U.S. patent application number 14/084593 was filed with the patent office on 2014-07-17 for humanized antibody compositions and methods for binding lysophosphatidic acid.
The applicant listed for this patent is Lpath, Inc.. Invention is credited to William A. GARLAND, Genevieve HANSEN, Steven Tarran JONES, Roger A. SABBADINI, David Gareth WILLIAMS.
Application Number | 20140199293 14/084593 |
Document ID | / |
Family ID | 42983157 |
Filed Date | 2014-07-17 |
United States Patent
Application |
20140199293 |
Kind Code |
A1 |
SABBADINI; Roger A. ; et
al. |
July 17, 2014 |
HUMANIZED ANTIBODY COMPOSITIONS AND METHODS FOR BINDING
LYSOPHOSPHATIDIC ACID
Abstract
Compositions and methods for making and using humanized anti-LPA
monoclonal antibodies, and fragments and derivatives thereof, are
described.
Inventors: |
SABBADINI; Roger A.;
(Lakeside, CA) ; HANSEN; Genevieve; (San Diego,
CA) ; GARLAND; William A.; (San Clemente, CA)
; JONES; Steven Tarran; (Hertfordshire, GB) ;
WILLIAMS; David Gareth; (Epsom, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Lpath, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
42983157 |
Appl. No.: |
14/084593 |
Filed: |
November 19, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12761584 |
Apr 16, 2010 |
8604172 |
|
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14084593 |
|
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61170595 |
Apr 17, 2009 |
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Current U.S.
Class: |
424/133.1 ;
424/172.1; 435/252.3; 435/252.31; 435/252.33; 435/252.34;
435/252.35; 435/254.11; 435/254.2; 435/254.21; 435/254.22;
435/254.23; 435/254.3; 435/254.4; 435/254.5; 435/320.1; 435/332;
435/419; 530/387.3; 530/389.8; 530/391.3; 530/391.7; 536/23.53 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 2317/24 20130101; C07K 2317/92 20130101; C07K 16/18 20130101;
A61P 27/02 20180101; C07K 16/44 20130101; C07K 2317/565 20130101;
C07K 2317/76 20130101; A61P 35/00 20180101; A61P 19/04 20180101;
C07K 2317/56 20130101; C07K 2317/73 20130101; C07K 2317/70
20130101; A61P 29/00 20180101 |
Class at
Publication: |
424/133.1 ;
530/389.8; 530/391.7; 530/391.3; 536/23.53; 424/172.1; 530/387.3;
435/320.1; 435/254.2; 435/252.3; 435/252.33; 435/252.31;
435/252.34; 435/252.35; 435/254.11; 435/254.21; 435/254.23;
435/254.22; 435/254.3; 435/254.4; 435/254.5; 435/332; 435/419 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Claims
1. A compound selected from the group consisting of: a. anti-LPA
agent, which agent specifically binds to LPA under physiological
conditions and comprises a peptide amino acid sequence selected
from the group consisting of SEQ ID NOs: 162, 163, 164, 165, 166,
167, 168, 169, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,
182, 183, 184, 185, 186, 187, 188, and 189, optionally wherein said
anti-LPA agent is selected from the group consisting of an
antibody, an antibody derivative, and a non-antibody-derived
moiety, wherein the antibody may be a full-length antibody or an
antibody fragment, optionally wherein said anti-LPA agent is
conjugated to a moiety selected from the group consisting of a
polymer, a radionuclide, a chemotherapeutic agent, and a detection
agent; b. an isolated nucleic acid molecule that encodes an
immunoglobulin heavy chain variable domain that comprises an amino
acid sequence selected from the group consisting of SEQ ID NO: 172,
173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,
186, 187, 188 and 189, optionally wherein said isolated nucleic
acid molecule encodes a fragment of an immunoglobulin heavy chain
or a full length immunoglobulin heavy chain; c an isolated nucleic
acid molecule that encodes an immunoglobulin light chain variable
domain that comprises an amino acid sequence selected from the
group consisting of SEQ ID NO: 162, 163, 164, 165, 166, 167, 168
and 169, optionally wherein said isolated nucleic acid molecule
encodes a fragment of an immunoglobulin light chain or a full
length immunoglobulin light chain; and d isolated polypeptide,
which polypeptide specifically binds LPA in a physiological context
and comprises an amino acid sequence that has a sequence identity
of at least 50 percent with a peptide amino acid sequence selected
from the group consisting of SEQ ID NO: 162, 163, 164, 165, 166,
167, 168, 169, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,
182, 183, 184, 185, 186, 187, 188 and 189, optionally wherein the
isolated polypeptide is selected from the group consisting of a
fragment of a variable domain of an animal immunoglobulin heavy
chain, a full length variable domain of an animal immunoglobulin
heavy chain, and a full length animal immunoglobulin heavy chain, a
fragment of a variable domain of an animal immunoglobulin light
chain, a full length variable domain of an animal immunoglobulin
light chain and a full length animal immunoglobulin light
chain.
2. An anti-LPA agent according to claim 1 that comprises an
isolated anti-LPA antibody comprising two immunoglobulin heavy
chains and two immunoglobulin light chains, wherein one or both of
the immunoglobulin heavy chains comprises a variable domain having
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,
184, 185, 186, 187, 188 and 189, and wherein said isolated anti-LPA
antibody is optionally a humanized anti-LPA antibody.
3. An anti-LPA agent according to claim 1 that comprises an
isolated anti-LPA antibody comprising two immunoglobulin heavy
chains and two immunoglobulin light chains, wherein one or both of
the immunoglobulin light chains comprises a variable domain having
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 162, 163, 164, 165, 166, 167, 168 and 169, and wherein said
isolated anti-LPA antibody is optionally a humanized anti-LPA
antibody.
4. An anti-LPA agent according to claim 1 that comprises an
isolated anti-LPA antibody comprising two immunoglobulin heavy
chains and two immunoglobulin light chains, wherein one or both of
the immunoglobulin heavy chains comprises a variable domain having
an amino acid sequence independently selected from the group
consisting of SEQ ID NOs: 172, 173, 174, 175, 176, 177, 178, 179,
180, 181, 182, 183, 184, 185, 186, 187, 188 and 189, and wherein
one or both of the immunoglobulin light chains comprises a variable
domain having an amino acid sequence independently selected from
the group consisting of SEQ ID NOs: 162, 163, 164, 165, 166, 167,
168 and 169, and wherein said isolated anti-LPA antibody is
optionally a humanized anti-LPA antibody, optionally wherein both
immunoglobulin heavy chains comprise a variable domain having the
same amino acid sequence selected from the group consisting of SEQ
ID NOs: 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,
184, 185, 186, 187, 188 and 189, optionally wherein both
immunoglobulin light chains comprise a variable domain having the
same amino acid sequence selected from the group consisting of SEQ
ID NOs: 162, 163, 164, 165, 166, 167, 168 and 169.
5. An anti-LPA agent according to claim 1 that comprises an
isolated anti-LPA antibody comprising two immunoglobulin heavy
chains and two immunoglobulin light chains, wherein each
immunoglobulin heavy chain comprises a variable domain having an
amino acid sequence independently selected from the group
consisting of SEQ ID NOs: 172, 179 and 180, and each immunoglobulin
light chain comprises a variable domain having an amino acid
sequence independently selected from the group consisting of SEQ ID
NOs: 162 and 166, and wherein said isolated anti-LPA antibody is
optionally a humanized anti-LPA antibody.
6. A anti-LPA agent according to claim 5 that comprises a humanized
anti-LPA antibody comprising two immunoglobulin heavy chains and
two immunoglobulin light chains, wherein each immunoglobulin heavy
chain comprises a variable domain having an amino acid sequence
consisting of SEQ ID NO: 172 and each immunoglobulin light chain
comprises a variable domain having an amino acid sequence
consisting of SEQ ID NO: 162.
7. An isolated polypeptide according to claim 1, which polypeptide
has a sequence identity of at least 65 percent, optionally at least
75 percent, at least 80 percent, at least 85 percent, at least 90
percent or at least 95 percent with a peptide amino acid sequence
selected from the group consisting of SEQ ID NO: 162, 163, 164,
165, 166, 167, 168, 169, 172, 173, 174, 175, 176, 177, 178, 179,
180, 181, 182, 183, 184, 185, 186, 187, 188 and 189, optionally
wherein the isolated polypeptide is selected from the group
consisting of a fragment of a variable domain of an animal
immunoglobulin heavy chain, a full length variable domain of an
animal immunoglobulin heavy chain, and a full length animal
immunoglobulin heavy chain, a fragment of a variable domain of an
animal immunoglobulin light chain, a full length variable domain of
an animal immunoglobulin light chain and a full length animal
immunoglobulin light chain.
8. A vector comprising an isolated nucleic acid molecule according
to claim 1, wherein optionally said isolated nucleic acid molecule
encodes (i) a fragment of an immunoglobulin heavy chain or a full
length immunoglobulin heavy chain, (ii) a fragment of an
immunoglobulin light chain or a full length immunoglobulin light
chain, or (iii) a fragment of an immunoglobulin heavy chain or a
full length immunoglobulin heavy chain and a fragment of an
immunoglobulin light chain or a full length immunoglobulin light
chain.
9. A host cell transfected with an isolated nucleic acid molecule
according to claim 1.
10. A host cell transfected with a vector according to claim 8.
11. A composition comprising a pharmaceutically acceptable carrier
and an anti-LPA agent selected from the group consisting of: a. an
isolated anti-LPA antibody which comprises two immunoglobulin heavy
chains and two immunoglobulin light chains, wherein one or both of
the immunoglobulin heavy chains comprises a variable domain having
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,
184, 185, 186, 187, 188 and 189, and wherein said isolated anti-LPA
antibody is optionally a humanized anti-LPA antibody; b. an
isolated anti-LPA antibody which comprises two immunoglobulin heavy
chains and two immunoglobulin light chains, wherein one or both of
the immunoglobulin light chains comprises a variable domain having
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 162, 163, 164, 165, 166, 167, 168 and 169, and wherein said
isolated anti-LPA antibody is optionally a humanized anti-LPA
antibody; c. an isolated anti-LPA antibody molecule which comprises
two immunoglobulin heavy chains and two immunoglobulin light
chains, wherein each immunoglobulin heavy chain comprises a
variable domain having an amino acid sequence independently
selected from the group consisting of SEQ ID NOs: 172, 173, 174,
175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,
188 and 189, and each immunoglobulin light chain comprises a
variable domain having an amino acid sequence independently
selected from the group consisting of SEQ ID NOs: 162, 163, 164,
165, 166, 167, 168 and 169, optionally wherein both immunoglobulin
heavy chains comprise a variable domain having the same amino acid
sequence selected from the group consisting of SEQ ID NOs: 172,
173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,
186, 187, 188 and 189, optionally wherein both immunoglobulin light
chains comprise a variable domain having the same amino acid
sequence selected from the group consisting of SEQ ID NOs: 162,
163, 164, 165, 166, 167, 168 and 169, and wherein said isolated
anti-LPA antibody is optionally a humanized anti-LPA antibody; d.
an anti-LPA agent, which agent is reactive against LPA under
physiological conditions and comprises a peptide amino acid
sequence selected from the group consisting of SEQ ID NOs: 162,
163, 164, 165, 166, 167, 168, 169, 172, 173, 174, 175, 176, 177,
178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188 and 189, and
e. an anti-LPA agent that comprises an anti-LPA antibody which
comprises two immunoglobulin heavy chains and two immunoglobulin
light chains, wherein each immunoglobulin heavy chain comprises the
same variable domain amino acid sequence selected from the group
consisting of SEQ ID NOs: 172, 173, 174, 175, 176, 177, 178, 179,
180, 181, 182, 183, 184, 185, 186, 187, 188 and 189, and each
immunoglobulin light chain comprises the same variable domain amino
acid sequence selected from the group consisting of 162, 163, 164,
165, 166, 167, 168 and 169; optionally wherein said composition is
packaged in a container, and optionally further comprising
instructions for use of the composition.
12. A method selected from the group consisting of: a.
administering to a subject, optionally a human subject in need of
such administration, an agent selected from the group consisting
of: (i) an anti-LPA agent which agent specifically binds LPA under
physiological conditions and comprises a peptide amino acid
sequence selected from the group consisting of SEQ ID NOs: 162,
163, 164, 165, 166, 167, 168, 169, 172, 173, 174, 175, 176, 177,
178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188 and 189; (ii)
an isolated polypeptide which specifically binds LPA in a
physiological context and comprises an amino acid sequence that has
a sequence identity of at least 65 percent, optionally at least 75
percent, at least 80 percent, at least 85 percent, at least 90
percent or at least 95 percent with a peptide amino acid sequence
selected from the group consisting of SEQ ID NO: 162, 163, 164,
165, 166, 167, 168, 169, 172, 173, 174, 175, 176, 177, 178, 179,
180, 181, 182, 183, 184, 185, 186, 187, 188 and 189; (iii) an
isolated antibody molecule which specifically binds LPA in a
physiological context, comprising: A. two immunoglobulin heavy
chains, wherein each immunoglobulin heavy chain comprises a
variable domain having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 172, 173, 174, 175, 176, 177, 178,
179, 180, 181, 182, 183, 184, 185, 186, 187, 188 and 189; B. two
immunoglobulin light chains, wherein each immunoglobulin light
chain comprises a variable domain having an amino acid sequence
selected from the group consisting of SEQ ID NOs: 162, 163, 164,
165, 166, 167, 168 and 169; (iv) a multivalent binding molecule
that comprises at least first and second ligand binding elements,
wherein the first ligand binding element specifically binds LPA and
comprises a peptide amino acid sequence selected from the group
consisting of SEQ ID NO: 162, 163, 164, 165, 166, 167, 168, 169,
172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,
185, 186, 187, 188 and 189, wherein the second ligand binding
element optionally also specifically binds LPA. (v) an isolated
anti-LPA antibody comprising a heavy chain which comprises a
variable domain having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 172, 173, 174, 175, 176, 177, 178,
179, 180, 181, 182, 183, 184, 185, 186, 187, 188 and 189; (vi) an
isolated anti-LPA antibody comprising a light chain which comprises
a variable domain having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 162, 163, 164, 165, 166, 167, 168
and 169; (vii) an isolated anti-LPA antibody, wherein each
immunoglobulin heavy chain comprises a variable domain having an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,
184, 185, 186, 187, 188 and 189, and each immunoglobulin light
chain comprises a variable domain having an amino acid sequence
selected from the group consisting of SEQ ID NOs: 162, 163, 164,
165, 166, 167, 168 and 169; and (viii) a humanized anti-LPA
antibody molecule which comprises two immunoglobulin heavy chains
and two immunoglobulin light chains, wherein each immunoglobulin
heavy chain comprises a variable domain having an amino acid
sequence consisting of SEQ ID NO: 172 and each immunoglobulin light
chain comprises a variable domain having an amino acid sequence
consisting of SEQ ID NO: 162; in an amount sufficient to have a
desired effect, wherein the method consists of administering said
agent to said subject systemically, parenterally, intravenously,
intrathecally, epidurally, intramuscularly, subcutaneously,
transdermally, intradermally, transmucosally, intraocularly,
periocularly, mucosally, topically or by inhalation, wherein said
desired effect is selected from the group consisting of decreasing
the effective concentration of LPA in one or more bodily fluids or
tissues and treating or preventing a disease or disorder correlated
with aberrant levels of LPA, optionally elevated levels, of LPA; b.
treating or preventing a disease or disorder correlated with
elevated levels of LPA, comprising administering to a subject,
optionally a human subject in need of such treatment an agent
selected from the group consisting of: (i) an anti-LPA agent which
agent specifically binds LPA under physiological conditions and
comprises a peptide amino acid sequence selected from the group
consisting of SEQ ID NOs: 162, 163, 164, 165, 166, 167, 168, 169,
172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,
185, 186, 187, 188 and 189; (ii) an isolated polypeptide which
specifically binds LPA in a physiological context and comprises an
amino acid sequence that has a sequence identity of at least 65
percent, optionally at least 75 percent, at least 80 percent, at
least 85 percent, at least 90 percent or at least 95 percent with a
peptide amino acid sequence selected from the group consisting of
SEQ ID NO: 162, 163, 164, 165, 166, 167, 168, 169, 172, 173, 174,
175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,
188 and 189; (iii) an isolated antibody molecule which specifically
binds LPA in a physiological context, comprising: A. two
immunoglobulin heavy chains, wherein each immunoglobulin heavy
chain comprises a variable domain having an amino acid sequence
selected from the group consisting of SEQ ID NOs: 172, 173, 174,
175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,
188 and 189; B. two immunoglobulin light chains, wherein each
immunoglobulin light chain comprises a variable domain having an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 162, 163, 164, 165, 166, 167, 168 and 169; (iv) a multivalent
binding molecule that comprises at least first and second ligand
binding elements, wherein the first ligand binding element
specifically binds LPA and comprises a peptide amino acid sequence
selected from the group consisting of SEQ ID NO: 162, 163, 164,
165, 166, 167, 168, 169, 172, 173, 174, 175, 176, 177, 178, 179,
180, 181, 182, 183, 184, 185, 186, 187, 188 and 189, wherein the
second ligand binding element optionally also specifically binds
LPA. (v) an isolated anti-LPA antibody comprising a heavy chain
which comprises a variable domain having an amino acid sequence
selected from the group consisting of SEQ ID NOs: 172, 173, 174,
175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,
188 and 189; (vi) an isolated anti-LPA antibody comprising a light
chain which comprises a variable domain having an amino acid
sequence selected from the group consisting of SEQ ID NOs: 162,
163, 164, 165, 166, 167, 168 and 169; and (vii) an isolated
anti-LPA antibody, wherein each immunoglobulin heavy chain
comprises a variable domain having an amino acid sequence selected
from the group consisting of SEQ ID NOs: 172, 173, 174, 175, 176,
177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188 and 189,
and each immunoglobulin light chain comprises a variable domain
having an amino acid sequence selected from the group consisting of
SEQ ID NOs: 162, 163, 164, 165, 166, 167, 168 and 169; and (viii) a
humanized anti-LPA antibody molecule which comprises two
immunoglobulin heavy chains and two immunoglobulin light chains,
wherein each immunoglobulin heavy chain comprises a variable domain
having an amino acid sequence consisting of SEQ ID NO: 172 and each
immunoglobulin light chain comprises a variable domain having an
amino acid sequence consisting of SEQ ID NO: 162; in an amount
effective to reduce in vivo the effective concentration of LPA,
thereby effecting treatment or prevention of the disease or
disorder, optionally wherein the disease or disorder is selected
from the group consisting of cancer, an inflammatory disorder, a
cerebrovascular disease, a cardiovascular disease, an ocular
disorder, a disease and disorder associated with excessive
fibrogenesis, a disease or disorder associated with metastasis, a
disease or disorder associated with tumor growth, and a disease or
disorder associated with pathologic angiogenesis, and optionally
wherein the anti-LPA agent, isolated polypeptide, isolated antibody
or multivalent binding molecule is administered in combination with
another therapeutic agent to effect treatment or prevention of the
disease or disorder; c. decreasing the effective concentration of
LPA in at least one bodily fluid or tissue of a subject, optionally
a human subject, comprising administering to a subject, optionally
a human subject in need of such treatment, an agent selected from
the group consisting of: (i) an anti-LPA agent which specifically
binds LPA under physiological conditions and comprises a peptide
amino acid sequence selected from the group consisting of SEQ ID
NOs: 162, 163, 164, 165, 166, 167, 168, 169, 172, 173, 174, 175,
176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188 and
189; (ii) an isolated polypeptide which specifically binds LPA in a
physiological context and comprises an amino acid sequence that has
a sequence identity of at least 65 percent, optionally at least 75
percent, at least 80 percent, at least 85 percent, at least 90
percent or at least 95 percent with a peptide amino acid sequence
selected from the group consisting of SEQ ID NO: 162, 163, 164,
165, 166, 167, 168, 169, 172, 173, 174, 175, 176, 177, 178, 179,
180, 181, 182, 183, 184, 185, 186, 187, 188 and 189; (iii) an
isolated antibody molecule which specifically binds LPA in a
physiological context, comprising: A. two immunoglobulin heavy
chains, wherein each immunoglobulin heavy chain comprises a
variable domain having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 172, 173, 174, 175, 176, 177, 178,
179, 180, 181, 182, 183, 184, 185, 186, 187, 188 and 189; B. two
immunoglobulin light chains, wherein each immunoglobulin light
chain comprises a variable domain having an amino acid sequence
selected from the group consisting of SEQ ID NOs: 162, 163, 164,
165, 166, 167, 168 and 169; (iv) a multivalent binding molecule
that comprises at least first and second ligand binding elements,
wherein the first ligand binding element specifically binds LPA and
comprises a peptide amino acid sequence selected from the group
consisting of SEQ ID NO: 162, 163, 164, 165, 166, 167, 168, 169,
172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,
185, 186, 187, 188 and 189, wherein the second ligand binding
element optionally also specifically binds LPA. (v) an isolated
anti-LPA antibody comprising a heavy chain which comprises a
variable domain having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 172, 173, 174, 175, 176, 177, 178,
179, 180, 181, 182, 183, 184, 185, 186, 187, 188 and 189; (vi) an
isolated anti-LPA antibody comprising a light chain which comprises
a variable domain having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 162, 163, 164, 165, 166, 167, 168
and 169; and (vii) an isolated anti-LPA antibody, wherein each
immunoglobulin heavy chain comprises a variable domain having an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,
184, 185, 186, 187, 188 and 189, and each immunoglobulin light
chain comprises a variable domain having an amino acid sequence
selected from the group consisting of SEQ ID NOs: 162, 163, 164,
165, 166, 167, 168 and 169; and (viii) a humanized anti-LPA
antibody molecule which comprises two immunoglobulin heavy chains
and two immunoglobulin light chains, wherein each immunoglobulin
heavy chain comprises a variable domain having an amino acid
sequence consisting of SEQ ID NO: 172 and each immunoglobulin light
chain comprises a variable domain having an amino acid sequence
consisting of SEQ ID NO: 162; in an amount effective to reduce the
effective concentration of LPA in at least one bodily fluid or
tissue of the subject; and d. A method for detecting LPA or an LPA
metabolite that comprises a native LPA epitope comprising exposing
a sample suspected of containing LPA or an LPA metabolite that
comprises a native LPA epitope to a compound which specifically
binds a native LPA epitope, wherein said compound is selected from
the group consisting of: (i) an anti-LPA agent which agent
specifically binds LPA under physiological conditions and comprises
a peptide amino acid sequence selected from the group consisting of
SEQ ID NOs: 162, 163, 164, 165, 166, 167, 168, 169, 172, 173, 174,
175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,
188 and 189; (ii) an isolated polypeptide which specifically binds
LPA in a physiological context and comprises an amino acid sequence
that has a sequence identity of at least 65 percent, optionally at
least 75 percent, at least 80 percent, at least 85 percent, at
least 90 percent or at least 95 percent with a peptide amino acid
sequence selected from the group consisting of SEQ ID NO: 162, 163,
164, 165, 166, 167, 168, 169, 172, 173, 174, 175, 176, 177, 178,
179, 180, 181, 182, 183, 184, 185, 186, 187, 188 and 189; (iii) an
isolated antibody molecule which specifically binds LPA in a
physiological context, comprising: A. two immunoglobulin heavy
chains, wherein each immunoglobulin heavy chain comprises a
variable domain having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 172, 173, 174, 175, 176, 177, 178,
179, 180, 181, 182, 183, 184, 185, 186, 187, 188 and 189; B. two
immunoglobulin light chains, wherein each immunoglobulin light
chain comprises a variable domain having an amino acid sequence
selected from the group consisting of SEQ ID NOs: 162, 163, 164,
165, 166, 167, 168 and 169; (iv) a multivalent binding molecule
that comprises at least first and second ligand binding elements,
wherein the first ligand binding element specifically binds LPA and
comprises a peptide amino acid sequence selected from the group
consisting of SEQ ID NOs: 162, 163, 164, 165, 166, 167, 168, 169,
172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,
185, 186, 187, 188 and 189; wherein the second ligand binding
element is optionally also specifically binds LPA. (v) an isolated
anti-LPA antibody comprising a heavy chain which comprises a
variable domain having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 172, 173, 174, 175, 176, 177, 178,
179, 180, 181, 182, 183, 184, 185, 186, 187, 188 and 189; (vi) an
isolated anti-LPA antibody comprising a light chain which comprises
a variable domain having an amino acid sequence selected from the
group consisting of SEQ ID NOs: 162, 163, 164, 165, 166, 167, 168
and 169; and (vii) an isolated anti-LPA antibody, wherein each
immunoglobulin heavy chain comprises a variable domain having an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,
184, 185, 186, 187, 188 and 189, and each immunoglobulin light
chain comprises a variable domain having an amino acid sequence
selected from the group consisting of SEQ ID NO: 162, 163, 164,
165, 166, 167, 168 and 169; and determining binding of the compound
which specifically binds LPA to the sample, optionally wherein
detecting binding results from exposing a sample known or suspected
to contain the LPA or LPA metabolite with the compound which
specifically binds LPA, under conditions that allow the compound
which specifically binds LPA to bind to the LPA or LPA metabolite,
if present in the sample, wherein the sample is optionally a tissue
or liquid sample, optionally selected from the group consisting of
whole blood, plasma, serum, urine, semen, bile, aqueous humor,
vitreous humor, mucus, and sputum; wherein the compound which
specifically binds LPA or LPA metabolite is optionally selected
from the group consisting of a monoclonal antibody, an antibody
fragment, an antibody variant, and an antibody derivative; wherein
the compound which specifically binds LPA or LPA metabolite is
optionally attached to a solid support; and wherein the method is
optionally an ELISA assay.
Description
RELATED APPLICATION
[0001] This application claims the benefit of and priority to
commonly owned U.S. provisional patent application Ser. No.
61/170,595, filed 17 Apr. 2009, and U.S. utility patent application
Ser. No. 12/761,584, filed 16 Apr. 2010, which are herein
incorporated by reference in entirety for any and all purposes.
TECHNICAL FIELD
[0002] The present invention relates to agents that bind
lysophosphatidic acid (LPA) and its variants, particularly to
monoclonal antibodies, antibody fragments, and antibody derivatives
specifically reactive to LPA under physiological conditions. Such
agents can be used in the treatment and/or prevention of various
diseases or disorders through the delivery of pharmaceutical
compositions that contain such agents.
[0003] LPA is a bioactive lipid mediating multiple cellular
responses including proliferation, differentiation, angiogenesis,
motility, and protection from apoptosis in a variety of cell
types.
[0004] LPA is involved in the establishment and progression of
cancer by providing a pro-growth tumor microenvironment and
promoting angiogenesis. In addition, LPA has been implicated in
fibrosis, ocular diseases such as macular degeneration, and
pain-related disorders. Therefore, an antibody-based approach to
the neutralization of LPA offers the potential to increase the
arsenal of current therapies for these indications.
[0005] Applicants have invented a family of high-affinity, specific
monoclonal antibodies to LPA, one of which is known as Lpathomab or
LT3000. The efficacy of Lpathomab in various animal models of
cancer, fibrosis, and ocular disorders highlights the utility of
this class of anti-LPA antibodies (and molecules derived
therefrom), for example, in the treatment of malignancies,
angiogenesis, and fibrosis-related disorders.
BACKGROUND OF THE INVENTION
[0006] 1. Introduction
[0007] The following description includes information that may be
useful in understanding the present invention. It is not an
admission that any of the information provided herein, or any
publication specifically or implicitly referenced herein, is prior
art, or even particularly relevant, to the presently claimed
invention.
[0008] 2. Background
[0009] A. Bioactive Signaling Lipids
[0010] Lipids and their derivatives are now recognized as important
targets for medical research, not as just simple structural
elements in cell membranes or as a source of energy for
.beta.-oxidation, glycolysis or other metabolic processes. In
particular, certain bioactive lipids function as signaling
mediators important in animal and human disease. Although most of
the lipids of the plasma membrane play an exclusively structural
role, a small proportion of them are involved in relaying
extracellular stimuli into cells. These lipids are referred to as
"bioactive lipids" or, alternatively, "bioactive signaling lipids."
"Lipid signaling" refers to any of a number of cellular signal
transduction pathways that use cell membrane lipids as second
messengers, as well as referring to direct interaction of a lipid
signaling molecule with its own specific receptor. Lipid signaling
pathways are activated by a variety of extracellular stimuli,
ranging from growth factors to inflammatory cytokines, and regulate
cell fate decisions such as apoptosis, differentiation and
proliferation. Research into bioactive lipid signaling is an area
of intense scientific investigation as more and more bioactive
lipids are identified and their actions characterized.
[0011] Examples of bioactive lipids include the eicosanoids
(including the cannabinoids, leukotrienes, prostaglandins,
lipoxins, epoxyeicosatrienoic acids, and isoeicosanoids),
non-eicosanoid cannabinoid mediators, phospholipids and their
derivatives such as phosphatidic acid (PA) and phosphatidylglycerol
(PG), platelet activating factor (PAF) and cardiolipins as well as
lysophospholipids such as lysophosphatidyl choline (LPC) and
various lysophosphatidic acids (LPA). Bioactive signaling lipids
also include the sphingolipids such as sphingomyelin, ceramide,
ceramide-1-phosphate, sphingosine, sphingosylphosphoryl choline,
sphinganine, sphinganine-1-phosphate (dihydro-S1P) and
sphingosine-1-phosphate. Sphingolipids and their derivatives
represent a group of extracellular and intracellular signaling
molecules with pleiotropic effects on important cellular processes.
Other examples of bioactive signaling lipids include
phosphatidylinositol (PI), phosphatidylethanolamine (PEA),
diacylglyceride (DG), sulfatides, gangliosides, and
cerebrosides.
[0012] 1. Lysolipids
[0013] Lysophospholipids (LPLs), also known as lysolipids, are low
molecular weight (typically less than about 500 dalton) lipids that
contain a single hydrocarbon backbone and a polar head group
containing a phosphate group. Some lysolipids are bioactive
signaling lipids. Two particular examples of medically important
bioactive lysolipids are LPA (glycerol backbone) and S1P (sphingoid
backbone). The structures of selected LPAs, S1P, and dihydro S1P
are presented below.
##STR00001## ##STR00002## ##STR00003##
[0014] The structural backbone of LPA is derived from
glycerol-based phospholipids such as phosphatidylcholine (PC) or
phosphatidic acid (PA). In the case of lysosphingolipids such as
S1P, the fatty acid of the ceramide backbone is missing. The
structural backbone of S1P, dihydro S1P (DHS1P), and
sphingosylphosphorylcholine (SPC) is based on sphingosine, which is
derived from sphingomyelin.
[0015] LPA and SIP regulate various cellular signaling pathways by
binding to the same class of multiple transmembrane domain G
protein-coupled (GPCR) receptors. The S1P receptors are designated
as S1P1, S1P2, S1P3, S1P4 and SIPS (formerly EDG-1, EDG-5/AGR16,
EDG-3, EDG-6 and EDG-8) and the LPA receptors designated as LPA1,
LPA2, LPA3 (formerly, EDG-2, EDG-4, and EDG-7). A fourth LPA
receptor of this family has been identified for LPA (LPA4), and
other putative receptors for these lysophospholipids have also been
reported.
[0016] LPA and SIP have been shown to play a role in the immune
response through modulation of immune-related cells such as T- and
B-lymphocytes. These lipids promote T-cell migration to sites of
immune response and regulate proliferation of T cells as well as
secretion of various cytokines. In particular, SIP is thought to
control egress of lymphocytes into the peripheral circulation. Thus
agents which bind LPA and SIP are believed to be useful in methods
for decreasing an undesired, excessive or aberrant immune response,
and for treating diseases and conditions, including certain
hematological cancers and autoimmune disorders, that are associated
with an undesired, excessive or aberrant involvement of lymphocytes
and or an aberrant immune response.
[0017] a. Lysophosphatic Acid (LPA)
[0018] Lysophosphatidic acid (mono-acylglycerol-3-phosphate,
<500 Dalton) consists of a single hydrocarbon backbone and a
polar head group containing a phosphate group. LPA is not a single
molecular entity but a collection of endogenous structural variants
with fatty acids of varied lengths and degrees of saturation.
Biologically relevant variants of LPA include 18:2, 18:1, 18:0,
16:0 and 20:4. LPA species with both saturated fatty acids (16:0
and 18:0) and unsaturated fatty acids (16:1, 18:1, 18:2, and 20:4)
have been detected in serum and plasma. The 16:0, 18:1, 18:2 and
20:4 LPA isoforms are the predominant species in blood. Significant
levels (>1 .mu.M) of bioactive LPA are detectable in various
body fluids, including serum, saliva, follicular fluid and
malignant effusions.
[0019] The present invention provides among its aspects anti-LPA
agents that are useful for treating or preventing
hyperproliferative disorders and various other disorders, as
described in greater detail below. In particular, certain
embodiments of the invention is drawn to antibodies targeted to LPA
including but not limited to 18:2, 18:1, 18:0, 16:0, and 20:4
variants of LPA.
[0020] LPAs have long been known as precursors of phospholipid
biosynthesis in both eukaryotic and prokaryotic cells, but LPAs
have emerged only recently as signaling molecules that are rapidly
produced and released by activated cells, notably platelets, to
influence target cells by acting on specific cell-surface receptor.
Besides being synthesized and processed to more complex
phospholipids in the endoplasmic reticulum, LPA can be generated
through the hydrolysis of pre-existing phospholipids following cell
activation; for example, the sn-2 position is commonly missing a
fatty acid residue due to de-acylation, leaving only the sn-3
hydroxyl esterified to a fatty acid. Moreover, a key enzyme in the
production of LPA, autotaxin (lysoPLD/NPP2), may be the product of
an oncogene, as many tumor types up-regulate autotoxin. The
concentrations of LPA in human plasma and serum have been reported,
including determinations made using sensitive and specific LC/MS
procedures. For example, in freshly prepared human serum allowed to
sit at 25.degree. C. for one hour, LPA concentrations have been
estimated to be approximately 1.2 mM, with the LPA analogs 16:0,
18:1, 18:2, and 20:4 being the predominant species. Similarly, in
freshly prepared human plasma allowed to sit at 25.degree. C. for
one hour, LPA concentrations have been estimated to be
approximately 0.7 mM, with 18:1 and 18:2 LPA being the predominant
species.
[0021] LPA mediates its biological functions predominantly by
binding to a class of multiple transmembrane G protein-coupled
receptors (GPCR). Five LPA-specific GPCRs, termed LPA1-5, have been
identified to date; they show both overlapping and distinct
signaling properties and tissue expression. The LPA1-3 receptors
belong to the so-called EDG subfamily (EGD2/LPA1, EDG4/LPA2, and
EDG7/LPA3) of GPCRs with 50% sequence similarity to each other.
Their closest relative is the cannabinoid CB1 receptor, which binds
the bioactive lipids 2-arachidonoyl-glycerol (2-AG) and
arachidonoyl-ethanolamine. Two newly identified LPA receptors,
termed LPA4 (formerly GPR23/p2y9) and LPA5 (formerly GPR92) are
more closely related to the P2Y nucleotide receptors. In addition,
LPA recognizes the intracellular receptor, PPRgamma.
[0022] LPA1 is expressed in a wide range of tissues and organs
whereas LPA2 and LPA3 show more restricted expression profile.
However, LPA2 and LPA3 expressions were shown to be increased in
ovarian and colon cancers and inflammation, suggesting that the
main role of LPA2 and LPA3 is in pathophysiological conditions.
[0023] The role of these receptors has been in part elucidated by
receptor knockout studies in mice. LPA1-deficient mice show partial
postnatal lethality due to a suckling defect resulting from
impaired olfaction. LPA1-deficient mice are also protected from
lung fibrosis in response to bleomycin-induced lung injury.
Furthermore, mice lacking the LPA1 receptor gene lose the nerve
injury-induced neuropathic pain behaviors and phenomena.
[0024] In contrast, mice lacking LPA2 receptors appear to be
normal. LPA3 receptor knockout mice have reduced litter size due to
delayed blastocyst implantation and altered embryo spacing, and
LPA3-deficient uteri show reduced cyclooxygenase-2 (COX-2)
expression and prostaglandin synthesis; while exogenous
administration of PGE2 into LPA3-deficient female mice has been
reported to rescue the implantation defect.
[0025] LPAs influence a wide range of biological responses,
including induction of cell proliferation, stimulation of cell
migration and neurite retraction, gap junction closure, and even
slime mold chemotaxis. The body of knowledge about the biology of
LPA continues to grow as more and more cellular systems are tested
for LPA responsiveness. The major physiological and
pathophysiological effects of LPA include, for example:
[0026] Wound healing: It is now known that, in addition to
stimulating cell growth and proliferation, LPA promote cellular
tension and cell-surface fibronectin binding, which are important
events in wound repair and regeneration.
[0027] Apoptosis: Recently, anti-apoptotic activity has also been
ascribed to LPA, and it has recently been reported that peroxisome
proliferation receptor gamma is a receptor/target for LPA.
[0028] Blood vessel maturation: Autotaxin, a secreted
lysophospholipase D responsible for producing LPAs, is essential
for blood vessel formation during development. In addition,
unsaturated LPAs were identified as major contributors to the
induction of vascular smooth muscle cell dedifferentiation.
[0029] Edema and vascular permeability: LPA induces plasma
exudation and histamine release in mice.
[0030] Pain: Initiation of neuropathic pain has been found to
require lysophosphatidic acid receptor signaling. Nature Med.
(2004) 10:712-718. As described above, mice lacking LPA1 receptors
show decreases in neuropathic pain behaviors.
[0031] Inflammation: LPA acts as inflammatory mediator in human
corneal epithelial cells. LPA participates in corneal wound healing
and stimulates the release of ROS in lens. LPA can also re-activate
HSV-1 in rabbit cornea.
[0032] The bite of the venomous spider, Loxosceles reclusa (brown
recluse spider), causes necrotic ulcers that can cause serious and
long lasting tissue damage, and occasionally death. The pathology
of wounds generated from the bite of this spider consists of an
intense inflammatory response mediated by AA and prostaglandins.
The major component of the L. reclusa spider venom is the
phospholipase D enzyme often referred to as sphingomyelinase D
(SMase D), which hydrolyzes sphingomyelin to produce C1P. It has
been found, however, that lysophospholipids with a variety of
headgroups are hydrolysed by the L. reclusa enzyme to release LPA.
It is believed that anti-LPA agents such as those of the invention
will be useful in reducing or treating inflammation of various
types, including but not limited to inflammation resulting from L.
reclusa envenomation.
[0033] Fibrosis and scar formation: LPA inhibits TGF-mediated
stimulation of type I collagen mRNA stability via an ERK-dependent
pathway in dermal fibroblasts. Moreover, LPA have some direct
fibrogenic effects by stimulating collagen gene expression and
proliferation of fibroblasts.
[0034] Immune response: LPA, like S1P, has been shown to play a
role in the immune response through modulation of immune-related
cells. These lipids promote T-cell migration to sites of immune
response and regulate proliferation of T cells as well as secretion
of various cytokines.
[0035] Thus agents that reduce the effective concentration of LPA,
such as Lpath's anti-LPA mAb, are believed to be useful in methods
for treating diseases and conditions such as those associated with
wound healing and fibrosis, apoptosis, angiogenesis and
neovascularizaion, vascular permeability and inflammation, that are
associated with an undesired, excessive or aberrant level of
LPA.
[0036] Recently, the applicants have developed several monoclonal
antibodies against LPAs. These anti-LPA antibodies can neutralize
various LPAs and mitigate their biologic and pharmacologic action.
Anti-LPA antibodies are, therefore, believed to be useful in
prevention and/or treatment of various diseases and conditions
associated with excessive, unwanted or aberrant levels of LPA.
[0037] Rapid and specific methods of detecting LPA are also
desired. Methods for separating and semi-quantitatively measuring
phospholipids such as LPA using techniques such as thin-layer
chromatography (TLC) followed by gas chromatography (GC) and/or
mass spectrometry (MS) are known. For example, lipids may be
extracted from the test sample of bodily fluid. Alternatively,
thin-layer chromatography may be used to separate various
phospholipids. Phospholipids and lysophospholipids can then be
visualized on plates, for example, using ultraviolet light.
Alternatively, lysophospholipid concentrations can be identified by
NMR or HPLC following isolation from phospholipids or as part of
the phospholipid. LPA levels have also been determined in ascites
from ovarian cancer patients using an assay that relies on
LysoPA-specific effects on eukaryotic cells in culture. However,
these prior procedures are time-consuming, expensive and variable
and typically only semi-quantitative. Enzymatic methods for
detecting lysophospholipids such as LPA in biological fluids, and
for correlating and detecting conditions associated with altered
levels of lysophospholipids, are also known. U.S. Pat. Nos.
6,255,063 and 6,248,553, originally assigned to Atairgin
Technologies, Inc. and now commonly owned with the instant
invention.
[0038] 3. Definitions
3. Definitions
[0039] Before describing the instant invention in detail, several
terms used in the context of the present invention will be defined.
In addition to these terms, others are defined elsewhere in the
specification, as necessary. Unless otherwise expressly defined
herein, terms of art used in this specification will have their
art-recognized meanings.
[0040] "Aberrant" means excessive or unwanted, for example in
reference to levels or effective concentrations of a cellular
target such as a protein or bioactive lipid.
[0041] The term "antibody" ("Ab") or "immunoglobulin" (Ig) refers
to any form of a peptide, polypeptide derived from, modeled after
or encoded by, an immunoglobulin gene, or fragment thereof, that is
capable of binding an antigen or epitope. See, e.g., IMMUNOBIOLOGY,
Fifth Edition, C. A. Janeway, P. Travers, M., Walport, M. J.
Shlomchiked., ed. Garland Publishing (2001). The term "antibody" is
used herein in the broadest sense, and encompasses monoclonal,
polyclonal or multispecific antibodies, minibodies,
heteroconjugates, diabodies, triabodies, chimeric, antibodies,
synthetic antibodies, antibody fragments, and binding agents that
employ the complementarity determining regions (CDRs) of the parent
antibody, or variants thereof that retain antigen binding activity.
Antibodies are defined herein as retaining at least one desired
activity of the parent antibody. Desired activities can include the
ability to bind the antigen specifically, the ability to inhibit
proleration in vitro, the ability to inhibit angiogenesis in vivo,
and the ability to alter cytokine profile(s) in vitro.
[0042] Native antibodies (native immunoglobulins) are usually
heterotetrameric glycoproteins of about 150,000 Daltons, typically
composed of two identical light (L) chains and two identical heavy
(H) chains. The heavy chain is approximately 50 kD in size, and the
light chain is approximately 25 kDa. Each light chain is typically
linked to a heavy chain by one covalent disulfide bond, while the
number of disulfide linkages varies among the heavy chains of
different immunoglobulin isotypes. Each heavy and light chain also
has regularly spaced intrachain disulfide bridges. Each heavy chain
has at one end a variable domain (V.sub.H) followed by a number of
constant domains. Each light chain has a variable domain at one end
(V.sub.L) and a constant domain at its other end; the constant
domain of the light chain is aligned with the first constant domain
of the heavy chain, and the light-chain variable domain is aligned
with the variable domain of the heavy chain. Particular amino acid
residues are believed to form an interface between the light- and
heavy-chain variable domains.
[0043] The light chains of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (.kappa.) and lambda (.lamda.), based on the
amino acid sequences of their constant domains. The ratio of the
two types of light chain varies from species to species. As a way
of example, the average .kappa. to .lamda. ratio is 20:1 in mice,
whereas in humans it is 2:1 and in cattle it is 1:20.
[0044] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
The heavy-chain constant domains that correspond to the different
classes of immunoglobulins are called alpha, delta, epsilon, gamma,
and mu, respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known.
[0045] An "antibody derivative" is an immune-derived moiety, i.e.,
a molecule that is derived from an antibody peptide or from nucleic
acid encoding an antibody peptide. This includes any antibody (Ab)
or immunoglobulin (Ig), and refers to any form of a peptide,
polypeptide derived from, modeled after or encoded by, an
immunoglobulin gene, or a fragment of such peptide or polypeptide
that is capable of binding an antigen or epitope. This comprehends,
for example, antibody variants, antibody fragments, chimeric
antibodies, humanized antibodies, multivalent antibodies, antibody
conjugates and the like, which retain a desired level of binding
activity for antigen.
[0046] As used herein, "antibody fragment" refers to a portion of
an intact antibody that includes the antigen binding site or
variable regions of an intact antibody, wherein the portion can be
free of the constant heavy chain domains (e.g., CH2, CH3, and CH4)
of the Fc region of the intact antibody. Alternatively, portions of
the constant heavy chain domains (e.g., CH2, CH3, and CH4) can be
included in the "antibody fragment". Antibody fragments retain
antigen-binding and include Fab, Fab', F(ab').sub.2, Fd, and Fv
fragments; diabodies; triabodies; single-chain antibody molecules
(sc-Fv); minibodies, nanobodies, and multispecific antibodies
formed from antibody fragments. Papain digestion of antibodies
produces two identical antigen-binding fragments, called "Fab"
fragments, each with a single antigen-binding site, and a residual
"Fc" fragment, whose name reflects its ability to crystallize
readily. Pepsin treatment yields an F(ab').sub.2 fragment that has
two antigen-combining sites and is still capable of cross-linking
antigen. By way of example, a Fab fragment also contains the
constant domain of a light chain and the first constant domain
(CH1) of a heavy chain. "Fv" is the minimum antibody fragment that
contains a complete antigen-recognition and -binding site. This
region consists of a dimer of one heavy chain and one light chain
variable domain in tight, non-covalent association. It is in this
configuration that the three hypervariable regions of each variable
domain interact to define an antigen-binding site on the surface of
the V.sub.H-V.sub.L dimer. Collectively, the six hypervariable
regions confer antigen-binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising
only three hypervariable regions specific for an antigen) has the
ability to recognize and bind antigen, although at a lower affinity
than the entire binding site. "Single-chain Fv" or "sFv" antibody
fragments comprise the V.sub.H and V.sub.L domains of antibody,
wherein these domains are present in a single polypeptide chain.
Generally, the Fv polypeptide further comprises a polypeptide
linker between the V.sub.H and V.sub.L domains that enables the sFv
to form the desired structure for antigen binding. For a review of
sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies,
vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp.
269-315 (1994).
[0047] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxyl terminus of the heavy chain CH1 domain
including one or more cysteine(s) from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0048] An "antibody variant," in this case generally an anti-LPA
antibody variant, refers herein to a molecule which differs in
amino acid sequence from a native anti-LPA antibody amino acid
sequence by virtue of addition, deletion and/or substitution of one
or more amino acid residue(s) in the antibody sequence and which
retains at least one desired activity of the parent anti-binding
antibody. Desired activities can include the ability to bind the
antigen specifically, the ability to inhibit proliferation in
vitro, the ability to inhibit angiogenesis in vivo, and the ability
to alter cytokine profile in vitro. The amino acid change(s) in an
antibody variant may be within a variable region or a constant
region of a light chain and/or a heavy chain, including in the Fc
region, the Fab region, the CH.sub.1 domain, the CH.sub.2 domain,
the CH.sub.3 domain, and the hinge region. In one embodiment, the
variant comprises one or more amino acid substitution(s) in one or
more hypervariable region(s) of the parent antibody. For example,
the variant may comprise at least one, e.g. from about one to about
ten, and preferably from about two to about five, substitutions in
one or more hypervariable regions of the parent antibody.
Ordinarily, the variant will have an amino acid sequence having at
least 50% amino acid sequence identity with the parent antibody
heavy or light chain variable domain sequences, more preferably at
least 65%, more preferably at 80%, more preferably at least 85%,
more preferably at least 90%, and most preferably at least 95%.
Identity or homology with respect to this sequence is defined
herein as the percentage of amino acid residues in the candidate
sequence that are identical with the parent antibody residues,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity. None of N-terminal,
C-terminal, or internal extensions, deletions, or insertions into
the antibody sequence shall be construed as affecting sequence
identity or homology. The variant retains the ability to bind LPA
and preferably has desired activities which are superior to those
of the parent antibody. For example, the variant may have a
stronger binding affinity, enhanced ability to reduce angiogenesis
and/or halt tumor progression. To analyze such desired properties
(for example les immunogenic, longer half-life, enhanced stability,
enhanced potency), one should compare a Fab form of the variant to
a Fab form of the parent antibody or a full length form of the
variant to a full length form of the parent antibody, for example,
since it has been found that the format of the anti-sphingolipid
antibody impacts its activity in the biological activity assays
disclosed herein. The variant antibody of particular interest
herein can be one which displays at least about 10 fold, preferably
at least about % 5, 25, 59, or more of at least one desired
activity. The preferred variant is one that has superior
biophysical properties as measured in vitro or superior activities
biological as measured in vitro or in vivo when compared to the
parent antibody.
