U.S. patent application number 17/501430 was filed with the patent office on 2022-08-25 for compositions and methods for treatment of thyroid eye disease.
The applicant listed for this patent is Viridian Therapeutics, Inc.. Invention is credited to Vahe Bedian, Peter Harwin, Tomas Kiselak, Angela She, Jonathan Violin, Yang Zhao.
Application Number | 20220267451 17/501430 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-25 |
United States Patent
Application |
20220267451 |
Kind Code |
A1 |
Bedian; Vahe ; et
al. |
August 25, 2022 |
COMPOSITIONS AND METHODS FOR TREATMENT OF THYROID EYE DISEASE
Abstract
Antibodies and compositions against IGF-1R and uses thereof are
provided herein.
Inventors: |
Bedian; Vahe; (Waltham,
MA) ; Harwin; Peter; (Philadelphia, PA) ;
Kiselak; Tomas; (Philadelphia, PA) ; She; Angela;
(Waltham, MA) ; Violin; Jonathan; (Waltham,
MA) ; Zhao; Yang; (Waltham, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Viridian Therapeutics, Inc. |
Waltham |
MA |
US |
|
|
Appl. No.: |
17/501430 |
Filed: |
October 14, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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63091839 |
Oct 14, 2020 |
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63201978 |
May 21, 2021 |
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63260130 |
Aug 10, 2021 |
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63261742 |
Sep 28, 2021 |
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International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 27/02 20060101 A61P027/02 |
Claims
1-47. (canceled)
48. A pharmaceutical composition comprising an antibody for
treating thyroid eye disease in a subject, wherein the antibody
comprises: a light chain variable region having the amino acid
sequence of SEQ ID NO: 13; a heavy chain variable region having the
amino acid sequence of SEQ ID NO: 14; and a Fc region comprising a
M428L and N434S substitutions and/or M252Y, S254T, and T256E
substitutions.
49. The pharmaceutical composition of claim 48, wherein the
antibody comprises a Fc region with M428L and N434S
substitutions.
50. The pharmaceutical composition of claim 48, wherein the
antibody comprises a Fc region with M252Y, S254T, and T256E
substitutions.
51. A method of treating thyroid eye disease in a subject, the
method comprising administering the pharmaceutical composition
comprising the antibody of claim 48.
52. The method of claim 51, wherein the pharmaceutical composition
is administered intravenously or subcutaneously.
53. A method of treating thyroid eye disease in a subject, the
method comprising administering the pharmaceutical composition
comprising the antibody of claim 49.
54. The method of claim 53, wherein the pharmaceutical composition
is administered subcutaneously or intravenously.
55. A method of treating thyroid eye disease in a subject, the
method comprising administering the pharmaceutical composition
comprising the antibody of claim 50.
56. A method of treating or reducing the severity of,
thyroid-associated ophthalmopathy (TAO), or a symptom thereof, the
method comprising administering to a subject the pharmaceutical
composition of claim 48.
57. A method of treating or reducing the severity of,
thyroid-associated ophthalmopathy (TAO), or a symptom thereof, the
method comprising administering to a subject the pharmaceutical
composition of claim 49.
58. A method of reducing proptosis in an eye in a subject with
thyroid-associated ophthalmopathy (TAO), the method comprising
administering to a subject the pharmaceutical composition of claim
48.
59. A method of reducing proptosis in an eye in a subject with
thyroid-associated ophthalmopathy (TAO), the method comprising
administering to a subject the pharmaceutical composition of claim
49.
60. A method of reducing Clinical Activity Score (CAS) of
thyroid-associated ophthalmopathy (TAO) in a subject, the method
comprising administering to a subject the pharmaceutical
composition of claim 48.
61. A method of reducing Clinical Activity Score (CAS) of
thyroid-associated ophthalmopathy (TAO) in a subject, the method
comprising administering to a subject the pharmaceutical
composition of claim 49.
62. A method of a) reducing proptosis by at least 2 mm and b)
reducing the clinical activity score (CAS) in a subject with
thyroid-associated ophthalmopathy (TAO), the method comprising
administering to a subject the pharmaceutical composition of claim
48.
63. A method of a) reducing proptosis by at least 2 mm and b)
reducing the clinical activity score (CAS) in a subject with
thyroid-associated ophthalmopathy (TAO), the method comprising
administering to a subject the pharmaceutical composition of claim
49.
64. A method of treating or reducing the severity of diplopia in a
subject with thyroid-associated ophthalmopathy (TAO), the method
comprising administering to a subject the pharmaceutical
composition of claim 48.
65. A method of treating or reducing the severity of diplopia in a
subject with thyroid-associated ophthalmopathy (TAO), the method
comprising administering to a subject the pharmaceutical
composition of claim 49.
66. The method of claim 51, wherein said pharmaceutical composition
is administered at a dosage of about 1 .mu.g/kg to about 5
.mu.g/kg, about 5 .mu.g/kg to about 10 .mu.g/kg, about 10 .mu.g/kg
to about 20 .mu.g/kg, about 20 .mu.g/kg to about 30 .mu.g/kg, about
5 .mu.g/kg, about 10 .mu.g/kg, about 15 .mu.g/kg, about 20
.mu.g/kg, about 25 .mu.g/kg, or about 30 .mu.g/kg of the antibody
as a first dose.
67. A method of treating thyroid eye disease in a subject, the
method comprising administering the pharmaceutical composition of
claim 48 to the subject, wherein the antibody has a serum
concentration in the subject of at least, or about, 10 .mu.g/ml or
20 .mu.g/ml or 50 .mu.g/ml, 70 .mu.g/ml, 75 .mu.g/ml, 80 .mu.g/ml,
85 .mu.g/ml, 90 .mu.g/ml, 95 .mu.g/ml, 100 .mu.g/ml, or 105
.mu.g/ml at least 1, 2, or 3 weeks after administration.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 63/091,839, filed Oct. 14, 2020, U.S. Provisional
Application No. 63/201,978, filed May 21, 2021, U.S. Provisional
Application No. 63/260,130, filed Aug. 10, 2021, and U.S.
Provisional Application No. 63/261,742, filed Sep. 28, 2021, each
of which is hereby incorporated by reference in its entirety.
BACKGROUND
[0002] Thyroid-associated ophthalmopathy (TAO), also known as
thyroid eye disease (TED), Graves' ophthalmopathy or orbitopathy
(GO), thyrotoxic exophthalmos, dysthyroid ophthalmopathy, and
several other terms, is orbitopathy associated with thyroid
dysfunction. TAO is divided into two types. Active TAO, which
typically lasts 1-3 years, is characterized by an ongoing
autoimmune/inflammatory response in the soft tissues of the orbit.
Active TAO is responsible for the expansion and remodeling of the
ocular soft tissues. The autoimmune/inflammatory response of active
TAO spontaneously resolves and the condition transitions into
inactive TAO. Inactive TAO is the term used to describe the
long-term/permanent sequelae of active TAO. The cause of TAO is
unknown. TAO is typically associated with Graves' hyperthyroidism,
but can also occur as part of other autoimmune conditions that
affect the thyroid gland and produce pathology in orbital and
periorbital tissue, and, rarely, the pretibial skin (pretibial
myxedema) or digits (thyroid acropachy). TAO is an autoimmune
orbitopathy in which the orbital and periocular soft tissues are
primarily affected with secondary effects on the eye and vision. In
TAO, as a result of inflammation and expansion of orbital soft
tissues, primarily eye muscles and adipose, the eyes are forced
forward (bulge) out of their sockets--a phenomenon termed proptosis
or exophthalmos. Although most cases of TAO do not result in loss
of vision, this condition can cause vision-threatening exposure
keratopathy, troublesome diplopia (double vision), and compressive
dysthyroid optic neuropathy. TAO may precede, coincide with, or
follow the systemic complications of dysthyroidism. The ocular
manifestations of TAO include upper eyelid retraction, lid lag,
swelling, redness (erythema), conjunctivitis, and bulging eyes
(exophthalmos or proptosis), chemosis, periorbital edema, and
altered ocular motility with significant functional, social, and
cosmetic consequences. Many of the signs and symptoms of TAO,
including proptosis and ocular congestion, result from expansion of
the orbital adipose tissue and periocular muscles. The adipose
tissue volume increases owing in part to new fat cell development
(adipogenesis) within the orbital fat. The accumulation of
hydrophilic glycosaminoglycans, primarily hyaluronic acid, within
the orbital adipose tissue and the perimysial connective tissue
between the extraocular muscle fibers, further expands the fat
compartments and enlarges the extraocular muscle bodies. Hyaluronic
acid is produced by fibroblasts residing within the orbital fat and
extraocular muscles, and its synthesis in vitro is stimulated by
several cytokines and growth factors, including IL-1beta,
interferon-gamma, platelet-derived growth factor, thyroid
stimulating hormone (TSH) and insulin-like growth factor I
(IGF-I).
[0003] Antibodies that activate the insulin-like growth factor I
receptor (IGF-IR) have also been detected and implicated in active
TAO. Without being bound to any theory, it is believed that TSHR
and IGF-IR form a physical and functional complex in orbital
fibroblasts, and that blocking IGF-IR appears to attenuate both
IGF-1 and TSH-dependent signaling. It has been suggested that
blocking IGF-IR using an antibody antagonist might reduce both
TSHR- and IGF-I-dependent signaling and therefore interrupt the
pathological activities of autoantibodies acting as agonists on
either receptor.
[0004] IGF-IR is a widely expressed heterotetrameric protein
involved in the regulation of proliferation and metabolic function
of many cell types. It is a tyrosine kinase receptor comprising two
subunits. IGF-IRalpha contains a ligand-binding domain while
IGF-IRbeta is involved in signaling and contains tyrosine
phosphorylation sites.
[0005] Current therapies for hyperthyroidism due to Graves' disease
are imperfect because therapies targeting the specific underlying
pathogenic autoimmune mechanisms of the disease are lacking. Even
more complex is the treatment of moderate-to-severe active TAO.
Although recent years have witnessed a better understanding of its
pathogenesis, TAO remains a therapeutic challenge and dilemma.
There are no approved drugs to treat active TAO. Intravenous
glucocorticoids (ivGCs) and oral glucocorticoids are used to treat
patients with moderate-to-severe active TAO, but results are seldom
satisfactory. Partial responses are frequent and relapses (rebound)
after drug withdrawal are not uncommon. Adverse events do occur and
many patients eventually require rehabilitative surgery conducted
when their condition has transitioned to inactive TAO. Accordingly,
there is still a need to provide alternative therapies for TAO and
its related symptoms.
SUMMARY
[0006] The embodiments relate generally to IGF-1R antibodies, and
antigen binding fragments thereof. Certain IGF-1R antibodies and
antigen-binding fragments inhibit IGF-1R function or block the
biological functions of IGF-I mediated IGF-1R signaling.
Additionally, the invention generally relates to methods for
treating thyroid-associated ophthalmopathy (TAO), also known as
thyroid eye disease (TED), Graves' ophthalmopathy or orbitopathy
(GO), thyrotoxic exophthalmos, dysthyroid ophthalmopathy, and other
thyroid eye disorders associated with IGF-1R signaling.
[0007] In some embodiments, an antibody, or antigen binding
fragment thereof, comprising a sequence as provided for herein is
provided. In some embodiments, the antibody comprises a VL sequence
as set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 79, or 86;
and a VH sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12,
14, 16, 80, or 83. In some embodiments, the antibody comprises a
LCDR sequence as set forth in SEQ ID NO: 17, 18, 19, 23, 24, 25,
29, 30, 31, 35, 36, 37, 41, 42, 43, 47, 48, 49, 53, 54, 55, 59, 60,
61, or 81, and a HCDR sequence as set forth in SEQ ID NO: 20, 21,
22, 26, 27, 28, 32, 33, 34, 38, 39, 40, 44, 45, 46, 50, 51, 52, 56,
57, 58, 62, 63, or 64; or any combination or variant thereof.
[0008] In some embodiments, the antibody, or antigen binding
fragment thereof, comprises a V.sub.L peptide as set forth in SEQ
ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 79, or 86, or any variant
thereof. In some embodiments, the antibody, or antigen binding
fragment thereof, comprises a V.sub.H peptide as set forth in SEQ
ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 80, or 83, or any variant
thereof.
[0009] In some embodiments, the antibody or antibody fragment
comprises: (i) a heavy chain variable region comprising heavy chain
CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1
sequence has the amino acid sequence of SEQ ID NO: 20, 26, 32, 38,
44, 50, or 56; the heavy chain CDR2 has the amino acid sequence of
SEQ ID NO: 21, 27, 33, 39, 45, 51, or 57; and the heavy chain CDR3
sequence has the amino acid sequence of SEQ ID NO: 22, 28, 34, 40,
46, 52, or 58; or variants of any of the foregoing; and (ii) a
light chain variable region comprising light chain CDR1, CDR2, and
CDR3 sequences, wherein the light chain CDR1 sequence has the amino
acid sequence SEQ ID NO: 17, 23, 29, 35, 41, 47, or 53; the light
chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 18,
24, 30, 36, 42, 48, or 54; and the light chain CDR3 sequence has
the amino acid sequence of SEQ ID NO: 19, 25, 31, 37, 43, 49, 55,
or 81; or variants of any of the foregoing.
[0010] In some embodiments, the antibody or antibody fragment
comprises: (i) a heavy chain variable region comprising heavy chain
CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1
sequence has the amino acid sequence of SEQ ID NO: 20; the heavy
chain CDR2 has the amino acid sequence of SEQ ID NO: 21; and the
heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:
22; or variants of any of the foregoing; and (ii) a light chain
variable region comprising light chain CDR1, CDR2, and CDR3
sequences, wherein the light chain CDR1 sequence has the amino acid
sequence SEQ ID NO: 17; the light chain CDR2 sequence has the amino
acid sequence of SEQ ID NO: 18; and the light chain CDR3 sequence
has the amino acid sequence of SEQ ID NO: 19; or variants of any of
the foregoing.
[0011] In some embodiments, the antibody or antibody fragment
comprises: (i) a heavy chain variable region comprising heavy chain
CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1
sequence has the amino acid sequence of SEQ ID NO: 26; the heavy
chain CDR2 has the amino acid sequence of SEQ ID NO: 27; and the
heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:
28; or variants of any of the foregoing; and (ii) a light chain
variable region comprising light chain CDR1, CDR2, and CDR3
sequences, wherein the light chain CDR1 sequence has the amino acid
sequence SEQ ID NO: 23; the light chain CDR2 sequence has the amino
acid sequence of SEQ ID NO: 24; and the light chain CDR3 sequence
has the amino acid sequence of SEQ ID NO: 25; or variants of any of
the foregoing.
[0012] In some embodiments, the antibody or antibody fragment
comprises: (i) a heavy chain variable region comprising heavy chain
CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1
sequence has the amino acid sequence of SEQ ID NO: 32; the heavy
chain CDR2 has the amino acid sequence of SEQ ID NO: 33; and the
heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:
34; or variants of any of the foregoing; and (ii) a light chain
variable region comprising light chain CDR1, CDR2, and CDR3
sequences, wherein the light chain CDR1 sequence has the amino acid
sequence SEQ ID NO: 29; the light chain CDR2 sequence has the amino
acid sequence of SEQ ID NO: 30; and the light chain CDR3 sequence
has the amino acid sequence of SEQ ID NO: 31; or variants of any of
the foregoing.
[0013] In some embodiments, the antibody or antibody fragment
comprises: (i) a heavy chain variable region comprising heavy chain
CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1
sequence has the amino acid sequence of SEQ ID NO: 38; the heavy
chain CDR2 has the amino acid sequence of SEQ ID NO: 39; and the
heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:
40; or variants of any of the foregoing; and (ii) a light chain
variable region comprising light chain CDR1, CDR2, and CDR3
sequences, wherein the light chain CDR1 sequence has the amino acid
sequence SEQ ID NO: 35; the light chain CDR2 sequence has the amino
acid sequence of SEQ ID NO: 36; and the light chain CDR3 sequence
has the amino acid sequence of SEQ ID NO: 37; or variants of any of
the foregoing.
[0014] In some embodiments, the antibody or antibody fragment
comprises: (i) a heavy chain variable region comprising heavy chain
CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1
sequence has the amino acid sequence of SEQ ID NO: 44; the heavy
chain CDR2 has the amino acid sequence of SEQ ID NO: 45; and the
heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:
46; or variants of any of the foregoing; and (ii) a light chain
variable region comprising light chain CDR1, CDR2, and CDR3
sequences, wherein the light chain CDR1 sequence has the amino acid
sequence SEQ ID NO: 41; the light chain CDR2 sequence has the amino
acid sequence of SEQ ID NO: 42; and the light chain CDR3 sequence
has the amino acid sequence of SEQ ID NO: 43; or variants of any of
the foregoing.
[0015] In some embodiments, the antibody or antibody fragment
comprises: (i) a heavy chain variable region comprising heavy chain
CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1
sequence has the amino acid sequence of SEQ ID NO: 50; the heavy
chain CDR2 has the amino acid sequence of SEQ ID NO: 51; and the
heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:
52; or variants of any of the foregoing; and (ii) a light chain
variable region comprising light chain CDR1, CDR2, and CDR3
sequences, wherein the light chain CDR1 sequence has the amino acid
sequence SEQ ID NO: 47; the light chain CDR2 sequence has the amino
acid sequence of SEQ ID NO: 48; and the light chain CDR3 sequence
has the amino acid sequence of SEQ ID NO: 49; or variants of any of
the foregoing.
[0016] In some embodiments, the antibody or antibody fragment
comprises: (i) a heavy chain variable region comprising heavy chain
CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1
sequence has the amino acid sequence of SEQ ID NO: 56; the heavy
chain CDR2 has the amino acid sequence of SEQ ID NO: 57; and the
heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:
58; or variants of any of the foregoing; and (ii) a light chain
variable region comprising light chain CDR1, CDR2, and CDR3
sequences, wherein the light chain CDR1 sequence has the amino acid
sequence SEQ ID NO: 53; the light chain CDR2 sequence has the amino
acid sequence of SEQ ID NO: 54; and the light chain CDR3 sequence
has the amino acid sequence of SEQ ID NO: 55; or variants of any of
the foregoing.
[0017] In some embodiments, the antibody or antibody fragment
comprises: (i) a heavy chain variable region comprising heavy chain
CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1
sequence has the amino acid sequence of SEQ ID NO: 62; the heavy
chain CDR2 has the amino acid sequence of SEQ ID NO: 63; and the
heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:
64; or variants of any of the foregoing; and (ii) a light chain
variable region comprising light chain CDR1, CDR2, and CDR3
sequences, wherein the light chain CDR1 sequence has the amino acid
sequence SEQ ID NO: 59; the light chain CDR2 sequence has the amino
acid sequence of SEQ ID NO: 60; and the light chain CDR3 sequence
has the amino acid sequence of SEQ ID NO: 61; or variants of any of
the foregoing.
[0018] In some embodiments, the antibody or antibody fragment
comprises: (i) a heavy chain variable region comprising heavy chain
CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1
sequence has the amino acid sequence of SEQ ID NO: 38; the heavy
chain CDR2 has the amino acid sequence of SEQ ID NO: 39; and the
heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:
40; or variants of any of the foregoing; and (ii) a light chain
variable region comprising light chain CDR1, CDR2, and CDR3
sequences, wherein the light chain CDR1 sequence has the amino acid
sequence SEQ ID NO: 35; the light chain CDR2 sequence has the amino
acid sequence of SEQ ID NO: 36; and the light chain CDR3 sequence
has the amino acid sequence of SEQ ID NO: 81; or variants of any
foregoing.
[0019] In some embodiments, the antibody comprises a V.sub.L
sequence as set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 79,
or 86, or a variant thereof. In some embodiments, the antibody
comprises a V.sub.H sequence as set forth in SEQ ID NO: 2, 4, 6, 8,
10, 12, 14, 16, 80, or 83, or a variant thereof.
[0020] In some embodiments, the antibody comprises a sequence of
SEQ ID NO: 65-72, 78, 82, or 85, or a variant thereof.
[0021] In some embodiments, the antibody comprises a light chain
having the amino acid sequence of SEQ ID NO: 3 and a heavy chain
comprising the amino acid sequence of SEQ ID NO: 83. In some
embodiments, the antibody comprising a light chain variable region
having the amino acid sequence of SEQ ID NO: 13 and a heavy chain
variable region having the amino acid sequence of SEQ ID NO:
14.
[0022] In some embodiments, the antibody comprises a light chain
having a an amino acid sequence of SEQ ID NO: 93 and a heavy chain
amino acid sequence of SEQ ID NO: 92.
[0023] In some embodiments, the antibody comprises a light chain
having a an amino acid sequence of SEQ ID NO: 93 and a heavy chain
amino acid sequence of SEQ ID NO: 94.
[0024] In some embodiments, the antibody comprises a light chain
having a an amino acid sequence of SEQ ID NO: 93 and a heavy chain
amino acid sequence of SEQ ID NO: 95.
[0025] In some embodiments, the a variant of any antibodies
provided herein are provided so long as the CDRs remain constant as
compared to the parental (non-variant) sequence provided for
herein.
[0026] In some embodiments, the antibody comprises a Fc region. In
some embodiments, the Fc region is as set forth in SEQ ID NO:
75-77, 84, 87, 88, 89, or 90. In some embodiments, the Fc region is
as set forth in SEQ ID NO: 75. In some embodiments, the Fc region
is as set forth in SEQ ID NO: 76. In some embodiments, the Fc
region is as set forth in SEQ ID NO: 77. In some embodiments, the
Fc region is as set forth in SEQ ID NO: 84. In some embodiments,
the Fc region is as set forth in SEQ ID NO: 87. In some
embodiments, the Fc region is as set forth in SEQ ID NO: 88. In
some embodiments, the Fc region is as set forth in SEQ ID NO: 89.
In some embodiments, the Fc region is as set forth in SEQ ID NO:
90.
[0027] In some embodiments, pharmaceutical compositions comprising
an antibody as provided for herein is provided.
[0028] In some embodiments, methods of treating or reducing the
severity of, thyroid-associated ophthalmopathy (TAO), or a symptom
thereof are provided, the methods comprising administering to a
subject an antibody as provided for herein or a pharmaceutical
composition comprising the same.
[0029] In some embodiments, methods of treating thyroid eye disease
in a subject are provided, the methods comprising administering to
a subject an antibody as provided for herein or a pharmaceutical
composition comprising the same.
[0030] In some embodiments, methods of reducing Clinical Activity
Score (CAS) of thyroid-associated ophthalmopathy (TAO) in a subject
are provided, the methods comprising administering to a subject an
antibody as provided for herein or a pharmaceutical composition
comprising the same.
[0031] In some embodiments, methods of a) reducing proptosis by at
least 2 mm and b) reducing the clinical activity score (CAS) in a
subject with thyroid-associated ophthalmopathy (TAO) are provided,
the methods comprising administering to a subject an antibody as
provided for herein or a pharmaceutical composition comprising the
same.
[0032] In some embodiments, methods of treating or reducing the
severity of thyroid-associated ophthalmopathy (TAO) in a subject
are provided, the methods comprising administering to a subject an
antibody as provided for herein, or a pharmaceutical composition
comprising the same, wherein treatment with said antibody (i)
reduces proptosis by at least 2 mm in an eye; (ii) is not
accompanied by a deterioration of 2 mm or more in the other (or
fellow eye); and (iii) reduces the CAS in said subject to either
one (1) or zero (0).
[0033] In some embodiments, methods of improving the quality of
life in a subject with thyroid-associated ophthalmopathy (TAO, also
called Graves' Ophthalmopathy/Graves' Orbitopathy) are provided,
the methods comprising administering to a subject an antibody as
provided for herein, or a pharmaceutical composition comprising the
same.
[0034] In some embodiments, methods of treating or reducing the
severity of diplopia in a subject with thyroid-associated
ophthalmopathy (TAO) are provided, the methods comprising
administering to a subject an antibody as provided for herein, or a
pharmaceutical composition comprising the same.
[0035] In some embodiments, methods of increasing the
internalization of IGF-1R on a cell are provided, the methods
comprising contacting the cell with an antibody as provided for
herein or a pharmaceutical composition comprising the same.
[0036] In some embodiments, methods of inhibiting IGF-1 stimulated
receptor phosphorylation on a cell are provided, the methods
comprising contacting the cell with an as provided for herein, or a
pharmaceutical composition comprising the same.
[0037] In some embodiments, methods of treating thyroid eye disease
in a subject are provided, the methods comprising administering an
as provided for herein, or a pharmaceutical composition comprising
the same to the subject, wherein the antibody has a serum
concentration in the subject of at least, or about, 70 .mu.g/ml, 75
.mu.g/ml, 80 .mu.g/ml, 85 .mu.g/ml, 90 .mu.g/ml, 95 .mu.g/ml, 100
.mu.g/ml, or 105 .mu.g/ml at least 1, 2, or 3 week after
administration.
[0038] In some embodiments, methods of inhibiting IGF-1 induced
receptor autophosphorylation in a cell by at least 95%, 96%, 97%,
98%, or 99% or by 100% are provided, the method comprising
contacting the cell with an antibody as provided for herein, or a
pharmaceutical composition comprising the same.
[0039] In some embodiments, embodiments are provided for any of the
methods provided for herein, wherein the antibody, or an antigen
binding fragment thereof, is administered in a pharmaceutical
composition that additionally comprises a pharmaceutically
acceptable diluent or excipient or carrier. In some embodiments,
the pharmaceutical composition further comprises one or more
pharmaceutically active compounds for the treatment of TAO. In some
embodiments, the pharmaceutical composition further comprises
corticosteroids; rituximab or other anti-CD20 antibodies;
tocilizumab or other anti-IL-6 antibodies; or selenium, infliximab
or other anti-TNFalpha antibodies or a thyroid-stimulating hormone
receptor (TSHR) inhibitor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 illustrates the NHP (non-human primates) serum
concentration of various antibodies and embodiments as provided for
herein.
[0041] FIG. 2A and FIG. 2B illustrate various properties of
antibodies as provided for herein.
[0042] FIG. 3A-F illustrate various properties of antibodies as
provided for herein.
[0043] FIG. 4A-C illustrate various properties of antibodies as
provided for herein.
[0044] FIG. 5A and FIG. 5B illustrate various properties of
antibodies as provided for herein.
[0045] FIG. 6A and FIG. 6B illustrate various properties of
antibodies as provided for herein.
[0046] FIG. 7 illustrates various properties of antibodies as
provided for herein.
[0047] FIG. 8 illustrates various properties of antibodies as
provided for herein.
DETAILED DESCRIPTION
[0048] Provided herein are antibodies that bind and modulate the
activity of IGF-1R. The antibodies can be used, for example, to
treat thyroid eye disease.
[0049] As used herein, "Thyroid-associated Ophthalmopathy" (TAO),
"Thyroid Eye Disease" (TED), "Graves' Ophthalmopathy" or "Graves'
Orbitopathy" (GO) refer to the same disorder or condition and are
used interchangeably. They all refer to the inflammatory orbital
pathology associated with some autoimmune thyroid disorders, most
commonly with "Graves' Disease" (GD), but sometimes with other
diseases, e.g. Hashimoto's thyroiditis.
[0050] The terms "proptosis" and "exophthalmos" (also known as
exophthalmos, exophthalmia, or exorbitism) refer to the forward
projection, displacement, bulging, or protrusion of an organ. As
used herein, the terms refer to the forward projection,
displacement, bulging, or protrusion of the eye anteriorly out of
the orbit. Proptosis and exophthalmos are considered by some of
skill in the art to have the same meaning and are often used
interchangeably, while others attribute subtle differences to their
meanings. Exophthalmos is used by some to refer to severe
proptosis; or to refer to endocrine-related proptosis. Yet others
use the term exophthalmos when describing proptosis associated with
the eye, in, for example, subjects with TAO (TED or GO).
[0051] As used herein, the terms "proptosis" and "exophthalmos" are
used interchangeably and refer to the forward projection,
displacement, bulging, or protrusion of the eye anteriorly out of
the orbit. Owing to the rigid bony structure of the orbit with only
anterior opening for expansion, any increase in orbital soft tissue
contents taking place from the side or from behind will displace
the eyeball forward. Proptosis or exophthalmos can be the result of
a several disease processes including infections, inflammations,
tumors, trauma, metastases, endocrine lesions, vascular diseases
& extra orbital lesions. TAO (TED or GO) is currently
recognized as the most common cause of proptosis in adults.
Exophthalmos can be either bilateral, as is often seen in TAO (TED
or GO), or unilateral (as is often seen in an orbital tumor).
[0052] Measurement of the degree of exophthalmos can be performed
using, for example, an exophthalmometer, an instrument used for
measuring the degree of forward displacement of the eye. The device
allows measurement of the forward distance of the lateral orbital
rim to the front of the cornea. Computed tomography (CT) scanning
and Magnetic resonance imaging (MRI) may also be used in evaluating
the degree of exophthalmos or proptosis. CT scanning is an
excellent imaging modality for the diagnosis of TAO. In addition to
allowing visualization of the enlarged extraocular muscles, CT
scans provide the surgeon or clinician with depictions of the bony
anatomy of the orbit when an orbital decompression is required.
MRI, with its multi-planar and inherent contrast capabilities,
provides excellent imaging of the orbital contents without the
radiation exposure associated with CT scan studies. MRI provides
better imaging of the optic nerve, orbital fat, and extraocular
muscle, but CT scans provide better views of the bony architecture
of the orbit. Orbital ultrasonography can also be a used for the
diagnosis and evaluation of TAO, because it can be performed
quickly and with a high degree of confidence. High reflectivity and
enlargement of the extraocular muscles are assessed easily, and
serial ultrasonographic examinations can also be used to assess
progression or stability of the ophthalmopathy. Based on the
technologies currently available, or that will become available in
the future, one of skill in the art would be capable of determining
the best modality for diagnosing and evaluating the extent of
proptosis or exophthalmos.
[0053] As used herein, the term "antibody" refers to any form of
antibody that exhibits the desired biological activity. Thus, it is
used in the broadest sense and specifically covers, but is not
limited to, monoclonal antibodies (including full length monoclonal
antibodies), polyclonal antibodies, multispecific antibodies (e.g.,
bispecific antibodies), humanized, fully human antibodies, chimeric
antibodies and camelized single domain antibodies. "Parental
antibodies" are antibodies obtained by exposure of an immune system
to an antigen prior to modification of the antibodies for an
intended use, such as humanization of an antibody for use as a
human therapeutic antibody.
[0054] As used herein, unless otherwise indicated, "antibody
fragment" or "antigen binding fragment" refers to antigen binding
fragments of antibodies, i.e. antibody fragments that retain the
ability to bind specifically to the antigen bound by the
full-length antibody, e.g. fragments that retain one or more CDR
regions. Examples of antibody binding fragments include, but are
not limited to, Fab, Fab', F(ab').sub.2, and Fv fragments;
diabodies; linear antibodies; single-chain antibody molecules,
e.g., sc-Fv; nanobodies and multispecific antibodies formed from
antibody fragments.
[0055] A "Fab fragment" is comprised of one light chain and the
C.sub.H1 and variable regions of one heavy chain. The heavy chain
of a Fab molecule cannot form a disulfide bond with another heavy
chain molecule.
[0056] An "Fc" region contains two heavy chain fragments comprising
the C.sub.H1 and C.sub.H2 domains of an antibody. The two heavy
chain fragments are held together by two or more disulfide bonds
and by hydrophobic interactions of the C.sub.H3 domains.
[0057] In some embodiments, the antibodies, or antigen fragments
herein, comprise a Fc region. In some embodiments, the Fc region
comprises a mutation that extends the half-life of the antibody
when linked to the Fc region. In some embodiments, the Fc region
comprises a S228P, L235E, M252Y, S254T, T256E, M428L, N434S, L234F,
P331S mutation, or any combination thereof. In some embodiments,
the Fc region comprises a M252Y, S254T, and T256E mutations. A
non-limiting example of a Fc region comprising the M252Y, S254T,
and T256E mutations (collectively, "YTE Mutations") can be found in
a sequence of SEQ ID NO: 89. In some embodiments, the Fc region
comprising the YTE Mutations comprises a sequence of SEQ ID NO: 90,
which differs from SEQ ID NO: 89 by the presence of a C-terminal
lysine (K) residue. The numbering of the Fc region can be according
to the Kabat numbering system for the Fc region.
[0058] In some embodiments, the Fc region comprises a S228P and a
L235E mutation. In some embodiments, the antibody comprises a
L234F, L235E, and P331S mutation. In some embodiments, the Fc
region comprises M252Y, S254T, T256E, S228P and L235E mutations. In
some embodiments, the Fc region comprises S228P, L235E, M428L, and
N434S mutations. In some embodiments, the Fc region comprises the
M428L and N434S mutations. In some embodiments, the Fc region
comprises the L234F, L235E, P331S, M252Y, S254T, and T256E
mutations. Mutations in the Fc region are also described in
US2007041972A1, EP2235059B1, U.S. Pat. No. 8,394,925, and Mueller
et al, Mol Immunol 1997 April; 34(6):441-52, each of which is
incorporated by reference in its entirety. The numbering referenced
herein refers to the Kabat numbering system for the Fc region.
[0059] In some embodiments, the Fc region comprises the sequence
selected from:
TABLE-US-00001 (SEQ ID NO: 75)
APELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS-
VLT
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW-
ESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK;
(SEQ ID NO: 76)
APELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS-
VLT
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW-
ESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK; or
(SEQ ID NO: 77)
APPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSV-
LTV
VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE-
SNG QPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPG;
or (SEQ ID NO: 84)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS-
SLG
TQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLYITREPEVTCVVVDVSH-
EDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE-
PQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV-
ESC SVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 87)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS-
SLG
TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH-
EDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE-
PQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV-
ESC SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 88)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS-
SLG
TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH-
EDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE-
PQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV-
ESC SVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 89)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS-
SLG
TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSH-
EDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE-
PQV
YTLPPSRDELTKNOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNV-
ESC SVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 90)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS-
SLG
TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSH-
EDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE-
PQV
YTLPPSRDELTKNOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNV-
ESC SVMHEALHNHYTQKSLSLSPGK
[0060] A "Fab' fragment" contains one light chain and a portion or
fragment of one heavy chain that contains the VH domain and the
C.sub.H1 domain and also the region between the C.sub.H1 and
C.sub.H2 domains, such that an interchain disulfide bond can be
formed between the two heavy chains of two Fab' fragments to form a
F(ab') 2 molecule.
[0061] A "F(ab').sub.2 fragment" contains two light chains and two
heavy chains containing a portion of the constant region between
the C.sub.H1 and C.sub.H.sup.2 domains, such that an interchain
disulfide bond is formed between the two heavy chains. A
F(ab').sub.2 fragment thus is composed of two Fab' fragments that
are held together by a disulfide bond between the two heavy
chains.
[0062] The "Fv region" comprises the variable regions from both the
heavy and light chains, but lacks the constant regions.
[0063] The term "single-chain Fv" or "scFv" antibody refers to
antibody fragments comprising the V.sub.H and V.sub.L domains of an
antibody, wherein these domains are present in a single polypeptide
chain. Generally, the Fv polypeptide further comprises a
polypeptide linker between the V.sub.H and V.sub.L domains which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv, see Pluckthun (1994) THE PHARMACOLOGY OF
MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds.
