U.S. patent application number 13/933453 was filed with the patent office on 2014-03-20 for humanized antibodies against monocyte chemotactic proteins.
This patent application is currently assigned to Biogen Idec MA Inc.. The applicant listed for this patent is Biogen Idec MA Inc.. Invention is credited to Antonin R. DE FOUGEROLLES, Ellen GARBER, Victor E. KOTELIANSKI, Carl REID, Jose W. SALDANHA, Herman VAN VLIJMEN.
Application Number | 20140079697 13/933453 |
Document ID | / |
Family ID | 32469404 |
Filed Date | 2014-03-20 |
United States Patent
Application |
20140079697 |
Kind Code |
A1 |
DE FOUGEROLLES; Antonin R. ;
et al. |
March 20, 2014 |
HUMANIZED ANTIBODIES AGAINST MONOCYTE CHEMOTACTIC PROTEINS
Abstract
The invention provides humanized antibodies that bind to a
plurality of b-chemokines, particularly monocyte chemotactic
proteins MCP-1, MCP-2 and MCP-3. The invention also provides
therapeutic reagents and methods of treating disorders associated
with detrimental MCP activity.
Inventors: |
DE FOUGEROLLES; Antonin R.;
(Brookline, MA) ; KOTELIANSKI; Victor E.; (Boston,
MA) ; GARBER; Ellen; (Cambridge, MA) ; REID;
Carl; (Mattapan, MA) ; SALDANHA; Jose W.;
(Enfield, GB) ; VAN VLIJMEN; Herman; (Mechelen,
BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Biogen Idec MA Inc. |
Weston |
MA |
US |
|
|
Assignee: |
Biogen Idec MA Inc.
Weston
MA
|
Family ID: |
32469404 |
Appl. No.: |
13/933453 |
Filed: |
July 2, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13027190 |
Feb 14, 2011 |
8519112 |
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13933453 |
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10536067 |
Aug 8, 2006 |
7888479 |
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PCT/US03/37834 |
Nov 25, 2003 |
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13027190 |
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60430007 |
Nov 27, 2002 |
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Current U.S.
Class: |
424/134.1 ;
424/133.1; 424/178.1 |
Current CPC
Class: |
C07K 2317/56 20130101;
C07K 2317/565 20130101; C07K 2317/24 20130101; C07K 16/24 20130101;
A61K 2039/505 20130101; C07K 2317/92 20130101 |
Class at
Publication: |
424/134.1 ;
424/133.1; 424/178.1 |
International
Class: |
C07K 16/24 20060101
C07K016/24 |
Claims
1-69. (canceled)
70. A method of treating a subject suffering from a disorder
selected from glomerulonephritis, scleroderma, cirrhosis, multiple
sclerosis, lupus nephritis, atherosclerosis, inflammatory bowel
disease or rheumatoid arthritis, comprising administering to the
subject a humanized immunoglobulin or antigen-binding fragment
thereof comprising a) heavy chain complementarity determining
regions as set forth in SEQ ID NO:29, SEQ ID NO:30, and SEQ ID
NO:31, and b) light chain complementarity determining regions as
set forth in SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34, wherein
the remainder of the heavy and light chains are from a human
immunoglobulin, wherein the immunoglobulin or antigen-binding
fragment thereof binds to MCP-1, MCP-2, or MCP-3, and wherein the
humanized immunoglobulin or antigen-binding fragment thereof is
administered in an amount sufficient to reduce the activity of at
least one of MCP-1, MCP-2, or MCP-3.
71. The method of claim 70, wherein the heavy chain further
comprises at least one variable region framework residue selected
from L27, I29, and T73 (Kabat numbering convention) from the
monoclonal antibody 11K2 heavy chain set forth as SEQ ID NO:27.
72. The method of claim 70, wherein the heavy chain further
comprises variable region framework residues L27, I29, and T73
(Kabat numbering convention) from the monoclonal antibody 11K2
heavy chain set forth as SEQ ID NO:27.
73. The method of claim 70, wherein the heavy chain further
comprises at least one variable region framework residue selected
from N28, K30, I48, and A67 (Kabat numbering convention) from the
monoclonal antibody 11K2 heavy chain as set forth in SEQ ID
NO:27.
74. The method of claim 70, wherein the heavy chain further
comprises variable region framework residues N28, K30, I48, and A67
(Kabat numbering convention) from the monoclonal antibody 11K2
heavy chain as set forth in SEQ ID NO:27.
75. The method of claim 70, wherein the light chain further
comprises at least one variable region framework residue selected
from S49 and Y71 (Kabat numbering convention) from the monoclonal
antibody 11K2 light chain set forth as SEQ ID NO: 28.
76. The method of claim 70, wherein the light chain further
comprises variable region framework residues S49 and Y71 (Kabat
numbering convention) from the monoclonal antibody 11K2 light chain
as set forth in SEQ ID NO:28.
77. The method of claim 76, wherein the light chain further
comprises variable region framework residue K69 (Kabat numbering
convention) from the monoclonal antibody 11K2 light chain as set
forth in SEQ ID NO:28.
78. The method of claim 70, wherein the heavy chain further
comprises at least one variable region framework residue from the
monoclonal antibody 11K2 heavy chain set forth as SEQ ID NO: 27,
wherein the residue is selected from the group consisting of L27,
N28, I29, K30, I48, A67, and T73 (Kabat numbering).
79. The method of claim 78, wherein the heavy chain comprises
variable region framework residues L27, N28, I29, K30, I48, A67,
and T73 (Kabat numbering convention) from the monoclonal antibody
11K2 heavy chain as set forth in SEQ ID NO:27.
80. The method of claim 76, wherein the heavy chain comprises at
least one variable region framework residues L27, I29, and T73
(Kabat numbering convention) from the monoclonal antibody 11K2
heavy chain as set forth in SEQ ID NO:27.
81. The method of claim 79, wherein the light chain comprises at
least one variable region framework residue selected from S49, K69,
and Y71 (Kabat numbering convention) from the monoclonal antibody
11K2 light chain set forth as SEQ ID NO: 28.
82. The method of claim 70, wherein the heavy chain comprises at
least one variable region framework residue selected from L27, N28,
I29, K30, I48, A67 and T73 (Kabat numbering convention) from the
monoclonal antibody 11K2 heavy chain set forth as SEQ ID NO: 27,
and wherein the light chain comprises at least one variable region
framework residue selected from S49, K69, and Y71 (Kabat numbering
convention) from the monoclonal antibody 11K2 light chain set forth
as SEQ ID NO: 28, wherein the remainder of the heavy and light
chains are from a human immunoglobulin.
83. The method of claim 82, wherein the heavy chain comprises
variable region framework residues L27, I29, and T73, and the light
chain comprises variable region framework residues S49 and Y71.
84. The method of claim 83, wherein the light chain further
comprises variable region framework residue K69.
85. The method of claim 82, wherein the heavy chain comprises
variable region framework residues L27, N28, I29, K30, I48, A67 and
T73, and the light chain comprises variable region framework
residues S49 and Y71.
86. The method of claim 70, wherein the humanized immunoglobulin or
antigen-binding fragment thereof is modified by reducing or
eliminating at least one potential glycosylation site.
87. The method of claim 70, wherein the immunoglobulin or
antigen-binding fragment thereof specifically binds to MCP-1 with a
binding affinity of at least 10.sup.-9 M.
88. The method of claim 70, wherein the immunoglobulin or
antigen-binding fragment thereof specifically binds to MCP-1 with a
binding affinity of at least 10.sup.-10 M.
89. The method of claim 70, wherein the immunoglobulin or
antigen-binding fragment thereof specifically binds to MCP-1 with a
binding affinity of at least 10.sup.-10 M.
90. The method of claim 70, wherein the immunoglobulin or
antigen-binding fragment thereof specifically binds to MCP-1 with a
binding affinity of 1 pM or less or 0.4 pM to about 0.7 pM.
91. The method of claim 87, wherein the immunoglobulin or
antigen-binding fragment thereof further binds to MCP-2 with a
binding affinity of at least 10.sup.-7 M.
92. The method of claim 87, wherein the immunoglobulin or
antigen-binding fragment of thereof further binds to MCP-2 with a
binding affinity of at least 10.sup.-8 M.
93. The method of claim 87, wherein the immunoglobulin or
antigen-binding fragment thereof further binds to MCP-2 with a
binding affinity of at least 10.sup.-9 M.
94. The method of claim 70, wherein the immunoglobulin or
antigen-binding fragment thereof binds to MCP-2.
95. The method of claim 70, wherein the immunoglobulin or
antigen-binding fragment thereof specifically binds to MCP-2 with a
binding affinity of at least 10.sup.-7 M.
96. The method of claim 70, wherein the immunoglobulin or
antigen-binding fragment thereof specifically binds to MCP-2 with a
binding affinity of at least 10.sup.-8 M.
97. The method of claim 70, wherein the immunoglobulin or
antigen-binding fragment thereof specifically binds to MCP-2 with a
binding affinity of at least 10.sup.-9 M.
98. The method of claim 70, wherein the immunoglobulin or
antigen-binding fragment thereof specifically binds to MCP-1 and
MCP-2.
99. The method of claim 70, wherein the immunoglobulin or
antigen-binding fragment thereof binds to an epitope within MCP-1,
MCP-2, and MCP-3.
100. The method of claim 70, wherein the immunoglobulin comprises
the same heavy and light chain polypeptide sequences as an antibody
produced by clone 3F2 (ATCC patent deposit designation
PTA-5308).
101. The method of claim 70, wherein the immunoglobulin heavy chain
isotype is gamma 1.
102. The method of claim 70, wherein the immunoglobulin is modified
by reducing or eliminating at least one potential glycosylation
site.
103. The method of claim 70, wherein the immunoglobulin or
antigen-binding fragment thereof is modified by conjugation to a
carrier selected from polyethylene glycol and albumen.
104. The method of claim 70, wherein the constant region of the
immunoglobulin or antigen-binding fragment thereof is modified to
reduce at least one constant region-mediated biological effector
function relative to an unmodified antibody.
105. The method of claim 70, wherein the antigen-binding fragment
is selected from an Fab, an Fab', an F(ab).sub.2, and an Fv.
106. The method of claim 105, wherein the antigen-binding fragment
is an Fab.
107. The method of claim 70, wherein the immunoglobulin or
antigen-binding fragment thereof inhibits MCP-induced chemotaxis in
the subject.
108. The method of claim 107, wherein the immunoglobulin or
antigen-binding fragment thereof inhibits MCP-1-induced chemotaxis,
MCP-2-induced chemotaxis, or both MCP-1-induced and MCP-2-induced
chemotaxis in the subject.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application under 37
C.F.R. .sctn.1.52(b) of application Ser. No. 13/027,190, filed Feb.
14, 2011, now U.S. Pat. No. 8,519,112, which is a divisional of
application Ser. No. 10/536,067, filed Aug. 8, 2006, now U.S. Pat.
No. 7,888,479, which is a national phase application of
International Application No. PCT/US03/37834, filed Nov. 25, 2003,
and claims the benefit of U.S. Provisional Application No.
60/430,007, filed Nov. 27, 2002, all of which are incorporated
herein by reference. This application is related to PCT application
no. PCT/US02/38229, filed Nov. 27, 2002. This application is also
related to U.S. Provisional Application No. 60/343,391, filed Nov.
30, 2001. This application is also related to U.S. Provisional
Application No. 60/383,277, filed May 24, 2002. This application is
also related to U.S. Provisional Application No. 60/400,469, filed
Aug. 1, 2002. The entire contents of each of these patents and
patent applications are hereby incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] "Chemokines," which take their name from chemotactic
cytokines, are small secreted polypeptides that regulate movement
of immune cells into tissues (Baggiolini et al. (1994) Adv.
Immunol. 55:97-179; Oppenheim et al. (1991) Ann Rev. Immunol.
9:617-648). Chemokines are assigned to three different families
based on the number and position of conserved cysteine residues
(Van Coillie et al. (1999) Cytokine & Growth Factor Rev.
10:61-86). The a and b chemokines each contain four conserved
cysteine residues. The first two cysteines of the a chemokines are
separated by a single amino acid, thus containing a CXC amino acid
motif. The first two conserved cysteines of the b chemokines are
adjacent. Thus, the b chemokines are also known as C-C chemokines.
By contrast, lymphotactin is the sole member of the third family of
chemokines, and contains only the second and fourth conserved
cysteine residues. Interestingly, in humans, a chemokines are all
encoded by genes on chromosome 4, b chemokines are all encoded by
genes on chromosome 17, and lymphotaxin is encoded by genes on
chromosome 1.
[0003] The b-chemokines form a gradient that serves as a
chemoattractant and potential proliferation signal for immune and
other cells such as monocytes, macrophages, basophils, eosinophils,
T lymphocytes and fibroblasts. MCP-1, MCP-2 and MCP-3 share
sequence homology with one another at the amino acid level. Through
interaction with specific receptors, termed C-C chemokine receptors
(CCR) which are G-protein coupled, seven transmembrane receptors
(Rossi and Zlotnik (2000) Ann. Rev. Immunol. 18:217-242), the
b-chemokines regulate the expression of adhesion molecules on
endothelial cells and thereby indirectly affect diapedesis and
extravasation of immune cells from the circulation into tissues.
There are ten different CCRs (CCR1 through CCR10). CCR2 acts as a
receptor for MCP-1, MCP-2, MCP-3, and MCP-4 (Rossi and Zlotnik
(2000) Ann. Rev. Immunol. 18:217-242). However, all human MCPs have
been shown to interact with more than one receptor (Van Coillie et
al. (1999) Cytokine & Growth Factor Rev. 10:61-86).
[0004] Human MCP-1, MCP-2 and MCP-3 all have chemotactic activity
for a variety of cell types, including T lymphocytes and monocytes
(Van Coillie et al. (1999) Cytokine & Growth Factor Rev.
10:61-86). Other shared functions of MCP-1, MCP-2, and MCP-3
include induction of N-acetyl b-D-glucosaminidase release,
gelatinase B release, and granzyme A release which are believed to
help the cells digest the extracellular matrix components necessary
to enable them to migrate into tissues (Van Coillie et al. (1999)
Cytokine & Growth Factor Rev. 10:61-86). In addition, MCP-1 and
MCP-3 share various functions, such as induction of arachidonic
acid release and stimulation of a respiratory burst (Van Coillie et
al. (1999) Cytokine & Growth Factor Rev. 10:61-86).
[0005] MCP-1-specific antibodies have previously been described in
the literature (WO 01/89582, WO 01/89565, Luo et al. (1994) J
Immunol 153:3708-16; Traynor, et al (2002) J Immunol 168:4659-66).
Certain MCP-1 antibodies have been described as binding MCP-1 and
MCP-3, specifically the MRHAS domain of MCP-1 and MCP-3 (WO
95/09232). In addition, a human anti-MCP-1 antibody has also been
described (WO 02/02640). There is a need in the art to identify
antibodies which can be used to manipulate b-chemokines in general,
and to specifically modulate the activity of multiple chemokines,
e.g., MCP-1 and MCP-2 or MCP-3.
SUMMARY OF THE INVENTION
[0006] The present invention features new immunological reagents,
in particular, therapeutic antibody reagents for the prevention and
treatment of disorders associated with detrimental MCP activity.
The invention is based, at least in part, on the identification and
characterization of two monoclonal pan-antibodies that specifically
bind to MCPs and are effective at binding MCPs, including MCP-1 and
MCP-2, with high affinity and at inhibiting MCP-induced chemotaxis.
Structural and functional analysis of these antibodies leads to the
design of various humanized antibodies for prophylactic and/or
therapeutic use. In particular, the invention features humanization
of the variable regions of these antibodies and, accordingly
provides for humanized immunoglobulin or antibody chains, intact
humanized immunoglobulins or antibodies, and functional
immunoglobulin or antibody fragments, in particular, antigen
binding fragments, of the featured antibodies.
[0007] Polypeptides comprising the complementarity determining
regions of the featured monoclonal antibodies are also disclosed,
as are polynucleotide reagents, vectors and host suitable for
encoding said polypeptides.
[0008] Methods of treatment of disorders associated with
detrimental MCP activity are disclosed, as are pharmaceutical
compositions and kits for use in such applications.
[0009] Also featured are methods of identifying residues within the
featured monoclonal antibodies which are important for proper
immunologic function and for identifying residues which are
amenable to substitution in the design of humanized antibodies
having improved binding affinities and/or reduced immunogenicity,
when used as therapeutic reagents.
[0010] In one embodiment, the invention features a humanized
immunoglobulin heavy chain or antigen-binding fragment thereof
comprising variable region complementary determining regions (CDRs)
from the 11K2 immunoglobulin heavy chain variable region sequence
set forth as SEQ ID NO: 27, and variable framework regions from a
human acceptor immunoglobulin heavy chain sequence, provided that
at least one framework residue is substituted with the
corresponding amino acid residue from the mouse 11K2 heavy chain
variable region sequence, wherein the framework residue is selected
from the group consisting of:
[0011] a residue that non-covalently binds antigen directly;
[0012] a residue adjacent to a CDR;
[0013] a CDR-interacting residue; and
[0014] a residue participating in the VL-VH interface.
[0015] In another embodiment, the invention features a humanized
immunoglobulin light chain or antigen-binding fragment thereof
comprising variable region complementary determining regions (CDRs)
from the 11K2 immunoglobulin light chain variable region sequence
set forth as SEQ ID NO: 28 and variable framework regions from a
human acceptor immunoglobulin light chain, provided that at least
one framework residue is substituted with the corresponding amino
acid residue from the mouse 11K2 light chain variable region
sequence, wherein the framework residue is selected from the group
consisting of:
[0016] a residue that non-covalently binds antigen directly;
[0017] a residue adjacent to a CDR;
[0018] a CDR-interacting residue; and
[0019] a residue participating in the VL-VH interface.
[0020] In some embodiments, a CDR-interacting residue is identified
by modeling the 11K2 heavy chain based on the solved structure of a
murine immunoglobulin heavy chain that shares at least 70%, 80%, or
90% sequence identity with the 11K2 heavy chain. In other
embodiments, a CDR-interacting residue is identified by modeling
the 11K2 light chain based on the solved structure of a murine
immunoglobulin light chain that shares at least 70%, 80%, or 90%
sequence identity with the 11K2 light chain.
[0021] Another embodiment of the invention features a humanized
immunoglobulin heavy chain or antigen-binding fragment thereof
comprising variable region complementary determining regions (CDRs)
from the 11K2 immunoglobulin heavy chain variable region sequence
set forth as SEQ ID NO: 27, and variable framework regions from a
human acceptor immunoglobulin heavy chain sequence, provided that
at least one framework residue is substituted with the
corresponding amino acid residue from the mouse 11K2 heavy chain
variable region sequence, wherein the framework residue is a
residue capable of affecting heavy chain variable region
conformation or function as identified by analysis of a
three-dimensional model of the variable region.
[0022] In yet other embodiments, the invention describes a
humanized immunoglobulin light chain or antigen-binding fragment
thereof comprising variable region complementary determining
regions (CDRs) from the 11K2 immunoglobulin light chain variable
region sequence set forth as SEQ ID NO: 28, and variable framework
regions from a human acceptor immunoglobulin light chain, provided
that at least one framework residue is substituted with the
corresponding amino acid residue from the mouse 11K2 light chain
variable region sequence, wherein the framework residue is a
residue capable of affecting light chain variable region
conformation or function as identified by analysis of a
three-dimensional model of the variable region.
[0023] In some embodiments, the framework residue is selected from
the group consisting of a residue capable of interacting with
antigen, a residue proximal to the antigen-binding site, a residue
capable of interacting with a CDR, a residue adjacent to a CDR, a
residue within 6 .DELTA. of a CDR residue, a canonical residue, a
vernier zone residue, an interchain packing residue, and a rare
residue. In other embodiments, the framework residue is selected
from the group consisting of a residue capable of interacting with
antigen, a residue proximal to the antigen-binding site, a residue
capable of interacting with a CDR, a residue adjacent to a CDR, a
residue within 6 .DELTA. of a CDR residue, a canonical residue, a
vernier zone residue, an interchain packing residue, and an unusual
residue.
[0024] In still other embodiments, the framework residue is
identified by modeling the 11K2 heavy chain based on the solved
structure of a murine immunoglobulin heavy chain that shares at
least 70%, 80%, or 90% sequence identity with the 11K2 heavy chain.
In still other embodiments, the framework residue is identified by
modeling the 11K2 light chain based on the solved structure of a
murine immunoglobulin light chain that shares at least 70%, 80%, or
90% sequence identity with the 11K2 light chain.
[0025] In one embodiment, the invention features a humanized
antibody or antigen-binding fragment thereof comprising the
complementary determining regions (CDR1, CDR2 and CDR3) of the 11K2
variable heavy chain sequence set forth as SEQ ID NO: 27. The
invention also features a humanized antibody comprising the
complementary determining regions (CDR1, CDR2 and CDR3) of the 11K2
variable light chain sequence set forth as SEQ ID NO: 28. In still
another embodiment, the invention features a humanized antibody, or
antigen-binding fragment thereof, which specifically binds to MCP-1
comprising variable region comprising complementary determining
regions (CDRs) corresponding to CDRs from the mouse 11K2 antibody.
In some embodiments, the fragment of the invention is a Fab
fragment.
[0026] In yet another embodiment, the invention features a chimeric
immunoglobulin comprising a variable region sequence substantially
as set forth in SEQ ID NO: 27 or SEQ ID NO: 28, and constant region
sequences from a human immunoglobulin.
[0027] In one embodiment, the invention features a humanized
antibody comprising the complementary determining regions (CDR1,
CDR2 and CDR3) of the 11K2 variable heavy chain sequence set forth
as SEQ ID NO: 27. In another embodiment, the invention features a
humanized antibody comprising the complementary determining regions
(CDR1, CDR2 and CDR3) of the 11K2 variable light chain sequence set
forth as SEQ ID NO: 28.
[0028] In another embodiment, the invention features a humanized
antibody, or antigen-binding fragment thereof, which specifically
binds to MCP-1 comprising variable region comprising complementary
determining regions (CDRs) corresponding to CDRs from the mouse
11K2 antibody. The invention also describes a chimeric
immunoglobulin comprising a variable region sequence substantially
as set forth in SEQ ID NO: 27 or SEQ ID NO: 28, and constant region
sequences from a human immunoglobulin.
[0029] In yet another embodiment, the invention features a method
for identifying residues amenable to substitution in a humanized
11K2 immunoglobulin variable framework region, comprising modeling
the three-dimensional structure of the 11K2 variable region based
on a solved immunoglobulin structure and analyzing said model for
residues capable of affecting 11K2 immunoglobulin variable region
conformation or function, such that residues amenable to
substitution are identified. The invention also features use of the
variable region sequence set forth as SEQ ID NO: 27 or SEQ ID NO:
28, or any portion thereof, in producing a three-dimensional image
of a 11K2 immunoglobulin, 11K2 immunoglobulin chain, or domain
thereof.
[0030] In still another embodiment, the invention features a method
of treating a disorder associated with detrimental MCP activity in
a subject by administering a nucleic acid molecule that encodes an
immunoglobulin heavy chain comprising the amino acid sequence of
SEQ ID NO: 47 or the amino acid sequence of SEQ ID NO: 48 and a
nucleic acid molecule that encodes an immunoglobulin light chain
comprising the amino acid sequence of SEQ ID NO: 49 or the amino
acid sequence of SEQ ID NO: 50, under conditions such that said
immunoglobulin chains are expressed, thereby treating the
subject.
[0031] In one embodiment, the invention features a humanized
immunoglobulin comprising the heavy chain set forth in SEQ ID NO:
47. In another embodiment, the invention features a humanized
immunoglobulin comprising the heavy chain set forth in SEQ ID NO:
48. In still another embodiment, the invention features a humanized
immunoglobulin comprising the light chain set forth in SEQ ID NO:
49. In yet another embodiment, the invention features a humanized
immunoglobulin comprising the light chain set forth in SEQ ID NO:
50.
[0032] In one embodiment, the invention features a heavy chain
comprising a complementarity determining region (CDR) and at least
one variable region framework residue from the monoclonal antibody
11K2 heavy chain set forth as SEQ ID NO: 27, wherein the residue is
selected from the group consisting of L27, I29, and T73 (Kabat
numbering convention), and wherein the remainder of the heavy chain
is from a human immunoglobulin. In one embodiment, the heavy chain
comprises variable region framework residues L27, I29, and T73. In
another embodiment, the heavy chain further comprises at least one
variable region framework residue selected from the group
consisting of N28, K30, I48, and A67 (Kabat numbering convention).
In still another embodiment of the invention, the heavy chain
comprises variable region framework residues N28, K30, I48, and
A67.
[0033] In another embodiment, the invention features a light chain
comprising a complementarity determining region (CDR) and at least
one variable region framework residue from the monoclonal antibody
11K2 light chain set forth as SEQ ID NO: 28, wherein the residue is
selected from the group consisting of S49 and Y71 (Kabat numbering
convention), and wherein the remainder of the light chain is from a
human immunoglobulin. In one embodiment, the light chain comprises
variable region framework residues S49 and Y71. In another
embodiment, the light chain further comprises variable region
framework residue K69 (Kabat numbering convention).
[0034] In another embodiment, the invention features a humanized
immunoglobulin or antigen-binding fragment thereof comprising heavy
chain complementary determining regions as set forth in SEQ ID NO:
29, SEQ ID NO: 30, and SEQ ID NO: 31, and at least one variable
region framework residue from the monoclonal antibody 11K2 heavy
chain set forth as SEQ ID NO: 27, wherein the residue is selected
from the group consisting of L27, N28, I29, K30, I48, A67, and T73
(Kabat numbering). In one embodiment, the heavy chain comprises
variable region framework residues L27, N28, I29, K30, I48, A67,
and T73. In another embodiment, the heavy chain comprises variable
region framework residues L27, I29, and T73.
[0035] In yet another embodiment, the invention features a
humanized immunoglobulin or antigen-binding fragment thereof
comprising light chain complementary determining regions as set
forth in SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, and at
least one variable region framework residues from the monoclonal
antibody 11K2 light chain set forth as SEQ ID NO: 28, wherein the
residue is selected from the group consisting of 849, K69, and Y71
(Kabat numbering). In one embodiment, the light chain comprises
variable region framework residues 849, K69, and Y71. In another
embodiment, the light chain further comprises variable region
framework residues S49 and Y71.
[0036] In still another embodiment, the invention describes a
humanized immunoglobulin or antigen-binding fragment comprising the
light chain of the invention and the heavy chain of the
invention.
[0037] In one embodiment, the invention features a humanized
immunoglobulin or antigen-binding fragment comprising
[0038] a) a heavy chain comprising a complementarity determining
region (CDR) and at least one variable region framework residue
from the monoclonal antibody 11K2 heavy chain set forth as SEQ ID
NO: 27, wherein the residue is selected from the group consisting
of L27, N28, I29, K30, I48, A67 and T73 (Kabat numbering
convention), and b) a light chain comprising a complementarity
determining region (CDR) and at least one variable region framework
residue from the monoclonal antibody 11K2 light chain set forth as
SEQ ID NO: 28, wherein the residue is selected from the group
consisting of S49, K69, and Y71 (Kabat numbering convention),
wherein the remainder of the heavy and light chains are from a
human immunoglobulin. In a certain embodiment, the heavy chain
comprises variable region framework residues L27, I29, and T73, and
the light chain comprises variable region framework residues S49
and Y71. In another embodiment, the heavy chain comprises variable
region framework residues L27, N28, I29, K30, I48, A67 and T73, and
the light chain comprises variable region framework residues S49,
K69, and Y71. In yet another embodiment, the heavy chain comprises
variable region framework residues L27, I29, and T73, and the light
chain comprises variable region framework residues S49, K69, and
Y71. In yet a further embodiment, the heavy chain comprises
variable region framework residues L27, N28, I29, K30, I48, A67 and
T73, and the light chain comprises variable region framework
residues S49 and Y71.
[0039] In one embodiment, the invention features a humanized
immunoglobulin or antigen-binding fragment comprising
[0040] a) heavy chain complementary determining regions as set
forth in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, and
variable region framework residues L27, N28, I29, K30, I48, A67,
and T73 (Kabat numbering) from the monoclonal antibody 11K2 heavy
chain set forth as SEQ ID NO: 27, and
[0041] b) light chain complementary determining regions as set
forth in SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, and
variable region framework residues S49, K69, and Y71 (Kabat
numbering), from the monoclonal antibody 11K2 light chain set forth
as SEQ ID NO: 28, wherein the remainder of the heavy and light
chains are from a human immunoglobulin. In one embodiment of the
invention, the inmlunoglobulin is modified by reducing or
eliminating at least one potential glycosylation site.
[0042] In one embodiment, the immunoglobulin or antigen-binding
fragment of the invention, binds to MCP-1. In one embodiment, the
immunoglobulin or antigen-binding fragment of the invention,
specifically binds to MCP-1 with a binding affinity of at least
10.sup.-9 M. In another embodiment, the immunoglobulin or
antigen-binding fragment of the invention, specifically binds to
MCP-1 with a binding affinity of at least 10.sup.-10 M. In still
another embodiment, the immunoglobulin or antigen-binding fragment
of the invention specifically binds to MCP-1 with a binding
affinity of at least 10.sup.-11 M. In still another embodiment, the
immunoglobulin or antigen-binding fragment of the invention,
further binds to MCP-2 with a binding affinity of at least
10.sup.-7 M. In another embodiment, the immunoglobulin or
antigen-binding fragment of the invention, further binds to MCP-2
with a binding affinity of at least 10.sup.-8 M. In yet another
embodiment, the immunoglobulin or antigen-binding fragment of the
invention, further binds to MCP-2 with a binding affinity of at
least 10.sup.-9 M.
[0043] In one embodiment, the immunoglobulin or antigen-binding
fragment of the invention binds to MCP-2. In one embodiment, the
immunoglobulin or antigen-binding fragment of the invention
specifically binds to MCP-2 with a binding affinity of at least
10.sup.-7 M. In yet another embodiment, the immunoglobulin or
antigen-binding fragment of the invention specifically binds to
MCP-2 with a binding affinity of at least 10.sup.-8 M. In yet
another embodiment, the immunoglobulin or antigen-binding fragment
of the invention specifically binds to MCP-2 with a binding
affinity of at least 10.sup.-9 M.
[0044] In one embodiment, the immunoglobulin or antigen-binding
fragment of the invention binds to MCP-1 and MCP-2. In another
embodiment, the immunoglobulin or antigen-binding fragment of the
invention binds to an epitope within MCP-1, MCP-2, and MCP-3.
[0045] Another embodiment of the invention features a method of
treating a disorder associated with detrimental MCP activity
comprising administering to a subject having said disorder, a
nucleic acid molecule that encodes an immunoglobulin heavy chain
comprising the amino acid sequence of SEQ ID NO: 53 or the amino
acid sequence of SEQ ID NO: 54 and a nucleic acid molecule that
encodes an immunoglobulin light chain that comprises the amino acid
sequence of SEQ ID NO: 55 or the amino acid sequence of SEQ ID NO:
56, under conditions such that said immunoglobulin chains are
expressed, such that a beneficial therapeutic response in said
subject is generated.
[0046] In one embodiment, the invention features an antibody
comprising the same heavy and light chain polypeptide sequences as
an antibody produced by a CHO cell line secreting humanized 11K2
(version H2L1) clone 3F2 (ATCC patent deposit designation
PTA-5308).
[0047] In another embodiment, the invention describes an isolated
nucleic acid molecule encoding the heavy chain of the
immunoglobulin or antigen-binding fragment of the invention. In
another embodiment, the invention features an isolated nucleic acid
molecule encoding the light chain of immunoglobulin or
antigen-binding fragment of the invention. In still another
embodiment, the invention features an isolated nucleic acid
molecule encoding the immunoglobulin or antigen-binding fragment of
the invention.
[0048] In one embodiment, the invention features an isolated
nucleic acid molecule comprising a nucleotide sequence
corresponding to the amino acid sequence selected from the group
consisting of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, and SEQ
ID NO: 50.
[0049] In still another embodiment, the invention features an
isolated nucleic acid comprising a coding sequence for the heavy
chain of an antibody produced by a CHO cell line secreting
humanized 11K2 (version H2L1) clone 3F2 (ATCC patent deposit
designation PTA-5308). In another embodiment, the invention
features an isolated nucleic acid comprising a coding sequence for
the light chain of an antibody produced by a CHO cell line
secreting humanized 11K2 (version H2L1) clone 3F2 (ATCC patent
deposit designation PTA-5308).
[0050] In another embodiment, the invention features a cell line of
humanized 11K2 (version H2L1) clone 3F2 (ATCC patent deposit
designation PTA-5308).
[0051] One embodiment of the invention features a humanized
immunoglobulin heavy chain or antigen-binding portion thereof
comprising variable region complementary determining regions (CDRs)
from the 1A1 immunoglobulin heavy chain variable region sequence
set forth as SEQ ID NO: 11, and variable framework regions from a
human acceptor immunoglobulin heavy chain sequence, provided that
at least one framework residue is substituted with the
corresponding amino acid residue from the mouse 1A1 heavy chain
variable region sequence, wherein the framework residue is selected
from the group consisting of:
[0052] a residue that non-covalently binds antigen directly;
[0053] a residue adjacent to a CDR;
[0054] a CDR-interacting residue; and
[0055] a residue participating in the VL-VH interface.
[0056] The invention features a humanized immunoglobulin heavy
chain or antigen-binding portion thereof, wherein a CDR-interacting
residue is identified by modeling the 1A1 heavy chain based on the
solved structure of a murine immunoglobulin heavy chain that shares
at least 70% sequence identity with the 1A1 heavy chain. The
invention also features a humanized immunoglobulin heavy chain or
antigen-binding portion thereof, wherein a CDR-interacting residue
is identified by modeling the 1A1 heavy chain based on the solved
structure of a murine immunoglobulin heavy chain that shares at
least 80% sequence identity with the 1A1 heavy chain. The invention
further features a humanized immunoglobulin heavy chain or
antigen-binding portion thereof, wherein a CDR-interacting residue
is identified by modeling the 1A1 heavy chain based on the solved
structure of a murine immunoglobulin heavy chain that shares at
least 90% sequence identity with the 1A1 heavy chain.
[0057] In another embodiment, the invention features a humanized
immunoglobulin light chain or antigen-binding portion thereof
comprising variable region complementary determining regions (CDRs)
from the 1A1 immunoglobulin light chain variable region sequence
set forth as SEQ ID NO: 12 and variable framework regions from a
human acceptor immunoglobulin light chain, provided that at least
one framework residue is substituted with the corresponding amino
acid residue from the mouse 1A1 light chain variable region
sequence, wherein the framework residue is selected from the group
consisting of:
[0058] a residue that non-covalently binds antigen directly;
[0059] a residue adjacent to a CDR;
[0060] a CDR-interacting residue; and
[0061] a residue participating in the VL-VH interface.
[0062] The invention features a humanized immunoglobulin light
chain or antigen-binding portion thereof, wherein a CDR-interacting
residue is identified by modeling the 1A1 light chain based on the
solved structure of a murine immunoglobulin light chain that shares
at least 70% sequence identity with the 1A1 light chain. The
invention also features a humanized immunoglobulin light chain,
wherein a CDR-interacting residue is identified by modeling the 1A1
light chain based on the solved structure of a murine
immunoglobulin light chain that shares at least 80% sequence
identity with the 1A1 light chain. The invention features a
humanized immunoglobulin light chain, wherein a CDR-interacting
residue is identified by modeling the 1A1 light chain based on the
solved structure of a murine immunoglobulin light chain that shares
at least 90% sequence identity with the 1A1 light chain.
[0063] In another embodiment, the invention features a humanized
immunoglobulin heavy chain or antigen-binding portion thereof
comprising variable region complementary determining regions (CDRs)
from the 1A1 immunoglobulin heavy chain variable region sequence
set forth as SEQ ID NO: 11, and variable framework regions from a
human acceptor immunoglobulin heavy chain sequence, provided that
at least one framework residue is substituted with the
corresponding amino acid residue from the mouse 1A1 heavy chain
variable region sequence, wherein the framework residue is a
residue capable of affecting heavy chain variable region
conformation or function as identified by analysis of a
three-dimensional model of the variable region. 70. The invention
also features a humanized immunoglobulin of a heavy chain or
antigen-binding portion thereof, wherein the framework residue is
selected from the group consisting of a residue capable of
interacting with antigen, a residue proximal to the antigen binding
site, a residue capable of interacting with a CDR, a residue
adjacent to a CDR, a residue within 6 .DELTA. of a CDR residue, a
canonical residue, a vernier zone residue, an interchain packing
residue, and a rare residue.
[0064] In another embodiment, the invention features a humanized
immunoglobulin light chain or antigen-binding portion thereof,
comprising variable region complementary determining regions (CDRs)
from the 1A1 immunoglobulin light chain variable region sequence
set forth as SEQ ID NO: 12, and variable framework regions from a
human acceptor immunoglobulin light chain, provided that at least
one framework residue is substituted with the corresponding amino
acid residue from the mouse 1A1 light chain variable region
sequence, wherein the framework residue is a residue capable of
affecting light chain variable region conformation or function as
identified by analysis of a three-dimensional model of the variable
region. The invention also features a humanized immunoglobulin of a
light chain or antigen-binding portion thereof, wherein the
framework residue is selected from the group consisting of a
residue capable of interacting with antigen, a residue proximal to
the antigen binding site, a residue capable of interacting with a
CDR, a residue adjacent to a CDR, a residue within 6 .DELTA. of a
CDR residue, a canonical residue, a vernier zone residue, an
interchain packing residue, and an unusual residue.
[0065] In one embodiment the invention features a heavy and/or
light chain, wherein the framework residue is identified by
modeling the 1A1 heavy chain based on the solved structure of a
murine immunoglobulin heavy chain that shares at least 70% sequence
identity with the 1A1 heavy and/or light chain. In another
embodiment the invention features a heavy and/or light chain,
wherein the framework residue is identified by modeling the 1A1
heavy or light chain based on the solved structure of a murine
immunoglobulin heavy and/or light chain that shares at least 80%
sequence identity with the 1A1 heavy or light chain. In another
embodiment the invention features a heavy and/or light chain,
wherein the framework residue is identified by modeling the 1A1
heavy chain based on the solved structure of a murine immunoglobul
in heavy and/or light chain that shares at least 90% sequence
identity with the 1A1 heavy or light chain.
[0066] In still another embodiment, the invention features a heavy
chain comprising the complementarity determining regions (CDRs) and
variable region framework residue H29, H30, H73, H91, H93, and H94
(Kabat numbering convention) from the monoclonal antibody 1A1 heavy
chain, wherein the remainder of the heavy chain is from a human
immunoglobulin. In another embodiment, the heavy chain of the
invention further comprises at least one variable framework residue
from the monoclonal antibody 1A1 heavy chain selected from the
group consisting of H27, H28, H66, H69, and H76 (Kabat numbering
convention).
[0067] In still another embodiment, the invention includes a light
chain comprising the complementarity determining regions (CDRs) and
variable framework residues L2 and L36 (Kabat numbering convention)
from the monoclonal antibody 1A1 light chain, wherein the remainder
of the light chain is from a human immunoglobulin. The invention
also features a light chain further comprising the variable
framework residue from the monoclonal antibody 1A1 heavy chain L45
(Kabat numbering convention).
[0068] In one embodiment, the invention features a humanized
immunoglobulin comprising the heavy chain set forth in SEQ ID NO:
53. In still another embodiment, the invention includes a humanized
immunoglobulin comprising the heavy chain set forth in SEQ ID NO:
54. In a further embodiment, the invention features a humanized
immunoglobulin comprising the light chain set forth in SEQ ID NO:
55. The invention also features a humanized immunoglobulin
comprising the light chain set forth in SEQ ID NO: 56. The
invention also features a humanized immunoglobulin comprising a
heavy chain comprising SEQ ID NO: 53 or SEQ ID NO: 54 and a light
chain comprising SEQ ID NO: 53 or SEQ ID NO: 56.
