U.S. patent application number 17/008941 was filed with the patent office on 2021-03-25 for anti-transferrin receptor antibodies and methods of use.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Genentech, Inc.. Invention is credited to Jasvinder Atwal, Jessica Couch, Mark S. Dennis, James A. Ernst, Gregory A. Lazar, Ryan J. Watts, Yin Zhang, Joy Yu Zuchero.
Application Number | 20210087288 17/008941 |
Document ID | / |
Family ID | 1000005253337 |
Filed Date | 2021-03-25 |
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United States Patent
Application |
20210087288 |
Kind Code |
A1 |
Zhang; Yin ; et al. |
March 25, 2021 |
ANTI-TRANSFERRIN RECEPTOR ANTIBODIES AND METHODS OF USE
Abstract
The present invention relates to anti-transferrin receptor
antibodies and methods of their use.
Inventors: |
Zhang; Yin; (Fremont,
CA) ; Zuchero; Joy Yu; (South San Francisco, CA)
; Atwal; Jasvinder; (San Carlos, CA) ; Couch;
Jessica; (San Francisco, CA) ; Dennis; Mark S.;
(South San Francisco, CA) ; Ernst; James A.; (San
Francisco, CA) ; Watts; Ryan J.; (San Mateo, CA)
; Lazar; Gregory A.; (South San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
1000005253337 |
Appl. No.: |
17/008941 |
Filed: |
September 1, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15614149 |
Jun 5, 2017 |
10808036 |
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17008941 |
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14283038 |
May 20, 2014 |
9708406 |
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15614149 |
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61825477 |
May 20, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
C07K 2317/31 20130101; C07K 2317/56 20130101; C07K 2317/52
20130101; C07K 2317/94 20130101; C07K 2317/565 20130101; C07K
2317/72 20130101; A61K 47/6803 20170801; C07K 2317/24 20130101;
C07K 16/2881 20130101; A61K 39/3955 20130101; C07K 2317/732
20130101; C07K 2317/55 20130101; C07K 2317/33 20130101; C07K 16/40
20130101; C07K 2317/92 20130101; C07K 2317/41 20130101; C07K
2317/71 20130101; C07K 2317/567 20130101; A61K 2039/505 20130101;
A61K 47/6849 20170801 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/40 20060101 C07K016/40; A61K 47/68 20060101
A61K047/68; A61K 39/395 20060101 A61K039/395; A61K 45/06 20060101
A61K045/06 |
Claims
1. An isolated antibody that binds to human transferrin receptor
(TfR) and primate TfR, wherein the antibody does not inhibit the
binding of transferrin to TfR, wherein the antibody comprises
HVR-H1, HVR-H2 and HVR-H3, and HVR-L1, HVR-L2 and HVR-L3,
respectively, comprising the amino acid sequences of: a) SEQ ID
NOs: 53, 54 and 55, and SEQ ID NOs: 50, 51 and 52; b) SEQ ID NOs:
53, 157 and 55, and SEQ ID NOs: 50, 51 and 52: or c) SEQ ID NOs:
53, 54 and 55, and SEQ ID NOs: 50, 51 and 155.
2. An isolated antibody that binds to human TfR and primate TfR,
wherein the antibody does not inhibit the binding of transferrin to
TfR, and wherein one or more properties of the antibody have been
modified to reduce or eliminate the impact of the antibody on
reticulocytes and/or reduce the severity or presence of acute
clinical symptoms in a subject or mammal treated with the antibody,
wherein the antibody comprises HVR-H1, HVR-H2 and HVR-H3, and
HVR-L1, HVR-L2 and HVR-L3, respectively, comprising the amino acid
sequences of: a) SEQ ID NOs: 53, 54 and 55, and SEQ ID NOs: 50, 51
and 52; b) SEQ ID NOs: 53, 157 and 55, and SEQ ID NOs: 50, 51 and
52: or c) SEQ ID NOs: 53, 54 and 55, and SEQ ID NOs: 50, 51 and
155.
3. The antibody of claim 1, which is a monoclonal antibody.
4. The antibody of claim 1, which is a human, humanized, or
chimeric antibody.
5. The antibody of claim 1, which is an antibody fragment that
binds human TfR and primate TfR.
6.-10. (canceled)
11. The antibody of claim 1, comprising (a) a VH sequence having at
least 95% sequence identity to the amino acid sequence of SEQ ID
NO: 108 or 154; (b) a VL sequence having at least 95% sequence
identity to the amino acid sequence of SEQ ID NO: 105 or 151 or (c)
a VH sequence as in (a) and a VL sequence as in (b).
12. The antibody of claim 11, comprising a VH sequence of a. SEQ ID
NO: 108; b. SEQ ID NO: 154.
13. The antibody of claim 11, comprising a VL sequence of a. SEQ ID
NO:105; b. SEQ ID NO: 151.
14. An antibody comprising: a. a VH sequence of SEQ ID NO:108 and a
VL sequence of SEQ ID NO:105; b. a VH sequence of SEQ ID NO: 154
and a VL sequence of SEQ ID NO: 105; or c. a VH sequence of SEQ ID
NO: 108 and a VL sequence of SEQ ID NO: 151.
15.-20. (canceled)
21. The antibody of claim 2, wherein the one or more properties are
selected from the effector function of the antibody Fc region, the
complement activation function of the antibody, the half-life of
the antibody and the affinity of the antibody for TfR.
22. The antibody of claim 21, 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.
23. The antibody of claim 22, 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.
24. The antibody of claim 23, 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.
25. The antibody of claim 24, wherein the antibody is produced in a
non-mammalian cell production system, or where the antibody is
produced synthetically.
26. The antibody of claim 24, 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.
27. The antibody of claim 23, wherein the effector function is
reduced or eliminated by at least one modification of the Fc
region.
28. The antibody of claim 27, 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.
29. The antibody of claim 27, 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: 234, 235,
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.
30. The antibody of claim 29, wherein the modification is at least
one point mutation of the Fc region to impair binding to one or
more Fc receptors selected from 234, 235, 265, 297 and 329.
31. The antibody of claim 30, wherein the modification is at
positions 297 or 265 and 297.
32. The antibody of claim 30, wherein the modification is at
positions 234, 235 and 329.
33. The antibody of claim 31, wherein the modification is N297G;
D265A and N297A or D265A and N297G.
34. The antibody of claim 31, wherein the modification is L234A,
L235A, and P329G.
35. The antibody of claim 21, wherein the one or more properties is
the half-life of the antibody.
36. The antibody of claim 35 wherein the half-life is increased by
a modification in the FcRn binding domain of the antibody at a
position selected from: 252, 254, 256, 434 and 436.
37. The antibody of claim 36, wherein the modification is at
positions 252, 254 and 256.
38. The antibody of claim 36, wherein the modification is at
positions 434 and 436.
39. The antibody of claim 37, wherein the modification is M252Y,
S254T and T256E.
40. The antibody of claim 38, wherein the modification is N434A and
Y436I.
41.-44. (canceled)
45. The antibody of claim 1, wherein the antibody has a KD or IC50
for TfR of about 1 pM to about 100 .mu.M.
46. The antibody of claim 1, wherein the antibody is coupled to a
therapeutic compound.
47. The antibody of claim 46, wherein the antibody is a
multispecific antibody and the therapeutic compound optionally
forms one portion of the multispecific antibody.
48. The antibody of claim 47, wherein the multispecific antibody
comprises a first antigen binding site which binds TfR and a second
antigen binding site which binds a brain antigen.
49. The antibody of claim 48, 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 E (ApoE),
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.
50. The antibody of claim 49, wherein the multispecific antibody
binds both TfR and BACE1.
51. The antibody of claim 49, wherein the multispecific antibody
binds both TfR and Abeta.
52. The antibody of claim 46, wherein the therapeutic compound is a
neurological disorder drug.
53. An isolated nucleic acid encoding the antibody of claim 1.
54. A host cell comprising the nucleic acid of claim 53.
55. A method of producing an antibody comprising culturing the host
cell of claim 54 so that the antibody is produced and optionally
further comprising recovering the antibody from the host cell.
56. A pharmaceutical formulation comprising the antibody of claim 1
and a pharmaceutically acceptable carrier.
57.-63. (canceled)
64. A method of transporting a compound across the BBB in a subject
comprising exposing an antibody of claim 46 to the BBB such that
the antibody transports the compound coupled thereto across the
BBB.
65. A method of increasing exposure of the CNS of a subject to a
compound, comprising exposing an antibody of claim 46 to the BBB
such that the antibody transports the compound coupled thereto
across the BBB.
66. A method of increasing retention in the CNS of a compound
administered to a subject, comprising exposing an antibody of claim
46 to the BBB such that the retention in the CNS of the compound is
increased.
67. A method of treating a neurological disorder in a mammal
comprising treating the mammal with an antibody of claim 46.
68. The method of claim 67, wherein the neurological disorder is
selected from the group consisting of a neuropathy disorder, a
neurodegenerative disease, cancer, an ocular disease disorder, a
seizure disorder, a lysosomal storage disease, amyloidosis, a viral
or microbial disease, ischemia, a behavioral disorder, and CNS
inflammation.
69. The method of any claim 64, wherein the BBB or neurological
disorder is in a human subject.
70. The method of claim 69, 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.
71. The method of claim 69, further comprising the step of
monitoring the subject for depletion of red blood cells.
72. The method of claim 69, wherein the antibody coupled to the
compound is administered at a therapeutic dose.
73. The method of claim 72, wherein the therapeutic dose is
TfR-saturating.
74. The method of claim 69, wherein administration of the antibody
is at a dose and/or dose frequency calibrated to minimize acute
clinical symptoms of the antibody administration.
75. The antibody of claim 14, wherein the antibody comprises a
heavy chain variable region (VH) sequence having the amino acid
sequence of SEQ ID NO: 108 and a light chain variable region (VL)
sequence having the amino acid sequence of SEQ ID NO: 105.
76. The antibody of claim 14, wherein the antibody comprises a
heavy chain variable region (VH) sequence having the amino acid
sequence of SEQ ID NO: 154 and a light chain variable region (VL)
sequence having the amino acid sequence of SEQ ID NO: 105.
77. The antibody of claim 14, wherein the antibody comprises a
heavy chain variable region (VH) sequence having the amino acid
sequence of SEQ ID NO: 108 and a light chain variable region (VL)
sequence having the amino acid sequence of SEQ ID NO: 151.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 15/614,149, filed Jun. 5, 2017, which is a divisional of U.S.
application Ser. No. 14/283,038, filed May 20, 2014, issued as U.S.
Pat. No. 9,708,406 on Jul. 18, 2017, which claims the benefit of
U.S. Provisional Application No. 61/825,477 filed on May 20, 2013,
each of which is incorporated by reference herein in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to anti-transferrin receptor
antibodies and methods of using the same.
SEQUENCE LISTING
[0003] A sequence listing is submitted concurrently with the
specification as an ASCII formatted text file via EFS-Web, with a
file name of "2020-08-31_01146-0046-02US_SeqList_ST25.txt", a
creation date of Aug. 31, 2020, and a size of 144 KB. The sequence
listing filed via EFS-Web is part of the specification and is
hereby incorporated by reference in its entirety herein.
BACKGROUND
[0004] 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, those antibodies
did not specifically bind to human and primate TfR.
SUMMARY
[0005] 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 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.
[0006] 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)).
[0007] 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 from Atwal et
al. and Yu et al., supra) 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.
[0008] 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. 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. It was found 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.
[0009] These prior studies utilized mouse antibodies which bound
specifically to mouse TfR, but which did not specifically recognize
primate or human TfR. Accordingly, the invention provides
antibodies and functional parts thereof which do specifically
recognize both primate and human TfR, in order to facilitate safety
and efficacy studies in primates with the antibodies prior to
therapeutic or diagnostic use in humans. In vitro studies using a
human erythroblast cell line and primary bone marrow cells treated
with the anti-human TfR antibodies of the invention demonstrated
that a robust depletion of TfR-positive erythroid cells is also
observable in human/primate cellular systems as it is in mice (see,
e.g., Example 4). Accordingly, also provided herein are
modifications to the antibodies of the invention to greatly reduce
or eliminate the unwanted reduction in the TfR-expressing
reticulocyte population upon anti-TfR administration while still
enabling the enhanced BBB transport, increased CNS distribution and
CNS retention provided by the anti-human/primate TfR antibodies
administered at therapeutic concentrations. Several general
approaches to mitigate the observed effect of the anti-TfR
antibodies of the invention on both the primary and residual
reticulocyte depletion are provided herein, and may be used singly
or in combination.
[0010] In one approach, the effector function of the
anti-human/cyno TfR antibody is reduced or eliminated in order to
reduce or eliminate ADCC activity. In another approach, the
affinity of the anti-human/cyno TfR antibody for human or primate
TfR 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-human/cyno TfR 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 in
circulation or in bone marrow by administration of the
anti-human/cyno TfR antibody is avoided, lessened, or
mitigated.
[0011] 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-human/cyno TfR 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).
[0012] Decreasing antibody complement activation, as described
herein, may be accomplished by reduction or elimination of the C1q
binding capability of the anti-human/cyno TfR antibody (for
example, by mutation of, deletion within or elimination of the Fc
region, or by modifying the non-Fc portion of the anti-human/cyno
TfR 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).
[0013] When binding of anti-human/cyno TfR antibody to human or
cyno TfR on reticulocytes or other cell types expressing high
levels of TfR triggers their depletion, as with the anti-human/cyno
TfR antibodies exemplified herein, reduction of binding of the
antibodies to the human or cyno TfR on the reticulocytes or other
cell types should in turn decrease the amount of reticulocyte or
other cell type depletion in circulation or in bone marrow observed
upon antibody administration. The affinity of the anti-human/cyno
TfR antibody for primate or human TfR may be modified using any of
the methods described herein and as shown in the Examples.
[0014] Reducing the amount of anti-human/cyno TfR 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, and in turn potentially
permitting a lower dose of the antibody with concomitantly lesser
risk of side effects.
[0015] Protecting, stabilizing and/or replenishing reticulocyte
populations may be accomplished using pharmaceutical or physical
methods. In addition to the anti-human/cyno TfR 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-human/cyno
TfR antibody is administered.
[0016] 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 primate or human TfR
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-human/cyno 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-human/cynoTfR
antibody; (iv) the lability of the anti-TfR/conjugated compound at
low pH to facilitate release of the conjugated compound on the
CNS/brain side of the BBB, and (v) the impact on reticulocyte
populations.
[0017] 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-human/cyno 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.
[0018] 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.
[0019] Accordingly, in a first embodiment, the invention provides
an isolated antibody that binds to human transferrin receptor (TfR)
and primate TfR, wherein the antibody does not inhibit the binding
of transferrin to TfR. In one aspect, the binding is specific
binding. In another aspect, the antibody further does not inhibit
the binding of human hemachromatosis protein ("IFE") to TfR. In one
aspect, the binding is specific binding. In one aspect, the
antibody is a monoclonal antibody. In another aspect, the antibody
is a human antibody. In another aspect, the antibody is a humanized
antibody. In another aspect, the antibody is a chimeric antibody.
In another aspect, the antibody is an antibody fragment that binds
human TfR and primate TfR. In another aspect, the primate TfR is
from cynomolgous monkey.
[0020] In one aspect of the above embodiment, the antibody
comprises HVR-L3, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 31, 33 and 34. In another
aspect of the above embodiment, the antibody comprises HVR-L3,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 36, 38 and 39. In another aspect of the above
embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 36,
40 and 34. In another aspect of the above embodiment, the antibody
comprises HVR-L3, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 42, 43 and 44. In another
aspect of the above embodiment, the antibody comprises HVR-L3,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 31, 33 and 34. In another aspect of the above
embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 46,
48 and 49. In another aspect of the above embodiment, the antibody
comprises HVR-L3, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 52, 54 and 55. In another
aspect of the above embodiment, the antibody comprises HVR-L3,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 52, 58 and 59. In another aspect of the above
embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 62,
63 and 55. In another aspect of the above embodiment, the antibody
comprises HVR-L3, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 52, 65 and 55. In another
aspect of the above embodiment, the antibody comprises HVR-L3,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 68, 69 and 70. In another aspect of the above
embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 73,
75 and 76. In another aspect of the above embodiment, the antibody
comprises HVR-L3, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 79, 81 and 82. In another
aspect of the above embodiment, the antibody comprises HVR-L3,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 79, 83 and 84. In another aspect of the above
embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 87,
89 and 90. In another aspect of the above embodiment, the antibody
comprises HVR-L3, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 93, 95 and 96. In another
aspect of the above embodiment, the antibody comprises HVR-L3,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 99, 101 and 102. In another aspect of the above
embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs:
127, 33 and 34. In another aspect of the above embodiment, the
antibody comprises HVR-L3, HVR-H2, and HVR-H3 respectively
comprising the amino acid sequences of SEQ ID NOs: 52, 156 and 55.
In another aspect of the above embodiment, the antibody comprises
HVR-L3, HVR-H2, and HVR-H3 respectively comprising the amino acid
sequences of SEQ ID NOs: 52, 157 and 55. In another aspect of the
above embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs:
155, 54 and 55
[0021] In one aspect of the above embodiment, the antibody
comprises HVR-H1, HVR-H2 and HVR-H3, respectively comprising the
amino acid sequences of SEQ ID NOs: 32, 33 and 34. In another
aspect of the above embodiment, the antibody comprises HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 37, 38 and 39. In another aspect of the above
embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 32,
40 and 34. In another aspect of the above embodiment, the antibody
comprises HVR-H1, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 37, 43 and 44. In another
aspect of the above embodiment, the antibody comprises HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 32, 33 and 34. In another aspect of the above
embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 47,
48 and 49. In another aspect of the above embodiment, the antibody
comprises HVR-H1, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 53, 54 and 55. In another
aspect of the above embodiment, the antibody comprises HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 53, 58 and 59. In another aspect of the above
embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 53,
63 and 55. In another aspect of the above embodiment, the antibody
comprises HVR-H1, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 53, 65 and 55. In another
aspect of the above embodiment, the antibody comprises HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 53, 69 and 70. In another aspect of the above
embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 74,
75 and 76. In another aspect of the above embodiment, the antibody
comprises HVR-H1, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 80, 81 and 82. In another
aspect of the above embodiment, the antibody comprises HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 80, 83 and 84. In another aspect of the above
embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 88,
89 and 90. In another aspect of the above embodiment, the antibody
comprises HVR-H1, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 94, 95 and 96. In another
aspect of the above embodiment, the antibody comprises HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 100, 101 and 102. In another aspect of the above
embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 53,
156 and 55. In another aspect of the above embodiment, the antibody
comprises HVR-H1, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 53, 157 and 55.
[0022] In one aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 29, 30 and 31. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 35, 30 and 36. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 41,
30 and 42. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 29, 30 and 31. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 45, 30 and 46. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 50,
51 and 52. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 56, 57 and 52. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 60, 61 and 62. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 60,
64 and 52. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 66, 67 and 68. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 71, 72 and 73. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs:77,
78 and 79. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 85, 86 and 87. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 91, 92 and 93. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 97,
98 and 99. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 29, 30 and 127. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 50, 51 and 155.
[0023] In one aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 29, 30 and 31 and HVR-H1,
HVR-H2 and HVR-H3, respectively comprising the amino acid sequences
of SEQ ID NOs: 32, 33 and 34. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 35,
30 and 36 and HVR-H1, HVR-H2, and HVR-H3 respectively comprising
the amino acid sequences of SEQ ID NOs: 37, 38 and 39. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 35, 30 and 36 and HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 32,
40 and 34. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 41, 30 and 42 and HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 37, 43 and 44 In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 29,
30 and 31 and HVR-H1, HVR-H2, and HVR-H3 respectively comprising
the amino acid sequences of SEQ ID NOs: 32, 33 and 34. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 45, 30 and 46 and HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 47,
48 and 49. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 50, 51 and 52 and HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 53, 54 and 55. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 56,
57 and 52 and HVR-H1, HVR-H2, and HVR-H3 respectively comprising
the amino acid sequences of SEQ ID NOs: 53, 58 and 59. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 60, 61 and 62 and HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 53,
63 and 55. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 60, 64 and 52 and HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 53, 65 and 55. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 66,
67 and 68 and HVR-H1, HVR-H2, and HVR-H3 respectively comprising
the amino acid sequences of SEQ ID NOs: 53, 69 and 70. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 71, 72 and 73 and HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 74,
75 and 76. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs:77, 78 and 79 and HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 80, 81 and 82. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 77,
78 and 79, and HVR-H1, HVR-H2, and HVR-H3 respectively comprising
the amino acid sequences of SEQ ID NOs: 80, 83 and 84. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 85, 86 and 87 and HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 88,
89 and 90. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 91, 92 and 93 and HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 94, 95 and 96. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 97,
98 and 99 and HVR-H1, HVR-H2, and HVR-H3 respectively comprising
the amino acid sequences of SEQ ID NOs: 100, 101 and 102. In
another aspect of the above embodiment, the antibody comprises
HVR-L1, HVR-L2, and HVR-L3 respectively comprising the amino acid
sequences of SEQ ID NOs: 29, 30 and 127 and HVR-H1, HVR-H2 and
HVR-H3 respectively comprising the amino acid sequences of SEQ ID
NOs: 32, 33 and 34. In another aspect of the above embodiment, the
antibody comprises HVR-L1, HVR-L2, and HVR-L3 respectively
comprising the amino acid sequences of SEQ ID NOs: 50, 51 and 52
and HVR-H1, HVR-H2 and HVR-H3 respectively comprising the amino
acid sequences of SEQ ID NOs: 53, 156 and 55. In another aspect of
the above embodiment, the antibody comprises HVR-L1, HVR-L2, and
HVR-L3 respectively comprising the amino acid sequences of SEQ ID
NOs: 50, 51 and 52 and HVR-H1, HVR-H2 and HVR-H3 respectively
comprising the amino acid sequences of SEQ ID NOs: 53, 157 and 55.
In another aspect of the above embodiment, the antibody comprises
HVR-L1, HVR-L2, and HVR-L3 respectively comprising the amino acid
sequences of SEQ ID NOs: 50, 51 and 155 and HVR-H1, HVR-H2 and
HVR-H3 respectively comprising the amino acid sequences of SEQ ID
NOs: 53, 54 and 55.
[0024] In one aspect of the above embodiment, the antibody
comprises at least one HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, or
HVR-L3 sequence selected from the group of sequences consisting of
an HVR-H1 sequence of SEQ ID NO: 47, 53, or 100; an HVR-H2 sequence
of SEQ ID NO: 48, 69, 101, 156 or 157; an HVR-H3 sequence of SEQ ID
NO: 49, 76, or 102; an HVR-L1 sequence of SEQ ID NO: 45, 66 or 97;
an HVR-L2 sequence of SEQ ID NOs: 30, 67 or 98; and an HVR-L3
sequence of SEQ ID NOs: 46, 68 or 102.
[0025] In one aspect of the above embodiment, the antibody
comprises (a) a VH sequence having at least 95% sequence identity
to the amino acid sequence of SEQ ID NO: 7, 8, 9, 10, 15, 16, 17,
18, 20, 25, 26, 27, 28, 108, 114, 120, 126, 153 or 154; (b) a VL
sequence having at least 95% sequence identity to the amino acid
sequence of SEQ ID NO:4, 5, 6, 11, 12, 13, 14, 19, 21, 22, 23, 24,
105, 111, 117, 123 or 151; or (c) a VH sequence as in (a) and a VL
sequence as in (b). In one such aspect, the antibody comprises a VL
sequence of SEQ ID NO: 4 and a VH sequence of SEQ ID NO: 7. In
another such aspect, the antibody comprises a VL sequence of SEQ ID
NO: 5 and a VH sequence of SEQ ID NO: 8. In another such aspect,
the antibody comprises a VL sequence of SEQ ID NO: 5 and a VH
sequence of SEQ ID NO: 9. In another such aspect, the antibody
comprises a VL sequence of SEQ ID NO: 6 and a VH sequence of SEQ ID
NO: 10. In another such aspect, the antibody comprises a VL
sequence of SEQ ID NO: 11 and a VH sequence of SEQ ID NO: 15. In
another such aspect, the antibody comprises a VL sequence of SEQ ID
NO: 12 and a VH sequence of SEQ ID NO: 16. In another such aspect,
the antibody comprises a VL sequence of SEQ ID NO: 13 and a VH
sequence of SEQ ID NO: 17. In another such aspect, the antibody
comprises a VL sequence of SEQ ID NO: 14 and a VH sequence of SEQ
ID NO: 18. In another such aspect, the antibody comprises a VL
sequence of SEQ ID NO: 19 and a VH sequence of SEQ ID NO: 20. In
another such aspect, the antibody comprises a VL sequence of SEQ ID
NO: 21 and a VH sequence of SEQ ID NO: 25. In another such aspect,
the antibody comprises a VL sequence of SEQ ID NO: 22 and a VH
sequence of SEQ ID NO: 26. In another such aspect, the antibody
comprises a VL sequence of SEQ ID NO: 23 and a VH sequence of SEQ
ID NO: 27. In another such aspect, the antibody comprises a VL
sequence of SEQ ID NO: 24 and a VH sequence of SEQ ID NO: 28. In
another such aspect, the antibody comprises a VL sequence of SEQ ID
NO: 105 and a VH sequence of SEQ ID NO: 108. In another such
aspect, the antibody comprises a VL sequence of SEQ ID NO: 111 and
a VH sequence of SEQ ID NO: 114. In another such aspect, the
antibody comprises a VL sequence of SEQ ID NO: 117 and a VH
sequence of SEQ ID NO: 120. In another such aspect, the antibody
comprises a VL sequence of SEQ ID NO: 123 and a VH sequence of SEQ
ID NO: 126. In another such aspect, the antibody comprises a VL
sequence of SEQ ID NO: 105 and a VH sequence of SEQ ID NO: 153. In
another such aspect, the antibody comprises a VL sequence of SEQ ID
NO: 105 and a VH sequence of SEQ ID NO: 154. In another such
aspect, the antibody comprises a VL sequence of SEQ ID NO: 151 and
a VH sequence of SEQ ID NO: 108. In another such aspect, the
antibody comprises a VH sequence of (a) SEQ ID NO: 108, (b) SEQ ID
NO: 114, (c) SEQ ID NO: 120 or (d) SEQ ID NO: 126. In another such
aspect, the antibody comprises a VL sequence of (a) SEQ ID NO: 105,
(b) SEQ ID NO: 111, (c) SEQ ID NO: 117, or (d) SEQ ID NO: 123.
[0026] In one aspect of the embodiment above, the antibody is
selected from the group consisting of antibodies 7A4, 8A2, 15D2,
10D11, 7B10, 15G11, 16G5, 13C3, 16G4, 16F6, 7G7, 4C2, 1B12, and
13D4. In one such aspect, the antibody is 7A4. In another such
aspect, the antibody is 8A2. In another such aspect, the antibody
is 15D2. In another such aspect, the antibody is 10D11. In another
such aspect, the antibody is 7B10. In another such aspect, the
antibody is 15G11. In another such aspect, the antibody is 16G5. In
another such aspect, the antibody is 13C3. In another such aspect,
the antibody is 16G4. In another such aspect, the antibody is 16F6.
In another such aspect, the antibody is 7G7. In another such
aspect, the antibody is 4C2. In another such aspect, the antibody
is 1B12. In another such aspect, the antibody is 13D4.
[0027] In one aspect of any of the foregoing, the antibody is
further affinity matured. In one such aspect, the antibody is
selected from the group consisting of 15G11.v1, 15G11.v2, 15G11.v3,
15G11.v4, 15G11.v5, 7A4.v1; 7A4.v2, 7A4.v3, 7A4.v4, 7A4.v5, 7A4.v6,
7A4.v7, 7A4.v8, 7A4.v9, 7A4.v10, 7A4.v11, 7A4.v12, 7A4.v13,
7A4.v14, 7A4.v15, 7G7.v1, 16F6.v1, 16F6.v2, 16F6.v3, 16F6.v4,
15G11.N52A, 15G11.T53A and 15G11.W92A. In one such aspect, the
antibody is 15G11.v1. In another such aspect, the antibody is
15G11.v2. In another such aspect, the antibody is 15G11.v3. In
another such aspect, the antibody is 15G11.v4. In another such
aspect, the antibody is 15G11.v5. In another such aspect, the
antibody is 7A4.v1. In another such aspect, the antibody is 7A4.v2.
In another such aspect, the antibody is 7A4.v3. In another such
aspect, the antibody is 7A4.v4. In another such aspect, the
antibody is 7A4.v5. In another such aspect, the antibody is 7A4.v6.
In another such aspect, the antibody is 7A4.v7. In another such
aspect, the antibody is 7A4.v8. In another such aspect, the
antibody is 7A4.v9. In another such aspect, the antibody is
7A4.v10. In another such aspect, the antibody is 7A4.v11. In
another such aspect, the antibody is 7A4.v12. In another such
aspect, the antibody is 7A4.v13. In another such aspect, the
antibody is 7A4.v14. In another such aspect, the antibody is
7A4.v15. In another such aspect, the antibody is 7G7.v1. In another
such aspect, the antibody is 16F6.v1. In another such aspect, the
antibody is 16F6.v2. In another such aspect, the antibody is
16F6.v3. In another such aspect, the antibody is 16F6.v4. In
another such aspect, the antibody is 15G11.N52A. In another such
aspect, the antibody is 15G11.T53A. In another such aspect, the
antibody is 15G11.W92A.
[0028] In one aspect of the above embodiment, the antibody is
modified at one or more amino acid positions in the VH or VL to the
amino acid indicated for that position in FIGS. 4E-1 and 4E-2. In
another aspect of the above embodiment, the antibody comprises a
sequence or one or more HVR sequences corresponding to that of or
one or more of those set forth for any one of the clones in FIGS. 3
and 4 and FIGS. 4E-1 and 4E-2. In another aspect of the above
embodiment, the antibody comprises a VH or VL sequence
corresponding to that of set forth for any one of the clones in
FIGS. 3 and 4 and FIGS. 4E-1 and 4E-2. In another aspect of the
above embodiment, the antibody comprises one or more HVR sequences
corresponding to one or more of those set forth for any one of the
clones in FIGS. 3 and 4 and FIGS. 4E-1 and 4E-2.
[0029] In one aspect of the above embodiment, the antibody is
coupled to a therapeutic compound. In another aspect of the above
embodiment, the antibody is coupled to an imaging agent or a label.
In one such aspect, the antibody is a multispecific antibody and
the therapeutic compound optionally forms one portion of the
multispecific antibody. In one such aspect, the multispecific
antibody comprises a first antigen binding site which binds TfR and
a second antigen binding site which binds a brain antigen. In one
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 E (ApoE), 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. In another such aspect, the
multispecific antibody binds both TfR and BACEL. In another such
aspect, the multispecific antibody binds both TfR and Abeta. In
another such aspect, the therapeutic compound is a neurological
disorder drug.
[0030] In one aspect of the above embodiment, the invention
provides an isolated nucleic acid encoding any of the foregoing
antibodies. In another aspect, the invention provides a host cell
comprising such nucleic acid. In another aspect, the invention
provides a method of producing any of the foregoing antibodies
comprising culturing such host cell so that the antibody is
produced and optionally further comprising recovering the antibody
from the host cell.
[0031] In one aspect of the above embodiment, the invention
provides a pharmaceutical formulation comprising any of the
foregoing antibodies and a pharmaceutically acceptable carrier.
[0032] In one aspect of the above embodiment, the invention
provides any of the foregoing antibodies for use as a medicament.