Springer-Verlag, New York, pp. 269-315. See also, International
Patent Application Publication No. WO 88/01649 and U.S. Pat. Nos.
4,946,778 and 5,260,203.
[0064] A "domain antibody" is an immunologically functional
immunoglobulin fragment containing only the variable region of a
heavy chain or the variable region of a light chain. In some
instances, two or more V.sub.H regions are covalently joined with a
peptide linker to create a bivalent domain antibody. The two
V.sub.H regions of a bivalent domain antibody may target the same
or different antigens.
[0065] A "bivalent antibody" comprises two antigen binding sites.
In some instances, the two binding sites have the same antigen
specificities. However, bivalent antibodies may be bispecific (see
below).
[0066] In certain embodiments, monoclonal antibodies herein also
include camelized single domain antibodies. See, e.g., Muyldermans
et al. (2001) Trends Biochem. Sci. 26:230; Reichmann et al. (1999)
J. Immunol. Methods 231:25; WO 94/04678; WO 94/25591; U.S. Pat. No.
6,005,079). In one embodiment, the present invention provides
single domain antibodies comprising two V.sub.H domains with
modifications such that single domain antibodies are formed.
[0067] As used herein, the term "diabodies" refers to small
antibody fragments with two antigen-binding sites, which fragments
comprise a heavy chain variable domain (V.sub.H) connected to a
light chain variable domain (V.sub.L) in the same polypeptide chain
(V.sub.H-V.sub.L or V.sub.L-V.sub.H). By using a linker that is too
short to allow pairing between the two domains on the same chain,
the domains are forced to pair with the complementary domains of
another chain and create two antigen-binding sites. Diabodies are
described more fully in, e.g., EP 404,097; WO 93/11161; and
Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448.
For a review of engineered antibody variants generally see Holliger
and Hudson (2005) Nat. Biotechnol. 23:1126-1136.
[0068] Typically, a variant antibody or antigen binding fragment of
the antibodies provided herein retain at least 10% of its IGF-1R
binding activity (when compared to a parental antibody that is
modified) when that activity is expressed on a molar basis. In some
embodiments, a variant antibody (or antigen fragment thereof), or
antigen binding fragment of an antibody provided herein, retains at
least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the IGF-1R
binding affinity as the parental antibody. As described herein, it
is also intended that an antibody or antigen binding fragment of
the invention can include conservative or non-conservative amino
acid substitutions, which can also be referred to as "conservative
variants" or "function conserved variants" of the antibody, that do
not substantially alter its biologic activity.
[0069] "Isolated antibody" refers to the purification status of a
binding compound and in such context means the molecule is
substantially free of other biological molecules such as nucleic
acids, proteins, lipids, carbohydrates, or other material such as
cellular debris and growth media. Generally, the term "isolated" is
not intended to refer to a complete absence of such material or to
an absence of water, buffers, or salts, unless they are present in
amounts that substantially interfere with experimental or
therapeutic use of the binding compound as described herein.
[0070] The term "monoclonal antibody", as used herein, refers to
population of substantially homogeneous antibodies, i.e., the
antibody molecules comprising the population are identical in amino
acid sequence except for possible naturally occurring mutations
that may be present in minor amounts. In contrast, conventional
(polyclonal) antibody preparations typically include a multitude of
different antibodies having different amino acid sequences in their
variable domains, particularly their CDRs, that are often specific
for different epitopes. The modifier "monoclonal" indicates the
character of the antibody as being obtained from a substantially
homogeneous population of antibodies, and is not to be construed as
requiring production of the antibody by any particular method. For
example, the monoclonal antibodies to be used in accordance with
the present invention may be made by the hybridoma method first
described by Kohler et al. (1975) Nature 256: 495, or may be made
by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).
The "monoclonal antibodies" may also be isolated from phage
antibody libraries using the techniques described in Clackson et
al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol.
Biol. 222: 581-597, for example. See also Presta (2005) J. Allergy
Clin. Immunol. 116:731.
[0071] As used herein, a "chimeric antibody" is an antibody having
the variable domain from a first antibody and constant domain from
a second antibody, where the first and second antibodies are from
different species. (U.S. Pat. No. 4,816,567; and Morrison et al.,
(1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855). Typically the
variable domains are obtained from an antibody from an experimental
animal (the "parental antibody"), such as a rodent, and the
constant domain sequences are obtained from human antibodies, so
that the resulting chimeric antibody will be less likely to elicit
an adverse immune response in a human subject than the parental
(e.g. rodent) antibody.
[0072] As used herein, the term "humanized antibody" refers to
forms of antibodies that contain sequences from both human and
non-human (e.g., murine, rat) antibodies. In general, the humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the hypervariable loops correspond to those of a non-human
immunoglobulin, and all or substantially all of the framework (FR)
regions are those of a human immunoglobulin sequence. The humanized
antibody may optionally comprise at least a portion of a human
immunoglobulin constant region (Fc).
[0073] The term "fully human antibody" refers to an antibody that
comprises human immunoglobulin protein sequences only. A fully
human antibody may contain murine carbohydrate chains if produced
in a mouse, in a mouse cell, or in a hybridoma derived from a mouse
cell. Similarly, "mouse antibody" refers to an antibody that
comprises mouse immunoglobulin sequences only. Alternatively, a
fully human antibody may contain rat carbohydrate chains if
produced in a rat, in a rat cell, or in a hybridoma derived from a
rat cell. Similarly, "rat antibody" refers to an antibody that
comprises rat immunoglobulin sequences only.
[0074] In general, the basic antibody structural unit comprises a
tetramer. Each tetramer includes two identical pairs of polypeptide
chains, each pair having one "light" (about 25 kDa) and one "heavy"
chain (about 50-70 kDa). The amino-terminal portion of each chain
includes a variable region of about 100 to 110 or more amino acids
primarily responsible for antigen recognition. The carboxy-terminal
portion of the heavy chain may define a constant region primarily
responsible for effector function. Typically, human light chains
are classified as kappa and lambda light chains. Furthermore, human
heavy chains are typically classified as mu, delta, gamma, alpha,
or epsilon, and define the antibody's isotype as IgM, IgD, IgG,
IgA, and IgE, respectively. Within light and heavy chains, the
variable and constant regions are joined by a "J" region of about
12 or more amino acids, with the heavy chain also including a "D"
region of about 10 more amino acids. See generally, Fundamental
Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.
(1989).
[0075] The variable regions of each light/heavy chain pair form the
antibody binding site. Thus, in general, an intact antibody has two
binding sites. Except in bifunctional or bispecific antibodies, the
two binding sites are, in general, the same.
[0076] Typically, the variable domains of both the heavy and light
chains comprise three hypervariable regions, also called
complementarity determining regions (CDRs), located within
relatively conserved framework regions (FR). The CDRs are usually
aligned by the framework regions, enabling binding to a specific
epitope. In general, from N-terminal to C-terminal, both light and
heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3,
CDR3 and FR4. The assignment of amino acids to each domain is,
generally, in accordance with the definitions of Sequences of
Proteins of Immunological Interest, Kabat, et al.; National
Institutes of Health, Bethesda, Md.; 5.sup.th ed.; NIH Publ. No.
91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et
al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J
Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature
342:878-883.
[0077] As used herein, the term "hypervariable region" refers to
the amino acid residues of an antibody that are responsible for
antigen-binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR" (i.e.
residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the
light chain variable domain and residues 31-35 (CDRH1), 50-65
(CDRH2) and 95-102 (CDRH3) in the heavy chain variable domain;
Kabat et al. (1991) Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md.) and/or those residues from a "hypervariable
loop" (i.e. residues 26-32 (CDRL1), 50-52 (CDRL2) and 91-96 (CDRL3)
in the light chain variable domain and 26-32 (CDRH1), 53-55 (CDRH2)
and 96-101 (CDRH3) in the heavy chain variable domain; Chothia and
Lesk (1987) J. Mol. Biol. 196: 901-917). As used herein, the term
"framework" or "FR" residues refers to those variable domain
residues other than the hypervariable region residues defined
herein as CDR residues. CDRs provide the majority of contact
residues for the binding of the antibody to the antigen or epitope.
CDRs of interest can be derived from donor antibody variable heavy
and light chain sequences, and include analogs of the naturally
occurring CDRs, which analogs also share or retain the same antigen
binding specificity and/or neutralizing ability as the donor
antibody from which they were derived.
[0078] Additionally, in some embodiments, the antibodies can take
the form of a full length antibody, single-domain antibody, a
recombinant heavy-chain-only antibody (VHH), a single-chain
antibody (scFv), a shark heavy-chain-only antibody (VNAR), a
microprotein (cysteine knot protein, knottin), a DARPin; a
Tetranectin; an Affibody; a Transbody; an Anticalin; an AdNectin;
an Affilin; a Microbody; a peptide aptamer; an alterase; a plastic
antibody; a phylomer; a stradobody; a maxibody; an evibody; a
fynomer, an armadillo repeat protein, a Kunitz domain, an avimer,
an atrimer, a probody, an immunobody, a triomab, a troybody; a
pepbody; a vaccibody, a UniBody; Affimers, a DuoBody, a Fv, a Fab,
a Fab', a F(ab')2, a peptide mimetic molecule, or a synthetic
molecule, as described in US Patent Nos. or Patent Publication Nos.
U.S. Pat. No. 7,417,130, US 2004/132094, U.S. Pat. No. 5,831,012,
US 2004/023334, U.S. Pat. Nos. 7,250,297, 6,818,418, US
2004/209243, U.S. Pat. Nos. 7,838,629, 7,186,524, 6,004,746,
5,475,096, US 2004/146938, US 2004/157209, U.S. Pat. Nos.
6,994,982, 6,794,144, US 2010/239633, U.S. Pat. No. 7,803,907, US
2010/119446, and/or U.S. Pat. No. 7,166,697, the contents of each
of which are hereby incorporated by reference in their entireties.
See also, Storz MAbs. 2011 May-June; 3(3): 310-317, which is hereby
incorporated by reference.
[0079] The term "antigen" as used herein means any molecule that
has the ability to generate antibodies either directly or
indirectly or that binds to antibody. Included within the
definition of "antigen" is a protein-encoding nucleic acid. An
"antigen" can also refer to the binding partner of an antibody. In
some embodiments, the antigen is the IGF-1R protein expressed on
the surface of a cell. In some embodiments, the cell is an intact
cell. An intact cell is a cell that has not been lysed or broken
open with the use of detergents or other reagents. A cell that has
been treated with detergents or other reagents that breaks up the
cellular membrane or punches holes in a cellular membrane is not an
intact cell. For example, methods are provided herein for
generating an antibody that binds to a IGF-1R protein, the method
comprising culturing a cell comprising a nucleic acid molecule
encoding the IGF-1R antibody.
[0080] As used herein, "specific binding" or "immunospecific
binding" or "binds immuno specifically" refer to antibody binding
to a predetermined antigen (e.g. IGF-1R) or epitope present on the
antigen. In some embodiments, the antibody binds with a
dissociation constant (K.sub.D) of 10.sup.-7 M or less, and binds
to the predetermined antigen with a K.sub.D that is at least
two-fold less than its K.sub.D for binding to a non-specific
antigen (e.g., BSA, casein, or another non-specific polypeptide)
other than the predetermined antigen. The phrases "an antibody
recognizing IGF-1R" and "an antibody specific for IGF-1R" are used
interchangeably herein with the term "an antibody which binds
immunospecifically to IGF-1R." Reference in the present disclosure
may be made to IGF-1R. The degree of specificity necessary for an
anti-IGF-1R antibody may depend on the intended use of the
antibody, and at any rate is defined by its suitability for use for
an intended purpose. In some embodiments, the antibody, or binding
compound derived from the antigen-binding site of an antibody, of
the contemplated method binds to its antigen (IGF-1R), with an
affinity that is at least two fold greater, at least ten times
greater, at least 20-times greater, or at least 100-times greater
than the affinity with any other antigen.
[0081] Methods for determining mAb specificity and affinity by
competitive inhibition can be found in Harlow, et al., Antibodies:
A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1988), Colligan et al., eds., Current
Protocols in Immunology, Greene Publishing Assoc. and Wiley
Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589
601 (1983), which references are entirely incorporated herein by
reference.
[0082] The term "homolog" means protein sequences having between
40% and 100% sequence homology or identity to a reference sequence.
Percent identity between two peptide chains can be determined by
pair wise alignment using the default settings of the AlignX module
of Vector NTI v.9.0.0 (Invitrogen Corp., Carslbad, Calif.). In some
embodiments, the antibody, or antigenic binding fragment thereof
has, at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98,
or 99% homology or identity to a sequence described herein. In some
embodiments, the antibody has conservative substitutions as
compared to a sequence described herein. Exemplary conservative
substitutions are illustrated in Table 1 and are encompassed within
the scope of the disclosed subject matter. The conservative
substitution may reside in the framework regions, or in
antigen-binding sites, as long they do not adversely affect the
properties of the antibody. Substitutions may be made to improve
antibody properties, for example stability or affinity.
Conservative substitutions will produce molecules having functional
and chemical characteristics similar to those molecules into which
such modifications are made. Exemplary amino acid substitutions are
shown in the table below.
TABLE-US-00002 Table: Exemplary Conservative Substitutions:
Original Residue Exemplary Conservative Substitutions Ala Val, Leu,
Ile Arg Lys, Gln, Asn Asn Gln Asp Glu Cys Ser, Ala Gln Asn Gly
Pro,Ala His Asn, Gln, Lys, Arg Ile Leu, Val, Met, Ala, Phe Leu Ile,
Val, Met, Ala, Phe Lys Arg, Gln, Asn Met Leu, Phe, Ile Phe Leu,
Val, Ile, Ala, Tyr Pro Ala Ser Thr, Ala, Cys Thr Ser Trp Tyr, Phe
Tyr Trp, Phe, Thr, Ser Val Ile, Met, Leu, Phe, Ala
[0083] In some embodiments, variants of the proteins and peptides
provided herein are provided. In some embodiments, a variant
comprises a substitution, deletions, or insertion. In some
embodiments, the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
(e.g., 1-10) substitutions. As described herein, the substitutions
can be conservative substitutions. In some embodiments, the
substitution is non-conservative. In some embodiments, the variant
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 1-10) deletions.
In some embodiments, the variant comprises 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10 (e.g., 1-10) insertions. In some embodiments, the
substitutions, deletions, or insertions are present in the CDRs
provided for herein. In some embodiments, the substitutions,
deletions, or insertions are not present in the CDRs provided for
herein.
[0084] The term "in combination with" as used herein means that the
described agents can be administered to an animal or subject
together in a mixture, concurrently as single agents or
sequentially as single agents in any order.
[0085] The techniques to raise antibodies to small peptide
sequences that recognize and bind to those sequences in the free or
conjugated form or when presented as a native sequence in the
context of a large protein are well known in the art. Such
antibodies include murine, murine-human and human-human antibodies
produced by hybridoma or recombinant techniques known in the art.
Antibodies can also be produced in human, a mouse, sheep, a rat, a
rabbit, a shark, a llama, or a chicken. In some embodiments, the
antibody is produced in a chicken. The antibodies can also be
produced in or other small animals.
[0086] The term "epitope" is meant to refer to that portion of any
molecule capable of being recognized by and bound by an antibody at
one or more of the Ab's antigen binding regions. Epitopes usually
consist of chemically active surface groupings of molecules such as
amino acids or sugar side chains and have specific three
dimensional structural characteristics as well as specific charge
characteristics. Example of epitopes include, but are not limited
to, the residues described herein that form IGF-1R epitopes. In
some embodiments, the epitope is only present in a non-denatured
protein. In some embodiments, the epitope is only present in a
denatured protein.
[0087] In some embodiments, the source for the DNA encoding a
non-human antibody include cell lines which produce antibody, such
as hybrid cell lines commonly known as hybridomas.
[0088] The hybrid cells are formed by the fusion of a non-human
antibody-producing cell, typically a spleen cell of an animal
immunized against either natural or recombinant antigen, or a
peptide fragment of the antigen protein sequence. Alternatively,
the non-human antibody-producing cell can be a B lymphocyte
obtained from the blood, spleen, lymph nodes or other tissue of an
animal immunized with the antigen.
[0089] The second fusion partner, which provides the immortalizing
function, can be a lymphoblastoid cell or a plasmacytoma or myeloma
cell, which is not itself an antibody producing cell, but is
malignant. Fusion partner cells include, but are not limited to,
the hybridoma SP2/0-Ag14, abbreviated as SP2/0 (ATCC CRL1581) and
the myeloma P3X63Ag8 (ATCC TIB9), or its derivatives. See, e.g.,
Ausubel infra, Harlow infra, and Colligan infra, the contents of
which references are incorporated entirely herein by reference.
[0090] The antibodies can be generated according the examples
provided herein. Once the sequences are known, the antibodies can
also be generated according to known methods. The antibodies can
also be converted to different types, such as being converted to
Human IgGs and the like. By converting the antibodies to a human
antibody, a human subject should not identify the antibodies as
foreign. The conversion of a non-human IgG antibody to a human IgG
antibody is well known and can routinely be done once the native
sequence is known. As discussed herein, the antibodies can be
modified according to known methods. Such methods are described in,
for example, Riechmann L, Clark M, Waldmann H, Winter G (1988).
Reshaping human antibodies for therapy". Nature 332 (6162):
332-323; Tsurushita N, Park M, Pakabunto K, Ong K, Avdalovic A, Fu
H, Jia A, Vasquez M, Kumar S. (2004). The antibody-producing cell
contributing the nucleotide sequences encoding the antigen-binding
region of the chimeric antibody can also be produced by
transformation of a non-human, such as a primate, or a human cell.
For example, a B lymphocyte which produces the antibody can be
infected and transformed with a virus such as Epstein-Barr virus to
yield an immortal antibody producing cell (Kozbor et al., Immunol.
Today 4:72 79 (1983)). Alternatively, the B lymphocyte can be
transformed by providing a transforming gene or transforming gene
product, as is well-known in the art. See, e.g., Ausubel infra,
Harlow infra, and Colligan infra, the contents of which references
are incorporated entirely herein by reference. The cell fusions are
accomplished by standard procedures well known to those skilled in
the field of immunology. Fusion partner cell lines and methods for
fusing and selecting hybridomas and screening for mAbs are well
known in the art. See, e.g., Ausubel infra, Harlow infra, and
Colligan infra, the contents of which references are incorporated
entirely herein by reference.
[0091] In some embodiments, the antibody is a MAb which binds to
IGF-1R. In some embodiments, the antibody binds to amino acids of
an epitope of the IGF-1R.
[0092] In some embodiments, the antibody comprises a sequence as
provided for herein.
[0093] The sequences of the antibodies can be modified to yield
human IgG antibodies. The conversion of the sequences provided
herein can be modified to yield other types of antibodies. The CDRs
can also be linked to other antibodies, proteins, or molecules to
create antibody fragments that bind to IGF-1R. This can be in the
form of an antibody drug conjugate ("ADC"), a multi-specific
molecule, or a chimeric antigen receptor. The CDRs and antibody
sequences provided herein also be humanized or made fully human
according to known methods. The sequences can also be made into
chimeric antibodies as described herein.
[0094] In some embodiments, the antibody comprises an amino acid
sequence comprising a sequence provided for herein or a fragment
thereof. In some embodiments, the antibody comprises one or more
amino acid sequences as provided herein, an antigen binding
fragments, thereof, or a human IgG variant thereof. "A human IgG
variant thereof" refers to an antibody that has been modified to be
a human IgG when the starting antibody is not a human IgG
antibody.
[0095] As described herein the production of antibodies with a
known sequence is routine and can be done by any method.
Accordingly, in some embodiments, a nucleic acid encoding an
antibody or fragment thereof is provided. In some embodiments, the
nucleic acid encodes a sequence provided for herein. The antibodies
can also be modified to be chimeric antibodies or human antibodies.
The antibodies can also be used in injectable pharmaceutical
compositions. As also described herein, the antibodies can be
isolated antibodies or engineered antibodies.
[0096] In some embodiments, "derivatives" of the antibodies,
fragments, regions or derivatives thereof, which term includes
those proteins encoded by truncated or modified genes to yield
molecular species functionally resembling the immunoglobulin
fragments are provided. The modifications include, but are not
limited to, addition of genetic sequences coding for cytotoxic
proteins such as plant and bacterial toxins. The modification can
also include a reporter protein, such as a fluorescent or
chemiluminescent tag. The fragments and derivatives can be produced
in any manner.
[0097] The identification of these antigen binding region and/or
epitopes recognized by Abs described herein provide the information
necessary to generate additional monoclonal antibodies with similar
binding characteristics and therapeutic or diagnostic utility that
parallel the embodiments of this application.
[0098] The nucleic acid sequence encoding an antibody described
herein can be genomic DNA or cDNA, or RNA (e.g. mRNA) which encodes
at least one of the variable regions described herein. A convenient
alternative to the use of chromosomal gene fragments as the source
of DNA encoding the V region antigen-binding segment is the use of
cDNA for the construction of chimeric immunoglobulin genes, e.g.,
as reported by Liu et al. (Proc. Natl. Acad. Sci., USA 84:3439
(1987) and J. Immunology 139:3521 (1987), which references are
hereby entirely incorporated herein by reference. The use of cDNA
requires that gene expression elements appropriate for the host
cell be combined with the gene in order to achieve synthesis of the
desired protein. The use of cDNA sequences is advantageous over
genomic sequences (which contain introns), in that cDNA sequences
can be expressed in bacteria or other hosts which lack appropriate
RNA splicing systems.
[0099] For example, a cDNA encoding a V region antigen-binding
segment able to detect, bind, to or neutralize a IGF-1R antigen can
be provided using known methods based on the use of the amino acid
sequences provided herein. Because the genetic code is degenerate,
more than one codon can be used to encode a particular amino acid
(Watson, et al., infra). Using the genetic code, one or more
different oligonucleotides can be identified, each of which would
be capable of encoding the amino acid. The probability that a
particular oligonucleotide will, in fact, constitute the actual
XXX-encoding sequence can be estimated by considering abnormal base
pairing relationships and the frequency with which a particular
codon is actually used (to encode a particular amino acid) in
eukaryotic or prokaryotic cells expressing an antibody or fragment.
Such "codon usage rules" are disclosed by Lathe, et al., J. Molec.
Biol. 183:1 12 (1985). Using the "codon usage rules" of Lathe, a
single oligonucleotide, or a set of oligonucleotides, that contains
a theoretical "most probable" nucleotide sequence capable of
encoding an antibody variable or constant region sequences is
identified.
[0100] The variable regions described herein can be combined with
any type of constant region including a human constant region or
murine constant region. Human genes which encode the constant (C)
regions of the antibodies, fragments and regions can be derived
from a human fetal liver library, by known methods. Human C regions
genes can be derived from any human cell including those which
express and produce human immunoglobulins. The human C.sub.H region
can be derived from any of the known classes or isotypes of human H
chains, including gamma, .mu., .alpha., .delta. or .epsilon., and
subtypes thereof, such as G1, G2, G3 and G4. Since the H chain
isotype is responsible for the various effector functions of an
antibody, the choice of C.sub.H region will be guided by the
desired effector functions, such as complement fixation, or
activity in antibody-dependent cellular cytotoxicity (ADCC).
Preferably, the C.sub.H region is derived from gamma 1 (IgG1),
gamma 3 (IgG3), gamma 4 (IgG4), or .mu. (IgM). The human C.sub.L
region can be derived from either human L chain isotype, kappa or
lambda. In some embodiments, the antibody comprises a Fc domain. In
some embodiments, the Fc domain comprises a mutation to extend the
half-life of the antibody. In some embodiments, the Fc domain
comprises a mutation such as those described in U.S. Pat. No.
7,670,600, which is hereby incorporated by reference in its
entirety. In some embodiment, the constant region comprises a
mutation at position at amino acid residue 428 relative to a
wild-type human IgG constant domain, numbered according to the EU
numbering index of Kabat. Without being bound to any particular
theory, an antibody comprising a mutation that corresponds to
residue 428 can have an increased half-life compared to the
half-life of an IgG having the wild-type human IgG constant domain.
In some embodiments, the mutation is a substitution of the native
residue with a threonine, leucine, phenylalanine or serine. In some
embodiments, the antibody further comprises one or more amino acid
substitutions relative to the corresponding wild-type human IgG
constant domain at one or more of amino acid residues 251-256,
285-290, 308-314, 385-389, and 429-436, numbered according to the
Kabat EU numbering index. The specific mutations or substitutions
at these positions are described in U.S. Pat. No. 7,670,600, which
is hereby incorporated by reference in its entirety.
[0101] Genes encoding human immunoglobulin C regions can be
obtained from human cells by standard cloning techniques (Sambrook,
et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et
al., eds. Current Protocols in Molecular Biology (1987 1993)).
Human C region genes are readily available from known clones
containing genes representing the two classes of L chains, the five
classes of H chains and subclasses thereof. Chimeric antibody
fragments, such as F(ab').sub.2 and Fab, can be prepared by
designing a chimeric H chain gene which is appropriately truncated.
For example, a chimeric gene encoding an H chain portion of an
F(ab').sub.2 fragment would include DNA sequences encoding the
CH.sub.1 domain and hinge region of the H chain, followed by a
translational stop codon to yield the truncated molecule.
[0102] In some embodiments, the antibodies, murine, human,
humanized, or chimeric antibodies, fragments and regions of the
antibodies described herein are produced by cloning DNA segments
encoding the H and L chain antigen-binding regions of a IGF-1R
antigen specific antibody, and joining these DNA segments to DNA
segments encoding C.sub.H and C.sub.L regions, respectively, to
produce murine, human or chimeric immunoglobulin-encoding
genes.
[0103] Thus, in some embodiments, a fused chimeric gene is created
which comprises a first DNA segment that encodes at least the
antigen-binding region of non-human origin, such as a functionally
rearranged V region with joining (J) segment, linked to a second
DNA segment encoding at least a part of a human C region.
[0104] Therefore, cDNA encoding the antibody V and C regions, the
method of producing the antibody according to some of the
embodiments described herein involve several steps, as exemplified
below: 1. isolation of messenger RNA (mRNA) from the cell line
producing an anti-IGF-1R antigen antibody and from optional
additional antibodies supplying heavy and light constant regions;
cloning and cDNA production therefrom; 2. preparation of a full
length cDNA library from purified mRNA from which the appropriate V
and/or C region gene segments of the L and H chain genes can be:
(i) identified with appropriate probes, (ii) sequenced, and (iii)
made compatible with a C or V gene segment from another antibody
for a chimeric antibody; 3. Construction of complete H or L chain
coding sequences by linkage of the cloned specific V region gene
segments to cloned C region gene, as described above; 4. Expression
and production of L and H chains in selected hosts, including
prokaryotic and eukaryotic cells to provide murine-murine,
human-murine, human-human or human murine antibodies.
[0105] Two coding DNA sequences are said to be "operably linked" if
the linkage results in a continuously translatable sequence without
alteration or interruption of the triplet reading frame. A DNA
coding sequence is operably linked to a gene expression element if
the linkage results in the proper function of that gene expression
element to result in expression of the coding sequence.
[0106] As used herein and unless otherwise indicated, the term
"about" is intended to mean.+-.5% of the value it modifies. Thus,
about 100 means 95 to 105.
[0107] In some embodiments, the antibodies described herein are
used to detect the presence of the antigen. The present antibody
can be used in any device or method to detect the presence of the
antigen.
[0108] The term "purified" with referenced to an antibody refers to
an antibody that is substantially free of other material that
associates with the molecule in its natural environment. For
instance, a purified protein is substantially free of the cellular
material or other proteins from the cell or tissue from which it is
derived. The term refers to preparations where the isolated protein
is sufficiently pure to be analyzed, or at least 70% to 80% (w/w)
pure, at least 80%-90% (w/w) pure, 90-95% pure; and, at least 95%,
96%, 97%, 98%, 99%, or 100% (w/w) pure. In some embodiments, the
antibody is purified.
[0109] As an alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal antibody to a polypeptide may be
identified and isolated by screening a recombinant combinatorial
immunoglobulin library (e.g., an antibody phage display library)
with a polypeptide described herein to thereby isolate
immunoglobulin library members that bind to the polypeptide.
Techniques and commercially available kits for generating and
screening phage display libraries are well known to those skilled
in the art. Additionally, examples of methods and reagents
particularly amenable for use in generating and screening antibody
or antigen binding protein display libraries can be found in the
literature. Thus, the epitopes described herein can be used to
screen for other antibodies that can be used therapeutically,
diagnostically, or as research tools.
[0110] Antibody Conjugates
[0111] The antibodies provided for herein may also be conjugated to
a chemical moiety. The chemical moiety may be, inter alia, a
polymer, a radionuclide or a cytotoxic factor. In some embodiments,
this can be referred to as an antibody drug conjugate. In some
embodiments, the chemical moiety is a polymer which increases the
half-life of the antibody molecule in the body of a subject.
Suitable polymers include, but are not limited to, polyethylene
glycol (PEG) (e.g., PEG with a molecular weight of 2 kDa, 5 kDa, 10
kDa, 12 kDa, 20 kDa, 30 kDa or 40 kDa), dextran and
monomethoxypolyethylene glycol (mPEG). Lee, et al., (1999)
(Bioconj. Chem. 10:973-981) discloses PEG conjugated single-chain
antibodies. Wen, et al., (2001) (Bioconj. Chem. 12:545-553)
disclose conjugating antibodies with PEG which is attached to a
radiometal chelator (diethylenetriaminpentaacetic acid (DTPA)).
Examples of chemical moieties include, but are not limited to,
anti-mitotics, such as calicheamicins (e.g. ozogamicin), monomethyl
auristatin E, mertansine, and the like. Other examples include, but
are not limited to, biologically active anti-microtubule agents,
alkylating agents and DNA minor groove binding agents. Other
examples of are provided herein and below. The chemical moiety can
be linked to the antibody through a linking group (maleimide), a
cleaveble linker, such as a cathepsin cleavable linkers
(valine-citrulline), and in some embodiments, one or more spacers
(e.g. para-aminobenzylcarbamate). Without being bound to any
particular theory, once the antibody conjugate binds IGF-1R it can
be internalized and the chemical moiety can kill the cell or
otherwise inhibit its growth. In some embodiments, the cell is a
thyroid cell.
[0112] The antibodies and antibody fragments of the invention may
also be conjugated with labels such as .sup.99Tc, .sup.90Y,
.sup.111In, .sup.32P, .sup.14C, .sup.125I, .sup.3H, .sup.131I,
.sup.11C, .sup.15O, .sup.13N, .sup.18F, .sup.35S, .sup.51Cr,
.sup.57To, .sup.226Ra, .sup.60Co, .sup.59Fe, .sup.57Se, .sup.152Eu,
.sup.67CU, .sup.217Ci, .sup.211At, .sup.212Pb, .sup.47Sc,
.sup.109Pd, .sup.234Th, and .sup.40K, .sup.157Gd, .sup.55Mn,
.sup.52Tr and .sup.56Fe.
[0113] The antibodies and antibody fragments may also be conjugated
with fluorescent or chemiluminescent labels, including fluorophores
such as rare earth chelates, fluorescein and its derivatives,
rhodamine and its derivatives, isothiocyanate, phycoerythrin,
phycocyanin, allophycocyanin, o-phthaladehyde, fluorescamine,
.sup.152Eu, dansyl, umbelliferone, luciferin, luminal label,
isoluminal label, an aromatic acridinium ester label, an imidazole
label, an acridimium salt label, an oxalate ester label, an
aequorin label, 2,3-dihydrophthalazinediones, biotin/avidin, spin
labels and stable free radicals.
[0114] The antibody molecules may also be conjugated to a cytotoxic
factor such as diptheria toxin, Pseudomonas aeruginosa exotoxin A
chain, ricin A chain, abrin A chain, modeccin A chain,
alpha-sarcin, Aleurites fordii proteins and compounds (e.g., fatty
acids), dianthin proteins, Phytoiacca americana proteins PAPI,
PAPII, and PAP-S, Momordica charantia inhibitor, curcin, crotin,
Saponaria officinalis inhibitor, mitogellin, restrictocin,
phenomycin, and enomycin.
[0115] Any method known in the art for conjugating the antibody
molecules of the invention to the various moieties may be employed,
including those methods described by Hunter, et al., (1962) Nature
144:945; David, et al., (1974) Biochemistry 13:1014; Pain, et al.,
(1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem.
and Cytochem. 30:407. Methods for conjugating antibodies are
conventional and very well known in the art.
[0116] Chimeric Antigen Receptors
[0117] The antibodies provided herein can also be incorporated into
a chimeric antigen receptor ("CAR") that can be used, for example,
in a CAR-T cell. In some embodiments, the extracellular domain of
the CAR can be an antibody as provided for herein. In some
embodiments, the antibody is in a scFv format. CAR-T cells are a
type of treatment in which a patient's T cells are modified so they
will attack the cells that are expressing IGF-1R. T cells are taken
from a patient's blood. Then the gene for a special receptor that
binds to a certain protein on the patient's cells is added in the
laboratory. In some embodiments, the receptor binds to IGF-1R using
the binding regions of the antibodies provided for herein. The
CAR-T cells comprising the IGF-1R antibody can then be used to
treat a condition, such as those provided for herein.
[0118] In some embodiments, antibodies (e.g. an anti-IGF-1R
antibody) are provided herein. In some embodiments, the antibody is
a recombinant antibody that binds to a IGF-1R protein. In some
embodiments, the IGF-1R protein is a human IGF-1R protein. In some
embodiments, the IGF-1R protein that is recognized by the
antibodies is in its native conformation (non-denatured)
conformation. In some embodiments, the antibody does not
specifically binds to a denatured IGF-1R protein. As used herein,
the term "recombinant antibody" refers to an antibody that is not
naturally occurring. In some embodiments, the term "recombinant
antibody" refers to an antibody that is not isolated from a human
subject.