[0069] In one embodiment, the invention features an immunoglobulin
or antigen binding fragment, which specifically binds to MCP-1 with
a binding affinity of at least 10.sup.-9 M. In another embodiment,
the immunoglobulin or antigen binding fragment of the invention
specifically binds to MCP-1 with a binding affinity of at least
10.sup.-10 M. In still a further embodiment, the immunoglobulin or
antigen binding fragment of the invention specifically binds to
MCP-1 with a binding affinity of at least 10.sup.-11 M. In one
embodiment, the immunoglobulin or antigen-binding fragment of the
invention binds to MCP-2. In another embodiment, the immunoglobulin
or antigen-binding fragment of the invention binds to MCP-1 and
MCP-2. In still another embodiment, the immunoglobulin or
antigen-binding fragment of the invention binds to an epitope
within MCP-1, MCP-2, and MCP-3.
[0070] In still another embodiment, the invention features a method
for identifying residues amenable to substitution in a humanized
1A1 immunoglobulin variable framework region, comprising modeling
the three-dimensional structure of the 1A1 variable region based on
a solved immunoglobulin structure and analyzing said model for
residues capable of affecting 1A1 immunoglobulin variable region
conformation or function, such that residues amenable to
substitution are identified. The invention also includes use of the
variable region sequence set forth as SEQ ID NO: 11 or SEQ ID NO:
12, or any portion thereof, in producing a three-dimensional image
of a 1A1 immunoglobulin, 1A1 immunoglobulin chain, or domain
thereof.
[0071] In one embodiment, the immunoglobulin or antigen-binding
fragment of the invention is modified by reducing or eliminating at
least one potential glycosylation site. In another embodiment, the
immunoglobulin or antigen-binding fragment of the invention is
modified by conjugation to a carrier selected from polyethylene
glycol and albumen. In yet another embodiment, the immunoglobulin
or antigen-binding fragment of the invention is modified to reduce
at least one constant region-mediated biological effector function
relative to an unmodified antibody.
[0072] In one embodiment, the heavy chain isotype of the
immunoglobulin or antigen-binding fragment of the invention is
gamma 1.
[0073] In one embodiment, the fragment of the invention is a Fab
fragment.
[0074] In one embodiment, the invention features an immunoglobulin
or antigen-binding fragment which inhibits MCP-induced chemotaxis.
In a certain embodiment, the immunoglobulin or antigen-binding
fragment of the invention inhibits MCP-1-induced chemotaxis,
MCP-2-induced chemotaxis, or both MCP-1-induced and MCP-2-induced
chemotaxis.
[0075] In one embodiment, the immunoglobulin or antigen-binding
fragment of the invention inhibits MCP-induced collagen expression.
In another embodiment, the invention features an immunoglobulin or
antigen-binding fragment, wherein the immunoglobulin or
antigen-binding fragment inhibits MCP-1-induced collagen
expression, MCP-2-induced collagen expression, or both
MCP-1-induced and MCP-2-induced collagen expression.
[0076] In one embodiment, the invention features an immunoglobulin
or antigen-binding fragment which inhibits MCP-1-induced
angiogenesis. In certain embodiments, an immunoglobulin or
antigen-binding fragment of the invention inhibits MCP-1-induced
angiogenesis, MCP-2-induced angiogenesis, or both MCP-1-induced and
MCP-2-induced angiogenesis.
[0077] In one embodiment, the immunoglobulin or antigen-binding
fragment of any the invention reduces inflammation in a subject. In
one embodiment, the inflammation is associated with a disorder
selected from the group consisting of arthritis, multiple
sclerosis, cirrhosis, atherosclerosis, and breast carcinoma.
[0078] In one embodiment, an immunoglobulin or antigen-binding
fragment of the invention reduces fibrosis in a subject.
[0079] In one embodiment, the invention features a pharmaceutical
composition comprising the immunoglobulin or antigen-binding
fragment of the invention and a pharmaceutical carrier.
[0080] In another embodiment, the invention features a host cell
comprising the nucleic acid molecule of a immunoglobulin or
antigen-binding fragment of the invention. In one embodiment, the
host cell of the invention is mammalian. In another embodiment, the
host cell of the invention is bacterial. In still another
embodiment, the invention describes a method of producing an
antibody or antigen binding fragment thereof of the invention,
comprising culturing the host cell under conditions such that the
antibody or fragment is produced and isolating said antibody from
the host cell or culture.
[0081] In still another embodiment, the invention provides a method
of preventing or treating a disorder associated with detrimental
MCP activity in a subject, comprising administering to the subject
an effective amount of an immunoglobulin or antigen binding
fragment of the invention.
[0082] In one embodiment, the effective amount of immunoglobulin or
antigen binding fragment thereof is 1-10 mg/kg body weight. In
another embodiment, the disorder is selected from the group
consisting of glomerulonephritis, scleroderma, cirrhosis, multiple
sclerosis, lupus nephritis, atherosclerosis, inflammatory bowel
diseases or rheumatoid arthritis.
[0083] In yet another embodiment, the invention provides a method
of preventing or treating MCP-associated inflammation in a subject,
comprising administering to the subject an effective amount of an
immunoglobulin or antigen binding fragment of the invention.
[0084] In yet another embodiment of the invention, a method of
preventing or treating MCP-associated inflammation in a subject is
described, comprising administering to the subject an effective
amount of the humanized immunoglobulin or antigen-binding portion
of the invention. Effective amounts of humanized immunoglobulin or
antigen-binding portion described in the invention include, for
example, 1 mg/kg body weight to 10 mg/kg body weight.
[0085] In yet another embodiment, the invention provides a method
of preventing or treating a fibrotic disorder in a subject
comprising administering to the subject an effective amount of an
immunoglobulin or antigen binding fragment of the invention.
[0086] In yet another embodiment, the invention provides a method
of preventing or treating cancer in a subject comprising
administering to the subject an effective amount of an
immunoglobulin or antigen binding fragment of the invention.
[0087] In yet another embodiment, the invention provides a method
of preventing or treating an immunopathologic disorder comprising
administering to the subject an effective amount of an
immunoglobulin or antigen binding fragment of the invention.
[0088] Another embodiment of the invention features use of the
antibodies or antigen-binding fragments of the invention for
preventing or treating an inflammatory disorder, e.g., Alzheimer's,
severe asthma, atopic dermatitis, cachexia, CHF-ischemia, coronary
restinosis, Crohn's disease, diabetic nephropathy, lymphoma,
psoriasis, fibrosis/radiation-induced, juvenile arthritis, stroke,
inflammation of the brain or central nervous system caused by
trauma, and ulcerative colitis, inflammation due to corneal
transplantation, chronic obstructive pulmonary disease, hepatitis
C, multiple myeloma, and osteoarthritis.
[0089] Yet another embodiment of the invention features used of the
antibodies or antigen-binding fragments of the invention for
preventing or treating a neurodegenerative disorder.
Neurodegenerative disorders which can be treated by the antibodies
or antigen-binding fragments thereof, include, but are not limited
to, Alzheimer's, stroke, traumatic brain or central nervous system
injuries, ALS/motor neuron disease, diabetic peripheral neuropathy,
diabetic retinopathy, Huntington's disease, macular degeneration,
and Parkinson's disease.
[0090] In yet another embodiment, the invention includes a heavy
chain of an anti-MCP antibody which contacts residues R30, T32,
S34, K38, E39, V41, P55, K56, Q61, M64 of MCP-1. The invention
further includes a light chain anti-MCP antibody which contacts
residues D65, D68, K69 of MCP-1.
BRIEF DESCRIPTION OF DRAWINGS
[0091] FIG. 1 graphically depicts results of a chemotaxis assay
using purified 11K2, 1A1, D9, and 2024 to inhibit chemotaxis in
response to MCP-1, MCP-2, and a combination of MCP-1/MCP-2. The
results show that chemotaxis to a combination of MCP-1 and MCP-2 is
inhibited by 11K2 and 1A1.
[0092] FIGS. 2A and 2B graphically depict the results of a
chemotaxis assay of cells in response to MCP-1. FIG. 2A graphically
depicts results using monoclonal antibodies 5D3-F7 (BD Biosciences,
Pharmingen, San Diego, Calif.), 1M11, 3N10, 5J23, and 11K2 in
response to 20 ng/mL of MCP-1. FIG. 2B graphically depicts results
using 20 ng/mL of murine MCP-1 (JE) and monoclonal antibodies 2H5
(BD Biosciences, Pharmingen, San Diego, Calif.), 1M11, 3N10, 5J23,
and 11K2.
[0093] FIG. 3 graphically depicts results of a chemotaxis assay
which demonstrates that monocyte chemotaxis mediated by cytokines
secreted from stimulated rheumatoid arthritis (RA) fibroblasts is
inhibited by pan-MCP mAbs (1A1 and 11K2) and MCP-1 mAb D9.
[0094] FIGS. 4A, 4B, and 4C graphically depict results from a
calcium flux assay using 11K2 (mAb and Fab fragments thereof) at
various concentrations, including none (FIG. 4A), 20 nM mAb (FIG.
4B), and 60 nM Fab (FIG. 4C).
[0095] FIGS. 5A and 5B graphically depict results from a chemotaxis
assay using pan-MCP antibodies demonstrating pan-MCP antibodies
11K2 and 1A1 increase MCP-2 mediated chemotaxis at low mAb
concentrations (FIG. 5A). Blocking is also observed with MCP-2 mAb
281 (RD Systems, Minneapolis, Minn.). FIG. 5B graphically depicts a
chemotaxis assay using the pan-MCP mAb 11K2 and the Fab fragment of
11K2.
[0096] FIGS. 6A, 6B, 6C, and 6D graphically depict results from a
MCP-2 calcium flux assay which depicts results from 55.5 nM of
MCP-2 alone, depicts results which demonstrate that the 11K2
monoclonal antibody shows agonistic activity (6B), and, as compared
to 55.5 nM of MCP-2 alone (6A), FIGS. 6C and 6D depict results
which demonstrate that Fab and F(ab')2 fragments of 11K2 are
inhibitory in this assay.
[0097] FIGS. 7A and 7B show the amino acid and nucleotide sequences
of the variable heavy region of the murine version of the 1A1
antibody (FIG. 7A), as well as the amino acid and nucleotide
sequences of the 1A1 variable light region (FIG. 7B). CDR regions
are underlined.
[0098] FIGS. 8A and 8B show the amino acid and nucleotide sequences
of the murine 11K2 variable heavy region (FIG. 8A), and the amino
acid and nucleotide sequences of murine 11K2 variable light region
(FIG. 8B). CDR regions are underlined.
[0099] FIGS. 9A and 9B show the nucleotide and amino acid sequences
of a heavy chain chimera (variable and constant regions) of the
11K2 antibody (FIG. 9A). The variable region is set forth at
nucleotides 1-351 (amino acids 1-117) of the heavy chain. FIG. 9B
shows the DNA/amino acid comparison of the 11K2 light chain chimera
(variable and constant regions). The variable region is set forth
at nucleotides 1-321 (amino acids 1-107) of the light chain. All
CDRs are underlined.
[0100] FIGS. 10A and 10B show the nucleotide and amino acid
sequences of the light and heavy chains of humanized 11K2 antibody.
FIG. 10A shows the sequence of humanized version 1 including heavy
chain variable and constant regions. FIG. 10B shows the sequence of
humanized version 2 including heavy chain variable and constant
regions. FIG. 10C shows the sequence of humanized version 1 light
chain variable and constant regions. FIG. 10D shows the sequence of
humanized version 2 light chain variable and constant regions. All
CDR regions are underlined, and all back mutations are highlighted
in bold.
[0101] FIGS. 11A and 11B show an alignment of the murine 11K2
antibody and the humanized 11K2 (versions 1 and 2) for the variable
heavy chain region (FIG. 11A) and the variable light chain region
(FIG. 11B). All CDRs are underlined and in bold.
[0102] FIGS. 12A and 12B graphically-depict results from a
neutralization assay using mAb 11K2, chimeric 11K2, a glycosylated
chimeric 11K2, H1/L1 humanized 11K2, H2/L2 humanized 11K2, H1/L2
humanized 11K2, and H2/L1 humanized 11K2 antibodies (whole and Fab
fragments). FIG. 12A graphically depicts results from a
neutralization assay using 2.3 nM MCP-1. FIG. 12B graphically
depicts results from a neutralization assay using 56 nM MCP-2. All
of the antibodies tested exhibit an agonist activity for MCP-2 at
low concentrations.
[0103] FIG. 13 graphically depicts results from an experiment which
demonstrates that PEGylation of 11K2 Fab retains in vitro
activity.
[0104] FIG. 14A graphically depicts results from an ELISA
experiment, which show reactivity of the humanized and chimeric
11K2 antibodies with huMCP-1, primate MCP-1, and muMCP-1. FIG. 14B
graphically depicts results from an ELISA experiment, which show
the reactivity of humanized and chimeric 11K2 antibodies with
MCP-2.
[0105] FIG. 15 graphically depicts the therapeutic effect of mouse
monoclonal antibody 11K2 treatment on survival of mice afflicted by
TNBS-induced colitis.
[0106] FIG. 16 graphically depicts the reduction of MCP-1 levels
seen in TNBS-induced colitis mice treated with monoclonal antibody
11K2.
[0107] FIG. 17 graphically depicts the results of experiments
demonstrating the efficacy of hu11K2 and pegylated 11K2-Fab (11K2
PEG-Fab) to inhibit colitis in a TNBS-induced mice.
[0108] FIG. 18 graphically depicts the therapeutic effect of
monoclonal antibody 11K2 administration to TNBS-induced mice. At
day 7 post-TNBS-induction 11K2 treatment resulted in elevated body
weight, reduced MCP and TNF.A-inverted. levels, and inhibition of
myeloperoxidase (MPO) activity, as compared with untreated and
control mAb-treated colitis model mice.
[0109] FIG. 19 graphically depicts results demonstrating a
reduction in atherosclerotic plaque size (total plaque area) in
apoE-deficient mice treated with mouse monoclonal antibody
11K2.
DETAILED DESCRIPTION OF THE INVENTION
[0110] The present invention features new immunological reagents
and methods for preventing or treating disorders associated with
detrimental MCP activity. The invention is based, at least in part,
on the characterization of two monoclonal immunoglobulins, 11K2 and
1A1, effective at binding MCPs (A.beta.) (e.g., MCP-1, MCP-2, and
MCP-3). The invention is further based on the determination and
structural characterization of the primary and secondary structure
of the variable light and heavy chains of these immunoglobulins and
the identification of residues important for activity and
immunogenicity.
[0111] Immunoglobulins are featured which include a variable light
and/or variable heavy chain of the preferred monoclonal
immunoglobulins described herein. Preferred immunoglobulins, e.g.,
therapeutic immunoglobulins, are featured which include a humanized
variable light and/or humanized variable heavy chain. Preferred
variable light and/or variable heavy chains include a
complementarity determining region (CDR) from the monoclonal
immunoglobulin (e.g., donor immunoglobulin) and variable framework
regions substantially from a human acceptor immunoglobulin. The
phrase "substantially from a human acceptor immunoglobulin" means
that the majority or key framework residues are from the human
acceptor sequence, allowing however, for substitution of residues
at certain positions with residues selected to improve activity of
the humanized immunoglobulin (e.g., alter activity such that it
more closely mimics the activity of the donor immunoglobulin) or
selected to decrease the immunogenicity of the humanized
immunoglobulin.
[0112] In one embodiment, the invention features a humanized
immunoglobulin light or heavy chain that includes 11K2 variable
region complementarity determining regions (CDRs) (i.e., includes
one, two or three CDRs from the light chain variable region
sequence set forth as SEQ ID NO: 28 or includes one, two or three
CDRs from the heavy chain variable region sequence set forth as SEQ
ID NO: 27), and includes a variable framework region substantially
from a human acceptor immunoglobulin light or heavy chain sequence,
provided that at least one residue of the framework residue is
backmutated to a corresponding murine residue, wherein said
backmutation does not substantially affect the ability of the chain
to direct MCP binding.
[0113] In another embodiment, the invention features a humanized
immunoglobulin light or heavy chain that includes 11K2 variable
region complementarity determining regions (CDRs) (e.g., includes
one, two or three CDRs from the light chain variable region
sequence set forth as SEQ ID NO: 28 and/or includes one, two or
three CDRs from the heavy chain variable region sequence set forth
as SEQ ID NO: 27), and includes a variable framework region
substantially from a human acceptor immunoglobulin light or heavy
chain sequence, provided that at least one framework residue is
substituted with the corresponding amino acid residue from the
mouse 11K2 light or heavy chain variable region sequence, where the
framework residue is selected from the group consisting of (a) a
residue that non-covalently binds antigen directly; (b) a residue
adjacent to a CDR; (c) a CDR-interacting residue (e.g., identified
by modeling the light or heavy chain on the solved structure of a
homologous known immunoglobulin chain); and (d) a residue
participating in the VL-VH interface.
[0114] In another embodiment, the invention features a humanized
immunoglobulin light or heavy chain that includes 11K2 variable
region CDRs and variable framework regions from a human acceptor
immunoglobulin light or heavy chain sequence, provided that at
least one framework residue is substituted with the corresponding
amino acid residue from the mouse 11K2 light or heavy chain
variable region sequence, where the framework residue is a residue
capable of affecting light chain variable region conformation or
function as identified by analysis of a three-dimensional model of
the variable region, for example a residue capable of interacting
with antigen, a residue proximal to the antigen binding site, a
residue capable of interacting with a CDR, a residue adjacent to a
CDR, a residue within 6 .DELTA. of a CDR residue, a canonical
residue, a vernier zone residue, an interchain packing residue, or
an unusual residue.
[0115] In another embodiment, the invention features a humanized
immunoglobulin light chain that includes 11K2 variable region CDRs
(e.g., from the 11K2 light chain variable region sequence set forth
as SEQ ID NO: 28), and includes a human acceptor immunoglobulin
variable framework region, provided that at least one framework
residue selected from the group consisting of L49, L69 and L71
(Kabat numbering convention) is substituted with the corresponding
amino acid residue from the mouse 11K2 light chain variable region
sequence. In another embodiment, the invention features a humanized
immunoglobulin heavy chain that includes 11K2 variable region CDRs
(e.g., from the 11K2 heavy chain variable region sequence set forth
as SEQ ID NO: 27), and includes a human acceptor immunoglobulin
variable framework region, provided that at least one framework
residue selected from the group consisting of H27, H28, H29, H30,
H48, H67, and H73 (Kabat numbering convention) is substituted with
the corresponding amino acid residue from the mouse 11K2 heavy
chain variable region sequence.
[0116] Preferred light chains include framework regions of the
subtype kappa 1 (Kabat convention), for example, framework regions
from the acceptor immunoglobulin GI-486875. Preferred heavy chains
include framework regions of the subtype 1 (Kabat convention), for
example, framework regions from the acceptor immunoglobulin Kabat
ID 000054.
[0117] In one embodiment, the invention features a humanized
immunoglobulin light or heavy chain that includes 1A1 variable
region complementarity determining regions (CDRs) (i.e., includes
one, two or three CDRs from the light chain variable region
sequence set forth as SEQ ID NO: 12 or includes one, two or three
CDRs from the heavy chain variable region sequence set forth as SEQ
ID NO: 11), and includes a variable framework region substantially
from a human acceptor immunoglobulin light or heavy chain sequence,
provided that at least one residue of the framework residue is
backmutated to a corresponding murine residue, wherein said
backmutation does not substantially affect the ability of the chain
to direct MCP binding.
[0118] In another embodiment, the invention features a humanized
immunoglobulin light or heavy chain that includes 1A1 variable
region complementarity determining regions (CDRs) (e.g., includes
one, two or three CDRs from the light chain variable region
sequence set forth as SEQ ID NO: 12 and/or includes one, two or
three CDRs from the heavy chain variable region sequence set forth
as SEQ ID NO: 11), and includes a variable framework region
substantially from a human acceptor immunoglobulin light or heavy
chain sequence, provided that at least one framework residue is
substituted with the corresponding amino acid residue from the
mouse 1A1 light or heavy chain variable region sequence, where the
framework residue is selected from the group consisting of (a) a
residue that non-covalently binds antigen directly; (b) a residue
adjacent to a CDR; (c) a CDR-interacting residue (e.g., identified
by modeling the light or heavy chain on the solved structure of a
homologous known immunoglobulin chain); and (d) a residue
participating in the VL-VH interface.
[0119] In another embodiment, the invention features a humanized
immunoglobulin light or heavy chain that includes 1A1 variable
region CDRs and variable framework regions from a human acceptor
immunoglobulin light or heavy chain sequence, provided that at
least one framework residue is substituted with the corresponding
amino acid residue from the mouse 1A1 light or heavy chain variable
region sequence, where the framework residue is a residue capable
of affecting light chain variable region conformation or function
as identified by analysis of a three-dimensional model of the
variable region, for example a residue capable of interacting with
antigen, a residue proximal to the antigen binding site, a residue
capable of interacting with a CDR, a residue adjacent to a CDR, a
residue within 6 .DELTA. of a CDR residue, a canonical residue, a
vernier zone residue, an interchain packing residue, or an unusual
residue.
[0120] In another embodiment, the invention features a humanized
immunoglobulin that includes a light chain and a heavy chain, as
described above, or an antigen-binding fragment of said
immunoglobulin. In an exemplary embodiment, the humanized
immunoglobulin binds (e.g., specifically binds) to MCP-1 with a
binding affinity of at least 10.sup.7 M.sup.-1, 10.sup.8 M.sup.-1,
or 10.sup.9 M.sup.-1.
[0121] In another embodiment, the invention features chimeric
immunoglobulins that include 11K2 variable regions (e.g., the
variable region sequences set forth as SEQ ID NO: 27 or SEQ ID NO:
28). In yet another embodiment, the invention features an
immunoglobulin, or antigen-binding fragment thereof, including a
variable heavy chain region as set forth in SEQ ID NO: 47 or SEQ ID
NO: 48 and a variable light chain region as set forth in SEQ ID NO:
49 or SEQ ID NO: 50.
[0122] In another embodiment, the invention features chimeric
immunoglobulins that include 1A1 variable regions (e.g., the
variable region sequences set forth as SEQ ID NO: 11 or SEQ ID NO:
12). In yet another embodiment, the invention features an
immunoglobulin, or antigen-binding fragment thereof, including a
variable heavy chain region as set forth in SEQ ID NO: 53 or SEQ ID
NO: 54 and a variable light chain region as set forth in SEQ ID NO:
55 or SEQ ID NO: 56. In yet another embodiment, the immunoglobulin,
or antigen-binding fragment thereof, further includes constant
regions from IgG1.
[0123] The immunoglobulins described herein are particularly suited
for use in therapeutic methods aimed at preventing or treating
disorders associated with detrimental MCP activity. In one
embodiment, the invention features a method of preventing or
treating a disorder associated with detrimental MCP activity that
involves administering to the subject an effective dosage of a
humanized immunoglobulin as described herein. In another
embodiment, the invention features pharmaceutical compositions that
include a humanized immunoglobulin as described herein and a
pharmaceutical carrier. Also featured are isolated nucleic acid
molecules, vectors and host cells for producing the immunoglobulins
or immunoglobulin fragments or chains described herein, as well as
methods for producing said immunoglobulins, immunoglobulin
fragments or immunoglobulin chains.
[0124] The present invention further features a method for
identifying 1A1 or 11K2 residues amenable to substitution when
producing a humanized 1A1 or 11K2 immunoglobulin, respectively. For
example, a method for identifying variable framework region
residues amenable to substitution involves modeling the
three-dimensional structure of the 1A1 or 11K2 variable region on a
solved homologous immunoglobulin structure and analyzing said model
for residues capable of affecting 1A1 or 11K2 immunoglobulin
variable region conformation or function, such that residues
amenable to substitution are identified. The invention further
features use of the variable region sequence set forth as SEQ ID
NO: 27 or SEQ ID NO: 28, or any portion thereof, in producing a
three-dimensional image of a 11K2 immunoglobulin, 11K2
immunoglobulin chain, or domain thereof. Also featured is the use
of the variable region sequence set forth as SEQ ID NO: 11 or SEQ
ID NO: 12, or any portion thereof, in producing a three-dimensional
image of a 1A1 immunoglobulin, 1A1 immunoglobulin chain, or domain
thereof.
[0125] Prior to describing the invention, it may be helpful to an
understanding thereof to set forth definitions of certain terms to
be used hereinafter.
[0126] The term "immunoglobulin" or "antibody" (used
interchangeably herein) refers to an antigen-binding protein having
a basic four-polypeptide chain structure consisting of two heavy
and two light chains, said chains being stabilized, for example, by
interchain disulfide bonds, which has the ability to specifically
bind antigen. Both heavy and light chains are folded into domains.
The term "domain" refers to a globular region of a heavy or light
chain polypeptide comprising peptide loops (e.g., comprising 3 to 4
peptide loops) stabilized, for example, by 3-pleated sheet and/or
intrachain disulfide bond. Domains are further referred to herein
as "constant" or "variable", based on the relative lack of sequence
variation within the domains of various class members in the case
of a "constant" domain, or the significant variation within the
domains of various class members in the case of a "variable"
domain. "Constant" domains on the light chain are referred to
interchangeably as "light chain constant regions", "light chain
constant domains", "CL" regions or "CL" domains). "Constant"
domains on the heavy chain are referred to interchangeably as
"heavy chain constant regions", "heavy chain constant domains",
"CH" regions or "CH" domains). "Variable" domains on the light
chain are referred to interchangeably as "light chain variable
regions", "light chain variable domains", "VL" regions or "VL"
domains). "Variable" domains on the heavy chain are referred to
interchangeably as "heavy chain constant regions", "heavy chain
constant domains", "CH" regions or "CH" domains).
[0127] The term "region" refers to a part or portion of an antibody
chain and includes constant or variable domains as defined herein,
as well as more discrete parts or portions of said domains. For
example, light chain variable domains or regions include
"complementarity determining regions" or "CDRs" interspersed among
"framework regions" or "FRs", as defined herein.
[0128] Immunoglobulins or antibodies can exist in monomeric or
polymeric form. The term "antigen-binding fragment" refers to a
polypeptide fragment of an immunoglobulin or antibody binds antigen
or competes with intact antibody (i.e., with the intact antibody
from which they were derived) for antigen binding (i.e., specific
binding). The term "conformation" refers to the tertiary structure
of a protein or polypeptide (e.g., an antibody, antibody chain,
domain or region thereof). For example, the phrase "light (or
heavy) chain conformation" refers to the tertiary structure of a
light (or heavy) chain variable region, and the phrase "antibody
conformation" or "antibody fragment conformation" refers to the
tertiary structure of an antibody or fragment thereof.
[0129] "Specific binding" of an antibody mean that the antibody
exhibits appreciable affinity for antigen or a preferred epitope
and, preferably, does not exhibit significant crossreactivity.
"Appreciable" or preferred binding include binding with an affinity
of at least 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9 M.sup.-1, or
10.sup.10 M.sup.-1. Affinities greater than 10.sup.7 M.sup.-1,
preferably greater than 10.sup.8 M.sup.-1 are more preferred.
Values intermediate of those set forth herein are also intended to
be within the scope of the present invention and a preferred
binding affinity can be indicated as a range of affinities, for
example, 10.sup.6 to 10.sup.13 M.sup.-1, preferably 10.sup.7 to
10.sup.13 M.sup.-1, more preferably 10.sup.8 to 10.sup.13 M.sup.-1.
An antibody that "does not exhibit significant crossreactivity" is
one that will not appreciably bind to an undesirable entity (e.g.,
an undesirable proteinaceous entity). An antibody specific for a
preferred epitope will, for example, not significantly crossreact
with remote epitopes on the same protein or peptide. Specifie
binding can be determined according to any art-recognized means for
determining such binding. Preferably, specific binding is
determined according to Scatchard analysis and/or competitive
binding assays.
[0130] In one embodiment, the invention provides for antibodies,
antigen-binding fragments and/or antibody fragments of the
invention that have high binding affinity for b-chemokines. High
binding affinity refers to binding affinities of, for example,
about 10.times.10.sup.-12 M (i.e. 10 .mu.M) or less. In one
embodiment the immunoglobulins or antigen-binding fragments have a
Kd for binding to the b-chemokines (either binding a plurality of
MCPs selected from MCP-1, MCP-2, and MCP-3 or that bind the
individual MCPs, i.e. an antibody or antigen-binding fragment that
binds to MCP-1, MCP-2 or MCP-3) between about 10.times.10.sup.-12 M
(8 pM) and about 8.times.10.sup.-12 M (8 pM) including
9.times.10.sup.-1 M (9 pM); alternatively between about
9.times.10.sup.-12 M (9 pM) and about 7.times.10.sup.-12 M (7 pM)
including 8.times.10.sup.-12 M (8 pM); alternatively between about
8.times.10.sup.-12 M (8 pM) and about 6.times.10.sup.-12 M (6 pM)
including 7.times.10.sup.-12 M (7 pM); alternativelybetween about
7.times.10.sup.-12 M (7 pM) and about 5.times.10.sup.-12 M (pM)
including 6.times.10.sup.-12 M (6 pM), alternatively between about
6.times.10.sup.-12 M (6 pM) and about 4.times.10.sup.-12 M (4 pM)
including 5.times.10.sup.-12 M (5 pM), alternatively between about
5.times.10.sup.-12 M (pM) and about 3.times.10.sup.-12 M (3 pM)
including 4.times.10.sup.-12 M (4 pM); alternatively between about
4.times.10.sup.-12 M (4 pM) and about 2.times.10.sup.-12 M (2 pM)
including 3.times.10.sup.-12 M (3 pM); alternatively between about
3.times.10.sup.-12 M (3 pM) and about 1.times.10.sup.-12 M (1 pM)
including 2.times.10.sup.-12 M (2 pM); alternatively about
1.times.10.sup.-12 M (1 pM) and about 8.times.10.sup.-13 M (0.8 pM)
including 9.times.10.sup.-13 M (0.9 pM); alternatively between
about 9.times.10.sup.-13 M (0.9 pM) and about 7.times.10.sup.-13 M
(0.7 pM) including 8.times.10.sup.-13 M (0.8 pM); alternatively
between about 8.times.10.sup.-13 M (0.8 pM) and about
6.times.10.sup.-13 M (0.6 pM) including 7.times.10.sup.-13 M (0.7
pM); alternatively between about 7.times.10.sup.-13 M (0.7 pM) and
about 5.times.10.sup.-13 M (0.5 pM) including 6.times.10.sup.-13 M
(0.6 pM), alternatively between about 6.times.10.sup.-13 M (0.6 pM)
and about 4.times.10.sup.-13 M (0.4 pM) including
5.times.10.sup.-13 M (0.5 pM), alternatively between about
5.times.10.sup.-13 M (0.5 pM) and about 3.times.10.sup.-13 M (0.3
pM) including 4.times.10.sup.-13 M (0.4 pM); alternatively between
about 4.times.10.sup.-13 M (0.4 pM) and about 2.times.10.sup.-13 M
(0.2 pM) including 3.times.10.sup.-13 M (0.3 pM); alternatively
between about 3.times.10.sup.-13 M (0.3 pM) and about
1.times.10.sup.-13 M (0.1 pM) including 2.times.10.sup.-13 M (0.2
pM). The invention would include for example an antibody or
antigen-binding fragment thereof that binds to MCP-1, MCP-2 or
MCP-3 wherein the antibody or antigen-binding fragment thereof has
a Kd for binding to MCP-1, MCP-2 or MCP-3 selected from the
following Kd's: about 10.times.10.sup.-13 M (1 pM),
9.times.10.sup.-13 M (0.9 pM), 8.times.10.sup.-13 M (0.8 pM),
7.times.10.sup.-13 M (0.7 pM), 6.times.10.sup.-13 M (0.6 pM),
5.times.10.sup.-13 M (0.5 pM), 4.times.10.sup.-13 M (0.4 pM),
3.times.10.sup.-13 M (0.3 pM), 2.times.10.sup.-13 M (0.2 pM) or
1.times.10.sup.-13 M (0.1 pM). (An example of such an antibody
would include for example 11K2 in which the antibody has a binding
affinity for human MCP-1 of about 0.4 pM.) The invention would also
include for example an antibody or antigen-binding fragment thereof
that binds to a plurality of MCP's (i.e. MCP-1 and MCP-2 or MCP-1
and MCP-3 or MCP-1, MCP-2 and MCP-3 or MCP-2 and MCP-3) wherein the
antibody or antigen-binding fragment thereof has a Kd for binding
to at least one of the MCP's (i.e. MCP-1, MCP-2 or MCP-3) selected
from the following Kd's: about .times.10.sup.-13 M (1 pM),
9.times.10.sup.-13 M (0.9 pM), 8.times.10.sup.-13 M (0.8 pM),
7.times.10.sup.-13 M (0.7 pM), 6.times.10.sup.-13 M (0.6 pM),
5.times.10.sup.-13 M (0.5 pM), 4.times.10.sup.-13 M (0.4 pM),
3.times.10.sup.-13 M (0.3 pM), 2.times.10.sup.-13 M (0.2 pM) or
1.times.10.sup.-13 M (0.1 pM). (An example of such an antibody
would also include for example 11K2 in which the antibody has a
binding affinity for human MCP-1 of about 0.4 pM. and also binds
MCP-2 and MCP-3). Methods for measuring the binding affinity of the
antibody, antigen-binding fragment and or antibody fragment for the
various b-chemokine(s) are known to those of skill in the art and
include, for example, the kinetic exclusion assay illustrated in
the Examples as well.
[0131] The invention also provides for immunoglobulins and
antigen-binding fragments comprising a Fab fragment wherein the Fab
fragment has a Kd for binding MCP-1, MCP-2 or MCP-3 of, for
example, about 1.5.times.10.sup.-11 M (i.e. 15 pM) or less. The
invention would include for example an antibody, antigen-binding
fragment and/or antibody fragment thereof wherein the Fab fragment
has a Kd for binding to MCP-1, MCP-2 or MCP-3 selected from the
following Kd's: about 1.8.times.10.sup.-11 M (18 pM), about
1.7.times.10.sup.-11 M (17 pM), about 1.6.times.10.sup.-11 M (15
pM), about 1.5.times.10.sup.-11 M (15 pM), 1.4.times.10.sup.-11 M
(14 pM), 1.3.times.10.sup.-11 M (13 pM), 1.2.times.10.sup.-11 M (12
pM), 1.1.times.10.sup.-11 M (11 pM), 1.times.10.sup.-11 M (10 pM),
0.9.times.10.sup.-11 M (9 pM), 0.8.times.10.sup.-11 M (8 pM),
0.7.times.10.sup.-11 M (7 pM), 0.6.times.10.sup.-11 M (6 pM),
0.5.times.10.sup.-11 M (5 pM), 0.4.times.10.sup.-11 M (4 pM),
0.3.times.10.sup.-11 M (3 pM), 0.2.times.10.sup.-11 M (2 pM) or
0.1.times.10.sup.-11 M (1 pM). Methods for measuring the binding
affinity of the antibody, antigen-binding fragment and or antibody
fragment are known to those of skill in the art and include, for
example, the kinetic exclusion assay illustrated in Example 4
herewith.
[0132] The invention also provides for immunoglobulins and
antigen-binding fragments that have the following binding affinity
for the b-chemokines (either binding a plurality of MCP's selected
from MCP-1, MCP-2, and MCP-3 or that bind the individual MCPs, i.e.
an antibody or antigen-binding fragment that binds to MCP-1, MCP-2
or MCP-3). In one embodiment the binding affinity is between about
5.times.10.sup.-4 M and about 5.times.10.sup.-12 M, in some
embodiments the binding affinity is about .times.10.sup.-9 M to
about 5.times.10.sup.-11 M, in some embodiments the binding
affinity is about .times.10.sup.-7 M to about .times.10.sup.-8 M,
in some embodiments the binding affinity is about .times.10.sup.-8
M to about .times.10.sup.-9 M, in some embodiments the binding
affinity is about 5.times.10.sup.-9 M to about 5.times.10.sup.-10
M, in some embodiments the binding affinity is about 5.times.10-M
to about 5.times.10.sup.-11 M.
[0133] Binding fragments are produced by recombinant DNA
techniques, or by enzymatic or chemical cleavage of intact
immunoglobulins. Binding fragments include Fab, Fab', F(ab').sub.2,
Fabc, Fv, single chains, and single-chain antibodies. Other than
"bispecific" or "bifunctional" immunoglobulins or antibodies, an
immunoglobulin or antibody is understood to have each of its
binding sites identical. A "bispecific" or "bifunctional antibody"
is an artificial hybrid antibody having two different heavy/light
chain pairs and two different binding sites. Bispecific antibodies
can be produced by a variety of methods including fusion of
hybridomas or linking of Fab' fragments. See, e.g., Songsivilai
& Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et
al., J. Immunol. 148, 1547-1553 (1992).
[0134] The term "humanized immunoglobulin" or "humanized antibody"
refers to an immunoglobulin or antibody that includes at least one
humanized immunoglobulin or antibody chain (i.e., at least one
humanized light or heavy chain). The term "humanized immunoglobulin
chain" or "humanized antibody chain" (i.e., a "humanized
immunoglobulin light chain" or "humanized immunoglobulin heavy
chain") refers to an immunoglobulin or antibody chain (i.e., a
light or heavy chain, respectively) having a variable region that
includes a variable framework region substantially from a human
immunoglobulin or antibody and complementarity determining regions
(CDRs) (e.g., at least one CDR, preferably two CDRs, more
preferably three CDRs) substantially from a non-human
immunoglobulin or antibody, and further includes constant regions
(e.g., at least one constant region or portion thereof, in the case
of a light chain, and preferably three constant regions in the case
of a heavy chain). The term "humanized variable region" (e.g.,
"humanized light chain variable region" or "humanized heavy chain
variable region") refers to a variable region that includes a
variable framework region substantially from a human immunoglobulin
or antibody and complementarity determining regions (CDRs)
substantially from a non-human immunoglobulin or antibody.
[0135] The phrase "substantially from a human immunoglobulin or
antibody" or "substantially human" means that, when aligned to a
human immunoglobulin or antibody amino sequence for comparison
purposes, the region shares at least 80-90%, preferably 90-95%,
more preferably 95-99% identity (i.e., local sequence identity)
with the human framework or constant region sequence, allowing, for
example, for conservative substitutions, consensus sequence
substitutions, germline substitutions, backmutations, and the like.
The introduction of conservative substitutions, consensus sequence
substitutions, germline substitutions, backmutations, and the like,
is often referred to as "optimization" of a humanized antibody or
chain. The phrase "substantially from a non-human immunoglobulin or
antibody" or "substantially nonhuman" means having an
immunoglobulin or antibody sequence at least 80-95%, preferably
90-95%, more preferably, 96%, 97%, 98%, or 99% identical to that of
a non-human organism, e.g., a non-human mammal.
[0136] Accordingly, all regions or residues of a humanized
immunoglobulin or antibody, or of a humanized immunoglobulin or
antibody chain, except possibly the CDRs, are substantially
identical to the corresponding regions or residues of one or more
native human immunoglobulin sequences. The term "corresponding
region" or "corresponding residue" refers to a region or residue on
a second amino acid or nucleotide sequence which occupies the same
(i.e., equivalent) position as a region or residue on a first amino
acid or nucleotide sequence, when the first and second sequences
are optimally aligned for comparison purposes.
[0137] The terms "humanized immunoglobulin" or "humanized antibody"
are not intended to encompass chimeric immunoglobulins or
antibodies, as defined infra. Although humanized immunoglobulins or
antibodies are chimeric in their construction (i.e., comprise
regions from more than one species of protein), they include
additional features (i.e., variable regions comprising donor CDR
residues and acceptor framework residues) not found in chimeric
immunoglobulins or antibodies, as defined herein.
[0138] The term "significant identity" means that two polypeptide
sequences, when optimally aligned, such as by the programs GAP or
BESTFIT using default gap weights, share at least 50-60% sequence
identity, preferably 60-70% sequence identity, more preferably
70-80% sequence identity, more preferably at least 80-90% identity,
even more preferably at least 90-95% identity, and even more
preferably at least 95% sequence identity or more (e.g., 99%
sequence identity or more). The term "substantial identity" means
that two polypeptide sequences, when optimally aligned, such as by
the programs GAP or BESTFIT using default gap weights, share at
least 80-90% sequence identity, preferably 90-95% sequence
identity, and more preferably at least 95% sequence identity or
more (e.g., 99% sequence identity or more). For sequence
comparison, typically one sequence acts as a reference sequence, to
which test sequences are compared. When using a sequence comparison
algorithm, test and reference sequences are input into a computer,
subsequence coordinates are designated, if necessary, and sequence
algorithm program parameters are designated. The sequence
comparison algorithm then calculates the percent sequence identity
for the test sequence(s) relative to the reference sequence, based
on the designated program parameters. The terms "sequence identity"
and "sequence identity" are used interchangeably herein.