[0049] An "anti-LPA agent" refers to any therapeutic agent that
binds LPA, and includes antibodies, antibody variants,
antibody-derived molecules or non-antibody-derived moieties that
bind LPA and its variants. An "anti-LPA antibody" or an
"immune-derived moiety reactive against LPA" refers to any antibody
or antibody-derived molecule that binds LPA. As will be understood
from these definitions, antibodies or immune-derived moieties may
be polyclonal or monoclonal and may be generated through a variety
of means, and/or may be isolated from an animal, including a human
subject.
[0050] An "anti-S1P agent" refers to any therapeutic agent that
binds S1P, and includes antibodies, antibody variants,
antibody-derived molecules or non-antibody-derived moieties that
bind LPA and its variants.
[0051] An "anti-S1P antibody" or an "immune-derived moiety reactive
against S1P" refers to any antibody or antibody-derived molecule
that binds S1P. As will be understood from these definitions,
antibodies or immune-derived moieties may be polyclonal or
monoclonal and may be generated through a variety of means, and/or
may be isolated from an animal, including a human subject.
[0052] A "bioactive lipid" refers to a lipid signaling molecule.
Bioactive lipids are distinguished from structural lipids (e.g.,
membrane-bound phospholipids) in that they mediate extracellular
and/or intracellular signaling and thus are involved in controlling
the function of many types of cells by modulating differentiation,
migration, proliferation, secretion, survival, and other processes.
In vivo, bioactive lipids can be found in extracellular fluids,
where they can be complexed with other molecules, for example serum
proteins such as albumin and lipoproteins, or in "free" form, i.e.,
not complexed with another molecule species. As extracellular
mediators, some bioactive lipids alter cell signaling by activating
membrane-bound ion channels or GPCRs or enzymes or factors that, in
turn, activate complex signaling systems that result in changes in
cell function or survival. As intracellular mediators, bioactive
lipids can exert their actions by directly interacting with
intracellular components such as enzymes, ion channels or
structural elements such as actin.
[0053] Examples of bioactive lipids include sphingolipids such as
ceramide, ceramide-1-phosphate (C1P), sphingosine, sphinganine,
sphingosylphosphorylcholine (SPC) and sphingosine-1-phosphate
(S1P). Sphingolipids and their derivatives and metabolites are
characterized by a sphingoid backbone (derived from sphingomyelin).
Sphingolipids and their derivatives and metabolites represent a
group of extracellular and intracellular signaling molecules with
pleiotropic effects on important cellular processes. They include
sulfatides, gangliosides and cerebrosides. Other bioactive lipids
are characterized by a glycerol-based backbone; for example,
lysophospholipids such as lysophosphatidyl choline (LPC) and
various lysophosphatidic acids (LPA), as well as
phosphatidylinositol (PI), phosphatidylethanolamine (PEA),
phosphatidic acid, platelet activating factor (PAF), cardiolipin,
phosphatidylglycerol (PG) and diacylglyceride (DG). Yet other
bioactive lipids are derived from arachidonic acid; these include
the eicosanoids (including the eicosanoid metabolites such as the
HETEs, cannabinoids, leukotrienes, prostaglandins, lipoxins,
epoxyeicosatrienoic acids, and isoeicosanoids), non-eicosanoid
cannabinoid mediators. Other bioactive lipids, including other
phospholipids and their derivatives, may also be used according to
the instant invention.
[0054] In some embodiments of the invention it may be preferable to
target glycerol-based bioactive lipids (those having a
glycerol-derived backbone, such as the LPAs) for antibody
production, as opposed to sphingosine-based bioactive lipids (those
having a sphingoid backbone, such as sphingosine and S1P). In other
embodiments it may be desired to target arachidonic acid-derived
bioactive lipids for antibody generation, and in other embodiments
arachidonic acid-derived and glycerol-derived bioactive lipids but
not sphingoid-derived bioactive lipids are preferred. Together the
arachidonic acid-derived and glycerol-derived bioactive lipids may
be referred to in the context of this invention as "non-sphingoid
bioactive lipids."
[0055] Specifically excluded from the class of bioactive lipids
according to the invention are phosphatidylcholine and
phosphatidylserine, as well as their metabolites and derivatives
that function primarily as structural members of the inner and/or
outer leaflet of cellular membranes.
[0056] The term "biologically active," in the context of an
antibody or antibody fragment or variant, refers to an antibody or
antibody fragment or antibody variant that is capable of binding
the desired epitope and in some ways exerting a biologic effect.
Biological effects include, but are not limited to, the modulation
of a growth signal, the modulation of an anti-apoptotic signal, the
modulation of an apoptotic signal, the modulation of the effector
function cascade, and modulation of other ligand interactions.
[0057] A "biomarker" is a specific biochemical in the body which
has a particular molecular feature that makes it useful for
measuring the progress of disease or the effects of treatment. For
example, S1P is a biomarker for certain hyperproliferative and/or
cardiovascular conditions.
[0058] The term "cardiotherapeutic agent" refers to an agent that
is therapeutic to diseases and diseases caused by or associated
with cardiac and myocardial diseases and disorders.
[0059] "Cardiovascular therapy" encompasses cardiac therapy
(treatment of myocardial ischemia and/or heart failure) as well as
the prevention and/or treatment of other diseases associated with
the cardiovascular system, such as heart disease. The term "heart
disease" encompasses any type of disease, disorder, trauma or
surgical treatment that involves the heart or myocardial tissue. Of
particular interest are conditions associated with tissue
remodeling. The term "cardiotherapeutic agent" refers to an agent
that is therapeutic to diseases and diseases caused by or
associated with cardiac and myocardial diseases and disorders.
[0060] A "carrier" refers to a moiety adapted for conjugation to a
hapten, thereby rendering the hapten immunogenic. A representative,
non-limiting class of carriers is proteins, examples of which
include albumin, keyhole limpet hemocyanin, hemaglutanin, tetanus,
and diptheria toxoid. Other classes and examples of carriers
suitable for use in accordance with the invention are known in the
art. These, as well as later discovered or invented naturally
occurring or synthetic carriers, can be adapted for application in
accordance with the invention.
[0061] As used herein, the expressions "cell," "cell line," and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived there
from without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Mutant progeny
that have the same function or biological activity as screened for
in the originally transformed cell are included. Where distinct
designations are intended, it will be clear from the context.
[0062] "Cerebrovascular therapy" refers to therapy directed to the
prevention and/or treatment of diseases and disorders associated
with cerebral ischemia and/or hypoxia. Of particular interest is
cerebral ischemia and/or hypoxia resulting from global ischemia
resulting from a heart disease, including without limitation heart
failure.
[0063] The term "chemotherapeutic agent" means anti-cancer and
other anti-hyperproliferative agents. Thus chemotherapeutic agents
are a subset of therapeutic agents in general. Chemotherapeutic
agents include, but are not limited to: DNA damaging agents and
agents that inhibit DNA synthesis: anthracyclines (doxorubicin,
donorubicin, epirubicin), alkylating agents (bendamustine,
busulfan, carboplatin, carmustine, chlorambucil, cyclophosphamide,
dacarbazine, hexamethylmelamine, ifosphamide, lomustine,
mechlorethamine, melphalan, mitotane, mytomycin, pipobroman,
procarbazine, streptozocin, thiotepa, and triethylenemelamine),
platinum derivatives (cisplatin, carboplatin, cis
diammine-dichloroplatinum), and topoisomerase inhibitors
(Camptosar); anti-metabolites such as capecitabine,
chlorodeoxyadenosine, cytarabine (and its activated form, ara-CMP),
cytosine arabinoside, dacabazine, floxuridine, fludarabine,
5-fluorouracil, 5-DFUR, gemcitabine, hydroxyurea, 6-mercaptopurine,
methotrexate, pentostatin, trimetrexate, 6-thioguanine);
anti-angiogenics (bevacizumab, thalidomide, sunitinib,
lenalidomide, TNP-470, 2-methoxyestradiol, ranibizumab, sorafenib,
erlotinib, bortezomib, pegaptanib, endostatin); vascular disrupting
agents (flavonoids/flavones, DMXAA, combretastatin derivatives such
as CA4DP, ZD6126, AVE8062A, etc.); biologics such as antibodies
(Herceptin, Avastin, Panorex, Rituxin, Zevalin, Mylotarg, Campath,
Bexxar, Erbitux); endocrine therapy: aromatase inhibitors
(4-hydroandrostendione, exemestane, aminoglutehimide, anastrazole,
letozole), anti-estrogens (Tamoxifen, Toremifine, Raoxifene,
Faslodex), steroids such as dexamethasone; immuno-modulators:
cytokines such as IFN-beta and IL2), inhibitors to integrins, other
adhesion proteins and matrix metalloproteinases); histone
deacetylase inhibitors like suberoylanilide hydroxamic acid;
inhibitors of signal transduction such as inhibitors of tyrosine
kinases like imatinib (Gleevec); inhibitors of heat shock proteins
like 17-N-allylamino-17-demethoxygeldanamycin; retinoids such as
all trans retinoic acid; inhibitors of growth factor receptors or
the growth factors themselves; anti-mitotic compounds and/or
tubulin-depolymerizing agents such as the taxoids (paclitaxel,
docetaxel, taxotere, BAY 59-8862), navelbine, vinblastine,
vincristine, vindesine and vinorelbine; anti-inflammatories such as
COX inhibitors and cell cycle regulators, e.g., check point
regulators and telomerase inhibitors.
[0064] The term "chimeric" antibody (or immunoglobulin) refers to a
molecule comprising a heavy and/or light chain which is identical
with or homologous to corresponding sequences in antibodies derived
from a particular species or belonging to a particular antibody
class or subclass, while the remainder of the chain(s) is identical
with or homologous to corresponding sequences in antibodies derived
from another species or belonging to another antibody class or
subclass, as well as fragments of such antibodies, so long as they
exhibit the desired biological activity (Cabilly, et al., infra;
Morrison et al., Proc. Natl. Acad. Sci. U.S.A., vol. 81:6851
(1984)).
[0065] The term "combination therapy" refers to a therapeutic
regimen that involves the provision of at least two distinct
therapies to achieve an indicated therapeutic effect. For example,
a combination therapy may involve the administration of two or more
chemically distinct active ingredients, for example, a fast-acting
chemotherapeutic agent and an anti-lipid antibody, or two different
antibodies. Alternatively, a combination therapy may involve the
administration of an anti-lipid antibody together with the delivery
of another treatment, such as radiation therapy and/or surgery.
Further, a combination therapy may involve administration of an
anti-lipid antibody together with one or more other biological
agents (e.g., anti-VEGF, TGF.beta., PDGF, or bFGF agent),
chemotherapeutic agents and another treatment such as radiation
and/or surgery. In the context of the administration of two or more
chemically distinct active ingredients, it is understood that the
active ingredients may be administered as part of the same
composition or as different compositions. When administered as
separate compositions, the compositions comprising the different
active ingredients may be administered at the same or different
times, by the same or different routes, using the same of different
dosing regimens, all as the particular context requires and as
determined by the attending physician. Similarly, when one or more
anti-lipid antibody species, for example, an anti-LPA antibody,
alone or in conjunction with one or more chemotherapeutic agents
are combined with, for example, radiation and/or surgery, the
drug(s) may be delivered before or after surgery or radiation
treatment.
[0066] The term "constant domain" refers to the C-terminal region
of an antibody heavy or light chain. Generally, the constant
domains are not directly involved in the binding properties of an
antibody molecule to an antigen, but exhibit various effector
functions, such as participation of the antibody in
antibody-dependent cellular toxicity. Here, "effector functions"
refer to the different physiological effects of antibodies (e.g.,
opsonization, cell lysis, mast cell, basophil and eosinophil
degranulation, and other processes) mediated by the recruitment of
immune cells by the molecular interaction between the Fc domain and
proteins of the immune system. The isotype of the heavy chain
determines the functional properties of the antibody. Their
distinctive functional properties are conferred by the
carboxy-terminal portions of the heavy chains, where they are not
associated with light chains.
[0067] The expression "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0068] A "derivatized bioactive lipid" is a bioactive lipid, e.g.,
LPA, which has a polar head group and at least one hydrocarbon
chain, wherein a carbon atom within the hydrocarbon chain is
derivatized with a reactive group [e.g., a sulfhydryl (thiol)
group, a carboxylic acid group, a cyano group, an ester, a hydroxy
group, an alkene, an alkyne, an acid chloride group or a halogen
atom] that may or may not be protected. This derivatization serves
to activate the bioactive lipid for reaction with a molecule, e.g.,
for conjugation to a carrier.
[0069] A "derivatized bioactive lipid conjugate" refers to a
derivatized bioactive lipid that is covalently conjugated to a
carrier. The carrier may be a protein molecule or may be a moiety
such as polyethylene glycol, colloidal gold, adjuvants or silicone
beads. A derivatized bioactive lipid conjugate may be used as an
immunogen for generating an antibody response according to the
instant invention, and the same or a different bioactive lipid
conjugate may be used as a detection reagent for detecting the
antibody thus produced. In some embodiments the derivatized
bioactive lipid conjugate is attached to a solid support when used
for detection.
[0070] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains are forced to pair with
the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA 90:6444-6448 (1993).
[0071] "Effective concentration" refers to the absolute, relative,
and/or available concentration and/or activity, for example of
certain undesired bioactive lipids. In other words, the effective
concentration of a bioactive lipid is the amount of lipid
available, and able, to perform its biological function. In the
present invention, an immune-derived moiety such as, for example, a
monoclonal antibody directed to a bioactive lipid (such as, for
example, C1P) is able to reduce the effective concentration of the
lipid in at least one fluid or milieu, possibly a physiological
fluid or milieu, by binding to the lipid and rendering it unable to
perform its biological function. In this example, the lipid itself
is still present (it is not degraded by the antibody, in other
words) but can no longer bind its receptor or other targets to
cause a downstream effect, so "effective concentration" rather than
absolute concentration is the appropriate measurement. Methods and
assays exist for directly and/or indirectly measuring the effective
concentration of bioactive lipids.
[0072] An "epitope" or "antigenic determinant" refers to that
portion of an antigen that reacts with an antibody antigen-binding
portion derived from an antibody.
[0073] The term "expression cassette" refers to a nucleotide
molecule capable of affecting expression of a structural gene
(i.e., a protein coding sequence, such as an antibody of the
invention) in a host compatible with such sequences. Expression
cassettes include at least a promoter operably linked with the
polypeptide-coding sequence, and, optionally, with other sequences,
e.g., transcription termination signals. Additional regulatory
elements necessary or helpful in effecting expression may also be
used, e.g., enhancers. Thus, expression cassettes include plasmids,
expression vectors, recombinant viruses, any form of recombinant
"naked DNA" vector, and the like.
[0074] A "fully human antibody" can refer to an antibody produced
in a genetically engineered (i.e., transgenic) mouse (e.g. from
Medarex) that, when presented with an immunogen, can produce a
human antibody that does not necessarily require CDR grafting.
These antibodies are fully human (100% human protein sequences)
from animals such as mice in which the non-human antibody genes are
suppressed and replaced with human antibody gene expression. The
applicants believe that antibodies could be generated against
bioactive lipids when presented to these genetically engineered
mice or other animals who might be able to produce human frameworks
for the relevant CDRs.
[0075] A "hapten" is a substance that is non-immunogenic but can
react with an antibody or antigen-binding portion derived from an
antibody. In other words, haptens have the property of antigenicity
but not immunogenicity. A hapten is generally a small molecule that
can, under most circumstances, elicit an immune response (i.e., act
as an antigen) only when attached to a carrier, for example, a
protein, polyethylene glycol (PEG), colloidal gold, silicone beads,
or the like. The carrier may be one that also does not elicit an
immune response by itself. A representative, non-limiting class of
hapten molecules is proteins, examples of which include albumin,
keyhole limpet hemocyanin, hemaglutanin, tetanus, and diphtheria
toxoid. Other classes and examples of hapten molecules are known in
the art. These, as well as later discovered or invented naturally
occurring or synthetic haptens, can be adapted for application in
accordance with the invention.
[0076] The term "heteroconjugate antibody" can refer to two
covalently joined antibodies. Such antibodies can be prepared using
known methods in synthetic protein chemistry, including using
crosslinking agents. As used herein, the term "conjugate" refers to
molecules formed by the covalent attachment of one or more antibody
fragment(s) or binding moieties to one or more polymer
molecule(s).
[0077] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. Or, looked at another way, a humanized
antibody is a human antibody that also contains selected sequences
from non-human (e.g., murine) antibodies in place of the human
sequences. A humanized antibody can include conservative amino acid
substitutions or non-natural residues from the same or different
species that do not significantly alter its binding and/or biologic
activity. Such antibodies are chimeric antibodies that contain
minimal sequence derived from non-human immunoglobulins. For the
most part, humanized antibodies are human immunoglobulins
(recipient antibody) in which residues from a
complementary-determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat, camel, bovine, goat, or rabbit having
the desired properties. In some instances, framework region (FR)
residues of the human immunoglobulin are replaced by corresponding
non-human residues.
[0078] Furthermore, humanized antibodies can comprise residues that
are found neither in the recipient antibody nor in the imported CDR
or framework sequences. These modifications are made to further
refine and maximize antibody performance. Thus, in general, a
humanized antibody will comprise all of at least one, and in one
aspect two, variable domains, in which all or all of the
hypervariable loops correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin sequence. The humanized antibody
optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), or that of a human
immunoglobulin. See, e.g., Cabilly, et al., U.S. Pat. No.
4,816,567; Cabilly, et al., European Patent No. 0,125,023 B1; Boss,
et al., U.S. Pat. No. 4,816,397; Boss, et al., European Patent No.
0,120,694 B1; Neuberger, et al., WO 86/01533; Neuberger, et al.,
European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539;
Winter, European Patent No. 0,239,400 B1; Padlan, et al., European
Patent Application No. 0,519,596 A1; Queen, et al. (1989), Proc.
Nat'l Acad. Sci. USA, vol. 86:10029-10033). For further details,
see Jones et al., Nature 321:522-525 (1986); Reichmann et al.,
Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.
2:593-596 (1992) and Hansen, WO2006105062.
[0079] The term "hyperproliferative disorder" refers to diseases
and disorders associated with, the uncontrolled proliferation of
cells, including but not limited to uncontrolled growth of organ
and tissue cells resulting in cancers and benign tumors.
Hyperproliferative disorders associated with endothelial cells can
result in diseases of angiogenesis such as angiomas, endometriosis,
obesity, age-related macular degeneration and various
retinopathies, as well as the proliferation of endothelial cells
and smooth muscle cells that cause restenosis as a consequence of
stenting in the treatment of atherosclerosis. Hyperproliferative
disorders involving fibroblasts (i.e., fibrogenesis) include but
are not limited to disorders of excessive scarring (i.e., fibrosis)
such as age-related macular degeneration, cardiac remodeling and
failure associated with myocardial infarction, excessive wound
healing such as commonly occurs as a consequence of surgery or
injury, keloids, and fibroid tumors and stenting.
[0080] An "immune-derived moiety" includes any antibody (Ab) or
immunoglobulin (Ig), and refers to any form of a peptide,
polypeptide derived from, modeled after or encoded by, an
immunoglobulin gene, or a fragment of such peptide or polypeptide
that is capable of binding an antigen or epitope (see, e.g.,
Immunobiology, 5th Edition, Janeway, Travers, Walport, Shlomchiked.
(editors), Garland Publishing (2001)). In the present invention,
the antigen is a lipid molecule, such as a bioactive lipid
molecule.
[0081] An "immunogen" is a molecule capable of inducing a specific
immune response, particularly an antibody response in an animal to
whom the immunogen has been administered. In the instant invention,
the immunogen is a derivatized bioactive lipid conjugated to a
carrier, i.e., a "derivatized bioactive lipid conjugate". The
derivatized bioactive lipid conjugate used as the immunogen may be
used as capture material for detection of the antibody generated in
response to the immunogen. Thus the immunogen may also be used as a
detection reagent. Alternatively, the derivatized bioactive lipid
conjugate used as capture material may have a different linker
and/or carrier moiety from that in the immunogen.
[0082] The phrase "in silico" refers to computer simulations that
model natural or laboratory processes
[0083] To "inhibit," particularly in the context of a biological
phenomenon, means to decrease, suppress or delay. For example, a
treatment yielding "inhibition of tumorigenesis" may mean that
tumors do not form at all, or that they form more slowly, or are
fewer in number than in the untreated control.
[0084] An "isolated" antibody is one that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0085] The word "label" when used herein refers to a detectable
compound or composition, such as one that is conjugated directly or
indirectly to the antibody. The label may itself be detectable by
itself (e.g., radioisotope labels or fluorescent labels) or, in the
case of an enzymatic label, may catalyze chemical alteration of a
substrate compound or composition that is detectable.
[0086] A "ligand" is a substance that is able to bind to and form a
complex with a biomolecule to serve a biological purpose. Thus an
antigen may be described as a ligand of the antibody to which it
binds.
[0087] A "liposome" is a small vesicle composed of various types of
lipids, phospholipids and/or surfactant that is useful for delivery
of a drug (such as the anti-sphingolipid antibodies disclosed
herein and, optionally, a chemotherapeutic agent) to a mammal. The
components of the liposome are commonly arranged in a bilayer
formation, similar to the lipid arrangement of biological
membranes. An "isolated" nucleic acid molecule is a nucleic acid
molecule that is identified and separated from at least one
contaminant nucleic acid molecule with which it is ordinarily
associated in the natural source of the antibody nucleic acid. An
isolated nucleic acid molecule is other than in the form or setting
in which it is found in nature. Isolated nucleic acid molecules
therefore are distinguished from the nucleic acid molecule as it
exists in natural cells. However, an isolated nucleic acid molecule
includes a nucleic acid molecule contained in cells that ordinarily
express the antibody where, for example, the nucleic acid molecule
is in a chromosomal location different from that of natural
cells.
[0088] In the context of this invention, a "liquid composition"
refers to one that, in its filled and finished form as provided
from a manufacturer to an end user (e.g., a doctor or nurse), is a
liquid or solution, as opposed to a solid. Here, "solid" refers to
compositions that are not liquids or solutions. For example, solids
include dried compositions prepared by lyophilization,
freeze-drying, precipitation, and similar procedures.
[0089] The expression "linear antibodies" when used throughout this
application refers to the antibodies described in Zapata et al.
Protein Eng. 8(10):1057-1062 (1995). Briefly, these antibodies
comprise a pair of tandem Fd segments
(V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) that form a pair of antigen
binding regions. Linear antibodies can be bispecific or
monospecific.
[0090] The term "metabolites" refers to compounds from which a
given bioactive lipid is made, as well as those that result from
the degradation of the bioactive lipid; that is, compounds that are
involved in the respective metabolic pathways of each bioactive
lipid. The term "metabolic precursors" may be used to refer to
compounds from which each bioactive lipid is made.
[0091] The term "monoclonal antibody" (mAb) as used herein refers
to an antibody obtained from a population of substantially
homogeneous antibodies, or to said population of antibodies. The
individual antibodies comprising the population are essentially
identical, except for possible naturally occurring mutations that
may be present in minor amounts. Monoclonal antibodies are highly
specific, being directed against a single antigenic site.
Furthermore, in contrast to conventional (polyclonal) antibody
preparations that typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. The
modifier "monoclonal" indicates the character of the antibody as
being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler et al.,
Nature 256:495 (1975), or may be made by recombinant DNA methods
(see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the
techniques described in Clackson et al., Nature 352:624-628 (1991)
and Marks et al., J. Mol. Biol. 222:581-597 (1991), for example, or
by other methods known in the art. The monoclonal antibodies herein
specifically include chimeric antibodies in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (U.S. Pat. No. 4,816,567; and Morrison
et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
[0092] "Monotherapy" refers to a treatment regimen based on the
delivery of one therapeutically effective compound, whether
administered as a single dose or several doses over time.
[0093] The term "multispecific antibody" can refer to an antibody,
or a monoclonal antibody, having binding properties for at least
two different epitopes. In one embodiment, the epitopes are from
the same antigen. In another embodiment, the epitopes are from two
or more different antigens. Methods for making multispecific
antibodies are known in the art. Multispecific antibodies include
bispecific antibodies (having binding properties for two epitopes),
trispecific antibodies (three epitopes) and so on. For example,
multispecific antibodies can be produced recombinantly using the
co-expression of two or more immunoglobulin heavy chain/light chain
pairs. Alternatively, multispecific antibodies can be prepared
using chemical linkage. One of skill can produce multispecific
antibodies using these or other methods as may be known in the art.
Multispecific antibodies include multispecific antibody fragments.
One example of a multispecific (in this case, bispecific) antibody
comprehended by this invention is an antibody having binding
properties for an S1P epitope and a C1P epitope, which thus is able
to recognize and bind to both S1P and C1P. Another example of of a
bispecific antibody comprehended by this invention is an antibody
having binding properties for an epitope from a bioactive lipid and
an epitope from a cell surface antigen. Thus the antibody is able
to recognize and bind the bioactive lipid and is able to recognize
and bind to cells, e.g., for targeting purposes.
[0094] "Neoplasia" or "cancer" refers to abnormal and uncontrolled
cell growth. A "neoplasm", or tumor or cancer, is an abnormal,
unregulated, and disorganized proliferation of cell growth, and is
generally referred to as cancer. A neoplasm may be benign or
malignant. A neoplasm is malignant, or cancerous, if it has
properties of destructive growth, invasiveness, and metastasis.
Invasiveness refers to the local spread of a neoplasm by
infiltration or destruction of surrounding tissue, typically
breaking through the basal laminas that define the boundaries of
the tissues, thereby often entering the body's circulatory system.
Metastasis typically refers to the dissemination of tumor cells by
lymphatics or blood vessels. Metastasis also refers to the
migration of tumor cells by direct extension through serous
cavities, or subarachnoid or other spaces. Through the process of
metastasis, tumor cell migration to other areas of the body
establishes neoplasms in areas away from the site of initial
appearance.
[0095] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0096] The "parent" antibody herein is one that is encoded by an
amino acid sequence used for the preparation of the variant. The
parent antibody may be a native antibody or may already be a
variant, e.g., a chimeric antibody. For example, the parent
antibody may be a humanized or human antibody.
[0097] A "patentable" composition, process, machine, or article of
manufacture according to the invention means that the subject
matter satisfies all statutory requirements for patentability at
the time the analysis is performed. For example, with regard to
novelty, non-obviousness, or the like, if later investigation
reveals that one or more claims encompass one or more embodiments
that would negate novelty, non-obviousness, etc., the claim(s),
being limited by definition to "patentable" embodiments,
specifically exclude the non-patentable embodiment(s). Also, the
claims appended hereto are to be interpreted both to provide the
broadest reasonable scope, as well as to preserve their validity.
Furthermore, the claims are to be interpreted in a way that (1)
preserves their validity and (2) provides the broadest reasonable
interpretation under the circumstances, if one or more of the
statutory requirements for patentability are amended or if the
standards change for assessing whether a particular statutory
requirement for patentability is satisfied from the time this
application is filed or issues as a patent to a time the validity
of one or more of the appended claims is questioned.
[0098] The term "pharmaceutically acceptable salt" refers to a
salt, such as used in formulation, which retains the biological
effectiveness and properties of the agents and compounds of this
invention and which are is biologically or otherwise undesirable.
In many cases, the agents and compounds of this invention are
capable of forming acid and/or base salts by virtue of the presence
of charged groups, for example, charged amino and/or carboxyl
groups or groups similar thereto. Pharmaceutically acceptable acid
addition salts may be prepared from inorganic and organic acids,
while pharmaceutically acceptable base addition salts can be
prepared from inorganic and organic bases. For a review of
pharmaceutically acceptable salts (see Berge, et al. (1977) J.
Pharm. Sci., vol. 66, 1-19).
[0099] A "plurality" means more than one.
[0100] The term "promoter" includes all sequences capable of
driving transcription of a coding sequence in a cell. Thus,
promoters used in the constructs of the invention include
cis-acting transcriptional control elements and regulatory
sequences that are involved in regulating or modulating the timing
and/or rate of transcription of a gene. For example, a promoter can
be a cis-acting transcriptional control element, including an
enhancer, a promoter, a transcription terminator, an origin of
replication, a chromosomal integration sequence, 5' and 3'
untranslated regions, or an intronic sequence, which are involved
in transcriptional regulation. Transcriptional regulatory regions
suitable for use in the present invention include but are not
limited to the human cytomegalovirus (CMV) immediate-early
enhancer/promoter, the SV40 early enhancer/promoter, the E. coli
lac or trp promoters, and other promoters known to control
expression of genes in prokaryotic or eukaryotic cells or their
viruses.
[0101] The term "recombinant DNA" refers to nucleic acids and gene
products expressed therefrom that have been engineered, created, or
modified by man. "Recombinant" polypeptides or proteins are
polypeptides or proteins produced by recombinant DNA techniques,
for example, from cells transformed by an exogenous DNA construct
encoding the desired polypeptide or protein. "Synthetic"
polypeptides or proteins are those prepared by chemical
synthesis.
[0102] The terms "separated", "purified", "isolated", and the like
mean that one or more components of a sample contained in a
sample-holding vessel are or have been physically removed from, or
diluted in the presence of, one or more other sample components
present in the vessel. Sample components that may be removed or
diluted during a separating or purifying step include, chemical
reaction products, non-reacted chemicals, proteins, carbohydrates,
lipids, and unbound molecules.
[0103] By "solid phase" is meant a non-aqueous matrix such as one
to which the antibody of the present invention can adhere. Examples
of solid phases encompassed herein include those formed partially
or entirely of glass (e.g. controlled pore glass), polysaccharides
(e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol
and silicones. In certain embodiments, depending on the context,
the solid phase can comprise the well of an assay plate; in others
it is a purification column (e.g. an affinity chromatography
column). This term also includes a discontinuous solid phase of
discrete particles, such as those described in U.S. Pat. No.
4,275,149.
[0104] The term "species" is used herein in various contexts, e.g.,
a particular species of chemotherapeutic agent. In each context,
the term refers to a population of chemically indistinct molecules
of the sort referred in the particular context.
[0105] The term "specific" or "specificity" in the context of
antibody-antigen interactions refers to the selective, non-random
interaction between an antibody and its target epitope. Here, the
term "antigen" refers to a molecule that is recognized and bound by
an antibody molecule or other immune-derived moiety. The specific
portion of an antigen that is bound by an antibody is termed the
"epitope". This interaction depends on the presence of structural,
hydrophobic/hydrophilic, and/or electrostatic features that allow
appropriate chemical or molecular interactions between the
molecules. Thus an antibody is commonly said to "bind" (or
"specifically bind") or be "reactive with" (or "specifically
reactive with), or, equivalently, "reactive against" (or
"specifically reactive against") the epitope of its target antigen.
Antibodies are commonly described in the art as being "against" or
"to" their antigens as shorthand for antibody binding to the
antigen. Thus an "antibody that binds C1P," an "antibody reactive
against C1P," an "antibody reactive with C1P," an "antibody to C1P"
and an "anti-C1P antibody" all have the same meaning in the art.
Antibody molecules can be tested for specificity of binding by
comparing binding to the desired antigen to binding to unrelated
antigen or analogue antigen or antigen mixture under a given set of
conditions. Preferably, an antibody according to the invention will
lack significant binding to unrelated antigens, or even analogs of
the target antigen. "Specifically associate" and "specific
association" and the like refer to a specific, non-random
interaction between two molecules, which interaction depends on the
presence of structural, hydrophobic/hydrophilic, and/or
electrostatic features that allow appropriate chemical or molecular
interactions between the molecules.
[0106] The term "sphingolipid" as used herein refers to the class
of compounds in the art known as sphingolipids, including, but not
limited to the following compounds (see http//www.lipidmaps.org for
chemical formulas, structural information, etc. for the
corresponding compounds):
[0107] Sphingoid bases [SP01] [0108] Sphing-4-enines (Sphingosines)
[SP0101] [0109] Sphinganines [SP0102] [0110] 4-Hydroxysphinganines
(Phytosphingosines) [SP0103] [0111] Sphingoid base homologs and
variants [SP0104] [0112] Sphingoid base 1-phosphates [SP0105]
[0113] Lysosphingomyelins and lysoglycosphingolipids [SP0106]
[0114] N-methylated sphingoid bases [SP0107] [0115] Sphingoid base
analogs [SP0108]
[0116] Ceramides [SP02] [0117] N-acylsphingosines (ceramides)
[SP0201] [0118] N-acylsphinganines (dihydroceramides) [SP0202]
[0119] N-acyl-4-hydroxysphinganines (phytoceramides) [SP0203]
[0120] Acylceramides [SP0204] [0121] Ceramide 1-phosphates
[SP0205]
[0122] Phosphosphingolipids [SP03] [0123] Ceramide phosphocholines
(sphingomyelins) [SP0301] [0124] Ceramide phosphoethanolamines
[SP0302] [0125] Ceramide phosphoinositols [SP0303]
[0126] Phosphonosphingolipids [SP04]
[0127] Neutral glycosphingolipids [SP05] [0128] Simple Glc series
(GlcCer, LacCer, etc) [SP0501] [0129] GalNAcb1-3Gala1-4Galb1-4Glc-
(Globo series) [SP0502] [0130] GalNAcb1-4Galb1-4Glc- (Ganglio
series) [SP0503] [0131] Galb1-3GlcNAcb1-3Galb1-4Glc- (Lacto series)
[SP0504] [0132] Galb1-4GlcNAcb1-3Galb1-4Glc- (Neolacto series)
[SP0505] [0133] GalNAcb1-3Gala1-3Galb1-4Glc- (Isoglobo series)
[SP0506] [0134] GlcNAcb1-2Mana1-3Manb1-4Glc- (Mollu series)
[SP0507] [0135] GalNAcb1-4GlcNAcb1-3Manb1-4Glc- (Arthro series)
[SP0508] [0136] Gal- (Gala series) [SP0509] [0137] Other
[SP0510]
[0138] Acidic glycosphingolipids [S1P06] [0139] Gangliosides
[SP0601] [0140] Sulfoglycosphingolipids (sulfatides) [SP0602]
[0141] Glucuronosphingolipids [SP0603] [0142]
Phosphoglycosphingolipids [SP0604] [0143] Other [SP0600]
[0144] Basic glycosphingolipids [SP07]
[0145] Amphoteric glycosphingolipids [SP08]
[0146] Arsenosphingolipids [SP09]
[0147] The term "sphingolipid metabolite" refers to a compound from
which a sphingolipid is made, as well as a that results from the
degradation of a particular sphingolipid. In other words, a
"sphingolipid metabolite" is a compound that is involved in the
sphingolipid metabolic pathways. Metabolites include metabolic
precursors and metabolic products. The term "metabolic precursors"
of sphingolipids refers to compounds from which sphingolipids are
made. Metabolic precursors of particular interest include but are
not limited to SPC, sphingomyelin, dihydrosphingosine,
dihydroceramide, and 3-ketosphinganine. The term "metabolic
products" refers to compounds that result from the degradation of
sphingolipids, such as phosphorylcholine (e.g., phosphocholine,
choline phosphate), fatty acids, including free fatty acids, and
hexadecanal (e.g., palmitaldehyde).
[0148] Herein, "stable" refers to an interaction between two
molecules (e.g., a peptide and a TLR molecule) that is sufficiently
stable such that the molecules can be maintained for the desired
purpose or manipulation. For example, a "stable" interaction
between a peptide and a TLR molecule refers to one wherein the
peptide becomes and remains associated with a TLR molecule for a
period sufficient to achieve the desired effect.
[0149] A "subject" or "patient" refers to an animal in need of
treatment that can be effected by molecules of the invention.
Animals that can be treated in accordance with the invention
include vertebrates, with mammals such as bovine, canine, equine,
feline, ovine, porcine, and primate (including humans and non-human
primates) animals being particularly preferred examples.
[0150] A "surrogate marker" refers to laboratory measurement of
biological activity within the body that indirectly indicates the
effect of treatment on disease state. Examples of surrogate markers
for hyperproliferative and/or cardiovascular conditions include
SPHK and/or S1PRs.
[0151] A "therapeutic agent" refers to a drug or compound that is
intended to provide a therapeutic effect including, but not limited
to: anti-inflammatory drugs including COX inhibitors and other
NSAIDS, anti-angiogenic drugs, chemotherapeutic drugs as defined
above, cardiovascular agents, immunomodulatory agents, agents that
are used to treat neurodegenerative disorders, opthalmic drugs,
anti-fibrotics, etc.
[0152] A "therapeutically effective amount" (or "effective amount")
refers to an amount of an active ingredient, e.g., an agent
according to the invention, sufficient to effect treatment when
administered to a subject in need of such treatment. Accordingly,
what constitutes a therapeutically effective amount of a
composition according to the invention may be readily determined by
one of ordinary skill in the art. In the context of cancer therapy,
a "therapeutically effective amount" is one that produces an
objectively measured change in one or more parameters associated
with cancer cell survival or metabolism, including an increase or
decrease in the expression of one or more genes correlated with the
particular cancer, reduction in tumor burden, cancer cell lysis,
the detection of one or more cancer cell death markers in a
biological sample (e.g., a biopsy and an aliquot of a bodily fluid
such as whole blood, plasma, serum, urine, etc.), induction of
induction apoptosis or other cell death pathways, etc. Of course,
the therapeutically effective amount will vary depending upon the
particular subject and condition being treated, the weight and age
of the subject, the severity of the disease condition, the
particular compound chosen, the dosing regimen to be followed,
timing of administration, the manner of administration and the
like, all of which can readily be determined by one of ordinary
skill in the art. It will be appreciated that in the context of
combination therapy, what constitutes a therapeutically effective
amount of a particular active ingredient may differ from what
constitutes a therapeutically effective amount of the active
ingredient when administered as a monotherapy (i.e., a therapeutic
regimen that employs only one chemical entity as the active
ingredient).
[0153] The compositions of the invention are used in methods of
bioactive lipid-based therapy. As used herein, the terms "therapy"
and "therapeutic" encompasses the full spectrum of prevention
and/or treatments for a disease, disorder or physical trauma. A
"therapeutic" agent of the invention may act in a manner that is
prophylactic or preventive, including those that incorporate
procedures designed to target individuals that can be identified as
being at risk (pharmacogenetics); or in a manner that is
ameliorative or curative in nature; or may act to slow the rate or
extent of the progression of at least one symptom of a disease or
disorder being treated; or may act to minimize the time required,
the occurrence or extent of any discomfort or pain, or physical
limitations associated with recuperation from a disease, disorder
or physical trauma; or may be used as an adjuvant to other
therapies and treatments.
[0154] The term "treatment" or "treating" means any treatment of a
disease or disorder, including preventing or protecting against the
disease or disorder (that is, causing the clinical symptoms not to
develop); inhibiting the disease or disorder (i.e., arresting,
delaying or suppressing the development of clinical symptoms;
and/or relieving the disease or disorder (i.e., causing the
regression of clinical symptoms). As will be appreciated, it is not
always possible to distinguish between "preventing" and
"suppressing" a disease or disorder because the ultimate inductive
event or events may be unknown or latent. Those "in need of
treatment" include those already with the disorder as well as those
in which the disorder is to be prevented. Accordingly, the term
"prophylaxis" will be understood to constitute a type of
"treatment" that encompasses both "preventing" and "suppressing".
The term "protection" thus includes "prophylaxis".
[0155] The term "therapeutic regimen" means any treatment of a
disease or disorder using chemotherapeutic and cytotoxic agents,
radiation therapy, surgery, gene therapy, DNA vaccines and therapy,
siRNA therapy, anti-angiogenic therapy, immunotherapy, bone marrow
transplants, aptamers and other biologics such as antibodies and
antibody variants, receptor decoys and other protein-based
therapeutics.
[0156] The "variable" region of an antibody comprises framework and
complementarity determining regions (CDRs, otherwise known as
hypervariable regions). The variability is not evenly distributed
throughout the variable domains of antibodies. It is concentrated
in six CDR segments, three in each of the light chain and the heavy
chain variable domains. The more highly conserved portions of
variable domains are called the framework region (FR). The variable
domains of native heavy and light chains each comprise four FRs
(FR1, FR2, FR3 and FR4, respectively), largely adopting a n-sheet
configuration, connected by three hypervariable regions, which form
loops connecting, and in some cases forming part of, the beta-sheet
structure. The term "hypervariable region" when used herein refers
to the amino acid residues of an antibody which are responsible for
antigen binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR" (for
example residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light
chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in
the heavy chain variable domain; Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) and/or those
residues from a "hypervariable loop" (for example residues 26-32
(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain
and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain
variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917
(1987)). "Framework" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined.
[0157] The hypervariable regions in each chain are held together in
close proximity by the FRs and, with the hypervariable regions from
the other chain, contribute to the formation of the antigen-binding
site of antibodies (see Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991), pages 647-669). The
constant domains are not involved directly in binding an antibody
to an antigen, but exhibit various effector functions, such as
participation of the antibody in antibody-dependent cellular
toxicity.
[0158] A "vector" or "plasmid" or "expression vector" refers to a
nucleic acid that can be maintained transiently or stably in a cell
to effect expression of one or more recombinant genes. A vector can
comprise nucleic acid, alone or complexed with other compounds. A
vector optionally comprises viral or bacterial nucleic acids and/or
proteins, and/or membranes. Vectors include, but are not limited,
to replicons (e.g., RNA replicons, bacteriophages) to which
fragments of DNA may be attached and become replicated. Thus,
vectors include, but are not limited to, RNA, autonomous
self-replicating circular or linear DNA or RNA and include both the
expression and non-expression plasmids. Plasmids can be
commercially available, publicly available on an unrestricted
basis, or can be constructed from available plasmids as reported
with published protocols. In addition, the expression vectors may
also contain a gene to provide a phenotypic trait for selection of
transformed host cells such as dihydrofolate reductase or neomycin
resistance for eukaryotic cell culture, or such as tetracycline or
ampicillin resistance in E. coli.
SUMMARY OF THE INVENTION
[0159] The instant application provides compositions and methods
relating to anti-LPA agents, particularly anti-LPA antibodies,
including humanized anti-LPA antibodies. Anti-LPA agents comprising
a variable domain having an amino acid sequence selected from a set
of selected sequences are provided, as are anti-LPA agents
comprising a heavy chain and a light chain, wherein each
immunoglobulin heavy chain comprises a variable domain having an
amino acid sequence selected from a set of selected heavy chain
variable domain sequences, and each immunoglobulin light chain
comprises a variable domain having an amino acid sequence selected
from a group of selected light chain sequences. In some embodiments
the anti-LPA agent comprises two immunoglobulin heavy chains and
two immunoglobulin light chains, wherein one or both of the heavy
chains comprise an amino acid sequence from a set of selected heavy
chain variable domain sequences, and wherein one or both of the
light chains comprise an amino acid sequence from a set of selected
light chain variable domain sequences. The two light chain variable
domain sequences may be the same or different, as may the two heavy
chain variable domain sequences.
[0160] The anti-LPA agent may be an antibody, an antibody
derivative, or a non-antibody-derived moiety, and may be a
full-length antibody or an antibody fragment.
[0161] The anti-LPA agent may be conjugated to a polymer, a
radionuclide, a chemotherapeutic agent, or a detection agent.
Pharmaceutical compositions comprising a pharmaceutically
acceptable carrier and an anti-LPA agent are also provided.
[0162] Further provided are isolated nucleic acid molecules that
encode an immunoglobulin heavy chain variable domain or an
immunoglobulin light chain variable domain that comprises an amino
acid sequence according specific sequences provided. Vectors and
host cells are also provided.
[0163] In addition, isolated polypeptides reactive with LPA in a
physiological context are provided; these comprise an amino acid
sequence that has a sequence identity of at least 65 percent with a
peptide amino acid sequence selected from a specified group. In
some embodiments thes polypeptides are fragments of a variable
domain of an animal immunoglobulin heavy or light chain, a full
length variable domain of an immunoglobulin heavy or light chain or
a full length immunoglobulin heavy or light chain.
[0164] Methods of administering an anti-LPA agent, isolated
polypeptide which specifically binds LPA or an isolated antibody
molecule are provided. Methods of treating or preventing a disease
or disorder correlated with elevated levels of LPA are also
provided, wherein a composition such as the antibodies, peptides or
anti-LPA agents of the invention are administered to a subject in
an amount effective to reduce in vivo the effective concentration
of LPA, thereby effecting treatment or prevention of the disease or
disorder. In some embodiments the disease or disorder may be
cancer, an inflammatory disorder, a cerebrovascular disease, a
cardiovascular disease, an ocular disorder, a disease and disorder
associated with excessive fibrogenesis, a disease or disorder
associated with metastasis, a disease or disorder associated with
tumor growth, and a disease or disorder associated with pathologic
angiogenesis, and the anti-LPA agent, isolated polypeptide,
isolated antibody or multivalent binding molecule may be
administered in combination with another therapeutic agent to
effect treatment or prevention of the disease or disorder. Also
provided are methods of decreasing the effective concentration of
LPA in a bodily fluid or tissue of a subject, including a human
subject.
[0165] Methods for detecting LPA or an LPA metabolite are also
provided, utilizing the compositions of the invention; these may be
attached to a solid support and the method may be an ELISA
assay.
BRIEF DESCRIPTION OF THE DRAWINGS
[0166] A brief summary of each of the figures and tables described
in this specification are provided below, as is a list of various
nucleotide and amino acid sequences described herein.
[0167] FIGS. 1a, 1b and 1c. Organic synthesis scheme for making of
a typical thiolated-S1P analog that was used as a key component of
an immunogen, as well as a key component of the laydown material
for the ELISA and BiaCore assays.
[0168] FIGS. 2a and 2b. Organic synthesis scheme for making the
thiolated-related fatty acid used in the synthesis of the
thiolated-LPA analog of FIG. 3.
[0169] FIGS. 3a and 3b. Organic synthesis scheme for making the
thiolated-LPA analog that is a key component of an immunogen, as
well as a key component of the laydown material for the ELISA and
other assays.
DETAILED DESCRIPTION OF THE INVENTION
[0170] A. Derivatized and/or Conjugated LPA
[0171] 1. Compositions
[0172] LPA can be derivatized in such a way as to facilitate the
immunogenic response (i.e., antibody production). In one
embodiment, the LPA may be derivatized in order to allow
conjugation of the LPA molecule to a carrier molecule. In one
embodiment, a carbon atom within the hydrocarbon chain of the LPA
is derivatized with a pendant reactive group [e.g., a sulfhydryl
(thiol) group, a carboxylic acid group, a cyano group, an ester, a
hydroxy group, an alkene, an alkyne, an acid chloride group or a
halogen atom] that may or may not be protected. This derivatization
serves to activate the bioactive lipid for reaction with a
molecule, e.g., for conjugation to a carrier. In one embodiment,
the derivatized LPA is thiolated LPA. In one embodiment, the
derivatized LPA is derivatized C12 or C18 LPA. In one embodiment,
the thiolated LPA is conjugated via a crosslinker, e.g., a
bifunctional crosslinker such as IOA or SMCC, to a carrier, which
may be a protein. It may be useful to conjugate the LPA in this way
to a protein or other carrier that is immunogenic in the species to
be immunized, e.g., keyhole limpet hemocyanin (KLH), serum albumin
(including bovine serum albumin or BSA), bovine thyroglobulin, or
soybean trypsin inhibitor, using a bifunctional or derivatizing
agent, for example, maleimidobenzoyl sulfosuccinimide ester
(conjugation through cysteine residues), N-hydroxysuccinimide
(through lysine residues), glutaraldehyde, succinic anhydride,
SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R and R.sup.1 are
different alkyl groups. Non-protein carriers (e.g., colloidal gold)
are also known in the art for use in antibody production.
[0173] The derivatized or derivatized and conjugated LPA may be
used to generate anti-LPA antibodies (polyclonal and/or
monoclonal). The derivatized or derivatized and conjugated LPA may
also be used in the methods of the invention, particularly in
diagnostic methods.
[0174] 2. Research and Diagnostic Uses for Derivatized LPA
[0175] The derivatized LPAs may be used to detect and/or purify
anti-LPA antibodies and may be conjugated to a carrier as described
above. The derivatives and conjugates are preferably conjugated to
a solid support for use in diagnostic methods, including clinical
diagnostic methods. For example, detection and/or quantitation of
LPA antibodies may be used in diagnosing various medical conditions
in LPA plays a role. Quantitation of LPA antibodies is also useful
in a clinical setting to evaluate dosing, halflife and drug levels
after treatment with, e.g., an anti-LPA antibody such as LT3000
described herein.
[0176] In one embodiment, the derivatized LPA conjugate (e.g.,
thiolated LPA conjugated to BSA or KLH) is used as laydown material
in ELISAs which are used to detect anti-LPA antibodies. In one
embodiment the LPA is thiolated C12 LPA or thiolated C18 LPA
conjugated to BSA. This embodiment is useful, for example, as
laydown material (to coat the plate) in ELISA assays for detection
of LPA. For example, in an LPA competitive ELISA, the plate is
coated with derivatized and/or derivatized and conjugated LPA. A
set of one or more LPA standards and one or more samples (e.g.,
serum or cell culture supernatant) is mixed with the mouse anti-LPA
antibody of the invention and added to the derivitized-LPA-coated
plate. The antibody competes for binding to either plate-bound LPA
or LPA in the sample or standard. Following incubation and several
ELISA steps, the absorbance at 450 nm is measured and the LPA
concentration in the samples is determined by comparison to the
standard curve.