In another aspect of the above embodiment, the invention provides
the use of any of the foregoing antibodies in the manufacture of a
medicament for treating a neurological disorder. In one such
aspect, the neurological disorder is selected from the group
consisting of a neuropathy disorder, a neurodegenerative disease,
cancer, an ocular disease disorder, a seizure disorder, a lysosomal
storage disease, amyloidosis, a viral or microbial disease,
ischemia, a behavioral disorder, and CNS inflammation.
[0033] In another aspect of the above embodiment, the invention
provides any of the foregoing antibodies for use in treating a
neurological disorder. In one such aspect, the neurological
disorder is selected from the group consisting of a neuropathy
disorder, a neurodegenerative disease, cancer, an ocular disease
disorder, a seizure disorder, a lysosomal storage disease,
amyloidosis, a viral or microbial disease, ischemia, a behavioral
disorder, and CNS inflammation.
[0034] In another aspect of the above embodiment, the invention
provides any of the foregoing antibodies for use in transporting
one or more compounds across the BBB. In another aspect of the
above embodiment, use of any of the foregoing antibodies in the
manufacture of a medicament for transporting one or more compounds
across the BBB is provided.
[0035] In one aspect of the above embodiment, a method of
transporting a compound across the BBB in a subject is provided,
comprising exposing any of the foregoing antibodies to the BBB such
that the antibody transports the compound coupled thereto across
the BBB. In another such aspect, the BBB is in a human subject. In
another such aspect, the dose amount and/or frequency of
administration is modulated to reduce the concentration of antibody
to which red blood cells are exposed. In another such aspect, the
method further comprises the step of monitoring the subject for
depletion of red blood cells. In another such aspect, the antibody
coupled to the compound is administered at a therapeutic dose. In
one such aspect, the therapeutic dose is TfR-saturating. In another
such aspect, administration of the antibody is at a dose and/or
dose frequency calibrated to minimize acute clinical symptoms of
the antibody administration.
[0036] In another aspect of the above embodiment, a method of
increasing exposure of the CNS of a subject to a compound is
provided, comprising exposing any of the foregoing antibodies to
the BBB such that the antibody transports the compound coupled
thereto across the BBB. In another such aspect, the BBB is in a
human subject. In another such aspect, the dose amount and/or
frequency of administration is modulated to reduce the
concentration of antibody to which red blood cells are exposed. In
another such aspect, the method further comprises the step of
monitoring the subject for depletion of red blood cells. In another
such aspect, the antibody coupled to the compound is administered
at a therapeutic dose. In one such aspect, the therapeutic dose is
TfR-saturating. In another such aspect, administration of the
antibody is at a dose and/or dose frequency calibrated to minimize
acute clinical symptoms of the antibody administration.
[0037] In one aspect of the above embodiment, a method of
increasing retention in the CNS of a compound administered to a
subject is provided, comprising exposing any of the foregoing
antibodies to the BBB such that the retention in the CNS of the
compound is increased. In another such aspect, the BBB is in a
human subject. In another such aspect, the dose amount and/or
frequency of administration is modulated to reduce the
concentration of antibody to which red blood cells are exposed. In
another such aspect, the method further comprises the step of
monitoring the subject for depletion of red blood cells. In another
such aspect, the antibody coupled to the compound is administered
at a therapeutic dose. In one such aspect, the therapeutic dose is
TfR-saturating. In another such aspect, administration of the
antibody is at a dose and/or dose frequency calibrated to minimize
acute clinical symptoms of the antibody administration.
[0038] In one aspect of the above embodiment, a method of treating
a neurological disorder in a mammal is provided, comprising
treating the mammal with any of the foregoing antibodies. In one
such aspect, the neurological disorder is selected from the group
consisting of a neuropathy disorder, a neurodegenerative disease,
cancer, an ocular disease disorder, a seizure disorder, a lysosomal
storage disease, amyloidosis, a viral or microbial disease,
ischemia, a behavioral disorder, and CNS inflammation. In another
such aspect, the neurological disorder is in a human subject. In
another such aspect, the dose amount and/or frequency of
administration is modulated to reduce the concentration of antibody
to which the re blood cells are exposed. In another such aspect,
the method further comprises the step of monitoring the subject for
depletion of red blood cells. In another such aspect, the antibody
coupled to the compound is administered at a therapeutic dose. In
one such aspect, the therapeutic dose is TfR-saturating. In another
such aspect, administration of the antibody is at a dose and/or
dose frequency calibrated to minimize acute clinical symptoms of
the antibody administration.
[0039] In another embodiment, the invention provides an isolated
antibody that binds to human TfR and primate TfR, wherein the
antibody does not inhibit the binding of transferrin to TfR, and
wherein one or more properties of the antibody have been modified
to reduce or eliminate the impact of the antibody on reticulocytes
and/or reduce the severity or presence of acute clinical symptoms
in a subject or mammal treated with the antibody. In one aspect,
the binding is specific binding. In another aspect, the antibody
further does not inhibit the binding of HFE to TfR. In one aspect,
the antibody is a monoclonal antibody. In another aspect, the
antibody is a human antibody. In another aspect, the antibody is a
humanized antibody. In another aspect, the antibody is a chimeric
antibody. In another aspect, the antibody is an antibody fragment
that binds human TfR and primate TfR. In another aspect, the
primate TfR is from cynomolgous monkey.
[0040] In one aspect of the above embodiment, the antibody
comprises HVR-L3, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 31, 33 and 34. In another
aspect of the above embodiment, the antibody comprises HVR-L3,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 36, 38 and 39. In another aspect of the above
embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 36,
40 and 34. In another aspect of the above embodiment, the antibody
comprises HVR-L3, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 42, 43 and 44. In another
aspect of the above embodiment, the antibody comprises HVR-L3,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 31, 33 and 34. In another aspect of the above
embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 46,
48 and 49. In another aspect of the above embodiment, the antibody
comprises HVR-L3, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs:52, 54 and 55. In another aspect
of the above embodiment, the antibody comprises HVR-L3, HVR-H2, and
HVR-H3 respectively comprising the amino acid sequences of SEQ ID
NOs: 52, 58 and 59. In another aspect of the above embodiment, the
antibody comprises HVR-L3, HVR-H2, and HVR-H3 respectively
comprising the amino acid sequences of SEQ ID NOs: 62, 63 and 55.
In another aspect of the above embodiment, the antibody comprises
HVR-L3, HVR-H2, and HVR-H3 respectively comprising the amino acid
sequences of SEQ ID NOs:52, 65 and 55. In another aspect of the
above embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 68,
69 and 70. In another aspect of the above embodiment, the antibody
comprises HVR-L3, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 73, 75 and 76. In another
aspect of the above embodiment, the antibody comprises HVR-L3,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 79, 81 and 82. In another aspect of the above
embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 79,
83 and 84. In another aspect of the above embodiment, the antibody
comprises HVR-L3, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs:87, 89 and 90. In another aspect
of the above embodiment, the antibody comprises HVR-L3, HVR-H2, and
HVR-H3 respectively comprising the amino acid sequences of SEQ ID
NOs: 93, 95 and 96. In another aspect of the above embodiment, the
antibody comprises HVR-L3, HVR-H2, and HVR-H3 respectively
comprising the amino acid sequences of SEQ ID NOs: 99, 101 and 102.
In another aspect of the above embodiment, the antibody comprises
HVR-L3, HVR-H2, and HVR-H3 respectively comprising the amino acid
sequences of SEQ ID NOs: 127, 33 and 34.
[0041] In one aspect of the above embodiment, the antibody
comprises HVR-H1, HVR-H2 and HVR-H3, respectively comprising the
amino acid sequences of SEQ ID NOs: 32, 33 and 34. In another
aspect of the above embodiment, the antibody comprises HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 37, 38 and 39. In another aspect of the above
embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 32,
40 and 34. In another aspect of the above embodiment, the antibody
comprises HVR-H1, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 37, 43 and 44. In another
aspect of the above embodiment, the antibody comprises HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 32, 33 and 34. In another aspect of the above
embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 47,
48 and 49. In another aspect of the above embodiment, the antibody
comprises HVR-H1, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 53, 54 and 55. In another
aspect of the above embodiment, the antibody comprises HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 53, 58 and 59. In another aspect of the above
embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 53,
63 and 55. In another aspect of the above embodiment, the antibody
comprises HVR-H1, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 53, 65 and 55. In another
aspect of the above embodiment, the antibody comprises HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 53, 69 and 70. In another aspect of the above
embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 74,
75 and 76. In another aspect of the above embodiment, the antibody
comprises HVR-H1, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 80, 81 and 82. In another
aspect of the above embodiment, the antibody comprises HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 80, 83 and 84. In another aspect of the above
embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 88,
89 and 90. In another aspect of the above embodiment, the antibody
comprises HVR-H1, HVR-H2, and HVR-H3 respectively comprising the
amino acid sequences of SEQ ID NOs: 94, 95 and 96. In another
aspect of the above embodiment, the antibody comprises HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 100, 101 and 102.
[0042] In one aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 29, 30 and 31. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 35, 30 and 36. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 41,
30 and 42. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 29, 30 and 31. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 45, 30 and 46. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 50,
51 and 52. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 56, 57 and 52. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 60, 61 and 62. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 60,
64 and 52. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 66, 67 and 68. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 71, 72 and 73. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs:77,
78 and 79. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 85, 86 and 87. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 91, 92 and 93. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 97,
98 and 99. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 29, 30 and 127.
[0043] In one aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 29, 30 and 31 and HVR-H1,
HVR-H2 and HVR-H3, respectively comprising the amino acid sequences
of SEQ ID NOs: 32, 33 and 34. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 35,
30 and 36 and HVR-H1, HVR-H2, and HVR-H3 respectively comprising
the amino acid sequences of SEQ ID NOs: 37, 38 and 39. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 35, 30 and 36 and HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 32,
40 and 34. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 41, 30 and 42 and HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 37, 43 and 44 In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 29,
30 and 31 and HVR-H1, HVR-H2, and HVR-H3 respectively comprising
the amino acid sequences of SEQ ID NOs: 32, 33 and 34. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 45, 30 and 46 and HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 47,
48 and 49. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 50, 51 and 52 and HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 53, 54 and 55. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 56,
57 and 52 and HVR-H1, HVR-H2, and HVR-H3 respectively comprising
the amino acid sequences of SEQ ID NOs: 53, 58 and 59. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 60, 61 and 62 and HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 53,
63 and 55. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 60, 64 and 52 and HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 53, 65 and 55. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 66,
67 and 68 and HVR-H1, HVR-H2, and HVR-H3 respectively comprising
the amino acid sequences of SEQ ID NOs: 53, 69 and 70. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 71, 72 and 73 and HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 74,
75 and 76. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs:77, 78 and 79 and HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 80, 81 and 82. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 77,
78 and 79, and HVR-H1, HVR-H2, and HVR-H3 respectively comprising
the amino acid sequences of SEQ ID NOs: 80, 83 and 84. In another
aspect of the above embodiment, the antibody comprises HVR-L1,
HVR-L2, and HVR-L3 respectively comprising the amino acid sequences
of SEQ ID NOs: 85, 86 and 87 and HVR-H1, HVR-H2, and HVR-H3
respectively comprising the amino acid sequences of SEQ ID NOs: 88,
89 and 90. In another aspect of the above embodiment, the antibody
comprises HVR-L1, HVR-L2, and HVR-L3 respectively comprising the
amino acid sequences of SEQ ID NOs: 91, 92 and 93 and HVR-H1,
HVR-H2, and HVR-H3 respectively comprising the amino acid sequences
of SEQ ID NOs: 94, 95 and 96. In another aspect of the above
embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3
respectively comprising the amino acid sequences of SEQ ID NOs: 97,
98 and 99 and HVR-H1, HVR-H2, and HVR-H3 respectively comprising
the amino acid sequences of SEQ ID NOs: 100, 101 and 102. In
another aspect of the above embodiment, the antibody comprises
HVR-L1, HVR-L2, and HVR-L3 respectively comprising the amino acid
sequences of SEQ ID NOs: 29, 30 and 127 and HVR-H1, HVR-H2 and
HVR-H3 respectively comprising the amino acid sequences of SEQ ID
NOs: 32, 33 and 34.
[0044] In one aspect of the above embodiment, the antibody
comprises at least one HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, or
HVR-L3 sequence selected from the group of sequences consisting of
an HVR-H1 sequence of SEQ ID NO: 47, 53, or 100; an HVR-H2 sequence
of SEQ ID NO: 48, 69, or 101; an HVR-H3 sequence of SEQ ID NO: 49,
76, or 102; an HVR-L1 sequence of SEQ ID NO: 45, 66 or 97; an
HVR-L2 sequence of SEQ ID NOs: 30, 67 or 98; and an HVR-L3 sequence
of SEQ ID NOs: 46, 68 or 102.
[0045] In one aspect of the above embodiment, the antibody
comprises (a) a VH sequence having at least 95% sequence identity
to the amino acid sequence of SEQ ID NO: 7, 8, 9, 10, 15, 16, 17,
18, 20, 25, 26, 27, 28, 108, 114, 120 or 126; (b) a VL sequence
having at least 95% sequence identity to the amino acid sequence of
SEQ ID NO:4, 5, 6, 11, 12, 13, 14, 19, 21, 22, 23, 24, 105, 111,
117 or 123; or (c) a VH sequence as in (a) and a VL sequence as in
(b). In one such aspect, the antibody comprises a VL sequence of
SEQ ID NO: 4 and a VH sequence of SEQ ID NO: 7. In another such
aspect, the antibody comprises a VL sequence of SEQ ID NO: 5 and a
VH sequence of SEQ ID NO: 8. In another such aspect, the antibody
comprises a VL sequence of SEQ ID NO: 5 and a VH sequence of SEQ ID
NO: 9. In another such aspect, the antibody comprises a VL sequence
of SEQ ID NO: 6 and a VH sequence of SEQ ID NO: 10. In another such
aspect, the antibody comprises a VL sequence of SEQ ID NO: 11 and a
VH sequence of SEQ ID NO: 15. In another such aspect, the antibody
comprises a VL sequence of SEQ ID NO: 12 and a VH sequence of SEQ
ID NO: 16. In another such aspect, the antibody comprises a VL
sequence of SEQ ID NO: 13 and a VH sequence of SEQ ID NO: 17. In
another such aspect, the antibody comprises a VL sequence of SEQ ID
NO: 14 and a VH sequence of SEQ ID NO: 18. In another such aspect,
the antibody comprises a VL sequence of SEQ ID NO: 19 and a VH
sequence of SEQ ID NO: 20. In another such aspect, the antibody
comprises a VL sequence of SEQ ID NO: 21 and a VH sequence of SEQ
ID NO: 25. In another such aspect, the antibody comprises a VL
sequence of SEQ ID NO: 22 and a VH sequence of SEQ ID NO: 26. In
another such aspect, the antibody comprises a VL sequence of SEQ ID
NO: 23 and a VH sequence of SEQ ID NO: 27. In another such aspect,
the antibody comprises a VL sequence of SEQ ID NO: 24 and a VH
sequence of SEQ ID NO: 28. In another such aspect, the antibody
comprises a VL sequence of SEQ ID NO: 105 and a VH sequence of SEQ
ID NO: 108. In another such aspect, the antibody comprises a VL
sequence of SEQ ID NO: 111 and a VH sequence of SEQ ID NO: 114. In
another such aspect, the antibody comprises a VL sequence of SEQ ID
NO: 117 and a VH sequence of SEQ ID NO: 120.
[0046] In another such aspect, the antibody comprises a VL sequence
of SEQ ID NO: 123 and a VH sequence of SEQ ID NO: 126. In another
such aspect, the antibody comprises a VH sequence of (a) SEQ ID NO:
108, (b) SEQ ID NO: 114, (c) SEQ ID NO: 120 or (d) SEQ ID NO: 126.
In another such aspect, the antibody comprises a VL sequence of (a)
SEQ ID NO: 105, (b) SEQ ID NO: 111, (c) SEQ ID NO: 117, or (d) SEQ
ID NO: 123.
[0047] In one aspect of the embodiment above, the antibody is
selected from the group consisting of antibodies 7A4, 8A2, 15D2,
10D11, 7B10, 15G11, 16G5, 13C3, 16G4, 16F6, 7G7, 4C2, 1B12, and
13D4. In one such aspect, the antibody is 7A4. In another such
aspect, the antibody is 8A2. In another such aspect, the antibody
is 15D2. In another such aspect, the antibody is 10D11. In another
such aspect, the antibody is 7B10. In another such aspect, the
antibody is 15G11. In another such aspect, the antibody is 16G5. In
another such aspect, the antibody is 13C3. In another such aspect,
the antibody is 16G4. In another such aspect, the antibody is 16F6.
In another such aspect, the antibody is 7G7. In another such
aspect, the antibody is 4C2. In another such aspect, the antibody
is 1B12. In another such aspect, the antibody is 13D4.
[0048] In one aspect of any of the foregoing, the antibody is
further affinity matured. In one such aspect, the antibody is
selected from the group consisting of 15G11.v1, 15G11.v2, 15G11.v3,
15G11.v4, 15G11.v5, 7A4.v1; 7A4.v2, 7A4.v3, 7A4.v4, 7A4.v5, 7A4.v6,
7A4.v7, 7A4.v8, 7A4.v9, 7A4.v10, 7A4.v11, 7A4.v12, 7A4.v13,
7A4.v14, 7A4.v15, 7G7.v1, 16F6.v1, 16F6.v2, 16F6.v3 and 16F6.v4. In
one such aspect, the antibody is 15G11.v1. In another such aspect,
the antibody is 15G11.v2. In another such aspect, the antibody is
15G11.v3. In another such aspect, the antibody is 15G11.v4. In
another such aspect, the antibody is 15G11.v5. In another such
aspect, the antibody is 7A4.v1. In another such aspect, the
antibody is 7A4.v2. In another such aspect, the antibody is 7A4.v3.
In another such aspect, the antibody is 7A4.v4. In another such
aspect, the antibody is 7A4.v5. In another such aspect, the
antibody is 7A4.v6. In another such aspect, the antibody is 7A4.v7.
In another such aspect, the antibody is 7A4.v8. In another such
aspect, the antibody is 7A4.v9. In another such aspect, the
antibody is 7A4.v10. In another such aspect, the antibody is
7A4.v11. In another such aspect, the antibody is 7A4.v12. In
another such aspect, the antibody is 7A4.v13. In another such
aspect, the antibody is 7A4.v14. In another such aspect, the
antibody is 7A4.v15. In another such aspect, the antibody is
7G7.v1. In another such aspect, the antibody is 16F6.v1. In another
such aspect, the antibody is 16F6.v2. In another such aspect, the
antibody is 16F6.v3. In another such aspect, the antibody is
16F6.v4. In another such aspect, the antibody is 15G11.N52A. In
another such aspect, the antibody is 16F6.v4. In another such
aspect, the antibody is 15G11.T53A. In another such aspect, the
antibody is 16F6.v4. In another such aspect, the antibody is
15G11.W92A. In another such aspect, the antibody is 16F6.v4.
[0049] In one aspect of the above embodiment, the antibody is
modified at one or more amino acid positions in the VH or VL to the
amino acid indicated for that position in FIGS. 4E-1 and 4E-2. In
another aspect of the above embodiment, the antibody comprises a
sequence or one or more HVR sequences corresponding to that of or
one or more of those set forth for any one of the clones in FIGS. 3
and 4 and FIGS. 4E-1 and 4E-2. In another aspect of the above
embodiment, the antibody comprises a VH or VL sequence
corresponding to that of set forth for any one of the clones in
FIGS. 3 and 4 and FIGS. 4E-1 and 4E-2. In another aspect of the
above embodiment, the antibody comprises one or more HVR sequences
corresponding to one or more of those set forth for any one of the
clones in FIGS. 3 and 4 and FIGS. 4E-1 and 4E-2.
[0050] In one aspect of the above embodiment, the one or more
properties of the antibody 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 TfR. In one such
aspect, the property is the effector function of the antibody Fc
region. In another such aspect, the property is the complement
activation function of the antibody. In another such aspect, the
property is the affinity of the antibody for TfR. In one such
aspect, the effector function or complement activation function has
been reduced or eliminated relative to a wild-type antibody of the
same isotype. In one aspect, 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.
[0051] In one such aspect, the effector function is reduced or
eliminated by reduction of glycosylation of the antibody. In one
such aspect, 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. In one such aspect, the glycosylation of the antibody is
reduced by a production of the antibody in an environment that does
not permit wild-type glycosylation, such as production in a
non-mammalian cell production system or where the antibody is
produced synthetically. 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
modification of the antibody such that wild-type glycosylation does
not occur, such as 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.
[0052] In another such aspect, the effector function is reduced or
eliminated by at least one modification of the Fc region. In one
such aspect, 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. In another such aspect,
the at least one modification of the Fc region 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. 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).
[0053] In one aspect, the antibody the half-life of the antibody is
increased by a modification in the FcRn binding region. In one
aspect, the modification is a substitution in an amino acid
selected from the following positions: 251 256, 285, 290, 308, 314,
385, 389, 428, 434, 436, 238, 265, 272, 286, 303, 305, 307, 311,
312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434.
In one aspect the modification is a substitution selected from the
following: M252Y, S254T, T256E, N434A and Y436I.
[0054] In one aspect, the antibody is combined with a further
compound that mitigates or contributes to the reduction of impact
on reticulocyte levels or acute clinical symptoms. In one such
aspect, the further compound protects reticulocytes from
antibody-related depletion or supports the growth, development, or
reestablishment of reticulocytes. In another such aspect, the
further compound is selected from erythropoietin (EPO), an iron
supplement, vitamin C, folic acid, and vitamin B12, or is red blood
cells or reticulocytes.
[0055] In one aspect of the above embodiment, the affinity of the
antibody for TfR is decreased, as measured relative to a wild-type
antibody of the same isotype not having lowered affinity for TfR.
In one such aspect, the antibody has a KD or IC50 for TfR of about
1 pM to about 100 .mu.M. 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.
[0056] In one aspect of the above embodiment, the antibody is
coupled to a therapeutic compound. In another aspect of the above
embodiment, the antibody is coupled to an imaging agent or a label.
In one such aspect, the antibody is a multispecific antibody and
the therapeutic compound optionally forms one portion of the
multispecific antibody. In one such aspect, the multispecific
antibody comprises a first antigen binding site which binds TfR and
a second antigen binding site which binds a brain antigen. In one
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 E (ApoE), 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. 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 therapeutic compound is a neurological
disorder drug.
[0057] In one aspect of the above embodiment, the invention
provides an isolated nucleic acid encoding any of the foregoing
antibodies. In another aspect, the invention provides a host cell
comprising such nucleic acid. In another aspect, the invention
provides a method of producing any of the foregoing antibodies
comprising culturing such host cell so that the antibody is
produced and optionally further comprising recovering the antibody
from the host cell.
[0058] In one aspect of the above embodiment, the invention
provides a pharmaceutical formulation comprising any of the
foregoing antibodies and a pharmaceutically acceptable carrier.
[0059] In one aspect of the above embodiment, the invention
provides any of the foregoing antibodies for use as a medicament.
In another aspect of the above embodiment, the invention provides
the use of any of the foregoing antibodies in the manufacture of a
medicament for treating a neurological disorder. In one such
aspect, the neurological disorder is selected from the group
consisting of a neuropathy disorder, a neurodegenerative disease,
cancer, an ocular disease disorder, a seizure disorder, a lysosomal
storage disease, amyloidosis, a viral or microbial disease,
ischemia, a behavioral disorder, and CNS inflammation.
[0060] In another aspect of the above embodiment, the invention
provides any of the foregoing antibodies for use in treating a
neurological disorder. In one such aspect, the neurological
disorder is selected from the group consisting of a neuropathy
disorder, a neurodegenerative disease, cancer, an ocular disease
disorder, a seizure disorder, a lysosomal storage disease,
amyloidosis, a viral or microbial disease, ischemia, a behavioral
disorder, and CNS inflammation.
[0061] In another aspect of the above embodiment, the invention
provides any of the foregoing antibodies for use in transporting
one or more compounds across the BBB. In another aspect of the
above embodiment, use of any of the foregoing antibodies in the
manufacture of a medicament for transporting one or more compounds
across the BBB is provided.
[0062] In one aspect of the above embodiment, a method of
transporting a compound across the BBB in a subject is provided,
comprising exposing any of the foregoing antibodies to the BBB such
that the antibody transports the compound coupled thereto across
the BBB. In another such aspect, the BBB is in a human subject. In
another such aspect, the dose amount and/or frequency of
administration is modulated to reduce the concentration of antibody
to which red blood cells are exposed. In another such aspect, the
method further comprises the step of monitoring the subject for
depletion of red blood cells. In another such aspect, the antibody
coupled to the compound is administered at a therapeutic dose. In
one such aspect, the therapeutic dose is TfR-saturating. In another
such aspect, administration of the antibody is at a dose and/or
dose frequency calibrated to minimize acute clinical symptoms of
the antibody administration.
[0063] In another aspect of the above embodiment, a method of
increasing exposure of the CNS of a subject to a compound is
provided, comprising exposing any of the foregoing antibodies to
the BBB such that the antibody transports the compound coupled
thereto across the BBB. In another such aspect, the BBB is in a
human subject. In another such aspect, the dose amount and/or
frequency of administration is modulated to reduce the
concentration of antibody to which red blood cells are exposed. In
another such aspect, the method further comprises the step of
monitoring the subject for depletion of red blood cells. In another
such aspect, the antibody coupled to the compound is administered
at a therapeutic dose. In one such aspect, the therapeutic dose is
TfR-saturating. In another such aspect, administration of the
antibody is at a dose and/or dose frequency calibrated to minimize
acute clinical symptoms of the antibody administration.
[0064] In one aspect of the above embodiment, a method of
increasing rentention in the CNS of a compound administered to a
subject is provided, comprising exposing any of the foregoing
antibodies to the BBB such that the retention in the CNS of the
compound is increased. In another such aspect, the BBB is in a
human subject. In another such aspect, the dose amount and/or
frequency of administration is modulated to reduce the
concentration of antibody to which red blood cells are exposed. In
another such aspect, the method further comprises the step of
monitoring the subject for depletion of red blood cells. In another
such aspect, the antibody coupled to the compound is administered
at a therapeutic dose. In one such aspect, the therapeutic dose is
TfR-saturating. In another such aspect, administration of the
antibody is at a dose and/or dose frequency calibrated to minimize
acute clinical symptoms of the antibody administration.
[0065] In one aspect of the above embodiment, a method of treating
a neurological disorder in a mammal is provided, comprising
treating the mammal with any of the foregoing antibodies. In one
such aspect, the neurological disorder is selected from the group
consisting of a neuropathy disorder, a neurodegenerative disease,
cancer, an ocular disease disorder, a seizure disorder, a lysosomal
storage disease, amyloidosis, a viral or microbial disease,
ischemia, a behavioral disorder, and CNS inflammation. In another
such aspect, the neurological disorder is in a human subject. In
another such aspect, the dose amount and/or frequency of
administration is modulated to reduce the concentration of antibody
to which the re blood cells are exposed. In another such aspect,
the method further comprises the step of monitoring the subject for
depletion of red blood cells. In another such aspect, the antibody
coupled to the compound is administered at a therapeutic dose. In
one such aspect, the therapeutic dose is TfR-saturating. In another
such aspect, administration of the antibody is at a dose and/or
dose frequency calibrated to minimize acute clinical symptoms of
the antibody administration.
[0066] In another embodiment, the invention provides an isolated
antibody that binds to the same epitope on TfR as an antibody
selected from the group consisting of antibodies 7A4, 8A2, 15D2,
101D11, 7B10, 15G11, 16G5, 13C3, 16G4, 16F6, 7G7, 4C2, 1B12, 13D4,
15G11.v1, 15G11.v2, 15G11.v3, 15G11.v4, 15G11.v5, 7A4.v1; 7A4.v2,
7A4.v3, 7A4.v4, 7A4.v5, 7A4.v6, 7A4.v7, 7A4.v8, 7A4.v9, 7A4.v10,
7A4.v11, 7A4.v12, 7A4.v13, 7A4.v14, 7A4.v15, 7G7.v1, 16F6.v1,
16F6.v2, 16F6.v3, 16F6.v4 15G11.N52A, 15G11.T53A and 15G11W92A.
[0067] In another embodiment, a method of decreasing clearance of a
compound administered to a subject is provided, wherein the
compound is coupled to an antibody which binds with low affinity to
TfR, 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.
[0068] In another embodiment, a method of optimizing the
pharmcokinetics and/or pharmacodynamics of a compound to be
efficacious in the CNS in a subject is provided, wherein the
compound is coupled to an antibody which binds with low affinity to
TfR, and the antibody is selected such that its affinity for TfR
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.
[0069] 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
[0070] FIG. 1 depicts a three-dimensional crystal structure of a
TfR dimer in complex with Tf, based on the pdb file 3SM9. The
non-Tf-binding apical region of TfR is labeled.
[0071] FIGS. 2A and 2B depict FACS analysis of mouse hybridoma
parental clone supernatants binding to human and cynomolgus TfR
transiently expressed in 293 cells in the presence of 1 .mu.M human
holo-Tf Unless otherwise indicated, the filled grey trace in each
graph is background from the detection antibody, the medium grey
trace is binding to 293 cells that endogenously express basal
levels of human TfR, the bold black trace represents binding to
transiently expressed human TfR and the thin grey trace represents
binding to transiently expressed cyno TfR.
[0072] FIG. 2C depicts the results of human/cynomolgous
cross-reactive antibody competition assays as described in Example
1. Nine of the fourteen clones were found to block binding of the
apical binding antibody displayed on phage.
[0073] FIGS. 3A-1, 3A-2, 3B-1, 3B-2, 3C-1, 3C-2, 3D-land 3D-2
depict the heavy and light chain variable region sequences of
hybridoma clones that bind to apical and non-apical regions of TfR.
The sequences can be further subdivided by epitope and sequence
similarity into class I-III (apical binders) and class IV
(non-apical binders). The HVRs according to Kabat are indicated by
underlining.
[0074] FIGS. 4A-1, 4A-2, 4B-1, 4B-2, 4C-1, 4C-2, 4D-1 and 4D-2
depict alignments of humanized sequences for (A) 15G11, (B)
7A4/8A2, (C) 7G7 and (D) 16F6. Each mouse light or heavy variable
domain sequence (second line) is aligned to the closest human
germline or consensus variable domain (first line). The humanized
version for each antibody is shown at the bottom (third line).
Differences from the human germline or consensus sequences are
shaded. HVR sequences that were grafted into the human framework
are boxed. CDR definitions according to Kabat are indicated.
[0075] FIGS. 4E-1 and 4E-2 show that for the class I-III groups of
antibodies, variant forms of the antibodies with modifications at
one or more residues of an FR retained affinity and binding
specifity.
[0076] FIG. 5 depicts the binding of hu7A4.v15, hu15G11.v5 and
hu7G7.v1 to huTfR in the presence of 6.3 .mu.M holo-Tf Antibody
binding to immobilized huTfR is shown in the presence (open sybols
and dashed lines) or absence (filled symbols and solid lines) of
6.3 .mu.M holo-Tf.
[0077] FIGS. 6A and 6B depict the results of the HFE-HuTfR binding
and the HFE blocking assays described in Example 1. FIG. 6A shows
the binding of antibody to increasing concentrations of huTfR
captured via immobilized HFE. FIG. 6B shows the binding of huTfR to
immobilized HFE in the presence of increasing concentrations of
antibody.