[0119] In some embodiments, the antibody comprises one or more
peptides having the following sequences, or a variant thereof:
TABLE-US-00003 AB ID NO. AB Sequence LC and HC LC Sequence HC
Sequence VRDN-03100 EIVLTQSPATLSLSPGERATLSC EIVLTQSPATLSLSP
QVELVESGGGVVQPGRSQRLSC RASQSVSSYLAWYQQKPGQAPRL GERATLSCRASQSVS
AASGFTFSSYGMHWVRQAPGKG LIYDASKRATGIPARFSGSGSGT SYLAWYQQKPGQAPR
LEWVAIIWFDGSSTYYADSVRG DFTLTISSLEPEDFAVYYCQQRS LLIYDASKRATGIPA
RFTISRDNSKNTLYLQMNSLRA KWPPWTFGQGTKVESKRTVAAPS RFSGSGSGTDFTLTI
EDTAVYFCARELGRRYFDLWGR VFIFPPSDEQLKSGTASVVCLLN SSLEPEDFAVYYCQQ
GTLVSVSSASTKGPSVFPLAPS NFYPREAKVQWKVDNALQSGNSQ RSKWPPWTFGQGTKV
SKSTSGGTAALGCLVKDYFPEP ESVTEQDSKDSTYSLSSTLTLSK ESKRTVAAPSVFIFP
VTVSWNSGALTSGVHTFPAVLQ ADYEKHKVYACEVTHQGLSSPVT PSDEQLKSGTASVVC
SSGLYSLSSVVTVPSSSLGTQT KSFNRGEC LLNNFYPREAKVQWK
YICNVNHKPSNTKVDKKVEPKS QVELVESGGGVVQPGRSQRLSCA VDNALQSGNSQESVT
CDKTHTCPPCPAPELLGGPSVF ASGFTFSSYGMHWVRQAPGKGLE EQDSKDSTYSLSSTL
LFPPKPKDTLMISRTPEVTCVV WVAIIWFDGSSTYYADSVRGRFT TLSKADYEKHKVYAC
VDVSHEDPEVKFNWYVDGVEVH ISRDNSKNTLYLQMNSLRAEDTA EVTHQGLSSPVTKSF
NAKTKPREEQYNSTYRVVSVLT VYFCARELGRRYFDLWGRGTLVS NRGEC (SEQ ID
VLHQDWLNGKEYKCKVSNKALP VSSASTKGPSVFPLAPSSKSTSG NO: 1)
APIEKTISKAKGQPREPQVYTL GTAALGCLVKDYFPEPVTVSWNS
PPSRDELTKNQVSLTCLVKGFY GALTSGVHTFPAVLQSSGLYSLS
PSDIAVEWESNGQPENNYKTTP SVVTVPSSSLGTQTYICNVNHKP
PVLDSDGSFFLYSKLTVDKSRW SNTKVDKKVEPKSCDKTHTCPPC
QQGNVFSCSVMHEALHNHYTQK PAPELLGGPSVFLFPPKPKDTLM SLSLSPGK (SEQ ID NO:
ISRTPEVTCVVVDVSHEDPEVKF 2) NWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPR
EPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID
NO: 65) VRDN-02100 DIVMTQSPLSLPVTPGEPASISC DIVMTQSPLSLPVTP
QVQLQESGPGLVKPSETLSLTC RSSQSIVHSNGNTYLQWYLQKPG GEPASISCRSSQSIV
TVSGYSITGGYLWNWIRQPPGK QSPQLLIYKVSNRLYGVPDRFSG HSNGNTYLQWYLQKP
GLEWIGYISYDGTNNYKPSLKD SGSGTDFTLKISRVEAEDVGVYY GQSPQLLIYKVSNRL
RVTISRDTSKNQFSLKLSSVTA CFQGSHVPWTFGQGTKVEIKRTV YGVPDRFSGSGSGTD
ADTAVYYCARYGRVFFDYWGQG AAPSVFIFPPSDEQLKSGTASVV FTLKISRVEAEDVGV
TLVTVSSASTKGPSVFPLAPSS CLLNNFYPREAKVQWKVDNALQS YYCFQGSHVPWTFGQ
KSTSGGTAALGCLVKDYFPEPV GNSQESVTEQDSKDSTYSLSSTL GTKVEIKRTVAAPSV
TVSWNSGALTSGVHTFPAVLQS TLSKADYEKHKVYACEVTHQGLS FIFPPSDEQLKSGTA
SGLYSLSSVVTVPSSSLGTQTY SPVTKSFNRGEC SVVCLLNNFYPREAK
ICNVNHKPSNTKVDKRVEPKSC QVQLQESGPGLVKPSETLSLTCT VQWKVDNALQSGNSQ
DKTHTCPPCPAPELLGGPSVFL VSGYSITGGYLWNWIRQPPGKGL ESVTEQDSKDSTYSL
FPPKPKDTLMISRTPEVTCVVV EWIGYISYDGTNNYKPSLKDRVT SSTLTLSKADYEKHK
DVSHEDPEVKFNWYVDGVEVHN ISRDTSKNQFSLKLSSVTAADTA VYACEVTHQGLSSPV
AKTKPREEQYNSTYRVVSVLTV VYYCARYGRVFFDYWGQGTLVTV TKSFNRGEC (SEQ
LHQDWLNGKEYKCKVSNKALPA SSASTKGPSVFPLAPSSKSTSGG ID NO: 3)
PIEKTISKAKGQPREPQVYTLP TAALGCLVKDYFPEPVTVSWNSG
PSREEMTKNQVSLTCLVKGFYP ALTSGVHTFPAVLQSSGLYSLSS
SDIAVEWESNGQPENNYKTTPP VVTVPSSSLGTQTYICNVNHKPS
VLDSDGSFFLYSKLTVDKSRWQ NTKVDKRVEPKSCDKTHTCPPCP
QGNVFSCSVMHEALHNHYTQKS APELLGGPSVFLFPPKPKDTLMI LSLSPGK (SEQ ID NO:
4) SRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID
NO: 66) VRDN-02200 SSELTQDPAVSVALGQTVRITCQ SSELTQDPAVSVALG
EVQLVQSGAEVKKPGSSVKVSC GDSLRSYYATWYQQKPGQAPILV QTVRITCQGDSLRSY
KASGGTFSSYAISWVRQAPGQG IYGENKRPSGIPDRFSGSSSGNT YATWYQQKPGQAPIL
LEWMGGIIPIFGTANYAQKFQG ASLTITGAQAEDEADYYCKSRDG VIYGENKRPSGIPDR
RVTITADKSTSTAYMELSSLRS SGQHLVFGGGTKLTVLGQPKAAP FSGSSSGNTASLTIT
EDTAVYYCARAPLRFLEWSTQD SVTLFPPSSEELQANKATLVCLI GAQAEDEADYYCKSR
HYYYYYMDVWGKGTTVTVSSAS SDFYPGAVTVAWKADSSPVKAGV DGSGQHLVFGGGTKL
TKGPSVFPLAPSSKSTSGGTAA ETTTPSKQSNNKYAASSYLSLTP TVLGQPKAAPSVTLF
LGCLVKDYFPEPVTVSWNSGAL EQWKSHRSYSCQVTHEGSTVEKT PPSSEELQANKATLV
TSGVHTFPAVLQSSGLYSLSSV VAPAECS CLISDFYPGAVTVAW
VTVPSSSLGTQTYICNVNHKPS EVQLVQSGAEVKKPGSSVKVSCK KADSSPVKAGVETTT
NTKVDKKVEPKSCDKTHTCPPC ASGGTFSSYAISWVRQAPGQGLE PSKQSNNKYAASSYL
PAPELLGGPSVFLFPPKPKDTL WMGGIIPIFGTANYAQKFQGRVT SLTPEQWKSHRSYSC
MISRTPEVTCVVVDVSHEDPEV ITADKSTSTAYMELSSLRSEDTA QVTHEGSTVEKTVAP
KFNWYVDGVEVHNAKTKPREEQ VYYCARAPLRFLEWSTQDHYYYY AECS (SEQ ID
YNSTYRVVSVLTVLHQDWLNGK YMDVWGKGTTVTVSSASTKGPSV NO: 5)
EYKCKVSNKALPAPIEKTISKA FPLAPSSKSTSGGTAALGCLVKD
KGQPREPQVYTLPPSREEMTKN YFPEPVTVSWNSGALTSGVHTFP
QVSLTCLVKGFYPSDIAVEWES AVLQSSGLYSLSSVVTVPSSSLG
NGQPENNYKTTPPVLDSDGSFF TQTYICNVNHKPSNTKVDKKVEP
LYSKLTVDKSRWQQGNVFSCSV KSCDKTHTCPPCPAPELLGGPSV MHEALHNHYTQKSLSLSPGK
FLFPPKPKDTLMISRTPEVTCVV (SEQ ID NO: 6) VDVSHEDPEVKFNWYVDGVEVHN
AKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPI
EKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 67) VRDN-02300
DIQMTQFPSSLSASVGDRVTITC DIQMTQFPSSLSASV EVQLLESGGGLVQPGGSLRLSC
RASQGIRNDLGWYQQKPGKAPKR GDRVTITCRASQGIR TASGFTFSSYAMNWVRQAPGKG
LIYAASRLHRGVPSRFSGSGSGT NDLGWYQQKPGKAPK LEWVSAISGSGGTTFYADSVKG
EFTLTISSLQPEDFATYYCLQHN RLIYAASRLHRGVPS RFTISRDNSRTTLYLQMNSLRA
SYPCSFGQGTKLEIKRTVAAPSV RFSGSGSGTEFTLTI EDTAVYYCAKDLGWSDSYYYYY
FIFPPSDEQLKSGTASVVCLLNN SSLQPEDFATYYCLQ GMDVWGQGTTVTVSSASTKGPS
FYPREAKVQWKVDNALQSGNSQE HNSYPCSFGQGTKLE VFPLAPCSRSTSESTAALGCLV
SVTEQDSKDSTYSLSSTLTLSKA IKRTVAAPSVFIFPP KDYFPEPVTVSWNSGALTSGVH
DYEKHKVYACEVTHQGLSSPVTK SDEQLKSGTASVVCL TFPAVLQSSGLYSLSSVVTVPS
SFNRGEC LNNFYPREAKVQWKV SNFGTQTYTCNVDHKPSNTKVD
EVQLLESGGGLVQPGGSLRLSCT DNALQSGNSQESVTE KTVERKCCVECPPCPAPPVAGP
ASGFTFSSYAMNWVRQAPGKGLE QDSKDSTYSLSSTLT SVFLFPPKPKDTLMISRTPEVT
WVSAISGSGGTTFYADSVKGRFT LSKADYEKHKVYACE CVVVDVSHEDPEVQFNWYVDGV
ISRDNSRTTLYLQMNSLRAEDTA VTHQGLSSPVTKSFN EVHNAKTKPREEQFNSTFRVVS
VYYCAKDLGWSDSYYYYYGMDVW RGEC (SEQ ID VLTVVHQDWLNGKEYKCKVSNK
GQGTTVTVSSASTKGPSVFPLAP NO: 7) GLPAPIEKTISKTKGQPREPQV
CSRSTSESTAALGCLVKDYFPEP YTLPPSREEMTKNQVSLTCLVK
VTVSWNSGALTSGVHTFPAVLQS GFYPSDIAVEWESNGQPENNYK
SGLYSLSSVVTVPSSNFGTQTYT TTPPMLDSDGSFFLYSKLTVDK
CNVDHKPSNTKVDKTVERKCCVE SRWQQGNVFSCSVMHEALHNHY
CPPCPAPPVAGPSVFLFPPKPKD TQKSLSLSPG (SEQ ID NO:
TLMISRTPEVTCVVVDVSHEDPE 8) VQFNWYVDGVEVHNAKTKPREEQ
FNSTFRVVSVLTVVHQDWLNGKE YKCKVSNKGLPAPIEKTISKTKG
QPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPMLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG (SEQ
ID NO: 68) VRDN-02400 DVVMTQSPLSLPVTPGEPASISC DVVMTQSPLSLPVTP
QVQLQESGPGLVKPSGTLSLTC RSSQSLLHSNGYNYLDWYLQKPG GEPASISCRSSQSLL
AVSGGSISSSNWWSWVRQPPGK QSPQLLIYLGSNRASGVPDRFSG HSNGYNYLDWYLQKP
GLEWIGEIYHSGSTNYNPSLKS SGSGTDFTLKISRVEAEDVGVYY GQSPQLLIYLGSNRA
RVTISVDKSKNQFSLKLSSVTA CMQGTHWPLTFGQGTKVEIKRTV SGVPDRFSGSGSGTD
ADTAVYYCARWTGRTDAFDIWG AAPSVFIFPPSDEQLKSGTASVV FTLKISRVEAEDVGV
QGTMVTVSSASTKGPSVFPLAP CLLNNFYPREAKVQWKVDNALQS YYCMQGTHWPLTFGQ
SSKSTSGGTAALGCLVKDYFPE GNSQESVTEQDSKDSTYSLSSTL GTKVEIKRTVAAPSV
PVTVSWNSGALTSGVHTFPAVL TLSKADYEKHKVYACEVTHQGLS FIFPPSDEQLKSGTA
QSSGLYSLSSVVTVPSSSLGTQ SPVTKSFNRGEC SVVCLLNNFYPREAK
TYICNVNHKPSNTKVDKKVEPK QVQLQESGPGLVKPSGTLSLTCA VQWKVDNALQSGNSQ
SCDKTHTCPPCPAPELLGGPSV VSGGSISSSNWWSWVRQPPGKGL ESVTEQDSKDSTYSL
FLFPPKPKDTLMISRTPEVTCV EWIGEIYHSGSTNYNPSLKSRVT SSTLTLSKADYEKHK
VVDVSHEDPEVKFNWYVDGVEV ISVDKSKNQFSLKLSSVTAADTA VYACEVTHQGLSSPV
HNAKTKPREEQYNSTYRVVSVL VYYCARWTGRTDAFDIWGQGTMV TKSFNRGEC (SEQ
TVLHQDWLNGKEYKCKVSNKAL TVSSASTKGPSVFPLAPSSKSTS ID NO: 9)
PAPIEKTISKAKGQPREPQVYT GGTAALGCLVKDYFPEPVTVSWN
LPPSRDELTKNQVSLTCLVKGF SGALTSGVHTFPAVLQSSGLYSL
YPSDIAVEWESNGQPENNYKTT SSVVTVPSSSLGTQTYICNVNHK
PPVLDSDGSFFLYSKLTVDKSR PSNTKVDKKVEPKSCDKTHTCPP
WQQGNVFSCSVMHEALHNHYTQ CPAPELLGGPSVFLFPPKPKDTL KSLSLSPGK (SEQ ID
NO: MISRTPEVTCVVVDVSHEDPEVK 10) FNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ
ID NO: 69) VRDN-02500 EIVLTQSPGTLSVSPGERATLSC EIVLTQSPGTLSVSP
EVQLVQSGGGLVKPGGSLRLSC RASQSIGSSLHWYQQKPGQAPRL GERATLSCRASQSIG
AASGFTFSSFAMHWVRQAPGKG LIKYASQSLSGIPDRFSGSGSGT SSLHWYQQKPGQAPR
LEWISVIDTRGATYYADSVKGR DFTLTISRLEPEDFAVYYCHQSS LLIKYASQSLSGIPD
FTISRDNAKNSLYLQMNSLRAE RLPHTFGQGTKVEIKRTVAAPSV RFSGSGSGTDFTLTI
DTAVYYCARLGNFYYGMDVWGQ FIFPPSDEQLKSGTASVVCLLNN SRLEPEDFAVYYCHQ
GTTVTVSSASTKGPSVFPLAPS FYPREAKVQWKVDNALQSGNSQE SSRLPHTFGQGTKVE
SKSTSGGTAALGCLVKDYFPEP SVTEQDSKDSTYSLSSTLTLSKA IKRTVAAPSVFIFPP
VTVSWNSGALTSGVHTFPAVLQ DYEKHKVYACEVTHQGLSSPVTK SDEQLKSGTASVVCL
SSGLYSLSSVVTVPSSSLGTQT SFNRGEC LNNFYPREAKVQWKV
YICNVNHKPSNTKVDKKVEPKS EVQLVQSGGGLVKPGGSLRLSCA DNALQSGNSQESVTE
CDKTHTCPPCPAPELLGGPSVF ASGFTFSSFAMHWVRQAPGKGLE QDSKDSTYSLSSTLT
LFPPKPKDTLMISRTPEVTCVV WISVIDTRGATYYADSVKGRFTI LSKADYEKHKVYACE
VDVSHEDPEVKFNWYVDGVEVH SRDNAKNSLYLQMNSLRAEDTAV VTHQGLSSPVTKSFN
NAKTKPREEQYNSTYRVVSVLT YYCARLGNFYYGMDVWGQGTTVT RGEC (SEQ ID
VLHQDWLNGKEYKCKVSNKALP VSSASTKGPSVFPLAPSSKSTSG NO: 11)
APIEKTISKAKGQPREPQVYTL GTAALGCLVKDYFPEPVTVSWNS
PPSRDELTKNQVSLTCLVKGFY GALTSGVHTFPAVLQSSGLYSLS
PSDIAVEWESNGQPENNYKTTP SVVTVPSSSLGTQTYICNVNHKP
PVLDSDGSFFLYSKLTVDKSRW SNTKVDKKVEPKSCDKTHTCPPC
QQGNVFSCSVMHEALHNHYTQK PAPELLGGPSVFLFPPKPKDTLM SLSLSPGK (SEQ ID NO:
ISRTPEVTCVVVDVSHEDPEVKF 12) NWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPR
EPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID
NO: 70) VRDN-02700 DIVMTQSPLSLPVTPGEPASISC DIVMTQSPLSLPVTP
QVQLQESGPGLVKPSETLSLTC RSSQSIVHSNGNTYLQWYLQKPG GEPASISCRSSQSIV
TVSGYSITGGYLWNWIRQPPGK QSPQLLIYKVSNRLYGVPDRFSG HSNGNTYLQWYLQKP
GLEWIGYISYDGTNNYKPSLKD SGSGTDFTLKISRVEAEDVGVYY GQSPQLLIYKVSNRL
RVTISRDTSKNQFSLKLSSVTA CFQGSHVPWTFGQGTKVEIKRTV YGVPDRFSGSGSGTD
ADTAVYYCARYGRVFFDYWGQG AAPSVFIFPPSDEQLKSGTASVV FTLKISRVEAEDVGV
TLVTVSSASTKGPSVFPLAPSS CLLNNFYPREAKVQWKVDNALQS YYCFQGSHVPWTFGQ
KSTSGGTAALGCLVKDYFPEPV GNSQESVTEQDSKDSTYSLSSTL GTKVEIKRTVAAPSV
TVSWNSGALTSGVHTFPAVLQS TLSKADYEKHKVYACEVTHQGLS FIFPPSDEQLKSGTA
SGLYSLSSVVTVPSSSLGTQTY SPVTKSFNRGEC SVVCLLNNFYPREAK
ICNVNHKPSNTKVDKRVEPKSC QVQLQESGPGLVKPSETLSLTCT VQWKVDNALQSGNSQ
DKTHTCPPCPAPELLGGPSVFL VSGYSITGGYLWNWIRQPPGKGL ESVTEQDSKDSTYSL
FPPKPKDTLYITREPEVTCVVV EWIGYISYDGTNNYKPSLKDRVT SSTLTLSKADYEKHK
DVSHEDPEVKFNWYVDGVEVHN ISRDTSKNQFSLKLSSVTAADTA VYACEVTHQGLSSPV
AKTKPREEQYNSTYRVVSVLTV VYYCARYGRVFFDYWGQGTLVTV TKSFNRGEC (SEQ
LHQDWLNGKEYKCKVSNKALPA SSASTKGPSVFPLAPSSKSTSGG ID NO: 3)
PIEKTISKAKGQPREPQVYTLP TAALGCLVKDYFPEPVTVSWNSG
PSREEMTKNQVSLTCLVKGFYP ALTSGVHTFPAVLQSSGLYSLSS
SDIAVEWESNGQPENNYKTTPP VVTVPSSSLGTQTYICNVNHKPS
VLDSDGSFFLYSKLTVDKSRWQ NTKVDKRVEPKSCDKTHTCPPCP
QGNVFSCSVMHEALHNHYTQKS APELLGGPSVFLFPPKPKDTLYI LSLSPG (SEQ ID NO:
83) TREPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG (SEQ ID
NO: 82)
[0120] In some embodiments, the antibody comprises one or more
peptides having the following sequences, or a variant thereof:
TABLE-US-00004 AB ID NO. AB Sequence of LC and HC VL Sequence VH
Sequence VRDN- DVVMTQTPLSLPVSLGDPASISC DVVMTQTPLSLPVSL
QVQLVQSGAEVVKPGASVKLSC 01100 RSSQSIVHSNVNTYLEWYLQKPG
GDPASISCRSSQSIV KASGYTFTSYWMHWVKQRPGQG QSPRLLIYKVSNRFSGVPDRFSG
HSNVNTYLEWYLQKP LEWIGEINPSNGRTNYNQKFQG SGAGTDFTLRISRVEAEDLGIYY
GQSPRLLIYKVSNRF KATLTVDKSSSTAYMQLSSLTS CFQGSHVPPTFGGGTKLEIKRTV
SGVPDRFSGSGAGTD EDSAVYYFARGRPDYYGSSKWY AAPSVFIFPPSDEQLKSGTASVV
FTLRISRVEAEDLGI FDVWGQGTTVTVSS (SEQ ID CLLNNFYPREAKVQWKVDNALQS
YYCFQGSHVPPTFGG NO: 14) GNSQESVTEQDSKDSTYSLSSTL GTKLEIKR (SEQ
TLSKADYEKHKVYACEVTHQGLS ID NO: 13) SPVTKSFNRGEC
QVQLVQSGAEVVKPGASVKLSCK ASGYTFTSYWMHWVKQRPGQGLE
WIGEINPSNGRTNYNQKFQGKAT LTVDKSSSTAYMQLSSLTSEDSA
VYYFARGRPDYYGSSKWYFDVWG QGTTVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK (SEQ ID NO: 71) VRDN-
DIQMTQSPLSLSASVGDRVTITC DIQMTQSPLSLSASV EVQLLESGGGLVQPGGSLRLSC
02600 QASRDIRNYLNWYQQKPGKAPKL GDRVTITCQASRDIR
AASGFTFSIYRMQWVRQAPGKG LIYDASSLQTGVPSRFGGSGSGT NYLNWYQQKPGKAPK
LEWVSGISPSGGTTWYADSVKG DFSFTIGSLQPEDIATYYCQQFD LLIYDASSLQTGVPS
RFTISRDNSKNTLYLQMNSLRA SLPHTFGQGTKLEIK RFGGSGSGTDFSFTI
EDTAVYYCARWSGGSGYAFDIW EVQLLESGGGLVQPGGSLRLSCA GSLQPEDIATYYCQQ
GQGTMVTVSS (SEQ ID NO: ASGFTFSIYRMQWVRQAPGKGLE FDSLPHTFGQGTKLE 16)
WVSGISPSGGTTWYADSVKGRFT IK (SEQ ID NO: ISRDNSKNTLYLQMNSLRAEDTA 15)
VYYCARWSGGSGYAFDIWGQGTM VTVSS (SEQ ID NO: 72) VRDN-
DIQMTQFPSSLSASVGDRVTITC DIQMTQFPSSLSASV EVQLLESGGGLVQPGGSLRLSC
02301 RASQGIRNDLGWYQQKPGKAPKR GDRVTITCRASQGIR
TASGFTFSSYAMNWVRQAPGKG LIYAASRLHRGVPSRFSGSGSGT NDLGWYQQKPGKAPK
LEWVSAISGSGGTTFYADSVKG EFTLTISSLQPEDFATYYCLQHN RLIYAASRLHRGVPS
RFTISRDNSRTTLYLQMNSLRA SYPSSFGQGTKLEIKEVQLLESG RFSGSGSGTEFTLTI
EDTAVYYCAKDLGWSDSYYYYY GGLVQPGGSLRLSCTASGFTFSS SSLQPEDFATYYCLQ
GMDVWGQGTTVTVSS (SEQ YAMNWVRQAPGKGLEWVSAISGS HNSYPSSFGQGTKLE ID NO:
80) GGTTFYADSVKGRFTISRDNSRT IK (SEQ ID NO: TLYLQMNSLRAEDTAVYYCAKDL
79) GWSDSYYYYYGMDVWGQGTTVTV SS (SEQ ID NO: 78) VRDN-
DVVMTQTPLSLPVSLGDPASISC DVVMTQTPLSLPVSL QVQLVQSGAEVVKPGASVKLSC
01101 RSSQSIVHSNVNTYLEWYLQKPG GDPASISCRSSQSIV
KASGYTFTSYWMHWVKQRPGQG QSPKLLIYKVSNRFSGVPDRFSG HSNVNTYLEWYLQKP
LEWIGEINPSNGRTNYNQKFQG SGAGTDFTLRISRVEAEDLGIYY GQSPKLLIYKVSNRF
KATLTVDKSSSTAYMQLSSLTS CFQGSHVPPTFGGGTKLEIKRTV SGVPDRFSGSGAGTD
EDSAVYYFARGRPDYYGSSKWY AAPSVFIFPPSDEQLKSGTASVV FTLRISRVEAEDLGI
FDVWGQGTTVTVSS (SEQ ID CLLNNFYPREAKVQWKVDNALQS YYCFQGSHVPPTFGG NO:
14) GNSQESVTEQDSKDSTYSLSSTL GTKLEIKR (SEQ TLSKADYEKHKVYACEVTHQGLS
ID NO: 86) SPVTKSFNRGEC (Light Chain) QVQLVQSGAEVVKPGASVKLSCK
ASGYTFTSYWMHWVKQRPGQGLE WIGEINPSNGRTNYNQKFQGKAT
LTVDKSSSTAYMQLSSLTSEDSA VYYFARGRPDYYGSSKWYFDVWG
QGTTVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK
(SEQ ID NO: 85; heavy chain) VRDN- DIVMTQSPLSLPVTPGEPASISC
DIVMTQSPLSLPVTP QVQLQESGPGLVKPSETLSLTC 2700 RSSQSIVHSNGNTYLQWYLQKPG
GEPASISCRSSQSIV TVSGYSITGGYLWNWIRQPPGK QSPQLLIYKVSNRLYGVPDRFSG
HSNGNTYLQWYLQKP GLEWIGYISYDGTNNYKPSLKD SGSGTDFTLKISRVEAEDVGVYY
GQSPQLLIYKVSNRL RVTISRDTSKNQFSLKLSSVTA CFQGSHVPWTFGQGTKVEIKRTV
YGVPDRFSGSGSGTD ADTAVYYCARYGRVFFDYWGQG AAPSVFIFPPSDEQLKSGTASVV
FTLKISRVEAEDVGV TLVTVSS (SEQ ID NO: CLLNNFYPREAKVQWKVDNALQS
YYCFQGSHVPWTFGQ 99) GNSQESVTEQDSKDSTYSLSSTL GTKVEIKR (SEQ
TLSKADYEKHKVYACEVTHQGLS ID NO: 98) SPVTKSFNRGEC (Light Chain)
QVQLQESGPGLVKPSETLSLTCT VSGYSITGGYLWNWIRQPPGKGL
EWIGYISYDGTNNYKPSLKDRVT ISRDTSKNQFSLKLSSVTAADTA
VYYCARYGRVFFDYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGG
TAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCDKTHTCPPCP
APELLGGPSVFLFPPKPKDTLYI TREPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG (Heavy Chain)(SEQ ID NO: 82)
[0121] The column that is indicated as the antibody sequence
comprises the VH and VL chains of the antibody. In instances where
the VH chain is illustrated with a Fc sequence, the Fc sequence can
be modified or substituted for a different Fc region as provided
for herein. However, in some embodiments, the antibody can comprise
the VH and VL sequence as provided for in the tables provided for
herein. For example, in some embodiments, the antibody comprises
one or more VH, HC, LC, or VL (those sequence that have a constant
domain are the complete light or heavy chain) having the following
sequences, or a variant thereof:
TABLE-US-00005 VL or LC AB ID NO. Sequence VH or HC Sequence
VRDN-03100 EIVLTQSPATLSLSP QVELVESGGGVVQPGRSQRLSC GERATLSCRASQSVS
AASGFTFSSYGMHWVRQAPGKG SYLAWYQQKPGQAPR LEWVAIIWFDGSSTYYADSVRG
LLIYDASKRATGIPA RFTISRDNSKNTLYLQMNSLRA RFSGSGSGTDFTLTI
EDTAVYFCARELGRRYFDLWGR SSLEPEDFAVYYCQQ GTLVSVSSASTKGPSVFPLAPS
RSKWPPWTFGQGTKV SKSTSGGTAALGCLVKDYFPEP ESKRTVAAPSVFIFP
VTVSWNSGALTSGVHTFPAVLQ PSDEQLKSGTASVVC SSGLYSLSSVVTVPSSSLGTQT
LLNNFYPREAKVQWK YICNVNHKPSNTKVDKKVEPKS VDNALQSGNSQESVT
CDKTHTCPPCPAPELLGGPSVF EQDSKDSTYSLSSTL LFPPKPKDTLMISRTPEVTCVV
TLSKADYEKHKVYAC VDVSHEDPEVKFNWYVDGVEVH EVTHQGLSSPVTKSF
NAKTKPREEQYNSTYRVVSVLT NRGEC (SEQ ID VLHQDWLNGKEYKCKVSNKALP NO: 1)
APIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 2) VRDN-02100
DIVMTQSPLSLPVTP QVQLQESGPGLVKPSETLSLTC GEPASISCRSSQSIV
TVSGYSITGGYLWNWIRQPPGK HSNGNTYLQWYLQKP GLEWIGYISYDGTNNYKPSLKD
GQSPQLLIYKVSNRL RVTISRDTSKNQFSLKLSSVTA YGVPDRFSGSGSGTD
ADTAVYYCARYGRVFFDYWGQG FTLKISRVEAEDVGV TLVTVSSASTKGPSVFPLAPSS
YYCFQGSHVPWTFGQ KSTSGGTAALGCLVKDYFPEPV GTKVEIKRTVAAPSV
TVSWNSGALTSGVHTFPAVLQS FIFPPSDEQLKSGTA SGLYSLSSVVTVPSSSLGTQTY
SVVCLLNNFYPREAK ICNVNHKPSNTKVDKRVEPKSC VQWKVDNALQSGNSQ
DKTHTCPPCPAPELLGGPSVFL ESVTEQDSKDSTYSL FPPKPKDTLMISRTPEVTCVVV
SSTLTLSKADYEKHK DVSHEDPEVKFNWYVDGVEVHN VYACEVTHQGLSSPV
AKTKPREEQYNSTYRVVSVLTV TKSFNRGEC (SEQ LHQDWLNGKEYKCKVSNKALPA ID NO:
3) PIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 4) VRDN-02200
SSELTQDPAVSVALG EVQLVQSGAEVKKPGSSVKVSC QTVRITCQGDSLRSY
KASGGTFSSYAISWVRQAPGQG YATWYQQKPGQAPIL LEWMGGIIPIFGTANYAQKFQG
VIYGENKRPSGIPDR RVTITADKSTSTAYMELSSLRS FSGSSSGNTASLTIT
EDTAVYYCARAPLRFLEWSTQD GAQAEDEADYYCKSR HYYYYYMDVWGKGTTVTVSSAS
DGSGQHLVFGGGTKL TKGPSVFPLAPSSKSTSGGTAA TVLGQPKAAPSVTLF
LGCLVKDYFPEPVTVSWNSGAL PPSSEELQANKATLV TSGVHTFPAVLQSSGLYSLSSV CLI
SDFYPGAVTVAW VTVPSSSLGTQTYICNVNHKPS KADSSPVKAGVETTT
NTKVDKKVEPKSCDKTHTCPPC PSKQSNNKYAASSYL PAPELLGGPSVFLFPPKPKDTL
SLTPEQWKSHRSYSC MISRTPEVTCVVVDVSHEDPEV QVTHEGSTVEKTVAP
KFNWYVDGVEVHNAKTKPREEQ AECS (SEQ ID YNSTYRVVSVLTVLHQDWLNGK NO: 5)
EYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO: 6)
VRDN-02300 DIQMTQFPSSLSASV EVQLLESGGGLVQPGGSLRLSC GDRVTITCRASQGIR
TASGFTFSSYAMNWVRQAPGKG NDLGWYQQKPGKAPK LEWVSAISGSGGTTFYADSVKG
RLIYAASRLHRGVPS RFTISRDNSRTTLYLQMNSLRA RFSGSGSGTEFTLTI
EDTAVYYCAKDLGWSDSYYYYY SSLQPEDFATYYCLQ GMDVWGQGTTVTVSSASTKGPS
HNSYPCSFGQGTKLE VFPLAPCSRSTSESTAALGCLV IKRTVAAPSVFIFPP
KDYFPEPVTVSWNSGALTSGVH SDEQLKSGTASVVCL TFPAVLQSSGLYSLSSVVTVPS
LNNFYPREAKVQWKV SNFGTQTYTCNVDHKPSNTKVD DNALQSGNSQESVTE
KTVERKCCVECPPCPAPPVAGP QDSKDSTYSLSSTLT SVFLFPPKPKDTLMISRTPEVT
LSKADYEKHKVYACE CVVVDVSHEDPEVQFNWYVDGV VTHQGLSSPVTKSFN
EVHNAKTKPREEQFNSTFRVVS RGEC (SEQ ID VLTVVHQDWLNGKEYKCKVSNK NO: 7)
GLPAPIEKTISKTKGQPREPQV YTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYK TTPPMLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG (SEQ ID NO: 8) VRDN-02400
DVVMTQSPLSLPVTP QVQLQESGPGLVKPSGTLSLTC GEPASISCRSSQSLL
AVSGGSISSSNWWSWVRQPPGK HSNGYNYLDWYLQKP GLEWIGEIYHSGSTNYNPSLKS
GQSPQLLIYLGSNRA RVTISVDKSKNQFSLKLSSVTA SGVPDRFSGSGSGTD
ADTAVYYCARWTGRTDAFDIWG FTLKISRVEAEDVGV QGTMVTVSSASTKGPSVFPLAP
YYCMQGTHWPLTFGQ SSKSTSGGTAALGCLVKDYFPE GTKVEIKRTVAAPSV
PVTVSWNSGALTSGVHTFPAVL FIFPPSDEQLKSGTA QSSGLYSLSSVVTVPSSSLGTQ
SVVCLLNNFYPREAK TYICNVNHKPSNTKVDKKVEPK VQWKVDNALQSGNSQ
SCDKTHTCPPCPAPELLGGPSV ESVTEQDSKDSTYSL FLFPPKPKDTLMISRTPEVTCV
SSTLTLSKADYEKHK VVDVSHEDPEVKFNWYVDGVEV VYACEVTHQGLSSPV
HNAKTKPREEQYNSTYRVVSVL TKSFNRGEC (SEQ TVLHQDWLNGKEYKCKVSNKAL ID NO:
9) PAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK (SEQ ID NO: 10) VRDN-02500
EIVLTQSPGTLSVSP EVQLVQSGGGLVKPGGSLRLSC GERATLSCRASQSIG
AASGFTFSSFAMHWVRQAPGKG SSLHWYQQKPGQAPR LEWISVIDTRGATYYADSVKGR
LLIKYASQSLSGIPD FTISRDNAKNSLYLQMNSLRAE RFSGSGSGTDFTLTI
DTAVYYCARLGNFYYGMDVWGQ SRLEPEDFAVYYCHQ GTTVTVSSASTKGPSVFPLAPS
SSRLPHTFGQGTKVE SKSTSGGTAALGCLVKDYFPEP IKRTVAAPSVFIFPP
VTVSWNSGALTSGVHTFPAVLQ SDEQLKSGTASVVCL SSGLYSLSSVVTVPSSSLGTQT
LNNFYPREAKVQWKV YICNVNHKPSNTKVDKKVEPKS DNALQSGNSQESVTE
CDKTHTCPPCPAPELLGGPSVF QDSKDSTYSLSSTLT LFPPKPKDTLMISRTPEVTCVV
LSKADYEKHKVYACE VDVSHEDPEVKFNWYVDGVEVH VTHQGLSSPVTKSFN
NAKTKPREEQYNSTYRVVSVLT RGEC (SEQ ID VLHQDWLNGKEYKCKVSNKALP NO: 11)
APIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 12) VRDN-02700
DIVMTQSPLSLPVTP QVQLQESGPGLVKPSETLSLTC GEPASISCRSSQSIV
TVSGYSITGGYLWNWIRQPPGK HSNGNTYLQWYLQKP GLEWIGYISYDGTNNYKPSLKD
GQSPQLLIYKVSNRL RVTISRDTSKNQFSLKLSSVTA YGVPDRFSGSGSGTD
ADTAVYYCARYGRVFFDYWGQG FTLKISRVEAEDVGV TLVTVSSASTKGPSVFPLAPSS
YYCFQGSHVPWTFGQ KSTSGGTAALGCLVKDYFPEPV GTKVEIKRTVAAPSV
TVSWNSGALTSGVHTFPAVLQS FIFPPSDEQLKSGTA SGLYSLSSVVTVPSSSLGTQTY
SVVCLLNNFYPREAK ICNVNHKPSNTKVDKRVEPKSC VQWKVDNALQSGNSQ
DKTHTCPPCPAPELLGGPSVFL ESVTEQDSKDSTYSL FPPKPKDTLYITREPEVTCVVV
SSTLTLSKADYEKHK DVSHEDPEVKFNWYVDGVEVHN VYACEVTHQGLSSPV
AKTKPREEQYNSTYRVVSVLTV TKSFNRGEC (SEQ LHQDWLNGKEYKCKVSNKALPA ID NO:
3) PIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKS LSLSPG (SEQ ID NO: 83) VRDN-01100
DVVMTQTPLSLPVSL QVQLVQSGAEVVKPGASVKLSC GDPASISCRSSQSIV
KASGYTFTSYWMHWVKQRPGQG HSNVNTYLEWYLQKP LEWIGEINPSNGRTNYNQKFQG
GQSPRLLIYKVSNRF KATLTVDKSSSTAYMQLSSLTS SGVPDRFSGSGAGTD
EDSAVYYFARGRPDYYGSSKWY FTLRISRVEAEDLGI FDVWGQGTTVTVSS (SEQ ID
YYCFQGSHVPPTFGG NO: 14) GTKLEIKR (SEQ ID NO: 13) VRIN-02600
DIQMTQSPLSLSASV EVQLLESGGGLVQPGGSLRLSC GDRVTITCQASRDIR
AASGFTFSIYRMQWVRQAPGKG NYLNWYQQKPGKAPK LEWVSGISPSGGTTWYADSVKG
LLIYDASSLQTGVPS RFTISRDNSKNTLYLQMNSLRA RFGGSGSGTDFSFTI
EDTAVYYCARWSGGSGYAFDIW GSLQPEDIATYYCQQ GQGTMVTVSS (SEQ ID NO:
FDSLPHTFGQGTKLE 16) IK (SEQ ID NO: 15) VRIN-02301 DIQMTQFPSSLSASV
EVQLLESGGGLVQPGGSLRLSC GDRVTITCRASQGIR TASGFTFSSYAMNWVRQAPGKG
NDLGWYQQKPGKAPK LEWVSAISGSGGTTFYADSVKG RLIYAASRLHRGVPS
RFTISRDNSRTTLYLQMNSLRA RFSGSGSGTEFTLTI EDTAVYYCAKDLGWSDSYYYYY
SSLQPEDFATYYCLQ GMDVWGQGTTVTVSS (SEQ HNSYPSSFGQGTKLE ID NO: 80) IK
(SEQ ID NO: 79) VRIN-01101 DVVMTQTPLSLPVSL QVQLVQSGAEVVKPGASVKLSC
GDPASISCRSSQSIV KASGYTFTSYWMHWVKQRPGQG HSNVNTYLEWYLQKP
LEWIGEINPSNGRTNYNQKFQG GQSPKLLIYKVSNRF KATLTVDKSSSTAYMQLSSLTS
SGVPDRFSGSGAGTD EDSAVYYFARGRPDYYGSSKWY FTLRISRVEAEDLGI
FDVWGQGTTVTVSS (SEQ ID YYCFQGSHVPPTFGG NO: 14) GTKLEIKR (SEQ ID NO:
86) VRDN- DVVMTQTPLSLPVSL QVQLVQSGAEVVKPGASVKLSS 01100A or
GDPASISCRSSQSIV KASGYTFTSYWMHWVKQRPGQG 01110B HSNVNTYLEWYLQKP
LEWIGEINPSNGRTNYNQKFQG GQSPKLLIYKVSNRF KATLTVDKSSSTAYMQLSSLTS
SGVPDRFSGSGAGTD EDSAVYYFARGRPDYYGSSKWY FTLRISRVEAEDLGI
FDVWGQGTTVTVSS (SEQ ID YYCFQGSHVPPTFGG NO: 91) GTKLEIKR (SEQ ID NO:
86)
[0122] In some embodiments, the variable light chain as set forth
in SEQ ID NO: 13 does not have the C-terminal arginine residue.
