U.S. patent application number 16/040945 was filed with the patent office on 2018-11-15 for antagonist anti-il-7 receptor antibodies and methods.
This patent application is currently assigned to RINAT NEUROSCIENCE CORP.. The applicant listed for this patent is RINAT NEUROSCIENCE CORP.. Invention is credited to LI-FEN LEE, CHIA-YANG LIN, WENWU ZHAI.
Application Number | 20180327503 16/040945 |
Document ID | / |
Family ID | 44476676 |
Filed Date | 2018-11-15 |
United States Patent
Application |
20180327503 |
Kind Code |
A1 |
LIN; CHIA-YANG ; et
al. |
November 15, 2018 |
ANTAGONIST ANTI-IL-7 RECEPTOR ANTIBODIES AND METHODS
Abstract
The present invention provides antagonizing antibodies that bind
to interleukin-7 receptor (IL-7R). The invention further provides a
method of obtaining such antibodies and antibody-encoding nucleic
acids. The invention further relates to therapeutic methods for use
of these antibodies and antigen-binding portions thereof for the
treatment and/or prevention of type 2 diabetes and immunological
disorders, including type 1 diabetes, multiple sclerosis,
rheumatoid arthritis, graft-versus-host disease, and lupus.
Inventors: |
LIN; CHIA-YANG; (PALO ALTO,
CA) ; LEE; LI-FEN; (PALO ALTO, CA) ; ZHAI;
WENWU; (REDWOOD CITY, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RINAT NEUROSCIENCE CORP. |
NEW YORK |
NY |
US |
|
|
Assignee: |
RINAT NEUROSCIENCE CORP.
NEW YORK
NY
|
Family ID: |
44476676 |
Appl. No.: |
16/040945 |
Filed: |
July 20, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15136584 |
Apr 22, 2016 |
10059772 |
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16040945 |
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14109267 |
Dec 17, 2013 |
9346885 |
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15136584 |
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13627601 |
Sep 26, 2012 |
8637273 |
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14109267 |
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13033491 |
Feb 23, 2011 |
8298535 |
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13627601 |
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61307670 |
Feb 24, 2010 |
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61438205 |
Jan 31, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 19/02 20180101; A61P 25/00 20180101; A61K 2039/505 20130101;
A61P 3/10 20180101; C07K 2317/51 20130101; C07K 2317/34 20130101;
A61P 37/02 20180101; C07K 2317/52 20130101; C07K 2317/565 20130101;
C07K 2317/515 20130101; A61P 19/04 20180101; C07K 2317/76 20130101;
C07K 16/2866 20130101; C07K 2317/92 20130101; A61P 37/00 20180101;
A61P 37/06 20180101; C07K 2317/14 20130101; A61P 29/00
20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1. An isolated interleukin-7 receptor (IL-7R) antibody which
specifically binds to interleukin-7 receptor alpha (IL-7R.alpha.)
and comprises an antigen binding region that cross-competes with a
monoclonal antibody selected from the group consisting of P3A9,
P4B3, P2D2, P2E11, HAL403a, HAL403b, C1GM and C2M3, for binding to
IL-7R.alpha..
2. The antibody of claim 1, wherein the antibody binds to an
epitope comprising residues I82, K84, K100, T105, and Y192 of human
IL-7R.alpha..
3. An isolated antibody which specifically binds to interleukin-7
receptor alpha (IL-7R.alpha.), wherein the antibody comprises a
heavy chain variable region (VH) complementary determining region
one (CDR1) having the amino acid sequence X.sub.1X.sub.2VMH,
wherein X.sub.1 is D or N; X.sub.2 is S or Y (SEQ ID NO: 50), a VH
CDR2 having the amino acid sequence
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5GX.sub.6X.sub.7TYYADSVKG,
wherein X.sub.1 is L or A; X.sub.2 is V or I; X.sub.3 is G or S,
X.sub.4 is W or G, X.sub.5 is D or S, X.sub.6 is F, G or S, X.sub.7
is F, A or S (SEQ ID NO: 51), and a VH CDR3 having the amino acid
sequence X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8,
wherein X.sub.1 is Q or D; X.sub.2 is G or I; X.sub.3 is D or S,
X.sub.4 is Y or G, X.sub.5 is M, V or G; X.sub.6 is G or F; X.sub.7
is N, D or M; X.sub.8 is N, Y or D (SEQ ID NO: 52), a light chain
variable region (VL) CDR1 having the amino acid sequence
TX.sub.1SSGX.sub.2IX.sub.3SSYVQ wherein X.sub.1 is R or G; X.sub.2
is S or R X.sub.3 is D or A (SEQ ID NO: 53), a VL CDR2 having the
amino acid sequence EDX.sub.1QRPS wherein X.sub.1 is D or N (SEQ ID
NO: 54), and a VL CDR3 having the amino acid sequence
X.sub.1X.sub.2YX.sub.3X.sub.4X.sub.5X.sub.6LX.sub.7 wherein X.sub.1
is Q or M; X.sub.2 is S or Q; X.sub.3 is D or A; X.sub.4 is F or S,
X.sub.5 is H or S, X.sub.6 is H or S, X.sub.7 is V or W (SEQ ID NO:
55), wherein the antibody blocks STAT5 phosphorylation in a STAT5
activation assay.
4. An isolated antibody which specifically binds to interleukin-7
receptor alpha (IL-7R.alpha.), wherein the antibody comprises: a
heavy chain variable region (VH) comprising the following
complementarity determining regions (CDRs): a VH CDR1 that is a VH
CDR1 in SEQ ID NO: 40; a VH CDR2 that is a VH CDR2 in SEQ ID NO:
40; and a VH CDR3 that is a VH CDR3 in SEQ ID NO: 40; and light
chain variable region (VL) comprising the following CDRs: a VL CDR1
that is a VL CDR1 in SEQ ID NO: 41; a VL CDR2 that is a VL CDR2 in
SEQ ID NO: 41; and a VL CDR3 that is a VL CDR3 in SEQ ID NO:
41.
5. The isolated antibody of claim 4, wherein the VH region
comprises a VH CDR1 having the amino acid sequence DSVMH (SEQ ID
NO: 19), GFTFDDS (SEQ ID NO: 46), or GFTFDDSVMH (SEQ ID NO: 47), a
VH CDR2 having the amino acid sequence LVGWDGFFTYYADSVKG (SEQ ID
NO: 23) or GWDGFF (SEQ ID NO: 48), and a VH CDR3 having the amino
acid sequence QGDYMGNN (SEQ ID NO: 49).
6. The isolated antibody of claim 5, wherein the VL region
comprises a VL CDR1 having the amino acid sequence TRSSGSIDSSYVQ
(SEQ ID NO: 29), a VL CDR2 having the amino acid sequence EDDQRPS
(SEQ ID NO: 31), and a VL CDR3 having the amino acid sequence
QSYDFHHLV (SEQ ID NO: 36).
7. The antibody of claim 4, wherein the VH region comprises the
amino acid sequence shown in SEQ ID NO: 40 and the VL region
comprises the amino acid sequence shown in SEQ ID NO: 41.
8. The antibody of claim 7, wherein said antibody comprises a light
chain having the amino acid sequence shown in SEQ ID NO: 43 and a
heavy chain having the amino acid sequence shown in SEQ ID NO: 42,
with or without the C-terminal lysine of SEQ ID NO: 42.
9. The isolated antibody of claim 4, wherein each CDR is defined in
accordance with the Kabat definition, the Chothia definition, the
combination of the Kabat definition and the Chothia definition, the
AbM definition, or the contact definition of CDR.
10. The antibody of any claim 4, wherein the antibody further
comprises a constant region.
11. The antibody of claim 10, wherein the antibody is of the human
IgG1 or IgG2.DELTA.a subclass.
12. A pharmaceutical composition comprising the antibody of claim
4.
13. A cell line that recombinantly produces the antibody of claim
4.
14. A nucleic acid encoding the antibody of claim 4.
15. A method for treating and/or preventing an autoimmune disorder
in an individual, the method comprising administering a
therapeutically effective amount of an antagonist IL-7R antibody to
an individual suffering from or at risk for an autoimmune disorder,
thereby ameliorating and/or preventing one or more symptoms of the
autoimmune disorder, wherein the autoimmune disorder is selected
from the group consisting of type 1 diabetes, rheumatoid arthritis,
lupus and multiple sclerosis.
16. The method of claim 15, wherein administration of the
antagonist IL-7R antibody results in reduced naive and activated T
cell populations in the individual compared to before
administration.
17. The method of claim 16, wherein the reduced T cell populations
in the individual comprise T.sub.H1 and/or T.sub.H17 cells.
18. A method of treating and/or preventing type 2 diabetes in an
individual, the method comprising administering a therapeutically
effective amount of an IL-7R antagonist to an individual suffering
from or at risk for type 2 diabetes, thereby ameliorating and/or
preventing one or more symptoms of type 2 diabetes.
19. The method of claim 18, wherein the IL-7R antagonist is an
antagonist IL-7R antibody.
20. A method for treating and/or preventing graft-versus-host
disease (GVHD) in an individual, comprising administering a
therapeutically effective amount of an antagonist IL-7R antibody to
an individual suffering from GVHD, thereby ameliorating one or more
symptoms of GVHD.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/136,584, filed Apr. 22, 2016, which is a divisional of
U.S. patent application Ser. No. 14/109,267, filed Dec. 17, 2013,
now U.S. Pat. No. 9,346,885, issued on May 24, 2016, which is a
divisional of U.S. patent application Ser. No. 13/627,601, filed
Sep. 26, 2012, now U.S. Pat. No. 8,637,273, issued on Jan. 28,
2014, which is a divisional of U.S. patent application Ser. No.
13/033,491, filed Feb. 23, 2011, now U.S. Pat. No. 8,298,535,
issued on Oct. 30, 2012, which claims priority, under 35 USC .sctn.
119(e), to U.S. Provisional Application Ser. No. 61/307,670, filed
Feb. 24, 2010, and U.S. Provisional Application Ser. No.
61/438,205, filed Jan. 31, 2011, each of which is hereby
incorporated by reference in its entirety.
REFERENCE TO SEQUENCE LISTING
[0002] This application is being filed electronically via EFS-Web
and includes an electronically submitted sequence listing in .txt
format. The .txt file contains a sequence listing entitled
"PC33990E_SequenceListing_ST25.txt" created on Jul. 19, 2018 and
having a size of 41 KB. The sequence listing contained in this .txt
file is part of the specification and is herein incorporated by
reference in its entirety.
FIELD
[0003] The present invention relates to antibodies, e.g., full
length antibodies or antigen-binding portions thereof, that
antagonize the activity of interleukin-7 receptor (IL-7R),
including its interaction with IL-7. The invention further relates
to compositions comprising an IL-7R antagonist, such as an
antagonist IL-7R antibody, and methods of using the IL-7R
antagonist as a medicament. The IL-7R antagonist can be used in the
prevention and/or treatment of type 2 diabetes, graft-versus-host
disease (GVHD), and autoimmune disorders, including type 1
diabetes, multiple sclerosis, rheumatoid arthritis, and lupus.
BACKGROUND
[0004] The IL-7R complex is a heterodimeric receptor made up of the
IL-7R alpha chain (IL-7R.alpha.) and the common gamma chain
(.gamma.c) (Mazzucchelli et al., Nat Rev Immunol., 2007, 7:144-54).
IL-7R is bound by interleukin-7 (IL-7), a cytokine essential to the
development and homeostatic maintenance of T and B lymphocytes (Fry
et al., J Immunol., 2005, 174:6571-6). Binding of IL-7 to IL-7R
activates multiple pathways that regulate lymphocyte survival,
glucose uptake, proliferation and differentiation.
[0005] IL-7R is expressed on both dendritic cells and monocytes and
appears to act in multiple hematopoietic lineages (Reche P A, et
al., J Immunol., 2001, 167:336-43). In dendritic cells, IL-7R plays
an immunomodulatory role, whereas lymphocytes require IL-7R
signaling for survival, proliferation and differentiation. Both the
Jak-Stat and PI3K-Akt pathways are activated by the binding of IL-7
to IL-7R (Jian et al., Cytokine Growth Factor Rev., 2005,
16:513-533). These pathways involve signaling crosstalk, shared
interaction domains, feedback loops, integrated gene regulation,
mulitimerization and ligand competition. Some targets of IL-7
signaling, including Bcl2 and Pyk2, contribute to cellular
survival. Other targets, such as PI3 kinase, src family kinases
(Ick and fyn) and STAT5, contribute to cellular proliferation. The
transcription factor STAT5 contributes to activation of multiple
different downstream genes in B and T cells and may contribute to
VDJ recombination through alteration of chromatin structure. The
cell survival and cell proliferation signals induced by IL-7
combine to induce normal T cell development. Details of the complex
IL-7 signaling network and its interaction with other signaling
cascades in cells of the immune system have not yet been fully
elucidated.
[0006] From the information available in the art, and prior to the
present invention, it remained unclear whether the introduction of
an antagonist IL-7R antibody into the blood circulation to
selectively antagonize IL-7R would be effective to treat type 2
diabetes, type 1 diabetes, GVHD, lupus and rheumatoid arthritis,
and, if so, what properties of an IL-7R antibody are needed for
such in vivo effectiveness.
SUMMARY
[0007] Antagonist antibodies that selectively interact with and
inhibit IL-7R function are provided. It is demonstrated for the
first time that certain antagonist IL-7R antibodies are effective
in vivo to treat type 1 diabetes, type 2 diabetes, rheumatoid
arthritis, GVHD and lupus.
[0008] In some embodiments, antagonist antibodies that selectively
interact with and inhibit IL-7R function are provided. In some
embodiments, the antibody specifically binds to IL-7R and comprises
an antigen binding region that competes with a monoclonal antibody
selected from the group consisting of C1GM, C2M3, P3A9, P4B3, P2D2,
P2E11, HAL403a and HAL403b, for binding to IL-7R. In some
embodiments, the antibody comprises a polypeptide having the amino
acid sequence shown in SEQ ID NO: 42 or SEQ ID NO: 43. In other
embodiments, the antibody specifically binds to IL-7R and
recognizes an epitope which overlaps an epitope of IL-7R that is
recognized by a monoclonal antibody selected from the group
consisting of C1GM, C2M3, P3A9, P4B3, P2D2, P2E11, HAL403a and
HAL403b. In some embodiments, the antibody the antibody binds to an
epitope comprising residues I82, K84, K100, T105, and Y192 of
interleukin-7 receptor alpha (IL-7R.alpha.). In some embodiments,
the epitope further comprises one or more additional residues
selected from the group consisting of residues D190, H191, and K194
of human IL-7R.alpha..
[0009] In some embodiments, the IL-7R is human IL-7R.
[0010] In some embodiments, the antibody specifically binds to
interleukin-7 receptor alpha (IL-7R.alpha.) and comprises a heavy
chain variable region (VH) complementary determining region one
(CDR1) having the amino acid sequence X.sub.1X.sub.2VMH, wherein
X.sub.1 is D or N; X.sub.2 is S or Y (SEQ ID NO: 50), a VH CDR2
having the amino acid sequence
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5GX.sub.6X.sub.7TYYADSVKG,
wherein X.sub.1 is L or A; X.sub.2 is V or I, X.sub.3 is G or 5,
X.sub.4 is W or G; X.sub.5 is D or 5, X.sub.6 is F, G or 5, X.sub.7
is F, A or S (SEQ ID NO: 51), and a VH CDR3 having the amino acid
sequence X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8,
wherein X.sub.1 is Q or ID, X.sub.2 is G or I; X.sub.3 is D or S,
X.sub.4 is Y or G, X.sub.5 is M, V or G, X.sub.6 is G or F, X.sub.7
is N, D or M; X.sub.8 is N, Y or D (SEQ ID NO: 52), a light chain
variable region (VL) CDR1 having the amino acid sequence
TX.sub.1SSGX.sub.2IX.sub.3SSYVQ wherein X.sub.1 is R or G; X.sub.2
is S or R X.sub.3 is D or A (SEQ ID NO: 53), a VL CDR2 having the
amino acid sequence EDX.sub.1QRPS wherein X.sub.1 is D or N (SEQ ID
NO: 54), and a VL CDR3 having the amino acid sequence
X.sub.1X.sub.2YX.sub.3X.sub.4X.sub.5X.sub.6LX.sub.7 wherein X.sub.1
is Q or M; X.sub.2 is S or Q; X.sub.3 is D or A; X.sub.4 is F or 5,
X.sub.5 is H or 5, X.sub.6 is H or 5, X.sub.7 is V or W (SEQ ID NO:
55), wherein the antibody blocks STAT5 phosphorylation in a STAT5
activation assay. In some embodiments, the framework region between
VH CDR2 and VH CDR3 comprises the amino acid sequence
alanine-arginine, wherein the arginine is adjacent to the first
amino acid residue of VH CDR3. In some embodiments, the framework
region between VH CDR2 and VH CDR3 comprises the amino acid
sequence cysteine-alanine-arginine, wherein the arginine is
adjacent to the first amino acid residue of VH CDR3.
[0011] In some embodiments, the antibody specifically binds to
IL-7R.alpha. and comprises a heavy chain variable region (VH)
comprising the following complementarity determining regions
(CDRs): a VH CDR1 that is a VH CDR1 in SEQ ID NO: 40; a VH CDR2
that is a VH CDR2 in SEQ ID NO: 40; and a VH CDR3 that is a VH CDR3
in SEQ ID NO: 40. In some embodiments, the antibody specifically
binds to IL-7R.alpha. and comprises a light chain variable region
(VL) comprising the following CDRs: a VL CDR1 that is a VL CDR1 in
SEQ ID NO: 41; a VL CDR2 that is a VL CDR2 in SEQ ID NO: 41; and a
VL CDR3 that is a VL CDR3 in SEQ ID NO: 41. In some embodiments,
the antibody specifically binds to IL-7R.alpha. and comprises: a
heavy chain variable region (VH) comprising the following
complementarity determining regions (CDRs): a VH CDR1 that is a VH
CDR1 in SEQ ID NO: 40; a VH CDR2 that is a VH CDR2 in SEQ ID NO:
40; and a VH CDR3 that is a VH CDR3 in SEQ ID NO: 40; and a light
chain variable region (VL) comprising the following CDRs: a VL CDR1
that is a VL CDR1 in SEQ ID NO: 41; a VL CDR2 that is a VL CDR2 in
SEQ ID NO: 41; and a VL CDR3 that is a VL CDR3 in SEQ ID NO: 41. In
some embodiments, each CDR is defined in accordance with the Kabat
definition, the Chothia definition, the combination of the Kabat
definition and the Chothia definition, the AbM definition, or the
contact definition of CDR.
[0012] In some embodiments, the antibody comprises a VH CDR1 having
the amino acid sequence X.sub.1X.sub.2VMH, wherein X.sub.1 is D or
N; X.sub.2 is S or Y (SEQ ID NO: 50), a VH CDR2 having the amino
acid sequence GWDGFF (SEQ ID NO: 57), and a VH CDR3 having the
amino acid sequence ARX.sub.1X.sub.2X.sub.3X.sub.4 (SEQ ID NO: 58),
a VL CDR1 having the amino acid sequence SGSIDSSY (SEQ ID NO: 59),
a VL CDR2 having the amino acid sequence EDDQRPSGV (SEQ ID NO: 60),
and a VL CDR3 having the amino acid sequence FHHL (SEQ ID NO: 61),
wherein the antibody blocks STAT5 phosphorylation in a STAT5
activation assay.
[0013] In some embodiments, the antibody specifically binds to
IL-7R.alpha. and comprises a heavy chain variable region (VH)
complementary determining region one (CDR1) having the amino acid
sequence DSVMH (SEQ ID NO: 19), GFTFDDS (SEQ ID NO: 46), or
GFTFDDSVMH (SEQ ID NO: 47), a VH CDR2 having the amino acid
sequence LVGWDGFFTYYADSVKG (SEQ ID NO: 23) or GWDGFF (SEQ ID NO:
48), and a VH CDR3 having the amino acid sequence QGDYMGNN (SEQ ID
NO: 49), or a variant thereof having one or more conservative amino
acid substitutions in CDR1, CDR2, and/or CDR3.
[0014] In some embodiments, the antibody comprises a light chain
variable region (VL) CDR1 having the amino acid sequence
TRSSGSIDSSYVQ (SEQ ID NO: 29), a VL CDR2 having the amino acid
sequence EDDQRPS (SEQ ID NO: 31), and/or VL CDR3 having the amino
acid sequence QSYDFHHLV (SEQ ID NO: 36), or a variant thereof
having one or more conservative amino acid substitutions in CDR1,
CDR2, and/or CDR3. In some embodiments, the antibody further
comprises a VH CDR1 having the amino acid sequence shown in SEQ ID
NO: 19, 46 or 47, a VH CDR2 having the amino acid sequence shown in
SEQ ID NO: 23, or 48, and a VH CDR3 having the amino acid sequence
shown in SEQ ID NO: 49, or a variant thereof having one or more
conservative amino acid substitutions in CDR1, CDR2, and/or
CDR3.
[0015] In some embodiments, the antibody specifically binds to
IL-7R.alpha. and comprises a heavy chain variable region (VH)
complementary determining region one (CDR1) having the amino acid
sequence shown in SEQ ID NO: 19, 46 or 47, a VH CDR2 having the
amino acid sequence shown in SEQ ID NO: 23, or 48, and a VH CDR3
having the amino acid sequence shown in SEQ ID NO: 49, a light
chain variable region (VL) CDR1 having the amino acid sequence
shown in SEQ ID NO: 29, a VL CDR2 having the amino acid sequence
shown in SEQ ID NO: 31, and a VL CDR3 having the amino acid
sequence shown in SEQ ID NO: 36. In some embodiments, the VH region
comprises the amino acid sequence
EVQLVESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWVSLVGWDG
FFTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGDYMGNNWGQGTL VTVSS
(SEQ ID NO: 40) and the VL region comprises the amino acid sequence
NFMLTQPHSVSESPGKTVTISCTRSSGSIDSSYVQWYQQRPGSSPTTVIYEDDQRPSG
VPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDFHHLVFGGGTKLTVL (SEQ ID NO:
41). In some embodiments, the antibody comprises a light chain
having the amino acid sequence
NFMLTQPHSVSESPGKTVTISCTRSSGSIDSSYVQWYQQRPGSSPTTVIYEDDQRPSG
VPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDFHHLVFGGGTKLTVLQPKAAPS
VTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKY
AASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 43) and a
heavy chain having the amino acid sequence
EVQLVESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWVSLVGWDG
FFTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGDYMGNNWGQGTL
VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVAPELLGGPSVFLFP
PKPKDTLMISRTPEVTCWVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 42), with or without the
C-terminal lysine of SEQ ID NO: 42.
[0016] In some embodiments, the antibody can be a human antibody, a
humanized antibody, or a chimeric antibody. In some embodiments,
the antibody is a monoclonal antibody.
[0017] In some embodiments, the antibody comprises a constant
region. In some embodiments, the antibody is of the human IgG1 or
IgG2.DELTA.a subclass. In some embodiments, the antibody comprises
a glycosylated constant region. In some embodiments, the antibody
comprises a constant region having increased binding affinity to a
human Fc gamma receptor. In some embodiments, the antibody
comprises an aglycosylated constant region.
[0018] In some embodiments, a pharmaceutical composition comprising
an antibody that selectively interacts with and inhibits IL-7R
function is provided.
[0019] In some embodiments, a cell line that recombinantly produces
an antibody that selectively interacts with and inhibits IL-7R
function is provided.
[0020] In some embodiments, a nucleic acid encoding an antibody
that selectively interacts with and inhibits IL-7R function is
provided.
[0021] In some embodiments, methods of lowering blood glucose
levels in an individual are provided. In some embodiments, the
method comprises administering a therapeutically effective amount
of an antagonist IL-7R antibody to an individual in need of such
treatment, thereby lowering blood glucose levels.
[0022] In some embodiments, methods of improving glucose tolerance
in an individual are provided. In some embodiments, the method
comprises administering a therapeutically effective amount of an
antagonist IL-7R antibody to an individual in need of such
treatment, thereby improving glucose tolerance.
[0023] In some embodiments, methods of preventing or treating type
1 diabetes in an individual are provided. In some embodiments, the
method comprises administering a therapeutically effective amount
of an antagonist IL-7R antibody to an individual in need of such
treatment, thereby preventing or treating one or more symptoms of
type 1 diabetes.
[0024] In some embodiments, methods of preventing or treating type
2 diabetes in an individual are provided. In some embodiments, the
method comprises administering a therapeutically effective amount
of an IL-7R antagonist to an individual in need of such treatment,
thereby preventing or treating one or more symptoms of type 2
diabetes. In some embodiments, the IL-7R antagonist is an
antagonist IL-7R antibody.
[0025] In some embodiments, methods of preventing or treating
rheumatoid arthritis in an individual are provided. In some
embodiments, the method comprises administering a therapeutically
effective amount of an antagonist IL-7R antibody to an individual
in need of such treatment, thereby preventing or treating one or
more symptoms of rheumatoid arthritis.
[0026] In some embodiments, methods of preventing or treating
graft-versus-host disease (GVHD) in an individual are provided. In
some embodiments, the method comprises administering a
therapeutically effective amount of an antagonist IL-7R antibody to
an individual in need of such treatment, thereby preventing or
treating one or more symptoms of GVHD.
[0027] In some embodiments, the GVHD is chronic GVHD or acute
GVHD.
[0028] In some embodiments, methods of preventing or treating lupus
in an individual are provided. In some embodiments, the method
comprises administering a therapeutically effective amount of an
antagonist IL-7R antibody to an individual in need of such
treatment, thereby preventing or treating one or more symptoms of
lupus.
[0029] In some embodiments, the lupus is cutaneous lupus
erythematosus, systemic lupus erythematosus, drug-induced
erythematosus or neonatal lupus.
[0030] In some embodiments, methods of preventing or treating
multiple sclerosis in an individual are provided. In some
embodiments, the method comprises administering a therapeutically
effective amount of an antagonist IL-7R antibody to an individual
in need of such treatment, thereby preventing or treating one or
more symptoms of multiple sclerosis and reducing and/or depleting
the naive and/or activated T cell populations in the individual. In
some embodiments, the reduced or depleted T cell populations in the
individual comprise T.sub.H1 and/or T.sub.H17 cells. In some
embodiments, administration of the antagonist IL-7R antibody does
not result in expansion of T.sub.H17 cell population in the
individual.
[0031] In some embodiments, the antibody can be administered
parenterally. In some embodiments, the individual is a human.
BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS
[0032] FIG. 1 depicts the dose-dependent effect of antagonist IL-7R
monoclonal antibodies P2D2, P2E11 and HAL403a on IL-7-mediated
STAT5 phosphorylation in human peripheral blood mononuclear cell
(PBMCs). The x-axis indicates the percentage of CD4+ cells
expressing phospho-STAT5 (p-STAT).
[0033] FIG. 2 depicts the effect of antagonist IL-7R monoclonal
antibody 28G9 on development of diabetes in non-obese diabetic
(NOD) mice.
[0034] FIG. 3A depicts the effect of antagonist IL-7R monoclonal
antibody 28G9 on blood glucose levels (mg/dL) in NOD mice.
[0035] FIG. 3B depicts the effect of antagonist IL-7R monoclonal
antibody 28G9 on body weight (g) in NOD mice.
[0036] FIG. 4A depicts the effect of antagonist IL-7R monoclonal
antibody 28G9 on naive CD8+ T cell populations in NOD mice. For the
x-axis, the total CD8+ T cell population was set as 100%.
[0037] FIG. 4B depicts the effect of antagonist IL-7R monoclonal
antibody 28G9 on memory CD8+ T cell populations in NOD mice. For
the x-axis, the total CD8+ T cell population was set as 100%.
[0038] FIG. 5 depicts the effect of antagonist IL-7R monoclonal
antibody 28G9 on naive CD4+ T cell population in NOD mice. For the
x-axis, the total CD4+ T cell population was set as 100%.
[0039] FIG. 6 depicts the effect of antagonist IL-7R monoclonal
antibodies 28B6 and 28G9 on clinical severity of EAE animals.
Clinical severity of EAE was assessed daily with a 0 to 5 point
scoring system: 0, normal; 1, flaccid tail; 2, partial hind-limb
paralysis; 3, total hind-limb paralysis; 4, quadriplegia; 5,
moribund state or dead.
[0040] FIG. 7 depicts the dose-dependent effect of antagonist IL-7R
monoclonal antibody 28G9 on clinical severity of EAE animals.
[0041] FIG. 8 depicts the effect of antagonist IL-7R monoclonal
antibody 28G9 on clinical severity of EAE animals.
[0042] FIG. 9 depicts the effect of antagonist IL-7R monoclonal
antibody 28G9 in animals with established EAE.
[0043] FIG. 10 depicts the effect of antagonist IL-7R monoclonal
antibody 28G9 at lower dose in animals with established EAE.
