarium bromide,
apraclonidine, brimonidine, brinzolamide, levobunolol, timolol,
betaxolol, dorzolamide, bimatoprost, carteolol, metipranolol,
dipivefrin, travoprost and latanoprost), a carbonic anhydrase
inhibitor (i.e., methazolamide and acetazolamide), an ophthalmic
antihistamine (i.e., naphazoline, phenylephrine and
tetrahydrozoline), an ocular lubricant, an ophthalmic steroid
(i.e., fluorometholone, prednisolone, loteprednol, dexamethasone,
difluprednate, rimexolone, fluocinolone, medrysone and
triamcinolone), an ophthalmic anesthetic (i.e., lidocaine,
proparacaine and tetracaine), an ophthalmic anti-infective (i.e.,
levofloxacin, gatifloxacin, ciprofloxacin, moxifloxacin,
chloramphenicol, bacitracin/polymyxin b, sulfacetamide, tobramycin,
azithromycin, besifloxacin, norfloxacin, sulfisoxazole, gentamicin,
idoxuridine, erythromycin, natamycin, gramicidin, neomycin,
ofloxacin, trifluridine, ganciclovir, vidarabine), an ophthalmic
anti-inflammatory agent (i.e., nepafenac, ketorolac, flurbiprofen,
suprofen, cyclosporine, triamcinolone, diclofenac and bromfenac),
and an ophthalmic antihistamine or decongestant (i.e., ketotifen,
olopatadine, epinastine, naphazoline, cromolyn, tetrahydrozoline,
pemirolast, bepotastine, naphazoline, phenylephrine, nedocromil,
lodoxamide, phenylephrine, emedastine and azelastine).
[0218] For a seizure disorder, a neurological drug may be selected
that is an anticonvulsant or antiepileptic including, but not
limited to, barbiturate anticonvulsants (i.e., primidone,
metharbital, mephobarbital, allobarbital, amobarbital,
aprobarbital, alphenal, barbital, brallobarbital and
phenobarbital), benzodiazepine anticonvulsants (i.e., diazepam,
clonazepam, and lorazepam), carbamate anticonvulsants (i.e.
felbamate), carbonic anhydrase inhibitor anticonvulsants (i.e.,
acetazolamide, topiramate and zonisamide), dibenzazepine
anticonvulsants (i.e., rufinamide, carbamazepine, and
oxcarbazepine), fatty acid derivative anticonvulsants (i.e.,
divalproex and valproic acid), gamma-aminobutyric acid analogs
(i.e., pregabalin, gabapentin and vigabatrin), gamma-aminobutyric
acid reuptake inhibitors (i.e., tiagabine), gamma-aminobutyric acid
transaminase inhibitors (i.e., vigabatrin), hydantoin
anticonvulsants (i.e. phenytoin, ethotoin, fosphenytoin and
mephenytoin), miscellaneous anticonvulsants (i.e., lacosamide and
magnesium sulfate), progestins (i.e., progesterone),
oxazolidinedione anticonvulsants (i.e., paramethadione and
trimethadione), pyrrolidine anticonvulsants (i.e., levetiracetam),
succinimide anticonvulsants (i.e., ethosuximide and methsuximide),
triazine anticonvulsants (i.e., lamotrigine), and urea
anticonvulsants (i.e., phenacemide and pheneturide).
[0219] For a lysosomal storage disease, a neurological drug may be
selected that is itself or otherwise mimics the activity of the
enzyme that is impaired in the disease. Exemplary recombinant
enzymes for the treatment of lysosomal storage disorders include,
but are not limited to those set forth in e.g., U.S. Patent
Application publication no. 2005/0142141 (i.e.,
alpha-L-iduronidase, iduronate-2-sulphatase, N-sulfatase,
alpha-N-acetylglucosaminidase, N-acetyl-galactosamine-6-sulfatase,
beta-galactosidase, arylsulphatase B, beta-glucuronidase, acid
alpha-glucosidase, glucocerebrosidase, alpha-galactosidase A,
hexosaminidase A, acid sphingomyelinase, beta-galactocerebrosidase,
beta-galactosidase, arylsulfatase A, acid ceramidase,
aspartoacylase, palmitoyl-protein thioesterase 1 and tripeptidyl
amino peptidase 1).
[0220] For amyloidosis, a neurological drug may be selected that
includes, but is not limited to, an antibody or other binding
molecule (including, but not limited to a small molecule, a
peptide, an aptamer, or other protein binder) that specifically
binds to a target selected from: beta secretase, tau, presenilin,
amyloid precursor protein or portions thereof, amyloid beta peptide
or oligomers or fibrils thereof, death receptor 6 (DR6), receptor
for advanced glycation endproducts (RAGE), parkin, and huntingtin;
a cholinesterase inhibitor (i.e., galantamine, donepezil,
rivastigmine and tacrine); an NMDA receptor antagonist (i.e.,
memantine), a monoamine depletor (i.e., tetrabenazine); an ergoloid
mesylate; an anticholinergic antiparkinsonism agent (i.e.,
procyclidine, diphenhydramine, trihexylphenidyl, benztropine,
biperiden and trihexyphenidyl); a dopaminergic antiparkinsonism
agent (i.e., entacapone, selegiline, pramipexole, bromocriptine,
rotigotine, selegiline, ropinirole, rasagiline, apomorphine,
carbidopa, levodopa, pergolide, tolcapone and amantadine); a
tetrabenazine; an anti-inflammatory (including, but not limited to,
a nonsteroidal anti-inflammatory drug (i.e., indomethicin and other
compounds listed above); a hormone (i.e., estrogen, progesterone
and leuprolide); a vitamin (i.e., folate and nicotinamide); a
dimebolin; a homotaurine (i.e., 3-aminopropanesulfonic acid; 3APS);
a serotonin receptor activity modulator (i.e., xaliproden); an, an
interferon, and a glucocorticoid.
[0221] For a viral or microbial disease, a neurological drug may be
selected that includes, but is not limited to, an antiviral
compound (including, but not limited to, an adamantane antiviral
(i.e., rimantadine and amantadine), an antiviral interferon (i.e.,
peginterferon alfa-2b), a chemokine receptor antagonist (i.e.,
maraviroc), an integrase strand transfer inhibitor (i.e.,
raltegravir), a neuraminidase inhibitor (i.e., oseltamivir and
zanamivir), a non-nucleoside reverse transcriptase inhibitor (i.e.,
efavirenz, etravirine, delavirdine and nevirapine), a nucleoside
reverse transcriptase inhibitors (tenofovir, abacavir, lamivudine,
zidovudine, stavudine, entecavir, emtricitabine, adefovir,
zalcitabine, telbivudine and didanosine), a protease inhibitor
(i.e., darunavir, atazanavir, fosamprenavir, tipranavir, ritonavir,
nelfinavir, amprenavir, indinavir and saquinavir), a purine
nucleoside (i.e., valacyclovir, famciclovir, acyclovir, ribavirin,
ganciclovir, valganciclovir and cidofovir), and a miscellaneous
antiviral (i.e., enfuvirtide, foscarnet, palivizumab and
fomivirsen)), an antibiotic (including, but not limited to, an
aminopenicillin (i.e., amoxicillin, ampicillin, oxacillin,
nafcillin, cloxacillin, dicloxacillin, flucoxacillin, temocillin,
azlocillin, carbenicillin, ticarcillin, mezlocillin, piperacillin
and bacampicillin), a cephalosporin (i.e., cefazolin, cephalexin,
cephalothin, cefamandole, ceftriaxone, cefotaxime, cefpodoxime,
ceftazidime, cefadroxil, cephradine, loracarbef, cefotetan,
cefuroxime, cefprozil, cefaclor, and cefoxitin), a carbapenem/penem
(i.e., imipenem, meropenem, ertapenem, faropenem and doripenem), a
monobactam (i.e., aztreonam, tigemonam, norcardicin A and
tabtoxinine-beta-lactam, a beta-lactamase inhibitor (i.e.,
clavulanic acid, tazobactam and sulbactam) in conjunction with
another beta-lactam antibiotic, an aminoglycoside (i.e., amikacin,
gentamicin, kanamycin, neomycin, netilmicin, streptomycin,
tobramycin, and paromomycin), an ansamycin (i.e., geldanamycin and
herbimycin), a carbacephem (i.e., loracarbef), a glycopeptides
(i.e., teicoplanin and vancomycin), a macrolide (i.e.,
azithromycin, clarithromycin, dirithromycin, erythromycin,
roxithromycin, troleandomycin, telithromycin and spectinomycin), a
monobactam (i.e., aztreonam), a quinolone (i.e., ciprofloxacin,
enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin,
norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin
and temafloxacin), a sulfonamide (i.e., mafenide,
sulfonamidochrysoidine, sulfacetamide, sulfadiazine,
sulfamethizole, sulfanilamide, sulfasalazine, sulfisoxazole,
trimethoprim, trimethoprim and sulfamethoxazole), a tetracycline
(i.e., tetracycline, demeclocycline, doxycycline, minocycline and
oxytetracycline), an antineoplastic or cytotoxic antibiotic (i.e.,
doxorubicin, mitoxantrone, bleomycin, daunorubicin, dactinomycin,
epirubicin, idarubicin, plicamycin, mitomycin, pentostatin and
valrubicin) and a miscellaneous antibacterial compound (i.e.,
bacitracin, colistin and polymyxin B)), an antifungal (i.e.,
metronidazole, nitazoxanide, tinidazole, chloroquine, iodoquinol
and paromomycin), and an antiparasitic (including, but not limited
to, quinine, chloroquine, amodiaquine, pyrimethamine, sulphadoxine,
proguanil, mefloquine, atovaquone, primaquine, artemesinin,
halofantrine, doxycycline, clindamycin, mebendazole, pyrantel
pamoate, thiabendazole, diethylcarbamazine, ivermectin, rifampin,
amphotericin B, melarsoprol, efornithine and albendazole). For
ischemia, a neurological drug may be selected that includes, but is
not limited to, a thrombolytic (i.e., urokinase, alteplase,
reteplase and tenecteplase), a platelet aggregation inhibitor
(i.e., aspirin, cilostazol, clopidogrel, prasugrel and
dipyridamole), a statin (i.e., lovastatin, pravastatin,
fluvastatin, rosuvastatin, atorvastatin, simvastatin, cerivastatin
and pitavastatin), and a compound to improve blood flow or vascular
flexibility, including, e.g., blood pressure medications.
[0222] For a behavioral disorder, a neurological drug may be
selected from a behavior-modifying compound including, but not
limited to, an atypical antipsychotic (i.e., risperidone,
olanzapine, apripiprazole, quetiapine, paliperidone, asenapine,
clozapine, iloperidone and ziprasidone), a phenothiazine
antipsychotic (i.e., prochlorperazine, chlorpromazine,
fluphenazine, perphenazine, trifluoperazine, thioridazine and
mesoridazine), a thioxanthene (i.e., thiothixene), a miscellaneous
antipsychotic (i.e., pimozide, lithium, molindone, haloperidol and
loxapine), a selective serotonin reuptake inhibitor (i.e.,
citalopram, escitalopram, paroxetine, fluoxetine and sertraline), a
serotonin-norepinephrine reuptake inhibitor (i.e., duloxetine,
venlafaxine, desvenlafaxine, a tricyclic antidepressant (i.e.,
doxepin, clomipramine, amoxapine, nortriptyline, amitriptyline,
trimipramine, imipramine, protriptyline and desipramine), a
tetracyclic antidepressant (i.e., mirtazapine and maprotiline), a
phenylpiperazine antidepressant (i.e., trazodone and nefazodone), a
monoamine oxidase inhibitor (i.e., isocarboxazid, phenelzine,
selegiline and tranylcypromine), a benzodiazepine (i.e.,
alprazolam, estazolam, flurazeptam, clonazepam, lorazepam and
diazepam), a norepinephrine-dopamine reuptake inhibitor (i.e.,
bupropion), a CNS stimulant (i.e., phentermine, diethylpropion,
methamphetamine, dextroamphetamine, amphetamine, methylphenidate,
dexmethylphenidate, lisdexamfetamine, modafinil, pemoline,
phendimetrazine, benzphetamine, phendimetrazine, armodafinil,
diethylpropion, caffeine, atomoxetine, doxapram, and mazindol), an
anxiolytic/sedative/hypnotic (including, but not limited to, a
barbiturate (i.e., secobarbital, phenobarbital and mephobarbital),
a benzodiazepine (as described above), and a miscellaneous
anxiolytic/sedative/hypnotic (i.e. diphenhydramine, sodium oxybate,
zaleplon, hydroxyzine, chloral hydrate, aolpidem, buspirone,
doxepin, eszopiclone, ramelteon, meprobamate and ethclorvynol)), a
secretin (see, e.g., Ratliff-Schaub et al. Autism 9: 256-265
(2005)), an opioid peptide (see, e.g., Cowen et al., J. Neurochem.
89:273-285 (2004)), and a neuropeptide (see, e.g., Hethwa et al.