This is illustrated for example, in the following sequence:
TABLE-US-00006 (SEQ ID NO: 97)
DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPR
LLIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVP
PTFGGGTKLEIK.
[0123] Thus, in some embodiments, where the variable light chain
comprises the sequence of SEQ ID NO: 13, it can be substituted with
a sequence of SEQ ID NO: 97.
[0124] In some embodiments, the heavy chain variable region as set
forth in SEQ ID NO: 14 can comprises a C22S substitution. This is
illustrated in the following sequence:
TABLE-US-00007 (SEQ ID NO: 96)
QVQLVQSGAEVVKPGASVKLSSKASGYTFTSYWMHWVKQRPGQGLEWIGE
INPSNGRTNYNQKFQGKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGR
PDYYGSSKWYFDVWGQGTTVTVSS.
[0125] Accordingly, in some embodiments, the antibody comprises a
VH sequence of SEQ ID NO: 96 and a VL sequence of SEQ ID NO: 13 or
SEQ ID NO: 97.
[0126] In some embodiments, the antibody comprises a VH of SEQ ID
NO: 14 and a VL sequence of SEQ ID NO: 97.
[0127] In some embodiments, the antibody comprises a VL of SEQ ID
NO: 98 and a VH of SEQ ID NO: 99. In some embodiments, the antibody
comprises a VL of SEQ ID NO: 98 and a VH of SEQ ID NO: 99 with a Fc
region comprising the M252Y, S254T, and T256E mutations. In some
embodiments, the antibody comprises a VL of SEQ ID NO: 98 and a VH
of SEQ ID NO: 99 with a Fc region comprising the M428L and N434S
mutations.
[0128] As provided for herein, the heavy chain can be linked to a
Fc region, including those with mutations that can affect the
half-life of the antibody. Non-limiting mutations in the Fc region
are provided for herein.
[0129] In the tables provided for herein, the LC and HC may be
illustrated with the VH and VL domains with or without constant
regions. The constant regions can be replaced as provided for
herein. The VH and VL regions can be used to form an antibody as
provided for herein. The VH and the VL sequences can be in any
format, including, but not limited to a scFv format where the VH
and VL regions are linked with a peptide linker. Examples of
peptide linkers that can be used to link various peptides provided
for herein include, but are not limited to: (GGGGS).sub.n (SEQ ID
NO: 73); (GGGGA).sub.n (SEQ ID NO: 74), or any combination thereof,
wherein each n is independently 1-5. In some embodiments, the
variable regions are not linked with a peptide linker. In some
embodiments, the antibody comprises SEQ ID NO: 1 and SEQ ID NO: 2,
or the CDR regions thereof. In some embodiments, the antibody
comprises SEQ ID NO: 3 and SEQ ID NO: 4, or the CDR regions
thereof. In some embodiments, the antibody comprises SEQ ID NO: 5
and SEQ ID NO: 6, or the CDR regions thereof. In some embodiments,
the antibody comprises SEQ ID NO: 7 and SEQ ID NO: 8, or the CDR
regions thereof. In some embodiments, the antibody comprises SEQ ID
NO: 9 and SEQ ID NO: 10, or the CDR regions thereof. In some
embodiments, the antibody comprises SEQ ID NO: 11 and SEQ ID NO:
12, or the CDR regions thereof. In some embodiments, the antibody
comprises SEQ ID NO: 13 and SEQ ID NO: 14, or the CDR regions
thereof. In some embodiments, the antibody comprises SEQ ID NO: 15
and SEQ ID NO: 16, or the CDR regions thereof.
[0130] In some embodiments, an antibody, or antigen binding
fragment thereof is provided, wherein the antibody or antibody
fragment comprises a peptide selected from the following table.
TABLE-US-00008 Ab ID No LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 VRDN-
RASQSV DASKRAT QQRSKWPPWT SYGMH IIWFDGSSTYYADS ELGRRYFDL 03100
SSYLA (SEQ ID (SEQ ID (SEQ ID VRG (SEQ ID (SEQ ID (SEQ NO: 18) NO:
19) NO: 20) NO: 21) NO: 22) ID NO: 17) VRDN- RSSQSI KVSNRLY
FQGSHVPWT GGYLWN YISYDGTNNYKPSL YGRVFFDY 02100/ VHSNGN (SEQ ID (SEQ
ID (SEQ ID KD (SEQ ID NO: (SEQ ID 2700 TYLQWY NO: 24) NO: 25) NO:
26) 27) NO: 28) LQ (SEQ ID NO: 23) VRDN- QGDSLR GENKRPS KSRDGSGQHL
SYAIS GIIPIFGTANYAQK APLRFLEWST 02200 SYYAT (SEQ ID V (SEQ ID (SEQ
ID FQG (SEQ ID QDHYYYYYMD (SEQ NO: 30) NO: 31) NO: 32) NO: 33) V
(SEQ ID ID NO: NO: 34) 29) VRDN- RASQGI AASRLHR LQHNSYPCS SYAMN
AISGSGGTTFYADS DLGWSDSYYY 02300 RNDLG (SEQ ID (SEQ ID (SEQ ID VKG
(SEQ ID YYGMDV (SEQ NO: 36) NO: 37) NO: 38) NO: 39) (SEQ ID ID NO:
NO: 40) 35) VRDN- RSSQSL LGSNRA MQGTHWPLT SSSNWWS EIYHSGSTNYNPSL
WTGRTDAFDI 02400 LHSNGY (SEQ ID (SEQ ID (SEQ ID KS (SEQ ID NO: (SEQ
ID NYLD NO: 42) NO: 43) NO: 44) 45) NO: 46) (SEQ ID NO: 41) VRDN-
RASQSI YASQSLS HQSSRLPHT SFAMH VIDTRGATYYADSV LGNFYYGMDV 02500
GSSLH (SEQ ID (SEQ ID (SEQ ID KG (SEQ ID NO: (SEQ ID (SEQ NO: 48)
NO: 49) NO: 50) 51) NO: 52) ID NO: 47) VRDN- RSSQSI KVSNRFS
FQGSHVPPT SYWMH GEINPSNGRTNYNQ GRPDYYGSSK 1100/ VHSNVN (SEQ ID (SEQ
ID (SEQ ID KFQG (SEQ ID WYFDV (SEQ 1100A/ TYLE NO: 54) NO: 55) NO:
56) NO: 57) ID NO: 58) 1100B (SEQ ID NO: 53) VRDN- 4ASRDI DASSLQT
QQFDSLPHT IYRMQ GISPSGGTTWYADS WSGGSGYAFD 2600 RNYLN (SEQ ID (SEQ
ID (SEQ ID VK (SEQ ID NO: I (SEQ ID (SEQ NO: 60) NO: 61) NO: 62)
63) NO: 64) ID NO: 59) VRDN- RASQGI AASRLHR LQHNSYPSS SYAMN
AISGSGGTTFYADS DLGWSDSYYY 2301 RNDLG (SEQ ID (SEQ ID (SEQ ID VKG
(SEQ ID YYGMDV (SEQ NO: 36) NO: 81) NO: 38) NO: 39) (SEQ ID ID NO:
NO: 40) 35)
[0131] In some embodiments, an antibody, or antibody binding
fragment thereof, comprises a heavy or light chain CDR having a
sequence of SEQ ID NOs: 17-64 and 81. In some embodiments, an
antibody, or antibody binding fragment thereof, comprises a light
chain CDR having a sequence of SEQ ID NO: 17, 18, 19, 23, 24, 25,
29, 30, 31, 35, 36, 37, 41, 42, 43, 47, 48, 49, 53, 54, 55, 59, 60,
61, or 81. In some embodiments, an antibody, or antibody binding
fragment thereof, comprises a heavy chain CDR having a sequence of
SEQ ID NO: 20, 21, 22, 26, 27, 28, 32, 33, 34, 38, 39, 40, 44, 45,
46, 50, 51, 52, 56, 57, 58, 62, 63, or 64.
[0132] In some embodiments, an antibody, or antibody binding
fragment thereof, comprises a light chain having a LCDR1, a LCDR2,
and a LCDR3, wherein the LCDR1 has a sequence of SEQ ID NO: 17, 23,
29, 35, 41, 47, 53, or 59 the LCDR2 has a sequence of SEQ ID NO:
18, 24, 30, 36, 42, 48, 54, or 60 and the LCDR3 has a sequence of
SEQ ID NO: 19, 25, 31, 37, 43, 49, 55, 61, or 81.
[0133] In some embodiments, an antibody, or antibody binding
fragment thereof, comprises a heavy chain having a HCDR1, a HCDR2,
and a HCDR3, wherein the HCDR1 has a sequence of SEQ ID NO: 20, 26,
32, 38, 44, 50, 56, or 62 the HCDR2 has a sequence of SEQ ID NO:
21, 27, 33, 39, 45, 51, 57, or 63 and the HCDR3 has a sequence of
SEQ ID NO: 22, 28, 34, 40, 46, 52, 58, or 64.
[0134] The different CDR motifs can be combined in any combination
including those not depicted in the table above. For example, the
following embodiments are provided as non-limiting examples of such
combinations.
[0135] In some embodiments, an antibody, or antigen binding
fragment thereof, comprises: (i) a light chain variable region
comprising light chain CDR1, CDR2, and CDR3 sequences, wherein the
light chain CDR1 sequence has the amino acid sequence of SEQ ID NO:
17; the light chain CDR2 has the amino acid sequence of SEQ ID NO:
18; and the light chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 19; and (ii) a heavy chain variable region comprising
heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain
CDR1 sequence has the amino acid sequence of SEQ ID NO: 20; the
heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:
21; and the heavy chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 22; or variants of any of the foregoing.
[0136] In some embodiments, an antibody, or antigen binding
fragment thereof, comprises: (i) a light chain variable region
comprising light chain CDR1, CDR2, and CDR3 sequences, wherein the
light chain CDR1 sequence has the amino acid sequence of SEQ ID NO:
23; the light chain CDR2 has the amino acid sequence of SEQ ID NO:
24; and the light chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 25; and (ii) a heavy chain variable region comprising
heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain
CDR1 sequence has the amino acid sequence of SEQ ID NO: 26; the
heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:
27; and the heavy chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 28; or variants of any of the foregoing.
[0137] In some embodiments, an antibody, or antigen binding
fragment thereof, comprises: (i) a light chain variable region
comprising light chain CDR1, CDR2, and CDR3 sequences, wherein the
light chain CDR1 sequence has the amino acid sequence of SEQ ID NO:
29; the light chain CDR2 has the amino acid sequence of SEQ ID NO:
30; and the light chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 31; and (ii) a heavy chain variable region comprising
heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain
CDR1 sequence has the amino acid sequence of SEQ ID NO: 32; the
heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:
33; and the heavy chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 34; or variants of any of the foregoing.
[0138] In some embodiments, an antibody, or antigen binding
fragment thereof, comprises: (i) a light chain variable region
comprising light chain CDR1, CDR2, and CDR3 sequences, wherein the
light chain CDR1 sequence has the amino acid sequence of SEQ ID NO:
35; the light chain CDR2 has the amino acid sequence of SEQ ID NO:
36; and the light chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 37; and (ii) a heavy chain variable region comprising
heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain
CDR1 sequence has the amino acid sequence of SEQ ID NO: 38; the
heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:
39; and the heavy chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 40; or variants of any of the foregoing.
[0139] In some embodiments, an antibody, or antigen binding
fragment thereof, comprises: (i) a light chain variable region
comprising light chain CDR1, CDR2, and CDR3 sequences, wherein the
light chain CDR1 sequence has the amino acid sequence of SEQ ID NO:
41; the light chain CDR2 has the amino acid sequence of SEQ ID NO:
42; and the light chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 43; and (ii) a heavy chain variable region comprising
heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain
CDR1 sequence has the amino acid sequence of SEQ ID NO: 44; the
heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:
45; and the heavy chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 46; or variants of any of the foregoing.
[0140] In some embodiments, an antibody, or antigen binding
fragment thereof, comprises: (i) a light chain variable region
comprising light chain CDR1, CDR2, and CDR3 sequences, wherein the
light chain CDR1 sequence has the amino acid sequence of SEQ ID NO:
47; the light chain CDR2 has the amino acid sequence of SEQ ID NO:
48; and the light chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 49; and (ii) a heavy chain variable region comprising
heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain
CDR1 sequence has the amino acid sequence of SEQ ID NO: 50; the
heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:
51; and the heavy chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 52; or variants of any of the foregoing.
[0141] In some embodiments, an antibody, or antigen binding
fragment thereof, comprises: (i) a light chain variable region
comprising light chain CDR1, CDR2, and CDR3 sequences, wherein the
light chain CDR1 sequence has the amino acid sequence of SEQ ID NO:
53; the light chain CDR2 has the amino acid sequence of SEQ ID NO:
54; and the light chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 55; and (ii) a heavy chain variable region comprising
heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain
CDR1 sequence has the amino acid sequence of SEQ ID NO: 56; the
heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:
57; and the heavy chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 58; or variants of any of the foregoing.
[0142] In some embodiments, an antibody, or antigen binding
fragment thereof, comprises: (i) a light chain variable region
comprising light chain CDR1, CDR2, and CDR3 sequences, wherein the
light chain CDR1 sequence has the amino acid sequence of SEQ ID NO:
59; the light chain CDR2 has the amino acid sequence of SEQ ID NO:
60; and the light chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 61; and (ii) a heavy chain variable region comprising
heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain
CDR1 sequence has the amino acid sequence of SEQ ID NO: 62; the
heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:
63; and the heavy chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 64; or variants of any of the foregoing.
[0143] In some embodiments, an antibody, or antigen binding
fragment thereof, comprises: (i) a light chain variable region
comprising light chain CDR1, CDR2, and CDR3 sequences, wherein the
light chain CDR1 sequence has the amino acid sequence of SEQ ID NO:
35; the light chain CDR2 has the amino acid sequence of SEQ ID NO:
36; and the light chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 81; and (ii) a heavy chain variable region comprising
heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain
CDR1 sequence has the amino acid sequence of SEQ ID NO: 38; the
heavy chain CDR2 sequence has the amino acid sequence of SEQ ID NO:
39; and the heavy chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 40; or variants of any of the foregoing.
[0144] In some embodiments, the light chain variable region CDR1 is
replaced with any of the other light chain CDR1 sequences. In some
embodiments, the light chain variable region CDR2 is replaced with
any of the other light chain CDR2 sequences. In some embodiments,
the light chain variable region CDR3 is replaced with any of the
other light chain CDR3 sequences. In some embodiments, the heavy
chain variable region CDR1 is replaced with any of the other light
chain CDR1 sequences. In some embodiments, the heavy chain variable
region CDR2 is replaced with any of the other light chain CDR2
sequences. In some embodiments, the heavy chain variable region
CDR3 is replaced with any of the other light chain CDR3
sequences.
[0145] In some embodiments, the antibody, or antigen binding
fragment thereof, or protein is provided that comprises a peptide
having a sequence as set forth in any of SEQ ID NOs: 1, 3, 5, 7, 9,
11, 13, 15, 79, or 86, and 2, 4, 6, 8, 10, 12, 14, 16, 80, or
83.
[0146] In some embodiments, the antibody, or antigen binding
fragment thereof, comprises a sequence of, or a variant of any of
the foregoing.
[0147] In some embodiments, the antibody, or antigen binding
fragment thereof, comprises a sequence of SEQ ID NOs: 65, or a
variant of any of the foregoing. In some embodiments, the antibody,
or antigen binding fragment thereof, comprises a sequence of SEQ ID
NOs: 66, or a variant of any of the foregoing. In some embodiments,
the antibody, or antigen binding fragment thereof, comprises a
sequence of SEQ ID NOs: 67, or a variant of any of the foregoing.
In some embodiments, the antibody, or antigen binding fragment
thereof, comprises a sequence of SEQ ID NOs: 68, or a variant of
any of the foregoing. In some embodiments, the antibody, or antigen
binding fragment thereof, comprises a sequence of SEQ ID NOs: 69,
or a variant of any of the foregoing. In some embodiments, the
antibody, or antigen binding fragment thereof, comprises a sequence
of SEQ ID NOs: 70, or a variant of any of the foregoing. In some
embodiments, the antibody, or antigen binding fragment thereof,
comprises a sequence of SEQ ID NOs: 71, or a variant of any of the
foregoing. In some embodiments, the antibody, or antigen binding
fragment thereof, comprises a sequence of SEQ ID NOs: 72, or a
variant of any of the foregoing. In some embodiments, the antibody,
or antigen binding fragment thereof, comprises a sequence of SEQ ID
NOs: 78, or a variant of any of the foregoing. In some embodiments,
the antibody, or antigen binding fragment thereof, comprises a
sequence of SEQ ID NOs: 82, or a variant of any of the foregoing.
In some embodiments, the antibody, or antigen binding fragment
thereof, comprises a sequence of SEQ ID NOs: 85, or a variant of
any of the foregoing.
[0148] In some embodiment, the V.sub.L and/or V.sub.H sequences are
as provided herein. In some embodiments, the V.sub.L sequences are
provided as elements of the light chain (LC). In some embodiments,
the V.sub.L sequences that are provided as elements of the light
chain (LC) are underlined in the LC sequence. In some embodiments,
the V.sub.H sequences that are provided as elements of the heavy
chain (LC) are underlined in the HC sequence.
[0149] In some embodiments, an antibody, or antigen binding
fragment thereof, comprises a V.sub.L peptide as set forth in SEQ
ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 79, or 86, or any combination
thereof. The V.sub.L peptide can comprise a variant of any of these
sequences as provided for herein.
[0150] In some embodiments, an antibody, or antigen binding
fragment thereof, comprises a V.sub.H peptide as set forth in SEQ
ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 80, or 83, or any combination
thereof. The V.sub.H peptide can comprise a variant of any of these
sequences as provided for herein.
[0151] In some embodiments, an antibody, or antigen binding
fragment thereof, comprises a V.sub.H peptide and a V.sub.L
peptide, wherein the wherein the V.sub.H peptide comprises a
sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 80,
or 83 and the V.sub.L peptide comprises a sequence as set forth in
SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 79, or 86.
[0152] In some embodiments, an antibody, or antigen binding
fragment thereof, comprises a V.sub.H peptide and a V.sub.L
peptide, wherein the V.sub.H peptide comprises a sequence as set
forth in SEQ ID NO: 2 and the V.sub.L peptide comprises a sequence
as set forth in SEQ ID NO: 1. In some embodiments, an antibody, or
antigen binding fragment thereof, comprises a V.sub.H peptide and a
V.sub.L peptide, wherein the V.sub.H peptide comprises a sequence
as set forth in SEQ ID NO: 4 and the V.sub.L peptide comprises a
sequence as set forth in SEQ ID NO: 3. In some embodiments, an
antibody, or antigen binding fragment thereof, comprises a V.sub.H
peptide and a V.sub.L peptide, wherein the V.sub.H peptide
comprises a sequence as set forth in SEQ ID NO: 6 and the V.sub.L
peptide comprises a sequence as set forth in SEQ ID NO: 5. In some
embodiments, an antibody, or antigen binding fragment thereof,
comprises a V.sub.H peptide and a V.sub.L peptide, wherein the
V.sub.H peptide comprises a sequence as set forth in SEQ ID NO: 8
and the V.sub.L peptide comprises a sequence as set forth in SEQ ID
NO: 7. In some embodiments, an antibody, or antigen binding
fragment thereof, comprises a V.sub.H peptide and a V.sub.L
peptide, wherein the V.sub.H peptide comprises a sequence as set
forth in SEQ ID NO: 10 and the V.sub.L peptide comprises a sequence
as set forth in SEQ ID NO: 9. In some embodiments, an antibody, or
antigen binding fragment thereof, comprises a V.sub.H peptide and a
V.sub.L peptide, wherein the V.sub.H peptide comprises a sequence
as set forth in SEQ ID NO: 12 and the V.sub.L peptide comprises a
sequence as set forth in SEQ ID NO: 11. In some embodiments, an
antibody, or antigen binding fragment thereof, comprises a V.sub.H
peptide and a V.sub.L peptide, wherein the V.sub.H peptide
comprises a sequence as set forth in SEQ ID NO: 14 and the V.sub.L
peptide comprises a sequence as set forth in SEQ ID NO: 13. In some
embodiments, an antibody, or antigen binding fragment thereof,
comprises a V.sub.H peptide and a V.sub.L peptide, wherein the
V.sub.H peptide comprises a sequence as set forth in SEQ ID NO: 16
and the V.sub.L peptide comprises a sequence as set forth in SEQ ID
NO: 15. In some embodiments, an antibody, or antigen binding
fragment thereof, comprises a V.sub.H peptide and a V.sub.L
peptide, wherein the V.sub.H peptide comprises a sequence as set
forth in SEQ ID NO: 80 and the V.sub.L peptide comprises a sequence
as set forth in SEQ ID NO: 79. In some embodiments, an antibody, or
antigen binding fragment thereof, comprises a V.sub.H peptide and a
V.sub.L peptide, wherein the V.sub.H peptide comprises a sequence
as set forth in SEQ ID NO: 83 and the V.sub.L peptide comprises a
sequence as set forth in SEQ ID NO: 3. In some embodiments, an
antibody, or antigen binding fragment thereof, comprises a V.sub.H
peptide and a V.sub.L peptide, wherein the V.sub.H peptide
comprises a sequence as set forth in SEQ ID NO: 14 and the V.sub.L
peptide comprises a sequence as set forth in SEQ ID NO: 86.
[0153] In some embodiments, an antibody, or antigen binding
fragment thereof, comprises a LC peptide as set forth in SEQ ID
NOs: 1, 3, 5, 7, 9, or 11, or any combination thereof. The LC
peptide can comprise a variant of any of these sequences as
provided for herein.
[0154] In some embodiments, an antibody, or antigen binding
fragment thereof, comprises a HC peptide as set forth in SEQ ID
NOs: 2, 4, 6, 8, 10, 12, or 83, or any combination thereof. The HC
peptide can comprise a variant of any of these sequences as
provided for herein.
[0155] In some embodiments, an antibody, or antigen binding
fragment thereof, comprises a HC peptide and a LC peptide, wherein
the wherein the HC peptide comprises a sequence as set forth in SEQ
ID NO: 2, 4, 6, 8, 10, 12, or 83 and the LC peptide comprises a
sequence as set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11. In some
embodiments, an antibody, or antigen binding fragment thereof,
comprises a HC peptide and a LC peptide, wherein the HC peptide
comprises a sequence as set forth in SEQ ID NO: 2 and the LC
peptide comprises a sequence as set forth in SEQ ID NO: 1. In some
embodiments, an antibody, or antigen binding fragment thereof,
comprises a HC peptide and a LC peptide, wherein the HC peptide
comprises a sequence as set forth in SEQ ID NO: 4 and the LC
peptide comprises a sequence as set forth in SEQ ID NO: 3. In some
embodiments, the HC peptide comprising the sequence as set forth in
SEQ ID NO: 4 has an additional C terminal lysine (K) residue. In
some embodiments, an antibody, or antigen binding fragment thereof,
comprises a HC peptide and a LC peptide, wherein the HC peptide
comprises a sequence as set forth in SEQ ID NO: 6 and the LC
peptide comprises a sequence as set forth in SEQ ID NO: 5. In some
embodiments, an antibody, or antigen binding fragment thereof,
comprises a HC peptide and a LC peptide, wherein the HC peptide
comprises a sequence as set forth in SEQ ID NO: 8 and the LC
peptide comprises a sequence as set forth in SEQ ID NO: 7. In some
embodiments, an antibody, or antigen binding fragment thereof,
comprises a HC peptide and a LC peptide, wherein the HC peptide
comprises a sequence as set forth in SEQ ID NO: 10 and the LC
peptide comprises a sequence as set forth in SEQ ID NO: 9. In some
embodiments, an antibody, or antigen binding fragment thereof,
comprises a HC peptide and a LC peptide, wherein the HC peptide
comprises a sequence as set forth in SEQ ID NO: 12 and the LC
peptide comprises a sequence as set forth in SEQ ID NO: 11. In some
embodiments, an antibody, or antigen binding fragment thereof,
comprises a HC peptide and a LC peptide, wherein the HC peptide
comprises a sequence as set forth in SEQ ID NO: 83 and the LC
peptide comprises a sequence as set forth in SEQ ID NO: 3.
[0156] In addition to these specific combinations any of the
V.sub.H peptides and the V.sub.L peptides can be combined with one
another.
[0157] In addition to these specific combinations any of the HC
peptides and the LC peptides can be combined with one another.
[0158] In some embodiments, the antibody comprises a sequence, or
antigen binding fragment of ATCC clone PTA-7444. The sequence of
the antibody produced by ATCC clone PTA-7444 is hereby incorporated
by reference in its entirety, which includes the antigen binding
fragments thereof.
[0159] Additionally, as provided for herein, the antibodies can be
multi-specific antibodies, in that the antibodies have multiple
binding regions that target different proteins or the same protein
at different epitopes. In some embodiments, the antibody is a
bi-specific antibody.
[0160] As provided for herein, the different peptides (V.sub.H or
V.sub.L) described herein can be linked with a peptide linker or
not linked with a peptide linker and instead for a contiguous
sequence. In some embodiments, the peptide linker comprises a
sequence of: (GGGGS).sub.n (SEQ ID NO: 73); (GGGGA).sub.n (SEQ ID
NO: 74), or any combination thereof, wherein each n is
independently 1-5. The linked peptide format can be represented by
a formula of V.sub.H-Z-V.sub.L or V.sub.L-Z-V.sub.H, wherein Z is
the peptide linker. In some embodiments, Z is (GGGGS).sub.n (SEQ ID
NO: 73); (GGGGA).sub.n (SEQ ID NO: 74), or any combination thereof,
wherein each n is independently 1-5.
[0161] As provided for herein, the antibodies, or antigen binding
fragments thereof can be variants of the sequences.
[0162] Other examples of antibodies include, but are not limited
to, those provided in US20160096894A1, EP1399483B1, EP2194067B1,
US20040202651A1, US20110229933A1, U.S. Pat. No. 8,137,933B2, U.S.
Pat. No. 8,951,790B2, US20190270820A1, U.S. Pat. No. 7,572,897B2,
US20090275126A1, EP1959014B1, US20080014203A1, US20080226635A1,
US20120076778A1, US20190153071A1, WO2011161119A1, U.S. Ser. No.
10/611,825B2, US20120237507A1, EP2681240B1, U.S. Pat. No.
9,982,036B2, US20180312573A1, EP2681239B1, US20160151487A1,
US20190225696A1, WO2017011773A2, US20200023076A1, US20190153471A1,
US20190194713A1, WO2020006486A1, US20080112888A1, US20150168424A1,
EP2032989B2, U.S. Pat. No. 9,045,536B2, each of which is hereby
incorporated by reference in its entirety. Other examples of
antibodies include, but are not limited to, those provided in U.S.
Pat. No. 8,153,121B2, EP1469879B1, WO2016064716A1, US20190270820A1,
US20180280527A1, US20190225696A1, U.S. Pat. No. 7,998,681B2,
US20040202651A1, US20050136063A1, US20090285824A1, US20150274829A1,
EP2322550B1, US20060286103A1, US20070071675A1, US20100047239A1,
US20130004416A1, US20080112888A1, US20150168424A1, US20100143340A1,
US20110014117A1, US20100260668A1, US20100074900A1, US20150017168A1,
US20110044980A1, US20130330323A1, US20120263722A1, US20120201746A1,
U.S. Ser. No. 10/519,245B2, US20180243432A1, US20170218091A1,
US20200115460A1, US20100104645A1, US20120065380A1, EP2970433B1,
US20160289341A1, US20160289343A1, US20190293656A1, each of which is
hereby incorporated by reference in its entirety.
[0163] In some embodiments, the antibody comprises a heavy and a
light chain, wherein the heavy chain comprises a sequence of:
TABLE-US-00009 (SEQ ID NO: 92)
QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGE
INPSNGRTNYNQKFQGKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGR
PDYYGSSKWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK;
and the light chain comprises a sequence of:
TABLE-US-00010 (SEQ ID NO: 93)
DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPR
LLIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVP
PTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC.
[0164] In some embodiments, the antibody comprises a heavy and a
light chain, wherein the heavy chain comprises a sequence of:
TABLE-US-00011 (SEQ ID NO: 94)
QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGE
INPSNGRTNYNQKFQGKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGR
PDYYGSSKWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG;
and the light chain comprises a sequence of
TABLE-US-00012 (SEQ ID NO: 93)
DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPR
LLIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVP
PTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC.
[0165] In some embodiments, the heavy chain of SEQ ID NO: 94
comprises a C-terminal lysine residue that is added to the
C-terminus of SEQ ID NO: 94.
[0166] In some embodiments, the antibody comprises a heavy and a
light chain, wherein the heavy chain comprises a sequence of:
TABLE-US-00013 (SEQ ID NO: 95)
QVQLVQSGAEVVKPGASVKLSSKASGYTFTSYWMHWVKQRPGQGLEWIGE
INPSNGRTNYNQKFQGKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGR
PDYYGSSKWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLE
PPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSL SPG;
[0167] and the light chain comprises a sequence of SEQ ID NO:
93
[0168] In some embodiments, the heavy chain of SEQ ID NO: 95
comprises a C-terminal lysine residue that is added to the
C-terminus of SEQ ID NO: 95.
[0169] In some embodiments, the antibody comprises a heavy chain
and light chain, wherein the heavy chain comprises a sequence of
SEQ ID NO: 83 and the and the light chain comprises a sequence of
SEQ ID NO: 3.
[0170] In some embodiments, the antibody comprises a VH sequence of
SEQ ID NO: 96 and a VL sequence of SEQ ID NO: 13 or SEQ ID NO: 97.
In some embodiments, the antibody comprises a VH of SEQ ID NO: 14
and a VL sequence of SEQ ID NO: 97.
[0171] Pharmaceutical Compositions
[0172] In some embodiments, to prepare pharmaceutical or sterile
compositions of the anti-IGF-1R antibodies or other proteins
provided herein, the antibody or antigen binding fragment thereof
or other proteins provided herein are admixed with a
pharmaceutically acceptable carrier or excipient. See, e.g.,
Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National
Formulary, Mack Publishing Company, Easton, Pa. (1984).
[0173] Formulations of therapeutic and diagnostic agents may be
prepared by mixing with acceptable carriers, excipients, or
stabilizers in the form of, e.g., lyophilized powders, slurries,
aqueous solutions or suspensions (see, e.g., Hardman, et al. (2001)
Goodman and Gilman's The Pharmacological Basis of Therapeutics,
McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science
and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New
York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms:
Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.)
(1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY;
Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:
Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000)
Excipient Toxicity and Safety, Marcel Dekker, Inc., New York,
N.Y.). In some embodiments embodiment, the antibodies are diluted
to an appropriate concentration in a sodium acetate solution pH
5-6, and NaCl or sucrose is added for tonicity. Additional agents,
such as polysorbate 20 or polysorbate 80, may be added to enhance
stability.