[0177] The derivatized or derivatized and conjugated LPA may also
be coupled to a solid support (e.g., resin or other column matrix,
beads, membrane, plate) and used to isolate and/or purify anti-LPA
antibodies, e.g., from blood or serum. Such anti-LPA antibodies may
be newly generated antibodies such as those of the invention (e.g.,
mammalian monoclonal or polyclonal antibodies to LPA) or may be
native human anti-LPA antibodies.
[0178] Thus the derivatized LPA and derivatized and conjugated LPA
of the invention are useful both for research and in clinical
diagnostics.
[0179] 3. Diagnostic Kits Incorporating Derivatized LPA
[0180] As a matter of convenience, the derivatized LPAs of the
present invention can be provided in a kit, for example, a packaged
combination of reagents in predetermined amounts with instructions
for performing the diagnostic assay.
[0181] As described above, In one embodiment, the derivatized LPA
conjugate (e.g., thiolated LPA conjugated to BSA or KLH) is used as
laydown material (to coat the plate) in ELISA kits which are used
to detect anti-LPA antibodies. Such kits are useful for detection
of LPA. For example, in an LPA competitive ELISA kit, the plate
(provided) is coated with derivatized and/or derivatized and
conjugated LPA. A set of one or more LPA standards (generally
provided in the kit) and one or more samples (e.g., serum or cell
culture supernatant) is mixed with the mouse anti-LPA antibody of
the invention and added to the derivitized-LPA-coated plate. The
antibody competes for binding to either plate-bound LPA or LPA in
the sample or standard. Following incubation and several ELISA
steps (instructions and reagents for which are provided in the
kit), the absorbance at 450 nm is measured and the LPA
concentration in the samples is determined by comparison to the
standard curve. In one embodiment the LPA used for laydown material
in the ELISA kit is thiolated C12 LPA or thiolated C18 LPA
conjugated to BSA. The antibody used in the kit may be polyclonal
or monoclonal antibody, preferably a monoclonal antibody.
[0182] A kit incorporating an Lpath derivatized and conjugated LPA
of the invention and an Lpath anti-LPA antibody of the invention,
is commercially available from Echelon Biosciences, Inc., Salt Lake
City, Utah (Lysophosphatidic Assay Kit, Cat. No. K-2800).
[0183] B. Anti-LPA Agents, Including Anti-LPA Antibodies
[0184] 1. Introduction
[0185] The use of monoclonal antibodies (mAbs) as a therapeutic
treatment for a variety of diseases and disorders is rapidly
increasing because they have been shown to be safe and efficacious
therapeutic agents. Approved therapeutic monoclonal antibodies
include Avastin.TM., Erbitux.TM., and Rituxan.TM.. Additional
monoclonal antibodies are in various phases of clinical development
for a variety of diseases with the majority targeting various forms
of cancer. In general, monoclonal antibodies are generated in
non-human mammals. The therapeutic utility of murine monoclonal
antibodies may be improved with chimerization or humanization of
non-human mammalian antibodies. Humanization greatly lessens the
development of an immune response against the administered
therapeutic monoclonal antibodies and thereby avoids the reduction
of half-life and therapeutic efficacy consequent on such a
response. For the most part, the humanization process consists of
grafting the murine complementary determining regions (CDRs) into
the framework region (FR) of a human immunoglobulin. Backmutation
to murine amino acid residues of selected residues in the FR is
often required to improve or regain affinity that is lost in the
initial grafted construct.
[0186] The manufacture of monoclonal antibodies is a complex
process that stems from the variability of the immunoglobulin
protein itself. The heterogeneity can be attributed to the
formation of alternative disulfide pairings, deamidation and the
formation of isoaspartyl residues, methionine and cysteine
oxidation, cyclization of N-terminal glutamine residues to
pyroglutamate and partial enzymatic cleavage of C-terminal lysines
by mammalian carboxypeptidases. Engineering is commonly applied to
antibody molecules to improve their properties, such as enhanced
stability, resistance to proteases, aggregation behavior and
enhance the expression level in heterologous systems.
[0187] 2. Disease Associations of LPA and Therapeutic Uses for
Anti-LPA Agents
[0188] LPA has been associated with a number of diseases and
disorders. For review, see Gardell et al., (2006) Trends Mol Med.
12(2):65-75 and Chun J. and Rosen, H., (2006) Curr. Pharma. Design
12:161-171. These include autoimmune disorders such as diabetes,
multiple sclerosis and scleroderma; hyperproliferative disorders
including cancer; disorders associated with angiogenesis and
neovascularization; obesity; neurodegenerative diseases including
Alzheimer's disease; schizophrenia, immune-related disorders such
as transplant rejection and graft-vs.-host disease, and others.
[0189] a. Hyperproliferative Disorders
[0190] One aspect of the invention concerns methods for treating
hyperproliferative disorders. These methods comprise administering
to a mammal (e.g., a bovine, canine, equine, ovine, or porcine
animal, particularly a human) known or suspected to suffer from an
LPA-associated hyperproliferative disorder a therapeutically
effective amount of a composition comprising an agent that
interferes with LPA concentration and/or activity, preferably in a
pharmaceutically or veterinarily acceptable carrier, as the
intended application may require. LPA-associated hyperproliferative
disorders include neoplasias, disorders associated with endothelial
cell proliferation, and disorders associated with fibrogenesis.
Most often, the neoplasia will be a cancer. Typical disorders
associated with endothelial cell proliferation are
angiogenesis-dependent disorders, for example, cancers caused by a
solid tumors, hematological tumors, and age-related macular
degeneration. Disorders associated with fibrogenesis include those
than involve aberrant cardiac remodeling, such as cardiac
failure.
[0191] There are many known hyperproliferative disorders, in which
cells of various tissues and organs exhibit aberrant patterns of
growth, proliferation, migration, signaling, senescence, and death.
While a number of treatments have been developed to address some of
these diseases, many still remain largely untreatable with existing
technologies, while in other cases, while treatments are available,
they are frequently less than optimal and are seldom curative.
[0192] Cancer represents perhaps the most widely recognized class
of hyperproliferative disorders. Cancers are a devastating class of
diseases, and together, they have a mortality rate second only to
cardiovascular disease. Many cancers are not fully understood on a
molecular level. As a result, cancer is a major focus of research
and development programs for both the United States government and
pharmaceutical companies. The result has been an unprecedented
R&D effort and the production of many valuable therapeutic
agents to help in the fight against cancer.
[0193] Unfortunately the enormous amount of cancer research has not
been enough to overcome the significant damage caused by cancer.
There are still over one million new cases of cancer diagnosed
annually and over five hundred thousand deaths in the United States
alone. This is a dramatic demonstration that even though an
enormous effort has been put forth to discover new therapeutics for
cancer, effective therapeutic agents to combat the disease remain
elusive.
[0194] Cancer is now primarily treated with one or a combination of
three types of therapies, surgery, radiation, and chemotherapy.
Surgery involves the bulk removal of diseased tissue. While surgery
is sometimes effective in removing tumors located at certain sites,
for example, in the breast, colon, and skin, it cannot be used in
the treatment of tumors located in other areas, such as the
backbone, nor in the treatment of disseminated neoplastic
conditions such as leukemia. Radiation therapy involves the
exposure of living tissue to ionizing radiation causing death or
damage to the exposed cells. Side effects from radiation therapy
may be acute and temporary, while others may be irreversible.
Chemotherapy involves the disruption of cell replication or cell
metabolism.
[0195] Further insult is that current therapeutic agents usually
involve significant drawbacks for the patient in the form of
toxicity and severe side effects. Therefore, many groups have
recently begun to look for new approaches to fighting the war
against cancer. These new so-called "innovative therapies" include
gene therapy and therapeutic proteins such as monoclonal
antibodies.
[0196] The first monoclonal antibody used in the clinic for the
treatment of cancer was Rituxan (rituximab) which was launched in
1997, and has demonstrated the utility of monoclonal antibodies as
therapeutic agents. Thus, not surprisingly, twenty monoclonal
antibodies have since been approved for use in the clinic,
including nine that are prescribed for cancer. The success of these
products, as well as the reduced cost and time to develop
monoclonal antibodies as compared with small molecules has made
monoclonal antibody therapeutics the second largest category of
drug candidates behind small molecules. Further, the exquisite
specificity of antibodies as compared to small molecule
therapeutics has proven to be a major advantage both in terms of
efficacy and toxicity. For cancer alone there are currently more
than 270 industry antibody R&D projects with more than 50
companies involved in developing new cancer antibody therapeutics.
Consequently, monoclonal antibodies are poised to become a major
player in the treatment of cancer and they are estimated to capture
an increasing share of the cancer therapeutic market. Generally
therapeutic mAbs are targeted to proteins; only recently has it
been feasible to raise mAbs to bioactive lipids (for example,
antibodies to S1P, see Applicants' US Application Serial No.
20070148168).
[0197] The identification of extracellular mediators that promote
tumor growth and survival is a critical step in discovering
therapeutic interventions that will reduce the morbidity and
mortality of cancer. As described below, LPA is considered to be a
pleiotropic, tumorigenic growth factor. LPA promotes tumor growth
by stimulating cell proliferation, cell survival, and metastasis.
LPA also promotes tumor angiogenesis by supporting the migration
and survival of endothelial cells as they form new vessels within
tumors. Taken together, LPA initiates a proliferative,
pro-angiogenic, and anti-apoptotic sequence of events contributing
to cancer progression. Thus, therapies that modulate, and, in
particular, reduce LPA levels in vivo will be effective in the
treatment of cancer.
[0198] Typically, the methods of the invention for treating or
preventing a hyperproliferative disorder such as cancer involve
administering to a subject suffering from a hyperproliferative
disorder an effective amount of each of an agent (or a plurality of
different agent species) according to the invention and a cytotoxic
agent. Cytotoxic agents include chemotherapeutic drugs.
[0199] A related aspect concerns methods of reducing toxicity of a
therapeutic regimen for treatment or prevention of a
hyperproliferative disorder. Such methods comprise administering to
a subject suffering from a hyperproliferative disorder an effective
amount of an agent (or a plurality of different agent species)
according to the invention before, during, or after administration
of a therapeutic regimen intended to treat or prevent the
hyperproliferative disorder. It is believed that by sensitizing
cells, e.g., cancer cells, to chemotherapeutic drugs, efficacy can
be achieved at lower doses and hence lower toxicity due to
chemotherapeutic drugs.
[0200] Yet another aspect of the invention concerns methods of
enhancing a survival probability of a subject treated for a
hyperproliferative disorder by administering to a subject suffering
from a hyperproliferative disorder an agent (or a plurality of
different agent species) according to the invention before, during,
or after administration of a therapeutic regimen intended to treat
or prevent the hyperproliferative disorder to enhance the subject's
survival probability.
[0201] 3. Fibrosis, Wound Healing and Scar Formation
[0202] Fibroblasts, particularly myofibroblasts, are key cellular
elements in scar formation in response to cellular injury and
inflammation (Tomasek et al. (2002), Nat Rev Mol Cell Biol, vol 3:
349-63, and Virag and Murry (2003), Am J Pathol, vol 163: 2433-40).
Collagen gene expression by myofibroblasts is a hallmark of
remodeling and necessary for scar formation (Sun and Weber (2000),
Cardiovasc Res, vol 46: 250-6, and Sun and Weber (1996), J Mol Cell
Cardiol, vol 28: 851-8).
[0203] Fibrosis can be described as the formation or development of
excess or aberrant fibrous connective tissue in an organ or tissue
as part of a pathological reparative or reactive process, in
contrast to normal wound healing or development. The most common
forms of fibrosis are: liver, lung, kidney, skin, uterine and
ovarian fibroses. Some conditions, such as scleroderma, sarcoidosis
and others, are characterized by fibrosis in multiple organs and
tissues.
[0204] Recently, the bioactive lysophospholipid lysophosphatidic
acid (LPA) has been recognized for its role in tissue repair and
wound healing. Watterson et al., Wound Repair Regen. (2007)
15:607-16. As a biological mediator, LPA has been recognized for
its role in tissue repair and wound healing (Watterson, 2007). In
particular, LPA is linked to pulmonary and renal inflammation and
fibrosis. LPA is detectable in human bronchioalveolar lavage (BAL)
fluids at baseline and its expression increases during allergic
inflammation Georas, S. N. et al. (2007) Clin Exp Allergy. (2007)
37: 311-22. Furthermore, LPA promotes inflammation in airway
epithelial cells. Barekzi, E. et al (2006) Prostaglandins Leukot
Essent Fatty Acids. 74:357-63. Recently, pulmonary and renal
fibrosis have been linked to increased LPA release and signaling
though the LPA type 1 receptor (LPA.sub.1). LPA levels were
elevated in bronchialveolar lavage (BAL) samples from IPF patients
and bleomycin-induced lung fibrosis in mice was dependent on
activation of LPA.sub.1. Tager et al., (2008) Proc Am Thorac Soc.
5: 363. (2008) Following unilateral ureteral obstruction in mice,
tubulointerstitial fibrosis was reduced in LPA.sub.1 knock-out mice
and pro-fibrotic cytokine expression was attenuated in wild-type
mice treated with an LPA.sub.1 antagonist. J. P. Pradere et al.,
(2007) J. Am. Soc. Nephrol. 18:3110-3118. LPA has been shown to
have direct fibrogenic effects in cardiac fibroblasts by
stimulating collagen gene expression and proliferation. Chen, et
al. (2006) FEBS Lett. 580:4737-45. Combined, these studies
demonstrate a role for LPA in tissue repair and fibrosis, and
identify bioactive lipids as a previously unrecognized class of
targets in the treatment of fibrotic disorders.
[0205] a. Scleroderma
[0206] The compositions and methods of the invention will be useful
in treating disorders and diseases characterized, at least in part,
by aberrant neovascularization, angiogenesis, fibrogenesis,
fibrosis, scarring, inflammation, and immune response. One such
disease is scleroderma, which is also referred to as systemic
sclerosis.
[0207] Scleroderma is an autoimmune disease that causes scarring or
thickening of the skin, and sometimes involves other areas of the
body, including the lungs, heart, and/or kidneys. Scleroderma is
characterized by the formation of scar tissue (fibrosis) in the
skin and organs of the body, which can lead to thickening and
firmness of involved areas, with consequent reduction in function.
Today, about 300,000 Americans have scleroderma, according to the
Scleroderma Foundation. One-third or less of those affected have
widespread disease, while the remaining two-thirds primarily have
skin symptoms. When the disease affects the lungs and causing
scarring, breathing can become restricted because the lungs can no
longer expand as they should. To measure breathing capability,
doctors use a device that assesses forced vital capacity (FVC). In
people with an FVC of less than 50 percent of the expected reading,
the 10-year mortality rate from scleroderma-related lung disease is
about 42 percent. One reason the mortality rate is so high is that
no effective treatment is currently available.
[0208] Without wishing to be bound by any particular theory, it is
believed that inappropriate concentrations of lipids such as S1P
and/or LPA, and/or their metabolites, cause or contribute to the
development of scleroderma. As such, the compositions and methods
of the invention can be used to treat scleroderma, particularly by
decreasing the effective in vivo concentration of a particular
target lipid, for example, LPA.
[0209] Evidence indicates that LPA is a pro-fibrotic growth factor
that can contribute to fibroblast activation, proliferation, and
the resulting increased fibroblast activity associated with
maladaptive scarring and remodeling. Moreover, potential roles for
LPA in skin fibroblast activity have been demonstrated. For
example, it has been shown that LPA stimulates the migration of
murine skin fibroblasts (Hama et al., J Biol Chem. 2004 Apr. 23;
279(17):17634-9).
[0210] b. Pulmonary Fibrosis
[0211] Pulmonary fibrosis, sometimes referred to as interstitial
lung disease or ILD, affects more than 5 million people worldwide.
Within the USA the prevalence of the disease seems to be
under-estimated and vary from 3 to 6 cases for 100,000 inhabitants
to 28 per 100,000. Within Europe; the numbers vary depending on the
countries, and is reported around 1 to 24 cases per 100,000 without
a clear gender effect. The disease is usually diagnosed between 40
and 70 years of age. The median survival is 3 to 5 years. Despite
its prevalence, there are no therapies available to halt or reverse
the progression of IPF and there are no FDA-approved courses of
treatment. Thus, there is an unmet need for new therapeutic
strategies to treat IPF as well as other diseases that involve
pathological tissue fibrosis.
[0212] Interstitial lung disease, or ILD, includes more than 180
chronic lung disorders, which are chronic, nonmalignant and
noninfectious. Interstitial lung diseases are named for the tissue
between the air sacs of the lungs called the interstitium--the
tissue affected by fibrosis (scarring). Interstitial lung diseases
may also be called interstitial pulmonary fibrosis or pulmonary
fibrosis. The symptoms and course of these diseases may vary from
person to person, but the common link between the many forms of ILD
is that they all begin with an inflammation, e.g.:
bronchiolitis--inflammation that involves the bronchioles (small
airways); alveolitis--inflammation that involves the alveoli (air
sacs); vasculitis--inflammation that involves the small blood
vessels (capillaries)
[0213] More than 80% of interstitial lung diseases are diagnosed as
pneumoconiosis, drug-induced disease, or hypersensitivity
pneumonitis. The other types are:
[0214] Occupational and environmental exposures: Many jobs,
particularly those that involve working with asbestos, ground
stone, or metal dust, can cause pulmonary fibrosis. The small
particles are inhaled, damage the alveoli, and cause fibrosis. Some
organic substances, such as moldy hay can also initiate pulmonary
fibrosis; this is known as farmer's lung.
[0215] Asbestosis is usually caused when small needle-like
particles of asbestos are inhaled into the lungs. This can cause
lung scarring (pulmonary fibrosis) and in addition can lead to lung
cancer. The key to asbestosis is prevention. In manufacturing
asbestos products, both employer and employee must be aware of
government standards and should take all precautions against
inhaling the particles. The paramount danger in working with
asbestos comes when old, friable (crumbly) asbestos-containing
products are replaced or destroyed. In those circumstances,
particles can be released into the air and breathed into the lungs.
Today however, the asbestos fibres usually are "locked in" by
binders such as cement, rubber or plastics, thus preventing the
particles from floating free in the air. Cigarette smoking has an
interactive relationship with asbestos--the asbestos worker who
smokes has a much higher chance of developing lung cancer than does
the non-smoker.
[0216] Silicosis is another disease producing pulmonary fibrosis in
which the cause is known. It is a disease that results from
breathing in free crystalline silica dust. All types of mining in
which the ore is extracted from quartz rock can produce silicosis
if precautions are not taken. This includes the mining of gold,
lead, zinc, copper, iron, anthracite (hard) coal, and some
bituminous (soft) coal. Workers in foundries, sandstone grinding,
tunneling, sandblasting, concrete breaking, granite carving, and
china manufacturing also encounter silica.
[0217] Large silica particles are stopped in the upper airways. But
the tiniest specks of silica can be carried down to the alveoli
where they lead to pulmonary fibrosis. Silicosis can be either mild
or severe, in direct proportion to the percentage and concentration
of silica in the air and the duration of exposure. Silicosis can be
prevented by measures specifically designed for each industry and
each job. Dust control is essential. Sometimes this is accomplished
by the wetting down of mines, improved ventilation, or the wearing
of masks.
[0218] Idiopathic pulmonary fibrosis: Although a number of separate
diseases can initiate pulmonary fibrosis, many times the cause is
unknown. When this is so, the condition is called "idiopathic (of
unknown origin) pulmonary fibrosis". In idiopathic pulmonary
fibrosis, careful examination of the patient's environmental and
occupational history gives no clues to the cause. Some physicians
and scientists believe that the disease is an infectious or
allergic condition, however bacteria and other microorganisms are
not routinely found in the lungs of such patients. On the other
hand, the condition does sometimes appear to follow a viral-like
illness. Thus, although the cause of pulmonary fibrosis is known in
many cases, the idiopathic variety still remains a mystery.
[0219] Sarcoidosis is disease characterized by the formation of
granulomas (areas of inflammatory cells), which can attack any area
of the body but most frequently affects the lungs.
[0220] Certain medicines may have the undesirable side effect of
causing pulmonary fibrosis; for example, Nitrofurantoin (sometimes
used for urinary tract infections); Amiodarone (sometimes
prescribed for an irregular heart rate); Bleomycin,
cyclophosphamide, and methotrexate (sometimes prescribed to fight
cancer).
[0221] Radiation, such as given as treatment for breast cancer, may
also cause pulmonary fibrosis. Other diseases characterized, at
least in part, by pulmonary fibrosis include tuberculosis,
rheumatoid arthritis, systemic lupus erythematosis, systemic
sclerosis, grain handler's lung, mushroom worker's lung,
bagassosis, detergent worker's lung, maple bark stripper's lung,
malt worker's lung, paprika splitter's lung, bird breeder's lung
and Hermansky Pudlak syndrome. Pulmonary fibrosis can also be
genetically inherited.
[0222] Clinical Features:
[0223] Breathlessness is the hallmark of pulmonary fibrosis. Many
lung diseases show breathlessness as the main symptom--a fact that
can complicate and confuse diagnosis. Usually the breathlessness
idiopathic pulmonary fibrosis first appears during exercise. The
condition may progress to the point where any exertion is
impossible. A dry cough is a common symptom. The fingertips may
enlarge at the ends and take on a bulbous appearance. This is often
referred to as "clubbing".
[0224] Additional symptoms may include: shortness of breath,
especially with exertion, fatigue and weakness, loss of appetite,
loss of weight, dry cough that does not produce phlegm, discomfort
in chest, labored breathing and hemorrhage in lungs.
[0225] Diagnosis
[0226] In addition to a complete medical history and physical
examination, the following tests maybe required to refine and/or
confirm the diagnosis of pulmonary fibrosis: pulmonary function
tests--to determine characteristics and capabilities of the lungs;
spirometry--to measure the amount of air that can be forced out;
peak flow meter--to evaluate changes in breathing and response to
medications; blood tests--to analyze the amount of carbon dioxide
and oxygen in the blood; X-ray; computerized axial tomography (CAT)
scan; bronchoscopy--to examine the lung using a long, narrow tube
called a bronchoscope; bronchoalveolar lavage--to remove cells from
lower respiratory tract to help identify inflammation and exclude
certain causes; and lung biopsy--to remove tissue from the lung for
examination in the pathology laboratory.
[0227] Treatment
[0228] If one of the known causes of pulmonary fibrosis exists,
then treatment of that underlying disease or removal of the patient
from the environment causing the disease can be effective. This may
include treatment with: oral medications, including
corticosteroids; influenza vaccine; pneumococcal pneumonia vaccine,
oxygen therapy from portable tanks and/or lung transplantation.
[0229] Many times treatment is limited only to treating the
inflammatory response that occurs in the lungs. This is done in the
hope that stopping the inflammation will prevent the laying down of
scar tissue or fibrosis in the lungs and thus stop the progression
of the disease.
[0230] Corticosteroids are the drugs which are usually administered
in an attempt to stop the inflammation. The advantage of this
treatment has not been proven in every case, although it does
appear that if the drugs are given early on in the course of the
disease, there is a better chance of improvement. Corticosteroid
medications can have various side effects and so patients taking
these medications must be frequently reassessed by their physicians
in order to judge the safety and benefit of this therapy.
[0231] Other drugs have been tried but convincing evidence of their
efficacy is lacking Drug therapy of pulmonary fibrosis may not
always be successful, and so supportive (non-medication) therapy
may be used to ease the breathlessness that accompanies this
condition.
[0232] LPA and Pulmonary Fibrosis
[0233] Although the exact etiology is not known, IPF is believed to
result from an aberrant wound healing response following pulmonary
injury. Scotton, C. J. and Chambers, R. C. (2007) Chest,
132:1311-21. In particular, increased proliferation and migration
of lung fibroblasts as well as the formation of scar
tissue-producing myofibroblasts are key events in the pathogenesis
of IPF. Myofibroblasts are smooth muscle-like fibroblasts that
express alpha-smooth muscle actin (.alpha.-SMA) and contain a
contractile apparatus composed of actin filaments and associated
proteins that are organized into prominent stress fibers. In
addition to their normal role in tissue homeostasis and repair,
myofibroblasts are pathological mediators in numerous fibrotic
disorders. Hinz, B. (2007) J Invest Dermatol. 127:526-37. Increased
number and density of myofibroblasts has been demonstrated in the
fibrotic foci of animal models of lung fibrosis. Myofibroblasts are
formed following tissue injury whereby increased levels of growth
factors, cytokines and mechanical stimuli promote transformation of
resident tissue fibroblasts into contractile, scar tissue-producing
myofibroblasts. In the lung and other tissues, persistent, elevated
levels of biochemical mediators including TGF.beta., CTGF, PDGF and
various inflammatory cytokines, promotes myofibroblast formation
and exaggerated scar tissue production which leads to tissue
fibrosis (Scotton, 2007). Thus, current clinical strategies for
treating IPF and other fibrotic disorders have targeted biochemical
factors that promote myofibroblast formation and subsequent fibrous
tissue production.
[0234] Recently, the bioactive lysophospholipid lysophosphatidic
acid (LPA) has been recognized for its role in tissue repair and
wound healing (Watterson, 2007). LPA is a bioactive
lysophospholipid (<500 Dalton) with a single hydrocarbon
backbone and a polar head group containing a phosphate group. LPA
elicits numerous cellular effects through the interaction with
specific G protein-coupled receptors (GPCR), designated
EGD2/LPA.sub.1, EDG4/LPA.sub.2, EDG7/LPA.sub.3, and LPA.sub.4.
Anliker B. and J. Chun, (2004) Seminars in Cell & Developmental
Biology, 15: 457-465. As a biological mediator, LPA has been
recognized for its role in tissue repair and wound healing
(Watterson, 2007). In particular, LPA is linked to pulmonary and
renal inflammation and fibrosis. LPA is detectable in human
bronchioalveolar lavage (BAL) fluids at baseline and its expression
increases during allergic inflammation (Georas, 2007). Furthermore,
LPA promotes inflammation in airway epithelial cells (Barekzi,
2006). Recently, pulmonary and renal fibrosis have been linked to
increased LPA release and signaling though the LPA type 1 receptor
(LPA.sub.1). LPA levels were elevated in bronchialveolar lavage
(BAL) samples from IPF patients and bleomycin-induced lung fibrosis
in mice was dependent on activation of LPA.sub.1 (Tager, 2008).
Following unilateral ureteral obstruction in mice,
tubulointerstitial fibrosis was reduced in LPA.sub.1 knock-out mice
and pro-fibrotic cytokine expression was attenuated in wild-type
mice treated with an LPA.sub.1 antagonist (Pradere, 2007).
Combined, these studies demonstrate a role for LPA in tissue repair
and fibrosis, and identify bioactive lipids as a previously
unrecognized class of targets in the treatment of IPF and other
fibrotic disorders.
[0235] c. Hepatic (Liver) Fibrosis
[0236] The liver possesses a remarkable regenerative capacity,
therefore the process of repair by regeneration proceeds to
complete restitutio ad integrum (full restoration). If however the
damage has affected the reticular framework, the repair will occur
by scar formation (fibrosis) which may lead to rearrangement of the
blood circulation and to cirrhosis.
[0237] The reaction to injury proceeds as is follows: Damage
(necrosis), accompanied by cellular changes and tissue changes;
inflammatory reaction; and repair (either by regeneration
(restitutio ad integrum) or by scarring (fibrosis).
[0238] Chronic liver diseases lead to fibrosis which leads to
disturbance of the architecture, portal hypertension and may
produce such an irreversible rearrangement of the circulation as to
cause cirrhosis. There is a fine line between fibrosis and
cirrhosis. Fibrosis is not only the result of necrosis, collapse
and scar formation but also the result of disturbances in the
synthesis and degradation of matrix by injured mesenchymal cells
that synthesize the various components of the matrix which in the
liver are the following categories: collagens, glycoproteins and
proteoglycans.
[0239] Evaluation of Liver Fibrosis
[0240] Evaluation of liver fibrosis can be histological, e.g., with
Masson trichrome stain, silver reticulin stain, specific antibodies
for collagen types, desmin and vimentin for lipocytes, or vimentin
for myofibroblasts, or may be biochemical, e.g, by: determination
of various enzymes in matrix or of serum laminin in benign
fibrosis.
[0241] Classifications of Liver Fibrosis
[0242] There are 2 main types, congenital and acquired liver
fibrosis. The former is a genetic disorder, which causes polycystic
liver diseases. The latter has many different categories and is
mainly caused by liver cell injuries. Pathologically, fibrosis can
be classified as:
[0243] Portal area fibrosis: There is fibroblasts proliferation and
fibers expansion from the portal areas to the lobule. Finally,
these fibers connected to form bridging septa. This kind of
fibrosis is mainly seen in viral hepatitis and malnutritional liver
fibrosis.
[0244] Intra-lobular fibrosis: There is almost no fibroblast found
in normal lobule. When large numbers of liver cells degenerate and
undergo necrosis, the reticular fiber frame collapses and becomes
thick collagen fibers. At the same time, intra lobule fibrotic
tissue proliferates and surrounds the liver cells.
[0245] Central fibrosis: Proliferated fibrotic tissue mainly
surrounds the center vein and causes the thickening of the wall of
the center vein.
[0246] Peri-micro-bile-duct fibrosis: Type fibrosis mainly caused
by long-term bile retention and mainly happens around the bile
ducts. Microscopically, there are connective tissues surrounding
the newly formed bile canaliulus and bile-plugs. The base-membrane
of the bile canaliulus becomes fibrotic.
[0247] Immunologically, liver fibrosis can be classified as:
[0248] Passive fibrosis: There is extensive necrosis of the liver
cells and secondary liver structure collapse and scar formation,
which causes connective tissue proliferation.
[0249] Active fibrosis: Lymph cells and other inflammatory cells
infiltration and recurrent and consistent inflammation promote the
connective tissue to invade the lobule.
[0250] Causally, liver fibrosis can be classified as:
[0251] Viral hepatitis fibrosis: Usually caused by chronic
hepatitis B, C, and D. Worldwide, there are three hundred fifty
million of hepatitis B virus carriers, and one hundred seventy
million of hepatitis C infected people. About 15% of HBV and 85% of
HCV infected persons will develop chronic hepatitis and lead to
fibrosis. In which, the liver shows peri-portal area inflammation
and piecemeal necrosis and fibrosis. With such large population
being affected, this is the most important category of the liver
fibrosis.
[0252] Parasitic infection fibrosis: This kind of liver fibrosis is
mainly happening in developing countries and is caused by
schistosomiasis. There are two hundred and twenty million people in
Asia, Africa, South and Center America suffering from this
infection. The recurrent infection and the eggs of schistosome
accumulated in the liver can cause liver fibrosis and
cirrhosis.
[0253] Alcoholic fibrosis: It is mainly caused by the oxidized
metabolite of alcohol, acetaldehyde. In western countries, the
incidence of this disorder is positively related to the amount of
alcohol consumption. The total cases of alcoholic fibrosis in the
USA is about three times higher than the number of hepatitis C.
Alcoholic fibrosis causes two morphological changes in the liver:
fatty liver and cellular organelles deterioration. The fibrosis
first appears around the center veins and at the same time, the
liver parenchymal inflammation. Gradually the fibrosis expends to
the whole liver.
[0254] Biliary fibrosis: There is primary and secondary biliary
fibrosis. Primary biliary hepatic fibrosis (PBHF) is an autoimmune
disorder in which chronic intra-liver bile retention caused the
liver fibrosis. It is more often affect female around the age 40 to
60. In serum tests, elevated gamma globulin and positive for the
anti-mitochondria antibody. Pathological studies found that the
fibrosis mainly around the micro-bile ducts and peri-portal area
fibrosis and inflammation. Secondary biliary fibrosis happens
following the obstruction of the bile ducts, which causes
peri-portal inflammation and progressive fibrosis.
[0255] Metabolic fibrosis: This category is not common and has
fewer cases. Wilson's disease or liver lenticular degeneration and
hemochromatosis are the main disorders that cause metabolic
fibrosis. The former is a genetic disorder and causes cooper
metabolism disorder and deposits in the liver. The latter is an
iron metabolic disorder and causes hemoglobin deposits in the
liver. Both of these metabolic disorders can cause liver fibrosis
and cirrhosis.
[0256] Intoxication fibrosis: When long-term contact with
liver-toxic substances, such as carbon-tetrachloride,
organophosphorus, dimethyl nitrosamine, thioacetamide, or taking
liver toxic medications, such as isoniazid, thio-oxidizing
pyrimidine, wintermin, tetracycline, acetaminophen etc. can all
cause various degrees of liver cell injuries, necrosis, bile
retention, or allergic inflammation and cause liver fibrosis.
[0257] Malnutritional fibrosis: This type is mainly caused by
insufficient or imbalanced nutritional intake. A long-term low
protein or high fat diet can cause fatty liver and lead to
fibrosis.
[0258] Cardiogenic fibrosis: Chronic congestive heart failure can
cause long lasting liver vein stagnancy causing ischemic
degeneration of the liver cells. In this type of liver fibrosis,
the connective tissue hypertrophy starts at the center of the liver
lobule and gradually expands to rest of the lobule.
[0259] Diagnosis and Staging of Liver Fibrosis
[0260] The gold standard for assessing the health of the liver is
the liver biopsy. However since the procedure requires that a
needle be inserted through the skin there is a potential for
complications even though the incidence of complications is
extremely low. The complications of a liver biopsy can include
internal bleeding, and puncturing another organ such as the lungs,
stomach, intestines, or any other organs that are close to the
liver. In regards to accuracy of the biopsy the sample liver tissue
size is important for correctly staging and grading a liver biopsy.
Another problem is that the tissue taken from one part of the liver
may not be 100% representative of the entire liver. Once the liver
tissue sample is collected it is graded and staged by a specialist
(pathologist), which could lead to possible human error in
interpreting the results. In addition there is no standardized
interpretation protocol so it is difficult to compare the results
of different biopsies read by different pathologists. Price is also
an issue since a typical liver biopsy can cost between $1,500 and
$2,000.
[0261] Given these potential problems it is not surprising that
there is a lot of research that is being conducted on the
development of non-invasive tests. The tests that have been
developed so far have had mixed results in accuracy when compared
to the results of a liver biopsy. There have been few prospective
clinical trials that have compared the results from various
non-invasive markers to the results from a liver biopsy.
[0262] In order to objectively evaluate the stage of fibrosis,
liver biopsy, especially a series of biopsies, is the main method
used today. From the biopsy, it is possible to diagnose the liver
inflammation grade and also the stage of the fibrosis. The most
commonly used scoring system is Kanel scoring system, which stages
the fibrosis from 0 to 5. (At the same time the biopsy diagnosis
also give a ranking of inflammation grade, which is from 0 to 4)
Stage 0: normal; Stage 1: portal expansion with fibrosis (<1/3
tracts with wisps of bridging.); Stage 2: bridging fibrosis; Stage
3: marked bridging fibrosis or early cirrhosis (with thin septa
fibrosis); Stage 4: definite cirrhosis with <50% of biopsy
fibrosis; Stage 5: definite cirrhosis with >50% of biopsy
fibrosis.
[0263] Blood tests to diagnose liver fibrosis: Because biopsy is an
invasive procedure, many patients are wary of the procedure. Blood
tests are being studied as a method to evaluate the fibrosis
progression. The most commonly used serum chemical analysis method
is by measuring the amount of HA (hyaluronic acid), LN (Laminin),
CIV (collagen IV), PCIII (procollagen type III) in the serum. They
can be used as a reference index of fibrosis activities. From the
blood tests, the ratio of AST/ALT is found and when it is greater
than 1, it often shows that the degree of fibrosis is relatively
advanced. Combined with whether is there an enlarged spleen and
depletion of platelets count and albumin level, we can also
estimate the stage of the fibrosis. In advanced fibrosis, the
spleen is usually enlarged with platelets counts lower than 100 and
albumin lower than 3.5. With blood test results, the evaluation of
the severity of fibrosis is only useful to access the stage 0, 1
and 3, 4, and 5. It is not able to distinguish the stages between 2
and 3.
[0264] Medical imagery diagnosis B-ultrasonic, CT, and MRI can also
be used to evaluate the liver fibrosis. The B-ultrasonic image is
often used to check the size of the spleen, measure the diameter of
the main stern of the portal vein, the diameters of right and left
portal vein branches, the diameter of vein at the portal of the
spleen, and the blood flow speed of the portal vein. GI endoscopies
can be used to see whether varices exists in the stomach and
esophagus. These can be used as a reference for the hepatologist to
evaluate the stage of fibrosis.
[0265] In general, the term fibrosis refers to the abnormal
formation of fibrous (scar) tissue. For hepatitis patients,
fibrosis means that the liver has been under assault by the
hepatitis for some time. Early stages of fibrosis are identified by
discrete, localized areas of scarring in one portal (zone) of the
liver. Later stages of fibrosis are identified by "bridging"
fibrosis, which is scar tissue that crosses across zones of the
liver. The rate at which people progress from inflammation to
fibrosis, and eventually to cirrhosis seems to vary tremendously,
but in most people the progression is very slow. There is a growing
body of evidence that people who respond to interferon therapy for
HCV infection may experience a decrease in the amount of tissue
scarring. This speaks to the liver's ability to regenerate itself.
If fibrosis advances far enough, it is described as Cirrhosis.
Liver biopsy is conducted to assess the degree of inflammation
(grade) and degree of scarring (stage). Diagnosis: One of the major
clinical problems facing the hepatology and gastroenterology
community is how best to evaluate and manage the increasing numbers
of patients identified with hepatitis C virus (HCV). In the last
decade, advances in serologic and virologic testing for HCV and
improvements in therapy have led more patients to be identified and
to seek treatment. However, little progress has been made in
improving either our ability to determine the degree of hepatic
injury, particularly fibrosis, or to predict the risk of disease
progression for the individual patient.
[0266] The clinician relies on the biopsy results for both
prognostic and therapeutic decision making, which can have a major
impact on the patient's life. A single-pass liver biopsy is able to
correctly diagnose the stage of fibrosis or presence of cirrhosis
in 80% of patients. Factors that improve the diagnostic accuracy of
liver biopsy include the presence of a uniform disease throughout
the liver such as HCV, multiple passes, type of needle used, and an
unfragmented biopsy core of 2 cm or greater in length. Even with
experienced physicians performing the liver biopsy and expert
pathologists interpreting the biopsy, this gold standard has up to
a 20% error rate in staging disease.
[0267] d. Renal (Kidney) Fibrosis
[0268] LPA is linked to renal inflammation and fibrosis. Recently,
renal fibrosis has been linked to increased LPA release and
signaling though the LPA type 1 receptor (LPA.sub.1). Following
unilateral ureteral obstruction in mice, tubulointerstitial
fibrosis was reduced in LPA.sub.1 knock-out mice and pro-fibrotic
cytokine expression was attenuated in wild-type mice treated with
an LPA.sub.1 antagonist (Pradere, 2007).
[0269] e. Other Fibroses
[0270] Uterine fibroses are non-malignant tumors known as uterine
leiomyomata (commonly called fibroids). They can be isolated or
grow in clusters, with sizes varying from the size of an apple seed
to the size of a grapefruit or larger. Diagnosis of uterine
fibroids is generally achieved by ultrasound, X-rays, CAT scan,
laparoscopy and/or hysteroscopy. Treatment of uterine fibroids can
be either medical (drug treatment, e.g., non-steroid
anti-inflammatory drugs or gonadotropin release hormone agonists)
or surgical (e.g., myomectomy, hysterectomy, endometrial ablation
or myolysis, with recent development of less invasive methods such
as uterine fibroid embolization and thermal ultrasound
ablation.
[0271] Fibrosis of the skin can be described as a thickening or
hardening of the skin, and occurs in scleroderma and other fibrotic
skin diseases. When severe, fibrosis can limit movement and normal
function. A keloid is an excessive scar that forms in response to
trauma, sometimes minor trauma such as ear piercing or acne. Unlike
normal scar formation, keloids have disproportionate proliferation
of fibroblasts resulting in masses of collagenous tissue. The scar
therefore protrudes above the surface of the surrounding skin and
infiltrates skin which was not originally traumatized. Roles for
LPA in skin fibroblast activity have been demonstrated. For
example, it has been shown that LPA stimulates the migration of
murine skin fibroblasts (Hama et al., J Biol Chem. 2004 Apr. 23;
279(17):17634-9). Thus it is believed that anti-LPA agents such as
antibodies are useful for treatment of aberrant skin fibrosis such
as keloids or skin fibrosis.
[0272] Cardiac Fibrosis
[0273] LPA has also been shown to have direct fibrogenic effects in
cardiac fibroblasts by stimulating collagen gene expression and
fibroblast proliferation. Chen, et al. (2006) FEBS Lett.
580:4737-45. Thus anti-LPA agents such as antibodies are expected
to have anti-fibrotic effects in cardiac cells as well, and thus to
be effective in treatment of cardiac fibrosis.
[0274] Agents that reduce the effective concentration of LPA, such
as Lpath's anti-LPA mAb, are believed to be useful in methods for
treating diseases and conditions characterized by aberrant
fibrosis.
[0275] 4. Cardiovascular and Cerebrovascular Disorders
[0276] Because LPA is involved in fibrogenesis and wound healing of
liver tissue (Davaille et al., J. Biol. Chem. 275:34268-34633,
2000; Ikeda et al., Am J. Physiol. Gastrointest. Liver Physiol
279:G304-G310, 2000), healing of wounded vasculatures (Lee et al.,
Am. J. Physiol. Cell Physiol. 278:C612-C618, 2000), and other
disease states, or events associated with such diseases, such as
cancer, angiogenesis and inflammation (Pyne et al., Biochem. J.
349:385-402, 2000), the compositions and methods of the disclosure
may be applied to treat not only these diseases but cardiac
diseases as well, particularly those associated with tissue
remodeling. LPA have some direct fibrogenic effects by stimulating
collagen gene expression and proliferation of cardiac fibroblasts.
Chen, et al. (2006) FEBS Lett. 580:4737-45.
[0277] 5. Obesity and Diabetes
[0278] Autotaxin, a phospholipase D responsible for LPA synthesis,
has been found to be secreted by adipocytes and its expression is
up-regulated in adipocytes from obese-diabetic db/db mice as well
as in massively obese women subjects and human patients with type 2
diabetes, independently of obesity (Ferry et al. (2003) JBC
278:18162-18169; Boucher et al. (2005) Diabetologia 48:569-577,
cited in Pradere et al. (2007) BBA 1771:93-102. LPA itself has been
shown to influence proliferation and differentiation of
preadipocytes. Pradere et al., 2007. Together this suggests a role
for anti-LPA agents in treatment of obesity and diabetes.
[0279] 3. Antibody Generation and Characterization
[0280] The examples hereinbelow describe the production of anti-LPA
agents, particularly anti-LPA antibodies, with desirable properties
from a therapeutic perspective including: (a) binding affinity for
LPA and/or its variants, including 18:2, 18:1, 18:0, 16:0, 12:0 and
20:4 LPA. Antibody affinities may be determined as described in the
examples herein below. Preferably antibodies bind LPA with a high
affinity, e.g., a K.sub.d value of no more than about
1.times.10.sup.-7 M; possibly no more than about 1.times.10.sup.-8
M; and possibly no more than about 5.times.10.sup.-9 M. In a
physiological context, it is preferable for an antibody to bind LPA
with an affinity that is higher than the LPA's affinity for an LPA
receptor. It will be understood that this need not necessarily be
the case in a nonphysiological context such as a diagnostic
assay.
[0281] Aside from antibodies with strong binding affinity for LPA,
it is also desirable to select chimeric, humanized or variant
antibodies which have other beneficial properties from a
therapeutic perspective. For example, the antibody may be one that
reduces scar formation or alters tumor progression. One assay for
determining the activity of the anti-LPA antibodies of the
invention is ELISA. Preferably the humanized or variant antibody
fails to elicit an immunogenic response upon administration of a
therapeutically effective amount of the antibody to a human
patient. If an immunogenic response is elicited, preferably the
response will be such that the antibody still provides a
therapeutic benefit to the patient treated therewith.
[0282] According to one embodiment of the invention, humanized
anti-LPA antibodies bind the epitope as herein defined. To screen
for antibodies that bind to the epitope on an LPA bound by an
antibody of interest (e.g., those that block binding of the
antibody to LPA), a routine cross-blocking assay such as that
described in Antibodies, A Laboratory Manual, Cold Spring Harbor
Laboratory, Ed Harlow and David Lane (1988), can be performed.
Alternatively, epitope mapping, e.g. as described in Champe et al.,
J. Biol. Chem. 270:1388-1394 (1995), can be performed to determine
whether the antibody binds an epitope of interest.
[0283] The antibodies of the invention have a heavy chain variable
domain comprising an amino acid sequence represented by the
formula: FR1-CDRH1-FR2-CDRH2-FR3-CDRH3-FR4, wherein "FR1-4"
represents the four framework regions and "CDRH1-3" represents the
three hypervariable regions of an anti-LPA antibody variable heavy
domain. FR1-4 may be derived from a consensus sequence (for example
the most common amino acids of a class, subclass or subgroup of
heavy or light chains of human immunoglobulins) or may be derived
from an individual human antibody framework region or from a
combination of different framework region sequences. Many human
antibody framework region sequences are compiled in Kabat et al.,
supra, for example. In one embodiment, the variable heavy FR is
provided by a consensus sequence of a human immunoglobulin subgroup
as compiled by Kabat et al., supra.
[0284] The human variable heavy FR sequence may have substitutions
therein, e.g. wherein the human FR residue is replaced by a
corresponding nonhuman residue (by "corresponding nonhuman residue"
is meant the nonhuman residue with the same Kabat positional
numbering as the human residue of interest when the human and
nonhuman sequences are aligned), but replacement with the nonhuman
residue is not necessary. For example, a replacement FR residue
other than the corresponding nonhuman residue may be selected by
phage display.
[0285] The antibodies of the preferred embodiment herein have a
light chain variable domain comprising an amino acid sequence
represented by the formula: FR1-CDRL1-FR2-CDRL2-FR3-CDRL3-FR4,
wherein "FR1-4" represents the four framework regions and "CDRL1-3"
represents the three hypervariable regions of an anti-LPA antibody
variable light domain. FR1-4 may be derived from a consensus
sequence (for example the most common amino acids of a class,
subclass or subgroup of heavy or light chains of human
immunoglobulins) or may be derived from an individual human
antibody framework region or from a combination of different
framework region sequences. In one preferred embodiment, the
variable light FR is provided by a consensus sequence of a human
immunoglobulin subgroup as compiled by Kabat et al., supra.
[0286] The human variable light FR sequence may have substitutions
therein, e.g. wherein the human FR residue is replaced by a
corresponding mouse residue, but replacement with the nonhuman
residue is not necessary. For example, a replacement residue other
than the corresponding nonhuman residue may be selected by phage
display. Methods for generating humanized anti-LPA antibodies of
interest herein are elaborated in more detail below.
[0287] a. Antibody Preparation
[0288] Methods for generating anti-LPA antibodies and variants of
anti-LPA antibodies are described in the Examples below. Humanized
anti-LPA antibodies may be prepared, based on a nonhuman anti-LPA
antibody. Fully human antibodies may also be prepared, e.g, in a
genetically engineered (i.e., transgenic) mouse (e.g. from Medarex)
that, when presented with an immunogen, can produce a human
antibody that does not necessarily require CDR grafting. These
antibodies are fully human (100% human protein sequences) from
animals such as mice in which the non-human antibody genes are
suppressed and replaced with human antibody gene expression. The
applicants believe that antibodies could be generated against
bioactive lipids when presented to these genetically engineered
mice or other animals that might be able to produce human
frameworks for the relevant CDRs.
[0289] Where a variant is to be generated, the parent antibody is
prepared. Exemplary techniques for generating such nonhuman
antibody and parent antibodies will be described in the following
sections.
[0290] (i) Antigen Preparation.
[0291] The antigen to be used for production of antibodies may be,
e.g., intact LPA or a portion of an LPA (e.g. an LPA fragment
comprising the epitope). Other forms of antigens useful for
generating antibodies will be apparent to those skilled in the art.
It has been found that derivatized LPA conjugated to a carrier is
particularly useful as an immunogen for generation of anti-LPA
antibodies.
[0292] (ii) Polyclonal Antibodies.
