U.S. patent application number 16/796652 was filed with the patent office on 2020-10-29 for activatable antibodies that bind interleukin-6 receptor and methods of use thereof.
The applicant listed for this patent is CytomX Therapeutics, Inc.. Invention is credited to Jeanne Grace Flandez, Daniel Robert Hostetter, Stephen Moore, Margaret Thy Luu Nguyen, Jason Gary Sagert, Olga Vasiljeva, James William West.
Application Number | 20200339694 16/796652 |
Document ID | / |
Family ID | 1000004945865 |
Filed Date | 2020-10-29 |
View All Diagrams
United States Patent
Application |
20200339694 |
Kind Code |
A1 |
West; James William ; et
al. |
October 29, 2020 |
ACTIVATABLE ANTIBODIES THAT BIND INTERLEUKIN-6 RECEPTOR AND METHODS
OF USE THEREOF
Abstract
The invention relates generally to activatable antibodies that
include a masking moiety (MM), a cleavable moiety (CM), and an
antibody (AB) that specifically binds to interleukin-6 receptor
(IL-6R), and to methods of making and using these anti-IL-6R
activatable antibodies in a variety of therapeutic, diagnostic and
prophylactic indications.
Inventors: |
West; James William; (Bend,
OR) ; Sagert; Jason Gary; (San Mateo, CA) ;
Hostetter; Daniel Robert; (Rocklin, CA) ; Moore;
Stephen; (Danville, CA) ; Nguyen; Margaret Thy
Luu; (San Francisco, CA) ; Vasiljeva; Olga;
(Cupertino, CA) ; Flandez; Jeanne Grace; (Oakland,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CytomX Therapeutics, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
1000004945865 |
Appl. No.: |
16/796652 |
Filed: |
February 20, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15271897 |
Sep 21, 2016 |
10611845 |
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16796652 |
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14036973 |
Sep 25, 2013 |
9487590 |
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15271897 |
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61749232 |
Jan 4, 2013 |
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61705581 |
Sep 25, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/56 20130101;
C07K 2317/90 20130101; C07K 16/2866 20130101; C07K 2317/51
20130101; C07K 2317/92 20130101; C07K 2319/50 20130101; A61K
2039/505 20130101; C07K 2317/55 20130101; C07K 2317/515 20130101;
C07K 2317/94 20130101; C07K 2317/565 20130101; A61K 47/65
20170801 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 47/65 20060101 A61K047/65 |
Claims
1-39. (canceled)
40. A method of preventing, delaying the progression of, treating,
alleviating a symptom of, or otherwise ameliorating inflammation or
an inflammatory disorder comprising: administering a
therapeutically effective amount of an activatable antibody to a
subject in need thereof, wherein the activatable antibody
comprises: an antibody or an antigen binding fragment thereof (AB)
that specifically binds to IL-6R; a masking moiety (MM) that
inhibits the binding of the AB to IL-6R in an uncleaved state; and
a cleavable moiety (CM) coupled to the AB, wherein the CM is a
polypeptide that functions as a substrate for a protease.
41. (canceled)
42. A method of treating, preventing, delaying the progression of,
alleviating a symptom of, or otherwise ameliorating an
IL-6R-mediated disorder or disease comprising: administering a
therapeutically effective amount of an activatable antibody to a
subject in need thereof, wherein the activatable antibody
comprises: an antibody or an antigen binding fragment thereof (AB)
that specifically binds to IL-6R; a masking moiety (MM) that
inhibits the binding of the AB to IL-6R in an uncleaved state; and
a cleavable moiety (CM) coupled to the AB, wherein the CM is a
polypeptide that functions as a substrate for a protease.
43. A method of preventing, delaying the progression of, treating,
alleviating a symptom of, or otherwise ameliorating an autoimmune
disease or disorder in a subject comprising: administering a
therapeutically effective amount of an activatable antibody to a
subject in need thereof, wherein the activatable antibody
comprises: an antibody or an antigen binding fragment thereof (AB)
that specifically binds to IL-6R; a masking moiety (MM) that
inhibits the binding of the AB to IL-6R in an uncleaved state; and
a cleavable moiety (CM) coupled to the AB, wherein the CM is a
polypeptide that functions as a substrate for a protease.
44. The activatable antibody of claim 40, wherein the AB comprises
a VH CDR1 sequence that comprises the VH CDR1 sequence of SEQ ID
NO: 1, a VH CDR2 sequence that comprises the VH CDR2 sequence of
SEQ ID NO: 1, a VH CDR3 sequence that comprises the VH CDR3
sequence of SEQ ID NO: 1, a VL CDR1 sequence that comprises the VL
CDR1 sequence of SEQ ID NO: 2, a VL CDR2 sequence that comprises
the VL CDR2 sequence of SEQ ID NO: 2, and a VL CDR3 sequence that
comprises the VL CDR3 sequence of SEQ ID NO: 2.
45. The method of claim 40, wherein the AB comprises a VH CDR1
sequence that comprises the amino acid sequence SDHAWS (SEQ ID NO:
175); a VH CDR2 sequence that comprises the amino acid sequence
YISYSGITTYNPSLKSRVT (SEQ ID NO: 176); a VH CDR3 sequence that
comprises the amino acid sequence SLARTTAMDY (SEQ ID NO: 177); a VL
CDR1 sequence that comprises the amino acid sequence RASQDISS (SEQ
ID NO: 178); a VL CDR2 sequence that comprises the amino acid
sequence TISSLQP (SEQ ID NO: 179); and a VL CDR3 sequence that
comprises the amino acid sequence QQGNTLPY (SEQ ID NO: 180).
46. The method of claim 40, wherein the activatable antibody has
the structural arrangement from N-terminus to C-terminus as follows
in the uncleaved state: MM-CM-AB or AB-CM-MM.
47. The method of claim 40, wherein the activatable antibody
comprises a linking peptide between the MM and the CM.
48. The method of claim 40, wherein the activatable antibody
comprises a linking peptide between the CM and the AB.
49. The method of claim 40, wherein the activatable antibody
comprises a first linking peptide (LP1) and a second linking
peptide (LP2), and wherein the activatable antibody has the
structural arrangement from N-terminus to C-terminus as follows in
the uncleaved state: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.
50. The method of claim 49, wherein the two linking peptides need
not be identical to each other.
51. The method of claim 49, wherein at least one of LP1 or LP2
comprises an amino acid sequence selected from the group consisting
of (GS).sub.n, (GGS).sub.n, (GSGGS).sub.n (SEQ ID NO: 93) and
(GGGS).sub.n (SEQ ID NO: 94), where n is an integer of at least
one.
52. The method of claim 49, wherein at least one of LP1 or LP2
comprises an amino acid sequence selected from the group consisting
of GGSG (SEQ ID NO: 95), GGSGG (SEQ ID NO: 96), GSGSG (SEQ ID NO:
97), GSGGG (SEQ ID NO: 98), GGGSG (SEQ ID NO: 99), and GSSSG (SEQ
ID NO: 100).
53. The method of claim 49, wherein LP1 comprises the amino acid
sequence GSSGGSGGSGGSG (SEQ ID NO: 101), GSSGGSGGSGG (SEQ ID NO:
112), GSSGGSGGSGGS (SEQ ID NO: 113), GSSGGSGGSGGSGGGS (SEQ ID NO:
169), GSSGGSGGSG (SEQ ID NO: 170), or GSSGGSGGSGS (SEQ ID NO:
171).
54. The method of claim 49, wherein LP2 comprises the amino acid
sequence GSS, GGS, GGGS (SEQ ID NO: 172), GSSGT (SEQ ID NO: 102) or
GSSG (SEQ ID NO: 103).
55. The method of claim 49, wherein the AB has an equilibrium
dissociation constant of about 100 nM or less for binding to
IL-6R.
56. The method of claim 40, wherein the antigen binding fragment
thereof is selected from the group consisting of a Fab fragment, a
F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy
chain antibody, and a single domain light chain antibody.
57. The method of claim 40, wherein the AB comprises a heavy chain
variable region comprising the variable region of the heavy chain
amino acid sequence SEQ ID NO: 1.
58. The method of claim 40, wherein the AB comprises a light chain
variable region comprising the variable region of the light chain
amino acid sequence SEQ ID NO: 2.
59. The method of claim 40, wherein the AB comprises a heavy chain
variable region comprising the variable region of the heavy chain
amino acid sequence SEQ ID NO: 1 and a light chain variable region
comprising the variable region of the light chain amino acid
sequence SEQ ID NO: 2.
60. The method of claim 40, wherein the AB comprises a heavy chain
amino acid sequence comprising SEQ ID NO: 1 and a light chain amino
acid sequence comprising SEQ ID NO: 2.
61. The method of claim 40, wherein the activatable antibody
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOs: 6-32, 109-111, 163-168, 181, and 182.
62. The method of claim 40, wherein the activatable antibody
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOs: 6-32, and 163-168.
63. The method of claim 40, wherein the MM has an equilibrium
dissociation constant for binding to the AB which is greater than
the equilibrium dissociation constant of the AB to IL-6R.
64. The method of claim 40, wherein the MM does not interfere or
compete with the AB for binding to IL-6R in a cleaved state.
65. The method of claim 40, wherein the MM is a polypeptide of up
to 40 amino acids in length.
66. The method of claim 40, wherein the MM polypeptide sequence is
different from that of IL-6R.
67. The method of claim 40, wherein the MM polypeptide sequence is
no more than 50% identical to any natural binding partner of the
AB.
68. The method of claim 40, wherein the MM comprises an amino acid
sequence selected from the group consisting of SEQ ID NOs:
33-89.
69. The method of claim 40, wherein the protease is co-localized
with IL-6R in a tissue, and wherein the protease cleaves the CM in
the activatable antibody when the activatable antibody is exposed
to the protease.
70. The method of claim 40, wherein the CM is a polypeptide of up
to 15 amino acids in length.
71. The method of claim 40, wherein the CM is a substrate for an
enzyme selected from the group consisting of a matrix
metalloprotease (MMP), thrombin, a neutrophil elastase, a cysteine
protease, legumain, matriptase, and uPA.
72. The method of claim 40, wherein the CM comprises an amino acid
sequence selected from the group consisting of SEQ ID NOs: 90-92,
104, 105, 107, 116-128, 157-162, 173, 174, and 183-193.
73. The method of claim 40, wherein the activatable antibody
comprises a spacer, wherein the spacer is joined directly to the MM
and has the structural arrangement from N-terminus to C-terminus of
spacer-MM-CM-AB.
74. The method of claim 40 comprising an agent conjugated to the
AB.
75. The method of claim 74, wherein the agent is a therapeutic
agent, an antineoplastic agent, or a toxin or fragment thereof.
76. The method of claim 74, wherein the agent is conjugated to the
AB via a linker.
77. The method of claim 76, wherein the linker is a cleavable
linker.
78. The method of claim 40, wherein the activatable antibody
comprises a detectable moiety.
79. The method of claim 78, wherein the detectable moiety is a
diagnostic agent.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 15/271,897, filed Sep. 21, 2016, which
is a continuation application of U.S. patent application Ser. No.
14/036,973, filed Sep. 25, 2013, now U.S. Pat. No. 9,487,590,
issued Nov. 8, 2016, which claims the benefit of U.S. Provisional
Application No. 61/749,232, filed Jan. 4, 2013, and U.S.
Provisional Application No. 61/705,581, filed Sep. 25, 2012, each
of which is incorporated herein by reference in its entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] The contents of the text file named
"CYTM021C02US_SeqList.txt," which was created on Feb. 19, 2020 and
is 128 KB in size, are hereby incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0003] The invention relates generally to activatable antibodies
that specifically bind to interleukin-6 receptor (IL-6R), and
methods of making and using these anti-IL-6R activatable antibodies
in a variety of therapeutic, diagnostic and prophylactic
indications.
BACKGROUND OF THE INVENTION
[0004] Antibody-based therapies have proven effective treatments
for several diseases but in some cases, toxicities due to broad
target expression have limited their therapeutic effectiveness. In
addition, antibody-based therapeutics have exhibited other
limitations such as rapid clearance from the circulation following
administration.
[0005] In the realm of small molecule therapeutics, strategies have
been developed to provide prodrugs of an active chemical entity.
Such prodrugs are administered in a relatively inactive (or
significantly less active) form. Once administered, the prodrug is
metabolized in vivo into the active compound. Such prodrug
strategies can provide for increased selectivity of the drug for
its intended target and for a reduction of adverse effects.
[0006] Accordingly, there is a continued need in the field of
antibody-based therapeutics for antibodies that mimic the desirable
characteristics of the small molecule prodrug.
SUMMARY OF THE INVENTION
[0007] The invention provides activatable antibodies that include
an antibody or antigen-binding fragment thereof that specifically
binds interleukin-6 receptor (IL-6R) coupled to a masking moiety
(MM), such that coupling of the MM reduces the ability of the
antibody or antigen-binding fragment thereof to bind IL-6R. In some
embodiments, the MM is coupled via a sequence that includes a
substrate for a protease, for example, a protease that is
co-localized with IL-6R at a treatment site in a subject. The
activatable anti-IL-6R antibodies provided herein are stable in
circulation, activated at intended sites of therapy and/or
diagnosis but not in normal, e.g., healthy tissue or other tissue
not targeted for treatment and/or diagnosis, and, when activated,
exhibit binding to IL-6R that is at least comparable to the
corresponding, unmodified antibody.
[0008] The invention provides methods of treating, preventing
and/or delaying the onset or progression of, or alleviating a
symptom associated with aberrant expression and/or activity of
IL-6R in a subject using activatable antibodies that bind IL-6R,
particularly activatable antibodies that bind and neutralize or
otherwise inhibit at least one biological activity of IL-6R and/or
IL-6R-mediated signaling.
[0009] The activatable antibodies described herein in an activated
state bind IL-6R and include (i) an antibody or an antigen binding
fragment thereof (AB) that specifically binds to IL-6R; (ii) a
masking moiety (MM) that inhibits the binding of the AB to IL-6R in
an uncleaved state; and (c) a cleavable moiety (CM) coupled to the
AB, wherein the CM is a polypeptide that functions as a substrate
for a protease.
[0010] In some embodiments, the activatable antibody has the
structural arrangement from N-terminus to C-terminus as follows in
the uncleaved state: MM-CM-AB or AB-CM-MM.
[0011] In some embodiments, the activatable antibody comprises a
linking peptide between the MM and the CM.
[0012] In some embodiments, the activatable antibody comprises a
linking peptide between the CM and the AB.
[0013] In some embodiments, the activatable antibody comprises a
first linking peptide (LP1) and a second linking peptide (LP2), and
wherein the activatable antibody has the structural arrangement
from N-terminus to C-terminus as follows in the uncleaved state:
MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the two
linking peptides need not be identical to each other.
[0014] In some embodiments, at least one of LP1 or LP2 comprises an
amino acid sequence selected from the group consisting of
(GS).sub.n, (GGS).sub.n, (GSGGS).sub.n (SEQ ID NO: 93) and
(GGGS).sub.n (SEQ ID NO: 94), where n is an integer of at least
one.
[0015] In some embodiments, at least one of LP1 or LP2 comprises an
amino acid sequence selected from the group consisting of GGSG (SEQ
ID NO: 95), GGSGG (SEQ ID NO: 96), GSGSG (SEQ ID NO: 97), GSGGG
(SEQ ID NO: 98), GGGSG (SEQ ID NO: 99), and GSSSG (SEQ ID NO:
100).
[0016] In some embodiments, LP1 comprises the amino acid sequence
GSSGGSGGSGGSG (SEQ ID NO: 101), GSSGGSGGSGG (SEQ ID NO: 112),
GSSGGSGGSGGS (SEQ ID NO: 113), GSSGGSGGSGGSGGGS (SEQ ID NO: 169),
GSSGGSGGSG (SEQ ID NO: 170), or GSSGGSGGSGS (SEQ ID NO: 171).
[0017] In some embodiments, LP2 comprises the amino acid sequence
GSS, GGS, GGGS (SEQ ID NO: 172), GSSGT (SEQ ID NO: 102) or GSSG
(SEQ ID NO: 103).
[0018] In some embodiments, the AB has an equilibrium dissociation
constant of about 100 nM or less for binding to IL-6R.
[0019] In some embodiments, the activatable antibody includes an
antibody or antigen-binding fragment thereof that specifically
binds IL-6R. In some embodiments, the antibody or immunologically
active fragment thereof that binds IL-6R is a monoclonal antibody,
domain antibody, single chain, Fab fragment, a F(ab')2 fragment, a
scFv, a scab, a dAb, a single domain heavy chain antibody, or a
single domain light chain antibody. In some embodiments, such an
antibody or immunologically active fragment thereof that binds
IL-6R is a mouse, other rodent, chimeric, humanized or fully human
monoclonal antibody.
[0020] In some embodiments, the activatable antibody comprises a
heavy chain amino acid sequence comprising SEQ ID NO: 1. In some
embodiments, the activatable antibody comprises a light chain amino
acid sequence comprising SEQ ID NO: 2. In some embodiments, the
activatable antibody comprises a heavy chain amino acid sequence
comprising SEQ ID NO: 1, and a light chain amino acid sequence
comprising SEQ ID NO: 2.
[0021] In some embodiments, the activatable antibody comprises a
heavy chain amino acid sequence that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid
sequence of SEQ ID NO: 1. In some embodiments, the activatable
antibody comprises a light chain amino acid that is at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the
amino acid sequence of SEQ ID NO: 2. In some embodiments, the
activatable antibody comprises a heavy chain amino acid sequence
that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the amino acid sequence of SEQ ID NO: 1, and a light
chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID
NO: 2.
[0022] In some embodiments, the activatable antibody comprises a
combination of a variable heavy chain complementarity determining
region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a
variable heavy chain complementarity determining region 2 (VH CDR2,
also referred to herein as CDRH2) sequence, a variable heavy chain
complementarity determining region 3 (VH CDR3, also referred to
herein as CDRH3) sequence, a variable light chain complementarity
determining region 1 (VL CDR1, also referred to herein as CDRL1)
sequence, a variable light chain complementarity determining region
2 (VL CDR2, also referred to herein as CDRL2) sequence, and a
variable light chain complementarity determining region 3 (VL CDR3,
also referred to herein as CDRL3) sequence, wherein at least one
CDR sequence is selected from the group consisting of a VH CDR1
sequence that includes at least the amino acid sequence SDHAWS (SEQ
ID NO: 175); a VH CD2 sequence that includes at least the amino
acid sequence YISYSGITTYNPSLKSRVT (SEQ ID NO: 176); a VH CDR3
sequence that includes at least the amino acid sequence SLARTTAMDY
(SEQ ID NO: 177); a VL CDR1 sequence that includes at least the
amino acid sequence RASQDISS (SEQ ID NO: 178); a VL CDR2 sequence
that includes at least the amino acid sequence TISSLQP (SEQ ID NO:
179); and a VL CDR3 sequence that includes at least the amino acid
sequence QQGNTLPY (SEQ ID NO: 180).
[0023] In some embodiments, the activatable antibody comprises a
combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3
sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3
sequence, wherein at least one CDR sequence is selected from the
group consisting of a VH CDR1 sequence that includes a sequence
that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
or more identical to the amino acid sequence SDHAWS (SEQ ID NO:
175); a VH CD2 sequence that includes a sequence that is at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical
to the amino acid sequence YISYSGITTYNPSLKSRVT (SEQ ID NO: 176); a
VH CDR3 sequence that includes a sequence that is at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to
the amino acid sequence SLARTTAMDY (SEQ ID NO: 177); a VL CDR1
sequence that includes a sequence that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino
acid sequence RASQDISS (SEQ ID NO: 178); a VL CDR2 sequence that
includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence
TISSLQP (SEQ ID NO: 179); and a VL CDR3 sequence that includes a
sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more identical to the amino acid sequence QQGNTLPY (SEQ
ID NO: 180).
[0024] [In some embodiments, the activatable antibody comprises a
combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3
sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3
sequence, wherein the VH CDR1 sequence comprises at least the amino
acid sequence SDHAWS (SEQ ID NO: 175); the VH CD2 sequence
comprises at least the amino acid sequence YISYSGITTYNPSLKSRVT (SEQ
ID NO: 176); the VH CDR3 sequence comprises at least the amino acid
sequence SLARTTAMDY (SEQ ID NO: 177); the VL CDR1 sequence
comprises at least the amino acid sequence RASQDISS (SEQ ID NO:
178); the VL CDR2 sequence comprises at least the amino acid
sequence TISSLQP (SEQ ID NO: 179); and the VL CDR3 sequence
comprises at least the amino acid sequence QQGNTLPY (SEQ ID NO:
180).
[0025] In some embodiments, the activatable antibody comprises a
combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3
sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3
sequence, wherein the VH CDR1 sequence comprises a sequence that is
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to the amino acid sequence SDHAWS (SEQ ID NO: 175); a VH
CD2 sequence comprises a sequence that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino
acid sequence YISYSGITTYNPSLKSRVT (SEQ ID NO: 176); the VH CDR3
sequence comprises a sequence that is at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid
sequence SLARTTAMDY (SEQ ID NO: 177); the VL CDR1 sequence
comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence
RASQDISS (SEQ ID NO: 178); the VL CDR2 sequence comprises a
sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more identical to the amino acid sequence TISSLQP (SEQ
ID NO: 179); and the VL CDR3 sequence comprises a sequence that is
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to the amino acid sequence QQGNTLPY (SEQ ID NO: 180).
[0026] In some embodiments, the activatable antibody comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOs 6-32, 109, 110 and 111. In some embodiments, the activatable
antibody comprises an amino acid sequence selected from the group
consisting of SEQ ID NOs 6-32. In some embodiments, the activatable
antibody comprises an amino acid sequence that is at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino
acid sequence selected from the group consisting of SEQ ID NOs
6-32, 109, 110 and 111. In some embodiments, the activatable
antibody comprises an amino acid that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid
sequence selected from the group consisting of SEQ ID NOs 6-32.
[0027] In some embodiments, the activatable antibody is encoded by
a nucleic acid sequence that comprises a nucleic acid sequence
encoding a heavy chain amino acid sequence comprising SEQ ID NO: 1.
In some embodiments, the activatable antibody is encoded by a
nucleic acid sequence that comprises a nucleic acid sequence that
comprises a nucleic acid sequence encoding a light chain amino acid
sequence comprising SEQ ID NO: 2. In some embodiments, the
activatable antibody is encoded by a nucleic acid sequence that
comprises a nucleic acid sequence encoding a heavy chain amino acid
sequence comprising SEQ ID NO: 1, and a nucleic acid sequence that
comprises a nucleic acid sequence encoding a light chain amino acid
sequence comprising SEQ ID NO: 2.
[0028] In some embodiments, the activatable antibody is encoded by
a nucleic acid sequence that comprises a nucleic acid sequence
encoding a heavy chain amino acid sequence that is at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the
amino acid sequence of SEQ ID NO: 1. In some embodiments, the
activatable antibody is encoded by a nucleic acid sequence that
comprises a nucleic acid sequence that comprises a nucleic acid
sequence encoding a light chain amino acid that is at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the
amino acid sequence of SEQ ID NO: 2. In some embodiments, the
activatable antibody is encoded by a nucleic acid sequence that
comprises a nucleic acid sequence encoding a heavy chain amino acid
sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identical to the amino acid sequence of SEQ ID NO: 1,
and a nucleic acid sequence that comprises a nucleic acid sequence
encoding a light chain amino acid that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid
sequence of SEQ ID NO: 2.
[0029] In some embodiments, the activatable antibody is encoded by
a nucleic acid sequence that encodes a combination of a VH CDR1
sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1
sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at
least one CDR sequence is selected from the group consisting of a
VH CDR1 sequence that includes at least the amino acid sequence
SDHAWS (SEQ ID NO: 175); a VH CD2 sequence that includes at least
the amino acid sequence YISYSGITTYNPSLKSRVT (SEQ ID NO: 176); a VH
CDR3 sequence that includes at least the amino acid sequence
SLARTTAMDY (SEQ ID NO: 177); a VL CDR1 sequence that includes at
least the amino acid sequence RASQDISS (SEQ ID NO: 178); a VL CDR2
sequence that includes at least the amino acid sequence TISSLQP
(SEQ ID NO: 179); and a VL CDR3 sequence that includes at least the
amino acid sequence QQGNTLPY (SEQ ID NO: 180).
[0030] In some embodiments, the activatable antibody is encoded by
a nucleic acid sequence that encodes a combination of a VH CDR1
sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1
sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at
least one CDR sequence is selected from the group consisting of a
VH CDR1 sequence that includes a sequence that is at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to
the amino acid sequence SDHAWS (SEQ ID NO: 175); a VH CD2 sequence
that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or more identical to the amino acid
sequence YISYSGITTYNPSLKSRVT (SEQ ID NO: 176); a VH CDR3 sequence
that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or more identical to the amino acid
sequence SLARTTAMDY (SEQ ID NO: 177); a VL CDR1 sequence that
includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence
RASQDISS (SEQ ID NO: 178); a VL CDR2 sequence that includes a
sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more identical to the amino acid sequence TISSLQP (SEQ
ID NO: 179); and a VL CDR3 sequence that includes a sequence that
is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
more identical to the amino acid sequence QQGNTLPY (SEQ ID NO:
180).
[0031] In some embodiments, the activatable antibody is encoded by
a nucleic acid sequence that encodes a combination of a VH CDR1
sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1
sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the
VH CDR1 sequence comprises at least the amino acid sequence SDHAWS
(SEQ ID NO: 175); the VH CD2 sequence comprises at least the amino
acid sequence YISYSGITTYNPSLKSRVT (SEQ ID NO: 176); the VH CDR3
sequence comprises at least the amino acid sequence SLARTTAMDY (SEQ
ID NO: 177); the VL CDR1 sequence comprises at least the amino acid
sequence RASQDISS (SEQ ID NO: 178); the VL CDR2 sequence comprises
at least the amino acid sequence TISSLQP (SEQ ID NO: 179); and the
VL CDR3 sequence comprises at least the amino acid sequence
QQGNTLPY (SEQ ID NO: 180).
[0032] In some embodiments, the activatable antibody is encoded by
a nucleic acid sequence that encodes a combination of a VH CDR1
sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1
sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the
VH CDR1 sequence comprises a sequence that is at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the
amino acid sequence SDHAWS (SEQ ID NO: 175); a VH CD2 sequence
comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence
YISYSGITTYNPSLKSRVT (SEQ ID NO: 176); the VH CDR3 sequence
comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence
SLARTTAMDY (SEQ ID NO: 177); the VL CDR1 sequence comprises a
sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more identical to the amino acid sequence RASQDISS (SEQ
ID NO: 178); the VL CDR2 sequence comprises a sequence that is at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to the amino acid sequence TISSLQP (SEQ ID NO: 179); and
the VL CDR3 sequence comprises a sequence that is at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to
the amino acid sequence QQGNTLPY (SEQ ID NO: 180).
[0033] In some embodiments, the activatable antibody is encoded by
a nucleic acid sequence that encodes an amino acid sequence
selected from the group consisting of SEQ ID NOs 6-32, 109, 110 and
111. In some embodiments, the activatable antibody comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOs 6-32. In some embodiments, the activatable antibody is encoded
by a nucleic acid sequence that encodes an amino acid sequence that
is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs 6-32, 109, 110 and 111. In some
embodiments, the activatable antibody comprises an amino acid that
is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs 6-32.
[0034] In some embodiments, the MM has an equilibrium dissociation
constant for binding to the AB which is greater than the
equilibrium dissociation constant of the AB to IL-6R.
[0035] In some embodiments, the MM has an equilibrium dissociation
constant for binding to the AB which is no more than the
equilibrium dissociation constant of the AB to IL-6R.
[0036] In some embodiments, the MM does not interfere or compete
with the AB for binding to IL-6R when the activatable antibody is
in a cleaved state.
[0037] In some embodiments, the MM is a polypeptide of about 2 to
40 amino acids in length. In some embodiments, the MM is a
polypeptide of up to about 40 amino acids in length.
[0038] In some embodiments, the MM polypeptide sequence is
different from that of IL-6R. In some embodiments, the MM
polypeptide sequence is no more than 50% identical to any natural
binding partner of the AB. In some embodiments, the MM polypeptide
sequence is different from that of IL-6R and is no more than 40%,
30%, 25%, 20%, 15%, or 10% identical to any natural binding partner
of the AB.
[0039] In some embodiments, the MM comprises an amino acid sequence
selected from the group consisting of SEQ ID NOs: 33-89. In some
embodiments, the MM comprises an amino acid sequence that is at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical
to an amino acid sequence selected from the group consisting of SEQ
ID NOs: 33-89.
[0040] In some embodiments, the MM comprises an amino acid sequence
selected from the group consisting of SEQ ID NO: 49, SEQ ID NO: 74,
and SEQ ID NO: 78. In some embodiments, the MM comprises an amino
acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identical to an amino acid sequence selected from
the group consisting of SEQ ID NO: 49, SEQ ID NO: 74, and SEQ ID
NO: 78.
[0041] In some embodiments, the MM comprises the amino acid
sequence of SEQ ID NO: 49. In some embodiments, the MM comprises an
amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID
NO: 49.
[0042] In some embodiments, the MM comprises the amino acid
sequence of SEQ ID NO: 74. In some embodiments, the MM comprises an
amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID
NO: 74.
[0043] In some embodiments, the MM comprises the amino acid
sequence of SEQ ID NO: 78. In some embodiments, the MM comprises an
amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID
NO: 78.
[0044] In some embodiments, the coupling of the MM to the AB
reduces the ability of the AB to bind IL-6R such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM
towards IL-6R is at least 20 times greater than the K.sub.d of the
AB when not coupled to the MM towards IL-6R.
[0045] In some embodiments, the coupling of the MM to the AB
reduces the ability of the AB to bind IL-6R such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM
towards IL-6R is at least 40 times greater than the K.sub.d of the
AB when not coupled to the MM towards IL-6R.
[0046] In some embodiments, the coupling of the MM to the AB
reduces the ability of the AB to bind IL-6R such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM
towards IL-6R is at least 100 times greater than the K.sub.d of the
AB when not coupled to the MM towards IL-6R.
[0047] In some embodiments, the coupling of the MM to the AB
reduces the ability of the AB to bind IL-6R such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM
towards IL-6R is at least 1000 times greater than the K.sub.d of
the AB when not coupled to the MM towards IL-6R.
[0048] In some embodiments, the coupling of the MM to the AB
reduces the ability of the AB to bind IL-6R such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM
towards IL-6R is at least 10,000 times greater than the K.sub.d of
the AB when not coupled to the MM towards IL-6R.
[0049] In some embodiments, in the presence of IL-6R, the MM
reduces the ability of the AB to bind IL-6R by at least 90% when
the CM is uncleaved, as compared to when the CM is cleaved when
assayed in vitro using a target displacement assay such as, for
example, the assay described in PCT Publication Nos. WO 2009/025846
and WO 2010/081173.
[0050] In some embodiments, the protease is co-localized with IL-6R
in a tissue, and the protease cleaves the CM in the activatable
antibody when the activatable antibody is exposed to the
protease.
[0051] In some embodiments, the CM is positioned in the activatable
antibody such that in the uncleaved state, binding of the
activatable antibody to IL-6R is reduced to occur with an
equilibrium dissociation constant that is at least 20-fold greater
than the equilibrium dissociation constant of an unmodified AB
binding to IL-6R, whereas in the cleaved state (i.e., when the
activatable antibody is in the cleaved state), the AB binds
IL-6R.
[0052] In some embodiments, the CM is positioned in the activatable
antibody such that in the uncleaved state, binding of the
activatable antibody to IL-6R is reduced to occur with an
equilibrium dissociation constant that is at least 40-fold greater
than the equilibrium dissociation constant of an unmodified AB
binding to IL-6R, whereas in the cleaved state, the AB binds
IL-6R.
[0053] In some embodiments, the CM is positioned in the activatable
antibody such that in the uncleaved state, binding of the
activatable antibody to IL-6R is reduced to occur with an
equilibrium dissociation constant that is at least 50-fold greater
than the equilibrium dissociation constant of an unmodified AB
binding to IL-6R, whereas in the cleaved state, the AB binds
IL-6R.
[0054] In some embodiments, the CM is positioned in the activatable
antibody such that in the uncleaved state, binding of the
activatable antibody to IL-6R is reduced to occur with an
equilibrium dissociation constant that is at least 100-fold greater
than the equilibrium dissociation constant of an unmodified AB
binding to IL-6R, whereas in the cleaved state, the AB binds
IL-6R.
[0055] In some embodiments, the CM is positioned in the activatable
antibody such that in the uncleaved state, binding of the
activatable antibody to IL-6R is reduced to occur with an
equilibrium dissociation constant that is at least 200-fold greater
than the equilibrium dissociation constant of an unmodified AB
binding to IL-6R, whereas in the cleaved state, the AB binds
IL-6R.
[0056] In some embodiments, the CM is a polypeptide of up to 15
amino acids in length.
[0057] Exemplary substrates include but are not limited to
substrates cleavable by one or more of the following enzymes or
proteases listed in Table 12.
[0058] In some embodiments, the CM is selected for use with a
specific protease, for example a protease that is known to be
co-localized with the target of the activatable antibody.
[0059] In some embodiments, the CM is a substrate for at least one
protease that is or is believed to be up-regulated in inflammation.
In some embodiments, the CM is a substrate for at least one
protease selected from the group consisting of a matrix
metalloprotease (MMP), thrombin, a neutrophil elastase, a cysteine
protease, legumain, matriptase (MT-SP1), and urokinase (uPA).
Without being bound by theory, it is believed that these proteases
are up-regulated in inflammation.
[0060] In some embodiments, the CM is a substrate for at least one
protease that is or is believed to be up-regulated in cancer. In
some embodiments, the CM is a substrate for at least one protease
selected from the group consisting of a matrix metalloprotease
(MMP), MT-SP1, uPA, legumain, and a neutrophil elastase. Without
being bound by theory, it is believed that these proteases are
up-regulated in cancer.
[0061] In some embodiments, the CM is a substrate for at least one
MMP. Examples of MMPs include the MMPs listed in the Table 12. In
some embodiments, the CM is a substrate for a protease selected
from the group consisting of MMP 9, MMP14, MMP1, MMP3, MMP13,
MMP17, MMP11, and MMP19. In some embodiments the CM is a substrate
for MMP9. In some embodiments, the CM is a substrate for MMP14.
[0062] In some embodiments, the CM is a substrate for an MMP and
includes the sequence ISSGLSS (SEQ ID NO: 157); QNQALRMA (SEQ ID
NO: 158); AQNLLGMV (SEQ ID NO: 159); STFPFGMF (SEQ ID NO: 160);
PVGYTSSL (SEQ ID NO: 161); DWLYWPGI (SEQ ID NO: 162), ISSGLLSS (SEQ
ID NO: 183), LKAAPRWA (SEQ ID NO: 184); GPSHLVLT (SEQ ID NO: 185);
LPGGLSPW (SEQ ID NO: 186); MGLFSEAG (SEQ ID NO: 187); SPLPLRVP (SEQ
ID NO: 188); RMHLRSLG (SEQ ID NO: 189); LAAPLGLL (SEQ ID NO: 190);
AVGLLAPP (SEQ ID NO: 191); LLAPSHRA (SEQ ID NO: 192); and/or
PAGLWLDP (SEQ ID NO: 193).
[0063] In some embodiments, the CM comprises the amino acid
sequence ISSGLSS (SEQ ID NO: 157). In some embodiments, the CM
comprises the amino acid sequence QNQALRMA (SEQ ID NO: 158). In
some embodiments, the CM comprises the amino acid sequence AQNLLGMV
(SEQ ID NO: 159). In some embodiments, the CM comprises the amino
acid sequence STFPFGMF (SEQ ID NO: 160). In some embodiments, the
CM comprises the amino acid sequence PVGYTSSL (SEQ ID NO: 161). In
some embodiments, the CM comprises the amino acid sequence DWLYWPGI
(SEQ ID NO: 162). In some embodiments, the CM comprises the amino
acid sequence ISSGLLSS (SEQ ID NO: 183). In some embodiments, the
CM comprises the amino acid sequence LKAAPRWA (SEQ ID NO: 184). In
some embodiments, the CM comprises the amino acid sequence GPSHLVLT
(SEQ ID NO: 185). In some embodiments, the CM comprises the amino
acid sequence LPGGLSPW (SEQ ID NO: 186). In some embodiments, the
CM comprises the amino acid sequence MGLFSEAG (SEQ ID NO: 187). In
some embodiments, the CM comprises the amino acid sequence SPLPLRVP
(SEQ ID NO: 188). In some embodiments, the CM comprises the amino
acid sequence RMHLRSLG (SEQ ID NO: 189). In some embodiments, the
CM comprises the amino acid sequence LAAPLGLL (SEQ ID NO: 190). In
some embodiments, the CM comprises the amino acid sequence AVGLLAPP
(SEQ ID NO: 191). In some embodiments, the CM comprises the amino
acid sequence LLAPSHRA (SEQ ID NO: 192). In some embodiments, the
CM comprises the amino acid sequence PAGLWLDP (SEQ ID NO: 193).
[0064] In some embodiments, the CM is a substrate for thrombin. In
some embodiments, the CM is a substrate for thrombin and includes
the sequence GPRSFGL (SEQ ID NO: 173) or GPRSFG (SEQ ID NO: 174).
In some embodiments, the CM comprises the amino acid sequence
GPRSFGL (SEQ ID NO: 173). In some embodiments, the CM comprises the
amino acid sequence GPRSFG (SEQ ID NO: 174).
[0065] In some embodiments, the CM is a substrate a neutrophil
elastase. In some embodiments, the CM is a substrate for uPA. In
some embodiments, the CM is a substrate for legumain. In some
embodiments, the CM is a substrate for MT-SP1. In some embodiments,
the CM is a substrate for a cysteine protease. In some embodiments,
the CM is a substrate for a cysteine protease, such as a
cathepsin.
[0066] For example, suitable cleavable moieties for use in the
activatable anti-IL-6R antibodies of the disclosure are cleaved by
at least one protease and include the sequence TGRGPSWV (SEQ ID NO:
91); SARGPSRW (SEQ ID NO: 116); TARGPSFK (SEQ ID NO: 117); LSGRSDNH
(SEQ ID NO: 90); GGWHTGRN (SEQ ID NO: 118); HTGRSGAL (SEQ ID NO:
119); PLTGRSGG (SEQ ID NO: 92); AARGPAIH (SEQ ID NO: 120); RGPAFNPM
(SEQ ID NO: 121); SSRGPAYL (SEQ ID NO: 122); RGPATPIM (SEQ ID NO:
123); RGPA (SEQ ID NO: 124); GGQPSGMWGW (SEQ ID NO: 104);
FPRPLGITGL (SEQ ID NO: 105); VHMPLGFLGP (SEQ ID NO: 106); SPLTGRSG
(SEQ ID NO: 125); SAGFSLPA (SEQ ID NO: 126); LAPLGLQRR (SEQ ID NO:
127); SGGPLGVR (SEQ ID NO: 128); and/or PLGL (SEQ ID NO: 107).
[0067] In some embodiments, the CM comprises the amino acid
sequence LSGRSDNH (SEQ ID NO: 90). In some embodiments, the CM
comprises the amino acid sequence TGRGPSWV (SEQ ID NO: 91). In some
embodiments, the CM comprises the amino acid sequence PLTGRSGG (SEQ
ID NO: 92). In some embodiments, the CM comprises the amino acid
sequence GGQPSGMWGW (SEQ ID NO: 104). In some embodiments, the CM
comprises the amino acid sequence FPRPLGITGL (SEQ ID NO: 105). In
some embodiments, the CM comprises the amino acid sequence
VHMPLGFLGP (SEQ ID NO: 106). In some embodiments, the CM comprises
the amino acid sequence PLGL (SEQ ID NO: 107). In some embodiments,
the CM comprises the amino acid sequence SARGPSRW (SEQ ID NO: 116).
In some embodiments, the CM comprises the amino acid sequence
TARGPSFK (SEQ ID NO: 117). In some embodiments, the CM comprises
the amino acid sequence GGWHTGRN (SEQ ID NO: 118). In some
embodiments, the CM comprises the amino acid sequence HTGRSGAL (SEQ
ID NO: 119). In some embodiments, the CM comprises the amino acid
sequence AARGPAIH (SEQ ID NO: 120). In some embodiments, the CM
comprises the amino acid sequence RGPAFNPM (SEQ ID NO: 121). In
some embodiments, the CM comprises the amino acid sequence SSRGPAYL
(SEQ ID NO: 122). In some embodiments, the CM comprises the amino
acid sequence RGPATPIM (SEQ ID NO: 123). In some embodiments, the
CM comprises the amino acid sequence RGPA (SEQ ID NO: 124).
[0068] In some embodiments, the CM comprises an amino acid sequence
selected from the group consisting of SEQ ID NOs: 90-92, 104-107,
116-128, 157-162, 173, 174, and 183-193. In some embodiments, the
CM comprises an amino acid sequence that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 90-92,
104-107, 116-128, 157-162, 173, 174, and 183-193.
[0069] In some embodiments, the CM comprises an amino acid sequence
selected from the group consisting of SEQ ID NOs: 90-92, 104, 105,
107, 116-128, 157-162, 173, 174, and 183-193. In some embodiments,
the CM comprises an amino acid sequence that is at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 90-92,
104, 105, 107, 116-128, 157-162, 173, 174, and 183-193.
[0070] In some embodiments, the CM comprises an amino acid sequence
selected from the group consisting of SEQ ID NOs: 90-92, 107,
116-128, 157-162, 173, 174, and 183-193. In some embodiments, the
CM comprises an amino acid sequence that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 90-92,
107, 116-128, 157-162, 173, 174, and 183-193.
[0071] In some embodiments, the CM comprises an amino acid sequence
selected from the group consisting of SEQ ID NOs: 90-92, 104, 105,
107, 116-125, 157-162, 173, 174, and 183-193. In some embodiments,
the CM comprises an amino acid sequence that is at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 90-92,
104, 105, 107, 116-125, 157-162, 173, 174, and 183-193.
[0072] In some embodiments, the CM comprises an amino acid sequence
selected from the group consisting of SEQ ID NOs: 90-92, 107,
116-125, 157-162, 173, 174, and 183-193. In some embodiments, the
CM comprises an amino acid sequence that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 90-92,
107, 116-125, 157-162, 173, 174, and 183-193.
[0073] In some embodiments, the CM is a substrate for at least two
proteases. In some embodiments, each protease is selected from the
group consisting of those shown in Table 12. In some embodiments,
the CM is a substrate for at least two proteases, wherein one of
the proteases is selected from the group consisting of a MMP,
thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain
and MT-SP1 and the other protease is selected from the group
consisting of those shown in Table 12. In some embodiments, the CM
is a substrate for at least two proteases selected from the group
consisting of a MMP, thrombin, a neutrophil elastase, a cysteine
protease, uPA, legumain and MT-SP1.
[0074] In some embodiments, the activatable antibody includes at
least a first CM and a second CM. In some embodiments, the first CM
and the second CM are each polypeptides of no more than 15 amino
acids long. In some embodiments, the first CM and the second CM in
the activatable antibody have the structural arrangement from
N-terminus to C-terminus as follows in the uncleaved state:
MM-CM1-CM2-AB or AB-CM2-CM1-MM. In some embodiments, at least one
of the first CM and the second CM is a polypeptide that functions
as a substrate for a protease selected from the group consisting of
a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA,
legumain, and MT-SP1. In some embodiments, the first CM is cleaved
by a first cleaving agent selected from the group consisting of a
MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA,
legumain, and MT-SP1 in a target tissue and the second CM is
cleaved by a second cleaving agent in a target tissue. In some
embodiments, the other protease is selected from the group
consisting of those shown in Table 12. In some embodiments, the
first cleaving agent and the second cleaving agent are the same
protease selected from the group consisting of a MMP, thrombin, a
neutrophil elastase, a cysteine protease, uPA, legumain, and
MT-SP1, and the first CM and the second CM are different substrates
for the enzyme. In some embodiments, the first cleaving agent and
the second cleaving agent are the same protease selected from the
group consisting of those shown in Table 12. In some embodiments,
the first cleaving agent and the second cleaving agent are
different proteases. In some embodiments, the first cleaving agent
and the second cleaving agent are co-localized in the target
tissue. In some embodiments, the first CM and the second CM are
cleaved by at least one cleaving agent in the target tissue.
[0075] In some embodiments, the activatable antibody is exposed to
and cleaved by a protease such that, in the activated or cleaved
state, the activated antibody includes a light chain amino acid
sequence that includes at least a portion of LP2 and/or CM sequence
after the protease has cleaved the CM.
[0076] In some embodiments, the activatable antibody also includes
an agent conjugated to the AB. In some embodiments, the agent is a
therapeutic agent. In some embodiments, the agent is an
antineoplastic agent. In some embodiments, the agent is a toxin or
fragment thereof. In some embodiments, the agent is conjugated to
the AB via a linker. In some embodiments, the linker is a cleavable
linker. In some embodiments, the agent is an agent selected from
the group listed in Table 17. In some embodiments, the agent is a
dolastatin. In some embodiments, the agent is an auristatin or
derivative thereof. In some embodiments, the agent is auristatin E
or a derivative thereof. In some embodiments, the agent is
monomethyl auristatin E (MMAE). In some embodiments, the agent is a
maytansinoid or maytansinoid derivative. In some embodiments, the
agent is DM1 or DM4. In some embodiments, the agent is a
duocarmycin or derivative thereof. In some embodiments, the agent
is a calicheamicin or derivative thereof.
[0077] In some embodiments, the agent is an anti-inflammatory
agent.
[0078] In some embodiments, the activatable antibody also includes
a detectable moiety. In some embodiments, the detectable moiety is
a diagnostic agent.
[0079] In some embodiments, the activatable antibody also includes
a signal peptide. In some embodiments, the signal peptide is
conjugated to the activatable antibody via a spacer. In some
embodiments, the spacer is conjugated to the activatable antibody
in the absence of a signal peptide. In some embodiments, the spacer
is joined directly to the MM of the activatable antibody. In some
embodiments, the spacer is joined directly to the MM of the
activatable antibody in the structural arrangement from N-terminus
to C-terminus of spacer-MM-CM-AB. An example of a spacer joined
directly to the N-terminus of MM of the activatable antibody is
QGQSGQ (SEQ ID NO: 108). In some embodiments, the spacer includes
at least the amino acid sequence QGQSGQ (SEQ ID NO: 108).
[0080] In some embodiments, the AB of the activatable antibody
naturally contains one or more disulfide bonds. In some
embodiments, the AB can be engineered to include one or more
disulfide bonds.
[0081] In some embodiments, the activatable antibody includes an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 6-32, 109-110, and 163-168. In some embodiments, the
activatable antibody includes an amino acid sequence selected from
the group consisting of SEQ ID NOs: 6-32. In some embodiments, the
activatable antibody includes an amino acid that is at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino
acid sequence selected from the group consisting of SEQ ID NOs:
6-32, 109-110, and 163-168. In some embodiments, the activatable
antibody includes an amino acid that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid
sequence selected from the group consisting of SEQ ID NOs:
6-32.