[0174] Toxicity and therapeutic efficacy of the antibody
compositions, administered alone or in combination with another
agent, can be determined by standard pharmaceutical procedures in
cell cultures or experimental animals, e.g., for determining the
LD.sub.50 (the dose lethal to 50% of the population) and the
ED.sub.50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index (LD.sub.50/ ED.sub.50). In particular
aspects, antibodies exhibiting high therapeutic indices are
desirable. The data obtained from these cell culture assays and
animal studies can be used in formulating a range of dosage for use
in human. The dosage of such compounds lies preferably within a
range of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration.
[0175] In some embodiments, a composition of the invention is
administered to a subject in accordance with the Physicians' Desk
Reference 2003 (Thomson Healthcare; 57th edition (Nov. 1,
2002)).
[0176] The mode of administration can vary. Suitable routes of
administration include oral, rectal, transmucosal, intestinal,
parenteral; intramuscular, subcutaneous, intradermal,
intramedullary, intrathecal, direct intraventricular, intravenous,
intraperitoneal, intranasal, intraocular, inhalation, insufflation,
topical, cutaneous, transdermal, or intra-arterial.
[0177] In some embodiments, the antibody or antigen binding
fragment thereof can be administered by an invasive route such as
by injection. In some embodiments, the antibodies or antigen
binding fragment thereof, or pharmaceutical composition thereof, is
administered intravenously, subcutaneously, intramuscularly,
intraarterially, intra-articularly (e.g. in arthritis joints), or
by inhalation, aerosol delivery. Administration by non-invasive
routes (e.g., orally; for example, in a pill, capsule or tablet) is
also within the scope of the present embodiments.
[0178] In some embodiments, the antibody or antigen binding
fragment thereof can be administered directly to the eye, the
anterior chamber of the eye, the vitreous chamber of the eye, the
suprachoroidal space, or the retro-orbital sinus. In some
embodiments, administration to the eye, the anterior chamber of the
eye, the vitreous chamber of the eye, the suprachoroidal space, or
the retro-orbital sinus is via an injection. In some embodiments,
the injection is an intravitreal injection, intraorbital injection,
retro-orbital injection, suprachoroidal injection, or intracameral
injection. In some embodiments, the injection is an intravitreal
injection. In some embodiments, the injection is an, intraorbital
injection. In some embodiments, the injection is a retro-orbital
injection. In some embodiments, the injection is a suprachoroidal
injection. In some embodiments, the injection is an intracameral
injection.
[0179] In some embodiments, the anti-IGF-1R antibody, or antigen
binding fragment thereof, is administered in combination with at
least one additional therapeutic agent, such as, but not limited to
any therapeutic used to treat thyroid eye disease. For example, in
some embodiments, the anti-IGF-1R antibody, or antigen binding
fragment thereof, is administered in combination with at least one
additional therapeutic agent, such as, but not limited to a
therapeutic used to treat thyroid eye disease or a condition
related to the same. Examples of such treatments and therapeutics
include, but are not limited to anti-thyroid medications, diabetes
medications, beta-blockers, propylthiouracil, methimazole,
propranolol, atenolol, metoprolol, nadolol, cortico steroids,
metformin, sulfonylureas, meglitinides, thiazolidinediones, DPP-4
inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, regular
insulin, insulin aspart, insulin glulisine, insulin lispro, insulin
isophane, insulin degludec, insulin detemir, insulin glargine,
acerbose, miglitol, acebutolol, atenolol, betaxolol, bisoprolol,
cartelol, carvedilol, esmolol, labetalol, metoprolol, nadolol,
nebivolol, penbutolol, pindolol, propranolol, sotalol, timolol,
tomolol ophthalmic solution, sitagliptin, saxagliptin, linagliptin,
alogliptin, dulaglutide, exenatide, semaglutide, liraglutide,
lixisenatide, canagliflozin, dapagliflozin, empagliflozin, or any
combination thereof.
[0180] Compositions can be administered with medical devices known
in the art. For example, a pharmaceutical composition of the
invention can be administered by injection with a hypodermic
needle, including, e.g., a prefilled syringe or autoinjector.
[0181] The pharmaceutical compositions may also be administered
with a needleless hypodermic injection device; such as the devices
disclosed in U.S. Pat. Nos. 6,620,135; 6,096,002; 5,399,163;
5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or
4,596,556.
[0182] The pharmaceutical compositions may also be administered by
infusion. Examples of well-known implants and modules form
administering pharmaceutical compositions include: U.S. Pat. No.
4,487,603, which discloses an implantable micro-infusion pump for
dispensing medication at a controlled rate; U.S. Pat. No.
4,447,233, which discloses a medication infusion pump for
delivering medication at a precise infusion rate; U.S. Pat. No.
4,447,224, which discloses a variable flow implantable infusion
apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196,
which discloses an osmotic drug delivery system having
multi-chamber compartments. Many other such implants, delivery
systems, and modules are well known to those skilled in the
art.
[0183] Alternately, one may administer the antibody in a local
rather than systemic manner, for example, via injection of the
antibody directly into an arthritic joint or pathogen-induced
lesion characterized by immunopathology, often in a depot or
sustained release formulation. Furthermore, one may administer the
antibody in a targeted drug delivery system, for example, in a
liposome coated with a tissue-specific antibody, targeting, for
example, arthritic joint or pathogen-induced lesion characterized
by immunopathology. The liposomes will be targeted to and taken up
selectively by the afflicted tissue.
[0184] The administration regimen depends on several factors,
including the serum or tissue turnover rate of the therapeutic
antibody, the level of symptoms, the immunogenicity of the
therapeutic antibody, and the accessibility of the target cells in
the biological matrix. Preferably, the administration regimen
delivers sufficient therapeutic antibody to effect improvement in
the target disease state, while simultaneously minimizing undesired
side effects. Accordingly, the amount of biologic delivered depends
in part on the particular therapeutic antibody and the severity of
the condition being treated. Guidance in selecting appropriate
doses of therapeutic antibodies is available (see, e.g.,
Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd,
Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies,
Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.)
(1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune
Diseases, Marcel Dekker, New York, N.Y.; Baert, et al. (2003) New
Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med.
341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792;
Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et
al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New
Engl. J. Med. 343:1594-1602).
[0185] Determination of the appropriate dose is made by the
clinician, e.g., using parameters or factors known or suspected in
the art to affect treatment. Generally, the dose begins with an
amount somewhat less than the optimum dose and it is increased by
small increments thereafter until the desired or optimum effect is
achieved relative to any negative side effects. Important
diagnostic measures include those of symptoms of, e.g., the
inflammation or level of inflammatory cytokines produced. In
general, it is desirable that a biologic that will be used is
derived from the same species as the animal targeted for treatment,
thereby minimizing any immune response to the reagent. In the case
of human subjects, for example, chimeric, humanized and fully human
antibodies are may be desirable.
[0186] Antibodies or antigen binding fragments thereof can be
provided by continuous infusion, or by doses administered, e.g.,
daily, 1-7 times per week, weekly, bi-weekly, monthly, bimonthly,
quarterly, semiannually, annually etc. Doses may be provided, e.g.,
intravenously, subcutaneously, topically, orally, nasally,
rectally, intramuscular, intracerebrally, intraspinally, or by
inhalation. In some embodiments, the antibody is administered every
three weeks, every four weeks, every five weeks, every six weeks,
every seven weeks, or every eight weeks. In some embodiments, the
antibody is administered every four weeks. In some embodiments, the
antibody is administered every five weeks. In some embodiments, the
antibody is administered every seven weeks. In some embodiments,
the antibody is administered every six weeks. In some embodiments,
the antibody is administered every eight weeks. In some
embodiments, the antibody is administered for at least 21-52 weeks
or longer. In some embodiments, the antibody is administered on
such a schedule for at least 21 weeks. In some embodiments, the
antibody is administered on such a schedule for at least 24 weeks.
In some embodiments, the antibody is administered on such a
schedule for at least 32 weeks. In some embodiments, the antibody
is administered on such a schedule for at least 36 weeks. In some
embodiments, the antibody is administered on such a schedule for at
least 40 weeks. In some embodiments, the antibody is administered
on such a schedule for at least 42 weeks. In some embodiments, the
antibody is administered (e.g. infusion or subcutaneous injection)
once. In some embodiments, the antibody is administered (e.g.
infusion or subcutaneous injection) twice. In some embodiments, the
antibody is administered (e.g. infusion or subcutaneous injection)
three times. In some embodiments, the antibody is administered
(e.g. infusion or subcutaneous injection) four times. In some
embodiments, the antibody is administered (e.g. infusion or
subcutaneous injection) five times. In some embodiments, the
antibody is administered (e.g. infusion or subcutaneous injection)
six times. In some embodiments, the antibody is administered (e.g.
infusion or subcutaneous injection) seven times. In some
embodiments, the antibody is administered (e.g. infusion or
subcutaneous injection) eight times. In some embodiments, the
antibody is administered (e.g. infusion or subcutaneous injection)
nine times. In some embodiments, the antibody is administered (e.g.
infusion or subcutaneous injection) 10 times. In some embodiments,
the antibody is administered (e.g. infusion or subcutaneous
injection) 11 times. In some embodiments, the antibody is
administered (e.g. infusion or subcutaneous injection) 12 times. In
some embodiments, the antibody is administered (e.g. infusion or
subcutaneous injection) 13 times. In some embodiments, the antibody
is administered (e.g. infusion or subcutaneous injection) 14 times.
In some embodiments, the antibody is administered (e.g. infusion or
subcutaneous injection) 15 times. In some embodiments, the antibody
is administered (e.g. infusion or subcutaneous injection) 16 times.
In some embodiments, the antibody is administered (e.g. infusion or
subcutaneous injection) 17 times. In some embodiments, the antibody
is administered (e.g. infusion or subcutaneous injection) 18 times.
In some embodiments, the antibody is administered (e.g. infusion or
subcutaneous injection) 19 times. In some embodiments, the antibody
is administered (e.g. infusion or subcutaneous injection) 20 times.
When the antibody is administered more than once it can be
administered according to a schedule, such as the schedules
provided for herein.
[0187] A total weekly dose can be as provided for herein. In some
embodiments, the total weekly dose is at least 0.05 .mu.g/kg body
weight, more generally at least 0.2 .mu.g/kg, 0.5 .mu.g/kg, 1
.mu.g/kg, 10 .mu.g/kg, 100 .mu.g/kg, 0.25 .mu.g/kg, 1.0 .mu.g/kg,
2.0 .mu.g/kg, 5.0 .mu.g/ml, 10 .mu.g/kg, 25 .mu.g/kg, 50 .mu.g/kg
or more (see, e.g., Yang, et al. (2003) New Engl. J. Med.
349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:1692-1698;
Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456;
Portielji, et al. (20003) Cancer Immunol. Immunother. 52:133-144).
Doses may also be provided to achieve a pre-determined target
concentration of the antibody in the subject's serum, such as 0.1,
0.3, 1, 3, 10, 30, 100, 300 .mu.g/ml or more.
[0188] In some embodiments, the antibody has a serum concentration
in the subject of at least, or about, 10 .mu.g/ml or 20 .mu.g/ml or
50 .mu.g/ml, 70 .mu.g/ml, 75 .mu.g/ml, 80 .mu.g/ml, 85 .mu.g/ml, 90
.mu.g/ml, 95 .mu.g/ml, 100 .mu.g/ml, or 105 .mu.g/ml at least 1, 2,
or 3 weeks after administration.
[0189] In some embodiments, a dose of 20 .mu.g/kg IV is
administered. In some embodiments, a dosing is used to provide a
Cmin of 133 ug/mL after about 5 weeks. In some embodiments, the
dose of the antibody that is administered that provides a Cmin of
102 ug/mL after 6 weeks. In some embodiments, the dose of the
antibody is as provided for herein, such as 10 mg/mg as a loading
dose with subsequent doses being the same or lower. In some
embodiments, the antibody is administered as provided for herein at
a dose to achieve a Cmin of at least, or about, 100 ug/mL.
[0190] As used herein, "inhibit" or "treat" or "treatment" includes
a postponement of development of the symptoms associated with a
disorder and/or a reduction in the severity of the symptoms of such
disorder. The terms further include ameliorating existing
uncontrolled or unwanted symptoms, preventing additional symptoms,
and ameliorating or preventing the underlying causes of such
symptoms. Thus, the terms denote that a beneficial result has been
conferred on a vertebrate subject with a disorder, disease or
symptom, or with the potential to develop such a disorder, disease
or symptom.
[0191] As used herein, the terms "therapeutically effective
amount", "therapeutically effective dose" and "effective amount"
refer to an amount of the antibody, or antigen binding fragment
thereof, that, when administered alone or in combination with an
additional therapeutic agent to a cell, tissue, or subject, is
effective to cause a measurable improvement in one or more symptoms
of a disease or condition or the progression of such disease or
condition. A therapeutically effective dose further refers to that
amount of the binding compound sufficient to result in at least
partial amelioration of symptoms, e.g., treatment, healing,
prevention or amelioration of the relevant medical condition, or an
increase in rate of treatment, healing, prevention or amelioration
of such conditions. When applied to an individual active ingredient
administered alone, a therapeutically effective dose refers to that
ingredient alone. When applied to a combination, a therapeutically
effective dose refers to combined amounts of the active ingredients
that result in the therapeutic effect, whether administered in
combination, serially or simultaneously. An effective amount of a
therapeutic will result in an improvement of a diagnostic measure
or parameter by at least 10%; usually by at least 20%; preferably
at least about 30%; more preferably at least 40%, and most
preferably by at least 50%. An effective amount can also result in
an improvement in a subjective measure in cases where subjective
measures are used to assess disease severity. In some embodiments,
an amount is a therapeutically effective amount if it is an amount
that can be used to treat or ameliorate a condition as provided for
herein.
[0192] The term "subject" as used throughout includes any organism,
such as an animal, including a mammal (e.g., rat, mouse, dog, cat,
rabbit) and, for example, a human. A subject can be also be
referred to as a patient. In some embodiments, the subject is a
subject in need thereof. A subject that is "in need thereof" refers
to a subject that has been identified as requiring treatment for
the condition that is to be treated and is treated with the
specific intent of treating such condition. The conditions can be,
for example, any of the conditions described herein.
[0193] Whereas, an isolated antibody binds an epitope on a IGF-1R
protein, or other protein described herein, and displays in vitro
and/or in vivo IGF-1R inhibiting or therapeutic activities, the
antibodies or antigen binding fragments thereof, capable of
inhibiting IGF-1R function, are suitable both as therapeutic agents
for treating IGF-1R-associated conditions in humans and animals.
These conditions include thyroid eye disease. According, methods of
treating such conditions are also provided, wherein the method
comprises administering an antibody, or antigen binding fragment
thereof, to the subject with such a condition.
[0194] In some embodiments, the methods comprise administering a
therapeutically or prophylactically effective amount of one or more
monoclonal antibodies or antigen binding fragments of the
antibodies described herein to a susceptible subject or to one
exhibiting a condition in which IGF-1R is known or suspected to
have caused the pathology observed. Any active form of the antibody
can be administered, including, but not limited to scFV, Fab and
F(ab')2 fragments and other forms of antibodies provided for
herein.
[0195] As used herein, a IGF-1R associated pathology refers to
conditions that are caused by the modulation of IGF-1R. These
conditions include, but are not limited to, thyroid eye disease and
other conditions provided for herein.
[0196] In some embodiments, the antibodies used are compatible with
the recipient species such that the immune response to the MAbs
does not result in an unacceptably short circulating half-life or
induce an immune response to the MAbs in the subject.
[0197] Treatment of individuals may comprise the administration of
a therapeutically effective amount of the antibodies described
herein. The antibodies can be provided in a kit, such as those
provided herein. The antibodies can be used or administered alone
or in admixture with another therapeutic, analgesic, or diagnostic
agent, such as provided for herein. In providing a patient with an
antibody, or fragment thereof, capable of binding to IGF-1R, or an
antibody capable of protecting against IGF-1R, pathology in a
recipient patient, the dosage of administered agent will vary
depending upon such factors as the patient's age, weight, height,
sex, general medical condition, previous medical history, etc.
[0198] An antibody, capable treating a condition associated with
IGF-1R activity or use to treat a IGF-1R related pathology, is
intended to be provided to subjects in an amount sufficient to
affect a reduction, resolution, or amelioration in the IGF-1R
related symptom or pathology. Such a pathology includes, thyroid
eye disease and the like
[0199] Accordingly, in some embodiments, methods of treating a
subject with a IGF-1R mediated disorder are provided. In some
embodiments, the method comprises administering a pharmaceutical
composition comprising an antibody, or antigen binding fragment
thereof, as provided herein. In some embodiments, the disorder is
thyroid eye disease. As provided for herein, the antibodies, or
antigen binding fragments thereof, can be administered with other
therapeutics. These can be administered simultaneously or
sequentially.
[0200] In some embodiments, the antibodies, or antigen binding
fragments thereof, may be used to treat thyroid eye disease. In
some embodiments, the antibodies, or antigen binding fragments
thereof, may be used to treating or reduce the severity of,
thyroid-associated ophthalmopathy (TAO), or a symptom thereof.
[0201] In some embodiments, methods or uses are provided to reduce
proptosis in an eye in a subject with thyroid-associated
ophthalmopathy (TAO).
[0202] In some embodiments, the subject is a subject how has
previously been treated with a different antibody than those
provided herein.
[0203] In some embodiments, methods or uses are provided to
Clinical Activity Score (CAS) in subject who has or is suspected of
having thyroid-associated ophthalmopathy (TAO).
[0204] In some embodiments, methods or uses are provided to reduce
proptosis by at least 2 mm and b) reducing the clinical activity
score (CAS) in a subject with thyroid-associated ophthalmopathy
(TAO).
[0205] As used herein, the term Clinical Activity Score (CAS)
refers to the protocol described and scored according to Table 2.
According to this protocol, one point is given for the presence of
each of the parameters assessed in the Table below. The sum of all
points defines clinical activity and provides the CAS, where 0 or 1
constitutes inactive disease and 7 severe active
ophthalmopathy.
TABLE-US-00014 TABLE 2 Parameters for calculating Clinical Activity
Score Item No. Parameter 1 Spontaneous retrobulbar pain 2 Pain on
attempted eye movements (upward, Side to side, and downward gazes;
sometimes termed gaze evoked orbital pain 3 Eyelid swelling 4
Eyelid erythema (redness) 5 Conjunctival redness 6 Chemosis
(swelling/edema of the conjunctiva) 7 Swelling of caruncle or
pila
[0206] As provided in Table 2, the CAS consists of seven
components: spontaneous retrobulbar pain, pain on attempted eye
movements (upward, side-to-side, and downward gazes), conjunctival
redness, redness of the eyelids, chemosis, swelling of the
caruncle/plica, and swelling of the eyelids. Each component is
scored as present (1 point) or absent (0 points). The score at each
efficacy assessment is the sum of all items present; giving a range
of 0-7, where 0 or 1 constitutes inactive disease and 7 severe
active ophthalmopathy. A change of >2 points is considered
clinically meaningful.
[0207] Item 1, spontaneous orbital pain could be a painful, or
oppressive feeling on, or behind, the globe. This pain may be
caused by the rise in intraorbital pressure, when the orbital
tissues volume increases through excess synthesis of extracellular
matrix, fluid accumulation, and cellular infiltration and
expansion. Item 2, gaze evoked orbital pain, could be pain in the
eyes when looking, or attempting to look, up, down or sideways,
i.e., pain with upward, downward, or lateral eye movement, or when
attempting eye movement. This kind of pain could arise from the
stretching of the inflamed muscle(s), especially on attempted
upgaze. The `stretching pain` cannot be provoked by digital
pressing on the eyeball, as would be expected if it were a
manifestation of the raised intraorbital pressure. Both kinds of
pain can be reduced after anti-inflammatory treatment. These kinds
of pain are therefore considered to be directly related to
autoimmune inflammation in the orbit and thus useful in assessing
TAO activity.
[0208] Swelling in TAO is seen as chemosis (edema of the
conjunctiva), item no. 6 in Table 1, and swelling of the caruncle
and/or plica semilunaris. Both are signs of TAO activity. Swollen
eyelids can be caused by edema, fat prolapse through the orbital
septum, or fibrotic degeneration. In addition to swelling, other
symptoms indicative of active TAO include redness and/or pain of
the conjunctiva, eyelid, caruncle and/or plica semilunaris.
[0209] In some embodiments, the subject who is treated has the
proptosis is reduced by at least 2 mm. In some embodiments, the
subject who is treated has the proptosis is reduced by at least 3
mm. In some embodiments, the subject who is treated has the
proptosis is reduced by at least 4 mm.
[0210] In some embodiments, in the subjects who are treated the
clinical activity score (CAS) of the subject is reduced by at least
2 points. In some embodiments, the clinical activity score (CAS) of
the subject is reduced to one (1). In some embodiments, the
clinical activity score (CAS) of the subject is reduced to zero
(0).
[0211] In some embodiments, methods off treating or reducing the
severity of thyroid-associated ophthalmopathy (TAO) in a subject
are provided, wherein the treatment with said antibody (i) reduces
proptosis by at least 2 mm in an eye; (ii) is not accompanied by a
deterioration of 2 mm or more in the other (or fellow eye); and
(iii) reduces the CAS in said subject to either one (1) or zero
(0).
[0212] In some embodiments, methods of improving the quality of
life in a subject with thyroid-associated ophthalmopathy (TAO, also
called Graves' Ophthalmopathy/Graves' Orbitopathy) are provided. In
some embodiments, the quality of life is measured by the Graves'
Ophthalmopathy Quality of Life (GO-QoL) assessment, or either the
Visual Functioning or Appearance subscale thereof. In some
embodiments, the treatment results in an improvement of greater
than or equal to 8 points on the GO-QoL. In some embodiments, the
treatment results in an improvement on the Functioning subscale of
the GO-QoL. In some embodiments, the treatment results in an
improvement on the Appearance subscale of the GO-QoL.
[0213] In some embodiments, methods of treating or reducing the
severity of diplopia in a subject with thyroid-associated
ophthalmopathy (TAO) are provided. In some embodiments, the
diplopia is constant diplopia. In some embodiments, the diplopia is
inconstant diplopia. In some embodiments, the diplopia is
intermittent diplopia. In some embodiments, the improvement in or
reduction in severity of diplopia is sustained at least 20 weeks
after discontinuation of antibody administration. In some
embodiments, the improvement in or reduction in severity of
diplopia is sustained at least 50 weeks after discontinuation of
antibody administration.
[0214] The severity of the disease can be measured in the following
non-limiting embodiments. For example, for lid aperture, the
distance between the lid margins are measured (in mm) with the
patient looking in the primary position, sitting relaxed, and with
distant fixation. For swelling of the eyelids, the
measure/evaluation is either "absent/equivocal," "moderate," or
"severe." Redness of the eyelids is either absent or present.
Redness of the conjunctivae is either absent or present. In some
embodiments, conjunctival edema is either absent or present. In
some embodiments, inflammation of the caruncle or plica is either
absent or present. Exophthalmos is measured in millimeter using the
same Hertel exophthalmometer and same intercanthal distance for an
individual patient. Subjective diplopia is scored from 0 to 3 (0=no
diplopia; 1=intermittent, i.e., diplopia in primary position of
gaze, when tired or when first awakening; 2=inconstant, i.e.,
diplopia at extremes of gaze; 3=constant, i.e., continuous diplopia
in primary or reading position). For eye muscle involvement, the
ductions are measured in degrees. Corneal involvement is either
absent/punctate or keratopathy/ulcer. For optic nerve involvement,
i.e., best-corrected visual acuity, color vision, optic disc,
relative afferent pupillary defect, the condition is either absent
or present. In addition, visual fields are checked if optic nerve
compression is suspected. In some embodiments, the patient can be
classified according to the following severity classification. For
example, sight-Threatening Thyroid Eye Disease: Patients with
dysthyroid optic neuropathy (DON) and/or corneal breakdown. This
category warrants immediate intervention. Moderate-to-Severe
Thyroid Eye Disease: Patients without sight-threatening disease
whose eye disease have sufficient impact on daily life to justify
the risks of immunosuppression (if active) or surgical intervention
(if inactive). Patients with moderate-to-severe thyroid eye disease
usually have any one or more of the following: lid retraction
greater than or equal to 2 mm, moderate or severe soft tissue
involvement, exophthalmos greater than or equal to 3 mm above
normal for race and gender, inconstant or constant diplopia. Mild
Thyroid Eye Disease: Patients whose features of thyroid eye disease
have only a minor impact on daily life insufficient to justify
immunosuppressive or surgical treatment. They usually have only one
or more of the following: minor lid retraction (<2 mm), mild
soft tissue involvement, exophthalmos <3 mm above normal for
race and gender, transient or no diplopia, and corneal exposure
responsive to lubricants.
[0215] In some embodiments, a patient can be characterized by
Graves Ophthalmopathy Quality of Life (GO-QoL) score. In addition
to proptosis (or exophthalmos) and CAS, quality of life is also
evaluated with the use of the GO quality of life (GO-QoL)
questionnaire. This questionnaire is designed to determine the
improved quality of life after treatment with a method disclosed
herein. In some embodiments, questionnaire may determine the
decreased or lack of side effects after being treated with an
antibody, or an antigen binding fragment thereof, according to a
method disclosed herein as compared to treatment with
glucocorticoids. The GO-QoL is a 16-item self-administered
questionnaire divided into 2 subsets and used to assess the
perceived effects of TED by the subjects on (i) their daily
physical activity as it relates to visual function, and (ii)
psychosocial functioning. Quality of life is evaluated with the use
of the GO QoL questionnaire. The GO-QoL questionnaire [C. B. Terwee
et al, 1998] is completed on Day 1 and Weeks 6, 12, and 24 (or PW)
during the Treatment Period, and at Months 7 and 12 (or PW) during
the Follow-Up Period. The GO-QoL is a 16-item self-administered
questionnaire divided into two self-assessment subscales; one
covering impact of visual function on daily activities, the other
assesses the impact of self-perceived appearance. The visual
function subscale covers activities such as driving, walking
outdoors, reading, watching television. The appearance subscale
asks the subject questions such as whether ophthalmopathy has
altered the subject's appearance, caused other people to have a
negative reaction to the subject, caused social isolation, and
caused the subject to try to mask his or her appearance. Each
subscale has 8 questions which are answered with: yes--very much
so; yes--a little; or no--not at all. Each question is scored 0-2,
respectively, and the total raw score is then mathematically
transformed to a 0-100 scale, where 0 represents the most negative
impact on quality of life, and 100 represents no impact. A change
of > or greater than equal to 8 points on the 0-100 scale has
been shown to be clinically meaningful. The combined score takes
raw scores from both subscales and again transforms them to a
single 0-100 scale. The questionnaire has two self-assessment
subscales. Each subscale has 8 questions which are answered with:
(i) yes--very much so; (ii) yes--a little; or (iii) no--not at all.
Each question is scored 0-2, respectively, and the total raw score
is then mathematically transformed to a 0-100 scale, where 0
represents the most negative impact on quality of life, and 100
represents no impact. A change of >8 points on the 0-100 scale
is considered to be clinically meaningful. The combined score takes
raw scores from both subscales and again transforms them to a
single 0-100 scale.
[0216] Patients can also be assessed by the presence of absence of
Gorman Grading of Diplopia. The Gorman assessment of subjective
diplopia includes four categories: no diplopia (absent), diplopia
when the patient is tired or awakening (intermittent), diplopia at
extremes of gaze (inconstant), and continuous diplopia in the
primary or reading position (constant). Patients are scored
according to which grade of diplopia they are experiencing. An
improvement of greater than equal or to 1 grade is considered
clinically meaningful.
[0217] In some embodiments, the methods comprise administering an
antibody, such as those provided herein. In some embodiments, the
antibody is administered at a dosage of about 1 .mu.g/kg to about 5
.mu.g/kg antibody as a first dose. In some embodiments, the
antibody is administered at a dosage of about 5 .mu.g/kg to about
10 .mu.g/kg antibody as a first dose. In some embodiments, the
antibody is administered at a dosage of about 5 .mu.g/kg to about
20 .mu.g/kg antibody in subsequent doses. In some embodiments, the
antibody is administered in the following amounts: about 10
.mu.g/kg antibody as a first dose; and about 20 .mu.g/kg antibody
in subsequent doses. In some embodiments, the subsequent doses are
administered every three weeks for at least 21 weeks.
[0218] In some embodiments, the antibody is administered in a
pharmaceutical composition, such as those provided herein. In some
embodiments, the pharmaceutical composition further comprises one
or more pharmaceutically active compounds for the treatment of TAO.
In some embodiments, the pharmaceutical composition further
comprises corticosteroids; rituximab or other anti-CD20 antibodies;
tocilizumab or other anti-IL-6 antibodies; or selenium, infliximab
or other anti-TNFalpha antibodies or a thyroid-stimulating hormone
receptor (TSHR) inhibitor.
[0219] In some embodiments, the method provided herein comprise
administering to a subject an antibody, or an antigen binding
fragment thereof, that specifically binds to and inhibits IGF-IR.
In some embodiments, the antibody is as provided herein.
[0220] Kits are also provided which are useful for carrying out
embodiments described herein. The present kits comprise a first
container containing or packaged in association with the
above-described antibodies. The kit may also comprise another
container containing or packaged in association solutions necessary
or convenient for carrying out the embodiments. The containers can
be made of glass, plastic or foil and can be a vial, bottle, pouch,
tube, bag, etc. The kit may also contain written information, such
as procedures for carrying out the embodiments or analytical
information, such as the amount of reagent contained in the first
container means. The container may be in another container
apparatus, e.g. a box or a bag, along with the written
information.
[0221] Yet another aspect provided for herein is a kit for
detecting IGF-1R protein in a biological sample. The kit includes a
container holding one or more antibodies which binds an epitope of
IGF-1R protein and instructions for using the antibody for the
purpose of binding to IGF-1R protein to form an immunological
complex and detecting the formation of the immunological complex
such that the presence or absence of the immunological complex
correlates with presence or absence of IGF-1R protein in the
sample. Examples of containers include multiwell plates which allow
simultaneous detection of IGF-1R protein in multiple samples.
[0222] In some embodiments, antibodies that bind to a IGF-1R
protein are provided. In some embodiments, the antibody is
isolated. In some embodiments, the antibody binds specifically. In
some embodiments, the antibody binds to a IGF-1R protein that is
properly folded. In some embodiments, the antibody is specific for
a specific IGF-1R conformational state (open or closed). In some
embodiments, the antibody binds to a IGF-1R protein in a cell
membrane. In some embodiments, the antibody binds to a IGF-1R
protein that is in a cell membrane in an intact cell. In some
embodiments, the antibody inhibits or neutralizes the function of a
IGF-1R protein. As used herein, the term "neutralize" means that
the activity or function of the protein is inhibited. The
inhibition can be complete or partial. In some embodiments, the
activity or function of the protein is inhibited at least 10, 20,
30, 40, 50, 60, 70, 80, 90, 95, or 99%. The percent inhibition can
be based upon the function or activity of the protein in the
absence of the antibody. In some embodiments, the antibody inhibits
the glucose transport facilitated by IGF-1R. In some embodiments,
the antibody inhibits the internalization of the IGF-1R
protein.
[0223] In some embodiments, the antibody comprises a sequence as
provided for herein or antigen binding fragment thereof. In some
embodiments, the antibody comprises a heavy chain CDR or an antigen
binding fragment thereof described herein. The heavy chain may be
one or more of the heavy chains described herein. In some
embodiments, the antibody comprises a light chain, or an antigen
binding fragment thereof as described herein
[0224] In some embodiments, methods of treating, inhibiting or
ameliorating a IGF-1R, associated pathology are provided. In some
embodiments, the methods comprise administering an antibody
described herein or a pharmaceutical composition described herein
to a subject to treat, inhibit or ameliorate a IGF-1R associated
pathology. In some embodiments, the pathology is as described
herein.
[0225] In some embodiments, methods of detecting the presence or
absence of a IGF-1R in a sample are provided, the method comprising
contacting a sample with one or more antibodies described herein
detecting the binding to a IGF-1R antigen by the antibody. In some
embodiments, the detection of the binding indicates the presence
IGF-1R antigen; or the absence of the detection of the binding to
the IGF-1R antigen indicates the absence of the IGF-1R antigen. The
detecting can be done with any known method, such as using a
biosensor, ELISA, sandwich assay, and the like. However, in some
embodiments, the method comprises detecting the presence of the
protein in non-denaturing conditions. The non-denaturing conditions
can be used so that the protein of interest is detected in its
native, or properly folded form.
[0226] In some embodiments, methods of identifying a test antibody
that binds to an epitope on IGF-1R protein, are provided, the
method comprising contacting a test antibody with the epitope on
IGF-1R protein and determining whether the test antibody binds to
the epitope. In some embodiments, the determining comprises
determining whether the test antibody binds to the protein and is
competitively inhibited by an antibody comprising a sequence as
provided herein. In some embodiments, the determining comprises
mutating one or more residues of epitope or protein and determining
binding of the test antibody to the mutated epitope, wherein if the
mutation reduces binding of the test antibody as compared to the
non-mutated epitope, the test antibody is deemed to bind to that
epitope.
[0227] In some embodiments, methods of monitoring internalization
of IGF-1R from the surface of a cell are provided. In some
embodiments, the method comprising contacting the cell with an
anti-IGF-1R antibody as provided herein and detecting the presence
of IGF-1R in the cell or on the surface of the cell. The
differences in cell surface expression can be measured and the
internalization can be monitored and measured. This can be used,
for example, to measure the effect of another molecule, such as a
test agent, to modulate internalization of IGF-1R protein. Thus,
the antibodies provided for herein can be used to identify test
agents that modulate (increase or decrease) the internalization of
IGF-1R protein. Test molecules that increase the internalization,
which would be measured as a decrease in binding of an anti-IGF-1R
antibody to IGF-1R protein on the cell surface, can be identified
according to the methods provided herein. Test molecules that
decrease the internalization, which would be measured as an
increase in binding of an anti-IGF-1R antibody to IGF-1R protein on
the cell surface, can be identified according to the methods
provided herein. The surface expression can be measured by
fluorescence, which can be done through a secondary antibody that
recognized the IGF-1R antibodies or by labelling the anti-IGF-1R
antibodies provided for herein.
[0228] In some embodiments, methods of inhibiting IGF-1 stimulated
receptor phosphorylation on a cell are provided. In some
embodiments, the methods comprise contacting the cell with an
antibody as provided for herein, or a pharmaceutical composition
comprising the same. In some embodiments, the contacting comprises
administering to a subject the antibody or a pharmaceutical
composition comprising the same. In some embodiments, the cell is a
cell in the eye. In some embodiments, the subject has or is at risk
of thyroid eye disease (TED). In some embodiments, the antibody has
an IC50 of less than, or equal to, about 0.2 nm, 0.15 nm, 0.10 nm,
0.09 nm. In some embodiments, the IC50 is measured in an in vitro
assay, such as an assay as provided for herein, such as illustrated
in the Examples. In some embodiments, the IC50 is measured in an
cell that is an A549 cell or a HOCF cell.
[0229] In some embodiments, methods of treating thyroid eye disease
in a subject are provided, the method comprising administering an
antibody as provided for herein, or a pharmaceutical composition
comprising the same to the subject, wherein the antibody has a
serum concentration in the subject of at least, or about, 70
.mu.g/ml, 75 .mu.g/ml, 80 .mu.g/ml, 85 .mu.g/ml, 90 .mu.g/ml, 95
.mu.g/ml, 100 .mu.g/ml, or 105 .mu.g/ml at least 1, 2, or 3 weeks
after administration. In some embodiments, the serum concentration
is measured after one, two or three doses of the antibody, or the
pharmaceutical composition comprising the same, are administered to
the subject.