Am. J. Physiol. 289: E301-305 (2005)).
[0223] For CNS inflammation, a neurological drug may be selected
that addresses the inflammation itself (i.e., a nonsteroidal
anti-inflammatory agent such as ibuprofen or naproxen), or one
which treats the underlying cause of the inflammation (i.e., an
anti-viral or anti-cancer agent).
[0224] According to one embodiment of the invention, the "coupling"
is achieved by generating a multispecific antibody (e.g. a
bispecific antibody). Multispecific antibodies are monoclonal
antibodies that have binding specificities for at least two
different antigens or epitopes. In one embodiment, the
multispecific antibody comprises a first antigen binding site which
binds the BBB-R and a second antigen binding site which binds a
brain antigen, such as beta-secretase 1 (BACE1) or Abeta, and the
other brain antigens disclosed herein.
[0225] An exemplary brain antigen bound by such
multispecific/bispecific antibody is BACE1, and an exemplary
antibody binding thereto is the YW412.8.31 antibody in FIGS. 9A-B
herein.
[0226] In another embodiment, the brain antigen is Abeta, exemplary
such antibodies being described in WO2007068412, WO2008011348,
WO20080156622, and WO2008156621, expressly incorporated herein by
reference, with an exemplary Abeta antibody comprising the IgG4
MABT5102A antibody comprising the heavy and light chain amino acid
sequences in FIGS. 11A and 11B, respectively.
[0227] Techniques for making multispecific antibodies include, but
are not limited to, recombinant co-expression of two immunoglobulin
heavy chain-light chain pairs having different specificities (see
Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and
Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole"
engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific
antibodies may also be made by engineering electrostatic steering
effects for making antibody Fc-heterodimeric molecules (WO
2009/089004A1); cross-linking two or more antibodies or fragments
(see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science,
229: 81 (1985)); using leucine zippers to produce bi-specific
antibodies (see, e.g., Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992)); using "diabody" technology for making
bispecific antibody fragments (see, e.g., Hollinger et al., Proc.
Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain
Fv (sFv) dimers (see, e.g. Gruber et al., J. Immunol., 152:5368
(1994)); and preparing trispecific antibodies as described, e.g.,
in Tutt et al. J. Immunol. 147: 60 (1991).
[0228] Engineered antibodies with three or more functional antigen
binding sites, including "Octopus antibodies" or "dual-variable
domain immunoglobulins" (DVDs) are also included herein (see, e.g.
US 2006/0025576A1, and Wu et al. Nature Biotechnology (2007)).
[0229] The antibody or fragment herein also includes a "Dual Acting
FAb" or "DAF" comprising an antigen binding site that binds to the
BBB-R (e.g. TfR) as well as the brain antigen (e.g. BACE1) (see, US
2008/0069820, for example).
[0230] In one embodiment, the antibody is an antibody fragment,
various such fragments being disclosed above. In another
embodiment, the antibody is an intact or full-length antibody.
Depending on the amino acid sequence of the constant domain of
their heavy chains, intact antibodies can be assigned to different
classes. There are five major classes of intact antibodies: IgA,
IgD, IgE, IgG, and IgM, and several of these may be further divided
into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and
IgA2. The heavy chain constant domains that correspond to the
different classes of antibodies are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively. The subunit structures
and three-dimensional configurations of different classes of
immunoglobulins are well known. In one embodiment, the intact
antibody lacks effector function. In another embodiment, the inact
antibody has reduced effector function.
[0231] Techniques for generating antibodies are known and examples
provided above in the definitions section of this document. In one
embodiment, the antibody is a chimeric, humanized, or human
antibody or antigen-binding fragment thereof.
[0232] Various techniques are available for determining binding of
the antibody to the BBB-R. One such assay is an enzyme linked
immunosorbent assay (ELISA) for confirming an ability to bind to
human BBB-R (and brain antigen). According to this assay, plates
coated with antigen (e.g. recombinant BBB-R) are incubated with a
sample comprising the anti-BBB-R antibody and binding of the
antibody to the antigen of interest is determined.
[0233] In one aspect, an antibody of the invention is tested for
its antigen binding activity, e.g., by known methods such as ELISA,
Western blot, etc.
[0234] Assays for evaluating uptake of systemically administered
antibody and other biological activity of the antibody can be
performed as disclosed in the examples or as known for the anti-CNS
antigen antibody of interest.
[0235] Exemplary assays where the multispecific antibody binds
BACE1 shall now be described.
[0236] Competition assays may be used to identify an antibody that
competes with any of the anti-BACE1 antibodies or Fabs descried
herein, for example, YW412.8, YW412.8.31, YW412.8.30, YW412.8.2,
YW412.8.29, YW412.8.51, Fab12, LC6, LC9, LC10 for binding to BACE1.
In certain embodiments, such a competing antibody binds to the same
epitope (e.g., a linear or a conformational epitope) that is bound
by any of the anti-BACE1 antibodies or Fabs descried herein, for
example, YW412.8, YW412.8.31, YW412.8.30, YW412.8.2, YW412.8.29,
YW412.8.51, Fab12, LC6, LC9, LC10. Detailed exemplary methods for
mapping an epitope to which an antibody binds are provided in
Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular
Biology vol. 66 (Humana Press, Totowa, N.J.).
[0237] In an exemplary competition assay, immobilized BACE1 is
incubated in a solution comprising a first labeled antibody that
binds to BACE1 (e.g., YW412.8, YW412.8.31, YW412.8.30, YW412.8.2,
YW412.8.29, YW412.8.51, Fab12, LC6, LC9, LC10) and a second
unlabeled antibody that is being tested for its ability to compete
with the first antibody for binding to BACE1. The second antibody
may be present in a hybridoma supernatant. As a control,
immobilized BACE1 is incubated in a solution comprising the first
labeled antibody but not the second unlabeled antibody. After
incubation under conditions permissive for binding of the first
antibody to BACE1, excess unbound antibody is removed, and the
amount of label associated with immobilized BACE1 is measured. If
the amount of label associated with immobilized BACE1 is
substantially reduced in the test sample relative to the control
sample, then that indicates that the second antibody is competing
with the first antibody for binding to BACE1. See Harlow and Lane
(1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.).
[0238] In one aspect, assays are provided for identifying
anti-BACE1 antibodies thereof having biological activity.
Biological activity may include, e.g., inhibition of BACE1 aspartyl
protease activity. Antibodies having such biological activity in
vivo and/or in vitro are also provided, e.g. as evaluated by
homogeneous time-resolved fluorescence HTRF assay or a microfluidic
capillary electrophoretic (MCE) assay using synthetic substrate
peptides, or in vivo in cell lines which express BACE1 substrates
such as APP.
[0239] The antibody (including the multispecific antibody) herein
is optionally recombinantly produced in a host cell transformed
with nucleic acid sequences encoding its heavy and/or light chains
(e.g. where the host cell or host cells have been transformed by
one or more vectors with the nucleic acid therein). The host
cell(s) is optionally a mammalian cell, for example a Chinese
Hamster Ovary (CHO) cell.
[0240] B. Pharmaceutical Formulations
[0241] Therapeutic formulations of the antibodies used in
accordance with the present invention are prepared for storage by
mixing an antibody having the desired degree of purity with
optional pharmaceutically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980)), in the form of lyophilized formulations or
aqueous solutions. Acceptable carriers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0242] The formulation herein may also contain more than one active
compound as necessary, optionally those with complementary
activities that do not adversely affect each other. The type and
effective amounts of such medicaments depend, for example, on the
amount of antibody present in the formulation, and clinical
parameters of the subjects. Exemplary such medicaments are
discussed below.
[0243] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in, for example, Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). One or
more therapeutic agents may be encapsulated in liposomes that are
coupled to anti-BBB-R (see e.g., U.S. Patent Application
Publication No. 20020025313).
[0244] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semi-permeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
[0245] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0246] In one embodiment the formulation is isotonic.
[0247] C. Therapeutic Uses of Anti-BBB-R Antibodies
[0248] The anti-BBB-R antibodies (including multispecific
antibodies comprising them) of the invention may be utilized in a
variety of in vivo methods. For example, the invention provides a
method of transporting a therapeutic compound across the
blood-brain barrier with reduced or eliminated impact on red blood
cell populations comprising exposing the anti-BBB-R antibody
coupled to a therapeutic compound (e.g. a multispecific antibody
which binds both the BBB-R and a brain antigen) to the BBB such
that the antibody transports the therapeutic compound coupled
thereto across the BBB. In another example, the invention provides
a method of transporting a neurological disorder drug across the
blood-brain barrier comprising exposing an anti-BBB-R antibody of
the invention coupled to a brain disorder drug (e.g. a
multispecific antibody which binds both the BBB-R and a brain
antigen) to the BBB such that the antibody transports the
neurological disorder drug coupled thereto across the BBB with
reduced or eliminated impact on red blood cell populations. In one
embodiment, the BBB here is in a mammal (e.g. a human), e.g. one
which has a neurological disorder, including, without limitation:
Alzheimer's disease (AD), stroke, dementia, muscular dystrophy
(MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS),
cystic fibrosis, Angelman's syndrome, Liddle syndrome, Parkinson's
disease, Pick's disease, Paget's disease, cancer, traumatic brain
injury, etc.
[0249] In one embodiment, neurological disorder is selected from: a
neuropathy, an amyloidosis, cancer (e.g. involving the CNS or
brain), an ocular disease or disorder, a viral or microbial
infection, inflammation (e.g. of the CNS or brain), ischemia,
neurodegenerative disease, seizure, behavioral disorder, lysosomal
storage disease, etc.
[0250] Neuropathy disorders are diseases or abnormalities of the
nervous system characterized by inappropriate or uncontrolled nerve
signaling or lack thereof, and include, but are not limited to,
chronic pain (including nociceptive pain), pain caused by an injury
to body tissues, including cancer-related pain, neuropathic pain
(pain caused by abnormalities in the nerves, spinal cord, or
brain), and psychogenic pain (entirely or mostly related to a
psychological disorder), headache, migraine, neuropathy, and
symptoms and syndromes often accompanying such neuropathy disorders
such as vertigo or nausea.
[0251] Amyloidoses are a group of diseases and disorders associated
with extracellular proteinaceous deposits in the CNS, including,
but not limited to, secondary amyloidosis, age-related amyloidosis,
Alzheimer's Disease (AD), mild cognitive impairment (MCI), Lewy
body dementia, Down's syndrome, hereditary cerebral hemorrhage with
amyloidosis (Dutch type); the Guam Parkinson-Dementia complex,
cerebral amyloid angiopathy, Huntington's disease, progressive
supranuclear palsy, multiple sclerosis; Creutzfeld Jacob disease,
Parkinson's disease, transmissible spongiform encephalopathy,
HIV-related dementia, amyotropic lateral sclerosis (ALS),
inclusion-body myositis (IBM), and ocular diseases relating to
beta-amyloid deposition (i.e., macular degeneration, drusen-related
optic neuropathy, and cataract).
[0252] Cancers of the CNS are characterized by aberrant
proliferation of one or more CNS cell (i.e., a neural cell) and
include, but are not limited to, glioma, glioblastoma multiforme,
meningioma, astrocytoma, acoustic neuroma, chondroma,
oligodendroglioma, medulloblastomas, ganglioglioma, Schwannoma,
neurofibroma, neuroblastoma, and extradural, intramedullary or
intradural tumors.
[0253] Ocular diseases or disorders are diseases or disorders of
the eye, which for the purposes herein is considered a CNS organ
segregated by the BBB. Ocular diseases or disorders include, but
are not limited to, disorders of sclera, cornea, iris and ciliary
body (i.e., scleritis, keratitis, corneal ulcer, corneal abrasion,
snow blindness, arc eye, Thygeson's superficial punctate
keratopathy, corneal neovascularisation, Fuchs' dystrophy,
keratoconus, keratoconjunctivitis sicca, iritis and uveitis),
disorders of the lens (i.e., cataract), disorders of choroid and
retina (i.e., retinal detachment, retinoschisis, hypertensive
retinopathy, diabetic retinopathy, retinopathy, retinopathy of
prematurity, age-related macular degeneration, macular degeneration
(wet or dry), epiretinal membrane, retinitis pigmentosa and macular
edema), glaucoma, floaters, disorders of optic nerve and visual
pathways (i.e., Leber's hereditary optic neuropathy and optic disc
drusen), disorders of ocular muscles/binocular movement
accommodation/refraction (i.e., strabismus, ophthalmoparesis,
progressive external opthalmoplegia, esotropia, exotropia,
hypermetropia, myopia, astigmatism, anisometropia, presbyopia and
ophthalmoplegia), visual disturbances and blindness (i.e.,
amblyopia, Lever's congenital amaurosis, scotoma, color blindness,
achromatopsia, nyctalopia, blindness, river blindness and
micro-opthalmia/coloboma), red eye, Argyll Robertson pupil,
keratomycosis, xerophthalmia and andaniridia.