[0082] In some embodiments, the serum half-life of the activatable
antibody is at least 5 days when administered to an organism. In
some embodiments, the serum half-life of the activatable antibody
is at least 4 days when administered to an organism. In some
embodiments, the serum half-life of the activatable antibody is at
least 3 days when administered to an organism. In some embodiments,
the serum half-life of the activatable antibody is at least 2 days
when administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 24 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 20 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 18 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 16 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 14 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 12 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 10 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 8 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 6 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 4 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 3 hours when
administered to an organism.
[0083] In some embodiments, the activatable anti-IL-6R antibody
and/or conjugated activatable anti-IL-6R antibody is monospecific.
In some embodiments, the activatable anti-IL-6R antibody and/or
conjugated activatable anti-IL-6R antibody is multispecific, e.g.,
by way of non-limiting example, bispecific or trifunctional. In
some embodiments, the activatable anti-IL-6R antibody and/or
conjugated activatable anti-IL-6R antibody is formulated as part of
a pro-Bispecific T Cell Engager (BITE) molecule. In some
embodiments, the activatable anti-IL-6R antibody and/or conjugated
activatable anti-IL-6R antibody is formulated as part of a
pro-Chimeric Antigen Receptor (CAR) modified T cell or other
engineered receptor.
[0084] The disclosure also provides compositions and methods that
include an activatable anti-IL-6R antibody that includes an
antibody or antibody fragment (AB) that specifically binds IL-6R,
where the AB is coupled to a masking moiety (MM) that decreases the
ability of the AB to bind its target. In some embodiments, the
activatable anti-IL-6R antibody further includes a cleavable moiety
(CM) that is a substrate for a protease. The compositions and
methods provided herein enable the attachment of one or more agents
to one or more cysteine residues in the AB without compromising the
activity (e.g., the masking, activating or binding activity) of the
activatable anti-IL-6R antibody. In some embodiments, the
compositions and methods provided herein enable the attachment of
one or more agents to one or more cysteine residues in the AB
without reducing or otherwise disturbing one or more disulfide
bonds within the MM. The compositions and methods provided herein
produce an activatable anti-IL-6R antibody that is conjugated to
one or more agents, e.g., any of a variety of therapeutic,
diagnostic and/or prophylactic agents, for example, in some
embodiments, without any of the agent(s) being conjugated to the MM
of the activatable anti-IL-6R antibody. The compositions and
methods provided herein produce conjugated activatable anti-IL-6R
antibodies in which the MM retains the ability to effectively and
efficiently mask the AB of the activatable antibody in an uncleaved
state. The compositions and methods provided herein produce
conjugated activatable anti-IL-6R antibodies in which the
activatable antibody is still activated, i.e., cleaved, in the
presence of a protease that can cleave the CM.
[0085] The activatable anti-IL-6R antibodies have at least one
point of conjugation for an agent, but in the methods and
compositions provided herein less than all possible points of
conjugation are available for conjugation to an agent. In some
embodiments, the one or more points of conjugation are sulfur atoms
involved in disulfide bonds. In some embodiments, the one or more
points of conjugation are sulfur atoms involved in interchain
disulfide bonds. In some embodiments, the one or more points of
conjugation are sulfur atoms involved in interchain sulfide bonds,
but not sulfur atoms involved in intrachain disulfide bonds. In
some embodiments, the one or more points of conjugation are sulfur
atoms of cysteine or other amino acid residues containing a sulfur
atom. Such residues may occur naturally in the antibody structure
or may be incorporated into the antibody by site-directed
mutagenesis, chemical conversion, or mis-incorporation of
non-natural amino acids.
[0086] Also provided are methods of preparing a conjugate of an
activatable anti-IL-6R antibody having one or more interchain
disulfide bonds in the AB and one or more intrachain disulfide
bonds in the MM, and a drug reactive with free thiols is provided.
The method generally includes partially reducing interchain
disulfide bonds in the activatable antibody with a reducing agent,
such as, for example, TCEP; and conjugating the drug reactive with
free thiols to the partially reduced activatable antibody. As used
herein, the term partial reduction refers to situations where an
activatable anti-IL-6R antibody is contacted with a reducing agent
and less than all disulfide bonds, e.g., less than all possible
sites of conjugation are reduced. In some embodiments, less than
99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%,
50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or less than 5% of all
possible sites of conjugation are reduced.
[0087] In yet other embodiments, a method of reducing and
conjugating an agent, e.g., a drug, to an activatable anti-IL-6R
antibody resulting in selectivity in the placement of the agent is
provided. The method generally includes partially reducing the
activatable anti-IL-6R antibody with a reducing agent such that any
conjugation sites in the masking moiety or other non-AB portion of
the activatable antibody are not reduced, and conjugating the agent
to interchain thiols in the AB. The conjugation site(s) are
selected so as to allow desired placement of an agent to allow
conjugation to occur at a desired site. The reducing agent is, for
example, TCEP. The reduction reaction conditions such as, for
example, the ratio of reducing agent to activatable antibody, the
length of incubation, the temperature during the incubation, the pH
of the reducing reaction solution, etc., are determined by
identifying the conditions that produce a conjugated activatable
antibody in which the MM retains the ability to effectively and
efficiently mask the AB of the activatable antibody in an uncleaved
state. The ratio of reduction agent to activatable anti-IL-6R
antibody will vary depending on the activatable antibody. In some
embodiments, the ratio of reducing agent to activatable anti-IL-6R
antibody will be in a range from about 20:1 to 1:1, from about 10:1
to 1:1, from about 9:1 to 1:1, from about 8:1 to 1:1, from about
7:1 to 1:1, from about 6:1 to 1:1, from about 5:1 to 1:1, from
about 4:1 to 1:1, from about 3:1 to 1:1, from about 2:1 to 1:1,
from about 20:1 to 1:1.5, from about 10:1 to 1:1.5, from about 9:1
to 1:1.5, from about 8:1 to 1:1.5, from about 7:1 to 1:1.5, from
about 6:1 to 1:1.5, from about 5:1 to 1:1.5, from about 4:1 to
1:1.5, from about 3:1 to 1:1.5, from about 2:1 to 1:1.5, from about
1.5:1 to 1:1.5, or from about 1:1 to 1:1.5. In some embodiments,
the ratio is in a range of from about 5:1 to 1:1. In some
embodiments, the ratio is in a range of from about 5:1 to 1.5:1. In
some embodiments, the ratio is in a range of from about 4:1 to 1:1.
In some embodiments, the ratio is in a range from about 4:1 to
1.5:1.
[0088] In some embodiments, a method of reducing interchain
disulfide bonds in the AB of an activatable anti-IL-6R antibody and
conjugating an agent, e.g., a thiol-containing agent such as a
drug, to the resulting interchain thiols to selectively locate
agent(s) on the AB is provided. The method generally includes
partially reducing the AB with a reducing agent to form at least
two interchain thiols without forming all possible interchain
thiols in the activatable antibody; and conjugating the agent to
the interchain thiols of the partially reduced AB. For example, the
AB of the activatable antibody is partially reduced for about 1
hour at about 37.degree. C. at a desired ratio of reducing
agent:activatable antibody. In some embodiments, the ratio of
reducing agent to activatable antibody will be in a range from
about 20:1 to 1:1, from about 10:1 to 1:1, from about 9:1 to 1:1,
from about 8:1 to 1:1, from about 7:1 to 1:1, from about 6:1 to
1:1, from about 5:1 to 1:1, from about 4:1 to 1:1, from about 3:1
to 1:1, from about 2:1 to 1:1, from about 20:1 to 1:1.5, from about
10:1 to 1:1.5, from about 9:1 to 1:1.5, from about 8:1 to 1:1.5,
from about 7:1 to 1:1.5, from about 6:1 to 1:1.5, from about 5:1 to
1:1.5, from about 4:1 to 1:1.5, from about 3:1 to 1:1.5, from about
2:1 to 1:1.5, from about 1.5:1 to 1:1.5, or from about 1:1 to
1:1.5. In some embodiments, the ratio is in a range of from about
5:1 to 1:1. In some embodiments, the ratio is in a range of from
about 5:1 to 1.5:1. In some embodiments, the ratio is in a range of
from about 4:1 to 1:1. In some embodiments, the ratio is in a range
from about 4:1 to 1.5:1.
[0089] The thiol-containing reagent can be, for example, cysteine
or N-acetyl cysteine. The reducing agent can be, for example, TCEP.
In some embodiments, the reduced activatable antibody can be
purified prior to conjugation, using for example, column
chromatography, dialysis, or diafiltration. Alternatively, the
reduced antibody is not purified after partial reduction and prior
to conjugation.
[0090] The invention also provides partially reduced activatable
anti-IL-6R antibodies in which at least one interchain disulfide
bond in the activatable antibody has been reduced with a reducing
agent without disturbing any intrachain disulfide bonds in the
activatable antibody, wherein the activatable antibody includes an
antibody or an antigen binding fragment thereof (AB) that
specifically binds to IL-6R, a masking moiety (MINI) that inhibits
the binding of the AB of the activatable antibody in an uncleaved
state to the IL-6R target, and a cleavable moiety (CM) coupled to
the AB, wherein the CM is a polypeptide that functions as a
substrate for a protease. In some embodiments the MINI is coupled
to the AB via the CM. In some embodiments, one or more intrachain
disulfide bond(s) of the activatable antibody is not disturbed by
the reducing agent. In some embodiments, one or more intrachain
disulfide bond(s) of the MINI within the activatable antibody is
not disturbed by the reducing agent. In some embodiments, the
activatable antibody in the uncleaved state has the structural
arrangement from N-terminus to C-terminus as follows: MM-CM-AB or
AB-CM-MM. In some embodiments, reducing agent is TCEP.
[0091] In some embodiments, the activatable antibody includes an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 6-32, 109-110, and 163-168. In some embodiments, the
activatable antibody includes an amino acid sequence selected from
the group consisting of SEQ ID NOs: 6-32. In some embodiments, the
activatable antibody includes an amino acid that is at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino
acid sequence selected from the group consisting of SEQ ID NOs:
6-32, 109-110, and 163-168. In some embodiments, the activatable
antibody includes an amino acid that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid
sequence selected from the group consisting of SEQ ID NOs:
6-32.
[0092] In some embodiments, the activatable anti-IL-6R antibodies
and/or conjugated activatable anti-IL-6R antibodies described
herein are used in conjunction with one or more additional agents
or a combination of additional agents. Suitable additional agents
include current pharmaceutical and/or surgical therapies for an
intended application, such as, for example, cancer. For example,
the activatable anti-IL-6R antibodies and/or conjugated activatable
anti-IL-6R antibodies can be used in conjunction with an additional
chemotherapeutic or anti-neoplastic agent. Examples include agents
considered to be standard of care treatments such as, but are not
limited to, bortezimib or thalidomides to treat multiple myeloma or
abraxane, paclitaxel or Herceptin to treat breast cancer.
[0093] In some embodiments, the activatable anti-IL-6R antibody
and/or conjugated activatable anti-IL-6R antibodies and the
additional agent are formulated into a single therapeutic
composition, and the activatable anti-IL-6R antibody and additional
agent are administered simultaneously. Alternatively, the
activatable anti-IL-6R antibody and/or conjugated activatable
anti-IL-6R antibodies and additional agent are separate from each
other, e.g., each is formulated into a separate therapeutic
composition, and the activatable anti-IL-6R antibody and/or
conjugated activatable anti-IL-6R antibodies and the additional
agent are administered simultaneously, or the activatable
anti-IL-6R antibody and/or conjugated activatable anti-IL-6R
antibodies and the additional agent are administered at different
times during a treatment regimen. For example, the activatable
anti-IL-6R antibody and/or conjugated activatable anti-IL-6R
antibodies is administered prior to the administration of the
additional agent, the activatable anti-IL-6R antibody and/or
conjugated activatable anti-IL-6R antibodies is administered
subsequent to the administration of the additional agent, or the
activatable anti-IL-6R antibody and/or conjugated activatable
anti-IL-6R antibodies and the additional agent are administered in
an alternating fashion. As described herein, the activatable
anti-IL-6R antibody and/or conjugated activatable anti-IL-6R
antibodies and additional agent are administered in single doses or
in multiple doses.
[0094] In some embodiments, the activatable antibody includes an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 6-32, 109-110, and 163-168. In some embodiments, the
activatable antibody includes an amino acid sequence selected from
the group consisting of SEQ ID NOs: 6-32. In some embodiments, the
activatable antibody includes an amino acid that is at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino
acid sequence selected from the group consisting of SEQ ID NOs:
6-32, 109-110, and 163-168. In some embodiments, the activatable
antibody includes an amino acid that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid
sequence selected from the group consisting of SEQ ID NOs:
6-32.
[0095] The invention also provides an isolated nucleic acid
molecule encoding an activatable anti-IL-6R antibody described
herein, as well as vectors that include these isolated nucleic acid
sequences. In some embodiments, the isolated nucleic acid sequence
encodes an amino acid sequence selected from the group consisting
of SEQ ID NOs: 6-32, 109-110, and 163-168. The invention also
provides an isolated nucleic acid molecule encoding an activatable
anti-IL-6R antibody described herein, as well as vectors that
include these isolated nucleic acid sequences. In some embodiments,
the isolated nucleic acid sequence encodes an amino acid sequence
selected from the group consisting of SEQ ID NOs: 6-32. In some
embodiments, the isolated nucleic acid sequence encodes an amino
acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%
or 99% identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 6-32, 109-110, and 163-168. In some
embodiments, the isolated nucleic acid sequence encodes an amino
acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%
or 99% identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 6-32. The invention provides methods of
producing an activatable antibody by culturing a cell under
conditions that lead to expression of the activatable antibody,
wherein the cell comprises such a vector.
[0096] The invention also provides a method of manufacturing
activatable antibodies that in an activated state binds
Interleukin-6 Receptor (IL-6R) by (a) culturing a cell comprising a
nucleic acid construct that encodes the activatable antibody under
conditions that lead to expression of the activatable antibody,
wherein the activatable antibody comprises a masking moiety (MM), a
cleavable moiety (CM), and an antibody or an antigen binding
fragment thereof (AB) that specifically binds IL-6R, (i) wherein
the CM is a polypeptide that functions as a substrate for a
protease; and (ii) wherein the CM is positioned in the activatable
antibody such that, in an uncleaved state, the MM interferes with
specific binding of the AB to IL-6R and in a cleaved state the MM
does not interfere or compete with specific binding of the AB to
IL-6R; and (b) recovering the activatable antibody.
[0097] In some embodiments, the activatable antibody has the
structural arrangement from N-terminus to C-terminus as follows in
the uncleaved state: MM-CM-AB or AB-CM-MM.
[0098] In some embodiments, the activatable antibody comprises a
linking peptide between the MM and the CM.
[0099] In some embodiments, the activatable antibody comprises a
linking peptide between the CM and the AB.
[0100] In some embodiments, the activatable antibody comprises a
first linking peptide (LP1) and a second linking peptide (LP2), and
wherein the activatable antibody has the structural arrangement
from N-terminus to C-terminus as follows in the uncleaved state:
MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.
[0101] In some embodiments, the two linking peptides need not be
identical to each other.
[0102] In some embodiments, the AB has an equilibrium dissociation
constant of about 100 nM or less for binding to IL-6R.
[0103] In some embodiments, the activatable antibody includes an
antibody or antigen-binding fragment thereof that specifically
binds IL-6R. In some embodiments, the antibody or immunologically
active fragment thereof that binds IL-6R is a monoclonal antibody,
domain antibody, single chain, Fab fragment, a F(ab')2 fragment, a
scFv, a scab, a dAb, a single domain heavy chain antibody, or a
single domain light chain antibody. In some embodiments, such an
antibody or immunologically active fragment thereof that binds
IL-6R is a mouse, chimeric, humanized or fully human monoclonal
antibody.
[0104] In some embodiments, the activatable antibody comprises a
heavy chain amino acid sequence of SEQ ID NO: 1, and a light chain
amino acid sequence of SEQ ID NO: 2. In some embodiments, the
activatable antibody comprises a heavy chain amino acid sequence
that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the amino acid sequence of SEQ ID NO: 1, and a light
chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID
NO: 2.
[0105] In some embodiments, the activatable antibody comprises a
combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3
sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3
sequence, wherein at least one CDR sequence is selected from the
group consisting of a VH CDR1 sequence that includes at least the
amino acid sequence SDHAWS (SEQ ID NO: 175); a VH CD2 sequence that
includes at least the amino acid sequence YISYSGITTYNPSLKSRVT (SEQ
ID NO: 176); a VH CDR3 sequence that includes at least the amino
acid sequence SLARTTAMDY (SEQ ID NO: 177); a VL CDR1 sequence that
includes at least the amino acid sequence RASQDISS (SEQ ID NO:
178); a VL CDR2 sequence that includes at least the amino acid
sequence TISSLQP (SEQ ID NO: 179); and a VL CDR3 sequence that
includes at least the amino acid sequence QQGNTLPY (SEQ ID NO:
180).
[0106] In some embodiments, the activatable antibody comprises a
combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3
sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3
sequence, wherein at least one CDR sequence is selected from the
group consisting of a VH CDR1 sequence that includes a sequence
that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
or more identical to the amino acid sequence SDHAWS (SEQ ID NO:
175); a VH CD2 sequence that includes a sequence that is at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical
to the amino acid sequence YISYSGITTYNPSLKSRVT (SEQ ID NO: 176); a
VH CDR3 sequence that includes a sequence that is at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to
the amino acid sequence SLARTTAMDY (SEQ ID NO: 177); a VL CDR1
sequence that includes a sequence that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino
acid sequence RASQDISS (SEQ ID NO: 178); a VL CDR2 sequence that
includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence
TISSLQP (SEQ ID NO: 179); and a VL CDR3 sequence that includes a
sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more identical to the amino acid sequence QQGNTLPY (SEQ
ID NO: 180).
[0107] In some embodiments, the activatable antibody comprises a
combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3
sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3
sequence, wherein the VH CDR1 sequence comprises at least the amino
acid sequence SDHAWS (SEQ ID NO: 175); the VH CD2 sequence
comprises at least the amino acid sequence YISYSGITTYNPSLKSRVT (SEQ
ID NO: 176); the VH CDR3 sequence comprises at least the amino acid
sequence SLARTTAMDY (SEQ ID NO: 177); the VL CDR1 sequence
comprises at least the amino acid sequence RASQDISS (SEQ ID NO:
178); the VL CDR2 sequence comprises at least the amino acid
sequence TISSLQP (SEQ ID NO: 179); and the VL CDR3 sequence
comprises at least the amino acid sequence QQGNTLPY (SEQ ID NO:
180).
[0108] In some embodiments, the activatable antibody comprises a
combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3
sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3
sequence, wherein the VH CDR1 sequence comprises a sequence that is
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to the amino acid sequence SDHAWS (SEQ ID NO: 175); a VH
CD2 sequence comprises a sequence that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino
acid sequence YISYSGITTYNPSLKSRVT (SEQ ID NO: 176); the VH CDR3
sequence comprises a sequence that is at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid
sequence SLARTTAMDY (SEQ ID NO: 177); the VL CDR1 sequence
comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence
RASQDISS (SEQ ID NO: 178); the VL CDR2 sequence comprises a
sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more identical to the amino acid sequence TISSLQP (SEQ
ID NO: 179); and the VL CDR3 sequence comprises a sequence that is
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to the amino acid sequence QQGNTLPY (SEQ ID NO: 180).
[0109] In some embodiments, the activatable antibody comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOs 6-32, 109, 110 and 111. In some embodiments, the activatable
antibody comprises an amino acid sequence selected from the group
consisting of SEQ ID NOs 6-32. In some embodiments, the activatable
antibody comprises an amino acid sequence that is at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino
acid sequence selected from the group consisting of SEQ ID NOs
6-32, 109, 110 and 111. In some embodiments, the activatable
antibody comprises an amino acid that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid
sequence selected from the group consisting of SEQ ID NOs 6-32.
[0110] In some embodiments, the MM has an equilibrium dissociation
constant for binding to the AB which is greater than the
equilibrium dissociation constant of the AB to IL-6R.
[0111] In some embodiments, the MM has an equilibrium dissociation
constant for binding to the AB which is no more than the
equilibrium dissociation constant of the AB to IL-6R.
[0112] In some embodiments, the MM does not interfere or compete
with the AB for binding to IL-6R in a cleaved state.
[0113] In some embodiments, the MM is a polypeptide of about 2 to
40 amino acids in length. For example, the MM is a polypeptide of
up to about 40 amino acids in length.
[0114] In some embodiments, the MM polypeptide sequence is
different from that of IL-6R. In some embodiments, the MM
polypeptide sequence is no more than 50% identical to any natural
binding partner of the AB. In some embodiments, the MM polypeptide
sequence is different from that of IL-6R and is no more than 40%,
30%, 25%, 20%, 15%, or 10% identical to any natural binding partner
of the AB.
[0115] In some embodiments, the MM comprises an amino acid sequence
selected from the group consisting of SEQ ID NOs: 33-89. In some
embodiments, the MM comprises an amino acid sequence that is at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical
to an amino acid sequence selected from the group consisting of SEQ
ID NOs: 33-89.
[0116] In some embodiments, the MM comprises an amino acid sequence
selected from the group consisting of SEQ ID NO: 49, SEQ ID NO: 74,
and SEQ ID NO: 78. In some embodiments, the MM comprises an amino
acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identical to an amino acid sequence selected from
the group consisting of SEQ ID NO: 49, SEQ ID NO: 74, and SEQ ID
NO: 78.
[0117] In some embodiments, the MM comprises the amino acid
sequence of SEQ ID NO: 49. In some embodiments, the MM comprises an
amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID
NO: 49.
[0118] In some embodiments, the MM comprises the amino acid
sequence of SEQ ID NO: 74. In some embodiments, the MM comprises an
amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID
NO: 74.
[0119] In some embodiments, the MM comprises the amino acid
sequence of SEQ ID NO: 78. In some embodiments, the MM comprises an
amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID
NO: 78.
[0120] In some embodiments, the coupling of the MM to the AB
reduces the ability of the AB to bind IL-6R such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM
towards IL-6R is at least 20 times greater than the K.sub.d of the
AB when not coupled to the MM towards IL-6R.
[0121] In some embodiments, the coupling of the MM to the AB
reduces the ability of the AB to bind IL-6R such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM
towards IL-6R is at least 40 times greater than the K.sub.d of the
AB when not coupled to the MM towards IL-6R.
[0122] In some embodiments, the coupling of the MM to the AB
reduces the ability of the AB to bind IL-6R such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM
towards IL-6R is at least 100 times greater than the K.sub.d of the
AB when not coupled to the MM towards IL-6R.
[0123] In some embodiments, the coupling of the MM to the AB
reduces the ability of the AB to bind IL-6R such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM
towards IL-6R is at least 1000 times greater than the K.sub.d of
the AB when not coupled to the MM towards IL-6R.
[0124] In some embodiments, the coupling of the MM to the AB
reduces the ability of the AB to bind IL-6R such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM
towards IL-6R is at least 10,000 times greater than the K.sub.d of
the AB when not coupled to the MM towards IL-6R.
[0125] In some embodiments, the protease is co-localized with IL-6R
in a tissue, and wherein the protease cleaves the CM in the
activatable antibody when the activatable antibody is exposed to
the protease.
[0126] In some embodiments, in the presence of IL-6R, the MM
reduces the ability of the AB to bind IL-6R by at least 90% when
the CM is uncleaved, as compared to when the CM is cleaved when
assayed in vitro using a target displacement assay such as, for
example, the assay described in PCT Publication Nos. WO 2009/025846
and WO 2010/081173.
[0127] In some embodiments, the CM is positioned in the activatable
antibody such that in the uncleaved state, binding of the
activatable antibody to IL-6R is reduced to occur with an
equilibrium dissociation constant that is at least 20-fold greater
than the equilibrium dissociation constant of an unmodified AB
binding to IL-6R, whereas in the cleaved state, the AB binds
IL-6R.
[0128] In some embodiments, the CM is positioned in the activatable
antibody such that in the uncleaved state, binding of the
activatable antibody to IL-6R is reduced to occur with an
equilibrium dissociation constant that is at least 40-fold greater
than the equilibrium dissociation constant of an unmodified AB
binding to IL-6R, whereas in the cleaved state, the AB binds
IL-6R.
[0129] In some embodiments, the CM is positioned in the activatable
antibody such that in the uncleaved state, binding of the
activatable antibody to IL-6R is reduced to occur with an
equilibrium dissociation constant that is at least 50-fold greater
than the equilibrium dissociation constant of an unmodified AB
binding to IL-6R, whereas in the cleaved state, the AB binds
IL-6R.
[0130] In some embodiments, the CM is positioned in the activatable
antibody such that in the uncleaved state, binding of the
activatable antibody to IL-6R is reduced to occur with an
equilibrium dissociation constant that is at least 100-fold greater
than the equilibrium dissociation constant of an unmodified AB
binding to IL-6R, whereas in the cleaved state, the AB binds
IL-6R.
[0131] In some embodiments, the CM is positioned in the activatable
antibody such that in the uncleaved state, binding of the
activatable antibody to IL-6R is reduced to occur with an
equilibrium dissociation constant that is at least 200-fold greater
than the equilibrium dissociation constant of an unmodified AB
binding to IL-6R, whereas in the cleaved state, the AB binds
IL-6R.
[0132] In some embodiments, the CM is a polypeptide of up to 15
amino acids in length.
[0133] Exemplary substrates include but are not limited to
substrates cleavable by one or more of the enzymes listed in Table
12.
[0134] In some embodiments, the CM is a substrate for an enzyme
selected from the group consisting of a matrix metalloprotease
(MMP), thrombin, a neutrophil elastase, a cysteine protease,
legumain, MT-SP1, and uPA. In some embodiments, the CM is a
substrate for at least one protease selected from the group
consisting of a matrix metalloprotease (MMP), MT-SP1, uPA,
legumain, and a neutrophil elastase.
[0135] In some embodiments, the CM is a substrate for at least one
MMP. Examples of MMPs include the MMPs listed in the Table 12. In
some embodiments, the CM is a substrate for a protease selected
from the group consisting of MMP 9, MMP14, MMP1, MMP3, MMP13,
MMP17, MMP11, and MMP19. In some embodiments the CM is a substrate
for MMP9. In some embodiments, the CM is a substrate for MMP14.
[0136] In some embodiments, the CM is a substrate for an MMP and
includes the sequence ISSGLSS (SEQ ID NO: 157); QNQALRMA (SEQ ID
NO: 158); AQNLLGMV (SEQ ID NO: 159); STFPFGMF (SEQ ID NO: 160);
PVGYTSSL (SEQ ID NO: 161); DWLYWPGI (SEQ ID NO: 162), ISSGLLSS (SEQ
ID NO: 183), LKAAPRWA (SEQ ID NO: 184); GPSHLVLT (SEQ ID NO: 185);
LPGGLSPW (SEQ ID NO: 186); MGLFSEAG (SEQ ID NO: 187); SPLPLRVP (SEQ
ID NO: 188); RMHLRSLG (SEQ ID NO: 189); LAAPLGLL (SEQ ID NO: 190);
AVGLLAPP (SEQ ID NO: 191); LLAPSHRA (SEQ ID NO: 192); and/or
PAGLWLDP (SEQ ID NO: 193).
[0137] In some embodiments, the CM comprises the amino acid
sequence ISSGLSS (SEQ ID NO: 157). In some embodiments, the CM
comprises the amino acid sequence QNQALRMA (SEQ ID NO: 158). In
some embodiments, the CM comprises the amino acid sequence AQNLLGMV
(SEQ ID NO: 159). In some embodiments, the CM comprises the amino
acid sequence STFPFGMF (SEQ ID NO: 160). In some embodiments, the
CM comprises the amino acid sequence PVGYTSSL (SEQ ID NO: 161). In
some embodiments, the CM comprises the amino acid sequence DWLYWPGI
(SEQ ID NO: 162). In some embodiments, the CM comprises the amino
acid sequence ISSGLLSS (SEQ ID NO: 183). In some embodiments, the
CM comprises the amino acid sequence LKAAPRWA (SEQ ID NO: 184). In
some embodiments, the CM comprises the amino acid sequence GPSHLVLT
(SEQ ID NO: 185). In some embodiments, the CM comprises the amino
acid sequence LPGGLSPW (SEQ ID NO: 186). In some embodiments, the
CM comprises the amino acid sequence MGLFSEAG (SEQ ID NO: 187). In
some embodiments, the CM comprises the amino acid sequence SPLPLRVP
(SEQ ID NO: 188). In some embodiments, the CM comprises the amino
acid sequence RMHLRSLG (SEQ ID NO: 189). In some embodiments, the
CM comprises the amino acid sequence LAAPLGLL (SEQ ID NO: 190). In
some embodiments, the CM comprises the amino acid sequence AVGLLAPP
(SEQ ID NO: 191). In some embodiments, the CM comprises the amino
acid sequence LLAPSHRA (SEQ ID NO: 192). In some embodiments, the
CM comprises the amino acid sequence PAGLWLDP (SEQ ID NO: 193).
[0138] In some embodiments, the CM is a substrate a neutrophil
elastase. In some embodiments, the CM is a substrate for uPA. In
some embodiments, the CM is a substrate for legumain. In some
embodiments, the CM is a substrate for MT-SP1. In some embodiments,
the CM is a substrate for thrombin. In some embodiments, the CM is
a substrate for thrombin and includes the amino acid sequence
GPRSFGL (SEQ ID NO: 173) or GPRSFG (SEQ ID NO: 174). In some
embodiments, the CM is a substrate for a cysteine protease. In some
embodiments, the CM is a substrate for a cysteine protease such as
a cathepsin. In some embodiments, the CM is a substrate for a MMP.
Examples of MMPs include the MMPs listed in Table 12. In some
embodiments the CM is a substrate for MMP9. In some embodiments,
the CM is a substrate for MMP14.
[0139] In some embodiments, the CM is selected for use with a
specific protease, for example a protease that is known to be
co-localized with the target of the activatable antibody. For
example, suitable cleavable moieties for use in the activatable
anti-IL-6R antibodies of the disclosure are cleaved by at least one
protease and include the sequence TGRGPSWV (SEQ ID NO: 91);
SARGPSRW (SEQ ID NO: 116); TARGPSFK (SEQ ID NO: 117); LSGRSDNH (SEQ
ID NO: 90); GGWHTGRN (SEQ ID NO: 118); HTGRSGAL (SEQ ID NO: 119);
PLTGRSGG (SEQ ID NO: 92); AARGPAIH (SEQ ID NO: 120); RGPAFNPM (SEQ
ID NO: 121); SSRGPAYL (SEQ ID NO: 122); RGPATPIM (SEQ ID NO: 123);
RGPA (SEQ ID NO: 124); GGQPSGMWGW (SEQ ID NO: 104); FPRPLGITGL (SEQ
ID NO: 105); VHMPLGFLGP (SEQ ID NO: 106); SPLTGRSG (SEQ ID NO:
125); SAGFSLPA (SEQ ID NO: 126); LAPLGLQRR (SEQ ID NO: 127);
SGGPLGVR (SEQ ID NO: 128); and/or PLGL (SEQ ID NO: 107).
[0140] In some embodiments, the CM comprises the amino acid
sequence LSGRSDNH (SEQ ID NO: 90). In some embodiments, the CM
comprises the amino acid sequence TGRGPSWV (SEQ ID NO: 91). In some
embodiments, the CM comprises the amino acid sequence PLTGRSGG (SEQ
ID NO: 92). In some embodiments, the CM comprises the amino acid
sequence GGQPSGMWGW (SEQ ID NO: 104). In some embodiments, the CM
comprises the amino acid sequence FPRPLGITGL (SEQ ID NO: 105). In
some embodiments, the CM comprises the amino acid sequence
VHMPLGFLGP (SEQ ID NO: 106). In some embodiments, the CM comprises
the amino acid sequence PLGL (SEQ ID NO: 107). In some embodiments,
the CM comprises the amino acid sequence SARGPSRW (SEQ ID NO: 116).
In some embodiments, the CM comprises the amino acid sequence
TARGPSFK (SEQ ID NO: 117). In some embodiments, the CM comprises
the amino acid sequence GGWHTGRN (SEQ ID NO: 118). In some
embodiments, the CM comprises the amino acid sequence HTGRSGAL (SEQ
ID NO: 119). In some embodiments, the CM comprises the amino acid
sequence AARGPAIH (SEQ ID NO: 120). In some embodiments, the CM
comprises the amino acid sequence RGPAFNPM (SEQ ID NO: 121). In
some embodiments, the CM comprises the amino acid sequence SSRGPAYL
(SEQ ID NO: 122). In some embodiments, the CM comprises the amino
acid sequence RGPATPIM (SEQ ID NO: 123). In some embodiments, the
CM comprises the amino acid sequence RGPA (SEQ ID NO: 124). In some
embodiments, the CM comprises the amino acid sequence GPRSFGL (SEQ
ID NO: 173). In some embodiments, the CM comprises the amino acid
sequence GPRSFGL GPRSFG (SEQ ID NO: 174).
[0141] In some embodiments, at least one of LP1 or LP2 comprises an
amino acid sequence selected from the group consisting of
(GS).sub.n, (GGS).sub.n, (GSGGS).sub.n (SEQ ID NO: 93) and
(GGGS).sub.n (SEQ ID NO: 94), where n is an integer of at least
one.
[0142] In some embodiments, at least one of LP1 or LP2 comprises an
amino acid sequence selected from the group consisting of GGSG (SEQ
ID NO: 95), GGSGG (SEQ ID NO: 96), GSGSG (SEQ ID NO: 97), GSGGG
(SEQ ID NO: 98), GGGSG (SEQ ID NO: 99), and GSSSG (SEQ ID NO:
100).
[0143] In some embodiments, LP1 comprises the amino acid sequence
GSSGGSGGSGGSG (SEQ ID NO: 101), GSSGGSGGSGG (SEQ ID NO: 112),
GSSGGSGGSGGS (SEQ ID NO: 113), GSSGGSGGSGGSGGGS (SEQ ID NO: 169),
GSSGGSGGSG (SEQ ID NO: 170), or GSSGGSGGSGS (SEQ ID NO: 171).
[0144] In some embodiments, LP2 comprises the amino acid sequence
GSS, GGS, GGGS (SEQ ID NO: 172), GSSGT (SEQ ID NO: 102) or GSSG
(SEQ ID NO: 103).
[0145] In some embodiments, the activatable antibody also includes
an agent conjugated to the AB. In some embodiments, the agent is a
therapeutic agent. In some embodiments, the agent is an
antineoplastic agent. In some embodiments, the agent is a toxin or
fragment thereof. In some embodiments, the agent is conjugated to
the AB via a linker. In some embodiments, the linker is a cleavable
linker. In some embodiments, the agent is an agent selected from
the group listed in Table 17. In some embodiments, the agent is a
dolastatin. In some embodiments, the agent is an auristatin or
derivative thereof. In some embodiments, the agent is auristatin E
or a derivative thereof. In some embodiments, the agent is
monomethyl auristatin E (MMAE). In some embodiments, the agent is a
maytansinoid or maytansinoid derivative. In some embodiments, the
agent is DM1 or DM4. In some embodiments, the agent is a
duocarmycin or derivative thereof. In some embodiments, the agent
is a calicheamicin or derivative thereof.
[0146] In some embodiments, the agent is an anti-inflammatory
agent.
[0147] In some embodiments, the activatable antibody also includes
a detectable moiety. In some embodiments, the detectable moiety is
a diagnostic agent.
[0148] In some embodiments, the activatable antibody also includes
a signal peptide. In some embodiments, the signal peptide is
conjugated to the activatable antibody via a spacer. In some
embodiments, the spacer is conjugated to the activatable antibody
in the absence of a signal peptide. In some embodiments, the spacer
is joined directly to the MM of the activatable antibody. In some
embodiments, the spacer is joined directly to the MM of the
activatable antibody in the structural arrangement from N-terminus
to C-terminus of spacer-MM-CM-AB. An example of a spacer joined
directly to the N-terminus of MM of the activatable antibody is
QGQSGQ (SEQ ID NO: 108). In some embodiments, the spacer includes
at least the amino acid sequence QGQSGQ (SEQ ID NO: 108).
[0149] In some embodiments, the activatable antibody includes an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 6-32, 109-110, and 163-168. In some embodiments, the
activatable antibody includes an amino acid sequence selected from
the group consisting of SEQ ID NOs: 6-32. In some embodiments, the
activatable antibody includes an amino acid that is at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino
acid sequence selected from the group consisting of SEQ ID NOs:
6-32, 109-110, and 163-168. In some embodiments, the activatable
antibody includes an amino acid that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid
sequence selected from the group consisting of SEQ ID NOs:
6-32.
[0150] The invention provides methods of preventing, delaying the
progression of, treating, alleviating a symptom of, or otherwise
ameliorating an IL-6R mediated disease in a subject by
administering a therapeutically effective amount of an activatable
anti-IL-6R antibody described herein to a subject in need
thereof.
[0151] The invention provides methods of preventing, delaying the
progression of, treating, alleviating a symptom of, or otherwise
ameliorating inflammation and/or an inflammatory disorder in a
subject by administering a therapeutically effective amount of an
activatable anti-IL-6R antibody described herein to a subject in
need thereof. In some embodiments, the inflammation is associated
with and/or the inflammatory disorder is Crohn's disease. In some
embodiments, the inflammation is associated with and/or the
inflammatory disorder is polychondritis, including but not limited
to, relapsing polychondritis. In some embodiments, the inflammation
is associated with and/or the inflammatory disorder is rheumatoid
arthritis (RA). In some embodiments, the inflammation is associated
with and/or the inflammatory disorder is another rheumatoid
disease, such as, by way of non-limiting example, ankylosing
spondylitis, juvenile arthritis, and/or psoriatic arthritis. In
some embodiments, the inflammation is associated with and/or the
inflammatory disorder is ulcerative colitis.
[0152] The invention also provides methods of preventing, delaying
the progression of, treating, alleviating a symptom of, or
otherwise ameliorating cancer in a subject by administering a
therapeutically effective amount of an activatable anti-IL-6R
antibody described herein to a subject in need thereof. In some
embodiments, the cancer is breast cancer, including but not limited
to, triple negative breast cancer (TNBC). In some embodiments, the
cancer is Castleman's disease. In some embodiments, the cancer is
hepatocellular carcinoma. In some embodiments, the cancer is lung
cancer. In some embodiments, the cancer is multiple myeloma. In
some embodiments, the cancer is ovarian cancer. In some
embodiments, the cancer is prostate cancer.
[0153] The invention also provides methods of preventing, delaying
the progression of, treating, alleviating a symptom of, or
otherwise ameliorating an autoimmune disease in a subject by
administering a therapeutically effective amount of an activatable
anti-IL-6R antibody described herein to a subject in need
thereof.
[0154] An activatable anti-IL-6R antibody and/or conjugated
activatable anti-IL-6R antibody used in any of the embodiments of
these methods and uses can be administered at any stage of the
disease. For example, such an activatable anti-IL-6R antibody
and/or conjugated activatable anti-IL-6R antibody can be
administered to a patient suffering cancer of any stage, from early
to metastatic. The terms subject and patient are used
interchangeably herein.
[0155] In some embodiments, the subject is a mammal, such as a
human, non-human primate, companion animal (e.g., cat, dog, horse),
farm animal, work animal, or zoo animal. In some embodiments, the
subject is a human. In some embodiments, the subject is a companion
animal. In some embodiments, the subject is an animal in the care
of a veterinarian.
[0156] The activatable anti-IL-6R antibody and therapeutic
formulations thereof are administered to a subject suffering from
or susceptible to a disease or disorder associated with aberrant
IL-6R expression and/or activity. A subject suffering from or
susceptible to a disease or disorder associated with aberrant IL-6R
expression and/or activity is identified using any of a variety of
methods known in the art. For example, subjects suffering from
cancer or other neoplastic condition are identified using any of a
variety of clinical and/or laboratory tests such as, physical
examination and blood, urine and/or stool analysis to evaluate
health status. For example, subjects suffering from inflammation
and/or an inflammatory disorder are identified using any of a
variety of clinical and/or laboratory tests such as physical
examination and/or bodily fluid analysis, e.g., blood, urine and/or
stool analysis, to evaluate health status.
[0157] Administration of an activatable anti-IL-6R antibody to a
patient suffering from a disease or disorder associated with
aberrant IL-6R expression and/or activity is considered successful
if any of a variety of laboratory or clinical objectives is
achieved. For example, administration of an activatable anti-IL-6R
antibody to a patient suffering from a disease or disorder
associated with aberrant IL-6R expression and/or activity is
considered successful if one or more of the symptoms associated
with the disease or disorder is alleviated, reduced, inhibited or
does not progress to a further, i.e., worse, state. Administration
of an activatable anti-IL-6R antibody to a patient suffering from a
disease or disorder associated with aberrant IL-6R expression
and/or activity is considered successful if the disease or disorder
enters remission or does not progress to a further, i.e., worse,
state.
[0158] In some embodiments, the activatable anti-IL-6R antibody is
administered during and/or after treatment in combination with one
or more additional agents such as, for example, an
anti-inflammatory agent, an immunosuppressive agent, and/or a
chemotherapeutic agent. In some embodiments, the activatable
anti-IL-6R antibody and the additional agent(s) are administered
simultaneously. For example, the activatable anti-IL-6R antibody
and the additional agent(s) can be formulated in a single
composition or administered as two or more separate compositions.
In some embodiments, the activatable anti-IL-6R antibody and the
additional agent(s) are administered sequentially.
[0159] The invention also provides methods and kits for using the
activatable anti-IL-6R antibodies and/or conjugated activatable
anti-IL-6R antibodies in a variety of diagnostic and/or
prophylactic indications. For example, the invention provides
methods and kits for detecting the presence or absence of a
cleaving agent and a target of interest in a subject or a sample by
(i) contacting a subject or sample with an anti-IL-6R activatable
antibody, wherein the anti-IL-6R activatable antibody comprises a
masking moiety (MM), a cleavable moiety (CM) that is cleaved by the
cleaving agent, and an antigen binding domain or fragment thereof
(AB) that specifically binds the target of interest, wherein the
anti-IL-6R activatable antibody in an uncleaved, non-activated
state comprises a structural arrangement from N-terminus to
C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is
a peptide that inhibits binding of the AB to IL-6R, and wherein the
MM does not have an amino acid sequence of a naturally occurring
binding partner of the AB and is not a modified form of a natural
binding partner of the AB; and (b) wherein, when the AB is in an
uncleaved, non-activated state, the MM interferes with specific
binding of the AB to IL-6R, and when the AB is in a cleaved,
activated state the MM does not interfere or compete with specific
binding of the AB to IL-6R; and (ii) measuring a level of activated
anti-IL-6R activatable antibody in the subject or sample, wherein a
detectable level of activated anti-IL-6R activatable antibody in
the subject or sample indicates that the cleaving agent and IL-6R
are present in the subject or sample and wherein no detectable
level of activated anti-IL-6R activatable antibody in the subject
or sample indicates that the cleaving agent, IL-6R or both the
cleaving agent and IL-6R are absent in the subject or sample.
[0160] In some embodiments, the activatable anti-IL-6R antibody is
an activatable anti-IL-6R antibody to which a therapeutic agent is
conjugated. In some embodiments, the activatable anti-IL-6R
antibody is not conjugated to an agent. In some embodiments, the
activatable anti-IL-6R antibody comprises a detectable label. In
some embodiments, the detectable label is positioned on the AB. In
some embodiments, measuring the level of activatable anti-IL-6R
antibody in the subject or sample is accomplished using a secondary
reagent that specifically binds to the activated antibody, wherein
the reagent comprises a detectable label. In some embodiments, the
secondary reagent is an antibody comprising a detectable label.
[0161] In some embodiments of these methods and kits, the
activatable anti-IL-6R antibody includes a detectable label. In
some embodiments of these methods and kits, the detectable label
includes an imaging agent, a contrasting agent, an enzyme, a
fluorescent label, a chromophore, a dye, one or more metal ions, or
a ligand-based label. In some embodiments of these methods and
kits, the imaging agent comprises a radioisotope. In some
embodiments of these methods and kits, the radioisotope is indium
or technetium. In some embodiments of these methods and kits, the
contrasting agent comprises iodine, gadolinium or iron oxide. In
some embodiments of these methods and kits, the enzyme comprises
horseradish peroxidase, alkaline phosphatase, or
.beta.-galactosidase. In some embodiments of these methods and
kits, the fluorescent label comprises yellow fluorescent protein
(YFP), cyan fluorescent protein (CFP), green fluorescent protein
(GFP), modified red fluorescent protein (mRFP), red fluorescent
protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. In
some embodiments of these methods and kits, the luminescent label
comprises an N-methylacrydium derivative. In some embodiments of
these methods, the label comprises an Alexa Fluor.RTM. label, such
as Alex Fluor.RTM. 680 or Alexa Fluor.RTM. 750. In some embodiments
of these methods and kits, the ligand-based label comprises biotin,
avidin, streptavidin or one or more haptens.
[0162] In some embodiments of these methods and kits, the subject
is a mammal. In some embodiments of these methods, the subject is a
human. In some embodiments, the subject is a non-human mammal, such
as a non-human primate, companion animal (e.g., cat, dog, horse),
farm animal, work animal, or zoo animal. In some embodiments, the
subject is a rodent.
[0163] In some embodiments of these methods and kits, the method is
an in vivo method. In some embodiments of these methods, the method
is an in situ method. In some embodiments of these methods, the
method is an ex vivo method. In some embodiments of these methods,
the method is an in vitro method.
[0164] In some embodiments of the methods and kits, the method is
used to identify or otherwise refine a patient population suitable
for treatment with an anti-IL-6R activatable antibody of the
disclosure, followed by treatment by administering that activatable
anti-IL-6R antibody and/or conjugated activatable anti-IL-6R
antibody to a subject in need thereof. For example, patients that
test positive for both the target (e.g., IL-6R) and a protease that
cleaves the substrate in the cleavable moiety (CM) of the
anti-IL-6R activatable antibody being tested in these methods are
identified as suitable candidates for treatment with such an
anti-IL-6R activatable antibody comprising such a CM, and the
patient is then administered a therapeutically effective amount of
the activatable anti-IL-6R antibody and/or conjugated activatable
anti-IL-6R antibody that was tested. Likewise, patients that test
negative for either or both of the target (e.g., IL-6R) and the
protease that cleaves the substrate in the CM in the activatable
antibody being tested using these methods might be identified as
suitable candidates for another form of therapy. In some
embodiments, such patients can be tested with other anti-IL-6R
activatable antibodies until a suitable anti-IL-6R activatable
antibody for treatment is identified (e.g., an anti-IL-6R
activatable antibody comprising a CM that is cleaved by the patient
at the site of disease). In some embodiments, the patient is then
administered a therapeutically effective amount of the activatable
anti-IL-6R antibody and/or conjugated for which the patient tested
positive.