[0230] In some embodiments, methods of inhibiting IGF-1 induced
receptor autophosphorylation by at least 95%, 96%, 97%, 98%, or 99%
or by 100% in a subject in need thereof are provided. In some
embodiments, the methods comprise administering to the subject an
antibody as provided for herein, or a pharmaceutical composition
comprising the same. In some embodiments, the IGF-1 induced
receptor autophosphorylation is inhibited in the eye or orbital
region of the subject. In some embodiments, the IGF-1 induced
receptor autophosphorylation is inhibited thereby treating a
subject for thyroid eye disease or improving a symptom as described
herein.
ENUMERATED EMBODIMENTS
[0231] In some embodiments, embodiments provided herein also
include, but are not limited to:
1. An antibody, or antigen binding fragment thereof, comprising:
[0232] a VL sequence as set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11,
13, 15, 79, or 86; [0233] a VH sequence as set forth in SEQ ID NO:
2, 4, 6, 8, 10, 12, 14, 16, 80, or 83; [0234] a LCDR sequence as
set forth in SEQ ID NO: 17, 18, 19, 23, 24, 25, 29, 30, 31, 35, 36,
37, 41, 42, 43, 47, 48, 49, 53, 54, 55, 59, 60, 61, or 81, or
[0235] a HCDR sequence as set forth in SEQ ID NO: 20, 21, 22, 26,
27, 28, 32, 33, 34, 38, 39, 40, 44, 45, 46, 50, 51, 52, 56, 57, 58,
62, 63, or 64; and [0236] any combination or variant thereof. 2.
The antibody of embodiment 1, or antigen binding fragment thereof,
wherein the antibody binds to IGF-1R. 3. The antibody of embodiment
1, wherein the antibody is a monoclonal antibody. 4. The antibody
of embodiment 1, wherein the antibody is a humanized antibody. 5.
The antibody of embodiment 1, wherein the antibody is a scFv
antibody. 6. The antibody of any one of embodiments 1-5, wherein
the antibody, or antigen binding fragment thereof, comprises a
V.sub.L peptide as set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,
15, 79, or 86, or any variant thereof. 7. The antibody of any one
of embodiments 1-6, wherein the antibody, or antigen binding
fragment thereof, comprises a V.sub.H peptide as set forth in SEQ
ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 80, or 83, or any variant
thereof. 8. An antibody, or antigen binding fragment thereof,
wherein the antibody or antibody fragment comprises: (i) a heavy
chain variable region comprising heavy chain CDR1, CDR2, and CDR3
sequences, wherein the heavy chain CDR1 sequence has the amino acid
sequence of SEQ ID NO: 20, 26, 32, 38, 44, 50, or 56; the heavy
chain CDR2 has the amino acid sequence of SEQ ID NO: 21, 27, 33,
39, 45, 51, or 57; and the heavy chain CDR3 sequence has the amino
acid sequence of SEQ ID NO: 22, 28, 34, 40, 46, 52, or 58; or
variants of any of the foregoing; and (ii) a light chain variable
region comprising light chain CDR1, CDR2, and CDR3 sequences,
wherein the light chain CDR1 sequence has the amino acid sequence
SEQ ID NO: 17, 23, 29, 35, 41, 47, or 53; the light chain CDR2
sequence has the amino acid sequence of SEQ ID NO: 18, 24, 30, 36,
42, 48, or 54; and the light chain CDR3 sequence has the amino acid
sequence of SEQ ID NO: 19, 25, 31, 37, 43, 49, 55, or 81; or
variants of any of the foregoing. 9. An antibody, or antigen
binding fragment thereof, wherein the antibody or antibody fragment
comprises: (i) a heavy chain variable region comprising heavy chain
CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1
sequence has the amino acid sequence of SEQ ID NO: 20; the heavy
chain CDR2 has the amino acid sequence of SEQ ID NO: 21; and the
heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:
22; or variants of any of the foregoing; and (ii) a light chain
variable region comprising light chain CDR1, CDR2, and CDR3
sequences, wherein the light chain CDR1 sequence has the amino acid
sequence SEQ ID NO: 17; the light chain CDR2 sequence has the amino
acid sequence of SEQ ID NO: 18; and the light chain CDR3 sequence
has the amino acid sequence of SEQ ID NO: 19; or variants of any of
the foregoing. 10. An antibody, or antigen binding fragment
thereof, wherein the antibody or antibody fragment comprises: (i) a
heavy chain variable region comprising heavy chain CDR1, CDR2, and
CDR3 sequences, wherein the heavy chain CDR1 sequence has the amino
acid sequence of SEQ ID NO: 26; the heavy chain CDR2 has the amino
acid sequence of SEQ ID NO: 27; and the heavy chain CDR3 sequence
has the amino acid sequence of SEQ ID NO: 28; or variants of any of
the foregoing; and (ii) a light chain variable region comprising
light chain CDR1, CDR2, and CDR3 sequences, wherein the light chain
CDR1 sequence has the amino acid sequence SEQ ID NO: 23; the light
chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 24;
and the light chain CDR3 sequence has the amino acid sequence of
SEQ ID NO: 25; or variants of any of the foregoing. 11. An
antibody, or antigen binding fragment thereof, wherein the antibody
or antibody fragment comprises: (i) a heavy chain variable region
comprising heavy chain CDR1, CDR2, and CDR3 sequences, wherein the
heavy chain CDR1 sequence has the amino acid sequence of SEQ ID NO:
32; the heavy chain CDR2 has the amino acid sequence of SEQ ID NO:
33; and the heavy chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 34; or variants of any of the foregoing; and (ii) a
light chain variable region comprising light chain CDR1, CDR2, and
CDR3 sequences, wherein the light chain CDR1 sequence has the amino
acid sequence SEQ ID NO: 29; the light chain CDR2 sequence has the
amino acid sequence of SEQ ID NO: 30; and the light chain CDR3
sequence has the amino acid sequence of SEQ ID NO: 31; or variants
of any of the foregoing. 12. An antibody, or antigen binding
fragment thereof, wherein the antibody or antibody fragment
comprises: (i) a heavy chain variable region comprising heavy chain
CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1
sequence has the amino acid sequence of SEQ ID NO: 38; the heavy
chain CDR2 has the amino acid sequence of SEQ ID NO: 39; and the
heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:
40; or variants of any of the foregoing; and (ii) a light chain
variable region comprising light chain CDR1, CDR2, and CDR3
sequences, wherein the light chain CDR1 sequence has the amino acid
sequence SEQ ID NO: 35; the light chain CDR2 sequence has the amino
acid sequence of SEQ ID NO: 36; and the light chain CDR3 sequence
has the amino acid sequence of SEQ ID NO: 37; or variants of any of
the foregoing. 13. An antibody, or antigen binding fragment
thereof, wherein the antibody or antibody fragment comprises: (i) a
heavy chain variable region comprising heavy chain CDR1, CDR2, and
CDR3 sequences, wherein the heavy chain CDR1 sequence has the amino
acid sequence of SEQ ID NO: 44; the heavy chain CDR2 has the amino
acid sequence of SEQ ID NO: 45; and the heavy chain CDR3 sequence
has the amino acid sequence of SEQ ID NO: 46; or variants of any of
the foregoing; and (ii) a light chain variable region comprising
light chain CDR1, CDR2, and CDR3 sequences, wherein the light chain
CDR1 sequence has the amino acid sequence SEQ ID NO: 41; the light
chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 42;
and the light chain CDR3 sequence has the amino acid sequence of
SEQ ID NO: 43; or variants of any of the foregoing. 14. An
antibody, or antigen binding fragment thereof, wherein the antibody
or antibody fragment comprises: (i) a heavy chain variable region
comprising heavy chain CDR1, CDR2, and CDR3 sequences, wherein the
heavy chain CDR1 sequence has the amino acid sequence of SEQ ID NO:
50; the heavy chain CDR2 has the amino acid sequence of SEQ ID NO:
51; and the heavy chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 52; or variants of any of the foregoing; and (ii) a
light chain variable region comprising light chain CDR1, CDR2, and
CDR3 sequences, wherein the light chain CDR1 sequence has the amino
acid sequence SEQ ID NO: 47; the light chain CDR2 sequence has the
amino acid sequence of SEQ ID NO: 48; and the light chain CDR3
sequence has the amino acid sequence of SEQ ID NO: 49; or variants
of any of the foregoing. 15. An antibody, or antigen binding
fragment thereof, wherein the antibody or antibody fragment
comprises: (i) a heavy chain variable region comprising heavy chain
CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1
sequence has the amino acid sequence of SEQ ID NO: 56; the heavy
chain CDR2 has the amino acid sequence of SEQ ID NO: 57; and the
heavy chain CDR3 sequence has the amino acid sequence of SEQ ID NO:
58; or variants of any of the foregoing; and (ii) a light chain
variable region comprising light chain CDR1, CDR2, and CDR3
sequences, wherein the light chain CDR1 sequence has the amino acid
sequence SEQ ID NO: 53; the light chain CDR2 sequence has the amino
acid sequence of SEQ ID NO: 54; and the light chain CDR3 sequence
has the amino acid sequence of SEQ ID NO: 55; or variants of any of
the foregoing. 16. An antibody, or antigen binding fragment
thereof, wherein the antibody or antibody fragment comprises: (i) a
heavy chain variable region comprising heavy chain CDR1, CDR2, and
CDR3 sequences, wherein the heavy chain CDR1 sequence has the amino
acid sequence of SEQ ID NO: 62; the heavy chain CDR2 has the amino
acid sequence of SEQ ID NO: 63; and the heavy chain CDR3 sequence
has the amino acid sequence of SEQ ID NO: 64; or variants of any of
the foregoing; and (ii) a light chain variable region comprising
light chain CDR1, CDR2, and CDR3 sequences, wherein the light chain
CDR1 sequence has the amino acid sequence SEQ ID NO: 59; the light
chain CDR2 sequence has the amino acid sequence of SEQ ID NO: 60;
and the light chain CDR3 sequence has the amino acid sequence of
SEQ ID NO: 61; or variants of any of the foregoing. 17. An
antibody, or antigen binding fragment thereof, wherein the antibody
or antibody fragment comprises: (i) a heavy chain variable region
comprising heavy chain CDR1, CDR2, and CDR3 sequences, wherein the
heavy chain CDR1 sequence has the amino acid sequence of SEQ ID NO:
38; the heavy chain CDR2 has the amino acid sequence of SEQ ID NO:
39; and the heavy chain CDR3 sequence has the amino acid sequence
of SEQ ID NO: 40; or variants of any of the foregoing; and (ii) a
light chain variable region comprising light chain CDR1, CDR2, and
CDR3 sequences, wherein the light chain CDR1 sequence has the amino
acid sequence SEQ ID NO: 35; the light chain CDR2 sequence has the
amino acid sequence of SEQ ID NO: 36; and the light chain CDR3
sequence has the amino acid sequence of SEQ ID NO: 81; or variants
of any of the foregoing. 18. The antibody of any one of embodiments
6-17, wherein the heavy chain variable region and the light chain
variable region are not linked by a linker. 19. The antibody of any
one of embodiments 6-17, wherein the heavy chain variable region
and the light chain variable region are linked with a peptide
linker. 20. The antibody of embodiment 19, wherein the peptide
linker comprises a sequence of: (GGGGS).sub.n (SEQ ID NO: 73)
(GGGGA).sub.n (SEQ ID NO: 74), or any combination thereof, wherein
each n is independently 1-5. 21. The antibody of any one of
embodiments 1-20, wherein the antibody comprises a sequence of SEQ
ID NO: 65-72, 78, 82, or 85, or a variant thereof. 22. The antibody
of any one of embodiments 1-21, wherein the antibody comprises a
V.sub.L sequence as set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,
15, 79, or 86, or a variant thereof. 23. The antibody of any one of
embodiments 1-21, wherein the antibody comprises a V.sub.H sequence
as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 80, or 83,
or a variant thereof. 24. The isolated antibody of any one of
embodiments 1-21, wherein the antibody comprises a sequence of SEQ
ID NO: 65-72, 78, 82, or 85, or a variant thereof. 25. The antibody
of any one of embodiments 1-24, wherein the variant has 1-10
substitutions, deletions, or insertions. 26. The antibody of any
one of embodiments 1-24, wherein the variant has 1-10 conservative
substitutions. 27. The antibody of any one of embodiments 1-26,
wherein the variant has at least 85% homology to a sequence of SEQ
ID NO: 1-72, 78-83, or 85-86. 28. The antibody of any one of
embodiments 1-26, wherein the variant has at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to a sequence of SEQ
ID NO: 1-72, 78-83, or 85-86. 29. The antibody of any one of
embodiments 1-26, wherein the variant has at least 85% identity to
a sequence of SEQ ID NO: 1-72, 78-83, or 85-86. 30. The antibody of
any one of embodiments 1-26, wherein the variant has at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identify to a
sequence of SEQ ID NO: 1-72, 78-83, or 85-86. 31. The antibody of
any one of embodiments 1-26, wherein the antibody is a scFv
antibody. 32. The antibody of any one of embodiments 1-26, wherein
the antibody is a monoclonal antibody. 33. The antibody of any one
of embodiments 1-26, wherein the antibody is a humanized antibody.
34. The antibody of any one of the preceding embodiments, wherein
the antibody comprises a Fc region. 35. The antibody of embodiment
34, wherein the Fc region is as set forth in SEQ ID NO: 75-77, or
84. 36. The antibody of any one of the preceding embodiments,
wherein the Fc region comprises a mutation that extends the
half-life of the antibody when linked to the Fc region. 37. The
antibody of embodiment 36, wherein the Fc region comprises a S228P,
L235E, M252Y, S254T, T256E, M428L, N434S, L234F, P331S mutation, or
any combination thereof. 38. The antibody of embodiment 36, wherein
the Fc region comprises a M252Y, S254T, and T256E mutation. 39. The
antibody of embodiment 36, wherein the Fc region comprises a S228P
and a L235E mutation. 40. The antibody of embodiment 36, wherein
the Fc region comprises a L234F, L235E, and P331S mutation. 41. The
antibody of embodiment 36, wherein the Fc region comprises M252Y,
S254T, T256E, S228P and L235E mutations. 42. The antibody of
embodiment 36, wherein the Fc region comprises S228P, L235E, M428L,
and N434S mutations. 43. The antibody of embodiment 36, wherein the
Fc region comprises M428L and N434S mutations. 44. The antibody of
embodiment 36, wherein the Fc region comprises L234F, L235E, P331S,
M252Y, S254T, and T256E mutations. 45. A nucleic acid molecule
encoding an antibody, or antigen binding fragment thereof, of any
of the preceding embodiments. 46. A vector comprising the nucleic
acid molecule of embodiment 45. 47. A cell comprising the nucleic
comprising the nucleic acid molecule of embodiment 46 or the vector
of embodiment 46. 48. A pharmaceutical composition comprising the
antibody of any one of embodiments 1-44 or a nucleic acid molecule
encoding the same. 49. The pharmaceutical composition of embodiment
48, wherein the composition is an injectable pharmaceutical
composition. 50. A method of treating or reducing the severity of,
thyroid-associated ophthalmopathy (TAO), or a symptom thereof,
comprising administering to a subject an antibody of any one of
embodiments 1-44 or a pharmaceutical composition comprising the
same. 51. A method of reducing proptosis in an eye in a subject
with thyroid-associated ophthalmopathy (TAO) comprising
administering to a subject an antibody of any one of embodiments
1-44 or a pharmaceutical composition comprising the same. 52. A
method of treating thyroid eye disease in a subject comprising
administering to a subject an antibody of any one of embodiments
1-44 or a pharmaceutical composition comprising the same. 53. A
method of reducing Clinical Activity Score (CAS) of
thyroid-associated ophthalmopathy (TAO) in a subject comprising
administering to a subject an antibody of any one of embodiments
1-44 or a pharmaceutical composition comprising the same. 54. A
method of a) reducing proptosis by at least 2 mm and b) reducing
the clinical activity score (CAS) in a subject with
thyroid-associated ophthalmopathy (TAO) comprising administering to
a subject an antibody of any one of embodiments 1-44 or a
pharmaceutical composition comprising the same. 55. The method of
any of embodiments 50-54, wherein proptosis is reduced by at least
2 mm. 56. The method of any of embodiments 50-54, wherein proptosis
is reduced by at least 3 mm. 57. The method of any of embodiments
50-54, wherein proptosis is reduced by at least 4 mm. 58. The
method of any of embodiments 50-54, wherein the clinical activity
score (CAS) of the subject is reduced by at least 2 points. 59. The
method of any of embodiments 50-54, wherein the clinical activity
score (CAS) of the subject is reduced to one (1). 60. The method of
any of embodiments 50-54, wherein the clinical activity score (CAS)
of the subject is reduced to zero (0). 61. A method of treating or
reducing the severity of thyroid-associated ophthalmopathy (TAO) in
a subject comprising administering to a subject an antibody of any
one of embodiments 1-44 or a pharmaceutical composition comprising
the same, wherein treatment with said antibody (i) reduces
proptosis by at least 2 mm in an eye; (ii) is not accompanied by a
deterioration of 2 mm or more in the other (or fellow eye); and
(iii) reduces the CAS in said subject to either one (1) or zero
(0). 62. A method of improving the quality of life in a subject
with thyroid-associated ophthalmopathy (TAO, also called Graves'
Ophthalmopathy/Graves' Orbitopathy) comprising administering to a
subject an antibody of any one of embodiments 1-44 or a
pharmaceutical composition comprising the same. 63. The method of
embodiment 62, wherein the quality of life is measured by the
Graves' Ophthalmopathy Quality of Life (GO-QoL) assessment, or
either the Visual Functioning or Appearance subscale thereof. 64.
The method of embodiment 63, wherein the treatment results in an
improvement of greater than or equal to 8 points on the GO-QoL. 65.
The method of embodiment 63, wherein the treatment results in an
improvement on the Functioning subscale of the GO-QoL. 66. The
method of embodiment 63, wherein the treatment results in an
improvement on the Appearance subscale of the GO-QoL. 67. A method
of treating or reducing the severity of diplopia in a subject with
thyroid-associated ophthalmopathy (TAO) comprising administering to
a subject an antibody of any one of embodiments 1-44 or a
pharmaceutical composition comprising the same. 68. The method of
embodiment 67, wherein the diplopia is constant diplopia. 69. The
method of embodiment 67, wherein the diplopia is inconstant
diplopia. 70. The method of embodiment 67, wherein the diplopia is
intermittent diplopia. 71. The method of embodiment 67,
wherein the improvement in or reduction in severity of diplopia is
sustained at least 20 weeks after discontinuation of antibody
administration. 72. The method of embodiment 67, wherein the
improvement in or reduction in severity of diplopia is sustained at
least 50 weeks after discontinuation of antibody administration.
73. The method of any one of embodiments 50-72, wherein said
antibody is administered at a dosage of about 1 .mu.g/kg to about 5
.mu.g/kg antibody as a first dose. 74. The method of any one of
embodiments 50-72, wherein said antibody is administered at a
dosage of about 5 .mu.g/kg to about 10 .mu.g/kg antibody as a first
dose. 75. The method of any one of embodiments 50-72, wherein said
antibody is administered at a dosage of about 5 .mu.g/kg to about
20 .mu.g/kg antibody in subsequent doses. 76. The method of any one
of embodiments 50-72, wherein said antibody is administered in the
following amounts: about 10 .mu.g/kg antibody as a first dose; and
about 20 .mu.g/kg antibody in subsequent doses. 77. The method of
embodiment 76, wherein said subsequent doses are administered every
three weeks for at least 21 weeks. 78. The method of any one of
embodiments 50-77, wherein the antibody, or an antigen binding
fragment thereof, is a human antibody, a monoclonal antibody, a
human monoclonal antibody, a purified antibody, a diabody, a
single-chain antibody, a multi-specific antibody, Fab, Fab',
F(ab')2, Fv or scFv. 79. The method of any one of embodiments
50-78, wherein the antibody, or an antigen binding fragment
thereof, is administered in a pharmaceutical composition that
additionally comprises a pharmaceutically acceptable diluent or
excipient or carrier. 80. The method of embodiment 79, wherein the
pharmaceutical composition further comprises one or more
pharmaceutically active compounds for the treatment of TAO. 81. The
method of embodiment 79 or 80, wherein the pharmaceutical
composition further comprises corticosteroids; rituximab or other
anti-CD20 antibodies; tocilizumab or other anti-IL-6 antibodies; or
selenium, infliximab or other anti-TNFalpha antibodies or a
thyroid-stimulating hormone receptor (TSHR) inhibitor. 82. The
method of any of the preceding embodiments, wherein the antibody or
an antigen binding fragment thereof is administered directly to the
eye, the anterior chamber of the eye, the vitreous chamber of the
eye, the suprachoroidal space, or the retro-orbital sinus. 83. The
method of embodiment 82, wherein the antibody or an antigen binding
fragment thereof is administered via an injection. 84. The method
of embodiment 83, wherein the injection is a intravitreal
injection, intraorbital injection, retro-orbital injection,
suprachoroidal injection, or intracameral injection. 85. A method
of increasing the internalization of IGF-1R on a cell, the method
comprising contacting the cell with an antibody of any one of
embodiments 1-44 or a pharmaceutical composition comprising the
same. 86. The method of embodiment 85, wherein the contacting
comprises administering to a subject an antibody of any one of
embodiments 1-44 or a pharmaceutical composition comprising the
same. 87. The method of embodiment 86, wherein the subject has or
is at risk of thyroid eye disease (TED). 88. A method of inhibiting
IGF-1 stimulated receptor phosphorylation on a cell, the method
comprising contacting the cell with an antibody of any one of
embodiments 1-44 or a pharmaceutical composition comprising the
same. 89. The method of embodiment 88, wherein the contacting
comprises administering to a subject an antibody of any one of
embodiments 1-44 or a pharmaceutical composition comprising the
same. 90. The method of embodiment 89, wherein the subject has or
is at risk of thyroid eye disease (TED). 91. The method of any one
of embodiments 88-90, wherein the antibody has an IC50 of less
than, or equal to, about 0.2 nm, 0.15 nm, 0.10 nm, 0.09 nm. 92. The
method of embodiment 91, wherein the IC50 is measured in an in
vitro assay, such as an assay as provided for herein. 93. The
method of any one of embodiments 88-92, wherein the cell is an A549
cell or a HOCF cell. 94. A method of treating thyroid eye disease
in a subject, the method comprising administering an antibody of
any one of embodiments 1-44 or as otherwise provided for herein, or
a pharmaceutical composition comprising the same to the subject,
wherein the antibody has a serum concentration in the subject of at
least, or about, 70 .mu.g/ml, 75 .mu.g/ml, 80 .mu.g/ml, 85
.mu.g/ml, 90 .mu.g/ml, 95 .mu.g/ml, 100 .mu.g/ml, or 105 .mu.g/ml
at least 1, 2, or 3 week after administration. 95. The method of
embodiment 94, wherein the antibody or the pharmaceutical
composition is administered intravenously. 96. The method of
embodiments 94 or 96, wherein the antibody or the pharmaceutical
composition is administered at a dose of about 20 .mu.g/kg. 97. The
method of any one of embodiments 94-96, wherein the antibody or the
pharmaceutical composition is administered at least, or about, once
a week, once every two weeks, once every 3 weeks, or once every 4
weeks. 98. A method of inhibiting IGF-1 induced receptor
autophosphorylation in a cell by at least 95%, 96%, 97%, 98%, or
99% or by 100%, the method comprising contacting the cell with an
antibody of any one of embodiments 1-44 or as otherwise provided
for herein, or a pharmaceutical composition comprising the same.
99. The method of embodiment 98, wherein the inhibition of the
IGF-1 induced receptor autophosphorylation is measured as compared
to the induced receptor autophosphorylation in the absence of the
antibody or the pharmaceutical composition. 100. The method of
embodiments 98 or 99, wherein the contacting comprises
administering to a subject the antibody or the pharmaceutical
composition comprising the same. 101. The method of embodiment 100,
wherein the subject has or is at risk of thyroid eye disease (TED).
102. A method of inhibiting IGF-1 induced receptor
autophosphorylation by at least 95%, 96%, 97%, 98%, or 99% or by
100% in a subject in need thereof, the method comprising
administering to the subject an antibody of any one of embodiments
1-44 or as otherwise provided for herein, or a pharmaceutical
composition comprising the same. 103. The method of embodiment 102,
wherein the subject has or is at risk of thyroid eye disease (TED).
104. The method of any one of embodiments 102 or 103, wherein the
antibody or the pharmaceutical composition is administered
intravenously. 105. The method of any one of embodiments 98-104,
wherein the antibody comprises the CDRs of VRDN-1100. 106. The
method of any one of embodiments 98-104, wherein the antibody
comprises the CDRs of the antibody of VRDN-1100 or the CDRs of
VRDN-2700. 107. An isolated antibody comprising a light chain
having the amino acid sequence of SEQ ID NO: 3 and a heavy chain
comprising the amino acid sequence of SEQ ID NO: 83. 108. An
isolated antibody comprising a light chain variable region having
the amino acid sequence of SEQ ID NO: 13 and a heavy chain variable
region having the amino acid sequence of SEQ ID NO: 14. 109. The
isolated antibody of embodiment 108, wherein antibody comprises a
light chain having a an amino acid sequence of SEQ ID NO: 93 and a
heavy chain amino acid sequence of SEQ ID NO: 92. 110. The isolated
antibody of embodiment 108, wherein antibody comprises a light
chain having a an amino acid sequence of SEQ ID NO: 93 and a heavy
chain amino acid sequence of SEQ ID NO: 94. 111. The isolated
antibody of embodiment 108, wherein antibody comprises a light
chain having a an amino acid sequence of SEQ ID NO: 93 and a heavy
chain amino acid sequence of SEQ ID NO: 95. 112. A pharmaceutical
composition comprising the antibody of any one of embodiments
107-111. 113. A pharmaceutical composition suitable for intravenous
administration comprising the antibody of any one of embodiments
107-111. 114. A pharmaceutical composition suitable for
subcutaneous administration comprising the antibody of any one of
embodiments 107-111. 115. A method of treating thyroid eye disease
in a subject, the method comprising administering a pharmaceutical
composition comprising the antibody of any one of embodiments
107-111. 116. The method of embodiment 115, wherein the
pharmaceutical composition is administered intravenously. 117. The
method of embodiment 115, wherein the pharmaceutical composition is
administered subcutaneously. 118. A method of treating or reducing
the severity of, thyroid-associated ophthalmopathy (TAO), or a
symptom thereof, comprising administering to a subject an antibody
of any one of embodiments 107-111 or a pharmaceutical composition
comprising the same. 119. A method of reducing proptosis in an eye
in a subject with thyroid-associated ophthalmopathy (TAO)
comprising administering to a subject an antibody of an antibody of
any one of embodiments 107-111 or a pharmaceutical composition
comprising the same. 120. A method of treating thyroid eye disease
in a subject comprising administering to a subject an antibody of
any one of embodiments 107-111 or a pharmaceutical composition
comprising the same. 121. A method of reducing Clinical Activity
Score (CAS) of thyroid-associated ophthalmopathy (TAO) in a subject
comprising administering to a subject an antibody of any one of
embodiments 107-111 or a pharmaceutical composition comprising the
same. 122. A method of a) reducing proptosis by at least 2 mm and
b) reducing the clinical activity score (CAS) in a subject with
thyroid-associated ophthalmopathy (TAO) comprising administering to
a subject an antibody of any one of embodiments 107-111 or a
pharmaceutical composition comprising the same. 123. The method of
any of embodiments 118-122, wherein proptosis is reduced by at
least 2 mm. 124. The method of any of embodiments 118-122, wherein
proptosis is reduced by at least 3 mm. 125. The method of any of
embodiments 118-122, wherein proptosis is reduced by at least 4 mm.
126. The method of any of embodiments 118-122, wherein the clinical
activity score (CAS) of the subject is reduced by at least 2
points. 127. The method of any of embodiments 118-122, wherein the
clinical activity score (CAS) of the subject is reduced to one (1).
128. The method of any of embodiments 118-122, wherein the clinical
activity score (CAS) of the subject is reduced to zero (0). 129. A
method of treating or reducing the severity of thyroid-associated
ophthalmopathy (TAO) in a subject comprising administering to a
subject an antibody of any one of embodiments 107-111, or a
pharmaceutical composition comprising the same, wherein treatment
with said antibody (i) reduces proptosis by at least 2 mm in an
eye; (ii) is not accompanied by a deterioration of 2 mm or more in
the other (or fellow eye); and (iii) reduces the CAS in said
subject to either one (1) or zero (0). 130. A method of improving
the quality of life in a subject with thyroid-associated
ophthalmopathy (TAO, also called Graves' Ophthalmopathy/Graves'
Orbitopathy) comprising administering to a subject an antibody of
any one of embodiments 107-111, or a pharmaceutical composition
comprising the same. 131. The method of embodiment 130, wherein the
quality of life is measured by the Graves' Ophthalmopathy Quality
of Life (GO-QoL) assessment, or either the Visual Functioning or
Appearance subscale thereof. 132. The method of embodiment 130,
wherein the treatment results in an improvement of greater than or
equal to 8 points on the GO-QoL. 133. The method of embodiment 130,
wherein the treatment results in an improvement on the Functioning
subscale of the GO-QoL. 134. The method of embodiment 130, wherein
the treatment results in an improvement on the Appearance subscale
of the GO-QoL. 135. A method of treating or reducing the severity
of diplopia in a subject with thyroid-associated ophthalmopathy
(TAO) comprising administering to a subject an antibody of any one
of embodiments 107-111 or a pharmaceutical composition comprising
the same. 136. The method of embodiment 135, wherein the diplopia
is constant diplopia. 137. The method of embodiment 135, wherein
the diplopia is inconstant diplopia. 138. The method of embodiment
135, wherein the diplopia is intermittent diplopia. 139. The method
of embodiment 135, wherein the improvement in or reduction in
severity of diplopia is sustained at least 20 weeks after
discontinuation of antibody administration. 140. The method of
embodiment 135, wherein the improvement in or reduction in severity
of diplopia is sustained at least 50 weeks after discontinuation of
antibody administration. 141. The method of any one of embodiments
115-140, wherein said antibody is administered at a dosage of about
1 .mu.g/kg to about 5 .mu.g/kg antibody as a first dose. 142. The
method of any one of embodiments 115-140, wherein said antibody is
administered at a dosage of about 5 .mu.g/kg to about 10 .mu.g/kg
antibody as a first dose. 143. The method of any one of embodiments
115-140, wherein said antibody is administered at a dosage of about
5 .mu.g/kg to about 20 .mu.g/kg antibody in subsequent doses. 144.
The method of any one of embodiments 115-140, wherein said antibody
is administered in the following amounts: about 10 .mu.g/kg
antibody as a first dose; and about 20 .mu.g/kg antibody in
subsequent doses. 145. The method of embodiment 144, wherein said
subsequent doses are administered every three weeks for at least 21
weeks. 146. The method of any one of embodiments 115-140, wherein
the antibody is administered in a pharmaceutical composition that
comprises a pharmaceutically acceptable diluent, excipient, or
carrier. 147. The method of embodiment 146, wherein the
pharmaceutical composition further comprises one or more
pharmaceutically active compounds for the treatment of TAO. 148.
The method of embodiment 146 or 147, wherein the pharmaceutical
composition further comprises corticosteroids; rituximab or other
anti-CD20 antibodies; tocilizumab or other anti-IL-6 antibodies; or
selenium, infliximab or other anti-TNFalpha antibodies or a
thyroid-stimulating hormone receptor (TSHR) inhibitor. 149. A
method of increasing the internalization of IGF-1R on a cell, the
method comprising contacting the cell with an antibody of any one
of embodiments 107-111 or a pharmaceutical composition comprising
the same. 150. The method of embodiment 149, wherein the contacting
comprises administering to a subject the antibody, or a
pharmaceutical composition comprising the same. 151. The method of
embodiment 150, wherein the subject has or is at risk of thyroid
eye disease (TED). 152. A method of inhibiting IGF-1 stimulated
receptor phosphorylation on a cell, the method comprising
contacting the cell with an antibody of any one of embodiments
107-111, or a pharmaceutical composition comprising the same. 153.
The method of embodiment 152, wherein the contacting comprises
administering to a subject an antibody of any one of embodiments
1-44 or a pharmaceutical composition comprising the same. 154. The
method of embodiment 153, wherein the subject has or is at risk of
thyroid eye disease (TED). 155. The method of embodiments 153 or
154, wherein the antibody has an IC50 of less than, or equal to,
about 0.2 nm, 0.15 nm, 0.10 nm, 0.09 nm. 156. The method of
embodiment 155, wherein the IC50 is measured in an in vitro assay,
such as an assay as provided for herein. 157. The method of any one
of embodiments 152-157, wherein the cell is an A549 cell or a HOCF
cell. 158. A method of treating thyroid eye disease in a subject,
the method comprising administering an antibody of any one of
embodiments 107-111, or a pharmaceutical composition comprising the
same to the subject, wherein the antibody has a serum concentration
in the subject of at least, or about, 70 .mu.g/ml, 75 .mu.g/ml, 80
.mu.g/ml, 85 .mu.g/ml, 90 .mu.g/ml, 95 .mu.g/ml, 100 .mu.g/ml, or
105 .mu.g/ml at least 1, 2, or 3 weeks after administration. 159.
The method of embodiment 158, wherein the antibody or the
pharmaceutical composition is administered intravenously. 160. The
method of embodiments 158 or 159, wherein the antibody or the
pharmaceutical composition is administered at a dose of about 1
.mu.g/kg to about 5 .mu.g/kg (mg antibody/kg subject), of about 5
.mu.g/kg to about 10 .mu.g/kg antibody, or about 5 .mu.g/kg to
about 20 .mu.g/kg in a first dose or subsequent dose. 161. The
method of any one of embodiments 158-160, wherein said antibody is
administered in the following amounts: about 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 .mu.g/kg antibody as a first dose; and about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
.mu.g/kg antibody in subsequent doses. 162. The method of any one
of embodiments 158-161, wherein the antibody or the pharmaceutical
composition is administered at least, or about, once a week, once
every two weeks, once every 3 weeks, or once every 4 weeks. 163. A
method of inhibiting IGF-1 induced receptor autophosphorylation by
at least 95%, 96%, 97%, 98%, or 99% or by 100% in a subject in need
thereof, the method comprising administering to the subject an
antibody of any one of embodiments 107-111, or a pharmaceutical
composition comprising the same. 164. A pharmaceutical composition
comprising an antibody for treating thyroid eye disease in a
subject, wherein the antibody comprises a light chain variable
region having the amino acid sequence of SEQ ID NO: 13 and a heavy
chain variable region having the amino acid sequence of SEQ ID NO:
14. 165. The pharmaceutical composition of embodiment 164, wherein
the antibody comprises a Fc region with M428L and N434S
substitutions. 166. The
pharmaceutical composition of embodiment 164, wherein the antibody
comprises a Fc region with M428L, N434S, M252Y, S254T, and T256E
substitutions. 167. The pharmaceutical composition of embodiment
164, wherein the antibody comprises a Fc region with M252Y, S254T,
and T256E substitutions. 168. The pharmaceutical composition of
embodiment 164, wherein antibody comprises a light chain having an
amino acid sequence of SEQ ID NO: 93 and a heavy chain amino acid
sequence of SEQ ID NO: 92. 169. The pharmaceutical composition of
embodiment 164, wherein antibody comprises a light chain having a
an amino acid sequence of SEQ ID NO: 93 and a heavy chain amino
acid sequence of SEQ ID NO: 94. 170. The pharmaceutical composition
of embodiment 164, wherein antibody comprises a light chain having
a an amino acid sequence of SEQ ID NO: 93 and a heavy chain amino
acid sequence of SEQ ID NO: 95. 171. A method of treating thyroid
eye disease in a subject, the method comprising administering the
pharmaceutical composition comprising the antibody of any one of
embodiments 164-170. 172. The method of embodiment 171, wherein the
pharmaceutical composition is administered intravenously. 173. The
method of embodiment 171, wherein the pharmaceutical composition is
administered subcutaneously. 174. A method of treating or reducing
the severity of, thyroid-associated ophthalmopathy (TAO), or a
symptom thereof, the method comprising administering to a subject
the pharmaceutical composition of any one of embodiments 164-170.