[0139] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Watennan, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 57Science Dr., Madison, Wis.), or by visual
inspection (see generally Ausubel et al., Current Protocols in
Molecular Biology). One example of algorithm that is suitable for
determining percent sequence identity and sequence similarity is
the BLAST algorithm, which is described in Altschul et al., J. Mol.
Biol. 215:403 (1990). Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (publicly accessible through the National Institutes of
Health NCBI internet server). Typically, default program parameters
can be used to perform the sequence comparison, although customized
parameters can also be used. For amino acid sequences, the BLASTP
program uses as defaults a wordlength (W) of 3, an expectation (E)
of 10, and the BLOSUM62 scoring matrix (see Henikoff &
Henikoff, Proc. Natl. Acad. Sci. USA 89:1091 (1989)).
[0140] Preferably, residue positions which are not identical differ
by conservative amino acid substitutions. For purposes of
classifying amino acids substitutions as conservative or
nonconservative, amino acids are grouped as follows: Group I
(hydrophobic sidechains): leu, met, ala, val, leu, ile; Group II
(neutral hydrophilic side chains): cys, ser, thr; Group III (acidic
side chains): asp, glu; Group IV (basic side chains): asn, gln,
his, lys, arg; Group V (residues influencing chain orientation):
gly, pro; and Group VI (aromatic side chains): trp, tyr, phe.
Conservative substitutions involve substitutions between amino
acids in the same class. Non-conservative substitutions constitute
exchanging a member of one of these classes for a member of
another.
[0141] Preferably, humanized immunoglobulins or antibodies bind
antigen with an affinity that is within a factor of three, four, or
five of that of the corresponding nonhuman antibody. For example,
if the nonhuman antibody has a binding affinity of 10.sup.9
M.sup.-1, humanized antibodies will have a binding affinity of at
least 3.times.10.sup.9 M.sup.-1 4.times.10.sup.9 M.sup.-1 or
10.sup.9 M.sup.-1. When describing the binding properties of an
immunoglobulin or antibody chain, the chain can be described based
on its ability to "direct antigen (e.g., MCP-1) binding". A chain
is said to "direct antigen binding" when it confers upon an intact
immunoglobulin or antibody (or antigen binding fragment thereof) a
specific binding property or binding affinity. A mutation (e.g., a
backmutation) is said to substantially affect the ability of a
heavy or light chain to direct antigen binding if it affects (e.g.,
decreases) the binding affinity of an intact immunoglobulin or
antibody (or antigen binding fragment thereof) comprising said
chain by at least an order of magnitude compared to that of the
antibody (or antigen binding fragment thereof) comprising an
equivalent chain lacking said mutation. A mutation "does not
substantially affect (e.g., decrease) the ability of a chain to
direct antigen binding" if it affects (e.g., decreases) the binding
affinity of an intact immunoglobulin or antibody (or antigen
binding fragment thereof) comprising said chain by only a factor of
two, three, or four of that of the antibody (or antigen binding
fragment thereof) comprising an equivalent chain lacking said
mutation.
[0142] The term "chimeric immunoglobulin" or antibody refers to an
immunoglobulin or antibody whose light and heavy chains are derived
from different species. Chimeric immunoglobulins or antibodies can
be constructed, for example by genetic engineering, from
immunoglobulin gene segments belonging to different species.
[0143] An "antigen" is an entity (e.g., a proteinaceous entity or
peptide) to which an antibody specifically binds.
[0144] The term "epitope" or "antigenic determinant" refers to a
site on an antigen to which an immunoglobulin or antibody (or
antigen binding fragment thereof) specifically binds. Epitopes can
be formed both from contiguous amino acids or noncontiguous amino
acids juxtaposed by tertiary folding of a protein. Epitopes formed
from contiguous amino acids are typically retained on exposure to
denaturing solvents whereas epitopes formed by tertiary folding are
typically lost on treatment with denaturing solvents. An epitope
typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14
or 15 1amino acids in a unique spatial conformation. Methods of
determining spatial conformation of epitopes include, for ex ample,
x-ray crystallography and 2-dimensional nuclear magnetic resonance.
See, e.g., Epitope Mapping Protocols in Methods in Molecular
Biology, Vol. 66, G. E. Morris, Ed. (1996).
[0145] Antibodies that recognize the same epitope can be identified
in a simple immunoassay showing the ability of one antibody to
block the binding of another antibody to a target antigen, i.e., a
competitive binding assay. Competitive binding is determined in an
assay in which the immunoglobulin under test inhibits specific
binding of a reference antibody to a common antigen, such as MCP-1.
Numerous types of competitive binding assays are known, for
example: solid phase direct or indirect radioimmunoassay (RIA),
solid phase direct or indirect enzyme immunoassay (EIA), sandwich
competition assay (see Stahli et al., Methods in Enzymology 9:242
(1983)); solid phase direct biotin-avidin EIA (see Kirkland et al.,
J. Immunol. 137:3614 (1986)); solid phase direct labeled assay,
solid phase direct labeled sandwich assay (see Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988));
solid phase direct label RIA using 1-125 label (see Morel et al.,
Mol. Immunol. 25(1):7 (1988); solid phase direct biotin-avidin EIA
(Cheung et al., Virology 176:546 (1990); and direct labeled RIA.
(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990). Typically,
such an assay involves the use of purified antigen bound to a solid
surface or cells bearing either of these, an unlabeled test
immunoglobulin and a labeled reference immunoglobulin. Competitive
inhibition is measured by determining the amount of label bound to
the solid surface or cells in the presence of the test
immunoglobulin. Usually the test immunoglobulin is present in
excess. Usually, when a competing antibody is present in excess, it
will inhibit specific binding of a reference antibody to a common
antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% or
more.
[0146] An epitope is also recognized by immunologie cells, for
example, B cells and/or T cells. Cellular recognition of an epitope
can be determined by in vitro assays that measure antigen-dependent
proliferation, as determined by .sup.3H-thymidine incorporation, by
cytokine secretion, by antibody secretion, or by antigen-dependent
killing (cytotoxic T lymphocyte assay).
[0147] Exemplary epitopes or antigenic determinants can be found
within human MCP molecules, and are preferably within MCP-1, MCP-2,
and MCP-3. Other preferred epitopes are those that are commonly
found within MCP-1 and MCP-2, MCP-1 and MCP-3, MCP-1 and MCP-3, and
MCP-1, MCP-2 and MCP-3.
[0148] As used herein, the term "b-chemokine" refers to a
polypeptide containing four conserved cysteine residues
characteristic of b-chemokines (e.g., as described in inflammation
(Van Coillie et al. (1999) Cytokine & Growth Factor Rev.
10:61-86) wherein the first two conserved cysteines are
adjacent.
[0149] As used herein, the term "inhibiting the activity of
b-chemokines" refers to causing a decrease in the relative activity
of b-chemokines in the presence of the antibody or antigen-binding
fragment thereof in comparison with the activity observed in the
absence of the antibody or antigen-binding fragment thereof. The
term "inhibits MCP activity" is defined herein as reducing or
eliminating activity associated with MCPs, e.g. MCP-1, MCP-2, or
MCP-3, for example by, reducing or inhibiting MCP-induced
chemotaxis and/or by reducing or inhibiting MCP-induced collagen
expression and/or by reducing or inhibiting MCP-induced
angiogenesis. Activities associated with MCP-induction can be
assayed according to standard methods known in the art, and as
described herein.
[0150] As used herein, the term "sign of an inflammatory disorder"
refers to observable or measurable indications of pathological
inflammation, including, but not limited to edema, fever,
emigration of leukocytes, proliferation of blood vessels,
proliferation of connective tissue, redness, localized heat,
exudation, and other signs as described in ROBBINS PATHOLOGIC BASIS
OF DISEASE, 4t EDITION, R. S. Cotran et al., Eds. W.B. Saunders,
Co., 1989.
[0151] As used herein, the term "blocking chemotaxis" refers to a
decrease in the relative amount of chemotactic activity of cells in
the presence of the antibody or antigen-binding fragment thereof in
comparison with chemotactic activity observed in the absence of the
antibody or antigen-binding fragment thereof.
[0152] As used herein, the term "MCP MRHAS Motif" refers to an
amino acid motif in human MCP-1 and MCP-3 termed Meningitis Related
Homologous Antigenic Sequence. For human MCP-1, the MRHAS amino
acid motif is Gln-Thr-Gln-Thr-Pro-Lys-Thr (SEQ ID NO: 1); and for
human MCP-3, the MRHAS motif is Lys-Thr-Gln-Thr-Pro-Lys-Leu (SEQ ID
NO:2).
[0153] The term "effective dose" or "effective dosage" is defined
as an amount sufficient to achieve or at least partially achieve
the desired effect. The term "therapeutically effective dose" is
defined as an amount sufficient to cure or at least partially
arrest the disease and its complications in a subject already
suffering from the disease. Amounts effective for this use will
depend upon the severity of the infection and the general state of
the subject's own immune system.
[0154] The term "subject" includes human and other mammalian
subjects that receive either prophylactic or therapeutic
treatment.
I. Immunological and Therapeutic Reagents
[0155] Immunological and therapeutic reagents of the invention
comprise or consist of immunogens or antibodies, or functional or
antigen binding fragments thereof, as defined herein. The basic
antibody structural unit is known to comprise a tetramer of
subunits. Each tetramer is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kDa) and
one "heavy" chain (about 50-70 kDa). The amino-terminal portion of
each chain includes a variable region of about 100 to 110 more
amino acids primarily responsible for antigen recognition. The
carboxy-terminal portion of each chain defines a constant region
primarily responsible for effector function.
[0156] Light chains are classified as either kappa or lambda and
are about 230 residues in length. Heavy chains are classified as
gamma (.gamma.), mu (.mu.), alpha (.alpha.), delta (.delta.), or
epsilon (.epsilon.), are about 450-600 residues in length, and
define the antibody's isotype as IgG, IgM, IgA, IgD and IgE,
respectively. Both heavy and light chains are folded into domains.
The term "domain" refers to a globular region of a protein, for
example, an immunoglobulin or antibody. Immunoglobulin or antibody
domains include, for example, 3 or four peptide loops stabilized by
.beta.-pleated sheet and an interchain disulfide bond. Intact light
chains have, for example, two domains (V.sub.L and C.sub.L) and
intact heavy chains have, for example, four or five domains
(V.sub.H, C.sub.H1, C.sub.H2, and C.sub.H3).
[0157] Within light and heavy chains, the variable and constant
regions are joined by a "J" region of about 12 or more amino acids,
with the heavy chain also including a "D" region of about 10 more
amino acids. (See generally, Fundamental Immunology (Paul, W., ed.,
2nd ed. Raven Press, N.Y. (1989), Ch. 7, incorporated by reference
in its entirety for all purposes).
[0158] The variable regions of each light/heavy chain pair form the
antibody binding site. Thus, an intact antibody has two binding
sites. Except in bifunctional or bispecific antibodies, the two
binding sites are the same. The chains all exhibit the same general
structure of relatively conserved framework regions (FR) joined by
three hypervariable regions, also called complementarity
determining regions or CDRs. Naturally-occurring chains or
recombinantly produced chains can be expressed with a leader
sequence which is removed during cellular processing to produce a
mature chain. Mature chains can also be recombinantly produced
having a non-naturally occurring leader sequence, for example, to
enhance secretion or alter the processing of a particular chain of
interest.
[0159] The CDRs of the two mature chains of each pair are aligned
by the framework regions, enabling binding to a specifie epitope.
From N-terminal to C-terminal, both light and heavy chains comprise
the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. "FR4" also is
referred to in the art as the D/J region of the variable heavy
chain and the J region of the variable light chain. The assignment
of amino acids to each domain is in accordance with the definitions
of Kabat, Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md., 1987 and 1991). An alternative
structural definition has been proposed by Chothia et al., J. Mol.
Biol. 196:901 (1987); Nature 342:878 (1989); and J. Mol. Biol.
186:651 (1989) (hereinafter collectively referred to as "Chothia et
al." and incorporated by reference in their entirety for all
purposes).
A. MCP Antibodies
[0160] Therapeutic agents of the invention include antibodies that
specifically bind to MCPs or other b-chemokines. Such antibodies
can be monoclonal or polyclonal. Some such antibodies bind
specifically MCP-1. Some bind specifically to MCP-2. Some bind to
both MCP-1 and MCP-2. Some such antibodies bind to MCP-3.
Antibodies used in therapeutic methods preferably have an intact
constant region or at least sufficient of the constant region to
interact with an Fc receptor. Human isotype IgG1 is preferred
because of it having highest affinity of human isotypes for the
FcRI receptor on phagocytic cells. Bispecific Fab fragments can
also be used, in which one arm of the antibody has specificity for
MCP-1, MCP-2, MCP-3, or a combination thereof, and the other for an
Fc receptor. Preferred antibodies bind to MCP-1 with a binding
affinity greater than (or equal to) about 10.sup.6, 10.sup.7,
10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, or 10.sup.13
M.sup.-1 (including affinities intermediate of these values).
[0161] In certain embodiments the antibodies of the invention, and
fragments thereof, bind to regions in the b-chemokines (e.g.,
MCP-1, MCP-2 and MCP-3). In some embodiments, the antibodies or
antigen-binding fragments thereof bind MCP-2 and at least one other
b-chernokine (e.g., MCP-1 or MCP-3). Thus in some embodiments, the
antibodies or fragments thereof bind MCP-1 and MCP-2 and in other
embodiments the antibodies or fragments thereof bind MCP2 and
MCP-3. In other embodiments, the antibodies or antigen-binding
fragments thereof bind MCP-1, MCP-2 and MCP-3. In other
embodiments, the antibodies or antigen-binding fragments thereof
bind MCP-1 and MCP-3 other than to regions containing the MRHAS
motifs, QTQTPKT (MCP-1) and KTQTPKL (MCP-3).
[0162] In certain embodiments, the antibodies or antigen-binding
fragments thereof comprise antibodies selected from the group
consisting of 11K2.1 (ATCC Accession No. PTA-3987), 6D21.1 (ATCC
Accession No. PTA-3989), 4N4.1 (ATCC Accession No. PTA-3994),
5A13.1 (ATCC Accession No. PTA-3995), 7H1.1 (ATCC Accession No.
PTA-3985), 1A1.1 (ATCC Accession No. PTA-3990), 615.1 (ATCC
Accession No. PTA-3986), 2024.1 (ATCC Accession No. PTA-3993),
9B11.1 (ATCC Accession No. PTA-3992), 9B12.1 (ATCC Accession No.
PTA-3996), 9C11.1 (ATCC Accession No. PTA-3988), and 12F15.1 (ATCC
Accession No. PTA-3991), or antigen-binding fragments of these
antibodies.
[0163] Polyclonal sera typically contain mixed populations of
antibodies binding to several epitopes, including, for example,
MCP-1. Monoclonal antibodies bind to a specific epitope within
b-chemokines, e.g., MCP-1, that can be a conformational or
nonconformational epitope. Preferred monoclonal antibodies bind to
an epitope within MCP-1 or MCP-2. Some preferred monoclonal
antibodies bind to an epitope within MCP-1 and MCP-2, and some to
an epitope within MCP-1, MCP-2, and MCP-3. It is recommended that
such antibodies be screened for activity in the mouse models before
use.
[0164] When an antibody is said to bind to an epitope within
specified residues of a certain b-chemokine, such as MCP-1 for
example, what is meant is that the antibody specifically binds to a
polypeptide containing the specified residues. Such an antibody
does not necessarily contact every residue, nor does every single
amino acid substitution or deletion with the specified necessarily
significantly affect binding affinity. Epitope specificity of an
antibody can be determined, for example, by forming a phage display
library in which different members display different subsequences
of MCPs. The phage display library is then selected for members
specifically binding to an antibody under test. A family of
sequences is isolated. Typically, such a family contains a common
core sequence, and varying lengths of flanking sequences in
different members. The shortest core sequence showing specific
binding to the antibody defines the epitope bound by the antibody.
Antibodies can also be tested for epitope specificity in a
competition assay with an antibody whose epitope specificity has
already been determined. For example, antibodies that compete with
the 11K2 antibody for binding to MCP-1 bind to the same or similar
epitope as 11K2. Likewise antibodies that compete with the 1A1
antibody bind to the same or similar epitope. Screening antibodies
for epitope specificity is a useful predictor of therapeutic
efficacy. In one embodiment, the invention includes an anti-MCP
antibody with a heavy chain which contacts residues R30, T32, S34,
K38, E39, V41, P55, K56, Q61, M64 of MCP-1. The invention also
includes a an anti-MCP antibody with a light chain which contacts
residues D65, D68, K69 of MCP-1.
[0165] 1. Production of Nonhuman Antibodies
[0166] The present invention features non-human antibodies, for
example, antibodies having specificity for the preferred MCP
epitopes of the invention. Such antibodies can be used in
formulating various therapeutic compositions of the invention or,
preferably, provide complementarity determining regions for the
production of humanized or chimeric antibodies (described in detail
below). The production of nonhuman monoclonal antibodies, e.g.,
murine, guinea pig, primate, rabbit or rat, can be accompli shed
by, for example, immunizing the animal with at least one
b-chemokine. In producing the antibodies of the invention, the
immunogens maybe a preparation containing at least one b-chemokine,
preferably more than one b-chemokine. In some embodiments, the
b-chemokines are human monocyte chemotactic proteins (MCPs),
including MCP-1, MCP-2 and MCP-3. The b-chemokines may be native or
recombinantly produced b-chemokines. In some embodiments, the
immunogens may be antigenic fragments of b-chemokines, such as
fragments of MCPs which may optionally be conjugated to a carrier
molecule to impart a stronger immune response upon administration
to an animal. The b-chemokine immunogens, such as MCPs may contain
additions, deletions and/or substitutions of amino acids, provided
that the alterations do not ablate antigenicity of the mutated
b-chemokines such that antibodies against the mutant versions do
not bind native b-chemokines. Preferably, the amino acid
substitutions are conservative changes in the amino acid sequence,
provided the MCP molecules remain antigenic. See Harlow & Lane,
supra, incorporated by reference for all purposes).
[0167] Such an immunogen can be obtained from a natural source, by
peptide synthesis or by recombinant expression. Optionally, the
immunogen can be administered fused or otherwise complexed with a
carrier protein, as described below. Optionally, the immunogen can
be administered with an adjuvant. The term "adjuvant" refers to a
compound that when administered in conjunction with an antigen
augments the immune response to the antigen, but when administered
alone does not generate an immune response to the antigen.
Adjuvants can augment an immune response by several mechanisms
including lymphocyte recruitment, stimulation of B and/or T cells,
and stimulation of macrophages. Several types of adjuvant can be
used as described below. Complete Freund's adjuvant followed by
incomplete adjuvant is preferred for immunization of laboratory
animals.
[0168] Rabbits or guinea pigs are typically used for making
polyclonal antibodies. Exemplary preparation of polyclonal
antibodies, e.g., for passive protection, can be performed as
follows. 125 non-transgenic mice are immunized with 100 .mu.g of a
MCP-1/MCP-2 cocktail, plus CFA/IFA adjuvant, and euthanized at 4-5
months. Blood is collected from immunized mice. IgG is separated
from other blood components. Antibody specific for the immunogen
may be partially purified by affinity chromatography. An average of
about 0.5-1 mg of immunogen-specific antibody is obtained per
mouse, giving a total of 60-120 mg.
[0169] Mice are typically used for making monoclonal antibodies.
Monoclonals can be prepared against a fragment by injecting, for
example, a fragment of MCP-1 into a mouse, preparing hybridomas and
screening the hybridomas for an antibody that specifically binds to
MCP-1. Optionally, antibodies are screened for binding to a
specific region or desired fragment of MCP-1 without binding to
other nonoverlapping fragments of MCP-1. The latter screening can
be accomplished by determining binding of an antibody to a
collection of deletion mutants of a MCP-1 peptide and determining
which deletion mutants bind to the antibody. Binding can be
assessed, for example, by Western blot or ELISA. The smallest
fragment to show specific binding to the antibody defines the
epitope of the antibody. Alternatively, epitope specificity can be
determined by a competition assay is which a test and reference
antibody compete for binding to MCP-1. If the test and reference
antibodies compete, then they bind to the same epitope or epitopes
sufficiently proximal such that binding of one antibody interferes
with binding of the other. The preferred isotype for such
antibodies is mouse isotype IgG2a or equivalent isotype in other
species. Mouse isotype IgG2a is the equivalent of human isotype
IgG1.
[0170] 2. Chimeric and Humanized Antibodies
[0171] The present invention also features chimeric and/or
humanized antibodies (i.e., chimeric and/or humanized
immunoglobulins) specific for b-chemokines, including MCPs.
Chimeric and/or humanized antibodies have the same or similar
binding specificity and affinity as a mouse or other nonhuman
antibody that provides the starting material for construction of a
chimeric or humanized antibody.
[0172] In one embodiment, the CDRs of the 11K2 antibody can be used
to produce humanized and chimeric antibodies. The invention
features an isolated antibody, or antigen binding portion thereof,
which binds a plurality of b-chemokines, wherein said b-chemokines
comprise MCP-2 and at least one other b-chemokine, which comprises
at least one of the following CDRs: CDR1, CDR2, or CDR3, from the
11K2 heavy chain variable region described in SEQ ID NO: 27. In an
additional embodiment, the invention features an isolated antibody,
or antigen binding portion thereof, which binds a plurality of
b-chemokines, wherein said b-chemokines comprise MCP-2 and at least
one other b-chemokine, which comprises at least one of the
following CDR combinations: CDR1 and CDR2; CDR1 and CDR3; CDR2 and
CDR3; or CDR1, CDR2, and CDR3, from the 11K2 heavy chain variable
region described in SEQ ID NO: 27. In one embodiment, the antibody
of the invention is a chimeric antibody. In another embodiment, the
antibody of the invention is a humanized antibody.
[0173] The CDRs of the 1A1 antibody can be also used to produce
humanized and chimeric antibodies. In one embodiment, the invention
provides an isolated antibody, or antigen binding portion thereof,
which binds a plurality of b-chemokines, wherein said b-chemokines
comprise MCP-2 and at least one other b-chemokine, which comprises
at least one of the following CDRs: CDR1, CDR2, or CDR3, from the
1A1 heavy chain variable region described in SEQ ID NO: 11. In
another embodiment, the invention provides an isolated antibody, or
antigen binding portion thereof, which binds a plurality of
b-chemokines, wherein said b-chemokines comprise MCP-2 and at least
one other b-chemokine, which comprises at least one or the
following CDR combinations: CDR1 and CDR2; CDR1 and CDR3; CDR2 and
CDR3; and CDR1, CDR2, and CDR3, from the 1A1 heavy chain variable
region described in SEQ ID NO: 11. In one embodiment, the antibody
of the invention is a chimeric antibody. In another embodiment, the
antibody of the invention is a humanized antibody.
[0174] a. Production of Chimeric Antibodies
[0175] The term "chimeric antibody" refers to an antibody whose
light and heavy chain genes have been constructed, typically by
genetic engineering, from immunoglobulin gene segments belonging to
different species. For example, the variable (V) segments of the
genes from a mouse monoclonal antibody may be joined to human
constant (C) segments, such as IgG1 and IgG4. Human isotype IgG1 is
preferred. A typical chimeric antibody is thus a hybrid protein
consisting of the V or antigen-binding domain from a mouse antibody
and the C or effector domain from a human antibody.
[0176] b. Production of Humanized Antibodies
[0177] The term "humanized antibody" refers to an antibody
comprising at least one chain comprising variable region framework
residues substantially from a human antibody chain (referred to as
the acceptor immunoglobulin or antibody) and at least one
complementarity determining region substantially from a
mouse-antibody, (referred to as the donor immunoglobulin or
antibody). See, Queen et al., Proc. Natl. Acad. Sci. USA
86:10029-10033 (1989), U.S. Pat. No. 5,530,101, U.S. Pat. No.
5,585,089, U.S. Pat. No. 5,693,761, U.S. Pat. No. 5,693,762, Selick
et al., WO 90/07861, and Winter, U.S. Pat. No. 5,225,539
(incorporated by reference in their entirety for all purposes). The
constant region(s), if present, are also substantially or entirely
from a human immunoglobulin.
[0178] The substitution of mouse CDRs into a human variable domain
framework is most likely to result in retention of their correct
spatial orientation if the human variable domain framework adopts
the same or similar conformation to the mouse variable framework
from which the CDRs originated. This is achieved by obtaining the
human variable domains from human antibodies whose framework
sequences exhibit a high degree of sequence identity with the
murine variable framework domains from which the CDRs were derived.
The heavy and light chain variable framework regions can be derived
from the same or different human antibody sequences. The human
antibody sequences can be the sequences of naturally occurring
human antibodies or can be consensus sequences of several human
antibodies. See Kettleborough et al., Protein Engineering 4:773
(1991); Kolbinger et al., Protein Engineering 6:971 (1993) and
Carter et al., WO 92/22653.
[0179] Having identified the complementarity determining regions of
the murine donor immunoglobulin and appropriate human acceptor
immunoglobulins, the next step is to determine which, if any,
residues from these components should be substituted to optimize
the properties of the resulting humanized antibody. In general,
substitution of human amino acid residues with murine should be
minimized, because introduction of murine residues increases the
risk of the antibody eliciting a human-anti-mouse-antibody (HAMA)
response in humans. Art-recognized methods of determining immune
response can be performed to monitor a HAMA response in a
particular subject or during clinical trials. Subjects administered
humanized antibodies can be given an immunogenicity assessment at
the beginning and throughout the administration of said therapy.
The HAMA response is measured, for example, by detecting antibodies
to the humanized therapeutic reagent, in serum samples from the
subject using a method known to one in the art, including surface
plasmon resonance technology (BIACORE) and/or solid-phase ELISA
analysis.
[0180] Certain amino acids from the human variable region framework
residues are selected for substitution based on their possible
influence on CDR conformation and/or binding to antigen. The
unnatural juxtaposition of murine CDR regions with human variable
framework region can result in unnatural conformational restraints,
which, unless corrected by substitution of certain amino acid
residues, lead to loss of binding affinity.
[0181] The selection of amino acid residues for substitution is
determined, in part, by computer modeling. Computer hardware and
software are described herein for producing three-dimensional
images of immunoglobulin molecules. In general, molecular models
are produced starting from solved structures for immunoglobulin
chains or domains thereof. The chains to be modeled are compared
for amino acid sequence similarity with chains or domains of solved
three-dimensional structures, and the chains or domains showing the
greatest sequence similarity is/are selected as starting points for
construction of the molecular model. Chains or domains sharing at
least 50% sequence identity are selected for modeling, and
preferably those sharing at least 60%, 70%, 80%, 90% sequence
identity or more are selected for modeling. The solved starting
structures are modified to allow for differences between the actual
amino acids in the immunoglobulin chains or domains being modeled,
and those in the starting structure. The modified structures are
then assembled into a composite immunoglobulin. Finally, the model
is refined by energy minimization and by verifying that all atoms
are within appropriate distances from one another and that bond
lengths and angles are within chemically acceptable limits.
[0182] The selection of amino acid residues for substitution can
also be determined, in part, by examination of the characteristics
of the amino acids at particular locations, or empirical
observation of the effects of substitution or mutagenesis of
particular amino acids. For example, when an amino acid differs
between a murine variable region framework residue and a selected
human variable region framework residue, the human framework amino
acid should usually be substituted by the equivalent framework
amino acid from the mouse antibody when it is reasonably expected
that the amino acid: [0183] (1) noncovalently binds antigen
directly, [0184] (2) is adjacent to a CDR region, [0185] (3)
otherwise interacts with a CDR region (e.g., is within about 3-6
.ANG. of a CDR region as determined by computer modeling), or
[0186] (4) participates in the VL-VH interface.
[0187] Residues which "noncovalently bind antigen directly" include
amino acids in positions in framework regions which are have a good
probability of directly interacting with amino acids on the antigen
according to established chemical forces, for example, by hydrogen
bonding, Van der Waals forces, hydrophobic interactions, and the
like.
[0188] CDR and framework regions are as defined by Kabat et al. or
Chothia et al., supra. When framework residues, as defined by Kabat
et al., supra, constitute structural loop residues as defined by
Chothia et al., supra, the amino acids present in the mouse
antibody may be selected for substitution into the humanized
antibody. Residues which are "adjacent to a CDR region" include
amino acid residues in positions immediately adjacent to one or
more of the CDRs in the primary sequence of the humanized
immunoglobulin chain, for example, in positions immediately
adjacent to a CDR as defined by Kabat, or a CDR as defined by
Chothia (See e.g., Chothia and Lesk JMB 196:901 (1987)). These
amino acids are particularly likely to interact with the amino
acids in the CDRs and, if chosen from the acceptor, to distort the
donor CDRs and reduce affinity. Moreover, the adjacent amino acids
may interact directly with the antigen (Amit et al., Science,
233:747 (1986), which is incorporated herein by reference) and
selecting these amino acids from the donor may be desirable to keep
all the antigen contacts that provide affinity in the original
antibody.
[0189] Residues that "otherwise interact with a CDR region" include
those that are determined by secondary structural analysis to be in
a spatial orientation sufficient to effect a CDR region. In one
embodiment, residues that "otherwise interact with a CDR region"
are identified by analyzing a three-dimensional model of the donor
immunoglobulin (e.g., a computer-generated model). A
three-dimensional model, typically of the original donor antibody,
shows that certain amino acids outside of the CDRs are close to the
CDRs and have a good probability of interacting with amino acids in
the CDRs by hydrogen bonding, Van der Waals forces, hydrophobic
interactions, etc. At those amino acid positions, the donor
immunoglobulin amino acid rather than the acceptor immunoglobulin
amino acid may be selected. Amino acids according to this criterion
will generally have a side chain atom within about 3 angstrom units
(.ANG.) of some atom in the CDRs and must contain an atom that
could interact with the CDR atoms according to established chemical
forces, such as those listed above.
[0190] In the case of atoms that may form a hydrogen bond, the 3
.ANG. is measured between their nuclei, but for atoms that do not
form a bond, the 3 .ANG. is measured between their Van der Waals
surfaces. Hence, in the latter case, the nuclei must be within
about 6 .ANG. (3 .ANG. plus the sum of the Van der Waals radii) for
the atoms to be considered capable of interacting. In many cases
the nuclei will be from 4 or 5 to 6 .ANG. apart. In determining
whether an amino acid can interact with the CDRs, it is preferred
not to consider the last 8 amino acids of heavy chain CDR 2 as part
of the CDRs, because from the viewpoint of structure, these 8 amino
acids behave more as part of the framework.
[0191] Amino acids that are capable of interacting with amino acids
in the CDRs, may be identified in yet another way. The solvent
accessible surface area of each framework amino acid is calculated
in two ways: (1) in the intact antibody, and (2) in a hypothetical
molecule consisting of the antibody with its CDRs removed. A
significant difference between these numbers of about 10 square
angstroms or more shows that access of the framework amino acid to
solvent is at least partly blocked by the CDRs, and therefore that
the amino acid is making contact with the CDRs. Solvent accessible
surface area of an amino acid may be calculated based on a
three-dimensional model of an antibody, using algorithms known in
the art (e.g., Connolly, J. Appl. Cryst. 16:548 (1983) and Lee and
Richards, J. Mol. Biol. 55:379 (1971), both of which are
incorporated herein by reference). Framework amino acids may also
occasionally interact with the CDRs indirectly, by affecting the
conformation of another framework amino acid that in turn contacts
the CDRs.
[0192] The amino acids at several positions in the framework are
known to be capable of interacting with the CDRs in many antibodies
(Chothia and Lesk, supra, Chothia et al., supra and Tramontano et
al., J. Mol. Biol. 215:175 (1990), all of which are incorporated
herein by reference). Notably, the amino acids at positions 2, 48,
64 and 71 of the light chain and 26-30, 71 and 94 of the heavy
chain (numbering according to Kabat) are known to be capable of
interacting with the CDRs in many antibodies. The amino acids at
positions 35 in the light chain and 93 and 103 in the heavy chain
are also likely to interact with the CDRs. At all these numbered
positions, choice of the donor amino acid rather than the acceptor
amino acid (when they differ) to be in the humanized immunoglobulin
is preferred. On the other hand, certain residues capable of
interacting with the CDR region, such as the first amino acids of
the light chain, may sometimes be chosen from the acceptor
immunoglobulin without loss of affinity in the humanized
immunoglobulin.
[0193] Residues which "participate in the VL-VH interface" or
"packing residues" include those residues at the interface between
VL and VH as defined, for example, by Novotny and Haber, Proc.
Natl. Acad. Sci. USA, 82:4592-66 (1985) or Chothia et al, supra.
Generally, unusual packing residues should be retained in the
humanized antibody if they differ from those in the human
frameworks.
[0194] In general, one or more of the amino acids fulfilling the
above criteria is substituted. In some embodiments, all or most of
the amino acids fulfilling the above criteria are substituted.
Occasionally, there is some ambiguity about whether a particular
amino acid meets the above criteria, and alternative variant
immunoglobulins are produced, one of which has that particular
substitution, the other of which does not. Alternative variant
immunoglobulins so produced can be tested in any of the assays
described herein for the desired activity, and the preferred
immunoglobulin selected.
[0195] Usually the CDR regions in humanized antibodies are
substantially identical, and more usually, identical to the
corresponding CDR regions of the donor antibody. Although not
usually desirable, it is sometimes possible to make one or more
conservative amino acid substitutions of CDR residues without
appreciably affecting the binding affinity of the resulting
humanized immunoglobulin. By conservative substitutions is intended
combinations such as gly, ala; val, ile, leu; asp, glu; asn, gln;
ser, thr; lys, arg; and phc, tyr.
[0196] Additional candidates for substitution are acceptor human
framework amino acids that are "unusual" or "rare" for a human
immunoglobulin at that position. These amino acids can be
substituted with amino acids from the equivalent position of the
mouse donor antibody or from the equivalent positions of more
typical human immunoglobulins. For example, substitution may be
desirable when the amino acid in a human framework region of the
acceptor immunoglobulin is rare for that position and the
corresponding amino acid in the donor immunoglobulin is common for
that position in human immunoglobulin sequences; or when the amino
acid in the acceptor immunoglobulin is rare for that position and
the corresponding amino acid in the donor immunoglobulin is also
rare, relative to other human sequences. These criterion help
ensure that an atypical amino acid in the human framework does not
disrupt the antibody structure. Moreover, by replacing an unusual
human acceptor amino acid with an amino acid from the donor
antibody that happens to be typical for human antibodies, the
humanized antibody may be made less immunogenic.
[0197] The term "rare", as used herein, indicates an amino acid
occurring at that position in less than about 20% but usually less
than about 10% of sequences in a representative sample of
sequences, and the term "common", as used herein, indicates an
amino acid occurring in more than about 25% but usually more than
about 50% of sequences in a representative sample. For example, all
human light and heavy chain variable region sequences are
respectively grouped into "subgroups" of sequences that are
especially homologous to each other and have the same amino acids
at certain critical positions (Kabat et al., supra). When deciding
whether an amino acid in a human acceptor sequence is "rare" or
"common" among human sequences, it will often be preferable to
consider only those human sequences in the same subgroup as the
acceptor sequence.
[0198] Additional candidates for substitution are acceptor human
framework amino acids that would be identified as part of a CDR
region under the alternative definition proposed by Chothia et al.,
supra. Additional candidates for substitution are acceptor human
framework amino acids that would be identified as part of a CDR
region under the AbM and/or contact definitions. Notably, CDR1 in
the variable heavy chain is defined as including residues
26-32.
[0199] Additional candidates for substitution are acceptor
framework residues that correspond to a rare or unusual donor
framework residue. Rare or unusual donor framework residues are
those that are rare or unusual (as defined herein) for murine
antibodies at that position. For murine antibodies, the subgroup
can be determined according to Kabat and residue positions
identified which differ from the consensus. These donor specific
differences may point to somatic mutations in the murine sequence
which enhance activity. Unusual residues that are predicted to
affect binding are retained, whereas residues predicted to be
unimportant for binding can be substituted.
[0200] Additional candidates for substitution are non-germline
residues occurring in an acceptor framework region. For example,
when an acceptor antibody chain (i.e., a human antibody chain
sharing significant sequence identity with the donor antibody
chain) is aligned to a germline antibody chain (likewise sharing
significant sequence identity with the donor chain), residues not
matching between acceptor chain framework and the germline chain
framework can be substituted with corresponding residues from the
germline sequence.
[0201] Other than the specific amino acid substitutions discussed
above, the .framework regions of humanized immunoglobulins are
usually substantially identical, and more usually, identical to the
framework regions of the human antibodies from which they were
derived. Of course, many of the amino acids in the framework region
make little or no direct contribution to the specificity or
affinity of an antibody. Thus, many individual conservative
substitutions of framework residues can be tolerated without
appreciable change of the specificity or affinity of the resulting
humanized immunoglobulin. Thus, in one embodiment the variable
framework region of the humanized immunoglobulin shares at least
85% sequence identity to a human variable framework region sequence
or consensus of such sequences. In another embodiment, the variable
framework region of the humanized immunoglobulin shares at least
90%, preferably 95%, more preferably 96%, 97%, 98% or 99% sequence
identity to a human variable framework region sequence or consensus
of such sequences. In general, however, such substitutions are
undesirable.
[0202] The humanized antibodies preferably exhibit a specific
binding affinity for antigen of at least 10.sup.7, 10.sup.8,
10.sup.9 or 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.-13 M.sup.-1.
Usually the upper limit of binding affinity of the humanized
antibodies for antigen is within a factor of three, four or five of
that of the donor immunoglobulin. Often the lower limit of binding
affinity is also within a factor of three, four or five of that of
donor immunoglobulin. Alternatively, the binding affinity can be
compared to that of a humanized antibody having no substitutions
(e.g., an antibody having donor CDRs and acceptor FRs, but no FR
substitutions). In such instances, the binding of the optimized
antibody (with substitutions) is preferably at least two- to
three-fold greater, or three- to four-fold greater, than that of
the unsubstituted antibody. For making comparisons, activity of the
various antibodies can be determined, for example, by BIACORE
(i.e., surface plasmon resonance using unlabelled reagents) or
competitive binding assays.
c. Production of 11K2 Humanized Antibodies
[0203] A preferred embodiment of the present invention features a
humanized antibody to MCPs, in particular, for use in therapeutic
and/or diagnostic methodologies described herein. A particularly
preferred starting material for production of humanized antibodies
is 11K2. 11K2 is a pan-MCP antibody and is specific for MCP-1,
MCP-2 and MCP-3. 11K2 has been shown to inhibit MCP-induced
chemotaxis (see Examples 3 and 5). The cloning and sequencing of
cDNA encoding the 11K2 antibody heavy and light chains is described
in Example 10.
[0204] Suitable human acceptor antibody sequences are identified by
computer comparisons of the amino acid sequences of the mouse
variable regions with the sequences of known human antibodies. The
comparison is performed separately for heavy and light chains but
the principles are similar for each. In particular, variable
domains from human antibodies whose framework sequences exhibit a
high degree of sequence identity with the murine VL and VH
framework regions were identified by query of the Kabat Database
using NCBI BLAST (publicly accessible through the National
Institutes of Health NCBI internet server) with the respective
murine framework sequences. In one embodiment, acceptor sequences
sharing greater that 50% sequence identity with murine donor
sequences are selected. Preferably, acceptor antibody sequences
sharing 60%, 70%, 80%, 90% or more are selected.
[0205] A computer comparison of 11K2 revealed that the 11K2 light
chain shows the greatest sequence identity to human light chains of
subtype kappa 1, and that the 11K2 heavy chain shows greatest
sequence identity to human heavy chains of subtype 1, as defined by
Kabat et al., supra. Thus, light and heavy human framework regions
are preferably derived from human antibodies of these subtypes, or
from consensus sequences of such subtypes. The preferred light
chain human variable regions showing greatest sequence identity to
the corresponding region from 11K2 are from antibodies GI-486875
(Griffiths et al. (1993) EMBO J. 12(2), 725-734). The preferred
heavy chain human variable regions showing greatest sequence
identity to the corresponding region from 11K2 are from antibodies
having Kabat ID Number 0000554 (Kipps and Duffy (1991), J. Clin.