[0165] In some embodiments of these methods, the MM is a peptide
having a length from about 4 to 40 amino acids. In some embodiments
of these methods, the anti-IL-6R activatable antibody comprises a
linker peptide, wherein the linker peptide is positioned between
the MM and the CM. In some embodiments of these methods, the
anti-IL-6R activatable antibody comprises a linker peptide, where
the linker peptide is positioned between the AB and the CM. In some
embodiments of these methods, the anti-IL-6R activatable antibody
comprises a first linker peptide (L1) and a second linker peptide
(L2), wherein the first linker peptide is positioned between the MM
and the CM and the second linker peptide is positioned between the
AB and the CM. In some embodiments of these methods, each of L1 and
L2 is a peptide of about 1 to 20 amino acids in length, and wherein
each of L1 and L2 need not be the same linker. In some embodiments
of these methods, one or both of L1 and L2 comprises a
glycine-serine polymer. In some embodiments of these methods, at
least one of LP1 or LP2 comprises an amino acid sequence selected
from the group consisting of (GS).sub.n, (GGS).sub.n, (GSGGS).sub.n
(SEQ ID NO: 93) and (GGGS).sub.n (SEQ ID NO: 94), where n is an
integer of at least one.
[0166] In some embodiments of these methods, the AB comprises an
antibody or antibody fragment sequence selected from the
cross-reactive anti-IL-6R antibody sequences presented herein. In
some embodiments of these methods, the AB comprises a Fab fragment,
a scFv or a single chain antibody (SCAB).
[0167] In some embodiments of these methods, the cleaving agent is
a protease that is co-localized in the subject or sample with IL-6R
and the CM is a polypeptide that functions as a substrate for the
protease, wherein the protease cleaves the CM in the anti-IL-6R
activatable antibody when the anti-IL-6R activatable antibody is
exposed to the protease. In some embodiments of these methods, the
CM is a polypeptide of up to 15 amino acids in length. In some
embodiments of these methods, the CM is coupled to the N-terminus
of the AB. In some embodiments of these methods, the CM is coupled
to the C-terminus of the AB. In some embodiments of these methods,
the CM is coupled to the N-terminus of a VL chain of the AB.
[0168] In some embodiments of these methods, the cleaving agent is
an enzyme and the CM is a substrate for the enzyme. In some
embodiments of these methods, the enzyme is a protease disclosed
herein. In some embodiments of these methods, the protease is one
of the proteases disclosed in Table 12. In some embodiments of
these methods, the protease is selected from the group consisting
of a matrix metalloprotease (MMP), thrombin, a neutrophil elastase,
a cysteine protease, legumain, MT-SP1, and uPA. Examples of
suitable MMPs include, but are not limited to, MMP9, MMP14, MMP1,
MMP3, MMP-13, MMP17, MMP11, and MMP-19.
[0169] In some embodiments, the activatable antibody includes an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 6-32, 109-110, and 163-168. In some embodiments, the
activatable antibody includes an amino acid sequence selected from
the group consisting of SEQ ID NOs: 6-32. In some embodiments, the
activatable antibody includes an amino acid that is at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino
acid sequence selected from the group consisting of SEQ ID NOs:
6-32, 109-110, and 163-168. In some embodiments, the activatable
antibody includes an amino acid that is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid
sequence selected from the group consisting of SEQ ID NOs:
6-32.
[0170] Pharmaceutical compositions according to the invention can
include an antibody of the invention and a carrier. These
pharmaceutical compositions can be included in kits, such as, for
example, diagnostic kits.
BRIEF DESCRIPTION OF THE DRAWINGS
[0171] FIG. 1 is a schematic representation of a selection outline
for identifying anti-IL6R binding peptides from cellular display
scaffold libraries.
[0172] FIGS. 2A and 2B are a series of graphs depicting an example
of off-rate screening of cell populations that were labeled with 1
nM anti-IL6R-Fab Dylight, resuspended in PBS and incubated at
37.degree. C. for 3-4 minutes before sorting the brightest 0.1%.
Panel (A) demonstrates what the M1F4 population looked like before
incubation at 37.degree. C. and Panel (B) demonstrates what the
M1F4 population looked like after the incubation at 37.degree.
C.
[0173] FIG. 3 is a graph comparing clones that were sequenced from
the M1F4 and M1F5 pools at 10 and 100 nM Fab by expression
normalized binding with the parental peptide. All sequenced clones
had a higher on-cell affinity as measured by FACS than the
parental.
[0174] FIG. 4 is a schematic representation of the screening of a
directed library based on the anti-ILR6 binding peptide referred to
herein as Peptide 1.
[0175] FIG. 5 is a graph comparing peptides that were isolated from
the directed libraries with the parental peptide isolated from the
naive library (Peptide 1, also referred to herein as 4278 and
having SEQ ID NO: 33) and a peptide isolated from the soft
randomized library (SEQ ID NO: 49). Three of the peptides from the
directed library pool M1F2 had a higher affinity as measured by
FACS.
[0176] FIG. 6 is a schematic representation of the affinity
maturation for the anti-ILR6 binding peptide referred to herein as
Peptide 2.
[0177] FIG. 7 is a graph comparing clones from the directed library
screen, a clone from the M1F2 pools (SEQ ID NO: 78) at 10 and 100
nM Fab by expression normalized binding with the parental peptide
(Peptide 2, also referred to herein as 4280 and having SEQ ID NO:
34). The peptide of SEQ ID NO: 78 had a higher on-cell affinity as
measured by FACS than the parental peptide.
[0178] FIG. 8 is a schematic representation of the screening of a
directed library based on the anti-ILR6 binding peptide referred to
herein as Peptide 2.
[0179] FIG. 9 is a graph comparing clones from the directed library
screen with the parental peptide (Peptide 2) and a clone from the
M1F2 pools (SEQ ID NO: 78).
[0180] FIG. 10 is a graph depicting how first generation masking
moieties minimally shift the binding affinity of AV1-based
activatable anti-IL6R antibodies as compared to parental AV1
antibodies.
[0181] FIGS. 11 and 12 are graphs depicting the masking
efficiencies of activatable anti-IL6R antibodies that include the
masking moiety 4792 comprising SEQ ID NO: 49 (FIG. 11) or the
masking moiety 4749 comprising SEQ ID NO: 78 (FIG. 12).
[0182] FIG. 13 is a series of graphs depicting binding of AV1-Fab
to peptides displayed on a cellular peptide display scaffold (first
panel) or of peptides incorporated into activatable antibodies
(second panel).
[0183] FIG. 14 is a graph depicting IgG concentrations in plasma,
96 hours post 10 mg/Kg dose of various activatable anti-IL6R
antibodies of the invention.
[0184] FIG. 15 is a graph depicting antigen binding of human IgG in
plasma of mice treated with AV1 antibody or an uncleavable
anti-IL6R antibody of the invention.
[0185] FIG. 16 is a series of graphs depicting antigen binding of
human IgG in plasma of mice treated with AV1-based activatable
antibodies having a masking moiety that includes SEQ ID NO: 78.
[0186] FIG. 17 is a series of graphs depicting antigen binding of
human IgG in plasma of mice treated with AV1-based activatable
antibodies having a masking moiety that includes SEQ ID NO: 49 or
SEQ ID NO: 78.
[0187] FIG. 18 is a graph depicting a simulated gel from CE
analysis of synovial fluid (SyF) and serum (Ser) activatable
antibody reactions. From top to bottom donors, 2, 3, 6 and 7
respectively.
[0188] FIG. 19 is a graph depicting the extent and time course of
anti-IL-6R activatable antibody activation in rheumatoid arthritis
(RA) patients' synovial fluid (SyF).
[0189] FIG. 20 is a graph depicting cleavage kinetics for
anti-IL-6R activatable antibody activation by MMP-9.
[0190] FIG. 21A is a photograph depicting that a quenched probe
containing protease substrate 1203 was only activated (i.e.,
proteolytically cleaved) in the swollen paws of a mouse with
collagen induced arthritis (CIA mouse). FIG. 21B is a photograph
depicting an increase in MMP protease activity associated with the
inflammatory process in a CIA mouse that was administered an MMP
substrate-containing probe.
[0191] FIG. 22 is a graph depicting the significant detection of
totally hydrolyzed internally quenched (IQ) probe at concentrations
below 150 nM.
[0192] FIG. 23 is a graph depicting the progress curves for
conversion of an IQ probe containing the 1203 substrate sequence
when contacted with synovial fluid samples in the presence or
absence of a variety of protease inhibitors. Results are shown in
units of product conversion.
[0193] FIG. 24 is a graph depicting the slope analysis of the
progress curves shown in FIG. 23.
[0194] FIG. 25A-25C are a series of graphs depicting a summary of
IQ probe activation data in rheumatoid arthritis (RA) synovial
fluid samples. FIG. 25A depicts product conversion curves for an IQ
probe that contains substrate 1203. FIG. 25B depicts product
conversion curves for IQ probes that contain the non-cleavable
substrate referred to herein as "NSUB." FIG. 25C depicts slope
analysis for an IQ probe that contains substrate 1203, when the
probe is pre-treated with no inhibitor or is pre-treated with
either a broad spectrum protease inhibitor (PI), a serine PI, a
cysteine PI or a metalloprotease PI.
DETAILED DESCRIPTION OF THE INVENTION
[0195] The present invention provides activatable monoclonal
antibodies (mAbs) that specifically bind human interleukin-6
receptor (IL-6R). Interleukin 6 (IL-6) is a potent pleiotropic
cytokine that regulates cell growth and differentiation and is also
an important mediator of acute inflammatory responses. IL-6
exhibits its action via a receptor complex consisting of a specific
IL-6 receptor (IL-6R) and a signal transducing subunit (gp130). In
particular, binding of IL-6 to IL-6R leads to disulfide-linked
homodimerization of gp130 within a cell, which, in turn, leads to
the activation of a tyrosine kinase as the first step in signal
transduction. Dys-regulated IL-6 signaling has been implicated in
the pathogenesis of many diseases and disorders, such as autoimmune
diseases, inflammation, and cancer.
[0196] The activatable anti-IL-6R antibodies are used in methods of
treating, preventing, delaying the progression of, ameliorating
and/or alleviating a symptom of a disease or disorder associated
with aberrant IL-6R expression and/or activity. For example, the
activatable anti-IL-6R antibodies are used in methods of treating,
preventing, delaying the progression of, ameliorating and/or
alleviating a symptom of inflammation, an inflammatory disorder, an
autoimmune disease, and/or a cancer or other neoplastic
condition.
[0197] In some embodiments, the inflammation is associated with
and/or the inflammatory disorder is Crohn's disease. In some
embodiments, the inflammation is associated with and/or the
inflammatory disorder is polychondritis, including but not limited
to, relapsing polychondritis. In some embodiments, the inflammation
is associated with and/or the inflammatory disorder is rheumatoid
arthritis (RA). In some embodiments, the inflammation is associated
with and/or the inflammatory disorder is another rheumatoid
disease, such as, by way of non-limiting example, ankylosing
spondylitis, juvenile arthritis, and/or psoriatic arthritis. In
some embodiments, the inflammation is associated with and/or the
inflammatory disorder is ulcerative colitis.
[0198] In some embodiments, the cancer is breast cancer, including
but not limited to, triple negative breast cancer (TNBC). In some
embodiments, the cancer is Castleman's disease. In some
embodiments, the cancer is hepatocellular carcinoma. In some
embodiments, the cancer is lung cancer. In some embodiments, the
cancer is multiple myeloma. In some embodiments, the cancer is
ovarian cancer. In some embodiments, the cancer is prostate
cancer.
[0199] The activatable anti-IL-6R antibodies include an antibody or
antigen-binding fragment thereof that specifically binds
interleukin-6 receptor (IL-6R) coupled to a masking moiety (MM),
such that coupling of the MM reduces the ability of the antibody or
antigen-binding fragment thereof to bind IL-6R. In some
embodiments, the MM is coupled via a sequence that includes a
substrate for a protease, for example, a protease that is
co-localized with IL-6R at a treatment site in a subject.
[0200] In some embodiments, the antibody or antigen-binding
fragment thereof in the activatable anti-IL-6R antibody is derived
from the anti-IL-6R antibody tocilizumab. Tocilizumab, also known
as ACTEMRA.RTM. or atlizumab, is a humanized monoclonal antibody
that specifically binds IL-6R. Tocilizumab is currently used in the
treatment of rheumatoid arthritis (RA) and systemic juvenile
idiopathic arthritis, a severe form of RA in children.
[0201] In some embodiments, the activatable anti-IL-6R antibody
includes a heavy chain that is or is derived from the amino acid
sequence of SEQ ID NO: 1, and a light chain that is or is derived
from the amino acid sequence of SEQ ID NO: 2.
[0202] The activatable anti-IL-6R antibodies provided herein
include a masking moiety. In some embodiments, the masking moiety
is an amino acid sequence that is coupled or otherwise attached to
the anti-IL-6R antibody and is positioned within the activatable
anti-IL-6R antibody construct such that the masking moiety reduces
the ability of the anti-IL-6R antibody to specifically bind IL-6R.
Suitable masking moieties are identified using any of a variety of
known techniques. For example, peptide masking moieties are
identified using the methods described in U.S. Patent Application
Publication No. 2009/025846 by Daugherty et al., the contents of
which are hereby incorporated by reference in their entirety.
[0203] The activatable anti-IL-6R antibodies provided herein
include a cleavable moiety. In some embodiments, the cleavable
moiety includes an amino acid sequence that is a substrate for a
protease, usually an extracellular protease. Suitable substrates
are identified using any of a variety of known techniques. For
example, peptide substrates are identified using the methods
described in U.S. Pat. No. 7,666,817 by Daugherty et al., the
contents of which are hereby incorporated by reference in their
entirety. (See also Boulware et al. "Evolutionary optimization of
peptide substrates for proteases that exhibit rapid hydrolysis
kinetics." Biotechnol Bioeng. 106.3 (2010): 339-46).
[0204] The activatable anti-IL-6R antibodies described herein
overcome a limitation of antibody therapeutics, particularly
antibody therapeutics that are known to be toxic to at least some
degree in vivo. Target-mediated toxicity constitutes a major
limitation for the development of therapeutic antibodies. The
activatable anti-IL-6R antibodies provided herein are designed to
address the toxicity associated with the inhibition of the target
in normal tissues by traditional therapeutic antibodies. These
activatable anti-IL-6R antibodies remain masked until
proteolytically activated at the site of disease. Starting with an
anti-IL6R antibody as a parental therapeutic antibody, the
activatable anti-IL-6R antibodies of the invention were engineered
by coupling the antibody to an inhibitory mask through a linker
that incorporates a protease substrate.
[0205] Exemplary activatable anti-IL-6R antibodies of the invention
include, for example, activatable antibodies that include a heavy
chain and a light chain that are, or are derived from, the antibody
referred to herein as the"Av1" antibody, which binds interleukin-6
receptor (IL-6R). The amino acid sequences for the Av1 heavy chain
and the Av1 light chain are shown below in SEQ ID NO: 1 and SEQ ID
NO: 2, respectively.
TABLE-US-00001 Av1 Antibody Heavy Chain Amino Acid Sequence: (SEQ
ID NO: 1) QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGY
ISYSGITTYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARSLAR
TTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Av1 Antibody Light Chain
Amino Acid Sequence: (SEQ ID NO: 2)
DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYT
SRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGT
KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC
[0206] Exemplary activatable anti-IL-6R antibodies of the invention
include, for example, activatable antibodies that include a heavy
chain and a light chain that are, or are derived from, the Av1
antibody and a masking moiety. Exemplary activatable anti-IL-6R
antibodies of the invention include an amino acid sequence attached
to the N-terminus of the AV1 light chain. These N-terminal amino
acid sequences include, for example, YGSCSWNYVHIFMDC (SEQ ID NO:
49); QGDFDIPFPAHWVPIT (SEQ ID NO: 78); or MGVPAGCVWNYAHIFMDC (SEQ
ID NO: 74). In some embodiments, these N-terminal amino acid
sequences include, for example, QGQSGQYGSCSWNYVHIFMDC (SEQ ID NO:
[0207] 3); QGQSGQGDFDIPFPAHWVPIT (SEQ ID NO: 4); or
QGQSGQMGVPAGCVWNYAHIFMDC (SEQ ID NO: 5). It is also to be
appreciated that such amino acid sequences can be attached to the
N-terminus of the AV1 heavy chain or to the C-terminus of the AV1
heavy or light chain.
[0208] Examples of activatable anti-IL6R antibodies of the
invention include, but are not limited to activatable antibodies
comprising a light chain sequence listed below:
TABLE-US-00002 Av1 Lc 4792 NSub, also referred to herein as
S4792.sup.NSUBAV1, 4792.sup.NSUBAV1, S4792.sup.NSUB or
4792.sup.NSUB [Mask] [Linker 1 - Non-Cleavable Moiety - Linker 2]
[Av1 Light Chain Sequence] (SEQ ID NO: 6)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSGGGSGGGSGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4792 1203, also referred to herein as S4792.sup.1203AV1,
4792.sup.1203AV1, S4792.sup.1203 or 4792.sup.1203 [Mask] [Linker 1
- Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ ID
NO: 7)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGTGRGPSWVGGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4792 1204, also referred to herein as S4792.sup.1204AV1,
4792.sup.1204AV1, S4792.sup.1204 or 4792.sup.1204 [Mask] [Linker 1
- Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ ID
NO: 8)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGLSGRSDNHGGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4792 1214, also referred to herein as S4792.sup.1214AV1,
4792.sup.1214AV1, S4792.sup.1214 or 4792.sup.1214 [Mask] [Linker 1
- Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ ID
NO: 9)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSPLTGRSGGGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4792 PLGL, also referred to herein as S4792.sup.PLGLAV1,
4792.sup.PLGLAV1, S4792.sup.PLGL or 4792.sup.PLGL [Mask] [Linker 1
- Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ ID
NO: 10)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSGGGSPLGLGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4792 BV256, also referred to herein as S4792.sup.BV256AV1,
4792.sup.BV256AV1, S4792.sup.BV256 or 4792.sup.BV256 [Mask] [Linker
1 - Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ ID
NO: 11)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGGQPSGMWGWGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4792 BV285, also referred to herein as S4792.sup.BV285AV1,
4792.sup.BV285AV1, S4792.sup.BV285 or 4792.sup.BV285 [Mask] [Linker
1 - Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ ID
NO: 12)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGFPRPLGITGLGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4792 Pan-MMP, also referred to herein as
S4792.sup.Pan-MMPAV1, 4792.sup.Pan-MMPAV1, S4792.sup.Pan-MMP or
4792.sup.Pan-MMP [Mask] [Linker 1 - Cleavable Moiety - Linker 2]
[Av1 Light Chain Sequence] (SEQ ID NO: 13)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSGGPLGVRGGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4792 MMP-14, also referred to herein as S4792.sup.MMP-14AV1,
4792.sup.MMP-14AV1, S4792.sup.MMP-14 or 4792.sup.MMP-14 [Mask]
[Linker 1 - Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence]
(SEQ ID NO: 14)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGSLAPLGLQRRGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4792 BV726, also referred to herein as S4792.sup.BV726AV1,
4792.sup.BV276AV1, S4792.sup.BV276 or 4792.sup.BV276 [Mask] [Linker
1 - Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ ID
NO: 109)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGVHMPLGFLGPGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4749 NSub, also referred to herein as S4749.sup.NSUBAV1,
4749.sup.NSUBAV1, S4749.sup.NSUB or 4749.sup.NSUB [Mask] [Linker 1
- Non-Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ
ID NO: 15)
QGQSGQGDFDIPFPAHWVPITGSSGGSGGSGGSGGGSGGGSGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4749 1203, also referred to herein as S4749.sup.1203AV1,
4749.sup.1203AV1, S4749.sup.1203 or 4749.sup.1203 [Mask] [Linker 1
- Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ ID
NO: 16)
QGQSGQGDFDIPFPAHWVPITGSSGGSGGSGGTGRGPSWVGGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4749 1204, also referred to herein as S4749.sup.1204AV1,
4749.sup.1204AV1, S4749.sup.1204 or 4749.sup.1204 [Mask] [Linker 1
- Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ ID
NO: 17)
QGQSGQGDFDIPFPAHWVPITGSSGGSGGSGGLSGRSDNHGGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4749 1214, also referred to herein as S4749.sup.1214AV1,
4749.sup.1214AV1, S4749.sup.1214 or 4749.sup.1214 [Mask] [Linker 1
- Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ ID
NO: 18)
QGQSGQGDFDIPFPAHWVPITGSSGGSGGSGGSPLTGRSGGGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4749 PLGL, also referred to herein as S4749.sup.PLGLAV1,
4749.sup.PLGLAV1, S4749.sup.PLGL or 4749.sup.PLGL [Mask] [Linker 1
- Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ ID
NO: 19)
QGQSGQGDFDIPFPAHWVPITGSSGGSGGSGGSGGGSPLGLGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4749 BV256, also referred to herein as S4749.sup.BV256AV1,
4749.sup.BV256AV1, S4749.sup.BV256 or 4749.sup.BV256 [Mask] [Linker
1 - Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ ID
NO: 20)
QGQSGQGDFDIPFPAHWVPITGSSGGSGGSGGGQPSGMWGWGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4749 BV285, also referred to herein as S4749.sup.BV285AV1,
4749.sup.BV285AV1, S4749.sup.BV285 or 4749.sup.BV285 [Mask] [Linker
1 - Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ ID
NO: 21)
QGQSGQGDFDIPFPAHWVPITGSSGGSGGSGFPRPLGITGLGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4749 Pan-MMP, also referred to herein as
S4749.sup.Pan-MMPAV1, 4749.sup.Pan-MMPAV1, S4749.sup.Pan-MMP or
4749.sup.Pan-MMP [Mask] [Linker 1 - Cleavable Moiety - Linker 2]
[Av1 Light Chain Sequence] (SEQ ID NO: 22)
QGQSGQGDFDIPFPAHWVPITGSSGGSGGSGGSGGPLGVRGGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4749 MMP-14, also referred to herein as S4749.sup.MMP-14AV1,
4749.sup.MMP-14AM1, S4749.sup.MMP-14 or 4749.sup.MMP-14 [Mask]
[Linker 1 - Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence]
(SEQ ID NO: 23)
QGQSGQGDFDIPFPAHWVPITGSSGGSGGSGSLAPLGLQRRGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4749 BV726, also referred to herein as S4749.sup.BV726AV1,
4749.sup.BV726AV1, S4749.sup.BV726 or 4749.sup.BV726 [Mask] [Linker
1 - Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence (SEQ ID
NO: 110)
QGQSGQGDFDIPFPAHWVPITGSSGGSGGSGVHMPLGFLGPGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 5382 NSub, also referred to herein as S5382.sup.NSUBAV1,
5382.sup.NSUBAV1, S5382.sup.NSUB or 5382.sup.NSUB [Mask] [Linker 1
- Non-Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ
ID NO: 24)
QGQSGQMGVPAGCVWNYAHIFMDCGSSGGSGGSGGSGGGSGGGSGGSDIQMTQSPSSLSASVGD
RVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPE
DIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
NRGEC Av1 Lc 5382 1203, also referred to herein as
S5382.sup.1203AV1, 5382.sup.1203AV1, S5382.sup.1203 or
5382.sup.1203 [Mask] [Linker 1 - Cleavable Moiety - Linker 2] [Av1
Light Chain Sequence] (SEQ ID NO: 25)
QGQSGQMGVPAGCVWNYAHIFMDCGSSGGSGGSGGTGRGPSWVGGGSDIQMTQSPSSLSASVGD
RVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPE
DIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
NRGEC Av1 Lc 5382 1204, also referred to herein as
S5382.sup.1204AV1, 5382.sup.1204AV1, S5382.sup.1204 or
5382.sup.1204 [Mask] [Linker 1 - Cleavable Moiety - Linker 2] [Av1
Light Chain Sequence] (SEQ ID NO: 26)
QGQSGQMGVPAGCVWNYAHIFMDCGSSGGSGGSGGLSGRSDNHGGGSDIQMTQSPSSLSASVGD
RVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPE
DIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
NRGEC Av1 Lc 5382 1214, also referred to herein as
S5382.sup.1214AV1, 5382.sup.1214AV1, S5382.sup.1214 or
5382.sup.1214 [Mask] [Linker 1 - Cleavable Moiety - Linker 2] [Av1
Light Chain Sequence] (SEQ ID NO: 27)
QGQSGQMGVPAGCVWNYAHIFMDCGSSGGSGGSGGSPLTGRSGGGGSDIQMTQSPSSLSASVGD
RVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPE
DIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
NRGEC Av1 Lc 5382 PLGL, also referred to herein as
S5382.sup.PLGLAV1, 5382.sup.PLGLAV1, S5382.sup.PLGL or
5382.sup.PLGL [Mask] [Linker 1 - Cleavable Moiety - Linker 2] [Av1
Light Chain Sequence] (SEQ ID NO: 28)
QGQSGQMGVPAGCVWNYAHIFMDCGSSGGSGGSGGSGGGSPLGLGGSDIQMTQSPSSLSASVGD
RVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPE
DIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
NRGEC Av1 Lc 5382 BV256, also referred to herein as
S5382.sup.BV256AV1, 5382.sup.BV256AV1, S5382.sup.BV256 or
5382.sup.BV256 [Mask] [Linker 1 - Cleavable Moiety - Linker 2] [Av1
Light Chain Sequence] (SEQ ID NO: 29)
QGQSGQMGVPAGCVWNYAHIFMDCGSSGGSGGSGGGQPSGMWGWGGSDIQMTQSPSSLSASVGD
RVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPE
DIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
NRGEC Av1 Lc 5382 BV285, also referred to herein as
S5382.sup.BV285AV1, 5382.sup.BV285AV1, S53821.sup.BV285 or
5382.sup.BV285 [Mask] [Linker 1 - Cleavable Moiety - Linker 2] [Av1
Light Chain Sequence] (SEQ ID NO: 30)
QGQSGQMGVPAGCVWNYAHIFMDCGSSGGSGGSGFPRPLGITGLGGSDIQMTQSPSSLSASVGD
RVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPE
DIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
NRGEC Av1 Lc 5382 Pan-MMP, also referred to herein as
S5382.sup.Pan-MMPAV1, 5382.sup.Pan-MMPAV1, S5382.sup.Pan-MMP or
5382.sup.Pan-MMP [Mask] [Linker 1 - Cleavable Moiety - Linker 2]
[Av1 Light Chain Sequence] (SEQ ID NO: 31)
QGQSGQMGVPAGCVWNYAHIFMDCGSSGGSGGSGGSGGPLGVRGGGSDIQMTQSPSSLSASVGD
RVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPE
DIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
NRGEC Av1 Lc 5382 MMP-14, also referred to herein as
S5382.sup.MMP-14AV1, 5382.sup.MMP-14AV1, S5382.sup.MMP-14 or
5382.sup.MMP-14 [Mask] [Linker 1 - Cleavable Moiety - Linker 2]
[Av1 Light Chain Sequence] (SEQ ID NO: 32)
QGQSGQMGVPAGCVWNYAHIFMDCGSSGGSGGSGSLAPLGLQRRGGSDIQMTQSPSSLSASVGD
RVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPE
DIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
NRGEC Av1 Lc 5382 BV726, also referred to herein as
S5382.sup.BV726AV1, 5382.sup.BV726AV1, S5382.sup.BV726 or
5382.sup.BV726 [Mask] [Linker 1 - Cleavable Moiety - Linker 2] [Av1
Light Chain Sequence] (SEQ ID NO: 111)
QGQSGQMGVPAGCVWNYAHIFMDCGSSGGSGGSGVHMPLGFLGPGGSDIQMTQSPSSLSASVGD
RVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPE
DIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
NRGEC Av1 Lc 4792 10419, also referred to herein as
S4792.sup.10419AV1, 4792.sup.10419AV1, S4792.sup.10419 or
4792.sup.10419: [Mask] [Linker 1 - Cleavable Moiety - Linker 2]
[Av1 Light Chain Sequence] (SEQ ID NO: 163)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSGISSGLSSGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4792 559, also referred to herein as S4792.sup.559AV1,
4792.sup.559AV1, S4792.sup.559 or 4792.sup.559 [Mask] [Linker 1 -
Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ ID NO:
164)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSQNQALRMAGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4792 601, also referred to herein as S4792.sup.601AV1,
4792.sup.601AV1, S4792.sup.601 or 4792.sup.601 [Mask] [Linker 1 -
Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ ID NO:
165)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSAQNLLGMVGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4792 3457, also referred to herein as S4792.sup.3457AV1,
4792.sup.3457AV1, S4792.sup.3457 or 4792.sup.3457 [Mask] [Linker 1
- Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ ID
NO: 166)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSSTFPFGMFGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4792 3458, also referred to herein as S4792.sup.3458AV1,
4792.sup.3458AV1, S4792.sup.3458 or 4792.sup.3458 [Mask] [Linker 1
- Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ ID
NO: 167)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSPVGYTSSLGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4792 3463, also referred to herein as S4792.sup.3463AV1,
4792.sup.3463AV1, S4792.sup.3463 or 4792.sup.3463 [Mask] [Linker 1
- Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence] (SEQ ID
NO: 168)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSDWLYWPGIGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
Av1 Lc 4792 Thromb2, also referred to herein as
S4792.sup.Thromb2AV1, 4792.sup.Thromb2AV1, S4792.sup.Thromb2 or
4792.sup.Thromb2 [Mask] [Linker 1 - Cleavable Moiety - Linker 2]
[Av1 Light Chain Sequence] (SEQ ID NO: 181)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSGPRSFGLDIQMTQSPSSLSASVGDRVTITCR
ASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC
QQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN
ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Av1
Lc 4792 Thromb3, also referred to herein as S4792.sup.Thromb3AV1,
4792.sup.Thromb3AV1, S4792.sup.Thromb3 or 4792.sup.Thromb3 [Mask]
[Linker 1 - Cleavable Moiety - Linker 2] [Av1 Light Chain Sequence]
(SEQ ID NO: 182)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSGPRSFGDIQMTQSPSSLSASVGDRVTITCRA
SQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQ
QGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
[0209] Activatable Anti-IL-6R Antibodies
[0210] The activatable antibodies and activatable antibody
compositions provided herein contain at least an antibody or
antibody fragment thereof (collectively referred to as AB
throughout the disclosure) that specifically binds human IL-6R,
wherein the AB is modified by a masking moiety (MM).
[0211] When the AB is modified with a MM and is in the presence of
IL-6R, specific binding of the AB to its target is reduced or
inhibited, as compared to the specific binding of the AB not
modified with an MM or the specific binding of the parental AB to
the target.
[0212] The K.sub.d of the AB modified with a MM towards the target,
i.e., IL-6R, is at least 5, 10, 25, 50, 100, 250, 500, 1,000,
2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000,
5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10,
10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000,
10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000,
100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000,
1000-10,000,000, 10,000-100,000, 10,000-1,000,000,
10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times
greater than the K.sub.d of the AB not modified with an MM or the
parental AB towards the target. Conversely, the binding affinity of
the AB modified with a MM towards the target, i.e., IL-6R, is at
least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000,
50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000,
50,000,000 or greater, or between 5-10, 10-100, 10-1,000,
10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000,
100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000,
1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000,
10,000-100,000, 10,000-1,000,000, 10,000-10,000,000,
100,000-1,000,000, or 100,000-10,000,000 times lower than the
binding affinity of the AB not modified with an MM or the parental
AB towards the target.
[0213] The dissociation constant (K.sub.d) of the MM towards the AB
is generally greater than the K.sub.d of the AB towards the target,
i.e., IL-6R. The K.sub.d of the MM towards the AB can be at least
5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000,
1,000,000 or even 10,000,000 times greater than the K.sub.d of the
AB towards the target, i.e., IL-6R. Conversely, the binding
affinity of the MM towards the AB is generally lower than the
binding affinity of the AB towards the target, i.e., IL-6R. The
binding affinity of MM towards the AB can be at least 5, 10, 25,
50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000
or even 10,000,000 times lower than the binding affinity of the AB
towards the target, i.e., IL-6R.
[0214] When the AB is modified with a MM and is in the presence of
the target, i.e., IL-6R, specific binding of the AB to its target
is reduced or inhibited, as compared to the specific binding of the
AB not modified with an MM or the specific binding of the parental
AB to the target. When compared to the binding of the AB not
modified with an MM or the binding of the parental AB to the
target, i.e., IL-6R, the AB's ability to bind the target when
modified with an MM can be reduced by at least 50%, 60%, 70%, 80%,
90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at
least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours,
or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, or 12 months or more when measured in vivo
or in an in vitro assay.
[0215] The MM inhibits the binding of the AB to the target, i.e.,
IL-6R. The MM binds the antigen binding domain of the AB and
inhibits binding of the AB to IL-6R. The MM can sterically inhibit
the binding of the AB to the target, i.e., IL-6R. The MM can
allosterically inhibit the binding of the AB to its target. In
these embodiments when the AB is modified or coupled to a MM and in
the presence of target, i.e., IL-6R, there is no binding or
substantially no binding of the AB to the target, or no more than
0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, or 50% binding of the AB to the target, as
compared to the binding of the AB not modified with an MM, the
parental AB, or the AB not coupled to an MM to the target, for at
least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours,
or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, or 12 months or longer when measured in vivo
or in an in vitro assay.
[0216] When an AB is coupled to or modified by a MM, the MM `masks`
or reduces or otherwise inhibits the specific binding of the AB to
IL-6R. When an AB is coupled to or modified by a MM, such coupling
or modification can effect a structural change that reduces or
inhibits the ability of the AB to specifically bind its target.
[0217] An AB coupled to or modified with an MM can be represented
by the following formulae (in order from an amino (N) terminal
region to carboxyl (C) terminal region:
(MM)-(AB)
(AB)-(MM)
(MM)-L-(AB)
(AB)-L-(MM)
where MM is a masking moiety, the AB is an antibody or antibody
fragment thereof, and the L is a linker. In many embodiments, it
may be desirable to insert one or more linkers, e.g., flexible
linkers, into the composition so as to provide for flexibility.
[0218] In certain embodiments, the MM is not a natural binding
partner of the AB. In some embodiments the MM contains no or
substantially no homology to any natural binding partner of the AB.
In other embodiments the MM is no more than 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to
any natural binding partner of the AB. In some embodiments, the MM
is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding
partner of the AB. In some embodiments, the MM is no more than 25%
identical to any natural binding partner of the AB. In some
embodiments, the MM is no more than 50% identical to any natural
binding partner of the AB. In some embodiments, the MM is no more
than 20% identical to any natural binding partner of the AB. In
some embodiments, the MM is no more than 10% identical to any
natural binding partner of the AB.
[0219] In some embodiments, the activatable antibodies include an
AB that is modified by an MM and also includes one or more
cleavable moieties (CM). Such activatable antibodies exhibit
activatable/switchable binding, to the AB's target, i.e., IL-6R.
Activatable antibodies generally include an antibody or antibody
fragment (AB), modified by or coupled to a masking moiety (MM) and
a modifiable or cleavable moiety (CM). In some embodiments, the CM
contains an amino acid sequence that serves as a substrate for a
protease of interest.
[0220] The elements of the activatable antibodies are arranged so
that the MM and CM are positioned such that in a cleaved (or
relatively active) state and in the presence of a target, the AB
binds a target, i.e., IL-6R, while in an uncleaved (or relatively
inactive) state in the presence of the target, specific binding of
the AB to its target, i.e., IL-6R, is reduced or inhibited. The
specific binding of the AB to its target can be reduced due to the
inhibition or masking of the AB's ability to specifically bind its
target by the MM.
[0221] The K.sub.d of the AB modified with a MM and a CM towards
the target, i.e., IL-6R, is at least 5, 10, 25, 50, 100, 250, 500,
1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000,
5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10,
10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000,
10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000,
100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000,
1000-10,000,000, 10,000-100,000, 10,000-1,000,000,
10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times
greater than the K.sub.d of the AB not modified with an MM and a CM
or the parental AB towards the target, i.e., IL-6R. Conversely, the
binding affinity of the AB modified with a MM and a CM towards the
target, i.e., IL-6R, is at least 5, 10, 25, 50, 100, 250, 500,
1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000,
5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10,
10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000,
10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000,
100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000,
1000-10,000,000, 10,000-100,000, 10,000-1,000,000,
10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times
lower than the binding affinity of the AB not modified with an MM
and a CM or the parental AB towards the target, i.e., IL-6R.
[0222] When the AB is modified with a MM and a CM and is in the
presence of the target but not in the presence of a modifying agent
(for example a protease), specific binding of the AB to its target,
i.e., IL-6R, is reduced or inhibited, as compared to the specific
binding of the AB not modified with an MM and a CM or the parental
AB to the target. When compared to the binding of the parental AB
or the binding of an AB not modified with an MM and a CM to its
target, the AB's ability to bind the target when modified with an
MM and a CM can be reduced by at least 50%, 60%, 70%, 80%, 90%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least
2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours or 5,
10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, or 12 months or longer when measured in vivo or in
an in vitro assay.
[0223] As used herein, the term cleaved state refers to the
condition of the activatable antibodies following modification of
the CM by a protease. The term uncleaved state, as used herein,
refers to the condition of the activatable antibodies in the
absence of cleavage of the CM by a protease. As discussed above,
the term "activatable antibodies" is used herein to refer to an
activatable antibody in both its uncleaved (native) state, as well
as in its cleaved state. It will be apparent to the ordinarily
skilled artisan that in some embodiments a cleaved activatable
antibody may lack an MM due to cleavage of the CM by protease,
resulting in release of at least the MM (e.g., where the MM is not
joined to the activatable antibodies by a covalent bond (e.g., a
disulfide bond between cysteine residues).
[0224] By activatable or switchable is meant that the activatable
antibody exhibits a first level of binding to a target, i.e.,
IL-6R, when in a inhibited, masked or uncleaved state (i.e., a
first conformation), and a second level of binding to the target,
i.e., IL-6R, in the uninhibited, unmasked and/or cleaved state
(i.e., a second conformation), where the second level of target
binding is greater than the first level of binding. In general, the
access of target to the AB of the activatable antibody is greater
in the presence of a cleaving agent capable of cleaving the CM than
in the absence of such a cleaving agent. Thus, when the activatable
antibody is in the uncleaved state, the AB is inhibited from target
binding and can be masked from target binding (i.e., the first
conformation is such the AB cannot bind the target), and in the
cleaved state the AB is not inhibited or is unmasked to target
binding.
[0225] The CM and AB of the activatable antibodies are selected so
that the AB represents a binding moiety for IL-6R, and the CM
represents a substrate for a protease that is co-localized with
IL-6R at a treatment site or diagnostic site in a subject. The
activatable antibodies disclosed herein find particular use where,
for example, a protease capable of cleaving a site in the CM is
present at relatively higher levels in target-containing tissue of
a treatment site or diagnostic site than in tissue of non-treatment
sites (for example in healthy tissue).
[0226] In some embodiments activatable antibodies provide for
reduced toxicity and/or adverse side effects that could otherwise
result from binding of the AB at non-treatment sites if the AB were
not masked or otherwise inhibited from binding IL-6R.
[0227] In general, an activatable antibody can be designed by
selecting an AB of interest and constructing the remainder of the
activatable antibody so that, when conformationally constrained,
the MM provides for masking of the AB or reduction of binding of
the AB to its target. Structural design criteria can be to be taken
into account to provide for this functional feature.
[0228] Activatable antibodies exhibiting a switchable phenotype of
a desired dynamic range for target binding in an inhibited versus
an uninhibited conformation are provided. Dynamic range generally
refers to a ratio of (a) a maximum detected level of a parameter
under a first set of conditions to (b) a minimum detected value of
that parameter under a second set of conditions. For example, in
the context of an activatable antibody, the dynamic range refers to
the ratio of (a) a maximum detected level of target protein, i.e.,
IL-6R, binding to an activatable antibody in the presence of
protease capable of cleaving the CM of the activatable antibodies
to (b) a minimum detected level of target protein, i.e., IL-6R,
binding to an activatable antibody in the absence of the protease.
The dynamic range of an activatable antibody can be calculated as
the ratio of the equilibrium dissociation constant of an
activatable antibody cleaving agent (e.g., enzyme) treatment to the
equilibrium dissociation constant of the activatable antibodies
cleaving agent treatment. The greater the dynamic range of an
activatable antibody, the better the switchable phenotype of the
activatable antibody. Activatable antibodies having relatively
higher dynamic range values (e.g., greater than 1) exhibit more
desirable switching phenotypes such that target protein binding by
the activatable antibodies occurs to a greater extent (e.g.,
predominantly occurs) in the presence of a cleaving agent (e.g.,
enzyme) capable of cleaving the CM of the activatable antibodies
than in the absence of a cleaving agent.
[0229] Activatable antibodies can be provided in a variety of
structural configurations. Exemplary formulae for activatable
antibodies are provided below. It is specifically contemplated that
the N- to C-terminal order of the AB, MM and CM may be reversed
within an activatable antibody. It is also specifically
contemplated that the CM and MM may overlap in amino acid sequence,
e.g., such that the CM is contained within the MM.
[0230] For example, activatable antibodies can be represented by
the following formula (in order from an amino (N) terminal region
to carboxyl (C) terminal region:
(MM)-(CM)-(AB)
(AB)-(CM)-(MM)
where MM is a masking moiety, CM is a cleavable moiety, and AB is
an antibody or fragment thereof. It should be noted that although
MM and CM are indicated as distinct components in the formulae
above, in all exemplary embodiments (including formulae) disclosed
herein it is contemplated that the amino acid sequences of the MM
and the CM could overlap, e.g., such that the CM is completely or
partially contained within the MM. In addition, the formulae above
provide for additional amino acid sequences that may be positioned
N-terminal or C-terminal to the activatable antibodies
elements.
[0231] In certain embodiments, the MM is not a natural binding
partner of the AB. In some embodiments the MM contains no or
substantially no homology to any natural binding partner of the AB.
In other embodiments the MM is no more than 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to
any natural binding partner of the AB. In some embodiments, the MM
is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding
partner of the AB. In some embodiments, the MM is no more than 50%
identical to any natural binding partner of the AB. In some
embodiments, the MM is no more than 25% identical to any natural
binding partner of the AB. In some embodiments, the MM is no more
than 20% identical to any natural binding partner of the AB. In
some embodiments, the MM is no more than 10% identical to any
natural binding partner of the AB.
[0232] In many embodiments it may be desirable to insert one or
more linkers, e.g., flexible linkers, into the activatable antibody
construct so as to provide for flexibility at one or more of the
MM-CM junction, the CM-AB junction, or both. For example, the AB,
MM, and/or CM may not contain a sufficient number of residues
(e.g., Gly, Ser, Asp, Asn, especially Gly and Ser, particularly
Gly) to provide the desired flexibility. As such, the switchable
phenotype of such activatable antibody constructs may benefit from
introduction of one or more amino acids to provide for a flexible
linker. In addition, as described below, where the activatable
antibody is provided as a conformationally constrained construct, a
flexible linker can be operably inserted to facilitate formation
and maintenance of a cyclic structure in the uncleaved activatable
antibody.
[0233] For example, in certain embodiments an activatable antibody
comprises one of the following formulae (where the formula below
represent an amino acid sequence in either N- to C-terminal
direction or C- to N-terminal direction):
(MM)-L1-(CM)-(AB)
(MM)-(CM)-L2-(AB)
(MM)-L1-(CM)-L2-(AB)
wherein MM, CM, and AB are as defined above; wherein L1 and L2 are
each independently and optionally present or absent, are the same
or different flexible linkers that include at least 1 flexible
amino acid (e.g., Gly). In addition, the formulae above provide for
additional amino acid sequences that may be positioned N-terminal
or C-terminal to the activatable antibodies elements. Examples
include, but are not limited to, targeting moieties (e.g., a ligand
for a receptor of a cell present in a target tissue) and serum
half-life extending moieties (e.g., polypeptides that bind serum
proteins, such as immunoglobulin (e.g., IgG) or serum albumin
(e.g., human serum albumin (HAS)).
[0234] In some embodiments, the cleavable moiety (CM) of the
activatable antibody includes an amino acid sequence that can serve
as a substrate for a protease, usually an extracellular protease.
The CM may be selected based on a protease that is co-localized in
tissue with the desired target of the AB of the activatable
antibody. A variety of different conditions are known in which a
target of interest is co-localized with a protease, where the
substrate of the protease is known in the art. In the example of
cancer, the target tissue can be a cancerous tissue, particularly
cancerous tissue of a solid tumor. There are reports in the
literature of increased levels of proteases having known substrates
in a number of cancers, e.g., solid tumors. See, e.g., La Rocca et
al, (2004) British J. of Cancer 90(7): 1414-1421. Non-liming
examples of disease include: all types of cancers (breast, lung,
colorectal, prostate, melanomas, head and neck, pancreatic, etc.),
rheumatoid arthritis, Crohn's disuse, SLE, cardiovascular damage,
ischemia, etc. For example, indications would include leukemias,
including T-cell acute lymphoblastic leukemia (T-ALL),
lymphoblastic diseases including multiple myeloma, and solid
tumors, including lung, colorectal, prostate, pancreatic and
breast, including triple negative breast cancer. For example,
indications include bone disease or metastasis in cancer,
regardless of primary tumor origin; breast cancer, including by way
of non-limiting example, ER/PR+ breast cancer, Her2+ breast cancer,
triple-negative breast cancer; colorectal cancer; gastric cancer;
glioblastoma; head and neck cancer; lung cancer, such as by way of
non-limiting example, non-small cell lung cancer; multiple myeloma
ovarian cancer; pancreatic cancer; prostate cancer; sarcoma; renal
cancer, such as by way of nonlimiting example, renal cell
carcinoma; and/or skin cancer, such as by way of nonlimiting
example, squamous cell cancer, basal cell carcinoma, melanoma. In
addition to cancer, IL-6R-dependent notch signaling is critical to
epithelial and fibroblast differentiation to myofibroblasts, cells
with a central role in the development of fibrotic disease.
Inhibition of IL-6R dependent notch signaling, and therefore
inhibition of the emergence of myofibroblasts, would be an
effective treatment for fibrotic diseases of the kidney, liver,
lung, and skin. For example, indications would include a fibrotic
disorder, such as idiopathic pulmonary fibrosis (IPF); kidney
fibrotic disease, liver fibrotic disease, peritoneal
dialysis-induced fibrosis, and/or scleroderma. Other suitable
indications include, for example, a pathology such as, for example,
hearing loss.
[0235] The CM is specifically cleaved by an enzyme at a rate of
about 0.001-1500.times.10.sup.4 M.sup.-1S.sup.-1 or at least 0.001,
0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50,
75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or
1500.times.10.sup.4 M.sup.-1S.sup.-1.
[0236] For specific cleavage by an enzyme, contact between the
enzyme and CM is made. When the activatable antibody comprising an
AB coupled to a MM and a CM is in the presence of target and
sufficient enzyme activity, the CM can be cleaved. Sufficient
enzyme activity can refer to the ability of the enzyme to make
contact with the CM and effect cleavage. It can readily be
envisioned that an enzyme may be in the vicinity of the CM but
unable to cleave because of other cellular factors or protein
modification of the enzyme.