[0293] Polyclonal antibodies are preferably raised in animals
(vertebrate or invertebrates, including mammals, birds and fish,
including cartilaginous fish) by multiple subcutaneous (sc) or
intraperitoneal (ip) injections of the relevant antigen and an
adjuvant. It may be useful to conjugate the relevant antigen to a
protein or other carrier that is immunogenic in the species to be
immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine
thyroglobulin, or soybean trypsin inhibitor using a bifunctional or
derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide
ester (conjugation through cysteine residues), N-hydroxysuccinimide
(through lysine residues), glutaraldehyde, succinic anhydride,
SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R and R.sup.1 are
different alkyl groups. Non-protein carriers (e.g., colloidal gold)
are also known in the art for antibody production.
[0294] Animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 ug or 5 ug of
the protein or conjugate (for rabbits or mice, respectively) with
three volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later the
animals are boosted with one-fifth to one-tenth of the original
amount of peptide or conjugate in Freund's complete adjuvant by
subcutaneous injection at multiple sites. Seven to 14 days later
the animals are bled and the serum is assayed for antibody titer.
Animals are boosted until the titer plateaus. Preferably, the
animal is boosted with the conjugate of the same antigen, but
conjugated to a different protein and/or through a different
cross-linking reagent. Conjugates also can be made in recombinant
cell culture as protein fusions. Also, aggregating agents such as
alum are suitably used to enhance the immune response.
[0295] (iii) Monoclonal Antibodies.
[0296] Monoclonal antibodies may be made using the hybridoma method
first described by Kohler et al., Nature, 256:495 (1975), or may be
made by other methods such as recombinant DNA methods (U.S. Pat.
No. 4,816,567). In the hybridoma method, a mouse or other
appropriate host animal, such as a hamster or macaque monkey, is
immunized as hereinabove described to elicit lymphocytes that
produce or are capable of producing antibodies that will
specifically bind to the protein used for immunization.
Alternatively, lymphocytes may be immunized in vitro. Lymphocytes
then are fused with myeloma cells using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell (Goding,
Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)).
[0297] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0298] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOP-21 and M.C.-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from
the American Type Culture Collection, Rockville, Md. USA. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
(Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal
Antibody Production Techniques and Applications, pp. 51-63 (Marcel
Dekker, Inc., New York, 1987)).
[0299] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbant assay
(ELISA).
[0300] The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis of Munson et al.,
Anal. Biochem., 107:220 (1980).
[0301] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal.
[0302] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0303] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the monoclonal
antibodies). The hybridoma cells serve as a preferred source of
such DNA. Once isolated, the DNA may be placed into expression
vectors, which are well known in the art, and which are then
transfected into host cells such as E coli cells, simian COS cells,
Chinese hamster ovary (CHO) cells, or myeloma cells that do not
otherwise produce immunoglobulin protein, to obtain the synthesis
of monoclonal antibodies in the recombinant host cells. Recombinant
production of antibodies will be described in more detail
below.
[0304] (iv) Humanization and Amino Acid Sequence Variants.
[0305] General methods for humanization of antibodies are described
in update U.S. Pat. No. 5,861,155, US19960652558 19960606, U.S.
Pat. No. 6,479,284, US20000660169 20000912, U.S. Pat. No.
6,407,213, US19930146206 19931117, U.S. Pat. No. 6,639,055,
US20000705686 20001102, U.S. Pat. No. 6,500,931, US19950435516
19950504, U.S. Pat. No. 5,530,101, U.S. Pat. No. 5,585,089,
US19950477728 19950607, U.S. Pat. No. 5,693,761, US19950474040
19950607, U.S. Pat. No. 5,693,762, US19950487200 19950607, U.S.
Pat. No. 6,180,370, US19950484537 19950607, US2003229208,
US20030389155 20030313, U.S. Pat. No. 5,714,350, US19950372262
19950113, U.S. Pat. No. 6,350,861, US19970862871 19970523, U.S.
Pat. No. 5,777,085, US19950458516 19950517, U.S. Pat. No.
5,834,597, US19960656586 19960531, U.S. Pat. No. 5,882,644,
US19960621751 19960322, U.S. Pat. No. 5,932,448, US19910801798
19911129, U.S. Pat. No. 6,013,256, US19970934841 19970922, U.S.
Pat. No. 6,129,914, US19950397411 19950301, U.S. Pat. No.
6,210,671, v, U.S. Pat. No. 6,329,511, US19990450520 19991129,
US2003166871, US20020078757 20020219, U.S. Pat. No. 5,225,539,
US19910782717 19911025, U.S. Pat. No. 6,548,640, US19950452462
19950526, U.S. Pat. No. 5,624,821, and US19950479752 19950607. In
certain embodiments, it may be desirable to generate amino acid
sequence variants of these humanized antibodies, particularly where
these improve the binding affinity or other biological properties
of the antibody.
[0306] Amino acid sequence variants of the anti-LPA antibody are
prepared by introducing appropriate nucleotide changes into the
anti-LPA antibody DNA, or by peptide synthesis. Such variants
include, for example, deletions from, and/or insertions into and/or
substitutions of, residues within the amino acid sequences of the
anti-LPA antibodies of the examples herein. Any combination of
deletion, insertion, and substitution is made to arrive at the
final construct, provided that the final construct possesses the
desired characteristics. The amino acid changes also may alter
post-translational processes of the humanized or variant anti-LPA
antibody, such as changing the number or position of glycosylation
sites.
[0307] A useful method for identification of certain residues or
regions of the anti-LPA antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis," as described
by Cunningham and Wells Science, 244:1081-1085 (1989). Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
LPA antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, alanine scanning or random mutagenesis is
conducted at the target codon or region and the expressed anti-LPA
antibody variants are screened for the desired activity. Amino acid
sequence insertions include amino- and/or carboxyl-terminal fusions
ranging in length from one residue to polypeptides containing a
hundred or more residues, as well as intrasequence insertions of
single or multiple amino acid residues. Examples of terminal
insertions include an N-terminal methionyl residue or the antibody
fused to an epitope tag. Other insertional variants include the
fusion of an enzyme or a polypeptide which increases the serum
half-life of the antibody to the N- or C-terminus of the
antibody.
[0308] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue
removed from the antibody molecule and a different residue inserted
in its place. The sites of greatest interest for substitutional
mutagenesis include the hypervariable regions, but FR alterations
are also contemplated. Conservative substitutions are preferred,
but more substantial changes may be introduced and the products may
be screened. Examples of substitutions are listed below:
[0309] Exemplary Amino Acid Residue Substitutions [0310] Ala (A)
val; leu; ile val [0311] Arg (R) lys; gln; asn lys [0312] Asn (N)
gln; his; asp, lys; gln arg [0313] Asp (D) glu; asn glu [0314] Cys
(C) ser; ala ser [0315] Gln (Q) asn; glu asn [0316] Glu (E) asp;
gln asp [0317] Gly (G) ala ala [0318] His (H) asn; gln; lys; arg
arg [0319] Ile (I) leu; val; met; ala; leu phe; norleucine [0320]
Leu (L) norleucine; ile; val; ile met; ala; phe [0321] Lys (K) arg;
gln; asn arg [0322] Met (M) leu; phe; ile leu [0323] Phe (F) leu;
val; ile; ala; tyr tyr [0324] Pro (P) ala ala [0325] Ser (S) thr
thr [0326] Thr (T) ser ser [0327] Tip (W) tyr; phe tyr [0328] Tyr
(Y) trp; phe; thr; ser phe [0329] Val (V) ile; leu; met; phe; leu
ala; norleucine
[0330] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties:
[0331] (1) hydrophobic: norleucine, met, ala, val, leu, ile;
[0332] (2) neutral hydrophilic: cys, ser, thr;
[0333] (3) acidic: asp, glu;
[0334] (4) basic: asn, gln, his, lys, arg;
[0335] (5) residues that influence chain orientation: gly, pro;
and
[0336] (6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of
one of these classes for another class.
[0337] Any cysteine residue not involved in maintaining the proper
conformation of the antibody also may be substituted, to improve
the oxidative stability of the molecule and prevent aberrant
crosslinking Conversely, cysteine bond(s) may be added to the
antibody to improve its stability (particularly where the antibody
is an antibody fragment such as an Fv fragment).
[0338] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further development will have improved biological
properties relative to the parent antibody from which they are
generated. A convenient way for generating such substitutional
variants is affinity maturation using phage display. Briefly,
several hypervariable region sites (e.g. 6-7 sites) are mutated to
generate all possible amino substitutions at each site. The
antibody variants thus generated are displayed in a monovalent
fashion from filamentous phage particles as fusions to the gene III
product of M13 packaged within each particle. The phage-displayed
variants are then screened for their biological activity (e.g.
binding affinity) as herein disclosed. In order to identify
candidate hypervariable region sites for modification, alanine
scanning mutagenesis can be performed to identify hypervariable
region residues contributing significantly to antigen binding.
Alternatively, or in addition, it may be beneficial to analyze a
crystal structure of the antigen-antibody complex to identify
contact points between the antibody and antigen. Such contact
residues and neighboring residues are candidates for substitution
according to the techniques elaborated herein. Once such variants
are generated, the panel of variants is subjected to screening as
described herein and antibodies with superior properties in one or
more relevant assays may be selected for further development.
[0339] Another type of amino acid variant of the antibody alters
the original glycosylation pattern of the antibody. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, and/or adding one or more glycosylation sites that are
not present in the antibody.
[0340] Glycosylation of antibodies is typically either N-linked
and/or or O-linked. N-linked refers to the attachment of the
carbohydrate moiety to the side chain of an asparagine residue. The
tripeptide sequences asparagine-X-serine and
asparagine-X-threonine, where X is any amino acid except proline,
are the most common recognition sequences for enzymatic attachment
of the carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation
refers to the attachment of one of the sugars
N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid,
most commonly serine or threonine, although 5-hydroxyproline or
5-hydroxylysine may also be used.
[0341] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0342] Nucleic acid molecules encoding amino acid sequence variants
of the anti-sphingolipid antibody are prepared by a variety of
methods known in the art. These methods include, but are not
limited to, isolation from a natural source (in the case of
naturally occurring amino acid sequence variants) or preparation by
oligonucleotide-mediated (or site-directed) mutagenesis, PCR
mutagenesis, and cassette mutagenesis of an earlier prepared
variant or a non-variant version of the anti-sphingolipid
antibody.
[0343] (v) Human Antibodies.
[0344] As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array into such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258(1993); Bruggermann et al., Year in Immuno.,
7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and 5,545,807.
Human antibodies can also be derived from phage-display libraries
(Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J.
Mol. Biol., 222:581-597 (1991); and U.S. Pat. Nos. 5,565,332 and
5,573,905). Human antibodies may also be generated by in vitro
activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).
[0345] (vi) Antibody Fragments.
[0346] In certain embodiments, the anti-LPA agent is an antibody
fragment which retains at least one desired activity, including
antigen binding. Various techniques have been developed for the
production of antibody fragments. Traditionally, these fragments
were derived via proteolytic digestion of intact antibodies (see,
e.g., Morimoto et al., Journal of Biochemical and Biophysical
Methods 24:107-117(1992) and Brennan et al., Science 229:81
(1985)). However, these fragments can now be produced directly by
recombinant host cells. For example, Fab'-SH fragments can be
directly recovered from E. coli and chemically coupled to form
F(ab').sub.2 fragments (Carter et al., Bio/Technology 10:163-167
(1992)). In another embodiment, the F(ab').sub.2 is formed using
the leucine zipper GCN4 to promote assembly of the F(ab').sub.2
molecule. According to another approach, Fv, Fab or F(ab').sub.2
fragments can be isolated directly from recombinant host cell
culture. Other techniques for the production of antibody fragments
will be apparent to the skilled practitioner.
[0347] (vii) Multispecific Antibodies and Other Agents.
[0348] In some embodiments, the anti-LPA agent will comprise a
first binding moiety and a second binding moiety, wherein the first
binding moiety is specifically reactive with a first molecule that
is an LPA or LPA metabolite and the second binding moiety is
specifically reactive with a second molecule that is a molecular
species different from the first molecule. Such agents may comprise
a plurality of first binding moieties, a plurality of second
binding moieties, or a plurality of first binding moieties and a
plurality of second binding moieties. Preferably, the ratio of
first binding moieties to second binding moieties is about 1:1,
although it may range from about 1000:1 to about 1:1000, wherein
the ratio is preferably measured in terms of valency.
[0349] In those embodiments wherein the first moiety is an
antibody, the binding moiety may also be an antibody. In preferred
embodiments, the first and second moieties are linked via a linker
moiety, which may have two to many 100's or even thousand of
valencies for attachment of first and second binding moieties by
one or different chemistries. Examples of bispecific antibodies
include those which are reactive against two different epitopes; in
some embodiment one epitope is an LPA epitope and the second
epitope is another bioactive lipid, e.g., SIP. In other embodiments
the bispecific antibody is reactive against an epitope on LPA and
against an epitope found on the cell surface. This serves to target
the LPA-specific antibody moiety to the cell.
[0350] The compositions of the invention may also comprise a first
agent and a second agent, wherein the first agent comprises a first
binding moiety specifically reactive with a first molecule selected
from the group consisting of an LPA and an LPA metabolite and the
second agent comprises a second binding moiety specifically
reactive with a second molecule that is a molecular species
different than the first molecule. The first and/or second agent
may be an antibody. The ratio of first agent to second agent may
range from about 1,000:1 to 1:1,000, although the preferred ratio
is about 1:1. In preferred embodiments, the agent that interferes
with LPA activity is an antibody specifically reactive with LPA. In
some embodiments, it may be desirable to generate multispecific
(e.g. bispecific) anti-LPA antibodies having binding specificities
for at least two different epitopes. Exemplary bispecific
antibodies may bind to two different epitopes of the LPA.
Altematively, an anti-LPA arm (of the antibody) may be combined
with an arm which binds to a different molecule; for example, S1P
or a cell-surface specific antigen for localization of the antibody
to the cell surface. Bispecific antibodies can be prepared as full
length antibodies or antibody fragments (e.g., F(ab').sub.2
bispecific antibodies).
[0351] According to another approach for making bispecific
antibodies, the interface between a pair of antibody molecules can
be engineered to maximize the percentage of heterodimers that are
recovered from recombinant cell culture. The preferred interface
comprises at least a part of the C.sub.H3 domain of an antibody
constant domain. In this method, one or more small amino acid side
chains from the interface of the first antibody molecule are
replaced with larger side chains (e.g., tyrosine or tryptophan).
Compensatory "cavities" of identical or similar size to the large
side chain(s) are created on the interface of the second antibody
molecule by replacing large amino acid side chains with smaller
ones (e.g., alanine or threonine). This provides a mechanism for
increasing the yield of the heterodimer over other unwanted
end-products such as homodimers. See WO96/27011 published Sep. 6,
1996.
[0352] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0353] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science 229:81 (1985) describe a procedure
wherein intact antibodies are proteolytically cleaved to generate
F(ab').sub.2 fragments. These fragments are reduced in the presence
of the dithiol complexing agent sodium arsenite to stabilize
vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes. In yet a further embodiment,
Fab'-SH fragments directly recovered from E. coli can be chemically
coupled in vitro to form bispecific antibodies. Shalaby et al., J.
Exp. Med. 175:217-225 (1992).
[0354] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker that is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See Gruber et al., J. Immunol.
152:5368 (1994). Alternatively, the bispecific antibody may be a
"linear antibody" produced as described in Zapata et al. Protein
Eng. 8(10):1057-1062 (1995).
[0355] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147:60 (1991).
[0356] The antibody (or polymer or polypeptide) of the invention
comprising one or more binding sites per arm or fragment thereof
will be referred to herein as "multivalent" antibody. For example a
"bivalent" antibody of the invention comprises two binding sites
per Fab or fragment thereof whereas a "trivalent" polypeptide of
the invention comprises three binding sites per Fab or fragment
thereof. In a multivalent polymer of the invention, the two or more
binding sites per Fab may be binding to the same or different
antigens. For example, the two or more binding sites in a
multivalent polypeptide of the invention may be directed against
the same antigen, for example against the same parts or epitopes of
said antigen or against two or more same or different parts or
epitopes of said antigen; and/or may be directed against different
antigens; or a combination thereof. Thus, a bivalent polypeptide of
the invention for example may comprise two identical binding sites,
may comprise a first binding sites directed against a first part or
epitope of an antigen and a second binding site directed against
the same part or epitope of said antigen or against another part or
epitope of said antigen; or may comprise a first binding sites
directed against a first part or epitope of an antigen and a second
binding site directed against the a different antigen. However, as
will be clear from the description hereinabove, the invention is
not limited thereto, in the sense that a multivalent polypeptide of
the invention may comprise any number of binding sites directed
against the same or different antigens. In one embodiment the
multivalent polypeptide comprises at least two ligand binding
elements, one of which contains one or more CDR peptide sequences
shown herein. In another embodiment there multivalent polypeptide
comprises three ligand binding sites, each independently selected
from the CDR sequences disclosed herein.
[0357] At least one of the ligand binding elements binds LPA. In
one embodiment at least one of the ligand binding elements binds
another target; in another embodiment, all of the ligand binding
elements bind LPA. In one embodiment there are up to to 10,000
binding elements in a multivalent binding molecule, and the ligand
binding elements may be linked to a scaffold.
[0358] The antibody (or polymer or polypeptide) of the invention
that contains at least two binding sites per Fab or fragment
thereof, in which at least one binding site is directed against a
first antigen and a second binding site directed against a second
antigen different from the first antigen, will also be referred to
as "multispecific." Thus, a "bispecific" polymer comprises at least
one site directed against a first antigen and at least one a second
site directed against a second antigen, whereas a "trispecific" is
a polymer that comprises at least one binding site directed against
a first antigen, at least one further binding site directed against
a second antigen, and at least one further binding site directed
against a third antigen; etc. Accordingly, in their simplest form,
a bispecific polypeptide of the invention is a bivalent polypeptide
(per Fab) of the invention. However, as will be clear from the
description hereinabove, the invention is not limited thereto, in
the sense that a multispecific polypeptide of the invention may
comprise any number of binding sites directed against two or more
different antigens.
[0359] (viii) Other Modifications.
[0360] Other modifications of the anti-LPA antibody are
contemplated. For example, the invention also pertains to
immunoconjugates comprising the antibody described herein
conjugated to a cytotoxic agent such as a toxin (e.g., an
enzymatically active toxin of bacterial, fungal, plant or animal
origin, or fragments thereof), or a radioactive isotope (for
example, a radioconjugate). Conjugates are made using a variety of
bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
[0361] The anti-LPA antibodies disclosed herein may also be
formulated as immunoliposomes. Liposomes containing the antibody
are prepared by methods known in the art, such as described in
Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et
al., Proc. Natl Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos.
4,485,045 and 4,544,545. Liposomes with enhanced circulation time
are disclosed in U.S. Pat. No. 5,013,556. For example, liposomes
can be generated by the reverse phase evaporation method with a
lipid composition comprising phosphatidyl choline, cholesterol and
PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are
extruded through filters of defined pore size to yield liposomes
with the desired diameter. Fab' fragments of the antibody of the
present invention can be conjugated to the liposomes as described
in Martin et al., J. Biol. Chem. 257:286-288 (1982) via a disulfide
interchange reaction. Another active ingredient is optionally
contained within the liposome.
[0362] Enzymes or other polypeptides can be covalently bound to the
anti-LPA antibodies by techniques well known in the art such as the
use of the heterobifunctional crosslinking reagents discussed
above. Alternatively, fusion proteins comprising at least the
antigen binding region of an antibody of the invention linked to at
least a functionally active portion of an enzyme of the invention
can be constructed using recombinant DNA techniques well known in
the art (see, e.g., Neuberger et al., Nature 312:604-608
(1984)).
[0363] In certain embodiments of the invention, it may be desirable
to use an antibody fragment, rather than an intact antibody, to
increase penetration of target tissues and cells, for example. In
this case, it may be desirable to modify the antibody fragment in
order to increase its serum half life. This may be achieved, for
example, by incorporation of a salvage receptor binding epitope
into the antibody fragment (e.g., by mutation of the appropriate
region in the antibody fragment or by incorporating the epitope
into a peptide tag that is then fused to the antibody fragment at
either end or in the middle, e.g., by DNA or peptide synthesis).
See WO96/32478 published Oct. 17, 1996.
[0364] Covalent modifications of the anti-LPA antibody are also
included within the scope of this invention. They may be made by
chemical synthesis or by enzymatic or chemical cleavage of the
antibody, if applicable. Other types of covalent modifications of
the antibody are introduced into the molecule by reacting targeted
amino acid residues of the antibody with an organic derivatizing
agent that is capable of reacting with selected side chains or the
N- or C-terminal residues. Exemplary covalent modifications of
polypeptides are described in U.S. Pat. No. 5,534,615, specifically
incorporated herein by reference. A preferred type of covalent
modification of the antibody comprises linking the antibody to one
of a variety of nonproteinaceous polymers, e.g., polyethylene
glycol, polypropylene glycol, or polyoxyalkylenes, in the manner
set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;
4,670,417; 4,791,192 or 4,179,337.
[0365] b. Vectors, Host Cells and Recombinant Methods
[0366] The invention also provides isolated nucleic acid encoding
the anti-LPA antibody, vectors and host cells comprising the
nucleic acid, and recombinant techniques for the production of the
antibody.
[0367] For recombinant production of the antibody, the nucleic acid
encoding it may be isolated and inserted into a replicable vector
for further cloning (amplification of the DNA) or for expression.
In another embodiment, the antibody may be produced by homologous
recombination, e.g. as described in U.S. Pat. No. 5,204,244,
specifically incorporated herein by reference. DNA encoding the
monoclonal antibody is readily isolated and sequenced using
conventional procedures (e.g., by using oligonucleotide probes that
are capable of binding specifically to genes encoding the heavy and
light chains of the antibody). Many vectors are available. The
vector components generally include, but are not limited to, one or
more of the following: a signal sequence, an origin of replication,
one or more marker genes, an enhancer element, a promoter, and a
transcription termination sequence, e.g., as described in U.S. Pat.
No. 5,534,615 issued Jul. 9, 1996 and specifically incorporated
herein by reference.
[0368] Suitable host cells for cloning or expressing the DNA in the
vectors herein are the prokaryote, yeast, or higher eukaryote cells
described above. Suitable prokaryotes for this purpose include
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. One preferred E. coli cloning host is E. coli 294
(ATCC 31,446), although other strains such as E. coli B, E. coli
X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.
These examples are illustrative rather than limiting.
[0369] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for anti-sphingolipid antibody-encoding vectors. Saccharomyces
cerevisiae, or common baker's yeast, is the most commonly used
among lower eukaryotic host microorganisms. However, a number of
other genera, species, and strains are commonly available and
useful herein, such as Schizosaccharomyces pombe; Kluyveromyces
hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K.
bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii
(ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,
and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP
183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora
crassa; Schwanniomyces such as Schwanniomyces occidentalis; and
filamentous fungi such as, e.g., Neurospora, Penicillium,
Tolypocladium, and Aspergillus hosts such as A. nidulans and A.
niger.
[0370] Suitable host cells for the expression of glycosylated
anti-sphingolipid antibodies are derived from
multicellularorganisms. Examples of invertebrate cells include
plant and insect cells. Numerous baculoviral strains and variants
and corresponding permissive insect host cells from hosts such as
Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito),
Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly),
and Bombyx mori have been identified. A variety of viral strains
for transfection are publicly available, e.g., the L-1 variant of
Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,
and such viruses may be used as the virus herein according to the
present invention, particularly for transfection of Spodoptera
frugiperda cells. Plant cell cultures of cotton, corn, potato,
soybean, petunia, tomato, and tobacco can also be utilized as
hosts.
[0371] However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure. Examples of useful mammalian host cell
lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC
CRL 1651); human embryonic kidney line (293 or 293 cells subcloned
for growth in suspension culture, Graham et al., J. Gen Virol.
36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);
Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl.
Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather,
Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL
70); African green monkey kidney cells (VERO-76, ATCC CRL-1587);
human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney
cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC
CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells
(Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);
TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982));
MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
[0372] Host cells are transformed with the above-described
expression or cloning vectors for antibody production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0373] The host cells used to produce the antibody of this
invention may be cultured in a variety of media. Commercially
available media such as Ham's F10 (Sigma), Minimal Essential Medium
((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's
Medium ((DMEM), Sigma) are suitable for culturing the host cells.
In addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as
culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM.), trace elements
(defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0374] When using recombinant techniques, the antibody can be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. If the antibody is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, is removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology
10:163-167 (1992) describe a procedure for isolating antibodies
that are secreted to the periplasmic space of E. coli. Briefly,
cell paste is thawed in the presence of sodium acetate (pH 3.5),
EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
Cell debris can be removed by centrifugation. Where the antibody is
secreted into the medium, supernatants from such expression systems
are generally first concentrated using a commercially available
protein concentration filter, for example, an Amicon or Millipore
Pellicon ultrafiltration unit. A protease inhibitor such as PMSF
may be included in any of the foregoing steps to inhibit
proteolysis and antibiotics may be included to prevent the growth
of adventitious contaminants.
[0375] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human heavy chains (Lindmark et al., J. Immunol. Meth.
62:1-13 (1983)). Protein G is recommended for all mouse isotypes
and for human .gamma.3 (Guss et al., EMBO J. 5:15671575 (1986)).
The matrix to which the affinity ligand is attached is most often
agarose, but other matrices are available. Mechanically stable
matrices such as controlled pore glass or
poly(styrenedivinyl)benzene allow for faster flow rates and shorter
processing times than can be achieved with agarose. Where the
antibody comprises a C.sub.H3 domain, the Bakerbond ABX.TM. resin
(J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other
techniques for protein purification, such as fractionation on an
ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin SEPHAROSE.TM.,
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered.
[0376] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed
at low salt concentrations (e.g., from about 0-0.25M salt).
[0377] c. Pharmaceutical Formulations, Dosing and Routes of
Administration
[0378] The present invention provides anti-LPA antibodies and
related compositions and methods to reduce blood and tissue levels
of the bioactive lipid, LPA.
[0379] The therapeutic methods and compositions of the invention
are said to be "LPA-based" in order to indicate that these
therapies can change the relative, absolute or effective
concentration(s) of undesirable or toxic lipids "Undesirable
lipids" include toxic bioactive lipids, as well as metabolites,
particularly metabolic precursors, of toxic lipids. One example of
an undesirable bioactive lipid of particular interest is LPA.
[0380] One way to control the amount of undesirable LPA in a
patient is by providing a composition that comprises one or more
anti-LPA antibodies to bind one or more LPAs, thereby acting as
therapeutic "sponges" that reduce the level of free undesirable
LPA. When a compound is stated to be "free," the compound is not in
any way restricted from reaching the site or sites where it exerts
its undesirable effects. Typically, a free compound is present in
blood and tissue, which either is or contains the site(s) of action
of the free compound, or from which a compound can freely migrate
to its site(s) of action. A free compound may also be available to
be acted upon by any enzyme that converts the compound into an
undesirable compound.
[0381] Anti-LPA antibodies may be formulated in a pharmaceutical
composition that is useful for a variety of purposes, including the
treatment of diseases, disorders or physical trauma. Pharmaceutical
compositions comprising one or more anti-LPA antibodies of the
invention may be incorporated into kits and medical devices for
such treatment. Medical devices may be used to administer the
pharmaceutical compositions of the invention to a patient in need
thereof, and according to one embodiment of the invention, kits are
provided that include such devices. Such devices and kits may be
designed for routine administration, including self-administration,
of the pharmaceutical compositions of the invention. Such devices
and kits may also be designed for emergency use, for example, in
ambulances or emergency rooms, or during surgery, or in activities
where injury is possible but where full medical attention may not
be immediately forthcoming (for example, hiking and camping, or
combat situations).
[0382] Therapeutic formulations of the antibody are prepared for
storage by mixing the antibody having the desired degree of purity
with optional physiologically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980)), in the form of lyophilized formulations or
aqueous solutions. Acceptable carriers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0383] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0384] The active ingredients may also be entrapped in microcapsule
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0385] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished for instance by filtration
through sterile filtration membranes.
[0386] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsule. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the Lupron Depot.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies remain in
the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0387] For therapeutic applications, the anti-LPA agents, e.g.,
antibodies, of the invention are administered to a mammal,
preferably a human, in a pharmaceutically acceptable dosage form
such as those discussed above, including those that may be
administered to a human intravenously as a bolus or by continuous
infusion over a period of time, or by intramuscular,
intraperitoneal, intra-cerebrospinal, subcutaneous,
intra-articular, intrasynovial, intrathecal, oral, topical, or
inhalation routes.
[0388] For the prevention or treatment of disease, the appropriate
dosage of antibody will depend on the type of disease to be
treated, as defined above, the severity and course of the disease,
whether the antibody is administered for preventive or therapeutic
purposes, previous therapy, the patient's clinical history and
response to the antibody, and the discretion of the attending
physician. The antibody is suitably administered to the patient at
one time or over a series of treatments.
[0389] Depending on the type and severity of the disease, about 1
.mu.g/kg to about 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an
initial candidate dosage for administration to the patient,
whether, for example, by one or more separate administrations, or
by continuous infusion. A typical daily or weekly dosage might
range from about 1 .mu.g/kg to about 20 mg/kg or more, depending on
the factors mentioned above. For repeated administrations over
several days or longer, depending on the condition, the treatment
is repeated until a desired suppression of disease symptoms occurs.
However, other dosage regimens may be useful. The progress of this
therapy is easily monitored by conventional techniques and assays,
including, for example, radiographic imaging. Detection methods
using the antibody to determine LPA levels in bodily fluids or
tissues may be used in order to optimize patient exposure to the
therapeutic antibody.
[0390] According to another embodiment of the invention, the
composition comprising an agent, e.g, a mAb, that interferes with
LPA activity is administered as a monotherapy, while in other
preferred embodiments, the composition comprising the agent that
interferes with LPA activity is administered as part of a
combination therapy. In some cases the effectiveness of the
antibody in preventing or treating disease may be improved by
administering the antibody serially or in combination with another
agent that is effective for those purposes, such as a
chemotherapeutic drug for treatment of cancer. In other cases, the
anti-LPA agent may serve to enhance or sensitize cells to
chemotherapeutic treatment, thus permitting efficacy at lower doses
and with lower toxicity. Preferred combination therapies include,
in addition to administration of the composition comprising an
agent that interferes with LPA activity, delivering a second
therapeutic regimen selected from the group consisting of
administration of a chemotherapeutic agent, radiation therapy,
surgery, and a combination of any of the foregoing.
[0391] Such other agents may be present in the composition being
administered or may be administered separately. Also, the antibody
is suitably administered serially or in combination with the other
agent or modality, e.g., chemotherapeutic drug or radiation for
treatment of cancer.
[0392] Exemplary routes of administration of an immune-derived
moiety, preferably as part of a therapeutic composition, include
systemic administration, parenteral administration (e.g., via
injection via an intravenous, intramuscular, intrathecal, epidural
or subcutaneous route), transdermal, intradermal or transmucosal
delivery, intraocular or periocular injection, mucosal or topical
administration or by inhalation.
[0393] d. Research and Diagnostic, Including Clinical Diagnostic,
Uses for the Anti-LPA Agents of the Invention
[0394] The anti-LPA agents, e.g., antibodies, of the invention may
be used to detect and/or purify LPA, e.g., from bodily
fluid(s).
[0395] For use of anti-LPA antibodies as affinity purification
agents, the antibodies are immobilized on a solid support such as
beads, a Sephadex resin or filter paper, using methods well known
in the art. The immobilized antibody is contacted with a sample
containing the LPA to be purified, and thereafter the support is
washed with a suitable solvent that will remove substantially all
the material in the sample except the LPA, which is bound to the
immobilized antibody. Finally, the support is washed with another
suitable solvent, such as glycine buffer, for instance between pH 3
to pH 5.0, that will release the LPA from the antibody.
[0396] Anti-LPA antibodies may also be useful in diagnostic assays
for LPA, e.g., detecting its presence in specific cells, tissues,
or bodily fluids. Such diagnostic methods may be useful in
diagnosis, e.g., of a hyperproliferative disease or disorder. Thus
clinical diagnostic uses as well as research uses are comprehended
by the invention. In these methods, the anti-LPA antibody is
preferably attached to a solid support, e.g., bead, column, plate,
gel, filter, membrane, etc.
[0397] For diagnostic applications, the antibody may be labeled
with a detectable moiety. Numerous labels are available which can
be generally grouped into the following categories:
[0398] (a) Radioisotopes, such as .sup.35S, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I. The antibody can be labeled with the
radioisotope using the techniques described in Current Protocols in
Immunology, Volumes 1 and 2, Coligen et al., Ed.
Wiley-Interscience, New York, N.Y., Pubs. (1991), for example, and
radioactivity can be measured using scintillation counting.
[0399] (b) Fluorescent labels such as rare earth chelates (europium
chelates) or fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are
available. The fluorescent labels can be conjugated to the antibody
using the techniques disclosed in Current Protocols in Immunology,
supra, for example. Fluorescence can be quantified using a
fluorimeter.
[0400] (c) Various enzyme-substrate labels are available and U.S.
Pat. No. 4,275,149 provides a review of some of these. The enzyme
generally catalyzes a chemical alteration of the chromogenic
substrate that can be measured using various techniques. For
example, the enzyme may catalyze a color change in a substrate,
which can be measured spectrophotometrically. Alternatively, the
enzyme may alter the fluorescence or chemiluminescence of the
substrate. Techniques for quantifying a change in fluorescence are
described above. The chemiluminescent substrate becomes
electronically excited by a chemical reaction and may then emit
light that can be measured (using a chemiluminometer, for example)
or donates energy to a fluorescent acceptor. Examples of enzymatic
labels include luciferases (e.g., firefly luciferase and bacterial
luciferase; U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, malate dehydrogenase, urease,
peroxidase such as horseradish peroxidase (HRPO), alkaline
phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide
oxidases (e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase), heterocyclicoxidases (such as
uricase and xanthine oxidase), lactoperoxidase, microperoxidase,
and the like. Techniques for conjugating enzymes to antibodies are
described in O'Sullivan et al., Methods for the Preparation of
Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in
Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic
press, New York, 73:147-166 (1981).
[0401] Examples of enzyme-substrate combinations include, for
example:
[0402] (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase
as a substrate, wherein the hydrogen peroxidase oxidizes a dye
precursor (e.g., orthophenylene diamine (OPD) or
3,3',5,5'-tetramethyl benzidine hydrochloride (TMB));
[0403] (ii) alkaline phosphatase (AP) with para-Nitrophenyl
phosphate as chromogenic substrate; and (iii)
.beta.-D-galactosidase (.beta.-D-Gal) with a chromogenic substrate
(e.g., p-nitrophenyl-.beta.-D-galactosidase) or fluorogenic
substrate 4-methylumbelliferyl-.beta.-D-galactosidase.
[0404] Numerous other enzyme-substrate combinations are available
to those skilled in the art. For a general review of these, see
U.S. Pat. Nos. 4,275,149 and 4,318,980.
[0405] Sometimes, the label is indirectly conjugated with the
antibody. The skilled artisan will be aware of various techniques
for achieving this. For example, the antibody can be conjugated
with biotin and any of the three broad categories of labels
mentioned above can be conjugated with avidin, or vice versa.
Biotin binds selectively to avidin and thus, the label can be
conjugated with the antibody in this indirect manner.
Alternatively, to achieve indirect conjugation of the label with
the antibody, the antibody is conjugated with a small hapten (e.g.,
digoxin) and one of the different types of labels mentioned above
is conjugated with an anti-hapten antibody (e.g., anti-digoxin
antibody). Thus, indirect conjugation of the label with the
antibody can be achieved.
[0406] In another embodiment of the invention, the anti-LPA
antibody need not be labeled, and the presence thereof can be
detected, e.g., using a labeled antibody which binds to the
anti-LPA antibody.
[0407] The antibodies of the present invention may be employed in
any known assay method, such as competitive binding assays, direct
and indirect sandwich assays, and immunoprecipitation assays. Zola,
Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC
Press, Inc. 1987).
[0408] Competitive binding assays rely on the ability of a labeled
standard to compete with the test sample analyte for binding with a
limited amount of antibody. The amount of LPA in the test sample is
inversely proportional to the amount of standard that becomes bound
to the antibodies. To facilitate determining the amount of standard
that becomes bound, the antibodies generally are insoluble before
or after the competition, so that the standard and analyte that are
bound to the antibodies may conveniently be separated from the
standard and analyte that remain unbound.
[0409] Sandwich assays involve the use of two antibodies, each
capable of binding to a different immunogenic portion, or epitope,
of the protein to be detected. In a sandwich assay, the test sample
analyte is bound by a first antibody that is immobilized on a solid
support, and thereafter a second antibody binds to the analyte,
thus forming an insoluble three-part complex. See, e.g., U.S. Pat.
No. 4,376,110. The second antibody may itself be labeled with a
detectable moiety (direct sandwich assays) or may be measured using
an anti-immunoglobulin antibody that is labeled with a detectable
moiety (indirect sandwich assay). For example, one type of sandwich
assay is an ELISA assay, in which case the detectable moiety is an
enzyme.
[0410] For immunohistochemistry, the blood or tissue sample may be
fresh or frozen or may be embedded in paraffin and fixed with a
preservative such as formalin, for example.
[0411] The antibodies may also be used for in vivo diagnostic
assays. Generally, the antibody is labeled with a radionuclide
(such as .sup.111In, .sup.99Tc, .sup.14C, .sup.131I, .sup.125I,
.sup.3H, .sup.32P, or .sup.35S) so that the bound target molecule
can be localized using immunoscintillography.
[0412] e. Diagnostic Kits Incorporating the Anti-LPA Agents of the
Invention
[0413] As a matter of convenience, the antibody of the present
invention can be provided in a kit, for example, a packaged
combination of reagents in predetermined amounts with instructions
for performing the diagnostic assay. Where the antibody is labeled
with an enzyme, the kit will include substrates and cofactors
required by the enzyme (e.g., a substrate precursor which provides
the detectable chromophore or fluorophore). In addition, other
additives may be included such as stabilizers, buffers (e.g., a
block buffer or lysis buffer) and the like. The relative amounts of
the various reagents may be varied widely to provide for
concentrations in solution of the reagents which substantially
optimize the sensitivity of the assay. Particularly, the reagents
may be provided as dry powders, usually lyophilized, including
excipients which on dissolution will provide a reagent solution
having the appropriate concentration.
[0414] f. Articles of Manufacture
[0415] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment of the
disorders described above is provided. The article of manufacture
comprises a container and a label. Suitable containers include, for
example, bottles, vials, syringes, and test tubes. The containers
may be formed from a variety of materials such as glass or plastic.
The container holds a composition which is effective for treating
the condition and may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The active
agent in the composition is the anti-sphingolipid antibody. The
label on, or associated with, the container indicates that the
composition is used for treating the condition of choice. The
article of manufacture may further comprise a second container
comprising a pharmaceutically-acceptable buffer, such as
phosphate-buffered saline, Ringer's solution and dextrose solution.
It may further include other materials desirable from a commercial
and user standpoint, including other buffers, diluents, filters,
needles, syringes, and package inserts with instructions for
use.
[0416] The invention will be better understood by reference to the
following Examples, which are intended to merely illustrate the
best mode now known for practicing the invention. The scope of the
invention is not to be considered limited thereto.
EXAMPLES
[0417] The invention will be further described by reference to the
following detailed examples. These Examples are in no way to be
considered to limit the scope of the invention in any manner.
Example 1
Synthetic Scheme for Making a Representative Thiolated Analog of
S1P
[0418] The synthetic approach described in this example results in
the preparation of an antigen by serial addition of structural
elements using primarily conventional organic chemistry. A scheme
for the approach described in this example is provided in FIG. 1,
and the compound numbers in the synthetic description below refer
to the numbered structures in FIG. 1.
[0419] This synthetic approach began with the commercially
available 15-hydroxyl pentadecyne, 1, and activation by methyl
sulphonyl chloride of the 15-hydroxy group to facilitate hydroxyl
substitution to produce the sulphonate, 2. Substitution of the
sulphonate with t-butyl thiol yielded the protected thioether, 3,
which was condensed with Garner's aldehyde to produce 4. Gentle
reduction of the alkyne moiety to an alkene (5), followed by acid
catalyzed opening of the oxazolidene ring yielded S-protected and
N-protected thiol substituted sphingosine, 6. During this last
step, re-derivatization with di-t-butyl dicarbonate was employed to
mitigate loss of the N-BOC group during the acid-catalyzed ring
opening.
[0420] As will be appreciated, compound 6 can itself be used as an
antigen for preparing haptens to raise antibodies to sphingosine,
or, alternatively, as starting material for two different synthetic
approaches to prepare a thiolated S1P analog. In one approach,
compound 6 phosphorylation with trimethyl phosphate produced
compound 7. Treatment of compound 7 with trimethylsilyl bromide
removed both methyl groups from the phosphate and the
t-butyloxycarbonyl group from the primary amine, leaving compound 8
with the t-butyl group on the sulfur as the only protecting group.
To remove this group, the t-butyl group was displaced by NBS to
form the disulfide, 9, which was then reduced to form the thiolated
SIP analog, 10.
[0421] Another approach involved treating compound 6 directly with
NBSCl to form the disulfide, 11, which was then reduced to form the
N-protected thiolated S1P analog, 12. Treatment of this compound
with mild acid yielded the thiolated sphingosine analog, 13, which
can be phosphorylated enzymatically with, e.g., sphingosine kinase,
to yield the thiolated SIP analog, 10.
[0422] Modifications of the presented synthetic approach are
possible, particularly with regard to the selection of protecting
and de-protecting reagents, e.g., the use of trimethyl disulfide
triflate described in Example 3 to de-protect the thiol.
[0423] Compound 2.
[0424] DCM (400 mL) was added to a 500 mL RB flask charged with 1
(10.3 g, 45.89 mmol), and the resulting solution cooled to
0.degree. C. Next, TEA (8.34 g, 82.60 mmol, 9.5 mL) was added all
at once followed by MsCl (7.88 g, 68.84 mmol, 5.3 mL) added drop
wise over 10 min. The reaction was allowed to stir at RT for 0.5 h
or until the disappearance of starting material (R.sub.f=0.65, 5:1
hexanes: EtOAc). The reaction was quenched with NH.sub.4Cl (300 mL)
and extracted (2.times.200 mL) DCM. The organic layers were dried
over MgSO.sub.4, filtered and the filtrate evaporated to a solid
(13.86 g, 99.8% yield). .sup.1H NMR (CDCl.sub.3) .delta. 4.20 (t,
J=6.5 Hz, 2H), 2.98 (s, 3H), 2.59 (td, J=7 Hz, 3 Hz, 2H), 1.917 (t,
J=3 Hz, 1H), 1.72 (quintet, J=7.5 Hz, 2H), 1.505 (quintet, J=7.5
Hz, 2H), 1.37 (br s, 4H), 1.27 (br s, 14H). .sup.13C{.sup.1H} NMR
(CDCl.sub.3) .delta. 85.45, 70.90, 68.72, 46.69, 38.04, 30.22,
30.15, 30.14, 30.07, 29.81, 29.76, 29.69, 29.42, 29.17, 26.09,
19.06, 9.31. The principal ion observed in a HRMS analysis (ES-TOF)
of compound 2 was m/z=325.1804 (calculated for
C.sub.16H.sub.30O.sub.3S: M+Na.sup.+ 325.1808).
[0425] Compound 3.
[0426] A three-neck 1 L RB flask was charged with t-butylthiol
(4.54 g, 50.40 mmol) and THF (200 mL) and then placed into an ice
bath. n-BuLi (31.5 mL of 1.6 M in hexanes) was added over 30 min.
Next, compound 2 (13.86 g, 45.82 mmol), dissolved in THF (100 mL),
was added over 2 min. The reaction is allowed to stir for 1 hour or
until starting material disappeared (R.sub.f=0.7, 1:1
hexanes/EtOAc). The reaction was quenched with saturated NH.sub.4Cl
(500 mL) and extracted with EtO.sub.2 (2.times.250 mL), dried over
MgSO.sub.4, filtered, and the filtrate evaporated to yield a yellow
oil (11.67 g, 86% yield). .sup.1H NMR (CDCl.sub.3) .delta. 2.52 (t,
J=7.5 Hz, 2H), 2.18 (td, J=7 Hz, 2.5 Hz, 2H), 1.93 (t, J=2.5 Hz,
1H), 1.55 (quintet, J=7.5 Hz, 2H), 1.51 (quintet, J=7 Hz, 2H), 1.38
(br s, 4H), 1.33 (s, 9H), 1.26 (s, 14H). .sup.13C {.sup.1H} NMR
(CDCl.sub.3) .delta. 85.42, 68.71, 68.67, 54.07, 42.37, 31.68,
30.58, 30.28, 30.26, 30.19, 30.17, 29.98, 29.78, 29.44, 29.19,
29.02, 19.08.
[0427] Compound 4.
[0428] A 250 mL Schlenk flask charged with compound 3 (5.0 g, 16.85
mmol) was evacuated and filled with nitrogen three times before dry
THF (150 mL) was added. The resulting solution cooled to
-78.degree. C. Next, n-BuLi (10.5 mL of 1.6M in hexanes) was added
over 2 min. and the reaction mixture was stirred for 18 min. at
-78.degree. C. before the cooling bath was removed for 20 min. The
dry ice bath was returned. After 15 min., Garner's aldeyde (3.36 g,
14.65 mmol) in dry THF (10 mL) was then added over 5 min. After 20
min., the cooling bath was removed. Thin layer chromatography (TLC)
after 2.7 hr. showed that the Garner's aldehyde was gone. The
reaction was quenched with saturated aqueous NH.sub.4Cl (300 mL)
and extracted with Et.sub.2O (2.times.250 mL). The combined
Et.sub.2O phases were dried over Na.sub.2SO.sub.4, filtered, and
the filtrate evaporated to give crude compound 4 and its syn
diastereomer (not shown in FIG. 1) as a yellow oil (9.06 g). This
material was then used in the next step without further
purification.
[0429] Compound 5.
[0430] To reduce the triple bond in compound 4, the oil was
dissolved in dry Et.sub.2O (100 mL) under nitrogen. RED-Al (20 mL,
65% in toluene) was slowly added to the resulting solution at RT to
control the evolution of hydrogen gas (H.sub.2). The reaction was
allowed to stir at RT overnight or when TLC showed the
disappearance of the starting material (R.sub.f=0.6 in 1:1
EtOAc:hexanes) and quenched slowly with cold MeOH or aqueous
NH.sub.4Cl to control the evolution of H.sub.2. The resulting white
suspension was filtered through a Celite pad and the filtrate was
extracted with EtOAc (2.times.400 mL). Combined EtOAc extracts were
dried over MgSO.sub.4, filtered, and the filtrate evaporated to
leave crude compound 5 and its syn diastereomer (not shown in FIG.
1) as a yellow oil (7.59 g).
[0431] Compound 6.
[0432] The oil containing compound 5 was dissolved in MeOH (200
mL), PTSA hydrate (0.63 g) was added, and the solution stirred at
RT for 1 day and then at 50.degree. C. for 2 days, at which point
TLC suggested that all starting material (5) was gone. However,
some polar material was present, suggesting that the acid had
partially cleaved the BOC group. The reaction was worked up by
adding saturated aqueous NH.sub.4Cl (400 mL), and extracted with
ether (3.times.300 mL). The combined ether phases were dried over
Na.sub.2SO.sub.4, filtered, and the filtrate evaporated to dryness,
leaving 5.14 g of oil. In order to re-protect whatever amine had
formed, the crude product was dissolved in CH.sub.2Cl.sub.2 (150
mL), to which was added BOC.sub.2O (2.44 g) and TEA (1.7 g). When
TLC (1:1 hexanes/EtOAc) showed no more material remaining on the
baseline, saturated aqueous NH.sub.4Cl (200 mL) was added, and,
after separating the organic phase, the mixture was extracted with
CH.sub.2Cl.sub.2 (3.times.200 mL). Combined extracts were dried
over Na.sub.2SO.sub.4, filtered, and the filtrated concentrated to
dryness to yield a yellow oil (7.7 g) which was chromatographed on
a silica column using a gradient of hexanes/EtOAc (up to 1:1) to
separate the diastereomers. By TLC using 1:1 PE/EtOAc, the R.sub.f
for the anti isomer, compound 6, was 0.45. For the syn isomer (not
shown in FIG. 1) the R.sub.f was 0.40. The yield of compound 6 was
2.45 g (39% overall based on Garner's aldehyde). .sup.1H NMR of
anti isomer (CDCl.sub.3) .delta. 1.26 (br s, 20H), 1.32 (s, 9H),
1.45 (s, 9H), 1.56 (quintet, 2H, J=8 Hz), 2.06 (q, 2H, J=7 Hz),
2.52 (t, 2H, J=7 Hz), 2.55 (br s, 2H), 3.60 (br s, 1H), 3.72 (ddd,
1H, J=11.5 Hz, 7.0 Hz, 3.5 Hz), 3.94 (dt, 1H, J=11.5 Hz, 3.5 Hz),
4.32 (d, 1H, J=4.5 Hz), 5.28 (br s, 1H), 5.54 (dd, 1H, J=15.5 Hz,
6.5 Hz), 5.78 (dt, 1H, J=15.5 Hz, 6.5 Hz). .sup.13C {'H} NMR
(CDCl.sub.3) .delta. 156.95, 134.80, 129.66, 80.47, 75.46, 63.33,
56.17, 42.44, 32.98, 31.70, 30.58, 30.32, 30.31, 30.28, 30.20,
30.16, 30.00, 29.89, 29.80, 29.08, 29.03.