175. A method of reducing proptosis in an eye in a subject with
thyroid-associated ophthalmopathy (TAO), the method comprising
administering to a subject the pharmaceutical composition of any
one of embodiments 164-170. 176. A method of treating thyroid eye
disease in a subject, the method comprising administering to a
subject the pharmaceutical composition of any one of embodiments
2-4. 177. A method of reducing Clinical Activity Score (CAS) of
thyroid-associated ophthalmopathy (TAO) in a subject, the method
comprising administering to a subject the pharmaceutical
composition of any one of embodiments 164-170. 178. A method of a)
reducing proptosis by at least 2 mm and b) reducing the clinical
activity score (CAS) in a subject with thyroid-associated
ophthalmopathy (TAO), the method comprising administering to a
subject the pharmaceutical composition of any one of embodiments
164-170. 179. The method of any of embodiments 174-178, wherein
proptosis is reduced by at least 2 mm. 180. The method of any of
embodiments 174-178, wherein proptosis is reduced by at least 3 mm.
181. The method of any of embodiments 174-178, wherein proptosis is
reduced by at least 4 mm. 182. The method of any of embodiments
174-178, wherein the clinical activity score (CAS) of the subject
is reduced by at least 2 points. 183. The method of any of
embodiments 174-178, wherein the clinical activity score (CAS) of
the subject is reduced to one (1). 184. The method of any of
embodiments 174-178, wherein the clinical activity score (CAS) of
the subject is reduced to zero (0). 185. A method of treating or
reducing the severity of thyroid-associated ophthalmopathy (TAO) in
a subject comprising administering to a subject a pharmaceutical
composition of any one of embodiments 164-170, wherein treatment
with said antibody (i) reduces proptosis by at least 2 mm in an
eye; (ii) is not accompanied by a deterioration of 2 mm or more in
the other (or fellow eye); and (iii) reduces the CAS in said
subject to either one (1) or zero (0). 186. A method of improving
the quality of life in a subject with thyroid-associated
ophthalmopathy (TAO, also called Graves' Ophthalmopathy/Graves'
Orbitopathy), the method comprising administering to a subject the
pharmaceutical composition of any one of embodiments 164-170. 187.
The method of embodiment 186, wherein the quality of life is
measured by the Graves' Ophthalmopathy Quality of Life (GO-QoL)
assessment, or either the Visual Functioning or Appearance subscale
thereof. 188. The method of embodiment 186, wherein the treatment
results in an improvement of greater than or equal to 8 points on
the GO-QoL. 189. The method of embodiment 186, wherein the
treatment results in an improvement on the Functioning subscale of
the GO-QoL. 190. The method of embodiment 186, wherein the
treatment results in an improvement on the Appearance subscale of
the GO-QoL. 191. A method of treating or reducing the severity of
diplopia in a subject with thyroid-associated ophthalmopathy (TAO),
the method comprising administering to a subject the pharmaceutical
composition of any one of embodiments 164-170. 192. The method of
embodiment 191, wherein the diplopia is constant diplopia. 193. The
method of embodiment 191, wherein the diplopia is inconstant
diplopia. 194. The method of embodiment 191, wherein the diplopia
is intermittent diplopia. 195. The method of embodiment 191,
wherein the improvement in or reduction in severity of diplopia is
sustained at least 20 weeks after discontinuation of antibody
administration. 196. The method of embodiment 191, wherein the
improvement in or reduction in severity of diplopia is sustained at
least 50 weeks after discontinuation of antibody administration.
197. The method of any one of embodiments 171-196, wherein said
pharmaceutical composition is administered at a dosage of about 1
.mu.g/kg to about 5 .mu.g/kg, about 5 .mu.g/kg to about 10
.mu.g/kg, about 10 .mu.g/kg to about 20 .mu.g/kg, about 20 .mu.g/kg
to about 30 .mu.g/kg, about 5 .mu.g/kg, about 10 .mu.g/kg, about 15
.mu.g/kg, about 20 .mu.g/kg, about 25 .mu.g/kg, or about 30
.mu.g/kg of the antibody as a first dose. 198. The method of any
one of embodiments 171-196, wherein said pharmaceutical composition
is administered at a dosage of about 10 .mu.g/kg to about 20
.mu.g/kg of antibody as a first dose. 199. The method of any one of
embodiments 171-196, wherein said pharmaceutical composition is
administered at a dosage of about 1 .mu.g/kg to about 10 .mu.g/kg,
about 2 .mu.g/kg to about 5 .mu.g/kg, or about 5 .mu.g/kg to about
20 .mu.g/kg of antibody in subsequent doses. 200. The method of any
one of embodiments 171-196, wherein said pharmaceutical composition
is administered in the following amounts: about 10 .mu.g/kg
antibody as a first dose; and about 20 .mu.g/kg antibody in
subsequent doses. 201. The method of embodiment 200, wherein said
subsequent doses are administered every three weeks, every four
weeks, every five weeks, every six weeks, every seven weeks, or
every eight weeks for at least 21-52 weeks or longer. 202. A method
of increasing the internalization of IGF-1R on a cell, the method
comprising contacting the cell with the pharmaceutical composition
of any one of embodiments 164-170. 203. The method of embodiment
202, wherein the contacting comprises administering to a subject
the pharmaceutical composition of any one of embodiments 164-170.
204. The method of embodiment 203, wherein the subject has or is at
risk of thyroid eye disease (TED). 205. A method of inhibiting
IGF-1 stimulated receptor phosphorylation on a cell, the method
comprising contacting the cell with the pharmaceutical composition
of any one of embodiments 164-170. 206. The method of embodiment
205, wherein the contacting comprises administering to a subject
the pharmaceutical composition of any one of embodiments 164-170.
207. The method of embodiment 206, wherein the subject has or is at
risk of thyroid eye disease (TED). 208. The method of any one of
embodiments 205-207, wherein the antibody has an IC50 of less than,
or equal to, about 0.2 nm, 0.15 nm, 0.10 nm, 0.09 nm. 209. A method
of treating thyroid eye disease in a subject, the method comprising
administering the pharmaceutical composition of any one of
embodiments 164-170 to the subject, wherein the antibody has a
serum concentration in the subject of at least, or about, 10
.mu.g/ml or 20 .mu.g/ml or 50 .mu.g/ml, 70 .mu.g/ml, 75 .mu.g/ml,
80 .mu.g/ml, 85 .mu.g/ml, 90 .mu.g/ml, 95 .mu.g/ml, 100 .mu.g/ml,
or 105 .mu.g/ml at least 1, 2, or 3 weeks after administration.
210. The method of embodiment 209, wherein the pharmaceutical
composition is administered intravenously or subcutaneously. 211.
An isolated antibody comprising a light chain having the amino acid
sequence of SEQ ID NO: 3 and a heavy chain comprising the amino
acid sequence of SEQ ID NO: 83. 212. An isolated antibody
comprising variable light chain comprising the sequence of SEQ ID
NO: 98 and a variable heavy chain comprising the sequence of SEQ ID
NO: 99 and a Fc region comprising M252Y, S254T, and T256E
mutations. 213. An isolated antibody comprising variable light
chain comprising the sequence of SEQ ID NO: 98 and a variable heavy
chain comprising the sequence of SEQ ID NO: 99 and a Fc region
comprising M428L and N434S mutations. 214. A pharmaceutical
composition comprising the antibody of any one of embodiments
211-213. 215. A pharmaceutical composition suitable for intravenous
administration comprising the antibody of any one of embodiments
211-213. 216. A pharmaceutical composition suitable for
subcutaneous administration comprising the antibody of any one of
embodiments 211-213. 217. A method of treating thyroid eye disease
in a subject, the method comprising administering a pharmaceutical
composition comprising the antibody of any one of embodiments
211-213. 218. The method of embodiment 217, wherein the
pharmaceutical composition is administered intravenously. 219. The
method of embodiment 217, wherein the pharmaceutical composition is
administered subcutaneously. 220. A method of treating or reducing
the severity of, thyroid-associated ophthalmopathy (TAO), or a
symptom thereof, comprising administering to a subject an antibody
of any one of embodiments 211-213 or a pharmaceutical composition
comprising the same. 221. A method of reducing proptosis in an eye
in a subject with thyroid-associated ophthalmopathy (TAO)
comprising administering to a subject an antibody of any one of
embodiments 211-213, or a pharmaceutical composition comprising the
same. 222. A method of treating thyroid eye disease in a subject
comprising administering to a subject an antibody of any one of
embodiments 211-213, or a pharmaceutical composition comprising the
same. 223. A method of reducing Clinical Activity Score (CAS) of
thyroid-associated ophthalmopathy (TAO) in a subject comprising
administering to a subject an antibody of any one of embodiments
211-213, or a pharmaceutical composition comprising the same. 224.
A method of a) reducing proptosis by at least 2 mm and b) reducing
the clinical activity score (CAS) in a subject with
thyroid-associated ophthalmopathy (TAO) comprising administering to
a subject an antibody of any one of embodiments 211-213, or a
pharmaceutical composition comprising the same. 225. The method of
any of embodiments 220-224, wherein proptosis is reduced by at
least 2 mm. 226. The method of any of embodiments 220-224, wherein
proptosis is reduced by at least 3 mm. 227. The method of any of
embodiments 220-224, wherein proptosis is reduced by at least 4 mm.
228. The method of any of embodiments 220-224, wherein the clinical
activity score (CAS) of the subject is reduced by at least 2
points. 229. The method of any of embodiments 220-224, wherein the
clinical activity score (CAS) of the subject is reduced to one (1).
230. The method of any of embodiments 220-224, wherein the clinical
activity score (CAS) of the subject is reduced to zero (0). 231. A
method of treating or reducing the severity of thyroid-associated
ophthalmopathy (TAO) in a subject comprising administering to a
subject an antibody of any one embodiments 1-3, or a pharmaceutical
composition comprising the same, wherein treatment with said
antibody (i) reduces proptosis by at least 2 mm in an eye; (ii) is
not accompanied by a deterioration of 2 mm or more in the other (or
fellow eye); and (iii) reduces the CAS in said subject to either
one (1) or zero (0). 232. A method of improving the quality of life
in a subject with thyroid-associated ophthalmopathy (TAO, also
called Graves' Ophthalmopathy/Graves' Orbitopathy) comprising
administering to a subject an antibody of any one of embodiments
211-213, or a pharmaceutical composition comprising the same. 233.
The method of embodiment 232, wherein the quality of life is
measured by the Graves' Ophthalmopathy Quality of Life (GO-QoL)
assessment, or either the Visual Functioning or Appearance subscale
thereof. 234. The method of embodiment 232, wherein the treatment
results in an improvement of greater than or equal to 8 points on
the GO-QoL. 235. The method of embodiment 232, wherein the
treatment results in an improvement on the Functioning subscale of
the GO-QoL. 236. The method of embodiment 232, wherein the
treatment results in an improvement on the Appearance subscale of
the GO-QoL. 237. A method of treating or reducing the severity of
diplopia in a subject with thyroid-associated ophthalmopathy (TAO)
comprising administering to a subject an antibody of any one of
embodiments 211-213, or a pharmaceutical composition comprising the
same. 238. The method of embodiment 237, wherein the diplopia is
constant diplopia. 239. The method of embodiment 237, wherein the
diplopia is inconstant diplopia. 240. The method of embodiment 237,
wherein the diplopia is intermittent diplopia. 241. The method of
embodiment 237, wherein the improvement in or reduction in severity
of diplopia is sustained at least 20 weeks after discontinuation of
antibody administration. 242. The method of embodiment 237, wherein
the improvement in or reduction in severity of diplopia is
sustained at least 50 weeks after discontinuation of antibody
administration. 243. The method of any one of embodiments 217-242,
wherein said antibody is administered at a dosage of about 1
.mu.g/kg to about 5 .mu.g/kg, about 5 .mu.g/kg to about 10
.mu.g/kg, about 10 .mu.g/kg to about 20 .mu.g/kg, about 20 .mu.g/kg
to about 30 .mu.g/kg, about 5 .mu.g/kg, about 10 .mu.g/kg, about 15
.mu.g/kg, about 20 .mu.g/kg, about 25 .mu.g/kg, or about 30
.mu.g/kg of the antibody as a first dose. 244. The method of any
one of embodiments 217-242, wherein said antibody is administered
at a dosage of about 10 .mu.g/kg to about 20 .mu.g/kg of antibody
as a first dose. 245. The method of any one of embodiments 217-242,
wherein said antibody is administered at a dosage of about 1
.mu.g/kg to about 10 .mu.g/kg, about 2 .mu.g/kg to about 5
.mu.g/kg, or about 5 .mu.g/kg to about 20 .mu.g/kg of antibody in
subsequent doses. 246. The method of any one of embodiments
217-242, wherein said antibody is administered in the following
amounts: about 10 .mu.g/kg antibody as a first dose; and about 20
.mu.g/kg antibody in subsequent doses. 247. The method of
embodiment 246, wherein said subsequent doses are administered
every three weeks, every four weeks, every five weeks, every six
weeks, every seven weeks, or every eight weeks for at least 21-52
weeks or longer. 248. A method of increasing the internalization of
IGF-1R on a cell, the method comprising contacting the cell with an
antibody of any one of embodiments 211-213, or a pharmaceutical
composition comprising the same. 249. The method of embodiment 248,
wherein the contacting comprises administering to a subject the
antibody, or a pharmaceutical composition comprising the same. 250.
The method of embodiment 249, wherein the subject has or is at risk
of thyroid eye disease (TED). 251. A method of inhibiting IGF-1
stimulated receptor phosphorylation on a cell, the method
comprising contacting the cell with an antibody of any one of
embodiments 211-213, or a pharmaceutical composition comprising the
same. 252. The method of embodiment 251, wherein the contacting
comprises administering to a subject an antibody of any one of
embodiments 211-213 or a pharmaceutical composition comprising the
same. 253. The method of embodiment 252, wherein the subject has or
is at risk of thyroid eye disease (TED). 254. The method of any one
of embodiments 251-253, wherein the antibody has an IC50 of less
than, or equal to, about 0.2 nm, 0.15 nm, 0.10 nm, 0.09 nm. 255. A
method of treating thyroid eye disease in a subject, the method
comprising administering an antibody of any one of embodiments
211-213, or a pharmaceutical composition comprising the same to the
subject, wherein the antibody has a serum concentration in the
subject of at least, or about, 10 .mu.g/ml or 20 .mu.g/ml or 50
.mu.g/ml, 70 .mu.g/ml, 75 .mu.g/ml, 80 .mu.g/ml, 85 .mu.g/ml, 90
.mu.g/ml, 95 .mu.g/ml, 100 .mu.g/ml, or 105 .mu.g/ml at least 1, 2,
or 3 weeks after administration. 256. The method of embodiment 255,
wherein the antibody or the pharmaceutical composition is
administered intravenously or subcutaneously.
[0237] The subject matter is now described with reference to the
following examples. These examples are provided for the purpose of
illustration only and the claims should in no way be construed as
being limited to these examples, but rather should be construed to
encompass any and all variations which become evident as a result
of the teaching provided herein. Those of skill in the art will
readily recognize a variety of non-critical parameters that could
be changed or modified to yield essentially similar results.
EXAMPLES
Example 1: IGF-1R Antibodies Block IGF-1 Stimulation
[0238] Blockage of IGF-1 stimulation is measured by secretion of
hyaluronan, in the presence of IGF-1R antibodies VRDN-2700,
VRDN-03100, VRDN-02100, VRDN-02200, VRDN-02300, VRDN-02400,
VRDN-02500, VRDN-01100, VRDN-02600, and VRDN-02301, all of which
are disclosed herein. Immunoglobulins are purified from the sera of
patients with Graves' ophthalmopathy (GO) and tested for their
ability to activate TSHR and/or IGF-1R directly, and TSHR/IGF-1R
cross talk in primary cultures of GO fibroblasts. Cells are treated
with M22 or GO-Igs with or without IGF-1R inhibitory antibodies
such as those provided for herein, including but not limited to,
VRDN-2700, VRDN-03100, VRDN-02100, VRDN-02200, VRDN-02300,
VRDN-02400, VRDN-02500, VRDN-01100, VRDN-02600, and VRDN-02301, all
of which are disclosed herein. Hyaluronan (hyaluronic acid; HA)
secretion is measured as a major biological response for GO
fibroblast stimulation. IGF-1R autophosphorylation is used as a
measure of direct IGF-1R activation. TSHR activation is determined
through cyclic-AMP (cAMP) production. The IGF-1R antibodies, as
disclosed herein, are found to effectively block HA secretion and,
therefore, are found to block IGF stimulation.
Example 2: Treatment of Patients with Thyroid Eye Disease and
Clinical Assessment of IGF-1R Antibodies on Thyroid Eye Disease
[0239] Infusions of IGF-1R inhibitory antibodies such as those
provided for herein, including but not limited to, VRDN-2700,
VRDN-03100, VRDN-02100, VRDN-02200, VRDN-02300, VRDN-02400,
VRDN-02500, VRDN-01100, VRDN-02600, and VRDN-02301, all of which
are disclosed herein, are provided to the subjects. The number of
infusions is individualized for each subject and is based on the
investigator's clinical judgment. The Day 1 Visit occurs within 14
days after the final visit of the prior trial. Visit windows are
.+-.1 day for Weeks 1 and 4, .+-.3 days for Weeks 3, 6, 9, 12, 15,
18, 21, and 24. The Follow-up period is meant for subjects who were
proptosis non-responders in the prior trial only; subjects who
relapsed in the prior trial did not participate in the Follow-Up
Period. Visit windows during the Follow-up period are .+-.7
days.
[0240] Treatment Period is 24 weeks (6 months), during which 8
infusions of teprotumumab are administered.
[0241] Subjects who are proptosis non-responders are scheduled to
participate in a 6-month Follow-Up Period in this extension study;
subjects who relapsed in the lead-in study and are retreated in
this extension study will not participate in the Follow-Up
Period.
[0242] Efficacy assessments are performed for both eyes at each
assessment time point. The "study eye" (i.e., the more severely
affected eye) will remain the same as that identified at the
Baseline (Day 1) Visit of the prior study. Both eyes are assessed
for efficacy but the study eye is used to assess the primary
outcome measure.
[0243] Efficacy is assessed by proptosis (measured as exophthalmos
evaluation of the Clinical Measures of Severity using a Hertel
instrument for consistency in measurement), CAS (7-item scale),
diplopia (measured as part of the Clinical Measures of Severity)
and Clinical Measures of Severity (including motility restriction
assessments).
[0244] Quality of Life is Assessed Using the GO-QoL
Questionnaire.
[0245] Safety is assessed via AE and concomitant medication use
monitoring, immunogenicity testing, physical and ophthalmic
examinations, vital signs, clinical safety laboratory evaluations
(complete blood count, chemistry (including thyroid panel and
HbA1C), and urinalysis), pregnancy testing (if applicable), and
electrocardiograms (ECG). The study is also monitored by a Data
Safety Monitoring Board (DSMB).
[0246] Proptosis assessments is performed using a Hertel
exophthalmometer for consistency in measurement, and (except when
strictly unavoidable) the same Hertel instrument and same observer
is used at each evaluation for the full duration of the study.
Additionally, the same intercanthal distance (ICD) is used on each
occasion.
[0247] Proptosis is measured for each eye on Day 1 and Weeks 6, 12,
18, and 24 (or premature withdrawal (PW)) during the Treatment
Period, and at Months 7, 9, and 12 (or PW) during the Follow-Up
Period. Measurements is recorded on the Clinical Measures of
Severity eCRF under exophthalmos.
[0248] The antibodies are found to be effective in treating thyroid
eye disease and also improving quality of life as provided for
herein.
Example 3: Antibody with Increased pK
[0249] Cynomolgus monkeys were dosed with an antibody comprising
the CDRs of VRDN-2700 with the YTE mutation in the Fc domain in an
amount of 10 .mu.g/kg by either intravenous or subcutaneous route,
and samples were collected at 0.5 hr, 2 hr, 8 hr and days 1, 3, 7,
10, 14, 21, and 28 time points for PK analysis by ELISA.
Teprotumumab was also administered at 10 .mu.g/kg IV as a
comparator. The results illustrated in FIG. 1 demonstrate that the
antibody had a significantly higher PK as compared to
Teprotumumab.
[0250] This result demonstrates an antibody comprising the CDRs of
VRDN-2700 can likely be given at a lower dose as compared to
Teprotumumab, even when administered subcutaneously. These results
could not have been predicted.
Example 4
[0251] VRDN-1100 is an antagonist antibody to insulin-like growth
factor-1 receptor (IGF-1R) under development for treatment of
Thyroid Eye Disease (TED). TED is driven by Thyroid Stimulating
Hormone Receptor (TSHR) agonistic autoantibodies and crosstalk
between TSHR and IGF-1R. TED is characterized by recruitment of
fibrocytes that express IGF-1R and TSHR in orbital tissues, where
they mediate deposition of hyaluronan and expansion of orbital
muscle and fat1. IGF-1R antagonism has been found to reverse this
orbital tissue expansion and robustly relieve symptoms in TED
patients2.
[0252] VRDN-1100 is a humanized monoclonal antibody targeting
IGF-1R. The IGF-1R binding and antagonist characteristics of
VRDN-1100 was analyzed.
[0253] Methods
[0254] Surface plasmon resonance (SPR): Antibodies were captured by
immobilized anti-Fc, and recombinant IGF-1R extracellular domain
(ECD) was flowed as analyte. Association and dissociation rate
constants (ka and kd, respectively), and equilibrium dissociation
constant KD were derived by global fit of data to single site
model.
[0255] Epitope binning: VRDN-1100 was immobilized on a chip surface
by amine coupling and used to capture IGF-1R-ECD, after which
teprotumumab was flowed over the chip.
[0256] Cell binding: A549 human lung adenocarcinoma cells or
primary human ocular choroid fibroblasts (HOCF) were incubated with
varying concentrations of VRDN-1100 or teprotumumab. A single dose
50 nM IgG1 isotype control was used as negative control. Unbound
antibody was removed by washing, and the cells were incubated with
an Alexa Fluor 488-goat anti-human antibody and a cell impermeable
dye to gate live cells. The median fluorescence intensity (MFI) of
viable cells was measured by flow cytometry and the data were
analyzed using FlowJo software. Dose curves were fitted using a
non-linear regression model; log(agonist) vs response-variable
slope (four parameters).
[0257] Internalization: Cells were incubated with various
concentrations of antibodies of interest at 4.degree. C. and
37.degree. C. for 60 minutes. Cells were then washed 3.times. and
incubated with FITC-labeled goat anti-human Fc secondary antibody
for 30 minutes at 4.degree. C. The MFI of viable cells was measured
by flow cytometry and the data were analyzed using FlowJo
software.
[0258] Cell surface marker expression: HOCF cells were incubated
with directly labeled antibodies or IgG isotype control at 10
ug/mL. The median fluorescence intensity (MFI) was measured by flow
cytometry and the data were analyzed using FlowJo software.
[0259] Antagonism: Serum starved A549 or HOCF cells were
preincubated with varying concentrations of test antibody for one
hour at 37.degree. C., then stimulated by addition of 100 ng/mL
(A549s) or 200 ng/mL (HOCFs) IGF-1 for 7 minutes at 37.degree. C.
Phosphorylated IGF-1R (pIGF1R) of biological duplicates was
measured using the R&D Systems pIGF-1R ELISA according to the
manufacturer's protocol and pIGF-1R concentrations were normalized
to the lowest test antibody concentration. Dose curves were fit
using a non-linear regression model; log(inhibitor) vs
response-variable slope (four parameters)).
[0260] Results
[0261] VRDN-1100 Binds IGF-1R With Sub-Nanomolar Affinity. FIG. 2A
illustrates that increasing concentrations of IGF-1R-ECD bound to
anti-FC captured VRDN-1100 or teprotumumab reveal a stepwise
increase in SPR signal, enabling a global fit to a binding model.
Following IGF-1R washout, VRDN-1100 shows a more sustained binding
interaction. FIG. 2B illustrates IGF-1R-ECD bound robustly to
immobilized VRDN-1100. Teprotumumab showed no binding to the
IGF-1R:VRDN-1100 complex, suggesting that teprotumumab and
VRDN-1100 have overlapping epitopes. The data is also illustrated
in the table as shown in FIG. 2B.
[0262] VRDN-1100 Binds With High Affinity To IGF-1R On A549 Cells.
As illustrated in FIG. 3A-C, VRDN-1100 binding to A549 cells was
assessed by flow cytometry and found to have similar binding
distribution as teprotumumab at three different concentrations. As
also illustrated in FIG. 3D, in, the binding dose response curve
demonstrated VRDN-1100 EC50=0.1 nM. As illustrated in FIG. 3E-F,
VRDN-1100, VRDN-2700 with M252Y, S254T, and T256E mutation in the
Fc domain, and teprotumumab show comparable binding at temperatures
that block IGF-1R receptor internalization. FIG. 3F illustrates
that VRDN-1100, VRDN-2700 with a M252Y, S254T, and T256E mutation
in the Fc domain, and teprotumumab cause comparable levels of
internalization (.about.50%) measured by reduction in membrane
IGF-1R receptor levels at 37.degree. C. vs 4.degree. C. In FIG. 3F
bar graphs the left most bars are the isotype control, the second
to left set of bars are teprotumumab, the second from the right set
of bars are VRDN-1100 and the right most set of bars are
VRDN-2700.
[0263] HOCFs as an In Vitro Model for TED Pathology.
[0264] CD34+, Thy-1+ orbital fibroblasts are implicated in
extracellular matrix deposition and pathogenic fibrosis in TEDS. As
illustrated in FIG. 4A-C, HOFCs were shown to express (Panel A)
IGF-1R and (Panel B) TSHR, as well as (Panel C) CD34 and Thy-1,
which demonstrates their ability to be used as an in vitro model
system for IGF-1R function in TED.
[0265] VRDN-1100 Binds with High Affinity to IGF-1R on HOCF
Cells.
[0266] FIG. 5A and FIG. 5B illustrate VRDN-1100 binding to HOCF
cells, which was assessed by flow cytometry and found to have
largely similar binding as teprotumumab at three different
concentrations. The lower panel of FIG. 5B illustrates a binding
dose response curve, which demonstrated VRDN-1100 having an
EC50=0.4 nM.
[0267] VRDN-1100 Is A Sub-Nanomolar IGF-1R Antagonist. VRDN-1100
potently inhibits IGF-1 stimulated receptor phosphorylation on A549
cells (IC50=0.09 nM) and HOCF cells (IC50=0.09 nM), which is
illustrated in FIG. 6A and FIG. 6B.
[0268] These results demonstrate that VRDN-1100 and teprotumumab
epitopes on IGF-1R overlap, that VRDN-1100 binds to IGF-1R on cells
with sub-nanomolar EC50, VRDN-1100 promotes IGF-1R internalization,
and that VRDN-1100 inhibits IGF-1R phosphorylation with
sub-nanomolar IC50. Accordingly, VRDN-1100 binds, antagonizes, and
internalizes IGF-1R at sub-nanomolar concentrations, suggesting
that VRDN-1100 should be a able to be used for the potential,
potent inhibition of the pathophysiology driving TED.
Example 4
[0269] VRDN-2700, which has a M252Y, S254T, and T256E mutation in
the Fc domain is a novel anti-IGF-1R antibody incorporating
half-life extension modifications in its Fc region as described
herein and can be used for the treatment of Thyroid Eye Disease
(TED). The pharmacokinetic (PK) parameters of VRDN-2700 with such
Fc mutations was measured in cynomolgus monkeys to the marketed
IGF-1R antibody, teprotumumab, and a PK model was constructed to
project potential human dosing regimens.
[0270] TED is an autoimmune condition most commonly associated with
Graves' disease and hyperthyroidism but can also be found in
patients who are euthyroid or hypothyroid. Orbitopathy in TED is
driven by Thyroid Stimulating Hormone Receptor (TSHR) agonistic
autoantibodies and crosstalk between TSHR and IGF-1R. Pathological
remodeling of the orbit and periorbital tissues results in varied
presentations which may include dry eyes, increased lacrimation,
local irritation, eyelid retraction and eventually proptosis,
diplopia, and optic nerve compression, with ensuing vision
loss.
[0271] The underlying pathology of TED is the activation of an
inflammatory cascade within the orbit, primarily due to recruitment
of fibrocytes and immune cells. Over-expression of IGF-1R has been
demonstrated within the orbit of TED patients, and it has been
surmised that IGF-1R inhibitory antibodies may disrupt the IGF-1R
and TSHR cross-talk and dampen the inflammatory cascade. Indeed,
IGF-1R antagonism has been demonstrated to robustly relieve much of
the inflammatory symptomology that affects TED patients.
[0272] VRDN-2700 is a monoclonal antibody that inhibits IGF-1
mediated signaling via IGF-1R with subnanomolar potency and
incorporates clinically validated Fc modifications (M252Y, S254T,
and T256E) to extend half-life. This antibody was found to have a
more favorable PK profile with the potential for a less burdensome
treatment paradigm for patients than conventional IgG therapeutic
antibodies.
[0273] VRDN-2700 with the Fc mutations was administered to
cynomolgus monkeys by 30 min intravenous (IV) infusions at 2, 10,
and 50 .mu.g/kg, and by subcutaneous (SC) injection at 2 and 10
.mu.g/kg. Teprotumumab at 10 .mu.g/kg was likewise administered by
30 min IV infusion. VRDN-2700 and teprotumumab levels in serum were
measured using a human IgG specific ELISA assay. Data were analyzed
using the WinNonlin non-compartmental model. A semi-mechanistic
model incorporating target mediated drug disposition was
constructed using available human and cynomolgus data. The data is
illustrated below.
[0274] The table and graphs illustrate of FIG. 7 the more favorable
PK profile.
[0275] The table shows PK parameters+/-SD. Evidence of target
mediated drug disposition (TMDD) was observed at 2 .mu.g/kg, but
not at 10 and 50 .mu.g/kg doses, in line with teprotumumab and
other IGF-1R antibodies that have reported saturation of TMDD at
higher doses.
[0276] VRDN-2700 Half-Life Extension Modifications Prolong
Exposure
[0277] At equivalent doses, SC dosed VRDN-2700 with the YTE
mutations has greater exposure than intravenously infused
teprotumumab and achieves .about.2.times. half-life of teprotumumab
in NHPs Estimated 62% bioavailability (F) of VRDN-2700 from SC
dosing using preliminary discovery-stage formulation. Parameter
estimates+/-SD shown in FIG. 8.
[0278] Model simulations predict that dosing of VRDN-2700 at 10
.mu.g/kg every 3 weeks or 20 .mu.g/kg every 6 weeks will result in
Cmin of >100 ug/mL, similar to the approved teprotumumab regimen
(10 .mu.g/kg first dose followed by seven 20 .mu.g/kg doses q3w).
The 10 .mu.g/kg q3w regimen will with lower Cmax values. A longer
dosing interval would increase patient convenience and reduce
treatment costs, while lower dose and Cmax values may potentially
mitigate toxicities. Furthermore, the model predicts that weekly
subcutaneous dosing of VRDN-2700 at 300 mg fixed dose could achieve
a steady-state Cmin of .about.130 ug/mL, enabling at home
self-administration. In the event that lower Cmin values are
efficacious, subcutaneous administration of VRDN-2700 at 300 mg
fixed dose every other week is predicted to achieve .about.50 ug/mL
steady-state Cmin levels. Taken together, the extended half-life of
VRDN-2700 is predicted to provide patients with a wider range of
options for more convenient dosing interval and route of
administration.
Example 5
[0279] VRDN2700 Properties During the evaluation of the antibodies,
expression of VRDN-2700 was compared to other antibodies having
mutations in the Fc domain, such as the L/S mutations that are
described herein. Unexpectedly, the yield for the antibody with the
YTE mutation in the Fc domain (VRDN2700) was approximately 80%
higher than the yield of a similar antibody except that it has a
L/S mutation. This was surprising and unexpected as other
antibodies that have been tested that target IGF-1R with the YTE or
LS mutations had similar expressions regardless of the Fc
mutations. The YTE version had fewer lower molecular weight species
as compared to the LS version. Thus, indicating that the YTE
antibody has fewer impurities and is a more homogenous composition,
which provides advantages over the antibody with the LS mutation.
This was also not predictable as another antibody that was
evaluated showed the opposite effect on such species. Furthermore,
during purification, it was found that the LS mutant formed more
aggregates when being purified on a cation exchange column as
compared VRDN-2700. The aggregation of the LS mutant would cause
significant manufacturing issues, which were not observed for
VRDN-2700. Therefore, this difference in the Fc mutants for this
antibody could not have been predicted or expected and leads to
significant and unexpected advantages for the antibody that is
referenced herein as VRDN-2700.
[0280] The prolonged half-life of VRDN-2700 (YTE) demonstrates that
it can be used in a convenient SC injection, or as an IV infusion
requiring fewer and/or less frequent treatments vs. conventional
therapeutic IgG antibodies and has superior properties as compared
to other Fc mutant versions of the same antibody (same variable
regions).
Example 6: VRDN-1100 with YTE or YTE/C22S Mutations Bind to IGF-1R
and Inhibits IGF-1R Autophosphorylation
[0281] The binding of VRDN-1100 with the Fc YTE mutations in the
heavy chain (SEQ ID NO: 94) or C22S mutation and Fc YTE mutations
in the heavy chain (SEQ ID NO: 95) to IGF-1R was evaluated in a
cell based binding assay (A549 cells). The light chains have a
sequence of SEQ ID NO: 93. The YTE Fc mutatant version of VRDN1100
was found to bind to A549 cells with an EC50 of 0.30 nm and the
C22S and Fc YTE mutant had an EC50 of 0.36 nm. The antibodies were
also evaluated for their ability to inhibit IGF-1R
autophosphorylation. The YTE only mutant had an IC50 of 0.40 nm and
the C22S plus YTE mutations had an IC50 of 0.37 nm. Thus, the
antibodies were found to be able to both bind to IGF-1R and inhibit
its autophosphorylation.