[0237] Exemplary substrates include but are not limited to
substrates cleavable by one or more of the following enzymes or
proteases in Table 12:
TABLE-US-00003 TABLE 12 Exemplary proteases ADAMS, Cysteine Serine
ADAMTS, e.g. proteinases, e.g., proteases, e.g., ADAM8 Cruzipain
activated protein C ADAM9 Legumain Cathepsin A ADAM10 Otubain-2
Cathepsin G ADAM12 KLKs, e.g., Chymase ADAM15 KLK4 coagulation
factor proteases ADAM17/TACE KLK5 (e.g., FVIIa, FIXa, ADAMDEC1 KLK6
FXa, FXIa, FXIIa) ADAMTS1 KLK7 Elastase ADAMTS4 KLK8 Granzyme B
ADAMTS5 KLK10 Guanidinobenzoatase Aspartate KLK11 HtrA1 proteases,
e.g., KLK13 Human Neutrophil Elastase BACE KLK14 Lactoferrin Renin
Metallo- Marapsin Aspartic proteases, e.g., NS3/4A cathepsins,
e.g., Meprin PACE4 Cathepsin D Neprilysin Plasmin Cathepsin E PSMA
PSA Caspases, e.g., BMP-1 tPA Caspase 1 MMPs, e.g., Thrombin
Caspase 2 MMP-1 Tryptase Caspase 3 MMP-2 uPA Caspase 4 MMP-3 Type
II Transmembrane Caspase 5 MMP-7 Serine Proteases Caspase 6 MMP-8
(TTSPs), e.g., Caspase 7 MMP-9 DESC1 Caspase 8 MMP-10 DPP-4 Caspase
9 MMP-11 FAP Caspase 10 MMP-12 Hepsin Caspase 14 MMP-13
Matriptase-2 Cysteine MMP-14 MT-SP1/Matriptase cathepsins, e.g.,
MMP-15 TMPRSS2 Cathepsin B MMP-16 TMPRSS3 Cathepsin C MMP-17
TMPRSS4 Cathepsin K MMP-19 Cathepsin L MMP-20 Cathepsin S MMP-23
Cathepsin V/L2 MMP-24 Cathepsin X/Z/P MMP-26 MMP-27
[0238] For example, in some embodiments, the substrate is cleavable
by one or more of the following enzymes or proteases: uPA,
legumain, MT-SP1, ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-9, MMP-12,
MMP-13, and/or MMP-14. In some embodiments, the protease is
selected from the group of uPA, legumain, and MT-SP1. In some
embodiments, the protease is a matrix metalloproteinase.
[0239] Linkers suitable for use in compositions described herein
are generally ones that provide flexibility of the modified AB or
the activatable antibodies to facilitate the inhibition of the
binding of the AB to the target. Such linkers are generally
referred to as flexible linkers. Suitable linkers can be readily
selected and can be of any of a suitable of different lengths, such
as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino
acids to 15 amino acids, from 3 amino acids to 12 amino acids,
including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino
acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino
acids, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 amino acids in length.
[0240] Exemplary flexible linkers include glycine polymers
(G).sub.n, glycine-serine polymers (including, for example,
(GS).sub.n, (GSGGS).sub.n (SEQ ID NO: 93) and (GGGS).sub.n (SEQ ID
NO: 94), where n is an integer of at least one), glycine-alanine
polymers, alanine-serine polymers, and other flexible linkers known
in the art. Glycine and glycine-serine polymers are relatively
unstructured, and therefore may be able to serve as a neutral
tether between components. Glycine accesses significantly more
phi-psi space than even alanine, and is much less restricted than
residues with longer side chains (see Scheraga, Rev. Computational
Chem. 11173-142 (1992)). Exemplary flexible linkers include, but
are not limited to Gly-Gly-Ser-Gly (SEQ ID NO: 95),
Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 96), Gly-Ser-Gly-Ser-Gly (SEQ ID
NO: 97), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 98), Gly-Gly-Gly-Ser-Gly
(SEQ ID NO: 99), Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 100), and the
like. The ordinarily skilled artisan will recognize that design of
an activatable antibodies can include linkers that are all or
partially flexible, such that the linker can include a flexible
linker as well as one or more portions that confer less flexible
structure to provide for a desired activatable antibodies
structure.
[0241] In some embodiments, the activatable anti-IL-6R antibodies
described herein also include an agent conjugated to the
activatable antibody. In some embodiments, the conjugated agent is
a therapeutic agent, such as an anti-inflammatory and/or an
antineoplastic agent. In such embodiments, the agent is conjugated
to a carbohydrate moiety of the activatable antibody, for example,
in some embodiments, where the carbohydrate moiety is located
outside the antigen-binding region of the antibody or
antigen-binding fragment in the activatable antibody. In some
embodiments, the agent is conjugated to a sulfhydryl group of the
antibody or antigen-binding fragment in the activatable
antibody.
[0242] In some embodiments, the agent is a cytotoxic agent such as
a toxin (e.g., an enzymatically active toxin of bacterial, fungal,
plant, or animal origin, or fragments thereof), or a radioactive
isotope (i.e., a radioconjugate).
[0243] In some embodiments, the agent is a detectable moiety such
as, for example, a label or other marker. For example, the agent is
or includes a radiolabeled amino acid, one or more biotinyl
moieties that can be detected by marked avidin (e.g., streptavidin
containing a fluorescent marker or enzymatic activity that can be
detected by optical or calorimetric methods), one or more
radioisotopes or radionuclides, one or more fluorescent labels, one
or more enzymatic labels, and/or one or more chemiluminescent
agents. In some embodiments, detectable moieties are attached by
spacer molecules.
[0244] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a toxin (e.g.,
an enzymatically active toxin of bacterial, fungal, plant, or
animal origin, or fragments thereof), or a radioactive isotope
(i.e., a radioconjugate). Suitable cytotoxic agents include, for
example, dolastatins and derivatives thereof (e.g. auristatin E,
AFP, MMAF, MMAE). For example, the cytotoxic agent is monomethyl
auristatin E (MMAE). In some embodiments, the agent is an agent
selected from the group listed in Table 17. In some embodiments,
the agent is a dolastatin. In some embodiments, the agent is an
auristatin or derivative thereof. In some embodiments, the agent is
auristatin E or a derivative thereof. In some embodiments, the
agent is monomethyl auristatin E (MMAE). In some embodiments, the
agent is a maytansinoid or maytansinoid derivative. In some
embodiments, the agent is DM1 or DM4. In some embodiments, the
agent is a duocarmycin or derivative thereof. In some embodiments,
the agent is a calicheamicin or derivative thereof. Enzymatically
active toxins and fragments thereof that can be used include
diphtheria A chain, nonbinding active fragments of diphtheria
toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A
chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites
fordii proteins, dianthin proteins, Phytolaca americana proteins
(PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin,
crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin,
restrictocin, phenomycin, enomycin, and the tricothecenes. A
variety of radionuclides are available for the production of
radioconjugated antibodies. Examples include .sup.212Bi, .sup.131I,
.sup.131In, .sup.90Y, and .sup.186Re.
[0245] Conjugates of the antibody and cytotoxic agent are made
using a variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. (See WO94/11026).
[0246] Table 17 lists some of the exemplary pharmaceutical agents
that may be employed in the herein described invention but in no
way is meant to be an exhaustive list.
TABLE-US-00004 TABLE 17 Exemplary Pharmaceutical Agents for
Conjugation CYTOTOXIC AGENTS Auristatins Turbostatin Auristatin E
Phenstatins Monomethyl auristatin E (MMAE) Hydroxyphenstatin
Desmethyl auristatin E (DMAE) Spongistatin 5 Auristatin F
Spongistatin 7 Monomethyl auristatin F (MMAF) Halistatin 1
Desmethyl auristatin F (DMAF) Halistatin 2 Auristatin derivatives,
e.g., Halistatin 3 amides thereof Modified Bryostatins Auristatin
tyramine Halocomstatins Auristatin quinoline Pyrrolobenzimidazoles
(PBI) Dolastatins Cibrostatin6 Dolastatin derivatives Doxaliform
Dolastatin 16 DmJ Anthracyclins analogues Dolastatin 16 Dpv
Anthracyclins analogues Maytansinoids, Cemadotin analogue e.g.
DM-1; DM-4 (CemCH2-SH) Maytansinoid derivatives Pseudomonas toxin A
Duocarmycin (PE38) variant Duocarmycin derivatives Pseudomonas
toxin A Alpha-amanitin (ZZ-PE38) variant Anthracyclines ZJ-101
Doxorubicin OSW-1 Daunorubicin 4-Nitrobenzyloxycarbonyl Bryostatins
Derivatives of Camptothecin O6-Benzylguanine Camptothecin
derivatives Topoisomerase inhibitors 7-substituted Camptothecin
Hemiasterlin 10, 11- Cephalotaxine Difluoromethylenedioxy-
Homoharringtonine camptothecin Pyrrolobenzodiazepine
Combretastatins dimers (PBDs) Debromoaplysiatoxin Functionalized
pyrrolo- Kahalalide-F benzodiazepenes Discodermolide Calicheamicins
Ecteinascidins Podophyllotoxins ANTIVIRALS Taxanes Acyclovir Vinca
alkaloids Vira A CONJUGATABLE DETECTION Symmetrel REAGENTS
ANTIFUNGALS Fluorescein and derivatives Nystatin thereof ADDITIONAL
Fluorescein isothiocyanate ANTI-NEOPLASTICS (FITC) Adriamycin
RADIOPHARMA- Cerubidine CEUTICALS Bleomycin .sup.125I Alkeran
.sup.131I Velban .sup.89Zr Oncovin .sup.111In Fluorouracil
.sup.123I Methotrexate .sup.131I Thiotepa .sup.99mTc Bisantrene
.sup.201TI Novantrone .sup.133Xe Thioguanine .sup.11C Procarabizine
.sup.62Cu Cytarabine .sup.18F ANTI-BACTERIALS .sup.68Ga
Aminoglycosides .sup.13N Streptomycin .sup.15O Neomycin .sup.38K
Kanamycin .sup.82Rb Amikacin .sup.99mTc (Technetium) Gentamicin
HEAVY METALS Tobramycin Barium Streptomycin B Gold Spectinomycin
Platinum Ampicillin ANTI-MYCOPLASMALS Sulfanilamide Tylosine
Polymyxin Spectinomycin Chloramphenicol
[0247] Those of ordinary skill in the art will recognize that a
large variety of possible moieties can be coupled to the resultant
antibodies of the invention. (See, for example, "Conjugate
Vaccines", Contributions to Microbiology and Immunology, J. M.
Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989),
the entire contents of which are incorporated herein by
reference).
[0248] Coupling may be accomplished by any chemical reaction that
will bind the two molecules so long as the antibody and the other
moiety retain their respective activities. This linkage can include
many chemical mechanisms, for instance covalent binding, affinity
binding, intercalation, coordinate binding and complexation. In
some embodiments the binding is, however, covalent binding.
Covalent binding can be achieved either by direct condensation of
existing side chains or by the incorporation of external bridging
molecules. Many bivalent or polyvalent linking agents are useful in
coupling protein molecules, such as the antibodies of the present
invention, to other molecules. For example, representative coupling
agents can include organic compounds such as thioesters,
carbodiimides, succinimide esters, diisocyanates, glutaraldehyde,
diazobenzenes and hexamethylene diamines. This listing is not
intended to be exhaustive of the various classes of coupling agents
known in the art but, rather, is exemplary of the more common
coupling agents. (See Killen and Lindstrom, Jour. Immun.
133:1335-2549 (1984); Jansen et al., Immunological Reviews
62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987).
[0249] Suitable linkers are described in the literature. (See, for
example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984)
describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide
ester). See also, U.S. Pat. No. 5,030,719, describing use of
halogenated acetyl hydrazide derivative coupled to an antibody by
way of an oligopeptide linker. In some embodiments, suitable
linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)
carbodiimide hydrochloride; (ii) SMPT
(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene
(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6
[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat
#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6
[3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.
#2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce
Chem. Co., Cat. #24510) conjugated to EDC.
[0250] The linkers described above contain components that have
different attributes, thus leading to conjugates with differing
physio-chemical properties. For example, sulfo-NHS esters of alkyl
carboxylates are more stable than sulfo-NHS esters of aromatic
carboxylates. NETS-ester containing linkers are less soluble than
sulfo-NHS esters. Further, the linker SMPT contains a sterically
hindered disulfide bond, and can form conjugates with increased
stability. Disulfide linkages, are in general, less stable than
other linkages because the disulfide linkage is cleaved in vitro,
resulting in less conjugate available. Sulfo-NHS, in particular,
can enhance the stability of carbodimide couplings. Carbodimide
couplings (such as EDC) when used in conjunction with sulfo-NHS,
forms esters that are more resistant to hydrolysis than the
carbodimide coupling reaction alone.
[0251] In some embodiments, the linkers are cleavable. In some
embodiments, the linkers are non-cleavable. In some embodiments,
two or more linkers are present. The two or more linkers are all
the same, i.e., cleavable or non-cleavable, or the two or more
linkers are different, i.e., at least one cleavable and at least
one non-cleavable.
[0252] The present invention utilizes several methods for attaching
agents to ABs: (a) attachment to the carbohydrate moieties of the
AB, or (b) attachment to sulfhydryl groups of the AB, or (c)
attachment to amino groups of the AB, or (d) attachment to
carboxylate groups of the AB. According to the invention, ABs may
be covalently attached to an agent through an intermediate linker
having at least two reactive groups, one to react with AB and one
to react with the agent. The linker, which may include any
compatible organic compound, can be chosen such that the reaction
with AB (or agent) does not adversely affect AB reactivity and
selectivity. Furthermore, the attachment of linker to agent might
not destroy the activity of the agent. Suitable linkers for
reaction with oxidized antibodies or oxidized antibody fragments
include those containing an amine selected from the group
consisting of primary amine, secondary amine, hydrazine, hydrazide,
hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide
groups. Such reactive functional groups may exist as part of the
structure of the linker, or may be introduced by suitable chemical
modification of linkers not containing such groups.
[0253] According to the present invention, suitable linkers for
attachment to reduced ABs include those having certain reactive
groups capable of reaction with a sulfhydryl group of a reduced
antibody or fragment. Such reactive groups include, but are not
limited to: reactive haloalkyl groups (including, for example,
haloacetyl groups), p-mercuribenzoate groups and groups capable of
Michael-type addition reactions (including, for example, maleimides
and groups of the type described by Mitra and Lawton, 1979, J.
Amer. Chem. Soc. 101: 3097-3110).
[0254] According to the present invention, suitable linkers for
attachment to neither oxidized nor reduced Abs include those having
certain functional groups capable of reaction with the primary
amino groups present in unmodified lysine residues in the Ab. Such
reactive groups include, but are not limited to, NHS carboxylic or
carbonic esters, sulfo-NHS carboxylic or carbonic esters,
4-nitrophenyl carboxylic or carbonic esters, pentafluorophenyl
carboxylic or carbonic esters, acyl imidazoles, isocyanates, and
isothiocyanates.
[0255] According to the present invention, suitable linkers for
attachment to neither oxidized nor reduced Abs include those having
certain functional groups capable of reaction with the carboxylic
acid groups present in aspartate or glutamate residues in the Ab,
which have been activated with suitable reagents. Suitable
activating reagents include EDC, with or without added NHS or
sulfo-NHS, and other dehydrating agents utilized for carboxamide
formation. In these instances, the functional groups present in the
suitable linkers would include primary and secondary amines,
hydrazines, hydroxylamines, and hydrazides.
[0256] The agent may be attached to the linker before or after the
linker is attached to the AB. In certain applications it may be
desirable to first produce an AB-linker intermediate in which the
linker is free of an associated agent. Depending upon the
particular application, a specific agent may then be covalently
attached to the linker. In other embodiments the AB is first
attached to the MM, CM and associated linkers and then attached to
the linker for conjugation purposes.
[0257] Branched Linkers: In specific embodiments, branched linkers
that have multiple sites for attachment of agents are utilized. For
multiple site linkers, a single covalent attachment to an AB would
result in an AB-linker intermediate capable of binding an agent at
a number of sites. The sites may be aldehyde or sulfhydryl groups
or any chemical site to which agents can be attached.
[0258] Alternatively, higher specific activity (or higher ratio of
agents to AB) can be achieved by attachment of a single site linker
at a plurality of sites on the AB. This plurality of sites may be
introduced into the AB by either of two methods. First, one may
generate multiple aldehyde groups and/or sulfhydryl groups in the
same AB. Second, one may attach to an aldehyde or sulfhydryl of the
AB a "branched linker" having multiple functional sites for
subsequent attachment to linkers. The functional sites of the
branched linker or multiple site linker may be aldehyde or
sulfhydryl groups, or may be any chemical site to which linkers may
be attached. Still higher specific activities may be obtained by
combining these two approaches, that is, attaching multiple site
linkers at several sites on the AB.
[0259] Cleavable Linkers:
[0260] Peptide linkers that are susceptible to cleavage by enzymes
of the complement system, such as but not limited to urokinase,
tissue plasminogen activator, trypsin, plasmin, or another enzyme
having proteolytic activity may be used in one embodiment of the
present invention. According to one method of the present
invention, an agent is attached via a linker susceptible to
cleavage by complement. The antibody is selected from a class that
can activate complement. The antibody-agent conjugate, thus,
activates the complement cascade and releases the agent at the
target site. According to another method of the present invention,
an agent is attached via a linker susceptible to cleavage by
enzymes having a proteolytic activity such as a urokinase, a tissue
plasminogen activator, plasmin, or trypsin. These cleavable linkers
are useful in conjugated activatable antibodies that include an
extracellular toxin, e.g., by way of non-limiting example, any of
the extracellular toxins shown in Table 17.
[0261] Non-liming examples of cleavable linker sequences are
provided in Table 18.
TABLE-US-00005 TABLE 18 Exemplary Linker Sequences for Conjugation
Types of Cleavable Sequences Amino Acid Sequence Plasmin cleavable
sequences Pro-urokinase PRFKIIGG (SEQ ID NO: 129) PRFRIIGG (SEQ ID
NO: 130) TGF.beta. SSRHRRALD (SEQ ID NO: 131) Plasminogen
RKSSIIIRMRDVVL (SEQ ID NO: 132) Staphylokinase SSSFDKGKYKKGDDA (SEQ
ID NO: 133) SSSFDKGKYKRGDDA (SEQ ID NO: 134) Factor Xa cleavable
IEGR (SEQ ID NO: 135) sequences IDGR (SEQ ID NO: 136) GGSIDGR (SEQ
ID NO: 137) MMP cleavable sequences Gelatinase A PLGLWA (SEQ ID NO:
138) Collagenase cleavable sequences Calf skin collagen GPQGIAGQ
(SEQ ID NO: 139) (.alpha.1(I) chain) Calf skin collagen GPQGLLGA
(SEQ ID NO: 140) (.alpha.2(I) chain) Bovine cartilage GIAGQ (SEQ ID
NO: 141) collagen (.alpha.1(II) chain) Human liver collagen
GPLGIAGI (SEQ ID NO: 142) (.alpha.1(III) chain) Human
.alpha..sub.2M GPEGLRVG (SEQ ID NO: 143) Human PZP YGAGLGVV (SEQ ID
NO: 144) AGLGVVER (SEQ ID NO: 145) AGLGISST (SEQ ID NO: 146) Rat
.alpha..sub.1M EPQALAMS (SEQ ID NO: 147) QALAMSAI (SEQ ID NO: 148)
Rat .alpha..sub.2M AAYHLVSQ (SEQ ID NO: 149) MDAFLESS (SEQ ID NO:
150) Rat .alpha..sub.1I.sub.3(2J) ESLPVVAV (SEQ ID NO: 151) Rat
.alpha..sub.1I.sub.3(27J) SAPAVESE (SEQ ID NO: 152) Human
fibroblast DVAQFVLT (SEQ ID NO: 153) collagenase VAQFVLTE (SEQ ID
NO: 154) (autolytic cleavages) AQFVLTEG (SEQ ID NO: 155) PVQPIGPQ
(SEQ ID NO: 156)
[0262] In addition, agents may be attached via disulfide bonds (for
example, the disulfide bonds on a cysteine molecule) to the AB.
Since many tumors naturally release high levels of glutathione (a
reducing agent) this can reduce the disulfide bonds with subsequent
release of the agent at the site of delivery. In certain specific
embodiments the reducing agent that would modify a CM would also
modify the linker of the conjugated activatable antibody.
[0263] Spacers and Cleavable Elements: In still another embodiment,
it may be necessary to construct the linker in such a way as to
optimize the spacing between the agent and the AB of the
activatable antibody. This may be accomplished by use of a linker
of the general structure:
W--(CH.sub.2).sub.n-Q
wherein W is either --NH--CH.sub.2-- or --CH.sub.2--; Q is an amino
acid, peptide; and n is an integer from 0 to 20.
[0264] In still other embodiments, the linker may comprise a spacer
element and a cleavable element. The spacer element serves to
position the cleavable element away from the core of the AB such
that the cleavable element is more accessible to the enzyme
responsible for cleavage. Certain of the branched linkers described
above may serve as spacer elements.
[0265] Throughout this discussion, it should be understood that the
attachment of linker to agent (or of spacer element to cleavable
element, or cleavable element to agent) need not be particular mode
of attachment or reaction. Any reaction providing a product of
suitable stability and biological compatibility is acceptable.
[0266] Serum Complement and Selection of Linkers:
[0267] According to one method of the present invention, when
release of an agent is desired, an AB that is an antibody of a
class that can activate complement is used. The resulting conjugate
retains both the ability to bind antigen and activate the
complement cascade. Thus, according to this embodiment of the
present invention, an agent is joined to one end of the cleavable
linker or cleavable element and the other end of the linker group
is attached to a specific site on the AB. For example, if the agent
has an hydroxy group or an amino group, it may be attached to the
carboxy terminus of a peptide, amino acid or other suitably chosen
linker via an ester or amide bond, respectively. For example, such
agents may be attached to the linker peptide via a carbodimide
reaction. If the agent contains functional groups that would
interfere with attachment to the linker, these interfering
functional groups can be blocked before attachment and deblocked
once the product conjugate or intermediate is made. The opposite or
amino terminus of the linker is then used either directly or after
further modification for binding to an AB that is capable of
activating complement.
[0268] Linkers (or spacer elements of linkers) may be of any
desired length, one end of which can be covalently attached to
specific sites on the AB of the activatable antibody. The other end
of the linker or spacer element may be attached to an amino acid or
peptide linker.
[0269] Thus when these conjugates bind to antigen in the presence
of complement the amide or ester bond that attaches the agent to
the linker will be cleaved, resulting in release of the agent in
its active form. These conjugates, when administered to a subject,
will accomplish delivery and release of the agent at the target
site, and are particularly effective for the in vivo delivery of
pharmaceutical agents, antibiotics, antimetabolites,
antiproliferative agents and the like as presented in but not
limited to those in Table 17.
[0270] Linkers for Release without Complement Activation:
[0271] In yet another application of targeted delivery, release of
the agent without complement activation is desired since activation
of the complement cascade will ultimately lyse the target cell.
Hence, this approach is useful when delivery and release of the
agent should be accomplished without killing the target cell. Such
is the goal when delivery of cell mediators such as hormones,
enzymes, corticosteroids, neurotransmitters, genes or enzymes to
target cells is desired. These conjugates may be prepared by
attaching the agent to an AB that is not capable of activating
complement via a linker that is mildly susceptible to cleavage by
serum proteases. When this conjugate is administered to an
individual, antigen-antibody complexes will form quickly whereas
cleavage of the agent will occur slowly, thus resulting in release
of the compound at the target site.
[0272] Biochemical Cross Linkers:
[0273] In other embodiments, the activatable antibody may be
conjugated to one or more therapeutic agents using certain
biochemical cross-linkers. Cross-linking reagents form molecular
bridges that tie together functional groups of two different
molecules. To link two different proteins in a step-wise manner,
hetero-bifunctional cross-linkers can be used that eliminate
unwanted homopolymer formation.
[0274] Peptidyl linkers cleavable by lysosomal proteases are also
useful, for example, Val-Cit, Val-Ala or other dipeptides. In
addition, acid-labile linkers cleavable in the low-pH environment
of the lysosome may be used, for example: bis-sialyl ether. Other
suitable linkers include cathepsin-labile substrates, particularly
those that show optimal function at an acidic pH.
[0275] Exemplary hetero-bifunctional cross-linkers are referenced
in Table 19.
TABLE-US-00006 TABLE 19 Exemplary Hetero-Bifunctional Cross Linkers
HETERO-BIFUNCTIONAL CROSS-LINKERS Spacer Arm Length after
Advantages and cross-linking Linker Reactive Toward Applications
(Angstroms) SMPT Primary amines Greater stability 11.2 .ANG.
Sulfhydryls SPDP Primary amines Thiolation 6.8 .ANG. Sulfhydryls
Cleavable cross-linking LC-SPDP Primary amines Extended spacer arm
15.6 .ANG. Sulfhydryls Sulfo-LC- Primary amines Extender spacer arm
15.6 .ANG. SPDP Sulfhydryls Water-soluble SMCC Primary amines
Stable maleimide 11.6 .ANG. reactive group Sulfhydryls
Enzyme-antibody conjugation Hapten-carrier protein conjugation
Sulfo-SMCC Primary amines Stable maleimide 11.6 .ANG. reactive
group Sulfhydryls Water-soluble Enzyme-antibody conjugation MBS
Primary amines Enzyme-antibody 9.9 .ANG. conjugation Sulfhydryls
Hapten-carrier protein conjugation Sulfo-MBS Primary amines
Water-soluble 9.9 .ANG. Sulfhydryls SIAB Primary amines
Enzyme-antibody 10.6 .ANG. Sulfhydryls conjugation Sulfo-SIAB
Primary amines Water-soluble 10.6 .ANG. Sulfhydryls SMPB Primary
amines Extended spacer arm 14.5 .ANG. Sulfhydryls Enzyme-antibody
conjugation Sulfo-SMPB Primary amines Extended spacer arm 14.5
.ANG. Sulfhydryls Water-soluble EDE/Sulfo- Primary amines
Hapten-Carrier 0 NHS Carboxyl groups conjugation ABH Carbohydrates
Reacts with sugar 11.9 .ANG. Nonselective groups
[0276] Non-Cleavable Linkers or Direct Attachment:
[0277] In still other embodiments of the invention, the conjugate
may be designed so that the agent is delivered to the target but
not released. This may be accomplished by attaching an agent to an
AB either directly or via a non-cleavable linker.
[0278] These non-cleavable linkers may include amino acids,
peptides, D-amino acids or other organic compounds that may be
modified to include functional groups that can subsequently be
utilized in attachment to ABs by the methods described herein.
A-general formula for such an organic linker could be
W--(CH.sub.2)n-Q
wherein W is either --NH--CH.sub.2-- or --CH.sub.2--; Q is an amino
acid, peptide; and n is an integer from 0 to 20.
[0279] Non-Cleavable Conjugates:
[0280] Alternatively, a compound may be attached to ABs that do not
activate complement. When using ABs that are incapable of
complement activation, this attachment may be accomplished using
linkers that are susceptible to cleavage by activated complement or
using linkers that are not susceptible to cleavage by activated
complement.
[0281] The antibodies disclosed herein can also be formulated as
immunoliposomes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc.
Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045
and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556.
[0282] Particularly useful liposomes can be generated by the
reverse-phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange
reaction.
Definitions
[0283] Unless otherwise defined, scientific and technical terms
used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. The term "a" entity or "an" entity refers to one or more
of that entity. For example, a compound refers to one or more
compounds. As such, the terms "a", "an", "one or more" and "at
least one" can be used interchangeably. Further, unless otherwise
required by context, singular terms shall include pluralities and
plural terms shall include the singular. Generally, nomenclatures
utilized in connection with, and techniques of, cell and tissue
culture, molecular biology, and protein and oligo- or
polynucleotide chemistry and hybridization described herein are
those well-known and commonly used in the art. Standard techniques
are used for recombinant DNA, oligonucleotide synthesis, and tissue
culture and transformation (e.g., electroporation, lipofection).
Enzymatic reactions and purification techniques are performed
according to manufacturer's specifications or as commonly
accomplished in the art or as described herein. The foregoing
techniques and procedures are generally performed according to
conventional methods well known in the art and as described in
various general and more specific references that are cited and
discussed throughout the present specification. See e.g., Sambrook
et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The
nomenclatures utilized in connection with, and the laboratory
procedures and techniques of, analytical chemistry, synthetic
organic chemistry, and medicinal and pharmaceutical chemistry
described herein are those well-known and commonly used in the art.
Standard techniques are used for chemical syntheses, chemical
analyses, pharmaceutical preparation, formulation, and delivery,
and treatment of patients.
[0284] As utilized in accordance with the present disclosure, the
following terms, unless otherwise indicated, shall be understood to
have the following meanings:
[0285] As used herein, the term "antibody" refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
(Ig) molecules, i.e., molecules that contain an antigen binding
site that specifically binds (immunoreacts with) an antigen. By
"specifically bind" or "immunoreacts with" or "immunospecifically
bind" is meant that the antibody reacts with one or more antigenic
determinants of the desired antigen and does not react with other
polypeptides or binds at much lower affinity (K.sub.d>10').
Antibodies include, but are not limited to, polyclonal, monoclonal,
chimeric, domain antibody, single chain, Fab, and F(ab')2
fragments, scFvs, and an Fab expression library.
[0286] The basic antibody structural unit is known to comprise a
tetramer. Each tetramer is composed of 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 each chain defines a constant region
primarily responsible for effector function. In general, antibody
molecules obtained from humans relate to any of the classes IgG,
IgM, IgA, IgE and IgD, which differ from one another by the nature
of the heavy chain present in the molecule. Certain classes have
subclasses as well, such as IgG.sub.1, IgG.sub.2, and others.
Furthermore, in humans, the light chain may be a kappa chain or a
lambda chain.
[0287] The term "monoclonal antibody" (mAb) or "monoclonal antibody
composition", as used herein, refers to a population of antibody
molecules that contain only one molecular species of antibody
molecule consisting of a unique light chain gene product and a
unique heavy chain gene product. In particular, the complementarity
determining regions (CDRs) of the monoclonal antibody are identical
in all the molecules of the population. MAbs contain an antigen
binding site capable of immunoreacting with a particular epitope of
the antigen characterized by a unique binding affinity for it.
[0288] The term "antigen-binding site" or "binding portion" refers
to the part of the immunoglobulin molecule that participates in
antigen binding. The antigen binding site is formed by amino acid
residues of the N-terminal variable ("V") regions of the heavy
("H") and light ("L") chains. Three highly divergent stretches
within the V regions of the heavy and light chains, referred to as
"hypervariable regions," are interposed between more conserved
flanking stretches known as "framework regions," or "FRs". Thus,
the term "FR" refers to amino acid sequences which are naturally
found between, and adjacent to, hypervariable regions in
immunoglobulins. In an antibody molecule, the three hypervariable
regions of a light chain and the three hypervariable regions of a
heavy chain are disposed relative to each other in three
dimensional space to form an antigen-binding surface. The
antigen-binding surface is complementary to the three-dimensional
surface of a bound antigen, and the three hypervariable regions of
each of the heavy and light chains are referred to as
"complementarity-determining regions," or "CDRs." The assignment of
amino acids to each domain is in accordance with the definitions of
Kabat Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia
& Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature
342:878-883 (1989).
[0289] As used herein, the term "epitope" includes any protein
determinant capable of specific binding to an immunoglobulin, an
scFv, or a T-cell receptor. The term "epitope" includes any protein
determinant capable of specific binding to an immunoglobulin or
T-cell receptor. Epitopic determinants usually consist of
chemically active surface groupings of molecules such as amino
acids or sugar side chains and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics. For example, antibodies may be raised against
N-terminal or C-terminal peptides of a polypeptide. An antibody is
said to specifically bind an antigen when the dissociation constant
is <1 .mu.M; in some embodiments, <100 nM and in some
embodiments, <10 nM.
[0290] As used herein, the terms "specific binding," "immunological
binding," and "immunological binding properties" refer to the
non-covalent interactions of the type which occur between an
immunoglobulin molecule and an antigen for which the immunoglobulin
is specific. The strength, or affinity of immunological binding
interactions can be expressed in terms of the dissociation constant
(K.sub.d) of the interaction, wherein a smaller K.sub.d represents
a greater affinity. Immunological binding properties of selected
polypeptides can be quantified using methods well known in the art.
One such method entails measuring the rates of antigen-binding
site/antigen complex formation and dissociation, wherein those
rates depend on the concentrations of the complex partners, the
affinity of the interaction, and geometric parameters that equally
influence the rate in both directions. Thus, both the "on rate
constant" (K.sub.on) and the "off rate constant" (K.sub.off) can be
determined by calculation of the concentrations and the actual
rates of association and dissociation. (See Nature 361:186-87
(1993)). The ratio of K.sub.off/K.sub.on enables the cancellation
of all parameters not related to affinity, and is equal to the
dissociation constant K.sub.d. (See, generally, Davies et al.
(1990) Annual Rev Biochem 59:439-473). An antibody of the present
invention is said to specifically bind to IL-6R, when the
equilibrium binding constant (K.sub.d) is .mu.M, in some
embodiments 100 nM, in some embodiments 10 nM, and in some
embodiments 100 .mu.M to about 1 .mu.M, as measured by assays such
as radioligand binding assays or similar assays known to those
skilled in the art.
[0291] The term "isolated polynucleotide" as used herein shall mean
a polynucleotide of genomic, cDNA, or synthetic origin or some
combination thereof, which by virtue of its origin the "isolated
polynucleotide" (1) is not associated with all or a portion of a
polynucleotide in which the "isolated polynucleotide" is found in
nature, (2) is operably linked to a polynucleotide which it is not
linked to in nature, or (3) does not occur in nature as part of a
larger sequence. Polynucleotides in accordance with the invention
include the nucleic acid molecules encoding the heavy chain
immunoglobulin molecules shown herein, and nucleic acid molecules
encoding the light chain immunoglobulin molecules shown herein.
[0292] The term "isolated protein" referred to herein means a
protein of cDNA, recombinant RNA, or synthetic origin or some
combination thereof, which by virtue of its origin, or source of
derivation, the "isolated protein" (1) is not associated with
proteins found in nature, (2) is free of other proteins from the
same source, e.g., free of murine proteins, (3) is expressed by a
cell from a different species, or (4) does not occur in nature.
[0293] The term "polypeptide" is used herein as a generic term to
refer to native protein, fragments, or analogs of a polypeptide
sequence. Hence, native protein fragments, and analogs are species
of the polypeptide genus. Polypeptides in accordance with the
invention comprise the heavy chain immunoglobulin molecules shown
herein, and the light chain immunoglobulin molecules shown herein,
as well as antibody molecules formed by combinations comprising the
heavy chain immunoglobulin molecules with light chain
immunoglobulin molecules, such as kappa light chain immunoglobulin
molecules, and vice versa, as well as fragments and analogs
thereof.
[0294] The term "naturally-occurring" as used herein as applied to
an object refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism (including viruses) that can be isolated
from a source in nature and which has not been intentionally
modified by man in the laboratory or otherwise is
naturally-occurring.
[0295] The term "operably linked" as used herein refers to
positions of components so described are in a relationship
permitting them to function in their intended manner. A control
sequence "operably linked" to a coding sequence is ligated in such
a way that expression of the coding sequence is achieved under
conditions compatible with the control sequences.
[0296] The term "control sequence" as used herein refers to
polynucleotide sequences which are necessary to effect the
expression and processing of coding sequences to which they are
ligated. The nature of such control sequences differs depending
upon the host organism in prokaryotes, such control sequences
generally include promoter, ribosomal binding site, and
transcription termination sequence in eukaryotes, generally, such
control sequences include promoters and transcription termination
sequence. The term "control sequences" is intended to include, at a
minimum, all components whose presence is essential for expression
and processing, and can also include additional components whose
presence is advantageous, for example, leader sequences and fusion
partner sequences. The term "polynucleotide" as referred to herein
means nucleotides of at least 10 bases in length, either
ribonucleotides or deoxynucleotides or a modified form of either
type of nucleotide. The term includes single and double stranded
forms of DNA.
[0297] The term oligonucleotide referred to herein includes
naturally occurring, and modified nucleotides linked together by
naturally occurring, and non-naturally occurring oligonucleotide
linkages. Oligonucleotides are a polynucleotide subset generally
comprising a length of 200 bases or fewer. In some embodiments,
oligonucleotides are 10 to 60 bases in length and in some
embodiments, 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in
length. Oligonucleotides are usually single stranded, e.g., for
probes, although oligonucleotides may be double stranded, e.g., for
use in the construction of a gene mutant. Oligonucleotides of the
invention are either sense or antisense oligonucleotides.
[0298] The term "naturally occurring nucleotides" referred to
herein includes deoxyribonucleotides and ribonucleotides. The term
"modified nucleotides" referred to herein includes nucleotides with
modified or substituted sugar groups and the like. The term
"oligonucleotide linkages" referred to herein includes
oligonucleotide linkages such as phosphorothioate,
phosphorodithioate, phosphoroselerloate, phosphorodiselenoate,
phosphoroanilothioate, phoshoraniladate, phosphoronmidate, and the
like. See e.g., LaPlanche et al. Nucl. Acids Res. 14:9081 (1986);
Stec et al. J. Am. Chem. Soc. 106:6077 (1984), Stein et al. Nucl.
Acids Res. 16:3209 (1988), Zon et al. Anti Cancer Drug Design 6:539
(1991); Zon et al. Oligonucleotides and Analogues: A Practical
Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press,
Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;
Uhlmann and Peyman Chemical Reviews 90:543 (1990). An
oligonucleotide can include a label for detection, if desired.
[0299] As used herein, the twenty conventional amino acids and
their abbreviations follow conventional usage. See Immunology--A
Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer
Associates, Sunderland7 Mass. (1991)). Stereoisomers (e.g., D-amino
acids) of the twenty conventional amino acids, unnatural amino
acids such as a-, a-disubstituted amino acids, N-alkyl amino acids,
lactic acid, and other unconventional amino acids may also be
suitable components for polypeptides of the present invention.
Examples of unconventional amino acids include: 4 hydroxyproline,
y-carboxyglutamate, .epsilon.-N,N,N-trimethyllysine,
.epsilon.-N-acetyllysine, O-phosphoserine, N-acetylserine,
N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,
6-N-methylarginine, and other similar amino acids and imino acids
(e.g., 4-hydroxyproline). In the polypeptide notation used herein,
the left-hand direction is the amino terminal direction and the
right-hand direction is the carboxy-terminal direction, in
accordance with standard usage and convention.
[0300] Similarly, unless specified otherwise, the left-hand end of
single-stranded polynucleotide sequences is the 5' end the
left-hand direction of double-stranded polynucleotide sequences is
referred to as the 5' direction. The direction of 5' to 3' addition
of nascent RNA transcripts is referred to as the transcription
direction sequence regions on the DNA strand having the same
sequence as the RNA and which are 5' to the 5' end of the RNA
transcript are referred to as "upstream sequences", sequence
regions on the DNA strand having the same sequence as the RNA and
which are 3' to the 3' end of the RNA transcript are referred to as
"downstream sequences".
[0301] As applied to polypeptides, the term "substantial identity"
means that two peptide sequences, when optimally aligned, such as
by the programs GAP or BESTFIT using default gap weights, share at
least 80 percent sequence identity, in some embodiments, at least
90 percent sequence identity, in some embodiments, at least 95
percent sequence identity, and in some embodiments, at least 99
percent sequence identity.
[0302] In some embodiments, residue positions which are not
identical differ by conservative amino acid substitutions.
[0303] As discussed herein, minor variations in the amino acid
sequences of antibodies or immunoglobulin molecules are
contemplated as being encompassed by the present invention,
providing that the variations in the amino acid sequence maintain
at least 75%, in some embodiments, at least 80%, 90%, 95%, and in
some embodiments, 99%. In particular, conservative amino acid
replacements are contemplated. Conservative replacements are those
that take place within a family of amino acids that are related in
their side chains. Genetically encoded amino acids are generally
divided into families: (1) acidic amino acids are aspartate,
glutamate; (2) basic amino acids are lysine, arginine, histidine;
(3) non-polar amino acids are alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan, and (4) uncharged
polar amino acids are glycine, asparagine, glutamine, cysteine,
serine, threonine, tyrosine. The hydrophilic amino acids include
arginine, asparagine, aspartate, glutamine, glutamate, histidine,
lysine, serine, and threonine. The hydrophobic amino acids include
alanine, cysteine, isoleucine, leucine, methionine, phenylalanine,
proline, tryptophan, tyrosine and valine. Other families of amino
acids include (i) serine and threonine, which are the
aliphatic-hydroxy family; (ii) asparagine and glutamine, which are
the amide containing family; (iii) alanine, valine, leucine and
isoleucine, which are the aliphatic family; and (iv) phenylalanine,
tryptophan, and tyrosine, which are the aromatic family. For
example, it is reasonable to expect that an isolated replacement of
a leucine with an isoleucine or valine, an aspartate with a
glutamate, a threonine with a serine, or a similar replacement of
an amino acid with a structurally related amino acid will not have
a major effect on the binding or properties of the resulting
molecule, especially if the replacement does not involve an amino
acid within a framework site. Whether an amino acid change results
in a functional peptide can readily be determined by assaying the
specific activity of the polypeptide derivative. Assays are
described in detail herein. Fragments or analogs of antibodies or
immunoglobulin molecules can be readily prepared by those of
ordinary skill in the art. Suitable amino- and carboxy-termini of
fragments or analogs occur near boundaries of functional domains.
Structural and functional domains can be identified by comparison
of the nucleotide and/or amino acid sequence data to public or
proprietary sequence databases. In some embodiments, computerized
comparison methods are used to identify sequence motifs or
predicted protein conformation domains that occur in other proteins
of known structure and/or function. Methods to identify protein
sequences that fold into a known three-dimensional structure are
known. Bowie et al. Science 253:164 (1991). Thus, the foregoing
examples demonstrate that those of skill in the art can recognize
sequence motifs and structural conformations that may be used to
define structural and functional domains in accordance with the
invention.
[0304] Suitable amino acid substitutions are those which: (1)
reduce susceptibility to proteolysis, (2) reduce susceptibility to
oxidation, (3) alter binding affinity for forming protein
complexes, (4) alter binding affinities, and (4) confer or modify
other physicochemical or functional properties of such analogs.
Analogs can include various muteins of a sequence other than the
naturally-occurring peptide sequence. For example, single or
multiple amino acid substitutions (for example, conservative amino
acid substitutions) may be made in the naturally-occurring sequence
(for example, in the portion of the polypeptide outside the
domain(s) forming intermolecular contacts. A conservative amino
acid substitution should not substantially change the structural
characteristics of the parent sequence (e.g., a replacement amino
acid should not tend to break a helix that occurs in the parent
sequence, or disrupt other types of secondary structure that
characterizes the parent sequence). Examples of art-recognized
polypeptide secondary and tertiary structures are described in
Proteins, Structures and Molecular Principles (Creighton, Ed., W.
H. Freeman and Company, New York (1984)); Introduction to Protein
Structure (C. Branden and J. Tooze, eds., Garland Publishing, New
York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991).
[0305] The term "polypeptide fragment" as used herein refers to a
polypeptide that has an amino terminal and/or carboxy-terminal
deletion and/or one or more internal deletion(s), but where the
remaining amino acid sequence is identical to the corresponding
positions in the naturally-occurring sequence deduced, for example,
from a full length cDNA sequence. Fragments typically are at least
5, 6, 8 or 10 amino acids long, in some embodiments, at least 14
amino acids long, in some embodiments, at least 20 amino acids
long, usually at least 50 amino acids long, and in some
embodiments, at least 70 amino acids long. The term "analog" as
used herein refers to polypeptides which are comprised of a segment
of at least 25 amino acids that has substantial identity to a
portion of a deduced amino acid sequence and which has specific
binding to IL-6R, under suitable binding conditions. Typically,
polypeptide analogs comprise a conservative amino acid substitution
(or addition or deletion) with respect to the naturally-occurring
sequence. Analogs typically are at least 20 amino acids long, in
some embodiments, at least 50 amino acids long or longer, and can
often be as long as a full-length naturally-occurring
polypeptide.
[0306] The term "agent" is used herein to denote a chemical
compound, a mixture of chemical compounds, a biological
macromolecule, or an extract made from biological materials.
[0307] As used herein, the terms "label" or "labeled" refers to
incorporation of a detectable marker, e.g., by incorporation of a
radiolabeled amino acid or attachment to a polypeptide of biotinyl
moieties that can be detected by marked avidin (e.g., streptavidin
containing a fluorescent marker or enzymatic activity that can be
detected by optical or calorimetric methods). In certain
situations, the label or marker can also be therapeutic. Various
methods of labeling polypeptides and glycoproteins are known in the
art and may be used. Examples of labels for polypeptides include,
but are not limited to, the following: radioisotopes or
radionuclides (e.g. .sup.3H, .sup.14C, .sup.15N, .sup.35S,
.sup.90Y, .sup.99Tc, .sup.111In, .sup.125I, .sup.131I), fluorescent
labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic
labels (e.g., horseradish peroxidase, p-galactosidase, luciferase,
alkaline phosphatase), chemiluminescent, biotinyl groups,
predetermined polypeptide epitopes recognized by a secondary
reporter (e.g., leucine zipper pair sequences, binding sites for
secondary antibodies, metal binding domains, epitope tags). In some
embodiments, labels are attached by spacer arms of various lengths
to reduce potential steric hindrance. The term "pharmaceutical
agent or drug" as used herein refers to a chemical compound or
composition capable of inducing a desired therapeutic effect when
properly administered to a patient.
[0308] Other chemistry terms herein are used according to
conventional usage in the art, as exemplified by The McGraw-Hill
Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San
Francisco (1985)).
[0309] As used herein, "substantially pure" means an object species
is the predominant species present (i.e., on a molar basis it is
more abundant than any other individual species in the
composition), and in some embodiments, a substantially purified
fraction is a composition wherein the object species comprises at
least about 50 percent (on a molar basis) of all macromolecular
species present.
[0310] Generally, a substantially pure composition will comprise
more than about 80 percent of all macromolecular species present in
the composition, in some embodiments, more than about 85%, 90%,
95%, and 99%. In some embodiments, the object species is purified
to essential homogeneity (contaminant species cannot be detected in
the composition by conventional detection methods) wherein the
composition consists essentially of a single macromolecular
species.
[0311] The term patient includes human and veterinary subjects.
[0312] Antibodies
[0313] Activatable antibodies of the invention specifically bind
human interleukin-6 receptor (IL-6R). Also included in the
invention are activatable antibodies that bind to the same epitope
as the activatable anti-IL-6R antibodies described herein.
[0314] Those skilled in the art will recognize that it is possible
to determine, without undue experimentation, if a monoclonal
antibody (e.g., a murine monoclonal or humanized antibody) has the
same specificity as a monoclonal antibody used in the methods
described herein by ascertaining whether the former prevents the
latter from binding to IL-6R. If the monoclonal antibody being
tested competes with the monoclonal antibody of the invention, as
shown by a decrease in binding by the monoclonal antibody of the
invention, then the two monoclonal antibodies bind to the same, or
a closely related, epitope. An alternative method for determining
whether a monoclonal antibody has the specificity of a monoclonal
antibody of the invention is to pre-incubate the monoclonal
antibody of the invention with IL-6R and then add the monoclonal
antibody being tested to determine if the monoclonal antibody being
tested is inhibited in its ability to bind IL-6R. If the monoclonal
antibody being tested is inhibited then, in all likelihood, it has
the same, or functionally equivalent, epitopic specificity as the
monoclonal antibody of the invention.