[0044] FIG. 11A depicts the effect of antagonist IL-7R monoclonal
antibodies 28G9 and 28B6 on CD4 T cell populations from bone marrow
(BM), spleen, blood and lymph nodes (LNs) of EAE animals. For the
x-axis, the total lymphocyte population was set as 100%.
[0045] FIG. 11B depicts the effect of antagonist IL-7R monoclonal
antibodies 28G9 and 28B6 on CD8 T cell populations from bone marrow
(BM), spleen, blood and lymph nodes (LNs) of EAE animals. For the
x-axis, the total lymphocyte population was set as 100%.
[0046] FIG. 12A depicts the effect of antagonist IL-7R monoclonal
antibody 28G9 on naive T cell populations from bone marrow, spleen,
blood and lymph nodes of EAE animals. For the x-axis, the CD8+ T
cell population was set as 100%.
[0047] FIG. 12B depicts the effect of antagonist IL-7R monoclonal
antibody 28G9 on memory T cell populations from bone marrow,
spleen, blood and lymph nodes of EAE animals. For the x-axis, the
CD8+ T cell population was set as 100%.
[0048] FIG. 12C depicts the effect of antagonist IL-7R monoclonal
antibody 28G9 on activated T cell populations from bone marrow,
spleen, blood and lymph nodes of EAE animals. For the x-axis, the
CD8+ T cell population was set as 100%.
[0049] FIG. 13 depicts the effect of antagonist IL-7R monoclonal
antibody 28G9 on T.sub.eff cell (left graph) and T.sub.reg cell
(right graph) populations from bone marrow, spleen, blood and lymph
nodes of EAE animals. For the x-axis, the CD4+ T cell population
was set as 100%. "*" indicates P<0.05 as compared to
control.
[0050] FIG. 14 depicts the effect of antagonist IL-7R monoclonal
antibody 28G9 on blood glucose levels (mg/dL) in high fat
diet-induced obesity (DIO) mice.
[0051] FIG. 15 depicts the effect of antagonist IL-7R monoclonal
antibody 28G9 on glucose intolerance in high fat diet-induced
obesity (DIO) mice.
DETAILED DESCRIPTION
[0052] Disclosed herein are antibodies that antagonize the function
of IL-7R, including its interaction with IL-7. Methods of making
antagonist IL-7R antibodies, compositions comprising these
antibodies, and methods of using these antibodies as a medicament
are provided. IL-7R antagonists, e.g., antagonist IL-7R antibodies,
can be used to in the prevention and/or treatment of type 2
diabetes, GVHD and autoimmune disorders, including multiple
sclerosis (MS), rheumatoid arthritis, type 1 diabetes, and
lupus.
General Techniques
[0053] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature, such as,
Molecular Cloning: A Laboratory Manual, second edition (Sambrook et
al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.
J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press;
Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998)
Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987);
Introduction to Cell and Tissue Culture (J. P. Mather and P. E.
Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory
Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.,
1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic
Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and
C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells
(J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in
Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The
Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current
Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short
Protocols in Molecular Biology (Wiley and Sons, 1999);
Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.
Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL
Press, 1988-1989); Monoclonal antibodies: a practical approach (P.
Shepherd and C. Dean, eds., Oxford University Press, 2000); Using
antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring
Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.
D. Capra, eds., Harwood Academic Publishers, 1995).
Definitions
[0054] An "antibody" is an immunoglobulin molecule capable of
specific binding to a target, such as a carbohydrate,
polynucleotide, lipid, polypeptide, etc., through at least one
antigen recognition site, located in the variable region of the
immunoglobulin molecule. As used herein, the term encompasses not
only intact polyclonal or monoclonal antibodies, but also fragments
thereof (such as Fab, Fab', F(ab').sub.2, Fv), single chain (ScFv)
and domain antibodies (including, for example, shark and camelid
antibodies), and fusion proteins comprising an antibody, and any
other modified configuration of the immunoglobulin molecule that
comprises an antigen recognition site. An antibody includes an
antibody of any class, such as IgG, IgA, or IgM (or sub-class
thereof), and the antibody need not be of any particular class.
Depending on the antibody amino acid sequence of the constant
region of its heavy chains, immunoglobulins can be assigned to
different classes. There are five major classes of immunoglobulins:
IgA, IgD, IgE, IgG, and IgM, and several of these may be further
divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4,
IgA1 and IgA2. The heavy-chain constant regions that correspond to
the different classes of immunoglobulins are called alpha, delta,
epsilon, gamma, and mu, respectively. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known.
[0055] As used herein, "monoclonal antibody" refers to an antibody
obtained from a population of substantially homogeneous antibodies,
i.e., the individual antibodies comprising the population are
identical except for possible naturally-occurring mutations that
may be present in minor amounts. Monoclonal antibodies are highly
specific, being directed against a single antigenic site.
Furthermore, in contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler and Milstein, 1975,
Nature 256:495, or may be made by recombinant DNA methods such as
described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may
also be isolated from phage libraries generated using the
techniques described in McCafferty et al., 1990, Nature
348:552-554, for example.
[0056] As used herein, "humanized" antibody refers to forms of
non-human (e.g. murine) antibodies that are chimeric
immunoglobulins, immunoglobulin chains, or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding
subsequences of antibodies) that contain minimal sequence derived
from non-human immunoglobulin. Preferably, humanized antibodies are
human immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat, or rabbit having the desired
specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore, the
humanized antibody may comprise residues that are found neither in
the recipient antibody nor in the imported CDR or framework
sequences, but are included to further refine and optimize antibody
performance. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the CDR regions
correspond to those of a non-human immunoglobulin and all or
substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region or domain (Fc), typically that of a human immunoglobulin.
Preferred are antibodies having Fc regions modified as described in
WO 99/58572. Other forms of humanized antibodies have one or more
CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) which are
altered with respect to the original antibody, which are also
termed one or more CDRs "derived from" one or more CDRs from the
original antibody.
[0057] In certain embodiments, definitive delineation of a CDR and
identification of residues comprising the binding site of an
antibody is accomplished by solving the structure of the antibody
and/or solving the structure of the antibody-ligand complex. In
certain embodiments, that can be accomplished by any of a variety
of techniques known to those skilled in the art, such as X-ray
crystallography. In certain embodiments, various methods of
analysis can be employed to identify or approximate the CDR
regions. Examples of such methods include, but are not limited to,
the Kabat definition, the Chothia definition, the AbM definition
and the contact definition.
[0058] The Kabat definition is a standard for numbering the
residues in an antibody and is typically used to identify CDR
regions. See, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:
214-8. The Chothia definition is similar to the Kabat definition,
but the Chothia definition takes into account positions of certain
structural loop regions. See, e.g., Chothia et al., 1986, J. Mol.
Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83. The
AbM definition uses an integrated suite of computer programs
produced by Oxford Molecular Group that model antibody structure.
See, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA),
86:9268-9272; "AbM.TM., A Computer Program for Modeling Variable
Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM
definition models the tertiary structure of an antibody from
primary sequence using a combination of knowledge databases and ab
initio methods, such as those described by Samudrala et al., 1999,
"Ab Initio Protein Structure Prediction Using a Combined
Hierarchical Approach," in PROTEINS, Structure, Function and
Genetics Suppl., 3:194-198. The contact definition is based on an
analysis of the available complex crystal structures. See, e.g.,
MacCallum et al., 1996, J. Mol. Biol., 5:732-45.
[0059] As used herein, "human antibody" means an antibody having an
amino acid sequence corresponding to that of an antibody produced
by a human and/or which has been made using any of the techniques
for making human antibodies known to those skilled in the art or
disclosed herein. This definition of a human antibody includes
antibodies comprising at least one human heavy chain polypeptide or
at least one human light chain polypeptide. One such example is an
antibody comprising murine light chain and human heavy chain
polypeptides. Human antibodies can be produced using various
techniques known in the art. In one embodiment, the human antibody
is selected from a phage library, where that phage library
expresses human antibodies (Vaughan et al., 1996, Nature
Biotechnology, 14:309-314; Sheets et al., 1998, Proc. Natl. Acad.
Sci. (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol.
Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581). Human
antibodies can also be made by immunization of animals into which
human immunoglobulin loci have been transgenically introduced in
place of the endogenous loci, e.g., mice in which the endogenous
immunoglobulin genes have been partially or completely inactivated.
This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806;
5,569,825; 5,625,126; 5,633,425; and 5,661,016. Alternatively, the
human antibody may be prepared by immortalizing human B lymphocytes
that produce an antibody directed against a target antigen (such B
lymphocytes may be recovered from an individual or may have been
immunized in vitro). See, e.g., Cole et al. Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al.,
1991, J. Immunol., 147 (1):86-95; and U.S. Pat. No. 5,750,373.
[0060] As used herein, the term "IL-7R" refers to any form of IL-7R
and variants thereof that retain at least part of the activity of
IL-7R. Unless indicated differently, such as by specific reference
to human IL-7R, IL-7R includes all mammalian species of native
sequence IL-7R, e.g., human, canine, feline, equine, and
bovine.
[0061] As used herein, an "IL-7R antagonist" refers to an antibody
or molecule that is able to inhibit IL-7R biological activity
and/or downstream pathway(s) mediated by IL-7R signaling, including
binding to IL-7, phosphorylation of STAT5, Src kinases, PI3 kinase
and Pyk2, and upregulation of Bcl2 protein. Examples of IL-7R
antagonists include, without limitation, antagonist IL-7R
antibodies, IL-7R siRNA, IL-7R shRNA, and IL-7R antisense
oligonucleotides.
[0062] Antagonist IL-7R antibodies encompass antibodies that block,
antagonize, suppress or reduce (to any degree including
significantly) IL-7R biological activity, including downstream
pathways mediated by IL-7R signaling, such interaction with IL-7
and/or elicitation of a cellular response to IL-7. For purpose of
the present invention, it will be explicitly understood that the
term "antagonist IL-7R antibody" (interchangeably termed "IL-7R
antagonist antibody," "antagonist anti-IL-7R antibody" or
"anti-IL-7R antagonist antibody") encompasses all the previously
identified terms, titles, and functional states and characteristics
whereby the IL-7R itself, an IL-7R biological activity (including
but not limited to interaction with IL-7, its ability to mediate
any aspect of phosphorylation of STAT5,
phosphatidylinositol-3-kinase (PI3K)-Akt pathway activation,
p27.sup.Kip1 downregulation, Bcl-2 upregulation, Rb
hyperphosphorylation, and CXCR4 upregulation), or the consequences
of the biological activity, are substantially nullified, decreased,
or neutralized in any meaningful degree. In some embodiments, an
antagonist IL-7R antibody binds IL-7R and prevents interaction with
IL-7. Examples of antagonist IL-7R antibodies are provided
herein.
[0063] As used herein a "full antagonist" is an antagonist which,
at an effective concentration, essentially completely blocks a
measurable effect of IL-7R. By a partial antagonist is meant an
antagonist that is capable of partially blocking a measurable
effect, but that, even at a highest concentration is not a full
antagonist. By essentially completely is meant at least about 80%,
preferably, at least about 90%, more preferably, at least about
95%, and most preferably, at least about 98% of the measurable
effect is blocked.
[0064] The terms "polypeptide", "oligopeptide", "peptide" and
"protein" are used interchangeably herein to refer to chains of
amino acids of any length, preferably, relatively short (e.g.,
10-100 amino acids). The chain may be linear or branched, it may
comprise modified amino acids, and/or may be interrupted by
non-amino acids. The terms also encompass an amino acid chain that
has been modified naturally or by intervention; for example,
disulfide bond formation, glycosylation, lipidation, acetylation,
phosphorylation, or any other manipulation or modification, such as
conjugation with a labeling component. Also included within the
definition are, for example, polypeptides containing one or more
analogs of an amino acid (including, for example, unnatural amino
acids, etc.), as well as other modifications known in the art. It
is understood that the polypeptides can occur as single chains or
associated chains.
[0065] As known in the art, "polynucleotide," or "nucleic acid," as
used interchangeably herein, refer to chains of nucleotides of any
length, and include DNA and RNA. The nucleotides can be
deoxyribonucleotides, ribonucleotides, modified nucleotides or
bases, and/or their analogs, or any substrate that can be
incorporated into a chain by DNA or RNA polymerase. A
polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and their analogs. If present, modification
to the nucleotide structure may be imparted before or after
assembly of the chain. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after polymerization, such as by conjugation with
a labeling component. Other types of modifications include, for
example, "caps", substitution of one or more of the naturally
occurring nucleotides with an analog, internucleotide modifications
such as, for example, those with uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.)
and with charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), those containing pendant moieties, such
as, for example, proteins (e.g., nucleases, toxins, antibodies,
signal peptides, poly-L-lysine, etc.), those with intercalators
(e.g., acridine, psoralen, etc.), those containing chelators (e.g.,
metals, radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotide(s). Further, any of the hydroxyl groups ordinarily
present in the sugars may be replaced, for example, by phosphonate
groups, phosphate groups, protected by standard protecting groups,
or activated to prepare additional linkages to additional
nucleotides, or may be conjugated to solid supports. The 5' and 3'
terminal OH can be phosphorylated or substituted with amines or
organic capping group moieties of from 1 to 20 carbon atoms. Other
hydroxyls may also be derivatized to standard protecting groups.
Polynucleotides can also contain analogous forms of ribose or
deoxyribose sugars that are generally known in the art, including,
for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or
2'-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric
sugars, epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs
and abasic nucleoside analogs such as methyl riboside. One or more
phosphodiester linkages may be replaced by alternative linking
groups. These alternative linking groups include, but are not
limited to, embodiments wherein phosphate is replaced by
P(O)S("thioate"), P(S)S ("dithioate"), (O)NR.sub.2 ("amidate"),
P(O)R, P(O)OR', CO or CH.sub.2 ("formacetal"), in which each R or
R' is independently H or substituted or unsubstituted alkyl (1-20
C) optionally containing an ether (--O--) linkage, aryl, alkenyl,
cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a
polynucleotide need be identical. The preceding description applies
to all polynucleotides referred to herein, including RNA and
DNA.
[0066] A "variable region" of an antibody refers to the variable
region of the antibody light chain or the variable region of the
antibody heavy chain, either alone or in combination. As known in
the art, the variable regions of the heavy and light chain each
consist of four framework regions (FR) connected by three
complementarity determining regions (CDRs) also known as
hypervariable regions. The CDRs in each chain are held together in
close proximity by the FRs and, with the CDRs from the other chain,
contribute to the formation of the antigen-binding site of
antibodies. There are at least two techniques for determining CDRs:
(1) an approach based on cross-species sequence variability (i.e.,
Kabat et al. Sequences of Proteins of Immunological Interest, (5th
ed., 1991, National Institutes of Health, Bethesda Md.)); and (2)
an approach based on crystallographic studies of antigen-antibody
complexes (Al-lazikani et al., 1997, J. Molec. Biol. 273:927-948).
As used herein, a CDR may refer to CDRs defined by either approach
or by a combination of both approaches.
[0067] As known in the art a "constant region" of an antibody
refers to the constant region of the antibody light chain or the
constant region of the antibody heavy chain, either alone or in
combination.
[0068] As used herein, an antibody "interacts with" IL-7R when the
equilibrium dissociation constant is equal to or less than 20 nM,
preferably less than about 6 nM, more preferably less than about 1
nM, most preferably less than about 0.2 nM, as measured by the
methods disclosed herein in Example 2.
[0069] An epitope that "preferentially binds" or "specifically
binds" (used interchangeably herein) to an antibody or a
polypeptide is a term well understood in the art, and methods to
determine such specific or preferential binding are also well known
in the art. A molecule is said to exhibit "specific binding" or
"preferential binding" if it reacts or associates more frequently,
more rapidly, with greater duration and/or with greater affinity
with a particular cell or substance than it does with alternative
cells or substances. An antibody "specifically binds" or
"preferentially binds" to a target if it binds with greater
affinity, avidity, more readily, and/or with greater duration than
it binds to other substances. For example, an antibody that
specifically or preferentially binds to an IL-7R epitope is an
antibody that binds this epitope with greater affinity, avidity,
more readily, and/or with greater duration than it binds to other
IL-7R epitopes or non-IL-7R epitopes. It is also understood that by
reading this definition, for example, an antibody (or moiety or
epitope) that specifically or preferentially binds to a first
target may or may not specifically or preferentially bind to a
second target. As such, "specific binding" or "preferential
binding" does not necessarily require (although it can include)
exclusive binding. Generally, but not necessarily, reference to
binding means preferential binding.
[0070] As used herein, "substantially pure" refers to material
which is at least 50% pure (i.e., free from contaminants), more
preferably, at least 90% pure, more preferably, at least 95% pure,
yet more preferably, at least 98% pure, and most preferably, at
least 99% pure.
[0071] A "host cell" includes an individual cell or cell culture
that can be or has been a recipient for vector(s) for incorporation
of polynucleotide inserts. Host cells include progeny of a single
host cell, and the progeny may not necessarily be completely
identical (in morphology or in genomic DNA complement) to the
original parent cell due to natural, accidental, or deliberate
mutation. A host cell includes cells transfected in vivo with a
polynucleotide(s) of this invention.
[0072] As known in the art, the term "Fc region" is used to define
a C-terminal region of an immunoglobulin heavy chain. The "Fc
region" may be a native sequence Fc region or a variant Fc region.
Although the boundaries of the Fc region of an immunoglobulin heavy
chain might vary, the human IgG heavy chain Fc region is usually
defined to stretch from an amino acid residue at position Cys226,
or from Pro230, to the carboxyl-terminus thereof. The numbering of
the residues in the Fc region is that of the EU index as in Kabat.
Kabat et al., Sequences of Proteins of Immunological Interest, 5th
Ed. Public Health Service, National Institutes of Health, Bethesda,
Md., 1991. The Fc region of an immunoglobulin generally comprises
two constant regions, CH2 and CH3.
[0073] As used in the art, "Fc receptor" and "FcR" describe a
receptor that binds to the Fc region of an antibody. The preferred
FcR is a native sequence human FcR. Moreover, a preferred FcR is
one which binds an IgG antibody (a gamma receptor) and includes
receptors of the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof. FcRs are
reviewed in Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92;
Capel et al., 1994, Immunomethods, 4:25-34; and de Haas et al.,
1995, J. Lab. Clin. Med., 126:330-41. "FcR" also includes the
neonatal receptor, FcRn, which is responsible for the transfer of
maternal IgGs to the fetus (Guyer et al., 1976, J. Immunol.,
117:587; and Kim et al., 1994, J. Immunol., 24:249).
[0074] The term "compete", as used herein with regard to an
antibody, means that a first antibody, or an antigen-binding
portion thereof, binds to an epitope in a manner sufficiently
similar to the binding of a second antibody, or an antigen-binding
portion thereof, such that the result of binding of the first
antibody with its cognate epitope is detectably decreased in the
presence of the second antibody compared to the binding of the
first antibody in the absence of the second antibody. The
alternative, where the binding of the second antibody to its
epitope is also detectably decreased in the presence of the first
antibody, can, but need not be the case. That is, a first antibody
can inhibit the binding of a second antibody to its epitope without
that second antibody inhibiting the binding of the first antibody
to its respective epitope. However, where each antibody detectably
inhibits the binding of the other antibody with its cognate epitope
or ligand, whether to the same, greater, or lesser extent, the
antibodies are said to "cross-compete" with each other for binding
of their respective epitope(s). Both competing and cross-competing
antibodies are encompassed by the present invention. Regardless of
the mechanism by which such competition or cross-competition occurs
(e.g., steric hindrance, conformational change, or binding to a
common epitope, or portion thereof), the skilled artisan would
appreciate, based upon the teachings provided herein, that such
competing and/or cross-competing antibodies are encompassed and can
be useful for the methods disclosed herein.
[0075] A "functional Fc region" possesses at least one effector
function of a native sequence Fc region. Exemplary "effector
functions" include C1q binding; complement dependent cytotoxicity;
Fc receptor binding; antibody-dependent cell-mediated cytotoxicity;
phagocytosis; down-regulation of cell surface receptors (e.g. B
cell receptor), etc. Such effector functions generally require the
Fc region to be combined with a binding domain (e.g. an antibody
variable domain) and can be assessed using various assays known in
the art for evaluating such antibody effector functions.
[0076] A "native sequence Fc region" comprises an amino acid
sequence identical to the amino acid sequence of an Fc region found
in nature. A "variant Fc region" comprises an amino acid sequence
which differs from that of a native sequence Fc region by virtue of
at least one amino acid modification, yet retains at least one
effector function of the native sequence Fc region. In some
embodiments, the variant Fc region has at least one amino acid
substitution compared to a native sequence Fc region or to the Fc
region of a parent polypeptide, e.g. from about one to about ten
amino acid substitutions, and preferably, from about one to about
five amino acid substitutions in a native sequence Fc region or in
the Fc region of the parent polypeptide. The variant Fc region
herein will preferably possess at least about 80% sequence identity
with a native sequence Fc region and/or with an Fc region of a
parent polypeptide, and most preferably, at least about 90%
sequence identity therewith, more preferably, at least about 95%,
at least about 96%, at least about 97%, at least about 98%, at
least about 99% sequence identity therewith.
[0077] As used herein, "treatment" is an approach for obtaining
beneficial or desired clinical results. For purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, one or more of the following: enhancement of
glucose clearance, lowering blood glucose levels, improving glucose
tolerance, reducing incidence of high blood glucose levels
resulting from type 1 or type 2 diabetes, reducing incidence or
amelioration of one or more symptoms of rheumatoid arthritis,
reducing incidence or amelioration of one or more symptoms of GVHD,
reducing incidence or amelioration of one or more symptoms of
lupus, and reducing incidence or amerlioration of one or more
symptoms of multiple sclerosis.
[0078] "Reducing incidence" means any of reducing severity (which
can include reducing need for and/or amount of (e.g., exposure to)
other drugs and/or therapies generally used for this condition. As
is understood by those skilled in the art, individuals may vary in
terms of their response to treatment, and, as such, for example, a
"method of reducing incidence" reflects administering the IL-7R
antagonist based on a reasonable expectation that such
administration may likely cause such a reduction in incidence in
that particular individual.
[0079] "Ameliorating" means a lessening or improvement of one or
more symptoms as compared to not administering an IL-7R antagonist.
"Ameliorating" also includes shortening or reduction in duration of
a symptom.
[0080] As used herein, an "effective dosage" or "effective amount"
of drug, compound, or pharmaceutical composition is an amount
sufficient to effect any one or more beneficial or desired results.
For prophylactic use, beneficial or desired results include
eliminating or reducing the risk, lessening the severity, or
delaying the outset of the disease, including biochemical,
histological and/or behavioral symptoms of the disease, its
complications and intermediate pathological phenotypes presenting
during development of the disease. For therapeutic use, beneficial
or desired results include clinical results such as reducing blood
glucose levels, reducing incidence or amelioration of one or more
symptoms of type 1 diabetes, type 2 diabetes, rheumatoid arthritis,
GVHD, lupus or multiple sclerosis, decreasing the dose of other
medications required to treat the disease, enhancing the effect of
another medication, and/or delaying the progression of the disease
of patients. An effective dosage can be administered in one or more
administrations. For purposes of this invention, an effective
dosage of drug, compound, or pharmaceutical composition is an
amount sufficient to accomplish prophylactic or therapeutic
treatment either directly or indirectly. As is understood in the
clinical context, an effective dosage of a drug, compound, or
pharmaceutical composition may or may not be achieved in
conjunction with another drug, compound, or pharmaceutical
composition. Thus, an "effective dosage" may be considered in the
context of administering one or more therapeutic agents, and a
single agent may be considered to be given in an effective amount
if, in conjunction with one or more other agents, a desirable
result may be or is achieved.
[0081] An "individual" or a "subject" is a mammal, more preferably,
a human. Mammals also include, but are not limited to, farm
animals, sport animals, pets, primates, horses, dogs, cats, mice
and rats.
[0082] As used herein, "vector" means a construct, which is capable
of delivering, and, preferably, expressing, one or more gene(s) or
sequence(s) of interest in a host cell. Examples of vectors
include, but are not limited to, viral vectors, naked DNA or RNA
expression vectors, plasmid, cosmid or phage vectors, DNA or RNA
expression vectors associated with cationic condensing agents, DNA
or RNA expression vectors encapsulated in liposomes, and certain
eukaryotic cells, such as producer cells.
[0083] As used herein, "expression control sequence" means a
nucleic acid sequence that directs transcription of a nucleic acid.
An expression control sequence can be a promoter, such as a
constitutive or an inducible promoter, or an enhancer. The
expression control sequence is operably linked to the nucleic acid
sequence to be transcribed.
[0084] As used herein, "pharmaceutically acceptable carrier" or
"pharmaceutical acceptable excipient" includes any material which,
when combined with an active ingredient, allows the ingredient to
retain biological activity and is non-reactive with the subject's
immune system. Examples include, but are not limited to, any of the
standard pharmaceutical carriers such as a phosphate buffered
saline solution, water, emulsions such as oil/water emulsion, and
various types of wetting agents. Preferred diluents for aerosol or
parenteral administration are phosphate buffered saline (PBS) or
normal (0.9%) saline. Compositions comprising such carriers are
formulated by well known conventional methods (see, for example,
Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed.,
Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science
and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).
[0085] The term "k.sub.on", as used herein, refers to the rate
constant for association of an antibody to an antigen.
Specifically, the rate constants (k.sub.on and k.sub.off) and
equilibrium dissociation constants are measured using Fab antibody
fragments (i.e. univalent) and IL-7R.
[0086] The term "k.sub.off", as used herein, refers to the rate
constant for dissociation of an antibody from the antibody/antigen
complex.
[0087] The term "K.sub.D", as used herein, refers to the
equilibrium dissociation constant of an antibody-antigen
interaction.
[0088] Reference to "about" a value or parameter herein includes
(and describes) embodiments that are directed to that value or
parameter per se. For example, description referring to "about X"
includes description of "X." Numeric ranges are inclusive of the
numbers defining the range.
[0089] It is understood that wherever embodiments are described
herein with the language "comprising," otherwise analogous
embodiments described in terms of "consisting of" and/or
"consisting essentially of" are also provided.
[0090] Where aspects or embodiments of the invention are described
in terms of a Markush group or other grouping of alternatives, the
present invention encompasses not only the entire group listed as a
whole, but each member of the group individually and all possible
subgroups of the main group, but also the main group absent one or
more of the group members. The present invention also envisages the
explicit exclusion of one or more of any of the group members in
the claimed invention.
[0091] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. In case
of conflict, the present specification, including definitions, will
control. Throughout this specification and claims, the word
"comprise," or variations such as "comprises" or "comprising" will
be understood to imply the inclusion of a stated integer or group
of integers but not the exclusion of any other integer or group of
integers. Unless otherwise required by context, singular terms
shall include pluralities and plural terms shall include the
singular.
[0092] Exemplary methods and materials are described herein,
although methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the
present invention. The materials, methods, and examples are
illustrative only and not intended to be limiting.
Methods for Preventing or Treating Type 2 Diabetes, GVHD and
Autoimmune Disorders
[0093] In one aspect, the invention provides a method for treating
or preventing type 2 diabetes in an individual comprising
administering to the individual an effective amount of an IL-7R
antagonist such as, for example, an antagonist IL-7R antibody. In
another aspect, the invention provides a method for treating or
preventing an autoimmune disease, such as type 1 diabetes,
rheumatoid arthritis, lupus or multiple sclerosis, in an
individual, the method comprising administering to the individual
an effective amount of an IL-7R antagonist. In another aspect, the
invention provides a method for treating or preventing GVHD in an
individual comprising administering to the individual an effective
amount of an IL-7R antagonist.
[0094] In some embodiments, therapeutic administration of the IL-7R
antagonist advantageously results in lower blood glucose level and
improved glucose tolerance. In other embodiments, therapeutic
administration of the IL-7R antagonist advantageously maintains
blood glucose at desirable levels.
[0095] In some embodiments, therapeutic administration of the IL-7R
antagonist advantageously results in reduced incidence and/or
amelioration of one or more symptoms of rheumatoid arthritis
including, for example without limitation, joint stiffness, joint
swelling, joint pain, and joint redness and warmth.
[0096] In some embodiments, therapeutic administration of the IL-7R
antagonist advantageously results in reduced incidence and/or
amelioration of one or more symptoms of lupus including, for
example without limitation, fatigue, fever, weight loss, weight
gain, joint pain, joint stiffness, joint swelling, malar rash, skin
lesions, mouth sores, nose ulcers, hair loss, Raynaud's phenomenon,
shortness of breath, chest pain, dry eyes, bruising, anxiety,
depression and memory loss.
[0097] In some embodiments, therapeutic administration of the IL-7R
antagonist advantageously results in reduced incidence and/or
amelioration of one or more symptoms of multiple sclerosis
including, for example without limitation, limb paralysis, tremors,
difficulty walking, swallowing difficulties, blindness, blurring
vision, and muscle weakness.