[0433] Anal. Calculated for C.sub.27H.sub.53NO.sub.4S: C, 66.48; H,
10.95; N, 2.87. Found: C, 65.98; H, 10.46; N, 2.48.
[0434] Compound 7.
[0435] To a solution of the alcohol compound 6 (609.5 mg, 1.25
mmol) dissolved in dry pyridine (2 mL) was added CBr.sub.4 (647.2
mg, 1.95 mmol, 1.56 equiv). The flask was cooled in an ice bath and
P(OMe).sub.3 (284.7 mg, 2.29 mmol, 1.84 equiv) was added drop wise
over 2 min. After 4 min. the ice bath was removed and after 12 hr.
the mixture was diluted with ether (20 mL). The resulting mixture
washed with aqueous HCl (10 mL, 2 N) to form an emulsion which
separated on dilution with water (20 mL). The aqueous phase was
extracted with ether (2.times.10 mL), then EtOAc (2.times.10 mL).
The ether extracts and first EtOAc extract were combined and washed
with aqueous HCl (10 mL, 2 N), water (10 mL), and saturated aqueous
NaHCO.sub.3 (10 mL). The last EtOAc extract was used to
back-extract the aqueous washes. Combined organic phases were dried
over MgSO.sub.4, filtered, and the filtrate concentrated to leave
crude product (1.16 g), which was purified by flash chromatography
over silica (3.times.22 cm column) using CH.sub.2Cl.sub.2, then
CH.sub.2Cl.sub.2-EtOAc (1:20, 1:6, 1:3, and 1:1--product started to
elute, 6:4, 7:3). Early fractions contained 56.9 mg of oil. Later
fractions provided product (compound 7, 476.6 mg, 64%) as clear,
colorless oil.
[0436] Anal. Calculated for C.sub.29H.sub.58NO.sub.7PS (595.82): C,
58.46; H, 9.81; N, 2.35. Found: C, 58.09; H, 9.69; N, 2.41.
[0437] Compound 8.
[0438] A flask containing compound 7 (333.0 mg, 0.559 mmol) and a
stir bar was evacuated and filled with nitrogen. Acetonitrile (4
mL, distilled from CaH.sub.2) was injected by syringe and the flask
now containing a solution was cooled in an ice bath. Using a
syringe, (CH.sub.3).sub.3SiBr (438.7 mg, 2.87 mmol, 5.13 equiv.)
was added over the course of 1 min. After 35 min., the upper part
of the flask was rinsed with an additional portion of acetonitrile
(1 mL) and the ice bath was removed. After another 80 min., an
aliquot was removed, the solution dried by blowing nitrogen gas
over it, and the residue analyzed by .sup.1H NMR in CDCl.sub.3,
which showed only traces of peaks ascribed to P--OCH.sub.3
moieties. After 20 min., water (0.2 mL) was added to the reaction
mixture, followed by the CDCl.sub.3 solution used to analyze the
aliquot, and the mixture was concentrated to ca. 0.5 mL volume on a
rotary evaporator. Using acetone (3 mL) in portions the residue was
transferred to a tared test tube, forming a pale brown solution.
Water (3 mL) was added in portions. After addition of 0.3 mL,
cloudiness was seen. After a total of 1 mL, a gummy precipitate had
formed. As an additional 0.6 mL of water was added, more cloudiness
and gum separated, but the final portion of water seemed not to
change the appearance of the mixture. Overall, this process was
accomplished over a period of several hours. The tube was
centrifuged and the supernatant removed by pipet. The solid, no
longer gummy, was dried over P.sub.4O.sub.10 in vacuo, leaving
compound 8 (258.2 mg, 95%) as a monohydrate.
[0439] Anal. Calculated. for C.sub.22H.sub.46NO.sub.5PS+H.sub.2O
(485.66): C, 54.40; H, 9.96; N, 2.88. Found: C, 54.59; H, 9.84; N,
2.95.
[0440] Compound 9.
[0441] Compound 8 (202.6 mg, 0.417 mmol) was added in a glove box
to a test tube containing a stir bar, dry THF (3 mL) and glacial
HOAc (3 mL). NBSCl (90 mg, 0.475 mmol, 1.14 equiv) were added, and
after 0.5 hr., a clear solution was obtained. After a total of 9
hr., an aliquot was evaporated to dryness and the residue analyzed
by .sup.1H NMR in CDCl.sub.3. The peaks corresponding to
CH.sub.2StBu and CH.sub.2SSAr suggested that reaction was about 75%
complete, and comparison of the spectrum with that of pure NBSCl in
CDCl.sub.3 suggested that none of the reagent remained in the
reaction. Therefore, an additional portion (24.7 mg, 0.130 mmol,
0.31 equiv) was added, followed 3 hr. later by an additional
portion (19.5 mg, 0.103 mmol, 0.25 equiv). After another 1 hr., the
mixture was transferred to a new test tube using THF (2 mL) to
rinse and water (1 mL) was added.
[0442] Compound 10.
[0443] PMe.sub.3 (82.4 mg, 1.08 mmol, 1.52 times the total amount
of 2-nitrobenzenesulfenyl chloride added) was added to the clear
solution compound 9 described above. The mixture grew warm and
cloudy, with precipitate forming over time. After 4.5 hr., methanol
was added, and the tube centrifuged. The precipitate settled with
difficulty, occupying the bottom 1 cm of the tube. The clear yellow
supernatant was removed using a pipet. Methanol (5 mL, deoxygenated
with nitrogen) was added, the tube was centrifuged, and the
supernatant removed by pipet. This cycle was repeated three times.
When concentrated, the final methanol wash left only 4.4 mg of
residue. The bulk solid residue was dried over P.sub.4O.sub.10 in
vacuo, leaving compound 10 (118.2 mg, 68%) as a
monohydrochloride.
[0444] Anal. Calculated for C.sub.18H.sub.38NO.sub.5S+HCl (417.03):
C, 51.84; H, 9.43; N, 3.36. Found: C, 52.11; H, 9.12; N, 3.30.
[0445] Compound 11.
[0446] Compound 6 (1.45 g, 2.97 mmol) was dissolved in AcOH (20
mL), and NBSCl (0.56 g, 2.97 mmol) was added all at once. The
reaction was allowed to stir for 3 hr. or until the disappearance
of the starting material and appearance of the product was observed
by TLC [product R.sub.f=0.65, starting material R.sub.f=0.45, 1:1
EtOAc/hexanes]. The reaction was concentrated to dryness on a high
vacuum line and the residue dissolved in THF/H.sub.2O (100 mL of
10:1).
[0447] Compound 12.
[0448] Ph.sub.3P (0.2.33 g, 8.91 mmol) was added all at once to the
solution above that contained compound 11 and the reaction was
allowed to stir for 3 hr. or until the starting material
disappeared. The crude reaction mixture was concentrated to dryness
on a high vacuum line, leaving a residue that contained compound
12.
[0449] Compound 13.
[0450] The residue above containing compound 12 was dissolved in
DCM (50 mL) and TFA (10 mL). The mixture was stirred at RT for 5
hr. and concentrated to dryness. The residue was the loaded onto a
column with silica gel and chromatographed with pure DCM, followed
by DCM containing 5% MeOH, then 10% MeOH, to yield the final
product, compound 13, as a sticky white solid (0.45 g, 46% yield
from 5). .sup.1H NMR (CDCl.sub.3) .delta. 1.27 (s), 1.33 (br m,),
1.61 (p, 2H, J=7.5 Hz), 2.03 (br d, 2H, J=7 Hz), 2.53 (q, 2H, J=7.5
Hz), 3.34 (br s, 1H), 3.87 (br d, 2H, J=12 Hz), 4.48 (br s, 2H),
4.58 (br s, 2H), 5.42 (dd, 1H, J=15 Hz, 5.5 Hz), 5.82 (dt, 1H, J=15
Hz, 5.5 Hz), 7.91 (br s, 4H). .sup.13C{.sup.1H} NMR (CDCl.sub.3)
.delta. 136.85, 126.26, 57.08, 34.76, 32.95, 30.40, 30.36, 30.34,
30.25, 30.19, 30.05, 29.80, 29.62, 29.09, 25.34.
Example 2
Synthetic Schemes for Making Thiolated Fatty Acids
[0451] The synthetic approach described in this example details the
preparation of a thiolated fatty acid to be incorporated into a
more complex lipid structure that could be further complexed to a
protein or other carrier and administered to an animal to elicit an
immune response. The approach uses using conventional organic
chemistry. A scheme showing the approach taken in this example is
provided in FIG. 2, and the compound numbers in the synthetic
description below refer to the numbered structures in FIG. 2.
[0452] Two syntheses are described. The first synthesis, for a C-12
thiolated fatty acid, starts with the commercially available
12-dodecanoic acid, compound 14. The bromine is then displaced with
t-butyl thiol to yield the protected C-12 thiolated fatty acid,
compound 15. The second synthesis, for a C-18 thiolated fatty acid,
starts with the commercially available 9-bromo-nonanol (compound
16). The hydroxyl group in compound 16 is protected by addition of
a dihydroyran group and the resulting compound, 17, is dimerized
through activation of half of the brominated material via a
Grignard reaction, followed by addition of the other half. The
18-hydroxy octadecanol (compound 18) produced following
acid-catalyzed removal of the dihydropyran protecting group is
selectively mono-brominated to form compound 19. During this
reaction approximately half of the alcohol groups are activated for
nucleophilic substitution by formation of a methane sulfonic acid
ester. The alcohol is then oxidized to form the 18-bromocarboxylic
acid, compound 20, which is then treated with t-butyl thiol to
displace the bromine and form the protected, thiolated C-18 fatty
acid, compound 21.
[0453] The protected thiolated fatty acids, each a t-butyl
thioether, can be incorporated into a complex lipid and the
protecting group removed using, e.g., one of the de-protecting
approaches described in Examples 1 and 3. The resulting free thiol
then can be used to complex with a protein or other carrier prior
to inoculating animal with the hapten.
[0454] A. Synthesis of a C-12 Thiolated Fatty Acid
[0455] Compound 15.
[0456] t-Butyl thiol (12.93 g, 143 mmol) was added to a dry Schlenk
flask, and Schlenk methods were used to put the system under
nitrogen. Dry, degassed THF (250 mL) was added and the flask cooled
in an ice bath. n-BuLi (55 mL of 2.5 M in hexanes, 137.5 mmol) was
slowly added over 10 min by syringe. The mixture was allowed to
stir at 0.degree. C. for an hour. The bromoacid, compound 14 (10 g,
36 mmol), was added as a solid and the reaction heated and stirred
at 60.degree. C. for 24 hr. The reaction was quenched with 2 M HCl
(250 mL), and extracted with ether (2.times.300 mL). The combined
ethereal layers were dried with magnesium sulfate, filtered, and
the filtrate concentrated by rotary evaporation to yield the
thioether acid, compound 15 (10 g, 99% yield) as a beige powder.
.sup.1H NMR (CDCl.sub.3, 500 MHz) .delta. 1.25-1.35 (br s, 12H),
1.32 (s, 9H), 1.35-1.40 (m, 2H), 1.50-1.60 (m, 2H), 1.60-1.65 (m,
2H), 2.35 (t, 2H, J=7.5 Hz), 2.52 (t, 2H, J=7.5 Hz). Principal ion
in HRMS (ES-TOF) was observed at m/z 311.2020, calculated for
M+Na.sup.+ 311.2015.
[0457] B. Synthesis of a C-12 thiolated fatty acid
[0458] Compound 17.
[0459] A dry Schlenk flask was charged with compound 16 (50 g,
224.2 mmol) and dissolved in dry degassed THF (250 mL) distilled
from sodium/benzophenone. The flask was cooled in an ice bath and
then PTSA (0.5 g, 2.6 mmol) was added. Dry, degassed DHP (36 g,
42.8 mmol) was then added slowly over 5 min. The mixture was
allowed to warm up to RT and left to stir overnight and monitored
by TLC (10:1 PE: EtOAc) until the reaction was deemed done by the
complete disappearance of the spot for the bromoalcohol. TEA (1 g,
10 mmol) was then added to quench the PTSA. The mixture was then
washed with cold sodium bicarbonate solution and extracted with
EtOAc (3.times.250 mL). The organic layers were then dried with
magnesium sulfate and concentrated to yield 68.2 g of crude product
which was purified by column chromatography (10:1 PE: EtOAc) to
yield 60 g (99% yield) of a colorless oil. .sup.1H NMR (CDCl.sub.3,
500 MHz) .delta. 1.31 (br s, 6H), 1.41-1.44 (m, 2H), 1.51-1.62
(obscured multiplets, 6H), 1.69-1.74 (m, 1H), 1.855 (quintet, J=7.6
Hz, 2H), 3.41 (t, J=7 Hz, 2H), 3.48-3.52 (m, 2H), 3.73 (dt, 2H,
J=6.5 Hz), 3.85-3.90 (m, 2H), 4.57 (t, 2H, J=3 Hz).
[0460] Compound 18.
[0461] Magnesium shavings (2.98 g, 125 mmol) were added to a
flame-dried Schlenk flask along with a crystal of iodine. Dry THF
(200 mL) distilled from sodium was then added and the system was
degassed using Schlenk techniques. Compound 17 (30 g, 97 mmol) was
then slowly added to the magnesium over 10 min. and the solution
was placed in an oil bath at 65.degree. C. and allowed to stir
overnight. The reaction was deemed complete by TLC by quenching an
aliquot with acetone and observing the change in RF in a 10:1
PE:EtOAc mixture. The Grignard solution was then transferred by
cannula to a three-necked flask under nitrogen containing
additional compound 17 (30 g, 97 mmol). The flask containing the
resulting mixture was then cooled to 0.degree. C. in an ice bath
and a solution of Li.sub.2CuCl.sub.4 (3 mL of 1 M) was then added
via syringe. The reaction mixture turned a very dark blue within a
few minutes. This mixture was left to stir overnight. The next
morning the reaction was deemed complete by TLC (10:1 PE:EtOAc),
quenched with a saturated NH.sub.4Cl solution, and then extracted
into ether (3.times.250 mL). The ether layers were dried with
magnesium sulfate and concentrated to yield crude product (40 g),
which was dissolved in MeOH. Concentrated HCl (0.5 mL) was then
added, which resulted in the formation of a white emulsion, which
was left to stir for 3 hr. The white emulsion was then filtered to
yield 16 g (58% yield) of the pure diol, compound 18. .sup.1H NMR
(CDCl.sub.3, 200 MHz) .delta. 1.26 (br s, 24H), 1.41-1.42 (m, 4H),
1.51-1.68 (m, 4H), 3.65 (t, 4H, J=6.5 Hz).
[0462] Compound 19.
[0463] The symmetrical diol, compound 18 (11 g, 38.5 mmol), was
added to a dry Schlenk flask under nitrogen, then dry THF (700 mL)
distilled from sodium was added. The system was degassed and the
flask put in an ice bath.
[0464] Diisopropylethylamine (6.82 mL, 42.3 mmol) was added via
syringe, followed by MsCl (3.96 g, 34.4 mmol) added slowly, and the
mixture was left to stir for 1 hr. The reaction was quenched with
saturated NaH.sub.2PO.sub.4 solution (300 mL), and then extracted
with EtOAc (3.times.300 mL). The organic layers were then combined,
dried with MgSO.sub.4, and concentrated to yield 14 g of a mixture
of the diol, monomesylate, and dimesylate. NMR showed a 1:0.8
mixture of CH.sub.2OH:CH.sub.2OMs protons. The mixture was then
dissolved in dry THF (500 mL), deoxygenated, and to it was added
LiBr (3.5 g, 40.23 mmol). This mixture was allowed reflux
overnight, upon which the reaction was quenched with water (150
mL), and extracted with EtOAc (3.times.250 mL). The organic layer
was then dried with MgSO.sub.4, and concentrated to yield a mixture
of brominated products that were then purified by flash
chromatography (DCM) to yield compound 19 (3.1 g, 25% yield) as a
white powder. .sup.1H NMR (CDCl.sub.3, 500 MHz) .delta. 1.26 (br s,
26H), 1.38-1.46 (m, 2H), 1.55 (quintet, 2H, J=7.5 Hz), 1.85
(quintet, 2H, J=7.5 Hz), 3.403 (t, 2H, J=6.8 Hz), 3.66 (t. 2H,
J=6.8 Hz).
[0465] Compound 20.
[0466] A round bottom flask was charged with compound 19 (2.01 g,
5.73 mmol) and the solid dissolved in reagent grade acetone (150
mL). Simultaneously, Jones reagent was prepared by dissolving
CrO.sub.3 (2.25 g, 22 mmol) in H.sub.2SO.sub.4 (4 mL) and then
slowly adding 10 mL of cold water and letting the solution stir for
10 min. The cold Jones reagent was then added to the round bottom
flask slowly over 5 min., after which the solution stirred for 1
hr. The resulting orange solution turned green within several
minutes. The mixture was then quenched with water (150 mL)
extracted twice in ether (3.times.150 mL). The ether layers were
then dried with magnesium sulfate, and concentrated to yield
compound 20 (2.08 g, 98% yield) as a white powder. .sup.1H NMR
(CDCl.sub.3, 200 MHz) .delta. 1.27 (br s, 26H), 1.58-1.71 (m, 2H),
1.77-1.97 (m, 2H), 2.36 (t, 2H, J=7.4 Hz), 3.42 (t, 2H, J=7
Hz).
[0467] Compound 21.
[0468] t-Butylthiol (11.32 g, 125 mmol) was added to a dry Schlenk
flask and dissolved in dry THF (450 mL) distilled from sodium. The
solution was deoxygenated by bubbling nitrogen through it before
the flask was placed in an ice bath. n-BuLi solution in hexanes (70
mL of 1.6 M) was then added slowly via syringe over 10 min. This
mixture was allowed to stir for 1 hr., then compound 20 (5.5 g,
16.2 mmol) was added and the solution was left to reflux at
60.degree. C. overnight. The next morning an aliquot was worked up,
analyzed by NMR, and the reaction deemed complete. The reaction was
quenched with HCl (200 mL of 2 M) and extracted with ether
(3.times.250 mL). The ethereal layers were then dried with
magnesium sulfate, filtered, and the filtrate concentrated to yield
the product, compound 21, as a white solid (5 g, 90% yield).
.sup.1H NMR (CDCl.sub.3, 200 MHz) .delta. 1.26 (br s, 26H), 1.32
(br s, 9H), 1.48-1.70 (m, 4H), 2.35 (t, 2H, J=7.3 Hz), 2.52 (t, 2H,
J=7.3 Hz). .sup.13C NMR (CDCl.sub.3, 200 MHz) .delta. 24.69, 28.35,
29.05, 29.21, 29.28, 29.39, 29.55, 29.89, 31.02 (3C), 33.98, 41.75,
179.60.
Example 3
Synthetic Scheme for Making a Thiolated Analog of LPA
[0469] The synthetic approach described in this example results in
the preparation of thiolated LPA. The LPA analog can then be
further complexed to a carrier, for example, a protein carrier,
which can then be administered to an animal to elicit an immugenic
response to LPA. This approach uses both organic chemistry and
enzymatic reactions, the synthetic scheme for which is provided in
FIG. 3. The compound numbers in the synthetic description below
refer to the numbered structures in FIG. 3.
[0470] The starting materials were compound 15 in Example 2 and
enantiomerically pure glycerophoshocholine (compound 22). These two
chemicals combined to yield the di-acetylated product, compound 23,
using DCC to facilitate the esterification. In one synthetic
process variant, the resulting di-acylated glycerophosphocholine
was treated first with phospholipase-A2 to remove the fatty acid at
the sn-2 position of the glycerol backbone to produce compound 24.
This substance was further treated with another enzyme,
phospholipase-D, to remove the choline and form compound 26. In
another synthetic process variant, the phospholipase-D treatment
preceded the phospholipase-A2 treatment to yield compound 25, and
treatment of compound 25 with phospholipase-D then yields compound
26. Both variants lead to the same product, the phosphatidic acid
derivative, compound 26. The t-butyl protecting group in compound
26 is then removed, first using trimethyl disulfide triflate to
produce compound 27, followed by a disulfide reduction to produce
the desired LPA derivative, compound 28. As those in the art will
appreciate, the nitrobenzyl sulfenyl reaction sequence described in
Example 1 can also be used to produce compound 28.
[0471] Compound 23.
[0472] To a flame-dried Schlenk flask were added the thioether
acid, compound 15 (10 g, 35.8 mmol), compound 22
(glycerophosphocholine-CdCl.sub.2 complex, 4.25 g, 8.9 mmol), DCC
(7.32 g, 35.8 mmol), and DMAP (2.18 g, 17.8 mmol), after which the
flask was evacuated and filled with nitrogen. A minimal amount of
dry, degassed DCM was added (100 mL), resulting in a cloudy
mixture. The flask was covered with foil and then left to stir
until completed, as by TLC (silica, 10:5:1 DCM:MeOH:concentrated
NH.sub.4OH). The insolubility of compound 16 precluded monitoring
its disappearance by TLC, but the reaction was stopped when the
product spot of R.sub.f0.1 was judged not to be increasing in
intensity. This typically required 3 to 4 days, and in some cases,
addition of more DCC and DMAP. Upon completion, the reaction
mixture was filtered, and the filtrate concentrated to yield a
yellow oil, which was purified using flash chromatography using the
solvent system described above to yield 3.6 g (50% yield) of a
clear wax containing a mixture of compound 23 and monoacylated
products in a ratio of 5 to 1, as estimated from comparing the
integrals for the peaks for the (CH.sub.3).sub.3N--, --CH.sub.2StBu
and --CH.sub.2COO-- moieties. Analysis of the oil by HRMS (ESI-TOF)
produced a prominent ion at m/z 820.4972, calculated for
M+Na.sup.+.dbd.C.sub.40H.sub.80NNaO.sub.8PS.sub.2.sup.+
820.4960.
[0473] A. Synthesis Variant 1--Phospholipase-A2 Treatment
[0474] Compound 24.
[0475] A mixture of compound 23 and monoacetylated products as
described above (3.1 g, 3.9 mmol) was dissolved in Et.sub.2O (400
mL) and methanol (30 mL). Borate buffer (100 mL, pH 7.4 0.1M, 0.072
mM in CaCl.sub.2) was added, followed by phospholipase-A2 (from bee
venom, 130 units, Sigma). The resulting mixture was left to stir
for 10 hr., at which point TLC (silica, MeOH:water 4:1--the
previous solvent system 10:5:1 DCM:MeOH:concentrated NH.sub.4OH
proved ineffective) showed the absence of the starting material
(R.sub.f=0.7) and the appearance of a new spot (R.sub.f=0.2). The
organic and aqueous layers were separated and the aqueous layer was
washed with ether (2.times.250 mL). The product was extracted from
the aqueous layer with a mixture of DCM:MeOH (2:1, 2.times.50 mL).
The organic layers were then concentrated by rotary evaporation to
yield product as a white wax (1.9 g, 86% yield) that NMR showed to
be a pure product (compound 24). .sup.1H NMR (CDCl.sub.3, 500 MHz)
.delta. 1.25-1.27 (br s, 12H), 1.31 (s, 9H), 1.35-1.45 (m, 2H),
1.52-1.60 (m, 4H), 2.31 (t, 2H, J=7.5 Hz), 2.51 (t, 2H, J=7.5 Hz),
3.28 (br s, 9H) 3.25-3.33 (br s, 2H), 3.78-3.86 (m, 1H), 3.88-3.96
(m, 2H), 4.04-4.10 (m, 2H), 4.26-4.34 (m, 2H). Analysis of the wax
by HRMS (ESI-TOF) produced a prominent ion at m/z 550.2936,
calculated for M+Na.sup.+ 550.2943
(C.sub.24H.sub.50NNaO.sub.7PS.sub.2.sup.+), and an m/z at 528.3115,
calculated for MH.sup.+ 528.3124
(C.sub.24H.sub.51NO.sub.7PS.sub.2.sup.+).
[0476] Anal. Calculated. for C.sub.24H.sub.50NO.sub.7PS+2H.sub.2O
(563.73): C, 51.13; H, 9.66; N, 2.48. Found: C, 50.90; H, 9.37; N,
2.76.
[0477] Compound 26.
[0478] The lyso compound 24 (1.5 g, 2.7 mmol) was dissolved in a
mixture of sec-butanol (5 mL) and Et.sub.2O (200 mL), and the
resulting cloudy mixture was sonicated until the cloudiness
dissipated. Buffer (200 mL, pH 5.8, 0.2 M NaOAc, 0.08 M CaCl.sub.2)
was added, followed by cabbage extract (80 mL of extract from savoy
cabbage (which contains phospholipase-D), containing 9 mg of
protein/mL). The reaction was stirred for 1 day and monitored by
TLC (C.sub.18RP SiO.sub.2, 5:1 ACN: water), R.sub.f of starting
material and product=0.3 and 0.05, respectively. In order to push
the reaction to completion, as needed an additional portion of
cabbage extract (50 mL) was added and the reaction stirred a
further day. This process was repeated twice more, as needed to
complete the conversion. When the reaction was complete, the
mixture was concentrated on the rotary evaporator to remove the
ether, and then EDTA solution (0.5 M, 25 mL) was added and the
product extracted into a 5:4 mixture of MeOH: DCM (300 mL).
Concentration of the organic layer followed by recrystallization of
the residue from DCM and acetone afforded pure product (0.9 g, 75%
yield). .sup.1H NMR (CDCl.sub.3, 200 MHz) .delta. 1.25-1.27 (br s,
12H), 1.33 (s, 9H), 1.52-1.60 (m, 4H), 2.34 (t, 2H, J=7.5 Hz), 2.52
(t, 2H, J=7.5 Hz), 3.6-3.8 (br s, 1H), 3.85-3.97 (br s, 2H),
4.02-4.18 (m, 2H).
[0479] Compound 27.
[0480] The protected sample LPA, compound 26 (0.150 g, 0.34 mmol),
was methanol washed and added to a vial in the glove box. This was
then suspended in a mixture of AcOH:THF (1:1, 10 mL), which never
fully dissolved even after 1 hr. of sonication. Solid
[Me.sub.2SSMe]OTf (0.114 g, 0.44 mmol) was then added. This was
left to stir for 18 hr. The reaction was monitored by removing an
aliquot, concentrating it to dryness under vacuum, and
re-dissolving or suspending the residue in CD.sub.3OD for observing
the .sup.1H NMR shift of the CH.sub.2 peak closest to the sulfur.
The starting material had a peak at 2.52 ppm, whereas the
unsymmetrical disulfide formed at this juncture had a peak at
around 2.7 ppm. This material (compound 27) was not further
isolated or characterized.
[0481] Compound 28.
[0482] The mixture containing compound 27 was treated with water
(100 .mu.L) immediately followed by PMe.sub.3 (0.11 g, 1.4 mmol).
After stirring for 3 hr. the solvent was removed by vacuum to yield
an insoluble white solid. Methanol (5 mL) was added, the mixture
centrifuged, and the mother liquor decanted. Vacuum concentration
yielded 120 mg (91% yield) of compound 28, a beige solid. Compound
28 is a thiolated LPA hapten that can be conjugated to a carrier,
for example, albumin or KLH, via disulfide bond formation.
Characterization of compound 28: .sup.1H NMR (1:1
CD.sub.3OD:CD.sub.3CO.sub.2D, 500 MHz) .delta. 1.25-1.35 (br s,
12H), 1.32-1.4 (m, 2H), 1.55-1.6 (m, 4H), 2.34 (t, 2H, J=7), 2.47
(t, 2H, J=8.5), 3.89-3.97 (br s, 2H), 3.98-4.15 (m, 2H), 4.21 (m,
1H). Negative ion ES of the sample dissolved in methanol produced a
predominant ion at m/z=385.1.
Example 4
Antibodies to S1P
[0483] One type of therapeutic antibody specifically binds
undesirable sphingolipids to achieve beneficial effects such as,
e.g., (1) lowering the effective concentration of undesirable,
toxic sphingolipids (and/or the concentration of their metabolic
precursors) that would promote an undesirable effect such as a
cardiotoxic, tumorigenic, or angiogenic effect; (2) to inhibit the
binding of an undesirable, toxic, tumorigenic, or angiogenic
sphingolipids to a cellular receptor therefore, and/or to lower the
concentration of a sphingolipid that is available for binding to
such a receptor. Examples of such therapeutic effects include, but
are not limited to, the use of anti-S1P antibodies to lower the in
vivo serum concentration of available S1P, thereby blocking or at
least limiting S1P's tumorigenic and angiogenic effects and its
role in post-MI heart failure, cancer, or fibrogenic diseases.
[0484] Thiolated S1P (compound 10 of FIG. 1) was synthesized to
contain a reactive group capable of cross-linking the essential
structural features of S1P to a carrier moiety such as KLH. Prior
to immunization, the thio-S1P analog was conjugated via IOA or SMCC
cross-linking to protein carriers (e.g., KLH) using standard
protocols. SMCC is a heterobifunctional crosslinker that reacts
with primary amines and sulfhydryl groups, and represents a
preferred crosslinker.
[0485] Swiss Webster or BALB-C mice were immunized four times over
a two month period with 50 .mu.g of immunogen (SMCC facilitated
conjugate of thiolated-S1P and KLH) per injection. Serum samples
were collected two weeks after the second, third, and fourth
immunizations and screened by direct ELISA for the presence of
anti-S1P antibodies. Spleens from animals that displayed high
titers of the antibody were subsequently used to generate
hybridomas per standard fusion procedures. The resulting hybridomas
were grown to confluency, after which the cell supernatant was
collected for ELISA analysis. Of the 55 mice that were immunized, 8
were good responders, showing significant serum titers of
antibodies reactive to S1P. Fusions were subsequently carried out
using the spleens of these mice and myeloma cells according to
established procedures. The resulting 1,500 hybridomas were then
screened by direct ELISA, yielding 287 positive hybridomas. Of
these 287 hybridomas screened by direct ELISA, 159 showed
significant titers. Each of the 159 hybridomas was then expanded
into 24-well plates. The cell-conditioned media of the expanded
hybridomas were then re-screened to identify stable hybridomas
capable of secreting antibodies of interest. Competitive ELISAs
were performed on the 60 highest titer stable hybridomas.
[0486] Of the 55 mice and almost 1,500 hybridomas screened, one
hybridoma was discovered that displayed performance characteristics
that justified limited dilution cloning, as is required to
ultimately generate a true monoclonal antibody. This process
yielded 47 clones, the majority of which were deemed positive for
producing S1P antibodies. Of these 47 clones, 6 were expanded into
24-well plates and subsequently screened by competitive ELISA. From
the 4 clones that remained positive, one was chosen to initiate
large-scale production of the S1P monoclonal antibody. SCID mice
were injected with these cells and the resulting ascites was
protein A-purified (50% yield) and analyzed for endotoxin levels
(<3 EU/mg). For one round of ascites production, 50 mice were
injected, producing a total of 125 mL of ascites. The antibodies
were isotyped as IgG1 kappa, and were deemed >95% pure by HPLC.
The antibody was prepared in 20 mM sodium phosphate with 150 mM
sodium chloride (pH 7.2) and stored at -70.degree. C.
[0487] The positive hybridoma clone (designated as clone
306D326.26) was deposited with the ATCC (safety deposit storage
number SD-5362), and represents the first murine mAb
(Sphingomab.TM.) directed against S1P. The clone also contains the
variable regions of the antibody heavy and light chains that could
be used for the generation of a "humanized" antibody variant, as
well as the sequence information needed to construct a chimeric
antibody.
[0488] Screening of serum and cell supernatant for S1P-specific
antibodies was by direct ELISA using the thiolated SIP analog
described in Example 1 (i.e., compound 10) as the antigen. A
standard ELISA was performed, as described below, except that 50 ul
of sample (serum or cell supernatant) was diluted with an equal
volume of PBS/0.1% Tween-20 (PBST) during the primary incubation.
ELISAs were performed in 96-well high binding ELISA plates (Costar)
coated with 0.1 .mu.g of chemically-synthesized compound 10
conjugated to BSA in binding buffer (33.6 mM Na2CO3, 100 mM NaHCO3;
pH 9.5). The thiolated-S1P-BSA was incubated at 37.degree. C. for 1
hr. at 4.degree. C. overnight in the ELISA plate wells. The plates
were then washed four times with PBS (137 mM NaCl, 2.68 mM KCl,
10.14 mM Na2HPO4, 1.76 mM KH2PO4; pH 7.4) and blocked with PBST for
1 hr. at room temperature. For the primary incubation step, 75 uL
of the sample (containing the SIP to be measured), was incubated
with 25 uL of 0.1 ug/mL anti-S1P mAb diluted in PBST and added to a
well of the ELISA plate. Each sample was performed in triplicate
wells. Following a 1 hr. incubation at room temperature, the ELISA
plates were washed four times with PBS and incubated with 100 ul
per well of 0.1 ug/mL HRP goat anti-mouse secondary (Jackson
Immunoresearch) for 1 hr. at room temperature. Plates were then
washed four times with PBS and exposed to tetramethylbenzidine
(Sigma) for 1-10 minutes. The detection reaction was stopped by the
addition of an equal volume of 1M H2SO4. Optical density of the
samples was determined by measurement at 450 nm using an EL-X-800
ELISA plate reader (Bio-Tech).
[0489] For cross reactivity, a competitive ELISA was performed as
described above, except for the following alterations. The primary
incubation consisted of the competitor (SIP, SPH, LPA, etc.) and a
biotin-conjugated anti-S1P mAb. Biotinylation of the purified
monoclonal antibody was performed using the EZ-Link
Sulfo-NHS-Biotinylation kit (Pierce). Biotin incorporation was
determined as per kit protocol and ranged from 7 to 11 biotin
molecules per antibody. The competitor was prepared as follows:
lipid stocks were sonicated and dried under argon before
reconstitution in DPBS/BSA [1 mg/ml fatty acid-free BSA
(Calbiochem) in DPBS (Invitrogen 14040-133)]. Purified anti-S1P mAb
was diluted as necessary in PBS/0.5% Triton X-100. Competitor and
antibody solutions were mixed together so to generate 3 parts
competitor to 1 part antibody. A HRP-conjugated streptavidin
secondary antibody (Jackson Immunoresearch) was used to generate
signal.
[0490] Another aspect of the competitive ELISA data is that it
shows that the anti-SIP mAb was unable to distinguish the
thiolated-S1P analog (compound 10) from the natural SIP that was
added in the competition experiment. It also demonstrates that the
antibody does not recognize any oxidation products because the
analog was constructed without any double bonds (as is also also
true for the LPA analog described in Example 3). The anti-S1P mAb
was also tested against natural product containing the double bond
that was allowed to sit at room temperature for 48 hours. Reverse
phase HPLC of the natural SIP was performed according to methods
reported previously (Deutschman, et al. (July 2003), Am Heart J.,
vol. 146(1):62-8), and the results showed no difference in
retention time. Further, a comparison of the binding
characteristics of the monoclonal antibody to the various lipids
tested indicates that the epitope recognized by the antibody do not
involve the hydrocarbon chain in the region of the double bond of
natural S1P. On the other hand, the epitope recognized by the
monoclonal antibody is the region containing the amino alcohol on
the sphingosine base backbone plus the free phosphate. If the free
phosphate is linked with a choline (as is the case with SPC), then
the binding was somewhat reduced. If the amino group is esterified
to a fatty acid (as is the case with C1P), no antibody binding was
observed. If the sphingosine amino alcohol backbone was replaced by
a glycerol backbone (as is the case with LPA), there the
SIP-specific monoclonal exhibited no binding. These epitope mapping
data indicate that there is only one epitope on S1P recognized by
the monoclonal antibody, and that this epitope is defined by the
unique polar headgroup of SIP.
[0491] In a similar experiment using ELISA measurements, suitable
control materials were evaluated to ensure that this anti-S1P
monoclonal antibody did not recognize either the protein carrier or
the crosslinking agent. For example, the normal crosslinker SMCC
was exchanged for IOA in conjugating the thiolated-S1P to BSA as
the laydown material in the ELISA. When IOA was used, the
antibody's binding characteristics were nearly identical to when
BSA-SMCC-thiolated-S1P was used. Similarly, KLH was exchanged for
BSA as the protein that was complexed with thiolated-S1P as the
laydown material. In this experiment, there was also no significant
difference in the binding characteristics of the antibody.
[0492] Binding kinetics: The binding kinetics of SIP to its
receptor or other moieties has, traditionally, been problematic
because of the nature of lipids. Many problems have been associated
with the insolubility of lipids. For BIAcore measurements, these
problems were overcome by directly immobilizing SIP to a BIAcore
chip. Antibody was then flowed over the surface of the chip and
alterations in optical density were measured to determine the
binding characteristics of the antibody to S1P. To circumvent the
bivalent binding nature of antibodies, S1P was coated on the chip
at low densities. Additionally, the chip was coated with various
densities of S1P (7, 20, and 1000 RU) and antibody binding data was
globally fit to a 1:1 interaction model. Changes in optical density
resulted due to the binding of the monoclonal antibody to S1P at
three different densities of S1P. Overall, the affinity of the
monoclonal antibody to SIP was determined to be very high, in the
range of approximately 88 picomolar (pM) to 99 nM, depending on
whether a monovalent or bivalent binding model was used to analyze
the binding data.
Example 5
Chimeric mAb to SIP
[0493] A chimeric antibody to SIP was generated using the variable
regions (Fv) containing the active SIP binding regions of the
murine antibody from a particular hybridoma (ATCC safety deposit
storage number SD-5362) with the Fc region of a human IgG1
immunoglobulin. The Fc regions contained the CL, ChL, and Ch3
domains of the human antibody. Without being limited to a
particular method, chimeric antibodies could also have been
generated from Fc regions of human IgG1, IgG2, IgG3, IgG4, IgA, or
IgM. As those in the art will appreciate, "humanized" antibodies
can be generated by grafting the complementarity determining
regions (CDRs, e.g. CDR1-4) of the murine anti-S1P mAb with a human
antibody framework regions (e.g., Fr1, Fr4, etc.) such as the
framework regions of an IgG1. For the direct ELISA experiments, the
chimeric antibody to S1P had similar binding characteristics to the
fully murine monoclonal antibody. ELISAs were performed in 96-well
high-binding ELISA plates (Costar) coated with 0.1 ug of
chemically-synthesized, thiolated SIP conjugated to BSA in binding
buffer (33.6 mM Na2CO3, 100 mM NaHCO3; pH 9.5). The thiolated
S1P-BSA was incubated at 37.degree. C. for 1 hr. or at 4.degree. C.
overnight in the ELISA plate. Plates were then washed four times
with PBS (137 mM NaCl, 2.68 mM KCl, 10.14 mM Na2HPO4, 1.76 mM
KH2PO4; pH 7.4) and blocked with PBST for 1 hr. at room
temperature. For the primary incubation step, 75 uL of the sample
(containing the SIP to be measured), was incubated with 25 .mu.L of
0.1 .mu.g/mL anti-S1P monoclonal antibody diluted in PBST and added
to a well of the ELISA plate. Each sample was performed in
triplicate wells. Following a 1 hr incubation at room temperature,
the ELISA plates were washed four times with PBS and incubated with
100 ul per well of 0.1 ug/mL HRP goat anti-mouse secondary (Jackson
Immunoresearch) for 1 hr. at room temperature. Plates were then
washed four times with PBS and exposed to tetramethylbenzidine
(Sigma) for 1-10 minutes. The detection reaction was stopped by the
addition of an equal volume of 1M H2SO4. Optical density of the
samples was determined by measurement at 450 nm using an EL-X-800
ELISA plate reader (Bio-Tech).
[0494] The preferred method of measuring either antibody titer in
the serum of an immunized animal or in cell-conditioned media
(i.e., supernatant) of an antibody-producing cell such as a
hybridoma, involves coating the ELISA plate with a target ligand
(e.g., a thiolated analog of S1P, LPA, etc.) that has been
covalently linked to a protein carrier such as BSA.
Example 6
Monoclonal Antibodies to LPA
[0495] Antibody Production
[0496] Although polyclonal antibodies against naturally-occurring
LPA have been reported in the literature (Chen J H, et al., Bioorg
Med Chem Lett. 2000 Aug. 7; 10(15):1691-3), monoclonal antibodies
have not been described. Using an approach similar to that
described in Example 4, a C-12 thio-LPA analog (compound 28 in
Example 3) as the key component of a hapten formed by the
cross-linking of the analog via the reactive SH group to a protein
carrier (KLH) via standard chemical cross-linking using either IOA
or SMCC as the cross-linking agent, monoclonal antibodies against
LPA were generated. To do this, mice were immunized with the
thio-LPA-KLH hapten (in this case, thiolated-LPA:SMCC:KLH) using
methods described in Example 4 for the generation of anti-SIP
monoclonal antibodies. Of the 80 mice immunized against the LPA
analog, the five animals that showed the highest titers against LPA
(determined using an ELISA in which the same LPA analog (compound
28) as used in the hapten was conjugated to BSA using SMCC and laid
down on the ELISA plates) were chosen for moving to the hybridoma
phase of development.
[0497] The spleens from these five mice were harvested and
hybridomas were generated by standard techniques. Briefly, one
mouse yielded hybridoma cell lines (designated 504A). Of all the
plated hybridomas of the 504A series, 66 showed positive antibody
production as measured by the previously-described screening
ELISA.
[0498] Table 1, below, shows the antibody titers in cell
supernatants of hybridomas created from the spleens of two of mice
that responded to an LPA analog hapten in which the thiolated LPA
analog was cross-linked to KLH using heterobifunctional
cross-linking agents. These data demonstrate that the anti-LPA
antibodies do not react either to the crosslinker or to the protein
carrier. Importantly, the data show that the hybridomas produce
antibodies against LPA, and not against S1P.
TABLE-US-00001 TABLE 1 LPA hybridomas LPA S1P 3rd bleed binding
binding Cross mouse titer OD at Supernatants OD at OD at reactivity
# 1:312,500 from 24 well 1:20 1:20 w/S1P* 1 1.242 1.A.63 1.197
0.231 low 1.A.65 1.545 0.176 none 2 0.709 2.B.7 2.357 0.302 low
2.B.63 2.302 0.229 low 2.B.83 2.712 0.175 none 2.B.104 2.57 0.164
none 2.B.IB7 2.387 0.163 none 2.B.3A6 2.227 0.134 none *Cross
reactivity with S1P from 24 well supernatants: high = OD >
1.0-2.0 at [1:20]; mid = OD 0.4-1.0 at [1:20]; low = OD 0.4-0.2 at
[1:20]; none = OD < 0.2 OD at [1:20].
[0499] The development of anti-LPA mAbs in mice was monitored by
ELISA (direct binding to 12:0 and 18:1 LPA and competition ELISA).
A significant immunological response was observed in at least half
of the immunized mice and five mice with the highest antibody titer
were selected to initiate hybridoma cell line development following
spleen fusion.
[0500] After the initial screening of over 2000 hybridoma cell
lines generated from these 5 fusions, a total of 29 anti-LPA
secreting hybridoma cell lines exhibited high binding to 18:1 LPA.
Of these hybridoma cell lines, 24 were further subcloned and
characterized in a panel of ELISA assays. From the 24 clones that
remained positive, six hybridoma clones were selected for further
characterization. Their selection was based on their superior
biochemical and biological properties. Mouse hybridoma cell lines
504B3-6C2, 504B7.1, 504B58/3F8, 504A63.1 and 504B3A6 (corresponding
to clones referred to herein as B3, B7, B58, A63, and B3A6,
respectively) were received on May 8, 2007 by the American Type
Culture Collection (ATCC Patent Depository, 10801 University Blvd.,
Manassas, Va. 20110) for patent deposit purposes on behalf of LPath
Inc. and were granted deposit numbers PTA-8417, PTA-8420, PTA-8418,
PTA-8419 and PTA-8416, respectively.
[0501] All anti-LPA antibodies and portions thereof referred to
herein were derived from these cell lines.
[0502] Direct Binding Kinetics
[0503] The binding of 6 anti-LPA mAbs (B3, B7, B58, A63, B3A6, D22)
to 12:0 and 18:1 LPA (0.1 uM) was measured by ELISA. EC.sub.50
values were calculated from titration curves using 6 increasing
concentrations of purified mAbs (0 to 0.4 ug/ml). EC.sub.50
represents the effective antibody concentration with 50% of the
maximum binding. Max denotes the maximal binding (expressed as
OD450). Results are shown in Table 2.
TABLE-US-00002 TABLE 2 Direct Binding Kinetics of Anti-LPA mAbs B3
B7 B58 D22 A63 B3A6 12:0 LPA EC.sub.50 (nM) 1.420 0.413 0.554 1.307
0.280 0.344 Max (OD450) 1.809 1.395 1.352 0.449 1.269 1.316 18:1
LPA EC.sub.50 (nM) 1.067 0.274 0.245 0.176 0.298 0.469 Max (OD450)
1.264 0.973 0.847 0.353 1.302 1.027
[0504] The kinetics parameters k.sub.a (association rate constant),
k.sub.d (disassociation rate constant) and K.sub.D (association
equilibrium constant) were determined for the 6 lead candidates
using the BIAcore 3000 Biosensor machine. In this study, LPA was
immobilized on the sensor surface and the anti-LPA mAbs were flowed
in solution across the surface. As shown, all six mAbs bound LPA
with similar K.sub.D values ranging from 0.34 to 3.8 pM and similar
kinetic parameters.
[0505] The Anti-LPA Murine mAbs Exhibit High Affinity to LPA
[0506] LPA was immobilized to the sensor chip at densities ranging
150 resonance units. Dilutions of each mAb were passed over the
immobilized LPA and kinetic constants were obtained by nonlinear
regression of association/dissociation phases. Errors are given as
the standard deviation using at least three determinations in
duplicate runs. Results are shown in Table 3. Apparent affinities
were determined by K.sub.D=k.sub.a/k.sub.d.
[0507] k.sub.a=Association rate constant in M.sup.-1s.sup.-1k.sub.d
Dissociation rate constant in s.sup.-1
TABLE-US-00003 TABLE 3 Affinity of anti-LPA mAb for LPA mAbs
k.sub.a (M.sup.-1 s.sup.-1) k.sub.d (s.sup.-1) K.sub.D (pM) A63 4.4
.+-. 1.0 .times. 10.sup.5 1 .times. 10.sup.-6 2.3 .+-. 0.5 B3 7.0
.+-. 1.5 .times. 10.sup.5 1 .times. 10.sup.-6 1.4 .+-. 0.3 B7 6.2
.+-. 0.1 .times. 10.sup.5 1 .times. 10.sup.-6 1.6 .+-. 0.1 D22 3.0
.+-. 0.9 .times. 10.sup.4 1 .times. 10.sup.-6 33 .+-. 10 B3A6 1.2
.+-. 0.9 .times. 10.sup.6 1.9 .+-. 0.4 .times. 10.sup.-5 16 .+-.
1.2
[0508] Specificity Profile of Six Anti-LPA mAbs.
[0509] Many isoforms of LPA have been identified to be biologically
active and it is preferable that the mAb recognize all of them to
some extent to be of therapeutic relevance. The specificity of the
anti-LPA mAbs was evaluated utilizing a competition assay in which
the competitor lipid was added to the antibody-immobilized lipid
mixture.
[0510] Competition ELISA assays were performed with the anti-LPA
mAbs to assess their specificity. 18:1 LPA was captured on ELISA
plates. Each competitor lipid (up to 10 uM) was serially diluted in
BSA (1 mg/ml)-PBS and then incubated with the mAbs (3 nM). Mixtures
were then transferred to LPA coated wells and the amount of bound
antibody was measured with a secondary antibody. Data are
normalized to maximum signal (A.sub.450) and are expressed as
percent inhibition. Assays were performed in triplicate. IC.sub.50:
Half maximum inhibition concentration; MI: Maximum inhibition (% of
binding in the absence of inhibitor); - - - : not estimated because
of weak inhibition. A high inhibition result indicates recognition
of the competitor lipid by the antibody. As shown in Table 4, all
the anti-LPA mAbs recognized the different LPA isoforms.