[0315] Activatable Anti-IL-6R Antibodies Having Non-Binding Steric
Moieties or Binding Partners for Non-Binding Steric Moieties
[0316] The invention also provides activatable anti-IL-6R
antibodies that include non-binding steric moieties (NB) or binding
partners (BP) for non-binding steric moieties, where the BP
recruits or otherwise attracts the NB to the activatable anti-IL-6R
antibody. The activatable anti-IL-6R antibodies provided herein
include, for example, an activatable anti-IL-6R antibody that
includes a non-binding steric moiety (NB), a cleavable linker (CL)
and antibody or antibody fragment (AB) that binds IL-6R; an
activatable antibody that includes a binding partner for a
non-binding steric moiety (BP), a CL and an AB; and an activatable
anti-IL-6R antibody that includes a BP to which an NB has been
recruited, a CL and an AB that binds IL-6R. Activatable antibodies
in which the NB is covalently linked to the CL and AB of the
activatable anti-IL-6R antibody or is associated by interaction
with a BP that is covalently linked to the CL and AB of the
activatable anti-IL-6R antibody are referred to herein as
"NB-containing activatable anti-IL-6R antibodies." By activatable
or switchable is meant that the activatable antibody exhibits a
first level of binding to a target, i.e., IL-6R, when the
activatable antibody is in an inhibited, masked or uncleaved state
(i.e., a first conformation), and a second level of binding to the
target when the activatable antibody is in an uninhibited, unmasked
and/or cleaved state (i.e., a second conformation, i.e., activated
antibody), where the second level of target binding is greater than
the first level of target binding. The activatable antibody
compositions can exhibit increased bioavailability and more
favorable biodistribution compared to conventional antibody
therapeutics.
[0317] In some embodiments, activatable antibodies provide for
reduced toxicity and/or adverse side effects that could otherwise
result from binding of the anti-IL-6R AB at non-treatment sites
and/or non-diagnostic sites if the anti-IL-6R AB were not masked or
otherwise inhibited from binding to such a site.
[0318] In one embodiment, the activatable antibody includes a
non-binding steric moiety (NB); a cleavable linker (CL); and an
antibody or antibody fragment (AB) that binds specifically to
IL-6R, wherein the NB is a polypeptide that does not bind
specifically to the AB; the CL is a polypeptide that includes a
substrate (S) for an enzyme; the CL is positioned such that in an
uncleaved state, the NB interferes with binding of the AB to IL-6R
and in a cleaved state, the NB does not interfere with binding of
the AB to IL-6R; and the NB does not inhibit cleavage of the CL by
the enzyme. As used herein and throughout, the term polypeptide
refers to any polypeptide that includes at least two amino acid
residues, including larger polypeptides, full-length proteins and
fragments thereof, and the term polypeptide is not limited to
single-chain polypeptides and can include multi-unit, e.g.,
multi-chain, polypeptides. In cases where the polypeptide is of a
shorter length, for example, less than 50 amino acids total, the
terms peptide and polypeptide are used interchangeably herein, and
in cases where the polypeptide is of a longer length, e.g., 50
amino acids or greater, the terms polypeptide and protein are used
interchangeably herein.
[0319] In one embodiment, the activatable antibody includes a
non-binding steric moiety (NB); a cleavable linker (CL); and an
antibody or antibody fragment (AB) that binds specifically to
IL-6R, wherein (i) the NB includes a polypeptide that does not bind
specifically to the AB; (ii) CL is a polypeptide of up to 50 amino
acids in length that includes a substrate (S) for an enzyme; (iii)
the CL is positioned such that in an uncleaved state, the NB
interferes with binding of the AB to IL-6R and in a cleaved state,
the NB does not interfere with binding of the AB to IL-6R; and (iv)
the NB does not inhibit cleavage of the CL by the enzyme. For
example, the CL has a length of up to 15 amino acids, a length of
up to 20 amino acids, a length of up to 25 amino acids, a length of
up to 30 amino acids, a length of up to 35 amino acids, a length of
up to 40 amino acids, a length of up to 45 amino acids, a length of
up to 50 amino acids, a length in the range of 10-50 amino acids, a
length in the range of 15-50 amino acids, a length in the range of
20-50 amino acids, a length in the range of 25-50 amino acids, a
length in the range of 30-50 amino acids, a length in the range of
35-50 amino acids, a length in the range of 40-50 amino acids, a
length in the range of 45-50 amino acids, a length in the range of
10-40 amino acids, a length in the range of 15-40 amino acids, a
length in the range of 20-40 amino acids, a length in the range of
25-40 amino acids, a length in the range of 30-40 amino acids, a
length in the range of 35-40 amino acids, a length in the range of
10-30 amino acids, a length in the range of 15-30 amino acids, a
length in the range of 20-30 amino acids, a length in the range of
25-30 amino acids, a length in the range of 10-20 amino acids, or a
length in the range of 10-15 amino acids.
[0320] In one embodiment, the activatable antibody includes a
non-binding steric moiety (NB); a cleavable linker (CL); and an
antibody or antibody fragment (AB) that binds specifically to
IL-6R, wherein (i) the NB includes a polypeptide that does not bind
specifically to the AB; (ii) the CL is a polypeptide that includes
a substrate (S) for an enzyme; (iii) the CL is positioned such that
in an uncleaved state, the NB interferes with binding of the AB to
IL-6R and in a cleaved state, the NB does not interfere with
binding of the AB to IL-6R; (iv) the NB does not inhibit cleavage
of the CL by the enzyme; and (v) the activatable antibody has the
structural arrangement from N-terminus to C-terminus as follows in
the uncleaved state: NB-CL-AB or AB-CL-NB.
[0321] In one embodiment, the activatable antibody includes a
non-binding steric moiety (NB); a cleavable linker (CL); and an
antibody or antibody fragment (AB) that binds specifically to
IL-6R, wherein (i) the NB includes a polypeptide that does not bind
specifically to the AB; (ii) the CL is a polypeptide that includes
a substrate (S) for an enzyme; (iii) the CL is positioned such that
in an uncleaved state, the NB interferes with binding of the AB to
IL-6R and in a cleaved state, the NB does not interfere with
binding of the AB to IL-6R, and wherein the NB in the uncleaved
activatable antibody reduces the ability of the AB to bind IL-6R by
at least 50%, for example, by at least 60%, by at least 70%, by at
least 75%, by at least 80%, by at least 85%, by at least 90%, by at
least 95%, by at least 96%, by at least 97%, by at least 98%, by at
least 99%, by at least 100% as compared to the ability of the
cleaved AB to bind IL-6R; and (iv) the NB does not inhibit cleavage
of the CL by the enzyme. The reduction in the ability of the AB to
bind IL-6R is determined, e.g., using an assay as described herein
or an in vitro target displacement assay such as, for example, the
assay described in PCT Publication Nos. WO 2009/025846 and WO
2010/081173.
[0322] In one embodiment, the activatable antibody includes a
binding partner (BP) for a non-binding steric moiety (NB); a
cleavable linker (CL); and an antibody or antibody fragment (AB)
that binds specifically to IL-6R, wherein the BP is a polypeptide
that binds to the NB when exposed thereto; the NB does not bind
specifically to the AB; the CL is a polypeptide that includes a
substrate (S) for an enzyme; the CL is positioned such that in an
uncleaved state in the presence of the NB, the NB interferes with
binding of the AB to IL-6R and in a cleaved state, the NB does not
interfere with binding of the AB to IL-6R and the BP does not
interfere with binding of the AB to IL-6R; and the NB and the BP do
not inhibit cleavage of the CL by the enzyme. In some examples of
this embodiment, the BP of the activatable antibody is optionally
bound to the NB. In one embodiment, the NB is recruited by the BP
of the activatable antibody in vivo.
[0323] In some examples of any of these activatable anti-IL-6R
antibody embodiments, the activatable anti-IL-6R antibody is
formulated as a composition. In some of these embodiments, the
composition also includes the NB, where the NB is co-formulated
with the activatable anti-IL-6R antibody that includes the BP, the
CL, and the AB. In some examples of this embodiment, the BP is
selected from the group consisting of an albumin binding peptide, a
fibrinogen binding peptide, a fibronectin binding peptide, a
hemoglobin binding peptide, a transferrin binding peptide, an
immunoglobulin domain binding peptide, and other serum protein
binding peptides.
[0324] In some examples of any of these activatable anti-IL-6R
antibody embodiments, the NB is a soluble, globular protein. In
some examples of any of these activatable anti-IL-6R antibody
embodiments, the NB is a protein that circulates in the
bloodstream. In some examples of any of these activatable
anti-IL-6R antibody embodiments, the NB is selected from the group
consisting of albumin, fibrinogen, fibronectin, hemoglobin,
transferrin, an immunoglobulin domain, and other serum
proteins.
[0325] In some examples of any of these activatable anti-IL-6R
antibody embodiments, the CL is a polypeptide that includes a
substrate (S) for a protease. In some examples of any of these
activatable anti-IL-6R antibody embodiments, the protease is
co-localized with IL-6R in a tissue, and the protease cleaves the
CL in the activatable anti-IL-6R antibody when the activatable
antibody is exposed to the protease. In some examples of any of
these activatable anti-IL-6R antibody embodiments, the CL is a
polypeptide of up to 50 amino acids in length. In some examples of
any of these activatable anti-IL-6R antibody embodiments, the CL is
a polypeptide that includes a substrate (S) having a length of up
to 15 amino acids, e.g., 3 amino acids long, 4 amino acids long, 5
amino acids long, 6 amino acids long, 7 amino acids long, 8 amino
acids long, 9 amino acids long, 10 amino acids long, 11 amino acids
long, 12 amino acids long, 13 amino acids long, 14 amino acids
long, or 15 amino acids long.
[0326] In some examples of any of these activatable anti-IL-6R
antibody embodiments, the activatable antibody has the structural
arrangement from N-terminus to C-terminus as follows in the
uncleaved state: NB-CL-AB, AB-CL-NB, BP-CL-AB or AB-CL-BP. In
embodiments where the activatable anti-IL-6R antibody includes a BP
and the activatable antibody is in the presence of the
corresponding NB, the activatable antibody has a structural
arrangement from N-terminus to C-terminus as follows in the
uncleaved state: NB:BP-CM-AB or AB-CM-BP:NB, where ":" represents
an interaction, e.g., binding, between the NB and BP.
[0327] In some examples of any of these activatable anti-IL-6R
antibody embodiments, the activatable antibody includes an antibody
or antigen-binding fragment thereof that specifically binds IL-6R
and is a monoclonal antibody, domain antibody, single chain, Fab
fragment, a F(ab')2 fragment, a scFv, a scab, a dAb, a single
domain heavy chain antibody, or a single domain light chain
antibody. In some embodiments, such an antibody or immunologically
active fragment thereof that binds IL-6R is a mouse, chimeric,
humanized or fully human monoclonal antibody.
[0328] In some examples of any of these activatable anti-IL-6R
antibody embodiments, the activatable antibody includes a
combination of a variable heavy chain region comprising the amino
acid sequence of SEQ ID NO: 1 and a variable light chain region
comprising the amino acid sequence of SEQ ID NO: 2. In some
embodiments, the activatable antibody includes a combination of a
variable heavy chain region comprising an amino acid sequence that
is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
more identical to the amino acid sequence of SEQ ID NO: 1, and a
variable light chain region comprising an amino acid sequence that
is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
more identical to the amino acid sequence of SEQ ID NO: 2.
[0329] In some examples of any of these activatable anti-IL-6R
antibody embodiments, the activatable antibody also includes an
agent conjugated to the AB. In some embodiments, the agent is a
therapeutic agent. In some embodiments, the agent is an
antineoplastic agent. In some embodiments, the agent is a toxin or
fragment thereof. In some embodiments, the agent is conjugated to
the AB via a linker. In some embodiments, the linker is a cleavable
linker. In some embodiments, the agent is an agent selected from
the group listed in Table 30. In some embodiments, the agent is a
dolastatin. In some embodiments, the agent is an auristatin or
derivative thereof. In some embodiments, the agent is auristatin E
or a derivative thereof. In some embodiments, the agent is
monomethyl auristatin E (MMAE). In some embodiments, the agent is a
maytansinoid or maytansinoid derivative. In some embodiments, the
agent is DM1 or DM4. In some embodiments, the agent is a
duocarmycin or derivative thereof. In some embodiments, the agent
is a calicheamicin or derivative thereof.
[0330] In some examples of any of these activatable anti-IL-6R
antibody embodiments, the activatable antibody also includes a
detectable moiety. In some embodiments, the detectable moiety is a
diagnostic agent.
[0331] In some examples of any of these activatable anti-IL-6R
antibody embodiments, the activatable antibody also includes a
spacer. In some examples of any of these activatable anti-IL-6R
antibody embodiments, the activatable antibody also includes a
signal peptide. In some embodiments, the signal peptide is
conjugated to the activatable antibody via a spacer. In some
examples of any of these activatable anti-IL-6R antibody
embodiments, the spacer is joined directly to the MM of the
activatable antibody.
[0332] In some examples of any of these activatable anti-IL-6R
antibody embodiments, the serum half-life of the activatable
antibody is at least 5 days when administered to an organism. In
some embodiments, the serum half-life of the activatable antibody
is at least 4 days when administered to an organism. In some
embodiments, the serum half-life of the activatable antibody is at
least 3 days when administered to an organism. In some embodiments,
the serum half-life of the activatable antibody is at least 2 days
when administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 24 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 20 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 18 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 16 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 14 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 12 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 10 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 8 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 6 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 4 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 3 hours when
administered to an organism.
[0333] The invention also provides an isolated nucleic acid
molecule encoding any of these activatable anti-IL-6R antibodies,
as well as vectors that include these isolated nucleic acid
sequences. The invention provides methods of producing an
activatable antibody by culturing a cell under conditions that lead
to expression of the activatable antibody, wherein the cell
comprises such a nucleic acid sequence. In some embodiments, the
cell comprises such a vector.
[0334] The dissociation constant (K.sub.d) of the NB-containing
activatable antibody toward the target is greater than the K.sub.d
of the AB towards the target when it is not associated with the NB
or NB:BP. The dissociation constant (K.sub.d) of the NB-containing
activatable antibody toward the target is greater than the K.sub.d
of the parental AB towards the target. For example, the K.sub.d of
the NB-containing activatable antibody toward the target is at
least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000,
50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000,
50,000,000 or greater, or between 5-10, 10-100, 10-1,000,
10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000,
100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000,
1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000,
10,000-100,000, 10,000-1,000,000, 10,000-10,000,000,
100,000-1,000,000, or 100,000-10,000,000 times greater than the
K.sub.d of the AB when it is not associated with the NB or NB:BP or
the K.sub.d of the parental AB towards the target. Conversely, the
binding affinity of the NB-containing activatable antibody towards
the target is lower than the binding affinity of the AB when it is
not associated with the NB or NB:BP or lower than the binding
affinity of the parental AB towards the target. For example, the
binding affinity of the NB-containing activatable antibody toward
the target is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500,
5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000,
10,000,000, 50,000,000 or greater, or between 5-10, 10-100,
10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000,
100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000,
1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000,
10,000-100,000, 10,000-1,000,000, 10,000-10,000,000,
100,000-1,000,000, or 100,000-10,000,000 times lower than the
binding affinity of the AB when it is not associated with the NB or
NB:BP or lower than the binding affinity of the parental AB towards
the target.
[0335] When the NB-containing activatable antibody is in the
presence of IL-6R, specific binding of the AB to IL-6R is reduced
or inhibited, as compared to the specific binding of the AB when it
is not associated with the NB or NB:BP. When the NB-containing
activatable antibody is in the presence of IL-6R, specific binding
of the AB to IL-6R is reduced or inhibited, as compared to the
specific binding of the parental AB to IL-6R. When compared to the
binding of the AB not associated with an NB or NB:BP or the binding
of the parental AB to IL-6R, the ability of the NB-containing
activatable antibody to bind IL-6R is reduced, for example, by at
least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48,
60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or
180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or
longer when measured in vitro and/or in vivo.
[0336] When the NB-containing activatable antibody is in the
presence of IL-6R but not in the presence of a modifying agent (for
example a protease or other enzyme), specific binding of the AB to
IL-6R is reduced or inhibited, as compared to the specific binding
of the AB when it is not associated with the NB or NB:BP. When the
NB-containing activatable antibody is in the presence of IL-6R but
not in the presence of a modifying agent (for example a protease,
other enzyme, reduction agent, or light), specific binding of the
AB to IL-6R is reduced or inhibited, as compared to the specific
binding of the parental AB to IL-6R. When compared to the binding
of the AB not associated with an NB or NB:BP or the binding of the
parental AB to IL-6R, the ability of the NB-containing activatable
antibody to bind IL-6R is reduced, for example, by at least 50%,
60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even
100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84,
or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days,
or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer when
measured in vitro and/or in vivo.
[0337] In some examples of any of these activatable antibody
embodiments, the activatable antibody includes an agent conjugated
to the AB to produce an activatable antibody conjugate. In some
embodiments of the activatable antibody conjugate, the agent is a
therapeutic agent. In some embodiments, the agent is a diagnostic
agent. In some embodiments, the agent is a detectable marker. In
some embodiments of the activatable antibody conjugate, the agent
is an antineoplastic agent. In some embodiments of the activatable
antibody conjugate, the agent is a toxin or fragment thereof. In
some embodiments of the activatable antibody conjugate, the agent
is conjugated to the AB via a linker. In some embodiments of the
activatable antibody conjugate, the linker is a cleavable linker.
In some embodiments, the agent is an agent selected from the group
listed in Table 30. In some embodiments, the agent is a dolastatin.
In some embodiments, the agent is an auristatin or derivative
thereof. In some embodiments, the agent is auristatin E or a
derivative thereof. In some embodiments, the agent is monomethyl
auristatin E (MMAE). In some embodiments, the agent is a
maytansinoid or maytansinoid derivative. In some embodiments, the
agent is DM1 or DM4. In some embodiments, the agent is a
duocarmycin or derivative thereof. In some embodiments, the agent
is a calicheamicin or derivative thereof.
[0338] In some examples of any of these activatable antibody
embodiments, the activatable antibodies are dual-target binding
activatable antibodies. Such dual target binding activatable
antibodies contain two Abs that may bind the same or different
targets. In specific embodiments, dual-targeting activatable
antibodies contain bispecific antibodies or antibody fragments.
[0339] Dual target binding activatable antibodies are designed so
as to have a CL cleavable by a cleaving agent that is co-localized
in a target tissue with one or both of the targets capable of
binding to the ABs of the activatable antibodies. Dual target
binding activatable antibodies with more than one AB to the same or
different targets can be designed so as to have more than one CL,
wherein the first CL is cleavable by a cleaving agent in a first
target tissue and wherein the second CL is cleavable by a cleaving
agent in a second target tissue, with one or more of the targets
binding to the ABs of the activatable antibodies. In one
embodiment, the first and second target tissues are spatially
separated, for example, at different sites in the organism. In one
embodiment, the first and second target tissues are the same tissue
temporally separated, for example the same tissue at two different
points in time, for example the first time point is when the tissue
is an early stage tumor, and the second time point is when the
tissue is a late stage tumor.
[0340] The invention also provides nucleic acid molecules encoding
the activatable antibodies described herein. The invention also
provides vectors that include these nucleic acids. The activatable
antibodies described herein are produced by culturing a cell under
conditions that lead to expression of the activatable antibody,
wherein the cell includes these nucleic acid molecules or
vectors.
[0341] The invention also provides methods of manufacturing
activatable antibodies. In one embodiment, the method includes the
steps of (a) culturing a cell that includes a nucleic acid
construct that encodes the activatable antibody under conditions
that lead to expression of the activatable antibody, wherein the
activatable antibody includes (i) a non-binding steric moiety (NB);
(ii) a cleavable linker (CL); and (iii) an antibody or an antigen
binding fragment thereof (AB) that specifically binds a target,
wherein (1) the NB does not bind specifically to the AB; (2) the CL
is a polypeptide that includes a substrate (S) for an enzyme; (3)
the CL is positioned such that in an uncleaved state, the NB
interferes with binding of the AB to the target and in a cleaved
state, the NB does not interfere with binding of the AB to the
target; and (4) the NB does not inhibit cleavage of the CL by the
enzyme; and (b) recovering the activatable antibody.
[0342] In another embodiment, the method includes the steps of (a)
culturing a cell that includes a nucleic acid construct that
encodes the activatable antibody under conditions that lead to
expression of the activatable antibody, wherein the activatable
antibody includes (i) a binding partner (BP) for a non-binding
steric moiety (NB); (ii) a cleavable linker (CL); and (iii) an
antibody or an antigen binding fragment thereof (AB) that
specifically binds a target, wherein (1) the NB does not bind
specifically to the AB; (2) the CL is a polypeptide that includes a
substrate (S) for an enzyme; (3) the CL is positioned such that in
an uncleaved state in the presence of the NB, the NB interferes
with binding of the AB to the target and in a cleaved state, the NB
does not interfere with binding of the AB to the target and the BP
does not interfere with binding of the AB to the target; and (4)
the NB and the BP do not inhibit cleavage of the CL by the enzyme;
and (b) recovering the activatable antibody. In some examples of
this embodiment, the BP of the activatable antibody is bound to the
NB.
[0343] Use of Activatable Anti-IL-6R Antibodies
[0344] It will be appreciated that administration of therapeutic
entities in accordance with the invention will be administered with
suitable carriers, excipients, and other agents that are
incorporated into formulations to provide improved transfer,
delivery, tolerance, and the like. A multitude of appropriate
formulations can be found in the formulary known to all
pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th
ed, Mack Publishing Company, Easton, Pa. (1975)), particularly
Chapter 87 by Blaug, Seymour, therein. These formulations include,
for example, powders, pastes, ointments, jellies, waxes, oils,
lipids, lipid (cationic or anionic) containing vesicles (such as
Lipofectin.TM.), DNA conjugates, anhydrous absorption pastes,
oil-in-water and water-in-oil emulsions, emulsions carbowax
(polyethylene glycols of various molecular weights), semi-solid
gels, and semi-solid mixtures containing carbowax. Any of the
foregoing mixtures may be appropriate in treatments and therapies
in accordance with the present invention, provided that the active
ingredient in the formulation is not inactivated by the formulation
and the formulation is physiologically compatible and tolerable
with the route of administration. See also Baldrick P.
"Pharmaceutical excipient development: the need for preclinical
guidance." Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W.
"Lyophilization and development of solid protein pharmaceuticals."
Int. J. Pharm. 203(1-2):1-60 (2000), Charman W N "Lipids,
lipophilic drugs, and oral drug delivery-some emerging concepts." J
Pharm Sci. 89(8):967-78 (2000), Powell et al. "Compendium of
excipients for parenteral formulations" PDA J Pharm Sci Technol.
52:238-311 (1998) and the citations therein for additional
information related to formulations, excipients and carriers well
known to pharmaceutical chemists.
[0345] Therapeutic formulations of the invention, which include an
activatable anti-IL-6R antibody, are used to prevent, treat or
otherwise ameliorate a disease or disorder associated with aberrant
IL-6R expression and/or activity. For example, therapeutic
formulations of the invention, which include an activatable
anti-IL-6R antibody, are used to treat or otherwise ameliorate
inflammation, an inflammatory disorder, an autoimmune disease
and/or a cancer or other neoplastic condition.
[0346] In some embodiments, the inflammation is associated with
and/or the inflammatory disorder is Crohn's disease. In some
embodiments, the inflammation is associated with and/or the
inflammatory disorder is polychondritis, including but not limited
to, relapsing polychondritis. In some embodiments, the inflammation
is associated with and/or the inflammatory disorder is rheumatoid
arthritis (RA). In some embodiments, the inflammation is associated
with and/or the inflammatory disorder is another rheumatoid
disease, such as, by way of non-limiting example, ankylosing
spondylitis, juvenile arthritis, and/or psoriatic arthritis. In
some embodiments, the inflammation is associated with and/or the
inflammatory disorder is ulcerative colitis.
[0347] In some embodiments, the cancer is breast cancer, including
but not limited to, triple negative breast cancer (TNBC). In some
embodiments, the cancer is Castleman's disease. In some
embodiments, the cancer is hepatocellular carcinoma. In some
embodiments, the cancer is lung cancer. In some embodiments, the
cancer is multiple myeloma. In some embodiments, the cancer is
ovarian cancer. In some embodiments, the cancer is prostate
cancer.
[0348] Efficaciousness of prevention, amelioration or treatment is
determined in association with any known method for diagnosing or
treating the disease or disorder associated with aberrant IL-6R
expression and/or activity. Prolonging the survival of a subject or
otherwise delaying the progression of the disease or disorder
associated with aberrant IL-6R expression and/or activity in a
subject indicates that the activatable antibody confers a clinical
benefit.
[0349] Activatable anti-IL-6R antibodies can be administered in the
form of pharmaceutical compositions. Principles and considerations
involved in preparing such compositions, as well as guidance in the
choice of components are provided, for example, in Remington: The
Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et
al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption
Enhancement: Concepts, Possibilities, Limitations, And Trends,
Harwood Academic Publishers, Langhorne, Pa., 1994;
[0350] and Peptide And Protein Drug Delivery (Advances In
Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.
[0351] In some embodiments where activatable antibody fragments are
used, the smallest fragment that specifically binds to the binding
domain of the target protein is selected. For example, based upon
the variable-region sequences of an antibody, peptide molecules can
be designed that retain the ability to bind the target protein
sequence. Such peptides can be synthesized chemically and/or
produced by recombinant DNA technology. (See, e.g., Marasco et al.,
Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation
can also contain more than one active compounds as necessary for
the particular indication being treated, for example, in some
embodiments, those with complementary activities that do not
adversely affect each other. Alternatively, or in addition, the
composition can comprise an agent that enhances its function, such
as, for example, a cytotoxic agent, cytokine, chemotherapeutic
agent, or growth-inhibitory agent. Such molecules are suitably
present in combination in amounts that are effective for the
purpose intended.
[0352] The active ingredients can also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacrylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles, and nanocapsules) or in macroemulsions.
[0353] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0354] Sustained-release preparations can be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods.
[0355] In some embodiments, the activatable antibody contains a
detectable label. An intact antibody, or a fragment thereof (e.g.,
Fab, scFv, or F(ab).sub.2) is used. The term "labeled", with regard
to the probe or antibody, is intended to encompass direct labeling
of the probe or antibody by coupling (i.e., physically linking) a
detectable substance to the probe or antibody, as well as indirect
labeling of the probe or antibody by reactivity with another
reagent that is directly labeled. Examples of indirect labeling
include detection of a primary antibody using a
fluorescently-labeled secondary antibody and end-labeling of a DNA
probe with biotin such that it can be detected with
fluorescently-labeled streptavidin. The term "biological sample" is
intended to include tissues, cells and biological fluids isolated
from a subject, as well as tissues, cells and fluids present within
a subject. Included within the usage of the term "biological
sample", therefore, is blood and a fraction or component of blood
including blood serum, blood plasma, or lymph. That is, the
detection method of the invention can be used to detect an analyte
mRNA, protein, or genomic DNA in a biological sample in vitro as
well as in vivo. For example, in vitro techniques for detection of
an analyte mRNA include Northern hybridizations and in situ
hybridizations. In vitro techniques for detection of an analyte
protein include enzyme linked immunosorbent assays (ELISAs),
Western blots, immunoprecipitations, immunochemical staining, and
immunofluorescence. In vitro techniques for detection of an analyte
genomic DNA include Southern hybridizations. Procedures for
conducting immunoassays are described, for example in "ELISA:
Theory and Practice: Methods in Molecular Biology", Vol. 42, J. R.
Crowther (Ed.) Human Press, Totowa, N.J., 1995; "Immunoassay", E.
Diamandis and T. Christopoulus, Academic Press, Inc., San Diego,
Calif., 1996; and "Practice and Theory of Enzyme Immunoassays", P.
Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore,
in vivo techniques for detection of an analyte protein include
introducing into a subject a labeled anti-analyte protein antibody.
For example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques.
[0356] The activatable anti-IL-6R antibodies of the invention are
also useful in a variety of diagnostic and prophylactic
formulations. In one embodiment, an anti-IL-6R antibody and/or
activatable anti-IL-6R antibody is administered to patients that
are at risk of developing one or more of the aforementioned cancer
or fibrotic disorders. A patient's or organ's predisposition to one
or more of the aforementioned disorders can be determined using
genotypic, serological or biochemical markers.
[0357] In another embodiment of the invention, an anti-IL-6R
antibody and/or activatable anti-IL-6R antibody is administered to
human individuals diagnosed with a clinical indication associated
with one or more of the aforementioned disorders. Upon diagnosis,
an anti-IL-6R antibody and/or activatable anti-IL-6R antibody is
administered to mitigate or reverse the effects of the clinical
indication.
[0358] Antibodies and/or activatable antibodies of the invention
are also useful in the detection of IL-6R in patient samples and
accordingly are useful as diagnostics. For example, the activatable
anti-IL-6R antibodies of the invention are used in in vitro assays,
e.g., ELISA, to detect IL-6R levels in a patient sample.
[0359] In one embodiment, an anti-IL-6R antibody and/or activatable
anti-IL-6R antibody of the invention is immobilized on a solid
support (e.g., the well(s) of a microtiter plate). The immobilized
antibody and/or activatable antibody serves as a capture antibody
for any IL-6R that may be present in a test sample. Prior to
contacting the immobilized antibody with a patient sample, the
solid support is rinsed and treated with a blocking agent such as
milk protein or albumin to prevent nonspecific adsorption of the
analyte.
[0360] Subsequently the wells are treated with a test sample
suspected of containing the antigen, or with a solution containing
a standard amount of the antigen. Such a sample is, e.g., a serum
sample from a subject suspected of having levels of circulating
antigen considered to be diagnostic of a pathology. After rinsing
away the test sample or standard, the solid support is treated with
a second antibody that is detectably labeled. The labeled second
antibody serves as a detecting antibody. The level of detectable
label is measured, and the concentration of IL-6R antigen in the
test sample is determined by comparison with a standard curve
developed from the standard samples.
[0361] It will be appreciated that based on the results obtained
using the anti-IL-6R antibodies of the invention in an in vitro
diagnostic assay, it is possible to stage a disease in a subject
based on expression levels of the IL-6R antigen. For a given
disease, samples of blood are taken from subjects diagnosed as
being at various stages in the progression of the disease, and/or
at various points in the therapeutic treatment of the disease.
Using a population of samples that provides statistically
significant results for each stage of progression or therapy, a
range of concentrations of the antigen that may be considered
characteristic of each stage is designated.
[0362] Activatable anti-IL-6R antibodies can also be used in
diagnostic and/or imaging methods. In some embodiments, such
methods are in vitro methods. In some embodiments, such methods are
in vivo methods. In some embodiments, such methods are in situ
methods. In some embodiments, such methods are ex vivo methods. For
example, activatable anti-IL-6R antibodies having an enzymatically
cleavable CM can be used to detect the presence or absence of an
enzyme that is capable of cleaving the CM. Such activatable
anti-IL-6R antibodies can be used in diagnostics, which can include
in vivo detection (e.g., qualitative or quantitative) of enzyme
activity (or, in some embodiments, an environment of increased
reduction potential such as that which can provide for reduction of
a disulfide bond) through measured accumulation of activated
anti-IL-6R antibodies (i.e., antibodies resulting from cleavage of
an activatable anti-IL-6R antibody) in a given cell or tissue of a
given host organism. Such accumulation of activated anti-IL-6R
antibodies indicates not only that the tissue expresses enzymatic
activity (or an increased reduction potential depending on the
nature of the CM) but also that the tissue expresses target to
which the activated antibody binds.
[0363] For example, the CM can be selected to be a protease
substrate for a protease found at the site of a tumor, at the site
of a viral or bacterial infection at a biologically confined site
(e.g., such as in an abscess, in an organ, and the like), and the
like. The AB can be one that binds a target antigen. Using methods
familiar to one skilled in the art, a detectable label (e.g., a
fluorescent label or radioactive label or radiotracer) can be
conjugated to an AB or other region of an anti-IL-6R antibody
and/or activatable anti-IL-6R antibody. Suitable detectable labels
are discussed in the context of the above screening methods and
additional specific examples are provided below. Using an AB
specific to a protein or peptide of the disease state, along with a
protease whose activity is elevated in the disease tissue of
interest, activatable anti-IL-6R antibodies will exhibit an
increased rate of binding to disease tissue relative to tissues
where the CM specific enzyme is not present at a detectable level
or is present at a lower level than in disease tissue or is
inactive (e.g., in zymogen form or in complex with an inhibitor).
Since small proteins and peptides are rapidly cleared from the
blood by the renal filtration system, and because the enzyme
specific for the CM is not present at a detectable level (or is
present at lower levels in non-disease tissues or is present in
inactive conformation), accumulation of activated anti-IL-6R
antibodies in the disease tissue is enhanced relative to
non-disease tissues.
[0364] In another example, activatable anti-IL-6R antibodies can be
used to detect the presence or absence of a cleaving agent in a
sample. For example, where the activatable anti-IL-6R antibodies
contain a CM susceptible to cleavage by an enzyme, the activatable
anti-IL-6R antibodies can be used to detect (either qualitatively
or quantitatively) the presence of an enzyme in the sample. In
another example, where the activatable anti-IL-6R antibodies
contain a CM susceptible to cleavage by reducing agent, the
activatable anti-IL-6R antibodies can be used to detect (either
qualitatively or quantitatively) the presence of reducing
conditions in a sample. To facilitate analysis in these methods,
the activatable antibodies can be detectably labeled, and can be
bound to a support (e.g., a solid support, such as a slide or
bead). The detectable label can be positioned on a portion of the
activatable anti-IL-6R antibody that is not released following
cleavage, for example, the detectable label can be a quenched
fluorescent label or other label that is not detectable until
cleavage has occurred. The assay can be conducted by, for example,
contacting the immobilized, detectably labeled activatable
anti-IL-6R antibodies with a sample suspected of containing an
enzyme and/or reducing agent for a time sufficient for cleavage to
occur, then washing to remove excess sample and contaminants. The
presence or absence of the cleaving agent (e.g., enzyme or reducing
agent) in the sample is then assessed by a change in detectable
signal of the activatable anti-IL-6R antibodies prior to contacting
with the sample e.g., the presence of and/or an increase in
detectable signal due to cleavage of the activatable antibody by
the cleaving agent in the sample.
[0365] Such detection methods can be adapted to also provide for
detection of the presence or absence of a target that is capable of
binding the AB of the activatable anti-IL-6R antibodies when
cleaved. Thus, the assays can be adapted to assess the presence or
absence of a cleaving agent and the presence or absence of a target
of interest. The presence or absence of the cleaving agent can be
detected by the presence of and/or an increase in detectable label
of the activatable anti-IL-6R antibodies as described above, and
the presence or absence of the target can be detected by detection
of a target-AB complex e.g., by use of a detectably labeled
anti-target antibody.
[0366] Activatable anti-IL-6R antibodies are also useful in in situ
imaging for the validation of activatable antibody activation,
e.g., by protease cleavage, and binding to a particular target. In
situ imaging is a technique that enables localization of
proteolytic activity and target in biological samples such as cell
cultures or tissue sections. Using this technique, it is possible
to confirm both binding to a given target and proteolytic activity
based on the presence of a detectable label (e.g., a fluorescent
label).
[0367] These techniques are useful with any frozen cells or tissue
derived from a disease site (e.g. tumor tissue) or healthy tissues.
These techniques are also useful with fresh cell or tissue
samples.
[0368] In these techniques, an activatable anti-IL-6R antibody is
labeled with a detectable label. The detectable label may be a
fluorescent dye, (e.g. a fluorophore, Fluorescein Isothiocyanate
(FITC), Rhodamine Isothiocyanate (TRITC), an Alexa Fluor.RTM.
label), a near infrared (NIR) dye (e.g., Qdot.RTM. nanocrystals), a
colloidal metal, a hapten, a radioactive marker, biotin and an
amplification reagent such as streptavidin, or an enzyme (e.g.
horseradish peroxidase or alkaline phosphatase).
[0369] Detection of the label in a sample that has been incubated
with the labeled, activatable anti-IL-6R antibody indicates that
the sample contains the target, i.e., IL-6R, and contains a
protease that is specific for the CM of the activatable anti-IL-6R
antibody. In some embodiments, the presence of the protease can be
confirmed using broad spectrum protease inhibitors such as those
described herein, and/or by using an agent that is specific for the
protease, for example, an antibody such as All, which is specific
for the protease matriptase (MT-SP1) and inhibits the proteolytic
activity of MT-SP1; see e.g., International Publication Number WO
2010/129609, published 11 Nov. 2010. The same approach of using
broad spectrum protease inhibitors such as those described herein,
and/or by using a more selective inhibitory agent can be used to
identify a protease or class of proteases specific for the CM of
the activatable anti-IL-6R antibody. In some embodiments, the
presence of the target can be confirmed using an agent that is
specific for the target, e.g., another anti-IL-6R antibody, or the
detectable label can be competed with unlabeled IL-6R. In some
embodiments, unlabeled activatable anti-IL-6R antibody could be
used, with detection by a labeled secondary antibody or more
complex detection system.
[0370] Similar techniques are also useful for in vivo imaging where
detection of the fluorescent signal in a subject, e.g., a mammal,
including a human, indicates that the disease site contains the
target, i.e., IL-6R, and contains a protease that is specific for
the CM of the activatable anti-IL-6R antibody.
[0371] These techniques are also useful in kits and/or as reagents
for the detection, identification or characterization of protease
activity in a variety of cells, tissues, and organisms based on the
protease-specific CM in the activatable anti-IL-6R antibody.
[0372] The invention provides methods of using the activatable
anti-IL-6R antibodies in a variety of diagnostic and/or
prophylactic indications. For example, the invention provides
methods of detecting presence or absence of a cleaving agent and a
target of interest in a subject or a sample by (i) contacting a
subject or sample with an activatable anti-IL-6R antibody, wherein
the activatable anti-IL-6R antibody comprises a masking moiety
(MM), a cleavable moiety (CM) that is cleaved by the cleaving
agent, and an antigen binding domain or fragment thereof (AB) that
specifically binds the target of interest, wherein the activatable
anti-IL-6R antibody in an uncleaved, non-activated state comprises
a structural arrangement from N-terminus to C-terminus as follows:
MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits
binding of the AB to the IL-6R target, and wherein the MM does not
have an amino acid sequence of a naturally occurring binding
partner of the AB and is not a modified form of a natural binding
partner of the AB; and (b) wherein, in an uncleaved, non-activated
state, the MM interferes with specific binding of the AB to the
IL-6R target, and in a cleaved, activated state the MM does not
interfere or compete with specific binding of the AB to the IL-6R
target; and (ii) measuring a level of activated activatable
anti-IL-6R antibody in the subject or sample, wherein a detectable
level of activated activatable anti-IL-6R antibody in the subject
or sample indicates that the cleaving agent and IL-6R are present
in the subject or sample and wherein no detectable level of
activated activatable anti-IL-6R antibody in the subject or sample
indicates that the cleaving agent, IL-6R or both the cleaving agent
and IL-6R are absent and/or not sufficiently present in the subject
or sample. In some embodiments, the activatable anti-IL-6R antibody
is an activatable anti-IL-6R antibody to which a therapeutic agent
is conjugated. In some embodiments, the activatable anti-IL-6R
antibody is not conjugated to an agent. In some embodiments, the
activatable anti-IL-6R antibody comprises a detectable label. In
some embodiments, the detectable label is positioned on the AB. In
some embodiments, measuring the level of activatable anti-IL-6R
antibody in the subject or sample is accomplished using a secondary
reagent that specifically binds to the activated antibody, wherein
the reagent comprises a detectable label. In some embodiments, the
secondary reagent is an antibody comprising a detectable label.
[0373] The invention also provides methods of detecting presence or
absence of a cleaving agent in a subject or a sample by (i)
contacting a subject or sample with an activatable anti-IL-6R
antibody in the presence of a target of interest, e.g., IL-6R,
wherein the activatable anti-IL-6R antibody comprises a masking
moiety (MINI), a cleavable moiety (CM) that is cleaved by the
cleaving agent, and an antigen binding domain or fragment thereof
(AB) that specifically binds the target of interest, wherein the
activatable anti-IL-6R antibody in an uncleaved, non-activated
state comprises a structural arrangement from N-terminus to
C-terminus as follows: MIM-CM-AB or AB-CM-MM; (a) wherein the MM is
a peptide that inhibits binding of the AB to the IL-6R target, and
wherein the MM does not have an amino acid sequence of a naturally
occurring binding partner of the AB and is not a modified form of a
natural binding partner of the AB; and (b) wherein, in an
uncleaved, non-activated state, the MM interferes with specific
binding of the AB to the IL-6R target, and in a cleaved, activated
state the MM does not interfere or compete with specific binding of
the AB to the IL-6R target; and (ii) measuring a level of activated
activatable anti-IL-6R antibody in the subject or sample, wherein a
detectable level of activated activatable anti-IL-6R antibody in
the subject or sample indicates that the cleaving agent is present
in the subject or sample and wherein no detectable level of
activated activatable anti-IL-6R antibody in the subject or sample
indicates that the cleaving agent is absent and/or not sufficiently
present in the subject or sample. In some embodiments, the
activatable anti-IL-6R antibody is an activatable anti-IL-6R
antibody to which a therapeutic agent is conjugated. In some
embodiments, the activatable anti-IL-6R antibody is not conjugated
to an agent. In some embodiments, the activatable anti-IL-6R
antibody comprises a detectable label. In some embodiments, the
detectable label is positioned on the AB. In some embodiments,
measuring the level of activatable anti-IL-6R antibody in the
subject or sample is accomplished using a secondary reagent that
specifically binds to the activated antibody, wherein the reagent
comprises a detectable label. In some embodiments, the secondary
reagent is an antibody comprising a detectable label.
[0374] The invention also provides kits for use in methods of
detecting presence or absence of a cleaving agent and IL-6Rin a
subject or a sample, where the kits include at least an activatable
anti-IL-6R antibody comprises a masking moiety (MINI), a cleavable
moiety (CM) that is cleaved by the cleaving agent, and an antigen
binding domain or fragment thereof (AB) that specifically binds the
target of interest, wherein the activatable anti-IL-6R antibody in
an uncleaved, non-activated state comprises a structural
arrangement from N-terminus to C-terminus as follows: MIM-CM-AB or
AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of
the AB to the IL-6R target, and wherein the MM does not have an
amino acid sequence of a naturally occurring binding partner of the
AB and is not a modified form of a natural binding partner of the
AB; and (b) wherein, in an uncleaved, non-activated state, the MINI
interferes with specific binding of the AB to the IL-6R target, and
in a cleaved, activated state the MM does not interfere or compete
with specific binding of the AB to the IL-6R target; and (ii)
measuring a level of activated activatable anti-IL-6R antibody in
the subject or sample, wherein a detectable level of activated
activatable anti-IL-6R antibody in the subject or sample indicates
that the cleaving agent is present in the subject or sample and
wherein no detectable level of activated activatable anti-IL-6R
antibody in the subject or sample indicates that the cleaving agent
is absent and/or not sufficiently present in the subject or sample.
In some embodiments, the activatable anti-IL-6R antibody is an
activatable anti-IL-6R antibody to which a therapeutic agent is
conjugated. In some embodiments, the activatable anti-IL-6R
antibody is not conjugated to an agent. In some embodiments, the
activatable anti-IL-6R antibody comprises a detectable label. In
some embodiments, the detectable label is positioned on the AB. In
some embodiments, measuring the level of activatable anti-IL-6R
antibody in the subject or sample is accomplished using a secondary
reagent that specifically binds to the activated antibody, wherein
the reagent comprises a detectable label. In some embodiments, the
secondary reagent is an antibody comprising a detectable label.
[0375] The invention also provides methods of detecting presence or
absence of a cleaving agent in a subject or a sample by (i)
contacting a subject or sample with an activatable anti-IL-6R
antibody, wherein the activatable anti-IL-6R antibody comprises a
masking moiety (MINI), a cleavable moiety (CM) that is cleaved by
the cleaving agent, an antigen binding domain (AB) that
specifically binds IL-6R, and a detectable label, wherein the
activatable anti-IL-6R antibody in an uncleaved, non-activated
state comprises a structural arrangement from N-terminus to
C-terminus as follows: MIM-CM-AB or AB-CM-MM; wherein the MM is a
peptide that inhibits binding of the AB to the IL-6R target, and
wherein the MM does not have an amino acid sequence of a naturally
occurring binding partner of the AB and is not a modified form of a
natural binding partner of the AB; wherein, in an uncleaved,
non-activated state, the MM interferes with specific binding of the
AB to the IL-6R target, and in a cleaved, activated state the MM
does not interfere or compete with specific binding of the AB to
the IL-6R target; and wherein the detectable label is positioned on
a portion of the activatable anti-IL-6R antibody that is released
following cleavage of the CM; and (ii) measuring a level of
detectable label in the subject or sample, wherein a detectable
level of the detectable label in the subject or sample indicates
that the cleaving agent is absent and/or not sufficiently present
in the subject or sample and wherein no detectable level of the
detectable label in the subject or sample indicates that the
cleaving agent is present in the subject or sample. In some
embodiments, the activatable anti-IL-6R antibody is an activatable
anti-IL-6R antibody to which a therapeutic agent is conjugated. In
some embodiments, the activatable anti-IL-6R antibody is not
conjugated to an agent. In some embodiments, the activatable
anti-IL-6R antibody comprises a detectable label. In some
embodiments, the detectable label is positioned on the AB. In some
embodiments, measuring the level of activatable anti-IL-6R antibody
in the subject or sample is accomplished using a secondary reagent
that specifically binds to the activated antibody, wherein the
reagent comprises a detectable label. In some embodiments, the
secondary reagent is an antibody comprising a detectable label.
[0376] The invention also provides kits for use in methods of
detecting presence or absence of a cleaving agent and IL-6Rin a
subject or a sample, where the kits include at least an activatable
anti-IL-6R antibody and/or conjugated activatable anti-IL-6R
antibody (e.g., an activatable antibody to which a therapeutic
agent is conjugated) described herein for use in contacting a
subject or biological sample and means for detecting the level of
activated activatable anti-IL-6R antibody and/or conjugated
activatable anti-IL-6R antibody in the subject or biological
sample, wherein a detectable level of activated activatable
anti-IL-6R antibody in the subject or biological sample indicates
that the cleaving agent and the IL-6R target are present in the
subject or biological sample and wherein no detectable level of
activated activatable anti-IL-6R antibody in the subject or
biological sample indicates that the cleaving agent, the IL-6R
target or both the cleaving agent and the IL-6R target are absent
and/or not sufficiently present in the subject or biological
sample, such that IL-6R target binding and/or protease cleavage of
the activatable anti-IL-6R antibody cannot be detected in the
subject or biological sample.