[0254] Viral or microbial infections of the CNS include, but are
not limited to, infections by viruses (i.e., influenza, HIV,
poliovirus, rubella,), bacteria (i.e., Neisseria sp., Streptococcus
sp., Pseudomonas sp., Proteus sp., E. coli, S. aureus, Pneumococcus
sp., Meningococcus sp., Haemophilus sp., and Mycobacterium
tuberculosis) and other microorganisms such as fungi (i.e., yeast,
Cryptococcus neoformans), parasites (i.e., toxoplasma gondii) or
amoebas resulting in CNS pathophysiologies including, but not
limited to, meningitis, encephalitis, myelitis, vasculitis and
abscess, which can be acute or chronic.
[0255] Inflammation of the CNS includes, but is not limited to,
inflammation that is caused by an injury to the CNS, which can be a
physical injury (i.e., due to accident, surgery, brain trauma,
spinal cord injury, concussion) and an injury due to or related to
one or more other diseases or disorders of the CNS (i.e., abscess,
cancer, viral or microbial infection).
[0256] Ischemia of the CNS, as used herein, refers to a group of
disorders relating to aberrant blood flow or vascular behavior in
the brain or the causes therefor, and includes, but is not limited
to: focal brain ischemia, global brain ischemia, stroke (i.e.,
subarachnoid hemorrhage and intracerebral hemorrhage), and
aneurysm.
[0257] Neurodegenerative diseases are a group of diseases and
disorders associated with neural cell loss of function or death in
the CNS, and include, but are not limited to: adrenoleukodystrophy,
Alexander's disease, Alper's disease, amyotrophic lateral
sclerosis, ataxia telangiectasia, Batten disease, cockayne
syndrome, corticobasal degeneration, degeneration caused by or
associated with an amyloidosis, Friedreich's ataxia, frontotemporal
lobar degeneration, Kennedy's disease, multiple system atrophy,
multiple sclerosis, primary lateral sclerosis, progressive
supranuclear palsy, spinal muscular atrophy, transverse myelitis,
Refsum's disease, and spinocerebellar ataxia.
[0258] Seizure diseases and disorders of the CNS involve
inappropriate and/or abnormal electrical conduction in the CNS, and
include, but are not limited to epilepsy (i.e., absence seizures,
atonic seizures, benign Rolandic epilepsy, childhood absence,
clonic seizures, complex partial seizures, frontal lobe epilepsy,
febrile seizures, infantile spasms, juvenile myoclonic epilepsy,
juvenile absence epilepsy, Lennox-Gastaut syndrome, Landau-Kleffner
Syndrome, Dravet's syndrome, Otahara syndrome, West syndrome,
myoclonic seizures, mitochondrial disorders, progressive myoclonic
epilepsies, psychogenic seizures, reflex epilepsy, Rasmussen's
Syndrome, simple partial seizures, secondarily generalized
seizures, temporal lobe epilepsy, toniclonic seizures, tonic
seizures, psychomotor seizures, limbic epilepsy, partial-onset
seizures, generalized-onset seizures, status epilepticus, abdominal
epilepsy, akinetic seizures, autonomic seizures, massive bilateral
myoclonus, catamenial epilepsy, drop seizures, emotional seizures,
focal seizures, gelastic seizures, Jacksonian March, Lafora
Disease, motor seizures, multifocal seizures, nocturnal seizures,
photosensitive seizure, pseudo seizures, sensory seizures, subtle
seizures, sylvan seizures, withdrawal seizures, and visual reflex
seizures).
[0259] Behavioral disorders are disorders of the CNS characterized
by aberrant behavior on the part of the afflicted subject and
include, but are not limited to: sleep disorders (i.e., insomnia,
parasomnias, night terrors, circadian rhythm sleep disorders, and
narcolepsy), mood disorders (i.e., depression, suicidal depression,
anxiety, chronic affective disorders, phobias, panic attacks,
obsessive-compulsive disorder, attention deficit hyperactivity
disorder (ADHD), attention deficit disorder (ADD), chronic fatigue
syndrome, agoraphobia, post-traumatic stress disorder, bipolar
disorder), eating disorders (i.e., anorexia or bulimia), psychoses,
developmental behavioral disorders (i.e., autism, Rett's syndrome,
Aspberger's syndrome), personality disorders and psychotic
disorders (i.e., schizophrenia, delusional disorder, and the
like).
[0260] Lysosomal storage disorders are metabolic disorders which
are in some cases associated with the CNS or have CNS-specific
symptoms; such disorders include, but are not limited to: Tay-Sachs
disease, Gaucher's disease, Fabry disease, mucopolysaccharidosis
(types I, II, III, IV, V, VI and VII), glycogen storage disease,
GM1-gangliosidosis, metachromatic leukodystrophy, Farber's disease,
Canavan's leukodystrophy, and neuronal ceroid lipofuscinoses types
1 and 2, Niemann-Pick disease, Pompe disease, and Krabbe's
disease.
[0261] In another embodiment, diseases related to or caused by
inappropriate overproduction of red blood cells, or wherein the
overproduction of red blood cells is an effect of the disease, can
be prevented or treated by the reticulocyte-depleting effect
recognized herein of anti-TfR antibodies retaining at least partial
effector function. For example, in congenital or neoplastic
polycythemia vera, elevated red blood cell counts due to
hyperproliferation of, e.g., reticulocytes, results in thickening
of blood and concomitant physiological symptoms (d'Onofrio et al.,
Clin. Lab. Haematol. (1996) Suppl. 1: 29-34). Administration of an
anti-TfR antibody of the invention wherein at least with at least
partial effector function of the antibody was preserved would
permit selective removal of immature reticulocyte populations
without impacting normal transferrin transport into the CNS. Dosing
of such an antibody could be modulated such that acute clinical
symptoms could be minimized (ie, by dosing at a very low dose or at
widely-spaced intervals), as well-understood in the art.
[0262] In one aspect, an antibody of the invention is used to
detect a neurological disorder before the onset of symptoms and/or
to assess the severity or duration of the disease or disorder. In
one aspect, the antibody permits detection and/or imaging of the
neurological disorder, including imaging by radiography,
tomography, or magnetic resonance imaging (MRI).
[0263] In one aspect, a low affinity anti-BBB-R antibody of the
invention for use as a medicament is provided. In further aspects,
a low affinity anti-BBB-R antibody for use in treating a
neurological disease or disorder (e.g., Alzheimer's disease)
without depleting red blood cells (ie, reticulocytes) is provided.
In certain embodiments, a modified low affinity anti-BBB-R antibody
for use in a method of treatment as described herein is provided.
In certain embodiments, the invention provides a low affinity
anti-BBB-R antibody modified to improve its safety for use in a
method of treating an individual having a neurological disease or
disorder comprising administering to the individual an effective
amount of the anti-BBB-R antibody (optionally coupled to a
neurological disorder drug). In one such embodiment, the method
further comprises administering to the individual an effective
amount of at least one additional therapeutic agent. In further
embodiments, the invention provides an anti-BBB-R antibody modified
to improve its safety for use in reducing or inhibiting amlyoid
plaque formation in a patient at risk or suffering from a
neurological disease or disorder (e.g., Alzheimer's disease). An
"individual" according to any of the above embodiments is
optionally a human. In certain aspects, the anti-BBB-R antibody of
the invention for use in the methods of the invention improves
uptake of the neurological disorder drug with which it is
coupled.
[0264] In a further aspect, the invention provides for the use of a
low affinity anti-BBB-R antibody of the invention in the
manufacture or preparation of a medicament. In one embodiment, the
medicament is for treatment of neurological disease or disorder. In
a further embodiment, the medicament is for use in a method of
treating neurological disease or disorder comprising administering
to an individual having neurological disease or disorder an
effective amount of the medicament. In one such embodiment, the
method further comprises administering to the individual an
effective amount of at least one additional therapeutic agent.
[0265] In a further aspect, the invention provides a method for
treating Alzheimer's disease. In one embodiment, the method
comprises administering to an individual having Alzheimer's disease
an effective amount of a multispecific antibody of the invention
which binds both BACE1 and TfR or both Abeta and TfR. In one such
embodiment, the method further comprises administering to the
individual an effective amount of at least one additional
therapeutic agent. An "individual" according to any of the above
embodiments may be a human.
[0266] The anti-BBB-R antibodies of the invention can be used
either alone or in combination with other agents in a therapy. For
instance, the anti-BBB-R antibody of the invention may be
co-administered with at least one additional therapeutic agent. In
certain embodiments, an additional therapeutic agent is a
therapeutic agent effective to treat the same or a different
neurological disorder as the anti-BBB-R antibody is being employed
to treat. Exemplary additional therapeutic agents include, but are
not limited to: the various neurological drugs described above,
cholinesterase inhibitors (such as donepezil, galantamine,
rovastigmine, and tacrine), NMDA receptor antagonists (such as
memantine), amyloid beta peptide aggregation inhibitors,
antioxidants, .gamma.-secretase modulators, nerve growth factor
(NGF) mimics or NGF gene therapy, PPAR.gamma. agonists, HMS-CoA
reductase inhibitors (statins), ampakines, calcium channel
blockers, GABA receptor antagonists, glycogen synthase kinase
inhibitors, intravenous immunoglobulin, muscarinic receptor
agonists, nicrotinic receptor modulators, active or passive amyloid
beta peptide immunization, phosphodiesterase inhibitors, serotonin
receptor antagonists and anti-amyloid beta peptide antibodies. In
certain embodiments, the at least one additional therapeutic agent
is selected for its ability to mitigate one or more side effects of
the neurological drug.
[0267] As exemplified herein, certain anti-BBB-R antibodies may
have side effects that negatively impact reticulocyte populations
in a subject treated with the anti-BBB-R antibody. Thus, in certain
embodiments, at least one further therapeutic agent selected for
its ability to mitigate such negative side effect on reticulocyte
populations is coadministered with an anti-BBB-R antibody of the
invention. Examples of such therapeutic agents include, but are not
limited to, agents to increase red blood cell (ie, reticulocyte)
populations, agents to support growth and development of red blood
cells (ie, reticulocytes), and agents to protect red blood cell
populations from the effects of the anti-BBB-R antibody; such
agents include, but are not limited to, erythropoietin (EPO), iron
supplements, vitamin C, folic acid, and vitamin B12, as well as
physical replacement of red blood cells (ie, reticulocytes) by, for
example, transfusion with similar cells, which may be from another
individual of similar blood type or may have been previously
extracted from the subject to whom the anti-BBB-R antibody is
administered. It will be understood by one of ordinary skill in the
art that in some instances, agents intended to protect existing red
blood cells (ie, reticulocytes) are preferably administered to the
subject preceding or concurrent with the anti-BBB-R antibody
therapy, while agents intended to support or initiate the
regrowth/development of red blood cells or blood cell populations
(ie, reticulocytes or reticulocyte populations) are preferably
administered concurrent with or after the anti-BBB-R antibody
therapy such that such blood cells can be replenished after the
anti-BBB-R antibody treatment.
[0268] In certain other such embodiments, the at least one further
therapeutic agent is selected for its ability to inhibit or prevent
the activation of the complement pathway upon administration of the
anti-BBB-R antibody. Examples of such therapeutic agents include,
but are not limited to, agents that interfere with the ability of
the anti-BBB-R antibody to bind to or activate the complement
pathway and agents that inhibit one or more molecular interactions
within the complement pathway, and are described generally in
Mollnes and Kirschfink (2006) Molec. Immunol. 43:107-121, the
contents of which are expressly incorporated herein by
reference.
[0269] Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included
in the same or separate formulations), and separate administration,
in which case, administration of the antibody of the invention can
occur prior to, simultaneously, and/or following, administration of
the additional therapeutic agent and/or adjuvant. Antibodies of the
invention can also be used in combination with other interventional
therapies such as, but not limited to, radiation therapy,
behavioral therapy, or other therapies known in the art and
appropriate for the neurological disorder to be treated or
prevented.
[0270] The anti-BBB-R antibody of the invention (and any additional
therapeutic agent) can be administered by any suitable means,
including parenteral, intrapulmonary, and intranasal, and, if
desired for local treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration.
Dosing can be by any suitable route, e.g. by injections, such as
intravenous or subcutaneous injections, depending in part on
whether the administration is brief or chronic. Various dosing
schedules including but not limited to single or multiple
administrations over various time-points, bolus administration, and
pulse infusion are contemplated herein.
[0271] Antibodies of the invention are formulated, dosed, and
administered in a fashion consistent with good medical practice.
Factors for consideration in this context include the particular
disorder being treated, the particular mammal being treated, the
clinical condition of the individual patient, the cause of the
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners. The antibody need not be, but is
optionally formulated with one or more agents currently used to
prevent or treat the disorder in question or to prevent, mitigate
or ameliorate one or more side effects of antibody administration.
The effective amount of such other agents depends on the amount of
antibody present in the formulation, the type of disorder or
treatment, and other factors discussed above. These are generally
used in the same dosages and with administration routes as
described herein, or about from 1 to 99% of the dosages described
herein, or in any dosage and by any route that is
empirically/clinically determined to be appropriate.