TABLE-US-00004 TABLE 4 Specificity profile of anti-LPA mAbs. 14:0
LPA 16:0 LPA 18:1 LPA 18:2 LPA 20:4 LPA IC.sub.50 MI IC.sub.50 MI
IC.sub.50 MI IC.sub.50 MI IC.sub.50 MI uM % uM % uM % uM % uM % B3
0.02 72.3 0.05 70.3 0.287 83 0.064 72.5 0.02 67.1 B7 0.105 61.3
0.483 62.9 >2.0 100 1.487 100 0.161 67 B58 0.26 63.9 5.698
>100 1.5 79.3 1.240 92.6 0.304 79.8 B104 0.32 23.1 1.557 26.5
28.648 >100 1.591 36 0.32 20.1 D22 0.164 34.9 0.543 31 1.489
47.7 0.331 31.4 0.164 29.5 A63 1.147 31.9 5.994 45.7 -- -- -- --
0.119 14.5 B3A6 0.108 59.9 1.151 81.1 1.897 87.6 -- -- 0.131
44.9
[0511] Interestingly, the anti-LPA mAbs were able to discriminate
between 12:0 (lauroyl), 14:0 (myristoyl), 16:0 (palmitoyl), 18:1
(oleoyl), 18:2 (linoleoyl) and 20:4 (arachidonoyl) LPAs. A
desirable EC.sub.50 rank order for ultimate drug development is
18:2>18:1>20:4 for unsaturated lipids and
14:0>16:0>18:0 for the saturated lipids, along with high
specificity. The specificity of the anti-LPA mAbs was assessed for
their binding to LPA related biolipids such as
distearoyl-phosphatidic acid, lysophosphatidylcholine, S1P,
ceramide and ceramide-1-phosphate. None of the antibodies
demonstrated cross-reactivity to distearoyl PA and LPC, the
immediate metabolic precursor of LPA.
Example 7
Anti-Cancer Activities of Anti-LPA Monoclonal Antibodies
[0512] Cancer Cell Proliferation
[0513] LPA is a potent growth factor supporting cell survival and
proliferation by stimulation of G.sub.i, G.sub.q and G.sub.12/13
via GPCR-receptors and activation of downstream signaling events.
Cell lines were tested for their proliferative response to LPA
(0.01 mM to 10 mM). Cell proliferation was assayed by using the
cell proliferation assay kit from Chemicon (Temecula Calif.)
(Panc-1) and the Cell-Blue titer from Pierce (Caki-1). Each data
point is the mean of three independent experiments. LPA increased
proliferation of 7 human-derived tumor cell lines in a dose
dependent manner including SKOV3 and OVCAR3 (ovarian cancer),
Panc-1 (pancreatic cancer), Caki-1 (renal carcinoma cell), DU-145
(prostate cancer), A549 (lung carcinoma), and HCT-116 (colorectal
adenocarcinoma) cells and one rat-derived tumor cell line, RBL-2H3
(rat leukemia cells). Even though tumor-derived cells normally have
high basal levels of proliferation, LPA appears to further augment
proliferation in most tumor cell lines. Anti-LPA mAbs (B7 and B58)
were assessed for the ability to inhibit LPA-induced proliferation
in selected human cancer cell lines. The increase in proliferation
induced by LPA was shown to be mitigated by the addition of
anti-LPA mAb.
[0514] Anti-LPA mAb Sensitizes Tumor Cells to Chemotherapeutic
Agents
[0515] The ability of LPA to protect ovarian tumor cells against
apoptosis when exposed to clinically-relevant levels of the
chemotherapeutic agent, paclitaxel (Taxol) was investigated. SKVO3
cells were treated with 1% FBS (S), Taxol (0.5 mM), +/-anti-LPA
mAbs for 24 h. LPA protected SKOV3 cells from Taxol-induced
apoptosis. Apoptosis was assayed by measurement of the caspase
activity as recommended by the manufacturer (Promega). As
anticipated, LPA protected most of the cancer cell lines tested
from taxol-induced cell death. When the anti-LPA antibody B7 was
added to a selection of the LPA responsive cells, it blocked the
ability of LPA to protect cells from death induced by the cytotoxic
chemotherapeutic agent. Moreover, the anti-LPA antibody was able to
remove the protection provided by serum. Serum is estimated to
contain about 5-20 uM LPA. Taxol induced caspase-3,7 activation in
SKOV3 cells and the addition of serum to cells protected cells from
apoptosis. Taxol-induced caspase activation was enhanced by the
addition of LT3000 to the culture medium. This suggests that the
protective and anti-apoptotic effects of LPA were removed by the
selective antibody mediated neutralization of the LPA present in
serum.
[0516] Anti-LPA mAb Inhibits LPA-Mediated Migration of Tumor
Cells
[0517] An important characteristic of metastatic cancers is that
the tumor cells escape contact inhibition and migrate away from
their tissue of origin. LPA has been shown to promote metastatic
potential in several cancer cell types. Accordingly, we tested the
ability of anti-LPA mAb to block LPA-dependent cell migration in
several human cancer cell lines by using the cell monolayer scratch
assay. Cells were seeded in 96 well plates and grown to confluence.
After 24 h of starvation, the center of the wells was scratched
with a pipette tip. In this art-accepted "scratch assay," the cells
respond to the scratch wound in the cell monolayer in a
stereotypical fashion by migrating toward the scratch and close the
wound. Progression of migration and wound closure are monitored by
digital photography at 10.times. magnification at desired
timepoints. Cells were not treated (NT), treated with LPA (2.5 mM)
with or w/o mAb B7 (10 .mu.g/ml) or an isotype matching
non-specific antibody (NS) (10 .mu.g/ml). In untreated cells, a
large gap remains between the monolayer margins following the
scratch. LPA-treated cells in contrast, have only a small gap
remaining at the same timepoint, and a few cells are making contact
across the gap. In cells treated with both LPA and the anti-LPA
antibody B7, the gap at this timepoint was several fold larger than
the LPA-only treatment although not as large as the untreated
control cells. This shows that the anti-LPA antibody had an
inhibitory effect on the LPA-stimulated migration of renal cell
carcinoma (Caki-1) cells. Similar data were obtained with mAbs B3
and B58. This indicates that the anti-LPA mAb can reduce
LPA-mediated migration of cell lines originally derived from
metastatic carcinoma.
[0518] Anti-LPA mAbs Inhibit Release of Pro-Tumorigenic Cytokines
from Tumor Cells
[0519] LPA is involved in the establishment and progression of
cancer by providing a pro-growth tumor microenvironment and
promoting angiogenesis. In particular, increases of the pro-growth
factors such as IL-8 and VEGF have been observed in cancer cells.
IL-8 is strongly implicated in cancer progression and prognosis.
IL-8 may exert its effect in cancer through promoting
neovascularization and inducing chemotaxis of neutrophils and
endothelial cells. In addition, overexpression of IL-8 has been
correlated to the development of a drug resistant phenotype in many
human cancer types.
[0520] Three anti-LPA mAbs (B3, B7 and B58) were tested for their
abilities to reduce in vitro IL-8 production compared to a
non-specific antibody (NS). Caki-1 cells were seeded in 96 well
plates and grown to confluency. After overnight serum starvation,
cells were treated with 18:1 LPA (0.2 mM) with or without anti-LPA
mAb B3, B7, B58 or NS (Non-Specific). After 24 h, cultured
supernatants of renal cancer cells (Caki-1), treated with or
without LPA and in presence of increasing concentrations of the
anti-LPA mAbs B3, B7 and B58, were collected and analyzed for IL-8
levels using a commercially available ELISA kit (Human Quantikine
Kit, R&D Systems, Minneapolis, Minn.). In cells pre-treated
with the anti-LPA mAbs, IL-8 expression was significantly reduced
in a dose-dependent manner (from 0.1-30 .mu.g/mL mAb) whereas LPA
increased the expression of IL-8 by an average of 100% in
non-treated cells. The inhibition of IL-8 release by the anti-LPA
mAbs was also observed in other cancerous cell lines such as the
pancreatic cell line Panc-1. These data suggest that the blockade
of the pro-angiogenic factor release is an additional and
potentially important effect of these anti-LPA mAbs.
[0521] Anti-LPA mAbs Inhibit Angiogenesis In Vivo
[0522] One of the anti-LPA mAbs (B7) was tested for its ability to
mitigate angiogenesis in vivo using the Matrigel Plug assay. This
assay utilizes Matrigel, a proprietary mixture of tumor remnants
including basement membranes derived from murine tumors. When
Matrigel, or its derivate growth factor-reduced (GFR) Matrigel, is
injected sc into an animal, it solidifies and forms a `plug.` If
pro-angiogenic factors are mixed with the matrix prior to
placement, the plug will be invaded by vascular endothelial cells
which eventually form blood vessels. Matrigel can be prepared
either alone or mixed with recombinant growth factors (bFGF, VEGF),
or tumor cells and then injected sc in the flanks of 6-week old
nude (NCr Nu/Nu) female mice. In this example, Caki-1 (renal
carcinoma) cells were introduced inside the Matrigel and are
producing sufficient levels of VEGF and/or IL8 and LPA. Matrigel
plugs were prepared containing 5.times.10.sup.5 Caki-1 cells from
mice treated with saline or with 10 mg/kg of anti-LPA mAb-B7, every
3 days starting 1 day prior to Matrigel implantation. Plugs were
stained for endothelial CD31, followed by quantitation of the
micro-vasculature formed in the plugs. Quantitation data were
means+/-SEM of at least 16 fields/section from 3 plugs. The plugs
from mice treated with the anti-LPA mAb B7 demonstrated a prominent
reduction in blood vessel formation, as assayed by endothelial
staining for CD31, compared to the plugs from saline-treated mice.
Quantification of stained vessels demonstrates a greater than 50%
reduction in angiogenesis in Caki-1-containing plugs from animals
treated with mAb B7 compared to saline-treated animals. This was a
statistically significant reduction (p<0.05 for mAb B7 vs.
Saline as determined by Student's T-test) in tumor cell
angiogenesis as a result of anti-LPA mAb treatment.
[0523] Anti-LPA mAbs Reduces Tumor Progression in a Murine Model of
Metastasis
[0524] One important characteristic of tumor progression is the
ability of a tumor to metastasize and form secondary tumor nodules
at remote sites. In vitro studies described hereinabove have
demonstrated the ability of LPA to induce tumor cells to escape
contact inhibition and promote migration in a scratch assay for
cell motility. In these studies, the anti-LPA mAbs also inhibited
LPA's tumor growth promoting effectors. The efficacy of the
anti-LPA mAb to inhibit tumor metastasis in vivo was also
evaluated. The phenomenon of tumor metastasis has been difficult to
mimic in animal models. Many investigators utilize an
"experimental" metastasis model in which tumor cells are directly
injected into the blood stream.
[0525] Blood vessel formation is an integral process of metastasis
because an increase in the number of blood vessels means cells have
to travel a shorter distance to reach circulation. It is believed
that anti-LPA mAb will inhibit in vivo tumor cell metastasis, based
on the finding that the anti-LPA mAb can block several integral
steps in the metastatic process.
[0526] Study: The highly metastatic murine melanoma (B16-F10) was
used to examine the therapeutic effect of anti-LPA mAbs on
metastasis in vivo. This model has demonstrated to be highly
sensitive to cPA inhibitors of autotaxin. 4 week old female
(C57BL/6) mice received an injection of B16-F10 murine melanoma
tumor cells (100 uL of 5.times.10.sup.4 cells/animal) via the tail
vein. Mice (10 per group) were administered 25 mg/kg of the
anti-LPA mAb (either B3 or B7) or saline every three days by i.p.
injection. After 18 days, lungs were harvested and analyzed. The
pulmonary organs are the preferred metastatic site of the melanoma
cells, and were therefore closely evaluated for metastatic nodules.
The lungs were inflated with 10% buffered formalin via the trachea,
in order to inflate and fix simultaneously, so that even small foci
could be detectable on histological examination. Lungs were
separated into five lobes and tumors were categorized by dimension
(large.gtoreq.5 mm; medium 1-4 mm; small<1 mm) and counted under
a dissecting microscope. Upon examination of the lungs, the number
of tumors was clearly reduced in antibody-treated animals. For
animals treated with mAb B3, large tumors were reduced by 21%,
medium tumors by 17% and small tumors by 22%. Statistical analysis
by student's T-test gave a p<0.05 for number of small tumors in
animals treated with mAb B3 vs saline.
[0527] As shown in the above examples, it has now been shown that
the tumorigenic effects of LPA are extended to renal carcinoma
(e.g., Caki-1) and pancreatic carcinoma (Panc-1) cell lines. LPA
induces tumor cell proliferation, migration and release of
pro-angiogenic and/or pro-metastatic agents, such as VEGF and IL-8,
in both cell lines. It has now been shown that three high-affinity
and specific monoclonal anti-LPA antibodies demonstrate efficacy in
a panel of in vitro cell assays and in vivo tumor models of
angiogenesis and metastasis.
Example 8
Cloning of the Murine Anti-LPA Antibodies--Overview
[0528] Chimeric antibodies to LPA were generated using the variable
domains (Fv) containing the active LPA binding regions of one of
three murine antibodies from hybridomas with the Fc region of a
human IgG1 immunoglobulin. The Fc regions contained the CH1, CH2,
and CH3 domains of the human antibody. Without being limited to a
particular method, chimeric antibodies could also have been
generated from Fc regions of human IgG1, IgG2, IgG3, IgG4, IgA, or
IgM. As those in the art will appreciate, "humanized" antibodies
can be generated by grafting the complementarity determining
regions (CDRs, e.g. CDR1-4) of the murine anti-LPA mAbs with a
human antibody framework regions (e.g., Fr1, Fr4, etc.) such as the
framework regions of an IgG1.
[0529] The overall strategy for cloning of the murine mAb against
LPA consisted of cloning the murine variable domains of both the
light chain (VL) and the heavy chain (VH) from each antibody. The
consensus sequences of the genes show that the constant region
fragment is consistent with a gamma isotype and that the light
chain is consistent with a kappa isotype. The murine variable
domains were cloned together with the constant domain of the human
antibody light chain (CL) and with the constant domain of the human
heavy chain (CH1, CH2, and CH3), resulting in a chimeric antibody
construct.
[0530] The variable domains of the light chain and the heavy chain
were amplified by PCR. The amplified fragments were cloned into an
intermediate vector (pTOPO). After verification of the sequences,
the variable domains were then assembled together with their
respective constant domains. The variable domain of the light chain
was cloned into pCONkappa2 and the variable domain of the heavy
chain was cloned into pCONgamma1f. The cloning procedure included
the design of an upstream primer to include a signal peptide
sequence, a consensus Kozak sequence preceding the ATG start codon
to enhance translation initiation, and the 5' cut site, HindIII.
The downstream primer was designed to include the 3' cut site ApaI
for the heavy chain and BsiWI for the light chain.
[0531] The vectors containing the variable domains together with
their respective constant domains were transfected into mammalian
cells. Three days after transfections, supernatants were collected
and analyzed by ELISA for binding to LPA. Detailed methods for
cloning, expression and characterization of the anti-LPA antibody
variable domains are shown on the following pages.
[0532] Binding characteristics for the chimeric antibodies are
shown in Table 5. "HC" and "LC" indicate the identities of the
heavy chain and light chain, respectively.
TABLE-US-00005 TABLE 5 Binding characteristics of the chimeric
anti-LPA antibodies B3, B7, and B58. Titer EC50 Max HC x LC (ug/ml)
(ng/ml) OD 1 B7 B7 3.54 43.24 2.237 2 B7 B58 1.84 25.79 1.998 3 B7
B3 2.58 24.44 2.234 4 B58 B7 3.80 38.99 2.099 5 B58 B58 3.42 41.3
2.531 6 B58 B3 2.87 29.7 2.399 7 B3 B7 4.18 49.84 2.339 8 B3 B58
0.80 20.27 2.282 9 B3 B3 4.65 42.53 2.402
[0533] It can be seen from Table 5 that it is possible to optimize
antibody binding to LPA by recombining light chains and heavy
chains from different hybridomas (i.e., different clones) into
chimeric molecules.
[0534] Materials and Methods for the Cloning, Expression and
Characterization of the Anti-LPA Antibody Variable Domains
[0535] Cloning of the Variable Domains from Hybridoma Cell
Lines
[0536] Clones from the anti-LPA hybridoma cell lines were grown in
DMEM (Dulbecco's Dulbecco's Modified Eagle Medium with GlutaMAX.TM.
I, 4500 mg/L D-Glucose, Sodium Puruvate; Gibco/Invitrogen,
Carlsbad, Calif., 111-035-003), 10% FBS (Sterile Fetal Clone I,
Perbio Science), and 1.times. glutamine/Penicillin/Streptomycin
(Gibco/Invitrogen). Total RNA was isolated from 10.sup.7 hybridoma
cells using a procedure based on the RNeasy Mini kit (Qiagen,
Hilden Germany). The RNA was used to generate first strand cDNA
following the manufacturer's protocol for SMART RACE cDNA
Amplification Kit (Clonetech).
[0537] The immunoglobulin heavy chain variable domain (VH) cDNA was
amplified by PCR using primers listed in Table 6. Heavy Chain
variable domain PCR set-up was as follows: MHCG1 (known IgG1
constant region primer) combined with Group 1 and Group 2 V region
primers for all five antibodies. The product of each reaction was
ligated into the pCR2.1.RTM.-TOPO.RTM. vector (Invitrogen, Carlsbad
Calif.) using the TOPO-TA cloning.RTM. kit and sequence.
[0538] Similarly, the immunoglobulin light chain variable domains
(VK) were amplified using the primers listed in Table 7. The light
chain variable domain PCR set-up was as follows: Two constant
region primers were each combined with Group 1, Group 2 and Group 3
V region primers for all five antibodies. The product of each
reaction was ligated into the pCR2.1.RTM.-TOPO.RTM. vector using
the TOPO-TA cloning.RTM. kit and sequence.
[0539] The list of oligonucleotides was designed according to the
literature (Dattamajumdar, A. K., Jacobson, D. P., Hood, L. E. and
Osman, G. E. (1991) Rapid cloning of any rearranged mouse
immunoglobulin variable genes. Immunogenetics. 43(3):141-51;
Coloma, M. J., Hastings, A., Wims, L. A. and Morrison, S. L. (1992)
Novel vectors for the expression of antibody molecules using
variable domains generated by polymerase chain reaction. J Immunol
Methods, 152(1):89-104; Coronella, J. A., Telleman, P., Truong, T.
D., Ylera, F. and Junghans, R. P. (2000) Amplification of IgG VH
and VL (Fab) from single human plasma cells and B cells. Nucleic
Acids Res., 28(20):E85.).
TABLE-US-00006 TABLE 6 List of oligonucleotides for the cloning of
the heavy chain variable domains from the anti-LPA monoclonal
antibodies. SEQ ID Heavy Chain NO: Variable Group 1 MHV1
ATGAAATGCAGCTGGGGCATSTTCTTC 1 MHV2 ATGGGATGGAGCTRTATCATSYTCTT 2
MHV3 ATGAAGWTGTGGTTAAACTGGGTTTTT 3 MHV4 ATGRACTTTGGGYTCAGCTTGRTTT 4
MHV5 ATGGACTCCAGGCTCAATTTAGTTTTCCTT 5 MHV6
ATGGCTGTCYTRGSGCTRCTCTTCTGC 6 Group 2 MHV7
ATGGRATGGAGCKGGRTCTTTMTCTT 7 MHV8 ATGAGAGTGCTGATTCTTTTGTG 8 MHV9
ATGGMTTGGGTGTGGAMCTTGCTATTCCTG 9 MHV10 ATGGGCAGACTTACATTCTCATTCCTG
10 MHV11 ATGGATTTTGGGCTGATTTTTTTTATTG 11 MHV12
ATGATGGTGTTAAGTCTTCTGTACCTG 12 MH1: ATATCCACCA TGGRATGSAG 13
CTGKGTMATS CTCTT Constant MHCG1 CAGTGGATAGACAGATGGGGG 14 MHCG2a
CAGTGGATAGACCGATGGGGC 15 MHCG2b CAGTGGATAGACTGATGGGGG 16 MHCG3
CAAGGGATAGACAGATGGGGC 17 MVG1R 5'-GGCAGCACTAGTAGGGGCCAGTGGATA- 18
3'
TABLE-US-00007 TABLE 7 List of oligonucleotides used for the
cloning of the light chain variable domains from the anti-LPA
monoclonal antibodies. SEQ ID Light chain NO: Variable Group 1
MLALT1 GGGCACCATGGAGACAGACACACTCCTGCTAT 19 MLALT2
GGGCACCATGGATTTTCAAGTGCAGATTTTCAG 20 MLALT3
GGGCACCATGGAGWCACAKWCTCAGGTCTTTRTA 21 MLALT4
GGGCACCATGKCCCCWRCTCAGYTYCTKGT 22 MLALT5
5'-CACCATGAAGTTGCCTGTTAGGCTGTTG-3' 23 Group 2 MKV1a
ATGAAGTTGVVTGTTAGGCTGTTGGTGCTG 24 MKV2
ATGGAGWCAGACACACTCCTGYTATGGGTG 25 MKV3
ATGAGTGTGCTCACTCAGGTCCTGGSGTTG 26 MKV4
ATGAGGRCCCCTGCTCAGWTTYTTGGMWTCTTG 27 MKV5
ATGGATTTWAGGTGCAGATTWTCAGCTTC 28 MKV6 ATGAGGTKCKKTGKTSAGSTSCTGRGG
29 MKV7 ATGGGCWTCAAGATGGAGTCACAKWYYCWGG 30 MKV8
ATGTGGGGAYCTKTTTYCMMTTTTTCAATTG 31 MKV9 ATGGTRTCCWCASCTCAGTTCCTTG
32 MKV10 ATGTATATATGTTTGTTGTCTATTTCT 33 MKV11
ATGGAAGCCCCAGCTCAGCTTCTCTTCC 34 VK8 TGGGTATCTGGTRCSTGTG 35 MKV20
ATGGAGWCAGACACACTSCTG 36 Group 3 CL12A ATGRAGTYWCAGACCCAGGTCTTYRT
37 CL12B ATGGAGACACATTCTCAGGTCTTTGT 38 CL13
ATGGATTCACAGGCCCAGGTTCTTAT 39 CL14 ATGATGAGTCCTGCCCAGTTCCTCTT 40
CL15 ATGAATTTGCCTGTTCATCTCTTGGTGCT 41 CL16
ATGGATTTTCAATTGGTCCTCATCTCCTT 42 CL17A ATGAGGTGCCTARCTSAGTTCCTGRG
43 CL17B ATGAAGTACTCTGCTCAGTTTCTAGG 44 CL17C
ATGAGGCATTCTCTTCAATTCTTGGG 45 Constant MKC ACTGGATGGTGGGAAGATGG 46
33615: 5'GAAGATCTAGACTTACTA TGCAGCATCAGC-3' 47
[0540] TOP02.1 clones containing the heavy and light chain variable
domains were sequenced and CDR regions were determined. The
variable domain of the light chain was then amplified by PCR adding
a leader sequence and cut sites suggested by the manufacturer for
cloning into the Lonza light chain expression vector, pCONkappa2
(5' HindIII, 3' BsiWI, LC leader sequence: ATG TCT GTG CCT ACC CAG
GTG CTG GGA CTG CTG CTG CTG TGG CTG ACA GAC GCC CGC TGT, SEQ ID NO:
48). The variable domain of the heavy chain was then amplified by
PCR adding a leader sequence and cut sites suggested by Lonza for
cloning into the Lonza heavy chain expression vector, pCONgamma1f
(5' HindIII, 3' ApaI, HC leader sequence: ATG GAA TGG AGC TGG GTG
TTC CTG TTC TTT CTG TCC GTG ACC ACA GGC GTG CAT TCT, SEQ ID NO:
49). Final products were then inserted into light or heavy chain
expression vectors, containing the constant regions, with digestion
and ligation the Rapid Ligation Kit (Roche).
[0541] The heavy and light chain plasmids were transformed into One
Shot.RTM. TOP10 chemically competent bacterial cells (Invitrogen)
and stocked in glycerol. Large-scale plasmid DNA was prepared as
described by the manufacturer (Qiagen, endotoxin-free MAXIPREP.TM.
kit). DNA samples, purified using Qiagen's QIAprep Spin Miniprep
Kit or EndoFree Plasmid Mega/Maxi Kit, were sequenced using an ABI
3730xl automated sequencer, which also translates the fluorescent
signals into their corresponding nucleobase sequence. Primers were
designed at the 5' and 3' ends so that the sequence obtained would
overlap.
[0542] PCR Amplification of the Variable Domains
[0543] The Polymerase Chain Reactions (PCR) were performed using
Invitrogen's Pfx DNA polymerase kit with 10.times. buffer and 50 mM
MgSO4 (cat#11708-013) and 10 mM dNTPs (Invitrogen, cat#18427-013).
The reaction mixture consisted of 5 ul 10.times.pfx amplification
buffer, 1.5 ul 10 mM dNTPs, 1 ul 50 mM MgSO4, 1.5 ul
oligonucleotide 1, 1.5 ul oligonucleotide 2, 0.5 ul template
(.about.50 ng), 0.5 ul Pfx DNA polymerase, 38.5 ul sterile water.
All reagents were added minus Pfx and then Pfx was added
immediately before starting the thermocycler. After denaturation of
the templates at 95.degree. C. for 3 minutes, 35 cycles of
95.degree. C. for 30 seconds, annealing at 58.degree. C. with a
5.degree. C.+/-gradient and extension at 68.degree. C. for 30
seconds were performed. After a final extension at 68.degree. C.
for 5 minutes, the samples were kept at 4.degree. C.
[0544] Restriction Digest and Ligation Reactions to Clone the
Variable Domains
[0545] The restriction digests were performed on DNA to prepare
fragment for ligation or for cloning verification prior to checking
the molecular sequence. All restriction enzymes were purchased from
Invitrogen or New England Biolabs which come with the corresponding
buffers required for each enzyme. The DNA (usually 5-10 ul to check
for positive clones and 20-26 ul for DNA to be ligated) were mixed
with the enzyme buffer, 0.5 to 1.0 ul of the restriction enzyme,
and sterile water (to a total of 30 ul reaction). The reactions
were incubated at appropriate temperature for the enzyme for 1 hr.
Most enzymes were active at 37.degree. C. however the incubation
temperature could vary from room temperature to 55.degree. C.
depending on the enzymes. After adequate restriction enzyme digest,
the GeneClean kit was used to clean the insert fragment and vector
from agarose gel and any enzymes and buffers. Ligations were
performed using Roche Rapid Ligation Kit (catalog #11635379001)
that included T4 DNA 2.times. Ligation buffer, 5.times.DNA dilution
buffer, and T4 DNA ligase. Inserts and vectors were ligated in a
final 3:1 molar ratio for best results. Insert fragments were
diluted appropriately for efficient ligations. 5 to 7 ul of the
reaction was used to transformed E. coli TOP10 chemically competent
cells.
[0546] Quantitative ELISA
[0547] Microtiter ELISA plates (Costar, Cat No. 3361) were coated
with rabbit anti-mouse IgG, F(ab').sub.2 fragment specific antibody
(Jackson, 315-005-047) diluted in 1 M Carbonate Buffer (pH 9.5) at
37.degree. C. for 1 h. Plates were washed with PBS and blocked with
PBS/BSA/Tween-20 for 1 hr at 37.degree. C. For the primary
incubation, dilutions of non-specific mouse IgG or human IgG, whole
molecule (used for calibration curve) and samples to be measured
were added to the wells. Plates were washed and incubated with 100
ul per well of HRP conjugated anti-human diluted 1:50,000 (Jackson
109-035-003) for 1 hr at 37.degree. C. After washing, the enzymatic
reaction was detected with tetramethylbenzidine (Sigma, cat No
T0440) and stopped by adding 1 M H.sub.2SO.sub.4. The optical
density (OD) was measured at 450 nm using a Thermo Multiskan EX.
Raw data were transferred to GraphPad software for analysis.
[0548] Direct ELISA
[0549] Microtiter ELISA plates (Costar, Cat No. 3361) were coated
with LPA-BSA diluted in 1M Carbonate Buffer (pH 9.5) at 37.degree.
C. for 1 h. Plates were washed with PBS (137 mM NaCl, 2.68 mM KCl,
10.1 mM Na.sub.2HPO.sub.4, 1.76 mM KH.sub.2PO.sub.4; pH 7.4) and
blocked with PBS/BSA/Tween-20 for 1 h at room temperature or
overnight at 4.degree. C. The samples to be tested were diluted at
0.4 ug/mL, 0.2 ug/mL, 0.1 ug/mL, 0.05 ug/mL, 0.0125 ug/mL, and 0
ug/mL and 100 ul added to each well. Plates were washed and
incubated with 100 ul per well of HRP anti-human diluted 1:50,000
(Jackson 109-035-003) for 1 hr at 37.degree. C. After washing, the
enzymatic reaction was detected with tetramethylbenzidine (Sigma,
Cat No T0440) and stopped by adding 1 M H.sub.2SO.sub.4. The
optical density (OD) was measured at 450 nm using a Thermo
Multiskan EX. Raw data were transferred to GraphPad software for
analysis.
[0550] Transient Expression
[0551] The vectors were transfected into the human embryonic kidney
cell line 293F using 293fectin and using 293F-FreeStyle Media for
culture. Transfections were performed at a cell density of 10.sup.6
cells/mL with 0.5 .mu.g/mL. Supernatants were collected by
centrifugation at 1100 rpm for 5 minutes at 25.degree. C. 3 days
after transfection. The expression level was quantified by
quantitative ELISA and the binding was measured in a binding ELISA
as described above.
[0552] The mouse V.sub.H and V.sub.L domains were sequenced using
standard methods. Tables 8-17 show nucleic acid and amino acid
sequences for the complementarity-determining regions (CDRs) of the
variable (V.sub.H and V.sub.L) domains for five mouse anti-LPA
monoclonal antibody clones. For each CDRH1 amino acid sequence, the
CDR defined according to Kabat is the 10-amino acid sequence shown.
The five-amino acid portion of the Kabat sequence that is shown in
bold is the canonical CDRH1 sequence.
TABLE-US-00008 TABLE 8 Mouse LPA CDR nucleic acid sequences of the
mouse V.sub.H and V.sub.L domains for clone B3 of mouse anti-LPA
monoclonal antibody SEQ ID CLONE CDR NO: V.sub.H CDR B3
GGAGACGCCTTCACAAATTACTTA CDRH1 50 ATAGAG B3
CTGATTTATCCTGATAGTGGTTAC CDRH2 51 ATTAACTACAATGAGAACTTCAA GGGC B3
AGATTTGCTTACTACGGTAGTGGC CDRH3 52 TACTACTTTGACTAC V.sub.L CDR B3
AGATCTAGTCAGAGCCTTCTAAA CDRL1 53 AACTAATGGAAACACCTATTTAC AT B3
AAAGTTTCCAACCGATTTTCT CDRL2 54 B3 TCTCAAAGTACACATTTTCCATTC CDRL3 55
ACG
TABLE-US-00009 TABLE 9 Mouse LPA CDR amino acid sequences of the
mouse V.sub.H and V.sub.L domains for clone B3 of mouse anti-LPA
monoclonal antibody SEQ ID CLONE CDR NO: V.sub.H CDR B3 GDAFTNYLIE*
CDRH1 56 B3 LIYPDSGYINYNENFKG CDRH2 57 B3 RFAYYGSGYYFDY CDRH3 58
V.sub.L CDR B3 RSSQSLLKTNGNTYLH CDRL1 59 B3 KVSNRFS CDRL2 60 B3
SQSTHFPFT CDRL3 61 *The CDRH1 sequence defined according to
Chothia/AbM is the 10-amino acid sequence shown. The five-amino
acid portion of this sequence shown in bold (NYLIE; SEQ ID NO: 62)
is the CDRH1 sequence defined according to Kabat.
TABLE-US-00010 TABLE 10 Mouse LPA CDR nucleic acid sequences of the
mouse V.sub.H and V.sub.L domains for clone B7 of mouse anti-LPA
monoclonal antibody SEQ ID CLONE CDR NO: V.sub.H CDR B7
GGATACGGCTTCATTAATTACT CDRH1 63 TAATAGAG B7 CTGATTAATCCTGGAAGTGATT
CDRH2 64 ATACTAACTACAATGAGAACT TCAAGGGC B7 AGATTTGGTTACTACGGTAGC
CDRH3 65 GGCAACTACTTTGACTAC V.sub.L CDR B7 ACATCTGGTCAGAGCCTTGTCC
CDRL1 66 ACATTAATGGAAACACCTATT TACAT B7 AAAGTTTCCAACCTATTTTCT CDRL2
67 B7 TCTCAAAGTACACATTTTCCAT CDRL3 68 TCACG
TABLE-US-00011 TABLE 11 Mouse LPA CDR amino acid sequences of the
mouse V.sub.H and V.sub.L domains for clone B7 of mouse anti-LPA
monoclonal antibody SEQ ID CLONE CDR NO: V.sub.H CDR B7 GYGFINYLIE*
CDRH1 69 B7 LINPGSDYTNYNENFKG CDRH2 70 B7 RFGYYGSGNYFDY CDRH3 71
V.sub.L CDR B7 TSGQSLVHINGNTYLH CDRL1 72 B7 KVSNLFS CDRL2 73 B7
SQSTHFPFT CDRL3 74 *The CDRH1 sequence defined according to
Chothia/AbM is the 10-amino acid sequence shown. The five-amino
acid portion of this sequence shown in bold (NYLIE; SEQ ID NO: 62)
is the CDRH1 sequence defined according to Kabat.
TABLE-US-00012 TABLE 12 Mouse LPA CDR nucleic acid sequences of the
mouse V.sub.H and V.sub.L domains for clone B58 of mouse anti-LPA
monoclonal antibody SEQ ID CLONE CDR NO: V.sub.H CDR B58
GGAGACGCCTTCACTAATTACTTGATC CDRH1 75 GAG B58
CTGATTATTCCTGGAACTGGTTATACT CDRH2 76 AACTACAATGAGAACTTCAAGGGC B58
AGATTTGGTTACTACGGTAGTAGCAAC CDRH3 77 TACTTTGACTAC V.sub.L CDR B58
AGATCTAGTCAGAGCCTTGTACACAGT CDRL1 78 AATGGAAACACCTATTTACAT B58
AAAGTTTCCAACCGATTTTCT CDRL2 79 B58 TCTCAAAGTACACATTTTCCATTCACT
CDRL3 80
TABLE-US-00013 TABLE 13 Mouse LPA CDR amino acid sequences of the
mouse V.sub.H and V.sub.L domains for clone B58 of mouse anti-LPA
monoclonal antibody CLONE V.sub.H CDR CDR SEQ ID NO: B58
GDAFTNYLIE* CDRH1 81 B58 LIIPGTGYTNYNENFKG CDRH2 82 B58
RFGYYGSSNYFDY CDRH3 83 V.sub.L CDR B58 RSSQSLVHSNGNTYLH CDRL1 84
B58 KVSNRFS CDRL2 85 B58 SQSTHFPFT CDRL3 86 *The CDRH1 sequence
defined according to Chothia/AbM is the 10-amino acid sequence
shown. The five-amino acid portion of this sequence shown in bold
(NYLIE; SEQ ID NO: 62) is the CDRH1 sequence defined according to
Kabat.
TABLE-US-00014 TABLE 14 Mouse LPA CDR nucleic acid sequences of the
mouse V.sub.H and V.sub.L domains for clone 3A6 of mouse anti-LPA
monoclonal antibody SEQ ID CLONE V.sub.H CDR CDR NO: 3A6
GGAGACGCCTTCACTAATTACTTGATCG CDRH1 87 AG 3A6
CTGATTATTCCTGGAACTGGTTATACTA CDRH2 88 ACTACAATGAGAACTTCAAGGGC 3A6
AGATTTGGTTACTACGGTAGTGGCTACT CDRH3 89 ACTTTGACTAC V.sub.L CDR 3A6
AGATCTAGTCAGAGCCTTGTACACAGTA CDRL1 90 ATGGAAACACCTATTTACAT 3A6
AAAGTTTCCAACCGATTTTCT CDRL2 91 3A6 TCTCAAAGTACACATTTTCCATTCACG
CDRL3 92
TABLE-US-00015 TABLE 15 Mouse LPA CDR amino acid sequences of the
mouse V.sub.H and V.sub.L domains for clone 3A6 of mouse anti-LPA
monoclonal antibody SEQ ID CLONE V.sub.H CDR CDR NO: 3A6
GDAFTNYLIE* CDRH1 93 3A6 LIIPGTGYTNYNENFKG CDRH2 94 3A6
RFGYYGSGYYFDY CDRH3 95 V.sub.L CDR 3A6 RSSQSLVHSNGNTYLH CDRL1 96
3A6 KVSNRFS CDRL2 97 3A6 SQSTHFPFT CDRL3 98 *The CDRH1 sequence
defined according to Chothia/AbM is the 10-amino acid sequence
shown. The five-amino acid portion of this sequence shown in bold
(NYLIE; SEQ ID NO: 62) is the CDRH1 sequence defined according to
Kabat.
TABLE-US-00016 TABLE 16 Mouse LPA CDR nucleic acid sequences of the
mouse V.sub.H and V.sub.L domains for clone A63 of mouse anti-LPA
monoclonal antibody SEQ ID CLONE V.sub.H CDR CDR NO: A63
GGCTTCTCCATCACCAGTGGTTATTACTGGA CDRH1 99 CC A63
TACATAGGCTACGATGGTAGCAATGACTCC CDRH2 100 AACCCATCTCTCAAAAAT A63
GCGATGTTGCGGCGAGGATTTGACTAC CDRH3 101 V.sub.L CDR A63
AGTGCCAGCTCAAGTTTAAGTTACATGCAC CDRL1 102 A63 GACACATCCAAACTGGCTTCT
CDRL2 103 A63 CATCGGCGGAGTAGTTACACG CDRL3 104
TABLE-US-00017 TABLE 17 Mouse LPA CDR amino acid sequences of the
mouse V.sub.H and V.sub.L domains for clone A63 of mouse anti-LPA
monoclonal antibody CLONE V.sub.H CDR CDR SEQ ID NO: A63
GFSITSGYYWT* CDRH1 105 A63 YIGYDGSNDSNPSLKN CDRH2 106 A63 AMLRRGFDY
CDRH3 107 V.sub.L CDR A63 SASSSLSYMH CDRL1 108 A63 DTSKLAS CDRL2
109 A63 HRRSSYT CDRL3 110 *The CDRH1 sequence defined according to
Chothia/AbM is the 11-amino acid sequence shown. The six-amino acid
portion of this sequence shown in bold (SGYYWT; SEQ ID NO: 111) is
the CDRH1 sequence defined according to Kabat.
[0553] Tables 18-27 show nucleotide and amino acid sequences
(nucleotides in Tables 18, 20, 22, 24 and 26, amino acids in Tables
19, 21, 23, 25 and 27) of the variable domains (V.sub.H and
V.sub.L) of the anti-LPA antibodies. In each heavy chain amino acid
sequence in Tables 18-27, amino acids 1-2 (KL) represent enzymatic
cut sites recommended for use with the pCON expression vectors and
amino acids 2-5 (AAT) are Kozak sequences in the corresponding
nucleotide sequence. Amino acids 6-24 (SEQ ID NO: 49) are leader
sequences recommended for use with the pCON heavy chain expression
vector. The last five amino acids of the heavy chain sequences
shown (ASTKG) are the beginning of the constant region sequence
contained within the pCON heavy chain vector.
[0554] In each light chain amino acid sequence in Tables 18-27,
amino acids 1-2 (KL) are enzymatic cut sites recommended for use
with the pCON expression vectors and amino acids 2-5 (AAT) are
Kozak sequences in the corresponding nucleotide sequence. Amino
acids 6-25 (SEQ ID NO: 48) are leader sequences recommended for use
with the pCON light chain expression vector. The last two amino
acids (RT) of the light chain sequences shown are the cut site
recommended for use with the pCON light chain vector.
[0555] Thus the actual heavy chain sequence (minus Kozak sequences,
leaders and cut sites can be seen to be amino acids 25-146 of each
amino acid sequence in Tables 18-27 and the actual light chain
sequence (minus Kozak sequences, leaders and cut sites) can be seen
to be amino acids 26-137 of each amino acid sequence in Tables
18-27. One of ordinary skill can readily determine which of the
nucleic acid sequences in Tables 18-27 (even numbered tables)
correspond to these amino acid sequences.
TABLE-US-00018 TABLE 18 Clone B3 nucleic acid sequences with leader
sequence and cut sites added SEQ ID Sequence NO: B3 Heavy Chain
AAGCTTGCCGCCACCATGGAATGGAGCTGGGTGTTCCTGTTCT 112
TTCTGTCCGTGACCACAGGCGTGCATTCTCAGGTCAAGCTGCA
GCAGTCTGGACCTGAGCTGGTAAGGCCTGGGACTTCAGTGAA
GGTGTCCTGCACGGCTTCTGGAGACGCCTTCACAAATTACTTA
ATAGAGTGGGTAAAACAGAGGCCTGGACAGGGCCTTGAGTGG
ATTGGACTGATTTATCCTGATAGTGGTTACATTAACTACAATG
AGAACTTCAAGGGCAAGGCAACACTGACTGCAGACAGATCCT
CCAGCACTGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGA
CTCTGCGGTCTATTTCTGTGCAAGAAGATTTGCTTACTACGGTA
GTGGCTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCAC
AGTCTCCTCAGCCTCCACCAAGGGCCC B3 Light Chain
AAGCTTGCCGCCACCATGTCTGTGCCTACCCAGGTGCTGGGAC 113
TGCTGCTGCTGTGGCTGACAGACGCCCGCTGTGATGTTGTGAT
GACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAA
GCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTCTAAAAACTA
ATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCA
GTCTCCAAAACTCCTAATCTTCAAAGTTTCCAACCGATTTTCTG
GGGTCCCGGACAGGTTCAGTGGCAGTGGATCAGGGACAGACT
TCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAG
TTTATTTCTGCTCTCAAAGTACACATTTTCCATTCACGTTCGGC
ACGGGGACAAAATTGGAAATAAAACGTACG
TABLE-US-00019 TABLE 19 Clone B3 amino acid sequences with leader
sequence and cut sites added SEQ ID Sequence NO: B3 Heavy Chain
KLAATMEWSWVFLFFLSVTTGVHSQVKLQQSGPELVRPGTSVKV 114
SCTASGDAFTNYLIEWVKQRPGQGLEWIGLIYPDSGYINYNENFK
GKATLTADRSSSTAYMQLSSLTSEDSAVYFCARRFAYYGSGYYF DYWGQGTTLTVSSASTKG B3
Light Chain KLAATMSVPTQVLGLLLLWLTDARCDVVMTQTPLSLPVSLGDQ 115
ASISCRSSQSLLKTNGNTYLHWYLQKPGQSPKLLIFKVSNRFSGVP
DRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHFPFTFGTGTKLEI KRT
TABLE-US-00020 TABLE 20 Clone B7 nucleic acid sequences with leader
sequence and cut sites added SEQ ID Sequence NO: B7 Heavy Chain
AAGCTTGCCGCCACCATGGAATGGAGCTGGGTGTTCCTGTTCT 116
TTCTGTCCGTGACCACAGGCGTGCATTCTCAGGTCCAACTGCA
GCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAA
GGTGTCCTGCAAGGCTTCTGGATACGGCTTCATTAATTACTTA
ATAGAGTGGATAAAACAGAGGCCTGGACAGGGCCTTGAGTGG
ATTGGACTGATTAATCCTGGAAGTGATTATACTAACTACAATG
AGAACTTCAAGGGCAAGGCAACACTGACTGCAGACAAGTCCT
CCAGCACTGCCTACATGCACCTCAGCAGCCTGACATCTGAGGA
CTCTGCGGTCTATTTCTGTGCAAGAAGATTTGGTTACTACGGT
AGCGGCAACTACTTTGACTACTGGGGCCAAGGCACCACTCTCA
CAGTCTCCTCAGCCTCCACCAAGGGCCC B7 Light Chain
AAGCTTGCCGCCACCATGTCTGTGCCTACCCAGGTGCTGGGAC 117
TGCTGCTGCTGTGGCTGACAGACGCCCGCTGTGATGTTGTGAT
GACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAA
GCCTCCATCTCTTGCACATCTGGTCAGAGCCTTGTCCACATTAA
TGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAG
TCTCCAAAGCTCCTCATCTACAAAGTTTCCAACCTATTTTCTGG
GGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTT
CACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGT
TTATTTCTGCTCTCAAAGTACACATTTTCCATTCACGTTCGGCA
CGGGGACAAAATTGGAAATAAAACGTACG
TABLE-US-00021 TABLE 21 Clone B7 amino acid sequences with leader
sequence and cut sites added SEQ ID Sequence NO: B7 Heavy Chain
KLAATMEWSWVFLFFLSVTTGVHSQVQLQQSGAELVRPGTSVK 118
VSCKASGYGFINYLIEWIKQRPGQGLEWIGLINPGSDYTNYNENF
KGKATLTADKSSSTAYMHLSSLTSEDSAVYFCARRFGYYGSGNY FDYWGQGTTLTVSSASTKG B7
Light Chain KLAATMSVPTQVLGLLLLWLTDARCDVVMTQTPLSLPVSLGDQ 119
ASISCTSGQSLVHINGNTYLHWYLQKPGQSPKLLIYKVSNLFSGVP
DRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHFPFTFGTGTKLEI KRT
TABLE-US-00022 TABLE 22 Clone B58 nucleic acid sequences with
leader sequence and cut sites added SEQ ID Sequence NO: B58 Heavy
Chain AAGCTTGCCGCCACCATGGAATGGAGCTGGGTGTTCCTGTTCT 120
TTCTGTCCGTGACCACAGGCGTGCATTCTCAGGTCCAGCTGCA
GCAGTCTGGAGCTGAGCTGGTCAGGCCTGGGACTTCAGTGAA
GGTGTCCTGCAAGGCTTCTGGAGACGCCTTCACTAATTACTTG
ATCGAGTGGGTAAAGCAGAGGCCTGGACAGGGCCTTGAGTGG
ATTGGACTGATTATTCCTGGAACTGGTTATACTAACTACAATG
AGAACTTCAAGGGCAAGGCAACACTGACTGCAGACAAATCCT
CCAGCACTGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGA
CTCTGCGGTCTATTTCTGTGCAAGAAGATTTGGTTACTACGGT
AGTAGCAACTACTTTGACTACTGGGGCCAAGGCACCACTCTCA
CAGTCTCCTCAGCCTCCACCAAGGGCCC B58 Light Chain
AAGCTTGCCGCCACCATGTCTGTGCCTACCCAGGTGCTGGGAC 121
TGCTGCTGCTGTGGCTGACAGACGCCCGCTGTGATGTTGTGAT
GACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAA
GCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTA
ATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCA
GTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTG
GGGTCCCAGACAGGTTCAGTGGCAGTGGACCAGGGACAGATT
TCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAA
TTTATTTCTGCTCTCAAAGTACACATTTTCCATTCACTTTCGGC
ACGGGGACAAAATTGGAAATAAAACGTACG
TABLE-US-00023 TABLE 23 Clone B58 amino acid sequences with leader
sequence and cut sites added SEQ ID Sequence NO: B58 Heavy Chain
KLAATMEWSWVFLFFLSVTTGVHSQVQLQQSGAELVRPGTSVK 122
VSCKASGDAFTNYLIEWVKQRPGQGLEWIGLIIPGTGYTNYNENF
KGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARRFGYYGSSNY FDYWGQGTTLTVSSASTKG
B58 Light Chain KLAATMSVPTQVLGLLLLWLTDARCDVVMTQTPLSLPVSLGDQ 123
ASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGV
PDRFSGSGPGTDFTLKISRVEAEDLGIYFCSQSTHFPFTFGTGTKLE IKRT
TABLE-US-00024 TABLE 24 Clone 3A6 nucleic acid sequences with
leader sequence and cut sites added SEQ ID Sequence NO: 3A6 Heavy
Chain AAGCTTGCCGCCACCATGGAATGGAGCTGGGTGTTCCTGTTCT 124
TTCTGTCCGTGACCACAGGCGTGCATTCTCAGGTCCAGCTGCA
GCAGTCTGGAGCTGAGCTGGTCAGGCCTGGGACTTCAGTGAA
GTTGTCCTGCAAGGCTTCTGGAGACGCCTTCACTAATTACTTG
ATCGAGTGGGTAAAGCAGAGGCCTGGACAGGGCCTTGAGTGG
ATTGGACTGATTATTCCTGGAACTGGTTATACTAACTACAATG
AGAACTTCAAGGGCAAGGCAACACTGACTGCAGACAAGTCCT
CCAGCACTGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGA
CTCTGCGGTCTATTTCTGTGCAAGAAGATTTGGTTACTACGGT
AGTGGCTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCA
CAGTCTCCTCAGCCTCCACCAAGGGCCC 3A6 Light Chain
AAGCTTGCCGCCACCATGTCTGTGCCTACCCAGGTGCTGGGAC 125
TGCTGCTGCTGTGGCTGACAGACGCCCGCTGTGATGTTGTGAT
GACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAA
GCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTA
ATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCA
GTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTG
GGGTCCCAGACAGGTTCAGTGGCAGTGGACCAGGGACAGATT
TCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAG
TTTATTTCTGCTCTCAAAGTACACATTTTCCATTCACGTTCGGC
ACGGGCACAAAATTGGAAATAAAACGTACG
TABLE-US-00025 TABLE 25 Clone 3A6 amino acid sequences with leader
sequence and cut sites added SEQ ID Sequence NO: 3A6 Heavy Chain
KLAATMEWSWVFLFFLSVTTGVHSQVQLQQSGAELVRPGTSVKL 126
SCKASGDAFTNYLIEWVKQRPGQGLEWIGLIIPGTGYTNYNENFK
GKATLTADKSSSTAYMQLSSLTSEDSAVYFCARRFGYYGSGYYF DYWGQGTTLTVSSASTKG 3A6
Light Chain KLAATMSVPTQVLGLLLLWLTDARCDVVMTQTPLSLPVSLGDQ 127
ASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGV
PDRFSGSGPGTDFTLKISRVEAEDLGVYFCSQSTHFPFTFGTGTKL EIKRT
TABLE-US-00026 TABLE 26 Clone A63 nucleic acid sequences with
leader sequence and cut sites added SEQ ID Sequence NO: A63 Heavy
Chain AAGCTTGCCGCCACCATGGAATGGAGCTGGGTGTTCCTGTTCT 128
TTCTGTCCGTGACCACAGGCGTGCATTCTGATATACAGCTTCA
GGAGTCAGGACCTGGCCTCGTGAAACCTTCTCAGTCTCTGTCT
CTCACCTGCTCTGTCACTGGCTTCTCCATCACCAGTGGTTATTA
CTGGACCTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTG
GGTGGCCTACATAGGCTACGATGGTAGCAATGACTCCAACCCA
TCTCTCAAAAATCGAATCTCCATCACCCGTGACACATCTAAGA
ACCAGTTTTTCCTGAAGTTGAATTCTGTGACTACTGAGGACAC
AGCCACATATTACTGTGCAAGAGCGATGTTGCGGCGAGGATTT
GACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCT CCACCAAGGGCCC A63 Light
Chain AAGCTTGCCGCCACCATGTCTGTGCCTACCCAGGTGCTGGGAC 129
TGCTGCTGCTGTGGCTGACAGACGCCCGCTGTCAAATTGTTCT
CACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAG
GTCACCATGACCTGCAGTGCCAGCTCAAGTTTAAGTTACATGC
ACTGGTACCAGCAGAAGCCAGGCACCTCCCCCAAAAGATGGA
TTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTC
AGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGCA
GCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCATCGGCG
GAGTAGTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAA ACGTACG
TABLE-US-00027 TABLE 27 Clone A63 amino acid sequences with leader
sequence and cut sites added SEQ ID Sequence NO: A63 Heavy Chain
KLAATMEWSWVFLFFLSVTTGVHSDIQLQESGPGLVKPSQSLSLT 130
CSVTGFSITSGYYWTWIRQFPGNKLEWVAYIGYDGSNDSNPSLK
NRISITRDTSKNQFFLKLNSVTTEDTATYYCARAMLRRGFDYWG QGTTLTVSSASTKG A63
Light Chain KLAATMSVPTQVLGLLLLWLTDARCQIVLTQSPAIMSASPGEKVT 131
MTCSASSSLSYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGS
GSGTSYSLTISSMEAEDAATYYCHRRSSYTFGGGTKLEIKRT
[0556] For purposes of convenience, Tables 28-32 below are provided
to show the amino acid sequences of the anti-LPA antibody variable
domains shown in Tables 19-27 (odd numbered tables), without the
leader and cut sites.