[0078] FIGS. 7A and 7B depict binding analyses of 15G11.v5 and
7A4.v5 IgG and Fab Ala variants on cyno and human TfR,
demonstrating the effects on affinity of Ala mutations in CDR-L3
and CDR-H3 of each antibody assessed as IgG by ELISA binding and
IgG or Fab by SPR analysis to immobilized human or cyno TfR, as
described in Example 2.
[0079] FIGS. 8A and 8B and FIGS. 9A-B depict the results of
experiments assessing the impact of effector function status on
ADCC activity of anti-human TfR ("anti-hTFR") antibodies in primary
human bone marrow mononuclear cells or in a human erythroblast cell
line, as described in Example 4.
[0080] FIG. 10 depicts the dosing and sampling scheme for the
primate study described in Example 5.
[0081] FIGS. 11A and 11B depict the pharmacokinetic results of the
experiments described in Example 5, specifically individual and
group mean anti-TfR.sup.1/BACE1, anti-TfR.sup.2/BACE1 and anti-gD
serum concentrations versus time following a single IV bolus
administration at 30 mg/kg in cynomolgus monkeys in serum (FIG.
11A) and CSF (FIG. 11).
[0082] FIGS. 12A, 12B, 12C, 12D and 12E depict the pharmacodynamic
results of the experiments described in Example 5, specifically
individual and group mean anti-TfR.sup.1/BACE1,
anti-TfR.sup.2/BACE1 and anti-gD plasma (A) or CSF (B-E)
concentrations versus time following a single IV bolus
administration at 30 mg/kg in cynomolgus monkeys. The upper panels
show Abeta1-40 levels in plasma (FIG. 12A) and CSF (FIG. 12B),
while the lower panels show soluble APPa levels (FIG. 12C), soluble
APPP levels (FIG. 12D), and sAPP.beta./sAPP.alpha. ratio (FIG. 12E)
over time.
[0083] FIGS. 13A, 13B, 13C and 13D depict the results of
hematological sampling performed during the studies described in
Example 5. At each of the indicated time points, total
reticulocytes (FIG. 13A), red blood cells (FIG. 13B), hemoglobin
(FIG. 13D) and the percentage of immature reticulocytes in the
total reticulocyte pool (FIG. 13C) were measured using standard
techniques.
[0084] FIG. 14 depicts the dosing and sampling scheme for the
primate study described in Example 6.
[0085] FIGS. 15A and 15B depict the pharmacodynamic results (A) and
brain antibody concentrations (B) of the experiments described in
Example 6. Specifically, FIG. 15A shows individual and group mean
anti-TfR/BACE1, anti-TfR.sup.2/BACE1, anti-gD, and anti-BACE1
ration of sAPPB/sAPPa in CSF versus time following a single IV
bolus administration at 30 mg/kg in cynomolgus monkeys. FIG. 15B
show individual ani-TfR.sup.1/BACE1, anti-TfR.sup.2/BACE1, anti-gD,
and anti-BACE1 concentrations of antibody in various brain regions
at 24 hours post-dose.
[0086] FIGS. 16A and 16B 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. 16A depicts the variable
light (VL) sequence alignments (SEQ ID NOs. 132-137). FIG. 16B
depicts the variable heavy (VH) sequence alignments (SEQ ID Nos.
138-139). 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).
[0087] FIGS. 17A and 17B 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. 17A depicts the light chain
sequence alignments (SEQ ID NOs. 140-143). FIG. 17B depicts the
heavy chain sequence alignments (SEQ ID NO. 144). In both figures,
the HVR sequences for each clone are indicated by the boxed
regions, with the first box indicating HVR-L1 (FIG. 17A) or HVR-H1
(FIG. 17B), the second box indicating HVR-L2 (FIG. 17A) or HVR-H2
(FIG. 17B), and the third box indicating HVR-L3 (FIG. 17A) or
HVR-H3 (FIG. 17B).
[0088] FIGS. 18A and 18B depict the heavy chain (FIG. 18A; SEQ ID
NO. 145) and light chain (FIG. 18B; SEQ ID NO. 146) of an exemplary
anti-Abeta antibody.
[0089] FIG. 19 depicts the pharmacokinetic properties of
Anti-TfR.sup.1/BACE1, Anti-Tfr.sup.52A/BACE1 and
Anti-TfR.sup.53A/BACE1 as described in Example 5.
[0090] FIG. 20 depicts the pharmacokinetic properties of the murine
IgG2a Anti-TfR.sup.D/BACE1 and Anti-gD antibodies with the Fc
effector function LALAPG mutations as described in Example 7.
[0091] FIGS. 21A and 21B depict the total and immature reticulocyte
count in mice 24 hours after administration of a 50 mg/kg dose of
the murine IgG2a Anti-TfR.sup.D/BACE1 and Anti-gD antibodies with
the Fc effector function LALAPG mutations as described in Example
7.
[0092] FIG. 22 depicts the total reticulocyte count in mice 24
hours after administration of a 50 mg/kg dose of the
anti-TfR.sup.52A/BACE1 (N297G), anti-TfR.sup.52A/BACE1 (LALAPG),
anti-TfR.sup.52A/BACE1 (LALAPG/YTE), TfR.sup.52A/BACE1 (LALAPG/AI)
antibodies in human transferrin receptor knock-in mice as described
in Example 8.
[0093] FIG. 23 depicts the results of experiments assessing the
impact of effector function status on ADCC activity of anti-TfR/gD,
anti-TfR/BACE1 (N297G), anti-TfR/BACE1 (LALAPG), anti-TfR/BACE1
(N297G/434A/436I) and anti-TfR/BACE1 (LALAPG/YTE) antibodies in
primary human bone marrow mononuclear cells or in a human
erythroblast cell line, as described in Example 8.
[0094] FIGS. 24A and 24B depict the heavy and light chain variable
region sequences of 1511Gv.5 (light chain--SEQ ID NO: 105 and heavy
chain--SEQ ID NO: 108) and affinity variants 15G11.52A (light
chain--SEQ ID NO:105 and heavy chain--SEQ ID NO: 153), 15G11.53A
(light chain--SEQ ID NO: 105 and heavy chain--SEQ ID NO: 154) and
15G11.92A (light chain--SEQ ID NO: 151 and heavy chain--SEQ ID NO:
108). The HVRs according to Kabat are indicated by underlining.
[0095] FIG. 25 depicts a competition assay between 15G11v.5 and
anti-TfR.sup.C12 as described in Example 1.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
1. Definitions
[0096] "Affinity" refers to the strength of the sum total of
noncovalent interactions between a single binding site of a
molecule (e.g., and antibody) and its binding partner (e.g., an
antigen). Unless indicted otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (KD, which is
a ratio of the off-rate of X from Y (kd or Koff) to the on-rate of
X to Y (ka or kon)). A surrogate measurement for the affinity of
one or more antibodies for its target is its half maximal
inhibitory concentration (IC50), a measure of how much of the
antibody is needed to inhibit the binding of a known ligand to the
antibody target by 50%. Affinity can be measured by common methods
known in the art, including those described herein. Specific
illustrative and exemplary embodiments for measuring binding
affinity are described herein. 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.
[0097] The terms "amyloid beta," "beta-amyloid," "Abeta,"
"amyloido," and "Ap", used interchangeably herein, refer to the
fragment of amyloid precursor protein ("APP") that is produced upon
.beta.-secretase 1 ("BACE1") cleavage of APP, as well as
modifications, fragments and any functional equivalents thereof,
including, but not limited to, A.beta..sub.1-40, and
A.beta..sub.1-42. AP is known to exist in monomeric form, as well
as to associate to form oligomers and fibril structures, which may
be found as constituent members of amyloid plaque. The structure
and sequences of such A peptides are well known to one of ordinary
skill in the art and methods of producing said peptides or of
extracting them from brain and other tissues are described, for
example, in Glenner and Wong, Biochem Biophys Res. Comm. 129:
885-890 (1984). Moreover, A peptides are also commercially
available in various forms.
[0098] "Anti-Abeta immunoglobulin," "anti-Abeta antibody," and
"antibody that binds Abeta" are used interchangeably herein, and
refer to an antibody that specifically binds to human Abeta. A
nonlimiting example of an anti-Abeta antibody is crenezumab. Other
non-limiting examples of anti-Abeta antibodies are solanezumab,
bapineuzumab, gantenerumab, aducanumab, ponezumab and any
anti-Abeta antibodies disclosed in the following publications:
WO2000162801, WO2002046237, WO2002003911, WO2003016466,
WO2003016467, WO2003077858, WO2004029629, WO2004032868,
WO2004032868, WO2004108895, WO2005028511, WO2006039470,
WO2006036291, WO2006066089, WO2006066171, WO2006066049,
WO2006095041, WO2009027105.
[0099] The terms "crenezumab" and "MABT5102A" are used
interchangeably herein, and refer to a specific anti-Abeta antibody
that binds to monomeric, oligomeric, and fibril forms of Abeta, and
which is associated with CAS registry number 1095207. In one
embodiment, such antibody comprises sequences set forth in FIGS.
18A and 18B.
[0100] "Apolipoprotein E4 carrier" or "ApoE4 carrier," used
interchangeably herein with "apolipoprotein E4 positive" or "ApoE4
positive," refers to an individual having at least one
apolipoprotein E4 (or "ApoE4") allele. An individual with zero
ApoE4 alleles is referred to herein as being "ApoE4 negative" or an
"ApoE4 non-carrier." See also Prekumar, et al., 1996, Am. J Pathol.
148:2083-95.
[0101] The term "cerebral vasogenic edema" refers to an excess
accumulation of intravascular fluid or protein in the intracellular
or extracellular spaces of the brain. Cerebral vasogenic edema is
detectable by, e.g., brain MRI, including, but not limited to FLAIR
MRI, and can be asymptomatic ("asymptomatic vasogenic edema") or
associated with neurological symptoms, such as confusion,
dizziness, vomiting, and lethargy ("symptomatic vasogenic edema")
(see Sperling et al. Alzheimer's & Dementia, 7:367, 2011).
[0102] The term "cerebral macrohemorrhage" refers to an
intracranial hemorrhage, or bleeding in the brain, of an area that
is more than about 1 cm in diameter. Cerebral macrohemorrhage is
detectable by, e.g., brain MRI, including but not limited to
T2*-weighted GRE MRI, and can be asymptomatic ("asymptomatic
macrohemorrhage") or associated with symptoms such as transient or
permanent focal motor or sensory impairment, ataxia, aphasia, and
dysarthria ("symptomatic macrohemorrhage") (see, e.g., Chalela J A,
Gomes J. Expert Rev. Neurother. 2004 4:267, 2004 and Sperling et
al. Alzheimer's & Dementia, 7:367, 2011).
[0103] The term "cerebral microhemorrhage" refers to an
intracranial hemorrhage, or bleeding in the brain, of an area that
is less than about 1 cm in diameter. Cerebral microhemorrhage is
detectable by, e.g., brain MRI, including, but not limited to
T2*-weighted GRE MRI, and can be asymptomatic ("asymptomatic
microhemorrhage") or can potentially be associated with symptoms
such as transient or permanent focal motor or sensory impairment,
ataxia, aphasia, and dysarthria ("symptomatic microhemorrhage").
See, e.g., Greenberg, et al., 2009, Lancet Neurol. 8:165-74.
[0104] The term "sulcal effusion" refers to an effusion of fluid in
the furrows, or sulci, of the brain. Sulcal effusions are
detectable by, e.g., brain MRI, including but not limited to FLAIR
MRI. See Sperling et al. Alzheimer's & Dementia, 7:367,
2011.
[0105] The term "superficial siderosis of the central nervous
system" refers to bleeding or hemorrhage into the subarachnoid
space of the brain and is detectable by, e.g., brain MRI, including
but not limited to T2*-weighted GRE MRI. Symptoms indicative of
superficial siderosis of the central nervous system include
sensorineural deafness, cerebellar ataxia, and pyramidal signs. See
Kumara-N, Am J Neuroradiol. 31:5, 2010.
The term "amyloidosis," as used herein, refers to a group of
diseases and disorders caused by or associated with amyloid or
amyloid-like proteins and includes, but is not limited to, diseases
and disorders caused by the presence or activity of amyloid-like
proteins in monomeric, fibril, or polymeric state, or any
combination of the three, including by amyloid plaques. Such
diseases include, but are not limited to, secondary amyloidosis and
age-related amyloidosis, such as diseases including, but not
limited to, neurological disorders such as Alzheimer's Disease
("AD"), diseases or conditions characterized by a loss of cognitive
memory capacity such as, for example, mild cognitive impairment
(MCI), Lewy body dementia, Down's syndrome, hereditary cerebral
hemorrhage with amyloidosis (Dutch type), the Guam
Parkinson-Demential complex and other diseases which are based on
or associated with amyloid-like proteins such as progressive
supranuclear palsy, multiple sclerosis, Creutzfeld Jacob disease,
Parkinson's disease, HIV-related dementia, ALS (amyotropic lateral
sclerosis), inclusion-body myositis (IBM), adult onset diabetes,
endocrine tumor and senile cardiac amyloidosis, and various eye
diseases including macular degeneration, drusen-related optic
neuropathy, glaucoma, and cataract due to beta-amyloid deposition.
Glaucoma is a group of diseases of the optic nerve involving loss
of retinal ganglion cells (RGCs) in a characteristic pattern of
optic neuropathy. RGCs are the nerve cells that transmit visual
signals from the eye to the brain. Caspase-3 and Caspase-8, two
major enzymes in the apoptotic process, are activated in the
process leading to apoptosis of RGCs. Caspase-3 cleaves amyloid
precursor protein (APP) to produce neurotoxic fragments, including
Abeta. Without the protective effect of APP, Abeta accumulation in
the retinal ganglion cell layer results in the death of RGCs and
irreversible loss of vision. Glaucoma is often, but not always,
accompanied by an increased eye pressure, which may be a result of
blockage of the circulation of aqueous, or its drainage. Although
raised intraocular pressure is a significant risk factor for
developing glaucoma, no threshold of intraocular pressure can be
defined which would be determinative for causing glaucoma. The
damage may also be caused by poor blood supply to the vital optic
nerve fibers, a weakness in the structure of the nerve, and/or a
problem in the health of the nerve fibers themselves. Untreated
glaucoma leads to permanent damage of the optic nerve and resultant
visual field loss, which can progress to blindness. The term "mild
Alzheimer's Disease" or "mild AD" as used herein (e.g., a "patient
diagnosed with mild AD") refers to a stage of AD characterized by
an MMSE score of 20 to 26. The term "mild to moderate Alzheimer's
Disease" or "mild to moderate AD" as used herein encompasses both
mild and moderate AD, and is characterized by an MMSE score of 18
to 26.
[0106] The term "moderate Alzheimer's Disease" or "moderate AD" as
used herein (e.g., a "patient diagnosed with moderate AD") refers
to a stage of AD characterized by an MMSE score of 18 to 19.
[0107] The "central nervous system" or "CNS" refers to the complex
of nerve tissues that control bodily function, and includes the
brain and spinal cord.
[0108] 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 (Glutl) and heparin-binding epidermal growth factor-like growth
factor (HB-EGF). An exemplary BBB-R herein is transferrin receptor
(TfR).
[0109] The term "transferrin receptor" or "TfR", as used herein,
refers to any native TfR 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 TfR as well as any form of TfR that
results from processing in the cell. The term also encompasses
naturally occurring variants of TfR, e.g., splice variants or
allelic variants. 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 ("hTfR") comprising the
amino acid sequence as set forth in Schneider et al. Nature 311:
675-678 (1984), for example (SEQ ID NO: 1). In another embodiment,
the TfR herein is primate TfR ("pTfR") comprising the amino acid
sequence as set forth in Genbank reference AFD18260.1 (SEQ ID NO:
2). For comparison, the mouse TfR sequence may be found in Genbank
reference AAH54522.1 (SEQ ID NO: 3).
[0110] 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).
[0111] 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 (GDNF) Parkinson's disease
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 cancer pertuzumab,
etc.) Anti-VEGF antibody (e.g., bevacizumab) Recurrent or newly
diagnosed glioblastoma, recurrent malignant glioma, brain
metastasis
[0112] An "imaging agent" is acompound that has one or more
properties that permit its presence and/or location to bedetected
directly or indirectly. Examples of such imaging agents include
proteins and small molecule compounds incorporating alabeled moiety
that permits detection.
[0113] A "CNS antigen" or "brain antigen" is anantigen 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
(L6R), TNF receptor 1 (TNFR1), interleukin 1 beta (IL1.beta.), and
caspase 6. In one embodiment, the antigen is BACE1.
[0114] 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.
[0115] 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 0.001 nM (e.g. 10.sup.-8 M or less, e.g. from
10.sup.-8 M 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).
[0116] 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.
[0117] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g. bispecific antibodies), and antibody fragments so
long as they exhibit the desired antigen-binding activity.
[0118] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. 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', Fab'-SH, F(ab')2, and Fv; 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).
[0119] 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, e.g., containing naturally occurring mutations
or that may arise during production of the monoclonal antibody,
such variants generally being present in minor amounts. In contrast
to polyclonal antibody preparations, which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody of a monoclonal antibody
preparation is directed against a single determinant on the
antigen. 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 a variety of techniques, including but not
limited to the hybridoma method (see, e.g., Kohler et al., Nature,
256:495 (1975)), recombinant DNA methods (see, e.g., U.S. Pat. No.
4,816,567), phage-display methods (e.g., using the techniques
described in Clackson et al., Nature, 352:624-628 (1991) and Marks
et al., J. Mol. Biol., 222:581-597 (1991)), and methods utilizing
transgenic animals containing all or part of the human
immunoglobulin loci, such methods and other exemplary methods for
making monoclonal antibodies being described herein. Specific
examples of monoclonal antibodies herein include chimeric
antibodies, humanized antibodies, and human antibodies, including
antigen-binding fragments thereof.
[0120] 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)).
[0121] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a light chain
variable domain (VL) framework or a heavy chain variable domain
(VH) framework derived from a human immunoglobulin framework or a
human consensus framework, as defined below. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain amino acid sequence changes. In some
embodiments, the number of amino acid changes are 10 or less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less. In some embodiments, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin
framework sequence or human consensus framework sequence.
[0122] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residues in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, NIH
Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al., supra. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al., supra.
[0123] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human antibodies. For the most part, humanized antibodies are
human antibodies (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. For example, in certain
embodiments, a humanized antibody will comprise substantially all
of at least one, and typically two, variable domains, in which all
or substantially all of the HVRs (e.g., CDRs) correspond to those
of a non-human antibody, and all or substantially all of the
framework regions (FRs) correspond to those of a human antibody. In
some instances, 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
certain embodiments, a 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 antibody and all or
substantially all of the FRs are those of a human antibody, except
for FR substitution(s) as noted above. The humanized antibody
optionally also will comprise at least a portion of an antibody
constant region, typically that of a human antibody. A "humanized
form" of an antibody, e.g., a non-human antibody, refers to an
antibody that has undergone humanization. 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).
[0124] A "human antibody" herein is an antibody comprising an amino
acid sequence structure that corresponds with the amino acid
sequence structure of an antibody produced by a human or a human
cell or derived from a non-human source that utilizes human
antibody repertoires or other human antibody-encoding sequences.
This definition of a human antibody specifically excludes a
humanized antibody comprising non-human antigen-binding residues.
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.
[0125] A "multispecific antibody" herein is an antibody having
binding specificities for at least two different epitopes.
Exemplary multispecific antibodies may bind both a TfR and a brain
antigen. Multispecific antibodies can be prepared as full-length
antibodies or antibody fragments (e.g. F(ab')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.
[0126] 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.).
[0127] 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.
[0128] 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 TfR and conjugated
compound by one of ordinary skill in the art.
[0129] 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.
[0130] 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 (GcNAc) 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).
[0131] The term "hypervariable region" or "HVR" as used herein
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence ("complementarity determining
regions" or "CDRs") and/or form structurally defined loops
("hypervariable loops") and/or contain the antigen-contacting
residues ("antigen contact"). Generally, antibodies comprise six
HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2,
L3). Exemplary HVRs herein include:
[0132] (a) hypervariable loops occurring at amino acid residues
26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and
96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917
(1987));
[0133] (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56
(L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991));
[0134] (c) antigen contacts occurring at amino acid residues 27c-36
(L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101
(H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and
[0135] (d) combinations of (a), (b), and/or (c), including HVR
amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2),
26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102
(H3).
[0136] In one embodiment, HVR residues comprise those identified in
FIGS. 3A-D or 4A-D, Table 4 or Table 5 or elsewhere in the
specification.
[0137] Unless otherwise indicated, HVR residues and other residues
in the variable domain (e.g., FR residues) are numbered herein
according to Kabat et al., supra.
[0138] "Framework" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined. 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. In certain embodiments, one
or more FR residue may be modified to modulate the stability of the
antibody or to modulate the three-dimensional positioning of one or
more HVR of the antibody to, e.g., enhance binding.
[0139] 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.
[0140] The terms "full length antibody," "intact antibody," and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc region
as defined herein.
[0141] A "naked antibody" refers to an antibody that is not
conjugated to a heterologous moiety (e.g., a cytotoxic moiety or
radiolabel). The naked antibody may be present in a pharmaceutical
formulation.
[0142] "Native antibodies" refer to naturally occurring
immunoglobulin molecules with varying structures. For example,
native IgG antibodies are heterotetrameric glycoproteins of about
150,000 daltons, composed of two identical light chains and two
identical heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2 and CH3). Similarly,
from N- to C-terminus, each light chain has a variable region (VL),
also called a variable light domain or a light chain variable
domain, followed by a constant light (CL) domain. The light chain
of an antibody may be assigned to one of two types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequence of
its constant domain.
[0143] 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, 256, 265, 268, 269, 270,
272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301, 303, 311,
322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389,
414, 416, 419, 434, 435, 436, 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 and
D265A).
[0144] 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)).
[0145] 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 in the art.
[0146] 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.
[0147] The terms "host cell," "host cell line," and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cells and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein. Examples of "host
cells" for producing recombinant antibodies include: (1) mammalian
cells, for example, Chinese Hamster Ovary (CHO), COS, myeloma cells
(including YO and NSO 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.
[0148] 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.).
[0149] 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 forming one of the bispecific or
multispecific antibodies of the invention binds to the BACE1
epitope bound by YW412.8.31. An exemplary competition assay is
provided herein.
[0150] 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; and the various antitumor or anticancer
agents disclosed herein.
[0151] 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.
[0152] 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 ofImmunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.,
1991.
[0153] The term "FcRn receptor" or "FcRn" as used herein refers to
an Fc receptor ("n" indicates neonatal) which is known to be
involved in transfer of maternal IgGs to a fetus through the human
or primate placenta, or yolk sac (rabbits) and to a neonate from
the colostrum through the small intestine. It is also known that
FcRn is involved in the maintenance of constant serum IgG levels by
binding the IgG molecules and recycling them into the serum. "FcRn
binding region" or "FcRn receptor binding region" refers to that
portion an an antibody which interacts with the FcRn receptor.
Certain modifications in the FcRn binding region of an antibody
increase the affinity of the antibody or fragment thereof, for the
FcRn, and also increase the in vivo half-life of the molecule.
Amino acid substitutions in one or more of the following amino acid
positions 251256, 285, 290, 308, 314, 385, 389, 428, 434 and 436
increases the interaction of the antibody with the FcRn receptor.
Substitutions at the following positions also increases the
interaction of an antibody with the FcRn receptor 238, 265, 272,
286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378,
380, 382, 413, 424 or 434, e.g., substitution of (U.S. Pat. No.
7,371,826).
[0154] 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.
[0155] A "linker" as used herein is a structure that covalently or
non-covalently connects the anti-TfR antibody to heterologous
molecule. In certain embodiments, a linker is a peptide. In other
embodiments, a linker is a chemical linker.
[0156] 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 1123, 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.
[0157] 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.
[0158] 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).
[0159] An "isolated nucleic acid refers to a nucleic acid molecule
that has been separated from a component of its natural
environment. An isolated nucleic acid includes a nucleic acid
molecule contained in cells that ordinarily contain the nucleic
acid molecule, but the nucleic acid molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location.
[0160] "Isolated nucleic acid encoding an anti-TfR antibody" refers
to one or more nucleic acid molecules encoding antibody heavy and
light chains (or fragments thereof), including such nucleic acid
molecule(s) in a single vector or separate vectors, and such
nucleic acid molecule(s) present at one or more locations in a host
cell.
[0161] 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.
[0162] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, including digital UNIX
V4.OD. All sequence comparison parameters are set by the ALIGN-2
program and do not vary.
[0163] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby
Immunology, 6.sup.th ed., W.H. Freeman and Co., page 91 (2007).) A
single VH or VL domain may be sufficient to confer antigen-binding
specificity. Furthermore, antibodies that bind a particular antigen
may be isolated using a VH or VL domain from an antibody that binds
the antigen to screen a library of complementary VL or VH domains,
respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887
(1993); Clarkson et al., Nature 352:624-628 (1991).
[0168] The term "vector," as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors."
2. Compositions and Methods
[0169] A. Production of Anti-TfR Antibodies and Conjugates
Thereof
[0170] In one aspect, the invention is based, in part, on anti-TfR
antibodies that can be used to transport desired molecules across
the BBB. In certain embodiments, antibodies that bind to human TfR
are provided. In certain embodiments, antibodies that bind to both
human TfR and primate TfR are provided. Antibodies of the invention
are useful, e.g., for the diagnosis or treatment of diseases
affecting the brain and/or CNS.
[0171] A. Exemplary Anti-TfR Antibodies
[0172] In one aspect, the invention provides isolated antibodies
that bind to TfR. In certain embodiments, an anti-TfR antibody of
the invention binds specifically to both human TfR and primate TfR.
In certain such embodiments, an anti-TfR antibody of the invention
does not inhibit binding of transferrin to the TfR. In certain such
embodiments, an anti-TfR antibody of the invention binds to an
apical domain of TfR. In other certain such embodiments, an
anti-TfR antibody of the invention binds to a non-apical domain of
TfR. In certain aspects, the anti-TfR antibodies may be used to
transport one or more conjugated imaging or therapeutic compounds
across the BBB.
[0173] In one aspect, the invention provides an anti-TfR antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:32; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:33; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:34; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:29; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:30; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:31. In one aspect, the antibody comprises all six of the
above-recited HVR sequences. In another aspect, the antibody is
clone 7A4, as shown in FIG. 3A and Table 3.
[0174] In one aspect, the invention provides an anti-TfR antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:37; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:38; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:39; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:35; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:30; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:36. In one aspect, the antibody comprises all six of the
above-recited HVR sequences. In another aspect, the antibody is
clone 8A2, as shown in FIG. 3A and Table 3.
[0175] In one aspect, the invention provides an anti-TfR antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO: 32; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:40; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:
34; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35;
(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30; and
(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 36. In
one aspect, the antibody comprises all six of the above-recited HVR
sequences. In another aspect, the antibody is clone 15D2, as shown
in FIG. 3A and Table 3.
[0176] In one aspect, the invention provides an anti-TfR antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO: 37; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO: 43; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:
44; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 41;
(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30; and
(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In
one aspect, the antibody comprises all six of the above-recited HVR
sequences. In another aspect, the antibody is clone 10D11, as shown
in FIG. 3A and Table 3.
[0177] In one aspect, the invention provides an anti-TfR antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO: 32; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO: 33; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:
34; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 29;
(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30; and
(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:31. In
one aspect, the antibody comprises all six of the above-recited HVR
sequences. In another aspect, the antibody is clone 7B10, as shown
in FIG. 3A and Table 3.
[0178] In one aspect, the invention provides an anti-TfR antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:53; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:54; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:55; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:50; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:51; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:52. In one aspect, the antibody comprises all six of the
above-recited HVR sequences. In another aspect, the antibody is
clone 15G11, as shown in FIG. 3B and Table 3.
[0179] In one aspect, the invention provides an anti-TfR antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:53; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:58; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:59; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:56; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:57; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:52. In one aspect, the antibody comprises all six of the
above-recited HVR sequences. In another aspect, the antibody is
clone 16G5, as shown in FIG. 3B and Table 3.
[0180] In one aspect, the invention provides an anti-TfR antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:53; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:63; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:55; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:60; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:61; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:62. In one aspect, the antibody comprises all six of the
above-recited HVR sequences. In another aspect, the antibody is
clone 13C3, as shown in FIG. 3B and Table 3.
[0181] In one aspect, the invention provides an anti-TfR antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:53; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:65; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:55; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:60; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:64; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:52. In one aspect, the antibody comprises all six of the
above-recited HVR sequences. In another aspect, the antibody is
clone 16G4, as shown in FIG. 3B and Table 3.
[0182] In one aspect, the invention provides an anti-TfR antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:74; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:75; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:76; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:71; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:72; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:73. In one aspect, the antibody comprises all six of the
above-recited HVR sequences. In another aspect, the antibody is
clone 16F6, as shown in FIG. 3C and Table 3.
[0183] In one aspect, the invention provides an anti-TfR antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:80; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:81; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:82; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:77; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:78; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:79. In one aspect, the antibody comprises all six of the
above-recited HVR sequences. In another aspect, the antibody is
clone 7G7, as shown in FIG. 3D and Table 3.
[0184] In one aspect, the invention provides an anti-TfR antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO: 80; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:83; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:84; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:77; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:78; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:79. In one aspect, the antibody comprises all six of the
above-recited HVR sequences. In another aspect, the antibody is
clone 4C2, as shown in FIG. 3D and Table 3.
[0185] In one aspect, the invention provides an anti-TfR antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:88; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:89; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:90; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:85; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:86; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:87. In one aspect, the antibody comprises all six of the
above-recited HVR sequences. In another aspect, the antibody is
clone 1B12, as shown in FIG. 3D and Table 3.
[0186] In one aspect, the invention provides an anti-TfR antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:94; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:95; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:96; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:91; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:92; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:93. In one aspect, the antibody comprises all six of the
above-recited HVR sequences. In another aspect, the antibody is
clone 13D4, as shown in FIG. 3D and Table 3.
[0187] In one aspect, the invention provides an anti-TfR antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:32; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:33; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:34; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:29; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:30; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:127. In one aspect, the antibody comprises all six of the
above-recited HVR sequences. In another aspect, the antibody is
clone 7A4.v15, as shown in FIG. 4B and Table 4.
[0188] The clones above fall into four complementation groups, with
sequence similarity within the HVRs. As shown in Table 3, consensus
sequences are readily derivable from the provided antibody
sequences for each HVR. As one nonlimiting example, the class I
antibody consensus HVRs are as follows:
[0189] HVR-L1: Arg-Ala-Ser-Glu-Ser-Val-Asp-[Ser or
Asp]-Tyr-Gly-[Asn or Pro]-Ser-Phe-Met-His (SEQ ID NO: 45);
[0190] HVR-L2: Arg-Ala-Ser-Asn-Leu-Glu-Ser (SEQ ID NO: 30);
[0191] HVR-L3: Gln-[Gln or His]-Ser-Asn-Glu-[Ala, Gly or
Asp]-Pro-Pro-Thr (SEQ ID NO: 46);
[0192] HVR-H1: Asp-Tyr-[Ala or Gly]-Met-His (SEQ ID NO: 47);
[0193] HVR-H2: [Gly or Val]-Ile-Ser-[Thr, Phe or Pro]-Tyr-[Phe or
Ser]-Gly-[Arg or Lys]-Thr-Asn-Tyr-[Asn or Ser]-Gln-[Lys or
Asn]-Phe-[Lys or Met]-Gly (SEQ ID NO: 48);
[0194] HVR-H3: Gly-Leu-Ser-Gly-Asn-[Tyr or Phe]-Val-[Met or
Val]-Asp-[Tyr or Phe] (SEQ ID NO: 49). (see Table 4). The consensus
sequences for class II and IV are also provided in Table 4.