[0377] The invention also provides methods of detecting presence or
absence of a cleaving agent in a subject or a sample by (i)
contacting a subject or biological sample with an activatable
anti-IL-6R antibody in the presence of the IL-6R target, and (ii)
measuring a level of activated activatable anti-IL-6R antibody in
the subject or biological sample, wherein a detectable level of
activated activatable anti-IL-6R antibody in the subject or
biological sample indicates that the cleaving agent is present in
the subject or biological sample and wherein no detectable level of
activated activatable anti-IL-6R antibody in the subject or
biological sample indicates that the cleaving agent is absent
and/or not sufficiently present in the subject or biological sample
at a detectable level, such that protease cleavage of the
activatable anti-IL-6R antibody cannot be detected in the subject
or biological sample. Such an activatable anti-IL-6R antibody
includes a masking moiety (MM), a cleavable moiety (CM) that is
cleaved by the cleaving agent, and an antigen binding domain or
fragment thereof (AB) that specifically binds the IL-6R target,
wherein the activatable anti-IL-6R antibody in an uncleaved (i.e.,
non-activated) state comprises a structural arrangement from
N-terminus to C-terminus as follows: MIM-CM-AB or AB-CM-MM; (a)
wherein the MM is a peptide that inhibits binding of the AB to the
IL-6R target, and wherein the MM does not have an amino acid
sequence of a naturally occurring binding partner of the AB; and
(b) wherein the MM of the activatable anti-IL-6R antibody in an
uncleaved state interferes with specific binding of the AB to the
IL-6R target, and wherein the MM of an activatable anti-IL-6R
antibody in a cleaved (i.e., activated) state does not interfere or
compete with specific binding of the AB to the IL-6R target. In
some embodiments, the activatable anti-IL-6R antibody is an
activatable anti-IL-6R antibody to which a therapeutic agent is
conjugated. In some embodiments, the activatable anti-IL-6R
antibody is not conjugated to an agent. In some embodiments, the
detectable label is attached to the masking moiety. In some
embodiments, the detectable label is attached to the cleavable
moiety N-terminal to the protease cleavage site. In some
embodiments, a single antigen binding site of the AB is masked. In
some embodiments wherein an antibody of the disclosure has at least
two antigen binding sites, at least one antigen binding site is
masked and at least one antigen binding site is not masked. In some
embodiments all antigen binding sites are masked. In some
embodiments, the measuring step includes use of a secondary reagent
comprising a detectable label.
[0378] The invention also provides kits for use in methods of
detecting presence or absence of a cleaving agent and IL-6Rin a
subject or a sample, where the kits include at least an activatable
anti-IL-6R antibody and/or conjugated activatable anti-IL-6R
antibody described herein for use in contacting a subject or
biological sample with an activatable anti-IL-6R antibody in the
presence of the IL-6R target, and measuring a level of activated
activatable anti-IL-6R antibody in the subject or biological
sample, wherein a detectable level of activated activatable
anti-IL-6R antibody in the subject or biological sample indicates
that the cleaving agent is present in the subject or biological
sample and wherein no detectable level of activated activatable
anti-IL-6R antibody in the subject or biological sample indicates
that the cleaving agent is absent and/or not sufficiently present
in the subject or biological sample at a detectable level, such
that protease cleavage of the activatable anti-IL-6R antibody
cannot be detected in the subject or biological sample. Such an
activatable anti-IL-6R antibody includes a masking moiety (MINI), a
cleavable moiety (CM) that is cleaved by the cleaving agent, and an
antigen binding domain or fragment thereof (AB) that specifically
binds the IL-6R target, wherein the activatable anti-IL-6R antibody
in an uncleaved (i.e., non-activated) state comprises a structural
arrangement from N-terminus to C-terminus as follows: MIM-CM-AB or
AB-CM-MM; (a) wherein the MINI is a peptide that inhibits binding
of the AB to the IL-6R target, and wherein the MM does not have an
amino acid sequence of a naturally occurring binding partner of the
AB; and (b) wherein the MM of the activatable anti-IL-6R antibody
in an uncleaved state interferes with specific binding of the AB to
the IL-6R target, and wherein the MM of an activatable anti-IL-6R
antibody in a cleaved (i.e., activated) state does not interfere or
compete with specific binding of the AB to the IL-6R target. In
some embodiments, the activatable anti-IL-6R antibody is an
activatable anti-IL-6R antibody to which a therapeutic agent is
conjugated. In some embodiments, the activatable anti-IL-6R
antibody is not conjugated to an agent. In some embodiments, the
detectable label is attached to the masking moiety. In some
embodiments, the detectable label is attached to the cleavable
moiety N-terminal to the protease cleavage site. In some
embodiments, a single antigen binding site of the AB is masked. In
some embodiments wherein an antibody of the disclosure has at least
two antigen binding sites, at least one antigen binding site is
masked and at least one antigen binding site is not masked. In some
embodiments all antigen binding sites are masked. In some
embodiments, the measuring step includes use of a secondary reagent
comprising a detectable label.
[0379] The invention also provides kits for use in methods of
detecting presence or absence of a cleaving agent in a subject or a
sample, where the kits include at least an activatable anti-IL-6R
antibody and/or conjugated activatable anti-IL-6R antibody
described herein for use in contacting a subject or biological
sample and means for detecting the level of activated activatable
anti-IL-6R antibody and/or conjugated activatable anti-IL-6R
antibody in the subject or biological sample, wherein the
activatable anti-IL-6R antibody includes a detectable label that is
positioned on a portion of the activatable anti-IL-6R antibody that
is released following cleavage of the CM, wherein a detectable
level of activated activatable anti-IL-6R antibody in the subject
or biological sample indicates that the cleaving agent is absent
and/or not sufficiently present in the subject or biological sample
such that IL-6R target binding and/or protease cleavage of the
activatable anti-IL-6R antibody cannot be detected in the subject
or biological sample, and wherein no detectable level of activated
activatable anti-IL-6R antibody in the subject or biological sample
indicates that the cleaving agent is present in the subject or
biological sample at a detectable level.
[0380] The invention provides methods of detecting presence or
absence of a cleaving agent and the IL-6R target in a subject or a
sample by (i) contacting a subject or biological sample with an
activatable anti-IL-6R antibody, wherein the activatable anti-IL-6R
antibody includes a detectable label that is positioned on a
portion of the activatable anti-IL-6R antibody that is released
following cleavage of the CM and (ii) measuring a level of
activated activatable anti-IL-6R antibody in the subject or
biological sample, wherein a detectable level of activated
activatable anti-IL-6R antibody in the subject or biological sample
indicates that the cleaving agent, the IL-6R target or both the
cleaving agent and the IL-6R target are absent and/or not
sufficiently present in the subject or biological sample, such that
IL-6R target binding and/or protease cleavage of the activatable
anti-IL-6R antibody cannot be detected in the subject or biological
sample, and wherein a reduced detectable level of activated
activatable anti-IL-6R antibody in the subject or biological sample
indicates that the cleaving agent and the IL-6R target are present
in the subject or biological sample. A reduced level of detectable
label is, for example, a reduction of about 5%, about 10%, about
15%, about 20%, about 25%, about 30%, about 35%, about 40%, about
45%, about 50%, about 55%, about 60%, about 65%, about 70%, about
75%, about 80%, about 85%, about 90%, about 95% and/or about 100%.
Such an activatable anti-IL-6R antibody includes a masking moiety
(MM), a cleavable moiety (CM) that is cleaved by the cleaving
agent, and an antigen binding domain or fragment thereof (AB) that
specifically binds the IL-6R target, wherein the activatable
anti-IL-6R antibody in an uncleaved (i.e., non-activated) state
comprises a structural arrangement from N-terminus to C-terminus as
follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that
inhibits binding of the AB to the IL-6R target, and wherein the MM
does not have an amino acid sequence of a naturally occurring
binding partner of the AB; and (b) wherein the MM of the
activatable anti-IL-6R antibody in an uncleaved state interferes
with specific binding of the AB to the IL-6R target, and wherein
the MM of an activatable anti-IL-6R antibody in a cleaved (i.e.,
activated) state does not interfere or compete with specific
binding of the AB to the IL-6R target. In some embodiments, the
activatable anti-IL-6R antibody is an activatable anti-IL-6R
antibody to which a therapeutic agent is conjugated. In some
embodiments, the activatable anti-IL-6R antibody is not conjugated
to an agent. In some embodiments, the activatable anti-IL-6R
antibody comprises a detectable label. In some embodiments, the
detectable label is positioned on the AB. In some embodiments,
measuring the level of activatable anti-IL-6R antibody in the
subject or sample is accomplished using a secondary reagent that
specifically binds to the activated antibody, wherein the reagent
comprises a detectable label. In some embodiments, the secondary
reagent is an antibody comprising a detectable label.
[0381] The invention also provides kits for use in methods of
detecting presence or absence of a cleaving agent and IL-6R in a
subject or a sample, where the kits include at least an activatable
anti-IL-6R antibody and/or conjugated activatable anti-IL-6R
antibody described herein for use in contacting a subject or
biological sample and means for detecting the level of activated
activatable anti-IL-6R antibody and/or conjugated activatable
anti-IL-6R antibody in the subject or biological sample, wherein a
detectable level of activated activatable anti-IL-6R antibody in
the subject or biological sample indicates that the cleaving agent,
the IL-6R target or both the cleaving agent and the IL-6R target
are absent and/or not sufficiently present in the subject or
biological sample, such that IL-6R target binding and/or protease
cleavage of the activatable anti-IL-6R antibody cannot be detected
in the subject or biological sample, and wherein a reduced
detectable level of activated activatable anti-IL-6R antibody in
the subject or biological sample indicates that the cleaving agent
and the IL-6R target are present in the subject or biological
sample. A reduced level of detectable label is, for example, a
reduction of about 5%, about 10%, about 15%, about 20%, about 25%,
about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,
about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,
about 90%, about 95% and/or about 100%.
[0382] The invention also provides methods of detecting presence or
absence of a cleaving agent in a subject or a sample by (i)
contacting a subject or biological sample with an activatable
anti-IL-6R antibody, wherein the activatable anti-IL-6R antibody
includes a detectable label that is positioned on a portion of the
activatable anti-IL-6R antibody that is released following cleavage
of the CM; and (ii) measuring a level of detectable label in the
subject or biological sample, wherein a detectable level of the
detectable label in the subject or biological sample indicates that
the cleaving agent is absent and/or not sufficiently present in the
subject or biological sample at a detectable level, such that
protease cleavage of the activatable anti-IL-6R antibody cannot be
detected in the subject or biological sample, and wherein a reduced
detectable level of the detectable label in the subject or
biological sample indicates that the cleaving agent is present in
the subject or biological sample. A reduced level of detectable
label is, for example, a reduction of about 5%, about 10%, about
15%, about 20%, about 25%, about 30%, about 35%, about 40%, about
45%, about 50%, about 55%, about 60%, about 65%, about 70%, about
75%, about 80%, about 85%, about 90%, about 95% and/or about 100%.
Such an activatable anti-IL-6R antibody includes a masking moiety
(MM), a cleavable moiety (CM) that is cleaved by the cleaving
agent, and an antigen binding domain or fragment thereof (AB) that
specifically binds the IL-6R target, wherein the activatable
anti-IL-6R antibody in an uncleaved (i.e., non-activated) state
comprises a structural arrangement from N-terminus to C-terminus as
follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that
inhibits binding of the AB to the IL-6R target, and wherein the MM
does not have an amino acid sequence of a naturally occurring
binding partner of the AB; and (b) wherein the MM of the
activatable anti-IL-6R antibody in an uncleaved state interferes
with specific binding of the AB to the IL-6R target, and wherein
the MM of an activatable anti-IL-6R antibody in a cleaved (i.e.,
activated) state does not interfere or compete with specific
binding of the AB to the IL-6R target. In some embodiments, the
activatable anti-IL-6R antibody is an activatable anti-IL-6R
antibody to which a therapeutic agent is conjugated. In some
embodiments, the activatable anti-IL-6R antibody is not conjugated
to an agent. In some embodiments, the activatable anti-IL-6R
antibody comprises a detectable label. In some embodiments, the
detectable label is positioned on the AB. In some embodiments,
measuring the level of activatable anti-IL-6R antibody in the
subject or sample is accomplished using a secondary reagent that
specifically binds to the activated antibody, wherein the reagent
comprises a detectable label. In some embodiments, the secondary
reagent is an antibody comprising a detectable label.
[0383] The invention also provides kits for use in methods of
detecting presence or absence of a cleaving agent of interest in a
subject or a sample, where the kits include at least an activatable
anti-IL-6R antibody and/or conjugated activatable anti-IL-6R
antibody described herein for use in contacting a subject or
biological sample and means for detecting the level of activated
activatable anti-IL-6R antibody and/or conjugated activatable
anti-IL-6R antibody in the subject or biological sample, wherein
the activatable anti-IL-6R antibody includes a detectable label
that is positioned on a portion of the activatable anti-IL-6R
antibody that is released following cleavage of the CM, wherein a
detectable level of the detectable label in the subject or
biological sample indicates that the cleaving agent, the IL-6R
target, or both the cleaving agent and the IL-6R target are absent
and/or not sufficiently present in the subject or biological
sample, such that IL-6R target binding and/or protease cleavage of
the activatable anti-IL-6R antibody cannot be detected in the
subject or biological sample, and wherein a reduced detectable
level of the detectable label in the subject or biological sample
indicates that the cleaving agent and the IL-6R target are present
in the subject or biological sample. A reduced level of detectable
label is, for example, a reduction of about 5%, about 10%, about
15%, about 20%, about 25%, about 30%, about 35%, about 40%, about
45%, about 50%, about 55%, about 60%, about 65%, about 70%, about
75%, about 80%, about 85%, about 90%, about 95% and/or about
100%.
[0384] In some embodiments of these methods and kits, the
activatable anti-IL-6R antibody includes a detectable label. In
some embodiments of these methods and kits, the detectable label
includes an imaging agent, a contrasting agent, an enzyme, a
fluorescent label, a chromophore, a dye, one or more metal ions, or
a ligand-based label. In some embodiments of these methods and
kits, the imaging agent comprises a radioisotope. In some
embodiments of these methods and kits, the radioisotope is indium
or technetium. In some embodiments of these methods and kits, the
contrasting agent comprises iodine, gadolinium or iron oxide. In
some embodiments of these methods and kits, the enzyme comprises
horseradish peroxidase, alkaline phosphatase, or
.beta.-galactosidase. In some embodiments of these methods and
kits, the fluorescent label comprises yellow fluorescent protein
(YFP), cyan fluorescent protein (CFP), green fluorescent protein
(GFP), modified red fluorescent protein (mRFP), red fluorescent
protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. In
some embodiments of these methods and kits, the luminescent label
comprises an N-methylacrydium derivative. In some embodiments of
these methods, the label comprises an Alexa Fluor.RTM. label, such
as Alex Fluor.RTM. 680 or Alexa Fluor.RTM. 750. In some embodiments
of these methods and kits, the ligand-based label comprises biotin,
avidin, streptavidin or one or more haptens.
[0385] In some embodiments of these methods and kits, the subject
is a mammal. In some embodiments of these methods and kits, the
subject is a human. In some embodiments, the subject is a non-human
mammal, such as a non-human primate, companion animal (e.g., cat,
dog, horse), farm animal, work animal, or zoo animal. In some
embodiments, the subject is a rodent.
[0386] In some embodiments of these methods, the method is an in
vivo method. In some embodiments of these methods, the method is an
in situ method. In some embodiments of these methods, the method is
an ex vivo method. In some embodiments of these methods, the method
is an in vitro method.
[0387] In some embodiments, in situ imaging and/or in vivo imaging
are useful in methods to identify which patients to treat. For
example, in in situ imaging, the activatable anti-IL-6R antibodies
are used to screen patient samples to identify those patients
having the appropriate protease(s) and target(s) at the appropriate
location, e.g., at a tumor site.
[0388] In some embodiments in situ imaging is used to identify or
otherwise refine a patient population suitable for treatment with
an activatable anti-IL-6R antibody of the disclosure. For example,
patients that test positive for both the target (e.g., IL-6R) and a
protease that cleaves the substrate in the cleavable moiety (CM) of
the activatable anti-IL-6R antibody being tested (e.g., accumulate
activated antibodies at the disease site) are identified as
suitable candidates for treatment with such an activatable
anti-IL-6R antibody comprising such a CM. Likewise, patients that
test negative for either or both of the target (e.g., IL-6R) and
the protease that cleaves the substrate in the CM in the
activatable antibody being tested using these methods might be
identified as suitable candidates for another form of therapy. In
some embodiments, such patients that test negative with respect to
a first activatable anti-IL-6R antibody can be tested with other
activatable anti-IL-6R antibodies comprising different CMs until a
suitable activatable anti-IL-6R antibody for treatment is
identified (e.g., an activatable anti-IL-6R antibody comprising a
CM that is cleaved by the patient at the site of disease).
[0389] In some embodiments in vivo imaging is used to identify or
otherwise refine a patient population suitable for treatment with
an activatable anti-IL-6R antibody of the disclosure. For example,
patients that test positive for both the target (e.g., IL-6R) and a
protease that cleaves the substrate in the cleavable moiety (CM) of
the activatable anti-IL-6R antibody being tested (e.g., accumulate
activated antibodies at the disease site) are identified as
suitable candidates for treatment with such an activatable
anti-IL-6R antibody comprising such a CM. Likewise, patients that
test negative might be identified as suitable candidates for
another form of therapy. In some embodiments, such patients that
test negative with respect to a first activatable anti-IL-6R
antibody can be tested with other activatable anti-IL-6R antibodies
comprising different CMs until a suitable activatable anti-IL-6R
antibody for treatment is identified (e.g., an activatable
anti-IL-6R antibody comprising a CM that is cleaved by the patient
at the site of disease).
[0390] In some embodiments of the methods and kits, the method or
kit is used to identify or otherwise refine a patient population
suitable for treatment with an activatable anti-IL-6R antibody of
the disclosure. For example, patients that test positive for both
the target (e.g., IL-6R) and a protease that cleaves the substrate
in the cleavable moiety (CM) of the activatable anti-IL-6R antibody
being tested in these methods are identified as suitable candidates
for treatment with such an activatable anti-IL-6R antibody
comprising such a CM. Likewise, patients that test negative for
both of the targets (e.g., IL-6R) and the protease that cleaves the
substrate in the CM in the activatable antibody being tested using
these methods might be identified as suitable candidates for
another form of therapy. In some embodiments, such patients can be
tested with other activatable anti-IL-6R antibodies until a
suitable activatable anti-IL-6R antibody for treatment is
identified (e.g., an activatable anti-IL-6R antibody comprising a
CM that is cleaved by the patient at the site of disease). In some
embodiments, patients that test negative for either of the target
(e.g., IL-6R) are identified as suitable candidates for treatment
with such an activatable anti-IL-6R antibody comprising such a CM.
In some embodiments, patients that test negative for either of the
target (e.g., IL-6R) are identified as not being suitable
candidates for treatment with such an activatable anti-IL-6R
antibody comprising such a CM. In some embodiments, such patients
can be tested with other activatable anti-IL-6R antibodies until a
suitable activatable anti-IL-6R antibody for treatment is
identified (e.g., an activatable anti-IL-6R antibody comprising a
CM that is cleaved by the patient at the site of disease). In some
embodiments, the activatable anti-IL-6R antibody is an activatable
anti-IL-6R antibody to which a therapeutic agent is conjugated. In
some embodiments, the activatable anti-IL-6R antibody is not
conjugated to an agent. In some embodiments, the activatable
anti-IL-6R antibody comprises a detectable label. In some
embodiments, the detectable label is positioned on the AB. In some
embodiments, measuring the level of activatable anti-IL-6R antibody
in the subject or sample is accomplished using a secondary reagent
that specifically binds to the activated antibody, wherein the
reagent comprises a detectable label. In some embodiments, the
secondary reagent is an antibody comprising a detectable label.
[0391] In some embodiments, a method or kit is used to identify or
otherwise refine a patient population suitable for treatment with
an anti-IL-6R activatable antibody and/or conjugated activatable
anti-IL-6R antibody (e.g., activatable antibody to which a
therapeutic agent is conjugated) of the disclosure, followed by
treatment by administering that activatable anti-IL-6R antibody
and/or conjugated activatable anti-IL-6R antibody to a subject in
need thereof. For example, patients that test positive for both the
targets (e.g., IL-6R) and a protease that cleaves the substrate in
the cleavable moiety (CM) of the activatable anti-IL-6R antibody
and/or conjugated activatable anti-IL-6R antibody being tested in
these methods are identified as suitable candidates for treatment
with such antibody and/or such a conjugated activatable anti-IL-6R
antibody comprising such a CM, and the patient is then administered
a therapeutically effective amount of the activatable anti-IL-6R
antibody and/or conjugated activatable anti-IL-6R antibody that was
tested. Likewise, patients that test negative for either or both of
the target (e.g., IL-6R) and the protease that cleaves the
substrate in the CM in the activatable anti-IL-6R antibody being
tested using these methods might be identified as suitable
candidates for another form of therapy. In some embodiments, such
patients can be tested with other antibody and/or conjugated
activatable anti-IL-6R antibody until a suitable antibody and/or
conjugated activatable anti-IL-6R antibody for treatment is
identified (e.g., an activatable anti-IL-6R antibody and/or
conjugated activatable anti-IL-6R antibody comprising a CM that is
cleaved by the patient at the site of disease). In some
embodiments, the patient is then administered a therapeutically
effective amount of the activatable anti-IL-6R antibody and/or
conjugated for which the patient tested positive.
[0392] In some embodiments of these methods and kits, the MM is a
peptide having a length from about 4 to 40 amino acids. In some
embodiments of these methods and kits, the activatable anti-IL-6R
antibody comprises a linker peptide, wherein the linker peptide is
positioned between the MM and the CM. In some embodiments of these
methods and kits, the activatable anti-IL-6R antibody comprises a
linker peptide, where the linker peptide is positioned between the
AB and the CM. In some embodiments of these methods and kits, the
activatable anti-IL-6R antibody comprises a first linker peptide
(L1) and a second linker peptide (L2), wherein the first linker
peptide is positioned between the MM and the CM and the second
linker peptide is positioned between the AB and the CM. In some
embodiments of these methods and kits, each of L1 and L2 is a
peptide of about 1 to 20 amino acids in length, and wherein each of
L1 and L2 need not be the same linker. In some embodiments of these
methods and kits, one or both of L1 and L2 comprises a
glycine-serine polymer. In some embodiments of these methods and
kits, at least one of L1 and L2 comprises an amino acid sequence
selected from the group consisting of (GS).sub.n, (GSGGS).sub.n
(SEQ ID NO: 93) and (GGGS).sub.n (SEQ ID NO: 94), where n is an
integer of at least one. In some embodiments of these methods and
kits, at least one of L1 and L2 comprises an amino acid sequence
having the formula (GGS).sub.n, where n is an integer of at least
one. In some embodiments of these methods and kits, at least one of
L1 and L2 comprises an amino acid sequence selected from the group
consisting of Gly-Gly-Ser-Gly (SEQ ID NO: 95), Gly-Gly-Ser-Gly-Gly
(SEQ ID NO: 96), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 97),
Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 98), Gly-Gly-Gly-Ser-Gly (SEQ ID
NO: 99), and Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 100).
[0393] In some embodiments of these methods and kits, the AB
comprises an antibody or antibody fragment sequence selected from
the cross-reactive anti-IL-6R antibody sequences presented herein.
In some embodiments of these methods and kits, the AB comprises a
Fab fragment, a scFv or a single chain antibody (scAb).
[0394] In some embodiments of these methods and kits, the cleaving
agent is a protease that is co-localized in the subject or sample
with the IL-6R target and the CM is a polypeptide that functions as
a substrate for the protease, wherein the protease cleaves the CM
in the activatable anti-IL-6R antibody when the activatable
anti-IL-6R antibody is exposed to the protease. In some embodiments
of these methods and kits, the CM is a polypeptide of up to 15
amino acids in length. In some embodiments of these methods and
kits, the CM is coupled to the N-terminus of the AB. In some
embodiments of these methods and kits, the CM is coupled to the
C-terminus of the AB. In some embodiments of these methods and
kits, the CM is coupled to the N-terminus of a VL chain of the
AB.
[0395] In some embodiments of these methods and kits, the cleaving
agent is an enzyme and the CM is a substrate for the enzyme. In
some embodiments of these methods and kits, the enzyme is a
protease disclosed herein. In some embodiments of these methods and
kits, the protease is one of the proteases disclosed herein. In
some embodiments of these methods and kits, the protease is
selected from the group consisting of uPA, legumain, MT-SP1,
ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-9, MMP-12, MMP-13, and MMP-14.
In some embodiments, the protease is a cathepsin.
[0396] The activatable anti-IL-6R antibodies and/or conjugated
activatable anti-IL-6R antibodies of the invention are used in
diagnostic and prophylactic formulations. In one embodiment, an
activatable anti-IL-6R antibody is administered to patients that
are at risk of developing one or more of the aforementioned
inflammation, inflammatory disorders, cancer or other disorders. In
some embodiments, the inflammation is associated with and/or the
inflammatory disorder is Crohn's disease. In some embodiments, the
inflammation is associated with and/or the inflammatory disorder is
polychondritis, including but not limited to, relapsing
polychondritis. In some embodiments, the inflammation is associated
with and/or the inflammatory disorder is rheumatoid arthritis (RA).
In some embodiments, the inflammation is associated with and/or the
inflammatory disorder is another rheumatoid disease, such as, by
way of non-limiting example, ankylosing spondylitis, juvenile
arthritis, and/or psoriatic arthritis. In some embodiments, the
inflammation is associated with and/or the inflammatory disorder is
ulcerative colitis. In some embodiments, the cancer is breast
cancer, including but not limited to, triple negative breast cancer
(TNBC). In some embodiments, the cancer is Castleman's disease. In
some embodiments, the cancer is hepatocellular carcinoma. In some
embodiments, the cancer is lung cancer. In some embodiments, the
cancer is multiple myeloma. In some embodiments, the cancer is
ovarian cancer. In some embodiments, the cancer is prostate
cancer.
[0397] A patient's or organ's predisposition to one or more of the
aforementioned disorders can be determined using genotypic,
serological or biochemical markers.
[0398] In another embodiment of the invention, an activatable
anti-IL-6R antibody and/or conjugated activatable anti-IL-6R
antibodies is administered to human individuals diagnosed with a
clinical indication associated with one or more of the
aforementioned disorders.
[0399] Upon diagnosis, an activatable anti-IL-6R antibody and/or
conjugated activatable anti-IL-6R antibodies is administered to
mitigate or reverse the effects of the clinical indication.
[0400] Activatable antibodies and/or conjugated activatable
antibodies of the invention are also useful in the detection of
IL-6R in patient samples and accordingly are useful as diagnostics.
For example, the activatable anti-IL-6R antibodies and/or
conjugated activatable anti-IL-6R antibodies of the invention are
used in in vitro assays, e.g., ELISA, to detect IL-6R levels in a
patient sample.
[0401] In one embodiment, an activatable anti-IL-6R antibody of the
invention is immobilized on a solid support (e.g., the well(s) of a
microtiter plate). The immobilized activatable antibody serves as a
capture antibody for any IL-6R that may be present in a test
sample. Prior to contacting the immobilized antibody with a patient
sample, the solid support is rinsed and treated with a blocking
agent such as milk protein or albumin to prevent nonspecific
adsorption of the analyte.
[0402] Subsequently the wells are treated with a test sample
suspected of containing the antigen, or with a solution containing
a standard amount of the antigen. Such a sample is, e.g., a serum
sample from a subject suspected of having levels of circulating
antigen considered to be diagnostic of a pathology. After rinsing
away the test sample or standard, the solid support is treated with
a second antibody that is detectably labeled. The labeled second
antibody serves as a detecting antibody. The level of detectable
label is measured, and the concentration of IL-6R antigen in the
test sample is determined by comparison with a standard curve
developed from the standard samples.
[0403] It will be appreciated that based on the results obtained
using the anti-IL-6R antibodies of the invention in an in vitro
diagnostic assay, it is possible to stage a disease in a subject
based on expression levels of the IL-6R antigen. For a given
disease, samples of blood are taken from subjects diagnosed as
being at various stages in the progression of the disease, and/or
at various points in the therapeutic treatment of the disease.
Using a population of samples that provides statistically
significant results for each stage of progression or therapy, a
range of concentrations of the antigen that may be considered
characteristic of each stage is designated.
[0404] Activatable anti-IL-6R antibodies and/or conjugated
activatable anti-IL-6R antibodies can also be used in diagnostic
and/or imaging methods. In some embodiments, such methods are in
vitro methods. In some embodiments, such methods are in vivo
methods. In some embodiments, such methods are in situ methods. In
some embodiments, such methods are ex vivo methods. For example,
activatable anti-IL-6R antibodies having an enzymatically cleavable
CM can be used to detect the presence or absence of an enzyme that
is capable of cleaving the CM. Such activatable anti-IL-6R
antibodies can be used in diagnostics, which can include in vivo
detection (e.g., qualitative or quantitative) of enzyme activity
(or, in some embodiments, an environment of increased reduction
potential such as that which can provide for reduction of a
disulfide bond) through measured accumulation of activated
anti-IL-6R antibodies (i.e., antibodies resulting from cleavage of
an activatable anti-IL-6R antibody) in a given cell or tissue of a
given host organism. Such accumulation of activated anti-IL-6R
antibodies indicates not only that the tissue expresses enzymatic
activity (or an increased reduction potential depending on the
nature of the CM) but also that the tissue expresses target to
which the activated antibody binds.
[0405] For example, the CM can be selected to be a protease
substrate for a protease found at the site of a tumor, at the site
of a viral or bacterial infection at a biologically confined site
(e.g., such as in an abscess, in an organ, and the like), and the
like. The AB can be one that binds a target antigen. Using methods
familiar to one skilled in the art, a detectable label (e.g., a
fluorescent label or radioactive label or radiotracer) can be
conjugated to an AB or other region of an activatable anti-IL-6R
antibody. Suitable detectable labels are discussed in the context
of the above screening methods and additional specific examples are
provided below. Using an AB specific to a protein or peptide of the
disease state, along with a protease whose activity is elevated in
the disease tissue of interest, activatable anti-IL-6R antibodies
will exhibit an increased rate of binding to disease tissue
relative to tissues where the CM specific enzyme is not present at
a detectable level or is present at a lower level than in disease
tissue or is inactive (e.g., in zymogen form or in complex with an
inhibitor). Since small proteins and peptides are rapidly cleared
from the blood by the renal filtration system, and because the
enzyme specific for the CM is not present at a detectable level (or
is present at lower levels in non-disease tissues or is present in
inactive conformation), accumulation of activated anti-IL-6R
antibodies in the disease tissue is enhanced relative to
non-disease tissues.
[0406] In another example, activatable anti-IL-6R antibodies can be
used to detect the presence or absence of a cleaving agent in a
sample. For example, where the activatable anti-IL-6R antibodies
contain a CM susceptible to cleavage by an enzyme, the activatable
anti-IL-6R antibodies can be used to detect (either qualitatively
or quantitatively) the presence of an enzyme in the sample. In
another example, where the activatable anti-IL-6R antibodies
contain a CM susceptible to cleavage by reducing agent, the
activatable anti-IL-6R antibodies can be used to detect (either
qualitatively or quantitatively) the presence of reducing
conditions in a sample. To facilitate analysis in these methods,
the activatable antibodies can be detectably labeled, and can be
bound to a support (e.g., a solid support, such as a slide or
bead). The detectable label can be positioned on a portion of the
activatable anti-IL-6R antibody that is not released following
cleavage, for example, the detectable label can be a quenched
fluorescent label or other label that is not detectable until
cleavage has occurred. The assay can be conducted by, for example,
contacting the immobilized, detectably labeled activatable
anti-IL-6R antibodies with a sample suspected of containing an
enzyme and/or reducing agent for a time sufficient for cleavage to
occur, then washing to remove excess sample and contaminants. The
presence or absence of the cleaving agent (e.g., enzyme or reducing
agent) in the sample is then assessed by a change in detectable
signal of the activatable anti-IL-6R antibodies prior to contacting
with the sample e.g., the presence of and/or an increase in
detectable signal due to cleavage of the activatable antibody by
the cleaving agent in the sample.
[0407] Such detection methods can be adapted to also provide for
detection of the presence or absence of a target that is capable of
binding the AB of the activatable anti-IL-6R antibodies when
cleaved. Thus, the assays can be adapted to assess the presence or
absence of a cleaving agent and the presence or absence of a target
of interest. The presence or absence of the cleaving agent can be
detected by the presence of and/or an increase in detectable label
of the activatable anti-IL-6R antibodies as described above, and
the presence or absence of the target can be detected by detection
of a target-AB complex e.g., by use of a detectably labeled
anti-target antibody.
[0408] Activatable anti-IL-6R antibodies are also useful in in situ
imaging for the validation of activatable antibody activation,
e.g., by protease cleavage, and binding to a particular target. In
situ imaging is a technique that enables localization of
proteolytic activity and target in biological samples such as cell
cultures or tissue sections. Using this technique, it is possible
to confirm both binding to a given target and proteolytic activity
based on the presence of a detectable label (e.g., a fluorescent
label).
[0409] These techniques are useful with any frozen cells or tissue
derived from a disease site (e.g. tumor tissue) or healthy tissues.
These techniques are also useful with fresh cell or tissue
samples.
[0410] In these techniques, an activatable anti-IL-6R antibody is
labeled with a detectable label. The detectable label may be a
fluorescent dye, (e.g. Fluorescein Isothiocyanate (FITC), Rhodamine
Isothiocyanate (TRITC), a near infrared (NIR) dye (e.g., Qdot.RTM.
nanocrystals), a colloidal metal, a hapten, a radioactive marker,
biotin and an amplification reagent such as streptavidin, or an
enzyme (e.g. horseradish peroxidase or alkaline phosphatase).
[0411] Detection of the label in a sample that has been incubated
with the labeled, activatable anti-IL-6R antibody indicates that
the sample contains the target, i.e., IL-6R, and contains a
protease that is specific for the CM of the activatable anti-IL-6R
antibody. In some embodiments, the presence of the protease can be
confirmed using broad spectrum protease inhibitors such as those
described herein, and/or by using an agent that is specific for the
protease, for example, an antibody such as All, which is specific
for the protease matriptase (MT-SP1) and inhibits the proteolytic
activity of MT-SP1; see e.g., International Publication Number WO
2010/129609, published 11 Nov. 2010. The same approach of using
broad spectrum protease inhibitors such as those described herein,
and/or by using a more selective inhibitory agent can be used to
identify a protease or class of proteases specific for the CM of
the activatable anti-IL-6R antibody. In some embodiments, the
presence of the target can be confirmed using an agent that is
specific for the target, e.g., another anti-IL-6R antibody, or the
detectable label can be competed with unlabeled IL-6R. In some
embodiments, unlabeled activatable anti-IL-6R antibody could be
used, with detection by a labeled secondary antibody or more
complex detection system.
[0412] Similar techniques are also useful for in vivo imaging where
detection of the fluorescent signal in a subject, e.g., a mammal,
including a human, indicates that the disease site contains the
target, i.e., IL-6R, and contains a protease that is specific for
the CM of the activatable anti-IL-6R antibody.
[0413] These techniques are also useful in kits and/or as reagents
for the detection, identification or characterization of protease
activity in a variety of cells, tissues, and organisms based on the
protease-specific CM in the activatable anti-IL-6R antibody.
[0414] In some embodiments, in situ imaging and/or in vivo imaging
are useful in methods to identify which patients to treat. For
example, in in situ imaging, the activatable anti-IL-6R antibodies
are used to screen patient samples to identify those patients
having the appropriate protease(s) and target(s) at the appropriate
location, e.g., at a tumor site.
[0415] In some embodiments in situ imaging is used to identify or
otherwise refine a patient population suitable for treatment with
an anti-IL-6R activatable antibody of the disclosure. For example,
patients that test positive for both the target (e.g., IL-6R) and a
protease that cleaves the substrate in the cleavable moiety (CM) of
the anti-IL-6R activatable antibody being tested (e.g., accumulate
activated antibodies at the disease site) are identified as
suitable candidates for treatment with such an anti-IL-6R
activatable antibody comprising such a CM. Likewise, patients that
test negative for either or both of the target (e.g., IL-6R) and
the protease that cleaves the substrate in the CM in the
activatable antibody being tested using these methods are
identified as suitable candidates for another form of therapy
(i.e., not suitable for treatment with the anti-IL-6R activatable
antibody being tested). In some embodiments, such patients that
test negative with respect to a first anti-IL-6R activatable
antibody can be tested with other anti-IL-6R activatable antibodies
comprising different CMs until a suitable anti-IL-6R activatable
antibody for treatment is identified (e.g., an anti-IL-6R
activatable antibody comprising a CM that is cleaved by the patient
at the site of disease).
[0416] In some embodiments in vivo imaging is used to identify or
otherwise refine a patient population suitable for treatment with
an anti-IL-6R activatable antibody of the disclosure. For example,
patients that test positive for both the target (e.g., IL-6R) and a
protease that cleaves the substrate in the cleavable moiety (CM) of
the anti-IL-6R activatable antibody being tested (e.g., accumulate
activated antibodies at the disease site) are identified as
suitable candidates for treatment with such an anti-IL-6R
activatable antibody comprising such a CM. Likewise, patients that
test negative are identified as suitable candidates for another
form of therapy (i.e., not suitable for treatment with the
anti-IL-6R activatable antibody being tested). In some embodiments,
such patients that test negative with respect to a first anti-IL-6R
activatable antibody can be tested with other anti-IL-6R
activatable antibodies comprising different CMs until a suitable
anti-IL-6R activatable antibody for treatment is identified (e.g.,
an anti-IL-6R activatable antibody comprising a CM that is cleaved
by the patient at the site of disease).
[0417] Pharmaceutical Compositions
[0418] The activatable anti-IL-6R antibodies of the invention (also
referred to herein as "active compounds"), and derivatives,
fragments, analogs and homologs thereof, can be incorporated into
pharmaceutical compositions suitable for administration. Such
compositions typically comprise the activatable antibody and a
pharmaceutically acceptable carrier. As used herein, the term
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. Suitable
carriers are described in the most recent edition of Remington's
Pharmaceutical Sciences, a standard reference text in the field,
which is incorporated herein by reference. Suitable examples of
such carriers or diluents include, but are not limited to, water,
saline, ringer's solutions, dextrose solution, and 5% human serum
albumin. Liposomes and non-aqueous vehicles such as fixed oils may
also be used. The use of such media and agents for pharmaceutically
active substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0419] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (i.e., topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfate; chelating agents such as
ethylenediaminetetraacetic acid (EDTA); buffers such as acetates,
citrates or phosphates, and agents for the adjustment of tonicity
such as sodium chloride or dextrose. The pH can be adjusted with
acids or bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0420] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL' (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In some embodiments, it
will be desirable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0421] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, methods of preparation are vacuum
drying and freeze-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0422] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0423] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0424] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0425] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0426] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0427] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0428] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0429] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1. Materials and Methods for Anti-IL6R Antibody Masking
Moiety Discovery
[0430] The studies described herein are used to identify suitable
masking moieties for activatable anti-IL6R antibodies using an
initial round of magnetic activated cell sorting (MACS) followed by
sufficient fluorescent activated cell sorting (FACS) to gain a
population that shows reasonable enrichment of one or more peptides
that specifically bind the antibody and Fab of interest.
[0431] Magnetic Activated Cell Sorting (MACS).
[0432] A 500 mL LB-CM (chloramphenicol) culture is inoculated with
1E11 cells from a cellular peptide display library in which the
displayed peptides include a 15-mer randomized section
(approximately 2E10 diversity). When the culture reaches an OD600
of between 0.6 and 0.8, arabinose (the cellular peptide display
libraries are expressed from the pBAD promoter) is added to 0.2%
and the culture is grown for another 1.5 hrs before being placed on
ice for 20 minutes. Sufficient cells to cover the library size
(2E11) 10.times. are spun down and resuspended in PBS 0.5% BSA to a
final volume of 500 .mu.L.
[0433] The initial MACS is done one of two ways: (1) The induced
cells are added to 100 .mu.L blocked SA DynoBeads to remove
streptavidin binding peptides. The DynoBeads are incubated with the
cells for 1 hr on ice and subsequently removed with a strong
magnet. Biotinylated antibody is added to the remaining cells to a
concentration between 100-250 nM and incubated in ice with frequent
agitation for 1 hr. Labeled cells are then added to 100 .mu.L SA
Dynobeads and incubated on ice for another hour. Sufficient SA
DynoBeads are used so that the majority of the labeled antibody
will be captured. The DynoBeads are captured and washed
approximately 5.times. with PBS 0.5% BSA using a strong magnet.
Captured beads and cells are resuspended in 5 mLs LB-CM with 0.04%
glucose and grown overnight at 37.degree. C.
[0434] 2) Alternatively, a similar protocol to the first is
followed except Protein A DynoBeads and unlabeled target antibody
are used instead of Streptavidin DynoBeads. This method has the
advantage of significantly reducing the background Straptavidin
binding.
[0435] To quantify the number of cells collected, serial dilutions
of the resuspended beads are plated and the colonies are counted,
for example, the next morning.
[0436] Fluorescent Activated Cell Sorting (FACS):
[0437] Culture Growth and Induction:
[0438] Fifty to one hundred microliters of overnight culture is
subcultured into 5 mls of prewarmed LB-CM and grown for
approximately 1.5 hours and subsequently induced with arabinose
(0.04% final concentration) for between 45 minutes and 1.5 hours.
Early populations with low enrichment for binders are typically
induced for 1.5 hours while enriched populations are typically
induced for shorter periods of time. The overall expression level
of a population is monitored by Ypet-Mona binding (the c-terminal
tag on the cellular peptide display scaffold) and the induction
time is adjusted so as to achieve a moderate level of expression
above background.
[0439] Cell Labeling:
[0440] Typically 20 to 5 .mu.Ls of the culture is spun down for
labeling. The number of cells to label depends on the theoretical
diversity of the population, with the goal being to sample at least
5.times. the theoretical diversity by FACS. Labeling is done in PBS
0.5% BSA or PBS 0.5% BSA+1 to 5 .mu.M pooled human IgG (GammaGard,
Baxter International, Deerfield, Ill.) to block non-specific IgG
and FAB binders. Labeling volume, between 100 and 1000 .mu.ls,
depends on the concentration of the target antibody or Fab.
[0441] Target antibody or Fab labeling is done on ice for 1 hour.
Early rounds are typically labeled with target antibody but later
rounds are labeled with either antibody or Fab. After labeling, the
cells are pelleted, primary label is removed, and the cells are
then labeled with secondary (Streptavidin-PE (Life Technologies,
Inc., Grand Island, N.Y.) or anti-Biotin-PE (Miltenyi Biotec Inc.,
Auburn, Calif.)) for 1 hour. The initial round of FACS is typically
sorted using SA-PE and, to remove excess SA binding, subsequent
rounds are sorted using anti-Biotin-PE. After secondary labeling,
the cells are spun down, excess secondary label is removed, and
then the sample is resuspended in PBS and analyzed by FACS.
Expression level (Ypet-Mona binding) background (secondary alone)
binding are monitored for each round of FACS. Sorted cells are out
grown overnight in LB-CM 0.04% glucose.
[0442] Binding Specificity:
[0443] Both enriched populations and/or individual binding clones
are validated for their specificity for the target antibody in
several ways. Candidate peptides must bind both the antibody and
FAB in the presence of excess pooled human IgG and inhibited from
binding the target antibody and FAB in the presence of excess
target ligand.
[0444] Anti-IL6R Binding Peptide Discovery
[0445] For the initial round of MACS (MAC1, M1), 2E11 cells were
screened using Protein A DynoBeads and anti-IL6R antibody at 250
nM. The MACS round resulted in 2.5E5 cells being selected. The
cells that were selected in the MACS1 round were sorted by FACS
using 100 nM anti-IL6R biotin and SA-PE secondary. Greater than 1E7
cells were sorted and approximately 2000 cells were collected.
[0446] The cells that were selected in the MACS1FAC1 round were
sorted by FACS using 50 nM anti-IL6R Fab biotin and anti-biotin-PE
secondary (MACS1FACS2, M1F2). Greater than 5E6 cells were sorted
and approximately 1900 cells were collected. FACS analysis
demonstrated enrichment over that of the MAC1 round.
[0447] The MAC1FACS2 population showed significant enrichment for
anti-IL6R-FAB binding. The specificity of binding was determined by
FACS analysis. The population bound both the antibody and Fab in
the presence of excess pooled human IgG (5 .mu.M) and was inhibited
by soluble anti-IL6R-Fc. Thirty clones from the M1F2 population
were sequenced (5 failed sequences runs) and showed the population
to be significantly enriched for two peptides: YRSCNWNYVSIFLDC (SEQ
ID NO: 33, referred to herein as Peptide 1) and PGAFDIPFPAHWVPNT
(SEQ ID NO: 34, referred to herein as Peptide 2). Both peptides
were subsequently verified to bind both the anti-IL6R antibody and
Fab.
[0448] The peptides were then affinity matured using the following
process: 1) screening softly randomized libraries of the initial
peptide binders and 2) screening directed libraries designed with
information gained from the outcome of the softly randomized
library screens.
[0449] In order to identify the critical residues or consensuses
for binding, a soft randomization approach is used. This approach
introduces a low level of mutation across the entire peptide by
using the nucleotide rations found in Table 1 below. The diversity
of the soft randomized libraries is typically >5E9.
TABLE-US-00007 TABLE 1 Original Ratio of Bases in Soft Base
Randomized Library G G = 70%; T = 8%; A = 11%; C = 11% T T = 70%; G
= 8%; A = 11%; C = 11% A A = 80%; G = 5%; T = 6%; C = 9% C C = 80%;
G = 5%; T = 6%; A = 9%
[0450] Critical residues, or consensus sequences, are identified
from the softly randomized library screens. Directed libraries are
designed by holding critical residues constant and using codon
degeneracy to encode a subset or fully randomize further positions.
Libraries are designed so that the total diversity can nearly or
completely be covered by a library of 5E9 diversity.
[0451] Affinity maturation libraries were screened using the
following MACS and FACS processes:
[0452] MACS:
[0453] The labeling of affinity maturation libraries was done with
low concentrations of Fab-biotin (25-50 nM) and the amount of
Streptavidin Dynobeads was reduced accordingly. Cells are labeled
under constant rotation at 4.degree. C. Labeled cells are
subsequently added to the Streptavidin Dynobeads, rotated for
another hour at 4.degree. C., and then washed extensively at room
temperature with PBS 0.5% BSA.
[0454] FACS:
[0455] Populations from affinity maturation libraries were all
labeled with Alex-488 labeled Fab. Using a directly labeled Fab is
an avidity associated with using an antibody or secondary label.
When screening affinity maturation libraries, only the brightest
1.0-0.1% of positive cells were sorted. In order to insure
sufficient label for equilibrium binding, 100 .mu.M was the lower
limit for labeling cells, therefore, to more stringently screen
affinity maturation libraries, off-rate screens are commonly done
in the later rounds of FACS. This is done by labeling the cells per
usual, however, after resuspending for FACS analysis, the sample
was incubated at 37.degree. C. for approximately 10 minutes before
sorting the top 1.0 to 0.1% positive cells.
[0456] Binding peptides that have been isolated from libraries were
analyzed for their relative ability to bind the Fab by comparing
the expression normalized binding at a given concentration of
Fab.