TABLE-US-00028 TABLE 28 Clone B3 variable domain amino acid
sequences without leader sequence and cut sites SEQ ID Sequence NO:
B3 Heavy Chain QVKLQQSGPELVRPGTSVKVSCTASGDAFTNYLIEWVKQRPGQG 132
LEWIGLIYPDSGYINYNENFKGKATLTADRSSSTAYMQLSSLTSE
DSAVYFCARRFAYYGSGYYFDYWGQGTTLTVSS B3 Light Chain
DVVMTQTPLSLPVSLGDQASISCRSSQSLLKTNGNTYLHWYLQKP 133
GQSPKLLIFKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVY
FCSQSTHFPFTFGTGTKLEIK
TABLE-US-00029 TABLE 29 Clone B7 variable domain amino acid
sequences without leader sequence and cut sites SEQ ID Sequence NO:
B7 Heavy Chain QVQLQQSGAELVRPGTSVKVSCKASGYGFINYLIEWIKQRPGQGL 134
EWIGLINPGSDYTNYNENFKGKATLTADKSSSTAYMHLSSLTSED
SAVYFCARRFGYYGSGNYFDYWGQGTTLTVSS B7 Light Chain
DVVMTQTPLSLPVSLGDQASISCTSGQSLVHINGNTYLHWYLQKP 135
GQSPKLLIYKVSNLFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVY
FCSQSTHFPFTFGTGTKLEIK
TABLE-US-00030 TABLE 30 Clone B58 variable domain amino acid
sequences without leader sequence and cut sites SEQ ID Sequence NO:
B58 Heavy Chain QVQLQQSGAELVRPGTSVKVSCKASGDAFTNYLIEWVKQRPGQG 136
LEWIGLIIPGTGYTNYNENFKGKATLTADKSSSTAYMQLSSLTSE
DSAVYFCARRFGYYGSSNYFDYWGQGTTLTVSS B58 Light Chain
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQK 137
PGQSPKLLIYKVSNRFSGVPDRFSGSGPGTDFTLKISRVEAEDLGI
YFCSQSTHFPFTFGTGTKLEIK
TABLE-US-00031 TABLE 31 Clone 3A6 variable domain amino acid
sequences without leader sequence and cut sites SEQ ID Sequence NO:
3A6 Heavy Chain QVQLQQSGAELVRPGTSVKLSCKASGDAFTNYLIEWVKQRPGQG 138
LEWIGLIIPGTGYTNYNENFKGKATLTADKSSSTAYMQLSSLTSE
DSAVYFCARRFGYYGSGYYFDYWGQGTTLTVSS 3A6 Light Chain
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQK 139
PGQSPKLLIYKVSNRFSGVPDRFSGSGPGTDFTLKISRVEAEDLGV
YFCSQSTHFPFTFGTGTKLEIK
TABLE-US-00032 TABLE 32 Clone A63 variable domain amino acid
sequences without leader sequence and cut sites SEQ ID Sequence NO:
A63 Heavy Chain DIQLQESGPGLVKPSQSLSLTCSVTGFSITSGYYWTWIRQFPGNKL 140
EWVAYIGYDGSNDSNPSLKNRISITRDTSKNQFFLKLNSVTTEDT
ATYYCARAMLRRGFDYWGQGTTLTVSS A63 Light Chain
QIVLTQSPAIMSASPGEKVTMTCSASSSLSYMHWYQQKPGTSPKR 141
WIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHRR SSYTFGGGTKLEIK
Example 9
Lpath's Murine Antibody, Lpathomab.TM. (LT3000)-Overview
[0557] Murine antibody clone B7 was chosen for further study and
renamed Lpathomab.TM., also known as LT3000. As described above,
this murine anti-LPA mAb, was derived from a hybridoma cell line
following immunization of mice with a protein-derivatized LPA
immunogen. A hybridoma cell line with favorable properties was
identified and used to produce a monoclonal antibody using standard
hybridoma culture techniques.
[0558] A comprehensive series of pre-clinical efficacy studies were
performed to confirm the potential therapeutic utility of an
anti-LPA-antibody-based approach. It is believed that antibody
neutralization (e.g., reduction in effective concentration) of
extracellular LPA could result in a marked decrease in disease
progression in humans. For cancer, LPA neutralization could result
in inhibition of tumor proliferation and the growing vasculature
needed to support tumor growth. Furthermore, recent research
suggests that many angiogenesis inhibitors may also act as
anti-invasive and anti-metastatic compounds that could also
mitigate the spread of cancer to sites distant from the initial
tumor. For fibrosis, LPA neutralization could result in a reduction
of the inflammation and fibrosis associated with the aberrant
wound-healing response following tissue injury. Thus, Lpathomab.TM.
could have several mechanisms of action, including: [0559] A direct
effect on tumor cell growth, migration and susceptibility to
chemotherapeutic agents [0560] An indirect effect on tumors through
anti-angiogenic effects [0561] An additional indirect effect on
tumors by preventing the release and neutralization of synergistic
pro-angiogenic growth factors [0562] A direct effect on
proliferation, migration, and transformation of fibroblasts to the
myofibroblast phenotype and collagen production by myofibroblasts
[0563] An indirect effect on tissue fibrosis by preventing the
expression and release of synergistic pro-angiogenic,
pro-inflammatory and pro-fibrotic growth factors
Example 10
Biophysical Properties of Lpathomab/LT3000
[0564] Lpathomab/LT3000 has high affinity for the signaling lipid
LPA (K.sub.D of 1-50 pM as demonstrated by surface plasmon
resonance in the BiaCore assay, and in a direct binding ELISA
assay); in addition, LT3000 demonstrates high specificity for LPA,
having shown no binding affinity for over 100 different bioactive
lipids and proteins, including over 20 bioactive lipids, some of
which are structurally similar to LPA. The murine antibody is a
full-length IgG1k isotype antibody composed of two identical light
chains and two identical heavy chains with a total molecular weight
of 155.5 kDa. The biophysical properties are summarized in Table
33.
TABLE-US-00033 TABLE 33 General Properties of Lpathomab (LT3000)
Identity LT3000 Antibody isotype Murine IgG1k Specificity
Lysophosphatidic acid (LPA) Molecular weight 155.5 kDa OD of 1
mg/mL 1.35 (solution at 280 nm) K.sub.D 1-50 pM Apparent Tm
67.degree. C. at pH 7.4 Appearance Clear if dissolved in 1x PBS
buffer (6.6 mM phosphate, 154 mM sodium chloride, pH 7.4)
Solubility >40 mg/mL in 6.6 mM phosphate, 154 mM sodium
chloride, pH 7.4
[0565] Lpathomab has also shown biological activity in preliminary
cell based assays such as cytokine release, migration and invasion;
these are summarized in Table 34 along with data showing
specificity of LT3000 for LPA isoforms and other bioactive lipids,
and in vitro biological effects of LT3000.
TABLE-US-00034 TABLE 34 LT3000 (Lpathomab, B7 antibody) A.
Competitor Lipid 14:0 16:0 18:1 18:2 20:4 LPA LPA LPA LPA LPA
IC.sub.50 (mM) 0.105 0.483 >2.0 1.487 0.161 MI (%) 61.3 62.9 100
100 67 B. Competitor Lipid LPC S1P C1P Cer DSPA MI (%) 0 2.7 1.0 1
0 C. Cell based assay LPA isoform % Inhibition (over LPA taken as
100) Migration 18:1 35* Invasion 14:0 95* IL-8 Release 18:1 20 IL-6
Release 18:1 23* % Induction (over LPA + TAXOL taken as 100)
Apoptosis 18:1 79 A. Competition ELISA assay was performed with
Lpathomab and 5 LPA isoforms. 18:1 LPA was captured on ELISA
plates. Each competitor lipid (up to 10 .mu.M) was serially diluted
in BSA/PBS and incubated with 3 nM Lpathomab. Mixtures were then
transferred to LPA coated wells and the amount of bound antibody
was measured. B. Competition ELISA was performed to assess
specificity of Lpathomab. Data were normalized to maximum signal
(A.sub.450) and were expressed as percent inhibition (n = 3).
IC.sub.50: half maximum inhibition concentration; MI %: maximum
inhibition (% of binding in the absence of inhibitor). C. Migration
assay: Lpathomab (150 .mu.g/mL) reduced SKOV3 cell migration
triggered by 1 .mu.M LPA (n = 3); Invasion assay: Lpathomab (15
mg/mL) blocked SKOV3 cell invasion triggered by 2 .mu.M LPA (n =
2); Cytokine release of human IL-8 and IL-6: Lpathomab (300-600
.mu.g/mL, respectively) reduced 1 .mu.M LPA-induced release of
pro-angiogenic and metastatic IL-8 and IL-6 in SKOV3 conditioned
media (n = 3). Apoptosis: SKOV3 cells were treated with 1 .mu.M
Taxol; 1 .mu.M LPA blocked Taxol induced apoptosis. The addition to
Lpathomab (150 .mu.g/mL) blocked LPA-induced protection from
apoptosis (n = 1). Data Analysis: Student-t test, *denotes p <
0.05.
[0566] The potent and specific binding of Lpathomab/LT3000 to LPA
results in reduced availability of extracellular LPA with
potentially therapeutic effects against cancer-, angiogenic- and
fibrotic-related disorders.
[0567] A second murine anti-LPA antibody, B3, was also subjected to
binding analysis as shown in Table 35.
TABLE-US-00035 TABLE 35 Biochemical characteristics of B3 antibody
A. BIACORE High density surface Low density surface Lipid Chip 12:0
LPA 18:0 LPA K.sub.D (pM), site 1 (site2) 61(32) 1.6 (0.3) B.
Competition Lipid Cocktail
(C.sub.16:C.sub.18:C.sub.18:1:C.sub.18:2:C.sub.20:4, ratio
3:2:5:11:2) (.mu.M) IC.sub.50 0.263 C. Neutralization Assay B3
antibody (nmol) LPA (nmol) 0 0.16 0.5 0.0428 1 0.0148 2 under limit
of detection A. Biacore analysis for B3 antibody. 12:0 and 18:0
isoforms of LPA were immobilized onto GLC sensor chips; solutions
of B3 were passed over the chips and sensograms were obtained for
both 12:0 and 18:0 LPA chips. Resulted sensograms showed complex
binding kinetics of the antibody due to monovalent and bivalent
antibody binding capacities. K.sub.D values were calculated
approximately for both LPA 12 and LPA 18. B. Competition ELISA
assay was performed with B3 and a cocktail of LPA isoforms
(C.sub.16:C.sub.18:C.sub.18:1:C.sub.18:2:C.sub.20:4 in ratio
3:2:5:11:2). Competitor/Cocktail lipid (up to 10 .mu.M) was
serially diluted in BSA/PBS and incubated with 0.5 .mu.g/mL B3.
Mixtures were then transferred to a LPA coated well plate and the
amount of bound antibody was measured. Data were normalized to
maximum signal (A.sub.450) and were expressed as IC.sub.50 (half
maximum inhibition concentration). C. Neutralization assay:
Increasing concentrations of B3 were conjugated to a gel. Mouse
plasma was then activated to increase endogenous levels of LPA.
Activated plasma samples were then incubated with the increasing
concentrations of the antibody-gel complex. LPA leftover which did
not complex to the antibody was then determined by ELISA. LPA was
sponged up by B3 in an antibody concentration dependent way.
[0568] Selected studies conducted with Lpathomab/LT3000/B7 and B3
are described in the following examples.
Example 11
Lpathomab.TM. in Cancer and Angiogenesis Models
[0569] The pleiotropic effects of LPA suggest that reduced
availability (effective concentration) of extracellular LPA will
(i) reduce growth, metastasis and angiogenesis of primary tumors
and (ii) counter-act LPA's protective anti-apoptotic effect on
tumor. Because of Lpathomab.TM./LT3000's potent and specific
binding to LPA, we hypothesized that in vivo treatment of LT3000 in
preclinical models of cancer would result in various therapeutic
benefits.
[0570] Preclinical studies were conducted using a variety of in
vitro and in vivo systems, demonstrating that Lpathomab.TM./LT3000
(administered every 3 days at doses of 10-50 mg/kg) exhibits a
profile of activity that is consistent with various mechanisms of
action, including:
[0571] Inhibition of tumor growth in human tumor xenograft models
in vivo;
[0572] Reduction in LPA-dependent cell proliferation and invasion
of human tumor in vitro;
[0573] Reduction in angiogenesis, together with reductions in
circulating levels of tumorigenic/angiogenic growth factors
including IL6, IL8, GM-CSF, MMP2 in vivo;
[0574] Reduced metastatic potential; and
[0575] Neutralization of LPA-induced protection against tumor-cell
death.
[0576] In In Vitro Models:
[0577] Reduced proliferation of OVCAR3 ovarian cancer cells;
Neutralization of LPA-induced release of IL-8 from Caki-1, IL-8 and
IL-6 from SKOV3 (ovarian) tumor cells in vitro;
[0578] Mitigation of LPA's effects in protecting SKOV3 tumor cells
from apoptosis (which suggests enhanced efficacy when used in
combination with standard chemotherapeutic agents);
[0579] Inhibition of LPA-induced tumor cell migration and invasion
from chemotherapeutic agents.
[0580] In In Vivo Models:
[0581] Inhibition of metastasis and progression of orthotopic and
subcutaneous human tumors implanted in nude mice;
[0582] Reduction of tumor-associated angiogenesis in subcutaneous
SKOV3 xenograft models and in prostate DU145 cancer cells;
[0583] Neutralization of bFGF- and VEGF-induced angiogenesis in the
murine Matrigel plug assay; and
[0584] Reduced choroidal neovascularization in a model of
laser-induced injury of Bruch's membrane in the eye.
[0585] Reduced inflammation and fibrosis with modulation of
cytokines and growth factors following bleomycin lung injury;
[0586] Further details on efficacy of LT3000 in disease models can
be found in, e.g., WO 2008/150841 and corresponding US application
US-2009-0136483-A1, both of which are commonly assigned with the
instant application and incorporated herein by reference in their
entirety.
Example 12
Humanization of Lpathomab (LT3000)
[0587] The present example describes the generation of humanized
variants of LT3000 and their biochemical properties. A summary of
these variants and properties is in Table 41.
TABLE-US-00036 TABLE 41 Summary of humanization data K.sub.D
K.sub.D Anti-LPA mAb variants BackMut# Yield EC.sub.50 Tm (C12)
(C18) Variant LC HC LC HC ug IgG1 ng/ml .degree. C. pM pM LT 3000
Murine Murine n/a n/a -- 268 67 27 (7) 11 (5) LT3010 510 610 n/a
n/a -- 448 71.5 58 (66) 159 (151) LT3011 502 603 3 3 172 675 71 nd
nd LT3012 502 604 3 4 128 326 67.5 132 (102) 201 (170) LT3013 506
603 1 3 242 1302 71.5 nd nd LT3014 506 604 1 4 451 560 66 218 (76)
370 (340) LT3015 502 602 3 6 416 293 71.5 80 (94) 58 (60) LT3016
506 602 1 6 318 506 71.5 126 (87) 126 (108)
[0588] Materials
[0589] 3,3',5,5'-tetramethylbenzidine liquid substrate (TMB) was
from Sigma-Aldrich (St. Louis, Mo.). Fatty acid-free bovine serum
albumin (BSA) was from Calbiochem (La Jolla, Calif.). Immobilized
Protein A, Immobilized Papain and protein desalting spin column
were from Pierce (Rockford, Ill.). Anti-human IgG (Fc specific)
antibody was purchased from Bethyl (Montgomery, Tex.). Reference
IgGs (non-specific human IgG and mouse IgG), anti-human IgG
(H+L)-horseradish peroxidase conjugate and anti-mouse IgG
(H+L)-horseradish peroxidase conjugate were from Jackson
ImmunoResearch Laboratories (West Grove, Pa.). Lysophosphatidic
acid (LPA) and other lipids used in the competition ELISA were
purchased from Avanti Polar Lipids (Alabaster, Ala.). Biotinylated
LPA was purchased from Echelon Biosciences (Salt Lake City,
Utah).
[0590] Humanization
[0591] The variable domains V.sub.H and V.sub.L of the murine
anti-LPA monoclonal antibody, LT3000 (Lpathomab) were humanized by
grafting the murine CDRs into human framework regions (FR), with
the goal of producing an antibody that retains high affinity,
specificity and binding capacity for LPA. Lefranc, M. P, (2003).
Nucleic Acids Res, 31: 307-10; Martin, A. C. and J. M. Thornton,
(1996) J Mol Biol, 1996. 263: 800-15; Morea, V., A. M. Lesk, and A.
Tramontano (2000) Methods, 20: 267-79; Foote, J. and G. Winter,
(1992) J Mol Biol, 224: 487-99; Chothia, C., et al., (1985). J Mol
Biol, 186:651-63.
[0592] Suitable acceptor human FR sequences were selected from the
IMGT and Kabat databases based on a homology to LT3000 using a
sequence alignment and analysis program (SR v7.6). Lefranc, M. P.
(2003) Nucl. Acids Res. 31:307-310; Kabat, E. A. et al. (1991)
Sequences of Proteins of Immunological Interest, NIH National
Techn. Inform. Service, pp. 1-3242. Sequences with high identity at
FR, vernier, canonical and VH-VL interface residues (VCI) were
initially selected. From this subset, sequences with the most
non-conservative VCI substitutions, unusual proline or cysteine
residues and somatic mutations were excluded. AJ002773 was thus
selected as the human framework on which to base the humanized
version of LT3000 heavy chain variable domain and DQ187679 was thus
selected as the human framework on which to base the humanized
version of LT3000 light chain variable domain.
[0593] A three-dimensional (3D) model containing the humanized VL
and VH sequences was constructed to identify FR residues juxtaposed
to residues that form the CDRs. These FR residues potentially
influence the CDR loop structure and the ability of the antibody to
retain high affinity and specificity for the antigen. Based on this
analysis, 6 residues in AJ002773 and 3 residues in DQ187679 were
identified, deemed significantly different from LT3000, and
considered for mutation back to the murine sequence. Framework
selection and backmutation identification was conducted by
DataMabs, LLP, Radlett, Hertfordshire, UK. A list of the humanized
variants is summarized in Table 42. The I2V mutation, which is
present within the light chain of every variant studied, supports
the presentation of residues in the CDRL3. Other light chain back
mutations include Q45K, which is solvent exposed, and the
conservative Y87F mutation, located on the side of the variable
domain opposite the CDRs. Based on their position, the heavy chain
back mutations appear more likely to influence the stability and
LPA-binding properties of the mAb. I24A and V28G support residues
that form the CDRH1 and the cluster of back mutations (I37V, M48I,
V67A and I69L) form an elaborate network of hydrophobic
interactions that likely effect the stability of the folded
variable domain and the position of the CDRH2. The role of these
back mutations on LPA binding, thermostability and cytokine
released were investigated to identify the lead candidate for
development of a fully humanized, anti-LPA monoclonal antibody.
TABLE-US-00037 TABLE 42 Vector designation and expression level of
the chimcric and the humanized variants in HEK293 cells. Light
Chain Heavy Chain Culture V Expression mAb pATH Back mutations pATH
Back mutations ml (ug/ml) LT3010 510 none 610 None 30 8.44 LT3011
502 I2V, Q45K, Y87F 603 S24A, I28G, M48I 60 2.88 LT3012 502 I2V,
Q45K, Y87F 604 I28G, M48I, V67A, I69L 30 11.2 LT3013 506 I2V 603
S24A, I28G, M48I 60 5.33 LT3014 506 I2V 604 I28G, M48I, V67A, I69L
60 5.83 LT3015 502 I2V, Q45K, Y87F 602 S24A, I28G, V37I, M48I, 60
5.99 V67A, I69L LT3016 506 I2V 602 S24A, I28G, V37I, M48I, 60 3.74
V67A, I69L
Expression of the Humanized Variants
[0594] The humanized variants shown in the table above were
transiently expressed in HEK 293 cells in serum-free conditions,
purified and then characterized in a panel of assays. Plasmids
containing sequences of each light chain (pATH500 series) and heavy
chain (pATH600 series) were transfected into mammalian cells for
production. After 5 days of culture, the mAb titer was determined
using quantitative ELISA. All combinations of the heavy and light
chains yielded between 2-12 ug of antibody per ml of cell culture.
SDS-PAGE under reducing conditions revealed two bands at 25 kDa and
50 kDa with high purity (>98%), consistent with the expected
masses of the light and heavy chains. A single band was observed
under nonreducing conditions with the expected mass of .about.150
KDa.
[0595] Characterization of the Humanized Variants
[0596] The biophysical properties of the humanized variants were
characterized for their binding affinity, binding capacity, yield,
potency and stability. Table 41 presents the binding affinities of
the variants as determined by BiaCore analysis. All the humanized
anti-LPA mAb variants exhibited binding affinity in the low
picomolar range similar to the chimeric anti-LPA antibody (also
known as LT3010) and the murine antibody (LT3000). All of the
humanized variants exhibited a T.sub.M similar to or higher than
that of LT3000, and most had a Tm of approximately 71.degree. C.
With regard to specificity, the humanized variants demonstrated
similar specificity profiles to that of LT3000. For example, LT3000
demonstrated no cross-reactivity to lysophosphatidyl choline (LPC),
phosphatidic acid (PA), various isoforms of lysophosphatidic acid
(14:0 and 18:1 LPA, cyclic phosphatidic acid (cPA), and
phosphatidylcholine (PC).
[0597] Activity of the Humanized Variants
[0598] Five humanized variants (LT3011, LT3013, LT3014, LT3015 and
LT3016) were further assessed in in vitro cell assays. LPA is known
to play an important role in eliciting the release of interleukin-8
(IL-8) from cancer cells. LT3000 reduced IL-8 release from ovarian
cancer cells in a concentration-dependent manner. The humanized
variants exhibited a similar reduction of IL-8 release compared to
LT3000.
[0599] Some humanized variants were also tested for their effect on
microvessel density (MVD) in a Matrigel tube formation assay for
neovascularization. Both were shown to decrease MVD formation.
TABLE-US-00038 TABLE 43 Quantitation of microblood vessel density
using CD31 immunostain with H&E counterstaining in matrigel
plugs. Humanized Humanized Humanized LT3000 LT3000 variant #1
variant #1 variant #2 murine murine (LT3015) (LT3015) (LT3016)
Control (8 m g/kg) (2 mg/kg) (8 mg/kg) (2 mg/kg) (2 mg/kg) Average
64.2 41.5 34 34.4 49 50.8 S.E. 8.0 14.2 13.7 4.2 31.5 18.8 N = 5 4
5 5 5 6 Percent Inhibition 35.4 47.0 46.4 23.7 20.8
[0600] Humanized anti-LPA antibody LT3015 was chosen for further
characterization.
[0601] Antibody Expression and Production in Mammalian Cells
[0602] The murine antibody genes were cloned from hybridomas.
Synthetic genes containing the human framework sequences and the
murine CDRs were assembled from synthetic oligonucleotides and
cloned into pCR4Blunt-TOPO using blunt restriction sites. After
sequencing and observing 100% sequence congruence, the heavy and
light chains were cloned and expressed as a full length IgG1
chimeric antibody using the pConGamma vector for the heavy chain
gene and pConKappa vector for the light chain gene (Lonza
Biologics, Portsmouth N.H.). The expression cassette for each of
these genes contained a promoter, a kozak sequence, and a
terminator. These plasmids were transformed into E. coli (One Shot
Top 10 chemically competent E. coli cells, Invitrogen, Cat No.
C4040-10), grown in LB media and stocked in glycerol. Large scale
plasmid DNA was prepared as described by the manufacturer (Qiagen,
endotoxin-free MAXIPREP.TM. kit, Cat. No 12362). Plasmids were
transfected into the human embryonic kidney cell line 293F using
293fectin and using 293F-FreeStyle Media for culture. The
transfected cultures expressed approximately 2-12 mg/L of humanized
antibody.
[0603] Antibody Purification
[0604] Monoclonal antibodies were purified from culture
supernatants using protein A affinity chromatography. Aliquots
containing 0.5 ml of ProSep-vA-Ultra resin (Millipore, Cat. No
115115827) were added to gravity-flow disposable columns (Pierce,
Cat. No 29924) and equilibrated with 10-15 ml of binding buffer
(Pierce, Cat. No 21001). Culture supernatants containing
transiently expressed humanized antibody were diluted 1:1 with
binding buffer and passed over the resin. The antibody retained on
the column was washed with 15 ml of binding buffer, eluted with low
pH elution buffer (Pierce, Cat. No 21004) and collected in 1 ml
fractions containing 100 ul of binding buffer to neutralize the pH.
Fractions with absorbance (280 nm)>0.1 were dialyzed overnight
(Slide-A-Lyzer Cassettes, 3500 MWCO, Pierce, Cat. No 66382) against
1 liter of PBS buffer (Cellgro, Cat. No 021-030). The dialyzed
samples were concentrated using centricon-YM50 (Amicon, Cat. No
4225) concentrators and filtered through 0.22 uM cellulose acetate
membranes (Costar, Cat. No 8160). The purity of each preparation
was accessed using SDS-PAGE.
[0605] SDS-PAGE Electrophoresis
[0606] Each antibody sample was diluted to 0.5 ug/ul using gel
loading buffer with (reduced) or without (non-reduced)
2-mercaptoethanol (Sigma, Cat. No M-3148). The reduced samples were
heated at 95.degree. C. for 5 min while the non-reduced samples
were incubated at room temperature. A 4-12% gradient gel
(Invitrogen, Cat. No NP0322) was loaded with 2 ug of antibody per
lane and ran at 170 volts for 1 hour at room temperature in
1.times.NuPAGE MOPS SDS running buffer (Invitrogen, Cat. No
NP0001). After electrophoresis, the antibodies were fixed by
soaking the gel in 50% methanol, 10% acetic acid for .about.10 min.
The gel was then washed with 3.times.200 ml distilled water.
Finally, the bands were visualized by staining the gel overnight in
GelCode.RTM. Blue Stain (Pierce, Cat. No 2490) and destaining with
water.
[0607] Quantitative ELISA
[0608] The antibody titer was determined using a quantitative
ELISA. Goat-anti human IgG-Fc antibody (Bethyl A80-104A, 1 mg/ml)
was diluted 1:100 in carbonate buffer (100 mM NaHCO.sub.3, 33.6 mM
Na.sub.2CO.sub.3, pH 9.5). Plates were coated by incubating 100
ul/well of coating solution at 37.degree. C. for 1 hour. The plates
were washed 4.times. with TBS-T (50 mM Tris, 0.14 M NaCl, 0.05%
tween-20, pH 8.0) and blocked with 200 ul/well TBS/BSA (50 mM Tris,
0.14 M NaCl, 1% BSA, pH 8.0) for 1 hour at 37.degree. C. Samples
and standard were prepared on non-binding plates with enough volume
to run in duplicate. The standard was prepared by diluting human
reference serum (Bethyl RS10-110; 4 mg/ml) in TBS-T/BSA (50 mM
Tris, 0.14 NaCl, 1% BSA, 0.05% Tween-20, pH 8.0) to the following
concentrations: 500 ng/ml, 250 ng/ml, 125 ng/ml, 62.5 ng/ml, 31.25
ng/ml, 15.625 ng/ml, 7.8125 ng/ml, and 0.0 ng/ml. Samples were
prepared by making appropriate dilutions in TBS-T/BSA, such that
the optical density (OD) of the samples fell within the range of
the standard; the most linear range being from 125 ng/ml 15.625
ng/ml. After washing the plates 4.times. with TBS-T, 100 ul of the
standard/samples preparation was added to each well and incubated
at 37.degree. C. for 1 hour. Next the plates were washed 4.times.
with TBS-T and incubated for 1 hour at 37.degree. C. with 100
ul/well of HRP-goat anti-human IgG antibody (Bethyl A80-104P, 1
mg/ml) diluted 1:150,000 in TBS-T/BSA. The plates were washed
4.times. with TBS-T and developed using 100 ul/well of TMB
substrate chilled to 4.degree. C. After 7 minutes, the reaction was
stopped with 1M H.sub.2SO.sub.4 (100 ul/well). The OD was measured
at 450 nm, and the data was analyzed using Graphpad Prizm software.
The standard curve was fit using a four parameter equation and used
to calculate the human IgG content in the samples.
[0609] Direct Binding ELISA
[0610] The LPA-binding affinities of the humanized antibodies were
determined using a direct binding ELISA assay. Microtiter ELISA
plates (Costar) were coated overnight with 1.0 ug/ml C12:0 LPA
conjugated to Imj ect malieimide activated bovine serum albumin
(BSA) (Pierce Co.) diluted in 0.1 M carbonate buffer (pH 9.5) at
37.degree. C. for 1 h. Plates were washed with PBS (137 mM NaCl,
2.68 mM KCl, 10.1 mM Na.sub.2HPO.sub.4, 1.76 mM KH.sub.2PO.sub.4;
pH 7.4) and blocked with PBS/BSA/tween-20 for 1 hr at room temp or
overnight at 4.degree. C. For the primary incubation (1 hr at room
temperature), a dilution series of the anti-LPA antibodies (0.4
ug/mL, 0.2 ug/mL, 0.1 ug/mL, 0.05 ug/mL, 0.0125 ug/mL, and 0 ug/mL)
was added to the microplate (100 ml per well). Plates were washed
and incubated with 100 ul per well of HRP conjugated goat
anti-human (H+L) diluted 1:20,000 (Jackson, cat#109-035-003) for 1
hr at room temperature. After washing, the peroxidase was developed
with tetramethylbenzidine substrate (Sigma, cat No T0440) and
stopped by adding 1 M H.sub.2SO.sub.4. The optical density (OD) was
measured at 450 nm using a Thermo Multiskan EX. The EC.sub.50
(half-maximal binding concentration) was determined by a
least-squares fit of the dose-response curves with a four parameter
equation using the Graphpad Prism software.
[0611] The EC.sub.50 of the humanized antibody, LT3015, was
determined to be 75.6 ng/mL, as compared to the murine antibody,
LT3000, which had an EC.sub.50 of 65.3 ng/mL.
[0612] LPA Competition ELISA
[0613] The specificity of the humanized antibody was determined by
competition ELISA. C18:0 LPA coating material was diluted to 0.33
ug/ml with carbonate buffer (100 mM NaHCO3, 33.6 mM Na2CO3, pH
9.5). Plates were coated with 100 ul/well of coating solution and
incubated at 37.degree. C. for 1 hour. The plates were washed 4
times with PBS (100 mM Na2HPO4, 20 mM KH2PO4, 27 mM KCl, 1.37 mM
NaCl, pH 7.4) and blocked with 150 ul/well of PBS, 1% BSA, 0.1%
tween-20 for 1 h at room temperature. The humanized, anti-LPA
antibodies were tested against lipid competitors (14:0 LPA (Avanti,
Cat. No 857120), 18:1 LPA (Avanti, Cat. No 857130), 18:1 LPC
(Avanti, Cat. No 845875), cLPA (Avanti, Cat. No 857328), 18:1 PA
(Avanti, Cat. No 840875), PC (Avanti, Cat. No 850454) at 5 uM, 2.5
uM, 1.25 uM, 0.625 uM, and 0.0 uM. The antibody was diluted to 0.5
ug/ml in PBS, 0.1% tween-20 and combined with the lipid samples at
a 1:3 ratio of antibody to sample on a non-binding plate. The
plates were washed 4 times with PBS and incubated for 1 hour at
room temperature with 100 ul/well of the primary antibody/lipid
complex. Next the plates were washed 4 times with PBS and incubated
for 1 h at room temperature with 100 ul/well of HRP-conjugated goat
anti-human antibody diluted 1:20,000 in PBS, 1% BSA, 0.1% tween-20.
Again the plates were washed 4 times with PBS and developed using
TMB substrate (100 ul/well) at 4.degree. C. After 8 minutes, the
reaction was stopped with 100 ul/well of 1M H2SO4. The optical
density (OD) was measured at 450 nm using a Thermo Multiskan EX.
Raw data were transferred to GraphPad software for analysis.
[0614] The IC.sub.50 for the humanized mAb LT3015 was determined to
be 0.08 uM, whereas the IC.sub.50 for the corresponding murine
antibody, LT3000, was 0.28 uM.
[0615] Thermostability
[0616] The thermostability of the humanized antibodies were studied
by measuring their LPA-binding affinity (EC50) after heating using
the direct binding ELISA. Antibodies dissolved in PBS (Cellgo, Cat.
No 021-040) were diluted to 25 ug/ml and incubated at 60.degree.
C., 65.degree. C., 70.degree. C., 75.degree. C. and 80.degree. C.
for 10 min. Prior to increasing the temperature, 10 ul of each
sample was removed and diluted with 90 ul of PBS and stored on ice.
The samples were then vortexed briefly and the insoluble material
was removed by centrifugation for 1 min at 13,000 rpm. The binding
activity of the supernatant was determined using the direct
LPA-binding ELISA and compared to a control, which consisted of the
same sample without heat treatment.
[0617] The Tm for the humanized antibody, LT3015, was determined to
be 71.5.degree. C., higher than that of the murine parent antibody,
LT3000, which had a Tm of 67.degree. C.
[0618] Surface Plasmon Resonance
[0619] All binding data were collected on a ProteOn optical
biosensor (BioRad, Hercules Calif.). 12:0 LPA-thiol and 18:0
LPA-thiol were coupled to a maleimide modified GLC sensor chip
(Cat. No 176-5011). First, the GLC chip was activated with an equal
mixture of sulfo-NHS/EDC for seven minutes followed by a 7 minute
blocking step with ethyldiamine. Next sulfo-MBS (Pierce Co., cat
#22312) was passed over the surfaces at a concentration of 0.5 mM
in HBS running buffer (10 mM HEPES, 150 mM NaCl, 0.005% tween-20,
pH 7.4). LPA-thiol was diluted into the HBS running buffer to a
concentration of 10, 1 and 0.1 uM and injected for 7 minutes
producing 3 different density LPA surfaces (.about.100, .about.300
and .about.1400 RU). Next, binding data for the humanized
antibodies was collected using a 3-fold dilution series starting
with 25 nM as the highest concentration (original stocks were each
diluted 1 to 100). Surfaces were regenerated with a 10 second pulse
of 100 mM HCl. All data were collected at 25.degree. C. Controls
were processed using a reference surface as well as blank
injections. The response data from each surface showed complex
binding behavior which a likely caused by various degrees of
multivalent binding. In order to extract estimates of the binding
constants, data from the varying antibody concentrations were
globally fit using 1-site and 2-site models. This produced
estimates of the affinity for the bivalent (site 1) and monovalent
site (site 2).
[0620] LPA Molar Binding Capacity
[0621] The molar ratio of LPA:mAb was determined using a
displacement assay. Borosilicate tubes (Fisherbrand, Cat. No
14-961-26) were coated with 5 nanomoles of biotinylated LPA (50 ug
of lipid (Echelon Bioscienes, Cat. No L-012B, Lot No F-66-136 were
suspended in 705 ul of 1:1 chloroform:methanol yielding a 100 uM
solution) using a dry nitrogen stream. The coated tubes were
incubated with 75 ul (125 pmoles) of antibody dissolved in PBS
(Cellgro, Cat. No 021-030) at room temperature. After 3 hours of
incubation, the LPA:mAb complexes were separated from free lipid
using protein desalting columns (Pierce, Cat, No 89849), and the
molar concentration of bound biotinylated LPA was determined using
the HABA/Avidin displacement assay (Pierce, Cat. No 28010)
according to the manufacturer's instructions.
[0622] Measurement of LPA-Induced IL-6 and IL-8 Release in SKOV3
Cells
[0623] Anti-LPA antibodies inhibit the LPA-dependant release of
human CXCL8/IL-8 in conditioned media of SKOV3 ovarian cells. SKOV3
cells (Lot No 4255558, passage 14) were harvested with 2 ml of
1.times. Trypsin EDTA (Mediatech Inc, Cat. No 25-053-CV) and
resuspended in 8 ml of complete medium (10% FBS, Mediatech Inc.
Cat. no 35-011-CV). The cells were centrifuged for 5 min (11,000
rpm) and re-suspended in 5 ml of complete medium. Cells were
counted in duplicate with 0.4% Trypan blue (10 ul cells plus 90 ul
Trypan blue, Invitrogen, Cat. No 15250-061) using a hemocytometer.
In a 96-well plate, 1.times.10.sup.5 cells per well were seeded
(final volume 100 ul/well). The cells were allowed to attach and
form a confluent monolayer by incubating overnight at 37'C. On the
following day, cells were gently washed two times with minimum
media (1 mg/ml BSA in McCoy's medium with L-glutamine, Mediatech,
Cat. No 10-050-CV). The media was adjusted to 1%
penicillin/streptomycin (Mediatech, Cat. No 30-002 CI) and 2.2 g/L
sodium-bicarbonate (Mediatech, Cat. No 25-035-CI). Next, the cells
were serum-starved at 37'C for exactly 24 h, followed by cytokine
stimulation with 1 uM C18:1 LPA (Avanti, Cat. No 857130) dissolved
in 1 mg/ml BSA/PBS (Calbiochem, Cat. No 126575) which was
pre-incubated in presence or absence of humanized LPA antibody
LT3015 (150, 300 or 600 ug/mL) for one hour. Treatments were then
added to the cells. After 22 h of cytokine stimulation, the cells
were centrifuged for 5 min (13,500 rpm) at 4.degree. C. and the
supernatants (cell-conditioned media) were collected. The
CXCL8/IL-8 levels in each supernatant were measured using the
Quantikine human CXCL8/IL-8 ELISA kit according to vendor
instructions (R&D Systems, Minneapolis Minn., Cat. No D8000C).
The IL-6 levels were measured by ELISA using the Quantikine human
IL-6 immunoassay kit (R&D systems, Cat. No. D6050). Data were
analyzed by one-way ANOVA followed by Bonferroni's post test and
expressed as human IL-8 or human IL-6 fold increase. Data are shown
in Table 44 and Table 45 below.
TABLE-US-00039 TABLE 44 Inhibition of human IL-8 release by
humanized anti-LPA antibody LT3015 Stimulus condition Human IL-8
Fold Increase (approx). NT (no treatment) 1 1 uM LPA 7.1 ## LPA +
LT3015, 150 ug/mL 5.7 LPA + LT3015, 300 ug/mL 4.5 ** LPA + LT3015,
600 ug/mL 2.7 ** LT3015, 300 ug/mL 1.1 FBS (10%) 20.1 (* p <
0.05, ** p < 0.001 and ## p < 0.001, n = 3)
TABLE-US-00040 TABLE 45 Inhibition of human IL-6 release by
humanized anti-LPA antibody LT3015 Stimulus condition Human IL-6
Fold Increase (approx). NT (no treatment) 1 1 uM LPA 29 ## LPA +
LT3015, 150 ug/mL 22.1 LPA + LT3015, 300 ug/mL 15.7 * LPA + LT3015,
600 ug/mL 10.8 ** LT3015, 300 ug/mL 1.1 FBS (10%) 69.2 (* p <
0.05, ** p < 0.001 and ## p < 0.001, n = 3)
[0624] Measurement of Tumor Cell Migration in the Scratch Assay
[0625] SKOV3 cells were plated at 15,000 cells per well in a
96-well plate. The following day the cells were serum starved in
minimal media (McCoy's Media 5a, adjusted to contain L-Glutamine,
2.2 g/L Sodium Bicarbonate, 1% penicillin/streptomycin and 1 mg/ml
BSA) for 24 hrs. At time 0 cells were scratched with a p200 pipet
tip down the center of each well, washed with minimal media and
pictures were taken prior to treatment. Cells were then treated
with LPA (C18:1) at 0.2 uM, 1.0 uM and 10 uM concentrations which
were pre-incubated at 37.degree. C. with 1.0 uM LPA in the presence
or absence of antibody at 150 ug/ml. Positive control (10% FBS
treated cells) and antibody alone added to 1 uM LPA, were also
tested. Cells were stimulated for 17 hrs at 37.degree. C. in a 5%
CO.sub.2 incubator. Pictures were taken again 17 hr post-treatment
and % wound closure was measured by adjusting pictures to the same
size and measuring the width of the scratch at time 0 and time 17
hr with a ruler. Data were analyzed by Student's t-test. Results
are shown in Table 46:
TABLE-US-00041 TABLE 46 LT3015 prevents migration of ovarian cancer
cells Treatment Percent wound closure (approx.) NT (no treatment)
30 10% FBS 98 0.2 uM LPA 65 1.0 uM LPA 81 10 uM LPA 89 LPA + LT3015
(150 ug/mL) 59 LT3015 (150 ug/mL) 30
[0626] Intracellular Localization of LPA in Ovarian Cancer
Cells
[0627] The intracellular localization of LPA in SKOV3 cells was
determined by immunohistochemistry. SKOV3 cells were seeded on
coverslips overnight and then processed for LPA staining Cells were
fixed in formalin, blocked in 1% fatty acid-free BSA and then
stained using the murine anti-LPA mAb (LT3000, 0.1 mg/mL),
incubated overnight at 4.degree. C., as primary antibody. LPA
presence was observed as punctuate staining evenly distributed
across the cytoplasm. Controls labeled with secondary antibody only
showed no fluorescent signal. LPA presence in cells has been
confirmed by biochemical measurements (by ELISA).
[0628] Matrigel Assays
[0629] Female C57BL/6 mice around 8 to 10-weeks old and Matrigel
Matrix High Concentration purchased from BD BioSciences (Franklin
Lakes, N.J. (from BD) mixed with 50 ng/ml VEGF and 50 ng/ml bFGF,
heparin 3 ng/ml as angiogenic stimuli were used for this study.
There were five groups of mice, 10 Matrigel plugs were inoculated
into five mice for each group on Day 0. One mouse group served as a
control; four others receive drug treatment in four different doses
by ip injection every other day. All treatments start at Day -1 and
finish at Day 8.
[0630] Thirty C57b1/6 mice were implanted with Matrigel plugs in
order to obtain 25 healthy mice with two well-shaped Matrigel plugs
per mouse. On Day 0, 500 ul Matrigel at 40.degree. C. was
subcutaneously injected to each side of the mouse, injection area
was shaved. To increase the contact area of injected Matrigel into
subcutaneous tissues and form a round shape plug, a wide
subcutaneous pocket was formed by swaying the needlepoint right and
left after a routine subcutaneous insertion. The injection was done
rapidly with an appropriate size needle (21G-25G) to ensure the
entire content was delivered in one plug. The injected Matrigel
rapidly formed a single solid gel plug.
[0631] Animals were treated with 8 or 2 mg/kg of antibody or saline
beginning 1 day prior to the implantation of Matrigel plugs or with
the vehicle. Treatments were administered ip, on a q2d
schedule.
[0632] Plugs from each group were collected at Day 12. The mice
were euthanized and mouse skin was pulled back to expose the plug.
The plugs was dissected out and fixed for histological analysis.
Sections of 5 .mu.m from paraffin-embedded plugs were stained with
anti-CD-31 antibodies. Blood vessel density in a cross sectional
area of each Matrigel plugs were analyzed. For each treatment
group, at least six or more Matrigel plugs were quantitatively
analyzed to assess any statistical significant difference of
microvessel density between groups.
[0633] Reduction of Tumor Progression
[0634] Human mAb LT3015 reduced ovarian tumor SKOV3 progression and
circulating cytokines in biological fluids. Nude mice were
engrafted with either 10 mg/kg LT3015, vehicle, or 2 mg/kg
paclitaxel (Taxol). After 56 days, mice were sacrificed and the
peritoneal cavities were analyzed for tumor burden and ascites
fluid accumulation. Tumors were harvested and final tumor weights
were determined along with ascites volumes. Data were analyzed by
ANOVA and student's t-test analysis. A 32% reduction in tumor
burden was observed in LT3015-treated mice. Serum and ascites
levels of IL-6, IL-8, GM-CSF and VEGF were measured using ELISA
kits from R&D systems, Minneapolis Minn. (Cat. No. D6050,
D8000C, HSGMO and DVE00, respectively) and a reduction in all was
observed in LT3015-treated animals compared to vehicle controls, as
shown in Table 47. *=p<0.05.
TABLE-US-00042 TABLE 47 LT3015 reduces SKOV3 tumor progression and
circulating cytokines in vivo LT3015 Taxol Analysis# Vehicle 10
mg/kg 2 mg/kg Tumor Burden (mg) 1274 .+-. 209 861 .+-. 135 381 .+-.
73 % Reduction 100 32.4 70.1 Ascites presence 6/11 4/12 1/7 Ascites
volume 3.563 .+-. 1.144 1.619 .+-. 0.6113 0.2 .+-. 0.0 (mL)
IL-8-serum (pg/mL) 687.6 .+-. 114.1 324.3 .+-. 55.23* 234.7 .+-.
83.04* IL-6-serum (pg/mL) 84.56 .+-. 16.62 28.02 .+-. 6.212* 29.19
.+-. 9.568* GM-CSF-serum 320.5 .+-. 43.18 225.9 .+-. 54.15 340.2
.+-. 62.98 (pg/mL) IL-8-ascites 2097 .+-. 132.1 1292 .+-. 363.1
2187 .+-. 0## (pg/mL) IL-6 -ascites 1018 .+-. 103.6 400.0 .+-.