[0195] In one aspect, the invention provides an anti-TfR antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:47; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:48; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:49; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:45; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:30; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:46. In one aspect, the antibody comprises all six of the
above-recited HVR sequences.
[0196] In one aspect, the invention provides an anti-TfR antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:53; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:69; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:70; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:66; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:67; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:68. In one aspect, the antibody comprises all six of the
above-recited HVR sequences.
[0197] In one aspect, the invention provides an anti-TfR antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:101; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:97; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:98; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:99. In one aspect, the antibody comprises all six of the
above-recited HVR sequences.
[0198] In one aspect, the invention provides an antibody comprising
at least one, at least two, or all three VH HVR sequences of any of
the antibodies described above. In one embodiment, the antibody
comprises the HVR-H3 sequence of any one of the antibodies
described above. In another embodiment, the antibody comprises the
HVR-H3 and HVR-L3 sequences of any one of the antibodies described
above. In a further embodiment, the antibody comprises the HVR-H3,
HVR-L3 and HVR-H2 sequences of any one of the antibodies described
above. In another embodiment, the antibody comprises the HVR-H1,
HVR-H2 and HVR-H3 sequences of any one of the antibodies described
above. In another aspect, the invention provides an antibody
comprising at least one, at least two or all three VL HVR sequences
of any of the antibodies described above. In one embodiment, the
antibody comprises the HVR-L1, HVR-L2, and HVR-L3 sequences of any
one of the antibodies described above.
[0199] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from the HVR-H1, HVR-H2, and HVR-H3
sequences of any one of the antibodies described above; and (b) a
VL domain comprising at least one, at least two, or all three VL
HVR sequences selected from the HVR-L1, HVR-L2 and HVR-L3 sequences
of any one of the antibodies described above.
[0200] In any of the above embodiments, an anti-TfR antibody is
humanized. In one embodiment, an anti-TfR antibody comprises HVRs
as in any of the above embodiments, and further comprises an
acceptor human framework, e.g. a human immunoglobulin framework or
a human consensus framework. In another embodiment, an anti-TfR
antibody comprises HVRs as in any of the above embodiments, and
further comprises a VH or VL comprising one or more amino acid
substitutions in one or more FR regions. Per Example 2 herein,
Applicants performed alanine scanning on certain antibodies
selected from those above, and determined that similar or improved
binding was obtained despite amino acid modifications at selected
FR positions. As shown in FIGS. 6-1 and 6-2 and Example 2 herein,
for the class I-III groups of antibodies, variant forms of the
antibodies with modifications at one or more residues of an FR
retained affinity and binding specifity. For example, for antibody
15G11, positions 43 and 48 in the light chain FR2, position 48 in
the heavy chain FR2 and positions 67, 69, 71 and 73 in the heavy
chain FR3 could be modified as shown in FIGS. 6-1 and 6-2 and the
resulting antibody still retained specificity and strong binding
affinity for human/primate TfR. In another example, for antibody
7A4, positions 58 and 68 of the light chain FR3, position 24 in the
heavy chain FR1 and position 71 in the heavy chain FR3 could be
modified as shown in FIGS. 6-1 and 6-2 and the resulting antibody
still retained specificity and strong binding affinity for
human/primate TfR. In a third example, for antibody 16F6, positions
43 and 44 of the light chain FR2 and positions 71 and 78 of the
heavy chain FR3 could be modified as shown in FIGS. 6-1 and 6-2 and
the resulting antibody still retained specificity and strong
binding affinity for human/primate TfR. In another aspect, an
anti-TfR antibody comprises a heavy chain variable domain (VH)
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to the amino acid sequence of
any one of SEQ ID NOs: 7-10, 15-18, 20, 25-28, 108, 114, 120 and
126. In certain embodiments, a VH sequence having at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but an anti-TfR
antibody comprising that sequence retains the ability to bind to
TfR. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted and/or deleted in any one of SEQ ID
NOs:7-10, 15-18, 20, 25-28, 108, 114, 120 and 126. In certain
embodiments, substitutions, insertions, or deletions occur in
regions outside the HVRs (i.e., in the FRs). Optionally, the
anti-TfR antibody comprises the VH sequence of any one of SEQ ID
NOs: 7-10, 15-18, 20 25-28, 108, 114, 120 and 126, including
post-translational modifications of that sequence. In a particular
embodiment, the VH for a particular antibody comprises one, two or
three HVRs selected from: the HVRs set forth above and in Table 3
or 4 for that particular antibody. VH sequences for the antibodies
of the invention are shown in FIGS. 3 and 4 herein.
[0201] In another aspect, an anti-TfR antibody is provided, wherein
the antibody comprises a light chain variable domain (VL) having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of any one of SEQ ID
NOs:4-6, 11-14, 19, 21-24, 105, 111, 117, and 123. In certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,
conservative substitutions), insertions, or deletions relative to
the reference sequence, but an anti-TfR antibody comprising that
sequence retains the ability to bind to TfR. In certain
embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted in any one of SEQ ID NOs:4-6, 11-14, 19,
21-24, 105, 111, 117, and 123. In certain embodiments, the
substitutions, insertions, or deletions occur in regions outside
the HVRs (i.e., in the FRs). Optionally, the anti-TfR antibody
comprises the VL sequence in any of SEQ ID NOs:4-6, 11-14, 19,
21-24, 105, 111, 117, and 123, including post-translational
modifications of that sequence. In a particular embodiment, the VL
comprises one, two or three HVRs selected from the HVRs set forth
above and in Table 4 or 5 for that particular antibody. VL
sequences for the antibodies of the invention are shown in FIGS. 3
and 4 herein.
[0202] In another aspect, an anti-TfR antibody is provided, wherein
the antibody comprises a VH as in any of the embodiments provided
above, and a VL as in any of the embodiments provided above. In one
embodiment, the antibody comprises the VL and VH sequences,
respectively, in SEQ ID NOs: 4 and 7; 5 and 8; 5 and 9; 6 and 10; 4
and 7; 11 and 15; 12 and 16; 13 and 17; 14 and 18; 19 and 20; 21
and 25; 22 and 26; 23 and 27; 24 and 28; 105 and 108; 111 and 114;
117 and 120; and 123 and 126, including post-translational
modifications of those sequences.
[0203] In a further aspect, the invention provides an antibody that
binds to the same epitope as an anti-TfR antibody provided herein.
For example, in certain embodiments, an antibody is provided that
binds to the same epitope as an anti-TfR antibody comprising VL and
VH sequences, respectively, of SEQ ID NOs: 4 and 7; 5 and 8; 5 and
9; 6 and 10; 4 and 7; 11 and 15; 12 and 16; 13 and 17; 14 and 18;
19 and 20; 21 and 25; 22 and 26; 23 and 27; 24 and 28; 105 and 108;
111 and 114; 117 and 120; or 123 and 126. In one aspect, the
antibody competes with any of the antibodies in Class I (ie.,
clones 7A4, 8A2, 15D2, 10D11, or 7B10, or affinity-matured versions
of any of those antibodies) for binding to TfR. In another aspect,
the antibody competes with any of the antibodies in Class II (ie,
clones 15G11, 16G5, 13C3 or 16G, or affinity-matured versions of
any of those antibodies) for binding to TfR. In another aspect, the
antibody competes with clone 16F6 or affinity-matured versions
thereof for binding to TfR. In another aspect, the antibody
competes with any of the antibodies in Class IV (ie, clones 7G7,
4C2, 1B12 or 13D4, or affinity matured versions thereof) for
binding to TfR.
[0204] In a further aspect of the invention, an anti-TfR antibody
according to any of the above embodiments is a monoclonal antibody,
including a chimeric, humanized or human antibody. In one
embodiment, an anti-TfR antibody is an antibody fragment, e.g., a
Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another
embodiment, the antibody is a full length antibody, e.g., an intact
IgG1, IgG2, IgG3, or IgG4 antibody or other antibody class or
isotype as defined herein.
[0205] In a further aspect, an anti-TfR antibody according to any
of the above embodiments may incorporate any of the features,
singly or in combination, as described in Sections 1-7 below:
[0206] 1. Antibody Affinity
[0207] In certain embodiments, an antibody provided herein 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.-8 M or less, e.g. from 10.sup.-8 M
to 10.sup.-13 M, e.g., from 10.sup.-9 M to 10.sup.-13 M).
[0208] In certain aspects of the present invention, a "low
affinity" anti-TfR antibody of the invention is selected, based,
e.g., on the results in Example 5 and in Atwal et al., Sci. Transl.
Med. 3, 84ra43 (2011) and Yu et al., Sci. Transl. Med. 25 May 2011:
Vol. 3, Issue 84, p. 84ra44, showing that such lower-affinity
antibodies to TfR display improved CNS (for example, brain) uptake
and/or persistence in the brain/CNS. 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 human or primate 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 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 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..
[0209] 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 TfR because it has an affinity for TfR 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 M, or in the range from about 30
nM to about 30 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 anti-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.
[0210] In one embodiment, Kd is measured by a radiolabeled antigen
binding assay (RIA). In one embodiment, an RIA is performed with
the Fab version of an antibody of interest and its antigen. For
example, solution binding affinity of Fabs for antigen is measured
by equilibrating Fab with a minimal concentration of
(.sup.125I)-labeled antigen in the presence of a titration series
of unlabeled antigen, then capturing bound antigen with an anti-Fab
antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.
293:865-881(1999)). To establish conditions for the assay,
MICROTITER.RTM. multi-well plates (Thermo Scientific) are coated
overnight with 5 g/ml of a capturing anti-Fab antibody (Cappel
Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked
with 2% (w/v) bovine serum albumin in PBS for two to five hours at
room temperature (approximately 23.degree. C.). In a non-adsorbent
plate (Nunc #269620), 100 .mu.M or 26 .mu.M [.sup.125I]-antigen are
mixed with serial dilutions of a Fab of interest (e.g., consistent
with assessment of the anti-VEGF antibody, Fab-12, in Presta et
al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then
incubated overnight; however, the incubation may continue for a
longer period (e.g., about 65 hours) to ensure that equilibrium is
reached. Thereafter, the mixtures are transferred to the capture
plate for incubation at room temperature (e.g., for one hour). The
solution is then removed and the plate washed eight times with 0.1%
polysorbate 20 (TWEEN-20.RTM.) in PBS. When the plates have dried,
150 .mu.l/well of scintillant (MICROSCINT-20 .TM.; Packard) is
added, and the plates are counted on a TOPCOUNT.TM. gamma counter
(Packard) for ten minutes. Concentrations of each Fab that give
less than or equal to 20% of maximal binding are chosen for use in
competitive binding assays.
[0211] In one aspect, the RIA is a Scatchard analysis. For example,
the anti-TfR antibody of interest can be iodinated using the
lactoperoxidase method (Bennett and Horuk, Methods in Enzymology
288 pg. 134-148 (1997)). A radiolabeled anti-TfR 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 TfR 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.
[0212] 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 g/ml before injection at a flow rate of 5
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 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)
[0213] According to another embodiment, Kd is measured using
surface plasmon resonance assays with a BIACORE@-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 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-TfR antibody variants are injected in HBS-P to
reach about 220 RU, then two-fold serial dilutions of TfR-His (0.61
nM to 157 nM) are injected in HBS-P at 25.degree. C. at a flow rate
of approximately 30 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).
[0214] 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 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.
[0215] 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 a biotinylated known
anti-TfR antibody 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 known anti-TfR
(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 antibody
bound to the plate was detected using TMB substrate (BioFX
Laboratories, Owings Mills).
[0216] 2. Antibody Fragments
[0217] In certain embodiments, an antibody provided herein is an
antibody fragment. Antibody fragments include, but are not limited
to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, and other
fragments described below. For a review of certain antibody
fragments, see Hudson et al. Nat. Med 9:129-134 (2003). For a
review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology
of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
(Springer-Verlag, New York), pp. 269-315 (1994); see also WO
93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For
discussion of Fab and F(ab')2 fragments comprising salvage receptor
binding epitope residues and having increased in vivo half-life,
see U.S. Pat. No. 5,869,046.
[0218] Diabodies are antibody fragments with two antigen-binding
sites that may be bivalent or bispecific. See, for example, EP
404,097; WO 1993/01161; Hudson et al., Nat. Med 9:129-134 (2003);
and Hollinger et al., Proc. Nat. Acad Sci. USA 90: 6444-6448
(1993). Triabodies and tetrabodies are also described in Hudson et
al., Nat. Med 9:129-134 (2003).
[0219] Single-domain antibodies are antibody fragments comprising
all or a portion of the heavy chain variable domain or all or a
portion of the light chain variable domain of an antibody. In
certain embodiments, a single-domain antibody is a human
single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g.,
U.S. Pat. No. 6,248,516 B1).
[0220] Antibody fragments can be made by various techniques,
including but not limited to proteolytic digestion of an intact
antibody as well as production by recombinant host cells (e.g. E.
coli or phage), as described herein.
[0221] 3. Chimeric and Humanized Antibodies
[0222] In certain embodiments, an antibody provided herein is a
chimeric antibody. Certain chimeric antibodies are described, e.g.,
in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Nat. Acad.
Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody
comprises a non-human variable region (e.g., a variable region
derived from a mouse, rat, hamster, rabbit, or non-human primate,
such as a monkey) and a human constant region. 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). In a further example, a chimeric antibody is a
"class switched" antibody in which the class or subclass has been
changed from that of the parent antibody. Chimeric antibodies
include antigen-binding fragments thereof.
[0223] In certain embodiments, a chimeric antibody is a humanized
antibody. Typically, a non-human antibody is humanized to reduce
immunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which HVRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) are derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region. In some
embodiments, some FR residues in a humanized antibody are
substituted with corresponding residues from a non-human antibody
(e.g., the antibody from which the HVR residues are derived), e.g.,
to restore or improve antibody specificity or affinity.
[0224] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro and Fransson, Front. Biosci.
13:1619-1633 (2008), and are further described, e.g., in Riechmann
et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad.
Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,
7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods
36:25-34 (2005) (describing specificity determining region (SDR)
grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing
"resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005)
(describing "FR shuffling"); and Osbourn et al., Methods 36:61-68
(2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000)
(describing the "guided selection" approach to FR shuffling).
[0225] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296
(1993)); framework regions derived from the consensus sequence of
human antibodies of a particular subgroup of light or heavy chain
variable regions (see, e.g., Carter et al. Proc. Nat. Acad. Sci.
USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623
(1993)); human mature (somatically mutated) framework regions or
human germline framework regions (see, e.g., Almagro and Fransson,
Front. Biosci. 13:1619-1633 (2008)); and framework regions derived
from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem.
272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.
271:22611-22618 (1996)).
[0226] 4. Human Antibodies
[0227] In certain embodiments, an antibody provided herein is a
human antibody. Human antibodies can be produced using various
techniques known in the art. Human antibodies are described
generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:
368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459
(2008).
[0228] Human antibodies may be prepared by administering an
immunogen to a transgenic animal that has been modified to produce
intact human antibodies or intact antibodies with human variable
regions in response to antigenic challenge. Such animals typically
contain all or a portion of the human immunoglobulin loci, which
replace the endogenous immunoglobulin loci, or which are present
extrachromosomally or integrated randomly into the animal's
chromosomes. In such transgenic mice, the endogenous immunoglobulin
loci have generally been inactivated. For review of methods for
obtaining human antibodies from transgenic animals, see Lonberg,
Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos.
6,075,181 and 6,150,584 describing XENOMOUSE.TM. technology; U.S.
Pat. No. 5,770,429 describing HUMAB.RTM. technology; U.S. Pat. No.
7,041,870 describing K-M MOUSE.RTM. technology, and U.S. Patent
Application Publication No. US 2007/0061900, describing
VELOCIMOUSE.RTM. technology). Human variable regions from intact
antibodies generated by such animals may be further modified, e.g.,
by combining with a different human constant region.
[0229] Human antibodies can also be made by hybridoma-based
methods. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described.
(See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.
Immunol., 147: 86 (1991).) Huma antibodies generated via human
B-cell hybrdorna technology are also described in Li et al., Proc.
Nad. Acad Sci. USA, 103:3557-3562 (2006). Additional methods
include those described, for example, in U.S. Pat. No. 7,189,826
(describing production of monoclonal human IgM antibodies from
hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268
(2006) (describing human-human hybridomas). Human hybridoma
technology (Trioma technology) is also described in Vollmers and
Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and
Vollmers and Brandlein, Methods and Findings in Experimental and
Clinical Pharmacology, 27(3):185-91 (2005).
[0230] Human antibodies may also be generated by isolating Fv clone
variable domain sequences selected from human-derived phage display
libraries. Such variable domain sequences may then be combined with
a desired human constant domain. Techniques for selecting human
antibodies from antibody libraries are described below.
[0231] 5. Library-Derived Antibodies
[0232] Antibodies of the invention may be isolated by screening
combinatorial libraries for antibodies with the desired activity or
activities. For example, a variety of methods are known in the art
for generating phage display libraries and screening such libraries
for antibodies possessing the desired binding characteristics. Such
methods are reviewed, e.g., in Hoogenboom et al. in Methods in
Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press,
Totowa, N.J., 2001) and further described, e.g., in the McCafferty
et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628
(1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and
Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed.,
Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.
338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093
(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472
(2004); and Lee et al., J. Immunol. Methods 284(1-2):
119-132(2004).
[0233] In certain phage display methods, repertoires of VH and VL
genes are separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be screened
for antigen-binding phage as described in Winter et al., Ann. Rev.
Immunol., 12: 433-455 (1994). Phage typically display antibody
fragments, either as single-chain Fv (scFv) fragments or as Fab
fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a
wide range of non-self and also self antigens without any
immunization as described by Griffiths et al., EMBO J, 12: 725-734
(1993). Finally, naive libraries can also be made synthetically by
cloning unrearranged V-gene segments from stem cells, and using PCR
primers containing random sequence to encode the highly variable
CDR3 regions and to accomplish rearrangement in vitro, as described
by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
Patent publications describing human antibody phage libraries
include, for example: U.S. Pat. No. 5,750,373, and US Patent
Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000,
2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and
2009/0002360.
[0234] Antibodies or antibody fragments isolated from human
antibody libraries are considered human antibodies or human
antibody fragments herein.
[0235] 6. Multispecific Antibodies
[0236] In certain embodiments, an antibody provided herein is a
multispecific antibody, e.g. a bispecific antibody. Multispecific
antibodies are monoclonal antibodies that have binding
specificities for at least two different sites. In certain
embodiments, one of the binding specificities is for TfR and the
other is for any other antigen. In certain embodiments, bispecific
antibodies may bind to two different epitopes of TfR. Bispecific
antibodies may also be used to localize cytotoxic agents to cells
which express TfR. Bispecific antibodies can be prepared as full
length antibodies or antibody fragments.
[0237] 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.
Nat. 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).
[0238] The antibody or fragment herein also includes a "Dual Acting
FAb" or "DAF" comprising an antigen binding site that binds to TfR
as well as another, different antigen (see, US 2008/0069820, for
example).
[0239] 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 TfR 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.
[0240] 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. 16A-B
herein.
[0241] 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.
[0242] 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.
Nat. 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).
[0243] 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)).
[0244] 7. Antibody Variants
[0245] In certain embodiments, amino acid sequence variants of the
antibodies provided herein are contemplated. For example, it may be
desirable to improve the binding affinity and/or other biological
properties of the antibody. Amino acid sequence variants of an
antibody may be prepared by introducing appropriate modifications
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-binding.
[0246] a) Substitution, Insertion, and Deletion Variants
[0247] In certain embodiments, antibody variants having one or more
amino acid substitutions are provided. Sites of interest for
substitutional mutagenesis include the HVRs and FRs. Conservative
substitutions are shown in Table 2 under the heading of "preferred
substitutions." More substantial changes are provided in Table 2
under the heading of "exemplary substitutions," and as further
described below in reference to amino acid side chain classes.
Amino acid substitutions may be introduced into an antibody of
interest and the products screened for a desired activity, e.g.,
retained/improved antigen binding, decreased immunogenicity, or
improved ADCC or CDC.
TABLE-US-00002 TABLE 2 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met;
Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
Amino acids may be grouped according to common side-chain
properties:
[0248] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0249] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0250] (3) acidic: Asp, Glu;
[0251] (4) basic: His, Lys, Arg;
[0252] (5) residues that influence chain orientation: Gly, Pro;
[0253] (6) aromatic: Trp, Tyr, Phe.
[0254] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0255] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further study will have modifications (e.g.,
improvements) in certain biological properties (e.g., increased
affinity, reduced immunogenicity) relative to the parent antibody
and/or will have substantially retained certain biological
properties of the parent antibody. An exemplary substitutional
variant is an affinity matured antibody, which may be conveniently
generated, e.g., using phage display-based affinity maturation
techniques such as those described herein. Briefly, one or more HVR
residues are mutated and the variant antibodies displayed on phage
and screened for a particular biological activity (e.g. binding
affinity).
[0256] Alterations (e.g., substitutions) may be made in HVRs, e.g.,
to improve antibody affinity. Such alterations may be made in HVR
"hotspots," i.e., residues encoded by codons that undergo mutation
at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues
that contact antigen, with the resulting variant VH or VL being
tested for binding affinity. Affinity maturation by constructing
and reselecting from secondary libraries has been described, e.g.,
in Hoogenboom et al. in Methods in Molecular Biology 178:1-37
(O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) In some
embodiments of affinity maturation, diversity is introduced into
the variable genes chosen for maturation by any of a variety of
methods (e.g., error-prone PCR, chain shuffling, or
oligonucleotide-directed mutagenesis). A secondary library is then
created. The library is then screened to identify any antibody
variants with the desired affinity. Another method to introduce
diversity involves HVR-directed approaches, in which several HVR
residues (e.g., 4-6 residues at a time) are randomized. HVR
residues involved in antigen binding may be specifically
identified, e.g., using alanine scanning mutagenesis or modeling.
CDR-H3 and CDR-L3 in particular are often targeted.
[0257] In certain embodiments, substitutions, insertions, or
deletions may occur within one or more HVRs so long as such
alterations do not substantially reduce the ability of the antibody
to bind antigen. For example, conservative alterations (e.g.,
conservative substitutions as provided herein) that do not
substantially reduce binding affinity may be made in HVRs. Such
alterations may, for example, be outside of antigen contacting
residues in the HVRs. In certain embodiments of the variant VH and
VL sequences provided above, each HVR either is unaltered, or
contains no more than one, two or three amino acid
substitutions.
[0258] A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham and Wells (1989)
Science, 244:1081-1085. In this method, a residue or group of
target residues (e.g., charged residues such as arg, asp, his, lys,
and glu) are identified and replaced by a neutral or negatively
charged amino acid (e.g., alanine or polyalanine) to determine
whether the interaction of the antibody with antigen is affected.
Further substitutions may be introduced at the amino acid locations
demonstrating functional sensitivity to the initial substitutions.
Alternatively, or additionally, a crystal structure of an
antigen-antibody complex to identify contact points between the
antibody and antigen. Such contact residues and neighboring
residues may be targeted or eliminated as candidates for
substitution. Variants may be screened to determine whether they
contain the desired properties.
[0259] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g. for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
[0260] b) Glycosylation Variants
[0261] In certain embodiments, an antibody provided herein is
altered to increase or decrease the extent to which the antibody is
glycosylated. Addition or deletion of glycosylation sites to an
antibody may be conveniently accomplished by altering the amino
acid sequence such that one or more glycosylation sites is created
or removed.
[0262] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.
TIBTECH 15:26-32 (1997). The oligosaccharide may include various
carbohydrates, e.g., mannose, N-acetyl glucosamine (GcNAc),
galactose, and sialic acid, as well as a fucose attached to a
GlcNAc in the "stem" of the biantennary oligosaccharide structure.
In some embodiments, modifications of the oligosaccharide in an
antibody of the invention may be made in order to create antibody
variants with certain improved properties.
[0263] In one embodiment, antibody variants are provided having a
carbohydrate structure that lacks fucose attached (directly or
indirectly) to an Fc region. For example, the amount of fucose in
such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%
or from 20% to 40%. The amount of fucose is determined by
calculating the average amount of fucose within the sugar chain at
Asn297, relative to the sum of all glycostructures attached to Asn
297 (e. g. complex, hybrid and high mannose structures) as measured
by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for
example. Asn297 refers to the asparagine residue located at about
position 297 in the Fc region (Eu numbering of Fc region residues);
however, Asn297 may also be located about 3 amino acids upstream or
downstream of position 297, i.e., between positions 294 and 300,
due to minor sequence variations in antibodies. Such fucosylation
variants may have improved ADCC function. See, e.g., US Patent
Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621
(Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to
"defucosylated" or "fucose-deficient" antibody variants include: US
2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US
2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US
2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO
2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki
et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al.
Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of
producing defucosylated antibodies include Lec13 CHO cells
deficient in protein fucosylation (Ripka et al. Arch. Biochem.
Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 A1,
Presta, L; and WO 2004/056312 A1, Adams et al., especially at
Example 11), and knockout cell lines, such as
alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,
e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda,
Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and
WO2003/085107).
[0264] Antibodies variants are further provided with bisected
oligosaccharides, e.g., in which a biantennary oligosaccharide
attached to the Fc region of the antibody is bisected by GlcNAc.
Such antibody variants may have reduced fucosylation and/or
improved ADCC function. Examples of such antibody variants are
described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.
No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).
Antibody variants with at least one galactose residue in the
oligosaccharide attached to the Fc region are also provided. Such
antibody variants may have improved CDC function. Such antibody
variants are described, e.g., in WO 1997/30087 (Patel et al.); WO
1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
[0265] c) Fc Region Variants
[0266] In certain embodiments, one or more amino acid modifications
may be introduced into the Fc region of an antibody provided
herein, thereby generating an Fc region variant. The Fc region
variant may comprise a human Fc region sequence (e.g., a human
IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid
modification (e.g. a substitution) at one or more amino acid
positions.
[0267] In certain embodiments, the invention contemplates an
antibody variant that possesses some but not all effector
functions, which make it a desirable candidate for applications in
which the half life of the antibody in vivo is important yet
certain effector functions (such as complement and ADCC) are
unnecessary or deleterious. In vitro and/or in vivo cytotoxicity
assays can be conducted to confirm the reduction/depletion of CDC
and/or ADCC activities. For example, Fc receptor (FcR) binding
assays can be conducted to ensure that the antibody lacks
Fc.gamma.R binding (hence likely lacking ADCC activity), but
retains FcRn binding ability. The primary cells for mediating ADCC,
NK cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting
examples of in vitro assays to assess ADCC activity of a molecule
of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.
Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063
(1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA
82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et
al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively,
non-radioactive assays methods may be employed (see, for example,
ACTI.TM. non-radioactive cytotoxicity assay for flow cytometry
(CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96.RTM.
non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful
effector cells for such assays include peripheral blood mononuclear
cells (PBMC) and Natural Killer (NK) cells. Alternatively, or
additionally, ADCC activity of the molecule of interest may be
assessed in vivo, e.g., in a animal model such as that disclosed in
Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q
binding assays may also be carried out to confirm that the antibody
is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q
and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To
assess complement activation, a CDC assay may be performed (see,
for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163
(1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg,
M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding
and in vivo clearance/half life determinations can also be
performed using methods known in the art (see, e.g., Petkova, S. B.
et al., Int'l. Immunol. 18(12):1759-1769 (2006)).
[0268] Non-limiting examples of antibodies with reduced effector
function include those with substitution of one or more of Fc
region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No.
6,737,056). Such Fc mutants include Fc mutants with substitutions
at two or more of amino acid positions 265, 269, 270, 297 and 327,
including the so-called "DANA" Fc mutant with substitution of
residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).
[0269] Certain antibody variants with improved or diminished
binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056;
WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604
(2001).)
[0270] In certain embodiments, an antibody variant comprises an Fc
region with one or more amino acid substitutions which improve
ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the
Fc region (EU numbering of residues).
[0271] In some embodiments, alterations are made in the Fc region
that result in altered (i.e., either improved or diminished) C1q
binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as
described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et
al. J. Immunol. 164: 4178-4184 (2000).
[0272] Antibodies with increased half lives and improved binding to
the neonatal Fc receptor (FcRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.
117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are
described in US2005/0014934A1 (Hinton et al.). Those antibodies
comprise an Fc region with one or more substitutions therein which
improve binding of the Fc region to FcRn. Such, non-limiting, Fc
variants include those with substitutions at one or more of Fc
region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312,
317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g.,
substitution of Fc region residue 434 (U.S. Pat. No.
7,371,826).
[0273] See also Duncan & Winter, Nature 322:738-40 (1988); U.S.
Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning other
examples of Fc region variants.
[0274] d) Cysteine Engineered Antibody Variants
[0275] In certain embodiments, it may be desirable to create
cysteine engineered antibodies, e.g., "thioMAbs," in which one or
more residues of an antibody are substituted with cysteine
residues. In particular embodiments, the substituted residues occur
at accessible sites of the antibody. By substituting those residues
with cysteine, reactive thiol groups are thereby positioned at
accessible sites of the antibody and may be used to conjugate the
antibody to other moieties, such as drug moieties or linker-drug
moieties, to create an immunoconjugate, as described further
herein. In certain embodiments, any one or more of the following
residues may be substituted with cysteine: V205 (Kabat numbering)
of the light chain; A118 (EU numbering) of the heavy chain; and
S400 (EU numbering) of the heavy chain Fc region. Cysteine
engineered antibodies may be generated as described, e.g., in U.S.
Pat. No. 7,521,541.
[0276] e) Antibody Derivatives
[0277] In certain embodiments, an antibody provided herein may be
further modified to contain additional nonproteinaceous moieties
that are known in the art and readily available. The moieties
suitable for derivatization of the antibody include but are not
limited to water soluble polymers. Non-limiting examples of water
soluble polymers include, but are not limited to, polyethylene
glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer are attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
[0278] In another embodiment, conjugates of an antibody and
nonproteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the nonproteinaceous
moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA
102: 11600-11605 (2005)). The radiation may be of any wavelength,
and includes, but is not limited to, wavelengths that do not harm
ordinary cells, but which heat the nonproteinaceous moiety to a
temperature at which cells proximal to the
antibody-nonproteinaceous moiety are killed.
[0279] B. Recombinant Methods and Compositions
[0280] Antibodies may be produced using recombinant methods and
compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one
embodiment, isolated nucleic acid encoding an anti-TfR antibody
described herein is provided. Such nucleic acid may encode an amino
acid sequence comprising the VL and/or an amino acid sequence
comprising the VH of the antibody (e.g., the light and/or heavy
chains of the antibody). In a further embodiment, one or more
vectors (e.g., expression vectors) comprising such nucleic acid are
provided. In a further embodiment, a host cell comprising such
nucleic acid is provided. In one such embodiment, a host cell
comprises (e.g., has been transformed with): (1) a vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the VL of the antibody and an amino acid sequence
comprising the VH of the antibody, or (2) a first vector comprising
a nucleic acid that encodes an amino acid sequence comprising the
VL of the antibody and a second vector comprising a nucleic acid
that encodes an amino acid sequence comprising the VH of the
antibody. In one embodiment, the host cell is eukaryotic, e.g. a
Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0,
Sp20 cell). In one embodiment, a method of making an anti-TfR
antibody is provided, wherein the method comprises culturing a host
cell comprising a nucleic acid encoding the antibody, as provided
above, under conditions suitable for expression of the antibody,
and optionally recovering the antibody from the host cell (or host
cell culture medium).