Invest. 87 (6): 2087-2096).
[0206] Residues are next selected for substitution, as follows.
When an amino acid differs between a 11K2 variable framework region
and an equivalent human variable framework region, the human
framework amino acid should usually be substituted by the
equivalent mouse amino acid if it is reasonably expected that the
amino acid:
[0207] noncovalently binds antigen directly,
[0208] is adjacent to a CDR region, is part of a CDR region under
the alternative definition proposed by Chothia et al., supra, or
otherwise interacts with a
[0209] CDR region (e.g., is within about 3 of a CDR region) (e.g.
amino acids at positions H29, H73, L49 of 11K2), or
[0210] participates in the VL-VH interface
[0211] Computer modeling of the 11K2 antibody heavy and light chain
variable regions, and humanization of the 11K2 antibody is
described in Example 14. Briefly, a three-dimensional model was
generated based on the closest solved murine antibody structures
for the heavy and light chains. For this purpose, an antibody
designated 184.1 (Protein Data Bank (PDB) ID: 184.1) was chosen as
a template for modeling the 11K2 light chain, and an antibody
designated E8 (PDB ID: 1OPG) was chosen as the template for
modeling the heavy chain. The model was further refined by a series
of energy minimization steps to relieve unfavorable atomic contacts
and optimize electrostatic and van der Walls interactions.
[0212] Three-dimensional structural information for the antibodies
described herein is publicly available, for example, from the
Research Collaboratory for Structural Bioinformatics' Protein Data
Bank (PDB). The PDB is freely accessible via the World Wide Web
internet and is described by Berman et al. (2000) Nucleic Acids
Research, 28:235. Computer modeling allows for the identification
of CDR-interacting residues. The computer model of the structure of
11K2 can in turn serve as a starting point for predicting the
three-dimensional structure of an antibody containing the 11K2
complementarity determining regions substituted in human framework
structures. Additional models can be constructed representing the
structure as further amino acid substitutions are introduced.
[0213] In general, substitution of one, most or all of the amino
acids fulfilling the above criteria is desirable. Accordingly, the
humanized antibodies of the present invention will usually contain
a substitution of a human light chain framework residue with a
corresponding 11K2 residue in at least 1, 2, and more usually 3, of
the following positions: L49, L69 and L71. The humanized antibodies
also usually contain a substitution of a human heavy chain
framework residue with a corresponding 11K2 residue in at least 1,
2, 3, 4, 5, 6, and sometimes 7, of the following positions: H27,
H28, H29, H30, H48, H677, and H73.
[0214] In one embodiment, the humanized antibodies of the invention
are based on two versions of the humanized 11K2 variable heavy
chain (H1 and H2) and two versions of the humanized 11K2 variable
light chain (L1 and L2). The humanized antibody of the invention is
based on any combination of these heavy and light chains (e.g.
H1-L1, H1-L2, H2-L1, H2-L2). Version 1 of the humanized heavy and
light chains contains most backmutations (i.e. L49, L69, L71, H27,
H28, H29, H30, H48, H67, and H73), while version 2 contains the
fewest (i.e. L49, L71, H27, H29, and H73). The sequence of
humanized 11K2 variable heavy chain version 1 is set forth as SEQ
ID NO: 47, and version 2 is set forth as SEQ ID NO: 48. The
sequence of humanized 11K2 variable light chain version 1 is set
forth as SEQ ID NO: 49, and version 2 is set forth as SEQ ID NO:
50.
[0215] In one embodiment, the humanized antibody of the invention
contains a heavy chain comprising SEQ ID NO: 47 and a light chain
comprising SEQ ID NO: 49. In another embodiment, the humanized
antibody of the invention contains a heavy chain comprising SEQ ID
NO: 47 and a light chain comprising SEQ ID NO: 50. In yet another
embodiment, the humanized antibody of the invention contains a
heavy chain comprising SEQ ID NO: 48 and a light chain comprising
SEQ ID NO: 49. IN still another embodiment, the humanized antibody
of the invention contains a heavy chain comprising SEQ ID NO: 48
and a light chain comprising SEQ ID NO: 50.
[0216] Occasionally, however, there is some ambiguity about whether
a particular amino acid meets the above criteria, and alternative
variant immunoglobulins are produced, one of which has that
particular substitution, the other of which does not. In instances
where substitution with a murine residue would introduce a residue
that is rare in human immunoglobulins at a particular position, it
may be desirable to test the antibody for activity with or without
the particular substitution. If activity (e.g., binding affinity
and/or binding specificity) is about the same with or without the
substitution, the antibody without substitution may be preferred,
as it would be expected to elicit less of a HAHA response, as
described herein.
[0217] Other candidates for substitution are acceptor human
framework amino acids that are unusual for a human immunoglobulin
at that position. These amino acids can be substituted with amino
acids from the equivalent position of more typical human
immunoglobulins. Alternatively, amino acids from equivalent
positions in the mouse 11K2 can be introduced into the human
framework regions when such amino acids are typical of human
immunoglobulin at the equivalent positions.
[0218] In additional embodiments, when the human light chain
framework acceptor immunoglobulin is GI-486875, the light chain
contains substitutions in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more usually 11, of the following positions: L8, L11, L13, L15,
L42, LA5, L74, L76, L80, L83, or L104. In additional embodiments
when the human heavy chain framework acceptor immunoglobulin is
Kabat ID Number 000054, the heavy chain contains substitutions in
at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
or more usually 18, of the following positions: H1, H5, H11, H12,
H14, H16, H20, H23, H38, H40, H42, H66, H75, H76, H80, H81, H82C,
or H83. These positions are substituted with the amino acid from
the equivalent position of a human immunoglobulin having a more
typical amino acid residue. Examples of appropriate amino acids to
substitute are shown in FIGS. 11A and 11B. Table 1 summarizes the
sequence analysis of the 11K2 VH and VL regions.
TABLE-US-00001 TABLE 1 Summary of 11K2 V-region sequence Chain
Heavy Light Mouse subgroup 2C kappa 5 Human subgroup 1 kappa 1
Chothia canonical H1: 5residues, no class L1: 11 residues, class 2
CDR groupings H2: 17 residues, class 2 L2: 7 residues, class 1 H3:
8 residues, no class L3: 9 residues, class 1 Closest solved E8
184.1 mouse structure
[0219] Kabat ID sequences referenced herein are publicly available,
for example, from the Northwestern University Biomedical
Engineering Department's Kabat Database of Sequences of Proteins of
Immunological Interest. Three-dimensional structural information
for antibodies described herein is publicly available, for example,
from the Research Collaboratory for Structural Bioinformatics'
Protein Data Bank (PDB). The PDB is freely accessible via the World
Wide Web internet and is described by Berman et al. (2000) Nucleic
Acids Research, p 235-242. Germline gene sequences referenced
herein are publicly available, for example, from the National
Center for Biotechnology Information (NCBI) database of sequences
in collections of Igh, Ig kappa 2 and Ig lambda germline V genes
(as a division of the National Library of Medicine (NLM) at the
National Institutes of Health (NIH). Homology searching of the NCBI
"Ig Germline Genes" database is provided by IgG BLAST.TM..
[0220] d. Production of 1A1 Humanized Antibodies
[0221] Another preferred embodiment of the present invention
features a humanized antibody to MCPs, in particular, for use in
therapeutic and/or diagnostic methodologies described herein,
wherein the starting material for production of humanized
antibodies is 1A1. 1A1 is a pan-MCP antibody, and is specific for
MCP-1, MCP-2 and MCP-3. 1A1 has been shown to inhibit MCP-induced
chemotaxis (see Examples 3 and 5). The cloning and sequencing of
cDNA encoding the 1A1 antibody heavy and light chains is described
in Example 9.
[0222] Suitable human acceptor antibody sequences are identified by
computer comparisons of the amino acid sequences of the mouse
variable regions with the sequences of known human antibodies. The
comparison is performed separately for heavy and light chains but
the principles are similar for each. In particular, variable
domains from human antibodies whose framework sequences exhibit a
high degree of sequence identity with the murine VL and VH
framework regions were identified by query of the Kabat Database
using NCBI BLAST (publicly accessible through the National
Institutes of Health NCBI internet server) with the respective
murine framework sequences. In one embodiment, acceptor sequences
sharing greater that 50% sequence identity with murine donor
sequences are selected. Preferably, acceptor antibody sequences
sharing 60%, 70%, 80%, 90% or more are selected.
[0223] A computer comparison of 1A1 revealed that the 1A1 light
chain is a member of mouse subgroup kappa 2 and shows the greatest
sequence identity to human light chains of subtype kappa 2. The
comparison also revealed that the 1A1 heavy chain is a member of
mouse subgroup 2C and shows greatest sequence identity to human
heavy chains of subgroup 1, as defined by Kabat et al., supra.
Thus, light and heavy human framework regions are preferably
derived from human antibodies of these subtypes, or from consensus
sequences of such subtypes. The preferred light chain human
variable regions showing greatest sequence identity to the
corresponding region from 1A1 are from antibodies GI-284256
(Kennedy et al. (1991) J. Exp. Med. 173(4), 1033-1036). The
preferred heavy chain human variable regions showing greatest
sequence identity to the corresponding region from 1A1 are from
antibodies having Kabat ID Number 037655 (Bejeck et al. (1995),
Cancer Res. 55(11): 2346-2351).
[0224] Residues are next selected for substitution, as follows.
When an amino acid differs between a 1A1 variable framework region
and an equivalent human variable framework region, the human
framework amino acid should usually be substituted by the
equivalent mouse amino acid if it is reasonably expected that the
amino acid: [0225] (1) noncovalently binds antigen directly, [0226]
(2) is adjacent to a CDR region, is part of a CDR region under the
alternative definition proposed by Chothia et al., supra, or
otherwise interacts with a CDR region (e.g., is within about 3 of a
CDR region) (e.g. amino acids at positions H30, H73, or H93 of
1A1), or [0227] (3) participates in the VL-VH interface (e.g. amino
acids at positions L36 and H91 of 1A1).
[0228] Computer modeling of the 1A1 antibody heavy and light chain
variable regions, and humanization of the 1A1 antibody is described
in the Examples. Briefly, a three-dimensional model was generated
based on the closest solved murine antibody structures for the
heavy and light chains. For this purpose, an antibody designated
Fab1583 (1NLD; 2.9 .ANG.) and D2.5 (1YEE; 2.2 .ANG.) were chosen as
templates for modeling the 1A1 light chain, and antibodies
designated 2E8 (12E8; 1.9 .ANG.) and F9.13.7 (1FB1; 3.0 .ANG.) were
chosen as templates for modeling the heavy chain. The model was
further refined by a series of energy minimization steps to relieve
unfavorable atomic contacts and optimize electrostatic and van der
Walls interactions.
[0229] Three-dimensional structural information for the antibodies
described herein is publicly available, for example, from the
Research Collaboratory for Structural Bioinformatics' Protein Data
Bank (PDB). The PDB is freely accessible via the World Wide Web
internet and is described by Berman et al. (2000) Nucleic Acids
Research, 228:235. Computer modeling allows for the identification
of CDR-interacting residues. The computer model of the structure of
1A1 can in turn serve as a starting point for predicting the
three-dimensional structure of an antibody containing the 1A1
complementarity determining regions substituted in human framework
structures. Additional models can be constructed representing the
structure as further amino acid substitutions are introduced.
[0230] In general, substitution of one, most or all of the amino
acids fulfilling the above criteria is desirable. Accordingly, the
humanized antibodies of the present invention will usually contain
a substitution of a human light chain framework with a
corresponding 1A1 residue in at least 1, 2, and sometimes 3, of the
following positions: L2, L36, and LA5. The humanized antibodies
also usually contain a substitution of a human heavy chain
framework residue with a corresponding 1A1 residue in at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, and sometimes 11, of the following
positions: H27, H28, H29, H30, H66, H69, H73, H76, H91, H93, and
H94.
[0231] In one embodiment, the humanized antibodies of the invention
are based on two versions of the humanized 1A1 variable heavy chain
(H1 and H2) and two versions of the humanized 1A1 variable light
chain (L1 and L2). The humanized antibody of the invention is based
on any combination of the 1A1 humanized heavy and light chains
(e.g. H1-L1, H1-L2, H2-L1, H2-L2). Version 1 of the humanized heavy
and light chains contains most backmutations (i.e. L2, L36, LA5,
H27, H28, H29, H30, H66, H69, H73, H76, H91, H93, and H94), while
version 2 contains the fewest (i.e. L2, L36, H29, H30, H73, H91,
H93, and H94). The sequence of humanized 1A1 variable heavy chain
version 1 is set forth as SEQ ID NO: 53, and version 2 is set forth
as SEQ ID NO: 54. The sequence of humanized 1A1 variable light
chain version 1 is set forth as SEQ ID NO: 55, and version 2 is set
forth as SEQ ID NO: 56.
[0232] In one embodiment, the humanized antibody of the invention
contains a heavy chain comprising SEQ ID NO: 53 and a light chain
comprising SEQ ID NO: 55. In another embodiment, the humanized
antibody of the invention contains a heavy chain comprising SEQ ID
NO: 53 and a light chain comprising SEQ ID NO: 56. In yet another
embodiment, the humanized antibody of the invention contains a
heavy chain comprising SEQ ID NO: 54 and a light chain comprising
SEQ ID NO: 55. In still another embodiment, the humanized antibody
of the invention contains a heavy chain comprising SEQ ID NO: 554
and a light chain comprising SEQ ID NO: 56.
[0233] Occasionally, however, there is some ambiguity about whether
a particular amino acid meets the above criteria, and alternative
variant immunoglobulins are produced, one of which has that
particular substitution, the other of which does not. In instances
where substitution with a murine residue would introduce a residue
that is rare in human immunoglobulins at a particular position, it
may be desirable to test the antibody for activity with or without
the particular substitution. If activity (e.g., binding affinity
and/or binding specificity) is about the same with or without the
substitution, the antibody without substitution may be preferred,
as it would be expected to elicit less of a HAHA response, as
described herein.
[0234] Other candidates for substitution are acceptor human
framework amino acids that are unusual for a human immunoglobulin
at that position. These amino acids can be substituted with amino
acids from the equivalent position of more typical human
immunoglobulins. Alternatively, amino acids from equivalent
positions in the mouse 11K2 can be introduced into the human
framework regions when such amino acids are typical of human
immunoglobulin at the equivalent positions.
[0235] In additional embodiments, when the human light chain
framework acceptor immunoglobulin is GI-284256, the light chain
contains substitutions in at least 1, 2, 3, 4, 5, 6, 7, 8, or more
usually 9, of the following positions: L3, L8, L9, L11, L12, L14,
L37, L63, or L99. In additional embodiments when the human heavy
chain framework acceptor immunoglobulin is Kabat ID Number 037655,
the heavy chain contains substitutions in at least 1, 2, 3, 4, 5,
6, 7, 8, or more usually 9, of the following positions: H1, H6,
H14, H16, H23, H42, H80, H82B, or H87. These positions are
substituted with the amino acid from the equivalent position of a
human immunoglobulin having a more typical amino acid residue.
Examples of appropriate amino acids to substitute are shown in
FIGS. 11A and 11B. Table 2 summarizes the sequence analysis of the
1A1 VH and VL regions.
TABLE-US-00002 TABLE 2 Summary of 1A1 V-region sequence Chain Heavy
Light Mouse subgroup 2C kappa 2 Human subgroup 1 kappa 2 Chothia
canonical H1: 5 residues, class 1 L1: 16 residues, class 4 CDR
groupings H2: 17 residues, class 2 L2: 7 residues, class 1 H3: 13
residues, no class L3: 9 residues, class 1 Closest solved 2E8
(12E8; 1.9 .ANG.) Fab1583 (1NLD; 2.9 .ANG.) mouse structure F9.13.7
(1FB1; 3.0 .ANG. D2.5 (1YEE; 2.2 .ANG.)
[0236] Kabat ID sequences referenced herein are publicly available,
for example, from the Northwestern University Biomedical
Engineering Department's Kabat Database of Sequences of Proteins of
Immunological Interest. Three-dimensional structural information
for antibodies described herein is publicly available, for example,
from the Research Collaboratory for Structural Bioinformatics'
Protein Data Bank (PDB). The PDB is freely accessible via the World
Wide Web internet and is described by Berman et al. (2000) Nucleic
Acids Research, p 235-242. Germline gene sequences referenced
herein are publicly available, for example, from the National
Center for Biotechnology information (NCBI) database of sequences
in collections of gh, Ig kappa and Ig lambda germline V genes (as a
division of the National Library of Medicine (NLM) at the National
Institutes of Health (NIH). Homology searching of the NCBI "Ig
Germline Genes" database is provided by IgG BLAST.TM..
[0237] In another embodiment, a humanized antibody of the present
invention has structural features, as described herein, and
specifically binds to an epitope comprising MCP-1 and MCP-2.
[0238] 3. Human Antibodies
[0239] Human antibodies against MCPs are provided by a variety of
techniques described below. Some human antibodies are selected by
competitive binding experiments, or otherwise, to have the same
epitope specificity as a particular mouse antibody, such as one of
the mouse monoclonals described herein. Human antibodies can also
be screened for a particular epitope specificity by using only a
fragment of an MCP molecule as the immunogen, and/or by screening
antibodies against a collection of deletion mutants of MCP. Human
antibodies preferably have human IgG1 isotype specificity.
[0240] a. Trioma Methodology
[0241] The basic approach and an exemplary cell fusion partner,
SPAZ-4, for use in this approach have been described by Oestberg et
al., Hybridoma 2:361 (1983); Oestberg, U.S. Pat. No. 4,634,664; and
Engleman et al., U.S. Pat. No. 4,634,666 (each of which is
incorporated by reference in its entirety for an purposes). The
antibody-producing cell lines obtained by this method are called
triomas, because they are descended from three cells; two human and
one mouse. Initially, a mouse myeloma line is fused with a human
B-lymphocyte to obtain a non-antibody-producing xenogeneic hybrid
cell, such as the SPAZ-4 cell line described by Oestberg, supra.
The xenogeneic cell is then fused with an immunized human
B-lymphocyte to obtain an antibody-producing trioma cell line.
Triomas have been found to produce antibody more stably than
ordinary hybridomas made from human cells.
[0242] The immunized B-lymphocytes are obtained from the blood,
spleen, lymph nodes or bone marrow of a human donor. If antibodies
against a specific antigen or epitope are desired, it is preferable
to use that antigen or epitope thereof for immunization.
Immunization can be either in vivo or in vitro. For example, for in
vivo immunization, B cells are typically isolated from a human
immunized with MCP-1, a fragment thereof, larger polypeptide
containing MCP-1 or fragment, or an anti-idiotypic antibody to an
antibody to MCP-1. In some methods, B cells are isolated from the
same subject who is ultimately to be administered antibody therapy.
For in vitro immunization, B-lymphocytes are typically exposed to
antigen for a period of 7-14 days in a media such as RPMI-1640 (see
Engleman, supra) supplemented with 10% human plasma.
[0243] The immunized B-lymphocytes are fused to a xenogeneic hybrid
cell such as SPAZ-4 by well-known methods. For example, the cells
are treated with 40-50% polyethylene glycol of MW 1000-4000, at
about 37 degrees C., for about 5-min. Cells are separated from the
fusion mixture and propagated in media selective for the desired
hybrids (e.g., HAT or AH). Clones secreting antibodies having the
required binding specificity are identified by assaying the trioma
culture medium for the ability to bind to A.beta. or a fragment
thereof. Triomas producing human antibodies having the desired
specificity are subcloned by the limiting dilution technique and
grown in vitro in culture medium. The trioma cell lines obtained
are then tested for the ability to bind A.beta. or a fragment
thereof.
[0244] Although triomas are genetically stable they do not produce
antibodies at very high levels. Expression levels can be increased
by cloning antibody genes from the trioma into one or more
expression vectors, and transforming the vector into standard
mammalian, bacterial or yeast cellines.
[0245] b. Transgenic Non-Human Mammals
[0246] Human antibodies against MCPs can also be produced from
non-human transgenic mammals having transgenes encoding at least a
segment of the human immunoglobulin locus. Usually, the endogenous
immunoglobulin locus of such transgenic mammals is functionally
inactivated. Preferably, the segment of the human immunoglobulin
locus includes unrearranged sequences of heavy and light chain
components. Both inactivation of endogenous immunoglobulin genes
and introduction of exogenous immunoglobulin genes can be achieved
by targeted homologous recombination, or by introduction of YAC
chromosomes. The transgenic mammals resulting from this process are
capable of functionally rearranging the immunoglobulin component
sequences, and expressing a repertoire of antibodies of various
isotypes encoded by human immunoglobulin genes, without expressing
endogenous immunoglobulin genes. The production and properties of
mammals having these properties are described in detail by, e.g.,
Lonberg et al., WO93/12227 (1993); U.S. Pat. No. 5,877,397, U.S.
Pat. No. 5,874,299, U.S. Pat. No. 5,814,318, U.S. Pat. No.
5,789,650, U.S. Pat. No. 5,770,429, U.S. Pat. No. 5,661,016, U.S.
Pat. No. 5,633,425, U.S. Pat. No. 5,625,126, U.S. Pat. No.
5,569,825, U.S. Pat. No. 5,545,806, Nature 148:1547 (1994), Nature
Biotechnology 14:826 (1996), Kucherlapati, WO 91/10741 (1991) (each
of which is incorporated by reference in its entirety for all
purposes). Transgenic mice are particularly suitable. For example,
anti-MCP-1 antibodies are obtained by immunizing a transgenic
nonhuman mammal, such as described by Lonberg or Kucherlapati,
supra, with MCP-1 or a fragment thereof. Monoclonal antibodies are
prepared by, e.g., fusing B-cells from such mammals to suitable
myeloma cell lines using conventional Kohler-Milstein technology.
Human polyclonal antibodies can also be provided in the form of
serum from humans immunized with an immunogenic agent. Optionally,
such polyclonal antibodies can be concentrated by affinity
purification using MCP-1 or other MCP peptide as an affinity
reagent.
[0247] c. Phage Display Methods
[0248] A further approach for obtaining human anti-MCP antibodies
is to screen a DNA library from human B cells according to the
general protocol outlined by Huse et al., Science 246:1275-1281
(1989). As described for trioma methodology, such B cells can be
obtained from a human immunized with MCP-1, fragments, longer
polypeptides containing MCP-1 or fragments or anti-idiotypic
antibodies. Optionally, such B cells are obtained from a subject
who is ultimately to receive antibody treatment. Antibodies binding
to MCP-1 or a fragment thereof are selected. Sequences encoding
such antibodies (or a binding fragments) are then cloned and
amplified. The protocol described by Huse is rendered more
efficient in combination with phage-display technology. See, e.g.,
Dower et al., WO 91/17271, McCafferty et al., WO 92/01047, Herzig
et al., U.S. Pat. No. 5,877,218, Winter et al., U.S. Pat. No.
5,871,907, Winter et al., U.S. Pat. No. 5,858,657, Holliger et al.,
U.S. Pat. No. 5,837,242, Johnson et al., U.S. Pat. No. 5,733,743
and Hoogenboom et al., U.S. Pat. No. 5,565,332 (each of which is
incorporated by reference in its entirety for all purposes). In
these methods, libraries of phage are produced in which members
display different antibodies on their outer surfaces. Antibodies
are usually displayed as Fv or Fab fragments. Phage displaying
antibodies with a desired specificity are selected by affinity
enrichment to an MCP-1 peptide or fragment thereof.
[0249] In a variation of the phage-display method, human antibodies
having the binding specificity of a selected murine antibody can be
produced. See Winter, WO 92/20791. In this method, either the heavy
or light chain variable region of the selected murine antibody is
used as a starting material. If, for example, a light chain
variable region is selected as the starting material, a phage
library is constructed in which members display the same light
chain variable region (i.e., the murine starting material) and a
different heavy chain variable region. The heavy chain variable
regions are obtained from a library of rearranged human heavy chain
variable regions. A phage showing strong specific binding for
MCP-1, MCP-2, MCP-3, or a combination thereof (e.g., at least
10.sup.8 and preferably at least 10.sup.9 M.sup.-1) is selected.
The human heavy chain variable region from this phage then serves
as a starting material for constructing a further phage library. In
this library, each phage displays the same heavy chain variable
region (i.e., the region identified from the first display library)
and a different light chain variable region. The light chain
variable regions are obtained from a library of rearranged human
variable light chain regions. Again, phage showing strong specific
binding for MCP-1, MCP-2, MCP-3, or a combination thereof, are
selected. These phage display the variable regions of completely
human anti-MCP antibodies. These antibodies usually have the same
or similar epitope specificity as the murine starting material.
[0250] 4. Production of Variable Regions
[0251] Having conceptually selected the CDR and framework
components of humanized immunoglobulins, a variety of methods are
available for producing such immunoglobulins. Because of the
degeneracy of the code, a variety of nucleic acid sequences will
encode each immunoglobulin amino acid sequence. The desired nucleic
acid sequences can be produced by de nova solid-phase DNA synthesis
or by PCR mutagenesis of an earlier prepared variant of the desired
polynucleotide. Oligonucleotide-mediated mutagenesis is a preferred
method for preparing substitution, deletion and insertion variants
of target polypeptide DNA. See Adelman et al., DNA 2:183 (1983).
Briefly, the target polypeptide DNA is altered by hybridizing an
oligonucleotide encoding the desired mutation to a single-stranded
DNA template. After hybridization, a DNA polymerase is used to
synthesize an entire second complementary strand of the template
that incorporates the oligonucleotide primer, and encodes the
selected alteration in the target polypeptide DNA.
[0252] 5. Selection of Constant Regions
[0253] The variable segments of antibodies produced as described
supra (e.g., the heavy and light chain variable regions of
chimeric, humanized, or human antibodies) are typically linked to
at least a portion of an immunoglobulin constant region (Fc),
typically that of a human immunoglobulin. Human constant region DNA
sequences can be isolated in accordance with well known procedures
from a variety of human cells, but preferably immortalized B cells
(see Kabat et al., supra, and Liu et al., WO87/02671) (each of
which is incorporated by reference in its entirety for all
purposes). Ordinarily, the antibody will contain both light chain
and heavy chain constant regions. The heavy chain constant region
usually includes CHI, hinge, CH2, CH3, and CH4 regions. The
antibodies described herein include antibodies having all types of
constant regions, including IgM, IgG, IgD, IgA and IgE, and any
isotype, including IgG1, IgG2, IgG3 and IgG4. The choice of
constant region depends, in part, whether antibody-dependent
complement and/or cellular mediated toxicity is desired. For
example, isotopes IgG1 and IgG3 have complement activity and
isotypes IgG2 and IgG4 do not. When it is desired that the antibody
(e.g., humanized antibody) exhibit cytotoxic activity, the constant
domain is usually a complement fixing constant domain and the class
is typically IgG1. When such cytotoxic activity is not desirable,
the constant domain may be of the IgG2 class. Choice of isotype can
also affect passage of antibody into the brain. Human isotype IgG1
is preferred. Light chain constant regions can be lambda or kappa.
The humanized antibody may comprise sequences from more than one
class or isotype. Antibodies can be expressed as tetramers
containing two light and two heavy chains, as separate heavy
chains, light chains, as Fab, Fab' F(ab')2, and Fv, or as single
chain antibodies in which heavy and light chain variable domains
are linked through a spacer.
[0254] 6. Chemical Modifications
[0255] In some embodiments, the antibodies and antibody fragments
of the invention may be chemically modified to provide a desired
effect. For example, pegylation of antibodies and antibody
fragments of the invention may be carried out by any of the
pegylation reactions known in the art, as described, for example,
in the following references: Focus on Growth Factors 3:4-(1992); EP
0 154316; and EP 0 401 384 (each of which is incorporated by
reference herein in its entirety). Preferably, the pegylation is
carried out via an acylation reaction or an alkylation reaction
with a reactive polyethylene glycol molecule (or an analogous
reactive water-soluble polymer). A preferred water-soluble polymer
for pegylation of the antibodies and antibody fragments of the
invention is polyethylene glycol (PEG). As used herein,
"polyethylene glycol" is meant to encompass any of the forms of PEG
that have been used to derivatize other proteins, such as mono
(Cl-ClO) alkoxy- or aryloxy-polyethylene glycol.
[0256] Methods for preparing pegylated antibodies and antibody
fragments of the invention will generally comprise the steps of (a)
reacting the antibody or antibody fragment with polyethylene
glycol, such as a reactive ester or aldehyde derivative of PEG,
under conditions whereby the antibody or antibody fragment becomes
attached to one or more PEG groups, and (b) obtaining the reaction
products. It will be apparent to one of ordinary skill in the art
to select the optimal reaction conditions or the acylation
reactions based on known parameters and the desired result.
[0257] Pegylated antibodies and antibody fragments may generally be
used to treat conditions that may be alleviated or modulated by
administration of the antibodies and antibody fragments described
herein. Generally the pegylated antibodies and antibody fragments
have increased half-life, as compared to the nonpegylated
antibodies and antibody fragments. The pegylated antibodies and
antibody fragments may be employed alone, together, or in
combination with other pharmaceutical compositions. In one
embodiment, the invention describes pegylated Fab antibodies,
including pegylated humanized Fab-11K2 and pegylated murine
Fab-11K2.
[0258] In other embodiments of the invention the antibodies or
antigen-binding fragments thereof are conjugated to albumen using
art recognized techniques.
[0259] In another embodiment of the invention, antibodies, or
fragments thereof, are modified to reduce or eliminate potential
glycosylation sites. Such modified antibodies are often referred to
as "aglycosylated" antibodies. In order to improve the binding
affinity of an antibody or antigen-binding fragment thereof,
glycosylation sites of the antibody can be altered, for example, by
mutagenesis (e.g., site-directed mutagenesis). "Glycosylation
sites" refer to amino acid residues which are recognized by a
eukaryotic cell as locations for the attachment of sugar residues.
The amino acids where carbohydrate, such as oligosaccharide, is
attached are typically asparagine (N-linkage), serine (O-linkage),
and threonine (O-linkage) residues. In order to identify potential
glycosylation sites within an antibody or antigen-binding fragment,
the sequence of the antibody is examined, for example, by using
publicly available databases such as the website provided by the
Center for Biological Sequence Analysis (see
http://www.cbs.dtu.dk/services/NetNGlyc/ for predicting N-linked
glycoslyation sites) and http://www.cbs.dtu.dk/services/NetOGlyc/
for predicting O-linked glycoslyation sites). Additional methods
for altering glycosylation sites of antibodies are described in
U.S. Pat. Nos. 6,350,861 and 5,714,350.
[0260] In yet another embodiment of the invention, antibodies or
fragments thereof can be altered wherein the constant region of the
antibody is modified to reduce at least one constant
region-mediated biological effector function relative to an
unmodified antibody. To modify an antibody of the invention such
that it exhibits reduced binding to the Fc receptor (FcR), the
immunoglobulin constant region segment of the antibody can be
mutated at particular regions necessary for FcR interactions (see
e.g., Canfield, S. M. and S. L. Morrison (1991) J. Exp. Med.
173:1483-1491; and Lund, J. et al. (1991) J. of Immunol.
147:2657-2662). Reduction in FcR binding ability of the antibody
may also reduce other effector functions which rely on FcR
interactions, such as opsonization and phagocytosis and
antigen-dependent cellular cytotoxicity.
[0261] In a particular embodiment the invention further features
antibodies having altered effector function, such as the ability to
bind effector molecules, for example, complement or a receptor on
an effector cell. In particular, the humanized antibodies of the
invention have an altered constant region, e.g., Fc region, wherein
at least one amino acid residue in the Fc region has been replaced
with a different residue or side chain thereby reducing the ability
of the antibody to bind the FcR. Reduction in FcR binding ability
of the antibody may also reduce other effector functions which rely
on FcR interactions, such as opsonization and phagocytosis and
antigen-dependent cellular cytotoxicity. In one embodiment, the
modified humanized antibody is of the IgG class, comprises at least
one amino acid residue replacement in the Fc region such that the
humanized antibody has an altered effector function, e.g., as
compared with an unmodified humanized antibody. In particular
embodiments, the humanized antibody of the invention has an altered
effector function such that it is less immunogenic (e.g., does not
provoke undesired effector cell activity, lysis, or complement
binding), and/or has a more desirable half-life while retaining
specificity for MCP1, MCP-2, and/or MCP-3.
[0262] Alternatively, the invention features humanized antibodies
having altered constant regions to enhance FcR binding, e.g.,
Fc.gamma.R3 binding. Such antibodies are useful for modulating
effector cell function, e.g., for increasing ADCC activity, e.g.,
particularly for use in oncology applications.
[0263] As used herein, "Antibody-dependent cell-mediated
cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which
nonspecific cytotoxic cells that express FcRs (e.g. Natural Killer
(NK) cells, neutrophils, and macrophages) recognize bound antibody
on a target cell and subsequently cause lysis of the target cell.
The primary cells for mediating ADCC, NK cells, express
Fc.gamma.RIII only, whereas monocytes express Fc.gamma.RI,
Fc.gamma.RII and Fc.gamma.RIII of the antibody, e.g., a conjugate
of the antibody and another agent or antibody.
[0264] 7. Expression of Recombinant Antibodies
[0265] Chimeric, humanized and human antibodies are typically
produced by recombinant expression. Nucleic acids encoding
humanized light and heavy chain variable regions, optionally linked
to constant regions, are inserted into expression vectors. The
light and heavy chains can be cloned in the same or different
expression vectors. The DNA segments encoding immunoglobulin chains
are operably linked to control sequences in the expression
vector(s) that ensure the expression of immunoglobulin
polypeptides. Expression control sequences include, but are not
limited to, promoters (e.g., naturally-associated or heterologous
promoters), signal sequences, enhancer elements, and transcription
termination sequences. Preferably, the expression control sequences
are eukaryotic promoter systems in vectors capable of transforming
or transfecting eukaryotic host cells. Once the vector has been
incorporated into the appropriate host, the host is maintained
under conditions suitable for high level expression of the
nucleotide sequences, and the collection and purification of the
crossreacting antibodies.
[0266] These expression vectors are typically replicable in the
host organisms either as episomes or as an integral part of the
host chromosomal DNA. Commonly, expression vectors contain
selection markers (e.g., ampicillin-resistance,
hygromycin-resistance, tetracycline resistance or neomycin
resistance) to permit detection of those cells transformed with the
desired DNA sequences (see, e.g., Itakura et al., U.S. Pat. No.
4,704,362).
[0267] E. coli is one prokaryotic host particularly useful for
cloning the polynucleotides (e.g., DNA sequences) of the present
invention. Other microbial hosts suitable for use include bacilli,
such as Bacillus subtilus, and other enterobacteriaceae, such as
Salmonella, Serratia, and various Pseudomonas species. In these
prokaryotic hosts, one can also make expression vectors, which will
typically contain expression control sequences compatible with the
host cell (e.g., an origin of replication). In addition, any number
of a variety of well-known promoters will be present, such as the
lactose promoter system, a tryptophan (trp) promoter system, a
beta-lactamase promoter system, or a promoter system from phage
lambda. The promoters will typically control expression, optionally
with an operator sequence, and have ribosome binding site sequences
and the like, for initiating and completing transcription and
translation.
[0268] Other microbes, such as yeast, are also useful for
expression. Saccharomyces is a preferred yeast host, with suitable
vectors having expression control sequences (e.g., promoters), an
origin of replication, termination sequences and the like as
desired. Typical promoters include 3-phosphoglycerate kinase and
other glycolytic enzymes. Inducible yeast promoters include, among
others, promoters from alcohol dehydrogenase, isocytochrome C, and
enzymes responsible for maltose and galactose utilization.
[0269] In addition to microorganisms, mammalian tissue cell culture
may also be used to express and produce the polypeptides of the
present invention (e.g., polynucleotides encoding immunoglobulins
or fragments thereof). See Winnacker, From Genes to Clones, VCH
Publishers, N.Y., N.Y. (1987). Eukaryotic cells are actually
preferred, because a number of suitable host cell lines capable of
secreting heterologous proteins (e.g., intact immunoglobulins) have
been developed in the art, and include CHO cell lines, various Cos
cell lines, HeLa cells, preferably, myeloma cell lines, or
transformed B-cells or hybridomas. Preferably, the cells are
nonhuman. Expression vectors for these cells can include expression
control sequences, such as an origin of replication, a promoter,
and an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and
necessary processing information sites, such as ribosome binding
sites, RNA splice sites, polyadenylation sites, and transcriptional
terminator sequences. Preferred expression control sequences are
promoters derived from immunoglobulin genes, SV40, adenovirus,
bovine papilloma virus, cytomegalovirus and the like. See Co et
al., J. Immunol. 148:1149 (1992).
[0270] Alternatively, antibody-coding sequences can be incorporated
in transgenes for introduction into the genome of a transgenic
animal and subsequent expression in the milk of the transgenic
animal (see, e.g., Deboer et al., U.S. Pat. No. 5,741,957, Rosen,
U.S. Pat. No. 5,304,489, and Meade et al., U.S. Pat. No.
5,849,992). Suitable transgenes include coding sequences for light
and/or heavy chains in operable linkage with a promoter and
enhancer from a mammary gland specific gene, such as casein or beta
lactoglobulin.
[0271] The vectors containing the polynucleotide sequences of
interest (e.g., the heavy and light chain encoding sequences and
expression control sequences) can be transferred into the host cell
by well-known methods, which vary depending on the type of cellular
host. For example, calcium chloride transfection is commonly
utilized for prokaryotic cells, whereas calcium phosphate
treatment, electroporation, lipofection, biolistics or viral-based
transfection may be used for other cellular hosts. (See generally
Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold
Spring Harbor Press, 2nd ed., 1989) (incorporated by reference in
its entirety for all purposes). Other methods used to transform
mammalian cells include the use of polybrene, protoplast fusion,
liposomes, electroporation, and microinjection (see generally,
Sambrook et al., supra). For production of transgenic animals,
transgenes can be microinjected into fertilized oocytes, or can be
incorporated into the genome of embryonic stem cells, and the
nuclei of such cells transferred into enucleated oocytes.
[0272] When heavy and light chains are cloned on separate
expression vectors, the vectors are co-transfected to obtain
expression and assembly of intact immunoglobulins. Once expressed,
the whole antibodies, their dimers, individual light and heavy
chains, or other immunoglobulin forms of the present invention can
be purified according to standard procedures of the art, including
ammonium sulfate precipitation, affinity columns, column
chromatography, HPLC purification, gel electrophoresis and the like
(see generally Scopes, Protein Purification (Springer-Verlag, N.Y.,
(1982)). Substantially pure immunoglobulins of at least about 90 to
95% homogeneity are preferred, and 98 to 99% or more homogeneity
most preferred, for pharmaceutical uses.
[0273] The invention also includes aglycosylated antibodies, which
may be desirable for therapeutic treatment of human disease.
Humanized antibodies with altered glycosylation are produced by
expression of a nucleic acid encoding a human antibody in a cell
line that has altered ability to post-translationally modify
polypeptides, e.g., glycosylate polypeptides. For example, e.g, EP
1,176,195 describes a fucosyltransferase mutant, WO 99/54342
describes a CHO cell line engineered with regulatable GntIII
expression resulting in increased bisecting GlcNAc structures
having enhanced effector function, Shields et al. ((2002) J. Biol.
Chem. 277 26733) describes a hypofucosylated anti-HER2 hu4D5 mAb
made in mutant lec13 cells has improved ADCC, and Umana et al.
((1999) Nat. Biotechnol. 17: 176) describes a mAb with altered
bisecting glycoforms made in cells overexpressing rat GnTIII that
exhibits improved ADCC.
[0274] 8. Antibody Fragments
[0275] Also contemplated within the scope of the instant invention
are antibody fragments. In one embodiment, fragments of non-human,
chimeric and/or human antibodies are provided. In another
embodiment, fragments of humanized antibodies are provided.
Typically, these fragments exhibit specific binding to antigen with
an affinity of at least 10.sup.7, and more typically 10.sup.8 or
10.sup.9 M.sup.-1. Humanized antibody fragments include separate
heavy chains, light chains Fab, Fab' F(ab')2, Fabc, and Fv.