[0098] In some embodiments, therapeutic administration of the IL-7R
antagonist advantageously results in reduced incidence and/or
amelioration of one or more symptoms of GVHD including, for example
without limitation, abdominal pain, abdominal cramps, fever,
jaundice, skin rash, vomiting, weight loss, dry eyes, dry mouth,
hair loss, hepatitis, lung disorders, and digestive tract
disorders.
[0099] In some embodiments, therapeutic administration of the IL-7R
antagonist advantageously results in reduced incidence and/or
amelioration of one or more symptoms of acute GVHD including, for
example without limitation, pneumonitis, intestinal inflammation,
diarrhea, abdominal pain, abdominal cramps, fever, jaundice,
nausea, vomiting, liver damage, skin rash, skin damage, damage to
the mucosa, sloughing of the mucosal membrane, damage to the
gastrointestinal tract, weight loss, maculopapular rash, elevated
bilirubin levels, morbidity and mortality.
[0100] In some embodiments, therapeutic administration of the IL-7R
antagonist advantageously results in reduced incidence and/or
amelioration of one or more symptoms of chronic GVHD including, for
example without limitation, dry eyes, dry mouth, hair loss,
hepatitis, lung disorders, digestive tract disorders, skin rash,
oral ulcer, oral atrophy, onchodystrophy, sicca syndrome,
sclerosis, lichen-planus-like lesions, poikiloderma, esophageal
webs, fasciitis and bronchiolitis obliterans, and damage to the
liver, skin and mucosa, connective tissue, exocrine glands and/or
the gastrointestinal tract.
[0101] A diabetic individual requiring lower blood glucose levels
can be treated with an IL-7R antagonist such as, for example, an
antagonist IL-7R antibody. An individual suitable for antibody
therapy is selected using clinical criteria and prognostic
indicators of diabetes that are well known in the art. An
individual at risk of developing diabetes as assessed by known
prognostic indicators such as family history, fasting blood glucose
levels, or decreased glucose tolerance also warrants administration
of an IL-7R antagonist. One skilled in the art would recognize or
know how to diagnose an individual with diabetes or disregulated
glucose uptake and, depending upon the degree or severity of the
disease, can make the appropriate determination of when to
administer the antibody and can also select the most desirable mode
of administration.
[0102] An individual suffering from rheumatoid arthritis can be
treated with an IL-7R antagonist such as, for example, an
antagonist IL-7R antibody. An individual suitable for IL-7R
antagonist therapy is selected using clinical criteria and
prognostic indicators of rheumatoid arthritis that are well known
in the art. Diagnosis or assessment of rheumatoid arthritis is
well-established in the art. Assessment of severity may be
performed based on measures known in the art, such as the
rheumatoid arthritis severity scale (RASS). Bardwell et al.,
Rheumatology, 2002, 41:38-45. In some embodiments, ameliorating,
controlling, reducing incidence of, or delaying the development or
progression of rheumatoid arthritis and/or symptoms of rheumatoid
arthritis is measured by RASS.
[0103] An individual suffering from lupus can be treated with an
IL-7R antagonist such as, for example, an antagonist IL-7R
antibody. An individual suitable for IL-7R antagonist therapy is
selected using clinical criteria and prognostic indicators of lupus
that are well known in the art. One skilled in the art would
recognize or know how to diagnose an individual with lupus and,
depending upon the degree or severity of the disease, can make the
appropriate determination of when to administer the IL-7R
antagonist and can also select the most desirable mode of
administration.
[0104] An individual suffering from multiple sclerosis can be
treated with an IL-7R antagonist such as, for example, an
antagonist IL-7R antibody. An individual suitable for IL-7R
antagonist therapy is selected using clinical criteria and
prognostic indicators of multiple sclerosis that are well known in
the art. An individual at risk of developing multiple sclerosis as
assessed by known prognostic indicators such as family history or
symptom history also warrants administration of an IL-7R
antagonist. One skilled in the art would recognize or know how to
diagnose an individual with multiple sclerosis and, depending upon
the degree or severity of the disease, can make the appropriate
determination of when to administer the IL-7R antagonist and can
also select the most desirable mode of administration.
[0105] An individual suffering from GVHD can be treated with an
IL-7R antagonist such as, for example, an antagonist IL-7R
antibody. An individual suitable for IL-7R antagonist therapy is
selected using clinical criteria and prognostic indicators of GVHD
that are well known in the art. Diagnosis or assessment of GVHD is
well-established in the art. Tests for GVHD usually depend on the
symptoms, but may include gastrointesting endoscopy, with or
without a biopsy, liver functions tests (AST, ALP, and bilirubin
levels will be increased), livery biopsy, lung x-rays, and/or skin
biopsy. Features sufficient to establish the diagnosis of chronic
GVHD include, for example without limitation, sclerosis,
lichen-planus-like lesions, poikiloderma, esophageal webs,
fasciitis and bronchiolitis obliterans (see, e.g., Leet and
Flowers, Hematology, January 2008; 2008:134-141). Acute liver GVHD
may be measured by, for example, the bilirubin level in acute
patients. Acute skin GVHD may result in a diffuse maculopapular
rash. Assessment of GVHD severity may be performed based on
measures known in the art. In some embodiments, ameliorating,
controlling, reducing incidence of, or delaying the development or
progression of GVHD and/or symptoms of GVHD is measured by overall
grade (skin-liver-gut) with each organ staged individually from a
low of 1 to a high of 4. In some embodiments, ameliorating,
controlling, reducing incidence of, or delaying the development or
progression of GVHD and/or symptoms of GVHD is measured by
monitoring body weight.
[0106] With respect to all methods described herein, reference to
IL-7R antagonists also includes compositions comprising one or more
additional agents. These compositions may further comprise suitable
excipients, such as pharmaceutically acceptable excipients
including buffers, which are well known in the art. The present
invention can be used alone or in combination with other
conventional methods of treatment.
[0107] The IL-7R antagonist can be administered to an individual
via any suitable route. It should be apparent to a person skilled
in the art that the examples described herein are not intended to
be limiting but to be illustrative of the techniques available.
Accordingly, in some embodiments, the IL-7R antagonist is
administered to an individual in accord with known methods, such as
intravenous administration, e.g., as a bolus or by continuous
infusion over a period of time, by intramuscular, intraperitoneal,
intracerebrospinal, transdermal, subcutaneous, intra-articular,
sublingually, intrasynovial, via insufflation, intrathecal, oral,
inhalation or topical routes. Administration can be systemic, e.g.,
intravenous administration, or localized. Commercially available
nebulizers for liquid formulations, including jet nebulizers and
ultrasonic nebulizers are useful for administration. Liquid
formulations can be directly nebulized and lyophilized powder can
be nebulized after reconstitution. Alternatively, an IL-7R
antagonist can be aerosolized using a fluorocarbon formulation and
a metered dose inhaler, or inhaled as a lyophilized and milled
powder.
[0108] In one embodiment, an IL-7R antagonist is administered via
site-specific or targeted local delivery techniques. Examples of
site-specific or targeted local delivery techniques include various
implantable depot sources of the IL-7R antagonist or local delivery
catheters, such as infusion catheters, indwelling catheters, or
needle catheters, synthetic grafts, adventitial wraps, shunts and
stents or other implantable devices, site specific carriers, direct
injection, or direct application. See, e.g., PCT Publication No. WO
00/53211 and U.S. Pat. No. 5,981,568.
[0109] Various formulations of an IL-7R antagonist may be used for
administration. In some embodiments, the IL-7R antagonist may be
administered neat. In some embodiments, IL-7R antagonist and a
pharmaceutically acceptable excipient may be in various
formulations. Pharmaceutically acceptable excipients are known in
the art, and are relatively inert substances that facilitate
administration of a pharmacologically effective substance. For
example, an excipient can give form or consistency, or act as a
diluent. Suitable excipients include but are not limited to
stabilizing agents, wetting and emulsifying agents, salts for
varying osmolarity, encapsulating agents, buffers, and skin
penetration enhancers. Excipients as well as formulations for
parenteral and nonparenteral drug delivery are set forth in
Remington, The Science and Practice of Pharmacy 20th Ed. Mack
Publishing, 2000.
[0110] In some embodiments, these agents are formulated for
administration by injection (e.g., intraperitoneally,
intravenously, subcutaneously, intramuscularly, etc.). Accordingly,
these agents can be combined with pharmaceutically acceptable
vehicles such as saline, Ringer's solution, dextrose solution, and
the like. The particular dosage regimen, i.e., dose, timing and
repetition, will depend on the particular individual and that
individual's medical history.
[0111] An IL-7R antagonist can be administered using any suitable
method, including by injection (e.g., intraperitoneally,
intravenously, subcutaneously, intramuscularly, etc.). IL-7R
antibodies can also be administered via inhalation, as described
herein. Generally, for administration of IL-7R antibodies, an
initial candidate dosage can be about 2 mg/kg. For the purpose of
the present invention, a typical daily dosage might range from
about any of 3 .mu.g/kg to 30 .mu.g/kg to 300 .mu.g/kg to 3 mg/kg,
to 30 mg/kg, to 100 mg/kg or more, depending on the factors
mentioned above. For example, dosage of about 1 mg/kg, about 2.5
mg/kg, about 5 mg/kg, about 10 mg/kg, and about 25 mg/kg may be
used. For repeated administrations over several days or longer,
depending on the condition, the treatment is sustained until a
desired suppression of symptoms occurs or until sufficient
therapeutic levels are achieved, for example, to reduce blood
glucose levels. An exemplary dosing regimen comprises administering
an initial dose of about 2 mg/kg, followed by a weekly maintenance
dose of about 1 mg/kg of the IL-7R antibody, or followed by a
maintenance dose of about 1 mg/kg every other week. However, other
dosage regimens may be useful, depending on the pattern of
pharmacokinetic decay that the practitioner wishes to achieve. For
example, in some embodiments, dosing from one to four times a week
is contemplated. In other embodiments dosing once a month or once
every other month or every three months is contemplated. The
progress of this therapy is easily monitored by conventional
techniques and assays. The dosing regimen (including the IL-7R
antagonist(s) used) can vary overtime.
[0112] For the purpose of the present invention, the appropriate
dosage of an IL-7R antagonist will depend on the IL-7R antagonist
(or compositions thereof) employed, the type and severity of
symptoms to be treated, whether the agent is administered for
preventive or therapeutic purposes, previous therapy, the patient's
clinical history and response to the agent, the patient's clearance
rate for the administered agent, and the discretion of the
attending physician. Typically the clinician will administer an
IL-7R antagonist until a dosage is reached that achieves the
desired result. Dose and/or frequency can vary over course of
treatment. Empirical considerations, such as the half-life,
generally will contribute to the determination of the dosage. For
example, antibodies that are compatible with the human immune
system, such as humanized antibodies or fully human antibodies, may
be used to prolong half-life of the antibody and to prevent the
antibody being attacked by the host's immune system. Frequency of
administration may be determined and adjusted over the course of
therapy, and is generally, but not necessarily, based on treatment
and/or suppression and/or amelioration and/or delay of symptoms,
e.g., high blood glucose levels, joint pain, etc. Alternatively,
sustained continuous release formulations of antagonist IL-7R
antibodies may be appropriate. Various formulations and devices for
achieving sustained release are known in the art.
[0113] In one embodiment, dosages for an IL-7R antagonist may be
determined empirically in individuals who have been given one or
more administration(s) of an IL-7R antagonist. Individuals are
given incremental dosages of an IL-7R antagonist. To assess
efficacy, an indicator of the disease can be followed.
[0114] Administration of an IL-7R antagonist in accordance with the
method in the present invention can be continuous or intermittent,
depending, for example, upon the recipient's physiological
condition, whether the purpose of the administration is therapeutic
or prophylactic, and other factors known to skilled practitioners.
The administration of an IL-7R antagonist may be essentially
continuous over a preselected period of time or may be in a series
of spaced doses.
[0115] In some embodiments, more than one IL-7R antagonist may be
present. At least one, at least two, at least three, at least four,
at least five different, or more IL-7R antagonists can be present.
Generally, those IL-7R antagonists may have complementary
activities that do not adversely affect each other. For example,
one or more of the following IL-7R antagonists may be used: an
antagonist IL-7R antibody, an anti-sense molecule directed to an
IL-7R (including an anti-sense molecule directed to a nucleic acid
encoding IL-7R), an IL-7R inhibitory compound, and an IL-7R
structural analog. An IL-7R antagonist can also be used in
conjunction with other agents that serve to enhance and/or
complement the effectiveness of the agents.
[0116] Therapeutic formulations of the IL-7R antagonist used in
accordance with the present invention are prepared for storage by
mixing an antibody having the desired degree of purity with
optional pharmaceutically acceptable carriers, excipients or
stabilizers (Remington, The Science and Practice of Pharmacy 20th
Ed. Mack Publishing, 2000), in the form of lyophilized formulations
or aqueous solutions. Acceptable carriers, excipients, or
stabilizers are nontoxic to recipients at the dosages and
concentrations employed, and may comprise buffers such as
phosphate, citrate, and other organic acids; salts such as sodium
chloride; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0117] Liposomes containing the IL-7R antagonist 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. 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.
[0118] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacrylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington, The Science and Practice of
Pharmacy 20th Ed. Mack Publishing, 2000.
[0119] Sustained-release preparations may 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 7 ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), sucrose
acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
[0120] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by, for example,
filtration through sterile filtration membranes. Therapeutic IL-7R
antagonist compositions are generally placed into a container
having a sterile access port, for example, an intravenous solution
bag or vial having a stopper pierceable by a hypodermic injection
needle.
[0121] The compositions according to the present invention may be
in unit dosage forms such as tablets, pills, capsules, powders,
granules, solutions or suspensions, or suppositories, for oral,
parenteral or rectal administration, or administration by
inhalation or insufflation.
[0122] For preparing solid compositions such as tablets, the
principal active ingredient is mixed with a pharmaceutical carrier,
e.g. conventional tableting ingredients such as corn starch,
lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate,
dicalcium phosphate or gums, and other pharmaceutical diluents,
e.g. water, to form a solid preformulation composition containing a
homogeneous mixture of a compound of the present invention, or a
non-toxic pharmaceutically acceptable salt thereof. When referring
to these preformulation compositions as homogeneous, it is meant
that the active ingredient is dispersed evenly throughout the
composition so that the composition may be readily subdivided into
equally effective unit dosage forms such as tablets, pills and
capsules. This solid preformulation composition is then subdivided
into unit dosage forms of the type described above containing from
0.1 to about 500 mg of the active ingredient of the present
invention. The tablets or pills of the novel composition can be
coated or otherwise compounded to provide a dosage form affording
the advantage of prolonged action. For example, the tablet or pill
can comprise an inner dosage and an outer dosage component, the
latter being in the form of an envelope over the former. The two
components can be separated by an enteric layer that serves to
resist disintegration in the stomach and permits the inner
component to pass intact into the duodenum or to be delayed in
release. A variety of materials can be used for such enteric layers
or coatings, such materials including a number of polymeric acids
and mixtures of polymeric acids with such materials as shellac,
cetyl alcohol and cellulose acetate.
[0123] Suitable surface-active agents include, in particular,
non-ionic agents, such as polyoxyethylenesorbitans (e.g. Tween.TM.
20, 40, 60, 80 or 85) and other sorbitans (e.g. Span.TM. 20, 40,
60, 80 or 85). Compositions with a surface-active agent will
conveniently comprise between 0.05 and 5% surface-active agent, and
can be between 0.1 and 2.5%. It will be appreciated that other
ingredients may be added, for example mannitol or other
pharmaceutically acceptable vehicles, if necessary.
[0124] Suitable emulsions may be prepared using commercially
available fat emulsions, such as Intralipid.TM., Liposyn.TM.,
Infonutrol.TM., Lipofundin.TM. and Lipiphysan.TM.. The active
ingredient may be either dissolved in a pre-mixed emulsion
composition or alternatively it may be dissolved in an oil (e.g.
soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or
almond oil) and an emulsion formed upon mixing with a phospholipid
(e.g. egg phospholipids, soybean phospholipids or soybean lecithin)
and water. It will be appreciated that other ingredients may be
added, for example glycerol or glucose, to adjust the tonicity of
the emulsion. Suitable emulsions will typically contain up to 20%
oil, for example, between 5 and 20%. The fat emulsion can comprise
fat droplets between 0.1 and 1.0 .mu.m, particularly 0.1 and 0.5
.mu.m, and have a pH in the range of 5.5 to 8.0.
[0125] The emulsion compositions can be those prepared by mixing an
IL-7R antagonist with Intralipid.TM. or the components thereof
(soybean oil, egg phospholipids, glycerol and water).
[0126] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable excipients as set out above. In some embodiments, the
compositions are administered by the oral or nasal respiratory
route for local or systemic effect. Compositions in preferably
sterile pharmaceutically acceptable solvents may be nebulised by
use of gases. Nebulised solutions may be breathed directly from the
nebulising device or the nebulising device may be attached to a
face mask, tent or intermittent positive pressure breathing
machine. Solution, suspension or powder compositions may be
administered, preferably orally or nasally, from devices which
deliver the formulation in an appropriate manner.
IL-7R Antagonists
[0127] The methods of the invention use an IL-7R antagonist, which
refers to any protein, peptide or nucleic acid molecule that
blocks, suppresses or reduces (including significantly reduces)
IL-7R biological activity, including downstream pathways mediated
by IL-7R signaling, such as elicitation of a cellular response to
IL-7R. Examples of IL-7R antagonists include, without limitation,
antagonist IL-7R antibodies, IL-7R siRNA, IL-7R shRNA, and IL-7R
antisense oligonucleotides.
[0128] An IL-7R antagonist should exhibit any one or more of the
following characteristics: (a) bind to IL-7R; (b) block IL-7R
interaction with IL-7; (c) block or decrease IL-7-mediated STAT5
phosphorylation; (d) decrease blood glucose levels in vivo; (e)
increase glucose tolerance in vivo; (f) reduce disease severity in
experimental autoimmune encephalomyelitis (EAE); (g) block or
decrease PI3K phosphorylation; (h) block or decrease AKT
phosphorylation; and (i) block IL-7R interaction with other yet to
be identified factors.
[0129] In some embodiments, the IL-7R antagonist is an antagonist
IL-7R antibody. For purposes of this invention, the antagonist
IL-7R antibody preferably reacts with IL-7R.alpha. in a manner that
inhibits IL-7R signaling function and IL-7 interaction. In some
embodiments, the antagonist IL-7R antibody specifically recognizes
primate IL-7R. In some embodiments, the antagonist IL-7R antibody
binds primate and rodent IL-7R.
[0130] The antibodies useful in the present invention can encompass
monoclonal antibodies, polyclonal antibodies, antibody fragments
(e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies,
bispecific antibodies, heteroconjugate antibodies, single chain
(ScFv), mutants thereof, fusion proteins comprising an antibody
portion (e.g., a domain antibody), humanized antibodies, and any
other modified configuration of the immunoglobulin molecule that
comprises an antigen recognition site of the required specificity,
including glycosylation variants of antibodies, amino acid sequence
variants of antibodies, and covalently modified antibodies. The
antibodies may be murine, rat, human, or any other origin
(including chimeric or humanized antibodies).
[0131] In some embodiments, the antagonist IL-7R antibody is a
monoclonal antibody. The antagonist IL-7R antibody can also be
humanized. In other embodiments, the antibody is human.
[0132] In some embodiments, the antibody comprises a modified
constant region, such as, for example without limitation, a
constant region that has increased potential for provoking an
immune response. For example, the constant region may be modified
to have increased affinity to an Fc gamma receptor such as, e.g.,
Fc.gamma.RI or Fc.gamma.RIIA.
[0133] In some embodiments, the antibody comprises a modified
constant region, such as a constant region that is immunologically
inert, that is, having a reduced potential for provoking an immune
response. In some embodiments, the constant region is modified as
described in Eur. J. Immunol., 1999, 29:2613-2624; PCT Application
No. PCT/GB99/01441, and/or UK Patent Application No. 9809951.8. The
Fc can be human human IgG1, human IgG2 or human IgG4. The Fc can be
human IgG2 containing the mutation A330P331 to S330S331
(IgG2.DELTA.a), in which the amino acid residues are numbered with
reference to the wild type IgG2 sequence. Eur. J. Immunol., 1999,
29:2613-2624. In some embodiments, the antibody comprises a
constant region of IgG4 comprising the following mutations (Armour
et al., 2003, Molecular Immunology 40 585-593): E233F234L235 to
P233V234A235 (IgG4.DELTA.c), in which the numbering is with
reference to wild type IgG4. In yet another embodiment, the Fc is
human IgG4 E233F234L235 to P233V234A235 with deletion G236
(IgG4.DELTA.b). In another embodiment the Fc is any human IgG4 Fc
(IgG4, IgG4.DELTA.b or IgG4.DELTA.c) containing hinge stabilizing
mutation S228 to P228 (Aalberse et al., 2002, Immunology 105,
9-19). In another embodiment, the Fc can be aglycosylated Fc.
[0134] In some embodiments, the constant region is aglycosylated by
mutating the oligosaccharide attachment residue (such as Asn297)
and/or flanking residues that are part of the glycosylation
recognition sequence in the constant region. In some embodiments,
the constant region is aglycosylated for N-linked glycosylation
enzymatically. The constant region may be aglycosylated for
N-linked glycosylation enzymatically or by expression in a
glycosylation deficient host cell.
[0135] The binding affinity (K.sub.D) of an antagonist IL-7R
antibody to IL-7R (such as human IL-7R) can be about 0.002 to about
200 nM. In some embodiments, the binding affinity is any of about
200 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM, about
500 pM, about 100 pM, about 60 pM, about 50 pM, about 20 pM, about
15 pM, about 10 pM, about 5 pM, or about 2 pM. In some embodiments,
the binding affinity is less than any of about 250 nM, about 200
nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500
pM, about 100 pM, about 50 pM, about 20 pM, about 10 pM, about 5
pM, or about 2 pM.
[0136] One way of determining binding affinity of antibodies to
IL-7R is by measuring binding affinity of monofunctional Fab
fragments of the antibody. To obtain monofunctional Fab fragments,
an antibody (for example, IgG) can be cleaved with papain or
expressed recombinantly. The affinity of an IL-7R Fab fragment of
an antibody can be determined by surface plasmon resonance
(Biacore.TM.3000.TM. surface plasmon resonance (SPR) system,
Biacore.TM., INC, Piscataway N.J.) equipped with pre-immobilized
streptavidin sensor chips (SA) using HBS-EP running buffer (0.01M
HEPES, pH 7.4, 0.15 NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20).
Biotinylated human IL-7R (or any other IL-7R) can be diluted into
HBS-EP buffer to a concentration of less than 0.5 .mu.g/mL and
injected across the individual chip channels using variable contact
times, to achieve two ranges of antigen density, either 50-200
response units (RU) for detailed kinetic studies or 800-1,000 RU
for screening assays. Regeneration studies have shown that 25 mM
NaOH in 25% v/v ethanol effectively removes the bound Fab while
keeping the activity of IL-7R on the chip for over 200 injections.
Typically, serial dilutions (spanning concentrations of
0.1-10.times. estimated K.sub.D) of purified Fab samples are
injected for 1 min at 100 .mu.L/minute and dissociation times of up
to 2 hours are allowed. The concentrations of the Fab proteins are
determined by ELISA and/or SDS-PAGE electrophoresis using a Fab of
known concentration (as determined by amino acid analysis) as a
standard. Kinetic association rates (k.sub.on) and dissociation
rates (k.sub.off) are obtained simultaneously by fitting the data
globally to a 1:1 Langmuir binding model (Karlsson, R. Roos, H.
Fagerstam, L. Petersson, B. (1994). Methods Enzymology 6. 99-110)
using the BIAevaluation program. Equilibrium dissociation constant
(K.sub.D) values are calculated as k.sub.off/k.sub.on. This
protocol is suitable for use in determining binding affinity of an
antibody to any IL-7R, including human IL-7R, IL-7R of another
mammal (such as mouse IL-7R, rat IL-7R, primate IL-7R), as well as
different forms of IL-7R. Binding affinity of an antibody is
generally measured at 25.degree. C., but can also be measured at
37.degree. C.
[0137] The antagonist IL-7R antibodies may be made by any method
known in the art, including the method as provided in Example 1.
For the production of hybridoma cell lines, the route and schedule
of immunization of the host animal are generally in keeping with
established and conventional techniques for antibody stimulation
and production, as further described herein. General techniques for
production of human and mouse antibodies are known in the art
and/or are described herein.
[0138] It is contemplated that any mammalian subject including
humans or antibody producing cells therefrom can be manipulated to
serve as the basis for production of mammalian, including human,
hybridoma cell lines. Typically, the host animal is inoculated
intraperitoneally, intramuscularly, orally, subcutaneously,
intraplantar, and/or intradermally with an amount of immunogen,
including as described herein.
[0139] Hybridomas can be prepared from the lymphocytes and
immortalized myeloma cells using the general somatic cell
hybridization technique of Kohler, B. and Milstein, C., 1975,
Nature 256:495-497 or as modified by Buck, D. W., et al., In Vitro,
18:377-381, 1982. Available myeloma lines, including but not
limited to X63-Ag8.653 and those from the Salk Institute, Cell
Distribution Center, San Diego, Calif., USA, may be used in the
hybridization. Generally, the technique involves fusing myeloma
cells and lymphoid cells using a fusogen such as polyethylene
glycol, or by electrical means well known to those skilled in the
art. After the fusion, the cells are separated from the fusion
medium and grown in a selective growth medium, such as
hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate
unhybridized parent cells. Any of the media described herein,
supplemented with or without serum, can be used for culturing
hybridomas that secrete monoclonal antibodies. As another
alternative to the cell fusion technique, EBV immortalized B cells
may be used to produce the IL-7R monoclonal antibodies of the
subject invention. The hybridomas are expanded and subcloned, if
desired, and supernatants are assayed for anti-immunogen activity
by conventional immunoassay procedures (e.g., radioimmunoassay,
enzyme immunoassay, or fluorescence immunoassay).
[0140] Hybridomas that may be used as source of antibodies
encompass all derivatives, progeny cells of the parent hybridomas
that produce monoclonal antibodies specific for IL-7R, or a portion
thereof.
[0141] Hybridomas that produce such antibodies may be grown in
vitro or in vivo using known procedures. The monoclonal antibodies
may be isolated from the culture media or body fluids, by
conventional immunoglobulin purification procedures such as
ammonium sulfate precipitation, gel electrophoresis, dialysis,
chromatography, and ultrafiltration, if desired. Undesired
activity, if present, can be removed, for example, by running the
preparation over adsorbents made of the immunogen attached to a
solid phase and eluting or releasing the desired antibodies off the
immunogen. Immunization of a host animal with a human IL-7R.alpha.,
or a fragment containing the target amino acid sequence conjugated
to a protein that is immunogenic in the species to be immunized,
e.g., keyhole limpet hemocyanin, serum albumin, bovine
thyroglobulin, or soybean trypsin inhibitor using a bifunctional or
derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide
ester (conjugation through cysteine residues), N-hydroxysuccinimide
(through lysine residues), glutaraldehyde, succinic anhydride,
SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R and R.sup.1 are
different alkyl groups, can yield a population of antibodies (e.g.,
monoclonal antibodies).
[0142] If desired, the antagonist IL-7R antibody (monoclonal or
polyclonal) of interest may be sequenced and the polynucleotide
sequence may then be cloned into a vector for expression or
propagation. The sequence encoding the antibody of interest may be
maintained in vector in a host cell and the host cell can then be
expanded and frozen for future use. Production of recombinant
monoclonal antibodies in cell culture can be carried out through
cloning of antibody genes from B cells by means known in the art.
See, e.g. Tiller et al., 2008, J. Immunol. Methods 329, 112; U.S.
Pat. No. 7,314,622.
[0143] In an alternative, the polynucleotide sequence may be used
for genetic manipulation to "humanize" the antibody or to improve
the affinity, or other characteristics of the antibody. For
example, the constant region may be engineered to more nearly
resemble human constant regions to avoid immune response if the
antibody is used in clinical trials and treatments in humans. It
may be desirable to genetically manipulate the antibody sequence to
obtain greater affinity to IL-7R and greater efficacy in inhibiting
IL-7R. It will be apparent to one of skill in the art that one or
more polynucleotide changes can be made to the antagonist IL-7R
antibody and still maintain its binding ability to IL-7R.
[0144] There are four general steps to humanize a monoclonal
antibody. These are: (1) determining the nucleotide and predicted
amino acid sequence of the starting antibody light and heavy
variable domains (2) designing the humanized antibody, i.e.,
deciding which antibody framework region to use during the
humanizing process (3) the actual humanizing
methodologies/techniques and (4) the transfection and expression of
the humanized antibody. See, for example, U.S. Pat. Nos. 4,816,567;
5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762;
5,585,089; and 6,180,370.