[0281] For recombinant production of an anti-TfR antibody, nucleic
acid encoding an antibody, e.g., as described above, is isolated
and inserted into one or more vectors for further cloning and/or
expression in a host cell. Such nucleic acid may be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
antibody).
[0282] Suitable host cells for cloning or expression of
antibody-encoding vectors include prokaryotic or eukaryotic cells
described herein. For example, antibodies may be produced in
bacteria, in particular when glycosylation and Fc effector function
are not needed. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,
5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular
Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.,
2003), pp. 245-254, describing expression of antibody fragments in
E. coli.) After expression, the antibody may be isolated from the
bacterial cell paste in a soluble fraction and can be further
purified.
[0283] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors, including fungi and yeast strains
whose glycosylation pathways have been "humanized," resulting in
the production of an antibody with a partially or fully human
glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414
(2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
[0284] Suitable host cells for the expression of glycosylated
antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frugiperda cells.
[0285] Plant cell cultures can also be utilized as hosts. See,
e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,
and 6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants).
[0286] Vertebrate cells may also be used as hosts. For example,
mammalian cell lines that are adapted to grow in suspension may be
useful. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic
kidney line (293 or 293 cells as described, e.g., in Graham et al.,
J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse
sertoli cells (TM4 cells as described, e.g., in Mather, Biol.
Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African
green monkey kidney cells (VERO-76); human cervical carcinoma cells
(HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL
3A); human lung cells (W138); human liver cells (Hep G2); mouse
mammary tumor (MMT 060562); TRI cells, as described, e.g., in
Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5
cells; and FS4 cells. Other useful mammalian host cell lines
include Chinese hamster ovary (CHO) cells, including DHFR.sup.- CHO
cells (Urlaub et al., Proc. Nat. Acad. Sci. USA 77:4216 (1980));
and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of
certain mammalian host cell lines suitable for antibody production,
see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248
(B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268
(2003).
[0287] C. Assays
[0288] Anti-TfR antibodies provided herein may be identified,
screened for, or characterized for their physical/chemical
properties and/or biological activities by various assays known in
the art.
[0289] 1. Binding Assays and Other Assays
[0290] Various techniques are available for determining binding of
the antibody to the TfR. One such assay is an enzyme linked
immunosorbent assay (ELISA) for confirming an ability to bind to
human TfR (and brain antigen). According to this assay, plates
coated with antigen (e.g. recombinant TfR) are incubated with a
sample comprising the anti-TfR antibody and binding of the antibody
to the antigen of interest is determined.
[0291] 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.
[0292] In another aspect, competition assays may be used to
identify an antibody that competes with any of the antibodies of
the invention for binding to TfR. 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 antibodies of
the invention, more specifically, any of the epitopes specifically
bound by antibodies in class I, class II, class III or class IV as
described herein (see, e.g., Example 1 and Table 4. 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.).
[0293] In an exemplary competition assay, immobilized TfR is
incubated in a solution comprising a first labeled antibody that
binds to TfR (e.g., one or more of the antibodies disclosed herein)
and a second unlabeled antibody that is being tested for its
ability to compete with the first antibody for binding to TfR. The
second antibody may be present in a hybridoma supernatant. As a
control, immobilized TfR 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 TfR, excess unbound antibody is removed, and the amount
of label associated with immobilized TfR is measured. If the amount
of label associated with immobilized TfR 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 TfR. See Harlow and Lane (1988) Antibodies:
A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.).
[0294] 2. Activity Assays
[0295] In one aspect, assays are provided for identifying anti-TfR
antibodies thereof having biological activity. Biological activity
may include, e.g., transporting a compound associated
with/conjugated to the antibody across the BBB into the brain
and/or CNS. Antibodies having such biological activity in vivo
and/or in vitro are also provided.
[0296] In certain embodiments, an antibody of the invention is
tested for such biological activity.
[0297] D. Immunoconjugates
[0298] The invention also provides immunoconjugates comprising an
anti-TfR antibody herein conjugated to one or more cytotoxic
agents, such as chemotherapeutic agents or drugs, growth inhibitory
agents, toxins (e.g., protein toxins, enzymatically active toxins
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or radioactive isotopes.
[0299] In one embodiment, the anti-TfR antibody herein is coupled
with a neurological disorder drug, a chemotherapeutic agent and/or
an imaging agent in order to more efficiently transport the drug,
chemotherapeutic agent and/or the imaging agent across the BBB.
[0300] 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 anti-TfR antibody and e.g., 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-TfR antibody and a
corresponding group or acceptor on the, e.g., 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 the anti-TfR antibody and a disulfide bond formed with the
e.g., 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.
[0301] Conjugation may also be performed using a variety of
linkers. For example, an anti-TfR 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). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al., Science
238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. 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.
[0302] 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, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,
sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and
sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which
are commercially available (e.g., from Pierce Biotechnology, Inc.,
Rockford, Ill., U.S.A).
[0303] In one embodiment, an immunoconjugate is an antibody-drug
conjugate (ADC) in which an antibody is conjugated to one or more
drugs, including but not limited to a maytansinoid (see U.S. Pat.
Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an
auristatin such as monomethylauristatin drug moieties DE and DF
(MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and
7,498,298); a dolastatin; a calicheamicin or derivative thereof
(see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285,
5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al.,
Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res.
58:2925-2928 (1998)); an anthracycline such as daunomycin or
doxorubicin (see Kratz et al., Current Med Chem. 13:477-523 (2006);
Jeffrey et al., Bioorganic & Med Chem. Letters 16:358-362
(2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et
al., Proc. Natl. Acad Sci. USA 97:829-834 (2000); Dubowchik et al.,
Bioorg. & Med Chem. Letters 12:1529-1532 (2002); King et al.,
J. Med Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579);
methotrexate; vindesine; a taxane such as docetaxel, paclitaxel,
larotaxel, tesetaxel, and ortataxel; a trichothecene; and
CC1065.
[0304] In another embodiment, an immunoconjugate comprises an
antibody as described herein conjugated to an enzymatically active
toxin or fragment thereof, including but not limited to diphtheria
A chain, nonbinding active fragments of diphtheria toxin, exotoxin
A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A
chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,
dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and
PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes.
[0305] In another embodiment, an immunoconjugate comprises an
antibody as described herein conjugated to a radioactive atom to
form a radioconjugate. A variety of radioactive isotopes are
available for the production of radioconjugates. Examples include
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. When the radioconjugate is used for detection, it
may comprise a radioactive atom for scintigraphic studies, for
example tc99m or 1123, 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 or iron.
[0306] E. Methods and Compositions for Diagnostics and
Detection
[0307] In certain embodiments, any of the anti-TfR antibodies
provided herein is useful for detecting the presence of TfR in a
biological sample. The term "detecting" as used herein encompasses
quantitative or qualitative detection. In certain embodiments, a
biological sample comprises a cell or tissue, such as blood (i.e.,
immature red blood cells), CSF, and BBB-containing tissue.
[0308] In one embodiment, an anti-TfR antibody for use in a method
of diagnosis or detection is provided. In a further aspect, a
method of detecting the presence of TfR in a biological sample is
provided. In certain embodiments, the method comprises contacting
the biological sample with an anti-TfR antibody as described herein
under conditions permissive for binding of the anti-TfR antibody to
TfR, and detecting whether a complex is formed between the anti-TfR
antibody and TfR. Such method may be an in vitro or in vivo method.
In one embodiment, an anti-TfR antibody is used to select subjects
eligible for therapy with an anti-TfR antibody, e.g. where TfR is a
biomarker for selection of patients.
[0309] Exemplary disorders that may be diagnosed using an antibody
of the invention include disorders involving immature red blood
cells, due to the fact that TfR is expressed in reticulocytes and
is therefore detectable by any of the antibodies of the invention.
Such disorders include anemia and other disorders arising from
reduced levels of reticulocytes, or congenital polycythemia or
neoplastic polycythemia vera, where raised red blood cell counts
due to hyperproliferation of, e.g., reticulocytes, results in
thickening of blood and concomitant physiological symptoms.
[0310] In certain embodiments, labeled anti-TfR antibodies are
provided. Labels include, but are not limited to, labels or
moieties that are detected directly (such as fluorescent,
chromophoric, electron-dense, chemiluminescent, and radioactive
labels), as well as moieties, such as enzymes or ligands, that are
detected indirectly, e.g., through an enzymatic reaction or
molecular interaction. Exemplary labels include, but are not
limited to, the radioisotopes .sup.32P, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I, fluorophores such as rare earth chelates or
fluorescein and its derivatives, rhodamine and its derivatives,
dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and
bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),
alkaline phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,
saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as
uricase and xanthine oxidase, coupled with an enzyme that employs
hydrogen peroxide to oxidize a dye precursor such as HRP,
lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,
bacteriophage labels, stable free radicals, and the like.
[0311] In one embodiment, the intact antibody lacks effector
function. In another embodiment, the inact antibody has reduced
effector function. In another embodiment, the intact antibody is
engineered to have reduced effector function. In one aspect, the
antibody is a Fab. In another aspect, the antibody has one or more
Fc mutations reducing or eliminating effector function. In another
aspect, the antibody has modified glycosylation due, e.g., to
producing the antibody in a system lacking normal human
glycosylation enzymes. In another aspect, the Ig backbone is
modified to one which naturally possesses limited or no effector
function.
[0312] Various techniques are available for determining binding of
the antibody to the TfR. One such assay is an enzyme linked
immunosorbent assay (ELISA) for confirming an ability to bind to
human TfR (and brain antigen). According to this assay, plates
coated with antigen (e.g. recombinant TfR) are incubated with a
sample comprising the anti-TfR antibody and binding of the antibody
to the antigen of interest is determined.
[0313] 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.
[0314] In one aspect, assays are provided for identifying anti-TfR
antibodies conjugated (either covalently or non-covalently) to
anti-BACE1 antibodies 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.
[0315] F. Pharmaceutical Formulations
[0316] Pharmaceutical formulations of an anti-TfR as described
herein are prepared by mixing such antibody having the desired
degree of purity with one or more 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.
Pharmaceutically acceptable carriers, excipients, or stabilizers
are generally nontoxic to recipients at the dosages and
concentrations employed, and include, but are not limited to:
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
polyethylene glycol (PEG). Exemplary pharmaceutically acceptable
carriers herein further include insterstitial drug dispersion
agents such as soluble neutral-active hyaluronidase glycoproteins
(sHASEGP), for example, human soluble PH-20 hyaluronidase
glycoproteins, such as rHuPH20 (HYLENEX.RTM., Baxter International,
Inc.). Certain exemplary sHASEGPs and methods of use, including
rHuPH20, are described in US Patent Publication Nos. 2005/0260186
and 2006/0104968. In one aspect, a sHASEGP is combined with one or
more additional glycosaminoglycanases such as chondroitinases.
[0317] Exemplary lyophilized antibody formulations are described in
U.S. Pat. No. 6,267,958. Aqueous antibody formulations include
those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the
latter formulations including a histidine-acetate buffer.
[0318] The formulation herein may also contain more than one active
ingredient as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. For example, it may be desirable to
further provide one or more active ingredients for treating a
neuropathy disorder, a neurodegenerative disease, cancer, an ocular
disease disorder, a seizure disorder, a lysosomal storage disease,
an amyloidosis, a viral or microbial disease, ischemia, a
behavioral disorder or CNS inflammation. Exemplary such medicaments
are discussed hereinbelow. Such active ingredients are suitably
present in combination in amounts that are effective for the
purpose intended.
[0319] Active ingredients may 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 active ingredients may be
encapsulated in liposomes that are coupled to anti-TfR antibodies
described herein (see e.g., U.S. Patent Application Publication No.
20020025313).
[0320] 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. Nonlimiting 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 7 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.
[0321] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
[0322] G. Therapeutic Methods and Compositions
[0323] Any of the anti-TfR antibodies provided herein may be used
in therapeutic methods. In one aspect, an anti-TfR antibody for use
as a medicament is provided. 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-TfR antibody coupled
to a therapeutic compound (e.g. a multispecific antibody which
binds both the TfR 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-TfR antibody of the invention
coupled to a brain disorder drug (e.g. a multispecific antibody
which binds both the TfR 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 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.
[0324] In one embodiment, the 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. The antibodies of the invention are
particularly suited to treatment of such neurological disorders due
to their ability to transport one or more associated active
ingredients/coupled therapeutic compounds across the BBB and into
the CNS/brain where such disorders find their molecular, cellular,
or viral/microbial basis.
[0325] 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.
[0326] 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.
[0327] 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).
[0328] 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.
[0329] 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.
[0330] 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 antiobiotic
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.
[0331] 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.
[0332] 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.
[0333] 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.
[0334] 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).
[0335] 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.
[0336] 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).
[0337] 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).
[0338] 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). 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.
[0339] 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.
[0340] 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.
[0341] 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-ira), 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).
[0342] 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).
[0343] 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).
[0344] 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).
[0345] 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)).
[0346] 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.
[0347] 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).
[0348] 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 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.
[0349] 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).
[0350] In one aspect, a low affinity anti-TfR antibody of the
invention for use as a medicament is provided. In further aspects,
a low affinity anti-TfR 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-TfR antibody for use in a
method of treatment as described herein is provided. In certain
embodiments, the invention provides a low affinity anti-TfR
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-TfR 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-TfR 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-TfR antibody of the invention for use in the
methods of the invention improves uptake of the neurological
disorder drug with which it is coupled.
[0351] In a further aspect, the invention provides for the use of a
low affinity anti-TfR 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.
[0352] 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.
[0353] The anti-TfR antibodies of the invention can be used either
alone or in combination with other agents in a therapy. For
instance, the anti-TfR antibody of the invention may be
coadministered 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-TfR 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, 7-secretase modulators, nerve growth factor (NGF)
mimics or NGF gene therapy, PPARy 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.
[0354] As exemplified herein, certain anti-TfR antibodies may have
side effects that negatively impact reticulocyte populations in a
subject treated with the anti-TfR 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-TfR 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-TfR 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-TfR 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-TfR 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-TfR antibody therapy
such that such blood cells can be replenished after the anti-TfR
antibody treatment.
[0355] 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-TfR antibody. Examples of such therapeutic agents include, but
are not limited to, agents that interfere with the ability of the
anti-TfR 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.
[0356] Such combination therapies noted above and herein 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.
In one embodiment, administration of the anti-TfR antibody and
administration of an additional therapeutic agent occur within
about one month, or within about one, two or three weeks, or within
about one, two, three, four, five or six days, of each other.
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.
[0357] An anti-TfR 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.
[0358] Antibodies of the invention would be 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.
[0359] 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 40 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0
mg/kg, 4.0 mg/kg, 5.0 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, 20
mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg or 40 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.
[0360] 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-TfR
antibody.
[0361] H. Articles of Manufacture
[0362] 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.
[0363] 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-TfR antibody.
EXAMPLES
Example 1: Generation, Characterization and Humanization of
Human/Cyno Cross-Reactive Anti-TFR Antibodies
[0364] Initially, a naive antibody phage panning process was
performed in an attempt to identify antibodies cross-reactive with
both human TfR and TfR from cynomolgous ("cyno") monkeys that
further did not compete with Tf for binding to TfR (Lee et al. JMB
(2004) 1073-1093). No such cross-reactive, non-Tf-competing clone
was identified from this phage panning process. However, two
antibodies were identified that were useful in characterizing
subsequently generated hybridoma clones.
[0365] A species cross-reactive antibody was identified that
competes with Tf for binding to human or cyno TfR (Tf-competing
antibody). The epitope of another clone, specific for human TfR,
was mapped to the apical domain of huTfR using mouse/human chimeric
TfR receptors (FIG. 1). This apical domain-binding clone lost
binding to huTfR when the mouse TfR sequence in the apical domain
was substituted into huTfR.
[0366] Next, an immunization-based approach to generate
cross-reactive anti-human/cyno TfR antibodies was performed. Human
TfR extracellular domain ("ecd") containing an N-terminal His tag
and human hemachromatosis protein ("HFE") were expressed and
purified as described (Bennet et al, Nature (2000) 403, 46-53). An
analogous cyno TfR ecd construct was also made. Cyno TfR was
expressed and purified in a similar manner. Human and cyno
cross-reactive TfR antibodies were generated by immunizing 5 Balb/C
mice in the footpad with 6 doses (twice per week) containing 2
.mu.g each of cynoTfR and huTfR ecd. All mice sera were FACS
positive and all mice were fused. Of 1632 hybridomas screened, 111
were ELISA positive for binding to both human and cyno TfR.
[0367] The resulting ELISA-positive hybridomas were screened by
FACS in the presence of 1 .mu.M human holo-Tf for binding to 293
cells transiently expressing human or cyno TfR. Briefly, FACS
analysis was performed using 293 cells transfected with full length
human or cyno TfR using lipofectamin 2000 plus (Invitrogen) 48-72 h
before FACS analysis. Non-transfected (control) and transfected 293
cells were washed twice with FACS buffer (PBS containing 1% BSA),
50 .mu.l of hybridoma supernatant (normalized to 10 .mu.g/ml) was
added to 293 cells in the presence of 1 .mu.M human holo-Tf and
incubated on ice for 30 min. Cells were washed twice with FACS
buffer, 50 .mu.l of PE-Goat-anti-murine Fc7 (Jackson
ImmunoResearch) was added to cells and they were incubated on ice
for 30 min. Cells were washed with FACS buffer and resuspended in
100 .mu.l FACS buffer for analysis. 14 clones were positive for
binding to both human and cyno TfR (FIGS. 2A and 2B).
[0368] These clones were further subcloned and evaluated for
binding to both human and cyno TfR by ELISA, and epitope mapped on
huTfR using the apical binding phage clone identified above.
Briefly, the apical domain phage competition ELISA was performed in
maxisorp plates coated with 2 .mu.g/ml of purified human or cyno
TfR in PBS at 4.degree. C. overnight. Plates were washed with
PBS/0.05% Tween 20 and blocked using Superblock with casein (Thermo
Scientific, Hudson, N.H.). A 30 .mu.l aliquot of hybridoma
supernatant (normalized to 10 .mu.g/ml) was added to each well for
45 min. This was followed by the addition of 30 .mu.l apical
domain-binding phage at OD 0.05 for 15 min. Plates were washed with
PBS/0.05% Tween 20 and 1:1000 diluted HRP-Mouse-anti M13 (GE
healthcare) was added the plate and incubated for 1 h at room
temperature. Plates were washed with PBS/0.05% Tween 20 and bound
phage were detected using TMB substrate (BioFX Laboratories, Owings
Mills). Nine of the fourteen clones were found to block binding of
the apical binding antibody displayed on phage (see FIG. 2C).
[0369] Antibody affinities were measured using surface plasmon
resonance ("SPR") (Biacore.TM. GE Healthcare). Anti-His antibody
(Qiagen) was coupled onto four different flow cells of a
BIACORE.TM. CM5 sensor chip (Biacore, Inc., Piscataway, N.J.) at
between 6000 and 8000 RU. Immobilization was achieved by random
coupling through amino groups using a protocol provided by the
manufacturer. 10.times.HBS-P (Biacore, Inc., Piscataway, N.J.) was
diluted in water and served as the dilution and running buffer.
Purified human or cyno TfR was captured, followed by a 3-fold
dilution series of IgG or Fab that was injected at a flow rate of
30 ml/min using the single cycle kinetics method. Affinity
constants were determined using a simple 1:1 Langmuir binding model
or using a steady state model when k.sub.on or k.sub.off was beyond
the detection limit. The equilibrium dissociation constant
(K.sub.D) was calculated as the ratio of association rate constant
(k.sub.on) and dissociation rate constant (k.sub.off). The results
are shown in FIG. 2C.
[0370] Each hybridoma was cloned. Total RNA was isolated from
hybridoma using an RNeasy mini kit (Qiagen). cDNA was generated
using a SMART 5' RACE cDNA Amplification kit (Clontech) based on
the manufacturer's instructions. The variable region of each
antibody was amplified using UPM (5' oligo) provided in the kit and
a 3' oligo that anneals to the constant region. The entire PCR
product was then cloned into pCR4Blunt-TOPO vector (Invitrogen) for
sequencing. After sequence analysis, the hybridomas could be
further subdivided into 4 groups (FIGS. 3A-3D). Clones that
competed with the apical binding antibody fell into 3 related
sequence classes (FIG. 3 A-C). The 4 non-apical clones (FIG. 3D)
consisted of 2 related clones and 2 other unique sequences. The
light and heavy chain CDRs of each clone are provided in Table
3.
TABLE-US-00003 TABLE 3 Light and Heavy Chain CDRs of Cross-Reactive
Anti-Cyno/Human HR Antibodies SEQ SEQ SEQ Clone Heavy/ ID ID ID
name Light HVR1 # HVR2 # HVR3 # 7A4 Light RASESVDSYGNSFMH 29
RASNLES 30 QQSNEAPPT 31 Heavy DYAMH 32 GISTYFGRTNYNQKFKG 33
GLSGNYVM 34 DY 8A2 Light RASESVDSYGNSFMH 35 RASNLES 30 QQSNEGPPT 36
Heavy DYGMH 37 VISPYSGRTNYNQNFKG 38 GLSGNYVVD 39 Y 15D2 Light
RASESVDSYGNSFMH 35 RASNLES 30 QQSNEGPPT 36 Heavy DYAMH 32
VISFYSGKTNYNQKFMG 40 GLSGNYVM 34 DY 10D11 Light RASESVDSYGNSFMH 41
RASNLES 30 QHSNEDPPT 42 Heavy DYGMH 37 VISPYSGKTNYSQKFKG 43
GLSGNFVMD 44 F 7B10 Light RASESVDSYGNSFMH 29 RASNLES 30 QQSNEAPPT
31 Heavy DYAMH 32 GISTYFGRTNYNQKFKG 33 GLSGNYVM 34 DY Consensus
Class RASESVD(S/D)YG(N/P) 45 RASNLES 30 Q(Q/H)SNE(A/ 46 I Light
CDRs SFMH G/D)PPT Consensus Class DY(A/G)MH 47
(G/V)IS(T/F/P)Y(F/S)G(R/ 48 GLSGN(Y/F)V 49 I heavy CDRs
K)TNY(N/S)Q(K/N)F(K/M) (M/V)D(Y/F) G 15G11 Light RASDNLYSNLA 50
DATNLAD 51 QHFWGTPLT 52 Heavy SYWMH 53 EINPTNGRTNYIEKFKS 54 GTRAYHY
55 16G5 Light RASENIYSNLA 56 AATDLAD 57 QHFWGTPLT 52 Heavy SYWMH 53
EINPTNGRTNYNENFKS 58 GTRAYHF 59 13C3 Light RASDNIYSNLA 60 AATNLAD
61 QHFWGTPLM 62 Heavy SYWMH 53 EINPINGRTNYSEKFKK 63 GTRAYHY 55 16G4
Light RASDNIYSNLA 60 AVTNLAD 64 QHFWGTPLT 52 Heavy SYWMH 53
EINPSNGRTNYNETFKS 65 GTRAYHY 55 Consensus Class RAS(E/D)N(L/I)YSNLA
66 (D/A)(A/V)T(N/D)LAD 67 QHFWGTPL 68 II Light CDRs (T/M) Consensus
Class SYWMH 53 EINP(T/I/S)NGRTNY(I/N/ 69 GTRAYH(Y/F) 70 II Heavy
CDRs S)E(K/N/T)FK(S/K) 16F6 Light RASKSISKYLA 71 SGSTLQS 72
QQHNEYPWT 73 Heavy SEYAWN 74 YISYSGTTSYNPSLKS 75 YGYGNPATR 76 YFDV
7G7 Light RARQSVSTSSYSFMH 77 YASIQES 78 QHTWEIPFT 79 Heavy SYWMH 80
NIYPGSGSTKYDERFKS 81 GGYDSRAWF 82 AY 4C2 Light RARQSVSTSSYSFMH 77
YASIQES 78 QHTWEIPFT 79 Heavy SYWMH 80 NIYPGSGSTKYDEKFKS 83
GGYDSRAWF 84 AH 1B12 Light TTS SSVPSSYFH 85 STSNLAS 86 HQYHRSPFT 87
Heavy DYYMY 88 SISNGGDNTYYPDTVKG 89 QGALYDGYY 90 RGAMDY 13D4 Light
RAGQDITNYLN 91 YTSRLHS 92 QQANTLPYT 93 Heavy NYWIE 94
EILPGSGSTKYNEKFKG 95 RGGYGYDGEF 96 AY Consensus Class
(R/T)(A/T)(R/S/G)(Q/S) 97 (Y/S)(A/T)S(I/N/R)(Q/L) 98
(Q/H)(H/Q)(T/Y/ 99 IV Light CDRs (S/-)(V/-)(S/-)(T/-) (E/A/H)S
A)(W/H/N)(E/R/ (S/V/D)(S/P/I)(Y/S/T)(S/N) T)(I/S/L)P(F/Y)
(F/Y)(M/F/L)(H/N) T Consensus Class (S/D/N)Y(W/Y)(M/I)(H/Y/ 100
(N/S/E)I(Y/S/L)(P/N)G(S/G) 101 (G/Q/R)G(Y/A/ 102 IV Heavy CDRs E)
(G/D)(S/N)T(K/Y)Y(D/P/N)(E/ G)(D/L/Y)(S/Y/ D)(R/K/T)(F/V)K(S/G)
G)(R/D/Y)(A/G/ D)(W/Y/G)(F/Y/ E)(R/F/-)(G/-) (A/-)(M/-)
(A/D)(Y/H)
[0371] Representative clones from each class, (15G11, 7A4, 16F6 and
7G7), are exemplified herein for humanization and further
characterization. Humanization was achieved using HVR grafts along
with the inclusion of select vernier positions as outlined below
and FIGS. 4A-4E. 15G11 was humanized by grafting the HVRs into the
IGKV1-NL1*01 and IGHV1-3*01 human variable domains. Combinations of
different mouse vernier positions were included in the humanized
variants as outlined in FIG. 4E. Humanized 15G11 variant 15G11.v5
contains selected vernier positions in VL (positions 43 and 48) and
VH (positions 48, 67, 69, 71 and 73) as outlined in FIG. 4A. In
addition, The N-terminus of VH was changed from Q to E. For
humanization of 7A4, an HVR graft was made using the 7A4 heavy
chain and 8A2 light chain HVRs (7A4 and 8A2 are related clones,
FIG. 3A). HVRs were grafted into the IGKV4-1*01 and IGHV1-2*02
human variable domains. Combinations of different mouse vernier
positions were included in the humanized variants as outlined in
FIG. 4E. Humanized 7A4 variant, 7A4.v15 contains selected vernier
positions in VL (position 68) and VH (positions 24 and 71) and the
CDR-L3 change G94A, as outlined in FIG. 4B. 7G7 was humanized by
grafting the HVRs into the kappa 4 and subgroup I human consensus
variable domains along with selected vernier positions in VH
(position 93) as outlined in FIG. 4C. This humanized variant is
called 7G7.v1. 16F6 was humanized by grafting the HVRs into the
IGKV1-9*01 and IGHV4-59*01 human variable domains. Combinations of
different mouse vernier positions were included in the humanized
variants as outlined in FIG. 4E. Humanized 16F6 variant 16F6.v4
contains 2 changes in VL (I48L and F71Y) as well as selected
vernier positions in VL (positions 43 and 44) and VH (positions 71
and 78) as outlined in FIG. 4D.
TABLE-US-00004 TABLE 4 Light and Heavy Chain CDRs of Humanized
Antibodies/Fabs SEQ SEQ SEQ Clone Heavy/ ID ID ID name Light CDR1 #
CDR2 # CDR3 # 15G11.v5 Light RASDNLYSNLA 50 DATNLAD 51 QHFWGTPLT 52
Heavy SYWMH 53 EINPTNGRTNYIEKFKS 54 GTRAYHY 55 7A4.v15 Light
RASESVDSYGNSFMH 29 RASNLES 30 QQSNEAPPT 127 Heavy DYAMH 32
GISTYFGRTNYNQKFK 33 GLSGNYVM 34 G DY 7G7.v1 Light RARQSVSTSSYSFMH
77 YASIQES 78 QHTWEIPFT 79 Heavy SYWMH 80 NIYPGSGSTKYDERFKS 81
GGYDSRAWF 82 AY 16F6.v4 Light RASKSISKYLA 71 SGSTLQS 72 QQHNEYPWT
73 Heavy SEYAWN 74 YISYSGTTSYNPSLKS 75 YGYGNPATR 76 YFDV
[0372] The affinity of humanized variants for human and cyno TfR
was determined by SPR as IgG (FIG. 4E). Selected clones were also
analyzed by SPR as Fab to assess monovalent affinity (Table 7). In
both cases, the SPR experiments were performed as described
above.
TABLE-US-00005 TABLE 5 Biacore Binding Data for Selected
Fab-Formatted Variants HuTfR CynoTfR Cy/hu Sample Ka Kd KD Ka Kd KD
ratio Mu15G11.Fab 1.38E+06 4.65E-03 3.37E-09 1.07E+06 6.23E-03
5.81E-09 1.72 Mu15G11.Fab 6.34E+05 1.52E-03 2.41E-09 4.85E+05
3.68E-03 7.57E-09 3.15 Hu15G11.v1.Fab 6.38E+05 0.006986 1.09E-08
5.05E+05 0.0373 7.39E-08 Hu15G11.v3.Fab 6.42E+05 0.004657 7.26E-09
4.83E+05 0.0201 1.09E-08 Hu15G11.v5.Fab 4.56E+05 0.004063 8.91E-09
hu15G11.v5.Fab 7.76E+05 0.003643 4.70E-09 1.41E+06 0.02184 1.56E-08
3.4 Mu7A4.Fab 1.65E+06 3.13E-04 1.90E-10 1.14E+06 8.45E-04 7.41E-10
3.9 Hu7A4.v5.Fab 2.24E+05 1.53E-03 6.86E-10 1.18E+06 6.41E-03
5.44E-09 Hu7A4.v8.Fab 9.28E+05 1.07E-03 1.15E-09 7.97E+05 6.81E-03
8.55E-09 Hu7A4.v9.Fab 1.71E+06 6.86E-04 4.01E-10 8.08E+05 3.42E-03
4.23E-09 Hu7A4.v12.Fab 3.32E+06 8.44E-04 2.55E-10 1.74E+06 3.31E-03
1.90E-09 hu7A4.v15.Fab 9.10E+05 3.17E-04 3.48E-10 3.78E+05 0.001618
4.28E-09 11 Hu7G7.v1Fab 1.44E+05 0.006594 4.58E-08 3.84E+04
0.007231 1.88E-07 4.4 Mu16F6.Fab 6.07E+04 1.90E-04 3.13E-09
5.11E+04 1.37E-03 2.68E-08 8.56 Hu16F6.v4.Fab 1.31E+05 1.69E-04
1.29E-09 9.89E+04 2.44E-03 2.47E-08 19.1
[0373] The binding epitope of the antibodies were re-confirmed as
follows. A Tf-TfR blocking ELISA was performed in maxisorp plates
coated with 2 .mu.g/ml of purified human TfR 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,
NIH). 50 .mu.l of 12.5 M human holo-Tf (R&D Systems,
Minneapolis, Minn.) was added to the plates for 40 min. A 50 .mu.l
titration of hu7A4.v15, hu15G11.v5, Tf competiting antibody, and
hu7G7.v1 (beginning at 10 ug/ml, 1:3 serial dilution) was added to
the plate and incubated for 20 min. Plates were washed with
PBS/0.05% Tween 20 and 1:1000 diluted HRP-Goat-anti human Fey
(Jackson ImmunoResearch) was added to the plate and incubated for 1
h at room temperature. Plates were washed with PBS/0.05% Tween 20
and detected using TMB substrate (BioFX Laboratories, Owings
Mills).