Preferred fragments of the invention include humanized 11K2 Fab and
humanized 1A1 Fab antibodies. In yet another embodiment, the
invention includes murine 11K2 Fab fragments and 1A1 Fab fragments.
Fragments are produced by recombinant DNA techniques, or by
enzymatic or chemical separation of intact immunoglobulins.
B. Nucleic Acid Encoding Immunologic and Therapeutic Agents
[0276] Immune responses against MCPs can also be induced by
administration of nucleic acids encoding antibodies and their
component chains used for passive immunization. Such nucleic acids
can be DNA or RNA. A nucleic acid segment encoding an immunogen is
typically linked to regulatory elements, such as a promoter and
enhancer, that allow expression of the DNA segment in the intended
target cells of a subject. For expression in blood cells, as is
desirable for induction of an immune response, promoter and
enhancer elements from light or heavy chain immunoglobulin genes or
the CMV major intermediate early promoter and enhancer are suitable
to direct expression. The linked regulatory elements and coding
sequences are often cloned into a vector. For administration of
double-chain antibodies, the two chains can be cloned in the same
or separate vectors.
[0277] A number of viral vector systems are available including
retroviral systems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet.
Develop. 3:102-109 (1993)); adenoviral vectors (see, e.g., Bett et
al., J. Virol. 67:5911 (1993)); adeno-associated virus vectors
(see, e.g., Zhou et al., J. Exp. Med. 179:1867 (1994)), viral
vectors from the pox family including vaccinia virus and the avian
pox viruses, viral vectors from the alpha virus genus such as those
derived from Sindbis and Semliki Forest Viruses (see, e.g.,
Dubensky et al., J. Virol. 70:508 (1996)), Venezuelan equine
encephalitis virus (see Johnston et al., U.S. Pat. No. 5,643,576)
and rhabdoviruses, such as vesicular stomatitis virus (see Rose, WO
96/34625) and papillomaviruses (Ohe et al., Human Gene Therapy
6:325 (1995); Woo et al., WO 94/12629 and Xiao & Brandsma,
Nucleic Acids. Res. 24, 2630-2622 (1996)).
[0278] DNA encoding an immunogen, or a vector containing the same,
can be packaged into liposomes. Suitable lipids and related analogs
are described by Eppstein et al., U.S. Pat. No. 5,208,036, Felgner
et al., U.S. Pat. No. 5,264,618, Rose, U.S. Pat. No. 5,279,833, and
Epand et al., U.S. Pat. No. 5,283,185. Vectors and DNA encoding an
immunogen can also be adsorbed to or associated with particulate
carriers, examples of which include polymethyl methacrylate
polymers and polylactides and poly (lactide-co-glycolides), see,
e.g., McGee et al., J. Micro Encap. (1996).
[0279] Gene therapy vectors or naked polypeptides (e.g. DNA) can be
delivered in vivo by administration to an individual subject,
typically by systemic administration (e.g., intravenous,
intraperitoneal, nasal, gastric, intradermal, intramuscular,
subdermal, or intracranial infusion) or topical application (see
e.g., Anderson et al., U.S. Pat. No. 5,399,346). The term "naked
polynucleotide" refers to a polynucleotide not complexed with
colloidal materials. Naked polynucleotides are sometimes cloned in
a plasmid vector. Such vectors can further include facilitating
agents such as bupivacine (Attardo et al., U.S. Pat. No.
5,593,970). DNA can also be administered using a gene gun. See Xiao
& Brandsma, supra. The DNA encoding an immunogen is
precipitated onto the surface of microscopic metal beads. The
microprojectiles are accelerated with a shock wave or expanding
helium gas, and penetrate tissues to a depth of several cell
layers. For example, The Accel.TM. Gene Delivery Device
manufactured by Agacetus, Inc. Middleton Wis. is suitable.
Alternatively, naked DNA can pass through skin into the blood
stream simply by spotting the DNA onto skin with chemical or
mechanical irritation (see Howell et al., WO 95/05853).
[0280] In a further variation, vectors encoding immunogens can be
delivered to cells ex vivo, such as cells explanted from an
individual subject (e.g., lymphocytes, bone marrow aspirates,
tissue biopsy) or universal donor hematopoietic stem cells,
followed by reimplantation of the cells into a subject, usually
after selection for cells which have incorporated the vector.
II. Prophylactic and Therapeutic Methods
[0281] The present invention is directed inter alia to treatment of
diseases associated with MCP-associated inflammation, by
administration of therapeutic immunological reagents (e.g.,
humanized immunoglobulins) to specific epitopes within an MCP
protein to a subject under conditions that generate a beneficial
therapeutic response in a subject, for example, for the prevention
or treatment of a disorder associated with detrimental MCP
activity. The invention is also directed to use of the disclosed
immunological reagents (e.g., humanized immunoglobulins) in the
manufacture of a medicament for the treatment or prevention of an
MCP-associated disease.
[0282] The term "treatment" as used herein, is defined as the
application or administration of a therapeutic agent to a subject,
or application or administration of a therapeutic agent to an
isolated tissue or cell line from a subject, who has a disease, a
symptom of disease or a predisposition toward a disease, with the
purpose to cure, heal, alleviate, relieve, alter, remedy,
ameliorate, improve or affect the disease, the symptoms of disease
or the predisposition toward disease.
[0283] Therapeutic agents of the invention are typically
substantially pure from undesired contaminant. This means that an
agent is typically at least about 50% w/w (weight/weight) purity,
as well as being substantially free from interfering proteins and
contaminants. Sometimes the agents are at least about 80% w/w and,
more preferably at least 90 or about 95% w/w purity. However, using
conventional protein purification techniques, homogeneous peptides
of at least 99% w/w can be obtained.
[0284] The methods can be used on both asymptomatic subjects and
those currently showing symptoms of disease. The antibodies used in
such methods can be human, humanized, chimeric or nonhuman
antibodies, or fragments thereof (e.g., antigen binding fragments)
and can be monoclonal or polyclonal, as described herein. In yet
another aspect, the invention features administering antibodies
prepared from a human immunized with an MCP peptide, which human
can be the subject to be treated with antibody.
[0285] In another aspect, the invention features administering an
antibody with a pharmaceutical carrier as a pharmaceutical
composition. Alternatively, the antibody can be administered to a
subject by administering a polynucleotide encoding at least one
antibody chain. The polynucleotide is expressed to produce the
antibody chain in the subject. Optionally, the polynucleotide
encodes heavy and light chains of the antibody. The polynucleotide
is expressed to produce the heavy and light chains in the subject.
In exemplary embodiments, the subject is monitored for level of
administered antibody in the blood of the subject.
[0286] The invention thus fulfills a longstanding need for
therapeutic regimes for preventing or ameliorating inflammation
associated with MCPs.
A. Disorders Amenable to Treatment
[0287] As used herein, the terms "a disorder in which MCP activity
is detrimental" and "an MCP-associated disorder" are intended to
include diseases and other disorders in which the presence of MCP,
including MCP-1, MCP-2, and/or MCP-3, in a subject suffering from
the disorder has been shown to be or is suspected of being either
responsible for the pathophysiology of the disorder or a factor
that contributes to a worsening of the disorder. Accordingly, a
disorder in which MCP activity is detrimental is a disorder in
which inhibition of MCP activity is expected to alleviate the
symptoms and/or progression of the disorder. Such disorders may be
evidenced, for example, by an increase in the concentration of MCP
in a biological fluid of a subject suffering from the disorder
(e.g., an increase in the concentration of MCP-1 in serum, plasma,
synovial fluid, etc. of the subject), which can be detected, for
example, using an anti-MCP antibody as described above. There are
numerous examples of disorders in which MCP activity is
detrimental. The use of the antibodies and antibody portions of the
invention in the treatment of specific disorders is discussed
further below.
[0288] The b-chemokines, particularly MCP-1, MCP-2 and MCP-3 have
been shown to play a role in pathological conditions associated
with inflammation (Van Coillie et al. (1999) Cytokine & Growth
Factor Rev. 10:61-86). MCP-1, MCP-2, and MCP-3 have all been shown
to have potent chemotactic activity for leukocytes, especially
monocytes (van Coillie et al (1999) Cytokine & Growth Factor
Rev. 10:61-86). All three chemokines also share other functions
(e.g., glucosaminidase release, gelatinase B release, granzyme A)
which combined with their chemotactic activity enable leukocytes to
migrate into tissues and towards sites of inflammation. Recruitment
of leukocytes to inflammatory sites is thought to contribute
greatly to the inflammatory process. Inhibition of leukocyte
recruitment via MCP-1 antagonism (e.g., in MCP-1 knockout animals
and in MCP-1 depletion studies using anti-MCP-1 mAbs) has been
shown to reduce leukocyte infiltration (particularly monocyte
recruitment) and is correlated with reduction in disease (van
Coillie et al. (1999) Cytokine & Growth Factor Rev. 10:61-86).
Like MCP-1, MCP-2 and MCP-3 are also molecules with potent
chemotactic activity for monocytes, T lymphocytes, and basophils.
Given their overlapping activities and the increased expression of
all three chemokines (MCP-1, MCP-2, and MCP-3) in human disease,
blockade of all three MCP molecules would be expected to have a
greater beneficial effect than just inhibition of MCP-1 alone.
Blockade of multiple MCP molecules (MCP-1, MCP-2 and MCP-3) would
also more efficiently inhibit recruitment of certain cell types for
which MCP-1 is a poor chemotactic stimulus. Thus, while MCP-1 does
not efficiently induce migration of eosinophils or resting
neutrophils, MCP-2 is a potent chemotactic stimulus for
eosinophils, and MCP-3 shows activity against both eosinophils and
neutrophils (van Coillie et al. (1999) Cytokine & Growth Factor
Rev. 10:61-86). The antibodies and antibody fragments of the
invention may be used to modulate the activity of these chemokines
and affect the pathology of these disorders, and therefore, may be
used in therapeutic compositions for the treatment of inflammatory
conditions and pathological conditions associated with expression
of MCP molecules. In these embodiments, a subject is identified as
having one of the diseases to be treated, such as by exhibiting at
least one sign or symptom of the disease or disorder. At least one
antibody or antigen-binding fragment thereof of the invention or
compositions comprising at least one antibody or antigen-binding
fragment thereof of the invention is administered in a sufficient
amount to alleviate at least one symptom of the disease or
disorder, or to reduce the activity of at least one of MCP-1, MCP-2
or MCP-3.
1. Fibrotic Disease
[0289] In one embodiment of the invention, an antibody or
antigen-binding fragment thereof, having binding specificity for
MCP-1, MCP-2 and/or MCP-3, e.g., an antibody or antigen-binding
fragment comprising CDRs from either the 1A1 or 11K2 antibodies, is
used in a method of prevention or treatment of a subject suffering
from a fibrotic disease. A "fibrotic disease" as used herein
includes any condition marked by an increase of interstitial
fibrous tissue. MCPs are known to be associated with fibrotic
conditions. For example, MCP-1 is a potent chemoattractant for
monocytes and has been implicated in a variety of inflammatory and
fibrotic diseases, the pathogenesis of which is known to involve
infiltration and activation of monocytes (Zhang, et al (1994) J.
Immunol. 153:4733-4741). Along with increased TGF-.beta. and
collagen production, fibrotic diseases are also characterized by
increased levels of MCP-1 (Antoniades et al. (1992) J. Immunol.
89:5371-5375; Wada et al. (1996) FASEB J., 10:1418-1425; Saitoh et
al. (1998) J. Clin. Lab. Anal. 12:1-5; Hasegawa et al. (1999) Clin.
Exp. Immunol. 117:159-165; Wada et al. (1999) Kidney Int.
65:995-1003; Wada et al. (2000) Kidney Int. 58-1492-1499).
Increased expression of MCP-1 during fibrotic diseases has been
well characterized in both human and in rodent models. In humans,
MCP-1 is up-regulated in idiopathic pulmonary fibrosis (Antoniades
et al., supra), IgA nephropathy (Saitoh et al., supra), diabetic
nephropathy (Wada et al. (2000), supra), lupus nephritis (Wada et
al. (1996), supra), crescentic glomerulonephritis (Wada, 1999),
supra), and scleroderma (Hasegawa, supra). While not expressed in
normal tissues, MCP-1 was highly expressed in the fibrotic skin and
lungs of scleroderma subjects, and the elevated levels of MCP-1
found in subject serum correlated with the presence of fibrosis and
with earlier onset of scleroderma (Hasegawa, supra). MCP-1
expression also correlated positively with severity of renal
fibrosis in diseases such as IgA nephropathy, diabetic nephropathy,
lupus nephritis, and crescentic glomerulonephritis.
2. Oncogenic Disease
[0290] In another embodiment of the invention, an antibody or
antigen-binding fragment thereof, having binding specificity for
MCP-1, MCP-2 and/or MCP-3, e.g. an antibody or antigen-binding
fragment comprising CDRs from either the 1A1 or 11K2 antibodies, is
used in a method of prevention or treatment of a subject suffering
from an oncogenic disease or cancer. MCPs are known to be
associated with oncogenic conditions. For example, MCP-1 is a
potent inducer of angiogenesis and plays an important role in tumor
growth. Evidence for a role of MCP-1 in tumorigenesis involved
treatment of immunodeficient mice bearing MCP-1 producing human
breast carcinoma cells with neutralizing anti-MCP-1 mAb (Salcedo,
(2000) Blood 96:34-40). Treatment with anti-MCP-1 mAb resulted in
significant increases in animal survival (mean survival increased
from 45 days to 75 days) and marked inhibition of tumor metastasis
(60% decrease in lung metastatic index).
3. Immunopathologic Disease
[0291] In another embodiment of the invention, an antibody or
antigen-binding fragment thereof, having binding specificity for
MCP-1, MCP-2 and/or MCP-3, e.g., an antibody or antigen-binding
fragment comprising CDRs from either the 1A1 or 11K2 antibodies, is
used in a method of prevention or treatment of a subject suffering
from an immunopathologic disease. An "immunopathologic disease" as
used herein is defined as any condition associated with an immune
response which is related to a disease. MCPs have been associated
with immunopathologic conditions. For example, there is a strong
link between MCP-1 expression and immunopathologic disease in
humans. Experiments using genetically-engineered mice and in vivo
data using function-blocking antibodies to MCP-1 provide evidence
supporting the validity of MCP-1 antagonism in a variety of
diseases characterized by mononuclear infiltration. Included among
these diseases is: atherosclerosis (MCP-1 KO, CCR2KO), arthritis
(MCP-1 mAb), asthma (MCP-1 mAb), glomerulonephritis (MCP-1 KO,
MCP-1 mAb), lupus nephritis (MCP-1 KO) and multiple sclerosis
(MCP-1 KO, MCP-1 mAb, CCR2KO) (see, for example, Lu et al. (1998)
J. Exp. Med. 187601-608); Kurihara et al. (1997) J. Exp. Med.
186:1757-1762; Boring et al. (1997) J. Clin. Invest.
100:2552-2561); Kuziel et al. (1997) PNAS 94:12053-12058; Blease et
al. (2000) J. Immunol. 165:2603-2611; Traynor et al. (2000) J.
Immunol. 164:2021-2027; Boring et al. (1998) Nature 394:894-897;
Dawson et al. (1999) Atherosclerosis 143:205-211; Fife et al.
(2000) J. Exp. Med. 192: 899-905; Izikson et al. (2000) J. Exp.
Med. 192:1075-1080; Bird et al. (2000) Kidney Int. 57:129-136;
MacLean et al. (2000) J. Immunol. 165:165:6568-6575; Karpus et al.
(1997) J. Leukoc. Biol. 62:681-687; Gonzalo et al. (1998) J. Exp.
Med. 188:157-167). In all these cases, interference with the MCP-1
pathway resulted in dramatically reduced leukocyte infiltration,
with monocyte recruitment being particularly affected. This
dramatic reduction in monocyte recruitment correlated well with
reduction in disease.
4. Other Disorders
[0292] In certain embodiments, the antibodies or antigen-binding
fragments of the present invention are useful in the prevention or
treatment of glomerulonephritis, scleroderma, cirrhosis, multiple
sclerosis, lupus nephritis, atherosclerosis, inflammatory bowel
diseases or rheumatoid arthritis. In another embodiment, the
antibodies or antigen-binding fragments of the invention can be
used to treat or prevent inflammatory disorders, including, but not
limited to, Alzheimer's, severe asthma, atopic dermatitis,
cachexia, CHF-ischemia, coronary restinosis, Crohn's disease,
diabetic nephropathy, lymphoma, psoriasis,
fibrosis/radiation-induced, juvenile arthritis, stroke,
inflammation of the brain or central nervous system caused by
trauma, and ulcerative colitis. Other inflammatory disorders which
can be prevented or treated with the antibodies or antigen-binding
fragments of the invention include inflammation due to corneal
transplantation, chronic obstructive pulmonary disease, hepatitis
C, multiple myeloma, and osteoarthritis. In another embodiment, the
antibodies or antigen-binding fragments of the invention can be
used to prevent or treat neoplasia, including, but not limited to
bladder cancer, breast cancer, head and neck cancer, kaposi's
sarcoma, melanoma, ovarian cancer, small cell lung cancer, stomach
cancer, leukemia/lymphoma, and multiple myeloma. Additional
neoplasia conditions include, cervical cancer, colo-rectal cancer,
endometrial cancer, kidney cancer, non-squamous cell lung cancer,
and prostate cancer. In another embodiment, the antibodies or
antigen-binding fragments of the invention can be used to prevent
or treat fibrotic disorders, including, but not limited to
CHF-ischemia, coronary restenosis, diabetic vasculopathy,
myocardial infarction/unstable angina, and radiation fibrosis.
Additional examples of fibrotic disorders which can be treated in
accordance with the invention include diabetic nephropathy, and
impotence (Peyronie's). In another embodiment, the antibodies or
antigen-binding fragments of the invention can be used to prevent
or treat neurodegenerative disorders, including, but not limited to
Alzheimer's, stroke, and traumatic brain or central nervous system
injuries. Additional neurodegenerative disorders include ALS/motor
neuron disease, diabetic peripheral neuropathy, diabetic
retinopathy, Huntington's disease, macular degeneration, and
Parkinson's disease.
[0293] In clinical applications, a subject is identified as having
or at risk of developing a disease or disorder associated with
detrimental MCP activity, such as by exhibiting at least one sign
or symptom of the disease or disorder. At least one antibody or
antigen-binding fragment thereof of the invention or compositions
comprising at least one antibody or antigen-binding fragment
thereof of the invention is administered in a sufficient amount to
treat at least one symptom of the disease or disorder, or to reduce
the activity of at least one of MCP-1, MCP-2 or MCP-3.
B. Animal Model for Testing Efficacy of Antibodies
[0294] Moreover, an antibody of the invention can be administered
to a non-human mammal expressing a chemokine with which the
antibody cross-reacts (e.g., a primate, pig or mouse) for
veterinary purposes or as an animal model of human disease.
Regarding the latter, such animal models may be useful for
evaluating the therapeutic efficacy of antibodies of the invention
(e.g., testing of dosages and time courses of administration).
Examples of animal models which can be used for evaluating the
therapeutic efficacy of antibodies or antigen-binding fragments of
the invention for preventing or treating glomerulonephritis include
anti-GBM-induced glomerulonephritis (Wada et al. (1996) Kidney Int.
49:761-767) and anti-thy1-induced glomerulonephritis (Schneider et
al. (1999) Kidney Int. 56:135-144). Examples of animal models which
can be used for evaluating the therapeutic efficacy of antibodies
or antigen-binding fragments of the invention for preventing or
treating colitis include a mouse model where colitis is
TNBS-induced, as described in Neurath et al. (1995) J Exp Med.
182(5):1281. Examples of animal models which can be used for
evaluating the therapeutic efficacy of antibodies or
antigen-binding fragments of the invention for preventing or
treating cirrhosis include carbon tetrachloride-induced cirrhosis
and liver fibrosis (Sakadamis et al. (2001) Res Exp Med
200:137-54). Examples of animal models which can be used for
evaluating the therapeutic efficacy of antibodies or
antigen-binding fragments of the invention for preventing or
treating multiple sclerosis include experimental autoimmune
encephalomyelitis (EAE) (Link and Xiao (2001) Immunol. Rev.
184:117-128). Animal models can also be used for evaluating the
therapeutic efficacy of antibodies or antigen-binding fragments of
the invention for preventing or treating lupus, for example using
the MRL-Fas.sup.lpr mice (Schneider, supra; Tesch et al. (1999) J.
Exp. Med. 190). Examples of animal models which can be used for
evaluating the therapeutic efficacy of antibodies or
antigen-binding fragments of the invention for preventing or
treating atherosclerosis include using mice deficient in
apolipoprotein A, ApoE, and LDL R.sub.L (Dansky et al. (1999)
Arterioscler Thromb. Vasc. Biol. 19:1960-1968; Lou et al. (1998)
PNAS 95:12591-12595). Examples of animal models which can be used
for evaluating the therapeutic efficacy of antibodies or
antigen-binding fragments of the invention for preventing or
treating inflammatory bowel disease (IBD) include TNBS-induced IBD,
DSS-induced IBD, and (Padol et al. (2000) Eur. J. Gastrolenterol
Hepatol 12:257; Murthy et al. (1993) Dig. Dis. Sci. 38:1722).
Examples, of animal models which can be used for evaluating the
therapeutic efficacy of antibodies or antigen-binding fragments of
the invention for preventing or treating rheumatoid arthritis (RA)
include adjuvant-induced RA, collagen-induced RA, and collagen
mAb-induced RA (Holmdahl et al., (2001) Immunol. Rev. 184:184;
Holmdahl et al., (2002) Ageing Res. Rev. 1:135; Van den Berg (2002)
Curr. Rheumatol. Rep. 4:232).
[0295] In addition, animal models for evaluating the efficacy of
antibodies or antigen-binding fragments of the invention in
treating or preventing human fibrotic diseases, include rodent
models of pulmonary (Brieland et al. (1992) Am. J. Respir. Cell.
Mol. Biol. 7:134-139; Zhang et al. (1994) J. Immunol.
153:4733-4741; Johnston et al. (1998) Exp. Lung Res. 24:321-337),
vascular (Furukawa et al. (1999) Circ. Res. 84:306-314), and renal
(Lloyd et al. (1997) J. Exp. Med 185:1371-1380; Fujinaka et al.
(1997) J. Am. Soc. Nephrol. 8:1174-1178; Schneider, supra; Tesch et
al. (1999) J. Exp. Med. 190: 1813-1824; Tesch et al. (1999) J.
Clin. Invest. 103:73-80) fibrosis. Alport's model of renal fibrosis
can also be used to evaluate the efficacy of the antibodies or
antigen-binding fragments of the invention.
C. Treatment Regimes and Dosages
[0296] In prophylactic applications, pharmaceutical compositions or
medicaments are administered to a subject suffering from a disorder
in which MCP activity is detrimental, in an amount sufficient to
eliminate or reduce the risk, lessen the severity, or delay the
outset of the disorder, including biochemical, histologic and/or
behavioral symptoms of the disorder, its complications and
intermediate pathological phenotypes presenting during development
of the disorder. In therapeutic applications, compositions or
medicants are administered to a subject suspected of, or already
suffering from such a disorder in an amount sufficient to cure, or
at least partially arrest, the symptoms of the disorder
(biochemical, histologic and/or behavioral), including its
complications and intermediate pathological phenotypes in
development of the disorder.
[0297] In some methods, administration of agent reduces or
eliminates inflammation associated with MCPs. An amount adequate to
accomplish therapeutic or prophylactic treatment is defined as a
therapeutically- or prophylactically-effective dose. In both
prophylactic and therapeutic regimes, agents are usually
administered in several dosages until a sufficient immune response
has been achieved. The term "immune response" or "immunological
response" includes the development of a humoral (antibody mediated)
and/or a cellular (mediated by antigen-specific T cells or their
secretion products) response directed against an antigen in a
recipient subject. Such a response can be an active response, i.e.,
induced by administration of immunogen, or a passive response,
i.e., induced by administration of immunoglobulin or antibody or
primed T-cells.
[0298] An "immunogenic agent" or "immunogen" is capable of inducing
an immunological response against itself on administration to a
mammal, optionally in conjunction with an adjuvant. Typically, the
immune response is monitored and repeated dosages are given if the
immune response starts to wane.
[0299] Effective doses of the compositions of the present
invention, for the treatment of the above described conditions vary
depending upon many different factors, including means of
administration, target site, physiological state of the subject,
whether the subject is human or an animal, other medications
administered, and whether treatment is prophylactic or therapeutic.
Usually, the subject is a human but non-human mammals including
transgenic mammals can also be treated. Treatment dosages need to
be titrated to optimize safety and efficacy.
[0300] For passive immunization with an antibody, the dosage ranges
from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg,
of the host body weight. For example dosages can be 1 mg/kg body
weight or 10 mg/kg body weight or within the range of 1-10 mg/kg,
preferably at least 1 mg/kg. Subjects can be administered such
doses daily, on alternative days, weekly or according to any other
schedule determined by empirical analysis. An exemplary treatment
entails administration in multiple dosages over a prolonged period,
for example, of at least six months. Additional exemplary treatment
regimes entail administration once per every two weeks or once a
month or once every 3 to 6 months. Exemplary dosage schedules
include 1-10 mg/kg or 15 mg/kg on consecutive days, 30 mg/kg on
alternate days or 60 mg/kg weekly. In some methods, two or more
monoclonal antibodies with different binding specificities are
administered simultaneously, in which case the dosage of each
antibody administered falls within the ranges indicated.
[0301] Antibody is usually administered on multiple occasions.
Intervals between single dosages can be weekly, monthly or yearly.
Intervals can also be irregular as indicated by measuring blood
levels of antibody to MCPs in the subject. In some methods, dosage
is adjusted to achieve a plasma antibody concentration of 1-1000
.mu.g/ml and in some methods 25-300 .mu.g/ml. Alternatively,
antibody can be administered as a sustained release formulation, in
which case less frequent administration is required. Dosage and
frequency vary depending on the half-life of the antibody in the
subject. In general, human antibodies show the longest half-life,
followed by humanized antibodies, chimeric antibodies, and nonhuman
antibodies.
[0302] The dosage and frequency of administration can vary
depending on whether the treatment is prophylactic or therapeutic.
In prophylactic applications, compositions containing the present
antibodies or a cocktail thereof are administered to a subject not
already in the disease state to enhance the subject's resistance.
Such an amount is defined to be a "prophylactic effective dose." In
this use, the precise amounts again depend upon the subject's state
of health and general immunity, but generally range from 0.1 to 25
mg per dose, especially 0.5 to 2.5 mg per dose. A relatively low
dosage is administered at relatively infrequent intervals over a
long period of time. Some subjects continue to receive treatment
for the rest of their lives.
[0303] In therapeutic applications, a relatively high dosage (e.g.,
from about 1 to 200 mg of antibody per dose, with dosages of from 5
to 25 mg being more commonly used) at relatively short intervals is
sometimes required until progression of the disease is reduced or
terminated, and preferably until the subject shows partial or
complete amelioration of symptoms of disease. Thereafter, the
patent can be administered a prophylactic regime.
[0304] Doses for nucleic acids encoding antibodies range from about
10 ng to 1 g, 100 ng to 100 mg, 1 .mu.g to 10 mg, or 30-300 .mu.g
DNA per subject. Doses for infectious viral vectors vary from
10-100, or more, virions per dose.
[0305] Therapeutic agents can be administered by parenteral,
topical, intravenous, oral, subcutaneous, intraarterial,
intracranial, intraperitoneal, intranasal or intramuscular means
for prophylactic and/or therapeutic treatment. The most typical
route of administration of an immunogenic agent is subcutaneous
although other routes can be equally effective. The next most
common route is intramuscular injection. This type of injection is
most typically performed in the arm or leg muscles. In some
methods, agents are injected directly into a particular tissue
where deposits have accumulated, for example intracranial
injection. Intramuscular injection or intravenous infusion are
preferred for administration of antibody. In some methods,
particular therapeutic antibodies are injected directly into the
cranium. In some methods, antibodies are administered as a
sustained release composition or device, such as a Medipad.TM.
device.
[0306] Agents of the invention can optionally be administered in
combination with other agents that are at least partly effective in
treatment of MCP-associated disorders.
D. Pharmaceutical Compositions
[0307] The therapeutic compositions of the invention include at
least one antibody or antibody fragment of the invention in a
pharmaceutically acceptable carrier. A "pharmaceutically acceptable
carrier" refers to at least one component of a pharmaceutical
preparation that is normally used for administration of active
ingredients. As such, a carrier may contain any pharmaceutical
excipient used in the art and any form of vehicle for
administration. The compositions may be, for example, injectable
solutions, aqueous suspensions or solutions, non-aqueous
suspensions or solutions, solid and liquid oral formulations,
salves, gels, ointments, intradermal patches, creams, lotions,
tablets, capsules, sustained release formulations, and the like.
Additional excipients may include, for example, colorants,
taste-masking agents, solubility aids, suspension agents,
compressing agents, enteric coatings, sustained release aids, and
the like.
[0308] Agents of the invention are often administered as
pharmaceutical compositions comprising an active therapeutic agent,
i.e., and a variety of other pharmaceutically acceptable
components. See Remington's Pharmaceutical Science (15th ed., Mack
Publishing Company, Easton, Pa. (1980)). The preferred form depends
on the intended mode of administration and therapeutic application.
The compositions can also include, depending on the formulation
desired, pharmaceutically-acceptable, non-toxic carriers or
diluents, which are defined as vehicles commonly used to formulate
pharmaceutical compositions for animal or human administration. The
diluent is selected so as not to affect the biological activity of
the combination. Examples of such diluents are distilled water,
physiological phosphate-buffered saline, Ringer's solutions,
dextrose solution, and Hank's solution. In addition, the
pharmaceutical composition or formulation may also include other
carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic
stabilizers and the like.
[0309] Pharmaceutical compositions can also include large, slowly
metabolized macromolecules such as proteins, polysaccharides such
as chitosan, polylactic acids, polyglycolic acids and copolymers
(such as latex functionalized sepharose(TM), agarose, cellulose,
and the like), polymeric amino acids, amino acid copolymers, and
lipid aggregates (such as oil droplets or liposomes). Additionally,
these carriers can function as immunostimulating agents (i.e.,
adjuvants).
[0310] For parenteral administration, agents of the invention can
be administered as injectable dosages of a solution or suspension
of the substance in a physiologically acceptable diluent with a
pharmaceutical carrier that can be a sterile liquid such as water
oils, saline, glycerol, or ethanol. Additionally, auxiliary
substances, such as wetting or emulsifying agents, surfactants, pH
buffering substances and the like can be present in compositions.
Other components of pharmaceutical compositions are those of
petroleum, animal, vegetable, or synthetic origin, for example,
peanut oil, soybean oil, and mineral oil. In general, glycols such
as propylene glycol or polyethylene glycol are preferred liquid
carriers, particularly for injectable solutions. Antibodies can be
administered in the form of a depot injection or implant
preparation, which can be formulated in such a manner as to permit
a sustained release of the active ingredient. An exemplary
composition comprises monoclonal antibody at 5 mg/mL, formulated in
aqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl,
adjusted to pH 6.0 with HCl.
[0311] Typically, compositions are prepared as injectables, either
as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid vehicles prior to injection
can also be prepared. The preparation also can be emulsified or
encapsulated in liposomes or micro particles such as polylactide,
polyglycolide, or copolymer for enhanced adjuvant effect, as
discussed above (see Langer, Science 249: 1527 (1990) and Hanes,
Advanced Drug Delivery Reviews 28:97 (1997)). The agents of this
invention can be administered in the form of a depot injection or
implant preparation, which can be formulated in such a manner as to
permit a sustained or pulsatile release of the active
ingredient.
[0312] Additional formulations suitable for other modes of
administration include oral, intranasal, and pulmonary
formulations, suppositories, and transdermal applications. For
suppositories, binders and carriers include, for example,
polyalkylene glycols or triglycerides; such suppositories can be
formed from mixtures containing the active ingredient in the range
of 0.5% to 10%, preferably 1%-2%. Oral formulations include
excipients, such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, and
magnesium carbonate. These compositions take the form of solutions,
suspensions, tablets, pills, capsules, sustained release
formulations or powders and contain 10%-95% of active ingredient,
preferably 25%-70%.
[0313] Topical application can result in transdermal or intradermal
delivery. Topical administration can be facilitated by
co-administration of the agent with cholera toxin or detoxified
derivatives or subunits thereof or other similar bacterial toxins
(See Glenn et al., Nature 391, 851 (1998)). Co-administration can
be achieved by using the components as a mixture or as linked
molecules obtained by chemical crosslinking or expression as a
fusion protein.
[0314] Alternatively, transdermal delivery can be achieved using a
skin path or using transferosomes (Paul et al., Eur. J. Immunol.
25:3521 (1995); Cevc et al., Biochem. Biophys. Acta 1368:201-15
(1998)).
III. Monitoring the Course of Treatment
[0315] The invention provides methods of monitoring treatment in a
subject suffering from a disorder in which MCP activity is
detrimental, i.e., for monitoring a course of treatment being
administered to a subject. The methods can be used to monitor both
therapeutic treatment on symptomatic subjects and prophylactic
treatment on asymptomatic subjects. In particular, the methods are
useful for monitoring passive immunization (e.g., measuring level
of administered antibody).
[0316] Some methods entail determining a baseline value, for
example, of an antibody level or profile in a subject, before
administering a dosage of agent, and comparing this with a value
for the profile or level after treatment. A significant increase
(i.e., greater than the typical margin of experimental error in
repeat measurements of the same sample, expressed as one standard
deviation from the mean of such measurements) in value of the level
or profile signals a positive treatment outcome (i.e., that
administration of the agent has achieved a desired response). If
the value for immune response does not change significantly, or
decreases, a negative treatment outcome is indicated.
[0317] In other methods, a control value (i.e., a mean and standard
deviation) of level or profile is determined for a control
population. Typically the individuals in the control population
have not received prior treatment. Measured values of the level or
profile in a subject after administering a therapeutic agent are
then compared with the control value. A significant increase
relative to the control value (e.g., greater than one standard
deviation from the mean) signals a positive or sufficient treatment
outcome. A lack of significant increase or a decrease signals a
negative or insufficient treatment outcome. Administration of agent
is generally continued while the level is increasing relative to
the control value. As before, attainment of a plateau relative to
control values is an indicator that the administration of treatment
can be discontinued or reduced in dosage and/or frequency.
[0318] In other methods, a control value of the level or profile
(e.g. a mean and standard deviation) is determined from a control
population of individuals who have undergone treatment with a
therapeutic agent and whose levels or profiles have plateaued in
response to treatment. Measured values of levels or profiles in a
subject are compared with the control value. If the measured level
in a subject is not significantly different (e.g., more than one
standard deviation) from the control value, treatment can be
discontinued. If the level in a subject is significantly below the
control value, continued administration of agent is warranted. If
the level in the subject persists below the control value, then a
change in treatment may be indicated.
[0319] In other methods, a subject who is not presently receiving
treatment but has undergone a previous course of treatment is
monitored for antibody levels or profiles to determine whether a
resumption of treatment is required. The measured level or profile
in the subject can be compared with a value previously achieved in
the subject after a previous course of treatment. A significant
decrease relative to the previous measurement (i.e., greater than a
typical margin of error in repeat measurements of the same sample)
is an indication that treatment can be resumed. Alternatively, the
value measured in a subject can be compared with a control value
(mean plus standard deviation) determined in a population of
subjects after undergoing a course of treatment. Alternatively, the
measured value in a subject can be compared with a control value in
populations of prophylactically treated subjects who remain free of
symptoms of disease, or populations of therapeutically treated
subjects who show amelioration of disease characteristics. In all
of these cases, a significant decrease relative to the control
level (i.e., more than a standard deviation) is an indicator that
treatment should be resumed in a subject.
[0320] The tissue sample for analysis is typically blood, plasma,
serum, mucous fluid or cerebrospinal fluid from the subject. The
sample is analyzed, for example, for levels or profiles of
antibodies to A.beta. peptide, e.g., levels or profiles of
humanized antibodies. ELISA methods of detecting antibodies
specific to MCPs are described in the Examples section.
[0321] The antibody profile following passive immunization
typically shows an immediate peak in antibody concentration
followed by an exponential decay. Without a further dosage, the
decay approaches pretreatment levels within a period of days to
months depending on the half-life of the antibody administered. For
example the half-life of some human antibodies is of the order of
20 days.
[0322] In some methods, a baseline measurement of antibody to MCPs
in the subject is made before administration, a second measurement
is made soon thereafter to determine the peak antibody level, and
one or more further measurements are made at intervals to monitor
decay of antibody levels. When the level of antibody has declined
to baseline or a predetermined percentage of the peak less baseline
(e.g., 50%, 25% or 10%), administration of a further dosage of
antibody is administered. In some methods, peak or subsequent
measured levels less background are compared with reference levels
previously determined to constitute a beneficial prophylactic or
therapeutic treatment regime in other subjects. If the measured
antibody level is significantly less than a reference level (e.g.,
less than the mean minus one standard deviation of the reference
value in population of subjects benefiting from treatment)
administration of an additional dosage of antibody is
indicated.
[0323] Additional methods include monitoring, over the course of
treatment, any art-recognized physiologic symptom (e.g., physical
or mental symptom) routinely relied on by researchers or physicians
to diagnose or monitor disorders associated with detrimental MCP
activity.
[0324] The invention further provides kits for performing the
monitoring methods described above. Typically, such kits contain an
agent that specifically binds to antibodies to MCPs, including
MCP-1, MCP-2, and/or MCP-3. The kit can also include a label. For
detection of antibodies to MCPs, the label is typically in the form
of labeled anti-idiotypic antibodies. For detection of antibodies,
the agent can be supplied prebound to a solid phase, such as to the
wells of a microtiter dish. Kits also typically contain labeling
providing directions for use of the kit. The labeling may also
include a chart or other correspondence regime correlating levels
of measured label with levels of antibodies to MCPs. The term
labeling refers to any written or recorded material that is
attached to, or otherwise accompanies a kit at any time during its
manufacture, transport, sale or use. For example, the term labeling
encompasses advertising leaflets and brochures, packaging
materials, instructions, audio or videocassettes, computer discs,
as well as writing imprinted directly on kits.
[0325] The invention also provides diagnostic kits, for example,
research, detection and/or diagnostic kits (e.g., for performing in
vivo imaging). Such kits typically contain an antibody for binding
to an epitope of an MCP. Preferably, the antibody is labeled or a
secondary labeling reagent is included in the kit. Preferably, the
kit is labeled with instructions for performing the intended
application, for example, for performing an in vivo imaging assay.
Exemplary antibodies are those described herein.
[0326] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application, as well as the figures and the
Sequence Listing, are incorporated herein by reference.
EXAMPLES
I. Characterization of Anti-Chemokine Monoclonal Antibody
Suernatants
Example 1
ELISA Screening
[0327] MaxiSorp 384 well plates were coated with 15-20 .mu.l
antigen in PBS. Recombinant purified human antigens included:
MCP-1, MCP-2, MCP-3, MCP-4, IL-8, eotaxin, fractalkine, Gcp-2,
DC-CK (Gcp-2 and DC-CK are also chemokines). Plates were incubated
with antigen for 2 hours at 37.degree. C. or overnight at 4.degree.
C. Non-specific sites were blocked with 80 .mu.l/well of 1% BSA/PBS
for 1 hour at room temperature. Plates were washed and 15 .mu.l of
hybridoma supernatant was added to each well and incubated for 1
hour at room temperature. Plates were washed and wells were
incubated with 20 .mu.l/well of a 1:25 000 dilution of goat
anti-mouse IgG peroxidase conjugate (Jackson Catalog Number
515-036-003). Plates were incubated for 1 hour at room temperature,
washed, and 20 .mu.l/well of substrate (TMB, tetramethylbenzidine,
Jackson, Catalog Number 515-036-062) was added. Reaction was
allowed to proceed and stopped by addition of 20 .mu.l/well of 2M
H.sub.2SO.sub.4. Reactive clones were picked for further analysis.