[0145] A number of "humanized" antibody molecules comprising an
antigen-binding site derived from a non-human immunoglobulin have
been described, including chimeric antibodies having rodent or
modified rodent V regions and their associated CDRs fused to human
constant regions. See, for example, Winter et al. Nature
349:293-299, 1991, Lobuglio et al. Proc. Nat. Acad. Sci. USA
86:4220-4224, 1989, Shaw et al. J Immunol. 138:4534-4538, 1987, and
Brown et al. Cancer Res. 47:3577-3583, 1987. Other references
describe rodent CDRs grafted into a human supporting framework
region (FR) prior to fusion with an appropriate human antibody
constant region. See, for example, Riechmann et al. Nature
332:323-327, 1988, Verhoeyen et al. Science 239:1534-1536, 1988,
and Jones et al. Nature 321:522-525, 1986. Another reference
describes rodent CDRs supported by recombinantly engineered rodent
framework regions. See, for example, European Patent Publication
No. 0519596. These "humanized" molecules are designed to minimize
unwanted immunological response toward rodent anti-human antibody
molecules which limits the duration and effectiveness of
therapeutic applications of those moieties in human recipients. For
example, the antibody constant region can be engineered such that
it is immunologically inert (e.g., does not trigger complement
lysis). See, e.g. PCT Publication No. PCT/GB99/01441; UK Patent
Application No. 9809951.8. Other methods of humanizing antibodies
that may also be utilized are disclosed by Daugherty et al., Nucl.
Acids Res. 19:2471-2476, 1991, and in U.S. Pat. Nos. 6,180,377;
6,054,297; 5,997,867; 5,866,692; 6,210,671; and 6,350,861; and in
PCT Publication No. WO 01/27160.
[0146] It is apparent that although the above discussion pertains
to humanized antibodies, the general principles discussed are
applicable to customizing antibodies for use, for example, in dogs,
cats, primate, equines and bovines. It is further apparent that one
or more aspects of humanizing an antibody described herein may be
combined, e.g., CDR grafting, framework mutation and CDR
mutation.
[0147] In yet another alternative, fully human antibodies may be
obtained by using commercially available mice that have been
engineered to express specific human immunoglobulin proteins.
Transgenic animals that are designed to produce a more desirable
(e.g., fully human antibodies) or more robust immune response may
also be used for generation of humanized or human antibodies.
Examples of such technology are Xenomouse.TM. from Abgenix, Inc.
(Fremont, Calif.) and HuMAb-Mouse.RTM. and TC Mouse.TM. from
Medarex, Inc. (Princeton, N.J.).
[0148] In an alternative, antibodies may be made recombinantly and
expressed using any method known in the art. In another
alternative, antibodies may be made recombinantly by phage display
technology. See, for example, U.S. Pat. Nos. 5,565,332; 5,580,717;
5,733,743; and 6,265,150; and Winter et al., Annu. Rev. Immunol.
12:433-455, 1994. Alternatively, the phage display technology
(McCafferty et al., Nature 348:552-553, 1990) can be used to
produce human antibodies and antibody fragments in vitro, from
immunoglobulin variable (V) domain gene repertoires from
unimmunized donors. According to this technique, antibody V domain
genes are cloned in-frame into either a major or minor coat protein
gene of a filamentous bacteriophage, such as M13 or fd, and
displayed as functional antibody fragments on the surface of the
phage particle. Because the filamentous particle contains a
single-stranded DNA copy of the phage genome, selections based on
the functional properties of the antibody also result in selection
of the gene encoding the antibody exhibiting those properties.
Thus, the phage mimics some of the properties of the B cell. Phage
display can be performed in a variety of formats; for review see,
e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in
Structural Biology 3:564-571, 1993. Several sources of V-gene
segments can be used for phage display. Clackson et al., Nature
352:624-628, 1991, isolated a diverse array of anti-oxazolone
antibodies from a small random combinatorial library of V genes
derived from the spleens of immunized mice. A repertoire of V genes
from unimmunized human donors can be constructed and antibodies to
a diverse array of antigens (including self-antigens) can be
isolated essentially following the techniques described by Mark et
al., J. Mol. Biol. 222:581-597, 1991, or Griffith et al., EMBO J.
12:725-734, 1993. In a natural immune response, antibody genes
accumulate mutations at a high rate (somatic hypermutation). Some
of the changes introduced will confer higher affinity, and B cells
displaying high-affinity surface immunoglobulin are preferentially
replicated and differentiated during subsequent antigen challenge.
This natural process can be mimicked by employing the technique
known as "chain shuffling." (Marks et al., Bio/Technol. 10:779-783,
1992). In this method, the affinity of "primary" human antibodies
obtained by phage display can be improved by sequentially replacing
the heavy and light chain V region genes with repertoires of
naturally occurring variants (repertoires) of V domain genes
obtained from unimmunized donors. This technique allows the
production of antibodies and antibody fragments with affinities in
the pM-nM range. A strategy for making very large phage antibody
repertoires (also known as "the mother-of-all libraries") has been
described by Waterhouse et al., Nucl. Acids Res. 21:2265-2266,
1993. Gene shuffling can also be used to derive human antibodies
from rodent antibodies, where the human antibody has similar
affinities and specificities to the starting rodent antibody.
According to this method, which is also referred to as "epitope
imprinting", the heavy or light chain V domain gene of rodent
antibodies obtained by phage display technique is replaced with a
repertoire of human V domain genes, creating rodent-human chimeras.
Selection on antigen results in isolation of human variable regions
capable of restoring a functional antigen-binding site, i.e., the
epitope governs (imprints) the choice of partner. When the process
is repeated in order to replace the remaining rodent V domain, a
human antibody is obtained (see PCT Publication No. WO 93/06213).
Unlike traditional humanization of rodent antibodies by CDR
grafting, this technique provides completely human antibodies,
which have no framework or CDR residues of rodent origin.
[0149] Antibodies may be made recombinantly by first isolating the
antibodies and antibody producing cells from host animals,
obtaining the gene sequence, and using the gene sequence to express
the antibody recombinantly in host cells (e.g., CHO cells). Another
method which may be employed is to express the antibody sequence in
plants (e.g., tobacco) or transgenic milk. Methods for expressing
antibodies recombinantly in plants or milk have been disclosed.
See, for example, Peeters, et al. Vaccine 19:2756, 2001; Lonberg,
N. and D. Huszar Int. Rev. Immunol 13:65, 1995; and Pollock, et
al., J Immunol Methods 231:147, 1999. Methods for making
derivatives of antibodies, e.g., humanized, single chain, etc. are
known in the art.
[0150] Immunoassays and flow cytometry sorting techniques such as
fluorescence activated cell sorting (FACS) can also be employed to
isolate antibodies that are specific for IL-7R.
[0151] The antibodies can be bound to many different carriers.
Carriers can be active and/or inert. Examples of well-known
carriers include polypropylene, polystyrene, polyethylene, dextran,
nylon, amylases, glass, natural and modified celluloses,
polyacrylamides, agaroses and magnetite. The nature of the carrier
can be either soluble or insoluble for purposes of the invention.
Those skilled in the art will know of other suitable carriers for
binding antibodies, or will be able to ascertain such, using
routine experimentation. In some embodiments, the carrier comprises
a moiety that targets the myocardium.
[0152] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the monoclonal
antibodies). The hybridoma cells serve as a preferred source of
such DNA. Once isolated, the DNA may be placed into expression
vectors (such as expression vectors disclosed in PCT Publication
No. WO 87/04462), which are then transfected into host cells such
as E. coli cells, simian COS cells, Chinese hamster ovary (CHO)
cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, to obtain the synthesis of monoclonal
antibodies in the recombinant host cells. See, e.g., PCT
Publication No. WO 87/04462. The DNA also may be modified, for
example, by substituting the coding sequence for human heavy and
light chain constant regions in place of the homologous murine
sequences, Morrison et al., Proc. Nat. Acad. Sci. 81:6851, 1984, or
by covalently joining to the immunoglobulin coding sequence all or
part of the coding sequence for a non-immunoglobulin polypeptide.
In that manner, "chimeric" or "hybrid" antibodies are prepared that
have the binding specificity of an IL-7R monoclonal antibody
herein.
[0153] Antagonist IL-7R antibodies can be identified or
characterized using methods known in the art, whereby reduction,
amelioration, or neutralization of IL-7R biological activity is
detected and/or measured. In some embodiments, an antagonist IL-7R
antibody is identified by incubating a candidate agent with IL-7R
and monitoring binding and/or attendant reduction or neutralization
of a biological activity of IL-7R. The binding assay may be
performed with purified IL-7R polypeptide(s), or with cells
naturally expressing, or transfected to express, IL-7R
polypeptide(s). In one embodiment, the binding assay is a
competitive binding assay, where the ability of a candidate
antibody to compete with a known IL-7R antagonist for IL-7R binding
is evaluated. The assay may be performed in various formats,
including the ELISA format. In other embodiments, an antagonist
IL-7R antibody is identified by incubating a candidate agent with
IL-7R and monitoring binding and attendant inhibition of STAT5
phorphorylation.
[0154] Following initial identification, the activity of a
candidate antagonist IL-7R antibody can be further confirmed and
refined by bioassays, known to test the targeted biological
activities. Alternatively, bioassays can be used to screen
candidates directly. Some of the methods for identifying and
characterizing antagonist IL-7R antibodies are described in detail
in the Examples.
[0155] Antagonist IL-7R antibodies may be characterized using
methods well known in the art. For example, one method is to
identify the epitope to which it binds, or "epitope mapping." There
are many methods known in the art for mapping and characterizing
the location of epitopes on proteins, including solving the crystal
structure of an antibody-antigen complex, competition assays, gene
fragment expression assays, and synthetic peptide-based assays, as
described, for example, in Chapter 11 of Harlow and Lane, Using
Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1999. In an additional example,
epitope mapping can be used to determine the sequence to which an
antagonist IL-7R antibody binds. Epitope mapping is commercially
available from various sources, for example, Pepscan Systems
(Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitope
can be a linear epitope, i.e., contained in a single stretch of
amino acids, or a conformational epitope formed by a
three-dimensional interaction of amino acids that may not
necessarily be contained in a single stretch. Peptides of varying
lengths (e.g., at least 4-6 amino acids long) can be isolated or
synthesized (e.g., recombinantly) and used for binding assays with
an antagonist IL-7R antibody. In another example, the epitope to
which the antagonist IL-7R antibody binds can be determined in a
systematic screening by using overlapping peptides derived from the
IL-7R sequence and determining binding by the antagonist IL-7R
antibody. According to the gene fragment expression assays, the
open reading frame encoding IL-7R is fragmented either randomly or
by specific genetic constructions and the reactivity of the
expressed fragments of IL-7R with the antibody to be tested is
determined. The gene fragments may, for example, be produced by PCR
and then transcribed and translated into protein in vitro, in the
presence of radioactive amino acids. The binding of the antibody to
the radioactively labeled IL-7R fragments is then determined by
immunoprecipitation and gel electrophoresis. Certain epitopes can
also be identified by using large libraries of random peptide
sequences displayed on the surface of phage particles (phage
libraries). Alternatively, a defined library of overlapping peptide
fragments can be tested for binding to the test antibody in simple
binding assays. In an additional example, mutagenesis of an antigen
binding domain, domain swapping experiments and alanine scanning
mutagenesis can be performed to identify residues required,
sufficient, and/or necessary for epitope binding. For example,
domain swapping experiments can be performed using a mutant IL-7R
in which various fragments of the IL-7R polypeptide have been
replaced (swapped) with sequences from IL-7R from another species,
or a closely related, but antigenically distinct protein (such as
another member of the proprotein convertase family). By assessing
binding of the antibody to the mutant IL-7R, the importance of the
particular IL-7R fragment to antibody binding can be assessed.
[0156] Yet another method which can be used to characterize an
antagonist IL-7R antibody is to use competition assays with other
antibodies known to bind to the same antigen, i.e., various
fragments on IL-7R, to determine if the antagonist IL-7R antibody
binds to the same epitope as other antibodies. Competition assays
are well known to those of skill in the art.
[0157] An expression vector can be used to direct expression of an
antagonist IL-7R antibody. One skilled in the art is familiar with
administration of expression vectors to obtain expression of an
exogenous protein in vivo. See, e.g., U.S. Pat. Nos. 6,436,908;
6,413,942; and 6,376,471. Administration of expression vectors
includes local or systemic administration, including injection,
oral administration, particle gun or catheterized administration,
and topical administration. In another embodiment, the expression
vector is administered directly to the sympathetic trunk or
ganglion, or into a coronary artery, atrium, ventrical, or
pericardium.
[0158] Targeted delivery of therapeutic compositions containing an
expression vector, or subgenomic polynucleotides can also be used.
Receptor-mediated DNA delivery techniques are described in, for
example, Findeis et al., Trends Biotechnol., 1993, 11:202; Chiou et
al., Gene Therapeutics: Methods And Applications Of Direct Gene
Transfer, J. A. Wolff, ed., 1994; Wu et al., J. Biol. Chem., 1988,
263:621; Wu et al., J. Biol. Chem., 1994, 269:542; Zenke et al.,
Proc. Natl. Acad. Sci. USA, 1990, 87:3655; Wu et al., J. Biol.
Chem., 1991, 266:338. Therapeutic compositions containing a
polynucleotide are administered in a range of about 100 ng to about
200 mg of DNA for local administration in a gene therapy protocol.
Concentration ranges of about 500 ng to about 50 mg, about 1 .mu.g
to about 2 mg, about 5 .mu.g to about 500 .mu.g, and about 20 .mu.g
to about 100 .mu.g of DNA can also be used during a gene therapy
protocol. The therapeutic polynucleotides and polypeptides can be
delivered using gene delivery vehicles. The gene delivery vehicle
can be of viral or non-viral origin (see generally, Jolly, Cancer
Gene Therapy, 1994, 1:51; Kimura, Human Gene Therapy, 1994, 5:845;
Connelly, Human Gene Therapy, 1995, 1:185; and Kaplitt, Nature
Genetics, 1994, 6:148). Expression of such coding sequences can be
induced using endogenous mammalian or heterologous promoters.
Expression of the coding sequence can be either constitutive or
regulated.
[0159] Viral-based vectors for delivery of a desired polynucleotide
and expression in a desired cell are well known in the art.
Exemplary viral-based vehicles include, but are not limited to,
recombinant retroviruses (see, e.g., PCT Publication Nos. WO
90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO
93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; GB
Patent No. 2,200,651; and EP Patent No. 0 345 242),
alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki
forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC
VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus
(ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and
adeno-associated virus (AAV) vectors (see, e.g., PCT Publication
Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO
95/11984 and WO 95/00655). Administration of DNA linked to killed
adenovirus as described in Curiel, Hum. Gene Ther., 1992, 3:147 can
also be employed.
[0160] Non-viral delivery vehicles and methods can also be
employed, including, but not limited to, polycationic condensed DNA
linked or unlinked to killed adenovirus alone (see, e.g., Curiel,
Hum. Gene Ther., 1992, 3:147); ligand-linked DNA (see, e.g., Wu, J.
Biol. Chem., 1989, 264:16985); eukaryotic cell delivery vehicles
cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO
95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic
charge neutralization or fusion with cell membranes. Naked DNA can
also be employed. Exemplary naked DNA introduction methods are
described in PCT Publication No. WO 90/11092 and U.S. Pat. No.
5,580,859. Liposomes that can act as gene delivery vehicles are
described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO
95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional
approaches are described in Philip, Mol. Cell Biol., 1994, 14:2411,
and in Woffendin, Proc. Natl. Acad. Sci., 1994, 91:1581.
[0161] In some embodiments, the invention encompasses compositions,
including pharmaceutical compositions, comprising antibodies
described herein or made by the methods and having the
characteristics described herein. As used herein, compositions
comprise one or more antibodies that antagonize the interaction of
IL-7R with IL-7, and/or one or more polynucleotides comprising
sequences encoding one or more these antibodies. These compositions
may further comprise suitable excipients, such as pharmaceutically
acceptable excipients including buffers, which are well known in
the art.
[0162] The antagonist IL-7R antibodies of the invention are
characterized by any (one or more) of the following
characteristics: (a) bind to IL-7R; (b) block IL-7R interaction
with IL-7; (c) block or decrease IL-7-mediated STAT5
phosphorylation; (d) decrease blood glucose levels in vivo; (e)
improve glucose tolerance in vivo; and (f) reduce disease severity
in EAE. Preferably, antagonist IL-7R antibodies have two or more of
these features. More preferably, the antibodies have three or more
of the features. More preferably, the antibodies have four or more
of the features. More preferably, the antibodies have five or more
of the features. Most preferably, the antibodies have all six
characteristics.
[0163] Accordingly, the invention provides any of the following, or
compositions (including pharmaceutical compositions) comprising any
of the following: (a) an antibody having a partial light chain
sequence of
TABLE-US-00001 (SEQ ID NO: 1)
NFMLTQPHSVSGSPGKTVTISCTRSSGSIDSSYVQWYQQRPGNSPTTVIY
EDDQRPSGVPDRFSGSIDSSSNSASLTISGLVTEDEADYYCQSYDSSHLV FGGGTKLTVLC,
(SEQ ID NO: 3) NFMLTQPHSVSESPGKTVTISCTGSSGRIASSYVQWYQQRPGSAPTTVIY
EDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYASSSLW VFGGGTQLTVLS,
(SEQ ID NO: 5) NFMLTQPHSVSGSPGKTVTISCTRSSGSIDSSYVQWYQQRPGNSPTTVIY
EDDQRPSGVPDRFSGSIDSSSNSASLTISGLVTEDEADYYCMQYDSSHLV FGGGTKLTVLC,
(SEQ ID NO: 7) NFMLTQPHSVSGSPGKTVTISCTRSSGSIDSSYVQWYQQRPGNSPTTVIY
EDDQRPSGVPDRFSGSIDSSSNSASLTISGLVTEDEADYYCQSYDFHHLV FGGGTKLTVLC,
(SEQ ID NO: 9) NFMLTQPHSVSGSPGKTVTISCTRSSGSIDSSYVQWYQQRPGNSPTTVIY
EDDQRPSGVPDRFSGSIDSSSNSASLTISGLVTEDEADYYCQSYDFHHLV FGGGTKLTVLC,
(SEQ ID NO: 11) NFMLTQPHSVSGSPGKTVTISCTRSSGSIDSSYVQWYQQRPGNSPTTVIY
EDDQRPSGVPDRFSGSIDSSSNSASLTISGLVTEDEADYYCMQYDFHHLV FGGGTKLTVLC,
(SEQ ID NO: 44) NFMLTQPHSVSESPGKTVTISCTRSSGSIDSSYVQWYQQRPGSSPTTVIY
EDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCMQYDFHHLV FGGGTKLTVL, or
(SEQ ID NO: 41) NFMLTQPHSVSESPGKTVTISCTRSSGSIDSSYVQWYQQRPGSSPTTVIY
EDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDFHHLV FGGGTKLTVL; and
(b) an antibody having a partial heavy chain sequence of (SEQ ID
NO: 2) QVNLRESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWLSL
VGWDGSATYYADSVKGRFTISRDNTKNLLYLQMNSLRAEDTAVYYCARQG
DYVFDYWGQGTLVTVSS, (SEQ ID NO: 4)
QVTLKESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSA
ISGSGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRDEDTAVYYCARDI
SGGGMDVWGQGTTVTVSS, (SEQ ID NO: 6)
QVNLRESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWLSL
VGWDGFFTYYADSVKGRFTISRDNTKNLLYLQMNSLRAEDTAVYYCARQG
DYVFNNWGQGTLVTVSS, (SEQ ID NO: 8)
QVNLRESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWLSL
VGWDGFFTYYADSVKGRFTISRDNTKNLLYLQMNSLRAEDTAVYYCARQG
DYMGDYWGQGTLVTVSS, (SEQ ID NO: 10)
QVNLRESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWLSL
VGWDGFFTYYADSVKGRFTISRDNTKNLLYLQMNSLRAEDTAVYYCARQG
DYMGNNWGQGTLVTVSS, (SEQ ID NO: 12)
QVNLRESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWLSL
VGWDGFFTYYADSVKGRFTISRDNTKNLLYLQMNSLRAEDTAVYYCARQG DYMGNNWGQGTL
VTVSS, or (SEQ ID NO: 40)
EVQLVESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWVSL
VGWDGFFTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQG
DYMGNNWGQGTLVTVSS.
TABLE-US-00002 TABLE 1 mAb Light Chain Heavy Chain P3A9
NFMLTQPHSVSGSPGKTVTIS QVNLRESGGGLVKPGGSLRLSCAAS
CTRSSGSIDSSYVQWYQQRP GFTFDDSVMHWVRQAPGKGLEWLS GNSPTTVIYEDDQRPSGVPDR
LVGWDGSATYYADSVKGRFTISRDN FSGSIDSSSNSASLTISGLVTE
TKNLLYLQMNSLRAEDTAVYYCARQ DEADYYCQSYDSSHLVFGGG GDYVFDYWGQGTLVTVSS
(SEQ ID TKLTVLC (SEQ ID NO: 1) NO: 2) P4B3 NFMLTQPHSVSESPGKTVTIS
QVTLKESGGGLVQPGGSLRLSCAAS CTGSSGRIASSYVQWYQQRP
GFTFSNYGMHWVRQAPGKGLWVS GSAPTTVIYEDNQRPSGVPDR
AISGSGGSTYYADSVKGRFTISRDNS FSGSIDSSSNSASLTISGLKTE
KNTVYLQMNSLRDEDTAVYYCARDIS DEADYYCQSYASSSLWVFGG GGGMDVWGQGTTVTVSS
(SEQ ID GTQLTVLS (SEQ ID NO: 3) NO: 4) P2D2 NFMLTQPHSVSGSPGKTVTIS
QVNLRESGGGLVKPGGSLRLSCAAS CTRSSGSIDSSYVQWYQQRP
GFTFDDSVMHWVRQAPGKGLEWLS GNSPTTVIYEDDQRPSGVPDR
LVGWDGFFTYYADSVKGRFTISRDN FSGSIDSSSNSASLTISGLVTE
TKNLLYLQMNSLRAEDTAVYYCARQ DEADYYCMQYDSSHLVFGGG GDYVFNNWGQGTLVTVSS
(SEQ ID TKLTVLC (SEQ ID NO: 5) NO: 6) P2E11 NFMLTQPHSVSGSPGKTVTIS
QVNLRESGGGLVKPGGSLRLSCAAS CTRSSGSIDSSYVQWYQQRP
GFTFDDSVMHWVRQAPGKGLEWLS GNSPTTVIYEDDQRPSGVPDR
LVGWDGFFTYYADSVKGRFTISRDN FSGSIDSSSNSASLTISGLVTE
TKNLLYLQMNSLRAEDTAVYYCARQ DEADYYCQSYDFHHLVFGGG GDYMGDYWGQGTLVTVSS
(SEQ ID TKLTVLC (SEQ ID NO: 7) NO: 8) HAL NFMLTQPHSVSGSPGKTVTIS
QVNLRESGGGLVKPGGSLRLSCAAS 403a CTRSSGSIDSSYVQWYQQRP
GFTFDDSVMHWVRQAPGKGLEWLS GNSPTTVIYEDDQRPSGVPDR
LVGWDGFFTYYADSVKGRFTISRDN FSGSIDSSSNSASLTISGLVTE
TKNLLYLQMNSLRAEDTAVYYCARQ DEADYYCQSYDFHHLVFGGG GDYMGNNWGQGTLVTVSS
(SEQ ID TKLTVLC (SEQ ID NO: 9) NO: 10) HAL NFMLTQPHSVSGSPGKTVTIS
QVNLRESGGGLVKPGGSLRLSCAAS 403b CTRSSGSIDSSYVQWYQQRP
GFTFDDSVMHWVRQAPGKGLEWLS GNSPTTVIYEDDQRPSGVPDR
LVGWDGFFTYYADSVKGRFTISRDN FSGSIDSSSNSASLTISGLVTE
TKNLLYLQMNSLRAEDTAVYYCARQ DEADYYCMQYDFHHLVFGGG GDYMGNNWGQGTLVTVSS
(SEQ ID TKLTVLC (SEQ ID NO: 11) NO: 12) C1GM NFMLTQPHSVSESPGKTVTIS
EVQLVESGGGLVKPGGSLRLSCAAS CTRSSGSIDSSYVQWYQQRP
GFTFDDSVMHWVRQAPGKGLEWVS GSSPTTVIYEDDQRPSGVPDR
LVGWDGFFTYYADSVKGRFTISRDN FSGSIDSSSNSASLTISGLKTE
AKNSLYLQMNSLRAEDTAVYYCARQ DEADYYCQSYDFHHLVFGGG GDYMGNNWGQGTLVTVSS
(SEQ ID TKLTVL (SEQ ID NO: 41) NO: 40) C2M3 NFMLTQPHSVSESPGKTVTIS
EVQLVESGGGLVKPGGSLRLSCAAS CTRSSGSIDSSYVQWYQQRP
GFTFDDSVMHWVRQAPGKGLEWVS GSSPTTVIYEDDQRPSGVPDR
LVGWDGFFTYYADSVKGRFTISRDN FSGSIDSSSNSASLTISGLKTE
AKNSLYLQMNSLRAEDTAVYYCARQ DEADYYCMQYDFHHLVFGGG GDYMGNNWGQGTLVTVSS
(SEQ ID TKLTVL (SEQ ID NO: 44) NO: 40)
In Table 1, the underlined sequences are CDR sequences according to
Kabat and in bold according to Chothia.
[0164] The invention also provides CDR portions of antibodies to
IL-7R (including Chothia, Kabat CDRs, and CDR contact regions).
Determination of CDR regions is well within the skill of the art.
It is understood that in some embodiments, CDRs can be a
combination of the Kabat and Chothia CDR (also termed "combined
CRs" or "extended CDRs"). In some embodiments, the CDRs are the
Kabat CDRs. In other embodiments, the CDRs are the Chothia CDRs. In
other words, in embodiments with more than one CDR, the CDRs may be
any of Kabat, Chothia, combination CDRs, or combinations thereof.
Table 2 provides examples of CDR sequences provided herein.
TABLE-US-00003 TABLE 2 Heavy Chain mAb CDRH1 CDRH2 CDRH3 P3A9 DSVMH
(SEQ ID LVGWDGSATYYADSVKG QGDYVFDY (SEQ NO: 19) (SEQ ID NO: 21) ID
NO: 24) P4B3 NYGMH (SEQ ID AISGSGGSTYYADSVKG DISGGGMDV NO: 20) (SEQ
ID NO: 22) (SEQ ID NO: 25) P2D2 DSVMH (SEQ ID LVGWDGFFTYYADSVKG
QGDYVFNN (SEQ NO: 19) (SEQ ID NO: 23) ID NO: 26) P2E11 DSVMH (SEQ
ID LVGWDGFFTYYADSVKG QGDYMGDY (SEQ NO: 19) (SEQ ID NO: 23) ID NO:
27) HAL DSVMH (SEQ ID LVGWDGFFTYYADSVKG QGDYMGNN (SEQ 403a NO: 19)
(SEQ ID NO: 23) ID NO: 28) C1GM DSVMH (SEQ ID LVGWDGFFTYYADSVKG
QGDYMGNN (SEQ NO: 19) (Kabat), (SEQ ID NO: 23) (Kabat), ID NO: 49);
GFTFDDS (SEQ ID GWDGFF (SEQ ID NO: 48) NO: 46) (Chothia),
(Chothia), GFTFDDSVMH (SEQ ID NO: 47) (extended) C2M3 DSVMH (SEQ ID
LVGWDGFFTYYADSVKG QGDYMGNN (SEQ NO: 19) (SEQ ID NO: 23) ID NO: 49)
HAL DSVMH (SEQ ID LVGWDGFFTYYADSVKG QGDYMGNN (SEQ 403b NO: 19) (SEQ
ID NO: 23) ID NO: 28) Heavy X.sub.1X.sub.2VMH, wherein
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5GX.sub.6X.sub.7TYYADSV
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8, Chain
X.sub.1 is D or N; X.sub.2 is S KG, wherein X.sub.1 is L or A;
X.sub.2 wherein X.sub.1 is Q or consensus or Y (SEQ ID NO: is V or
I; X.sub.3 is G or S; X.sub.4 is W D; X.sub.2 is G or I; X.sub.3 is
50) or G; X.sub.5 is D or S; X.sub.6 is F, G D or S; X.sub.4 is Y
or G; or S; X.sub.7 is F, A or S (SEQ ID X.sub.5 is M, V or G;
X.sub.6 NO: 51) is G or F; X.sub.7 is N, D or M; X.sub.8 is N, Y or
D (SEQ ID NO: 52) Light Chain mAb CDRL1 CDRL2 CDRL3 P3A9
TRSSGSIDSSYVQ EDDQRPS (SEQ ID NO: QSYDSSHLV (SEQ (SEQ ID NO: 29)
31) ID NO: 33) P4B3 TGSSGRIASSYVQ EDNQRPS (SEQ ID NO: QSYASSSLWV
(SEQ ID NO: 30) 32) (SEQ ID NO: 34) P2D2 TRSSGSIDSSYVQ EDDQRPS (SEQ
ID NO: MQYDSSHLV (SEQ ID NO: 29) 31) (SEQ ID NO: 35) P2E11
TRSSGSIDSSYVQ EDDQRPS (SEQ ID NO: QSYDFHHLV (SEQ (SEQ ID NO: 29)
31) ID NO: 36) HAL TRSSGSIDSSYVQ EDDQRPS (SEQ ID NO: QSYDFHHLV (SEQ
403a (SEQ ID NO: 29) 31) ID NO: 36) C1GM TRSSGSIDSSYVQ EDDQRPS (SEQ
ID NO: QSYDFHHLV (SEQ (SEQ ID NO: 29) 31) ID NO: 36) C2M3
TRSSGSIDSSYVQ EDDQRPS (SEQ ID NO: MQYDFHHLV (SEQ ID NO: 29) 31)
(SEQ ID NO: 37) HAL TRSSGSIDSSYVQ EDDQRPS (SEQ ID NO: MQYDFHHLV
403b (SEQ ID NO: 29) 31) (SEQ ID NO: 37) Light
TX.sub.1SSGX.sub.2IX.sub.3SSYVQ EDX.sub.1QRPS wherein X.sub.1 is D
X.sub.1X.sub.2YX.sub.3X.sub.4X.sub.5X.sub.6LX.sub.7 Chain wherein
X.sub.1 is R or G; or N (SEQ ID NO: 54) wherein X.sub.1 is Q or
consensus X.sub.2 is S or R; X.sub.3 is D or M; X.sub.2 is S or Q;
X.sub.3 A (SEQ ID NO: 53) is D or A; X.sub.4 is F or S; X.sub.5 is
H or S; X.sub.6 is H or S; X.sub.7 is V or W (SEQ ID NO: 55)
[0165] CDR contact regions are regions of an antibody that imbue
specificity to the antibody for an antigen. In general, CDR contact
regions include the residue positions in the CDRs and Vernier zones
which are constrained in order to maintain proper loop structure
for the antibody to bind a specific antigen. See, e.g., Makabe et
al., 2007, "Thermodynamic Consequences of Mutations in Vernier Zone
Residues of a Humanized Anti-human Epidermal Growth Factor Receptor
Murine Antibody," Journal of Biological Chemistry, 283:1156-1166.