[0374] An HFE-TfR binding ELISA was performed in maxisorp plates
coated with 1 .mu.g/ml of HFE 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 human TfR (start at 100 ug/ml, 1:3 serial
dilution) was added to the plate and incubated for 1 h. 1 .mu.g/ml
of hu15G11.v5, hu7A4.v15 or hu7G7.v1 was then added to the plate
for 1 h. Plates were washed with PBS/0.05% Tween 20 and 1:1000
diluted HRP-Goat-anti human Fc.gamma. (Jackson ImmunoResearch) was
added the plate and incubated for 1 h at room temperature. Plates
were washed with PBS/0.05% Tween 20 and detected using TMB
substrate (BioFX Laboratories, Owings Mills). An HFE-TfR blocking
ELISA was performed in maxisorp plates coated with 1 .mu.g/ml HFE
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.). In a NUNC.TM. plate, a titration
of hu7A4.v15, hu15G11.v5, Tf competiting antibody, human holo-Tf
and control IgG (400 .mu.g for all antibody, 8000 .mu.g/ml for holo
transferrin, 1:3 serial dilution) was combined with 2 .mu.g/ml of
biotinylated human TfR and incubated for 1 h. The mixture was then
added to the HFE coated plate for 1 h at room temperature. Plates
were washed with PBS/0.05% Tween 20 and 1:1000 diluted
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 human TfR bound to the
plate was detected using TMB substrate (BioFX Laboratories, Owings
Mills).
[0375] Binding of these humanized variants to TfR was unaffected by
the presence of 6.3 .mu.M holo-Tf, whereas the binding of the Tf
competing antibody that binds to the Tf binding site on TfR was
inhibited (FIG. 5). Further, humanized 7A4.v15, 15G11.v5 and 7G7.v1
could still bind HFE captured huTfR, indicating that they did not
affect binding of huTfR to immobilized HFE (FIG. 6A). In a related
experiment, 7A4.v15 and 15G11.v5 did not block biotinylated TfR
from binding to immobilized HFE. In contrast, this interaction was
blocked by the Tf competing antibody and holo Tf (FIG. 6B). HFE and
Tf are known to share a similar epitope on TfR (Bennet et al,
Nature (2000) 403, 46-53).
[0376] Immobilized 15G11v.5 and anti-TfR.sup.C12 were evaluated for
binding to biotinylated human TfR ECD or monovalent M13 phage
displaying the human TfR apical domain. Anti-TfR.sup.C12 was
derived from a synthetic antibody phage library that was panned
against human TfR ECD and binds to a site on the human TfR which
competes with transferrin binding. Antibodies were coated at 1
.mu.g/ml in PBS on Maxisorp plates. Bound biotinylated human TfR
ECD or TfR-apical domain phage were detected with HRP-streptavidin
(GE health care, RPN 4401V) or HRP-anti-M13 (GE health care,
27-9421-01), respectively. FIG. 25 shows that 15G11v.5 binds to
human TfR apical domain. The 15G11v.5 binding site was mapped to
the apical domain, a site distant from the TfR ligand binding
sites.
Example 2: Affinity Engineering Human/Cyno Cross-Reactive Anti-TFR
Antibodies
[0377] In addition to the humanized variants described above,
additional affinity engineered variants were made. Exemplified
herein is affinity engineering of 15G11.v5 and 7A4.v15. Affinity
variants were generated by making individual alanine substitutions
in CDR-L3 or CDR-H3 using standard techniques. These variants were
screened as IgG by ELISA and SPR to identify positions important
for binding to human and cyno TfR; the monovalent affinity of
selected variants as Fab was also determined. Ala scan variant IgGs
or Fab were expressed in 293 cells and binding to human or cyno TfR
quantified by ELISA in maxisorp plates coated with 1.8 .mu.g/ml of
goat anti-human Fc.gamma. (Jackson ImmunoResearch) 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.). Supernatants containing expressed IgG
were diluted serially 1:5 and added to the plate for 1 h. Purified
hu15G1.v5 or hu7A4.v15 were used as standards (diluted 1:5
beginning at 1 ug/ml). Plates were washed with PBS/0.05% Tween 20
and 1:1000 diluted HRP-Goat-anti kappa (Southern Biotech) was added
the plate and incubated for 1 h at room temperature. Plates were
washed with PBS/0.05% Tween 20 and detected using TMB substrate
(BioFX Laboratories, Owings Mills). Binding was also assessed by
SPR, as described above. The results are shown in FIGS. 7A
(15G11.v5 variants) and 7B (7A4.v15 variants).
[0378] Further variants of 15G11.v5 with individual alanine
substitutions at positions in CDR-L1, CDR-L2, CDR-H1 and CDR-H2
were also generated, expressed and first screened for binding to
human and cyno TfR by ELISA (Table 6). The Hu/Cy binding ELISA was
performed in maxisorp plates coated with 2 .mu.g/ml of purified
human or cyno TfR 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.). Cell
culture supernatants containing the expressed Ala scan variant IgGs
were serially diluted 1:5 and added to the wells for 1 h. Plates
were washed with PBS/0.05% Tween 20 and 1:1000 diluted
HRP-Goat-anti human Fc.gamma. (Jackson ImmunoResearch) was added
the plate and incubated for 1 hour at room temperature. Plates were
washed with PBS/0.05% Tween 20 and detected using TMB substrate
(BioFX Laboratories, Owings Mills).
TABLE-US-00006 TABLE 6 ELISA Analysis of hu15G11.v5 IgG Ala
Variants CynoTfR HuTfR EC50 (ng/ml) EC50 (ng/ml) 15G11.v5 1.8 0.8
HVR-H1 G26A 6.1 1.1 Y27A 1467.1 21.1 T28A 0.8 0.4 F29A 12.5 1.5
T30A 0.9 0.4 S31A 0.1 0.05 Y32A 1.4 0.4 W33A 362.5 59.6 M34A 1.1
0.4 HVR-H2 G49A 1.1 0.3 E50A 409.7 20.6 I51A 0.6 0.2 N52A 0.9 0.3
P52aA 8.1 3.9 T53A 0.7 0.3 R56A 5405.4 55.1 N58A 80.4 6.3 Y59A 0.7
0.3 I60A 0.7 0.3 E61A 0.6 0.2 K62A 0.7 0.3 F63A 0.6 0.4 K64A 0.6
0.3 S65A 0.7 0.2 R R24A 0.4 0.1 S26A 0.6 0.2 D27A 0.8 0.2 N28A 0.8
0.2 L29A 0.9 0.3 Y30A 1.0 0.3 S31A 0.7 0.3 N32A 4.0 1.6 L33A 0.1
0.05 HVR-L2 D50A 0.5 0.2 T52A 0.3 0.2 N53A 0.6 0.3 L54A 0.5 0.4
D56A 0.5 0.3
Selected variants were then purified and their monovalent affinity
for human or cyno TfR assessed by SPR (Table 7).
TABLE-US-00007 TABLE 7 Monovalent SPR Analysis of Select 15G11.v5
Fab Alanine Variants HuTfR CynoTfR Cy/hu Ka Kd KD Ka Kd KD ratio
Hu15G11.v5 Fab 6.74E+05 4.74E-03 7.03E-09 4.51E+05 1.27E-02
2.82E-08 4.0 Hu15G11.HC32A Fab 2.38E+05 1.78E-03 7.47E-09 1.77E+05
8.51E-03 4.80E-08 6.4 Hu15G11.HC34A Fab 5.64E+05 4.03E-03 7.14E-09
3.04E+05 9.90E-03 3.26E-08 4.6 Hu15G11.HC52A Fab 5.30E+05 2.36E-02
4.44E-08 4.87E+05 5.67E-02 1.17E-07 2.6 Hu15G11.HC52A Fab 5.64E+05
1.96E-02 3.46E-08 ND ND ND ND Hu15G11.HC51A Fab 4.33E+05 1.04E-02
2.39E-08 3.13E+05 3.29E-02 1.05E-07 4.4 Hu15G11.HC53A Fab 8.90E+05
1.15E-02 1.29E-08 4.84E+05 2.50E-02 5.18E-08 4.0 Hu15G11.HC54A Fab
2.06E+05 8.71E-03 4.24E-08 2.80E+05 1.69E-02 6.02E-08 1.4
Example 3: Bispecific Anti-Human TFR Antibody Construction and In
Vitro Analysis
[0379] Certain of the foregoing antibody variants were reformatted
as bispecific antibodies with a second arm specifically binding to
BACE1. The anti-human TfR antibodies Hu15G11.v5, Hu15G11.LC92A,
Hu15G11.HC52A and Hu15G11.HC53A were used to engineer the TfR
binding arm of the bispecific using `knob in hole` bispecific
antibody construction technology (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). In addition to
the knob and hole mutations in the Fc for anti-TfR (hole) and
anti-BACE1 (knob), all half-antibodies contained mutations in the
Fc region that abrogated effector function (N297G) and Hu15G11.v5
and Hu15G11.LC92A contained an additional Fc mutation that
abrogated effector function (D265A). The knob and hole
half-antibodies were purified separately from E. coli and combined
at a 1:1.1 ratio of anti-TfR to prevent formation of anti-TfR
homodimers. Assembly of the bispecific antibody was completed by
reductive annealing for at least three days at room temperature in
a buffer containing reduced glutathione at a 100.times. ratio to
antibody and 200 mM arginine pH 8.0. Following assembly, bispecific
antibodies were purified by hydrophobic interaction chromatography.
The assembly was confirmed by liquid chromatography mass
spectroscopy and SDS-PAGE. The purified antibodies were confirmed
to be homogeneous and monodisperse by size exclusion and multi
angle laser light spectroscopy.
[0380] The resulting bispecific antibodies were called 15G11.v5
(anti-TfR.sup.1), 15G11.W92A (15G11.LC92A or anti-TfR.sup.2),
Hu15G11.N52A (anti-TfR.sup.52A) and Hu15G11.T53A (anti-TfR.sup.53A)
The monovalent affinity and kinetics for human and cyno TfR was
determined for 115G11.v5 and 115G11.W92A by SPR, as above (see
Table 9). Anti-TfR.sup.1 and anti-TfR.sup.2 possess similar
monovalent binding affinities as anti-TfR.sup.A and anti-TfR.sup.D
do for binding to mouse TfR (see Atwal et al., Sci. Transl. Med. 3,
84ra43 (2011); Yu et al., Sci. Transl. Med. 25 May 2011: Vol. 3,
Issue 84, p. 84ra44).
TABLE-US-00008 TABLE 8 Monovalent SPR Analysis of 15G11.v5
(TfR.sup.1) and 15G11.W92A (LC92A, TfR.sup.2) Cyno/ HuTfR CynoTfR
human Ka Kd KD Ka Kd KD ratio Hu15G11.v5 Fab 6.74E+05 4.74E-03
7.03E-09 4.51E+05 1.27E-02 2.82E-08 4.0 Hu15G11.W92A 1.28E+05
3.77E-02 2.95E-07 8.36E+04 5.20E-02 6.22E-07 2.1 Bispecific
[0381] Additionally, the binding affinity of the anti-TfR.sup.1,
anti-TfR.sup.2, Hu15G11.N52A and Hu15G11.T53A bispecific antibodies
were measured against human and cyno TfR by SPR as previously
described. As shown in Table 9 below, Anti-TfR.sup.52A and
anti-TfR.sup.53A have binding affinities to human and cyno TfR
between TfR1.sup.h15G11.v5 and TfR2.sup.LC92A.
TABLE-US-00009 TABLE 9 Anti-cyno/human TfR antibodies (nM) Human
TfR Cyno TfR TfR1.sup.h15G11.v5 10 37 TfR2.sup.LC92A 270 810
TfR.sup.52A 52 343 TfR.sup.53A 24 143
Example 4: Impact of Effector-Containing and Effectorless
Monospecific and Bispecific Antibodies on a Human Erythroleukemia
Cell Line and Primary Bone Marrow Mononuclear Cells
[0382] Prior studies in mice had determined that antibodies binding
murine TfR with effector function and/or complement binding
capabilities selectively depleted TfR-expressing reticulocytes. To
ascertain whether the depletion observed in the mouse studies was
unique to a murine system, further experiments were performed
utilizing anti-TfR that bind to human TfR.
[0383] 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. 8A-B). For the second set of
experiments (FIG. 9A-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.
[0384] 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 4 9 0 ( Sample ) - A 4 9 0 ( A I C C ) A 4 9
0 ( High Contro1 ) - A 4 9 0 ( Low Contro1 ) ##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.
[0385] 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 an anti-BACE1 arm in a human IgG1 format
containing D265A and N297G mutations to abrogate effector function
(see Example 3), 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. 8A and 8B. With either the HEL cells as targets
(FIG. 8A) or the bone marrow mononuclear cells as targets (FIG.
8B), the monospecific, effector positive 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 relative to 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 heterogeneous
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.
[0386] 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, on 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. 9A and 9B. 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. 9A to FIG. 9B). 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)).
Example 5: Assessment of Bispecific Anti-Human TFR/BACE1 Bispecific
Antibodies In Vivo
[0387] A. Pharmacokinetic, Pharmacodynamic and Safety Study
[0388] To evaluate the drug concentrations, pharmacodynamics
effects, and safety of the bispecific anti-human TfR antibodies in
vivo, cynomolgus monkeys (Macaca fascicularis) were dosed with
bispecific antibodies using anti-TfR antibody clone 15G11 paired
with the same anti-BACE1 arm used in prior examples
(anti-TfR.sup.1/BACE1), or clone 15G11.LC92A paired with the same
anti-BACE1 arm used in prior examples (anti-TfR.sup.2/BACE1) or
Hu15G11.N52A (anti-TfR.sup.52A/BACE1) and Hu15G11.T.sup.53A
(anti-TfR.sup.53A/BACE1). These bispecific antibodies were in a
human IgG1 format with N297G or D265A and N297G mutations
abrogating effector function, as described previously. As a
control, an anti-gD molecule on human IgG1 was used. This study was
performed in non-human primates because crossreactivity of these
anti-TfR antibodies is limited to non-human primates and humans. In
addition, studies have shown that the mechanisms of drug transport
between the cerebrospinal fluid (CSF) and plasma compartments may
be similar between humans and primates (Poplack et al, 1977). The
antibodies were administered by a single intravenous (IV) bolus
injection into the saphenous vein at a dose of 30 mg/kg to
conscious cynomolgus monkeys with indwelling cisterna magna
catheters. At various timepoints up to 60 days post-dose, plasma,
serum, and (CSF) were sampled. Sample analysis included hematology
(whole blood), clinical chemistry (serum), antibody concentrations
(serum and CSF), and pharmacodynamic response to the antibody
(plasma and CSF). See FIG. 10 for a detailed sampling scheme.
[0389] The concentrations of the dosed antibodies in cynomolgus
monkey serum and CSF were measured with an ELISA using a sheep
anti-human IgG monkey absorbed antibody coat, followed by adding
serum samples starting at a dilution of 1:100, and finished by
adding a goat anti-human IgG antibody conjugated to horseradish
peroxidase monkey adsorbed for detection. The assay had a standard
curve range of 0.78-50 ng/mL and a limit of detection of 0.08
.mu.g/mL. Results below this limit of detection were reported as
less than reportable (LTR).
[0390] FIGS. 11A-B shows the results of the pharmacokinetic
analysis for anti-TfR1/BACE1 and anti-TfR2/BACE1. The
pharmacokinetic profile for anti-gD was as expected for a typical
human IgG1 antibody in cynomolgus monkey with a mean clearance of
3.98 mL/day/kg. Both anti-TfR/BACE1 antibodies cleared faster than
anti-gD, likely due to peripheral target-mediated clearance.
Anti-TfR1/BACE1 had the fastest clearance, consistent with it
having the highest binding affinity to TfR, whereas anti-TfR2/BACE1
showed an improved pharmacokinetic profile (ie, prolonged exposure
in serum) as compared to anti-TfR1/BACE1, likely due to its reduced
affinity for TfR. The clearance for anti-TfR/BACE1 and
anti-TfR2/BACE1 were 18.9 mL/day/kg and 8.14 mL/day/kg,
respectively. All antibodies were detected in the CSF at
approximately one one-thousandth of the serum concentration.
However, there was high variability, and overall no detectable
difference in the CSF antibody concentrations across the
molecules.
TABLE-US-00010 TABLE 10 Mean (.+-.SD) PK parameter estimates for
all test antibodies following a single IV bolus dose administration
at 30 mg/kg in cynomolgus monkeys (n = 5) AUC.sub.all AUC.sub.inf
C.sub.max CL V.sub.ss Antibody (day*.mu.g/mL) (day*.mu.g/mL)
(.mu.g/mL) (mL/day/kg) (mL/kg) anti-gD 7640 .+-. 1790 7930 .+-.
1910 912 .+-. 141 3.98 .+-. 1.05 51.3 .+-. 10.2
anti-TfR.sup.1/BACE1 1610 .+-. 240 1610 .+-. 237 809 .+-. 132 18.9
.+-. 2.54 41.0 .+-. 8.18 anti-TfR.sup.2/BACE1 3750 .+-. 528 3750
.+-. 530 850 .+-. 69.2 8.14 .+-. 1.21 41.2 .+-. 6.06 SD = standard
deviation; IV = intravenous; AUC.sub.all = area under the
concentration-time curve from time 0 to the time of last measurable
concentration; AUC.sub.inf = area under the concentration-time
curve extrapolated to infinity; C.sub.max = observed maximum serum
concentration; CL = clearance; V.sub.ss = volume of distribution at
steady state; Min = minimum; Max = maximum.
[0391] FIG. 19 shows the results of the pharmacokinetic analysis
for anti-TfR.sup.1/BACE1, anti-TfR.sup.52A/BACE1 and
anti-TfR.sup.53A/BACE1. All anti-TfR/BACE1 antibodies cleared
faster than anti-gD, likely due to peripheral target-mediated
clearance. Anti-TfR.sup.1/BACE1 had the fastest clearance,
consistent with it having the highest binding affinity to TfR,
whereas anti-TfR.sup.52A/BACE1 and anti-TfR.sup.53A/BACE1 showed an
improved pharmacokinetic profile (ie, prolonged exposure in serum)
as compared to anti-TfR.sup.1/BACE1, likely due to the reduced
affinity for TfR of anti-TfR.sup.52A/BACE1 and
anti-TfR.sup.53A/BACE1.
[0392] To look at the pharmacodynamic effect in response to
anti-TfR/BACE1 dosing, we measured Abeta.sub.1-40 and sAPP.alpha.
and sAPP.beta. levels in cynomolgus monkey plasma and CSF.
Abeta.sub.1-40 was measured with an ELISA using an
anti-Abeta.sub.1-40 specific polyclonal antibody coat, followed by
adding samples, and finishing by adding a mouse anti-human
Abeta.sub.1-40 monoclonal antibody conjugated to horseradish
peroxidase for detection. The assay has a limit of detection of 60
.mu.g/mL for plasma and 140 .mu.g/mL for CSF. Results below this
concentration were reported as less than reportable (LTR). CSF
concentrations of sAPP.alpha. and sAPP.beta. were determined using
the sAPP.alpha./sAPP.beta. Multi-spot assay (Mesoscale Discovery
(Gaithersburg, Md.)). CSF was thawed on ice, then diluted 1:10 into
1% BSA in TBS-T (10 mM Tris buffer, pH 8.0, 150 mM NaCl, 0.1%
Tween-20). The assay was performed as per the manufacturer's
protocol. The assay had lower limit of quantification values of
0.05 ng/ml for sAPP.alpha. and 0.03 ng/mL for sAPP.beta..
[0393] FIGS. 12A-E summarize the pharmacodynamics behavior of the
antibodies. In the periphery, plasma Abeta.sub.1-40 levels remained
unchanged following anti-gD administration, but transiently
decreased following anti-TfR/BACE1 administration. Both variants
reduced plasma Abeta.sub.1-40 levels, with a maximal inhibition of
50% achieved 1 day post-dosing. Plasma Abeta.sub.1-40 levels
gradually recovered, with animals given anti-TfR.sup.1/BACE1
returning to baseline Abeta.sub.1-40 levels around 14 days
post-dose. Abeta.sub.1-40 levels returned to baseline levels
between 21 and 30 days post-dose in animals treated with
anti-TfR.sup.2/BACE1. Both anti-TfR/BACE1 antibodies reduced CSF
Abeta.sub.1-40 levels, with no change observed in anti-gD dosed
animals. Anti-TfR.sup.1/BACE1 administration resulted in a more
significant decrease in CSF Abeta.sub.1-40 levels (average maximal
inhibition 50% of baseline) than that of anti-TfR.sup.2/BACE1
(average maximal inhibition 20% of baseline). sAPP.beta. production
was inhibited in anti-TfR/BACE1 treated animals, but not in animals
who received anti-gD. Similar to results for A040,
anti-TfR.sup.1/BACE1 had a stronger inhibitory effect on sAPP.beta.
production than anti-TfR.sup.2/BACE1. sAPP.alpha. production was
stimulated during BACE1 inhibition by both anti-TfR.sup.1/BACE1 and
anti-TfR.sup.2/BACE1, and the response correlated inversely with
the level of inhibition observed for sAPP.beta. and Abeta.sub.1-40.
SAPP.alpha. and sAPP.beta. are the primary processing products of
amyloid precursor protein (APP), and their levels are highly
correlated. The ratio of sAPP.beta./sAPP.alpha. normalizes the
results to potential changes in basal APP expression or potential
preanalytical differences in CSF collection and handling over the
course of the study. The ratio of CSF sAPP.beta./sAPP.alpha. with
anti-TfR.sup.1/BACE1 demonstrated a more robust PD effect than
anti-TfR.sup.2/BACE1. Thus, these results support target (i.e.
BACE1) engagement by the anti-TfR/BACE1 antibodies.
[0394] The PD response for anti-TfR.sup.52A/BACE1 and
anti-TfR.sup.53A/BACE1 also correlates with the duration of
antibody exposure and a reduced affinity TfR arm shows increased
reduction in A.beta..sub.40 (data not shown). These data also
support target engagement by these bispecific antibodies.
[0395] Overall, these results suggest that a bispecific
anti-TfR/BACE1 antibody with an affinity for human TfR between that
of anti-TfR.sup.1/BACE1 and anti-TfR.sup.2/BACE1 would likely have
a desirable pharmacokinetic/pharmacodynamic balance.
[0396] No safety signals were observed in this study. There were no
evident effects on any hematology or clinical chemistry parameters
of monkeys given 30 mg/kg of any bispecific antibody administered
up to 60 days post-dose. Importantly, reticulocyte levels were
unaffected by treatment with either anti-TfR.sup.1/BACE1 or
anti-TfR.sup.2/BACE1 (FIG. 13), as expected since these antibodies
were effector-function-impaired and the overall level of
circulating early reticulocytes with high TfR levels is very low in
normal primates (see Example 4).
Example 6: Assessment of Bispecific Anti-Human TFR/BACE1 Bispecific
Antibodies In Vivo
[0397] To examine the relationship between antibody
pharmacodynamics in CSF and pharmacokinetics in brain, cynomolgus
monkeys (Macaca fascicularis) were dosed with bispecific antibodies
anti-TfR.sup.1/BACE1 or anti-TfR.sup.2/BACE1, as in the previous
example. These bispecific antibodies were in a human IgG1 format
with D265A and N297G mutations abrogating effector function. As a
control, an anti-gD molecule on human IgG1 was used. For
comparison, we also dosed with a bivalent anti-BACE1 antibody,
which is the same clone used for the bispecific antibodies. The
antibodies were administered by a single intravenous (IV) bolus
injection into the saphenous vein at a dose of 30 mg/kg to
conscious cynomolgus monkeys with indwelling cisterna magna
catheters. Baseline CSF samples were collected 24 and 48 hours
prior to dosing, and another CSF sample was collected 24 hours
post-dose (as shown schematically in FIG. 14). Following CSF
collection 24 hours post-dose, animals were perfused with saline
and brains were harvested for analysis of antibody concentrations.
Different brain regions were 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, using the ELISA method described in the previous
example. Blood was also collected to confirm peripheral exposure
and pharmacodynamics responses, which were similar to our
observations in Example 5.
[0398] The pharmacodynamic effects of anti-TfR.sup.1/BACE1 and
anti-TfR.sup.2/BACE1 as assessed in CSF were also similar to that
observed in the previous example. FIG. 15 demonstrates that the
ratio of CSF sAPP.beta./sAPP.alpha. decreased robustly following
dosing with anti-TfR.sup.1/BACE1. Anti-TfR.sup.2/BACE1 did not show
an evident decrease at 24 hours post-dose in this study. Anti-BACE1
also showed no effect. Analysis of brain concentrations of antibody
revealed that both the control IgG and the anti-BACE1 antibody had
limited uptake into the brain, at levels that were just above
detection in our assay (average .about.670 .mu.M).
Anti-TfR.sup.2/BACE1 had .about.3-fold improved brain uptake over
control IgG (average .about.2 nM), and anti-TfR.sup.1/BACE1 had the
best brain uptake, .about.15-fold greater than control IgG (average
-10 nM). The brain antibody concentrations for the different
antibodies correlated with the pharmacodynamics response seen in
CSF in our studies, with anti-TfR.sup.1/BACE1 having the best brain
uptake and most robust pharmacodynamics effect, and
anti-TfR.sup.2/BACE1 having less brain uptake and a more modest
effect.
[0399] These results extend our previous findings to demonstrate
that TfR-binding bispecific antibodies improve uptake in brain of
non-human primates. In primates, as in mice, there is likely an
optimum affinity to TfR that best balances brain uptake and
TfR-mediated clearance. In our example, the higher affinity
anti-TfR.sup.1/BACE1 demonstrates good brain uptake, and is
affected by peripheral target-mediated clearance. The reduced
affinity TfR.sup.2/BACE1 has improved clearance properties, but
appears to have such low binding for TfR as to not be able to be
efficiently transported by TfR (much in the same way that the
lowest-affinity anti-TfR antibody TfR.sup.E in US2012/0171120
passes some affinity threshold beyond which the affinity is too low
to permit sufficient interaction between the antibody and TfR such
that the antibody would remain associated with TfR as TfR begins
the translocation process). From the results of this experiment, an
anti-human/cyno TfR/BACE1 bispecific antibody having affinity for
TfR between that of TfR.sup.1 and TfR.sup.2 would be predicted to
have improved uptake and clearance properties over either
anti-TfR.sup.1/BACE1 or anti-TfR.sup.2/BACE1 in this system.
Example 7: Creation of Additional Effectorless Mutations in the
Context of a Bispecific Transferrin Receptor Antibody
[0400] Other mutations in the Fc region, which abrogate effector
function in addition to N297G and D265A were tested for their
ability to reduce or prevent depletion of TfR-expressing
reticulocytes. Specifically, the Fc mutations L234A, L235A and
P329G ("LALAPG") which are described in US Application Publication
No 2012/0251531, which is incorporated herein by reference, were
incorporated into the anti-TfR.sup.D/BACE1 antibody (which is
described in International Application Publication No. WO
2013/177062, and which is incorporated by reference herein in its
entirety).
[0401] Pharmacokinetic analysis and reticulocyte count following a
single antibody administration in mice were 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 dosed intravenously with a single 50 mg/kg
dose of either an anti-gD antibody (murine IgG2a) with the LALAPG
mutations, an anti-TfR.sup.D/BACE1 antibody (rat/murine chimera)
with the LALAPG mutations. Total injection volume did not exceed
250 .mu.L and antibodies were diluted in D-PBS when necessary
(Invitrogen). After 24 hours, 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 measurements.
[0402] Total antibody concentrations in mouse plasma was measured
using an anti-mouse IgG2a (allotype a) anti-mouse IgG2a (allotype
a) ELISA. NUNC 384-well Maxisorp immunoplates (Neptune, N.J.) were
coated with mouse anti-mouse IgG2a allotype A, an allotype A
specific antibody (BD/Phannigen San Jose, Calif.), overnight at
4.degree. C. Plates were blocked with PBS, 0.5% BSA for 1 hour at
25.degree. C. Each antibody (anti-gD and the anti-TfR/BACE
bispecitic 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, 0.2% BgG, 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
biotin-conjugated mouse anti-mouse IgG2a allotype A, an allotype A
specific antibody (BD/Pharmigen San Jose, Calif.). Bound
biotin-conjugated antibody was detected with horseradish
peroxidase-conjugated streptavidin (GE Ielathcare Life Sciences,
Pittsburgh, Pa.). 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). 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 78.13 ng/ml in plasma. Statistical analysis of differences
between experimental groups was performed using a two-tailed
unpaired t-test.
[0403] Upon administration of the anti-TfR.sup.D/BACE1 antibodies
containing the Fc LALAPG mutations, the mice displayed no clinical
symptoms as had been previously observed using antibodies with full
effector function. See Couch et al., Sci. Trans. Med. 5:183ra57
(2013). FIG. 20 shows the results of the pharmacokinetic
analysis.
[0404] Additionally, immature and total reticulocyte counts were
determined using the Sysmex XT2000iV (Sysmex, Kobe, Japan)
according to manufacturer's instructions. At 24 hours post dose,
there was no observed difference in the immature reticulocyte
fraction or the total reticulocyte count with any antibody tested
as seen in FIG. 21. These results suggest that the LALAPG mutation
not only abrogates antibody effector function but also reduces
complement binding and complement-mediated reticulocyte clearance
seen even with an effectorless antibody framework (Couch et al.
2013). This is consistent with another report that incorporation of
the LALA mutation on a human IgG1 can limit complement binding
(Hessell et al. Nature 449:101-104 (2007)).
Example 8--Creation of FcRn.sup.HIGH Bispecific Variants
[0405] In order to increase the half-life of the bispecific
antibodies, and thereby potentially increase the concentration of
the antibody in the brain, bispecific variants were made containing
mutations in the IgG constant domain and specifically in the Fc
Receptor-neonate (FcRn) binding domain (FcRn.sup.HIGH mutations).
The FcRn binding domain has been implicated in the maternal-fetal
transfer of antibodies. See Story et al., J. Exp. Med., 180:2377
2381, 1994. The amino acid substitutions in the FcRn binding domain
increase the affinity of the constant domain for the FcRn thereby
increasing the half-life of the antibody.
[0406] FcRn binding domain mutations M252Y, S254T and T256E (YTE)
have been described to increase FcRn binding and thus increase the
half-life of antibodies. See U.S. Published Patent Application No.
2003/0190311 and Dall'Acqua et al., J. Biol. Chem. 281:23514-23524
(2006). Additionally, FcRn binding domain mutations N434A and Y436I
(AI) have been described to also increase FcRn binding. See Yeung
et al., J. Immunol. 182: 7663-7671 (2009). The YTE
(M252Y/S254T/T256E) and AI (N434/Y436I) mutations were incorporated
into both anti-TfR.sup.52A/BACE1 and anti-TfR.sup.2/BACE1
bispecific antibodies containing either WT human IgG1 or
effectorless LALAPG or N297G mutations. In addition, FcRn.sup.HIGH
mutation were made in the anti-gD hIgG1 antibody as a control.