Expression normalized binding = Mean Fab fluorescence Mean YpetMona
fluorescence ##EQU00001##
where Mean YpetMona fluorescence=peptide expression level
Example 2. Affinity Maturation of Anti-IL-6R Binding Peptides
[0457] The two anti-IL6R binding peptides isolated from the naive
library were both affinity matured. The peptide of SEQ ID NO: 33
(Peptide 1) is a cysteine constrained peptide that was the most
enriched member in the M1F2 pool, while the peptide of SEQ ID NO:
34 (Peptide 2) is a linear peptide. Although the peptide of SEQ ID
NO: 34 was isolated from an X.sub.15 library, a mutation in the
linker resulted in the peptide being 16 amino acids. The affinity
maturation for both peptides followed the standard methods,
beginning with the screening of a soft randomized library followed
by screening directed libraries.
[0458] Affinity Maturation for Peptide 1
[0459] A softly randomized library of the peptide of SEQ ID NO: 33
(Peptide 1) containing approximately 4E9 members was built and
screened as shown in FIG. 1.
[0460] For the initial round of MACS (MACS1, M1) for the soft
randomization library based on Peptide 1, 1E11 cells were screened
using Streptavidin DynoBeads and anti-IL6R Fab at 25 nM. The MACS
round resulted in 1E6 cells being selected.
[0461] The cells that were selected in the MACS1 round were sorted
by FACS using 10 nM anti-IL6R Fab-dylight (MACS1FACS1, M1F1). For
the first round of FACS, the entire positive population was sorted.
Greater than 1E7 cells were sorted and approximately 5000 cells
were collected.
[0462] The cells that were selected in the MACS1FACS1 round were
sorted by FACS using 1 nM anti-IL6R Fab-dylight (MACS1FACS2, M1F2).
Only the top 0.2% of positive cells were sorted. Greater than 5E6
cells were sorted and approximately 372 cells were collected.
[0463] The M1F2 population was sorted at 100 .mu.M (M1F3), however,
no further enrichment was observed over the M1F2 population. The
sequencing of 24 clones from the M1F2 populations showed that there
was still significant diversity in the population. The sequences
are shown below in Table 2:
TABLE-US-00008 TABLE 2 Sequences from the Peptide 1 Soft
Randomization M1F2 population 0verrepesentation SEQ ID in
population NO: Sequence sequenced 35 ESSCVWNYVHIYMDC 36
YPGCKWNYDRIFLDC 37 YRTCSWNYVGIFLDC 38 YGSCSWNYVHIFMDC 5.times. 39
YGSCSWNYVHIFLDC 3.times. 40 YGSCNWNYVHIFLDC 41 YTSCNWNYVHIFMDC 42
YPGCKWNYDRIFLDC 43 WRSCNWNYAHIFLDC 44 WSNCHWNYVHIFLDC 2.times. 45
DRSCTWNYVRISYDC 46 SGSCKWDYVHIFLDC 47 SRSCIWNYAHIHLDC 48
SMSCYWQYERIFLDC
[0464] Off-rate screening was used to more stringently screen the
population. Three sequential rounds of off-rate screening were done
in a similar manner to each other. Populations were labeled with 1
nM anti-IL6R-Fab Dylight, resuspended in PBS and incubated at
37.degree. C. for 3-4 minutes before sorting the brightest 0.1%. An
example of what a M1F4 population looked like (A) before and (B)
after the incubation at 37.degree. C. is shown in FIGS. 2A and 2B.
Twenty-four clones from the M1F4 and M1F5 populations were
sequenced. The sequences are shown below in Table 3:
TABLE-US-00009 TABLE 3 A summary of sequences from Peptide 1 SR
M1F4 and M1F5 pools. Overrepesentation SEQ ID in population NO:
Sequence sequenced 33 YRSCNWNYVSIFLDC Parental Pep. 49
YGSCSWNYVHIFMDC 20.times. 50 SGSCKWDYVHIFLDC 10.times. 51
YKSCHWDYVHIFLDC 3.times. 52 YGSCTWNYVHIFMEC 2.times. 53
FSSCNWNYVHIFLDC 2.times. 54 WRSCNWNYAHIFLDC 55 YGSCQWNYVHIFLDC 56
YRSCNWNYVHIFLDC 57 NMSCHWDYVHIFLDC 58 FGPCTWNYARISWDC 59 sC W YvhIf
dC (Consensus)
[0465] The clones that were sequenced from the M1F4 and M1F5 pools
were compared at 10 and 100 nM Fab by expression normalized binding
with the parental peptide (FIG. 3). All sequenced clones had a
higher on-cell affinity as measured by FACS than the parental.
[0466] The consensus information gained from the soft randomized
libraries was used to design three directed libraries shown in
Table 4 below. The general strategy is to extend the peptide from
both the N and C-terminus of the consensus determined from the soft
randomization library. All libraries had diversities of
approximately 5E9.
TABLE-US-00010 TABLE 4 Library design Lib. 1 (SEQ ID NO: 60)
xxxxxxCxW(n/d)Y(v/a)HIF(m/1)(d/e)C Lib. 2 (SEQ ID NO: 61)
CxW(n/d)Y(v/a)HIF(m/1)(d/e)Cxxxxxx Lib. 3 (SEQ ID NO: 62)
xxxCxW(n/d)Y(v/a)HIF(m/1)(d/e)Cxxx
[0467] The three directed libraries were pooled and screened as
shown in FIG. 4. For the initial round of MACS (M1), 1E11 cells
were screened using Streptavidin DynoBeads and anti-IL6R Fab at 25
nM. The Dynobeads were washed extensively at room temperature. The
MACS round resulted in approximately 1E6 cells being selected.
[0468] The cells that were selected in the MACS1 round were sorted
by FACS using 1 nM anti-IL6R Fab-dylight (MACS1FACS1, M1F1). Only
the brightest 0.2% were sorted. Greater than 1E7 total cells were
sorted and approximately 3000 cells were collected. Twenty clones
were sequenced and the results are shown below in Table 5. The
population was still relatively diverse.
TABLE-US-00011 TABLE 5 Peptide 1 directed library M1F1 sequences
0verrepesentation SEQ ID in population NO: Sequence sequenced 63
MGVPAGCVWNYAHIFMDC 4.times. 64 RDTGGQCRWDYVHIFMDC 65
AGVPAGCTWNYVHIFMEC 5.times. 66 VGVPNGCVWNYAHIFMEC 3.times. 67
DGGPAGCSWNYVHIFMEC 68 AVGPAGCWWNYVHIFMEC 69 CTWNYVHIFMDCGEGEGP
2.times. 70 GGVPEGCTWNYAHIFMEC 71 AEVPAGCWWNYVHIFMEC 2.times.
[0469] The cells that were selected in the MACS round were sorted
by FACS using 100 .mu.M anti-IL6R Fab-dylight (MACS1FACS2, M1F2).
Only the top 0.2% of positive cells were sorted. Greater than 5E6
cells were sorted and approximately 500 cells were collected.
Twenty clones were sequenced. The population was enriched for three
sequences shown below in Table 6.
TABLE-US-00012 TABLE 6 Peptide 1 directed libraries M1F2 sequences
0verrepesentation SEQ ID in population NO: Sequence sequenced 72
AGVPAGCTWNYVHIFMEC 7.times. 73 SGASGGCKWNYVHIFMDC 74
MGVPAGCVWNYAHIFMDC 9.times. 75 TPGCRWNYVHIFMECEAL 2.times. 76
VGVPNGCVWNYAHIFMEC
[0470] Peptides that were isolated from the directed libraries were
compared, as described above, with the parental peptide isolated
from the naive library (SEQ ID NO: 33) and with the peptide
isolated from the soft randomized library (SEQ ID NO: 49). Three of
the peptides from the directed library pool M1F2 had a higher
affinity as measured by FACS (FIG. 5).
[0471] Affinity Maturation for Peptide 2
[0472] A softly randomized library of Peptide 2 (SEQ ID NO: 34)
containing approximately 4E9 members was built and screened as
shown in FIG. 6.
[0473] For the initial round of MACS (MACS1, M1), 1E11 cells were
screened using Streptavidin DynoBeads and anti-IL6R Fab at 25 nM.
The MACS round resulted in 1E6 cells being selected.
[0474] The cells that were selected in the MACS1 round were sorted
by FACS using 10 nM anti-IL6R Fab-dylight (MACS1FACS1, M1F1). For
the first round of FACS, the entire positive population was sorted.
Greater than 1E7 cells were sorted and approximately 3000 cells
were collected.
[0475] The cells that were selected in the MACS round were sorted
by FACS using 1 nM anti-IL6R Fab-dylight (MACS1FACS2, M1F2). Only
the top 0.2% of positive cells were sorted. Greater than 5E6 cells
were sorted and approximately 381 cells were collected.
[0476] The M1F2 population was sorted at 100 .mu.M (M1F3), however,
only slight enrichment was observed over the M1F2 population. The
sequencing of 24 clones from the M1F2 population resulted in only
two peptide sequences. The sequences are shown below in Table
7.
TABLE-US-00013 TABLE 7 Sequences from the Peptide 2 soft randomized
M1F2 pool 0verrepesentation SEQ ID in population NO: Sequence
sequenced 34 PGAFDIPFPAHWVPNT Parental 77 RGACDIPFPAHWIPNT 6x 78
QGDFDIPFPAHWVPIT 13x 79 GafDIPFPAHWvPnT (Consensus)
[0477] The clone of SEQ ID NO: 78 from the M1F2 pools was compared
at 10 and 100 nM Fab by expression normalized binding with the
parental peptide. The clone of SEQ ID NO: 77 was excluded from this
analysis because of an undesirable unpaired cysteine. The clone of
SEQ ID NO: 78 had a higher on-cell affinity as measured by FACS
than the parental as shown in FIG. 7.
[0478] Although the consensus information gained from the soft
randomized libraries was limited in the case of Peptide 2, the
three directed libraries shown in Table 8 below were built. Again,
the general strategy was to extend the peptide from both the N and
C-terminus of the consensus determined from the soft randomization
library. All libraries had diversities of approximately 5E9.
TABLE-US-00014 TABLE 8 Library design Lib. 1 (SEQ ID NO: 80)
XXXXXXXDIPFPAHW(I/M/V)PXT Lib. 2 (SEQ ID NO: 81)
DIPFPAHW(I/M/V)PXTXXXXXXX Lib. 3 (SEQ ID NO: 82)
XXXXDIPFPAHW(I/M/V)PXTXXXX
[0479] The three directed libraries were pooled and screened as
shown in FIG. 8.
[0480] For the initial round of MACS (MACS1, M1), 1E11 cells were
screened using Streptavidin DynoBeads and anti-IL6R Fab at 50 nM.
The MACS round resulted in 9.1E6 cells being selected.
[0481] The cells that were selected in the MACS1 round were sorted
by FACS using 1 nM anti-IL6R Fab-dylight (MACS1FACS1, M1F1). For
the first round of FACS, the entire positive population was sorted.
Greater than 1E7 cells were sorted.
[0482] The cells that were selected in the MACS round were sorted
by FACS using 100 .mu.M anti-IL6R Fab-dylight (MACS1FACS2, M1F2).
Greater than 1E7 were sorted.
[0483] Twenty four clones were sequenced from the M1F2 pool. One
sequence, SEQ ID NO: 83, was significantly enriched. Several of the
peptides were compared with the peptide from the naive library and
soft randomization library. The sequences are shown below in Table
9. All peptides compared had an increased on cell affinity measured
by FACS as shown in FIG. 9.
TABLE-US-00015 TABLE 9 Sequences from the Peptide 2 directed
library sort M1F2 0verrepesentation SEQ ID in population NO:
Sequence sequenced 83 RGDGNDSDIPFPAHWVPRT 14x 84
SGVGRDRDIPFPAHWVPRT 85 WAGGNDCDIPFPAHWIPNT 3x 86
WGDGMDVDIPFPAHWVPVT 87 AGSGNDSDIPFPAHWVPRT 2x 88
ESRSGYADIPFPAHWVPRT 89 RECGRCGDIPFPAHWVPRT
Example 3. Evaluation of Efficiency of Masking Moieties
[0484] Masking the ability of an antibody to bind to its antigen is
an example of inhibition of binding. The extent of inhibition is
dependent on the affinity of the antibody for its antigen, the
affinity of the inhibitor for the antibody and the concentration of
all reactants. Local concentrations of the tethered peptide mask
(inhibitor) is very high in the activatable antibody context, on
the order of 10 mM, therefore moderate affinity peptides would
effectively mask activatable antibody antigen binding.
[0485] However, since peptide affinity is largely determined by
off-rate, displacement of the mask by antigen with a high affinity,
would present as a slow on-rate antibody, therefore long term
binding measurements are necessary to accurately measure the
masking efficiency of a tethered peptide inhibitor. To this end, a
time and concentration dependent ELISA assay, run at 37.degree. C.,
has been developed to measure the equilibrium binding of an
activatable antibody to its antigen. By comparing the equilibrium
activatable antibody binding to parental antibody binding under the
same conditions, masking efficiency as expressed as a shift in
apparent K.sub.D for activatable antibody/parental antibody binding
can be calculated.
[0486] The general outline for this assay is as follows: Nunc,
Maxisorp plates are coated overnight at 4.degree. C. with 100
.mu.l/well of a 500 ng/mL solution of human IL6R (R and D systems
cat #227-SR/CF) in PBS, pH 7.4. Plates are washed 3.times.PBST
(PBS, pH 7.4, 0.05% Tween-20), and wells are blocked with 200
ml/well, 2% NFDM (non-fat dry milk) in PBST for 2 hours at RT.
Plates are washed 3.times.PBST (PBS, pH 7.4, 0.05% Tween-20).
Dilution curves are prepared as shown below in Table 10.
TABLE-US-00016 TABLE 10 Plate layout for Masking efficiency assay,
one plate for each time point. [activatable [activatable
[activatable [Antibody] = antibody antibody antibody nM 1] = nM 2]
= nM 3] = nM Columns 1-3 Columns 4-6 Columns 7-9 Columns 10-12 A
300 1000 1000 1000 B 100 333 333 333 C 33.3 111 111 111 D 11.1 37
37 37 E 3.7 12.3 12.3 12.3 F 1.23 4.1 4.1 4.1 G 0.41 1.34 1.34 1.34
H 0.134 0.45 0.45 Blank
[0487] Plates are incubated 3 hours at 37.degree. C., and then
washed 3.times.PBST (PBS, pH 7.4, 0.05% Tween-20). 100 ml/well
1:3000 dilution gt-anti-human IgG (Fab specific, Sigma cat # A0293)
in 2% NFDM-PBST is added, and the plate is incubated for 1 hour RT.
The plate is developed with TMB and 1N HCL.
[0488] Generation 1 masking peptides, as discovered from a naive
library, have low to moderate affinity, and when incorporated into
an activatable antibody would not be expected to strongly shift the
apparent affinity of the activatable antibody binding from that of
the parental antibody. However, affinity matured peptides in
Generations 2 and 3, when incorporated into activatable antibodies
would increasingly shift the apparent affinity of the activatable
antibody, as compared to the parental antibody, to the right. It
might also be expected that above some affinity, peptide masks,
when incorporated into an activatable antibody would not diminish
the affinity of the activatable antibody any further.
[0489] Shown in FIG. 10 are plots of binding isotherms for
anti-IL6R activatable antibodies that include the AV1 antibody
described above, where the activatable antibodies include
Generation 1, 2 and 3 masks, and selective protease substrates.
Generation 1 masks Peptide 1 and Peptide 2, described above in
Examples 1 and 2, were incorporated into activatable antibodies
with one substrate (MMP/BV728).
[0490] Second generation masks SEQ ID NO: 78 (based on Peptide 2)
and SEQ ID NO: 49 (based on Peptide 1) were isolated from high
stringency screening of softly randomized libraries based on
Peptide 2 and Peptide 1 respectively. As with generation 1 masks,
SEQ ID NO: 78 and SEQ ID NO: 49 were incorporated in activatable
antibodies with (S) and without (E) an amino terminal extension,
and with a number of protease substrates. Masking efficiency assays
led to three observations. First, both masks potently shifted the
apparent K.sub.D for the activatable antibodies as compared to the
parental antibody (FIG. 11 and FIG. 12). Second, as with the first
generation mask Peptide 1, inclusion of the amino terminal
extension improved the masking of SEQ ID NO: 49, which is derived
from Peptide 1 (data not shown).
[0491] Comparing the data in FIGS. 10 and 11 it is clear that
affinity maturation of masks Peptide 1 and Peptide 2 led to the
development of higher affinity peptides SEQ ID NO: 49 and SEQ ID
NO: 78, and better masked activatable antibodies. For activatable
antibodies containing mask SEQ ID NO: 78 the apparent affinity is
between 300 nM and 1 .mu.M. For activatable antibodies containing
mask SEQ ID NO: 49 the affinity is greater than 1 .mu.m. Neither
activatable antibody binds IL6R.alpha. at 10 nM, the typical
concentration of activatable antibody found in tissues following
treatment at 20 mg/Kg.
[0492] A second round of affinity maturation of SEQ ID NO: 49,
through random amino and carboxy extensions of the core consensus
resulted in discovery of the mask of SEQ ID NO: 74, which binds
AV1-Fab better than Peptide 1 or SEQ ID NO: 49 (FIG. 13 top panel).
However, when the mask of SEQ ID NO: 74 was incorporated into an
activatable antibody the masking efficiency is not improved over
SEQ ID NO: 49 (FIG. 13, bottom panel). These data suggest that the
maximum, non-covalent masking efficiency for AV1 activatable
antibodies has been reached.
Example 4. In Vivo Evaluation of Anti-IL6R Activatable
Antibodies
[0493] The studies presented herein are designed to evaluate the in
vivo stability of various anti-IL6R activatable antibodies that
include heavy and light chain sequences based on the AV1 antibody.
The AV1-based activatable anti-IL6R antibodies differ in substrate
and/or masking regions. AV1 is similar to ACTEMRA.RTM., a humanized
IgG1 that binds and blocks the human IL-6 receptor and has been
approved for use in the treatment of rheumatoid arthritis and
systemic juvenile idiopathic arthritis. AV1 does not cross bind
mouse IL-6R.
[0494] The treatment groups are as shown below in Table 11.
TABLE-US-00017 TABLE 11 Treatment Groups: SEQ Dose Dose Vol Group
Count Test Article ID NO: (mg/kg) (ml/kg) 1 3 Actemra 10 10 2 3
S4749.sup.NSUBAV1 15 10 10 3 3 S4749.sup.1203AV1 16 10 10 4 3
S4749.sup.1204AV1 17 10 10 5 3 S4749.sup.1214AV1 18 10 10 6 3
S4749.sup.PLGLAV1 19 10 10 7 3 S4749.sup.BV726AV1 110 10 10 8 3
S4792.sup.NSUBAV1 6 10 10 9 3 S4792.sup.1203AV1 7 10 10 10 3
S4792.sup.1204AV1 8 10 10 11 3 S4792.sup.1214AV1 9 10 10 12 3
S4792.sup.PLGLAV1 10 10 10 13 3 S4792.sup.BV726AV1 109 10 10
[0495] In these studies, mice received a single 10 mg/kg IP
administration of ACTEMRA.RTM. or an AV1-based activatable
anti-IL6R antibody. Mice were euthanized by CO2 asphyxiation at 96
hours post dose. Terminal blood (.about.1 mL) was collected using
K.sub.2EDTA as an anticoagulant. Samples were processed within 1
hour of collection and the plasma stored at -80.degree. C.
[0496] Total human IgG concentration was measured in by ELISA.
Total IgG concentration in plasma, 96 hours post-10 mg/kg dose for
each antibody and/or activatable antibody is shown in FIG. 14.
[0497] The concentration of active antibody or AV1-based
activatable anti-IL6R antibody in plasma was measured by antigen
(hIL6R) binding ELISA. The close agreement between the AV1 standard
curve and the binding of AV1 in the plasma of treated mice,
demonstrates the accuracy of the IgG ELISA for determining the
total human IgG concentration in mouse plasma. Two animals treated
with an uncleavable (NSUB) activatable antibody comprising the mask
of SEQ ID NO: 78 did not bind antigen showing the stability of the
mask of SEQ ID NO: 78, and its ability to inhibit AV1 binding
activity.
[0498] The data in FIG. 16 show that the 2.sup.nd generation mask
of SEQ ID NO: 78 potently inhibits the binding of AV1 activatable
antibodies to IL6R.alpha., and these activatable antibodies are
stable in vivo, in mice for 96 hours. In addition, the substrates
1203, 1204, 1214, and PLGL are stable to protease digestion in
vivo, demonstrated by the lack of binding to IL6R.alpha. in this
assay. The substrate MMP (BV726) (denoted MMP in the Figure) is
cleaved to about 20% of AV1.
[0499] The data in FIG. 17 show that the 2.sup.nd generation mask
of SEQ ID NO: 49 potently inhibits the binding of AV1 activatable
antibodies to IL6R.alpha. and these activatable antibodies are
stable in vivo, in mice, for 96 hours. As seen for activatable
antibodies containing the mask of SEQ ID NO: 78, the activatable
antibodies masked with the mask of SEQ ID NO: 49 are stable to
protease digestion in vivo, demonstrated by the lack of binding to
IL6R.alpha. in this assay. Also like the activatable antibodies
shown in FIG. 16, the substrate MMP (BV726) (denoted in the Figure
as MMP) is cleaved to about 20% of AV1 in these activatable
antibodies as well.
[0500] Incorporation of the mask of SEQ ID NO: 49 into AV1
activatable antibodies shifts the apparent affinity of the AV1
activatable antibody by >500 fold. These data show that the mask
of SEQ ID NO: 49 is stable in vivo, in mice, for up to 96 hours,
and plasma borne activatable antibodies do not bind IL6R.alpha. at
concentrations saturating for AV1.
Example 5. Ability of Synovial Fluid to Activate Activatable
Antibodies of the Disclosure
[0501] The following anti-IL6R antibodies and activatable anti-IL6R
antibodies were used in the studies described herein:
TABLE-US-00018 TABLE 13 Group Test Article SEQ ID NO: 1 Actemra 8
S4792.sup.NSUBAV1 6 9 S4792.sup.1203AV1 7 10 S4792.sup.1204AV1 8 11
S4792.sup.1214AV1 9 12 S4792.sup.PLGLAV1 10 13 S4792.sup.BV726AV1
109
[0502] ACTEMRA.RTM. reaches serum levels of 588-1220 nM in patients
following treatment, and HUMIRA.RTM. concentration in synovial
fluid (SyF) reaches 30-96% of serum levels. Based on these
literature values, the concentration of anti-IL-6R activatable
antibodies used in these SyF digests was 645 nM. The incubation of
activatable antibodies in SyF and sera were set up as described in
herein. 2.5 .mu.l of a 6.45 .mu.M solution of activatable antibody
was combined with 22.5 .mu.l of serum or SyF and incubated for 0,
8, 24 and 48 hours at 37.degree. C.
[0503] For this study, capillary electrophoresis (CE) was used to
evaluate activation. At each time point in the experiment, 5 .mu.l
of reaction was transferred to 100 .mu.l of CE running buffer with
.beta.-mercaptoethanol ((ME) and heated to 95.degree. C. for 15
minutes and then frozen at -20.degree. C. At the end of the study,
all samples were analyzed by CE. FIG. 18 depicts a simulated gel
from CE analysis of SyF and serum activatable antibody reactions
(from top to bottom donors, 2, 3, 6 and 7 respectively).
[0504] The area of each peak was calculated and used to calculate
the fraction of activated antibody (FIG. 19). From these data, it
can be seen that the rate and extent of activation is dependent on
substrate; there is also a correlation with the kinetics of
activation by purified MMP9 (FIG. 20).
[0505] These data show that anti-IL-6R activatable antibodies
containing substrates 1203, 1214, PLGL and BV726 are cleaved in the
synovial fluid of donor 7 but not in any of the other synovial
fluids. Anti-IL-6R activatable antibodies containing substrates
1203 and 1214 show cleavage after 24 hours and 30-50% activation in
48 hours. Anti-IL-6R activatable antibodies containing substrates
PLGL and BV726 show cleavage by 8 hours and are completely
activated by 48 hours. The rate and extent of activation correlates
with Kcat/Km for activatable antibody cleavage with purified MMP-9.
The anti-IL-6R activatable antibody containing substrate BV726 is
activated in the serum of donor 6 but not in other donors, and none
of the other anti-IL-6R activatable antibodies are activated in
sera from any donor.
Example 6. Activation of Substrates of the Disclosure at the Site
of Inflamed Joints in Rheumatoid Arthritis
[0506] The studies described herein were designed to evaluate how a
substrate included in activatable antibodies described herein is
activated in a widely known animal model for rheumatoid arthritis,
the collagen induced arthritis (CIA) mouse. The CIA mouse model is
the most commonly studied autoimmune model of rheumatoid arthritis.
Autoimmune arthritis is induced in this model by immunization with
an emulsion of complete Freund's adjuvant and type II collagen
(CII) (see e.g., Williams, "Collagen-induced arthritis as a model
for rheumatoid arthritis." Methods Mol Med. 98:207-16 (2004); Brand
et al., "Collagen-induced arthritis." Nat Protoc. 2(5):1269-75
(2007)).
[0507] The paws of CIA mice were scored for inflammation severity
based on the measure of swelling. CIA mice with different scores of
inflammation severity in each of their four paws were injected
intravenously with a substrate-containing quenched probe and imaged
at different time points. Upon cleavage of the substrate, the
quencher dissociated from the probe, leading to an increase of
fluorescent signal at the site of activation.
[0508] The CIA mouse in FIG. 21A has two normal non-inflamed paws
(front left and hind right paws) and two paws with swelling
indicating an inflammatory process (front right and hind left
paws). Two hours after the injection of a quenched probe containing
substrate 1203, a significant increase of fluorescent signal was
detected in the paws with swelling as compared to the paws without
inflammation, indicating that the increase in protease activity
required for substrate 1203 cleavage is associated with the
inflammatory process.
[0509] Similarly, the CIA mouse in FIG. 21B, which was administered
MMP substrate-containing probe MMPSense (Perkin Elmer), indicates
an increase in MMP protease activity associated with the
inflammatory process.
Example 7. Ability of Synovial Fluid to Activate Quenched Probes
Comprising Substrates of the Disclosure
[0510] This Example demonstrates the ability of synovial fluid
samples to cleave a quenched probe comprising the substrate
referred to herein as substrate 1203 but not to cleave a quenched
probe comprising NSUB, a non-cleavable moiety.
[0511] The studies described herein used the following reagents
shown below in Table 14.
TABLE-US-00019 TABLE 14 Reagents Buffers: 1 .times. TBST + 50 mM
Tris pH = 7.4, 150 mM Ca + Zn: NaCl, 0.05% Tween-20, 5 mM
CaCl.sub.2, 100 .mu.M ZnCl.sub.2 1 .times. TBST: 50 mM Tris pH =
7.4, 150 mM NaCl, 0.05% Tween-20 4 .times. 4 .times. Broad 820
.mu.L 1 .times. TBST, 80 .mu.L 0.5 The concentration of protease
Protease spectrum M EDTA (Teknova, Cat inhibitors at 1 .times. are:
500 .mu.M Inhibitor inhibitor E0302), 80 .mu.L 0.5 M o- AEB SF, 400
nM aprotinin, cocktails: cocktail phenanthroline (EMD, Cat 25 .mu.M
bestatin, 7.5 .mu.M E- 516705), 20 .mu.L 200 .times. Protease 64,
10 .mu.M leupeptin, 5 .mu.M Inhibitor Cocktail Set III, pepstatin
A, 10 mM EDTA, EDTA free (EMD, Cat 10 mM o-phenanthroline 539134) 4
.times. Serine 960 .mu.L 1 .times. TBST, 40 .mu.L 100 .times. The
concentration of protease protease Serine Protease Inhibitor
inhibitors at 1 .times. are: 500 .mu.M inhibitor Cocktail Set I
(EMD, Cat AEBSF, 420 nM aprotinin, cocktail 565000) 20 .mu.M
elastatinal, 1 .mu.M H- Glu-Gly-Arg-CMK 4 .times. Cysteine 950
.mu.L 1 .times. TBST, 40 .mu.L 100 .times. The concentration of
protease protease Protease Inhibitor Cocktail inhibitors at 1
.times. are: 15.6 .mu.M inhibitor Set VIII (EMD, Cat 539129),
Ac-Leu-Leu-Nle-CHO, 5 .mu.M cocktail 10 .mu.L 1 mg/ml human
Z-Phe-Gly-NHO-Bz, 15 .mu.M cystatin (EMD, Cat 240896) E-64, 10
.mu.g/ml cystatin 4 .times. metallo- 840 .mu.L 1 .times. TBST, 80
.mu.L 0.5 The concentration of protease protease M EDTA (Teknova,
Cat inhibitors at 1 .times. are: 10 mM inhibitor E0306), 80 .mu.L
0.5 M o- EDTA, 10 mM o- cocktail phenanthroline (EMD, Cat
phenanthroline 516705)
[0512] 2.times. IQ probe stocks: The internally quenched (IQ)
probes were composed of a peptide sequence flanked by an N-terminal
HiLyte Fluor.TM. 488 dye (Anaspec) conjugated to the alpha amine of
the most N-terminal amino acid and a C-terminal QXL 520 quencher
conjugated to the epsilon amine of lysine. The C-terminus of the
peptide was amidated. The modifications at the N- and C-terminus
made these IQ probes resistant to exo-peptidases. All HiLyte Fluor
488 contained IQ probes were custom synthesized by Anaspec. The
sequences of the 1203 and NSUB IQ probes are:
TABLE-US-00020 1203 IQ Probe: (SEQ ID NO: 114) HiLyte Fluor
488-Thr-Gly-Arg-Gly-Pro-Ser-Trp-Val- Lys(QXL520)-NH.sub.2 NSUB IQ
Probe: (SEQ ID NO: 115) HiLyte Fluor
488-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser- Lys(QXL520)-NH.sub.2
[0513] 300 nM stocks of the 1203 and NSUB probes were prepared in
1.times.TBST+Ca+Zn. The final concentration of substrate in the
assay was 150 nM. Both probes contained the HiLyte488 fluorophore,
and the plot in FIG. 22 demonstrated that product can be detected
significantly below 150 nM.
[0514] Synovial Fluid Preparation:
[0515] Synovial fluid samples were thawed at 37.degree. C. until a
small amount of frozen material remains and then transferred to
ice. Synovial fluid was then diluted to 80% (v/v) in
1.times.TBST+Ca+Zn and used for the pre-incubation step described
below. Any remaining synovial fluid was stored. Glycerol was added
to a final concentration of 10% (v/v), divided into aliquots,
frozen on dry ice, and then stored in the -80.degree. C.
[0516] The studies were run as follows. Equal volumes of 80%
synovial fluid and either 4.times. protease inhibitor cocktail or
1.times.TBST+Ca+Zn were combined, mixed, and incubated at RT for at
least 1 h.
[0517] A 2.times. substrate plate was prepared by adding 100 .mu.L
of 2.times. substrate stocks to B2-B7 and C2-C7 in a 96-well
polypropylene plate with V-shaped bottom. A black walled, flat
bottomed 96-well plate certified as non-binding was used as the
assay plate, and a schematic representation of the assay plate is
shown below in Table 15.
TABLE-US-00021 TABLE 15 Schematic representation of assay plate 1 2
3 4 5 6 7 8 9 10 11 12 A X X X X X X X X B X No PI Broad PI SPI CPI
MPI Buffer 50 .mu.L X 1203 TH C X No PI Broad PI SPI CPI MPI Buffer
50 .mu.L X NSUB TH D X 25 .mu.L 25 .mu.L 50 .mu.L X X X X X No PI +
MPI + Buffer 25 .mu.L 25 .mu.L Buffer Buffer E X X X X X F G H
Abbreviations: No PI = synovial fluid pre-treated with buffer Broad
PI = synovial fluid pre-treated with broad spectrum protease
inhibitor cocktail SPI = synovial fluid pre-treated with serine
protease inhibitor cocktail CPI = synovial fluid pre-treated with
cysteine spectrum protease inhibitor cocktail MPI = synovial fluid
pre-treated with metalloprotease inhibitor cocktail Buffer = 1
.times. TBST + Zn + Ca TH = totally hydrolyzed
[0518] The wells marked with an "X" in Table 15 were filled with
200 .mu.L buffer/well to protect samples from edge effects. Other
wells were filled with 25 .mu.L/well, unless otherwise indicated.
Synovial fluid alone controls (i.e., no substrate added) were
prepared to account for auto-fluorescence. Previous data showed the
primary determinant of synovial fluid auto-fluorescence was
inclusion of the chelators, o-phenanthroline and EDTA. The
fluorescence intensity of synovial fluid treated with the chelators
was lower than non-treated samples, possibly due to chelation of an
auto-fluorescent compound. Therefore, the auto-fluorescence of all
synovial samples (No PI, Broad PI, SPI, CPI, MPI) can be accounted
for with plus and minus chelator controls.
[0519] Assay Initiation and Fluorimeter Settings:
[0520] The assay was initiated by transferring 25 .mu.L of 2.times.
IQ probe stock from substrate plate to assay plate. All
fluorescence intensity measurements were made on Tecan Infinite 200
plate reader at an excitation wavelength of 485 nm and emission
wavelength of 535 nm. Temperature was maintained at 37.degree. C.
The same batch of 2.times. substrate was used to interrogate all
patient samples; therefore, the gain was optimized to totally
hydrolyzed samples once and then manually entered for all
subsequent assays.
[0521] Data Analysis:
[0522] To calculate product conversion accurately, synovial fluid
auto-fluorescence and fluorescence from uncut substrate was
subtracted from all progress curves. The fluorescence of uncut
substrate in the synovial fluid was estimated by using a buffer
subtracted value. This avoided over-correcting the synovial fluid
with buffer auto-fluorescence.
[ P ] t [ P ] .infin. = ( FI progress curve ) t - [ ( FI synovial
fluid alone ) t + { FI IQ probe alone - Buffer } t ] ( FI totally
hydrolyzed ) t - ( FI IQ probe alone ) t ##EQU00002## [0523] where
Fl is Fluorescent Intensity
[0524] Progress curves shown in units of product conversion are
shown in FIG. 23 for the 1203 substrate. To quantify the impact of
pre-treatment with protease inhibitors, the slope of the linear
portion of the progress curve was obtained by fitting a line to the
data collected 1 hour after substrate addition.
[0525] Analysis of the slopes from the progress curve is shown in
the plot in FIG. 24. Without intending to be bound by theory, the
analysis shown in FIG. 24 appears to indicate a significant
contribution from metalloproteases.
Example 8. Ability of Synovial Fluid to Activate Quenched Probes
Comprising Substrates of the Disclosure
[0526] This Example demonstrates the ability of additional synovial
fluid samples to cleave activatable antibodies of the
disclosure.
[0527] Activatable antibodies S4792.sup.1203AV1 (having amino acid
sequence SEQ ID NO: 7) and S4792.sup.PLGLAV1 (having amino acid
sequence SEQ ID NO: 10) were incubated with synovial fluid as
described in the Examples. The extent of activatable antibody
activation was determined by an ELISA format that measured the
ability of the activatable antibody, following incubation in
synovial fluid, to bind to human IL6R as compared to the binding of
anti-IL6R parental antibody to IL6R. Briefly, Nunc Maxisorp plates
were coated overnight at 4.degree. C. with 100 .mu.l/well of a
500-ng/mL solution of human IL6R (R and D Systems, Cat No.
227-SR/CF) in PBS, pH 7.4. Plates were washed 3 times with PBST
(PBS, pH 7.4, 0.05% Tween-20). Wells were then blocked with 200
.mu.l/well, 2% NFDM (non-fat dry milk) in PB ST for 2 hours at room
temperature. The IL6R-coated plates were washed 3 times with PBST
(PBS, pH 7.4, 0.05% Tween-20). A dilution series of each
activatable antibody--synovial fluid reaction mixture, as well as a
dilution series of the parental anti-IL6R antibody, was added to
appropriate wells of the IL6R-coated ELISA plate. The plates were
incubated 1 hour at room temperature, and then washed 3 times with
PBST (PBS, pH 7.4, 0.05% Tween-20). One hundred .mu.l/well 1:3000
dilution goat-anti-human IgG (Fab specific, Sigma Cat No. A0293) in
2% NFDM-PBST was added, and the plate incubated for 1 hour at room
temperature. The plates were washed 6 times with PBST (PBS, pH 7.4,
0.05% Tween-20) and then developed with TMB and 1N HCl.
[0528] Table 16 provides the results of this experiment in
combination with those reported in Example 5, e.g., FIG. 18.
"Activation in vivo" refers to the percent of activated activatable
antibody found in the plasma of mice administered the activatable
antibody. "Activation in SyF" refers to the percent activation of
the activatable antibodies in synovial fluid using the assay
described above. "Incidence in SyF" refers to the number of
synovial fluid samples tested that demonstrated cleavage of
activatable antibody. The data indicate that anti-IL6R activatable
antibodies comprising substrate 1203 or substrate PLGL are cleaved
by at least some synovial fluid samples (SyF) obtained from RA
patients.
TABLE-US-00022 TABLE 16 Activation Activation Incidence Substrate
in vivo in SyF in SyF 1203 <10% 20% 5/10 PLGL <5% 20-100%
4/10
[0529] The following additional activatable antibodies were
incubated with synovial fluid as described in the Examples:
S4792.sup.10419AV1 (SEQ ID NO: 163); S4792.sup.559AV1 (SEQ ID NO:
164); S4792.sup.601AV1 (SEQ ID NO: 165); S4792.sup.3457AV1 (SEQ ID
NO: 166); S4792.sup.3458AV1 (SEQ ID NO: 167); S4792.sup.3463AV1
(SEQ ID NO: 168); S4792.sup.Throm2 AV1 (SEQ ID NO: 181); and
S4792.sup.Throm3 AV1 (SEQ ID NO: 182).
[0530] The extent of activatable antibody activation was determined
by an ELISA format that measured the ability of the activatable
antibody, following incubation in synovial fluid, to bind to human
IL6R as compared to the binding of anti-IL6R parental antibody to
IL6R. Briefly, Nunc Maxisorp plates were coated overnight at
4.degree. C. with 100 .mu.l/well of a 500-ng/mL solution of human
IL6R (R and D Systems, Cat No. 227-SR/CF) in PBS, pH 7.4. Plates
were washed 3 times with PBST (PBS, pH 7.4, 0.05% Tween-20). Wells
were then blocked with 200 .mu.l/well, 2% NFDM (non-fat dry milk)
in PBST for 2 hours at room temperature. The IL6R-coated plates
were washed 3 times with PBST (PBS, pH 7.4, 0.05% Tween-20). A
dilution series of each activatable antibody--synovial fluid
reaction mixture, as well as a dilution series of the parental
anti-IL6R antibody, was added to appropriate wells of the
IL6R-coated ELISA plate. The plates were incubated 1 hour at room
temperature, and then washed 3 times with PBST (PBS, pH 7.4, 0.05%
Tween-20). One hundred .mu.l/well 1:3000 dilution goat-anti-human
IgG (Fab specific, Sigma Cat No. A0293) in 2% NFDM-PBST was added,
and the plate incubated for 1 hour at room temperature. The plates
were washed 6 times with PBST (PBS, pH 7.4, 0.05% Tween-20) and
then developed with TMB and 1N HCl.
[0531] Table 20 provides the results of this experiment. The data
indicate that anti-IL6R activatable antibodies comprising the
substrates in Table 20 are cleaved by at least some synovial fluid
samples (SyF) obtained from RA patients.
TABLE-US-00023 TABLE 20 Activation Activation Incidence Substrate
in vivo in SyF in SyF 10419 <5% >30% 3/3 559 <5% 20% 3/3
601 <5% >30% 3/3 3457 10% >50% 3/3 3458 10% 20% 3/3 3463
<5% >30% 3/3 Thromb2 <5% 40% 3/3 Thromb3 <5% 40%
3/3
Other Embodiments
[0532] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following.