Determination of CDR contact regions is well within the skill of
the art. In some embodiments, an antagonist IL-7R antibody
comprises one or more CDR contact regions comprising an amino acid
sequence selected from the group consisting of FTFDDSVM (SEQ ID NO:
56), GWDGFF (SEQ ID NO: 57), ARX.sub.1X.sub.2X.sub.3X.sub.4 wherein
X.sub.1, X.sub.2, X.sub.3, and X.sub.4 can be any amino acid, (SEQ
ID NO: 58), SGSIDSSY (SEQ ID NO: 59), EDDQRPSGV (SEQ ID NO: 60),
and FHHL (SEQ ID NO: 61).
[0166] For any given embodiment containing more than one CDR, the
CDRs may be any of Kabat, Chothia, extended, AbM, and/or
contact.
[0167] The binding affinity (K.sub.D) of an antagonist IL-7R
antibody to IL-7R can be about 0.002 to about 200 nM. In some
embodiments, the binding affinity is any of about 200 nM, 100 nM,
about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM,
about 60 pM, about 50 pM, about 20 pM, about 15 pM, about 10 pM,
about 5 pM, or about 2 pM. In some embodiments, the binding
affinity is less than any of about 250 nM, about 200 nM, about 100
nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100
pM, or about 50 pM.
[0168] The invention also provides methods of making any of these
antibodies. The antibodies of this invention can be made by
procedures known in the art. The polypeptides can be produced by
proteolytic or other degradation of the antibodies, by recombinant
methods (i.e., single or fusion polypeptides) as described above or
by chemical synthesis. Polypeptides of the antibodies, especially
shorter polypeptides up to about 50 amino acids, are conveniently
made by chemical synthesis. Methods of chemical synthesis are known
in the art and are commercially available. For example, an antibody
could be produced by an automated polypeptide synthesizer employing
the solid phase method. See also, U.S. Pat. Nos. 5,807,715;
4,816,567; and 6,331,415.
[0169] In another alternative, the antibodies can be made
recombinantly using procedures that are well known in the art. In
one embodiment, a polynucleotide comprises a sequence encoding the
heavy chain and/or the light chain variable regions of antibody
P3A9, P4B3, P2D2, P2E11, HAL403a, HAL403b, C1GM, or C2M3. The
sequence encoding the antibody of interest may be maintained in a
vector in a host cell and the host cell can then be expanded and
frozen for future use. Vectors (including expression vectors) and
host cells are further described herein.
[0170] The invention also encompasses scFv of antibodies of this
invention. Single chain variable region fragments are made by
linking light and/or heavy chain variable regions by using a short
linking peptide (Bird et al., 1988, Science 242:423-426). An
example of a linking peptide is (GGGGS).sub.3 (SEQ ID NO: 13),
which bridges approximately 3.5 nm between the carboxy terminus of
one variable region and the amino terminus of the other variable
region. Linkers of other sequences have been designed and used
(Bird et al., 1988, supra). Linkers should be short, flexible
polypeptides and preferably comprised of less than about 20 amino
acid residues. Linkers can in turn be modified for additional
functions, such as attachment of drugs or attachment to solid
supports. The single chain variants can be produced either
recombinantly or synthetically. For synthetic production of scFv,
an automated synthesizer can be used. For recombinant production of
scFv, a suitable plasmid containing polynucleotide that encodes the
scFv can be introduced into a suitable host cell, either
eukaryotic, such as yeast, plant, insect or mammalian cells, or
prokaryotic, such as E. coli. Polynucleotides encoding the scFv of
interest can be made by routine manipulations such as ligation of
polynucleotides. The resultant scFv can be isolated using standard
protein purification techniques known in the art.
[0171] Other forms of single chain antibodies, such as diabodies
are also encompassed. Diabodies are bivalent, bispecific antibodies
in which heavy chain variable (VH) and light chain variable (VL)
domains are expressed on a single polypeptide chain, but using a
linker that is too short to allow for pairing between the two
domains on the same chain, thereby forcing the domains to pair with
complementary domains of another chain and creating two antigen
binding sites (see e.g., Holliger, P., et al., 1993, Proc. Natl.
Acad Sci. USA 90:6444-6448; Poljak, R. J., et al., 1994, Structure
2:1121-1123).
[0172] For example, bispecific antibodies, monoclonal antibodies
that have binding specificities for at least two different
antigens, can be prepared using the antibodies disclosed herein.
Methods for making bispecific antibodies are known in the art (see,
e.g., Suresh et al., 1986, Methods in Enzymology 121:210).
Traditionally, the recombinant production of bispecific antibodies
was based on the coexpression of two immunoglobulin heavy
chain-light chain pairs, with the two heavy chains having different
specificities (Millstein and Cuello, 1983, Nature 305,
537-539).
[0173] According to one approach to making bispecific antibodies,
antibody variable domains with the desired binding specificities
(antibody-antigen combining sites) are fused to immunoglobulin
constant region sequences. The fusion preferably is with an
immunoglobulin heavy chain constant region, comprising at least
part of the hinge, CH2 and CH3 regions. It is preferred to have the
first heavy chain constant region (CH1), containing the site
necessary for light chain binding, present in at least one of the
fusions. DNAs encoding the immunoglobulin heavy chain fusions and,
if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are cotransfected into a suitable
host organism. This provides for great flexibility in adjusting the
mutual proportions of the three polypeptide fragments in
embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yields. It is,
however, possible to insert the coding sequences for two or all
three polypeptide chains in one expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance.
[0174] In one approach, the bispecific antibodies are composed of a
hybrid immunoglobulin heavy chain with a first binding specificity
in one arm, and a hybrid immunoglobulin heavy chain-light chain
pair (providing a second binding specificity) in the other arm.
This asymmetric structure, with an immunoglobulin light chain in
only one half of the bispecific molecule, facilitates the
separation of the desired bispecific compound from unwanted
immunoglobulin chain combinations. This approach is described in
PCT Publication No. WO 94/04690.
[0175] Heteroconjugate antibodies, comprising two covalently joined
antibodies, are also within the scope of the invention. Such
antibodies have been used to target immune system cells to unwanted
cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection
(PCT Publication Nos. WO 91/00360 and WO 92/200373; EP 03089).
Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents and techniques
are well known in the art, and are described in U.S. Pat. No.
4,676,980.
[0176] Chimeric or hybrid antibodies also may be prepared in vitro
using known methods of synthetic protein chemistry, including those
involving cross-linking agents. For example, immunotoxins may be
constructed using a disulfide exchange reaction or by forming a
thioether bond. Examples of suitable reagents for this purpose
include iminothiolate and methyl-4-mercaptobutyrimidate.
[0177] Humanized antibodies can be made using any methods know in
the art. For example, four general steps may be used to humanize a
monoclonal antibody. These are: (1) determining the nucleotide and
predicted amino acid sequence of the starting antibody light and
heavy variable domains (2) designing the humanized antibody, i.e.,
deciding which antibody framework region to use during the
humanizing process (3) the actual humanizing
methodologies/techniques and (4) the transfection and expression of
the humanized antibody. See, for example, U.S. Pat. Nos. 4,816,567;
5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762;
5,585,089; and 6,180,370.
[0178] In the recombinant humanized antibodies, the Fc.gamma.
portion can be modified to avoid interaction with
Fc.gamma..quadrature. receptor and the complement and immune
systems. The techniques for preparation of such antibodies are
described in WO 99/58572. For example, the constant region may be
engineered to more resemble human constant regions to avoid immune
response if the antibody is used in clinical trials and treatments
in humans. See, for example, U.S. Pat. Nos. 5,997,867 and
5,866,692.
[0179] The invention encompasses modifications to the antibodies
and polypeptides of the invention variants shown in Table 1,
including functionally equivalent antibodies which do not
significantly affect their properties and variants which have
enhanced or decreased activity and/or affinity. For example, the
amino acid sequence may be mutated to obtain an antibody with the
desired binding affinity to IL-7R. Modification of polypeptides is
routine practice in the art and need not be described in detail
herein. Examples of modified polypeptides include polypeptides with
conservative substitutions of amino acid residues, one or more
deletions or additions of amino acids which do not significantly
deleteriously change the functional activity, or which mature
(enhance) the affinity of the polypeptide for its ligand, or use of
chemical analogs.
[0180] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to an epitope
tag. Other insertional variants of the antibody molecule include
the fusion to the N- or C-terminus of the antibody of an enzyme or
a polypeptide which increases the half-life of the antibody in the
blood circulation.
[0181] Substitution variants have at least one amino acid residue
in the antibody molecule removed and a different residue inserted
in its place. The sites of greatest interest for substitutional
mutagenesis include the hypervariable regions, but FR alterations
are also contemplated. Conservative substitutions are shown in
Table 3 under the heading of "conservative substitutions." If such
substitutions result in a change in biological activity, then more
substantial changes, denominated "exemplary substitutions" in Table
3, or as further described below in reference to amino acid
classes, may be introduced and the products screened.
TABLE-US-00004 TABLE 3 Amino Acid Substitutions Conservative
Original Residue Substitutions Exemplary Substitutions Ala (A) Val
Val; Leu; Ile Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp,
Lys; Arg Asp (D) Glu Glu; Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn;
Glu Glu (E) Asp Asp; Gln Gly (G) Ala Ala His (H) Arg Asn; Gln; Lys;
Arg Ile (I) Leu Leu; Val; Met; Ala; Phe; Norleucine Leu (L) Ile
Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met
(M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val; Ile; Ala; Tyr Pro (P)
Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr; Phe Tyr
(Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met; Phe; Ala;
Norleucine
[0182] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties: [0183]
(1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile, [0184] (2)
Polar without charge: Cys, Ser, Thr, Asn, Gin; [0185] (3) Acidic
(negatively charged): Asp, Glu; [0186] (4) Basic (positively
charged): Lys, Arg; [0187] (5) Residues that influence chain
orientation: Gly, Pro; and [0188] (6) Aromatic: Trp, Tyr, Phe,
His.
[0189] Non-conservative substitutions are made by exchanging a
member of one of these classes for another class.
[0190] Any cysteine residue not involved in maintaining the proper
conformation of the antibody also may be substituted, generally
with serine, to improve the oxidative stability of the molecule and
prevent aberrant cross-linking. Conversely, cysteine bond(s) may be
added to the antibody to improve its stability, particularly where
the antibody is an antibody fragment such as an Fv fragment.
[0191] Amino acid modifications can range from changing or
modifying one or more amino acids to complete redesign of a region,
such as the variable region. Changes in the variable region can
alter binding affinity and/or specificity. In some embodiments, no
more than one to five conservative amino acid substitutions are
made within a CDR domain. In other embodiments, no more than one to
three conservative amino acid substitutions are made within a CDR
domain. In still other embodiments, the CDR domain is CDR H3 and/or
CDR L3.
[0192] Modifications also include glycosylated and nonglycosylated
polypeptides, as well as polypeptides with other post-translational
modifications, such as, for example, glycosylation with different
sugars, acetylation, and phosphorylation. Antibodies are
glycosylated at conserved positions in their constant regions
(Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and
Morrison, 1997, TibTECH 15:26-32). The oligosaccharide side chains
of the immunoglobulins affect the protein's function (Boyd et al.,
1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard, 1990, Biochem.
29:4175-4180) and the intramolecular interaction between portions
of the glycoprotein, which can affect the conformation and
presented three-dimensional surface of the glycoprotein (Jefferis
and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech.
7:409-416). Oligosaccharides may also serve to target a given
glycoprotein to certain molecules based upon specific recognition
structures. Glycosylation of antibodies has also been reported to
affect antibody-dependent cellular cytotoxicity (ADCC). In
particular, CHO cells with tetracycline-regulated expression of
.beta.(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a
glycosyltransferase catalyzing formation of bisecting GlcNAc, was
reported to have improved ADCC activity (Umana et al., 1999, Mature
Biotech. 17:176-180).
[0193] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine, asparagine-X-threonine, and
asparagine-X-cysteine, where X is any amino acid except proline,
are the recognition sequences for enzymatic attachment of the
carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation
refers to the attachment of one of the sugars
N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid,
most commonly serine or threonine, although 5-hydroxyproline or
5-hydroxylysine may also be used.
[0194] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0195] The glycosylation pattern of antibodies may also be altered
without altering the underlying nucleotide sequence. Glycosylation
largely depends on the host cell used to express the antibody.
Since the cell type used for expression of recombinant
glycoproteins, e.g. antibodies, as potential therapeutics is rarely
the native cell, variations in the glycosylation pattern of the
antibodies can be expected (see, e.g. Hse et al., 1997, J. Biol.
Chem. 272:9062-9070).
[0196] In addition to the choice of host cells, factors that affect
glycosylation during recombinant production of antibodies include
growth mode, media formulation, culture density, oxygenation, pH,
purification schemes and the like. Various methods have been
proposed to alter the glycosylation pattern achieved in a
particular host organism including introducing or overexpressing
certain enzymes involved in oligosaccharide production (U.S. Pat.
Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation, or certain
types of glycosylation, can be enzymatically removed from the
glycoprotein, for example, using endoglycosidase H (Endo H),
N-glycosidase F, endoglycosidase F1, endoglycosidase F2,
endoglycosidase F3. In addition, the recombinant host cell can be
genetically engineered to be defective in processing certain types
of polysaccharides. These and similar techniques are well known in
the art.
[0197] Other methods of modification include using coupling
techniques known in the art, including, but not limited to,
enzymatic means, oxidative substitution and chelation.
Modifications can be used, for example, for attachment of labels
for immunoassay. Modified polypeptides are made using established
procedures in the art and can be screened using standard assays
known in the art, some of which are described below and in the
Examples.
[0198] In some embodiments of the invention, the antibody comprises
a modified constant region, such as a constant region that has
increased affinity to a human Fc gamma receptor, is immunologically
inert or partially inert, e.g., does not trigger complement
mediated lysis, does not stimulate antibody-dependent cell mediated
cytotoxicity (ADCC), or does not activate microglia; or has reduced
activities (compared to the unmodified antibody) in any one or more
of the following: triggering complement mediated lysis, stimulating
antibody-dependent cell mediated cytotoxicity (ADCC), or activating
microglia. Different modifications of the constant region may be
used to achieve optimal level and/or combination of effector
functions. See, for example, Morgan et al., Immunology 86:319-324,
1995; Lund et al., J. Immunology 157:4963-9 157:4963-4969, 1996;
Idusogie et al., J. Immunology 164:4178-4184, 2000; Tao et al., J.
Immunology 143: 2595-2601, 1989; and Jefferis et al., Immunological
Reviews 163:59-76, 1998. In some embodiments, the constant region
is modified as described in Eur. J. Immunol., 1999, 29:2613-2624;
PCT Application No. PCT/GB99/01441, and/or UK Patent Application
No. 9809951.8. In other embodiments, the antibody comprises a human
heavy chain IgG2 constant region comprising the following
mutations: A330P331 to S330S331 (amino acid numbering with
reference to the wild type IgG2 sequence). Eur. J. Immunol., 1999,
29:2613-2624. In still other embodiments, the constant region is
aglycosylated for N-linked glycosylation. In some embodiments, the
constant region is aglycosylated for N-linked glycosylation by
mutating the glycosylated amino acid residue or flanking residues
that are part of the N-glycosylation recognition sequence in the
constant region. For example, N-glycosylation site N297 may be
mutated to A, Q, K, or H. See, Tao et al., J. Immunology 143:
2595-2601, 1989; and Jefferis et al., Immunological Reviews
163:59-76, 1998. In some embodiments, the constant region is
aglycosylated for N-linked glycosylation. The constant region may
be aglycosylated for N-linked glycosylation enzymatically (such as
removing carbohydrate by enzyme PNGase), or by expression in a
glycosylation deficient host cell.
[0199] Other antibody modifications include antibodies that have
been modified as described in PCT Publication No. WO 99/58572.
These antibodies comprise, in addition to a binding domain directed
at the target molecule, an effector domain having an amino acid
sequence substantially homologous to all or part of a constant
region of a human immunoglobulin heavy chain. These antibodies are
capable of binding the target molecule without triggering
significant complement dependent lysis, or cell-mediated
destruction of the target. In some embodiments, the effector domain
is capable of specifically binding FcRn and/or Fc.gamma.RIIb. These
are typically based on chimeric domains derived from two or more
human immunoglobulin heavy chain C.sub.H2 domains. Antibodies
modified in this manner are particularly suitable for use in
chronic antibody therapy, to avoid inflammatory and other adverse
reactions to conventional antibody therapy.
[0200] The invention includes affinity matured embodiments. For
example, affinity matured antibodies can be produced by procedures
known in the art (Marks et al., 1992, Bio/Technology, 10:779-783;
Barbas et al., 1994, Proc Nat. Acad. Sci, USA 91:3809-3813; Schier
et al., 1995, Gene, 169:147-155; Yelton et al., 1995, J. Immunol.,
155:1994-2004; Jackson et al., 1995, J. Immunol., 154(7):3310-9;
Hawkins et al., 1992, J. Mol. Biol., 226:889-896; and PCT
Publication No. WO2004/058184).
[0201] The following methods may be used for adjusting the affinity
of an antibody and for characterizing a CDR. One way of
characterizing a CDR of an antibody and/or altering (such as
improving) the binding affinity of a polypeptide, such as an
antibody, termed "library scanning mutagenesis". Generally, library
scanning mutagenesis works as follows. One or more amino acid
positions in the CDR are replaced with two or more (such as 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino
acids using art recognized methods. This generates small libraries
of clones (in some embodiments, one for every amino acid position
that is analyzed), each with a complexity of two or more members
(if two or more amino acids are substituted at every position).
Generally, the library also includes a clone comprising the native
(unsubstituted) amino acid. A small number of clones, e.g., about
20-80 clones (depending on the complexity of the library), from
each library are screened for binding affinity to the target
polypeptide (or other binding target), and candidates with
increased, the same, decreased, or no binding are identified.
Methods for determining binding affinity are well-known in the art.
Binding affinity may be determined using Biacore.TM. surface
plasmon resonance analysis, which detects differences in binding
affinity of about 2-fold or greater. Biacore.TM. is particularly
useful when the starting antibody already binds with a relatively
high affinity, for example a K.sub.D of about 10 nM or lower.
Screening using Biacore.TM. surface plasmon resonance is described
in the Examples, herein.
[0202] Binding affinity may be determined using Kinexa Biocensor,
scintillation proximity assays, ELISA, ORIGEN immunoassay (IGEN),
fluorescence quenching, fluorescence transfer, and/or yeast
display. Binding affinity may also be screened using a suitable
bioassay.
[0203] In some embodiments, every amino acid position in a CDR is
replaced (in some embodiments, one at a time) with all 20 natural
amino acids using art recognized mutagenesis methods (some of which
are described herein). This generates small libraries of clones (in
some embodiments, one for every amino acid position that is
analyzed), each with a complexity of 20 members (if all 20 amino
acids are substituted at every position).
[0204] In some embodiments, the library to be screened comprises
substitutions in two or more positions, which may be in the same
CDR or in two or more CDRs. Thus, the library may comprise
substitutions in two or more positions in one CDR. The library may
comprise substitution in two or more positions in two or more CDRs.
The library may comprise substitution in 3, 4, 5, or more
positions, said positions found in two, three, four, five or six
CDRs. The substitution may be prepared using low redundancy codons.
See, e.g., Table 2 of Balint et al., 1993, Gene 137(1):109-18.
[0205] The CDR may be CDRH3 and/or CDRL3. The CDR may be one or
more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and/or CDRH3. The CDR
may be a Kabat CDR, a Chothia CDR, or an extended CDR.
[0206] Candidates with improved binding may be sequenced, thereby
identifying a CDR substitution mutant which results in improved
affinity (also termed an "improved" substitution). Candidates that
bind may also be sequenced, thereby identifying a CDR substitution
which retains binding.
[0207] Multiple rounds of screening may be conducted. For example,
candidates (each comprising an amino acid substitution at one or
more position of one or more CDR) with improved binding are also
useful for the design of a second library containing at least the
original and substituted amino acid at each improved CDR position
(i.e., amino acid position in the CDR at which a substitution
mutant showed improved binding). Preparation, and screening or
selection of this library is discussed further below.
[0208] Library scanning mutagenesis also provides a means for
characterizing a CDR, in so far as the frequency of clones with
improved binding, the same binding, decreased binding or no binding
also provide information relating to the importance of each amino
acid position for the stability of the antibody-antigen complex.
For example, if a position of the CDR retains binding when changed
to all 20 amino acids, that position is identified as a position
that is unlikely to be required for antigen binding. Conversely, if
a position of CDR retains binding in only a small percentage of
substitutions, that position is identified as a position that is
important to CDR function. Thus, the library scanning mutagenesis
methods generate information regarding positions in the CDRs that
can be changed to many different amino acids (including all 20
amino acids), and positions in the CDRs which cannot be changed or
which can only be changed to a few amino acids.
[0209] Candidates with improved affinity may be combined in a
second library, which includes the improved amino acid, the
original amino acid at that position, and may further include
additional substitutions at that position, depending on the
complexity of the library that is desired, or permitted using the
desired screening or selection method. In addition, if desired,
adjacent amino acid position can be randomized to at least two or
more amino acids. Randomization of adjacent amino acids may permit
additional conformational flexibility in the mutant CDR, which may
in turn, permit or facilitate the introduction of a larger number
of improving mutations. The library may also comprise substitution
at positions that did not show improved affinity in the first round
of screening.
[0210] The second library is screened or selected for library
members with improved and/or altered binding affinity using any
method known in the art, including screening using Biacore.TM.
surface plasmon resonance analysis, and selection using any method
known in the art for selection, including phage display, yeast
display, and ribosome display.
[0211] The invention also encompasses fusion proteins comprising
one or more fragments or regions from the antibodies of this
invention. In one embodiment, a fusion polypeptide is provided that
comprises at least 10 contiguous amino acids of the variable light
chain region shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 41 or 44
and/or at least 10 amino acids of the variable heavy chain region
shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12 or 40. In other
embodiments, a fusion polypeptide is provided that comprises at
least about 10, at least about 15, at least about 20, at least
about 25, or at least about 30 contiguous amino acids of the
variable light chain region and/or at least about 10, at least
about 15, at least about 20, at least about 25, or at least about
30 contiguous amino acids of the variable heavy chain region. In
another embodiment, the fusion polypeptide comprises a light chain
variable region and/or a heavy chain variable region, as shown in
any of the sequence pairs selected from among SEQ ID NOs: 1 and 2,
3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 41 and 40, and 44
and 40. In another embodiment, the fusion polypeptide comprises one
or more CDR(s). In still other embodiments, the fusion polypeptide
comprises CDR H3 (VH CDR3) and/or CDR L3 (VL CDR3). For purposes of
this invention, a fusion protein contains one or more antibodies
and another amino acid sequence to which it is not attached in the
native molecule, for example, a heterologous sequence or a
homologous sequence from another region. Exemplary heterologous
sequences include, but are not limited to a "tag" such as a FLAG
tag or a 6His tag. Tags are well known in the art.
[0212] A fusion polypeptide can be created by methods known in the
art, for example, synthetically or recombinantly. Typically, the
fusion proteins of this invention are made by preparing an
expressing a polynucleotide encoding them using recombinant methods
described herein, although they may also be prepared by other means
known in the art, including, for example, chemical synthesis.
[0213] This invention also provides compositions comprising
antibodies conjugated (for example, linked) to an agent that
facilitate coupling to a solid support (such as biotin or avidin).
For simplicity, reference will be made generally to antibodies with
the understanding that these methods apply to any of the IL-7R
binding and/or antagonist embodiments described herein. Conjugation
generally refers to linking these components as described herein.
The linking (which is generally fixing these components in
proximate association at least for administration) can be achieved
in any number of ways. For example, a direct reaction between an
agent and an antibody is possible when each possesses a substituent
capable of reacting with the other. For example, a nucleophilic
group, such as an amino or sulfhydryl group, on one may be capable
of reacting with a carbonyl-containing group, such as an anhydride
or an acid halide, or with an alkyl group containing a good leaving
group (e.g., a halide) on the other.
[0214] An antibody or polypeptide of this invention may be linked
to a labeling agent such as a fluorescent molecule, a radioactive
molecule or any others labels known in the art. Labels are known in
the art which generally provide (either directly or indirectly) a
signal.
[0215] The invention also provides compositions (including
pharmaceutical compositions) and kits comprising, as this
disclosure makes clear, any or all of the antibodies and/or
polypeptides described herein.
[0216] The invention also provides isolated polynucleotides
encoding the antibodies of the invention, and vectors and host
cells comprising the polynucleotide.
[0217] Accordingly, the invention provides polynucleotides (or
compositions, including pharmaceutical compositions), comprising
polynucleotides encoding any of the following: the antibodies C1GM,
C2M3, P3A9, P4B3, P2D2, P2E11, HAL403a and HAL403b, or any fragment
or part thereof having the ability to antagonize IL-7R.
[0218] In another aspect, the invention provides polynucleotides
encoding any of the antibodies (including antibody fragments) and
polypeptides described herein, such as antibodies and polypeptides
having impaired effector function. Polynucleotides can be made and
expressed by procedures known in the art.
[0219] In another aspect, the invention provides compositions (such
as a pharmaceutical compositions) comprising any of the
polynucleotides of the invention. In some embodiments, the
composition comprises an expression vector comprising a
polynucleotide encoding the antibody as described herein. In other
embodiment, the composition comprises an expression vector
comprising a polynucleotide encoding any of the antibodies
described herein. In still other embodiments, the composition
comprises either or both of the polynucleotides shown in SEQ ID NO:
38 and SEQ ID NO: 39 below:
TABLE-US-00005 C1GM heavy chain variable region (SEQ ID NO: 38)
GAGGTCCAGTTAGTGGAGTCTGGGGGAGGCCTGGTCAAGCCGGGGGGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTCTGTCA
TGCACTGGGTCCGTCAAGCTCCGGGGAAGGGTCTGGAGTGGGTTTCTCTT
GTTGGTTGGGATGGTTTTTTTACATACTATGCAGACTCAGTGAAGGGCCG
ATTCACCATCTCCAGAGACAACGCGAAGAACTCTCTGTATCTGCAAATGA
ACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGACAAGGG
GATTACATGGGGAACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTC A C2GM light
chain variable region (SEQ ID NO: 39)
AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAATCTCCGGGAAAGAC
GGTGACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGACAGTTCCTATG
TGCAGTGGTACCAGCAGCGCCCGGGCAGCTCCCCCACCACTGTGATCTAT
GAGGATGACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCAT
CGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAAACTG
AGGACGAGGCTGACTACTACTGTCAGTCTTATGATTTTCATCATCTGGTG
TTCGGCGGAGGGACCAAGCTGACCGTCCTA.