[0272] For the prevention or treatment of disease, the appropriate
dosage of an antibody of the invention (when used alone or in
combination with one or more other additional therapeutic agents)
will depend on the type of disease to be treated, the type of
antibody, the severity and course of the disease, whether the
antibody is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the antibody, and the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg)
of antibody can be an initial candidate dosage for administration
to the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. One typical daily
dosage might range from about 1 .mu.g/kg to 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment would generally be sustained until a
desired suppression of disease symptoms occurs. One exemplary
dosage of the antibody would be in the range from about 0.05 mg/kg
to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0
mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be
administered to the patient. Such doses may be administered
intermittently, e.g. every week or every three weeks (e.g. such
that the patient receives from about two to about twenty, or e.g.
about six doses of the antibody). An initial higher loading dose,
followed by one or more lower doses may be administered. However,
other dosage regimens may be useful. It will be appreciated that
one method to reduce impact on reticulocyte populations by
administration of anti-TfR antibodies is to modify the amount or
timing of the doses such that overall lower quantities of
circulating antibody are present in the bloodstream to interact
with reticulocytes. In one nonlimiting example, a lower dose of the
anti-TfR antibodies may be administered with greater frequency than
a higher dose would be. The dosage used may be balanced between the
amount of antibody necessary to be delivered to the CNS (itself
related to the affinity of the CNS antigen-specific portion of the
antibody), the affinity of that antibody for TfR, and whether or
not red blood cell (ie, reticulocyte)-protecting, growth and
development-stimulating, or complement pathway-inhibiting
compound(s) are being co- or serially administered with the
antibody. The progress of this therapy is easily monitored by
conventional techniques and assays as described herein and as known
in the art.
[0273] It is understood that any of the above formulations or
therapeutic methods may be carried out using an immunoconjugate of
the invention in place of or in addition to an anti-BBB-R
antibody.
[0274] D. Articles of Manufacture
[0275] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is by itself or combined with another composition
effective for treating, preventing and/or diagnosing the condition
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an antibody of the invention. The label
or package insert indicates that the composition is used for
treating the condition of choice. Moreover, the article of
manufacture may comprise (a) a first container with a composition
contained therein, wherein the composition comprises an antibody of
the invention; and (b) a second container with a composition
contained therein, wherein the composition comprises a further
cytotoxic or otherwise therapeutic agent. The article of
manufacture in this embodiment of the invention may further
comprise a package insert indicating that the compositions can be
used to treat a particular condition. Alternatively, or
additionally, the article of manufacture may further comprise a
second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0276] It is understood that any of the above articles of
manufacture may include an immunoconjugate of the invention in
place of or in addition to an anti-BBB-R antibody.
[0277] The article of manufacture optionally further comprises a
package insert with instructions for treating a neurological
disorder in a subject, wherein the instructions indicate that
treatment with the antibody as disclosed herein treats the
neurological disorder, and optionally indicates that the antibody
has improved uptake across the BBB due to its low affinity for the
BBB-R.
EXAMPLES
Example 1
Generation and Characterization of Low-Affinity Anti-TfR
Antibodies
[0278] The field has recognized that the natural ability of the
transferrin receptor (TfR) to transport transferrin across the
blood-brain barrier (BBB) may be exploited to permit the transport
of heterologous molecules into the brain from the bloodstream (see,
e.g., WO9502421). Applicants previously developed an important
modification to this system, (Sci. Transl. Med. 3, 84ra43 (2011))
namely that transport into the brain and retention in the brain of
a heterologous molecule conjugated to an anti-transferrin receptor
antibody (anti-TfR) was substantially enhanced by decreasing the
affinity of the anti-TfR for transferrin receptor, within a certain
range.
[0279] A panel of anti-TfR antibodies was generated with
progressively lessening affinities for murine TfR, three of which
(designated anti-TfR.sup.A, anti-TfR.sup.D, and anti-TfR.sup.E)
were further modified into a bispecific format with the other
antibody arm being specific for BACE1. Each monospecific and
bispecific antibody was assessed in a competition ELISA assay for
its affinity for murine TfR. Briefly, the assay was performed in
maxisorp plates (Neptune, N. J) coated with 2.5 .mu.g/ml of
purified muTfR tagged with a hexahistidine tag (muTfR-His) in PBS
at 4.degree. C. overnight. Plates were washed with PBS/0.05% Tween
20 and blocked using Superblock blocking buffer in PBS (Thermo
Scientific, Hudson, N.H.). A 1:3 serial titrated bivalent IgG
(anti-TfR.sup.A, anti-TfR.sup.D, anti-TfR.sup.E) or bi-specific Ab
(anti-TfR.sup.A/BACE1, anti-TfR.sup.D/BACE1, or
anti-TfR.sup.E/BACE1) was combined with 1 nM biotinylated
anti-TfR.sup.A and added to the plate for 1 hour at room
temperature. Plates were washed with PBS/0.05% Tween 20 and
HRP-streptavidin (SouthernBiotech, Birmingham) was added the plate
and incubated for 1 hour at room temperature. Plates were washed
with PBS/0.05% Tween 20 and biotinylated anti-TfR.sup.A bound to
the plate was detected using TMB substrate (BioFX Laboratories,
Owings Mills). (FIG. 1A). The observed IC50 values for the binding
of each monospecific or bispecific antibody to murine TfR in the
assay are shown in Table 2.
TABLE-US-00002 TABLE 2 IC.sub.50 values for antibody binding by
competition ELISA Antibody IC.sub.50 TfR.sup.A 1 nM TfR.sup.D 66 nM
TfR.sup.E 20 .mu.M TfR.sup.A/BACE1 14 nM TfR.sup.D/BACE1 1.6 .mu.M
TfR.sup.E/BACE1 95 .mu.M
[0280] Antibody distribution post a single administration in mice
was performed as follows. Wild type female C57B/6 mice ages 6-8
weeks were used for all studies. The animals' care was in
accordance with institutional guidelines. Mice were intravenously
injected with 50 mg/kg of either a control IgG, anti-BACE1 or an
anti-TfR/BACE1 variant. Total injection volume did not exceed 250
uL and antibodies were diluted in D-PBS when necessary
(Invitrogen). After the indicated time, mice were perfused with
D-PBS at a rate of 2 mL/min for 8 minutes. Brains were extracted
and the cortex and hippocampus was isolated, homogenized in 1%
NP-40 (Cal-Biochem) in PBS containing Complete Mini EDTA-free
protease inhibitor cocktail tablets (Roche Diagnostics).
Homogenized brain samples were rotated at 4.degree. C. for 1 hour
before spinning at 14,000 rpm for 20 minutes. The supernatant was
isolated for brain antibody measurement. Whole blood was collected
prior to perfusion in EDTA microtainer tubes (BD Diagnostics),
allowed to sit for 30 minutes at room temperature, and spun down at
5000.times. g for 10 minutes. The top layer of plasma was
transferred to new tubes for antibody and mouse A.beta..sub.1-40
measurements.
[0281] Total antibody concentrations in mouse plasma and brain
samples were measured using an anti-huFc/anti-huFc ELISA. NUNC
384-well Maxisorp immunoplates (Neptune, N.J.) were coated with the
F(ab').sub.2 fragment of donkey anti-human IgG, an Fc
fragment-specific polyclonal antibody (Jackson ImmunoResearch, West
Grove, Pa.), overnight at 4.degree. C. Plates were blocked with
PBS, 0.5% BSA for 1 hour at 25.degree. C. Each antibody (control
IgG, anti-BACE1, and anti-TfR/BACE1 bispecific variants) was used
as a standard to quantify respective antibody concentrations.
Plates were washed with PBS, 0.05% Tween-20 using a microplate
washer (Bio-Tek Instruments, Inc., Winooski, Vt.), and standards
and samples diluted in PBS containing 0.5% BSA, 0.35 M NaCl, 0.25%
CHAPS, 5 mM EDTA, 0.05% Tween-20 and 15 ppm Proclin.RTM.
(Sigma-Aldrich) were added for two hours at 25.degree. C. Bound
antibody was detected with horseradish peroxidase-conjugated
F(ab').sub.2 goat anti-human IgG, an Fc specific polyclonal
antibody (Jackson ImmunoResearch). Samples were developed using
3,3',5,5'-tetramethyl benzidine (TMB) (KPL, Inc., Gaithersburg,
Md.) and absorbance measured at 450 nm on a Multiskan Ascent reader
(Thermo Scientific, Hudson, N.H.). Concentrations were determined
from the standard curve using a four-parameter non-linear
regression program. The assay had lower limit of quantification
(LLOQ) values of 3.12 ng/ml in serum and 12.81 ng/g in brain.
Statistical analysis of differences between experimental groups was
performed using a two-tailed unpaired t-test.
[0282] The results are shown in FIGS. 1B and 1D. Both the control
IgG and the anti-BACE1 antibody had limited uptake into the brain
that persisted over the 10-day measurement period, while their
plasma concentrations were the highest of any of the tested
molecules at all time points, despite gradual clearance over time.
Of the three anti-TfR/BACE1 variants assessed, anti-TfR.sup.A/BACE1
and anti-TfR.sup.D/BACE1 both showed between 35 and 40 nM
concentrations in the brain at 1 day post-dose (7-8-fold greater
than the control IgG; FIG. 1D). However, the concentration of
anti-TfR.sup.A/BACE1 in the brain decreased rapidly after day 2 and
returned to control levels by day 6. Anti-TfR.sup.D/BACE1 persisted
longer in the brain than anti-TfR.sup.A/BACE1, with a more gradual
decline in brain concentrations; however, by day 10 the
concentration matched that of the control. Anti-TfR.sup.E/BACE1 had
a much more moderate entry into the brain (2-3-fold control), but
the decline over subsequent days was much less than that of the
other two antibody variants. Plasma levels of all three antibody
variants (FIG. 1B) declined over time. Anti-TfR.sup.A/BACE1 was
completely cleared from the plasma by day 4, while
anti-TfR.sup.D/BACE1 was fully cleared by day 10, and
anti-TfR.sup.E/BACE1 still persisted in the plasma at a level
comparable to that of the control IgG or anti-BACE1.
[0283] Taken together, these findings were consistent with the
previous discovery that a reduction in the affinity of an antibody
for TFR actually improves its retention in the brain, since the
highest affinity antibody used (anti-TfR.sup.A/BACE1) was the most
rapidly cleared from the brain and the lowest affinity antibody
used (anti-TfR.sup.E/BACE1) persisted the longest in the brain.
However, it was also clear from the data that the total amount of
anti-TfR.sup.D/BACE1 that was transported into the brain over time
was much greater than that of anti-TfR.sup.E/BACE1, suggesting that
there is an optimum affinity between anti-TfR.sup.D/BACE1 and
anti-TfR.sup.E/BACE1 to maximize both transport across the BBB and
persistence in the brain.
[0284] The presence and persistence of the transported molecule in
the brain and plasma is only one measure of potential efficacy; of
further interest is the activity of the molecule in those
compartments. Accordingly, the BACE1 enzyme activity was assessed
in both compartments by measuring the amount of A.beta..sub.1-40 (a
cleavage byproduct of BACE1 enzymatic activity on amyloid precursor
protein (APP)). Briefly, antibody treatment and perfusions were
performed in wild type mice as stated above. For A.beta..sub.1-40
measurements, hemi-brains were homogenized in 5M guanidine
hydrochloride buffer and samples rotated for 3 hours at room
temperature prior to diluting (1:10) in 0.25% casein, 5 mM EDTA (pH
8.0) in PBS containing freshly added aprotinin (20 mg/mL) and
leupeptin (10 mg/mL). Diluted homogenates were spun at 14,000 rpm
for 20 min. and supernatants were isolated for A.beta..sub.1-40
measurement. Plasma was prepared as described above. The
concentrations of total mouse A.beta..sub.1-40 in plasma and brain
were determined using a sandwich ELISA following similar procedures
described above. Hemi-brains for A.beta..sub.1-40 measurement were
homogenized in 1% NP-40 (Cal-Biochem) and rotated for 1 hour at
room temperature prior to spinning at 14,000 rpm for 20 minutes.
Rabbit polyclonal antibody specific for the C-terminus of
A.beta..sub.1-40 (Millipore, Bedford, Mass.) was coated onto
plates, and biotinylated anti-mouse A.beta. monoclonal antibody
M3.2 (Covance, Dedham, Mass.) was used for detection. The assay had
LLOQ values of 1.96 pg/ml in plasma and 39.1 pg/g in brain.
Statistical analysis of differences between experimental groups was
performed using a two-tailed unpaired t-test.
[0285] The results for plasma and brain are shown in FIGS. 1C and
1E, respectively, and are consistent with the amount of antibody
present in each compartment at the indicated time (see FIGS. 1B and
1D). Importantly, the amount of A.beta..sub.1-40 observed in the
brain over time was lowest over the longest period in the mice
treated with anti-TfR.sup.D/BACE1.