Sequence CWU 1
1
1931449PRTArtificial Sequencechemically synthesized 1Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln1 5 10 15Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30His
Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40
45Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu
50 55 60Lys Ser Arg Val 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 Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr
Trp Gly Gln Gly 100 105 110Ser Leu 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 Glu Glu Met 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 445Lys2214PRTArtificial Sequencechemically synthesized 2Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr
20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly
Asn Thr Leu Pro Tyr 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 210321PRTArtificial
Sequencechemically synthesized 3Gln Gly Gln Ser Gly Gln Tyr Gly Ser
Cys Ser Trp Asn Tyr Val His1 5 10 15Ile Phe Met Asp Cys
20421PRTArtificial Sequencechemically synthesized 4Gln Gly Gln Ser
Gly Gln Gly Asp Phe Asp Ile Pro Phe Pro Ala His1 5 10 15Trp Val Pro
Ile Thr 20524PRTArtificial Sequencechemically synthesized 5Gln Gly
Gln Ser Gly Gln Met Gly Val Pro Ala Gly Cys Val Trp Asn1 5 10 15Tyr
Ala His Ile Phe Met Asp Cys 206258PRTArtificial Sequencechemically
synthesized 6Gln Gly Gln Ser Gly Gln Tyr Gly Ser Cys Ser Trp Asn
Tyr Val His1 5 10 15Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly
Gly Ser Gly Gly 20 25 30Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
Ser Asp Ile Gln Met 35 40 45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly Asp Arg Val Thr 50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile
Ser Ser Tyr Leu Asn Trp Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr
Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr
Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln 130 135
140Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val
Phe145 150 155 160Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
Thr Ala Ser Val 165 170 175Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala Lys Val Gln Trp 180 185 190Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln Glu Ser Val Thr 195 200 205Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 210 215 220Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val225 230 235 240Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly 245 250
255Glu Cys7258PRTArtificial Sequencechemically synthesized 7Gln Gly
Gln Ser Gly Gln Tyr Gly Ser Cys Ser Trp Asn Tyr Val His1 5 10 15Ile
Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25
30Thr Gly Arg Gly Pro Ser Trp Val Gly Gly Gly Ser Asp Ile Gln Met
35 40 45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val
Thr 50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn
Trp Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
Tyr Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser
Gly Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr Phe Thr Ile Ser Ser
Leu Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr Tyr Cys Gln Gln Gly
Asn Thr Leu Pro Tyr Thr Phe Gly Gln 130 135 140Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe145 150 155 160Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 165 170
175Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
180 185 190Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr 195 200 205Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr 210 215 220Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val225 230 235 240Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser Phe Asn Arg Gly 245 250 255Glu
Cys8258PRTArtificial Sequencechemically synthesized 8Gln Gly Gln
Ser Gly Gln Tyr Gly Ser Cys Ser Trp Asn Tyr Val His1 5 10 15Ile Phe
Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30Leu
Ser Gly Arg Ser Asp Asn His Gly Gly Gly Ser Asp Ile Gln Met 35 40
45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp
Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu
Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr Tyr Cys Gln Gln Gly Asn
Thr Leu Pro Tyr Thr Phe Gly Gln 130 135 140Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala Pro Ser Val Phe145 150 155 160Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 165 170 175Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp 180 185
190Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
195 200 205Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr
Leu Thr 210 215 220Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
Ala Cys Glu Val225 230 235 240Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser Phe Asn Arg Gly 245 250 255Glu Cys9258PRTArtificial
Sequencechemically synthesized 9Gln Gly Gln Ser Gly Gln Tyr Gly Ser
Cys Ser Trp Asn Tyr Val His1 5 10 15Ile Phe Met Asp Cys Gly Ser Ser
Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30Ser Pro Leu Thr Gly Arg Ser
Gly Gly Gly Gly Ser Asp Ile Gln Met 35 40 45Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly Asp Arg Val Thr 50 55 60Ile Thr Cys Arg Ala
Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp Tyr65 70 75 80Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser 85 90 95Arg Leu
His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 100 105
110Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala
115 120 125Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe
Gly Gln 130 135 140Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
Pro Ser Val Phe145 150 155 160Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val 165 170 175Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp 180 185 190Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr 195 200 205Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 210 215 220Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val225 230
235 240Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
Gly 245 250 255Glu Cys10258PRTArtificial Sequencechemically
synthesized 10Gln Gly Gln Ser Gly Gln Tyr Gly Ser Cys Ser Trp Asn
Tyr Val His1 5 10 15Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly
Gly Ser Gly Gly 20 25 30Ser Gly Gly Gly Ser Pro Leu Gly Leu Gly Gly
Ser Asp Ile Gln Met 35 40 45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly Asp Arg Val Thr 50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile
Ser Ser Tyr Leu Asn Trp Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr
Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr
Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln 130 135
140Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val
Phe145 150 155 160Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
Thr Ala Ser Val 165 170 175Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala Lys Val Gln Trp 180 185 190Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln Glu Ser Val Thr 195 200 205Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 210 215 220Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val225 230 235 240Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly 245 250
255Glu Cys11258PRTArtificial Sequencechemically synthesized 11Gln
Gly Gln Ser Gly Gln Tyr Gly Ser Cys Ser Trp Asn Tyr Val His1 5 10
15Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly
20 25 30Gly Gln Pro Ser Gly Met Trp Gly Trp Gly Gly Ser Asp Ile Gln
Met 35 40 45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg
Val Thr 50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu
Asn Trp Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile Tyr Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr Phe Thr Ile Ser
Ser Leu Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr Tyr Cys Gln Gln
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln 130 135 140Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe145 150 155 160Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 165 170
175Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
180 185 190Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr 195 200 205Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr 210 215 220Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val225 230 235 240Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser Phe Asn Arg Gly 245 250 255Glu
Cys12258PRTArtificial Sequencechemically synthesized 12Gln Gly Gln
Ser Gly Gln Tyr Gly Ser Cys Ser Trp Asn Tyr Val His1 5 10 15Ile Phe
Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Phe 20 25 30Pro
Arg Pro Leu Gly Ile Thr Gly Leu Gly Gly Ser Asp Ile Gln Met 35 40
45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp
Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly 100 105
110Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala
115 120 125Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe
Gly Gln 130 135 140Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
Pro Ser Val Phe145 150 155 160Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val 165 170 175Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp 180 185 190Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr 195 200 205Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 210 215 220Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val225 230
235 240Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
Gly 245 250 255Glu Cys13258PRTArtificial Sequencechemically
synthesized 13Gln Gly Gln Ser Gly Gln Tyr Gly Ser Cys Ser Trp Asn
Tyr Val His1 5 10 15Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly
Gly Ser Gly Gly 20 25 30Ser Gly Gly Pro Leu Gly Val Arg Gly Gly Gly
Ser Asp Ile Gln Met 35 40 45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly Asp Arg Val Thr 50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile
Ser Ser Tyr Leu Asn Trp Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr
Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr
Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln 130 135
140Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val
Phe145 150 155 160Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
Thr Ala Ser Val 165 170 175Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala Lys Val Gln Trp 180 185 190Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln Glu Ser Val Thr 195 200 205Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 210 215 220Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val225 230 235 240Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly 245 250
255Glu Cys14258PRTArtificial Sequencechemically synthesized 14Gln
Gly Gln Ser Gly Gln Tyr Gly Ser Cys Ser Trp Asn Tyr Val His1 5 10
15Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Ser
20 25 30Leu Ala Pro Leu Gly Leu Gln Arg Arg Gly Gly Ser Asp Ile Gln
Met 35 40 45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg
Val Thr 50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu
Asn Trp Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile Tyr Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr Phe Thr Ile Ser
Ser Leu Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr Tyr Cys Gln Gln
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln 130 135 140Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe145 150 155 160Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 165 170
175Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
180 185 190Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr 195 200 205Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr 210 215 220Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val225 230 235 240Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser Phe Asn Arg Gly 245 250 255Glu
Cys15258PRTArtificial Sequencechemically synthesized 15Gln Gly Gln
Ser Gly Gln Gly Asp Phe Asp Ile Pro Phe Pro Ala His1 5 10 15Trp Val
Pro Ile Thr Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30Ser
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Ser Asp Ile Gln Met 35 40
45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp
Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu
Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr Tyr Cys Gln Gln Gly Asn
Thr Leu Pro Tyr Thr Phe Gly Gln 130 135 140Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala Pro Ser Val Phe145 150 155 160Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 165 170 175Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp 180 185
190Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
195 200 205Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr
Leu Thr 210 215 220Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
Ala Cys Glu Val225 230 235 240Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser Phe Asn Arg Gly 245 250 255Glu Cys16258PRTArtificial
Sequencechemically synthesized 16Gln Gly Gln Ser Gly Gln Gly Asp
Phe Asp Ile Pro Phe Pro Ala His1 5 10 15Trp Val Pro Ile Thr Gly Ser
Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30Thr Gly Arg Gly Pro Ser
Trp Val Gly Gly Gly Ser Asp Ile Gln Met 35 40 45Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 50 55 60Ile Thr Cys Arg
Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp Tyr65 70 75 80Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser 85 90 95Arg
Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 100 105
110Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala
115 120 125Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe
Gly Gln 130 135 140Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
Pro Ser Val Phe145 150 155 160Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val 165 170 175Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp 180 185 190Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr 195 200 205Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 210 215 220Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val225 230
235 240Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
Gly 245 250 255Glu Cys17258PRTArtificial Sequencechemically
synthesized 17Gln Gly Gln Ser Gly Gln Gly Asp Phe Asp Ile Pro Phe
Pro Ala His1 5 10 15Trp Val Pro Ile Thr Gly Ser Ser Gly Gly Ser Gly
Gly Ser Gly Gly 20 25 30Leu Ser Gly Arg Ser Asp Asn His Gly Gly Gly
Ser Asp Ile Gln Met 35 40 45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly Asp Arg Val Thr 50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile
Ser Ser Tyr Leu Asn Trp Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr
Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr
Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln 130 135
140Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val
Phe145 150 155 160Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
Thr Ala Ser Val 165 170 175Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala Lys Val Gln Trp 180 185 190Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln Glu Ser Val Thr 195 200 205Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 210 215 220Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val225 230 235 240Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly 245 250
255Glu Cys18258PRTArtificial Sequencechemically synthesized 18Gln
Gly Gln Ser Gly Gln Gly Asp Phe Asp Ile Pro Phe Pro Ala His1 5 10
15Trp Val Pro Ile Thr Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly
20 25 30Ser Pro Leu Thr Gly Arg Ser Gly Gly Gly Gly Ser Asp Ile Gln
Met 35 40 45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg
Val Thr 50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu
Asn Trp Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile Tyr Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr Phe Thr Ile Ser
Ser Leu Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr Tyr Cys Gln Gln
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln 130 135 140Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe145 150 155 160Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 165 170
175Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
180 185 190Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr 195 200 205Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr 210 215 220Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val225 230 235 240Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser Phe Asn Arg Gly 245 250 255Glu
Cys19258PRTArtificial Sequencechemically synthesized 19Gln Gly Gln
Ser Gly Gln Gly Asp Phe Asp Ile Pro Phe Pro Ala His1 5 10 15Trp Val
Pro Ile Thr Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30Ser
Gly Gly Gly Ser Pro Leu Gly Leu Gly Gly Ser Asp Ile Gln Met 35 40
45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp
Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu
Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr Tyr Cys Gln Gln Gly Asn
Thr Leu Pro Tyr Thr Phe Gly Gln 130 135 140Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala Pro Ser Val Phe145 150 155 160Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 165 170 175Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp 180 185
190Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
195 200 205Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr
Leu Thr 210 215 220Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
Ala Cys Glu Val225 230 235 240Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser Phe Asn Arg Gly 245 250 255Glu Cys20258PRTArtificial
Sequencechemically synthesized 20Gln Gly Gln Ser Gly Gln Gly Asp
Phe Asp Ile Pro Phe Pro Ala His1 5 10 15Trp Val Pro Ile Thr Gly Ser
Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30Gly Gln Pro Ser Gly Met
Trp Gly Trp Gly Gly Ser Asp Ile Gln Met 35 40 45Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 50 55 60Ile Thr Cys Arg
Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp Tyr65 70 75 80Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser 85 90 95Arg
Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 100 105
110Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala
115 120 125Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe
Gly Gln 130 135 140Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
Pro Ser Val Phe145 150 155 160Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val 165 170 175Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp 180 185 190Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr 195 200 205Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 210 215 220Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val225 230
235 240Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
Gly 245 250 255Glu Cys21258PRTArtificial Sequencechemically
synthesized 21Gln Gly Gln Ser Gly Gln Gly Asp Phe Asp Ile Pro Phe
Pro Ala His1 5 10 15Trp Val Pro Ile Thr Gly Ser Ser Gly Gly Ser Gly
Gly Ser Gly Phe 20 25 30Pro Arg Pro Leu Gly Ile Thr Gly Leu Gly Gly
Ser Asp Ile Gln Met 35 40 45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly Asp Arg Val Thr 50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile
Ser Ser Tyr Leu Asn Trp Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr
Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr
Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln 130 135
140Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val
Phe145 150 155 160Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
Thr Ala Ser Val 165 170 175Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala Lys Val Gln Trp 180 185
190Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
195 200 205Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr
Leu Thr 210 215 220Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
Ala Cys Glu Val225 230 235 240Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser Phe Asn Arg Gly 245 250 255Glu Cys22258PRTArtificial
Sequencechemically synthesized 22Gln Gly Gln Ser Gly Gln Gly Asp
Phe Asp Ile Pro Phe Pro Ala His1 5 10 15Trp Val Pro Ile Thr Gly Ser
Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30Ser Gly Gly Pro Leu Gly
Val Arg Gly Gly Gly Ser Asp Ile Gln Met 35 40 45Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 50 55 60Ile Thr Cys Arg
Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp Tyr65 70 75 80Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser 85 90 95Arg
Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 100 105
110Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala
115 120 125Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe
Gly Gln 130 135 140Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
Pro Ser Val Phe145 150 155 160Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val 165 170 175Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp 180 185 190Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr 195 200 205Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 210 215 220Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val225 230
235 240Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
Gly 245 250 255Glu Cys23258PRTArtificial Sequencechemically
synthesized 23Gln Gly Gln Ser Gly Gln Gly Asp Phe Asp Ile Pro Phe
Pro Ala His1 5 10 15Trp Val Pro Ile Thr Gly Ser Ser Gly Gly Ser Gly
Gly Ser Gly Ser 20 25 30Leu Ala Pro Leu Gly Leu Gln Arg Arg Gly Gly
Ser Asp Ile Gln Met 35 40 45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly Asp Arg Val Thr 50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile
Ser Ser Tyr Leu Asn Trp Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr
Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr
Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln 130 135
140Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val
Phe145 150 155 160Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
Thr Ala Ser Val 165 170 175Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala Lys Val Gln Trp 180 185 190Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln Glu Ser Val Thr 195 200 205Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 210 215 220Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val225 230 235 240Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly 245 250
255Glu Cys24261PRTArtificial Sequencechemically synthesized 24Gln
Gly Gln Ser Gly Gln Met Gly Val Pro Ala Gly Cys Val Trp Asn1 5 10
15Tyr Ala His Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly
20 25 30Ser Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Ser
Asp 35 40 45Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly Asp 50 55 60Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser
Ser Tyr Leu65 70 75 80Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile Tyr 85 90 95Tyr Thr Ser Arg Leu His Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser 100 105 110Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Gln Pro Glu 115 120 125Asp Ile Ala Thr Tyr Tyr
Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr 130 135 140Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro145 150 155 160Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 165 170
175Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
180 185 190Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln Glu 195 200 205Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser Ser 210 215 220Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr Ala225 230 235 240Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser Phe 245 250 255Asn Arg Gly Glu Cys
26025261PRTArtificial Sequencechemically synthesized 25Gln Gly Gln
Ser Gly Gln Met Gly Val Pro Ala Gly Cys Val Trp Asn1 5 10 15Tyr Ala
His Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly 20 25 30Ser
Gly Gly Thr Gly Arg Gly Pro Ser Trp Val Gly Gly Gly Ser Asp 35 40
45Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp
50 55 60Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr
Leu65 70 75 80Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile Tyr 85 90 95Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser 100 105 110Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile
Ser Ser Leu Gln Pro Glu 115 120 125Asp Ile Ala Thr Tyr Tyr Cys Gln
Gln Gly Asn Thr Leu Pro Tyr Thr 130 135 140Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala Pro145 150 155 160Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 165 170 175Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 180 185
190Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
195 200 205Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser Ser 210 215 220Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr Ala225 230 235 240Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe 245 250 255Asn Arg Gly Glu Cys
26026261PRTArtificial Sequencechemically synthesized 26Gln Gly Gln
Ser Gly Gln Met Gly Val Pro Ala Gly Cys Val Trp Asn1 5 10 15Tyr Ala
His Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly 20 25 30Ser
Gly Gly Leu Ser Gly Arg Ser Asp Asn His Gly Gly Gly Ser Asp 35 40
45Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp
50 55 60Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr
Leu65 70 75 80Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile Tyr 85 90 95Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser 100 105 110Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile
Ser Ser Leu Gln Pro Glu 115 120 125Asp Ile Ala Thr Tyr Tyr Cys Gln
Gln Gly Asn Thr Leu Pro Tyr Thr 130 135 140Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala Pro145 150 155 160Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 165 170 175Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 180 185
190Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
195 200 205Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser Ser 210 215 220Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr Ala225 230 235 240Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe 245 250 255Asn Arg Gly Glu Cys
26027261PRTArtificial Sequencechemically synthesized 27Gln Gly Gln
Ser Gly Gln Met Gly Val Pro Ala Gly Cys Val Trp Asn1 5 10 15Tyr Ala
His Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly 20 25 30Ser
Gly Gly Ser Pro Leu Thr Gly Arg Ser Gly Gly Gly Gly Ser Asp 35 40
45Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp
50 55 60Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr
Leu65 70 75 80Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile Tyr 85 90 95Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser 100 105 110Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile
Ser Ser Leu Gln Pro Glu 115 120 125Asp Ile Ala Thr Tyr Tyr Cys Gln
Gln Gly Asn Thr Leu Pro Tyr Thr 130 135 140Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala Pro145 150 155 160Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 165 170 175Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 180 185
190Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
195 200 205Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser Ser 210 215 220Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr Ala225 230 235 240Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe 245 250 255Asn Arg Gly Glu Cys
26028261PRTArtificial Sequencechemically synthesized 28Gln Gly Gln
Ser Gly Gln Met Gly Val Pro Ala Gly Cys Val Trp Asn1 5 10 15Tyr Ala
His Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly 20 25 30Ser
Gly Gly Ser Gly Gly Gly Ser Pro Leu Gly Leu Gly Gly Ser Asp 35 40
45Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp
50 55 60Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr
Leu65 70 75 80Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile Tyr 85 90 95Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser 100 105 110Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile
Ser Ser Leu Gln Pro Glu 115 120 125Asp Ile Ala Thr Tyr Tyr Cys Gln
Gln Gly Asn Thr Leu Pro Tyr Thr 130 135 140Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala Pro145 150 155 160Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 165 170 175Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 180 185
190Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
195 200 205Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser Ser 210 215 220Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr Ala225 230 235 240Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe 245 250 255Asn Arg Gly Glu Cys
26029261PRTArtificial Sequencechemically synthesized 29Gln Gly Gln
Ser Gly Gln Met Gly Val Pro Ala Gly Cys Val Trp Asn1 5 10 15Tyr Ala
His Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly 20 25 30Ser
Gly Gly Gly Gln Pro Ser Gly Met Trp Gly Trp Gly Gly Ser Asp 35 40
45Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp
50 55 60Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr
Leu65 70 75 80Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile Tyr 85 90 95Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser 100 105 110Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile
Ser Ser Leu Gln Pro Glu 115 120 125Asp Ile Ala Thr Tyr Tyr Cys Gln
Gln Gly Asn Thr Leu Pro Tyr Thr 130 135 140Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala Pro145 150 155 160Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 165 170 175Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 180 185
190Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
195 200 205Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser Ser 210 215 220Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr Ala225 230 235 240Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe 245 250 255Asn Arg Gly Glu Cys
26030261PRTArtificial Sequencechemically synthesized 30Gln Gly Gln
Ser Gly Gln Met Gly Val Pro Ala Gly Cys Val Trp Asn1 5 10 15Tyr Ala
His Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly 20 25 30Ser
Gly Phe Pro Arg Pro Leu Gly Ile Thr Gly Leu Gly Gly Ser Asp 35 40
45Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp
50 55 60Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr
Leu65 70 75 80Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile Tyr 85 90 95Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser 100 105 110Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile
Ser Ser Leu Gln Pro Glu 115 120 125Asp Ile Ala Thr Tyr Tyr Cys Gln
Gln Gly Asn Thr Leu Pro Tyr Thr 130 135 140Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala Pro145 150 155 160Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 165 170 175Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 180 185
190Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
195 200 205Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser Ser 210 215 220Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr Ala225 230 235 240Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe 245 250 255Asn Arg Gly Glu Cys
26031261PRTArtificial Sequencechemically synthesized
31Gln Gly Gln Ser Gly Gln Met Gly Val Pro Ala Gly Cys Val Trp Asn1
5 10 15Tyr Ala His Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly
Gly 20 25 30Ser Gly Gly Ser Gly Gly Pro Leu Gly Val Arg Gly Gly Gly
Ser Asp 35 40 45Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly Asp 50 55 60Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile
Ser Ser Tyr Leu65 70 75 80Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr 85 90 95Tyr Thr Ser Arg Leu His Ser Gly Val
Pro Ser Arg Phe Ser Gly Ser 100 105 110Gly Ser Gly Thr Asp Phe Thr
Phe Thr Ile Ser Ser Leu Gln Pro Glu 115 120 125Asp Ile Ala Thr Tyr
Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr 130 135 140Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro145 150 155
160Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
165 170 175Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala Lys 180 185 190Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln Glu 195 200 205Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser Ser 210 215 220Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr Ala225 230 235 240Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 245 250 255Asn Arg Gly
Glu Cys 26032261PRTArtificial Sequencechemically synthesized 32Gln
Gly Gln Ser Gly Gln Met Gly Val Pro Ala Gly Cys Val Trp Asn1 5 10
15Tyr Ala His Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly
20 25 30Ser Gly Ser Leu Ala Pro Leu Gly Leu Gln Arg Arg Gly Gly Ser
Asp 35 40 45Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly Asp 50 55 60Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser
Ser Tyr Leu65 70 75 80Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile Tyr 85 90 95Tyr Thr Ser Arg Leu His Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser 100 105 110Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Gln Pro Glu 115 120 125Asp Ile Ala Thr Tyr Tyr
Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr 130 135 140Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro145 150 155 160Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 165 170
175Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
180 185 190Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln Glu 195 200 205Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser Ser 210 215 220Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr Ala225 230 235 240Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser Phe 245 250 255Asn Arg Gly Glu Cys
2603315PRTArtificial Sequencechemically synthesized 33Tyr Arg Ser
Cys Asn Trp Asn Tyr Val Ser Ile Phe Leu Asp Cys1 5 10
153416PRTArtificial Sequencechemically synthesized 34Pro Gly Ala
Phe Asp Ile Pro Phe Pro Ala His Trp Val Pro Asn Thr1 5 10
153515PRTArtificial Sequencechemically synthesized 35Glu Ser Ser
Cys Val Trp Asn Tyr Val His Ile Tyr Met Asp Cys1 5 10
153615PRTArtificial Sequencechemically synthesized 36Tyr Pro Gly
Cys Lys Trp Asn Tyr Asp Arg Ile Phe Leu Asp Cys1 5 10
153715PRTArtificial Sequencechemically synthesized 37Tyr Arg Thr
Cys Ser Trp Asn Tyr Val Gly Ile Phe Leu Asp Cys1 5 10
153815PRTArtificial Sequencechemically synthesized 38Tyr Gly Ser
Cys Ser Trp Asn Tyr Val His Ile Phe Met Asp Cys1 5 10
153915PRTArtificial Sequencechemically synthesized 39Tyr Gly Ser
Cys Ser Trp Asn Tyr Val His Ile Phe Leu Asp Cys1 5 10
154015PRTArtificial Sequencechemically synthesized 40Tyr Gly Ser
Cys Asn Trp Asn Tyr Val His Ile Phe Leu Asp Cys1 5 10
154115PRTArtificial Sequencechemically synthesized 41Tyr Thr Ser
Cys Asn Trp Asn Tyr Val His Ile Phe Met Asp Cys1 5 10
154215PRTArtificial Sequencechemically synthesized 42Tyr Pro Gly
Cys Lys Trp Asn Tyr Asp Arg Ile Phe Leu Asp Cys1 5 10
154315PRTArtificial Sequencechemically synthesized 43Trp Arg Ser
Cys Asn Trp Asn Tyr Ala His Ile Phe Leu Asp Cys1 5 10
154415PRTArtificial Sequencechemically synthesized 44Trp Ser Asn
Cys His Trp Asn Tyr Val His Ile Phe Leu Asp Cys1 5 10
154515PRTArtificial Sequencechemically synthesized 45Asp Arg Ser
Cys Thr Trp Asn Tyr Val Arg Ile Ser Tyr Asp Cys1 5 10
154615PRTArtificial Sequencechemically synthesized 46Ser Gly Ser
Cys Lys Trp Asp Tyr Val His Ile Phe Leu Asp Cys1 5 10
154715PRTArtificial Sequencechemically synthesized 47Ser Arg Ser
Cys Ile Trp Asn Tyr Ala His Ile His Leu Asp Cys1 5 10
154815PRTArtificial Sequencechemically synthesized 48Ser Met Ser
Cys Tyr Trp Gln Tyr Glu Arg Ile Phe Leu Asp Cys1 5 10
154915PRTArtificial Sequencechemically synthesized 49Tyr Gly Ser
Cys Ser Trp Asn Tyr Val His Ile Phe Met Asp Cys1 5 10
155015PRTArtificial Sequencechemically synthesized 50Ser Gly Ser
Cys Lys Trp Asp Tyr Val His Ile Phe Leu Asp Cys1 5 10
155115PRTArtificial Sequencechemically synthesized 51Tyr Lys Ser
Cys His Trp Asp Tyr Val His Ile Phe Leu Asp Cys1 5 10
155215PRTArtificial Sequencechemically synthesized 52Tyr Gly Ser
Cys Thr Trp Asn Tyr Val His Ile Phe Met Glu Cys1 5 10
155315PRTArtificial Sequencechemically synthesized 53Phe Ser Ser
Cys Asn Trp Asn Tyr Val His Ile Phe Leu Asp Cys1 5 10
155415PRTArtificial Sequencechemically synthesized 54Trp Arg Ser
Cys Asn Trp Asn Tyr Ala His Ile Phe Leu Asp Cys1 5 10
155515PRTArtificial Sequencechemically synthesized 55Tyr Gly Ser
Cys Gln Trp Asn Tyr Val His Ile Phe Leu Asp Cys1 5 10
155615PRTArtificial Sequencechemically synthesized 56Tyr Arg Ser
Cys Asn Trp Asn Tyr Val His Ile Phe Leu Asp Cys1 5 10
155715PRTArtificial Sequencechemically synthesized 57Asn Met Ser
Cys His Trp Asp Tyr Val His Ile Phe Leu Asp Cys1 5 10
155815PRTArtificial Sequencechemically synthesized 58Phe Gly Pro
Cys Thr Trp Asn Tyr Ala Arg Ile Ser Trp Asp Cys1 5 10
155910PRTArtificial Sequencechemically synthesized 59Ser Cys Trp
Tyr Val His Ile Phe Asp Cys1 5 106018PRTArtificial
Sequencechemically synthesizedMISC_FEATURE(1)..(6)Xaa is any amino
acidMISC_FEATURE(8)..(8)Xaa is any amino
acidMISC_FEATURE(10)..(10)Xaa is Asn or
AspMISC_FEATURE(12)..(12)Xaa is Val or AlaMISC_FEATURE(16)..(16)Xaa
is Met or LeuMISC_FEATURE(17)..(17)Xaa is Asp or Glu 60Xaa Xaa Xaa
Xaa Xaa Xaa Cys Xaa Trp Xaa Tyr Xaa His Ile Phe Xaa1 5 10 15Xaa
Cys6118PRTArtificial Sequencechemically
synthesizedMISC_FEATURE(2)..(2)Xaa is any amino
acidMISC_FEATURE(4)..(4)Xaa is Asn or AspMISC_FEATURE(6)..(6)Xaa is
Val or AlaMISC_FEATURE(10)..(10)Xaa is Met or
LeuMISC_FEATURE(11)..(11)Xaa is Asp or GluMISC_FEATURE(13)..(18)Xaa
is any amino acid 61Cys Xaa Trp Xaa Tyr Xaa His Ile Phe Xaa Xaa Cys
Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa6218PRTArtificial Sequencechemically
synthesizedMISC_FEATURE(1)..(3)Xaa is any amino
acidMISC_FEATURE(5)..(5)Xaa is any amino
acidMISC_FEATURE(7)..(7)Xaa is Asn or AspMISC_FEATURE(9)..(9)Xaa is
Val or AlaMISC_FEATURE(13)..(13)Xaa is Met or
LeuMISC_FEATURE(14)..(14)Xaa is Asp or GluMISC_FEATURE(16)..(18)Xaa
is any amino acid 62Xaa Xaa Xaa Cys Xaa Trp Xaa Tyr Xaa His Ile Phe
Xaa Xaa Cys Xaa1 5 10 15Xaa Xaa6318PRTArtificial Sequencechemically
synthesized 63Met Gly Val Pro Ala Gly Cys Val Trp Asn Tyr Ala His
Ile Phe Met1 5 10 15Asp Cys6418PRTArtificial Sequencechemically
synthesized 64Arg Asp Thr Gly Gly Gln Cys Arg Trp Asp Tyr Val His
Ile Phe Met1 5 10 15Asp Cys6518PRTArtificial Sequencechemically
synthesized 65Ala Gly Val Pro Ala Gly Cys Thr Trp Asn Tyr Val His
Ile Phe Met1 5 10 15Glu Cys6618PRTArtificial Sequencechemically
synthesized 66Val Gly Val Pro Asn Gly Cys Val Trp Asn Tyr Ala His
Ile Phe Met1 5 10 15Glu Cys6718PRTArtificial Sequencechemically
synthesized 67Asp Gly Gly Pro Ala Gly Cys Ser Trp Asn Tyr Val His
Ile Phe Met1 5 10 15Glu Cys6818PRTArtificial Sequencechemically
synthesized 68Ala Val Gly Pro Ala Gly Cys Trp Trp Asn Tyr Val His
Ile Phe Met1 5 10 15Glu Cys6918PRTArtificial Sequencechemically
synthesized 69Cys Thr Trp Asn Tyr Val His Ile Phe Met Asp Cys Gly
Glu Gly Glu1 5 10 15Gly Pro7018PRTArtificial Sequencechemically
synthesized 70Gly Gly Val Pro Glu Gly Cys Thr Trp Asn Tyr Ala His
Ile Phe Met1 5 10 15Glu Cys7118PRTArtificial Sequencechemically
synthesized 71Ala Glu Val Pro Ala Gly Cys Trp Trp Asn Tyr Val His
Ile Phe Met1 5 10 15Glu Cys7218PRTArtificial Sequencechemically
synthesized 72Ala Gly Val Pro Ala Gly Cys Thr Trp Asn Tyr Val His
Ile Phe Met1 5 10 15Glu Cys7318PRTArtificial Sequencechemically
synthesized 73Ser Gly Ala Ser Gly Gly Cys Lys Trp Asn Tyr Val His
Ile Phe Met1 5 10 15Asp Cys7418PRTArtificial Sequencechemically
synthesized 74Met Gly Val Pro Ala Gly Cys Val Trp Asn Tyr Ala His
Ile Phe Met1 5 10 15Asp Cys7518PRTArtificial Sequencechemically
synthesized 75Thr Pro Gly Cys Arg Trp Asn Tyr Val His Ile Phe Met
Glu Cys Glu1 5 10 15Ala Leu7618PRTArtificial Sequencechemically
synthesized 76Val Gly Val Pro Asn Gly Cys Val Trp Asn Tyr Ala His
Ile Phe Met1 5 10 15Glu Cys7716PRTArtificial Sequencechemically
synthesized 77Arg Gly Ala Cys Asp Ile Pro Phe Pro Ala His Trp Ile
Pro Asn Thr1 5 10 157816PRTArtificial Sequencechemically
synthesized 78Gln Gly Asp Phe Asp Ile Pro Phe Pro Ala His Trp Val
Pro Ile Thr1 5 10 157915PRTArtificial Sequencechemically
synthesized 79Gly Ala Phe Asp Ile Pro Phe Pro Ala His Trp Val Pro
Asn Thr1 5 10 158019PRTArtificial Sequencechemically
synthesizedMISC_FEATURE(1)..(7)Xaa is any amino
acidMISC_FEATURE(16)..(16)Xaa is Ile or Met or
ValMISC_FEATURE(18)..(18)Xaa is any amino acid 80Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Asp Ile Pro Phe Pro Ala His Trp Xaa1 5 10 15Pro Xaa
Thr8119PRTArtificial Sequencechemically
synthesizedMISC_FEATURE(9)..(9)Xaa is Ile or Met or
Valmisc_feature(11)..(11)Xaa can be any naturally occurring amino
acidMISC_FEATURE(13)..(19)Xaa is any amino acid 81Asp Ile Pro Phe
Pro Ala His Trp Xaa Pro Xaa Thr Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa
Xaa8220PRTArtificial Sequencechemically
synthesizedMISC_FEATURE(1)..(4)Xaa is any amino
acidMISC_FEATURE(13)..(13)Xaa is Ile, Met or
ValMISC_FEATURE(15)..(15)Xaa is any amino
acidMISC_FEATURE(17)..(20)Xaa is any amino acid 82Xaa Xaa Xaa Xaa
Asp Ile Pro Phe Pro Ala His Trp Xaa Pro Xaa1 5 10 15Thr Xaa Xaa Xaa
Xaa 208319PRTArtificial Sequencechemically synthesized 83Arg Gly
Asp Gly Asn Asp Ser Asp Ile Pro Phe Pro Ala His Trp Val1 5 10 15Pro
Arg Thr8419PRTArtificial Sequencechemically synthesized 84Ser Gly
Val Gly Arg Asp Arg Asp Ile Pro Phe Pro Ala His Trp Val1 5 10 15Pro
Arg Thr8519PRTArtificial Sequencechemically synthesized 85Trp Ala
Gly Gly Asn Asp Cys Asp Ile Pro Phe Pro Ala His Trp Ile1 5 10 15Pro
Asn Thr8619PRTArtificial Sequencechemically synthesized 86Trp Gly
Asp Gly Met Asp Val Asp Ile Pro Phe Pro Ala His Trp Val1 5 10 15Pro
Val Thr8719PRTArtificial Sequencechemically synthesized 87Ala Gly
Ser Gly Asn Asp Ser Asp Ile Pro Phe Pro Ala His Trp Val1 5 10 15Pro
Arg Thr8819PRTArtificial Sequencechemically synthesized 88Glu Ser
Arg Ser Gly Tyr Ala Asp Ile Pro Phe Pro Ala His Trp Val1 5 10 15Pro
Arg Thr8919PRTArtificial Sequencechemically synthesized 89Arg Glu
Cys Gly Arg Cys Gly Asp Ile Pro Phe Pro Ala His Trp Val1 5 10 15Pro
Arg Thr908PRTArtificial Sequencechemically synthesized 90Leu Ser
Gly Arg Ser Asp Asn His1 5918PRTArtificial Sequencechemically
synthesized 91Thr Gly Arg Gly Pro Ser Trp Val1 5928PRTArtificial
Sequencechemically synthesized 92Pro Leu Thr Gly Arg Ser Gly Gly1
5935PRTArtificial Sequencechemically synthesized 93Gly Ser Gly Gly
Ser1 5944PRTArtificial Sequencechemically synthesized 94Gly Gly Gly
Ser1954PRTArtificial Sequencechemically synthesized 95Gly Gly Ser
Gly1965PRTArtificial Sequencechemically synthesized 96Gly Gly Ser
Gly Gly1 5975PRTArtificial Sequencechemically synthesized 97Gly Ser
Gly Ser Gly1 5985PRTArtificial Sequencechemically synthesized 98Gly
Ser Gly Gly Gly1 5995PRTArtificial Sequencechemically synthesized
99Gly Gly Gly Ser Gly1 51005PRTArtificial Sequencechemically
synthesized 100Gly Ser Ser Ser Gly1 510113PRTArtificial
Sequencechemically synthesized 101Gly Ser Ser Gly Gly Ser Gly Gly
Ser Gly Gly Ser Gly1 5 101025PRTArtificial Sequencechemically
synthesized 102Gly Ser Ser Gly Thr1 51034PRTArtificial
Sequencechemically synthesized 103Gly Ser Ser
Gly110410PRTArtificial Sequencechemically synthesized 104Gly Gly
Gln Pro Ser Gly Met Trp Gly Trp1 5 1010510PRTArtificial
Sequencechemically synthesized 105Phe Pro Arg Pro Leu Gly Ile Thr
Gly Leu1 5 1010610PRTArtificial Sequencechemically synthesized
106Val His Met Pro Leu Gly Phe Leu Gly Pro1 5 101074PRTArtificial
Sequencechemically synthesized 107Pro Leu Gly Leu11086PRTArtificial
Sequencechemically synthesized 108Gln Gly Gln Ser Gly Gln1
5109258PRTArtificial Sequencechemically synthesized 109Gln Gly Gln
Ser Gly Gln Tyr Gly Ser Cys Ser Trp Asn Tyr Val His1 5 10 15Ile Phe
Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Val 20 25 30His
Met Pro Leu Gly Phe Leu Gly Pro Gly Gly Ser Asp Ile Gln Met 35 40
45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp
Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu
Gln Pro Glu Asp Ile Ala 115 120
125Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln
130 135 140Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser
Val Phe145 150 155 160Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
Gly Thr Ala Ser Val 165 170 175Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu Ala Lys Val Gln Trp 180 185 190Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser Gln Glu Ser Val Thr 195 200 205Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 210 215 220Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val225 230 235
240Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
245 250 255Glu Cys110258PRTArtificial Sequencechemically
synthesized 110Gln Gly Gln Ser Gly Gln Gly Asp Phe Asp Ile Pro Phe
Pro Ala His1 5 10 15Trp Val Pro Ile Thr Gly Ser Ser Gly Gly Ser Gly
Gly Ser Gly Val 20 25 30His Met Pro Leu Gly Phe Leu Gly Pro Gly Gly
Ser Asp Ile Gln Met 35 40 45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly Asp Arg Val Thr 50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile
Ser Ser Tyr Leu Asn Trp Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr
Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr
Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln 130 135
140Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val
Phe145 150 155 160Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
Thr Ala Ser Val 165 170 175Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala Lys Val Gln Trp 180 185 190Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln Glu Ser Val Thr 195 200 205Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 210 215 220Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val225 230 235 240Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly 245 250
255Glu Cys111261PRTArtificial Sequencechemically synthesized 111Gln
Gly Gln Ser Gly Gln Met Gly Val Pro Ala Gly Cys Val Trp Asn1 5 10
15Tyr Ala His Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly
20 25 30Ser Gly Val His Met Pro Leu Gly Phe Leu Gly Pro Gly Gly Ser
Asp 35 40 45Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly Asp 50 55 60Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser
Ser Tyr Leu65 70 75 80Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile Tyr 85 90 95Tyr Thr Ser Arg Leu His Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser 100 105 110Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Gln Pro Glu 115 120 125Asp Ile Ala Thr Tyr Tyr
Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr 130 135 140Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro145 150 155 160Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 165 170
175Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
180 185 190Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln Glu 195 200 205Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser Ser 210 215 220Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr Ala225 230 235 240Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser Phe 245 250 255Asn Arg Gly Glu Cys
26011211PRTArtificial Sequencechemically synthesized 112Gly Ser Ser
Gly Gly Ser Gly Gly Ser Gly Gly1 5 1011312PRTArtificial
Sequencechemically synthesized 113Gly Ser Ser Gly Gly Ser Gly Gly
Ser Gly Gly Ser1 5 101149PRTArtificial Sequencechemically
synthesizedMISC_FEATURE(1)..(1)HiLyte Fluor 488 conjugated to the
alpha amino of ThrMISC_FEATURE(9)..(9)QXL 520 quencher conjugated
to the epsilon amino of Lys 114Thr Gly Arg Gly Pro Ser Trp Val Lys1
51159PRTArtificial Sequencechemically
synthesizedMISC_FEATURE(1)..(1)HiLyte Fluor 488 conjugated to the
alpha amino of GlyMISC_FEATURE(9)..(9)QXL 520 quencher conjugated
to epsilon amine of Lys 115Gly Gly Gly Ser Gly Gly Gly Ser Lys1
51168PRTArtificial Sequencechemically synthesized 116Ser Ala Arg
Gly Pro Ser Arg Trp1 51178PRTArtificial Sequencechemically
synthesized 117Thr Ala Arg Gly Pro Ser Phe Lys1 51188PRTArtificial
Sequencechemically synthesized 118Gly Gly Trp His Thr Gly Arg Asn1
51198PRTArtificial Sequencechemically synthesized 119His Thr Gly
Arg Ser Gly Ala Leu1 51208PRTArtificial Sequencechemically
synthesized 120Ala Ala Arg Gly Pro Ala Ile His1 51218PRTArtificial
Sequencechemically synthesized 121Arg Gly Pro Ala Phe Asn Pro Met1
51228PRTArtificial Sequencechemically synthesized 122Ser Ser Arg
Gly Pro Ala Tyr Leu1 51238PRTArtificial Sequencechemically
synthesized 123Arg Gly Pro Ala Thr Pro Ile Met1 51244PRTArtificial
Sequencechemically synthesized 124Arg Gly Pro Ala11258PRTArtificial
Sequencechemically synthesized 125Ser Pro Leu Thr Gly Arg Ser Gly1
51268PRTArtificial Sequencechemically synthesized 126Ser Ala Gly
Phe Ser Leu Pro Ala1 51279PRTArtificial Sequencechemically
synthesized 127Leu Ala Pro Leu Gly Leu Gln Arg Arg1
51288PRTArtificial Sequencechemically synthesized 128Ser Gly Gly
Pro Leu Gly Val Arg1 51298PRTArtificial Sequencechemically
synthesized 129Pro Arg Phe Lys Ile Ile Gly Gly1 51308PRTArtificial
Sequencechemically synthesized 130Pro Arg Phe Arg Ile Ile Gly Gly1
51319PRTArtificial Sequencechemically synthesized 131Ser Ser Arg
His Arg Arg Ala Leu Asp1 513214PRTArtificial Sequencechemically
synthesized 132Arg Lys Ser Ser Ile Ile Ile Arg Met Arg Asp Val Val
Leu1 5 1013315PRTArtificial Sequencechemically synthesized 133Ser
Ser Ser Phe Asp Lys Gly Lys Tyr Lys Lys Gly Asp Asp Ala1 5 10
1513415PRTArtificial Sequencechemically synthesized 134Ser Ser Ser
Phe Asp Lys Gly Lys Tyr Lys Arg Gly Asp Asp Ala1 5 10
151354PRTArtificial Sequencechemically synthesized 135Ile Glu Gly
Arg11364PRTArtificial Sequencechemically synthesized 136Ile Asp Gly
Arg11377PRTArtificial Sequencechemically synthesized 137Gly Gly Ser
Ile Asp Gly Arg1 51386PRTArtificial Sequencechemically synthesized
138Pro Leu Gly Leu Trp Ala1 51398PRTArtificial Sequencechemically
synthesized 139Gly Pro Gln Gly Ile Ala Gly Gln1 51408PRTArtificial
Sequencechemically synthesized 140Gly Pro Gln Gly Leu Leu Gly Ala1
51415PRTArtificial Sequencechemically synthesized 141Gly Ile Ala
Gly Gln1 51428PRTArtificial Sequencechemically synthesized 142Gly
Pro Leu Gly Ile Ala Gly Ile1 51438PRTArtificial Sequencechemically
synthesized 143Gly Pro Glu Gly Leu Arg Val Gly1 51448PRTArtificial
Sequencechemically synthesized 144Tyr Gly Ala Gly Leu Gly Val Val1
51458PRTArtificial Sequencechemically synthesized 145Ala Gly Leu
Gly Val Val Glu Arg1 51468PRTArtificial Sequencechemically
synthesized 146Ala Gly Leu Gly Ile Ser Ser Thr1 51478PRTArtificial
Sequencechemically synthesized 147Glu Pro Gln Ala Leu Ala Met Ser1
51488PRTArtificial Sequencechemically synthesized 148Gln Ala Leu
Ala Met Ser Ala Ile1 51498PRTArtificial Sequencechemically
synthesized 149Ala Ala Tyr His Leu Val Ser Gln1 51508PRTArtificial
Sequencechemically synthesized 150Met Asp Ala Phe Leu Glu Ser Ser1
51518PRTArtificial Sequencechemically synthesized 151Glu Ser Leu
Pro Val Val Ala Val1 51528PRTArtificial Sequencechemically
synthesized 152Ser Ala Pro Ala Val Glu Ser Glu1 51538PRTArtificial
Sequencechemically synthesized 153Asp Val Ala Gln Phe Val Leu Thr1
51548PRTArtificial Sequencechemically synthesized 154Val Ala Gln
Phe Val Leu Thr Glu1 51558PRTArtificial Sequencechemically
synthesized 155Ala Gln Phe Val Leu Thr Glu Gly1 51568PRTArtificial
Sequencechemically synthesized 156Pro Val Gln Pro Ile Gly Pro Gln1
51577PRTArtificial Sequencechemically synthesized 157Ile Ser Ser
Gly Leu Ser Ser1 51588PRTArtificial Sequencechemically synthesized
158Gln Asn Gln Ala Leu Arg Met Ala1 51598PRTArtificial
Sequencechemically synthesized 159Ala Gln Asn Leu Leu Gly Met Val1
51608PRTArtificial Sequencechemically synthesized 160Ser Thr Phe
Pro Phe Gly Met Phe1 51618PRTArtificial Sequencechemically
synthesized 161Pro Val Gly Tyr Thr Ser Ser Leu1 51628PRTArtificial
Sequencechemically synthesized 162Asp Trp Leu Tyr Trp Pro Gly Ile1
5163258PRTArtificial Sequencechemically synthesized 163Gln Gly Gln
Ser Gly Gln Tyr Gly Ser Cys Ser Trp Asn Tyr Val His1 5 10 15Ile Phe
Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30Ser
Gly Ile Ser Ser Gly Leu Ser Ser Gly Gly Ser Asp Ile Gln Met 35 40
45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp
Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu
Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr Tyr Cys Gln Gln Gly Asn
Thr Leu Pro Tyr Thr Phe Gly Gln 130 135 140Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala Pro Ser Val Phe145 150 155 160Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 165 170 175Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp 180 185
190Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
195 200 205Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr
Leu Thr 210 215 220Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
Ala Cys Glu Val225 230 235 240Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser Phe Asn Arg Gly 245 250 255Glu Cys164258PRTArtificial
Sequencechemically synthesized 164Gln Gly Gln Ser Gly Gln Tyr Gly
Ser Cys Ser Trp Asn Tyr Val His1 5 10 15Ile Phe Met Asp Cys Gly Ser
Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30Ser Gln Asn Gln Ala Leu
Arg Met Ala Gly Gly Ser Asp Ile Gln Met 35 40 45Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 50 55 60Ile Thr Cys Arg
Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp Tyr65 70 75 80Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser 85 90 95Arg
Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 100 105
110Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala
115 120 125Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe
Gly Gln 130 135 140Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
Pro Ser Val Phe145 150 155 160Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val 165 170 175Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp 180 185 190Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr 195 200 205Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 210 215 220Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val225 230
235 240Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
Gly 245 250 255Glu Cys165258PRTArtificial Sequencechemically
synthesized 165Gln Gly Gln Ser Gly Gln Tyr Gly Ser Cys Ser Trp Asn
Tyr Val His1 5 10 15Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly
Gly Ser Gly Gly 20 25 30Ser Ala Gln Asn Leu Leu Gly Met Val Gly Gly
Ser Asp Ile Gln Met 35 40 45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly Asp Arg Val Thr 50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile
Ser Ser Tyr Leu Asn Trp Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr
Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr
Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln 130 135
140Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val
Phe145 150 155 160Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
Thr Ala Ser Val 165 170 175Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala Lys Val Gln Trp 180 185 190Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln Glu Ser Val Thr 195 200 205Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 210 215 220Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val225 230 235 240Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly 245 250
255Glu Cys166258PRTArtificial Sequencechemically synthesized 166Gln
Gly Gln Ser Gly Gln Tyr Gly Ser Cys Ser Trp Asn Tyr Val His1 5 10
15Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly
20 25 30Ser Ser Thr Phe Pro Phe Gly Met Phe Gly Gly Ser Asp Ile Gln
Met 35 40 45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg
Val Thr 50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu
Asn Trp Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile Tyr Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr Phe Thr Ile Ser
Ser Leu Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr Tyr Cys Gln Gln
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln 130 135 140Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe145 150 155 160Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 165 170
175Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
180 185 190Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
Ser
Val Thr 195 200 205Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr 210 215 220Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val225 230 235 240Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser Phe Asn Arg Gly 245 250 255Glu
Cys167258PRTArtificial Sequencechemically synthesized 167Gln Gly
Gln Ser Gly Gln Tyr Gly Ser Cys Ser Trp Asn Tyr Val His1 5 10 15Ile
Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25
30Ser Pro Val Gly Tyr Thr Ser Ser Leu Gly Gly Ser Asp Ile Gln Met
35 40 45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val
Thr 50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn
Trp Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
Tyr Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser
Gly Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr Phe Thr Ile Ser Ser
Leu Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr Tyr Cys Gln Gln Gly
Asn Thr Leu Pro Tyr Thr Phe Gly Gln 130 135 140Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe145 150 155 160Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 165 170
175Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
180 185 190Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr 195 200 205Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr 210 215 220Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val225 230 235 240Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser Phe Asn Arg Gly 245 250 255Glu
Cys168258PRTArtificial Sequencechemically synthesized 168Gln Gly
Gln Ser Gly Gln Tyr Gly Ser Cys Ser Trp Asn Tyr Val His1 5 10 15Ile
Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25
30Ser Asp Trp Leu Tyr Trp Pro Gly Ile Gly Gly Ser Asp Ile Gln Met
35 40 45Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val
Thr 50 55 60Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn
Trp Tyr65 70 75 80Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
Tyr Tyr Thr Ser 85 90 95Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser
Gly Ser Gly Ser Gly 100 105 110Thr Asp Phe Thr Phe Thr Ile Ser Ser
Leu Gln Pro Glu Asp Ile Ala 115 120 125Thr Tyr Tyr Cys Gln Gln Gly
Asn Thr Leu Pro Tyr Thr Phe Gly Gln 130 135 140Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe145 150 155 160Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 165 170
175Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
180 185 190Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr 195 200 205Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr 210 215 220Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val225 230 235 240Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser Phe Asn Arg Gly 245 250 255Glu
Cys16916PRTArtificial Sequencechemically synthesized 169Gly Ser Ser
Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gly Ser1 5 10
1517010PRTArtificial Sequencechemically synthesized 170Gly Ser Ser
Gly Gly Ser Gly Gly Ser Gly1 5 1017111PRTArtificial
Sequencechemically synthesized 171Gly Ser Ser Gly Gly Ser Gly Gly
Ser Gly Ser1 5 101724PRTArtificial Sequencechemically synthesized
172Gly Gly Gly Ser11737PRTArtificial Sequencechemically synthesized
173Gly Pro Arg Ser Phe Gly Leu1 51746PRTArtificial
Sequencechemically synthesized 174Gly Pro Arg Ser Phe Gly1
51756PRTArtificial Sequencechemically synthesized 175Ser Asp His
Ala Trp Ser1 517619PRTArtificial Sequencechemically synthesized
176Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu Lys Ser1
5 10 15Arg Val Thr17710PRTArtificial Sequencechemically synthesized
177Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr1 5 101788PRTArtificial
Sequencechemically synthesized 178Arg Ala Ser Gln Asp Ile Ser Ser1
51797PRTArtificial Sequencechemically synthesized 179Thr Ile Ser
Ser Leu Gln Pro1 51808PRTArtificial Sequencechemically synthesized
180Gln Gln Gly Asn Thr Leu Pro Tyr1 5181254PRTArtificial
Sequencechemically synthesized 181Gln Gly Gln Ser Gly Gln Tyr Gly
Ser Cys Ser Trp Asn Tyr Val His1 5 10 15Ile Phe Met Asp Cys Gly Ser
Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30Ser Gly Pro Arg Ser Phe
Gly Leu Asp Ile Gln Met Thr Gln Ser Pro 35 40 45Ser Ser Leu Ser Ala
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 50 55 60Ala Ser Gln Asp
Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro65 70 75 80Gly Lys
Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser 85 90 95Gly
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 100 105
110Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys
115 120 125Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr
Lys Val 130 135 140Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro145 150 155 160Ser Asp Glu Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu 165 170 175Asn Asn Phe Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn 180 185 190Ala Leu Gln Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 195 200 205Lys Asp Ser
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 210 215 220Asp
Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly225 230
235 240Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 245
250182253PRTArtificial Sequencechemically synthesized 182Gln Gly
Gln Ser Gly Gln Tyr Gly Ser Cys Ser Trp Asn Tyr Val His1 5 10 15Ile
Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25
30Ser Gly Pro Arg Ser Phe Gly Asp Ile Gln Met Thr Gln Ser Pro Ser
35 40 45Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg
Ala 50 55 60Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys
Pro Gly65 70 75 80Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg
Leu His Ser Gly 85 90 95Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Phe 100 105 110Thr Ile Ser Ser Leu Gln Pro Glu Asp
Ile Ala Thr Tyr Tyr Cys Gln 115 120 125Gln Gly Asn Thr Leu Pro Tyr
Thr Phe Gly Gln Gly Thr Lys Val Glu 130 135 140Ile Lys Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser145 150 155 160Asp Glu
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 165 170
175Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
180 185 190Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys 195 200 205Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp 210 215 220Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu225 230 235 240Ser Ser Pro Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys 245 2501838PRTArtificial Sequencechemically
synthesized 183Ile Ser Ser Gly Leu Leu Ser Ser1 51848PRTArtificial
Sequencechemically synthesized 184Leu Lys Ala Ala Pro Arg Trp Ala1
51858PRTArtificial Sequencechemically synthesized 185Gly Pro Ser
His Leu Val Leu Thr1 51868PRTArtificial Sequencechemically
synthesized 186Leu Pro Gly Gly Leu Ser Pro Trp1 51878PRTArtificial
Sequencechemically synthesized 187Met Gly Leu Phe Ser Glu Ala Gly1
51888PRTArtificial Sequencechemically synthesized 188Ser Pro Leu
Pro Leu Arg Val Pro1 51898PRTArtificial Sequencechemically
synthesized 189Arg Met His Leu Arg Ser Leu Gly1 51908PRTArtificial
Sequencechemically synthesized 190Leu Ala Ala Pro Leu Gly Leu Leu1
51918PRTArtificial Sequencechemically synthesized 191Ala Val Gly
Leu Leu Ala Pro Pro1 51928PRTArtificial Sequencechemically
synthesized 192Leu Leu Ala Pro Ser His Arg Ala1 51938PRTArtificial
Sequencechemically synthesized 193Pro Ala Gly Leu Trp Leu Asp Pro1
5
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