[0220] In still other embodiments, the composition comprises either
or both of the polynucleotides shown in SEQ ID NO: 14 and SEQ ID
NO: 15 below:
TABLE-US-00006 HAL403a heavy chain variable region (SEQ ID NO: 14)
CAGGTCAACTTAAGGGAGTCTGGGGGAGGCCTGGTCAAGCCGGGGGGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTCTGTCA
TGCACTGGGTCCGTCAAGCTCCGGGGAAGGGTCTGGAGTGGCTCTCTCTT
GTTGGTTGGGATGGTTTTTTTACATACTATGCAGACTCAGTGAAGGGCCG
ATTCACCATCTCCAGAGACAACACCAAGAACTTACTGTATCTGCAAATGA
ACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGACAAGGG
GATTACATGGGGAACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTC A HAL403a light
chain variable region (SEQ ID NO: 15)
AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGGGTCTCCGGGAAAGAC
GGTGACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGACAGTTCCTATG
TGCAGTGGTACCAGCAGCGCCCGGGCAATTCCCCCACCACTGTGATCTAT
GAGGATGACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCAT
CGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGGTGACTG
AGGACGAGGCTGACTACTACTGTCAGTCTTATGATTTTCATCATCTGGTG
TTCGGCGGAGGGACCAAGCTGACCGTCCTATGT.
[0221] Expression vectors, and administration of polynucleotide
compositions are further described herein.
[0222] In another aspect, the invention provides a method of making
any of the polynucleotides described herein.
[0223] Polynucleotides complementary to any such sequences are also
encompassed by the present invention. Polynucleotides may be
single-stranded (coding or antisense) or double-stranded, and may
be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules
include HnRNA molecules, which contain introns and correspond to a
DNA molecule in a one-to-one manner, and mRNA molecules, which do
not contain introns. Additional coding or non-coding sequences may,
but need not, be present within a polynucleotide of the present
invention, and a polynucleotide may, but need not, be linked to
other molecules and/or support materials.
[0224] Polynucleotides may comprise a native sequence (i.e., an
endogenous sequence that encodes an antibody or a portion thereof)
or may comprise a variant of such a sequence. Polynucleotide
variants contain one or more substitutions, additions, deletions
and/or insertions such that the immunoreactivity of the encoded
polypeptide is not diminished, relative to a native immunoreactive
molecule. The effect on the immunoreactivity of the encoded
polypeptide may generally be assessed as described herein. Variants
preferably exhibit at least about 70% identity, more preferably, at
least about 80% identity, yet more preferably, at least about 90%
identity, and most preferably, at least about 95% identity to a
polynucleotide sequence that encodes a native antibody or a portion
thereof.
[0225] Two polynucleotide or polypeptide sequences are said to be
"identical" if the sequence of nucleotides or amino acids in the
two sequences is the same when aligned for maximum correspondence
as described below. Comparisons between two sequences are typically
performed by comparing the sequences over a comparison window to
identify and compare local regions of sequence similarity. A
"comparison window" as used herein, refers to a segment of at least
about 20 contiguous positions, usually 30 to about 75, or 40 to
about 50, in which a sequence may be compared to a reference
sequence of the same number of contiguous positions after the two
sequences are optimally aligned.
[0226] Optimal alignment of sequences for comparison may be
conducted using the Megalign program in the Lasergene suite of
bioinformatics software (DNASTAR, Inc., Madison, Wis.), using
default parameters. This program embodies several alignment schemes
described in the following references: Dayhoff, M. O., 1978, A
model of evolutionary change in proteins--Matrices for detecting
distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein
Sequence and Structure, National Biomedical Research Foundation,
Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990,
Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in
Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;
Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E.
W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971,
Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol.
4:406-425; Sneath, P. H. A. and Sokal, R. R., 1973, Numerical
Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman
Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J.,
1983, Proc. Natl. Acad. Sci. USA 80:726-730.
[0227] Preferably, the "percentage of sequence identity" is
determined by comparing two optimally aligned sequences over a
window of comparison of at least 20 positions, wherein the portion
of the polynucleotide or polypeptide sequence in the comparison
window may comprise additions or deletions (i.e., gaps) of 20
percent or less, usually 5 to 15 percent, or 10 to 12 percent, as
compared to the reference sequences (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The percentage is calculated by determining the number of positions
at which the identical nucleic acid bases or amino acid residue
occurs in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the reference sequence (i.e. the window size) and
multiplying the results by 100 to yield the percentage of sequence
identity.
[0228] Variants may also, or alternatively, be substantially
homologous to a native gene, or a portion or complement thereof.
Such polynucleotide variants are capable of hybridizing under
moderately stringent conditions to a naturally occurring DNA
sequence encoding a native antibody (or a complementary
sequence).
[0229] Suitable "moderately stringent conditions" include
prewashing in a solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH
8.0); hybridizing at 50.degree. C.-65.degree. C., 5.times.SSC,
overnight; followed by washing twice at 65.degree. C. for 20
minutes with each of 2.times., 0.5.times. and 0.2.times.SSC
containing 0.1% SDS.
[0230] As used herein, "highly stringent conditions" or "high
stringency conditions" are those that: (1) employ low ionic
strength and high temperature for washing, for example 0.015 M
sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate
at 50.degree. C.; (2) employ during hybridization a denaturing
agent, such as formamide, for example, 50% (v/v) formamide with
0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50
mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride,
75 mM sodium citrate at 42.degree. C.; or (3) employ 50% formamide,
5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times.Denhardt's
solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and
10% dextran sulfate at 42.degree. C., with washes at 42.degree. C.
in 0.2.times.SSC (sodium chloride/sodium citrate) and 50% formamide
at 55.degree. C., followed by a high-stringency wash consisting of
0.1.times.SSC containing EDTA at 55.degree. C. The skilled artisan
will recognize how to adjust the temperature, ionic strength, etc.
as necessary to accommodate factors such as probe length and the
like.
[0231] It will be appreciated by those of ordinary skill in the art
that, as a result of the degeneracy of the genetic code, there are
many nucleotide sequences that encode a polypeptide as described
herein. Some of these polynucleotides bear minimal homology to the
nucleotide sequence of any native gene. Nonetheless,
polynucleotides that vary due to differences in codon usage are
specifically contemplated by the present invention. Further,
alleles of the genes comprising the polynucleotide sequences
provided herein are within the scope of the present invention.
Alleles are endogenous genes that are altered as a result of one or
more mutations, such as deletions, additions and/or substitutions
of nucleotides. The resulting mRNA and protein may, but need not,
have an altered structure or function. Alleles may be identified
using standard techniques (such as hybridization, amplification
and/or database sequence comparison).
[0232] The polynucleotides of this invention can be obtained using
chemical synthesis, recombinant methods, or PCR. Methods of
chemical polynucleotide synthesis are well known in the art and
need not be described in detail herein. One of skill in the art can
use the sequences provided herein and a commercial DNA synthesizer
to produce a desired DNA sequence.
[0233] For preparing polynucleotides using recombinant methods, a
polynucleotide comprising a desired sequence can be inserted into a
suitable vector, and the vector in turn can be introduced into a
suitable host cell for replication and amplification, as further
discussed herein. Polynucleotides may be inserted into host cells
by any means known in the art. Cells are transformed by introducing
an exogenous polynucleotide by direct uptake, endocytosis,
transfection, F-mating or electroporation. Once introduced, the
exogenous polynucleotide can be maintained within the cell as a
non-integrated vector (such as a plasmid) or integrated into the
host cell genome. The polynucleotide so amplified can be isolated
from the host cell by methods well known within the art. See, e.g.,
Sambrook et al., 1989.
[0234] Alternatively, PCR allows reproduction of DNA sequences. PCR
technology is well known in the art and is described in U.S. Pat.
Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR:
The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer
Press, Boston, 1994.
[0235] RNA can be obtained by using the isolated DNA in an
appropriate vector and inserting it into a suitable host cell. When
the cell replicates and the DNA is transcribed into RNA, the RNA
can then be isolated using methods well known to those of skill in
the art, as set forth in Sambrook et al., 1989, supra, for
example.
[0236] Suitable cloning vectors may be constructed according to
standard techniques, or may be selected from a large number of
cloning vectors available in the art. While the cloning vector
selected may vary according to the host cell intended to be used,
useful cloning vectors will generally have the ability to
self-replicate, may possess a single target for a particular
restriction endonuclease, and/or may carry genes for a marker that
can be used in selecting clones containing the vector. Suitable
examples include plasmids and bacterial viruses, e.g., pUC18,
pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,
pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors
such as pSA3 and pAT28. These and many other cloning vectors are
available from commercial vendors such as BioRad, Strategene, and
Invitrogen.
[0237] Expression vectors generally are replicable polynucleotide
constructs that contain a polynucleotide according to the
invention. It is implied that an expression vector must be
replicable in the host cells either as episomes or as an integral
part of the chromosomal DNA. Suitable expression vectors include
but are not limited to plasmids, viral vectors, including
adenoviruses, adeno-associated viruses, retroviruses, cosmids, and
expression vector(s) disclosed in PCT Publication No. WO 87/04462.
Vector components may generally include, but are not limited to,
one or more of the following: a signal sequence; an origin of
replication; one or more marker genes; suitable transcriptional
controlling elements (such as promoters, enhancers and terminator).
For expression (i.e., translation), one or more translational
controlling elements are also usually required, such as ribosome
binding sites, translation initiation sites, and stop codons.
[0238] The vectors containing the polynucleotides of interest can
be introduced into the host cell by any of a number of appropriate
means, including electroporation, transfection employing calcium
chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or
other substances; microprojectile bombardment; lipofection; and
infection (e.g., where the vector is an infectious agent such as
vaccinia virus). The choice of introducing vectors or
polynucleotides will often depend on features of the host cell.
[0239] The invention also provides host cells comprising any of the
polynucleotides described herein. Any host cells capable of
over-expressing heterologous DNAs can be used for the purpose of
isolating the genes encoding the antibody, polypeptide or protein
of interest. Non-limiting examples of mammalian host cells include
but not limited to COS, HeLa, and CHO cells. See also PCT
Publication No. WO 87/04462. Suitable non-mammalian host cells
include prokaryotes (such as E. coli or B. subtillis) and yeast
(such as S. cerevisae, S. pombe; or K. lactis). Preferably, the
host cells express the cDNAs at a level of about 5 fold higher,
more preferably, 10 fold higher, even more preferably, 20 fold
higher than that of the corresponding endogenous antibody or
protein of interest, if present, in the host cells. Screening the
host cells for a specific binding to IL-7R or an IL-7R domain is
effected by an immunoassay or FACS. A cell overexpressing the
antibody or protein of interest can be identified.
Compositions
[0240] The compositions used in the methods of the invention
comprise an effective amount of an antagonist IL-7R antibody, an
antagonist IL-7R antibody derived polypeptide, or other IL-7R
antagonists described herein. Examples of such compositions, as
well as how to formulate, are also described in an earlier section
and below. In some embodiments, the composition comprises one or
more IL-7R antagonist antibodies. In other embodiments, the
antagonist IL-7R antibody recognizes human IL-7R.alpha.. In other
embodiments, the antagonist IL-7R antibody is a human antibody. In
other embodiments, the antagonist IL-7R antibody is a humanized
antibody. In some embodiments, the antagonist IL-7R antibody
comprises a constant region that is capable of triggering a desired
immune response, such as antibody-mediated lysis or ADCC. In other
embodiments, the antagonist IL-7R antibody comprises a constant
region that does not trigger an unwanted or undesirable immune
response, such as antibody-mediated lysis or ADCC. In other
embodiments, the antagonist IL-7R antibody comprises one or more
CDR(s) of the antibody (such as one, two, three, four, five, or, in
some embodiments, all six CDRs).
[0241] It is understood that the compositions can comprise more
than one antagonist IL-7R antibody (e.g., a mixture of antagonist
IL-7R antibodies that recognize different epitopes of IL-7R). Other
exemplary compositions comprise more than one antagonist IL-7R
antibody that recognize the same epitope(s), or different species
of antagonist IL-7R antibodies that bind to different epitopes of
IL-7R.
[0242] The composition used in the present invention can further
comprise pharmaceutically acceptable carriers, excipients, or
stabilizers (Remington: The Science and practice of Pharmacy 20th
Ed., 2000, Lippincott Williams and Wilkins, Ed. K. E. Hoover), in
the form of lyophilized formulations or aqueous solutions.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations, and may comprise
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrans; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g. Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
Pharmaceutically acceptable excipients are further described
herein.
[0243] The antagonist IL-7R antibody and compositions thereof can
also be used in conjunction with other agents that serve to enhance
and/or complement the effectiveness of the agents.
D. Kits
[0244] The invention also provides kits for use in the instant
methods. Kits of the invention include one or more containers
comprising an IL-7R antagonist (such as, for example, a human
antibody) described herein and instructions for use in accordance
with any of the methods of the invention described herein.
Generally, these instructions comprise a description of
administration of the IL-7R antagonist for the above described
therapeutic treatments.
[0245] In some embodiments, the IL-7R antagonist is an antagonist
IL-7R antibody. In some embodiments, the antibody is a human
antibody. In some embodiments, the antibody is a humanized
antibody. In some embodiments, the antibody is a monoclonal
antibody. The instructions relating to the use of an antagonist
IL-7R antibody generally include information as to dosage, dosing
schedule, and route of administration for the intended treatment.
The containers may be unit doses, bulk packages (e.g., multi-dose
packages) or sub-unit doses. Instructions supplied in the kits of
the invention are typically written instructions on a label or
package insert (e.g., a paper sheet included in the kit), but
machine-readable instructions (e.g., instructions carried on a
magnetic or optical storage disk) are also acceptable.
[0246] The kits of this invention are in suitable packaging.
Suitable packaging includes, but is not limited to, vials, bottles,
jars, flexible packaging (e.g., sealed Mylar or plastic bags), and
the like. Also contemplated are packages for use in combination
with a specific device, such as an inhaler, nasal administration
device (e.g., an atomizer) or an infusion device such as a
minipump. A kit may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The container
may also have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an antagonist IL-7R antibody. The
container may further comprise a second pharmaceutically active
agent.
[0247] Kits may optionally provide additional components such as
buffers and interpretive information. Normally, the kit comprises a
container and a label or package insert(s) on or associated with
the container.
Mutations and Modifications
[0248] To express the IL-7R antibodies of the present invention,
DNA fragments encoding VH and VL regions can first be obtained
using any of the methods described above. Various modifications,
e.g. mutations, deletions, and/or additions can also be introduced
into the DNA sequences using standard methods known to those of
skill in the art. For example, mutagenesis can be carried out using
standard methods, such as PCR-mediated mutagenesis, in which the
mutated nucleotides are incorporated into the PCR primers such that
the PCR product contains the desired mutations or site-directed
mutagenesis.
[0249] One type of substitution, for example, that may be made is
to change one or more cysteines in the antibody, which may be
chemically reactive, to another residue, such as, without
limitation, alanine or serine. For example, there can be a
substitution of a non-canonical cysteine. The substitution can be
made in a CDR or framework region of a variable domain or in the
constant region of an antibody. In some embodiments, the cysteine
is canonical.
[0250] The antibodies may also be modified, e.g. in the variable
domains of the heavy and/or light chains, e.g., to alter a binding
property of the antibody. For example, a mutation may be made in
one or more of the CDR regions to increase or decrease the K.sub.D
of the antibody for IL-7R, to increase or decrease k.sub.off, or to
alter the binding specificity of the antibody. Techniques in
site-directed mutagenesis are well-known in the art. See, e.g.,
Sambrook et al. and Ausubel et al., supra.
[0251] A modification or mutation may also be made in a framework
region or constant region to increase the half-life of an IL-7R
antibody. See, e.g., PCT Publication No. WO 00/09560. A mutation in
a framework region or constant region can also be made to alter the
immunogenicity of the antibody, to provide a site for covalent or
non-covalent binding to another molecule, or to alter such
properties as complement fixation, FcR binding and
antibody-dependent cell-mediated cytotoxicity. According to the
invention, a single antibody may have mutations in any one or more
of the CDRs or framework regions of the variable domain or in the
constant region.
[0252] In a process known as "germlining", certain amino acids in
the VH and VL sequences can be mutated to match those found
naturally in germline VH and V.sub.L sequences. In particular, the
amino acid sequences of the framework regions in the V.sub.H and VL
sequences can be mutated to match the germline sequences to reduce
the risk of immunogenicity when the antibody is administered.
Germline DNA sequences for human VH and VL genes are known in the
art (see e.g., the "Vbase" human germline sequence database; see
also Kabat, E. A., et al., 1991, Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242; Tomlinson et al.,
1992, J. Mol. Biol. 227:776-798; and Cox et al., 1994, Eur. J.
Immunol. 24:827-836.
[0253] Another type of amino acid substitution that may be made is
to remove potential proteolytic sites in the antibody. Such sites
may occur in a CDR or framework region of a variable domain or in
the constant region of an antibody. Substitution of cysteine
residues and removal of proteolytic sites may decrease the risk of
heterogeneity in the antibody product and thus increase its
homogeneity. Another type of amino acid substitution is to
eliminate asparagine-glycine pairs, which form potential
deamidation sites, by altering one or both of the residues. In
another example, the C-terminal lysine of the heavy chain of an
IL-7R antibody of the invention can be cleaved. In various
embodiments of the invention, the heavy and light chains of the
IL-7R antibodies may optionally include a signal sequence.
[0254] Once DNA fragments encoding the VH and VL segments of the
present invention are obtained, these DNA fragments can be further
manipulated by standard recombinant DNA techniques, for example to
convert the variable region genes to full-length antibody chain
genes, to Fab fragment genes, or to a scFv gene. In these
manipulations, a VL- or VH-encoding DNA fragment is operatively
linked to another DNA fragment encoding another protein, such as an
antibody constant region or a flexible linker. The term
"operatively linked", as used in this context, is intended to mean
that the two DNA fragments are joined such that the amino acid
sequences encoded by the two DNA fragments remain in-frame.
[0255] The isolated DNA encoding the VH region can be converted to
a full-length heavy chain gene by operatively linking the
VH-encoding DNA to another DNA molecule encoding heavy chain
constant regions (CH1, CH2 and CH3). The sequences of human heavy
chain constant region genes are known in the art (see e.g., Kabat,
E. A., et al., 1991, Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The heavy chain constant region can be an IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most
preferably is an IgG1 or IgG2 constant region.
[0256] The IgG constant region sequence can be any of the various
alleles or allotypes known to occur among different individuals,
such as Gm(1), Gm(2), Gm(3), and Gm(17). These allotypes represent
naturally occurring amino acid substitution in the IgG1 constant
regions. For a Fab fragment heavy chain gene, the V.sub.H-encoding
DNA can be operatively linked to another DNA molecule encoding only
the heavy chain CH1 constant region. The CH1 heavy chain constant
region may be derived from any of the heavy chain genes.
[0257] The isolated DNA encoding the VL region can be converted to
a full-length light chain gene (as well as a Fab light chain gene)
by operatively linking the VL-encoding DNA to another DNA molecule
encoding the light chain constant region, CL. The sequences of
human light chain constant region genes are known in the art (see
e.g., Kabat, E. A., et al., 1991, Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The light chain constant region can be a kappa or
lambda constant region. The kappa constant region may be any of the
various alleles known to occur among different individuals, such as
Inv(1), Inv(2), and Inv(3). The lambda constant region may be
derived from any of the three lambda genes.
[0258] To create a scFv gene, the VH- and VL-encoding DNA fragments
are operatively linked to another fragment encoding a flexible
linker, e.g., encoding the amino acid sequence
(Gly.sub.4-Ser).sub.3, (SEQ ID NO: 16) such that the VH and VL
sequences can be expressed as a contiguous single-chain protein,
with the VL and VH regions joined by the flexible linker (See e.g.,
Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc.
Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature
348:552-554. The single chain antibody may be monovalent, if only a
single VH and VL are used, bivalent, if two VH and VL are used, or
polyvalent, if more than two VH and VL are used. Bispecific or
polyvalent antibodies may be generated that bind specifically to
IL-7R and to another molecule.
[0259] In another embodiment, a fusion antibody or immunoadhesin
may be made that comprises all or a portion of an IL-7R antibody of
the invention linked to another polypeptide. In another embodiment,
only the variable domains of the IL-7R antibody are linked to the
polypeptide. In another embodiment, the VH domain of an IL-7R
antibody is linked to a first polypeptide, while the VL domain of
an IL-7R antibody is linked to a second polypeptide that associates
with the first polypeptide in a manner such that the VH and VL
domains can interact with one another to form an antigen binding
site. In another preferred embodiment, the VH domain is separated
from the VL domain by a linker such that the VH and VL domains can
interact with one another. The VH-linker-VL antibody is then linked
to the polypeptide of interest. In addition, fusion antibodies can
be created in which two (or more) single-chain antibodies are
linked to one another. This is useful if one wants to create a
divalent or polyvalent antibody on a single polypeptide chain, or
if one wants to create a bispecific antibody.
[0260] In other embodiments, other modified antibodies may be
prepared using IL-7R antibody encoding nucleic acid molecules. For
instance, "Kappa bodies" (Ill et al., 1997, Protein Eng.
10:949-57), "Minibodies" (Martin et al., 1994, EMBO J. 13:5303-9),
"Diabodies" (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA
90:6444-6448), or "Janusins" (Traunecker et al., 1991, EMBO J.
10:3655-3659 and Traunecker et al., 1992, Int. J. Cancer (Suppl.)
7:51-52) may be prepared using standard molecular biological
techniques following the teachings of the specification.
[0261] Bispecific antibodies or antigen-binding fragments can be
produced by a variety of methods including fusion of hybridomas or
linking of Fab' fragments. See, e.g., Songsivilai & Lachmann,
1990, Clin. Exp. Immunol. 79:315-321, Kostelny et al., 1992, J.
Immunol. 148:1547-1553. In addition, bispecific antibodies may be
formed as "diabodies" or "Janusins." In some embodiments, the
bispecific antibody binds to two different epitopes of IL-7R. In
some embodiments, the modified antibodies described above are
prepared using one or more of the variable domains or CDR regions
from a human IL-7R antibody provided herein.
Generation of Antigen-Specific Antibodies
[0262] Monoclonal antibodies raised against recombinant mouse
IL-7R.alpha./CD127/Fc chimera (R&D Systems Cat. No. 747-MR),
and human antibodies obtained by biopanning a human naive antibody
library with recombinant IL-7R.alpha. were evaluated for their
ability to bind mouse and human IL-7R. Antibodies were further
screened for their ability to block IL-7-mediated STAT5
phosphorylation in human peripheral blood mononuclear cells (PBMCs)
and/or monkey PBMCs. This manner of antibody preparation yielded
antagonist antibodies that show blocking of IL-7-mediated STAT5
phosphorylation, as shown in Example 1.
[0263] Representative materials of the present invention were
deposited in the American Type Culture Collection (ATCC) on Feb. 9,
2011. Vector C1GM-VH having ATCC Accession No. PTA- is a
polynucleotide encoding the C1GM heavy chain variable region, and
vector C1GM-VL having ATCC Accession No. PTA- is a polynucleotide
encoding the C1GM light chain variable region. The deposits were
made under the provisions of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purpose of Patent Procedure and Regulations thereunder (Budapest
Treaty). This assures maintenance of a viable curlture of the
deposit for 30 years from the date of deposit. The deposit will be
made available by ATCC under the terms of the Budapest Treaty, and
subject to an agreement between Pfizer, Inc. and ATCC, which
assures permanent and unrestricted availability of the progeny of
the culture of the deposit to the public upon issuance of the
pertinent U.S. patent or upon laying open to the public of any U.S.
or foreign patent application, whichever comes first, and assures
availability of the progeny to one determined by the U.S.
Commissioner of Patents and Trademarks to be entitled thereto
according to 35 U.S.C. Section 122 and the Commissioner's rules
pursuant thereto (including 37 C.F.R. Section 1.14 with particular
reference to 886 OG 638).
[0264] The assignee of the present application has agreed that if a
culture of the materials on deposit should die or be lost or
destroyed when cultivated under suitable conditions, the materials
will be promptly replaced on notification with another of the same.
Availability of the deposited material is not to be construed as a
license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws.
[0265] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
EXAMPLES
Example 1: Generating and Screening Antagonist IL-7R Antibodies
[0266] This example illustrates the generation and screening of
antagonist IL-7R antibodies.
General Procedures for Immunization of Animals for Generating
Monoclonal Antibodies:
[0267] A 2-month old female Sprague Dawley rat was immunized with
50 ug recombinant mouse IL-7R.alpha./CD127/Fc chimera, which
includes mouse IL-7R.alpha. (Glu21-Asp239), hCD33 signal peptide
(Met 1-Ala 16), and human IgG (Pro100-Lys330) (R&D Systems Cat.
No. 747-MR). The antigen was prepared for immunization by mixing 50
ug antigen in 100 ul PBS with 100 ul Sigma Adjuvant System (Cat.
No. S6322). The antigen mixture was vortexed and injected into the
hind footpads and peritoneum on days 0, 2, 5 and 7. On day 9, 50 ug
of antigen without adjuvant was injected intravenously in a total
volume of 150 ul in physiological saline. On day 13, the spleen
cells were prepared as a single cell suspension and fused with
P3x63Ag8.653 mouse myeloma cells following a standard fusion
protocol using 40% PEG 1500 (Boeringer Mannheim Biochemicals
#783641). The fused cells were resuspended in medium containing 18%
FBS, 2 mM L-glutamine, pen/strep, hypoxanthine, aminopterin and
thymidine (HAT) (Sigma H0262) and 10% hybridoma fusion and cloning
supplement (HFCS) (Cat. No. 11 363 735 001, Sigma), then plated out
in 54 96-well plates at 200 ul/well. At day 7 after fusion, 150 ul
of the medium was aspirated from each well, and the wells were
re-fed with 200 ul of fresh medium. At day 11-13, supernatant from
each well was tested for antibody to IL-7R and human Fc using ELISA
(described below).
ELISA Screening of Antibodies:
[0268] Supernatant media from growing hybridoma clones were
screened separately for their ability to bind the recombinant mouse
(rm) IL-7R. The assays were performed with 96-well plates coated
overnight with 50 .mu.l of a 1 .mu.g/ml solution of the antigen.
Fifty-five coated plates were washed 4 times with PBS with 0.05%
Tween and then 50 ul PBS with 0.5% BSA was added to each well. 5 ul
from each well of the hybridoma plates were added to the assay
plates, and the plates were incubated at room temperature for 2 hrs
to allow binding. Excess reagents were washed from the wells
between each step with PBS containing 0.05% Tween-20. 50 ul
horseradish peroxidase (HRP) conjugated goat-anti mouse, F(ab')2,
Fc specific (Jackson #115-036-008) was added to bind to the mouse
antibodies bound to the antigen. For detection, 50 ul ABTS,
2,2'-Azino-bis(3-ethyl benzothiazoline-6-sulfonic acid) diammonium
salt was added as substrate. The plates were read after 30 mins at
405 nm using a Molecular Devices THERMOmax.TM. instrument.
Hybridoma clones that secreted antibodies that were capable of
binding to mouse IL-7R were selected for further analysis. These
positive hybridoma supernatants were then collected from the
hybridoma plates and tested in ELISA assays against human Fc, goat
anti rat IgM, and recombinant human (rh) IL-7R. Purified antibodies
were then prepared for the antibodies that bound to rm IL-7R and
antibodies that bound to both rm IL-7R and rh IL-7R.
General Procedures for Generating Fully Human Monoclonal Antibodies
Using Phage Display:
[0269] Anti-human IL-7R.alpha. human antibodies were isolated from
a phage display human naive scFv antibody library (Glanville G. et
al., 2009, Proc Natl Acad Sci USA, 106(48):20216-20221) by a series
of four rounds of bio-panning against human IL-7R.alpha. (R&D
Systems.RTM.). For each round of panning, 1 ml IL-7R.alpha. (10
ug/ml in PBS) was coated on an immunotube at 4.degree. C.
overnight. The IL-7R.alpha. coated immunotube was washed three
times with PBST. 10.sup.13 phage (1 ml) were added to the
immunotube and incubated at room temperature for 1 hour to allow
binding. After binding, the immunotube was washed eight times with
PBST. Bound phage were eluted and used to infect freshly grown TG1
cells. After the fourth round of panning, the positive binders were
screened against both human IL-7R.alpha. and mouse IL-7R by ELISA.
The antibodies binding to both human and mouse IL-7R were further
studied for their affinities and blocking function, and antibodies
were selected for affinity maturation.
In Vitro Functional Assay:
[0270] Hybridoma clones secreting human or mouse IL-7R binding
antibodies were expanded and supernatants were harvested. Total
IgGs were purified from approximately 10 ml of the supernatant
using protein A beads, dialyzed into PBS buffer, and the final
volume reduced to yield solutions with 0.7-1 mg/ml of antibodies.