Isotyping of hybridoma supernatants was performed by
antigen-dependent ELISA. Briefly, wells were coated with 50 .mu.l
of human MCP-1 (5 .mu.g/ml) for 1 hour at 37.degree. C. Wells were
washed 4 times and blocked with PBS/1% BSA. Isotyping of hybridoma
supernatants was then performed using a mouse immunoglobulin
screening/isotyping kit (Zymed Laboratories, San Francisco, Calif.)
as recommended by the manufacturer. Specificities of the antibodies
and clones obtained are shown in Table 1.
TABLE-US-00003 TABLE 3 Panel of MCP mAbs Specificity Block
MCP-4/IL-8/ MCP-1 Eotaxin/Frac Affinity Sub- ligand Fusion
Immunogen Clone MCP-1 MCP-2 MCP-3 Gcp-2/DC-CK (Biacore) Type
binding IA1 MCP-1 1M-11 ++++ - - - ++++ IgG1 ++++ 3N10 ++++ - - -
++++ IgG1 ++++ IA7 MCP-1 11K2 ++++ ++++ ++++ - ++++ IgG1 ++++ MCP-2
7F7 ++++ - - - ++++ IgG1 +++ MCP-3 6D21 ++++ ++++ ++ - ++++ IgG1
++++ 6E11 ++ - + - +++ IgG1 +++ 1A1 +++ + + - ++++ IgG1 ++++ IA8
MCP-1 4N4 ++++ ++ ++ - ++++ IgG1 ++++ MCP-2 5A13 +++ ++ ++ - +++
IgG1 ++++ IA9 MCP-1 5J23 ++++ ++ - - ++++ IgG1 ++++ MCP-2 6I5 ++++
+++ +++ - ++++ IgG1 ++++ MCP-3 7H1 ++++ ++++ ++++ - ++++ IgG1 ++++
IA10 MCP-1 4N9 + - ++ - ++++ IgG1 +++ MCP-2 2O24 ++ - ++ - ++++
IgG1 +++ MCP-3 9H23 ++ - + - +++ IgG1 +++ 9B11 ++++ - ++++ - ++++
IgG1 +++ 9B12 + - +++ - ++++ IgG1 +++ 9C11 ++++ - ++++ - ++++ IgG1
+++ 10D18 ++++ - ++++ - ++++ IgG1 ++++ 12F15 +++ - +++ - ++++ IgG1
++++ MCP-1 D9 ++++ - - - (MCP-4, IL-8 +++ and eotaxin only tested)
+ and - indicate the relative amount of binding of the antibody to
the various immobilized ligands.
[0328] In addition, both 1A1 and 11K2 mAbs recognize primate MCP-1.
Plates were coated with 1 .mu.g/ml of chemically synthesized
chemokines (corresponding to the cynomolgus and rhesus MCP-1
sequences) and probed with 10 .mu.g/ml of monoclonal antibodies,
including MOPC21 (IgG1b control antisera), 11K2, 3N10, 1A1, D9, and
1M11, as described above. Results demonstrate that all of the above
mAbs, including 1A1 and 11K2, and with the exception of the isotype
control mAb MOPC21, also recognize primate MCP-1.
Example 2
Binding Assay
[0329] .sup.125I labeled MCP-1 (2200 Ci/mmol) was purchased from
NEN Life Sciences (Boston, Mass.). Hybridoma supernatants (50
.mu.l) were pre-incubated with 1 nM .sup.125I MCP-1 (50 .mu.l) for
60 minutes at room temperature prior to the addition of the
CCR2-expressing human monocyte cell line, THP-1. THP-1 cells
(1.times.10.sup.7 cells/ml; 501) were resuspended in binding buffer
(50 mM Hepes, 1 mM CaCl.sub.2, 5 mM MgCl.sub.2, 0.5% BSA) and added
to the combination of .sup.125I labeled MCP-1 and hybridoma
supernatant, and incubated at 4.degree. C. for 60 minutes. Cells
were then washed 3 times by centrifugation in wash buffer (50 mM
Hepes, 1 mM CaCl.sub.2, 5 mM MgCl.sub.2, 500 mM NaCl and 0.5% BSA).
Amount of bound .sup.125I labeled MCP-1 was then quantitated for
.gamma.-emission. Pre-incubation of THP-1 cells (1.times.10.sup.7
cells/ml; 501) with unlabeled MCP-1 (500 nM; 501) for 60 minutes at
4.degree. C. prior to addition of .sup.125I labeled MCP-1 (1 nM;
501) served as a negative control. The positive control represents
binding of .sup.125I labeled MCP-1 to THP-1 cells in the absence of
MCP-1 hybridoma supernatant. The results are shown in Table 2.
TABLE-US-00004 TABLE 4 Data of block ligand ([I.sup.125] MCP-1)
binding assay from .gamma. Counter CPM CPM Fusion Immunogen Clone #
(30/10/00) (22/11/00) IA1 MCP-1 1M-11 499 456 45 40 3N10 379 495 50
46 IA7 MCP-1 11K2 103 148 50 49 MCP-2 7F7 394 199 189 197 MCP-3
6D21 145 108 47 42 6E11 850 894 378 323 1A1 194 772 47 52 IA8 MCP-1
4N4 40 47 MCP-2 5A13 50 44 IA9 MCP-1 5J23 478 280 59 47 MCP-2 6I5
677 678 44 31 MCP-3 7H1 53 207 59 77 IA10 MCP-1 4N9 776 987 306 340
MCP-2 2O24 936 869 226 357 MCP-3 9H23 293 226 9B11 679 892 238 201
9B12 834 657 304 265 9C11 605 512 241 252 10D18 485 444 174 310
12F15 421 344 281 213 12K14 406 918 Negative control 836 461 68 143
Positive control 5861 3447 2084 2933
Example 3
Inhibition of Chemotaxis in Response to MCP-1
[0330] A 5 .mu.m pore size ChemoTX plate (Neuroprobe) was used to
assess the chemotactic response of THP-1 human monocytic cells.
Hybridoma supernatants containing MCP-1 at 10 ng/ml, or RPMI with
10% FBS with or without 10 ng/ml chemokine, was added to the lower
chamber of the plate. THP-1 cells at 2.times.10.sup.6 cells/ml were
layered on top. The plate was incubated for 2 hours at 37.degree.
C. in 5% CO2. The filter was removed and the number of cells that
migrated into the lower chamber was determined using Promega Cell
Titer reagent. The number of cells was calculated using a standard
curve (n=4, error bars=standard deviation). The results
demonstrated that antibodies 11K2, 7F7, 6D21 and 7H1 were all able
to inhibit MCP-1-induced chemotaxis, although 11K2 and 6D21 were
the most effective.
II. Characterization of Purified Anti-Chemokine Monoclonal
Antibodies
Example 4
Chemokine Specificity and Binding Assays
[0331] ELISA specificity assays were performed using purified
monoclonal antibodies to confirm the binding specificities of the
supernatant MCP-specific monoclonal antibodies described above.
Antibodies were purified by Protein A affinity column
chromatography, according to standard methods known in the art.
[0332] ELISA was performed as previously described in Example 1.
Briefly, MaxiSorp 384 well plates were coated with 15-20 .mu.l
antigen in PBS. Recombinant purified human antigens included:
MCP-1, MCP-2, MCP-3, MCP-4, IL-8, eotaxin, murine MCP-1 (JE),
murine MCP-3, murine MCP-5, and rat MCP-1. All antigens, including
MCP-3, were immobilized. Plates were washed and purified monoclonal
antibody (10 .mu.g/ml) was added to each well and incubated for 1
hour at room temperature. Plates were washed and wells were
incubated with 20 .mu.l/well of a 1:25,000 dilution of goat
anti-mouse IgG peroxidase conjugate (Jackson Catalog Number
515-036-003). Plates were incubated for 1 hour at room temperature,
washed, and 20 .mu.l/well of substrate (TMB, tetramethylbenzidine,
Jackson, Catalog Number 515-036-062) was added. Reaction was
allowed to proceed and stopped by addition of 20 .mu.l/well of 2M
H.sub.2SO.sub.4. Specificities of the purified antibodies and are
shown in Table 3. Antibodies 1A1 bound specifically to hMCP-1,
hMCP-2, hMCP-3, and mMCP-1. Antibodies 11K2, 4N4, 5A13, 6D21, 615,
and 7H1 bound specifically to hMCP-1, hMCP-2, hMCP-3, mMCP-1,
mMCP-3, and mMCP-5.
TABLE-US-00005 TABLE 5 ELISA performed using purified MCP mAbs
mMCP-1 hMCP-1 hMCP-2 hMCP-3 hMCP-4 (JE) mMCP-3 mMCP-5 rtMCP-1 hIL8
hEotaxin 1A1 + + + - + - - - - - 4N4 + + + - + + + - - - 5A13 + + +
- + + + - - - 6D21 + + + - + + + - - - 6I5 + + + - + + + - - - 7H1
+ + + - + + + - - - 11K2 + + + - + + + - - - D9 + - - - - - - - - -
1M11 + - - - - ND ND ND ND ND 3N10 + - - - - ND ND ND ND ND 2O24 +
- + - - ND ND ND ND ND 9B11 + - + - - ND ND ND ND ND 9B12 + - + - -
ND ND ND ND ND 9C11 + - + - - ND ND ND ND ND 5J23 + + +/- - + ND ND
- - -
[0333] Binding assays were performed using purified monoclonal
antibodies to confirm results obtained with the supernatants.
Binding assays were performed as described in Example 2. Briefly,
.sup.125I labeled MCP-1 (2200 Ci/mol) was purchased from NEN Life
Sciences (Boston, Mass.). Purified monoclonal antibodies at various
concentrations (33 nM, 3.3 nM and 0.33 nM) were pre-incubated with
1 nM .sup.125I MCP-1 (501) for 60 minutes at room temperature prior
to addition of the CCR2-expressing human monocyte cell line, THP-1.
THP-1 cells (1.times.10.sup.7 cells/ml; 50 .mu.l) were resuspended
in binding buffer (50 mM Hepes, 1 mM CaCl.sub.2, 5 mM MgCl.sub.2,
0.5% BSA) and added to the combination of .sup.125I labeled MCP-1
and purified mAb, and incubated at 4.degree. C. for 60 minutes.
Cells were then washed 3 times by centrifugation in wash buffer (50
mM Hepes, 1 mM CaCl.sub.2, 5 mM MgCl.sub.2, 500 mM NaCl, and 0.5%
BSA). Amount of bound .sup.125I labeled MCP-1 was then quantitated
for .gamma.-emission. Results from the binding assays are shown in
Table 4. This study demonstrates that many of the studied
monoclonal antibodies, including 1A1 and 11K2, were effective at
blocking hMCP-1 binding.
TABLE-US-00006 TABLE 6 Purified mAb binding assay Block hMCP-1 cell
Antibody binding 1A1 + 4N4 + 5A13 + 6D21 + 6I5 + 7H1 + 11K2 + D9 +
1M11 + 3N10 + 2O24 - 9B11 - 9B12 - 9C11 - 5J23 +
Example 5
Inhibition of Monocyte Chemotaxis by Anti-Chemokine Monoclonal
Antibodies
A. MCP-1 and MCP-2 Chemotaxis Assay
[0334] A 5 .mu.m pore size ChemoTX plate (Neuroprobe) was used to
assess the chemotactic response of THP-1 human monocytic cells.
Purified monoclonal antibodies (100 .mu.g/ml) 11K2, 1A1, D9, and
2024 were added in combination with and without MCP-1 (2.3 nM),
MCP-2 (56 nM), and MCP-1/MCP-2 (2.3 nM MCP-1 and 56 nM MCP-2), to
the lower chamber of the plate. THP-1 cells at 2.times.10.sup.6
cells/ml were layered on top. The plate was incubated for 4 hours
at 37.degree. C. in 5% CO.sub.2. The filter was removed and the
number of cells that migrated into the lower chamber was determined
using Promega Cell Titer reagent.
[0335] The results show that pan-monoclonal antibodies 11K2 and 1A1
were effective at inhibiting chemotaxis in the presence of both
MCP1 and MCP-2 (FIG. 1). The results also demonstrate that
antibodies D9 and 2024 can inhibit chemotaxis which is induced by
MCP-1 alone. Furthermore, as shown in FIGS. 2A and 2B, antibodies
1M11 and 3N10 can inhibit THP-1 chemotaxis induced by human MCP-1,
and antibody 5J23 can inhibit chemotaxis induced by human and mouse
MCP-1. In sum, chemotaxis to the combination of MCP-1 and MCP-2 is
inhibited by 11K2 and 1A1, but is not observed by antibodies D9 and
2024, which are MCP-1-specific mAb (D9 and 2024).
[0336] Within the pool of monoclonal antibodies studied, there are
three groups which arise based on their ability to recognize
certain MCP antigens. Monoclonal antibodies 1A1 and 11K2 recognize
MCP-1, MCP-2 and immobilized MCP-3. Monoclonal antibodies 1M11 and
3N10 recognize MCP-1, and antibody 2024 recognizes MCP-1 and MCP-3.
Antibody 5J23 recognizes mouse MCP-1 and recognizes only human
MCP-1 and human MCP-2.
[0337] Results from a separate experiment using the ChemoTX plate
(Neuroprobe) assay are shown below in Table 5. The protocol for
this experiment was the same as previously described, except a
titration of mAb was used in combination with fixed MCP
concentrations (concentrations of MCPs are shown below in Table 5).
The results described in Table 5 demonstrate that mAbs 11K2 and 1A1
are effective at inhibiting huMCP-1, huMCP-2, muMCP-1, and
muMCP-5-induced chemotaxis.
TABLE-US-00007 TABLE 7 11K2 and 1A1 inhibit THP-1 chemotaxis
towards human MCP-1, human MCP-2, mouse MCP-1 and mouse MCP-5 Human
MCP-1 MCP-2 MCP-3 MCP-4 ND.sub.50 (nM) (2.3 nM) (56 nM) (11.8 nM)
(58 nM) Commercial 10.0 33.0 2.6 11.5 1A1 1.4 47.5 No Inhib No
Inhib 11K2 1.3 52.0 No Inhib No Inhib D9 5.8 No Inhib No Inhib No
Inhib 2O24 1000.0 No Inhib 143.5 No Inhib Murine MCP-1 MCP-3 MCP-5
ND.sub.50 (nM) (1.4 nM) MCP-2 (59 nM) MCP-4 (0.54 nM) Commercial
3.2 ND 52.0 ND 0.1 1A1 1.7 ND No Inhib ND 13.5 11K2 2.1 ND No Inhib
ND 19.5 D9 No Inhib ND No Inhib ND No Inhib 2O24 No Inhib ND No
Inhib ND No Inhib No Inhib = less than 50% Neutralization at 3
uM
B. Inhibition of Chemotaxis by Cytokines Secreted from RA
Fibroblasts
[0338] Prior to studying the ability of purified monoclonal
antibodies 1A1, 11K2, D9, 2024, and 5D3-F7 (BD Biosciences,
Pharmingen, San Diego, Calif.), to inhibit chemotaxis from
chemokines secreted from stimulated RA fibroblasts, a study of the
different types of chemokines secreted by RA (rheumatoid arthritis)
fibroblasts in response to inflammatory chemokines was performed.
RA fibroblasts were exposed for 48 hours to 500 U/ml IFN-.gamma.,
IFN-.gamma. and 10 ng/ml of IL1.beta., or media (as a control).
Results showed that IFN-.gamma. alone induced low levels of MCP-1,
MCP-2, MCP-3, and very low levels of IP10. IFN-.gamma. exposure
alone did not induce expression of Rantes, IL-8, Mip 1.alpha., or
Mip1.beta.. In contrast, the combination of IFN-.gamma. and 10
ng/ml of IL1.beta. induced about 27 ng/ml of MCP-1, 31 ng/ml of
MCP-2, 9 ng/ml of MCP-3, and 55 ng/ml of IL-8. The combination of
IFN-.gamma. and 10 ng/ml of IL1.beta. also yielded low levels of
Rantes, IP10, and Mip1.alpha.. The media alone control did not
induce any chemokine secretion.
[0339] The ability of purified monoclonal antibodies to inhibit
monocyte chemotaxis to cytokines secreted from these stimulated RA
fibroblasts was then studied. Supernatant from RA fibroblasts which
were exposed to either media alone, IFN-.gamma. alone, or the
combination of IFN-.gamma. and IL-1.beta., were each tested for
their chemotactic ability using human THP-1 cells. As a control,
supernatant from unstimulated RA fibroblasts into which IFN-.gamma.
(500 U/ml) and IL1.beta. (10 ng/ml) was spiked was used. This
supernatant (spike) control was used to evaluate the direct effects
of IL1.beta. and IFN-.gamma. on chemotaxis. As shown in FIG. 3,
monocyte chemotaxis mediated by cytokines secreted from stimulated
RA fibroblasts was inhibited by MCP mAbs 1A1 and 11K2.
MCP-1-specific antibody D9 was also effective at inhibiting
chemotaxis in all experimental groups.
Example 6
MCP-1-Induced Calcium Flux Assay for Monoclonal 11K2
[0340] The MCP-1-induced calcium flux assay was performed according
to standard procedure. Briefly, monoclonal antibody 11K2 and a
chemokine (MCP-1 or MCP-2) were mixed at 200.times. concentration
and pre-incubated for one hour. This mixture was then added to
THP-1 cells stirring in a cuvette in a fluorimeter at t=30 sec.
Calcium flux was measured by a change in fluorescence of Indo-1.
Results show that MCP-1-induced calcium flux in THP-1 cells was
blocked by 11K2 (FIGS. 4A and 4B).
Example 7
Agonist Effect at Low Antibody Concentrations of 11K2 and 1A1
A. Chemotaxis Assay
[0341] Chemotaxis assays were performed as previously described
with recombinant MCP-2 and using low concentrations of monoclonal
antibodies 11K2 and 1A1. The results from the chemotaxis assay
showed that at a low concentration, monoclonal antibodies 11K2 and
1A1 increased MCP-2 mediated chemotaxis. As shown in FIG. 5A, there
was an increase in chemotaxis observed with low antibody
concentrations (ranging from about 1-15 nM) of 11K2 and 1A1, in
contrast to the MCP-2 mAb 281 (RD Systems, Minneapolis, Minn.). The
agonist effect was not seen with the Fab fragment of 11K2 or 1A1,
and was MCP-2-specific. As shown in FIG. 5B, low concentrations of
the 11K2 Fab fragment did not result in a chemotactic increase in
response to MCP-2 and showed only antagonist activity.
B. Calcium Flux Assay
[0342] A calcium flux assay was performed as previously described,
except a low concentration of 11K2 monoclonal antibody (16.5 nM)
was also included. The results (FIGS. 6A and 6B) demonstrate that
low concentrations of 11K2 exposure results in agonistic activity
in a MCP-2 calcium flux assay. In addition, however, 11K2 Fab and
F(ab).sub.2 fragments are inhibitory in the same assay (FIGS. 6C
and 6D).
Example 8
Binding Affinity Measurement of Monoclonal 11K2 and 1A1
[0343] To measure the affinity of MCP mAb and Fab molecules for
soluble MCP molecules, a kinetic exclusion assay was utilized and
affinity measured using a KinExA instrument (Sapidyne Instruments
Inc., Boise, Id.).
[0344] Polymethylmethacrylate beads activated with NHS were coated
with 10 .mu.g recombinant human MCP-1 in 1 ml buffer. The beads
were packed into a column in the KinExA instrument for each sample.
This packed bead column is able to capture free MCP mAb or Fab
flowed through the column. The amount of free mAb or Fab in
solution was determined using a secondary goat anti-mouse heavy and
light chain IgG-Cy5 conjugate.
[0345] A fixed amount of 11K2 mAb, 1A1 mAb, 11K2 Fab, or 1A1 Fab
was incubated with various amounts of human MCP-1, MCP-2 or MCP-3
for three hours. The amount of uncomplexed free antibody remaining
in solution was determined by flowing these mixtures over the
MCP-1-loaded bead column and labeling with the Cy5 secondary
antibody. The fluorescent signal was plotted against the MCP
concentration and the affinity was determined using a quadratic
curve fit
[0346] Affinities determined for both 11K2 and 1A1 mAb and Fab
molecules are listed in Table 6 below. Exact affinities of 11K2 mAb
and 1A1 mAb for human MCP-1 could not be determined, as the
affinity is much lower than the lowest amount of antibody that can
be detected by this method. In those cases, only an upper limit to
the affinity can be determined. Affinity of the 11K2 Fab and 1A1
Fab for human MCP-3 was not determined (ND).
TABLE-US-00008 TABLE 8 MCP binding affinity measurements in
solution Antibody MCP-1 MCP-2 MCP-3 11K2 mAb <4 .times.
10.sup.-13M 1.8 .times. 10.sup.-11M >5 .times. 10.sup.-8M 11K2
Fab 1.1 .times. 10.sup.-11M 4.3 .times. 10.sup.-10M ND 1A1 mAb
<7 .times. 10.sup.-13M 1.2 .times. 10.sup.-12M >5 .times.
10.sup.-8M 1A1 Fab 1.3 .times. 10.sup.-11M 3.2 .times. 10.sup.-10M
ND
[0347] In sum, monoclonal antibodies 1A1 and 11K2 recognize soluble
human MCP-1 and MCP-2 with a very high binding affinity which is in
the low pM range. Both 1A1 and 11K2 also recognize mouse MCP-1,
while neither recognizes soluble MCP-3.
III. Cloning and Sequencing of 1A1 and 11K2 Monoclonal
Antibodies
Example 9
Cloning and Sequencing of mu1A1 Variable Regions
[0348] Mouse monoclonal antibody 1A1 was cloned and sequenced
according to the following procedure. Total cellular RNA from 1A1
murine hybridoma cells was prepared using the Qiagen RNeasy mini
kit according to the manufacturer's recommended protocol.
[0349] cDNAs encoding the 1A1 variable regions of the heavy and
light chains were cloned by RT-PCR from total cellular RNA,
following standard procedures known to one of skill in the art.
Briefly, following the manufacturer's recommended protocols,
first-strand cDNAs (prepared with the Amersham First-Strand cDNA
Synthesis Kit) were amplified by PCR using the Clontech Advantage 2
PCR Kit. The following primers were used for first-strand synthesis
of the 1A1 heavy and light chain cDNAs (Y=C/T, and R=A/G): 1A1
Heavy Chain cDNA Primer: 5'-AGG TCT AGA AYC TCC ACA CAC AGG RRC CAG
TGG ATA GAC-3' (SEQ ID NO: 3) and 1A1 Light Chain cDNA Primer:
5'-GCG TCT AGA ACT GGA TGG TGG GAG ATG GA-3' (SEQ ID NO: 4).
[0350] Primers used for PCR amplification of the murine 1A1
immunoglobulin heavy chain variable domain were as follows: 5'-AGG
TSM ARC TGC AGS AGT CWG G-3' (SEQ ID NO: 5) and 5'-TGA GGA GAC GGT
GAC CGT GGT CCC TTG GCC CC-3' (SEQ ID NO: 6) (S=C/G, M=A/C, R=A/G,
and W=A/T). The primers used for PCR amplification of the murine
1A1 immunoglobulin light chain variable domain were: 5'-GAY ATH CAR
ATG ACN CAG-3' (SEQ ID NO: 7) and 5'-GCG TCT AGA ACT GGA TGG TGG
GAG ATG GA-3' (SEQ ID NO: 8) (Y=C/T, H=A/C/T, R=A/G, and
N=A/C/GT).
[0351] The PCR was performed at 30 cycles using Clontech's
Advantage 2 PCR Kit using the following PCR conditions: denature
0.5 min at 94.degree. C., anneal 1 min at 50.degree. C., and
elongate 1 min at 72.degree. C. The PCR products were gel-purified
using the Qiagen Qiaquick gel extraction kit following the
manufacturer's recommended protocol. Purified 1A1 heavy and light
chain PCR products were subcloned into Invitrogen's pCR2.1-TOPO
vector using their TOPO TA Cloning kit according to the
manufacturer's recommended protocol (pCR-049=1A1 heavy chain,
pcr-053=1A1 light chain). Inserts from multiple independent
subclones were sequenced according to methods known in the art and
those described in Sanger et al., PNAS 74, 5463-5467, incorporated
herein by reference, and subclones were found to be identical.
[0352] Sequence data was analyzed according to BLAST analysis (see
http://www.ncbi.nlm.nih.gov). Blast analyses of the variable domain
sequences confirmed their immunoglobulin identity. The 1A1 heavy
chain variable domain was determined to be a member of murine
subgroup II(C), while the 1 A1 light chain variable region was
determined to be a member of murine kappa subgroup II. The
predicted amino acid sequences of the mature 1A1 murine variable
domains, as well as the determined nucleotide sequences, are shown
below in Tables 7 and 8.
TABLE-US-00009 TABLE 9 Nucleotide sequence of mu1A1 variable
domains 1A1 Heavy Chain Variable Region 1
GAGGTCCAGCTGCAGCAGTCTGGGGCAGAACTTGTGAGGTCAGGGGCCTCAGTCAAGTTG 60 61
TCCTGCACAGCTTCTGGCTTCAACATTAAAGACAACTATATGCACTGGGTGAAGCAGAGG 120
121 CCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGGAGATACTGAATAT
180 181
GCCCCGAAGTTCCAGGGCAAGGCCACTATGACTGCAGACACATCCTCCAACACAGCCTAC 240
241 CTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTAATACATGGGCT
300 301
TACTACGGTACTAGCTACGGGGGATTTGCTTACTGGGGCCAAGGGACCACGGTCACCGTC 360
361 TCCTCA 366 (SEQ ID NO: 9) 1A1 Light Chain Variable Region 1
GATATCCAGATGACTCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCC 60 61
ATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGG 120
121 TCGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGAC
180 181
TCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATC 240
241 AGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCT
300 301 CAGACGTTCGGTGGAGGCACCAAGCTGGAGATCAAA 336 (SEQ ID NO:
10)
TABLE-US-00010 TABLE 10 Amino acid sequence of 1A1 variable domains
(CDR domains underlined) 1A1 Heavy Chain Variable Region
EVQLQQSGAELVRSGASVKLSCTASGFNIKDNYMHWVKQRPEQGLEWIGWIDPENGDTEYAPK
CDR1 CDR2
FQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNTWAYYGTSYGGFAYWGQGTTVTVSS CDR3
(SEQ ID NO: 11) 1A1 Light Chain Variable Region
DIQMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWSLQRPGQSPKRLIYLVSKLDSGV
CDR1 CDR2 PDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK CDR3
(SEQ ID NO: 12)
[0353] Nucleotide and amino acid comparisons of the 1A1 variable
heavy and light chains are also shown in FIGS. 7A and 7B. The
sequence of the CDR regions of the 1A1 antibody were determined to
be the following:
TABLE-US-00011 TABLE 11 CDRs of the 1A1 Antibody 1A1 Heavy Chain
Variable Region CDR1: DNYMH (SEQ ID NO: 13) CDR2: WIDPENGDTEYAPKFQG
(SEQ ID NO: 14) CDR3: WAYYGTSYGGFAY (SEQ ID NO: 15) 1A1 Light Chain
Variable Region CDR1: KSSQSLLDSDGKTYLN (SEQ ID NO: 16) CDR2:
LVSKLDS (SEQ ID NO: 17) CDR3: WQGTHFPQT (SEQ ID NO: 18)
Example 10
Cloning and Sequencing of mu11K2 Variable Regions
[0354] Mouse monoclonal antibody 11K2 was cloned and sequenced
according to the following procedure. Total cellular RNA from 11K2
murine hybridoma cells was prepared using the Qiagen RNeasy mini
kit according to the manufacturer's recommended protocol.
[0355] cDNAs encoding the variable regions of the heavy and light
chains were cloned by RT-PCR from total cellular RNA. Following the
manufacturers recommended protocols, first-strand cDNAs (prepared
with the Amersham First-Strand cDNA Synthesis Kit) were amplified
by PCR using the Clontech Advantage 2 PCR Kit. The following
primers were used for first-strand synthesis of the 11K2 heavy and
light chain cDNAs (Y=C/T, and R=A/G): 11K2 Heavy Chain cDNA Primer:
5'-AGG TCT AGA AYC TCC ACA CAC AGG RRC CAG TGG ATA GAC-3' (SEQ ID
NO: 19) and 11K2 Light Chain cDNA Primer: 5'-GCG TCT AGA ACT GGA
TGG TGG GAG ATG GA-3' (SEQ ID NO: 20).
[0356] The primers used for PCR amplification of the murine 11K2
immunoglobulin heavy chain variable domain were: 5'-GGG GAT ATC CAC
CAT GGR ATG SAG CTG KGT MAT SCT CT-3' (SEQ ID NO: 21) and 5'AGG TCT
AGA AYC TCC ACA CAC AGG RRC CAG TGG ATA GAC-3' (SEQ ID NO: 22)
(R=A/G, S=C/G, K=G/T, M=A/C, and Y.dbd.C/T). The primers used for
PCR amplification of the murine 11K2 immunoglobulin light chain
variable domain were: 5'-GAY ATH CAR ATG ACN CAG-3' (SEQ ID NO: 23)
and 5'-GCG TCT AGA ACT GGA TGG TGG GAG ATG GA-3' (SEQ ID NO: 24)
(Y=C/T, H=A/C/T, R=A/G, and N=A/C/G/T).
[0357] The PCR was performed at 30 cycles using Clontech's
Advantage 2 PCR Kit under the following PCR conditions: denature
0.5 min at 94.degree. C., anneal 1 min at 50.degree. C., and
elongate 1 min at 72.degree. C. The PCR products were gel-purified
using the Qiagen Qiaquick gel extraction kit following the
manufacturer's recommended protocol. Purified 11K2 heavy and light
chain PCR products were subcloned into Invitrogen's pCR2.1-TOPO
vector using their TOPO TA Cloning kit according to the
manufacturer's recommended protocol (pCR-008=11K2 heavy chain,
pcr-033=11K2 light chain. Inserts from multiple independent
subclones were sequenced according to methods known in the art and
those described in Sanger et al., PNAS 74, 5463-5467, incorporated
herein by reference, and the subclones were found to be
identical.
[0358] The variable light and heavy chains were compared with the
consensus sequences for mouse and human subgroups (Kabat et al,
1991) using the program FASTA and a database of consensus
sequences, which are publicly available (see
http://people.cryst.bbk.ac.uk/-ubcg07s/). The variable light chain
is a member of mouse subgroup Kappa 5 with a 68.8% identity in 113
aa overlap and the variable heavy chain is a member of mouse
subgroup 2C with a 85.6% identity in 125 aa overlap. The variable
light chain corresponds to human subgroup Kappa 1 with a 69.9%
identity in 113 aa overlap. The variable heavy chain corresponds to
human subgroup 1 with a 59.7% identity in 129 aa overlap. The
predicted amino acid sequences of the mature 11K2 murine variable
domains, as well as the determined nucleotide sequences, are shown
below in Tables 12 and 13.
TABLE-US-00012 TABLE 12 Nucleotide sequence of mu 1 1 K2 variable
domains 11K2 Heavy Chain Variable Region 1
GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGGCAGGGGCCTCAGTCAAGTTG 60 61
TCCTGCCCAGCTTCTGGCCTCAACATTAAAGACACCTATATGCACTGGGTGAAGCAGAGG 120
121 CCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTAAATTT
180 181
GACCCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTAC 240
241 CTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAGAGGCGTC
300 301 TTTGGCTTTTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 351
(SEQ ID NO: 25) 11K2 Light Chain Variable Region 1
GACATTCAGATGACTCAGTCTTCATCCTCCTTTTCTGTATCTCTAGGAGACAGAGTCACC 60 61
ATTACTTGCAAGGCAACTGAGGACATATATAATCGATTAGCCTGGTATCAGCAGAAACCA 120
121 GGAAGTGCTCCTAGGCTCTTAATTTCTGGTGCAACCAGTTTGGAGACTGGGGTTCCTTCA
180 181
AGATTCAGTGGCAGTGGATCTGGAAAAGATTACACTCTCAGCATTACCAGTCTTCAGACT 240
241 GAGGATGTTGCTACTTATTACTGTCAACAGTTTTGGAGTGCTCCGTACACGTTCGGAGGG
300 301 GGGACCAAGCTGGAGATCAAA 321 (SEQ ID NO: 26)
TABLE-US-00013 TABLE 13 Amino acid sequence of 11K2 variable
domains (CDR regions underlined) 11K2 heavy chain variable region
EVQLQQSGAELVKAGASVKLSCPASGLNIKDTYMHWVKQRPEQGLEWIGRIDPANGNTKFDPK
CDR1 CDR2 FQGKATITADTSSNTAYLQLSSLTSEDTAVYYCARGVFGFFDYWGQGTTLTVSS
CDR3 (SEQ ID NO: 27) 11K2 light chain variable region
DIQMTQSSSSFSVSLGDRVTITCKATEDIYNRLAWYQQKPGSAPRLLISGATSLETGVPSRFS
CDR1 CDR2 GSGSGKDYTLSITSLQTEDVATYYCQQFWSAPYTFGGGTKLEIK (SEQ ID NO:
28) CDR3
[0359] Nucleotide and amino acid comparisons of the 11K2 variable
heavy and light chains are also shown in FIGS. 8A and 8B. The
sequence of the CDR regions of the 11K2 antibody were determined to
be as follows:
TABLE-US-00014 TABLE 14 CDRs of 11K2 Antibody 11K2 Heavy Chain
Variable Region CDR1: DTYMH (SEQ ID NO: 29) CDR2: RIDPANGNTKFDPKFQG
(SEQ ID NO: 30) CDR3: GVFGFFDY (SEQ ID NO: 31) 11K2 Light Chain
Variable Region CDR1: KATEDIYNRLA (SEQ ID NO: 32) CDR2: GATSLET
(SEQ ID NO: 33) CDR3: QQFWSAPYT (SEQ ID NO: 34)
[0360] Based on the results described above, particularly Tables 3
and 5, antibodies were grouped according to their antigen-binding
specificity. The CDR region of mAbs which could recognize MCP-1,
MCP-2, and MCP-3, including 4N4, 5A13, 6D21, 615, 7H1, 11K2, and
1A1 were determined as described above. Sequencing revealed that
mAbs 4N4, 5A13, 6D21, 6I5, 7H1, and 11K2 all had identical
sequences. Monoclonal antibody 1A1 had a different sequence. Thus,
based on CDR cloning, as well as N-terminal sequencing, two
distinct pan-MCP monoclonals antibodies were found to exist.
Example 11
PEGylated 11K2 Fab
[0361] As depicted in FIG. 13, PEGylation of the 11K2 Fab fragment
does not interfere with 11K2's in vitro activity. PEGylated 11K2
fragments were tested in a chemotaxis inhibition assay (as
described previously), using 20 ngmL of MCP-1. PEGylated 11K2
fragments were as effective as the 11K2 Fab alone at inhibiting
migration of the cells.
Example 12
Chimeric 11K2 Antibody
[0362] The nucleotide and amino acid sequences of the 11K2 heavy
chain chimeric antibody are shown in FIG. 9A. The variable region
is defined as nucleotides 1-351 (amino acids 1-117) of the heavy
chain. The nucleotide and amino acid sequences of the 11K2 light
chain chimeric antibody are shown in FIG. 9B, where the variable
region is defined as nucleotides 1-321 and amino acids 1-107.
IV. Humanized 11K2 Antibody
Example 13
11K2 Humanization
[0363] Modeling the Structure of the Variable Regions
[0364] In order to identify key structural framework residues in
the murine 11K2 antibody, a three-dimensional model was generated
based on the closest murine antibodies for the heavy and light
chains. The 11K2 light and heavy chains were aligned against a
local copy of the most recent PDB database to determine structural
frames to be used to construct three dimensional models of the
light and heavy chains. Using FASTA the light chain was found to
have 90.7% sequence identity to monoclonal murine antibody Fab
184.1 (10SP; 1.8 .ANG.), and the heavy chain was found to have
89.7% sequence identity to murine E8 Fab fragment (1WEJ; 1.8
.ANG.).
[0365] Using the molecular modeling package Modeler 5.0 (Accelrys
Inc.) the three dimensional structures of the light and heavy
chains were built using antibodies 184.1 and E8, respectively. Five
homology models were created, and the best one in terms of Modeler
energy was selected. Procheck analysis showed that 1 residue (A51,
light chain) was in a disallowed region of the phi/psi map, but by
comparing phi/psi maps of the different models, the lowest energy
model was also the best in phi/psi map violations.
[0366] Design of the Reshaped Variable Regions
[0367] Human germline sequences were used as the acceptor
frameworks for humanized 11K2. To find the closest germline
sequences, the NCBI NR database and the Kabat database were
searched for the most homologous expressed human frameworks in. In
this search the CDR sequences were masked. The selection of the
most suitable expressed sequence includes checking for sequence
identity of the canonical and interface residues, and checking for
the similarity in CDR loop lengths. The source of the antibody was
also a determining factor. Previously humanized antibodies were
excluded. For the NCBI NR database search, a BLAST was used, and
for the Kabat database search, FASTA was used.
[0368] The most similar expressed light chain was found in the nr
database (GI-486875; Griffiths et al. (1993), supra), and the most
similar heavy chain was found in the Kabat database (Kabat ID
000054; Kipps. & Duffy (1991), supra). Both sequences were
searched against the database of germine sequences
(http://www.ncbi.nlm.nih.gov/igblast/), and resulted in the
following selected germlines: L11 for the light chain, and 1-69 for
the heavy chain.
[0369] As noted supra, the humanized antibodies of the invention
comprise variable framework regions substantially from a human
immunoglobulin (acceptor immunoglobulin) and complementarity
determining regions substantially from a mouse immunoglobulin
(donor immunoglobulin) termed 11K2. Having identified the
complementarity determining regions of 11K2 and appropriate human
acceptor immunoglobulins, the next step was to determine which, if
any, residues from these components to substitute to optimize the
properties of the resulting humanized antibody. The criteria
described supra were used to select residues for substitution. A
summary of the backmutations is shown below in Table 15:
TABLE-US-00015 TABLE 15 Summary of backmutations of humanized 11K2
Backmutations in Reshaped VL - Human germline L11 49 Y .fwdarw. S
This residue is close to a hypervariable loop and it retained in
both versions 69 T .fwdarw. K This residue is close to a
hypervariable loop but the sidechain is solvent exposed. Only
retained in the first version. 71 F .fwdarw. Y This is a canonical
residue and is retained in both versions Backmutations in Reshaped
VH - Human germline 1-69 27 G .fwdarw. L This is a canonical
residue. Retained in both versions. 28 T .fwdarw. N This residue is
close to a hypervariable loop. Retained in first version. 29 F
.fwdarw. I This residue is close to a hypervariable loop. Retained
in both versions. 30 S .fwdarw. K This residue is close to a
hypervariable loop. Retained in first version. 48 M .fwdarw. I This
residue is close to a hypervariable loop but is a fairly
conservative change. Retained in first version. 67 V .fwdarw. A
This residue is close to a hypervariable loop, and is close to
residue 48. Retained in first version. 73 K .fwdarw. T This residue
is close to a hypervariable loop. Moreover, the human and mouse
consensus is T. Retained in both versions.
[0370] FIGS. 11A and 11B depict alignments of the chimeric 11K2 VL
and VH, respectively (same as the original murine 11K2 sequence),
with the versions 1 and 2 of the humanized 11K2 antibody. Two
versions of the variable light reshaped chain and two versions of
the variable heavy reshaped chain were designed. The first version
contains the most backmutations and the second version contains the
fewest (i.e. the most "humanized"). The sequences of the two
versions of each variable and heavy chains of humanized 11K2 are
shown below:
Humanized 11K2 Heavy Chain (backmutations shown in lower case):
TABLE-US-00016 Version 1 (H1) (7 backmutations) (SEQ ID NO: 47)
QVQLVQSGAEVKKPGSSVKVSCKASGlnikDTYMHWVRQAPGQGLEWiGRIDPANGNTKF
DPKFQGRaTITADtSTSTAYMELSSLRSEDTA VYYCARGVFGFFDYWGQGTTVTVSS Version
2 (H2) (3 backmutations) (SEQ ID NO: 48)
QVQLVQSGAEVKKPGSSVKVSCKASGlTiSDTYMHWVRQAPGQGLEWMGRIDPANGNTKF
DPKFQGRVTITADtSTSTAYMELSSLRSEDTAVYYCARGVFGFFDYWGQGTTVTVSS Humanized
11K2 Light Chain (backmutations shown in lower case): Version 1
(L1) (3 backmutations) (SEQ ID NO: 49)
DIQMTQSPSSLSASAVGDRVTITCKATEDIYNRLAWYQQ KPGKAPKLLIsGATSLETGVPS
RFSGSGSGkDyTLTISSLQPEDFATYYCQQF WSAPYTFGGGTKVEIK Version 2 (L2) (2
backmutations) (SEQ ID NO: 50)
DIQMTQSPSSLSASVGDRVTITCKATEDIYNRLAWYQQKPGKAPKLLIsGATSLETGVPS
RFSGSGSGTDyTLTISSLQPEDFATYYCQQFW SAPYTFGGGTKVEIK
[0371] Tables 16 and 17 set forth Kabat numbering keys for the
various light and heavy chains of 11K2, respectively.