Example 2A
Effect of Anti-TfR Dosing on Reticulocytes
[0286] Unexpectedly, upon treatment of mice with monospecific
anti-TfR.sup.A or anti-TfR.sup.D at all dose levels of 1 mg/kg or
higher, unusual and acute clinical signs were observed that were
not observed in mice treated with bispecific anti-TfR.sup.A/BACE1
or anti-TfR.sup.D/BACE1 (see Table 3).
TABLE-US-00003 TABLE 3 SYMPTOMS OBSERVED IN MICE AFTER ANTIBODY
ADMINISTRATION Antibody Dose (mg/kg) Acute Clinical Signs Control
IgG 50* None (isotype matched) Anti-TfR.sup.D 0.01* None
(comprising effector 0.1 function) 1 Profound post-dose lethargy
within 5 minutes 5 Occasional spastic movements in few animals 25
Scruffy, hunched appearance by 20-25 minutes post-dose 50 Red urine
observed from some mice Completely reversible within hours
Anti-TfR.sup.D/Bace 1* None (not comprising 5* effector function)
25 50 200 *No reticulocyte decreases observed at these dose
levels
Specifically, the monospecific-treated mice displayed post-dose
lethargy within 5 minutes of the treatment, where they became
immobile and non-responsive (with occasional spastic movements in
some animals), followed by development of a scruffy, hunched
appearance by 20-25 minutes post-dose. All such observed effects
vanished within hours after the treatment. Certain monospecific
antibody-treated mice also appeared to present with occasional
presence of blood in the urine, as well as apparent hypotension at
1 hour post-dose based on difficult with terminal cardiac blood
collection compared to collection in bispecific-treated animals.
Because mouse immature red blood cells are known to express TfR
(see FIG. 2A), to exist in the peripheral bloodstream, and the
observed effects in mice may be explained if such blood cells were
injured, the impact of the antibody treatment on immature red blood
cells (reticulocytes) was investigated in mice.
[0287] Mice were dosed intravenously with a single 1 mg/kg, 5
mg/kg, or 50 mg/kg anti-TfR.sup.D or anti-TfR.sup.D/BACE1
injection, or with a single 50 mg/kg control IgG injection using
the same procedure as described in Example 1, and whole blood
samples were taken at 1 hour post-dose and placed into
potassium-EDTA-containing collection tubes. Red cell and
reticulocyte counts and indices were determined on these blood
samples using the Sysmex XT2000iV (Sysmex, Kobe, Japan) according
to the manufacturer's instructions. Briefly, the Sysmex detects and
classifies total reticulocytes as well as the immature reticulocyte
fraction (sum of high and middle/intermediate fluorescent
reticulocytes) by flow cytometry using a fluorescent polymethine
dye to bind cellular RNA and measure the resulting cell light
scatter characteristics.
[0288] At 1 hour post-dose, anti-TfR.sup.D reduced immature
reticulocyte levels at all dose levels tested, to approximately the
same extent regardless of dose. Treated mice in each anti-TfR.sup.D
dosage group also showed acute clinical signs of similar severity
and penetrance (see FIG. 2B). In contrast, blood samples from the 1
mg/kg and 5 mg/kg anti-TfR.sup.D/BACE1-treated mice had similar
fractions of immature reticulocytes as those from the
control-IgG-treated samples. The 50 mg/kg
anti-TfR.sup.D/BACE1-treated mice showed a marked reduction in
reticulocytes (to about 50% of control amounts) (FIG. 2B), but this
reduction was not accompanied by any acute clinical signs. Thus,
the bispecific anti-TfR.sup.D-containing antibody had a lesser
impact on reticulocyte levels than monospecific anti-TfR.sup.D, and
did not elicit acute adverse clinical signs.
[0289] The experiment was repeated, further including a second
bispecific antibody of a different affinity for TfR. Mice were
dosed intravenously with a single 5 mg/kg, 25 mg/kg or 50 mg/kg
anti-TfR.sup.A/BACE1 or anti-TfR.sup.D/BACE1 injection, or with a
single 50 mg/kg control IgG injection using the same procedure as
described in Example 1, and blood samples were taken at 24 hours
and 7 days post-dose. Reticulocyte counts were measured in whole
blood as described above. The results are shown in FIG. 2C. At 24
hours post-dose, all of the anti-TfR.sup.A/BACE1-treated mouse
samples showed similar marked reductions in total reticulocyte
count. The 25 mg/kg and 50 mg/kg anti-TfR.sup.D/BACE1-treated
samples showed similarly low reticulocyte counts as the
anti-TfR.sup.A/BACE1-treated samples. However, the 5 mg/kg
anti-TfR.sup.D/BACE1-treated samples showed only a modest reduction
in reticulocyte numbers relative to the IgG control sample at 24
hours post-dose. By 7 days post-dose, all groups showed normal
levels of reticulocytes (FIG. 2C) suggesting recovery from the
initial reticulocyte depletion, with the exception of the 50 mg/kg
anti-TfR.sup.D/BACE1 sample, which showed a sustained reduction in
reticulocyte levels (approximately 50%) relative to the control
amounts. Thus, only the lowest tested dose of anti-TfR.sup.D/BACE1
had a moderate impact on reticulocytes, while all other tested
doses led to an almost complete loss of reticulocytes at 24 hours
post-dose, indicating that reducing antibody affinity
(anti-TfR.sup.D relative to anti-TfR.sup.A) and dose attenuates
safety concerns related to reticulocyte loss. By 7 days post-dose,
however, only the highest dose of anti-TfR.sup.D/BACE1 had any
measurable impact on reticulocyte levels, whereas all other doses
tested showed a recovery of reticulocyte counts to levels similar
to those of the IgG control mice. Notably, the absolute affinity of
the antibody for TfR at 7 days post-dose was not as important as
the persistence of the antibody in the bloodstream for the longer
timepoints. Despite the much higher affinity of
anti-TfR.sup.A/BACE1 for TfR (Table A), mice treated with high-dose
anti-TfR.sup.A/BACE1 showed a recovery of reticulocyte numbers by 7
days that corresponded with the faster clearance of this antibody
from circulation relative to anti-TfR.sup.D/BACE1 (as seen in
Example 1, FIG. 1B).
[0290] Since a dose response was observed in reticulocyte
depletion, experiments were performed to determine whether it was
possible to correlate various dose levels with an associated
ability to reduce Abeta in brain. Briefly, wild type female C57B/6
mice ages 6-8 weeks were used for all studies. Mice were
intravenously injected with 50 mg/kg of either control IgG, or
anti-TfR/BACE1. Total injection volume did not exceed 250 .mu.L and
antibodies were diluted in D-PBS (Invitrogen) when necessary. After
the indicated time, mice were perfused with D-PBS at a rate of 2
mL/min for 8 minutes. Brains were extracted and the cortex and
hippocampus was isolated, homogenized in 1% NP-40 (Cal-Biochem) in
PBS containing Complete Mini EDTA-free protease inhibitor cocktail
tablets (Roche Doagnostics). Homogenized brain samples were rotated
at 4.degree. C. for 1 hour before spinning at 14,000 rpm for 20
minutes. The supernatant was isolated for brain antibody
measurement. Whole blood was collected prior to perfusion in EDTA
microtainer tubes (BD Diagnostics), allowed to site for 30 minutes
at room temperature, and spun down at 5000.times.g for 10 minutes.
The top layer of plasma was transferred to new tubes for antibody
and mouse Abeta.sub.1-40 measurements.
[0291] Total antibody concentrations in mouse plasma and brain
samples were measurements using an anti-Fc/anti-huFc ELISA. NUNC
384 well Maxisorp immunoplates (Neptune, N.J.) were coated with
F(ab').sub.2 fragment of donkey anti-human IgG, Fc fragment
specific polyclonal antibody (Jackson ImmunoResearch, West Grove,
Pa.) overnight at 4.degree. C. Plates were blocked with PBS, 0.5%
BSA for 1 hour at 25.degree. C. Each antibody was used as a
standard to quantify respective antibody concentrations. Plates
were washed with PBS, 0.05% Tween-20 using a microplate washer
(Bio-Tek Instruments Inc., Winooski, Vt.), standards and samples
filuted in PBS containing 0.5% BSA, 0.35M NaCl, 0.25% CHAPS, 5 mM
EDTA, 0.05% Tween-20 and 15 ppm Proclin were added for two hours at
25.degree. C. Bound antibody was detected with horseradish
peroxidase-conjugated F(ab').sub.2 goat anti-human IgG, Fc specific
polyclonal antibody (Jackson ImmunoResearch), developed using
3,3',5,5'-tetramethyl benzidine (TMB) (KPL, Inc., Gaithersburg,
Md.) and absorbance measured at 450 nm on a Multiskan Ascent reader
(Thermo Scientific, Hudson, N.H.). Concentrations were determined
from the standard curve using a four-parameter non-linear
regression program. The assay had a lower limit of quantification
(LLOQ) values of 3.12 ng/ml in serum and 12.81 ng/g in brain.
Statistical analysis of differences between experimental groups was
performed using two-tailed unpaired t-test.
[0292] Abeta.sub.1-40 was also detected in brain and plasma.
Briefly, mice were treated with antibody and perfused according to
the method described above. For Abeta.sub.1-40 measurements,
hemi-brains were homogenized in 5 M guanidine hydrochloride buffer
and samples rotated for 3 hours at room temperature prior to
diluting (1:10) in 0.25% casein, 5 mM EDTA (pH 8.0) in PBS
containing freshly added aprotinin (20 mg/mL) and leupeptin (10
mg/ml). Diluted homogenates were spun at 14,000 rpm for 20 min and
supernatants were isolated for Abeta.sub.1-40 measurement. Plasma
was prepared as described above. The concentrations of total mouse
Abeta.sub.1-40 in plasma and brain were determined using a sandwich
ELISA following similar procedures described above. Rabbit
polyclonal antibody specific for the C-terminus of Abeta.sub.1-40
(Millipore, Bedford, Mass.) was coated onto plates, and
biotinylated anti-mouse Abeta monoclonal antibody M3.2 (Covance,
Dedham, Mass.) was used for detection. The assay had LLOQ values of
1.96 pg/ml in plasma and 39.1 pg/g in brain. Statistical analysis
of differences between experimental groups was performed using
two-tailed unpaired t-test.
[0293] A robust and sustained reduction in brain Abeta at both 25
and 50 mg/kg dose levels for anti-TfR.sup.D/BACE1 was observed
(FIG. 2D), while anti-TfR.sup.A/BACE1 showed a robust, but acute
reduction in brain Abeta at all three dose levels (FIG. 2E). These
data were consistent with the observed pharmacokinetics of the
compounds in both the periphery and the brain (FIGS. 2F-2H). From
these data, it was apparent that a dosage level of 25 mg/kg of
anti-TfR.sup.D/BACE1 is sufficient to significantly reduce brain
Abeta levels in these studies.
[0294] Reticulocyte depletion by anti-TfR antibody species could be
due to a variety of different natural processes, including effector
function/antibody-dependent cell-mediated cytotoxicity (ADCC),
complement-dependent cytotoxicity (CDC), direct target-mediated
lysis/apoptosis, and/or phagocytosis of opsonized reticulocytes by
macrophages. A series of experiments was undertaken to better
understand the mechanisms responsible for the observed reticulocyte
depletion following anti-TfR antibody administration.
Example 2B
Impact of Modulating Effector Function
[0295] In addition to differing in affinity and valency for TfR,
the monospecific and bispecific anti-TfR antibodies used the
preceding experiments also differed in the degree of their effector
functions. The monospecific anti-TfR antibodies were produced in
CHO cells, and had mammalian-type glycosylation and wild-type
effector function. The bispecific anti-TfR/BACE1 antibodies had a
severely reduced or eliminated capacity to interact with
Fc.gamma.-receptors using one or more of the following methods
well-known in the art: abrogating glycosylation due to the presence
of the mutation N297G or N297A in the Fc region (Atwal et al., Sci.
Transl. Med. 3, 84ra43 (2011); Fares Al-Ejeh et al., Clin. Cancer
Res. (2007) 13:5519s-5527s), modifying the antibody Fc region to
contain an aspartic acid to alanine mutation at position 265
(D265A) known to completely abrogate effector function (see, e.g.,
U.S. Pat. No. 7,332,581), or producing the antibody in a manner
that prevented wild-type mammalian glycosylation, such as by
producing it in E. coli.
[0296] The mouse studies performed in Example 2A were repeated with
these Fc-modified antibodies and also in different mouse strains
lacking either Fc.gamma. receptors or complement C3, to evaluate
potential mechanisms of reticulocyte depletion including
effector-driven ADCC or CDC respectively; whole blood samples were
assessed for total reticulocyte counts 24 hours after intravenous
injection of the antibody. In a first experiment, administration of
monospecific 1 mg/kg or 25 mg/kg anti-TfR.sup.D lacking effector
function to wild-type mice had the same depletive effect on
reticulocyte counts as an anti-TfR.sup.D antibody with full
effector function (compare FIG. 3A with FIG. 2B). However, acute
clinical signs were not observed in mice treated with the
effectorless anti-TfR.sup.D antibody, in sharp contrast to those
treated with an effector-positive anti-TfR.sup.D antibody (Example
2A). Similarly, when effector-positive anti-TfR.sup.D was
administered to mice lacking Fc.gamma. receptor (to eliminate ADCC
mechanisms that may be triggered by effector function),
reticulocyte levels were reduced to near zero following a dose of
25 mg/kg, but no acute clinical signs were observed (FIG. 3B).