Mutations were constructed using Kunkel mutatgenesis, antibodies
were expressed transiently in CHO cells, and proteins were purified
using protein A chromatography followed by size exclusion
chromatography (SEC).
[0407] Binding of FcRn.sup.HIGH variant antibodies to FcRn was
measured using BIAcore. Human and cynomolgus monkey FcRn proteins
were expressed in CHO and purified using IgG affinity
chromatography. Data were acquired on a BIAcore T200 instrument. A
series S sensor chip CM5 (GE Healthcare, Cat. BR100530) was
activated with EDC and NHS reagents according to the supplier's
instructions, and anti-Fab antibody (Human Fab capture kit, GE
Health care Bio-science. AB SE-75184, upsala, Sweden) was coupled
to achieve approximately 10,000 response units (RU), followed by
blocking un-reacted groups with 1 Methanolamine. For affinity
measurements, antibodies were first injected at a 10 .mu.l/min flow
rate to capture approximately 1000 RU on 3 different flow cells
(FC), except for FC1 (reference), and then 2-fold serial dilutions
of human FcRn (or Cyno FcRn) in pH6 buffer (0.1M sodium phosphate),
from low (1 nM) to high (25 .mu.M) were injected (flow rate:30
.mu.l/min) one after the other in the same cycle with no
regeneration between injections. Sensograms were recorded and
subject to reference and buffer subtraction before evaluating by
using BIAcore T200 Evaluation Software (version 2.0). Affinities
were determined by analyzing the level of binding at steady state
based on a1:1 binding model. Binding affinities for LALAPG, N297G,
LALAPG.YTE, and LALAPG.AI variants of anti-TfR.sup.52A/BACE1 are
shown in Table 11 below. The data show that the FcRn.sup.HIGH
variants enhance affinity at endosomal (pH 6) to both human and
cyno FcRn.
TABLE-US-00011 TABLE 11 Human Cyno FcRn FcRn Effector KD at pH 6 KD
at pH 6 Antibody Funcion FcRn High (uM) (uM)
Anti-TfR.52A/BACE1.hIgG1 WT WT Anti-TfR.52A/BACE1.hIgG1.N297G N297G
WT 1.3 2.1 Anti-TfR.52A/BACE1.hIgG1.LALAPG LALAPG WT 0.8 1.2
Anti-TfR.52A/BACE1.hIgG1.N297G.YTE N297G YTE
Anti-TfR.52A/BACE1.hIgG1.LALAPG.YTE LALAPG YTE 0.2 0.2
Anti-TfR.52A/BACE1.hIgG1.N297G.AI N297G N434A/Y436I
Anti-TfR.52A/BACE1.hIgLALAPG.AI LALAPG N434A/Y436I 0.6 0.4
Anti-TfR.52A/BACE1.hIgG1.N297G.A N297G N434A
Anti-TfR2/BACE1.hIgG1.N297G N297G WT 1.7 2.1
Anti-TfR2/BACE1.hIgG1.LALAPG LALAPG WT 1.1 1.2
Anti-TfR2/BACE1.hIgG1.LALAPG.YTE LALAPG YTE 0.3 0.2 Anti-gD.hIgG1
WT WT 0.7 0.9 Anti-gD.hIgG1.YTE WT YTE Anti-gD.hIgG1.AI WT
N434A/Y436I 0.3 0.4 Anti-gD.hIgG1.A WT N434A 0.1 0.7
[0408] Select FcRnHIGH variants will be tested in cynomologus
monkeys to determine whether enhancement of FcRn affinity can
increase improve the pharmacokinetic properties and/or increase the
brain exposure of the anti-TfR/BACE1 antibodies.
[0409] To evaluate the safety of the effectorless and FcRn.sup.HIGH
mutations, certain bispecific antibodies were administered to human
transferrin receptor knock-in mice which express the human
transferrin receptor. The huTfR knock-in mice were generated as
follows. The construct for targeting human TFRC cDNA into the
C57BL/6 Tfrc locus in ES cells was made using a combination of
recombineering (Warming et al. Molecular and Cellular Biology vol.
26 (18) pp. 6913-22 2006; Liu et al Genome Research (2003) vol. 13
(3) pp. 476-84) and standard molecular cloning techniques.
[0410] Briefly, a cassette (human TFRC cDNA, SV40 pA, and
frt-PGK-em7-Neo-BGHpA-frt) flanked by short homologies to the mouse
Tfrc gene was used to modify a Tfrc C57BL/6J BAC (RP23 BAC library)
by recombineering. The human TFRC cDNA cassette was inserted at the
endogenous ATG and the remainder of Tfrc exon 2 plus the beginning
of intron 2 was deleted. The targeted region in the BAC was then
retrieved into pBlight-TK (Warming et al. Molecular and Cellular
Biology vol. 26 (18) pp. 6913-22 2006) along with flanking genomic
Tfrc sequences as homology arms for ES cell targeting.
Specifically, the 2950 bp 5' homology arm corresponds to (assembly
NCBI37/mm9): chr.16:32,610,333-32,613,282 and the 2599 bp 3'
homology arm corresponds to chr.16:32,613,320-32,615,918. The final
vector was confirmed by DNA sequencing.
[0411] The Tfrc/TFRC KI vector was linearized with NotI and
C57BL/6N C2 ES cells were targeted using standard methods (G418
positive and gancyclovir negative selection). Positive clones were
identified using PCR and taqman analysis, and confirmed by
sequencing of the modified locus. Correctly targeted ES cells were
transfected with a Flpe plasmid to remove Neo and ES cells were
then injected into blastocysts using standard techniques. Germline
transmission was obtained after crossing resulting chimaeras with
C57BL/6N females.
[0412] Specifically, the antibodies listed in the table below were
administered to huTfR knock-in mice in a single 50 mg/kg dose and
24 hours later blood was drawn and reticulocytes. huIg1, N297G
TABLE-US-00012 TABLE 12 Antibody Isotype Number of Mice Anti-gD
huIg1, N297G 6 anti-TfR.sup.52A/BACE1 huIgG1, N297G 6
anti-TfR.sup.52A/BACE1 huIgG1, LALAPG 6 anti-TfR.sup.52A/BACE1
huIgG1, LALAPG/YTE 6 anti-TfR.sup.52A/BACE1 huIgG1, LALAPG/AI 6
[0413] Following administration of the anti-TfR.sup.52A/BACE1
LALAPG, LALAPG/YTE or LALAPG/AI antibodies (Groups 3-5 in Table
12), human TfR knock-in mice displayed no clinical symptoms or
reticulocyte loss (FIG. 22) as previously observed using anti-TfR
antibodies with full effector function (Couch et al. 2013). These
results indicate that incorporation of the LALAPG mutation on the
human IgG1 framework also abrogates effector function, and further
suggest that addition of either the YTE or A FcRn.sup.HIGH
mutations do not interfere with the desired properties of the
LALAPG mutations to render the antibody effectorless.
[0414] ADCC assays were also carried out to confirm the
effectorless status of LALAPG, LALAPG/YTE, and LALAPG/AI mutation
combinations in a human-derived cell line. As previously, human
erythroleukemia cell line (HEL, ATCC) was used as 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.
[0415] 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 4 9 0 ( Sample ) - A 4 9 0 ( A I C C ) A 4 9
0 ( High Contro1 ) - A 4 9 0 ( Low Contro1 ) ##EQU00002##
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.
[0416] Results of the ADCC assay are shown in FIG. 23. As expected,
the effector positive anti-human TfR antibody (anti-TfR.sup.1/gD
IgG1 WT) elicited significant ADCC activity on the HEL cells. In
contrast, the anti-TfR.sup.52A/BACE1 antibody variants containing
LALAPG, LALAPG/YTE, or LALAPG/AI mutations did not display any ADCC
activity in HEL cells, similar to the negative control
anti-TfR.sup.52A/gD N297G antibody.
[0417] 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
1751760PRTHomo sapiens 1Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn
Leu Phe Gly Gly Glu1 5 10 15Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala
Arg Gln Val Asp Gly Asp 20 25 30Asn Ser His Val Glu Met Lys Leu Ala
Val Asp Glu Glu Glu Asn Ala 35 40 45Asp Asn Asn Thr Lys Ala Asn Val
Thr Lys Pro Lys Arg Cys Ser Gly 50 55 60Ser Ile Cys Tyr Gly Thr Ile
Ala Val Ile Val Phe Phe Leu Ile Gly65 70 75 80Phe Met Ile Gly Tyr
Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr 85 90 95Glu Cys Glu Arg
Leu Ala Gly Thr Glu Ser Pro Val Arg Glu Glu Pro 100 105 110Gly Glu
Asp Phe Pro Ala Ala Arg Arg Leu Tyr Trp Asp Asp Leu Lys 115 120
125Arg Lys Leu Ser Glu Lys Leu Asp Ser Thr Asp Phe Thr Ser Thr Ile
130 135 140Lys Leu Leu Asn Glu Asn Ser Tyr Val Pro Arg Glu Ala Gly
Ser Gln145 150 155 160Lys Asp Glu Asn Leu Ala Leu Tyr Val Glu Asn
Gln Phe Arg Glu Phe 165 170 175Lys Leu Ser Lys Val Trp Arg Asp Gln
His Phe Val Lys Ile Gln Val 180 185 190Lys Asp Ser Ala Gln Asn Ser
Val Ile Ile Val Asp Lys Asn Gly Arg 195 200 205Leu Val Tyr Leu Val
Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys 210 215 220Ala Ala Thr
Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys225 230 235
240Lys Asp Phe Glu Asp Leu Tyr Thr Pro Val Asn Gly Ser Ile Val Ile
245 250 255Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn
Ala Glu 260 265 270Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp
Gln Thr Lys Phe 275 280 285Pro Ile Val Asn Ala Glu Leu Ser Phe Phe
Gly His Ala His Leu Gly 290 295 300Thr Gly Asp Pro Tyr Thr Pro Gly
Phe Pro Ser Phe Asn His Thr Gln305 310 315 320Phe Pro Pro Ser Arg
Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr 325 330 335Ile Ser Arg
Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp 340 345 350Cys
Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Arg Met Val Thr Ser 355 360
365Glu Ser Lys Asn Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Ile
370 375 380Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu
Pro Asp385 390 395 400His Tyr Val Val Val Gly Ala Gln Arg Asp Ala
Trp Gly Pro Gly Ala 405 410 415Ala Lys Ser Gly Val Gly Thr Ala Leu
Leu Leu Lys Leu Ala Gln Met 420 425 430Phe Ser Asp Met Val Leu Lys
Asp Gly Phe Gln Pro Ser Arg Ser Ile 435 440 445Ile Phe Ala Ser Trp
Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr 450 455 460Glu Trp Leu
Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr465 470 475
480Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val
485 490 495Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met
Gln Asn 500 505 510Val Lys His Pro Val Thr Gly Gln Phe Leu Tyr Gln
Asp Ser Asn Trp 515 520 525Ala Ser Lys Val Glu Lys Leu Thr Leu Asp
Asn Ala Ala Phe Pro Phe 530 535 540Leu Ala Tyr Ser Gly Ile Pro Ala
Val Ser Phe Cys Phe Cys Glu Asp545 550 555 560Thr Asp Tyr Pro Tyr
Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu 565 570 575Ile Glu Arg
Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu 580 585 590Val
Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Val Glu Leu Asn 595 600
605Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Ser Phe Val Arg Asp
610 615 620Leu Asn Gln Tyr Arg Ala Asp Ile Lys Glu Met Gly Leu Ser
Leu Gln625 630 635 640Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg
Ala Thr Ser Arg Leu 645 650 655Thr Thr Asp Phe Gly Asn Ala Glu Lys
Thr Asp Arg Phe Val Met Lys 660 665 670Lys Leu Asn Asp Arg Val Met
Arg Val Glu Tyr His Phe Leu Ser Pro 675 680 685Tyr Val Ser Pro Lys
Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser 690 695 700Gly Ser His
Thr Leu Pro Ala Leu Leu Glu Asn Leu Lys Leu Arg Lys705 710 715
720Gln Asn Asn Gly Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala
725 730 735Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser
Gly Asp 740 745 750Val Trp Asp Ile Asp Asn Glu Phe 755
7602760PRTMacaca mulatta 2Met Met Asp Gln Ala Arg Ser Ala Phe Ser
Asn Leu Phe Gly Gly Glu1 5 10 15Pro Leu Ser Tyr Thr Arg Phe Ser Leu
Ala Arg Gln Val Asp Gly Asp 20 25 30Asn Ser His Val Glu Met Lys Leu
Gly Val Asp Glu Glu Glu Asn Thr 35 40 45Asp Asn Asn Thr Lys Pro Asn
Gly Thr Lys Pro Lys Arg Cys Gly Gly 50 55 60Asn Ile Cys Tyr Gly Thr
Ile Ala Val Ile Ile Phe Phe Leu Ile Gly65 70 75 80Phe Met Ile Gly
Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr 85 90 95Glu Cys Glu
Arg Leu Ala Gly Thr Glu Ser Pro Ala Arg Glu Glu Pro 100 105 110Glu
Glu Asp Phe Pro Ala Ala Pro Arg Leu Tyr Trp Asp Asp Leu Lys 115 120
125Arg Lys Leu Ser Glu Lys Leu Asp Thr Thr Asp Phe Thr Ser Thr Ile
130 135 140Lys Leu Leu Asn Glu Asn Leu Tyr Val Pro Arg Glu Ala Gly
Ser Gln145 150 155 160Lys Asp Glu Asn Leu Ala Leu Tyr Ile Glu Asn
Gln Phe Arg Glu Phe 165 170 175Lys Leu Ser Lys Val Trp Arg Asp Gln
His Phe Val Lys Ile Gln Val 180 185 190Lys Asp Ser Ala Gln Asn Ser
Val Ile Ile Val Asp Lys Asn Gly Gly 195 200 205Leu Val Tyr Leu Val
Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys 210 215 220Ala Ala Thr
Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys225 230 235
240Lys Asp Phe Glu Asp Leu Asp Ser Pro Val Asn Gly Ser Ile Val Ile
245 250 255Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn
Ala Glu 260 265 270Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp
Gln Thr Lys Phe 275 280 285Pro Ile Val Lys Ala Asp Leu Ser Phe Phe
Gly His Ala His Leu Gly 290 295 300Thr Gly Asp Pro Tyr Thr Pro Gly
Phe Pro Ser Phe Asn His Thr Gln305 310 315 320Phe Pro Pro Ser Gln
Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr 325 330 335Ile Ser Arg
Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp 340 345 350Cys
Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Lys Met Val Thr Ser 355 360
365Glu Asn Lys Ser Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Thr
370 375 380Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu
Pro Asp385 390 395 400His Tyr Val Val Val Gly Ala Gln Arg Asp Ala
Trp Gly Pro Gly Ala 405 410 415Ala Lys Ser Ser Val Gly Thr Ala Leu
Leu Leu Lys Leu Ala Gln Met 420 425 430Phe Ser Asp Met Val Leu Lys
Asp Gly Phe Gln Pro Ser Arg Ser Ile 435 440 445Ile Phe Ala Ser Trp
Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr 450 455 460Glu Trp Leu
Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr465 470 475
480Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val
485 490 495Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met
Gln Asp 500 505 510Val Lys His Pro Val Thr Gly Arg Ser Leu Tyr Gln
Asp Ser Asn Trp 515 520 525Ala Ser Lys Val Glu Lys Leu Thr Leu Asp
Asn Ala Ala Phe Pro Phe 530 535 540Leu Ala Tyr Ser Gly Ile Pro Ala
Val Ser Phe Cys Phe Cys Glu Asp545 550 555 560Thr Asp Tyr Pro Tyr
Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu 565 570 575Val Glu Arg
Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu 580 585 590Val
Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Thr Glu Leu Asn 595 600
605Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Leu Phe Leu Arg Asp
610 615 620Leu Asn Gln Tyr Arg Ala Asp Val Lys Glu Met Gly Leu Ser
Leu Gln625 630 635 640Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg
Ala Thr Ser Arg Leu 645 650 655Thr Thr Asp Phe Arg Asn Ala Glu Lys
Arg Asp Lys Phe Val Met Lys 660 665 670Lys Leu Asn Asp Arg Val Met
Arg Val Glu Tyr Tyr Phe Leu Ser Pro 675 680 685Tyr Val Ser Pro Lys
Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser 690 695 700Gly Ser His
Thr Leu Ser Ala Leu Leu Glu Ser Leu Lys Leu Arg Arg705 710 715
720Gln Asn Asn Ser Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala
725 730 735Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser
Gly Asp 740 745 750Val Trp Asp Ile Asp Asn Glu Phe 755
7603763PRTMus musculus 3Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn
Leu Phe Gly Gly Glu1 5 10 15Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala
Arg Gln Val Asp Gly Asp 20 25 30Asn Ser His Val Glu Met Lys Leu Ala
Ala Asp Glu Glu Glu Asn Ala 35 40 45Asp Asn Asn Met Lys Ala Ser Val
Arg Lys Pro Lys Arg Phe Asn Gly 50 55 60Arg Leu Cys Phe Ala Ala Ile
Ala Leu Val Ile Phe Phe Leu Ile Gly65 70 75 80Phe Met Ser Gly Tyr
Leu Gly Tyr Cys Lys Arg Val Glu Gln Lys Glu 85 90 95Glu Cys Val Lys
Leu Ala Glu Thr Glu Glu Thr Asp Lys Ser Glu Thr 100 105 110Met Glu
Thr Glu Asp Val Pro Thr Ser Ser Arg Leu Tyr Trp Ala Asp 115 120
125Leu Lys Thr Leu Leu Ser Glu Lys Leu Asn Ser Ile Glu Phe Ala Asp
130 135 140Thr Ile Lys Gln Leu Ser Gln Asn Thr Tyr Thr Pro Arg Glu
Ala Gly145 150 155 160Ser Gln Lys Asp Glu Ser Leu Ala Tyr Tyr Ile
Glu Asn Gln Phe His 165 170 175Glu Phe Lys Phe Ser Lys Val Trp Arg
Asp Glu His Tyr Val Lys Ile 180 185 190Gln Val Lys Ser Ser Ile Gly
Gln Asn Met Val Thr Ile Val Gln Ser 195 200 205Asn Gly Asn Leu Asp
Pro Val Glu Ser Pro Glu Gly Tyr Val Ala Phe 210 215 220Ser Lys Pro
Thr Glu Val Ser Gly Lys Leu Val His Ala Asn Phe Gly225 230 235
240Thr Lys Lys Asp Phe Glu Glu Leu Ser Tyr Ser Val Asn Gly Ser Leu
245 250 255Val Ile Val Arg Ala Gly Glu Ile Thr Phe Ala Glu Lys Val
Ala Asn 260 265 270Ala Gln Ser Phe Asn Ala Ile Gly Val Leu Ile Tyr
Met Asp Lys Asn 275 280 285Lys Phe Pro Val Val Glu Ala Asp Leu Ala
Leu Phe Gly His Ala His 290 295 300Leu Gly Thr Gly Asp Pro Tyr Thr
Pro Gly Phe Pro Ser Phe Asn His305 310 315 320Thr Gln Phe Pro Pro
Ser Gln Ser Ser Gly Leu Pro Asn Ile Pro Val 325 330 335Gln Thr Ile
Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Lys Met Glu 340 345 350Gly
Ser Cys Pro Ala Arg Trp Asn Ile Asp Ser Ser Cys Lys Leu Glu 355 360
365Leu Ser Gln Asn Gln Asn Val Lys Leu Ile Val Lys Asn Val Leu Lys
370 375 380Glu Arg Arg Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Tyr
Glu Glu385 390 395 400Pro Asp Arg Tyr Val Val Val Gly Ala Gln Arg
Asp Ala Leu Gly Ala 405 410 415Gly Val Ala Ala Lys Ser Ser Val Gly
Thr Gly Leu Leu Leu Lys Leu 420 425 430Ala Gln Val Phe Ser Asp Met
Ile Ser Lys Asp Gly Phe Arg Pro Ser 435 440 445Arg Ser Ile Ile Phe
Ala Ser Trp Thr Ala Gly Asp Phe Gly Ala Val 450 455 460Gly Ala Thr
Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys465 470 475
480Ala Phe Thr Tyr Ile Asn Leu Asp Lys Val Val Leu Gly Thr Ser Asn
485 490 495Phe Lys Val Ser Ala Ser Pro Leu Leu Tyr Thr Leu Met Gly
Lys Ile 500 505 510Met Gln Asp Val Lys His Pro Val Asp Gly Lys Ser
Leu Tyr Arg Asp 515 520 525Ser Asn Trp Ile Ser Lys Val Glu Lys Leu
Ser Phe Asp Asn Ala Ala 530 535 540Tyr Pro Phe Leu Ala Tyr Ser Gly
Ile Pro Ala Val Ser Phe Cys Phe545 550 555 560Cys Glu Asp Ala Asp
Tyr Pro Tyr Leu Gly Thr Arg Leu Asp Thr Tyr 565 570 575Glu Ala Leu
Thr Gln Lys Val Pro Gln Leu Asn Gln Met Val Arg Thr 580 585 590Ala
Ala Glu Val Ala Gly Gln Leu Ile Ile Lys Leu Thr His Asp Val 595 600
605Glu Leu Asn Leu Asp Tyr Glu Met Tyr Asn Ser Lys Leu Leu Ser Phe
610 615 620Met Lys Asp Leu Asn Gln Phe Lys Thr Asp Ile Arg Asp Met
Gly Leu625 630 635 640Ser Leu Gln Trp Leu Tyr Ser Ala Arg Gly Asp
Tyr Phe Arg Ala Thr 645 650 655Ser Arg Leu Thr Thr Asp Phe His Asn
Ala Glu Lys Thr Asn Arg Phe 660 665 670Val Met Arg Glu Ile Asn Asp
Arg Ile Met Lys Val Glu Tyr His Phe 675 680 685Leu Ser Pro Tyr Val
Ser Pro Arg Glu Ser Pro Phe Arg His Ile Phe 690 695 700Trp Gly Ser
Gly Ser His Thr Leu Ser Ala Leu Val Glu Asn Leu Lys705 710 715
720Leu Arg Gln Lys Asn Ile Thr Ala Phe Asn Glu Thr Leu Phe Arg Asn
725 730 735Gln Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly Val Ala Asn
Ala Leu 740 745 750Ser Gly Asp Ile Trp Asn Ile Asp Asn Glu Phe 755
7604111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 4Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu
Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser
Glu Ser Val Asp Asp Tyr 20 25 30Gly Asn Ser Phe Met His Trp Tyr Gln
Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala Ser
Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser
Arg Thr Asp Phe Thr Leu Thr Ile Asn65 70 75 80Pro Val Glu Ala Asp
Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Ala Pro Pro
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Arg 100 105
1105111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 5Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu
Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser
Glu Ser Val Asp Ser Tyr 20 25 30Gly Asn Ser Phe Met His Trp Tyr Gln
Gln Lys Pro Gly Gln Pro Pro 35
40 45Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro
Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr
Ile Asn65 70 75 80Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys
Gln Gln Ser Asn 85 90 95Glu Gly Pro Pro Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 1106111PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 6Asp Ile Val Leu Thr Gln
Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile
Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr 20 25 30Gly Pro Ser Phe
Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu
Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60Arg Phe
Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn65 70 75
80Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln His Ser Asn
85 90 95Glu Asp Pro Pro Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys
100 105 1107119PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 7Gln Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Val Arg Pro Gly Val1 5 10 15Ser Val Lys Ile Ser Cys Lys Gly
Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Ala Met His Trp Val Lys Gln
Ser His Ala Lys Ser Leu Glu Trp Ile 35 40 45Gly Gly Ile Ser Thr Tyr
Phe Gly Arg Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Ala Thr
Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ala Arg Leu Thr Ser Glu Asp Ser Ala Leu Tyr Tyr Cys 85 90 95Ala Arg
Gly Leu Ser Gly Asn Tyr Val Met Asp Tyr Trp Gly Gln Gly 100 105
110Thr Ser Val Thr Val Ser Ser 1158119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
8Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Val1 5
10 15Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30Gly Met His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu
Trp Ile 35 40 45Gly Val Ile Ser Pro Tyr Ser Gly Arg Thr Asn Tyr Asn
Gln Asn Phe 50 55 60Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser
Ser Thr Ala Tyr65 70 75 80Leu Glu Leu Ala Arg Leu Thr Ser Glu Asp
Ser Ala Ile Tyr Tyr Cys 85 90 95Ala Arg Gly Leu Ser Gly Asn Tyr Val
Val Asp Tyr Trp Gly Gln Gly 100 105 110Thr Ser Val Thr Val Ser Ser
1159119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 9Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Val Arg Pro Gly Val1 5 10 15Ser Val Lys Ile Ser Cys Lys Gly Ser Gly
Tyr Thr Val Thr Asp Tyr 20 25 30Ala Met His Trp Val Lys Gln Ser His
Ala Lys Ser Leu Glu Trp Ile 35 40 45Gly Val Ile Ser Phe Tyr Ser Gly
Lys Thr Asn Tyr Asn Gln Lys Phe 50 55 60Met Gly Lys Ala Thr Met Thr
Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ala Arg
Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95Ala Arg Gly Leu
Ser Gly Asn Tyr Val Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Ser
Val Thr Val Ser Ser 11510119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 10Gln Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Arg Pro Gly Val1 5 10 15Ala Val Lys Ile Ser
Cys Lys Gly Ser Gly Tyr Lys Phe Ile Asp Tyr 20 25 30Gly Met His Trp
Val Lys Gln Ser His Thr Lys Ser Leu Gln Trp Ile 35 40 45Gly Val Ile
Ser Pro Tyr Ser Gly Lys Thr Asn Tyr Ser Gln Lys Phe 50 55 60Lys Gly
Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95Ala Arg Gly Leu Ser Gly Asn Phe Val Met Asp Phe Trp Gly Gln
Gly 100 105 110Thr Ser Val Thr Val Ser Ser 11511107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
11Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly1
5 10 15Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Asp Asn Leu Tyr Ser
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu
Leu Val 35 40 45Tyr Asp Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn
Ser Leu Gln Ser65 70 75 80Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His
Phe Trp Gly Thr Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu
Leu Lys 100 10512107PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 12Asp Ile Gln Leu Thr Gln Thr Pro
Ala Ser Leu Ser Val Ser Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys
Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45Tyr Ala Ala Thr Asp
Leu Ala Asp Gly Val Pro Ser Arg Phe Arg Gly 50 55 60Ser Gly Ser Gly
Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser65 70 75 80Glu Asp
Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Leu 85 90 95Thr
Phe Gly Ala Gly Thr Lys Leu Glu Leu Ile 100 10513107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
13Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly1
5 10 15Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Asp Asn Ile Tyr Ser
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu
Leu Val 35 40 45Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn
Ser Leu Gln Ser65 70 75 80Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His
Phe Trp Gly Thr Pro Leu 85 90 95Met Phe Gly Ser Gly Thr Lys Leu Glu
Leu Lys 100 10514107PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 14Asp Ile Gln Met Thr Gln Ser Pro
Ala Ser Leu Ser Val Ser Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys
Arg Ala Ser Asp Asn Ile Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45Tyr Ala Val Thr Asn
Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser65 70 75 80Glu Asp
Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Leu 85 90 95Thr
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 10515116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
15Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala1
5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile
Glu Lys Phe 50 55 60Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser
Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Thr Arg Ala Tyr His Tyr
Trp Gly Gln Gly Thr Ser Val 100 105 110Thr Val Ser Ser
11516116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 16Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu
Val Arg Pro Gly Ala1 5 10 15Ala Val Lys Leu Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn Pro Thr Asn Gly
Arg Thr Asn Tyr Asn Glu Asn Phe 50 55 60Lys Ser Lys Ala Thr Leu Thr
Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser
Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Thr
Arg Ala Tyr His Phe Trp Gly Gln Gly Thr Ser Val 100 105 110Thr Val
Ser Ser 11517116PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 17Gln Val Gln Leu Gln Gln Pro Gly
Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Ala Ser Tyr 20 25 30Trp Met His Trp Val Lys
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn Pro
Ile Asn Gly Arg Thr Asn Tyr Ser Glu Lys Phe 50 55 60Lys Lys Lys Ala
Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Ser Val 100 105
110Thr Val Ser Ser 11518116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 18Gln Val Gln Leu Gln Gln
Pro Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp
Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile
Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Thr Phe 50 55 60Lys Ser
Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75
80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Ser
Val 100 105 110Thr Val Ser Ser 11519107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
19Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Thr Ala Ser Pro Gly1
5 10 15Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys
Tyr 20 25 30Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu
Leu Ile 35 40 45Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Asn Leu Glu Pro65 70 75 80Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln
His Asn Glu Tyr Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 10520122PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 20Asp Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Ser Leu Ser Leu Thr Cys Thr
Val Thr Gly Asn Ser Ile Thr Ser Glu 20 25 30Tyr Ala Trp Asn Trp Ile
Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45Met Gly Tyr Ile Ser
Tyr Ser Gly Thr Thr Ser Tyr Asn Pro Ser Leu 50 55 60Lys Ser Arg Ile
Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Leu Phe65 70 75 80Leu Gln
Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Ala
Arg Tyr Gly Tyr Gly Asn Pro Ala Thr Arg Tyr Phe Asp Val Trp 100 105
110Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115
12021111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 21Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu
Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Arg
Gln Ser Val Ser Thr Ser 20 25 30Ser Tyr Ser Phe Met His Trp Tyr Arg
Gln Lys Ala Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Lys Tyr Ala Ser
Ile Gln Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Asn Ile Leu65 70 75 80Pro Val Glu Glu Glu
Asp Thr Ala Thr Tyr Tyr Cys Gln His Thr Trp 85 90 95Glu Ile Pro Phe
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105
11022111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 22Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu
Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Arg
Gln Ser Val Ser Thr Ser 20 25 30Ser Tyr Ser Phe Met His Trp Tyr Gln
Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Lys Tyr Ala Ser
Ile Gln Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Asn Ile Leu65 70 75 80Pro Val Glu Glu Glu
Asp Thr Ala Thr Tyr Tyr Cys Gln His Thr Trp 85 90 95Glu Ile Pro Phe
Thr Phe Gly Ser Gly Thr Asn Leu Glu Ile Lys 100 105
11023108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 23Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met
Ser Ala Ser Leu Gly1 5 10 15Gly Arg Val Thr Met Thr Cys Thr Thr Ser
Ser Ser Val Pro Ser Ser 20 25 30Tyr Phe His Trp Tyr Gln Gln Lys Pro
Gly Ser Ser Pro Lys Leu Trp 35 40 45Ile Tyr Ser Thr Ser Asn Leu Ala
Ser Gly Val Pro Ala Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Ser
Tyr Ser Leu Thr Ile Ser Ser Met Glu65 70 75 80Ala Glu Asp Ala Ala
Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro 85 90 95Phe Thr Phe Gly
Ser Gly Thr Lys Leu Glu Ile Lys 100 10524107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
24Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly1
5 10 15Asp Arg Val Thr Ile Asn Cys Arg Ala Gly Gln Asp Ile Thr Asn
Tyr 20 25 30Leu Asn Trp Phe Gln Gln Lys Pro Asp Gly Thr Val Lys Leu
Leu Ile 35 40 45Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Thr
Asn Leu Glu Gln65 70 75 80Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
Ala Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 10525120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 25Gln Val Gln Leu Gln Gln Pro Gly
Ser Glu Leu Val Arg Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys
Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg His Gly Gln
Gly Leu Glu Trp Ile 35 40 45Gly Asn Ile Tyr Pro Gly Ser Gly Ser Thr
Lys Tyr Asp Glu Arg Phe 50 55 60Lys Ser Lys Gly Thr Leu Thr Val Asp
Thr Ser Ser Ser Thr Ala Tyr65 70 75 80Met His Leu Ser Ser Leu Thr
Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Thr Arg Gly Gly Tyr Asp
Ser Arg Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val
Thr Val Ser Ala 115 12026120PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 26Gln Val Gln Leu Gln Gln
Pro Gly Ser Glu Leu Val Arg Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp
Val Lys Gln Arg His Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Asn Ile
Tyr Pro Gly Ser Gly Ser Thr Lys Tyr Asp Glu Lys Phe 50 55 60Lys Ser
Lys Gly Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr65 70 75
80Met His Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Thr Arg Gly Gly Tyr Asp Ser Arg Ala Trp Phe Ala His Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ala 115
12027124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 27Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Phe Leu Lys Leu Ser Cys Ala Thr Ser Gly
Phe Thr Phe Ser Asp Tyr 20 25 30Tyr Met Tyr Trp Val Arg Gln Thr Pro
Glu Lys Arg Leu Glu Trp Val 35 40 45Ala Ser Ile Ser Asn Gly Gly Asp
Asn Thr Tyr Tyr Pro Asp Thr Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser Arg
Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Gln Gly
Ala Leu Tyr Asp Gly Tyr Tyr Arg Gly Ala Met Asp 100 105 110Tyr Trp
Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 12028121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
28Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala1
5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ala Gly Tyr Thr Phe Ser Asn
Tyr 20 25 30Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu
Trp Ile 35 40 45Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Lys Tyr Asn
Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser
Asn Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Gly Tyr Gly Tyr Asp
Gly Glu Phe Ala Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ala 115 1202915PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 29Arg Ala Ser Glu Ser Val Asp Ser Tyr
Gly Asn Ser Phe Met His1 5 10 15307PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 30Arg
Ala Ser Asn Leu Glu Ser1 5319PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 31Gln Gln Ser Asn Glu Ala Pro
Pro Thr1 5325PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 32Asp Tyr Ala Met His1
53317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 33Gly Ile Ser Thr Tyr Phe Gly Arg Thr Asn Tyr Asn
Gln Lys Phe Lys1 5 10 15Gly3410PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 34Gly Leu Ser Gly Asn Tyr Val
Met Asp Tyr1 5 103515PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 35Arg Ala Ser Glu Ser Val Asp
Ser Tyr Gly Asn Ser Phe Met His1 5 10 15369PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 36Gln
Gln Ser Asn Glu Gly Pro Pro Thr1 5375PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 37Asp
Tyr Gly Met His1 53817PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 38Val Ile Ser Pro Tyr Ser Gly
Arg Thr Asn Tyr Asn Gln Asn Phe Lys1 5 10 15Gly3910PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 39Gly
Leu Ser Gly Asn Tyr Val Val Asp Tyr1 5 104017PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 40Val
Ile Ser Phe Tyr Ser Gly Lys Thr Asn Tyr Asn Gln Lys Phe Met1 5 10
15Gly4115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 41Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser
Phe Met His1 5 10 15429PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 42Gln His Ser Asn Glu Asp Pro
Pro Thr1 54317PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 43Val Ile Ser Pro Tyr Ser Gly Lys Thr
Asn Tyr Ser Gln Lys Phe Lys1 5 10 15Gly4410PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 44Gly
Leu Ser Gly Asn Phe Val Met Asp Phe1 5 104515PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(8)..(8)Ser or AspMOD_RES(11)..(11)Asn or Pro 45Arg
Ala Ser Glu Ser Val Asp Xaa Tyr Gly Xaa Ser Phe Met His1 5 10
15469PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(2)..(2)Gln or HisMOD_RES(6)..(6)Ala, Gly
or Asp 46Gln Xaa Ser Asn Glu Xaa Pro Pro Thr1 5475PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(3)..(3)Ala or Gly 47Asp Tyr Xaa Met His1
54817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)Gly or ValMOD_RES(4)..(4)Thr, Phe
or ProMOD_RES(6)..(6)Phe or SerMOD_RES(8)..(8)Arg or
LysMOD_RES(12)..(12)Asn or SerMOD_RES(14)..(14)Lys or
AsnMOD_RES(16)..(16)Lys or Met 48Xaa Ile Ser Xaa Tyr Xaa Gly Xaa
Thr Asn Tyr Xaa Gln Xaa Phe Xaa1 5 10 15Gly4910PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(6)..(6)Tyr or PheMOD_RES(8)..(8)Met or
ValMOD_RES(10)..(10)Tyr or Phe 49Gly Leu Ser Gly Asn Xaa Val Xaa
Asp Xaa1 5 105011PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 50Arg Ala Ser Asp Asn Leu Tyr Ser Asn
Leu Ala1 5 10517PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 51Asp Ala Thr Asn Leu Ala Asp1
5529PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 52Gln His Phe Trp Gly Thr Pro Leu Thr1
5535PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 53Ser Tyr Trp Met His1 55417PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 54Glu
Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe Lys1 5 10
15Ser557PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 55Gly Thr Arg Ala Tyr His Tyr1 55611PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 56Arg
Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala1 5 10577PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 57Ala
Ala Thr Asp Leu Ala Asp1 55817PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 58Glu Ile Asn Pro Thr Asn Gly