TABLE-US-00017 TABLE 16 Key to Kabat Numbering for 11K2 Heavy Chain
Variable Region Mouse KABID Hum. Hum. Kabat # AA # Type 11K2 000054
11K2, v1 11K2, v2 Comment 1 1 FR1 E Q Q Q 2 2 V V V V 3 3 Q Q Q Q 4
4 L L L L 5 5 Q V V V 6 6 Q Q Q Q 7 7 S S S S 8 8 G G G G 9 9 A A A
A 10 10 E E E E 11 11 L V V V 12 12 V K K K 13 13 K K K K 14 14 A P
P P 15 15 G G G G 16 16 A S S S 17 17 S S S S 18 18 V V V V 19 19 K
K K K 20 20 L V V V 21 21 S S S S 22 22 C C C C 23 23 P K K K 24 24
A A A A 25 25 S S S S 26 26 G G G G 27 27 L G L L Canonical
residue, retained 28 28 N T N T Residue close to hyper variable
loop, retained in v1 (H1) 29 29 I F I I Residue close to hyper
variable loop, retained 30 30 K S K S Residue close to hyper
variable loop, retained in v1 (H1) 31 31 CDR1 D S D D 32 32 T Y T T
33 33 Y A Y Y 34 34 M I M M 35 35 H S H H 36 36 FR2 W W W W 37 37 V
V V V 38 38 K R R R 39 39 Q Q Q Q 40 40 R A A A 41 41 P P P P 42 42
E G G G 43 43 Q Q Q Q 44 44 G G G G 45 45 L L L L 46 46 E E E E 47
47 W W W W 48 48 I M I M Residue close to hypervariable loop,
retained in v1 (H1) 49 49 G G G G 50 50 CDR2 R G R R 51 51 I I I I
52 52 D I D D .sup. 52A 53 P P P P 53 54 A I A A 54 55 N F N N 55
56 G G G G 56 57 N T N N 57 58 T A T T 58 59 K N K K 59 60 F Y F F
60 61 D A D D 61 62 P Q P P 62 63 K K K K 63 64 F F F F 64 65 Q Q Q
Q 65 66 G G G G 66 67 FR3 K R R R 67 68 A V A V Residue close to
hypervariable loop, retained in v1 (H1) 68 69 T T T T 69 70 I I I I
70 71 T T T T 71 72 A A A A 72 73 D D D D 73 74 T E T T Residue
close to hypervariable loop, retained 74 75 S S S S 75 76 S T T T
76 77 N S S S 77 78 T T T T 78 79 A A A A 79 80 Y Y Y Y 80 81 L M M
M 81 82 Q E E E 82 83 L L L L .sup. 82A 84 S S S S .sup. 82B 85 S S
S S .sup. 82C 86 L L L L 83 87 T R R R 84 88 S S S S 85 89 E E E E
86 90 D D D D 87 91 T T T T 88 92 A A A A 89 93 V V V V 90 94 Y Y Y
Y 91 95 Y Y Y Y 92 96 C C C C 93 97 A A A A 94 98 R R R R 95 99
CDR3 G G G G 96 100 V S V V 97 101 F S F F 98 102 G W G G 99 103 F
T F F 100 104 F F F F 101 105 D D D D 102 106 Y Y Y Y 103 107 FR4 W
W W W 104 108 G G G G 105 109 Q Q Q Q 106 110 G G G G 107 111 T T T
T 108 112 T L T T 109 113 L V V V 110 114 T T T T 111 115 V V V V
112 116 S S S S 113 117 S S S S
TABLE-US-00018 TABLE 17 Key to Kabat Numbering for 11K2 Light Chain
Variable Region Mouse Hum. Hum. Kabat # AA # Type 11K2 GI-486875
11K2, v1 11K2, v2 Comment 1 1 FR1 D D D D 2 2 I I I I 3 3 Q Q Q Q 4
4 M M M M 5 5 T T T T 6 6 Q Q Q Q 7 7 S S S S 8 8 S P P P 9 9 S S S
S 10 10 S S S S 11 11 F L L L 12 12 S S S S 13 13 V A A A 14 14 S S
S S 15 15 L V V V 16 16 G G G G 17 17 D D D D 18 18 R R R R 19 19 V
V V V 20 20 T T T T 21 21 I I I I 22 22 T T T T 23 23 C C C C 24 24
CDR1 K R K K 25 25 A E A A 26 25 T S T T 27 27 E Q E E 28 28 D G D
D 29 29 I I I I 30 30 Y R Y Y 31 31 N N N N 32 32 R D R R 33 33 L L
L L 34 34 A G A A 35 35 FR2 W W W W 36 36 Y Y Y Y 37 37 Q Q Q Q 38
38 Q Q Q Q 39 39 K K K K 40 40 P P P P 41 41 G G G G 42 42 S K K K
43 43 A A A A 44 44 P P P P 45 45 R K K K 46 46 L L L L 47 47 L L L
L 48 48 I I I I 49 49 S Y S S Residue close to bypervariable loop,
retained. 50 50 CDR2 G G G G 51 51 A T A A 52 52 T S T T 53 53 S S
S S 54 54 L L L L 55 55 E Q E E 56 56 T S T T 57 57 FR3 G G G G 58
58 V V V V 59 59 P P P P 60 60 S S S S 61 61 R R R R 62 62 F F F F
63 63 S S S S 64 64 G G G G 65 65 S S S S 66 66 G G G G 67 67 S S S
S 68 68 G G G G 69 69 K T K T Residue close to hypervariable loop,
retained in v1 (L1) 70 70 D D D D 71 71 Y F Y Y Canonical residue,
retained. 72 72 T T T T 73 73 L L L L 74 74 S T T T 75 75 I I I I
76 76 T S S S 77 77 S S S S 78 78 L L L L 79 79 Q Q Q Q 80 80 T P P
P 81 81 E E E E 82 82 D D D D 83 83 V F F F 84 84 A A A A 85 85 T T
T T 86 86 Y Y Y Y 87 87 Y Y Y Y 88 88 C C C C 89 89 CDR3 Q Q Q Q 90
90 Q Q Q Q 91 91 F T F F 92 92 W T W W 93 93 S S S S 94 94 A F A A
95 95 P P P P 96 96 Y L Y Y 97 97 T T T T 98 98 FR4 F F F F 99 99 G
G G G 100 100 G G G G 101 101 G G G G 102 102 T T T T 103 103 K K K
K 104 104 L V V V 105 105 E E E E 106 106 I I I I 107 107 K K K
K
Example 14
Cloning and Sequencing of Humanized 11K2
[0372] Four different humanized versions of 11K2 were made based on
combinations of two different humanized versions of the 11K2 heavy
and light chains. Germline sequences were chosen as human acceptor
frameworks, including VK L11 for the light chain, and VH1-69 for
the heavy chain. The four combinations of humanized antibodies were
designated H1-L1, H1-L2, H2-L1, and H2-L2. Amino acid sequences of
the humanized versions of the 11K2 heavy and light chains
(nucleotide and amino acid) are shown in FIGS. 10A-10D. An
alignment of the heavy and light chains of the 11K2 chimeric
antibody and the humanized 11K2 antibody (versions 1 and 2) is
shown in FIGS. 11A and 11B. The first version contains the most
backmutations to the murine donor sequences, while the second
version contains the fewest (i.e., the most "humanized").
[0373] Hu11K2 variable regions were made by site-directed
mutagenesis using chimeric 11K2 variable domain plasmids as
starting templates. Following the manufacturer's recommended
protocol, the QuikChange Site-Directed Mutagenesis Kit (Stratagene
Cat. #200518) was used to systematically introduce (framework by
framework) the residue changes outlined in above in Tables 16 and
17, as well as FIGS. 11A and 11B. The mutagenic primers for the
framework (FR) changes are described below. The cDNA sequence of
the human acceptor frameworks (germline VK L11 for the light chain,
and germline VH1-69 for the heavy chain) were used, with silent
mutations introduced to produce restriction site changes to
facilitate identification of mutated plasmids. Mutated plasmids
were identified by screening for the introduced restriction site
changes. The variable region cDNA sequences in the resultant
plasmids were confirmed by DNA sequencing.
[0374] Hu11K2 light chain mutagenesis was performed according to
the following protocol. For the 11K2 version 1 light chain, plasmid
pCR060 was used as template in a PCR reaction with the following
primers: FR1 primer 5'CCC GCG GAG ACA TC AGA TGA CTC AGT CTC CAT
CCT CCC TGT CAG CAT CTG TGG GAG ACA GAG TCA CCA TA CT GCA AGG C3'
(SEQ ID NO: 57), which added an A1wn1 site; FR2 primer 5' GGT ATC
AGC AGA AAC CAG GAA AGG CCC CTA AGC TCT TAA TIT CTG GTG CAA CC 3'
(SEQ ID NO: 58), which added an EcoO109 I site; FR3 primer 5' GGA
AAA GAT TAC ACT CTC ACC ATT AGC AGT CTA CAG CCT GAG GAT TIT GCT ACT
TAT TAC TGT CAA CAG 3' (SEQ ID NO: 59), which added an AccI site;
and FR4 primer 5' CGT TCG GAG GGG GGA CCA AGG TGG AGA TCT AAA AAA
AGG GCG AAT TCT G 3' (SEQ ID NO: 60), which added a Styl site. The
resultant version 1 light chain plasmid was designated pCR067.
[0375] For the 11K2 version 2 light chain, plasmid pCR067 was used
as template in a PCR reaction with the following primer: FR3 primer
5' GAT TCA GTG GCA GTG GAT CCG GAA CAG ATT ACA CTC TCA CCA TTA GC
3' (SEQ ID NO: 61), which introduced a BspeI site. The resultant
version 2 light chain plasmid was designated pCR06.
[0376] Hu11K2 heavy chain mutagenesis was performed according to
the following protocol. For the 11K2 version 1 heavy chain, plasmid
pCR046 was used as template with the following primers: FR1 primers
5' GTG GTT ACA GGG GTC AAC TCA CAG GTT CAG CTG GTG CAG TCT GGG GCA
GAG CT G 3' (SEQ ID NO: 62), which added a Hinc2 site, and 5' GCA
GTC TGG GGC AGA GGT GAA GAA GCC CGG GTC CTC AGT CAA GGT CTC CTG CAA
GGC TC TGG CCT CAA CAT TAA AGA C3' (SEQ ID NO: 63), which added a
Sma1 site; FR2 primer 5' GAC ACC TAT ATG CAC TGG GTG CGA CAG GCG
CCT GGA CAG GGC CTG GAG TGG ATI GG 3' (SEQ ID NO: 64), which added
a Narl site; FR3 primer 5' CCC GAA GTT CCA GGG CAG AGC CAC TAT AAC
AGC AGA CAC ATC CAC GAG CAC AGC CTA CAT GGA GCT CAG CAG CCT GAG ATC
TGA GGA CAC TGC CG 3' (SEQ ID NO: 65), which added a Sac1 site; and
FR4 primer 5' GGG GCC AAG GGA CCA CTG TGA CAG TCT CCT CAG GTG AGT
CCT AAG CTT GGT ACC CGG G 3' (SEQ ID NO: 66), which added an Ava2
site. The resultant version 1 heavy chain plasmid was designated
pCR066.
[0377] For the 11K2 version 2 heavy chain, plasmid pCR066 was used
as template in a PCR reaction with the following primers: FR1
primer 5' GGT CTC CTG CAA GGC TIC AGG CCT CAC CAT TAG CGA CAC CTA
TAT GCA CTG GG 3' (SEQ ID NO: 67), which added a Stul site; FR2
primer 5' GGC GCC TGG ACA GGG CCT CGA GTG GAT GGG AAG GAT TGA TCC
TGC G 3' (SEQ ID NO: 68), which added an XhoI site; and FR3 primer
5' GAC CCG AAG TTC CAG GGC AGA GTC ACT ATA ACT GCA GAC ACA TCC ACG
AGC ACA GCC 3' (SEQ ID NO: 69), which added a PstI site. The
resultant version 2 heavy chain plasmid was designated pCR072.
Example 15
Expression of Humanized 11K2 Antibodies
[0378] Expression vectors for the hu11K2 light chains were made by
subcloning the 0.40 kb NotI-BglII light chain variable domain
fragments from pCR067, pCR069 or pCR037 (murine 11K2 light chain
variable domain bulk pcr reaction), and the 0.68 kb BclI-NotI
fragment from the plasmid pEAG963, containing a human kappa light
chain constant domain, into the NotI site of the pCEP4 EBV
expression vector-derived plasmid pCH274, producing light chain
expression vectors pCR068 (version 1), pCR077 (version 2), and
pCR045 (light chain chimera). NotI and BglII sites were engineered
onto pCR032 prior to digestion. Expression vectors for the hu11K2
heavy chains were made by subcloning the 0.49 kb NotI-HindIII heavy
chain variable domain fragments from pCR066, pCR072 or pCR032*
(murine 11K2 heavy chain variable domain), and the 1.21 kb
HindIII-NotI fragment from the plasmid pEAG964, containing a human
IgG1 constant region, into the NotI site of the pCEP4 EBV
expression vector-derived plasmid pCH274, producing heavy chain
expression vectors pCR073 (version 1), pCR075 (version 2) and
pCR054 (heavy chain chimera). NotI and HindIII sites were
engineered into plasmid pCR032 prior to digestion.
[0379] Hu11K2 heavy and light chain expression vectors were
co-transfected in all four (4) combinations (i.e. H1-L1, H1-L2,
H2-L1 and H2-L2) into 293-EBNA cells and transfected cells were
tested for antibody secretion and specificity. Western blot
analysis (developed with anti-human heavy and light chain
antibodies) of whole cell lysates and the conditioned culture media
indicated that hu11K2-transfected cells synthesized and efficiently
secreted heavy and light chains at levels similar to chimeric
11K2-transfected cells. All combinations retained reactivity to
MCP-1 (murine, primate, and human) and MCP-2 (human) immobilized on
ELISA plates.
Example 16
Characterization of Humanized 11K2 ELISA Screening
[0380] Binding specificities of chimeric 11K2 and the four versions
of humanized 11K2 (H1-L1, H1-L2, H2-L1, and H2-L2) were determined
by ELISA, according to the protocol described in Example 1. Human,
mouse, and primate MCP-1 were used as antigen, and antibody 3G9,
which does not bind MCP-1, was used as a negative control.
[0381] ELISA plates (Corning, Inc. Cat#3369) were coated with 50 ng
MCP-1 per well overnight at 4.degree. C. Plates were washed then
blocked with PBS/5% milk for 1.5 hrs at 25.degree. C. Another wash
was followed by incubation with the 11K2 mAbs (or 3G9 isotype
control) at the concentrations indicated for 1 hr at 25.degree. C.
After washing, the plates were incubated with secondary antibody
(anti-human IgG-HRP, Jackson Immunoresearch Cat#109-036-088) for 1
hr at 25.degree. C. Plates were then washed and developed using the
ABTS Peroxidase Substrate Kit (Vector Laboratories Cat#SK-4500). OD
(405 nm) values were plotted as a function of 11K2 mAb
concentration.
[0382] Results from the ELISA experiment are shown below in Table
18 and FIGS. 14A and 14B. These results demonstrate that each of
the humanized and chimeric antibodies retained MCP-1 reactivity for
each of the species tested.
Binding Affinity Measurement of Humanized 11K2
TABLE-US-00019 [0383] TABLE 18 Summary of humanised 11K2
reactivities MCP-1 MCP-2 MCP-3 Human Mouse Monkey Human Human
Chimera yes yes yes yes no H1-L1 yes yes yes yes no H1-L2 yes yes
yes yes no H2-L1 yes yes yes yes no H2-L2 yes yes yes yes no
[0384] Binding affinity studies were performed in order to compare
the four versions of the humanized 11K2 anti-MCP antibody to
chimeric and murine 11K2 antibodies. KinExA experiments were
performed, as previously described. Briefly, reagents that were
used included the following. Recombinant human MCP-1 (catalog
#279-MC) and MCP-2 (catalog #281-CP, R&D Systems, Inc.) were
reconstituted to 100 .mu.g/ml in PBS 0.1% BSA and stored at
4.degree. C. for the duration of the experiment. mu11K2 was
purified from hybridoma cell line supernatant. Humanized 11K2 was
purified from 293 cell supernatant. All Fab fragments were
generated according to standard methods. NHS Sepharose beads
(catalog #17-0906-01), Amersham Biosciences (Uppsula, Sweden) were
also used to determine affinity through the KinExA method. Goat
anti-human Cy-5 conjugate (catalog #109-177-003, Jackson
ImmunoResearch) was also used.
[0385] To determine the binding affinity of the hu 11K2,
NHS-Sepharose beads were washed six times in dH.sub.2O and once in
50 mM NaHCO.sub.3, pH 9.5. 1 ml of MCP-1 at a concentration of 10
.mu.g/ml in NaHCO.sub.3 was added to an amount of beads equivalent
to 1 ml of the original slurry. The beads were rocked overnight at
4.degree. C. Beads were spun down and washed once with 1M Tris, pH
8.0, then resuspended in 1M Tris pH 8.0 with 1% BSA, 0.02% azide.
The beads were rocked at room temperature for one hour then stored
at 4.degree. C. in this buffer for the duration of the
experiments.
[0386] The KinExA 3000 measures protein-protein interaction in
solution, and is ideally suited for measuring affinities of
antibodies to antigens. In the typical experiment, antigen (MCP) is
immobilized on a bead, antibody is flowed over a column of these
beads at a given concentration, and the antibody that binds to the
beads is detected using a fluorescent secondary. Antibody at a
fixed concentration is then incubated with different concentrations
of antigen in solution and the amount of free antibody is measured
by flowing the mixture over the bead column quickly enough that no
re-equilibration occurs between solution and solid phase. The
amount of antibody remaining free is plotted against the
concentration of antigen added and the data are fit to a quadratic
equation to determine the affinity of the interaction in
solution.
[0387] In these experiments, the only difference to the typical
experiment described above is that instead of using intact
antibody, a Fab fragment of 11K2 was used. Earlier KinExA
experiments using the intact murine 11K2 mAb binding to MCP-1 and
MCP-2 gave biphasic curves that the software could not interpret.
This may be due to these proteins acting as dimers, so to eliminate
the dimer/dimer interaction we generated Fab fragments. The binding
of the 11K2 Fab fragments to MCP-1 and MCP-2 produced curves that
the KinExA software could fit. Results from the KinExA experiment
are shown below in Table 19.
TABLE-US-00020 TABLE 19 MCP affinity measurements in solution of
humanized 11K2 Fab variants Anitbody MCP-1 MCP-2 MCP-3 mu11K2 Fab
11 pM 430 pM >50 nM Ch11K2 19.6 pM 226 pM ND H1L1 36.4 pM 725
pM* ND H2L2 77.5 pM* 3.2 pM* ND H1L2 62.3 pM 712 pM* ND H2L1 64.0
pM 940 pM* ND 1A1 Fab 12.9 pM 320 pM >50 nM D9 mAb 41.5 pM ND ND
S14 mAb (commercial) 5.6 pM ND ND *significant difference from
murine and chimeric
Example 17
Inhibition of THP-1 Chemotaxis by Chimeric and Humanized 11K2
[0388] Chemotaxis inhibition assays were performed as previously
described in Example 3, using MCP-1 to induce THP-1 cell migration.
MCP-1 was used at a concentration of 2.3 nM. The results, shown in
FIG. 12A, demonstrate that chimeric 11K2 and humanized versions
were as effective as monoclonal 11K2 at inhibiting THP-1 cell
chemotaxis. In additional experiments using MCP-2 (at a
concentration of 56 nM) to induce THP-1 migration, chimeric and all
humanized versions of 11K2 were effective at inhibiting chemotaxis
(FIGS. 12A and 12B). Interestingly, the agonist effect observed
with monoclonal 11K2 was also observed with chimeric and humanized
11K2 versions, as seen in FIG. 12B. In contrast to forms of 11K2
mAb having normal Fc region functionality, an aglycosylated form of
the chimeric 11K2 mAb no longer demonstrates agonist activity
towards human MCP-2. Thus, the aglycosylated form of 11K2 is acting
as a complete antagonist of both human MCP-1 and MCP-2.
V. Humanized 1A1 Antibody
Example 18
1A1 Humanization
[0389] Modeling the Structure of the Variable Regions
[0390] In order to identify key structural framework residues in
the murine 1A1 antibody, a three-dimensional model was generated
based on the closest murine antibodies for the heavy and light
chains. The 1A1 light and heavy chains were aligned against a local
copy of the most recent PDB database to determine structural frames
to be used to construct three dimensional models of the light and
heavy chains. Using FASTA the light chain was found to have 91.1%
sequence identity to monoclonal murine antibody Fab1583 (1NLD; 2.9
.ANG.), and have 87.4% sequence identity to the catalytic murine
antibody D2.5 (1YEE; 2.2 .ANG.). The heavy chain was found to have
85.7% sequence identity to murine 2E8 Fab fragment (12E8; 1.9
.ANG.), and 68.1% sequence identity to the murine monoclonal
antibody F9.13.7 (1FBI; 3.0 .ANG.). The reason for including 1FBI
with a relatively low sequence homology was that it has the same H3
loop length and also a relatively high sequence conservation in the
H3 loop.
[0391] Two composite antibody structures were created by aligning
the light chains of 1NLD and 1YEE onto the light chains of 12E8 and
1FBI, respectively. The atoms used to define the superposition were
the Ca atoms of the light chain interface residues. The two full
structural templates were obtained by saving the structural
combinations of 1NLD(light)/12E8(heavy) and 1
YEE(light)/1FBI(heavy).
[0392] Using the molecular modeling package Modeler 5.0 (Accelrys
Inc.) the three dimensional structures of the light and heavy
chains were built using the two composite structures. Five homology
models were created, and the best one in terms of Modeler energy
was selected. Procheck analysis showed that no residues were in a
disallowed region of the phi/psi map.
[0393] Design of the reshaped variable regions Human germline
sequences were used as the acceptor frameworks for humanized 1A1.
To find the closest germline sequences, the NCBI NR database and
the Kabat database were searched for the most homologous expressed
human frameworks in. In this search the CDR sequences were masked.
The selection of the most suitable expressed sequence included
checking for sequence identity of the canonical and interface
residues, and checking for the similarity in CDR loop lengths. The
source of the antibody was also a determining factor. Previously
humanized antibodies were excluded. BLAST was used for the NCBI NR
database search, and the Kabat database was used for the FASTA
search.
[0394] The most similar expressed light chain was found in the nr
database (GI-284256; Kennedy (1991), supra), and the most similar
heavy chain was found in the Kabat database (Kabat ID 037655;
Bejcek et al. (1995) supra). Both sequences were searched against
the database of germline sequences
(https://www.ncbi.nlm.nih.gov/igblast/), which resulted in the
following selected germlines: A17 for the light chain, and 5-51 for
the heavy chain. The light chain germline A17 was identical to the
expressed sequence GI-284256 in the framework regions. There were
many sequence differences between the 5-51 germline and the Kabat
ID 037655 expressed heavy chain, therefore the expressed sequence
(25C1) was used instead of the closest germline for the heavy
chain.
[0395] As noted supra, the humanized antibodies of the invention
comprise variable framework regions substantially from a human
immunoglobulin (acceptor immunoglobulin) and complementarity
determining regions substantially from a mouse immunoglobulin
(donor immunoglobulin) termed 1A1. Having identified the
complementarity determining regions of 1A1 and appropriate human
acceptor immunoglobulins, the next step was to determine which, if
any, residues from these components to substitute to optimize the
properties of the resulting humanized antibody. The criteria
described supra were used to select residues for substitution. A
summary of the backmutations is shown below in Table 20:
TABLE-US-00021 TABLE 20 Summary of backmutations of humanized 1A1
Backmutations in reshaped VL - Human germline A17 2 V -> I This
is a canonical residue and is retained in both versions. 36 F ->
L This is an interface residue and a significant change. Retained
in both versions. 45 R -> K This is a surface residue but is
close to a hypervariable loop. Retained in first version.
Backmutations in reshaped VH - Expressed sequence 25C1 (closest
germline 5-51) 27 Y .fwdarw. F This is a canonical residue, but a
conservative change. Retained in first version. 28 A .fwdarw. N
This residue is close to a hypervariable loop. Retained in first
version. 29 F .fwdarw. I This residue is a canonical residue.
Retained in both versions. 30 S .fwdarw. K This residue is close to
a hypervariable loop. Retained in both versions. 66 Q .fwdarw. K
This residue is close to a hypervariable loop and interacts with an
Asp. Retained in first version. 69 L .fwdarw. M This residue is
close to a hypervariable loop but is a conservative change.
Retained in first version. 73 K .fwdarw. T This residue is close to
a hypervariable loop, contacting an acidic residue. Retained in
both versions. 76 S .fwdarw. N This residue is close to a
hypervariable loop. Retained in first version. 91 S .fwdarw. Y This
residue is an interface residue and a big change. Retained in both
versions. 93 A .fwdarw. N This residue contacts a hypervariable
loop. Retained in both versions. 94 R .fwdarw. T This residue is a
canonical residue. Retained in both versions.
[0396] Two versions of the variable light reshaped chain and two
versions of the variable heavy reshaped chain were designed. The
first version contains the most backmutations and the second
version contains the fewest (i.e. the most "humanized"). The
sequences of the two versions of each variable and heavy chains of
humanized 1A1 are shown below:
TABLE-US-00022 Humanized 1A1 Heavy Chain (backmutations shown in
lower case): Version 1 (11 backmutations) (SEQ ID NO: 53)
QVQLLESGAELVRPGSSVKISCKASGfnikDNYMHWVKQRPGQGLEWIGWIDPENGDTEYAPKF
QGkATmTADtSSnTAYMQLSGLTSEDSAVYyCntWAYYGTSYGGFAYWGQGTTVT Version 2
(6 backmutations) (SEQ ID NO: 54)
QVQLLESGAELVRPGSSVKISCKASGYAikDNYMHWVKQRPGQGLEWIGWIDPENGDTEYAPKF
QGQATLTADtSSSTAYMQLSGLTSEDSAVYyCntWAYYGTSYGGFAYWGQGTTVT Humanized
1A1 Light Chain (backmutations shown in lower case): Version 1 (3
backmutations) (SEQ ID NO: 55)
DIVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWlQQRPGQSPkRLIYLVSKLDS
GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPQTFGQGTKLEIK Version 2 (2
backmutations) (SEQ ID NO: 56)
DiVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWlQQRPGQSPRRLIYLVSKLDS
GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPQTFGQGTKLEIK
[0397] Tables 21 and 22 set forth Kabat numbering keys for the
various light and heavy chains of 1A1, respectively.
TABLE-US-00023 TABLE 21 Key to Kabat Numbering for 1A1 Heavy Chain
Variable Region Mouse Hum. Hum. Kabat # AA # Type 1A1 25C1 1A1, v1
1A1, v2 Comment 1 1 FR1 E Q Q Q 2 V V V V 3 3 Q Q Q Q 4 4 L L L L 5
5 Q L L L 6 6 Q E E E 7 7 S S S 8 8 G G G G 9 9 A A A A 10 10 E E E
E 11 11 L L L L 12 12 V V V V 13 13 R R R R 14 14 S P P P 15 15 G G
G G 16 16 A S S S 17 17 S S S S 18 18 V V V V 19 19 K K K K 20 20 L
I I I 21 21 S S S S 22 22 C C C C 23 23 T K K K 24 24 A A A A 25 25
S S S S 26 26 G G G G 27 27 F Y Y F Canonical residue, retained in
v1 (H1) 28 28 N A A N Residue close to hyper variable loop,
retained in v1 (H1) 29 29 I F I I Canonical residue, retained 30 30
K S K K Residue close to hyper variable loop, retained 31 31 CDR1 D
S D D 32 32 N Y N N 33 33 Y W Y Y 34 34 M M M M 35 35 H N H H 36 36
FR2 W W W W 37 37 V V V V 38 38 K K K K 39 39 Q Q Q Q 40 40 R R R R
41 41 P P P P 42 42 E G G G 43 43 Q Q Q Q 44 44 G G G G 45 45 L L L
L 46 46 E E E E 47 47 W W W W 48 48 I I I I 49 49 G G G G 50 50
CDR2 W Q W W 51 51 I I I I 52 52 D Y D D .sup. 52A 53 P P P P 53 54
E G E E 54 55 N D N N 55 56 G G G G 56 57 D D D D 57 58 T T T T 58
59 E N E E 59 60 Y Y Y Y 60 61 A N A A 61 62 P G P P 62 63 K K K K
63 64 F F F F 64 65 Q K Q Q 65 66 G G G G 66 67 FR3 K Q Q K Residue
close to hypervariable loop, retained in v1 (H1) 67 68 A A A A 68
69 T T T T 69 70 M L L M Residue close to hypervariable loop,
retained in v1 (H1) 70 71 T T T T 71 72 A A A A 72 73 D D D D 73 74
T K T T Residue close to hypervariable loop, retained 74 75 S S S S
75 76 S S S S 76 77 N S S N Residue close to hypervariable loop,
retained in v1 (H1) 77 78 T T T T 78 79 A A A A 79 80 Y Y Y Y 80 81
L M M M 81 82 Q Q Q Q 82 83 L L L L .sup. 82A 84 S S S S .sup. 82B
85 S G G G .sup. 82C 86 L L L L 83 87 T T T T 84 88 S S S S 85 89 E
E E E 86 90 D D D D 87 91 T S S S 88 92 A A A A 89 93 V V V V 90 94
Y Y Y Y 91 95 Y S Y Y Residue is an interface residue, retained. 92
96 C C C C 93 97 N A N N Residue contacts hypervariable loop,
retained. 94 98 T R T Residue is canonical, retained 95 99 CDR3 W K
W W 96 100 A T A A 97 101 Y I Y Y 98 102 Y S Y Y 99 103 G S G G 100
104 T V T T .sup. 100A 105 S V S S .sup. 100B 106 Y D Y Y .sup.
100C 107 G F G G .sup. 100D 108 G YG G .sup. 100E 109 F F F F 101
110 A D A A 102 111 Y Y Y Y 103 112 FR4 W W W W 104 113 G G G G 105
114 Q Q Q Q 106 115 G G G G 107 116 T T T T 108 117 T T T T 109 118
V V V V 110 119 T T T T
TABLE-US-00024 TABLE 22 Key to Kabat Numbering for 1A1 Light Chain
Variable Region Mouse Hum. Hum. Kabat # AA # Type 1A1 25C1 1A1, v1
1A1, v2 Comment 1 1 FR1 D D D D 2 2 V I I I Canonical residue,
retained 3 3 Q V V V 4 4 M M M M 5 5 T T T T 6 6 Q Q Q Q 7 7 S S S
S 8 8 S P P P 9 9 S L L L 10 10 S S S S 11 11 F L L L 12 12 S P P P
13 13 V V V V 14 14 S T T T 15 15 L L L L 16 16 G G G G 17 17 Q Q Q
Q 18 18 P P P P 19 19 A A A A 20 20 S S S S 21 21 I I I I 22 22 S S
S S 23 23 C C C C 24 24 CDR1 K R K K 25 25 S S S S 26 25 S S S S 27
27 Q Q Q Q .sup. 27A 28 S S S S .sup. 27B 29 L L L L .sup. 27C 30 L
V L L .sup. 27D 31 D Y D D .sup. 27E 32 S S S S 28 33 D D D D 29 34
G G G G 30 35 K N K K 31 36 T T T T 32 37 Y H Y Y 33 38 L L L L 34
39 N N N N 35 40 FR2 W W W W 36 41 L F L L Interface residue,
retained. 37 42 L Q Q Q 38 43 Q Q Q Q 39 44 R R R R 40 45 P P P P
41 46 G G G G 42 47 Q Q Q Q 43 48 S S S S 44 49 P P P P 45 50 K R K
R Surface residue close to hypervariable loop, retained in v1. 46
51 R R R R 47 52 L L L L 48 53 I I I I 49 54 Y Y Y Y 50 55 CDR2 L K
L L 51 56 V V V V 52 57 S S S S 53 58 K N K K 54 59 L R L L 55 60 D
D D D 56 61 S S S S 57 62 FR3 G G G G 58 63 V V V V 59 64 P P P P
60 65 D D D D 61 66 R R R R 62 67 F F F F 63 68 T S S S 64 69 G G G
G 65 70 S S S S 66 71 G G G G 67 72 S S S S 68 73 G G G G 69 74 T T
T T 70 75 D D D D 71 76 F F F F 72 77 T T T T 73 78 L L L L 74 79 K
K K K 75 80 I I I I 76 81 S S S S 77 82 R R R R 78 83 V V V V 79 84
E E E E 80 85 A A A A 81 86 E E E E 82 87 D D D D 83 88 L V V V 84
89 G G G G 85 90 V V V V 86 91 Y Y Y Y 87 92 Y Y Y Y 88 93 C C C C
89 94 CDR3 W M W W 90 95 Q Q Q Q 91 96 G G G G 92 97 T T T T 93 98
H H H H 94 99 F W F F 95 100 P P P P 96 101 Q Y Q Q 97 102 T T T T
98 103 FR4 F F F F 99 104 G G G G 100 105 G Q Q Q 101 106 G G G G
102 107 T T T T 103 108 K K K K 104 109 L L L L 105 110 E E E E 106
111 I I I I 107 112 K K K K
Example 19
Efficacy of Anti-MCP Antibody Treatment in Tabs-Induced Murine
Colitis Model
[0398] To determine the efficacy of anti-MCP antibodies in treating
inflammatory disorders, a mouse model of colitis was selected.
Colitis was induced in Balb/c mice as previously described (Neurath
et al. (1995) J Exp Med. 182(5):1281). Briefly, 6-8 week old female
Balb/c mice (Charles River, Monza, Italy) were fasted for 1 day,
anesthetized, and a 3.5 F catheter was inserted into the colon such
that the tip was 4 cm proximal to the anus. To induce colitis in
the experimental mice, 1.0 mg of TNBS (Sigma Chemical Co, St Louis,
Mo.) in 50% ethanol was administered via catheter into the lumen
using a 1 ml syringe (injection volume of 1001). Control mice
received 50% ethanol alone.
[0399] Following induction of colitis, mice were monitored daily
for appearance of diarrhea, loss of body weight, and survival. At
the end of the experiment, surviving mice were sacrificed and blood
samples collected by cardiac puncture. A 7 cm segment of colon was
excised, weighed, and evaluated for macroscopic damage. Tissue
segments were then used for immunohistochemical studies, or were
homogenized in protein extraction buffer (Pierce, Rockford, II USA)
for use in cytokine and myeloperoxidase (MPO) activity measurements
as described (Fiorucci et al. (2002) Immunity 17: 769). Chemokine
measurements were performed using a commercially available ELISA
assay for MCP-1 (R+D Systems, Minneapolis, Minn. USA).
[0400] TNBS-induced colitis mice receiving 11K2 were studied for
physical changes (e.g., weight loss), reduction in proinflammatory
mediators, and reduction in circulating MCP-1 to determine the
efficacy of 11K2 at treating colitis. TNBS-induced colitis
experiments using different forms of the 11K2 antibody were
performed in parallel with control antibody mouse monoclonal
antibody MOPC21.
Anti-MCP Antibody Treatment Prevents Weight Loss and Enhances
Survival
[0401] Colitis was induced in experimental mice as described above.
Intraperitoneal (IP) injection of monoclonal antibody 11K2 and the
control antibody (IgG1b antisera MOPC21) was performed on days--1,
2 and 5. Mice were administered either 200 .mu.g of mouse
monoclonal antibody 11K2 or mouse control monoclonal antibody
MOPC21. Mice were monitored for weight gain/loss and survival for
seven days. On day 1, all mice were observed to weigh about 18
grams. Rapid weight gain was observed in control mice, which
reached a plateau weight of about 23 grams by day 4 of the trial.
While weight gain in colitis model mice administered monoclonal
antibody 11K2 was initially not as rapid as for control mice
(non-colitis induced mice), gradual weight gain was observed in the
mice over the course of the trial, reaching about 22 grams by day
7. In contrast, TNBS-induced colitis model mice that received
either control monoclonal antibody MOPC21 or no antibody failed to
gain significant weight between days 1 and 7 of the trial,
continuing to weigh about 18 grams on day 7. Thus, 11K2 treated
mice showed significant weight gain.
[0402] Treatment of colitis-induced mice with 11K2 also improved
survival over the seven day course of the trial, as compared to
mice treated with TNBS alone or the combination of TNBS and the
control monoclonal antibody MOPC21. As shown in FIG. 15, about 70%
of colitis model mice treated with 11K2 survived the seven day
trial, in contrast to a survival rate of about 40% for colitis
model mice administered either the control monoclonal antibody or
no antibody. Mouse monoclonal antibody 11K2 treatment therefore
reduced lethality associated with TNBS-induced colitis in model
mice.
Reduction of Proinflammatory Mediators in Anti-MCP Antibody-Treated
Mice with the Following Replacement Paragraph:
[0403] To determine the effect of anti-MCP antibodies at reducing
molecules associated with inflammation, colon tissues from
TNBS-induced colitis mice were dissected and assayed for
concentration of proinflammatory mediator cytokines, TNF.alpha.,
IFN-.gamma., and IL-2 according to manufacturer's protocols (R+D
Systems, Minneapolis, Minn. USA). Concentrations of the three
cytokines were observed to be less than 100 pg/mg in control mice
that were not administered TNBS. TNBS induction elevated colonic
levels of all three assayed cytokines in TNBS-induced mice and mice
administered the combination of TNBS and control monoclonal
antibody MOPC21, wherein about 600 pg/mg TNF.alpha., 750 pg/mg
IFN-.gamma., and 500 pg/mg IL 2 was observed. Lower levels of
TNF.alpha., IFN-.gamma., and IL-2 (about 200 pg/mg, 300 pg/mg and
200 pg/mg, respectively) were observed in colon tissues obtained
from TNBS-induced colitis mice injected with 11K2. Thus, 11K2
blockade of MCP-1 decreased production of proinflammatory mediators
in the inflamed colon.
Reduction of Circulating Mcp-1 Levels in Anti-MCP Antibody-Treated
Mice
[0404] To determine the effect of 11K2 on MCP-1 in TNBS-induced
colitis model mice, circulating levels of MCP-1 were analyzed
according to manufacturer's protocols (R+D Systems, Minneapolis,
Minn. USA). As shown in FIG. 16, MCP-1 was observed at about 500
pg/ml serum in control mice, whereas serum MCP-1 levels in
TNBS-induced colitis mice administered control monoclonal antibody
MOPC21 or no antibody were about 3000 pg/ml serum. MCP-1 levels in
TNBS-induced colitis mice treated with 11K2 were observed to be
about 1000 pg/ml serum, representing a significant reduction of
circulating MCP-1 levels relative to TNBS-induced colitis mice
administered the control monoclonal antibody or left untreated.
Thus, 11K2 treatment significantly reduced the level of MCP-1 in
circulation during colitis.
Dose-Dependent Analysis of Anti-MCP Antibody Treatment
[0405] Body weight was monitored for groups of TNBS-induced colitis
mice injected with varying amounts of mouse 11K2 monoclonal
antibody to determine dosage response. 11K2 was administered at
doses of 2, 50, 100, and 200 pg/mouse three times a week,
respectively. TNBS-induced colitis control mice that did not
receive antibody injections or colitis mice receiving only 2 .mu.g
doses of 11K2 showed a decline in weight about 25% relative to
starting weights over the course of the experiment. Improved body
weights were observed for mice administered 50 .mu.g doses of 11K2
(weight declines of about 15%), while improved body weights were
observed for mice administered 100 .mu.g and 200 .mu.g doses
(weights of these mice were observed to be about equal to starting
weights, with slight weight gain observed for those mice treated
with 200 .mu.g of 11K2). Uninduced and untreated control mice
gained about 15% of body weight over the course of the
experiment.