Example 7: VRDN-1100 with a C22S Mutation Binds to IGF-1R
[0282] A mutant of VRDN-1100 with a C22S mutation in the heavy
chain (SEQ ID NO: 96) and a VL comprising a sequence of SEQ ID NO:
97 was evaluated for its binding to IGF-1R in a surface plasma
resonance assay. Using this assay, the antibody was found to bind
to IGF-1R with a k.sub.a (1/Ms) of 1.04.times.10.sup.5, a k.sub.d
(1/s) of 2.18.times.10.sup.-5, and a K.sub.D(M) of
2.10.times.10.sup.-10 at a pH of 7.4.
[0283] Each of these examples and the embodiments provided herein
demonstrate that the antibodies provided for herein can be used to
treat TED and their associate symptoms.
[0284] All references cited herein are incorporated by reference to
the same extent as if each individual publication, database entry
(e.g. Genbank sequences or GeneID entries), patent application, or
patent, was specifically and individually indicated to be
incorporated by reference. This statement of incorporation by
reference is intended by Applicants, pursuant to 37 C.F.R. .sctn.
1.57(b)(1), to relate to each and every individual publication,
database entry (e.g. Genbank sequences or GeneID entries), patent
application, or patent, each of which is clearly identified in
compliance with 37 C.F.R. .sctn. 1.57(b)(2), even if such citation
is not immediately adjacent to a dedicated statement of
incorporation by reference. The inclusion of dedicated statements
of incorporation by reference, if any, within the specification
does not in any way weaken this general statement of incorporation
by reference. Citation of the references herein is not intended as
an admission that the reference is pertinent prior art, nor does it
constitute any admission as to the contents or date of these
publications or documents.
[0285] The present embodiments are not to be limited in scope by
the specific embodiments described herein. Indeed, various
modifications in addition to those described herein will become
apparent to those skilled in the art from the foregoing
description. Such modifications are intended to fall within the
scope of the embodiments and any appended claims.
[0286] The present specification is considered to be sufficient to
enable one skilled in the art to practice the embodiments. Various
modifications in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the present disclosure and
any appended claims.
Sequence CWU 1
1
991215PRTArtificial Sequencesynthetic sequence 1Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp
Ala Ser Lys Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Lys Trp Pro Pro
85 90 95Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ser Lys Arg Thr Val
Ala 100 105 110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser 115 120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg Glu 130 135 140Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser Gly Asn Ser145 150 155 160Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200
205Ser Phe Asn Arg Gly Glu Cys 210 2152448PRTArtificial
Sequencesynthetic sequence 2Gln Val Glu Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg1 5 10 15Ser Gln Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ile Ile Trp Phe Asp Gly
Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala Arg Glu
Leu Gly Arg Arg Tyr Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110Leu
Val Ser Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120
125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 4453219PRTArtificial
Sequencesynthetic sequence 3Asp 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 Ile Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu Gln Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys
Val Ser Asn Arg Leu Tyr 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 Phe Gln Gly 85 90 95Ser 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 2154447PRTArtificial Sequencesynthetic
sequence 4Gln 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 Tyr Ser Ile
Thr Gly Gly 20 25 30Tyr Leu Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp 35 40 45Ile Gly Tyr Ile Ser Tyr Asp Gly Thr Asn Asn
Tyr Lys Pro Ser Leu 50 55 60Lys Asp Arg Val Thr Ile Ser Arg Asp Thr
Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Tyr Gly Arg Val Phe
Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150
155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Arg Val Glu Pro
Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265
270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390
395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 435 440 4455214PRTArtificial Sequencesynthetic
sequence 5Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu
Gly Gln1 5 10 15Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser
Tyr Tyr Ala 20 25 30Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Ile
Leu Val Ile Tyr 35 40 45Gly Glu Asn Lys Arg Pro Ser Gly Ile Pro Asp
Arg Phe Ser Gly Ser 50 55 60Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile
Thr Gly Ala Gln Ala Glu65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Lys
Ser Arg Asp Gly Ser Gly Gln His 85 90 95Leu Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110Ala Ala Pro Ser Val
Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125Ala Asn Lys
Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140Ala
Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly145 150
155 160Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala
Ala 165 170 175Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
His Arg Ser 180 185 190Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr
Val Glu Lys Thr Val 195 200 205Ala Pro Ala Glu Cys Ser
2106460PRTArtificial Sequencesynthetic sequence 6Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr 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 Ala Pro Leu Arg Phe Leu Glu Trp Ser Thr Gln Asp
His Tyr 100 105 110Tyr Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr
Thr Val Thr Val 115 120 125Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser 130 135 140Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys145 150 155 160Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 165 170 175Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 180 185 190Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 195 200
205Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
210 215 220Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro225 230 235 240Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe 245 250 255Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val 260 265 270Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe 275 280 285Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 290 295 300Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr305 310 315
320Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
325 330 335Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala 340 345 350Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg 355 360 365Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly 370 375 380Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro385 390 395 400Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 405 410 415Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 420 425 430Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 435 440
445Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455
4607214PRTArtificial Sequencesynthetic sequence 7Asp Ile Gln Met
Thr Gln Phe Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr
Ala Ala Ser Arg Leu His Arg 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 Leu Gln His Asn Ser Tyr Pro
Cys 85 90 95Ser Phe Gly Gln Gly Thr Lys Leu 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 2108450PRTArtificial Sequencesynthetic
sequence 8Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe
Ser Ser Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Thr Thr Phe
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Arg Thr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Asp Leu Gly Trp Ser
Asp Ser Tyr Tyr Tyr Tyr Tyr Gly Met 100 105 110Asp Val Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 115 120 125Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu145 150
155 160Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His 165 170 175Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser 180 185 190Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr
Gln Thr Tyr Thr Cys 195 200 205Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys Thr Val Glu 210
215 220Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val
Ala225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 290 295 300Arg Val Val Ser
Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly305 310 315 320Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 325 330
335Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro385 390 395 400Met Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro
Gly 4509219PRTArtificial Sequencesynthetic sequence 9Asp Val Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30Asn
Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala 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
Met Gln Gly 85 90 95Thr His Trp Pro Leu 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
21510449PRTArtificial Sequencesynthetic sequence 10Gln Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly1 5 10 15Thr Leu Ser
Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30Asn Trp
Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile
Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu 50 55
60Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser65
70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Trp Thr Gly Arg Thr Asp Ala Phe Asp Ile Trp Gly
Gln Gly 100 105 110Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200
205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315
320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445Lys11214PRTArtificial Sequencesynthetic sequence 11Glu Ile Val
Leu Thr Gln Ser Pro Gly Thr Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Ser Ser 20 25 30Leu
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45Lys Tyr Ala Ser Gln Ser Leu Ser Gly Ile Pro Asp Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Ser Ser Arg
Leu Pro His 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 21012448PRTArtificial
Sequencesynthetic sequence 12Glu Val Gln Leu Val Gln Ser Gly Gly
Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Ala Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Ser Val Ile Asp Thr Arg
Gly Ala Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Leu
Gly Asn Phe Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr 100 105
110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser225 230
235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345
350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
44513113PRTArtificial Sequencesynthetic sequence 13Asp Val Val Met
Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Pro Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30Asn Val
Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro
Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55
60Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile65
70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln
Gly 85 90 95Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 105 110Arg14124PRTArtificial Sequencesynthetic sequence
14Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 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
Gln Lys Phe 50 55 60Gln Gly 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 Phe 85 90 95Ala Arg Gly Arg Pro Asp Tyr Tyr Gly
Ser Ser Lys Trp Tyr Phe Asp 100 105 110Val Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser 115 12015107PRTArtificial Sequencesynthetic
sequence 15Asp Ile Gln Met Thr Gln Ser Pro Leu Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Arg Asp Ile
Arg Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Ser Leu Gln Thr Gly Val Pro
Ser Arg Phe Gly Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Ser Phe Thr
Ile Gly Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys
Gln Gln Phe Asp Ser Leu Pro His 85 90 95Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 10516120PRTArtificial Sequencesynthetic
sequence 16Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ile Tyr 20 25 30Arg Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Gly Ile Ser Pro Ser Gly Gly Thr Thr Trp
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Ser Gly Gly Ser
Gly Tyr Ala Phe Asp Ile Trp Gly Gln 100 105 110Gly Thr Met Val Thr
Val Ser Ser 115 1201711PRTArtificial Sequencesynthetic sequence
17Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala1 5 10187PRTArtificial
Sequencesynthetic sequence 18Asp Ala Ser Lys Arg Ala Thr1
51910PRTArtificial Sequencesynthetic sequence 19Gln Gln Arg Ser Lys
Trp Pro Pro Trp Thr1 5 10205PRTArtificial Sequencesynthetic
sequence 20Ser Tyr Gly Met His1 52117PRTArtificial
Sequencesynthetic sequence 21Ile Ile Trp Phe Asp Gly Ser Ser Thr
Tyr Tyr Ala Asp Ser Val Arg1 5 10 15Gly229PRTArtificial
Sequencesynthetic sequence 22Glu Leu Gly Arg Arg Tyr Phe Asp Leu1
52320PRTArtificial Sequencesynthetic sequence 23Arg Ser Ser Gln Ser
Ile Val His Ser Asn Gly Asn Thr Tyr Leu Gln1 5 10 15Trp Tyr Leu Gln
20247PRTArtificial Sequencesynthetic sequence 24Lys Val Ser Asn Arg
Leu Tyr1 5259PRTArtificial Sequencesynthetic sequence 25Phe Gln Gly
Ser His Val Pro Trp Thr1 5266PRTArtificial Sequencesynthetic
sequence 26Gly Gly Tyr Leu Trp Asn1 52716PRTArtificial
Sequencesynthetic sequence 27Tyr Ile Ser Tyr Asp Gly Thr Asn Asn
Tyr Lys Pro Ser Leu Lys Asp1 5 10 15288PRTArtificial
Sequencesynthetic sequence 28Tyr Gly Arg Val Phe Phe Asp Tyr1
52911PRTArtificial Sequencesynthetic sequence 29Gln Gly Asp Ser Leu
Arg Ser Tyr Tyr Ala Thr1 5 10307PRTArtificial Sequencesynthetic
sequence 30Gly Glu Asn Lys Arg Pro Ser1 53111PRTArtificial
Sequencesynthetic sequence 31Lys Ser Arg Asp Gly Ser Gly Gln His
Leu Val1 5 10325PRTArtificial Sequencesynthetic sequence 32Ser Tyr
Ala Ile Ser1 53317PRTArtificial Sequencesynthetic sequence 33Gly
Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln1 5 10
15Gly3421PRTArtificial Sequencesynthetic sequence 34Ala Pro Leu Arg
Phe Leu Glu Trp Ser Thr Gln Asp His Tyr Tyr Tyr1 5 10 15Tyr Tyr Met
Asp Val 203511PRTArtificial Sequencesynthetic sequence 35Arg Ala
Ser Gln Gly Ile Arg Asn Asp Leu Gly1 5 10367PRTArtificial
Sequencesynthetic sequence 36Ala Ala Ser Arg Leu His Arg1
5379PRTArtificial Sequencesynthetic sequence 37Leu Gln His Asn Ser
Tyr Pro Cys Ser1 5385PRTArtificial Sequencesynthetic sequence 38Ser
Tyr Ala Met Asn1 53917PRTArtificial Sequencesynthetic sequence
39Ala Ile Ser Gly Ser Gly Gly Thr Thr Phe Tyr Ala Asp Ser Val Lys1
5 10 15Gly4016PRTArtificial Sequencesynthetic sequence 40Asp Leu
Gly Trp Ser Asp Ser Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val1 5 10
154116PRTArtificial Sequencesynthetic sequence 41Arg Ser Ser Gln
Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp1 5 10
15426PRTArtificial Sequencesynthetic sequence 42Leu Gly Ser Asn Arg
Ala1 5439PRTArtificial Sequencesynthetic sequence 43Met Gln Gly Thr
His Trp Pro Leu Thr1 5447PRTArtificial Sequencesynthetic sequence
44Ser Ser Ser Asn Trp Trp Ser1 54516PRTArtificial Sequencesynthetic
sequence 45Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu
Lys Ser1 5 10 154610PRTArtificial Sequencesynthetic sequence 46Trp
Thr Gly Arg Thr Asp Ala Phe Asp Ile1 5 104711PRTArtificial
Sequencesynthetic sequence 47Arg Ala Ser Gln Ser Ile Gly Ser Ser
Leu His1 5 10487PRTArtificial Sequencesynthetic sequence 48Tyr Ala
Ser Gln Ser Leu Ser1 5499PRTArtificial Sequencesynthetic sequence
49His Gln Ser Ser Arg Leu Pro His Thr1 5505PRTArtificial
Sequencesynthetic sequence 50Ser Phe Ala Met His1
55116PRTArtificial Sequencesynthetic sequence 51Val Ile Asp Thr Arg
Gly Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly1 5 10
155210PRTArtificial Sequencesynthetic sequence 52Leu Gly Asn Phe
Tyr Tyr Gly Met Asp Val1 5 105316PRTArtificial Sequencesynthetic
sequence 53Arg Ser Ser Gln Ser Ile Val His Ser Asn Val Asn Thr Tyr
Leu Glu1 5 10 15547PRTArtificial Sequencesynthetic sequence 54Lys
Val Ser Asn Arg Phe Ser1 5559PRTArtificial Sequencesynthetic
sequence 55Phe Gln Gly Ser His Val Pro Pro Thr1 5565PRTArtificial
Sequencesynthetic sequence 56Ser Tyr Trp Met His1
55718PRTArtificial Sequencesynthetic sequence 57Gly Glu Ile Asn Pro
Ser Asn Gly Arg Thr Asn Tyr Asn Gln Lys Phe1 5 10 15Gln
Gly5815PRTArtificial Sequencesynthetic sequence 58Gly Arg Pro Asp
Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp Val1 5 10
155911PRTArtificial Sequencesynthetic sequence 59Gln Ala Ser Arg
Asp Ile Arg Asn Tyr Leu Asn1 5 10607PRTArtificial Sequencesynthetic
sequence 60Asp Ala Ser Ser Leu Gln Thr1 5619PRTArtificial
Sequencesynthetic sequence 61Gln Gln Phe Asp Ser Leu Pro His Thr1
5625PRTArtificial Sequencesynthetic sequence 62Ile Tyr Arg Met Gln1
56316PRTArtificial Sequencesynthetic sequence 63Gly Ile Ser Pro Ser
Gly Gly Thr Thr Trp Tyr Ala Asp Ser Val Lys1 5 10
156411PRTArtificial Sequencesynthetic sequence 64Trp Ser Gly Gly
Ser Gly Tyr Ala Phe Asp Ile1 5 1065663PRTArtificial
Sequencesynthetic sequence 65Glu Ile Val Leu Thr Gln Ser Pro Ala
Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg
Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Lys Arg
Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Arg Ser Lys Trp Pro Pro 85 90 95Trp Thr
Phe Gly Gln Gly Thr Lys Val Glu Ser Lys Arg Thr Val Ala 100 105
110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu 130 135 140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205Ser Phe Asn
Arg Gly Glu Cys Gln Val Glu Leu Val Glu Ser Gly Gly 210 215 220Gly
Val Val Gln Pro Gly Arg Ser Gln Arg Leu Ser Cys Ala Ala Ser225 230
235 240Gly Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala
Pro 245 250 255Gly Lys Gly Leu Glu Trp Val Ala Ile Ile Trp Phe Asp
Gly Ser Ser 260 265 270Thr Tyr Tyr Ala Asp Ser Val Arg Gly Arg Phe
Thr Ile Ser Arg Asp 275 280 285Asn Ser Lys Asn Thr Leu Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu 290 295 300Asp Thr Ala Val Tyr Phe Cys
Ala Arg Glu Leu Gly Arg Arg Tyr Phe305 310 315 320Asp Leu Trp Gly
Arg Gly Thr Leu Val Ser Val Ser Ser Ala Ser Thr 325 330 335Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 340 345
350Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
355 360 365Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His 370 375 380Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser385 390 395 400Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys 405 410 415Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu 420 425 430Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 435 440 445Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 450 455 460Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val465 470
475 480Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp 485 490 495Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr 500 505 510Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp 515 520 525Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu 530 535 540Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg545 550 555 560Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 565 570 575Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 580 585
590Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
595 600 605Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser 610 615 620Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser625 630 635 640Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser 645 650 655Leu Ser Leu Ser Pro Gly Lys
66066666PRTArtificial Sequencesynthetic sequence 66Asp 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 Ile Val His Ser 20 25 30Asn Gly
Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro
Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu Tyr 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 Phe Gln
Gly 85 90 95Ser 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 Gln Val Gln Leu Gln
210 215 220Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser
Leu Thr225 230 235 240Cys Thr Val Ser Gly Tyr Ser Ile Thr Gly Gly
Tyr Leu Trp Asn Trp 245 250 255Ile Arg Gln Pro Pro Gly Lys Gly Leu
Glu Trp Ile Gly Tyr Ile Ser 260 265 270Tyr Asp Gly Thr Asn Asn Tyr
Lys Pro Ser Leu Lys Asp Arg Val Thr 275 280 285Ile Ser Arg Asp Thr
Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser 290 295 300Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Gly Arg305 310 315
320Val Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
325 330 335Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys 340 345 350Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr 355 360 365Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 370 375 380Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser385 390 395 400Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 405 410 415Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 420 425 430Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 435 440
445Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
450 455 460Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys465 470 475 480Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp 485 490 495Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 500 505 510Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 515 520 525His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 530 535 540Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly545 550 555
560Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
565 570 575Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr 580 585 590Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn 595 600 605Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe 610 615 620Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn625 630 635 640Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr 645 650 655Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 660 66567674PRTArtificial
Sequencesynthetic sequence 67Ser Ser Glu Leu Thr Gln Asp Pro Ala
Val Ser Val Ala Leu Gly Gln1 5 10 15Thr Val Arg Ile Thr Cys Gln Gly
Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30Thr Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Ile Leu Val Ile Tyr 35 40 45Gly Glu Asn Lys Arg Pro
Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60Ser Ser Gly Asn Thr
Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75 80Asp Glu Ala
Asp Tyr Tyr Cys Lys Ser Arg Asp Gly Ser Gly Gln His 85 90 95Leu Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105
110Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln
115 120 125Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr
Pro Gly 130 135 140Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
Val Lys Ala Gly145 150 155 160Val Glu Thr Thr Thr Pro Ser Lys Gln
Ser Asn Asn Lys Tyr Ala Ala 165 170 175Ser Ser Tyr Leu Ser Leu Thr
Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190Tyr Ser Cys Gln Val
Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205Ala Pro Ala
Glu Cys Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu 210 215 220Val
Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly225 230
235 240Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro
Gly 245 250 255Gln Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Phe
Gly Thr Ala 260 265 270Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr
Ile Thr Ala Asp Lys 275 280 285Ser Thr Ser Thr Ala Tyr Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp 290 295 300Thr Ala Val Tyr Tyr Cys Ala
Arg Ala Pro Leu Arg Phe Leu Glu Trp305 310 315 320Ser Thr Gln Asp
His Tyr Tyr Tyr Tyr Tyr Met Asp Val Trp Gly Lys 325 330 335Gly Thr
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 340 345
350Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
355 360 365Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser 370 375 380Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val385 390 395 400Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro 405 410 415Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys 420 425 430Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 435 440 445Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 450 455 460Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile465 470
475 480Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu 485 490 495Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His 500 505 510Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg 515 520 525Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys 530 535 540Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu545 550 555 560Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 565 570 575Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 580 585
590Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
595 600 605Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val 610 615 620Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp625 630 635 640Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His 645 650 655Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro 660 665 670Gly
Lys68664PRTArtificial Sequencesynthetic sequence 68Asp Ile Gln Met
Thr Gln Phe Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Arg Leu Ile 35 40 45Tyr Ala Ala Ser Arg Leu His Arg 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 Leu
Gln His Asn Ser Tyr Pro Cys 85 90 95Ser Phe Gly Gln Gly Thr Lys Leu
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 Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly 210 215 220Leu Val Gln Pro Gly Gly Ser Leu
Arg Leu Ser Cys Thr Ala Ser Gly225 230 235 240Phe Thr Phe Ser Ser
Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly 245 250 255Lys Gly Leu
Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Thr Thr 260 265 270Phe
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 275 280
285Ser Arg Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
290 295 300Thr Ala Val Tyr Tyr Cys Ala Lys Asp Leu Gly Trp Ser Asp
Ser Tyr305 310 315 320Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln
Gly Thr Thr Val Thr 325 330 335Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro 340 345 350Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val 355 360 365Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 370 375 380Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly385 390 395
400Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly
405 410 415Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
Thr Lys 420 425 430Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu
Cys Pro Pro Cys 435 440 445Pro Ala Pro Pro Val Ala Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys 450 455 460Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val465 470 475 480Val Val Asp Val Ser
His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 485 490 495Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 500 505 510Gln
Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His 515 520
525Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
530 535 540Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
Gly Gln545 550 555 560Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met 565 570 575Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro 580 585 590Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn 595 600 605Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 610 615 620Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val625 630 635
640Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
645 650 655Lys Ser Leu Ser Leu Ser Pro Gly 66069668PRTArtificial
Sequencesynthetic sequence 69Asp Val Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Leu His Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr
Leu Gly Ser Asn Arg Ala 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 Met Gln Gly 85 90 95Thr His
Trp Pro Leu 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 Gln Val Gln Leu Gln 210 215 220Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Gly Thr Leu Ser Leu Thr225 230
235 240Cys Ala Val Ser Gly Gly Ser Ile Ser Ser Ser Asn Trp Trp Ser
Trp 245 250 255Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly
Glu Ile Tyr 260 265 270His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu
Lys Ser Arg Val Thr 275 280 285Ile Ser Val Asp Lys Ser Lys Asn Gln
Phe Ser Leu Lys Leu Ser Ser 290 295 300Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Ala Arg Trp Thr Gly305 310 315 320Arg Thr Asp Ala
Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val 325 330 335Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 340 345
350Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
355 360 365Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu 370 375 380Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu385 390 395 400Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr 405 410 415Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val 420 425 430Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 435 440 445Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 450 455 460Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val465 470
475 480Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe 485 490 495Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro 500 505 510Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr 515 520 525Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val 530 535 540Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala545 550 555 560Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 565 570 575Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 580 585
590Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
595 600 605Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser 610 615 620Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln625 630 635 640Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His 645 650 655Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 660 66570662PRTArtificial Sequencesynthetic
sequence 70Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Val Ser
Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile
Gly Ser Ser 20 25 30Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu Ile 35 40 45Lys Tyr Ala Ser Gln Ser Leu Ser Gly Ile Pro
Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys
His Gln Ser Ser Arg Leu Pro His 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 Glu Val Gln
Leu Val Gln Ser Gly Gly Gly 210 215 220Leu Val Lys Pro Gly Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly225 230 235 240Phe Thr Phe Ser
Ser Phe Ala Met His Trp Val Arg Gln Ala Pro Gly 245 250 255Lys Gly
Leu Glu Trp Ile Ser Val Ile Asp Thr Arg Gly Ala Thr Tyr 260 265
270Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
275 280 285Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr 290 295 300Ala Val Tyr Tyr Cys Ala Arg Leu Gly Asn Phe Tyr
Tyr Gly Met Asp305 310 315 320Val Trp Gly Gln Gly Thr Thr Val Thr
Val Ser Ser Ala Ser Thr Lys 325 330 335Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly 340 345 350Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 355 360 365Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 370 375 380Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val385 390
395 400Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn 405 410 415Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro 420 425 430Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu 435 440 445Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 450 455 460Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp465 470 475 480Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 485 490 495Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 500 505
510Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
515 520 525Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro 530 535 540Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu545 550 555 560Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn 565 570 575Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile 580 585 590Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 595 600 605Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 610 615 620Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys625 630
635 640Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu 645 650 655Ser Leu Ser Pro Gly Lys 66071673PRTArtificial
Sequencesynthetic sequence 71Asp Val Val Met Thr Gln Thr Pro Leu
Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Ile Val His Ser 20 25 30Asn Val Asn Thr Tyr Leu Glu
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Arg Leu Leu Ile Tyr
Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly
Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile65 70 75 80Ser Arg Val
Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His
Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu 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 Gln Val Gln Leu Val 210 215 220Gln
Ser Gly Ala Glu Val Val Lys Pro Gly Ala Ser Val Lys Leu Ser225 230
235 240Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met His Trp
Val 245 250 255Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Glu
Ile Asn Pro 260 265 270Ser Asn Gly Arg Thr Asn Tyr Asn Gln Lys Phe
Gln Gly Lys Ala Thr 275 280 285Leu Thr Val Asp Lys Ser Ser Ser Thr
Ala Tyr Met Gln Leu Ser Ser 290 295 300Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Phe Ala Arg Gly Arg Pro305 310 315 320Asp Tyr Tyr Gly
Ser Ser Lys Trp Tyr Phe Asp Val Trp Gly Gln Gly 325 330 335Thr Thr
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 340 345
350Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
355 360 365Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp 370 375 380Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu385 390 395 400Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser 405 410 415Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro 420 425 430Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 435 440 445Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 450 455 460Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser465 470
475 480Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp 485 490 495Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 500 505 510Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 515 520 525Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu 530 535 540Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys545 550 555 560Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 565 570 575Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 580 585
590Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
595 600 605Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu 610 615 620Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys625 630 635 640Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 645 650 655Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 660 665
670Lys72227PRTArtificial Sequencesynthetic sequence 72Asp Ile Gln
Met Thr Gln Ser Pro Leu Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Gln Ala Ser Arg Asp Ile Arg Asn Tyr 20 25 30Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Asp Ala Ser Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Gly Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Ser Phe Thr Ile Gly Ser Leu Gln
Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Phe Asp Ser
Leu Pro His 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Glu
Val Gln Leu Leu 100 105 110Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly Ser Leu Arg Leu Ser 115 120 125Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ile Tyr Arg Met Gln Trp Val 130 135 140Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val Ser Gly Ile Ser Pro145 150 155 160Ser Gly Gly
Thr Thr Trp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 165 170 175Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 180 185
190Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Ser Gly
195 200 205Gly Ser Gly Tyr Ala Phe Asp Ile Trp Gly Gln Gly Thr Met
Val Thr 210 215 220Val Ser Ser225735PRTArtificial Sequencesynthetic
sequenceMISC_FEATURE(1)..(5)n=1-5 73Gly Gly Gly Gly Ser1
5745PRTArtificial Sequencesynthetic
sequenceMISC_FEATURE(1)..(5)n=1-5 74Gly Gly Gly Gly Ala1
575217PRTArtificial Sequencesynthetic sequence 75Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55
60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65
70 75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys 85 90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln 100 105 110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu 115 120 125Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro 130 135 140Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn145 150 155 160Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200
205Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 21576217PRTArtificial
Sequencesynthetic sequence 76Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His65 70 75 80Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105
110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro 130 135 140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn145 150 155 160Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu 165 170 175Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205Lys Ser Leu
Ser Leu Ser Pro Gly Lys 210 21577215PRTArtificial Sequencesynthetic
sequence 77Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro1 5 10 15Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val 20 25 30Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val 35 40 45Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln 50 55 60Phe Asn Ser Thr Phe Arg Val Val Ser Val
Leu Thr Val Val His Gln65 70 75 80Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly 85 90 95Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Thr Lys Gly Gln Pro 100 105 110Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 115 120 125Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 130 135 140Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr145 150
155 160Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr 165 170 175Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe 180 185 190Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys 195 200 205Ser Leu Ser Leu Ser Pro Gly 210
21578232PRTArtificial Sequencesynthetic sequence 78Asp Ile Gln Met
Thr Gln Phe Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr
Ala Ala Ser Arg Leu His Arg 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 Leu Gln His Asn Ser Tyr Pro
Ser 85 90 95Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Glu Val Gln
Leu Leu 100 105 110Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
Leu Arg Leu Ser 115 120 125Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser
Tyr Ala Met Asn Trp Val 130 135 140Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val Ser Ala Ile Ser Gly145 150 155 160Ser Gly Gly Thr Thr
Phe Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 165 170 175Ile Ser Arg
Asp Asn Ser Arg Thr Thr Leu Tyr Leu Gln Met Asn Ser 180 185 190Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp Leu Gly 195 200
205Trp Ser Asp Ser Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln
210 215 220Gly Thr Thr Val Thr Val Ser Ser225 23079107PRTArtificial
Sequencesynthetic sequence 79Asp Ile Gln Met Thr Gln Phe Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr Ala Ala Ser Arg Leu
His Arg 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 Leu Gln His Asn Ser Tyr Pro Ser 85 90 95Ser Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 10580125PRTArtificial
Sequencesynthetic sequence 80Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser
Gly Gly Thr Thr Phe Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Arg Thr Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys
Asp Leu Gly Trp Ser Asp Ser Tyr Tyr Tyr Tyr Tyr Gly Met 100 105
110Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
125819PRTArtificial Sequencesynthetic sequence 81Leu Gln His Asn
Ser Tyr Pro Ser Ser1 582665PRTArtificial Sequencesynthetic sequence
82Asp 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 Ile Val His
Ser 20 25 30Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu Tyr
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 Phe Gln Gly 85 90 95Ser 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 Gln Val Gln Leu Gln 210 215 220Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Glu Thr Leu Ser Leu Thr225 230 235 240Cys Thr Val Ser Gly
Tyr Ser Ile Thr Gly Gly Tyr Leu Trp Asn Trp 245 250 255Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Ser 260 265 270Tyr
Asp Gly Thr Asn Asn Tyr Lys Pro Ser Leu Lys Asp Arg Val Thr 275 280
285Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser
290 295 300Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr
Gly Arg305 310 315 320Val Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 325 330 335Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys 340 345 350Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 355 360 365Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 370 375 380Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser385 390 395
400Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
405 410 415Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 420 425 430Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 435 440 445Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 450 455 460Lys Pro Lys Asp Thr Leu Tyr Ile
Thr Arg Glu Pro Glu Val Thr Cys465 470 475 480Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 485 490 495Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 500 505 510Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 515 520
525His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
530 535 540Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly545 550 555 560Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu 565 570 575Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 580 585 590Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 595 600 605Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 610 615 620Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn625 630 635
640Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
645 650 655Gln Lys Ser Leu Ser Leu Ser Pro Gly 660
66583446PRTArtificial Sequencesynthetic sequence 83Gln 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 Tyr Ser Ile Thr Gly Gly 20 25 30Tyr Leu
Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile
Gly Tyr Ile Ser Tyr Asp Gly Thr Asn Asn Tyr Lys Pro Ser Leu 50 55
60Lys Asp Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser65
70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Tyr Gly Arg Val Phe Phe Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Arg Val Glu Pro
Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu 245 250 255Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265
270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390
395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly 435 440 44584329PRTArtificial Sequencesynthetic
sequence 84Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys
Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys 130 135 140Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150
155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
32585673PRTArtificial Sequencesynthetic sequence 85Asp Val Val Met
Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Pro Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30Asn Val
Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro
Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55
60Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile65
70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln
Gly 85 90 95Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu 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 Gln Val Gln Leu Val
210 215 220Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala Ser Val Lys
Leu Ser225 230 235 240Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
Trp Met His Trp Val 245 250 255Lys Gln Arg Pro Gly Gln Gly Leu Glu
Trp Ile Gly Glu Ile Asn Pro 260 265 270Ser Asn Gly Arg Thr Asn Tyr
Asn Gln Lys Phe Gln Gly Lys Ala Thr 275 280 285Leu Thr Val Asp Lys
Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser 290 295 300Leu Thr Ser
Glu Asp Ser Ala Val Tyr Tyr Phe Ala Arg Gly Arg Pro305 310 315
320Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp Val Trp Gly Gln Gly
325 330 335Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe 340 345 350Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu 355 360 365Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp 370 375 380Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu385 390 395 400Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 405 410 415Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 420 425 430Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 435 440
445Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
450 455 460Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser465 470 475 480Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp 485 490 495Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn 500 505 510Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val 515 520 525Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 530 535 540Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys545 550 555
560Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
565 570 575Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Thr 580 585 590Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu 595 600 605Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu 610 615 620Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys625 630 635 640Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu 645 650 655Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 660 665
670Lys86113PRTArtificial Sequencesynthetic sequence 86Asp Val Val
Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30Asn
Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg
Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys
Phe Gln Gly 85 90 95Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 110Arg87330PRTArtificial Sequencesynthetic
sequence 87Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150
155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 33088329PRTArtificial Sequencesynthetic sequence 88Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
32589329PRTArtificial Sequencesynthetic sequence 89Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65
70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg
Glu Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly 32590330PRTArtificial
Sequencesynthetic sequence 90Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 325 33091124PRTArtificial Sequencesynthetic
sequence 91Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro
Gly Ala1 5 10 15Ser Val Lys Leu Ser Ser 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 Gln Lys Phe 50 55 60Gln Gly 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 Phe 85 90 95Ala Arg Gly Arg Pro Asp Tyr
Tyr Gly Ser Ser Lys Trp Tyr Phe Asp 100 105 110Val Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Ser 115 12092454PRTArtificial
Sequencesynthetic sequence 92Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val 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 Gln Lys Phe 50 55 60Gln Gly 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 Phe 85 90 95Ala Arg
Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp 100 105
110Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly 130 135 140Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro145 150 155 160Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr 165 170 175Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 210 215 220Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu225 230
235 240Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp 245 250 255Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp 260 265 270Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly 275 280 285Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn 290 295 300Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp305 310 315 320Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345
350Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
355 360 365Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile 370 375 380Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr385 390 395 400Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys 405 410 415Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445Ser Leu Ser
Pro Gly Lys 45093219PRTArtificial Sequencesynthetic sequence 93Asp
Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10
15Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser
20 25 30Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr
Leu Arg Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr
Tyr Cys Phe Gln Gly 85 90 95Ser His Val Pro Pro Thr Phe Gly Gly Gly
Thr Lys Leu 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
21594453PRTArtificial Sequencesynthetic sequence 94Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val 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 Gln Lys Phe 50 55
60Gln Gly 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
Phe 85 90 95Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr
Phe Asp 100 105 110Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
Ala Ser Thr Lys 115 120 125Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly 130 135 140Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro145 150 155 160Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200
205Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
210 215 220Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu225 230 235 240Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp 245 250 255Thr Leu Tyr Ile Thr Arg Glu Pro Glu
Val Thr Cys Val Val Val Asp 260 265 270Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp305 310 315
320Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
325 330 335Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu 340 345 350Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn 355 360 365Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile 370 375 380Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr385 390 395 400Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440
445Ser Leu Ser Pro Gly 45095453PRTArtificial Sequencesynthetic
sequence 95Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro
Gly Ala1 5 10 15Ser Val Lys Leu Ser Ser 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 Gln Lys Phe 50 55 60Gln Gly 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 Phe 85 90 95Ala Arg Gly Arg Pro Asp Tyr
Tyr Gly Ser Ser Lys Trp Tyr Phe Asp 100 105 110Val Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro145 150
155 160Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr 165 170 175Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val 180 185 190Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn 195 200 205Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro 210 215 220Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu225 230 235 240Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255Thr Leu
Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp 260 265
270Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
275 280 285Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn 290 295 300Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp305 310 315 320Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro 325 330 335Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 355 360 365Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr385 390
395 400Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys 405 410 415Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys 420 425 430Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu 435 440 445Ser Leu Ser Pro Gly
45096124PRTArtificial Sequencesynthetic sequence 96Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala1 5 10 15Ser Val Lys
Leu Ser Ser 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 Gln Lys Phe 50 55
60Gln Gly 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
Phe 85 90 95Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr
Phe Asp 100 105 110Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 12097112PRTArtificial Sequencesynthetic sequence 97Asp Val Val
Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30Asn
Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg
Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys
Phe Gln Gly 85 90 95Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 11098113PRTArtificial Sequencesynthetic
sequence 98Asp 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 Ile
Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys
Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg
Leu Tyr 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 Phe Gln Gly 85 90 95Ser His Val Pro Trp Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
110Arg99117PRTArtificial Sequencesynthetic sequence 99Gln 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 Tyr Ser Ile Thr Gly Gly 20 25 30Tyr
Leu Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40
45Ile Gly Tyr Ile Ser Tyr Asp Gly Thr Asn Asn Tyr Lys Pro Ser Leu
50 55 60Lys Asp Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe
Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Tyr Gly Arg Val Phe Phe Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser 115
* * * * *