Arg Thr Asn Tyr Asn Glu Asn Phe Lys1 5 10 15Ser597PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 59Gly
Thr Arg Ala Tyr His Phe1 56011PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 60Arg Ala Ser Asp Asn Ile Tyr
Ser Asn Leu Ala1 5 10617PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 61Ala Ala Thr Asn Leu Ala
Asp1 5629PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 62Gln His Phe Trp Gly Thr Pro Leu Met1
56317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 63Glu Ile Asn Pro Ile Asn Gly Arg Thr Asn Tyr Ser
Glu Lys Phe Lys1 5 10 15Lys647PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 64Ala Val Thr Asn Leu Ala
Asp1 56517PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 65Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn
Glu Thr Phe Lys1 5 10 15Ser6611PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(4)..(4)Glu or
AspMOD_RES(6)..(6)Leu or Ile 66Arg Ala Ser Xaa Asn Xaa Tyr Ser Asn
Leu Ala1 5 10677PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(1)..(1)Asp or
AlaMOD_RES(2)..(2)Ala or ValMOD_RES(4)..(4)Asn or Asp 67Xaa Xaa Thr
Xaa Leu Ala Asp1 5689PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(9)..(9)Thr or Met
68Gln His Phe Trp Gly Thr Pro Leu Xaa1 56917PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(5)..(5)Thr, Ile or SerMOD_RES(12)..(12)Ile, Asn or
SerMOD_RES(14)..(14)Lys, Asn or ThrMOD_RES(17)..(17)Ser or Lys
69Glu Ile Asn Pro Xaa Asn Gly Arg Thr Asn Tyr Xaa Glu Xaa Phe Lys1
5 10 15Xaa707PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(7)..(7)Tyr or Phe 70Gly Thr Arg
Ala Tyr His Xaa1 57111PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 71Arg Ala Ser Lys Ser Ile Ser
Lys Tyr Leu Ala1 5 10727PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 72Ser Gly Ser Thr Leu Gln
Ser1 5739PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 73Gln Gln His Asn Glu Tyr Pro Trp Thr1
5746PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 74Ser Glu Tyr Ala Trp Asn1 57516PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 75Tyr
Ile Ser Tyr Ser Gly Thr Thr Ser Tyr Asn Pro Ser Leu Lys Ser1 5 10
157613PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 76Tyr Gly Tyr Gly Asn Pro Ala Thr Arg Tyr Phe Asp
Val1 5 107715PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 77Arg Ala Arg Gln Ser Val Ser Thr Ser
Ser Tyr Ser Phe Met His1 5 10 15787PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 78Tyr
Ala Ser Ile Gln Glu Ser1 5799PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 79Gln His Thr Trp Glu Ile Pro
Phe Thr1 5805PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 80Ser Tyr Trp Met His1
58117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 81Asn Ile Tyr Pro Gly Ser Gly Ser Thr Lys Tyr Asp
Glu Arg Phe Lys1 5 10 15Ser8211PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 82Gly Gly Tyr Asp Ser Arg Ala
Trp Phe Ala Tyr1 5 108317PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 83Asn Ile Tyr Pro Gly Ser Gly
Ser Thr Lys Tyr Asp Glu Lys Phe Lys1 5 10 15Ser8411PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 84Gly
Gly Tyr Asp Ser Arg Ala Trp Phe Ala His1 5 108512PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 85Thr
Thr Ser Ser Ser Val Pro Ser Ser Tyr Phe His1 5 10867PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 86Ser
Thr Ser Asn Leu Ala Ser1 5879PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 87His Gln Tyr His Arg Ser Pro
Phe Thr1 5885PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 88Asp Tyr Tyr Met Tyr1
58917PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 89Ser Ile Ser Asn Gly Gly Asp Asn Thr Tyr Tyr Pro
Asp Thr Val Lys1 5 10 15Gly9015PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 90Gln Gly Ala Leu Tyr Asp Gly
Tyr Tyr Arg Gly Ala Met Asp Tyr1 5 10 159111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 91Arg
Ala Gly Gln Asp Ile Thr Asn Tyr Leu Asn1 5 10927PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 92Tyr
Thr Ser Arg Leu His Ser1 5939PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 93Gln Gln Ala Asn Thr Leu Pro
Tyr Thr1 5945PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 94Asn Tyr Trp Ile Glu1
59517PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 95Glu Ile Leu Pro Gly Ser Gly Ser Thr Lys Tyr Asn
Glu Lys Phe Lys1 5 10 15Gly9612PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 96Arg Gly Gly Tyr Gly Tyr Asp
Gly Glu Phe Ala Tyr1 5 109715PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(1)..(1)Arg or
ThrMOD_RES(2)..(2)Ala or ThrMOD_RES(3)..(3)Arg, Ser or
GlyMOD_RES(4)..(4)Gln or SerMOD_RES(5)..(8)May or may not be
presentMOD_RES(9)..(9)Ser, Val or AspMOD_RES(10)..(10)Ser, Pro or
IleMOD_RES(11)..(11)Tyr, Ser or ThrMOD_RES(12)..(12)Ser or
AsnMOD_RES(13)..(13)Phe or TyrMOD_RES(14)..(14)Met, Phe or
LeuMOD_RES(15)..(15)His or Asn 97Xaa Xaa Xaa Xaa Ser Val Ser Thr
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15987PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(1)..(1)Tyr or SerMOD_RES(2)..(2)Ala or
ThrMOD_RES(4)..(4)Ile, Asn or ArgMOD_RES(5)..(5)Gln or
LeuMOD_RES(6)..(6)Glu, Ala or His 98Xaa Xaa Ser Xaa Xaa Xaa Ser1
5999PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)Gln or HisMOD_RES(2)..(2)His or
GlnMOD_RES(3)..(3)Thr, Tyr or AlaMOD_RES(4)..(4)Trp, His or
AsnMOD_RES(5)..(5)Glu, Arg or ThrMOD_RES(6)..(6)Ile, Ser or
LeuMOD_RES(8)..(8)Phe or Tyr 99Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Thr1
51005PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)Ser, Asp or AsnMOD_RES(3)..(3)Trp
or TyrMOD_RES(4)..(4)Met or IleMOD_RES(5)..(5)His, Tyr or Glu
100Xaa Tyr Xaa Xaa Xaa1 510117PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(1)..(1)Asn, Ser or
GluMOD_RES(3)..(3)Tyr, Ser or LeuMOD_RES(4)..(4)Pro or
AsnMOD_RES(6)..(6)Ser or GlyMOD_RES(7)..(7)Gly or
AspMOD_RES(8)..(8)Ser or AsnMOD_RES(10)..(10)Lys or
TyrMOD_RES(12)..(12)Asp, Pro or AsnMOD_RES(13)..(13)Glu or
AspMOD_RES(14)..(14)Arg, Lys or ThrMOD_RES(15)..(15)Phe or
ValMOD_RES(17)..(17)Ser or Gly 101Xaa Ile Xaa Xaa Gly Xaa Xaa Xaa
Thr Xaa Tyr Xaa Xaa Xaa Xaa Lys1 5 10 15Xaa10215PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(1)..(1)Gly, Gln or ArgMOD_RES(3)..(3)Tyr, Ala or
GlyMOD_RES(4)..(4)Asp, Leu or TyrMOD_RES(5)..(5)Ser, Tyr or
GlyMOD_RES(6)..(6)Arg, Asp or TyrMOD_RES(7)..(7)Ala, Gly or
AspMOD_RES(8)..(8)Trp, Tyr or GlyMOD_RES(9)..(9)Phe, Tyr or
GluMOD_RES(10)..(10)Arg, Phe or not presentMOD_RES(11)..(13)May or
may not be presentMOD_RES(14)..(14)Ala or AspMOD_RES(15)..(15)Tyr
or His 102Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Ala Met Xaa
Xaa1 5 10 15103107PRTHomo sapiens 103Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Ser Asn Ser 20 25 30Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45Tyr Ala Ala Ser
Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Tyr 85 90
95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105104107PRTMus
musculus 104Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser
Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Asp Asn Leu
Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro
Gln Leu Leu Val 35 40 45Tyr Asp Ala Thr Asn Leu Ala Asp Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys
Ile Asn Ser Leu Gln Ser65 70 75 80Glu Asp Phe Gly Thr Tyr Tyr Cys
Gln His Phe Trp Gly Thr Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Lys
Leu Glu Leu Lys 100 105105107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 105Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Asp Asn Leu Tyr Ser Asn 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val 35 40 45Tyr Asp
Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Leu
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105106112PRTHomo sapiens 106Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala
Pro Gly Gln Arg Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Ala Gly Asn
Gly Asn Thr Lys Tyr Ser Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile
Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Phe
Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 100 105
110107116PRTMus musculus 107Gln Val Gln Leu Gln Gln Pro Gly Ala Glu
Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg
Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn Pro Thr Asn
Gly Arg Thr Asn Tyr Ile Glu Lys Phe 50 55 60Lys Ser Lys Ala Thr Leu
Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110Thr
Val Ser Ser 115108116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 108Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His
Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45Gly Glu
Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe 50 55 60Lys
Ser Arg Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Met
Val 100 105 110Thr Val Ser Ser 115109113PRTHomo sapiens 109Asp Ile
Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu
Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25
30Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
Val 50 55 60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr65 70 75 80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr
Tyr Cys Gln Gln 85 90 95Tyr Tyr Ser Thr Pro Tyr Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile 100 105 110Lys110111PRTMus musculus 110Asp Ile
Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln
Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr 20 25
30Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro
Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr
Ile Asn65 70 75 80Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys
Gln Gln Ser Asn 85 90 95Glu Gly Pro Pro Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 110111111PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 111Asp Ile Val Met Thr
Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr
Ile Asn Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr 20 25 30Gly Asn Ser
Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu
Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60Arg
Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser65 70 75
80Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Asn
85 90 95Glu Ala Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110112112PRTHomo sapiens 112Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro
Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 100 105
110113119PRTMus musculus 113Gln Val Gln Leu Gln Gln Ser Gly Pro Glu
Leu Val Arg Pro Gly Val1 5 10 15Ser Val Lys Ile Ser Cys Lys Gly Ser
Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Ala Met His Trp Val Lys Gln Ser
His Ala Lys Ser Leu Glu Trp Ile 35 40 45Gly Gly Ile Ser Thr Tyr Phe
Gly Arg Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Ala Thr Met
Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ala
Arg Leu Thr Ser Glu Asp Ser Ala Leu Tyr Tyr Cys 85 90 95Ala Arg Gly
Leu Ser Gly Asn Tyr Val Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr
Ser Val Thr Val Ser Ser 115114119PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 114Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Ala Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly
Ile Ser Thr Tyr Phe Gly Arg Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys
Gly Arg Val Thr Met Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Leu Ser Gly Asn Tyr Val Met Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Thr Val Thr Val Ser Ser 115115113PRTHomo sapiens
115Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1
5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr
Ser 20 25 30Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu
Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala
Val Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr Ser Thr Pro Phe Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile 100 105 110Lys116111PRTMus musculus
116Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1
5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Arg Gln Ser Val Ser Thr
Ser 20 25 30Ser Tyr Ser Phe Met His Trp Tyr Arg Gln Lys Ala Gly Gln
Pro Pro 35 40 45Lys Leu Leu Ile Lys Tyr Ala Ser Ile Gln Glu Ser Gly
Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Asn Ile Leu65 70 75 80Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr
Tyr Cys Gln His Thr Trp 85 90 95Glu Ile Pro Phe Thr Phe Gly Ser Gly
Thr Lys Leu Glu Ile Lys 100 105 110117111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
117Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1
5 10 15Glu Arg Ala Thr Ile Asn Cys Arg Ala Arg Gln Ser Val Ser Thr
Ser 20 25 30Ser Tyr Ser Phe Met His Trp Tyr Gln Gln Pro Ala Gly Gln
Pro Pro 35 40 45Lys Leu Leu Ile Lys Tyr Ala Ser Ile Gln Glu Ser Gly
Val Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser65 70 75 80Ser Leu Gln Ala Glu Asp Val Ala Val Tyr
Tyr Cys Gln His Thr Trp 85 90 95Glu Ile Pro Phe Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 110118112PRTHomo sapiens 118Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45Gly Trp Ile Asn Pro Gly Ser Gly Asn Thr Asn Tyr Ala Gln Lys
Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr
Ala Tyr65 70 75 80Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 100 105 110119120PRTMus musculus 119Gln Val Gln
Leu Gln Gln Pro Gly Ser Glu Leu Val Arg Pro Gly Ala1 5 10 15Ser Val
Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp
Met His Trp Val Lys Gln Arg His Gly Gln Gly Leu Glu Trp Ile 35 40
45Gly Asn Ile Tyr Pro Gly Ser Gly Ser Thr Lys Tyr Asp Glu Arg Phe
50 55 60Lys Ser Lys Gly Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala
Tyr65 70 75 80Met His Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95Thr Arg Gly Gly Tyr Asp Ser Arg Ala Trp Phe Ala
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ala 115
120120120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 120Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Asn Ile Tyr Pro Gly Ser
Gly Ser Thr Lys Tyr Asp Glu Arg Phe 50 55 60Lys Ser Arg Val Thr Ile
Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Leu Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Gly
Gly Tyr Asp Ser Arg Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110Gly
Thr Leu Val Thr Val Ser Ser 115 120121107PRTHomo sapiens 121Asp Ile
Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn
Ser Tyr Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105122107PRTMus musculus 122Asp Val Gln Ile Thr Gln Ser Pro Ser
Tyr Leu Thr Ala Ser Pro Gly1 5 10 15Glu Thr Ile Thr Ile Asn Cys Arg
Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Leu Ala Trp Tyr Gln Glu Lys
Pro Gly Lys Thr Asn Lys Leu Leu Ile 35 40 45Tyr Ser Gly Ser Thr Leu
Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Asn Leu Glu Pro65 70 75 80Glu Asp Phe
Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Trp 85 90 95Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105123107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
123Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Thr Asn Lys
Leu Leu Leu 35 40 45Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile
Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln His Asn Glu Tyr Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105124111PRTHomo sapiens 124Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr
Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30Tyr Trp Ser Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile
Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser
100 105 110125122PRTMus musculus 125Asp Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Ser Leu Ser Leu Thr Cys Thr
Val Thr Gly Asn Ser Ile Thr Ser Glu 20 25 30Tyr Ala Trp Asn Trp Ile
Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45Met Gly Tyr Ile Ser
Tyr Ser Gly Thr Thr Ser Tyr Asn Pro Ser Leu 50 55 60Lys Ser Arg Ile
Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Leu Phe65 70 75 80Leu Gln
Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Ala
Arg Tyr Gly Tyr Gly Asn Pro Ala Thr Arg Tyr Phe Asp Val Trp 100 105
110Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115
120126122PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 126Glu Val Gln Leu Val Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Asn Ser Ile Thr Ser Glu 20 25 30Tyr Ala Trp Asn Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile Gly Tyr Ile Ser Tyr Ser
Gly Thr Thr Ser Tyr Asn Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Ile
Ser Arg Asp Thr Ser Lys Asn Gln Leu Ser65 70 75 80Leu Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Tyr
Gly Tyr Gly Asn Pro Ala Thr Arg Tyr Phe Asp Val Trp 100 105 110Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 1201279PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 127Gln
Gln Ser Asn Glu Ala Pro Pro Thr1 51289PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 128Gln
His Phe Trp Gly Thr Pro Leu Thr1 51297PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 129Gly
Thr Arg Ala Tyr His Tyr1 51309PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 130Gln Gln Ser Asn Glu Ala
Pro Pro Thr1 513110PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 131Gly Leu Ser Gly Asn Tyr Val Met Asp
Tyr1 5 10132108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 132Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe
Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro 85 90 95Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105133108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 133Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Phe Pro Thr Tyr Leu Pro 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105134108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
134Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr
Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Gly Tyr Asn Asp Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105135108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 135Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser
Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Thr Asp Pro Thr
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105136108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 136Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Val Val Ala Asn Ser 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Leu Ala Ser Phe Leu Tyr
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Asp Ala Thr Ser Pro Pro 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105137108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
137Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr
Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Ala Thr Asp Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105138119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 138Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30Ala Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Trp
Ile Ser Pro Ala Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Pro Phe Ser Pro Trp Val Met Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 115139119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
139Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Leu Gly
Tyr 20 25 30Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Gly Trp Ile Ser Pro Ala Gly Gly Ser Thr Asp Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Pro Phe Ser Pro Trp Val
Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115140108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 140Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Val Ser Ser Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Ser Leu Tyr
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Ser Tyr Ser Pro Phe 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105141108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
141Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser
Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Ser Trp Ala Ser Trp Leu Tyr Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Ser Tyr Ser Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105142108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 142Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala 20 25 30Val Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Trp Tyr
Ala Ser Trp Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Tyr Ser Pro Phe
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105143108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 143Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Val Ser Ser Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Trp Trp Ala Ser Ser Leu Tyr
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Ser Tyr Ser Pro Phe 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105144126PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
144Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Tyr Tyr
Ser 20 25 30Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Ser Ile Ser Pro Tyr Ser Gly Tyr Thr Ser Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gln Pro Thr His Tyr Tyr Tyr
Tyr Ala Lys Gly Tyr Lys Ala 100 105 110Met Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120 125145438PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
145Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Leu Val 35 40 45Ala Ser Ile Asn Ser Asn Gly Gly Ser Thr Tyr Tyr Pro
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Gly Asp Tyr Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Ser 100 105 110Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg 115 120 125Ser Thr Ser Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser145 150 155
160Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
165 170 175Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr 180 185 190Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys 195 200 205Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
Pro Pro Cys Pro Ala Pro 210 215 220Glu Phe Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys225 230 235 240Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 245 250 255Asp Val Ser
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 260 265 270Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 275 280
285Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
290 295 300Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Gly Leu305 310 315 320Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 325
330 335Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
Lys 340 345 350Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp 355 360 365Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys 370 375 380Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser385 390 395 400Arg Leu Thr Val Asp Lys
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 405 410 415Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 420 425 430Leu Ser
Leu Ser Leu Gly 435146219PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 146Asp Ile Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser
Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser 20 25 30Asn Gly Asp
Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln
Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser
85 90 95Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 110Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu 115 120 125Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln145 150 155 160Ser Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190Lys His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200
205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
21514710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 147Gly Tyr Thr Phe Thr Ser Tyr Trp Met His1 5
1014810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 148Gly Tyr Thr Phe Thr Asp Tyr Ala Met His1 5
101499PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 149Gly Tyr Thr Phe Ser Tyr Trp Met His1
5150107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 150Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Asp Asn Leu Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ser Pro Lys Leu Leu Val 35 40 45Tyr Asp Ala Thr Asn Leu Ala
Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Leu 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105151107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
151Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Asp Asn Leu Tyr Ser
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu
Leu Val 35 40 45Tyr Asp Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His
Phe Ala Gly Thr Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105152116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 152Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His
Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45Gly Glu
Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe 50 55 60Lys
Ser Arg Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Met
Val 100 105 110Thr Val Ser Ser 115153116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
153Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu
Trp Ile 35 40 45Gly Glu Ile Ala Pro Thr Asn Gly Arg Thr Asn Tyr Ile
Glu Lys Phe 50 55 60Lys Ser Arg Ala Thr Leu Thr Val Asp Lys Ser Ala
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Thr Arg Ala Tyr His Tyr
Trp Gly Gln Gly Thr Met Val 100 105 110Thr Val Ser Ser
115154116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 154Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Arg Gln Ala
Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn Pro Ala Asn
Gly Arg Thr Asn Tyr Ile Glu Lys Phe 50 55 60Lys Ser Arg Ala Thr Leu
Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Met Val 100 105 110Thr
Val Ser Ser 1151559PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 155Gln His Phe Ala Gly Thr Pro Leu Thr1
515617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 156Glu Ile Ala Pro Thr Asn Gly Arg Thr Asn Tyr
Ile Glu Lys Phe Lys1 5 10 15Ser15717PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 157Glu
Ile Asn Pro Ala Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe Lys1 5 10
15Ser158214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 158Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Asp Asn Leu Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ser Pro Lys Leu Leu Val 35 40 45Tyr Asp Ala Thr Asn Leu Ala
Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Leu 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 210159446PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 159Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Arg
Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn Pro
Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe 50 55 60Lys Ser Arg Ala
Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Met Val 100 105
110Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu 130 135 140Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly145 150 155 160Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser 165 170 175Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe225 230
235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro 245 250 255Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp
Pro Glu Val 260 265 270Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Tyr Gly Ser
Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345
350Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val
355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu Val Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445160108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
160Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Asp Asn Leu Tyr Ser
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu
Leu Val 35 40 45Tyr Asp Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His
Phe Ala Gly Thr Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 10516115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 161Arg Ala Ser Glu Ser Val
Asp Asp Tyr Gly Asn Ser Phe Met His1 5 10 1516215PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 162Arg
Ala Ser Glu Ser Val Asp Ser Tyr Gly Pro Ser Phe Met His1 5 10
15163112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 163Asp Ile Gln Met Thr Gln Ser Pro Ala Ser
Leu Ser Val Ser Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg Ala
Ser Asp Asn Leu Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Gln
Gly Lys Ser Pro Gln Leu Leu Val 35 40 45Tyr Asp Ala Thr Asn Leu Ala
Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln
Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser65 70 75 80Glu Asp Phe Gly
Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Leu 85 90 95Thr Phe Gly
Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala 100 105
110164125PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 164Gln Val Gln Leu Gln Gln Pro Gly Ala Glu
Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg
Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn Pro Thr Asn
Gly Arg Thr Asn Tyr Ile Glu Lys Phe 50 55 60Lys Ser Lys Ala Thr Leu
Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110Thr
Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr 115 120
125165113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 165Asp Ile Gln Leu Thr Gln Thr Pro Ala Ser
Leu Ser Val Ser Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg Ala
Ser Glu Asn Ile Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Gln
Gly Lys Ser Pro Gln Leu Leu Val 35 40 45Tyr Ala Ala Thr Asp Leu Ala
Asp Gly Val Pro Ser Arg Phe Arg Gly 50 55 60Ser Gly Ser Gly Thr Gln
Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser65 70 75 80Glu Asp Phe Gly
Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Leu 85 90 95Thr Phe Gly
Ala Gly Thr Lys Leu Glu Leu Ile Arg Ala Asp Ala Ala 100 105
110Pro166125PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 166Gln Val Gln Leu Gln Gln Pro Gly
Ala Glu Leu Val Arg Pro Gly Ala1 5 10 15Ala Val Lys Leu Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn Pro
Thr Asn Gly Arg Thr Asn Tyr Asn Glu Asn Phe 50 55 60Lys Ser Lys Ala
Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Thr Arg Ala Tyr His Phe Trp Gly Gln Gly Thr Ser Val 100
105
110Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr 115 120
125167113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 167Asp Ile Gln Met Thr Gln Ser Pro Ala Ser
Leu Ser Val Ser Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg Ala
Ser Asp Asn Ile Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Gln
Gly Lys Ser Pro Gln Leu Leu Val 35 40 45Tyr Ala Ala Thr Asn Leu Ala
Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln
Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser65 70 75 80Glu Asp Phe Gly
Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Leu 85 90 95Met Phe Gly
Ser Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala 100 105
110Pro168125PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 168Gln Val Gln Leu Gln Gln Pro Gly
Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Ala Ser Tyr 20 25 30Trp Met His Trp Val Lys
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn Pro
Ile Asn Gly Arg Thr Asn Tyr Ser Glu Lys Phe 50 55 60Lys Lys Lys Ala
Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Ser Val 100 105
110Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr 115 120
125169119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 169Gln Val Gln Leu Gln Gln Pro Gly Ala Glu
Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg
Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn Pro Ser Asn
Gly Arg Thr Asn Tyr Asn Glu Thr Phe 50 55 60Lys Ser Lys Ala Thr Leu
Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110Thr
Val Ser Ser Ala Lys Thr 115170113PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 170Asp Val Gln Ile Thr
Gln Ser Pro Ser Tyr Leu Thr Ala Ser Pro Gly1 5 10 15Glu Thr Ile Thr
Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30Leu Ala Trp
Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile 35 40 45Tyr Ser
Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Leu Glu Pro65 70 75
80Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Trp
85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala
Ala 100 105 110Pro171131PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 171Asp Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Ser Leu Ser Leu
Thr Cys Thr Val Thr Gly Asn Ser Ile Thr Ser Glu 20 25 30Tyr Ala Trp
Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45Met Gly
Tyr Ile Ser Tyr Ser Gly Thr Thr Ser Tyr Asn Pro Ser Leu 50 55 60Lys
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Leu Phe65 70 75
80Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95Ala Arg Tyr Gly Tyr Gly Asn Pro Ala Thr Arg Tyr Phe Asp Val
Trp 100 105 110Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr
Thr Pro Pro 115 120 125Ser Val Tyr 130172123PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
172Gln Val Gln Leu Gln Gln Pro Gly Ser Glu Leu Val Arg Pro Gly Ala1
5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg His Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Asn Ile Tyr Pro Gly Ser Gly Ser Thr Lys Tyr Asp
Glu Arg Phe 50 55 60Lys Ser Lys Gly Thr Leu Thr Val Asp Thr Ser Ser
Ser Thr Ala Tyr65 70 75 80Met His Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95Thr Arg Gly Gly Tyr Asp Ser Arg Ala
Trp Phe Ala Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ala Ala Lys Thr 115 120173123PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 173Gln Val Gln Leu Gln
Gln Pro Gly Ser Glu Leu Val Arg Pro Gly Ala1 5 10 15Ser Val Lys Leu
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His
Trp Val Lys Gln Arg His Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Asn
Ile Tyr Pro Gly Ser Gly Ser Thr Lys Tyr Asp Glu Lys Phe 50 55 60Lys
Ser Lys Gly Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr65 70 75
80Met His Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Thr Arg Gly Gly Tyr Asp Ser Arg Ala Trp Phe Ala His Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr 115
120174127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 174Glu Val Lys Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Phe Leu Lys Leu Ser Cys Ala Thr Ser
Gly Phe Thr Phe Ser Asp Tyr 20 25 30Tyr Met Tyr Trp Val Arg Gln Thr
Pro Glu Lys Arg Leu Glu Trp Val 35 40 45Ala Ser Ile Ser Asn Gly Gly
Asp Asn Thr Tyr Tyr Pro Asp Thr Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser
Arg Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Gln
Gly Ala Leu Tyr Asp Gly Tyr Tyr Arg Gly Ala Met Asp 100 105 110Tyr
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr 115 120
125175124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 175Gln Val Gln Leu Gln Gln Ser Gly Ala Glu
Leu Met Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ala
Gly Tyr Thr Phe Ser Asn Tyr 20 25 30Trp Ile Glu Trp Val Lys Gln Arg
Pro Gly His Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Leu Pro Gly Ser
Gly Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Phe
Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Met Gln Leu Ser
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg
Gly Gly Tyr Gly Tyr Asp Gly Glu Phe Ala Tyr Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr 115 120
* * * * *