Purified antibodies were then used to test their ability to block
IL-7-mediated STAT5 phosphorylation in human PBMCs. For PBMC
preparation, whole blood cells were collected through Ficoll
gradient. Cells were maintained at 37.degree. C. in 5% CO.sub.2 in
conical tubes (to prevent monocyte/macrophage adherence) for 1-2 h
before stimulation with IL-2.
[0271] For the functional screening, human PBMCs were preincubated
for 5 minutes with test antibodies (10 .mu.g/ml) prior to addition
of IL-7. A non-reactive isotype-matched antibody was used as a
negative control (isotype control). Cells were stimulated with
human IL-7 (0.1 ng/ml, R&D Systems for 15 minutes. To stop the
IL-7 stimulation, formaldehyde was added directly to the culture
medium to a final concentration of 1.6%. Cells were fixed for 15
min at room temperature. Methanol was then added directly to a
final concentration of 80%, and samples were stored at 4.degree. C.
for 30 minutes to 1 hour before being immunostained. Cells were
stained with anti-phospho-STAT5 (p-STAT) antibodies (BD Pharmingen,
Y694 clone 47) and anti-CD4 antibodies (BD Pharmingen, RPA-T4).
Using flow cytometry (LSRII, BD.TM. Biosciences), CD4+ gated cells
were analyzed for p-STAT5 staining. Isotype control was set as 100%
of p-STAT.
[0272] FIG. 1 illustrates the effect of antagonist IL-7R fully
human monoclonal antibodies P2D2 and P2E11, and HAL403a on
IL-7-mediated STAT5 phosphorylation in human PBMCs. A mouse
anti-human IL-7R monoclonal antibody, 13A2F4, was used as a
positive control, and a nonreactive isotype-matched antibody was
used as a negative control (isotype control). Human PBMCs were
preincubated for 5 minutes with each of the test antibodies or
13A2F4 at the following concentrations: 0.001, 0.01, 0.1, 1, and 10
.mu.g/ml. The isotype control antibody was used at the highest
concentration, 10 .mu.g/ml. Cells were stimulated with human IL-7
(0.1 ng/ml) for 15 minutes, then fixed and immunostained as
described above.
[0273] As measured by p-STAT5 staining, human antibodies P2D2,
P2E11, HAL403a C1GM, C1GM-2 and C2M3 block human IL-7 mediated
signaling in a dose-dependent manner (FIG. 1 and data not shown).
The isotype control was set as 100% p-STAT5 staining. At 10
.mu.g/ml antibody HAL403a blocked STAT5 phosophorylation very
effectively (FIG. 1). C1GM, C1GM-2 and C2M3 blocked STAT5
phosophorylation comparable to HAL403a (data not shown).
[0274] The amino acid sequence of antagonist IL-7R antibody C1GM
heavy chain (SEQ ID NO: 42) is shown below.
TABLE-US-00007 (SEQ ID NO: 42)
EVQLVESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEW
VSLVGWDGFFTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC
ARQGDYMGNNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTQTYICNVNHKPSNTKVDKKVAPELLGGPSVFLFPPKPKDTLMISRTP
EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0275] The amino acid sequence of antagonist IL-7R antibody C1GM
light chain (SEQ ID NO: 43) is shown below.
TABLE-US-00008 (SEQ ID NO: 43)
NFMLTQPHSVSESPGKTVTISCTRSSGSIDSSYVQVVYQQRPGSSPTTVI
YEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDFHH
LVFGGGTKLTVLQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVT
VAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSC
QVTHEGSTVEKTVAPTECS
[0276] The amino acid sequence of antagonist IL-7R antibody C1GM-2
heavy chain (SEQ ID NO: 45) is shown below.
TABLE-US-00009 (SEQ ID NO: 45)
EVQLVESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEW
VSLVGWDGFFTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC
ARQGDYMGNNWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSNF
GTQTYTCNVDHKPSNTKVDKTVAPPVAGPSVFLFPPKPKDTLMISRTPE
VTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRWSVL
TWHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0277] The amino acid sequence of antagonist IL-7R antibody C1GM-2
light chain (SEQ ID NO: 43) is shown below.
TABLE-US-00010 (SEQ ID NO: 43)
NFMLTQPHSVSESPGKTVTISCTRSSGSIDSSYVQWYQQRPGSSPTTVI
YEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDFHH
LVFGGGTKLTVLQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVT
VAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSC
QVTHEGSTVEKTVAPTECS
[0278] The amino acid sequence of antagonist IL-7R antibody HAL403a
heavy chain (SEQ ID NO: 17) is shown below.
TABLE-US-00011 (SEQ ID NO: 17)
QVNLRESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEW
LSLVGWDGFFTYYADSVKGRFTISRDNTKNLLYLQMNSLRAEDTAVYYC
ARQGDYMGNNWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSNF
GTQTYTCNVDHKPSNTKVDKTVAPPVAGPSVFLFPPKPKDTLMISRTPE
VTCWVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRWSVL
TWHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0279] The amino acid sequence of antagonist IL-7R antibody HAL403a
light chain (SEQ ID NO: 18) is shown below.
TABLE-US-00012 (SEQ ID NO: 18)
NFMLTQPHSVSGSPGKTVTISCTRSSGSIDSSYVQWYQQRPGNSPTTVI
YEDDQRPSGVPDRFSGSIDSSSNSASLTISGLVTEDEADYYCQSYDFHH
LVFGGGTKLTVLTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV
QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC
Example 2: Determining Antibody Binding Affinity
[0280] This example illustrates the determination of antibody
binding affinity for antagonist IL-7R antibodies.
[0281] The affinities of antagonist IL-7R antibodies to human IL-7R
were measured on a surface plasmon resonance Biacore.TM. 2000 or
3000 biosensor equipped with a research-grade CM5 sensor chip
(Biacore.TM. AB, Uppsala, Sweden--now GE Healthcare). Goat
polyclonal anti-human F(ab')2 fragments (Fc specific) were
amine-coupled at saturating levels onto all four flow cells using a
standard N-hydroxysuccinimide/ethyldimethylaminopropyl carbodiimide
(NHS/EDC) chemistry in HBS-P running buffer (from Biacore.TM.). The
buffer was switched to HBS-P containing 1 mg/mL BSA. Human
IL-7R-hFc antigen (R&D systems, Minneapolis, USA) was diluted
to about 30 .mu.g/mL and captured for 3 min at 5 .mu.L/min to give
levels of about 500-1000 RU per flow cell, leaving one blank to
serve as a reference channel. Fab, hIgG1, or hIgG2.DELTA.A formats
of the antibodies were injected in duplicates as a 5-membered
3-fold series starting at 2 .mu.M and a 5-membered 4-fold series
starting at 0.4 .mu.M for 3 min at 20-50 .mu.L/min. Dissociation
was monitored for 5 min. The capture chip was regenerated after the
last injection of each titration with two 30 sec pulses of 75 mM
phosphoric acid or 10 mM Glycine-HCl pH 1.7. Buffer cycles provided
blanks for double-referencing the binding response data, which were
then fit globally to a simple reversible binding model using
Biaevaluation software v.4.1 to deduce the kinetic association and
dissociation rate constants, respectively k.sub.a and k.sub.d.
Affinities were deduced from their ratio (K.sub.D=k.sub.d/k.sub.a).
The results in Table 4 show that these antibodies have picomolar or
nanomolar affinities for human IL-7R.
TABLE-US-00013 TABLE 4 K.sub.on for IL-7R K.sub.off for IL-7R mAb
(1/Ms) (1/S) K.sub.D for IL-7R (nM) P3A9* 5.60E+04 1.14E-02 204
P4B3* 1.56E+04 3.51E-03 225 P2D2* 7.17E+04 8.82E-04 12 P2E11*
1.55E+05 4.97E-04 3 HAL403a* 5.07E+06 2.86E-04 0.06 HAL403b*
1.39E+06 8.08E-05 0.06 C1GM* 4.85E+06 1.71E-04 0.04 C1GM** 1.42E+06
4.05E-04 0.286 C1GM-2*** 1.51E+06 4.07E-04 0.270 C2M3** 1.41E+06
3.07E-04 0.218 C2M3*** 1.55E+06 3.02E-04 0.195 *Fab; **hIgG1;
**hIgG2.DELTA.A
Example 4: Antagonist IL-7R Antibodies Reduce Disease Incidence in
Non-Obese Diabetic (NOD) Animals, a Mouse Model for Type 1
Diabetes
[0282] This example illustrates the effect of antagonist IL-7R
antibodies in a mouse model for type 1 diabetes.
[0283] To study the in vivo effect of antagonist IL-7R antibodies
on the diabetogenic process, a rat anti-mouse antagonist IL-7R
antibody, 28G9 (Rinat), was tested in NOD mice. NOD mice exhibit a
susceptibility to spontaneous development of automimmune insulin
dependent diabetes mellitus (IDDM, type 1 diabetes) (Kikutani et
al., 1992, Adv. Immunol. 51: 285-322). 28G9 blocks IL-7-mediated
STAT5 phosphorylation in mouse splenocytes and cross-competes with
antagonist IL-7R human antibodies C1GM, C2M3, HAL403a, HAL403b,
P3A9, P4B3, P2D2 and P2E11 in Biacore.TM..
[0284] 6-8 week old NOD female mice (The Jackson Laboratory) were
injected intraperitoneally (i.p.) weekly starting at 9 weeks old
(t=0) with either 3 or 10 mg/kg body weight of 28G9. PBS or
non-reactive isotype matched rat monoclonal antibody (isotype) were
used as negative controls. The isotype antibody was administered at
10 mg/kg body weight. Mice were monitored two times per week for
body weight and blood glucose. Diabetes was considered established
when blood glucose level was at or over positive readings, i.e.,
over 250 mg/dL for two consecutive monitorings. The onset of
diabetes was dated from the first of the sequential
measurements.
[0285] As shown in FIG. 2, none of the mice treated with 28G9 at 10
mg/kg developed diabetes even at 18 weeks of age. In contrast,
75-80% of the PBS and isotype-treated mice developed diabetes (FIG.
2). Although not all mice treated with 28G9 at 3 mg/kg were
diabetes-free at the end of the study, a significantly reduced
diabetes incidence compared to the PBS and isotype controls was
observed, demonstrating the inhibitory effect of 28G9 on diabetes
development was dose-dependent (FIG. 2). Treatment with 28G9 at 10
mg/kg significantly reduced blood glucose level compared to isotype
or PBS controls (FIG. 3A). Mouse development during antagonist
IL-7R antibody treatment was monitored by tracking body weight and
mortality. As shown in FIG. 3B, multiple dosing of 3 or 10 mg/kg
28G9 had no significant effect on mouse growth, and no mortality
was found at 10 mg/kg. Thus, antagonist IL-7R antibodies reduce
blood glucose levels and inhibit diabetes progression in NOD
animals. These results demonstrate that antagonist IL-7R antibodies
are effective in preventing and slowing the progression of type 1
diabetes.
[0286] To investigate the effect of antagonist IL-7R antibodies on
peripheral T cell regulation, CD4+ and CD8+ T cells were
immunostained for the activation markers CD44 and CD62L and
analyzed by flow cytometry. CD4+ and CD8+ T cells were isolated
from the peripheral blood of PBS-treated, 28G9-treated, or
isotype-treated mice. In comparison to the isotype control, the
percentage of naive CD8+ T cells (B220-CD8+CD44.sup.loCD62L.sup.hi)
in mice treated with 28G9 at 10 mg/kg was significantly lower, and
the percentage of memory CD8+ T cells
(B220-CD8+CD44.sup.hiCD62L.sup.hi) were significantly higher (FIGS.
4A and 4B). In contrast, naive CD4+ T cells
(B220-CD4+CD44.sup.loCD62L.sup.hi) were not significantly depleted
in antagonist IL-7R antibody treated mice compared to isotype
control (FIG. 5). These results indicate that antagonist IL-7R
antibodies reduce blood glucose levels through naive CD8+ T cell
depletion.
Example 5: Antagonist IL-7R Antibodies Delay Onset of Autoimmune
Disease
[0287] This example illustrates the effect of antagonist IL-7R
antibodies in a mouse model for multiple sclerosis, experimental
autoimmune encephalomyelitis (EAE).
[0288] The STAT5 activation assay was used to identify antagonist
IL-7R antibodies. Spleens from B6 or BALB/c were homogenized in PBS
and lysed in ACK lysis buffer (Invitrogen) for 2 min and then
filtered through 100-.mu.m pore size mesh, pelleted, and
resuspended at 5.times.10.sup.6 cells/ml in room temperature to
37.degree. C. RPMI 1640 containing 10% FBS, penicillin (100 U/ml),
streptomycin (100 .mu.g/ml), and L-glutamine. Cells were maintained
at 37.degree. C. in 5% CO.sub.2 in conical tubes (to prevent
monocyte/macrophage adherence) for 1-2 h before stimulation. Cells
were preincubated with test antibody for 5 minutes prior to
stimulation with IL-7. Cells were treated for 15 min with murine
IL-7 (mIL-7, 0.1 ng/ml). Formaldehyde was added directly to the
culture medium to a final concentration of 1.6%, and cells were
fixed for 15 min at room temperature. Methanol was then added
directly to a final concentration of 80%, and samples were stored
at 4.degree. C. for 30 min to 1 h before immunostaining. The
following antibodies were used for immunostaining: CD11b-FITC
(M1/70), B220-Cy5.5PerCP, TCR.beta.-FITC, p-STAT5 (Y694, clone
47)-Alexa 647 (BD.TM. Pharmingen). TCR.beta.+ and CD11b+ cells were
stained for phospho-STAT5. IL-7-stimulated STAT5 phosphorylation
was observed by gating with TCR.beta. in flow cytometry. A number
of antibodies that blocked STAT5 phosphorylation were identified,
including monoclonal antibodies 28B6 and 28G9.
[0289] Active EAE was induced in 6- to 8-week-old female B6 mice by
subcutaneous immunization with 100 .mu.g of MOG.sub.35-55 peptide
(MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 15)) emulsified in CFA
containing 1 mg/ml of heat-killed Mycobacterium tuberculosis H37RA
(Difco) on day 0 (see, Steinman and Zamvil, 2006). Additionally,
mice received 400 ng of pertussis toxin (Calbiochem) i.v. in 0.1 ml
of PBS on days 0 and 2. Starting on day 7 after MOG immunization,
animals were treated twice weekly with antagonist IL-7R antibody
28B6 (10 mg/kg), antagonist IL-7R antibody 28G9 (10 mg/kg), or
non-reactive isotype-matched antibody (10 mg/kg). Compared to
isotype control, treatment with either 28G9 or 28B6 significantly
reduced EAE activity as early as day 15 post immunization (FIG. 6).
This result demonstrates that antagonist IL-7R antibodies are
effective in slowing the progression of EAE.
[0290] To test whether the antagonist IL-7R antibodies are
efficacious in a dose-dependent manner, MOG immunized EAE animals
were treated with either 1 or 3 mg/kg of 28G9 at day 7 and day 14
post immunization. A non-reactive isotype-matched antibody (1
mg/kg) was used as a negative control. In comparison to the isotype
control, 28G9 treatment at both dosage levels reduced EAE severity
at disease peak (FIG. 7). This inhibitory effect of the antagonist
IL-7R antibody lasted for about a week. This result demonstrates
that antagonist IL-7R antibodies conferred protection at both 1 and
3 mg/kg. In a separate study, MOG immunized EAE animals were
treated weekly with 1, 3 or 10 mg/kg of 28G9 starting at day 7.
Mice treated with 28G9 at 1 mg/kg showed significant efficacy with
no mortality (FIG. 8). These results demonstrate that 1 mg/kg of
antagonist IL-7R antibody treatment is effective in slowing the
progression of EAE and is well-tolerated.
[0291] To investigate antagonist IL-7R antibody efficacy in
established disease, MOG immunized EAE animals were treated twice
weekly with 28G9 at 10 mg/kg starting day 14 after immunization. A
non-reactive isotype-matched antibody (10 mg/kg) was used as a
negative control. Compared to the control, treatment with
antagonist IL-7R antibody significantly reduced EAE severity (FIG.
9). No mortality with the antagonist IL-7R antibody observed. This
result demonstrates that antagonist IL-7R antibodies are effective
to ameliorate established, ongoing EAE.
[0292] To further determine whether antagonist IL-7R antibodies can
reduce EAE at late intervention at lower dose, MOG immunized EAE
animals were treated once weekly with 28G9 at 3 mg/kg starting day
14 after immunization. A non-reactive isotype-matched antibody (3
mg/kg) was used as a negative control. Compared to the control, a
highly significant reduction of disease severity was observed with
antagonist IL-7R antibody treatment (FIG. 10). This result
demonstrates that antagonist IL-7R antibody treatment is effective
to reduce disease activity even at late intervention and lower
dose.
Example 6: Immunological Changes after Antagonist IL-7R Antibody
Therapy in Autoimmune Disease
[0293] This example illustrates immunological changes in EAE mice
after antagonist IL-7R antibody treatment.
[0294] To gain insight into the mechanisms by which antagonist
IL-7R antibody acts to ameliorate EAE in the mouse model,
lymphocyte populations from treated and control animals were
analyzed by immunostaining and flow cytometry. For the
immunological studies in this example, MOG immunized EAE animals
were treated weekly with antagonist IL-7R antibody 28G9 (10 mg/kg),
28B6 (10 mg/kg) or vehicle (non-reactive isotype-matched antibody,
10 mg/kg). In selected studies, a group of MOG immunized EAE
animals were treated weekly with 28B6 (10 mg/kg). Animals were
sacrificed on day 21 after immunization, and central lymphoid
organs were collected. Lymphocytes were prepared from the organs
and stained as described below. Immunostained lymphocytes were
analyzed by flow cytometry.
[0295] T cell populations in the BM, spleen, blood and thymus from
EAE animals treated with antagonist IL-7R antibodies were
significantly reduced compared to vehicle controls. As shown in
FIG. 11, both CD4 T cell (FIG. 11A) and CD8 T cell (FIG. 11B)
populations from BM, spleen, blood and lymph nodes were
significantly reduced in antagonist IL-7R antibody treated EAE
animals. This is consistent with the role of IL-7R in both CD4 and
CD8 T cell development. However, B cell populations were not
significantly reduced in all of peripheral lymphoid organs. This
result differs from the mouse genetic data from the IL-7R knockout,
which lacks both T and B cells.
[0296] Because IL-7R signaling is critical for naive T cell
survival and for memory T cell proliferation, the effect of
antagonist IL-7R antibodies in the regulation of peripheral T cells
was analyzed by immunostaining using activation markers CD44 and
CD62L. CD44.sup.loCD62L.sup.hi represents naive T cells,
CD44.sup.hiCD62L.sup.lo represents activated T cells and
CD44.sup.hiCD62L.sup.hi represent memory T cells. Compared to
vehicle (nonreactive isotype-matched antibody) treated animals,
antagonist IL-7R antibody treated mice had significantly depleted
naive T cell and activated T cell populations (FIGS. 12A and 12C).
However, memory T cell populations were not significantly depleted
(FIG. 12B). This selective depletion of naive and activated T cell
populations may provide benefit in that naive T cell depletion can
block nascent autoAg-specific T cell activation, in turn preventing
EAE. Memory T cells are not depleted, and thus, anti-infection
immunity is preserved.
[0297] A reduction of NK cells in antagonist IL-7R antibody treated
EAE animals was not observed. A slight increase in the percent of
NK cells was observed, presumably due to the decreased percent of
CD4 and CD8 T cells. This data is consistent with the observation
that IL-7/IL-7R signaling regulates T cell, but not for NK cell,
development.
[0298] To determine the effect of antagonist IL-7R antibody
treatment on T.sub.reg cell population in EAE animals, lymphocytes
were stained for Foxp3 to identify T.sub.reg cells and MOG-MHC
class II (1-Ab) tetramer to detect MOG-specific T cells. The
population of MOG-specific CD4+T.sub.eff cells detected in lymph
nodes from 28G9 treated EAE animals was similar to that of control
(nonreactive isotype-matched antibody-treated) animals (FIG. 13,
left graph). However, an increase in T.sub.reg cell population was
observed with 28G9 treatment (FIG. 13, right graph). These results
demonstrate that treatment of EAE animals with antagonist IL-7R
antibody results in an increase of T.sub.reg cell population.
Advantageously, antagonist IL-7R antibody treatment may not develop
other inflammatory disease, a side effect observed with
IL-2R.alpha. antibody therapy.
Lymphocyte Preparation and Immunofluorescent Staining
[0299] For the above studies, single-cell leukocyte suspensions
from spleens, peripheral lymph nodes (paired axillary, bronchial
and inguinal), thymus and bilateral femurs bone marrow (BM) were
generated by gentle dissection. Mononuclear cells from the central
nervous system (CNS) were isolated after cardiac perfusion with
PBS. Briefly, CNS tissues were digested with collagenase D (2.5
mg/ml, Roche Diagnostics) and DNaseI (1 mg/ml, Roche Diagnostics)
at 37.degree. C. for 45 minutes. Mononuclear cells were isolated by
passing the tissue through 70-.mu.m cell strainers (BD
Biosciences), followed by Percoll gradient (70%/37%)
centrifugation. Lymphocytes were collected from the 37%/70%
interface and washed. The following antibodies were used for
immunostaining: FITC-, PE- or PE-Cy5-conjugated CD3 (17A2), CD4
(H129.19), CD8 (53-6.7), CD62L (MEL14), CD44 (IM7), B220 (H1.2F3),
IgM (II/41), DX5 (CD49b) (all from BD Biosciences). For
intracellular cytokine staining, lymphocytes were stimulated in
vitro with phorbol 12-myristate 13-acetate (20 ng/ml;
Sigma-Aldrich) and ionomycin (1 .mu.g/ml, Sigma-Aldrich) in the
presence of GolgiStop.TM. (monensin) (5 ug/ml) for 5 hours before
staining. MOG.sub.38-49/IAb tetramer and control tetramer
(CLIP/IAb) were constructed and supplied by the NIH Tetramer Core
Facility. Background staining was assessed using nonreactive,
isotype-matched control mAbs. For 2- or 3-color immunofluorescence
analysis, single-cell suspensions (10.sup.6 cells) were stained at
4.degree. C. using predetermined optimal concentrations of mAb for
20 minutes. For tetramer staining, lymphocytes were stained for 3
hours at 37.degree. C.
Example 7: Antagonist IL-7R Antibodies Ameliorate Glucose
Intolerance in Diet-Induced Obesity (DIO) Animals
[0300] This example illustrates the effect of antagonist IL-7R
antibodies in a mouse model for type 2 diabetes.
[0301] To study the in vivo effect of antagonist IL-7R antibodies
on pre-established adipose inflammation in DIO mice, C57BL/6 male
mice (The Jackson Laboratory) were fed a high fat diet (HFD,
D12492, 60 Kcal % fat, Research Diets) beginning at six weeks old.
After ten weeks of high fat diet, the 16-week-old obese mice were
randomly assigned to groups for i.p. administration of antagonist
IL-7R antibody 28G9 (10 mg/kg), PBS, or nonreactive isotype-matched
control antibody (10 mg/kg). Four days after antibody treatment,
the mice were subject to a glucose tolerance test (i.p. 1 g/kg,
after 16 hr fasting) to assess glucose intolerance. Table 5 shows
the average body weight and glucose levels for each of the treated
groups (PBS-, isotype- or 28G9-treated mice). The animals in each
group had similar body weight.
TABLE-US-00014 TABLE 5 Glucose (mg/dL) Treatment Body weight (g)
non-fasting/fasting PBS 44.4 232/152.6 isotype 42.3 233/183.6 28G9
41.4 229/122.2
[0302] The results of the glucose tolerance test are depicted in
FIG. 14. Glucose intolerance induced by high fat diet was
ameliorated by antagonist IL-7R antibody treatment. In the glucose
tolerance test, DIO mice treated with 28G9 had significantly lower
blood glucose levels compared to mice treated with isotype or PBS
(FIG. 14). This result demonstrates that antagonist IL-7R
antibodies are efficacious in an animal model for type 2
diabetes.
Example 8: Antagonist IL-7R Antibodies Reduce Disease Severity in a
Mouse Model for Rheumatoid Arthritis
[0303] This example illustrates the effect of antagonist IL-7R
antibodies in a mouse model for rheumatoid arthritis (RA).
[0304] Collagen induced arthritis (CIA) is a widely used animal
model sharing many pathological and histological similarities with
RA. To study the in vivo effect of antagonist IL-7R antibodies on
CIA, 6-8 week old male B10.RIII mice (stock #000457, The Jackson
Laboratory) were immunized with 150 ug of Type II collagen (Elastin
Products) emulsified in Freund's complete adjuvant containing 4
mg/ml heat-killed Mycobacterium tuberculosis H37RA (Difco) on day 0
and day 15. Mice were injected i.p. with 1, 3 or 10 mg/kg of
antagonist IL-7R antibody 28G9 or nonreactive isotype-matched
control antibodies on day -1, day 1, day 8, day 15 and day 22.
[0305] Clinical signs of CIA were assessed daily with a 0 to 5
point scoring system: 0, normal; 1, hind or fore paw joint affected
or minimal diffuse erythema and swelling; 2, hind or fore paw
joints affected or mild diffuse erythema and swelling; 3, hind or
fore paw joints affected or moderate diffuse erythema and swelling;
4, Marked diffuse erythema and swelling; 5, diffuse erythema and
severe swelling entire paw, unable to flex digits. Treatment with
28G9 at 3 mg/kg significantly reduced the severity of CIA in
CII-immunized mice as compared to isotype control (FIG. 15).
Treatment with 28G9 at 1 mg/kg did not show significant reduction.
This result demonstrates that antagonist IL-7R antibodies are
effective in slowing disease progession in an animal model for
rheumatoid arthritis.
Example 9: Antagonist IL-7R Antibodies Reduce Disease Severity in a
Mouse Model for Established EAE
[0306] This example illustrates efficicacy of antagonist IL-7R
antibodies in a mouse model for established EAE.
[0307] EAE was induced in SJL/J mice by immunization with 200 .mu.g
of PLP(p139-151) dissolved in complete Freund's adjuvant containing
4 mg/ml of heat-killed Mycobacterium tuberculosis H37Ra (Difco
Laboratories). Mice were examined daily for bodyweight measurements
and clinical signs of EAE and scored as follows: 0, no paralysis;
1, loss of tail tone; 2, hindlimb weakness; 3, hindlimb paralysis;
4, hindlimb and forelimb paralysis; 5, moribund or dead.
[0308] Mice having a EAE clinical score of 2-3 were treated with
28G9 (10 mg/kg, i.p.), SB/14 (10 mg/kg, i.p.) or control IgG (10
mg/kg, i.p.) once a week for 2 weeks (on days 0, 7 and 14). 28G9 is
rat IgG1 antibody and SB/14 (BD Biosciences) is a rat IgG2a
antibody. Clinical scores were monitored daily until day 61.
[0309] By day 7, mice treated with 28G9 had clinical scores lower
than 2 (N=7). The mice treated with 28G9 maintained clinical scores
of about 2 until the end of the study (day 61). In comparison, the
control IgG-treated animals had clinical scores between 3 and 4
throughout the study. No reduction of disease severity was observed
with SB/14 treatment compared to the control.
[0310] A highly significant reduction of disease severity was
observed in 28G9 antagonist IL-7R antibody treated animals. These
results demonstrate that antagonist IL-7R antibodies are effective
in reducing disease severity in established autoimmune disease.
Example 10: Antagonist IL-7R Antibodies Reduce Blood Glucose Levels
in Animals with Newly Onset Diabetes
[0311] This example illustrates the efficacy of antagonist IL-7R
antibodies in reversing newly onset diabetes in a mouse model for
type 1 diabetes.
[0312] A panel of antagonist IL-7R antibodies were tested in a
mouse model for type 1 diabetes. 28G9 is a rat IgG1 monoclonal
antibody, 28G9-mIgG2a is an antibody having the 28G9 variable
regions with mouse IgG2a constant region, and agly-28G9 is an
aglycosylated antibody having the 28G9 variable regions with mouse
IgG2a N297A. For construction and expression of 28G9-mIgG2a, the VH
and Vk gene of rat monoclonal antibody 28G9 were amplified by PCR,
cloned into pARC mouse IgG2a and pARC mouse kappa mammalian
expression vectors, and cotransfected into 293F cells by
Lipofectamin.TM. (Invitrogen.TM.). After 5 days of
post-transfection, the culture media was harvested and the 28G9
mouse IgG2a was purified by using Mabselect.TM. (GE) resin. For
construction and expression of agly-28G9, the VH of rat 28G9 was
cloned into an engineered pARC mouse IgG2a vector in which Asn-297
of the CH2 domain was replaced by Ala (pARC mouse IgG2a-N297A). An
aglycosylated m28G9 (agly-28G9) was obtained by cotransfection of
293F cells with pARC mouse IgG2a-N297A and pARC-28G9 mouse kappa
vector.