[0406] The effect of various doses of 11K2 was also studied by
analyzing myeloperoxidase (MPO) activity levels in TNBS-induced
colitis mice. To assess MPO activity levels, 50 .mu.l SureBlue TMB
(Kirkegaard & Perry Laboratories, Inc.) was added to 50 .mu.l
sample, e.g., serum or colon homogenate. This mixture was allowed
to incubate at room temperature for 5 minutes, with 100 .mu.l 0.18M
H.sub.2SO.sub.4 then added to the reaction mixture. Absorbance at
450 nm was detected on a plate reader for all samples, with a range
of 0.25 to 1 activity unit per sample. A standard curve was
generated using purified MPO (Sigma) in the peroxidase assay. MPO
activity levels were ascribed to samples by comparing detected
peroxidase activity values with the standard curve. MPO activity in
uninduced, untreated control mice was observed to be about 15 U/mg.
TNBS-induced mice left untreated exhibited about 35 U/mg MPO
activity. Reduced levels of MPO activity were observed in
TNBS-induced colitis model mice that had been treated with 50
.mu.g, 100 .mu.g and 200 .mu.g doses of 11K2, with MPO activity
levels observed to be about 25 U/mg, 18 U/mg, and 15 U/mg,
respectively. Efficacy of humanized anti-MCP antibody and pegylated
fab in treating colitis
[0407] To determine the efficacy of humanized 11K2 and 11K2
pegylated-Fab 11K2 PEG-Fab), TNBS-induced mice were treated via
intraperitoneal (IP) injection with mouse monoclonal antibody 11K2,
humanized 11K2 (h11K2) antibody, chimeric 11K2 antibody,
aglycosylated 11K2, or no antibody as a control. TNBS-induced
colitis model mice were monitored for MPO activity levels. As shown
in FIG. 17, administration of either hul 1K2 (FIG. 17A) or 11K2
PEG-Fab (FIG. 17B) to TNBS-induced colitis mice resulted in
significantly lower MPO levels than TNBS-induced colitis control
mice. Both hu 11K2- and 11K2 PEG-Fab-treated mouse MPO levels were
comparable to those observed for TNBS-induced colitis mice treated
with the mouse monoclonal antibody 11K2.
Therapeutic Treatment of Colitis Model Mice by Anti-MCP
Antibody
[0408] TNBS was administered to groups of mice and colitis was
allowed to progress for seven days. Mouse monoclonal antibody 11K2
or a control monoclonal antibody were then administered via IP
injection to the mice on day 7. Body weight was then measured on
days 10 and 14. In addition, MCP-1, TNF.alpha., and MPO activity
levels were assessed on day 14 for all mice. Elevated body weight,
and significantly inhibited levels of MCP-1, TNF.alpha. and MPO
activity were all observed for colitis-induced mice that had been
treated with 11K2 antibody (see FIG. 18), as compared to colitis
model mice left untreated or administered the non-therapeutic
control monoclonal antibody.
Example 20
Efficacy of Anti-MCP Antibodies in Atherosclerosis
[0409] To determine the efficacy of 11K2 at treating
atherosclerosis, the murine ApoE-deficient model was used.
Administration of mouse monoclonal antibody 11K2 to atherosclerotic
model mice (apoE-deficient) was performed according to the methods
of Lutgens et al. (2000) Proc. Natl. Acad. Sci. USA. 97:7464). ApoE
-/- mice (Iffa Credo) were fed normal chow diet, and administered
either 11K2 or a mouse control monoclonal antibody (IgG1b antisera
MOPC21) at 200 .mu.g per mouse by intraperitoneal injection twice a
week for 12 weeks. Injection of the early treatment group started
at 5 weeks of age (n=15 11K2 antibody, n=15 control antibody), when
hardly any atherosclerotic lesions are observed to be present.
Injections of the delayed treatment group (n=15 11K2 antibody; n=15
control antibody) started at 17 weeks of age, at which point
advanced atherosclerotic plaques are known to have developed. Mice
of both early and delayed treatment groups were sacrificed at the
end of 12 weeks of treatment for examination of plaque
development.
[0410] Atherosclerotic plaques were divided into initial and
advanced lesions. Initial lesions were defined as fatty streaks
containing macrophage-derived foam cells with intracellular lipid
accumulation (AHA type II) or pools of extracellular lipid (AHA
type III), whereas advanced lesions contained extracellular lipid,
a lipid core (AHA type IV), and/or a fibrous cap (AHA type Va-c)
(Stary et al. (1995) Arterioscler. Thromb. Vase. Biol. 15:1512).
Tissue processing, histological classification, and morphometry
were performed as described previously (Lutgens et al. (1999) Nat.
Med. 5:1313; Lutgens et al. (1999) Circulation 99:276). 11K2
antibody-treated apoE-1-mice were compared with control-treated
apoE-1-mice. 11K2 antibody-treated apoE-/- mice of the delayed
treatment group were also compared with control-treated 17-wk-old
apoE -/- mice to investigate plaque progression after treatment.
For all analyses, a nonparametric Mann Whitney U test was used. The
level of statistical significance was set at P=-0.05. As shown in
FIG. 19, significant reduction of total plaque area in the aortic
arch was observed for both early and advanced groups of mice
treated with 11K2.
[0411] Quantities of atherosclerotic plaques classified as initial
and advanced lesions were analyzed for all groups of mice. As shown
in FIG. 19, treatment of atherosclerotic mice with 11K2 reduced the
number of plaques. Reduction in the total number of plaques
observed in the early treatment group was comparable between
control and experimental groups. while about 5 plaques were
observed in the aortic arches of both MOPC21 (IgG1b control
antisera)- and 11K2-administered mice. Advanced lesion counts were
lower for delayed treatment group mice treated with 11K2 (about 3
advanced lesions per aortic arch observed), as compared to delayed
treatment group MOPC21-treated mice (about 4 advanced lesions per
aortic arch observed). Too few advanced lesion plaques were
observed in mice of the early treatment group to assess whether
11K2 treatment reduced advanced lesion formation in the early
treatment groups of mice.
[0412] All mice were also examined for both macrophage content and
CD45+ cell content. Sections were immunolabeled with ED-.sup.20
(1:10) for the detection of macrophages or anti-CD45 antibody.
Atherosclerosis model mice treated with 11K2 exhibited reductions
in macrophage content of both initial and advanced lesions, among
both early and delayed treatment groups of mice (about 75% versus
about 85% macrophage content for initial lesions in the early
treatment group; about 70% versus about 80% macrophage content for
initial lesions of the delayed treatment group; and about 45%
versus about 55% macrophage content for advanced lesions of the
delayed treatment group). When CD45+ cell content was examined,
advanced lesions of the delayed treatment group were significantly
reduced for CD45+ cell content in 11K2-treated versus MOPC21 (IgG1b
control antisera)-treated mice (50 cells/mm.sup.2 versus about 120
cells/mm.sup.2).
[0413] ApoE -/- mice treated with 11K2 showed systemic
abnormalities with either early (5-17 weeks) or late treatment
(17-29 weeks) with an anti-MCP antibody. On autopsy, mice treated
with 11K2 antisera exhibited no abnormalities relative to control
animals in the following tissues: heart, liver, kidneys, lung,
lymph nodes, brain, bone, skin, stomach, intestines, colon,
salivary glands, gall bladder, prostate, testis, thymus, adrenals,
pancreas, bladder, duodenum. No significant differences in CD3+,
CD4+ and CD8+ levels were observed in blood, spleen and lymph node
tissues of experimental apoE -/- mice treated with 11K2 antisera
relative to control-treated animals. Additionally, lipid profiles
of 11K2-treated mice were essentially identical to control-treated
mice when levels of total cholesterol, triglycerides, HDL
cholesterol, and LDL cholesterol were examined.
[0414] Collagen and .alpha.-smooth muscle actin (ASMA) content were
also assessed in plaques of 11K2-treated and control mice via
immunolabelling. Plaques from mice in both the early and late
treatment groups that were administered control antisera MOPC21
exhibited collagen content of about 3% for initial plaques, while
advanced plaques of delayed treatment group mice showed collagen
contents of about 30%. In contrast, plaques of early and late
treatment mice administered 11K2 antisera showed significantly
greater collagen content than control animals, as early and late
treatment group initial plaques from 11K2-treated mice had about 6%
collagen levels, and advanced plaques from delayed treatment group
mice treated with 11K2 exhibited about 40% collagen levels. Similar
effects were observed for .alpha.-smooth muscle actin (ASMA).
Plaques from control-treated mice in both early and delayed
treatment groups exhibited about 0.5% ASMA levels for initial
plaques, and advanced plaques from control-treated mice in the
delayed treatment group revealed about 2% ASMA content. In
contrast, plaques dissected from 11K2-treated mice exhibited ASMA
content of about 4% for early treatment mice, and about 3% ASMA
content for both initial and advanced plaques dissected from
delayed treatment mice.
Array-Based Detection of Differentially Expressed Genes in
Atherosclerotic Mice
[0415] Gene array was used to examine the gene expression patterns
of atheroclerotic mice. Groups of C57BU6 apoE -/- mice fed a diet
of normal chow for 3, 4.5, or 6 months, or a western type diet for
3, 4.5, or 6 months, were sacrificed at the end of the experimental
period, Following sacrifice, vascular tissue was dissected and
subjected to array-based expression profiling on mouse Unigene I
arrays (Incyte Genomics, Inc.). Genes that were either upregulated
or downregulated by greater than two-fold in comparisons of
apoE-1-mice on varying diets to apoE -/- mice on normal chow diet
for three months, were identified and examined. A preponderance of
the differentially expressed genes identified were involved in
inflammation and fibrosis, including: the small inducible
cytokines, such as MCP-1, MCP-2 and MIP; complement factors;
interleukins; cathepsins; MMP 2 and MMP 12; and TGF-.beta.. A
number of small inducible cytokines were examined in more detail,
including Fractalike. (SIC D1), MIP 1 (SIC A3), MCP-1, MCP-2 (SIC
A5), IL-8 like (SIC A6), MCP-3 (SIC A7), PDGF-inducible (SIC A2),
and RANTES (SIC A5). When relative array-based expression data was
examined for all of the supra listed cytokines, all cytokines other
than RANTES exhibited increases in relative expression levels as
atherosclerosis progressed in both normal chow diet and western
type diet apoE -/- mice.
[0416] Analysis of MCP-1 RNA and protein levels revealed a marked
increase in both levels in ApoE -/- mice fed Wester chow. Elevated
MCP-1 transcript levels during atherosclerosis progression produced
corresponding increases in levels of MCP-1 protein, as detected in
aortic arch tissue. While MCP-1 protein was not detectable in serum
for any mice examined via array-based expression profiling, MCP-1
levels of about 60 pg/ml were observed for apoE -/- mice fed a
western type diet for 4.5 months, and about 100 pg/ml levels of
MCP-1 protein were observed for apoE -/- mice fed a western type
diet for six months.
VI. Crystallization and Structural Determination of
MCP-1-11K2-Fab
Example 30
Crystal Structure of 11K2
[0417] A three-dimensional structure of acomplex of a Fab fragment
of murine 11K2 antibody with MCP-1 was determined by X-ray
crystallography. The Fab fragment was produced by proteolytic
cleavage. 1 mg of human MCP-1 and 1 mg of murine 11K2 Fab was mixed
and concentrated to 8 mg/ml. Equal volumes of protein and well
solution (10.sup.-15% PEG 4000, 100 mM HEPES pH=7.5, 30 mM
glycl-glycl-glycine) were combined and placed at room temperature
to equilibrate. Crystals appeared within 3 days and grew to full
size within 2 weeks. Crystals were flash-frozen for data
collection, using liquid nitrogen in a solution containing 100 mM
HEPES pH=7.5, 15% PEG 4000, 30 mM glycl-glycl-glycine and 25%
glycerol. Crystal transfer to the cryoprotection solution was
necessary to preserve the crystal during cooling to -180.degree. C.
for data collection, and also to allow compounds to bind under low
salt conditions, which is important to increase the solubility of
the compounds and to remove the sulfate ion bound in the active
site (placed by crystallization conditions, but removable upon
soaking for several days).
[0418] Data collection was performed on a rotating anode X-ray
generator for a duration of typically about 6 hours, using 1 degree
of oscillation and 5 minute exposure times. An oscillation range of
60 degrees was required for a complete data set. The space group of
the crystals was determined to be C222.sub.1 with unit cell
dimensions a=86.36 .ANG., b=89.10 .ANG., c=176.24 .ANG.. In 22,774
unique reflections (542,403 total reflections), resolution limits
were determined to be 50 to 2.5 .ANG. (2.59-2.50 .ANG.). Results
from the data collection are described below in Table 23.
TABLE-US-00025 TABLE 23 Data collection - phasing and model
refinement Resolution limits: 50-2.5 .ANG. (2.59-2.50 .ANG. Number
of total reflections: 542,403 Number of unique reflections: 22,774
Redundancy: 6.2 I/.sigma.: 13.2 (3.3) R.sub.merge: 0.090 (0.436)
Completeness: 99.8% (99.9%) R-factor/Rfree: 0.219/0.277 #
reflections for refinement: 23911
Structure was solved by molecular replacement with Fab model. After
an initial round of refinement, MCP-1 was clearly visible in 2Fo-Fc
maps. The final model contains residues 4-71 of MCP-1, 1-214 of the
light chain of 11K2, 1-217 of the heavy chain of 11K2, and 43 water
molecules. Crystal structure results revealed that the residue
contacts of MCP-1 which the heavy chain of 11K2 binds include R30,
T32, S34, K38, E39, V41, P55, K56, Q61, M64. Analysis of the
crystal structure also revealed that the light chain of 11K2
contacts residues D65, D68, K69 of MCP-1. Thus, 11K2 binds a
discontinuous sequence of MCP-1.
[0419] Forming part of the present disclosure is the appended
Sequence Listing, the contents of which are summarized in the table
below:
TABLE-US-00026 TABLE 24 Summary of sequences Sequence SEQ ID NO:
Description Type 1 MCP-1 MRHAS motif amino acid 2 MCP-3 MRHAS motif
amino acid 3 1A1 Heavy Chain cDNA Primer nucleic acid 4 1A1 Light
Chain cDNA Primer nucleic acid 5 1A1 Heavy Chain cDNA primer
nucleic acid 6 1A1 Heavy Chain cDNA primer nucleic acid 7 1A1 Light
Chain cDNA primer nucleic acid 8 1A1 Light Chain cDNA primer
nucleic acid 9 1A1 Heavy chain variable region nucleic acid 10 1A1
Light chain variable region nucleic acid 11 1A1 Heavy chain
variable region amino acid 12 1A1 Light chain variable region amino
acid 13 1A1 Heavy Chain Variable Region CDR1 amino acid 14 1A1
Heavy Chain Variable Region CDR2 amino acid 15 1A1 Heavy Chain
Variable Region CDR3 amino acid 16 1A1 Light Chain Variable Region
CDR1 amino acid 17 1A1 Light Chain Variable Region CDR2 amino acid
18 1A1 Light Chain Variable Region CDR3 amino acid 19 11K2 Heavy
Chain cDNA Primer nucleic acid 20 11K2 Light Chain cDNA Primer
nucleic acid 21 11K2 Heavy Chain cDNA Primer nucleic acid 22 11K2
Heavy Chain cDNA Primer nucleic acid 23 11K2 Light Chain cDNA
Primer nucleic acid 24 11K2 Light Chain cDNA Primer nucleic acid 25
11K2 Heavy Chain Variable Region nucleic acid 26 11K2 Light Chain
Variable Region nucleic acid 27 11K2 heavy chain variable region
amino acid 28 11K2 light chain variable region amino acid 29 11K2
Heavy Chain Variable Region amino acid CDR1 30 11K2 Heavy Chain
Variable Region amino acid CDR2 31 11K2 Heavy Chain Variable Region
amino acid CDR3 32 11K2 Light Chain Variable Region amino acid CDR1
33 11K2 Light Chain Variable Region CDR2 amino acid 34 11K2 Light
Chain Variable Region CDR3 amino acid 35 11K2 heavy chain chimera
nucleic acid 36 11K2 light chain chimera nucleic acid 37 11K2 heavy
chain chimera amino acid 38 11K2 light chain chimera amino acid 39
11K2 humanized heavy chain, version 1 nucleic acid (includes
constant region) 40 11K2 humanized heavy chain, version 1 amino
acid (includes constant region) 41 11K2 humanized heavy chain,
version 2 nucleic acid (includes constant region) 42 11K2 humanized
heavy chain, version 2 amino acid (includes constant region) 43
11K2 humanized light chain, version 1 nucleic acid (includes
constant region) 44 11K2 humanized light chain, version amino acid
1(includes constant region) 45 11K2 humanized light chain, version
2 nucleic acid (includes constant region) 46 11K2 humanized light
chain, version 2 amino acid (includes constant region) 47 Humanized
11K2 heavy chain, variable, amino acid version 1 48 Humanized 11K2
heavy chain, variable, amino acid v2 49 Humanized 11K2 light chain,
variable, v1 amino acid 50 Humanized 11K2 light chain, variable, v2
amino acid 51 Chimera, variable heavy chain 1A1 amino acid 52
Chimera, variable light chain 1A1 amino acid 53 Humanized 1A1 heavy
chain, variable v1 amino acid 54 Humanized 1A1 heavy chain,
variable v2 amino acid 55 Humanized 1A1 light chain, variable v1
amino acid 56 Humanized 1A1 light chain, variable v2 amino acid 57
v1 light chain primer nucleic acid 58 v1 light chain primer nucleic
acid 59 v1 light chain primer nucleic acid 60 v1 light chain primer
nucleic acid 61 v2 light chain primer nucleic acid 62 v1 heavy
chain primer nucleic acid 63 v1 heavy chain primer nucleic acid 64
v1 heavy chain primer nucleic acid 65 v1 heavy chain primer nucleic
acid 66 v1 heavy chain primer nucleic acid 67 v2 heavy chain primer
nucleic acid 68 v2 heavy chain primer nucleic acid 69 v2 heavy
chain primer nucleic acid
[0420] One of ordinary skill in the art will recognize that many
variations and changes may be made to the invention as described in
the Detailed Description without departing from the spirit and
scope of the invention. The examples provided herein are merely
illustrative, and should not be construed as limiting of the scope
of the invention, which is set forth in the appended claims.
Sequence CWU 1
1
6917PRTHomo sapiens 1Gln Thr Gln Thr Pro Lys Thr1 527PRTHomo
sapiens 2Lys Thr Gln Thr Pro Lys Leu1 5339DNAArtificial
SequencePrimer 3aggtctagaa yctccacaca caggrrccag tggatagac
39429DNAArtificial SequencePrimer 4gcgtctagaa ctggatggtg ggagatgga
29522DNAArtificial SequencePrimer 5aggtsmarct gcagsagtcw gg
22632DNAArtificial SequencePrimer 6tgaggagacg gtgaccgtgg tcccttggcc
cc 32718DNAArtificial SequencePrimer 7gayathcara tgacncag
18829DNAArtificial SequencePrimer 8gcgtctagaa ctggatggtg ggagatgga
299366DNAMus musculus 9gaggtccagc tgcagcagtc tggggcagaa cttgtgaggt
caggggcctc agtcaagttg 60tcctgcacag cttctggctt caacattaaa gacaactata
tgcactgggt gaagcagagg 120cctgaacagg gcctggagtg gattggatgg
attgatcctg agaatggaga tactgaatat 180gccccgaagt tccagggcaa
ggccactatg actgcagaca catcctccaa cacagcctac 240ctgcagctca
gcagcctgac atctgaggac actgccgtct attactgtaa tacatgggct
300tactacggta ctagctacgg gggatttgct tactggggcc aagggaccac
ggtcaccgtc 360tcctca 36610336DNAMus musculus 10gatatccaga
tgactcagac tccactcact ttgtcggtta ccattggaca accagcctcc 60atctcttgca
agtcaagtca gagcctctta gatagtgatg gaaagacata tttgaattgg
120tcgttacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc
taaactggac 180tctggagtcc ctgacaggtt cactggcagt ggatcaggga
cagatttcac actgaaaatc 240agcagagtgg aggctgagga tttgggagtt
tattattgct ggcaaggtac acattttcct 300cagacgttcg gtggaggcac
caagctggag atcaaa 33611122PRTMus musculus 11Glu Val Gln Leu Gln Gln
Ser Gly Ala Glu Leu Val Arg Ser Gly Ala1 5 10 15Ser Val Lys Leu Ser
Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asn 20 25 30Tyr Met His Trp
Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile
Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60Gln Gly
Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75
80Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Asn Thr Trp Ala Tyr Tyr Gly Thr Ser Tyr Gly Gly Phe Ala Tyr
Trp 100 105 110Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
12012112PRTMus musculus 12Asp Ile Gln Met Thr Gln Thr Pro Leu Thr
Leu Ser Val Thr Ile Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Lys Ser
Ser Gln Ser Leu Leu Asp Ser 20 25 30Asp Gly Lys Thr Tyr Leu Asn Trp
Ser Leu Gln Arg Pro Gly Gln Ser 35 40 45Pro Lys Arg Leu Ile Tyr Leu
Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Thr Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu
Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95Thr His Phe
Pro Gln Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
110135PRTMus musculus 13Asp Asn Tyr Met His 1 51417PRTMus musculus
14Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe Gln1
5 10 15Gly1513PRTMus musculus 15Trp Ala Tyr Tyr Gly Thr Ser Tyr Gly
Gly Phe Ala Tyr1 5 101616PRTMus musculus 16Lys Ser Ser Gln Ser Leu
Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn1 5 10 15177PRTMus musculus
17Leu Val Ser Lys Leu Asp Ser1 5189PRTMus musculus 18Trp Gln Gly
Thr His Phe Pro Gln Thr1 51939DNAArtificial SequencePrimer
19aggtctagaa yctccacaca caggrrccag tggatagac 392029DNAArtificial
SequencePrimer 20gcgtctagaa ctggatggtg ggagatgga
292139DNAArtificial SequencePrimer 21ggggatatcc accatggrat
gsagctgkgt matsctctt 392239DNAArtificial SequencePrimer
22aggtctagaa yctccacaca caggrrccag tggatagac 392318DNAArtificial
SequencePrimer 23gayathcara tgacncag 182429DNAArtificial
SequencePrimer 24gcgtctagaa ctggatggtg ggagatgga 2925351DNAMus
musculus 25gaggttcagc tgcagcagtc tggggcagag cttgtgaagg caggggcctc
agtcaagttg 60tcctgcccag cttctggcct caacattaaa gacacctata tgcactgggt
gaagcagagg 120cctgaacagg gcctggagtg gattggaagg attgatcctg
cgaatggtaa tactaaattt 180gacccgaagt tccagggcaa ggccactata
acagcagaca catcctccaa cacagcctac 240ctgcagctca gcagcctgac
atctgaggac actgccgtct attactgtgc tagaggcgtc 300tttggctttt
ttgactactg gggccaaggc accactctca cagtctcctc a 35126321DNAMus
musculus 26gacattcaga tgactcagtc ttcatcctcc ttttctgtat ctctaggaga
cagagtcacc 60attacttgca aggcaactga ggacatatat aatcgattag cctggtatca
gcagaaacca 120ggaagtgctc ctaggctctt aatttctggt gcaaccagtt
tggagactgg ggttccttca 180agattcagtg gcagtggatc tggaaaagat
tacactctca gcattaccag tcttcagact 240gaggatgttg ctacttatta
ctgtcaacag ttttggagtg ctccgtacac gttcggaggg 300gggaccaagc
tggagatcaa a 32127117PRTMus musculus 27Glu Val Gln Leu Gln Gln Ser
Gly Ala Glu Leu Val Lys Ala Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys
Pro Ala Ser Gly Leu Asn Ile Lys Asp Thr 20 25 30Tyr Met His Trp Val
Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp
Pro Ala Asn Gly Asn Thr Lys Phe Asp Pro Lys Phe 50 55 60Gln Gly Lys
Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Leu
Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Gly Val Phe Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr
100 105 110Leu Thr Val Ser Ser 11528107PRTMus musculus 28Asp Ile
Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile Tyr Asn Arg 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Arg Leu Leu Ile
35 40 45Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu
Gln Thr65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Phe Trp
Ser Ala Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105295PRTMus musculus 29Asp Thr Tyr Met His1 53017PRTMus
musculus 30Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Phe Asp Pro Lys
Phe Gln1 5 10 15Gly318PRTMus musculus 31Gly Val Phe Gly Phe Phe Asp
Tyr1 53211PRTMus musculus 32Lys Ala Thr Glu Asp Ile Tyr Asn Arg Leu
Ala1 5 10337PRTMus musculus 33Gly Ala Thr Ser Leu Glu Thr1
5349PRTMus musculus 34Gln Gln Phe Trp Ser Ala Pro Tyr Thr1
5351344DNAArtificial SequenceChimeric sequence 35gaggttcagc
tgcagcagtc tggggcagag cttgtgaagg caggggcctc agtcaagttg 60tcctgcccag
cttctggcct caacattaaa gacacctata tgcactgggt gaagcagagg
120cctgaacagg gcctggagtg gattggaagg attgatcctg cgaatggtaa
tactaaattt 180gacccgaagt tccagggcaa ggccactata acagcagaca
catcctccaa cacagcctac 240ctgcagctca gcagcctgac atctgaggac
actgccgtct attactgtgc tagaggcgtc 300tttggctttt ttgactactg
gggccaaggt accactctca cagtctcctc agcctccacc 360aagggcccat
cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg
420gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc
gtggaactca 480ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc
tacagtcctc aggactctac 540tccctcagca gcgtggtgac cgtgccctcc
agcagcttgg gcacccagac ctacatctgc 600aacgtgaatc acaagcccag
caacaccaag gtggacaaga aagttgagcc caaatcttgt 660gacaagactc
acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc
720ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc
tgaggtcaca 780tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca
agttcaactg gtacgtggac 840ggcgtggagg tgcataatgc caagacaaag
ccgcgggagg agcagtacaa cagcacgtac 900cgtgtggtca gcgtcctcac
cgtcctgcac caggactggc tgaatggcaa ggagtacaag 960tgcaaggtct
ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa
1020gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga
gctgaccaag 1080aaccaggtca gcctgacctg cctggtcaaa ggcttctatc
ccagcgacat cgccgtggag 1140tgggagagca atgggcagcc ggagaacaac
tacaagacca cgcctcccgt gttggactcc 1200gacggctcct tcttcctcta
cagcaagctc accgtggaca agagcaggtg gcagcagggg 1260aacgtcttct
catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc
1320ctctccctgt ctcccgggaa atga 134436645DNAArtificial
SequenceChimeric sequence 36gacattcaga tgactcagtc ttcatcctcc
ttttctgtat ctctaggaga cagagtcacc 60attacttgca aggcaactga ggacatatat
aatcgattag cctggtatca gcagaaacca 120ggaagtgctc ctaggctctt
aatttctggt gcaaccagtt tggagactgg ggttccttca 180agattcagtg
gcagtggatc tggaaaagat tacactctca gcattaccag tcttcagact
240gaggatgttg ctacttatta ctgtcaacag ttttggagtg ctccgtacac
gttcggaggg 300gggaccaagc tggagatcaa acgaactgtg gctgcaccat
ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc
tctgttgtgt gcctgctgaa taacttctat 420cccagagagg ccaaagtaca
gtggaaggtg gataacgccc tccaatcggg taactcccag 480gagagtgtca
cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg
540ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac
ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag
64537447PRTArtificial SequenceChimeric sequence 37Glu Val Gln Leu
Gln Gln Ser Gly Ala Glu Leu Val Lys Ala Gly Ala1 5 10 15Ser Val Lys
Leu Ser Cys Pro Ala Ser Gly Leu Asn Ile Lys Asp Thr 20 25 30Tyr Met
His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45Gly
Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Phe Asp Pro Lys Phe 50 55
60Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr65
70 75 80Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Gly Val Phe Gly Phe Phe Asp Tyr Trp Gly Gln Gly
Thr Thr 100 105 110Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200
205Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 340 345 350Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
44538214PRTArtificial SequenceChimeric sequence 38Asp Ile Gln Met
Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile Tyr Asn Arg 20 25 30Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Arg Leu Leu Ile 35 40 45Ser
Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr65
70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro
Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val
Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210391344DNAArtificial Sequencehumanized
antibody 39caggttcagc tggtgcagtc tggggcagag gtgaagaagc ccgggtcctc
agtcaaggtc 60tcctgcaagg cttctggcct caacattaaa gacacctata tgcactgggt
gcgacaggcg 120cctggacagg gcctggagtg gattggaagg attgatcctg
cgaatggtaa tactaaattt 180gacccgaagt tccagggcag agccactata
acagcagaca catccacgag cacagcctac 240atggagctca gcagcctgag
atctgaggac actgccgtct attactgtgc tagaggcgtc 300tttggctttt
ttgactactg gggccaaggg accactgtga cagtctcctc agcctccacc
360aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg
gggcacagcg 420gccctgggct gcctggtcaa ggactacttc cccgaaccgg
tgacggtgtc gtggaactca 480ggcgccctga ccagcggcgt gcacaccttc
ccggctgtcc tacagtcctc aggactctac 540tccctcagca gcgtggtgac
cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600aacgtgaatc
acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt
660gacaagactc acacatgccc accgtgccca gcacctgaac tcctgggggg
accgtcagtc 720ttcctcttcc ccccaaaacc caaggacacc ctcatgatct
cccggacccc tgaggtcaca 780tgcgtggtgg tggacgtgag ccacgaagac
cctgaggtca agttcaactg gtacgtggac 840ggcgtggagg tgcataatgc
caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900cgtgtggtca
gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag
960tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc
caaagccaaa 1020gggcagcccc gagaaccaca ggtgtacacc ctgcccccat
cccgggatga gctgaccaag 1080aaccaggtca gcctgacctg cctggtcaaa
ggcttctatc ccagcgacat cgccgtggag 1140tgggagagca atgggcagcc
ggagaacaac tacaagacca cgcctcccgt gttggactcc 1200gacggctcct
tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg
1260aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac
gcagaagagc 1320ctctccctgt ctcccgggaa atga 134440447PRTArtificial
Sequencehumanized antibody 40Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Leu Asn Ile Lys Asp Thr 20 25 30Tyr Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp Pro Ala
Asn Gly Asn Thr Lys Phe Asp Pro Lys Phe 50 55 60Gln Gly Arg Ala Thr
Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Gly Val Phe Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105
110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys 130 135
140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val225 230 235 240Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 435 440 445411344DNAArtificial
Sequencehumanized antibody 41caggttcagc tggtgcagtc tggggcagag
gtgaagaagc ccgggtcctc agtcaaggtc 60tcctgcaagg cttcaggcct caccattagc
gacacctata tgcactgggt gcgacaggcg 120cctggacagg gcctcgagtg
gatgggaagg attgatcctg cgaatggtaa tactaaattt 180gacccgaagt
tccagggcag agtcactata actgcagaca catccacgag cacagcctac
240atggagctca gcagcctgag atctgaggac actgccgtct attactgtgc
tagaggcgtc 300tttggctttt ttgactactg gggccaaggg accactgtga
cagtctcctc agcctccacc 360aagggcccat cggtcttccc cctggcaccc
tcctccaaga gcacctctgg gggcacagcg 420gccctgggct gcctggtcaa
ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480ggcgccctga
ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac
540tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac
ctacatctgc 600aacgtgaatc acaagcccag caacaccaag gtggacaaga
aagttgagcc caaatcttgt 660gacaagactc acacatgccc accgtgccca
gcacctgaac tcctgggggg accgtcagtc 720ttcctcttcc ccccaaaacc
caaggacacc ctcatgatct cccggacccc tgaggtcaca 780tgcgtggtgg
tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac
840ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa
cagcacgtac 900cgtgtggtca gcgtcctcac cgtcctgcac caggactggc
tgaatggcaa ggagtacaag 960tgcaaggtct ccaacaaagc cctcccagcc
cccatcgaga aaaccatctc caaagccaaa 1020gggcagcccc gagaaccaca
ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1080aaccaggtca
gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag
1140tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt
gttggactcc 1200gacggctcct tcttcctcta cagcaagctc accgtggaca
agagcaggtg gcagcagggg 1260aacgtcttct catgctccgt gatgcatgag
gctctgcaca accactacac gcagaagagc 1320ctctccctgt ctcccgggaa atga
134442447PRTArtificial Sequencehumanized antibody 42Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser Asp Thr 20 25 30Tyr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Phe Asp Pro Lys Phe 50 55
60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Gly Val Phe Gly Phe Phe Asp Tyr Trp Gly Gln Gly
Thr Thr 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200
205Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 340 345 350Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
44543645DNAArtificial Sequencehumanized antibody 43gacattcaga
tgactcagtc tccatcctcc ctgtcagcat ctgtgggaga cagagtcacc 60attacttgca
aggcaactga ggacatatat aatcgattag cctggtatca gcagaaacca
120ggaaaggccc ctaagctctt aatttctggt gcaaccagtt tggagactgg
ggttccttca 180agattcagtg gcagtggatc tggaaaagat tacactctca
ccattagcag tctacagcct 240gaggattttg ctacttatta ctgtcaacag
ttttggagtg ctccgtacac gttcggaggg 300gggaccaagg tggagatcaa
acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc
agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat
420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg
taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa
gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa
gagcttcaac aggggagagt gttag 64544214PRTArtificial Sequencehumanized
antibody 44Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile
Tyr Asn Arg 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Phe Trp Ser Ala Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150
155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
21045645DNAArtificial Sequencehumanized antibody 45gacattcaga
tgactcagtc tccatcctcc ctgtcagcat ctgtgggaga cagagtcacc 60attacttgca
aggcaactga ggacatatat aatcgattag cctggtatca gcagaaacca
120ggaaaggccc ctaagctctt aatttctggt gcaaccagtt tggagactgg
ggttccttca 180agattcagtg gcagtggatc tggaaaagat tacactctca
ccattagcag tctacagcct 240gaggattttg ctacttatta ctgtcaacag
ttttggagtg ctccgtacac gttcggaggg 300gggaccaagg tggagatcaa
acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc
agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat
420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg
taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa
gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa
gagcttcaac aggggagagt gttag 64546214PRTArtificial Sequencehumanized
antibody 46Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile
Tyr Asn Arg 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Phe Trp Ser Ala Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150
155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
21047117PRTArtificial Sequencehumanized 11k2 heavy chain 47Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Leu Asn Ile Lys Asp Thr 20 25
30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Phe Asp Pro Lys
Phe 50 55 60Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Gly Val Phe Gly Phe Phe Asp Tyr Trp
Gly Gln Gly Thr Thr 100 105 110Val Thr Val Ser Ser
11548117PRTArtificial Sequencehumanized 11k2 heavy chain 48Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser Asp Thr 20 25
30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Phe Asp Pro Lys
Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Gly Val Phe Gly Phe Phe Asp Tyr Trp
Gly Gln Gly Thr Thr 100 105 110Val Thr Val Ser Ser
11549107PRTArtificial Sequencehumanized 11k2 light chain 49Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile Tyr Asn Arg 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp
Ser Ala Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 10550107PRTArtificial Sequencehumanized 11k2 light chain 50Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile Tyr Asn Arg
20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe
Trp Ser Ala Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys 100 10551122PRTArtificial SequenceChimeric sequence 51Glu Val
Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala1 5 10 15Ser
Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asn 20 25
30Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys
Phe 50 55 60Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr
Ala Tyr65 70 75 80Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Asn Thr Trp Ala Tyr Tyr Gly Thr Ser Tyr Gly
Gly Phe Ala Tyr Trp 100 105 110Gly Gln Gly Thr Thr Val Thr Arg Leu
Leu 115 1205237PRTArtificial Sequencechimeric sequence 52Phe Thr
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr1 5 10 15Tyr
Cys Trp Gln Gly Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr 20 25
30Lys Leu Glu Ile Lys 3553119PRTArtificial SequenceHumanized
antibody 53Gln Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Arg Pro
Gly Ser1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile
Lys Asp Asn 20 25 30Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu
Tyr Ala Pro Lys Phe 50 55 60Gln Gly Lys Ala Thr Met Thr Ala Asp Thr
Ser Ser Asn Thr Ala Tyr65 70 75 80Met Gln Leu Ser Gly Leu Thr Ser
Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Asn Thr Trp Ala Tyr Tyr Gly
Thr Ser Tyr Gly Gly Phe Ala Tyr Trp 100 105 110Gly Gln Gly Thr Thr
Val Thr 11554119PRTArtificial SequenceHumanized antibody 54Gln Val
Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ser1 5 10 15Ser
Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Ile Lys Asp Asn 20 25
30Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp
Ile
35 40 45Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys
Phe 50 55 60Gln Gly Gln Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr
Ala Tyr65 70 75 80Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95Asn Thr Trp Ala Tyr Tyr Gly Thr Ser Tyr Gly
Gly Phe Ala Tyr Trp 100 105 110Gly Gln Gly Thr Thr Val Thr
11555112PRTArtificial SequenceHumanized antibody 55Asp Ile Val Met
Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Gln Pro Ala
Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30Asp Gly
Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser 35 40 45Pro
Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55
60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65
70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln
Gly 85 90 95Thr His Phe Pro Gln Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105 11056112PRTArtificial SequenceHumanized antibody
56Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1
5 10 15Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp
Ser 20 25 30Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly
Gln Ser 35 40 45Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Trp Gln Gly 85 90 95Thr His Phe Pro Gln Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 1105782DNAArtificial
Sequencesynthetic oligonucleotide for mutagenesis of hu11k2
57cccgcggaga cattcagatg actcagtctc catcctccct gtcagcatct gtgggagaca
60gagtcaccat tacttgcaag gc 825853DNAArtificial Sequencesynthetic
oligonucleotide for mutagenesis of hu11k2 58ggtatcagca gaaaccagga
aaggccccta agctcttaat ttctggtgca acc 535969DNAArtificial
Sequencesynthetic oligonucleotide for mutagenesis of hu11k2
59ggaaaagatt acactctcac cattagcagt ctacagcctg aggattttgc tacttattac
60tgtcaacag 696049DNAArtificial Sequencesynthetic oligonucleotide
for mutagenesis of hu11k2 60cgttcggagg ggggaccaag gtggagatct
aaaaaaaggg cgaattctg 496147DNAArtificial Sequencesynthetic
oligonucleotide for mutagenesis of hu11k2 61gattcagtgg cagtggatcc
ggaacagatt acactctcac cattagc 476255DNAArtificial Sequencesynthetic
oligonucleotide for mutagenesis of hu11k2 62gtggttacag gggtcaactc
acaggttcag ctggtgcagt ctggggcaga gcttg 556379DNAArtificial
Sequencesynthetic oligonucleotide for mutagenesis of hu11k2
63gcagtctggg gcagaggtga agaagcccgg gtcctcagtc aaggtctcct gcaaggcttc
60tggcctcaac attaaagac 796456DNAArtificial Sequencesynthetic
oligonucleotide for mutagenesis of hu11k2 64gacacctata tgcactgggt
gcgacaggcg cctggacagg gcctggagtg gattgg 566595DNAArtificial
Sequencesynthetic oligonucleotide for mutagenesis of hu11k2
65cccgaagttc cagggcagag ccactataac agcagacaca tccacgagca cagcctacat
60ggagctcagc agcctgagat ctgaggacac tgccg 956658DNAArtificial
Sequencesynthetic oligonucleotide for mutagenesis of hu11k2
66ggggccaagg gaccactgtg acagtctcct caggtgagtc ctaagcttgg tacccggg
586753DNAArtificial Sequencesynthetic oligonucleotide for
mutagenesis of hu11k2 67ggtctcctgc aaggcttcag gcctcaccat tagcgacacc
tatatgcact ggg 536846DNAArtificial Sequencesynthetic
oligonucleotide for mutagenesis of hu11k2 68ggcgcctgga cagggcctcg
agtggatggg aaggattgat cctgcg 466957DNAArtificial SequenceSynthetic
oligonucleotide for mutagenesis of hu11k2 69gacccgaagt tccagggcag
agtcactata actgcagaca catccacgag cacagcc 57
* * * * *
References