[0297] The impact of the bispecific anti-TfR.sup.D/BACE1 D265A
antibodies lacking effector function on reticulocyte levels was
also assessed in the Fc.gamma. knockout mice (FIG. 3B). Even the
full abrogation of antibody effector function and the absence of
the Fc.gamma. receptor in the mice did not mitigate reticulocyte
depletion when administered at a dose level of 25 mg/kg. Consistent
with other experiments using bispecific anti-TfR/BACE1 effectorless
antibodies in wild-type mice, adverse clinical signs were not
observed in treated Fc.gamma. knockout mice.
[0298] To determine whether the presence of effector function is
sufficient to drive acute clinical symptoms, and to further
characterize the contribution of effector function to reticulocyte
depletion, the experiments were repeated in wild-type mice
comparing a low dose (5 mg/kg) of effectorless anti-TfR.sup.D/BACE1
D265A with an equivalent dose of full effector-positive
anti-TfR.sup.D/BACE1 (FIG. 3C). Acute clinical signs were observed
upon introduction of effector function into the bispecific
antibody. Furthermore, robust reticulocyte depletion was observed
with the effector-positive antibodies at a lower dose level
relative to the effectorless version of the antibody (FIGS. 3C and
2C). From this combined data, effector function is not necessary to
drive reticulocyte depletion, but clearly contributes to this
depletion, particularly at lower dose levels. Importantly, the
acute clinical symptoms observed in mice are linked to the effector
status of the antibody, such that effectorless antibodies or
Fc.gamma.-knockout mice both completely mitigate these
symptoms.
[0299] To determine whether the complement cascade was involved in
either the clinical symptoms or the loss of reticulocytes, the
experiments were performed again in mice deficient in complement C3
(ie, mice lacking the normal complement cascade). As shown in FIG.
3D, effector-positive anti-TfR.sup.A caused both profound
reticulocyte depletion and robust acute clinical symptoms in these
mice, indicating that complement C3 and the associated complement
cascade do not play a major role in driving either of the observed
effects when the administered antibody possesses full effector
function. To test whether the same result would be obtained in the
absence of full effector function, C3 knockout mice were dosed with
effectorless anti-TfRD/BACE1 antibodies to determine if complement
mediates the residual reticulocyte depletion. The results are shown
in FIG. 3E. Indeed, residual reticulocyte depletion is rescued when
both effector function and the complement cascade are eliminated by
dosing C3 knockout mice with effectorless anti-TfR bispecific
antibodies at high therapeutic dose levels (50 mg/kg). Thus,
complement appears to act as a mechanism of reticulocyte depletion
following administration of effectorless anti-TfR antibodies in
mice.
[0300] An in vitro complement-dependent cytotoxicity (CDC) assay
was also performed.
[0301] Briefly, CDC assays were performed using primary mouse bone
marrow cells or mouse erythroleukemic lymphoblasts (HPA Cultures,
UK) as target cells and complement derived from rabbit serum (EMD
Chemicals, Gibbstown N.J.). Cells were counted and viability
determined by Vi-Cell.TM. (Beckman Coulter, Fullerton, Calif.).
Anti-TfR.sup.A/BACE1, anti-TfR.sup.A or negative or positive
control antibody (IgG or anti-H2Kb, respectively) were serially
diluted 1:4 in assay medium (RPMI-1640 medium supplemented with 20
mM HEPES, pH 7.2 and 1% FBS), and distributed into a white,
flat-bottom 96-well tissue culture plate (Costar; Corning, Acton
Mass.). Following the addition of serum complement diluted 1:3 in
assay medium and the target cells (2.times.10.sup.5 cells/well),
the plate was incubated with 5% CO.sub.2 for 2 hours at 37.degree.
C. The plates were then left at room temperature for 10 minutes
with constant shaking. The extent of cell lysis was quantified by
measuring luminescence intensity with a SpectroMax.TM. M5 plate
reader. Luminescence values of sample dilutions were plotted
against the antibody concentration, and the dose-response curves
were fitted to a four-parameter model using GraphPad.TM. (GraphPad
Software Inc.).
[0302] Interestingly, neither monospecific
effector-function-competent anti-TfR.sup.A nor effectorless
bispecific anti-TfR.sup.A/BACE1 treatment of mouse cells in the
presence of serum complement resulted in complement-mediated lysis
of the cells, while the anti-H2Kb positive control showed
significant cell lysis (FIG. 4A). Notably, the differing effector
activity of the antibodies did not appear to influence their
ability to elicit CDC activity. One nonlimiting explanation is that
complement may mediate reticulocyte depletion in vivo via
opsonization of circulating reticulocytes by splenic and liver
macrophages (Garratty (2008), Transfusion Med. 18(6): 321-334;
Mantovani et al, (1972) J. Exp. Med. 135: 780-792; Molina et al.,
(2002) Blood 100 (13): 4544-4549), a mechanism that must be intact
with anti-TfR F(ab').sub.2 fragments.
[0303] Similar in vitro experiments were also undertaken to confirm
the previously-described in vivo results supporting a link between
effector function-mediated antibody-dependent cell-mediated
cytotoxicity (ADCC), acute clinical symptoms, and reticulocyte
depletion. ADCC assays were carried out using freshly isolated
PBMCs from healthy donors as effector cells, and primary mouse bone
marrow cells or mouse erythroleukemic lymphoblasts (HPA Cultures,
UK) as target cells. To minimize donor variations derived from
allotypic differences at residue 158 position of Fc.gamma.RIIIA,
blood donors were limited to those carrying the heterozygous
Fc.gamma.RIIIA genotype (F/V158). Briefly, PBMCs were isolated by
density gradient centrifugation using a Uni-Sep blood separation
tube (Accurate Chemical & Scientific; Westbury, N.Y.). Target
cells were prelabled with 1.4 mM solution of calcein AM (Molecule
Probes) and were seeded in a 96-well, round-bottom plate (BD
Biosciences; Mississauga, Ontario; Canada) at
4.times.10.sup.4/well. Serial dilutions of anti-TfR/BACE1, anti-TfR
and control antibody were added to the plates containing the target
cells, followed by incubation at 37.degree. C. with 5% carbon
dioxide for 30 minutes to allow opsonization. The final
concentrations of antibodies ranged from 1,000 to 0.004 ng/mL
following 4-fold serial dilutions. After the incubation,
1.times.10.sup.6 PBMC effector cells in 100 .mu.L assay medium were
added to each well to give a ratio of 25:1 effector to target
cells, and the plates were incubated for an additional 3 hours. The
plates were centrifuged at the end of incubation, and fluorescent
signals in supernatants were measured using a SpectraMax.TM. M5
microplate reader, with excitation at 485 nm and emission at 520
nm. Signals of wells containing only the target cells represented
spontaneous release of the calcein AM from labeled cells
(spontaneous release), whereas wells containing target cells lysed
with Triton.TM. X-100 provided the maximum signal available
(maximum lysis). Antibody-independent cellular cytotoxicity (AICC)
was measured in wells containing target and effector cells without
the addition of antibody. The extent of specific ADCC was
calculated as follows:
%ADCC=100.times.(Sample signal-AICC)/(maximum lysis-spontaneous
release)
The ADCC values of sample dilutions were plotted against the
antibody concentration and the dose response curve fitted with a
four-parameter model using GraphPad.TM. (GraphPad Software
Inc.).
[0304] The anti-TfR.sup.A used in this assay had effector function,
while the anti-TfR.sup.A/BACE1 used in the assay had no effector
function. As shown in FIG. 4B, the antibody with effector function
induced ADCC while the anti-TfR.sup.A/BACE1 antibody lacking
effector function did not, correlating with the prior mouse
experiment results. These data further support the idea that acute
clinical signs in treated mice are due to ADCC actively elicited by
the effector--positive antibodies binding circulating
reticulocytes, and that effector-driven ADCC can also contribute to
reticulocyte depletion following antibody administration (FIG.
3C).
Example 2C
Impact of Modulating Fc or BACE1 Binding
[0305] The role of the Fc arm and the BACE1 arm were each
separately examined for their potential involvement in mediating
reticulocyte depletion. Monospecific and bispecific anti-TfR with
wild-type IgG1 Fc regions having full effector function and normal
glycosylation were generated. Briefly, TfR (hole) and IgG (knob)
half antibodies were expressed separately in CHO and annealed in
vitro as described (Carter, P. (2001) J. Immunol. Methods 248,
7-15; Ridgway, J. B., Presta, L. G., and Carter, P. (1996) Protein
Eng. 9, 617-621; Merchant, A. M., Zhu, Z., Yuan, J. Q., Goddard,
A., Adams, C. W., Presta, L. G., and Carter, P. (1998) Nat.
Biotechnol. 16, 677-681; Atwell, S., Ridgway, J. B., Wells, J. A.,
and Carter, P. (1997) J. Mol. Biol. 270, 26-35). F(ab').sub.2
fragments were generated from anti-TfR IgG, anti-TfR/IgG or
anti-TfR/BACE1 antibodies by digestion with immobilized pepsin. The
antibody was reconstituted in 100 mM sodium acetate, pH 4.2 and
incubated with immobilized pepsin resin (0.3 mL settled gel/mg IgG)
overnight at 37.degree. C. with rotation. After incubation, the
sample was centrifuged to separate the immobilized pepsin from the
F(ab').sub.2-digested mixture. The F(ab').sub.2 fragment was then
purified using an SP sepharose, strong cation-exchange resin (1 mL
HiTrap.TM. column (Supelco)). The sample was loaded in 50 mM NaOAc
pH 5.0 and eluted with a 0-0.5 M NaCl gradient over 20 column
volumes after which the sample was dialyzed against PBS, pH 7.4.
Mouse experiments were performed with these antibodies and
F(ab').sub.2 using the same procedures as above and an intravenous
25 mg/kg dose of monospecific F(ab').sub.2 or an intravenous 50
mg/kg dose of bispecific or control F(ab').sub.2 or antibody; whole
blood samples were assessed for total reticulocyte counts 24 hours
after intravenous injection of the antibody/F(ab').sub.2. The
results are shown in FIGS. 5A-5C.
[0306] Administration of the anti-TfR.sup.D F(ab').sub.2 had a very
similar reticulocyte depleting effect to administration of
anti-TfR.sup.D antibody (compare FIG. 5A to FIGS. 3A and 3B),
indicating that the Fc portion of the antibody is not necessary for
the observed reticulocyte depletion at the dose levels evaluated.
Although bispecific F(ab').sub.2 molecules showed a slight
attenuation of reticulocyte depletion relative to full-length
bispecific IgG antibodies (compare FIG. 5B to FIG. 2C), it should
be noted that this is most likely due to the general faster
clearance of F(ab').sub.2 relative to IgG (Covell et al., (1986)
Cancer Res. 46:3969-3978), leading to overall reduced antibody
exposure over the 24 hour post-dose interval. Nonetheless, the
reticulocyte depletion observed following administration of
bispecific F(ab').sub.2 antibodies further underscores the
conclusion that the Fc region is not necessary for reticulocyte
depletion to occur. Bispecific antibodies lacking the BACE1 arm
(anti-TfR.sup.D/control IgG) depleted reticulocytes to the same
degree as anti-TfR.sup.D/BACE1 (FIG. 5C), demonstrating that the
BACE1 arm also does not contribute to reticulocyte elimination.