[0313] Spontaneous new onset diabetic NOD mice (i.e., two
consecutive blood glucose concentrations over 250 mg/di) were
treated i.p. weekly with 28G9-mIgG2a (10 mg/kg, i.p.), 28G9 (10
mg/kg, i.p.), agly-28G9 (10 mg/kg, i.p.) or control IgG (10 mg/kg,
i.p.). Blood glucose levels were monitored daily for 140 days after
disease onset. In mice treated with 28G9-mIgG2a, 100% remission was
observed. In the 28G9-mIgG2a treated NOD mice, blood glucose levels
were maintained below 250 mg/dl with weekly 28G9-mIgG2a injections.
28G9 also showed some efficicacy in reducing blood glucose levels
compared to control IgG. Agly-28G9-treated and control IgG-treated
mice had blood glucose levels of greater than 250 mg/dl throughout
the study. These results demonstrate that 28G9-mIgG2a antagonist
IL-7R antibody is highly effective in reducing blood glucose levels
in mice with established type 1 diabetes.
[0314] In a separate study, spontaneous new onset diabetic NOD mice
were treated weekly, beginning at disease onset, with 28G9-mIgG2a
(10 mg/kg i.p.) for the number of doses indicated in Table 6. Blood
glucose levels were monitored daily.
TABLE-US-00015 TABLE 6 Age at disease Total # of Mouse onset (days)
doses Days diabetes-free 1 140 3 117 2 190 2 89 3 210 3 145
The results shown in Table 6 indicate that two doses of antagonist
IL-7R antibody were sufficient to maintain blood glucose levels
lower than 250 mg/dL for up to 89 days. Three doses of antagonist
IL-7R antibody were sufficient to maintain blood glucose levels
lower than 250 mg/dL for at least 117 days. In this study, blood
glucose levels maintained at lower than 250 mg/dL were observed for
up to five months post-antagonist IL-7R antibody treatment.
[0315] The results of the studies described above demonstrate that
antagonist IL-7R antibody 28G9-mIgG2a was highly effective in
reducing blood glucose levels in animals with newly onset diabetes.
Furthermore, mice treated with just two or three doses of
antagonist IL-7R antibodies maintained blood glucose levels lower
than 250 mg/dL for several months after antibody was
administered.
Example 11: Antagonist IL-7R Antibodies Reduce Disease Severity in
Mouse Models for Graft-Versus-Host Disease (GVHD)
[0316] This example illustrates the effect of antagonist IL-7R
antibodies in mouse models for acute and chronic graft-versus-host
disease (GVHD).
Acute GVHD
[0317] For the mouse model of acute GVHD, 10.times.10.sup.6 human
PBMC (freshly isolated) were injected into non-irradiated NOD.SCID
IL2R.gamma.-/- mice (The Jackson Laboratory, 8-12 weeks old). 14
days after injection, the mice are treated with 10 mg/kg antagonist
antagonist IL-7R fully human IgG1 antibody HAL403b (n=10) or
isotype control (n=10) once weekly. Clinical signs of GVHD and body
weight are monitored twice a week. Forty days post-treatment, 100%
of antagonist IL-7R antibody-treated animals remained alive, in
contrast to only 50% of isotype control-treated animals survived.
This result indicates that antagonist IL-7R antibodies are
effective in reducing mortality rate in an animal model for acute
GVHD.
Chronic GVHD
[0318] For the chronic GVHD mouse model, human cord blood cells
containing a small (1-5%) percentage of CD3+ T cells are
transplanted into newborn irradiated NOD.SCID IL2R.gamma.-/- mice.
Briefly, human CD34+ cord blood (AllCells, LLC, Emeryville, Calif.)
was depleted of CD3+ T cells using human CD3 selection beads
(Miltenyi Biotec GmBH, Germany, CAT #130-050-101) For the
transplantation, about 300,000 to 400,000 CD34+ cells containing
about 1-5% CD3+ T cells (in a volume of 50 .mu.l) are
intracardially injected per newborn irradiated NOD.SCID
IL2R.gamma.-/- mouse (The Jackson Laboratory). cGVHD develops 16-20
weeks post-transplantation.
[0319] Beginning at 24 weeks of age, mice with cGVHD are injected
with 10 mg/kg antagonist IL-7R fully human IgG1 antibody HAL403b
(n=4) or PBS (n=4) once weekly until sacrifice.
[0320] Mice are sacrificed at about 28-32 weeks old, after about 4
to 8 weeks of antagonist IL-7R antibody or PBS treatment. Mice
treated with antagonist IL-7R fully human IgG1 antibody had
significantly less hair loss than mice injected with PBS.
Histologic analysis showed kidneys of PBS-treated mice were
generally more severely affected than kidneys of antagonist IL-7R
antibody-treated mice. For example, kidneys of control
(PBS-treated) mice had markedly thickened capillary loops with
increased amounts of eosinophilic material. In contrast, kidneys of
mice treated with antagonist IL-7R antibody had mildly thickened
capillary loops with increased amount of eosinophilic material. In
addition, kidneys of mice treated with antagonist IL-7R antibody
had fewer dilated tubules compared to kidneys of mice treated with
isotype control, which showed many dilated tubules. Lung histology
revealed substantially reduced bronchial associated lymphoid tissue
(BALT) in lungs of mice treated with antagonist IL-7R antibody
compared to lungs of control mice, which had some BALT present.
Severe lymphoid atrophy was observed in spleen of mice treated with
antagonist IL-7 R antibody, compared to the mild to moderate change
in spleen of mice treated with PBS.
[0321] These results indicate that antagonist IL-7R antibodies are
effective in reducing disease severity in an animal model for
chronic GVHD.
Example 12: Antagonist IL-7R Antibodies Reduce Disease Severity in
a Mouse Model for Lupus
[0322] This example illustrates the effect of antagonist IL-7R
antibodies in a mouse model for lupus.
[0323] For the mouse model of lupus, MRL/MpJ-Fas.sup.lpr/J mice
(The Jackson Laboratory) were used. Commonly referred to as lpr
mutants, these mice are homozygous for the lymphoproliferation
spontaneous mutation (Fas.sup.lpr) and show systemic autoimmunity,
massive lymphadenopathy associated with proliferation of aberrant T
cells, arthritis, and immune complex glomerulonephrosis. As such,
the MRL/MpJ-Fas.sup.lpr/J mice are useful as a model for systemic
lupus erythematosus.
[0324] Beginning at the time of disease onset, mice were dosed i.p.
weekly with 1, 3, or 10 mg/kg 28G9-mIgG2a antagonist IL-7R antibody
(see Example 10), 1 mg/kg agly-28G9 antagonist IL-7R antibody, an
isotype control IgG (negative control) or cyclophosphamide
(positive control). For each treatment group, ten mice were used
(n=10). Disease severity was monitored by measuring proteinuria
levels, activity levels, and assessing the righting reflex. In
assessing the righting reflex, mice that failed to right themselves
within 30 seconds were sacrificed. Suvival rate is summarized in
Table 7 below.
TABLE-US-00016 TABLE 7 Survival rate at 8 weeks after Survival rate
at 12 weeks Treatment disease onset after disease onset 1 mg/kg
28G9-mIgG2a 60% 50% 3 mg/kg 28G9-mIgG2a 80% 70% 10 mg/kg
28G9-mIgG2a 90% 80% 1 mg/kg agly-28G9 100% 100% isotype control IgG
60% 60% (negative control) cyclophosphamide 80% 80% (positive
control)
[0325] As shown in Table 7, mice treated with 1 mg/kg agly-28G9, 3
mg/kg 28G9-mIgG2a or 10 mg/kg 28G9-mIgG2a had an increased survival
rate compared to mice treated with isotype control IgG. These
results indicate that antagonist IL-7R antibodies are effective in
reducing disease severity in an animal model for lupus.
Example 13: Epitope Mapping/Binding of Antagonist IL-7R
Antibodies
[0326] This example illustrates structure-guided mutagenesis to map
antibody binding epitopes.
[0327] Based on the crystal structure of the IL-7/IL-7R.alpha.
complex and the likely involvement of certain residues in IL-7
binding (McElroy et al., 2009, Structure, 17(1):54-65, twenty-three
IL-7R.alpha. surface-residue mutants (N29D, V58S, E59R, R66N, K775,
L805, I82S, K84S, K100S, T105A, N131S, Q136K, K138S, Y139F, K141S,
M144A, D1905, H191N, Y192A, Y192F, K194S, K194A and F1965) were
chosen for mutation to map the antibody binding epitopes. The
numbering for the mutants (i.e., N29D, V58S, E59R, etc.) follows
the convention of post-processed protein wherein the first 20 amino
acids are not counted.
[0328] A panel of IL-7R.alpha. single point mutants (his-tagged)
were prepared as follows. The twenty-three IL-7R.alpha. single
point mutants described above were generated from the previously
described wild-type DNA construct (McElroy et al., 2009, supra)
using standard DNA techniques. The mutant proteins were expressed
using transient transfection in HEK293T cells and secreted into the
cell media. The mutant proteins were purified by Ni.sup.2+ column
chromatography. Protein concentrations were measured by
spectrophotometry (NanoDrop.TM.).
[0329] Interaction analysis of IL-7R.alpha. was performed at
25.degree. C. using a surface-plasmon resonance-based ProteOn.TM.
XPR36 biosensor equipped with a GLM sensor chip (Bio-Rad, Hercules,
Calif., USA). HBST running buffer (10 mM Hepes pH7.4, 150 mM NaCl,
0.05% v/v Tween-20) was used throughout. Full-length IL-7R
antibodies (HAL403a or HAL403b) were amine-coupled onto separate
"vertical" channels of the chip via standard EDC/sulfo-NHS-mediated
chemistry to levels of about 2000-5000 RU. The panel of
IL-7R.alpha. mutants (including wild-type IL-7R.alpha.) was
screened in the "horizontal" direction at 100 nM using association
and dissociation phases of 3 and 10 mins respectively at 30 uL/min.
Surfaces were regenerated with 2/1 v/v Pierce immunopure elution
buffer (pH2.8)/4M NaCl. Most injections were duplicated to confirm
that the assay was reproducible.
[0330] Table 8 summarizes the impact of the single point mutations
in the IL-7R.alpha. mutants on antibody binding compared to
wild-type IL-7R.alpha..
TABLE-US-00017 TABLE 8 Impact on antibody (HAL403a or IL-7R.alpha.
mutant HAL403b) binding I82S Highly impaired K84S, K100S, T105A,
Impaired Y192A D190S, H191N, K194S Slightly impaired N29D, V58S,
E59R, R66N, None K77S, L80S, N131S, Q136K, K138S, Y139F, K141S,
M144A, Y192F, K194A, F196S, (wild-type)
[0331] The IL-7R.alpha. mutants displaying weakened antibody
binding compared to wild-type IL-7R.alpha. were identified as
having a point mutation at a residue involved in mAb binding. The
binding residues of IL-7R.alpha. to antibody HAL403a in descending
order of mutant effects were identified as follows: I82 (high
impact on binding), K84 (medium impact), K100 (medium impact), T105
(medium impact), Y192 (medium impact), D190 (small impact), H191
(small impact), and K194 (small impact). The binding residues of
IL-7R.alpha. to antibody HAL403b in descending order of mutant
effects were identified as follows: I82 (high impact on binding),
K84 (medium impact), K100 (medium impact), T105 (medium impact),
Y192 (medium impact), D190 (small impact), H191 (small impact), and
K194 (small impact).
[0332] Although the disclosed teachings have been described with
reference to various applications, methods, kits, and compositions,
it will be appreciated that various changes and modifications can
be made without departing from the teachings herein and the claimed
invention below. The foregoing examples are provided to better
illustrate the disclosed teachings and are not intended to limit
the scope of the teachings presented herein. While the present
teachings have been described in terms of these exemplary
embodiments, the skilled artisan will readily understand that
numerous variations and modifications of these exemplary
embodiments are possible without undue experimentation. All such
variations and modifications are within the scope of the current
teachings.
[0333] All references cited herein, including patents, patent
applications, papers, text books, and the like, and the references
cited therein, to the extent that they are not already, are hereby
incorporated by reference in their entirety. In the event that one
or more of the incorporated literature and similar materials
differs from or contradicts this application, including but not
limited to defined terms, term usage, described techniques, or the
like, this application controls.
[0334] The foregoing description and Examples detail certain
specific embodiments of the invention and describes the best mode
contemplated by the inventors. It will be appreciated, however,
that no matter how detailed the foregoing may appear in text, the
invention may be practiced in many ways and the invention should be
construed in accordance with the appended claims and any
equivalents thereof.
Sequence CWU 1
1
611111PRTHomo sapiens 1Asn Phe Met Leu Thr Gln Pro His Ser Val Ser
Gly Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser
Ser Gly Ser Ile Asp Ser Ser 20 25 30 Tyr Val Gln Trp Tyr Gln Gln
Arg Pro Gly Asn Ser Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asp
Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile
Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu
Val Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90
95 Ser His Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Cys 100
105 110 2117PRTHomo sapiens 2Gln Val Asn Leu Arg Glu Ser Gly Gly
Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Asp Asp Ser 20 25 30 Val Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Ser Leu Val
Gly Trp Asp Gly Ser Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Leu Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Gln Gly Asp Tyr Val Phe Asp Tyr Trp Gly Gln
Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 3112PRTHomo sapiens
3Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1
5 10 15 Thr Val Thr Ile Ser Cys Thr Gly Ser Ser Gly Arg Ile Ala Ser
Ser 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro
Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val
Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser
Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala
Asp Tyr Tyr Cys Gln Ser Tyr Ala Ser 85 90 95 Ser Ser Leu Trp Val
Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Ser 100 105 110 4118PRTHomo
sapiens 4Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asn Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Asp Ile Ser Gly Gly Gly Met Asp Val Trp Gly Gln Gly Thr 100 105 110
Thr Val Thr Val Ser Ser 115 5111PRTHomo sapiens 5Asn Phe Met Leu
Thr Gln Pro His Ser Val Ser Gly Ser Pro Gly Lys 1 5 10 15 Thr Val
Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Asp Ser Ser 20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Asn Ser Pro Thr Thr Val 35
40 45 Ile Tyr Glu Asp Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe
Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr
Ile Ser Gly 65 70 75 80 Leu Val Thr Glu Asp Glu Ala Asp Tyr Tyr Cys
Met Gln Tyr Asp Ser 85 90 95 Ser His Leu Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu Cys 100 105 110 6117PRTHomo sapiens 6Gln Val
Asn Leu Arg Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Ser 20
25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Leu 35 40 45 Ser Leu Val Gly Trp Asp Gly Phe Phe Thr Tyr Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr
Lys Asn Leu Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Asp Tyr Val
Phe Asn Asn Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser
115 7111PRTHomo sapiens 7Asn Phe Met Leu Thr Gln Pro His Ser Val
Ser Gly Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg
Ser Ser Gly Ser Ile Asp Ser Ser 20 25 30 Tyr Val Gln Trp Tyr Gln
Gln Arg Pro Gly Asn Ser Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp
Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80
Leu Val Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Phe 85
90 95 His His Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Cys
100 105 110 8117PRTHomo sapiens 8Gln Val Asn Leu Arg Glu Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Asp Asp Ser 20 25 30 Val Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Ser Leu
Val Gly Trp Asp Gly Phe Phe Thr Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Leu Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gln Gly Asp Tyr Met Gly Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 9111PRTHomo
sapiens 9Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Gly Ser Pro
Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser
Ile Asp Ser Ser 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly
Asn Ser Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asp Gln Arg Pro
Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser
Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Val Thr Glu
Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Phe 85 90 95 His His
Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Cys 100 105 110
10117PRTHomo sapiens 10Gln Val Asn Leu Arg Glu Ser Gly Gly Gly Leu
Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Asp Asp Ser 20 25 30 Val Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Ser Leu Val Gly Trp
Asp Gly Phe Phe Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Leu Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gln Gly Asp Tyr Met Gly Asn Asn Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser 115 11111PRTHomo sapiens 11Asn Phe
Met Leu Thr Gln Pro His Ser Val Ser Gly Ser Pro Gly Lys 1 5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Asp Ser Ser 20
25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Asn Ser Pro Thr Thr
Val 35 40 45 Ile Tyr Glu Asp Asp Gln Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser
Leu Thr Ile Ser Gly 65 70 75 80 Leu Val Thr Glu Asp Glu Ala Asp Tyr
Tyr Cys Met Gln Tyr Asp Phe 85 90 95 His His Leu Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu Cys 100 105 110 12117PRTHomo sapiens
12Gln Val Asn Leu Arg Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp
Ser 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Leu 35 40 45 Ser Leu Val Gly Trp Asp Gly Phe Phe Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Thr Lys Asn Leu Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Asp
Tyr Met Gly Asn Asn Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 1315PRTArtificial SequenceLinking peptide 13Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15
14351DNAHomo sapiens 14caggtcaact taagggagtc tgggggaggc ctggtcaagc
cgggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttgat gattctgtca
tgcactgggt ccgtcaagct 120ccggggaagg gtctggagtg gctctctctt
gttggttggg atggtttttt tacatactat 180gcagactcag tgaagggccg
attcaccatc tccagagaca acaccaagaa cttactgtat 240ctgcaaatga
acagcctgag agccgaggac acggctgtgt attactgtgc gagacaaggg
300gattacatgg ggaacaactg gggccaggga accctggtca ccgtctcctc a
35115333DNAHomo sapiens 15aattttatgc tgactcagcc ccactctgtg
tcggggtctc cgggaaagac ggtgaccatc 60tcctgcaccc gcagcagtgg cagcattgac
agttcctatg tgcagtggta ccagcagcgc 120ccgggcaatt cccccaccac
tgtgatctat gaggatgacc aaagaccctc tggggtccct 180gatcggttct
ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga
240ctggtgactg aggacgaggc tgactactac tgtcagtctt atgattttca
tcatctggtg 300ttcggcggag ggaccaagct gaccgtccta tgt
3331615PRTArtificial Sequencelinker peptide 16Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 17431PRTHomo
sapiens 17Gln Val Asn Leu Arg Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Asp Asp Ser 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Leu 35 40 45 Ser Leu Val Gly Trp Asp Gly Phe
Phe Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Thr Lys Asn Leu Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Gln Gly Asp Tyr Met Gly Asn Asn Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115
120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Asn 180 185 190 Phe Gly Thr Gln Thr Tyr
Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp
Lys Thr Val Ala Pro Pro Val Ala Gly Pro Ser Val 210 215 220 Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 225 230 235
240 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
245 250 255 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 260 265 270 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
Arg Val Val Ser 275 280 285 Val Leu Thr Val Val His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys 290 295 300 Cys Lys Val Ser Asn Lys Gly Leu
Pro Ser Ser Ile Glu Lys Thr Ile 305 310 315 320 Ser Lys Thr Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 325 330 335 Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 340 345 350 Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 355 360
365 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
370 375 380 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg 385 390 395 400 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu 405 410 415 His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 420 425 430 18216PRTHomo sapiens 18Asn Phe
Met Leu Thr Gln Pro His Ser Val Ser Gly Ser Pro Gly Lys 1 5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Asp Ser Ser 20
25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Asn Ser Pro Thr Thr
Val 35 40 45 Ile Tyr Glu Asp Asp Gln Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser
Leu Thr Ile Ser Gly 65 70 75 80 Leu Val Thr Glu Asp Glu Ala Asp Tyr
Tyr Cys Gln Ser Tyr Asp Phe 85 90 95 His His Leu Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu Thr Val 100 105 110 Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150
155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210
215 195PRTHomo sapiens 19Asp Ser Val Met His 1 5 205PRTHomo sapiens
20Asn Tyr Gly Met His 1 5 2117PRTHomo sapiens 21Leu Val Gly Trp Asp
Gly Ser Ala Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
2217PRTHomo sapiens 22Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr
Ala Asp Ser Val Lys 1 5 10 15 Gly 2317PRTHomo sapiens 23Leu Val Gly
Trp Asp Gly Phe Phe Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
248PRTHomo sapiens 24Gln Gly Asp Tyr Val Phe Asp Tyr 1 5 259PRTHomo
sapiens 25Asp Ile Ser Gly
Gly Gly Met Asp Val 1 5 268PRTHomo sapiens 26Gln Gly Asp Tyr Val
Phe Asn Asn 1 5 278PRTHomo sapiens 27Gln Gly Asp Tyr Met Gly Asp
Tyr 1 5 288PRTHomo sapiens 28Gln Gly Asp Tyr Met Gly Asn Asn 1 5
2913PRTHomo sapiens 29Thr Arg Ser Ser Gly Ser Ile Asp Ser Ser Tyr
Val Gln 1 5 10 3013PRTHomo sapiens 30Thr Gly Ser Ser Gly Arg Ile
Ala Ser Ser Tyr Val Gln 1 5 10 317PRTHomo sapiens 31Glu Asp Asp Gln
Arg Pro Ser 1 5 327PRTHomo sapiens 32Glu Asp Asn Gln Arg Pro Ser 1
5 339PRTHomo sapiens 33Gln Ser Tyr Asp Ser Ser His Leu Val 1 5
3410PRTHomo sapiens 34Gln Ser Tyr Ala Ser Ser Ser Leu Trp Val 1 5
10 359PRTHomo sapiens 35Met Gln Tyr Asp Ser Ser His Leu Val 1 5
369PRTHomo sapiens 36Gln Ser Tyr Asp Phe His His Leu Val 1 5
379PRTHomo sapiens 37Met Gln Tyr Asp Phe His His Leu Val 1 5
38351DNAHomo sapiens 38gaggtccagt tagtggagtc tgggggaggc ctggtcaagc
cgggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttgat gattctgtca
tgcactgggt ccgtcaagct 120ccggggaagg gtctggagtg ggtttctctt
gttggttggg atggtttttt tacatactat 180gcagactcag tgaagggccg
attcaccatc tccagagaca acgcgaagaa ctctctgtat 240ctgcaaatga
acagcctgag agccgaggac acggctgtgt attactgtgc gagacaaggg
300gattacatgg ggaacaactg gggccaggga accctggtca ccgtctcctc a
35139330DNAHomo sapiens 39aattttatgc tgactcagcc ccactctgtg
tcggaatctc cgggaaagac ggtgaccatc 60tcctgcaccc gcagcagtgg cagcattgac
agttcctatg tgcagtggta ccagcagcgc 120ccgggcagct cccccaccac
tgtgatctat gaggatgacc aaagaccctc tggggtccct 180gatcggttct
ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga
240ctgaaaactg aggacgaggc tgactactac tgtcagtctt atgattttca
tcatctggtg 300ttcggcggag ggaccaagct gaccgtccta 33040117PRTHomo
sapiens 40Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Asp Asp Ser 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Val Gly Trp Asp Gly Phe
Phe Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Gln Gly Asp Tyr Met Gly Asn Asn Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 41110PRTHomo sapiens 41Asn Phe Met Leu Thr
Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr
Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Asp Ser Ser 20 25 30 Tyr
Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val 35 40
45 Ile Tyr Glu Asp Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile
Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln
Ser Tyr Asp Phe 85 90 95 His His Leu Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 105 110 42432PRTHomo sapiens 42Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Ser 20 25 30
Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Leu Val Gly Trp Asp Gly Phe Phe Thr Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Asp Tyr Met Gly Asn
Asn Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165
170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Ala Pro Glu Leu
Leu Gly Gly Pro Ser 210 215 220 Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg 225 230 235 240 Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro 245 250 255 Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 260 265 270 Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 275 280 285
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 290
295 300 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr 305 310 315 320 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu 325 330 335 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys 340 345 350 Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser 355 360 365 Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 370 375 380 Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 385 390 395 400 Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 405 410
415 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
420 425 430 43215PRTHomo sapiens 43Asn Phe Met Leu Thr Gln Pro His
Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys
Thr Arg Ser Ser Gly Ser Ile Asp Ser Ser 20 25 30 Tyr Val Gln Trp
Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val 35 40 45 Ile Tyr
Glu Asp Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65
70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Asp Phe 85 90 95 His His Leu Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu Gln Pro 100 105 110 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro
Pro Ser Ser Glu Glu Leu 115 120 125 Gln Ala Asn Lys Ala Thr Leu Val
Cys Leu Ile Ser Asp Phe Tyr Pro 130 135 140 Gly Ala Val Thr Val Ala
Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 145 150 155 160 Gly Val Glu
Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165 170 175 Ala
Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 180 185
190 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr
195 200 205 Val Ala Pro Thr Glu Cys Ser 210 215 44110PRTHomo
sapiens 44Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro
Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser
Ile Asp Ser Ser 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly
Ser Ser Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asp Gln Arg Pro
Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser
Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu
Asp Glu Ala Asp Tyr Tyr Cys Met Gln Tyr Asp Phe 85 90 95 His His
Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110
45431PRTHomo sapiens 45Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Asp Asp Ser 20 25 30 Val Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Val Gly Trp
Asp Gly Phe Phe Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gln Gly Asp Tyr Met Gly Asn Asn Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn 180 185 190 Phe Gly Thr
Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr
Lys Val Asp Lys Thr Val Ala Pro Pro Val Ala Gly Pro Ser Val 210 215
220 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
225 230 235 240 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu 245 250 255 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys 260 265 270 Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr Phe Arg Val Val Ser 275 280 285 Val Leu Thr Val Val His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys 290 295 300 Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 305 310 315 320 Ser Lys
Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 325 330 335
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 340
345 350 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn 355 360 365 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met
Leu Asp Ser 370 375 380 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg 385 390 395 400 Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu 405 410 415 His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 420 425 430 467PRTHomo sapiens
46Gly Phe Thr Phe Asp Asp Ser 1 5 4710PRTHomo sapiens 47Gly Phe Thr
Phe Asp Asp Ser Val Met His 1 5 10 486PRTHomo sapiens 48Gly Trp Asp
Gly Phe Phe 1 5 498PRTHomo sapiens 49Gln Gly Asp Tyr Met Gly Asn
Asn 1 5 505PRTHomo sapiensMISC_FEATURE(1)..(1)Xaa can be Asp or
AsnMISC_FEATURE(2)..(2)Xaa can be Ser or Tyr 50Xaa Xaa Val Met His
1 5 5117PRTHomo sapiensMISC_FEATURE(1)..(1)Xaa can be Leu or
AlaMISC_FEATURE(2)..(2)Xaa can be Val or IleMISC_FEATURE(3)..(3)Xaa
can be Gly or SerMISC_FEATURE(4)..(4)Xaa can be Trp or
GlyMISC_FEATURE(5)..(5)Xaa can be Asp or SerMISC_FEATURE(7)..(7)Xaa
can be Phe, Gly or SerMISC_FEATURE(8)..(8)Xaa can be Phe, Ala or
Ser 51Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Thr Tyr Tyr Ala Asp Ser Val
Lys 1 5 10 15 Gly 528PRTHomo sapiensMISC_FEATURE(1)..(1)Xaa can be
Gln or AspMISC_FEATURE(2)..(2)Xaa can be Gly or
IleMISC_FEATURE(3)..(3)Xaa can be Asp or SerMISC_FEATURE(4)..(4)Xaa
can be Tyr or GlyMISC_FEATURE(5)..(5)Xaa can be Met, Val or
GlyMISC_FEATURE(6)..(6)Xaa can be Gly or PheMISC_FEATURE(7)..(7)Xaa
can be Asn, Asp or MetMISC_FEATURE(8)..(8)Xaa can be Asn, Tyr or
Asp 52Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 5313PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa can be Arg or
GlyMISC_FEATURE(6)..(6)Xaa can be Ser or ArgMISC_FEATURE(8)..(8)Xaa
can be Asp or Ala 53Thr Xaa Ser Ser Gly Xaa Ile Xaa Ser Ser Tyr Val
Gln 1 5 10 547PRTHomo sapiensMISC_FEATURE(3)..(3)Xaa can be Asp or
Asn 54Glu Asp Xaa Gln Arg Pro Ser 1 5 559PRTHomo
sapiensMISC_FEATURE(1)..(1)Xaa can be Gln or
MetMISC_FEATURE(2)..(2)Xaa can be Ser or GlnMISC_FEATURE(4)..(4)Xaa
can be Asp or AlaMISC_FEATURE(5)..(5)Xaa can be Phe or
SerMISC_FEATURE(6)..(6)Xaa can be His or SerMISC_FEATURE(7)..(7)Xaa
can be His or SerMISC_FEATURE(9)..(9)Xaa can be Val or Trp 55Xaa
Xaa Tyr Xaa Xaa Xaa Xaa Leu Xaa 1 5 568PRTHomo sapiens 56Phe Thr
Phe Asp Asp Ser Val Met 1 5 576PRTHomo sapiens 57Gly Trp Asp Gly
Phe Phe 1 5 586PRTHomo sapiensMISC_FEATURE(3)..(3)Xaa can be any
amino acidMISC_FEATURE(4)..(4)Xaa can be any amino
acidMISC_FEATURE(5)..(5)Xaa can be any amino
acidMISC_FEATURE(6)..(6)Xaa can be any amino acid 58Ala Arg Xaa Xaa
Xaa Xaa 1 5 598PRTHomo sapiens 59Ser Gly Ser Ile Asp Ser Ser Tyr 1
5 609PRTHomo sapiens 60Glu Asp Asp Gln Arg Pro Ser Gly Val 1 5
614PRTHomo sapiens 61Phe His His Leu 1
* * * * *