Example 3
Further Engineering Binding Affinity
[0307] Certain of the above results suggested that there was an
affinity and dose component to the observed degree of reticulocyte
depletion (FIG. 2C). To better understand how affinity and dose
impact reticulocyte depletion, the mouse dosing experiments
performed in Example 2 were repeated with additional lower-affinity
anti-TfR antibodies, specifically anti-TfR.sup.E/BACE1 at two
different dose levels (25 mg/kg and 50 mg/kg). Anti-TfR.sup.E at
either of the tested doses had essentially no impact on
reticulocytes (FIG. 6A), while similar doses of
anti-TfR.sup.A/BACE1 or anti-TfR.sup.D/BACE1 depleted
reticulocytes. From the results discussed in Example 1 it had been
observed that anti-TfR.sup.E/BACE1 had better sustained plasma
exposure and persistence in the brain, but less robust transport
across the blood-brain barrier than anti-TfR.sup.D/BACE1. Given
that the anti-TfR.sup.D/BACE1 administration resulted in
reticulocyte depletion but anti-TfR.sup.E/BACE1 administration did
not, variant anti-TfRs with affinities between that of
anti-TfR.sup.D and anti-TfR.sup.E for TfR were generated to see if
the safety profile of the antibody could be improved without
sacrificing BBB transport and persistence in brain. Briefly,
site-directed mutagenesis was employed to combine the two point
mutations representing the anti-TfR.sup.D and anti-TfR.sup.B
variants respectively into a single antibody designated
anti-TfR.sup.Db using standard mutagenesis techniques. Similarly,
the two point mutations representing the anti-TfR.sup.D and
anti-TfR.sup.c variants respectively into a single antibody
designated anti-TfR.sup.Dc. Both antibodies were made into a
bispecific format with anti-BACE1 using knob and hole technology as
described in Example 2C. The affinities of both antibodies were
between those of the anti-TfR.sup.D and anti-TfR.sup.E antibodies
for TfR, and anti-TfR.sup.Db/BACE1 antibody had approximately
three-fold greater affinity for TfR than did the
anti-TfR.sup.D/BACE1 antibody. The mouse
administration/reticulocyte depletion experiment was repeated with
these new variants, and the results are shown in FIG. 6B. Both
variants demonstrated markedly improved (ie, less) reticulocyte
depletion than that observed with the anti-TfR.sup.D/BACE1 antibody
at the same dose level, and reticulocyte levels approximated those
of control-treated mice at 24 hours post-dose. As expected, the
plasma antibody concentration of both new variant antibodies over
time, the brain antibody concentration (both the maximum value and
the decrease over time), and the reduction in A.beta..sub.1-40 was
between that of anti-TfR.sup.D/BACE1 and anti-TfR.sup.E/BACE1 when
administered at the same dose level.
[0308] The impact of affinity and dose on expression of TfR at the
blood-brain barrier was also examined. Mice were treated with a
single dose of anti-TfR.sup.A/BACE1 or anti-TfR.sup.D/BACE1 at 5,
25 or 50 mg/kg, and TfR expression in brain was evaluated at 4 days
post-dose via Western blot. Brains from antibody-treated mice were
PBS perfused before extraction, and isolated cortex and hippocampus
were homogenized in 1% NP-40 (Calbiochem) in PBS containing
Complete Mini EDTA-free protease inhibitor tables (Roche
Diagnostics). Homogenized brains were rotated at 4.degree. C. for 1
hour before spinning at 14,000 rpm for 20 minutes. Supernatant was
isolated and equal concentrations of protein were separated by
4-12% Novex Bis-Tris gels (Invitrogen). Membranes were incubated
with anti-TfR (Invitrogen) and anti-actin (Abcam) antibodies
overnight at 4.degree. C. followed by IRDye.RTM. (Li-Cor
Biosciences) secondary antibodies at room temperature for 2 hours.
Immunoblots were imaged and bands were quantified by densitometry
using Odyssey Infrared Imaging System.TM. software (Li-Cor
Biosciences, Lincoln, Nebr.). Four days post-dose, TfR expression
in all three of the anti-TfR.sup.D/BACE1 treated samples was
similar, although slightly depressed from control levels at higher
dose levels (FIG. 6C). In contrast, increasing doses of
anti-TfR.sup.A/BACE1 antibody resulted in a marked decrease in
expression of TfR at the blood-brain barrier 4 days post-dose.
Thus, reducing the affinity of the anti-TfR antibody also improves
the observed dose-dependent reduction in brain TfR expression,
potentially further contributing to improvement in the overall
safety profile of the antibody.
Example 4
Assessment of BBB Permeability
[0309] A concern of exploiting a blood-brain barrier transport
receptor for transport of heterologous molecules into the brain is
that the BBB itself might be impaired. Accordingly, the
permeability of the BBB to antibodies upon dosing with anti-TfR was
investigated. Wild-type mice were intravenously administered 50
mg/kg of control IgG, or 25 mg/kg of each of the indicated
co-injected antibody combinations. Mean antibody uptake in brain 24
hours after intravenous injection was assessed using a generic
human-Fc ELISA according to Example 1 or using an anti-BACE1
specific ELISA following similar procedures to those described in
Example 1. The BACE1 extracellular domain was used as the coat
protein and detection was performed with horseradish
peroxidase-conjugated F(ab').sub.2 goat anti-human IgG, Fc-specific
polyclonal antibody. This assay had LLOQ values of approximately
2.56 ng/g for anti-BACE1 and 12.8 ng/g for anti-TfR.sup.D/BACE1.
Brain A.beta..sub.1-40 levels were measured after administration
using the same procedure set forth in Example 1.
[0310] The results are shown in FIGS. 7A-7C. Brain antibody
exposure was highest in the control IgG+
anti-TfR.sup.D/BACE1-treated mice, but also substantial in the mice
treated with anti-TfR.sup.D-containing antibody combinations (FIG.
7A). This correlates with the results in Example 1 in that the
lower-affinity bispecific form of anti-TfR.sup.D is taken up and
persists in the brain longer than the higher-affinity monospecific
form of anti-TfR.sup.D. Antibodies coadministered with the anti-TfR
antibody were not taken up into the brain in substantial
quantities; the only anti-BACE1 observed in substantial quantity in
the brain was that directly conjugated to anti-TfR.sup.D (FIG. 7B).
Similarly, the only anti-BACE1 activity observed in the brain was
in the anti-TfR.sup.D/BACE1-treated mice (FIG. 7C). Taken together,
these data indicate that the blood-brain barrier permeability to
antibodies was not affected by anti-TfR treatment.
Example 5
Impact of Multiple Dosing on Reticulocyte Levels
[0311] The foregoing studies focused on a single dose of anti-TfR
antibody and the resulting impact on reticulocyte levels and
concomitant acute clinical symptoms. To ascertain whether different
effects were observed following multiple doses over a longer time
period, further studies were undertaken. The same protocols as
described in the preceding examples were used, with the exception
that instead of a single intravenous dose, mice were dosed
intravenously once per week with 25 mg/kg anti-TfR.sup.D/BACE1 or
an IgG control, for a total of four weeks. Tissue/blood was
collected at 1, 4 or 7 days post the second injection and post the
fourth injection, and processed using the above-described
protocols. In addition, direct bilirubin, serum iron, and total and
unsaturated iron binding capacity were determined for serum samples
by colorimetric assays using the Integra.TM. 400 (Roche,
Indianapolis, Ind.) according to the manufacturer's instructions.
Six mice were used for each time point and treatment group.
[0312] The serum antibody concentration for anti-TfR.sup.D/BACE1
was similar over time after 2 or 4 doses, suggesting that clearance
in the mouse bloodstream does not substantially differ after
repeated dosing (FIG. 8A). However, a slight decrease in overall
antibody exposure was apparent 4 days after the fourth dose
relative to the same time after the second dose, suggestive of the
occurrence of mouse anti-drug antibodies (ADAs) to the administered
human IgG antibodies. Similar to the serum antibody concentrations,
brain antibody concentrations were decreased by 4 days after the
fourth dose, although the persistence of the antibodies present in
the brain over time mirrored that observed after the second dose
(FIG. 8B). Plasma (FIG. 8C) and brain (FIG. 8D) levels of Abeta1-40
correlated well with the observed amounts of anti-TfR.sup.D/BACE1
present in the serum and brain after 2 or 4 doses.
[0313] Importantly, no exacerbation of reticulocyte toxicity was
observed in the multi-dose context. As shown in FIG. 8E, absolute
reticulocyte numbers improved dramatically from 1 day post-second
dose to 7 days post-fourth dose (where the values returned to or
exceeded control levels). There was no evidence of decreased red
cell mass or changes in serum iron and total iron binding capacity
(a surrogate parameter for serum transferrin) at four weeks. There
was also no evidence of histopathology changes or altered stainable
iron levels in any tissues evaluated. Without being bound by
theory, it is proposed that an enhanced bone marrow regenerative
response elicited by the initial dose administration and sustained
throughout the dosing period may be responsible for ameliorating
the overall reticulocyte decrease observed after the fourth dose.
Additionally, the suspected presence of ADAs further reduced
overall circulating antibody levels with repeated dosing, also
contributing to the mitigation of reticulocyte depletion observed
at week 4. Finally, brain expression of TfR did not differ between
anti-TfR.sup.D/BACE1 and control IgG treated mice at 1, 4, or 7
days post the fourth dose (FIG. 8F).
Example 6
Impact of Effector-Containing and Effectorless Bispecifics on
Erythroid Progenitor Cells in Blood and Bone Marrow
[0314] Additional experiments were performed to elucidate the
impact of antibody dosing on erythroid progenitor cell populations
in bone marrow. First, to examine the time course of reticulocyte
loss after anti-TfR/BACE1 dosing, blood and bone marrow were
isolated at 1, 4, 16, and 24 hours after wild-type mice were
intravenously injected with 50 mg/kg of control IgG or
anti-TfR.sup.D/BACE1 lacking effector function as a single bolus in
200 .mu.L in sterile PBS (n=6/group). Blood and bone marrow were
harvested from animals at the indicated time points post-dose.
Orbital bleeds were used for blood extraction after isofluorane
anesthesia, and bone marrow from one femur was harvested and single
cell suspensions were prepared. Cells were then filtered through a
70-micron cell strainer. Cells were washed and resuspended in a set
volume of PBS. A fixed volume of cell suspension was added to a
fixed concentration of FITC-labeled fluorescent beads and analyzed
on a flow cytometer, collecting 5000 bead events per sample to
obtain cell counts. Quantitative analysis of erythroid populations
was determined by flow cytometry. In both blood and bone marrow,
distinct populations of erythroid cells were gated by their
expression of the Ter119 marker (a marker that has been determined
to be expressed only on murine mature erythrocytes and erythroid
precursor cells), TfR expression, and side scatter profile (as
previously described in Paniga et al., "Expression of Prion Protein
in Mouse Erythroid Progenitors and Differentiating Murine
Erythroleukemia Cells." PLoS One 6, 9 (2011); FIGS. 9A and 9B).
Briefly, samples were incubated for 20 minutes on ice with
anti-mouse Ter119-PE (eBioscience) and biotinylated anti-mouse TfR,
followed by streptavidin-eFluor450 (eBioscience). Samples were
washed with PBS containing 0.5% BSA, 2 mM EDTA and run on a BD LSR
Fortessa multi-color flow cytometer and analyzed using FlowJo
software (Ashland, Oreg.).
[0315] Treatment with anti-TfR.sup.D/BACE1 lacking effector
function did not alter the total number of erythrocytes in blood
compared to control IgG (FIG. 9C), but nonetheless rapidly and
significantly reduced circulating TfR-expressing reticulocytes in
the blood (FIG. 9D). In contrast to the findings in blood,
effectorless anti-TfR.sup.D/BACE1 had no effect on any of the
erythroid progenitor populations in bone marrow (FIG. 10A-C),
including high TfR-expressing populations (EryA and EryB
populations) (FIG. 10B-C) and TfR-negative mature erythrocytes
(EryC population) (FIG. 10D). Together, these results demonstrated
that the effectorless anti-TfR.sup.D/BACE1 only depletes
TfR-expressing reticulocytes in blood in mice, without impacting
other subpopulations of erythroid cells in bone marrow after a
single dose.
[0316] To investigate the impact of full effector function
antibodies on erythrocyte subpopulations in both blood and bone
marrow, and to determine whether affinity plays a role in erythroid
cell depletion, wild-type mice were given a single IV dose of 25
mg/kg of anti-TfR.sup.A/BACE1 (Fc-), anti-TfR.sup.D/BACE1 (Fc-),
anti-TfR.sup.D/BACE1 (Fc+), or control IgG (where "Fc-" indicates
an effectorless antibody due to the presence of mutations D265A and
N297G or to lack of glycosylation and "Fc+" indicates an antibody
with wild-type effector function), following the same injection and
sample collection process as above. Neither presence of effector
function nor affinity for TfR affected the total number of mature
erythrocytes in circulating blood after antibody dosing, compared
to control IgG (FIG. 11A). Confirming the previous observation,
dosing with effectorless anti-TfR/BACE1 antibodies resulted in a
rapid and prolonged decrease in TfR-expressing reticulocytes in
blood (FIG. 11B, compare to FIG. 9D). Furthermore, affinity for TfR
did not alter the extent to which the bispecific antibodies drove
reticulocyte loss, as there were no significant differences in the
time course nor magnitude of reticulocyte decrease between animals
dosed with anti-TfR.sup.A/BACE1 (Fc-) or anti-TfR.sup.D/BACE1 (Fc-)
(FIG. 11B). However, dosing with full effector function
anti-TfR.sup.D/BACE1 (Fc+) resulted in a significant exacerbation
of reticulocyte loss, as compared with the effectorless bispecific
antibodies (FIG. 11B), suggesting that effector function plays an
important role in the severity of reticulocyte depletion after
antibody dosing.
[0317] In bone marrow, neither effectorless (Fc-) anti-TfR
bispecific antibody altered the total number of erythroid cells,
compared to control IgG (FIG. 11A). However, full effector function
anti-TfR.sup.D/BACE1 (Fc+) reduced the total number of erythroid
cells at 24 hrs post dose (FIG. 12A). Specifically, TfR positive
erythroid precursor cells (EryA and EryB populations) were
significantly and robustly reduced in the presence of a full
effector function, while effectorless anti-TfR/BACE1 antibodies had
no effect on TfR positive erythroid cell subpopulations compared to
control IgG (FIG. 12B-C) Interestingly, the number of mature
erythrocytes was transiently increased after dosing with full
effector function anti-TfR.sup.D/BACE1 (Fc+) at 4 and 16 hours
post-dose compared to the effectorless anti-TfR/BACE1 (Fc-)
antibodies and control IgG (FIG. 12D). In one nonlimiting
interpretation, this transient increase may be due to a secondary
compensatory mechanism driving accelerated erythrocyte maturation
in response to erythroid precursor cell depletion. Together, these
data suggest that an effectorless anti-TfR/BACE1 antibody mitigates
TfR-positive erythroid cell loss in bone marrow.
Example 7
Impact of Effector-Containing and Effectorless Monospecific and
Bispecific Antibodies on a Human Erythroleukemia Cell Line and
Primary Bone Marrow Mononuclear Cells
[0318] The foregoing examples used anti-murine TfR antibodies,
which do not specifically recognize human TfR. To ascertain whether
the reticulocyte depletion observed in the mouse studies was unique
to a murine system, further experiments were performed utilizing
anti-TfR that bind to human TfR.
[0319] ADCC assays were carried out using peripheral blood
mononuclear cells (PBMCs) from healthy human donors as effector
cells. A human erythroleukemia cell line (HEL, ATCC) and primary
human bone marrow mononuclear cells (AllCells, Inc.) were used as
target cells. To minimize inter-donor variability which could
potentially arise from allotypic differences at the residue 158
position in Fc.gamma.RIIIA, blood donors were limited to those
carrying the heterozygous Rc.gamma.RIIIA genotype (F/V158) in the
first set of experiments (FIG. 13A-B). For the second set of
experiments (FIG. 14A-B), only HEL cells were used as the target
cells, with PBMCs from healthy human donors carrying either the
F/V158 genotype or the Fc.gamma.RIIIA V/V158 genotype. The V/V158
genotype was also included in this assay due to the known
association with increased NK cell-mediated ADCC activity as well
as ability to bind IgG4 antibodies (Bowles and Weiner, 2005; Bruhns
et al. 2008). Cells were counted and viability was determined by
Vi-CELL.RTM. (Beckman Coulter; Fullerton, Calif.) following the
manufacturer's instructions.
[0320] PBMCs were isolated by density gradient centrifugation using
Uni-Sep.TM. blood separation tubes (Accurate Chemical &
Scientific Corp.; Westbury, N.Y.). Target cells in 50 .mu.L of
assay medium (RPMI-1640 with 1% BSA and 100 units/mL penicillin and
streptomycin) were seeded in a 96-well, round-bottom plate at
4.times.10.sup.4/well. Serial dilutions of test and control
antibodies (50 .mu.L/well) were added to the plates containing the
target cells, followed by incubation at 37.degree. C. with 5%
CO.sub.2 for 30 minutes to allow opsonization. The final
concentrations of antibodies ranged from 0.0051 to 10,000 ng/mL
following 5-fold serial dilutions for a total of 10 data points.
After the incubation, 1.0.times.10.sup.6 PBMC effector cells in 100
.mu.L of assay medium were added to each well to give a ratio of
25:1 effector: target cells, and the plates were incubated for an
additional 4 hours. The plates were centrifuged at the end of
incubation and the supernatants were tested for lactate
dehydrogenase (LDH) activity using a Cytotoxicity Detection Kit.TM.
(Roche Applied Science; Indianapolis, Ind.). The LDH reaction
mixture was added to the supernatants and the plates were incubated
at room temperature for 15 minutes with constant shaking. The
reaction was terminated with 1 M H.sub.3PO.sub.4, and absorbance
was measured at 490 nm (the background, measured at 650 nm was
subtracted for each well) using a SpectraMax Plus microplate
reader. Absorbance of wells containing only the target cells served
as the control for the background (low control), whereas wells
containing target cells lysed with Triton-X100 provided the maximum
signal available (high control). Antibody-independent cellular
cytotoxicity (AICC) was measured in wells containing target and
effector cells without the addition of antibody. The extent of
specific ADCC was calculated as follows:
% ADCC = 100 .times. A 490 ( Sample ) - A 490 ( AICC ) A 490 ( High
Control ) - A 490 ( Low Control ) ##EQU00001##
ADCC values of sample dilutions were plotted against the antibody
concentration, and the dose-response curves were fitted to a
four-parameter model using SoftMax Pro.
[0321] In a first set of experiments, the ADCC activity of various
anti-human TfR constructs were assessed using either a human
erythroleukemia cell line (HEL cells) or primary human bone marrow
mononuclear cells as the target cells. Bivalent IgG1 effector
function-competent anti-human TfR1 antibody 15G11 and a bispecific
form of this antibody with the same anti-BACE1 arm used in the
prior examples in a human IgG1 format with the D265A and N297G
mutations abrogating effector function (see Example 6) were tested
at various concentrations in the ADCC assay, using anti-gD WT IgG1
as a negative control and murine anti-human HLA (class I) as a
positive control. The results are shown in FIGS. 13A and 13B. With
either the HEL cells as targets (FIG. 13A) or the bone marrow
mononuclear cells as targets (FIG. 13B), the monospecific
anti-human TfR antibody 15G11 elicited significant ADCC activity.
This activity was similar to that of the positive control
anti-human HLA antibodies on the HEL cells, and at a robust yet
lower level than the positive control on the bone marrow
mononuclear cells. The somewhat lower level observed in the bone
marrow mononuclear cells experiment is likely due to the fact that
only a portion of the heterogenous mixture of myeloid and erythroid
lineage PBMC cells used in the experiment express high levels of
TfR, whereas the HEL cells have consistently high TfR expression
throughout the clonal cell population. In sharp contrast, the
bispecific effectorless anti-humanTfR/BACE1 antibody did not
display any ADCC activity in either HEL or bone marrow mononuclear
cells, similar to the negative control.
[0322] In a second set of experiments, the impact of switching the
antibody isotype in this assay system was assessed. The ADCC assay
procedure was identical to that described above, with the exception
that all target cells were HEL cells, and the effector cells were
PBMCs from healthy human donors either carrying the heterozygous
Fc.gamma.RIIIa-V/F158 genotype or the homozygous
Fc.gamma.RIIIa-V/V158 genotype. All anti-human TfR tested were
bispecific with anti-gD, with three different Ig backbones:
wild-type human IgG1, human IgG1 with the N297G mutation, and human
IgG4. An anti-Abeta antibody with a human IgG4 backbone was also
tested, and mouse anti-human HLA (class I) served as a positive
control. The results are shown in FIGS. 14A and 14B. As anticipated
based on the known association between effector cell activation and
the V/V158 genotype (Bowles and Weiner 2005), ADCC activity was
more robustly elicited by V/V158 donor PBMCs (.about.45% of target
cells impacted) relative to F/V158 donors (.about.25% of target
cells impacted) (compare FIG. 14A to FIG. 14B). Anti-TfR/gD with
the wild-type IgG1 induced robust ADCC in HEL cells, while the
anti-TfR/gD with the effectorless IgG1 did not show any ADCC
activity in HEL cells, replicating the results from the first set
of experiments. Notably, at concentrations of 100 ng/mL or higher,
anti-TfR/gD of the IgG4 isotype showed a mild ADCC activity. This
activity was not observed in the anti-Abeta IgG4 results,
indicating that TfR binding was required for the ADCC activity.
This finding correlates with previous reports that IgG4 has
minimal, but measurable, effector function (Adolffson et al., J.
Neurosci. 32(28):9677-9689 (2012); van der Zee et al. Clin Exp.
Immunol. 64: 415-422 (1986)); Tao et al., J. Exp. Med.
173:1025-1028 (1991)).
[0323] Thus, the findings herein that depletion of erythroid
lineage cells in mice occurs in a TfR- and
effector-function-dependent manner is directly translatable to the
human system. Although the foregoing invention has been described
in some detail by way of illustration and example for purposes of
clarity of understanding, the descriptions and examples should not
be construed as limiting the scope of the invention. The
disclosures of all patent and scientific literature cited herein
are expressly incorporated in their entirety by reference.
Sequence CWU 1
1
151108PRTArtificial sequencesequence is synthesized 1Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25 30Thr Ala Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Ser Tyr Thr
Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile Lys
Arg2108PRTArtificial sequencesequence is synthesized 2Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25 30Thr Ala Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Phe Pro Thr
Tyr Leu Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile Lys
Arg3108PRTArtificial sequencesequence is synthesized 3Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25 30Thr Ala Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Gly Tyr Asn
Asp Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile Lys
Arg4108PRTArtificial sequencesequence is synthesized 4Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25 30Thr Ala Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Ser Ser Thr
Asp Pro Thr Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile Lys
Arg5108PRTArtificial sequencesequence is synthesized 5Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Val Val Ala 20 25 30Asn Ser Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile
Tyr Leu Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Asp Ala Thr
Ser Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile Lys
Arg6108PRTArtificial sequencesequence is synthesized 6Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 20 25 30Thr Ala Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Tyr Ala Thr
Asp Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile Lys
Arg7119PRTArtificial sequencesequence is synthesized 7Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30Gly Tyr Ala
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val
Gly Trp Ile Ser Pro Ala Gly Gly Ser Thr Asp Tyr 50 55 60Ala Asp Ser
Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65 70 75Lys Asn Thr
Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val
Tyr Tyr Cys Ala Arg Gly Pro Phe Ser Pro Trp Val 95 100 105Met Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115
8119PRTArtificial sequencesequence is synthesized 8Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Leu 20 25 30Gly Tyr Gly Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly
Trp Ile Ser Pro Ala Gly Gly Ser Thr Asp Tyr 50 55 60Ala Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65 70 75Lys Asn Thr Ala
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr
Tyr Cys Ala Arg Gly Pro Phe Ser Pro Trp Val 95 100 105Met Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115
9108PRTArtificial sequencesequence is synthesized 9Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser 20 25 30Ser Ala Val Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile Tyr
Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu Gln
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Tyr Ser Tyr Ser
Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile Lys
Arg10108PRTArtificial sequencesequence is synthesized 10Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser 20 25 30Ser Ala Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile
Ser Trp Ala Ser Trp Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Tyr Ser Tyr
Ser Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile Lys
Arg11108PRTArtificial sequencesequence is synthesized 11Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser 20 25 30Ser Ala Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile
Trp Tyr Ala Ser Trp Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Tyr Ser Tyr
Ser Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile Lys
Arg12108PRTArtificial sequencesequence is synthesized 12Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser 20 25 30Ser Ala Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile
Trp Trp Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Tyr Ser Tyr
Ser Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile Lys
Arg13126PRTArtificial sequencesequence is synthesized 13Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Tyr 20 25 30Tyr Ser Ser
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val
Ala Ser Ile Ser Pro Tyr Ser Gly Tyr Thr Ser Tyr 50 55 60Ala Asp Ser
Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65 70 75Lys Asn Thr
Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val
Tyr Tyr Cys Ala Arg Gln Pro Thr His Tyr Tyr Tyr 95 100 105Tyr Ala
Lys Gly Tyr Lys Ala Met Asp Tyr Trp Gly Gln Gly Thr 110 115 120Leu
Val Thr Val Ser Ser 125 14438PRTArtificial sequencesequence is
synthesized 14Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser 20 25 30Ser Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 35 40 45Glu Leu Val Ala Ser Ile Asn Ser Asn Gly Gly Ser Thr
Tyr Tyr 50 55 60Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala 65 70 75Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Ser Gly Asp Tyr Trp Gly
Gln Gly 95 100 105Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val 110 115 120Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
Glu Ser Thr Ala 125 130 135Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr 140 145 150Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe 155 160 165Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val 170 175 180Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Lys Thr Tyr Thr Cys 185 190 195Asn Val Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys Arg Val 200 205 210Glu Ser Lys Tyr Gly
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu 215 220 225Phe Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 230 235 240Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255Val Asp
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 260 265 270Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280
285Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290
295 300Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
305 310 315Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser
Lys 320 325 330Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro 335 340 345Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu 350 355 360Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser 365 370 375Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu 380 385 390Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu Thr Val Asp 395 400 405Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser Val Met 410 415 420His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 425 430 435Ser Leu
Gly15219PRTArtificial sequencesequence is synthesized 15Asp Ile Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro1 5 10 15Gly Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val 20 25 30Tyr Ser Asn
Gly Asp Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro 35 40 45Gly Gln Ser
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe 50 55 60Ser Gly Val
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 65 70 75Phe Thr Leu
Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val 80 85 90Tyr Tyr Cys
Ser Gln Ser Thr His Val Pro Trp Thr Phe Gly Gln 95 100 105Gly Thr
Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val 110 115 120Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala 125 130
135Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 140
145 150Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
155 160 165Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu 170 175 180Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys 185 190 195Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val 200 205 210Thr Lys Ser Phe Asn Arg Gly Glu Cys 215
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