218.9* ND (pg/mL) GM-CSF-ascites 1200 .+-. 248.2 1289 .+-. 482.5*
2054 .+-. 0## (pg/mL) VEGF-ascites 3341 .+-. 202.5 2697 .+-. 255.6
3344 .+-. 0## (pg/mL) #Mean .+-. S.E. 1 way ANOVA; Bonferroni's
post test *p < 0.05 (Ascites IL-8 and IL-6, Student t test *p
< 0.05) ##Sample numbers for ascites measurements; N = 6
(Vehicle), N = 4 (hu-Ab), and N = 1(Taxol).
[0635] Thus from the foregoing examples it can be seen that
antibody inhibitors of LPA, particularly the humanized monoclonal
antibody LT3015, are well positioned for use in the treatment of
ovarian cancer, or to augment the efficacy of current ovarian
cancer therapy, by blocking the growth-promoting, angiogenic and/or
metastatic effects of LPA. For example: [0636] The half life of the
murine antibody in mice is .about.4 days when given by IV
administration, and the antibody is fully distributed to the blood
within 6-12 hours when given i.p.
[0637] Anti-LPA antibody (murine) significantly reduced SKOV3 tumor
progression along with lowering serum and ascites levels of human
IL-8, IL-6 and GM-CSF. [0638] Anti-LPA antibody (murine) inhibited
neovascularization in two classical angiogenesis models. [0639]
Anti-LPA antibody (murine) significantly reduced tumor size in a
xenograft CAM assay using a human colon cancer cell line
(COLO-205). [0640] Anti-LPA antibody (murine) reduced the
metastatic spread of B16-F10 cells to lungs. [0641] The murine
anti-LPA mAb, LT3000, was successfully humanized and the humanized
mAb retains the binding, specificity and thermostability of the
murine parent antibody.
Example 13
Preliminary Animal Pharmacokinetics of Lpathomab
[0642] Preliminary PK studies were conducted with Lpathomab. For IV
dosed groups, mice were injected with a single 30 mg/kg dose and
sacrificed at time points up to 15 days. Antibody was also given
via i.p. administration and animals were sacrificed during the
first 24 hrs to compare levels of mAb in the blood over this period
of time for different routes of delivery. Pharmacokinetic
parameters were assessed by WinNonlin. Three mice were sacrificed
at each time point and plasma samples were collected and analyzed
for mAb levels by ELISA. The half-life of Lpathomab in mice was
determined to be 102 hrs (4.25 days) by i.v. administration.
Moreover, the antibody is fully distributed to the blood within
6-12 hrs when given i.p., suggesting that the i.p. administration
is suitable for xenografts and other studies.
TABLE-US-00043 TABLE 48 Pharmacokinetic profile of Lpathomab in
mice Pharmacokinetic Parameters Treatment Group (mg/kg) Route
Estimate SD CV % 1 30 IV AUC 88.35 60.23 68.18 K10-HL 102.7 77.48
75.91 Cmax 0.6 0.13 21.71 Cl 0.34 0.23 68.24 AUMC 13009.8 18549.2
142.58 MRT 147.25 111.78 75.91 Vss 50 10.86 21.73 Software used to
calculate the parameters: WinNonlin V1.1 AUC Area under the curve
K10-HL Elimination half-life Cmax Dose related peak value Cl
Clearance AUMC Area under the first moment curve MRT Mean residence
time Vss Apparent volume of distribution, steady state
Example 14
Safety of Lpathomab Given by Intravenous Injection
[0643] Objective. This study assessed the safety of Lpathomab
following intravenous injection of the antibody. C57BL/6N mice
received Lpathomab (LT3000) for 7 consecutive days followed by a 7
day recovery period for selected animals of each treatment
(recovery groups). Blood samples were collected and processed for
multiple study parameters including classical hematology,
coagulation time and clinical chemistry. Selected organs were
weighed and compared with vehicle only controls.
[0644] Study design. Once a day, single iv bolus injections of
Lpathomab or vehicle control were given at the following doses: 0,
30, 60, 120, and 240 mg/kg. After 7 days of treatment, animals were
euthanized with the only exception of the recovery groups which
were observed for an additional 7 days (recovery period). For each
animal, necropsy consisted of an external examination, including
identification of all clinically recorded lesions, as well as a
detailed internal examination.
[0645] Results. There were no significant differences in the
hematology parameters of antibody-treated groups compared to the
control group. Almost all of the clinical chemistry parameters
tested showed no significant changes when compared to control
animals. There was, however a statistically significant reduction
in triglycerides in both female and male mice (female, mean.+-.SD:
vehicle 89.+-.17, mAb 120 mg/kg, 36.+-.8 p<0.003*; mAb 240 mg/kg
46.+-.18 p*<0.001; male, mean.+-.SD: vehicle 133.+-.24, mAb 240
mg/kg 50.+-.8 p<0.01*; Student t-test). However, there were no
statistically significant reductions in glucose, cholesterol, and
ALT (alanine aminotransferase) or other CBC parameters. No changes
were observed in the weights of mouse brains, hearts, lungs,
pituitary glands, ovaries, spleens, testes, thymus glands, thyroid
or uterus after Lpathomab treatment. There were, however,
significant reductions in liver weights for both genders at certain
doses. The highest treatment group of female mice showed
significant reduction in liver weights compared to controls
(mean.+-.SD: vehicle 1.2.+-.0.27, mAb 240 mg/kg 0.89.+-.0.26
p<0.014*), and the three highest treatment groups in male mice
(60, 120, and 240 mg/kg mAb) showed significant reductions when
compared to controls (mean.+-.SD: vehicle 1.28.+-.0.06, mAb
1.03.+-.0.07, p<0.0001; 1.08.+-.0.11 p<0.002, 1.11.+-.0.11
p<0.004 respectively; Student t-test).
Example 15
Humanized Anti-LPA Variable Region Sequences
[0646] Additional humanized anti-LPA variants of murine antibody B7
and murine antibody B3 heavy chains and of the B3 heavy chain were
generated, as described above. The nucleotide and amino acid
sequences of the variable regions of these variants are shown in
Tables 49-57 below.
TABLE-US-00044 TABLE 49 pATH608 humanized B7 heavy chain variant
(without leader sequence or cut sites) SEQ ID NO: DNA coding
sequence GAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCC 142
GGCGAGAGCCTGAAGATCAGCTGCCAGGCCTTCGGCTACGGC
TTCATCAACTACCTGATCGAGTGGATCCGGCAGATGCCCGGCC
AGGGCCTGGAATGGATCGGCGCAATCAACCCCGGCAGCGACT
ACACCAACTACAACGAGAACTTCAAGGGCCAGGCCACCCTGA
GCGCCGACAAGAGCAGCAGCACCGCCTACCTGCAGTGGAGCA
GCCTGAAGGCCAGCGACACCGCCATGTACTTTTGCGCCAGGCG
GTTCGGCTACTACGGCAGCGGCAACTACTTCGACTACTGGGGC
CAGGGCACCATGGTGACCGTGAGCAGC Translated amino acid sequence
EVQLVQSGAEVKKPGESLKISCQAFGYGFINYLIEWIRQMPGQGL 143
EWIGAINPGSDYTNYNENFKGQATLSADKSSSTAYLQWSSLKAS
DTAMYFCARRFGYYGSGNYFDYWGQGTMVTVSS
TABLE-US-00045 TABLE 50 pATH700 humanized B3 light chain variant
(without leader sequence or cut sites) SEQ ID NO: DNA coding
sequence GACGTGGTGATGACCCAGACCCCCCTGAGCCTGCCCGTGACCC 144
CAGGCGAACCCGCCAGCATCAGCTGTAGAAGCTCCCAGTCCCT
GCTGAAAACCAACGGCAACACCTATCTGCACTGGTATCTGCAG
AAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAAGGTGTCC
AACCGGTTCAGCGGCGTGCCCGACAGATTCAGCGGCAGCGGC
TCCGGCACCGACTTCACCCTGAAGATCAGCCGGGTGGAGGCC
GAGGACGTGGGCGTGTACTTCTGCAGCCAGTCCACCCACTTCC
CTTTCACCTTCGGCCAGGGCACAAAGCTGGAAATCAAG Translated amino acid
sequence DVVMTQTPLSLPVTPGEPASISCRSSQSLLKTNGNTYLHWYLQKP 145
GQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVY
FCSQSTHFPFTFGQGTKLEIK
TABLE-US-00046 TABLE 51 pATH701 humanized B3 light chain variant
(without leader sequence or cut sites) SEQ ID NO: DNA coding
sequence GACGTGGTGATGACCCAGACCCCCCTGAGCCTGCCCGTGACCC 146
CAGGCGAACCCGCCAGCATCAGCTGTAGAAGCTCCCAGTCCCT
GCTGAAAACCAACGGCAACACCTATCTGCACTGGTATCTGCAG
AAGCCCGGCCAGAGCCCCCAGCTGCTGATCTACAAGGTGTCCA
ACCGGTTCAGCGGCGTGCCCGACAGATTCAGCGGCAGCGGCT
CCGGCACCGACTTCACCCTGAAGATCAGCCGGGTGGAGGCCG
AGGACGTGGGCGTGTACTACTGCAGCCAGTCCACCCACTTCCC
TTTCACCTTCGGCCAGGGCACCAAGCTGGAAATCAAG Translated amino acid
sequence DVVMTQTPLSLPVTPGEPASISCRSSQSLLKTNGNTYLHWYLQKP 147
GQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVY
YCSQSTHFPFTFGQGTKLEIK
TABLE-US-00047 TABLE 52 pATH702 humanized B3 light chain variant
(without leader sequence or cut sites) SEQ ID NO: DNA coding
sequence GACGTGGTGATGACCCAGACCCCCCTGAGCCTGCCCGTGACCC 148
CAGGCGAACCCGCCAGCATCAGCTGTAGAAGCTCCCAGAGCC
TGCTGAAAACCAACGGCAACACCTATCTGCACTGGTATCTGCA
GAAGCCCGGCCAGAGCCCCAAGCTGCTGATTTTCAAGGTGTCC
AACCGGTTCAGCGGCGTGCCCGACAGATTCAGCGGCAGCGGC
TCCGGCACCGACTTCACCCTGAAGATCAGCCGGGTGGAGGCC
GAGGACGTGGGCGTGTACTTCTGCAGCCAGTCCACCCACTTCC
CTTTCACCTTCGGCCAGGGCACAAAGCTGGAAATCAAG Translated amino acid
sequence DVVMTQTPLSLPVTPGEPASISCRSSQSLLKTNGNTYLHWYLQKP 149
GQSPKLLIFKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVY
FCSQSTHFPFTFGQGTKLEIK
TABLE-US-00048 TABLE 53 pATH800 humanized B3 heavy chain variant
(without leader sequence or cut sites) SEQ ID NO: DNA coding
sequence GAGGTGCAGCTGGTGCAGAGCGGAGCCGAAGTGAAGAAGCCC 150
GGCGAGAGCCTGAAGATCAGCTGCCAGGCCTTCGGCTACGGC
TTCATCAACTACCTGATCGAGTGGATCCGGCAGATGCCCGGAC
AGGGCCTGGAATGGATCGGCCTGATCTACCCCGACAGCGGCT
ACATCAATTACAACGAGAACTTCAAGGGCCAGGCCACCCTGA
GCGCCGACAAGAGCAGCAGCACCGCCTATCTGCAGTGGAGCA
GCCTGAAGGCCAGCGACACCGCCATGTACTTTTGCGCCAGGCG
GTTCGCCTACTACGGCAGCGGCTACTACTTCGACTACTGGGGC
CAGGGCACAATGGTGACCGTGTCTAGC Translated amino acid sequence
EVQLVQSGAEVKKPGESLKISCQAFGYGFINYLIEWIRQMPGQGL 151
EWIGLIYPDSGYINYNENFKGQATLSADKSSSTAYLQWSSLKASD
TAMYFCARRFAYYGSGYYFDYWGQGTMVTVSS
TABLE-US-00049 TABLE 54 pATH801 humanized B3 heavy chain variant
(without leader sequence or cut sites) SEQ ID NO: DNA coding
sequence GAGGTGCAGCTGGTGCAGAGCGGCGCTGAAGTGAAGAAGCCC 152
GGCGAGAGCCTGAAGATCAGCTGCCAGGCCTTCGGCTACGCCT
TCACCAACTACCTGATCGAGTGGGTGCGCCAGATGCCCGGACA
GGGCCTGGAATGGATGGGCCTGATCTACCCCGACAGCGGCTA
CATCAACTACAACGAGAACTTCAAGGGCCAGGTGACCATCAG
CGCCGACAAGAGCAGCAGCACCGCCTATCTGCAGTGGAGCAG
CCTGAAGGCCAGCGACACCGCCATGTACTTTTGCGCCAGGCGG
TTCGCCTACTACGGCAGCGGCTACTACTTCGACTACTGGGGCC
AGGGCACAATGGTGACCGTGTCCAGC Translated amino acid sequence
EVQLVQSGAEVKKPGESLKISCQAFGYAFTNYLIEWVRQMPGQG 153
LEWMGLIYPDSGYINYNENFKGQVTISADKSSSTAYLQWSSLKAS
DTAMYFCARRFAYYGSGYYFDYWGQGTMVTVSS
TABLE-US-00050 TABLE 55 pATH802 humanized B3 heavy chain variant
(without leader sequence or cut sites) SEQ ID NO: DNA coding
sequence GAGGTGCAGCTGGTGCAGAGCGGCGCTGAAGTGAAGAAGCCC 154
GGCGAGAGCCTGAAGATCAGCTGCCAGGCCTTCGGCTACGCCT
TCACCAACTACCTGATCGAGTGGGTGCGCCAGATGCCCGGACA
GGGCCTGGAATGGATCGGCCTGATCTACCCCGACAGCGGCTAC
ATCAACTACAACGAGAACTTCAAGGGCCAGGCCACCCTGAGC
GCCGACAAGAGCAGCAGCACCGCCTATCTGCAGTGGAGCAGC
CTGAAGGCCAGCGACACCGCCATGTACTTTTGCGCCAGGCGGT
TCGCCTACTACGGCAGCGGCTACTACTTCGACTACTGGGGCCA
GGGCACAATGGTGACCGTGTCCAGC Translated amino acid sequence
EVQLVQSGAEVKKPGESLKISCQAFGYAFTNYLIEWVRQMPGQG 155
LEWIGLIYPDSGYINYNENFKGQATLSADKSSSTAYLQWSSLKAS
DTAMYFCARRFAYYGSGYYFDYWGQGTMVTVSS
TABLE-US-00051 TABLE 56 pATH803 humanized B3 heavy chain variant
(without leader sequence or cut sites) SEQ ID NO: DNA coding
sequence GAGGTGCAGCTGGTGCAGAGCGGAGCCGAAGTGAAGAAGCCC 156
GGCGAGAGCCTGAAGATCAGCTGCCAGGCCTTCGGCGACGCC
TTCACCAACTACCTGATCGAGTGGGTGCGCCAGATGCCCGGAC
AGGGCCTGGAATGGATGGGCCTGATCTACCCCGACAGCGGCT
ACATCAACTACAACGAGAACTTCAAGGGCCAGGTGACCATCA
GCGCCGACAGAAGCAGCAGCACCGCCTATCTGCAGTGGAGCA
GCCTGAAGGCCAGCGACACCGCCATGTACTTTTGCGCCAGGCG
GTTCGCCTACTACGGCAGCGGCTACTACTTCGACTACTGGGGC
CAGGGCACAATGGTGACCGTGTCCAGC Translated amino acid sequence
EVQLVQSGAEVKKPGESLKISCQAFGDAFTNYLIEWVRQMPGQG 157
LEWMGLIYPDSGYINYNENFKGQVTISADRSSSTAYLQWSSLKAS
DTAMYFCARRFAYYGSGYYFDYWGQGTMVTVSS
TABLE-US-00052 TABLE 57 pATH804 humanized B3 heavy chain variant
(without leader sequence or cut sites) SEQ ID NO: DNA coding
sequence GAGGTGCAGCTGGTGCAGAGCGGAGCCGAAGTGAAGAAGCCC 158
GGCGAGAGCCTGAAGATCAGCTGCCAGGCCTTCGGCGACGCC
TTCACCAACTACCTGATCGAGTGGGTGCGCCAGATGCCCGGAC
AGGGCCTGGAATGGATCGGCCTGATCTACCCCGACAGCGGCT
ACATCAACTACAACGAGAACTTCAAGGGCCAGGCCACCCTGA
GCGCCGACAGAAGCAGCAGCACCGCCTATCTGCAGTGGAGCA
GCCTGAAGGCCAGCGACACCGCCATGTACTTTTGCGCCAGGCG
GTTCGCCTACTACGGCAGCGGCTACTACTTCGACTACTGGGGC
CAGGGCACAATGGTGACCGTGTCCAGC Translated amino acid sequence
EVQLVQSGAEVKKPGESLKISCQAFGDAFTNYLIEWVRQMPGQG 159
LEWIGLIYPDSGYINYNENFKGQATLSADRSSSTAYLQWSSLKAS
DTAMYFCARRFAYYGSGYYFDYWGQGTMVTVSS
[0647] A comparison of the variant sequences is shown in Tables 58
and 60. Backmutations are shown in bold. CDR sequences are shown in
gray. Canonical residues are numbered according to the CDR (1, 2 or
3) which which they are associated.
TABLE-US-00053 TABLE 58 Sequences of the variable domains of
anti-LPA light chain humanized variants. CDRs are shaded,
backmutations are in bold. ##STR00004##
TABLE-US-00054 TABLE 59 LPA humanized antibody light chain variant
variable domain sequences and vectors containing them. Number
Identity Vector of back- of back- name Description mutations
mutations pATH500LC pCONkappa (Lonza vector alone) PATH501 B7
humanized light 0 -- chain RKA in vector pATH500LC pATH502 B7
humanized light 3 I2V, Q45K, Y87F chain RKB in vector pATH500
pATH503 B7 humanized light 2 Q45K, Y87F chain RKC in vector pATH500
pATH504 B7 humanized light 2 I2V, Y87F chain RKD in vector pATH500
pATH505 B7 humanized light 2 I2V, Q45K chain RKE in vector pATH500
pATH506 B7 humanized light 1 I2V chain RKF in vector pATH500
pATH700 B3 humanized light 9 I2V, T24R, G26S, chain B3-700 in
V27cL, H27dK, vector pATH500 I27eT, Q45K, L54R, Y87F pATH701 B3
humanized light 7 I2V, T24R, G26S, chain B3-701 in V27cL, H27dK,
vector pATH500 I27eT, L54R, pATH702 B3 humanized light 10 I2V,
T24R, G26S, chain B3-702 in V27cL, H27dK, vector pATH500 I27eT,
Q45K, Y49F, L54R, Y87F
TABLE-US-00055 TABLE 60 Sequences of the variable domains of
anti-LPA heavy chain humanized variants. CDRs are shaded,
backmutations are in bold. ##STR00005##
TABLE-US-00056 TABLE 61 LPA humanized antibody heavy chain variant
variable domain sequences and vectors containing them. Number
Identity Vector of back- of back- name Description mutations
mutations pATH600HC pCONgamma (Lonza vector alone) pATH601 B7
humanized heavy 0 -- chain RH0 in vector pATH600 pATH602 B7
humanized heavy 6 S24A, I28G, chain RH1 in vector V37I, M48I,
pATH600 V67A, I69L pATH603 B7 humanized heavy 3 S24A, I28G, chain
RH8 in vector M48I pATH600 pATH604 B7 humanized heavy 4 I28G, M48I,
chain RH9 in vector V67A, I69L pATH600 pATH605 B7 humanized heavy 2
I28G and M48I chain HX in vector pATH600 pATH606 B7 humanized heavy
2 S24A and M48I chain HY in vector pATH600 pATH607 B7 humanized
heavy 4 S24A, I28G, chain HZ in vector V37I, M48I pATH600 pATH608
B7 humanized heavy 7 S24A, I28G, chain B7-608 in V37I, M48I, vector
pATH600 L50A, V67A, I69L, pATH800 B3 humanized heavy 12 S24A, I28G,
chain B3-800 in V37I, M48I, vector pATH600 N52Y, G53D, D55G, T57I,
V67A, I69L, G97A, N100cY pATH801 B3 humanized heavy 9 S24A, I28A,
chain B3-801 in I30T, N52Y, vector pATH600 G53D, D55G, T57I, G97A,
N100cY pATH802 B3 humanized heavy 12 S24A, I28A, chain B3-802 in
I30T, M48I, vector pATH600 N52Y, G53D, D55G, T57I, V67A, I69L,
G97A, N100cY pATH803 B3 humanized heavy 11 S24A, Y27D, chain B3-803
in I28A, I30T, vector pATH600 N52Y, G53D, D55G, T57I, K73R, G97A,
N100cY pATH804 B3 humanized heavy 14 S24A, Y27D, chain B3-804 in
I28A, I30T, vector pATH600 M48I, N52Y, G53D, D55G, T57I, V67A,
I69L, K73R, G97A, N100cY
Example 16
Creation of the Vector pATH3015 for Cell Line Development
[0648] LT3015 is a recombinant, humanized, monoclonal antibody that
binds with high affinity to the bioactive lipid lysophosphatidic
acid (LPA). LT3015 is a full-length IgG1k isotype antibody composed
of two identical light chains and two identical heavy chains with a
total molecular weight of 150 kDa. The heavy chain contains an
N-linked glycosylation site. The two heavy chains are covalently
coupled to each other through two intermolecular disulfide bonds,
consistent with the structure of a human IgG1.
[0649] LT3015 was originally derived from a murine monoclonal
antibody which was produced using hybridomas generated from mice
immunized with LPA. The humanization of the murine antibody
involved the insertion of the six murine complementarity
determining regions (CDRs) in place of those of a human antibody
framework selected for its structure similarity to the murine
parent antibody. A series of substitutions were made in the
framework to engineer the humanized antibody. These substitutions
are called back mutations and replace human with murine residues
that are involved in the interaction with the antigen. The final
humanized version contains six murine back mutation in the human
framework of variable domain of the heavy chain (pATH602) and three
murine back mutations in the human framework of the variable domain
of the light chain (pATH502).
[0650] The variable domains of the humanized anti-LPA monoclonal
antibody were cloned into the vector IgG1k of the Lonza Biologics'
GS gene expression system to generate the vector pATH3015. This
expression system consists of an expression vector carrying the
constant domains of the antibody genes and the selectable marker
glutamine synthetase (GS). GS is the enzyme responsible for the
biosynthesis of glutamine from glutamate and ammonia. The vector
carrying both the antibody genes and the selectable marker were
transfected into the Chinese Hamster Ovary (CHOK1SV) cell line
providing sufficient glutamine for the cells to survive without
exogenous glutamine. In addition, the specific GS inhibitor,
methionine sulphoximine (MSX) was supplemented in the medium to
inhibit endogenous GS activity such that only the cell lines with
GS activity provided by the vector could survive. The transfected
cells were selected for their ability to grow in glutamine-free
medium in the presence of MSX.
[0651] This example illustrates the cloning steps of the variable
domains of the humanized anti-LPA monoclonal antibody into the
double gene vector IgG1.kappa. of the Lonza Biologic's GS gene
expression system to generate pATH3015.
[0652] Vectors
[0653] The humanized genes were cloned into the Lonza Biologics GS
gene expression system consisting of the GS expression pCON vector
with human antibody constant regions. The constant region genes of
the pCON vectors were isolated from genomic DNA of human peripheral
blood cells.
[0654] pCONgamma1f: Heavy chain vector containing IgG1f constant
region
[0655] pCONkappa2: Light chain vector containing the constant
region
[0656] Construction of Single-Gene Vectors
[0657] The humanized variable regions were assembled by synthetic
oligonucleotides and/or PCR products. Assembled products contained
the restriction sites for subcloning into pCONgammafl for the heavy
chain or pCONkappa2 for the light chain and included a kozak and
signal peptide sequences.
[0658] The heavy chain variable region was cloned into
pCR4Blunt-TOPO using blunt and blunt restriction sites. The plasmid
DNA was purified with Pure Yield Plasmid Midiprep (Promega) from
transformed bacteria. The final construct was verified by
sequencing which found 100% sequence match within the used
restriction sites. The heavy chain fragment was then subcloned into
pCONgamma1f using HindIII and ApaI restriction sites. The plasmid
DNA was purified with Pure Yield Plasmid Midiprep (Promega) from
transformed bacteria. The final construct was verified by
sequencing which found 100% sequence match within the used
restriction sites.
[0659] The light chain variable region was cloned into
pCR4Blunt-TOPO using blunt and blunt restriction sites. The plasmid
DNA was purified with Pure Yield Plasmid Midiprep (Promega) from
transformed bacteria. The final construct was verified by
sequencing which found 100% sequence match within the used
restriction sites. The light chain fragment was then subcloned into
pCONkappa2 using HindIII and BsiWI restriction sites. The plasmid
DNA was purified with Pure Yield Plasmid Midiprep (Promega) from
transformed bacteria. The final construct was verified by
sequencing which found 100% sequence match within the used
restriction sites.
Signal Peptide Design
[0660] To direct the protein towards the secretory pathway, a
signal sequence was introduced immediately upstream and in frame
with the variable region of both the light and heavy chains. The
signal peptide sequences used were the ones recommended by Lonza
Biologics and are from the murine monoclonal antibody B72.3:
TABLE-US-00057 Heavy Chain Leader - IgG (SEQ ID NO: 190) ATG GAA
TGG AGC TGG GTG TTC CTG TTC TTT CTG TCC GTG ACC ACA GGC GTG CAT TCT
Light Chain Leader - kappa (SEQ ID NO: 191) ATG TCT GTG CCT ACC CAG
GTG CTG GGA CTG CTG CTG CTG TGG CTG ACA GAC GCC CGC TGT
Construction of Double-Gene Vectors
[0661] The two single-gene vectors containing the full length heavy
chain gene sequence and the full length light chain gene sequence
were combined into one single vector carrying the GS selectable
marker. Each antibody gene in the double-gene vector is under
control of separate hCMV-MIE (human cytomegalovirus major immediate
early) promoter and has its own polyadenylation signal.
[0662] Cloning
[0663] The single-gene vectors were digested with the restriction
enzymes NotI and SalI releasing the heavy chain expression cassette
from pCONgamma and opening the light chain vector, pCONkappa, which
contains the light chain genes along with the GS selection marker.
De-phosphorylation of the digested vector pCONkappa was performed
to reduce background "vector-only" colonies. Digested products were
run on an agarose gel and bands of interest were cut out of the gel
and cleaned using Bio101 GeneClean kit. For ligation, high
efficiency ligase was used (Roche Rapid DNA Ligation Kit). To
obtain recombinant plasmid from ligation reactions, transformations
were performed using high efficiency E. coli from Invitrogen (One
Shot TOP10 Chemically Competent Cells).
[0664] Analysis of Double-Gene Vector Transformants
[0665] Bacterial colonies which contained successful ligations were
identified by growth of the bacterial colonies, DNA extraction
using Qiagen Miniprep Spin Kit, restriction enzyme digestion with
appropriate enzymes, and run on an agarose gel to visually identify
positive recombinants by the size of the insert and vector
products. Restriction enzyme HindIII was used to screen for
positive restriction digests to ensure both heavy and light chain
regions were present in the final construct. Sequencing was
performed on all positive clones.
[0666] Molecular Biology Protocols
[0667] Restriction Digest Restriction digests were performed on DNA
to prepare fragment for ligation or for cloning verification prior
to checking the molecular sequence. All restriction enzymes were
purchased from Invitrogen or New England Biolabs which come with
the corresponding buffers required for each enzyme.
[0668] Procedure: [0669] 1 DNA (usually 5-10 .mu.L to check for
positive clones and 20-26 .mu.L for DNA to be ligated). [0670] 2 3
.mu.L 10.times. enzyme buffer [0671] 3 0.5 to 1.0 .mu.L enzyme
[0672] 4 Sterile water (to a total of 30 .mu.L reaction volume)
[0673] 5 Reactions were incubated at correct temperature for the
enzyme for 1 hour. Most enzymes were active at 37.degree. C.
however the incubation temperature could vary from room temperature
to 55.degree. C. depending on the enzymes. The restriction enzymes
used for the cloning of pATH3015 were: NotI (Invitrogen 15441-025,
using buffer React 3 at 37.degree. incubation temperature); SalI
(Invitrogen 15217-011, using buffer React 10 at 37.degree.
incubation temperature), PvuI ((Invitrogen 25420-118, using buffer
React 7 at 37.degree. incubation temperature) and a combination of
NotI/SalI using buffer React 10 at 37.degree. incubation
temperature.
Ligation
[0674] Ligations were performed using Roche Rapid Ligation Kit
(cat. no 11635379001) that included T4 DNA 2.times. Ligation
buffer, 5.times.DNA dilution buffer, and T4 DNA ligase.
[0675] Procedure: After adequate restriction enzyme digest, the
GeneClean kit was used to clean the insert fragment and vector from
agarose gel and any enzymes and buffers. Inserts and vectors were
ligated in a final 3:1 molar ratio for best results. Insert
fragments were diluted appropriately for efficient ligations.
[0676] Ligation Reaction:
[0677] Volume ratios of inset to vector varied depending on
dilutions of DNA. Typically a final 3:1 insert to vector molar
ratio was used.
[0678] Standard Ligation Reaction: [0679] 1 X .mu.L INSERT (3:1
INSERT TO VECTOR MOLAR RATIO). [0680] 2 X .mu.L VECTOR (3:1 INSERT
TO VECTOR MOLAR RATIO). [0681] 3 VOLUME WAS BROUGHT UP TO 10 .mu.L
WITH 1.times. DILUTION BUFFER. [0682] 4 10 .mu.L 2.times. LIGATION
BUFFER WAS ADDED. [0683] 5 1 .mu.L T4 DNA LIGASE WAS ADDED. [0684]
6 The reaction was incubated at room temperature for 5 minutes.
[0685] 7 Transform 5 to 7 .mu.L into E. coli TOP10 chemically
competent cells.
Purification of DNA
[0686] The plasmid DNA was prepared using Qiagen Miniprep Kit
(Qiagen, cat. no 27106) according to the manufacturer's
protocol.
Procedure for the Purification of Small Bacterial Cultures for
Screening Recombinants
[0687] (Plasmid Purification with Qiagen Miniprep Kit (Qiagen, cat.
no 27106)) [0688] 1 Harvest bacterial cells by centrifugation in
epi tubes at 13.2 k rpm in microcentrifuge for 30 seconds. [0689] 2
Resuspend the bacterial pellet in 0.25 mL buffer P1 (resuspension
buffer with RNase A at 100 .mu.g/mL). [0690] 3 Add 0.25 mL buffer
P2 (lysis buffer), mix by inverting tube 4-6 times. [0691] 4 Add
0.35 ml, buffer N3 (neutralization buffer) to the lysate and mix
immediately by inverting vigorously 4-6 times. [0692] 5 Centrifuge
tubes at 13.2 k rpm in microcentrifuge for 10 minutes. [0693] 6
Pour supernatant into QIAprep spin column and centrifuge 13.2 k rpm
30 seconds to bind DNA to the column. [0694] 7 Discard
flow-through. [0695] 8 Wash the QIAprep spin column with 0.5 mL
buffer PB and centrifuge 30 seconds. Discard flow-through. [0696] 9
Wash QIAprep spin column with 0.75 mL buffer PE and centrifuge 30
seconds. Discard flow-through and centrifuge for an additional 1
minute. [0697] 10 Place the QIAprep spin column into a clean 1.5 mL
microcentrifuge tube. Add 50 .mu.L UltraPure DNase/RNase free water
(Gibco, cat. no 10977-015) to each spin column and let stand 1
minute. [0698] 11 Centrifuge 1 minute to elute DNA.
Procedure for the Large Scale DNA Purification of Final Double-Gene
Vector for Stable Cell Transfection
[0699] (Plasmid Purification with EndoFree Plasmid Purification Kit
from Qiagen, cat. no 12362) [0700] 1 Harvest bacterial cells by
centrifugation at 6000.times.g for 15 minutes at 4.degree. C.
[0701] 2 Resuspend the bacterial pellet in 10 mL buffer P1
(resuspension buffer with RNase A at 100 .mu.g/mL and LyseBlue
added at 1/1000 dilution). [0702] 3 Add 10 mL buffer P2 (lysis
buffer), mix by inverting tube 4-6 times and incubate at room
temperature for 5 minutes. [0703] 4 Place the QIAfilter cartridge,
with a cap on the outlet nozzle, into a 50 mL conical tube. [0704]
5 Add 10 mL chilled buffer P3 (neutralization buffer) to the lysate
and mix immediately by inverting vigorously 4-6 times. [0705] 6
Pour the lysate into the barrel of the QIAfilter cartridge and
incubate at room temperature for 10 minutes. [0706] 7 Remove the
cap from the QIAfilter cartridge outlet nozzle. Gently insert the
plunger into the QIAfilter cartridge and filter the cell lysate
into a 50 mL conical tube. [0707] 8 Add 2.5 mL buffer ER (endotoxin
removal buffer), mix by inverting the tube 10 times and incubate on
ice for 30 minutes. Buffer ER prevents LPS molecules from binding
to the resin in the QIAGEN-tips allowing purification of DNA
containing less than 0.1 endotoxin units per ug DNA. [0708] 9
Equilibrate a QIAGEN-tip 500 by applying 10 mL buffer QBT
(equilibration buffer) and allow the column to empty by gravity
flow. [0709] 10 Apply the filtered lysate to the QIAGEN-tip and
allow it to enter the resin by gravity flow. [0710] 11 Wash the
QIAGEN-tip with 2.times.30 mL buffer QC (wash buffer). [0711] 12
Elute DNA with 15 mL buffer QN (elution buffer). [0712] 13
Precipitate DNA by adding 10.5 mL room temperature isopropanol to
the eluted DNA. Mix and centrifuge immediately at 14000.times.g for
30 minutes at 4.degree. C. Carefully decant the supernatant. [0713]
14 Wash DNA pellet with 5 mL endotoxin-free room-temperature 70%
ethanol and centrifuge at 14000.times.g for 10 minutes. Carefully
decant the supernatant without disturbing the pellet. [0714] 15
Air-dry the pellet for 10-20 minutes. [0715] 16 Resuspend the DNA
pellet in 100 .mu.L UltraPure DNase/RNase free water (Gibco, cat.
no 10977-015).
Linearization of DNA
[0716] Prior to transfection by electroporation, the double-gene
vector pATH3015 was linearized with Pvu I.
[0717] Procedure: [0718] 1 Digest should contain 0.5 mg of DNA in
750 .mu.L final volume. [0719] 2 Add 75 .mu.L 10.times. restriction
enzyme buffer, 50 units of Pvu I and sterile water to a final
volume of 750 .mu.L. [0720] 3 Digest the DNA overnight at
37.degree. C.
Clean Linearized DNA
[0721] Proteins were removed from the digest by standard
phenol-chloroform extraction. The aqueous and non-aqueous phases
were separated using Phase Lock Gel by Eppendorf (cat. no
E0032005250).
[0722] Procedure: [0723] 1 Centrifuge Phase-Lock gel conicals at
1500 rpm for 1 minute to pellet gel. [0724] 2 Add sample to the
Phase-Lock gel. [0725] 3 Add equal volume Phenol/Chloroform/Isoamyl
alcohol (25:24:1), mix well, without vortexing. [0726] 4 Centrifuge
Phase-Lock gels at 1500 rpm for 5 minutes to separate aqueous and
non-aqueous phases. [0727] 5 Add equal aqueous phase volume of
chloroform and mix well without vortexing. [0728] 6 Centrifuge
Phase-Lock gels at 1500 rpm for 5 minutes to separate aqueous and
non-aqueous phases. [0729] 7 Remove aqueous phase into an Eppendorf
tubes for ethanol precipitation. Evaluation of pATH3015 for
Expression Linearized DNA
[0730] The plasmid pATH3015 linearized (a) or uncut (b) was
transfected into the human embryonic kidney cell line 293F using
293fectin and using 293F-FreeStyle Media for culture. Transfections
were performed at a cell density of 10.sup.6 cells/mL with 0.5
.mu.g/mL. Supernatants were collected by centrifugation at 1100 rpm
for 5 minutes at 25.degree. C. 3 days after transfection. The
expression level was quantified by quantitative ELISA and the
binding was measured in a binding ELISA. The linearized and the
uncut pATH3015 vector demonstrated expression and binding to LPA in
mammalian cells.
[0731] The variable domains of the humanized anti-LPA monoclonal
antibody were cloned into the Lonza Biologics GS gene expression
system to generate the vector pATH3015. The vector is of the
isotype IgG1K and allotype f. This expression system consists of an
expression vector carrying the constant domains of the light chain
and the heavy chain antibody genes and the selectable marker
Glutamine synthetase (GS). GS is the enzyme responsible for the
biosynthesis of glutamine from glutamate and ammonia. The vector
carrying both the antibody genes and the selectable marker is then
transfected into the Chinese Hamster Ovary (CHOK1SV) cell line
providing sufficient glutamine for the cells to survive without
exogenous glutamine. In addition, the cell line is adapted to grow
in serum-free medium.
[0732] The variable regions of the humanized anti-LPA monoclonal
antibody were amplified by PCR from the original vectors and
include a consensus Kozak sequence at the 5' end. The PCR fragments
were then ligated into the corresponding pCon vectors creating
single gene vector clones. After sequence verification and testing
of expression and binding to LPA by transient expression, the
expression cassette containing the heavy chain variable domain and
its corresponding constant domain was cloned as a Not I-Sal I
fragment into the light chain single gene vector generating the
doubling gene vector pATH3015. After sequencing the vector to
ensure that the cloning did not alter the DNA sequence, pATH3015
was linearized with a unique cutter-Pvu I in the .beta.-lactamase
gene. This vector was then tested by transient transfection and it
was observed that the cloning had not disrupted the expected
binding of the antibody protein LT3015 to LPA.
[0733] pATH3015 was introduced by electroporation into the Lonza
proprietary Chinese Hamster Ovary (CHOK1SV) host cell line adapted
for growth in serum-free medium. The cell line derived from this
transfection is designated LH2 and is used to produce drug
substance. The expressed drug LT3015 has the following
characteristics:
TABLE-US-00058 TABLE 62 Characteristics of LT3015 Drug Substance
LT3015 DNA pATH3015 Isotype IgG1.kappa. Molecular Substitutions 6
murine back mutation in the heavy chain 3 murine back mutations in
the light chain Specificity LPA Expression System Lonza Biologics'
GS gene expression system Potency in vitro and in vivo potency
[0734] pATH3016 was produced similarly to pATH3015. As described
above, the heavy chains of pATH3015 and 3016 are identical (derived
from pATH602, having six backmutations), but pATH3016 light chain
(derived from pATH506) contains only the single backmutation I2V.
The humanized monoclonal antibody produced from pATH3016 is LT3016.
Both pATH3015 and pATH3016 were deposited with the American Type
Culture Collection (Manassas Va.) and have ATCC Patent Deposit
Designations PTA-9219 and PTA-9220, respectively.
[0735] All of the compositions and methods described and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods. All such
similar substitutes and modifications apparent to those skilled in
the art are deemed to be within the spirit and scope of the
invention as defined by the appended claims.
[0736] All patents, patent applications, and publications mentioned
in the specification are indicative of the levels of those of
ordinary skill in the art to which the invention pertains. All
patents, patent applications, and publications, including those to
which priority or another benefit is claimed, are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0737] The invention illustratively described herein suitably may
be practiced in the absence of any element(s) not specifically
disclosed herein. Thus, for example, in each instance herein any of
the terms "comprising", "consisting essentially of", and
"consisting of" may be replaced with either of the other two terms.
The terms and expressions which have been employed are used as
terms of description and not of limitation, and there is no
intention that in the use of such terms and expressions of
excluding any equivalents of the features shown and described or
portions thereof, but it is recognized that various modifications
are possible within the scope of the invention claimed. Thus, it
should be understood that although the present invention has been
specifically disclosed by preferred embodiments and optional
features, modification and variation of the concepts herein
disclosed may be resorted to by those skilled in the art, and that
such modifications and variations are considered to be within the
scope of this invention as defined by the appended claims.
Sequence CWU 1
1
189127DNAartificialprimer 1atgaaatgca gctggggcat sttcttc
27226DNAartificialprimer 2atgggatgga gctrtatcat sytctt
26327DNAartificialprimer 3atgaagwtgt ggttaaactg ggttttt
27425DNAartificialprimer 4atgractttg ggytcagctt grttt
25530DNAartificialprimer 5atggactcca ggctcaattt agttttcctt
30627DNAartificialprimer 6atggctgtcy trgsgctrct cttctgc
27726DNAartificialprimer 7atggratgga gckggrtctt tmtctt
26823DNAartificialprimer 8atgagagtgc tgattctttt gtg
23930DNAartificialprimer 9atggmttggg tgtggamctt gctattcctg
301027DNAartificialprimer 10atgggcagac ttacattctc attcctg
271128DNAartificialprimer 11atggattttg ggctgatttt ttttattg
281227DNAartificialprimer 12atgatggtgt taagtcttct gtacctg
271335DNAartificialprimer 13atatccacca tggratgsag ctgkgtmats ctctt
351421DNAartificialprimer 14cagtggatag acagatgggg g
211521DNAartificialprimer 15cagtggatag accgatgggg c
211621DNAartificialprimer 16cagtggatag actgatgggg g
211721DNAartificialprimer 17caagggatag acagatgggg c
211827DNAartificialprimer 18ggcagcacta gtaggggcca gtggata
271932DNAartificialprimer 19gggcaccatg gagacagaca cactcctgct at
322033DNAartificialprimer 20gggcaccatg gattttcaag tgcagatttt cag
332134DNAartificialprimer 21gggcaccatg gagwcacakw ctcaggtctt trta
342230DNAartificialprimer 22gggcaccatg kccccwrctc agytyctkgt
302328DNAartificialprimer 23caccatgaag ttgcctgtta ggctgttg
282430DNAartificialprimer 24atgaagttgv vtgttaggct gttggtgctg
302530DNAartificialprimer 25atggagwcag acacactcct gytatgggtg
302630DNAartificialprimer 26atgagtgtgc tcactcaggt cctggsgttg
302733DNAartificialprimer 27atgaggrccc ctgctcagwt tyttggmwtc ttg
332829DNAartificialprimer 28atggatttwa ggtgcagatt wtcagcttc
292927DNAartificialprimer 29atgaggtkck ktgktsagst sctgrgg
273031DNAartificialprimer 30atgggcwtca agatggagtc acakwyycwg g
313131DNAartificialprimer 31atgtggggay ctktttycmm tttttcaatt g
313225DNAartificialprimer 32atggtrtccw casctcagtt ccttg
253327DNAartificialprimer 33atgtatatat gtttgttgtc tatttct
273428DNAartificialprimer 34atggaagccc cagctcagct tctcttcc
283519DNAartificialprimer 35tgggtatctg gtrcstgtg
193621DNAartificialprimer 36atggagwcag acacactsct g
213726DNAartificialprimer 37atgragtywc agacccaggt cttyrt
263826DNAartificialprimer 38atggagacac attctcaggt ctttgt
263926DNAartificialprimer 39atggattcac aggcccaggt tcttat
264026DNAartificialprimer 40atgatgagtc ctgcccagtt cctctt
264129DNAartificialprimer 41atgaatttgc ctgttcatct cttggtgct
294229DNAartificialprimer 42atggattttc aattggtcct catctcctt
294326DNAartificialprimer 43atgaggtgcc tarctsagtt cctgrg
264426DNAartificialprimer 44atgaagtact ctgctcagtt tctagg
264526DNAartificialprimer 45atgaggcatt ctcttcaatt cttggg
264620DNAartificialprimer 46actggatggt gggaagatgg
204730DNAartificialprimer 47gaagatctag acttactatg cagcatcagc
304860DNAartificialleader 48atgtctgtgc ctacccaggt gctgggactg
ctgctgctgt ggctgacaga cgcccgctgt 604957DNAartificialleader
49atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattct
575030DNAMus musculus 50ggagacgcct tcacaaatta cttaatagag
305151DNAMus musculus 51ctgatttatc ctgatagtgg ttacattaac tacaatgaga
acttcaaggg c 515239DNAMus musculus 52agatttgctt actacggtag
tggctactac tttgactac 395348DNAMus musculus 53agatctagtc agagccttct
aaaaactaat ggaaacacct atttacat 485421DNAMus musculus 54aaagtttcca
accgattttc t 215527DNAMus musculus 55tctcaaagta cacattttcc attcacg
275610PRTMus musculus 56Gly Asp Ala Phe Thr Asn Tyr Leu Ile Glu 1 5
10 5717PRTMus musculus 57Leu Ile Tyr Pro Asp Ser Gly Tyr Ile Asn
Tyr Asn Glu Asn Phe Lys 1 5 10 15 Gly 5813PRTMus musculus 58Arg Phe
Ala Tyr Tyr Gly Ser Gly Tyr Tyr Phe Asp Tyr 1 5 10 5916PRTMus
musculus 59Arg Ser Ser Gln Ser Leu Leu Lys Thr Asn Gly Asn Thr Tyr
Leu His 1 5 10 15 607PRTMus musculus 60Lys Val Ser Asn Arg Phe Ser
1 5 619PRTMus musculus 61Ser Gln Ser Thr His Phe Pro Phe Thr 1 5
625PRTMus musculus 62Asn Tyr Leu Ile Glu 1 5 6330DNAMus musculus
63ggatacggct tcattaatta cttaatagag 306451DNAMus musculus
64ctgattaatc ctggaagtga ttatactaac tacaatgaga acttcaaggg c
516539DNAMus musculus 65agatttggtt actacggtag cggcaactac tttgactac
396648DNAMus musculus 66acatctggtc agagccttgt ccacattaat ggaaacacct
atttacat 486721DNAMus musculus 67aaagtttcca acctattttc t
216827DNAMus musculus 68tctcaaagta cacattttcc attcacg 276910PRTMus
musculus 69Gly Tyr Gly Phe Ile Asn Tyr Leu Ile Glu 1 5 10
7017PRTMus musculus 70Leu Ile Asn Pro Gly Ser Asp Tyr Thr Asn Tyr
Asn Glu Asn Phe Lys 1 5 10 15 Gly 7113PRTMus musculus 71Arg Phe Gly
Tyr Tyr Gly Ser Gly Asn Tyr Phe Asp Tyr 1 5 10 7216PRTMus musculus
72Thr Ser Gly Gln Ser Leu Val His Ile Asn Gly Asn Thr Tyr Leu His 1
5 10 15 737PRTMus musculus 73Lys Val Ser Asn Leu Phe Ser 1 5
749PRTMus musculus 74Ser Gln Ser Thr His Phe Pro Phe Thr 1 5
7530DNAMus musculus 75ggagacgcct tcactaatta cttgatcgag 307651DNAMus
musculus 76ctgattattc ctggaactgg ttatactaac tacaatgaga acttcaaggg c
517739DNAMus musculus 77agatttggtt actacggtag tagcaactac tttgactac
397848DNAMus musculus 78agatctagtc agagccttgt acacagtaat ggaaacacct
atttacat 487921DNAMus musculus 79aaagtttcca accgattttc t
218027DNAMus musculus 80tctcaaagta cacattttcc attcact 278110PRTMus
musculus 81Gly Asp Ala Phe Thr Asn Tyr Leu Ile Glu 1 5 10
8217PRTMus musculus 82Leu Ile Ile Pro Gly Thr Gly Tyr Thr Asn Tyr
Asn Glu Asn Phe Lys 1 5 10 15 Gly 8313PRTMus musculus 83Arg Phe Gly
Tyr Tyr Gly Ser Ser Asn Tyr Phe Asp Tyr 1 5 10 8416PRTMus musculus
84Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1
5 10 15 857PRTMus musculus 85Lys Val Ser Asn Arg Phe Ser 1 5
869PRTMus musculus 86Ser Gln Ser Thr His Phe Pro Phe Thr 1 5
8730DNAMus musculus 87ggagacgcct tcactaatta cttgatcgag 308851DNAMus
musculus 88ctgattattc ctggaactgg ttatactaac tacaatgaga acttcaaggg c
518939DNAMus musculus 89agatttggtt actacggtag tggctactac tttgactac
399048DNAMus musculus 90agatctagtc agagccttgt acacagtaat ggaaacacct
atttacat 489121DNAMus musculus 91aaagtttcca accgattttc t
219227DNAMus musculus 92tctcaaagta cacattttcc attcacg 279310PRTMus
musculus 93Gly Asp Ala Phe Thr Asn Tyr Leu Ile Glu 1 5 10
9417PRTMus musculus 94Leu Ile Ile Pro Gly Thr Gly Tyr Thr Asn Tyr
Asn Glu Asn Phe Lys 1 5 10 15 Gly 9513PRTMus musculus 95Arg Phe Gly
Tyr Tyr Gly Ser Gly Tyr Tyr Phe Asp Tyr 1 5 10 9616PRTMus musculus
96Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1
5 10 15 977PRTMus musculus 97Lys Val Ser Asn Arg Phe Ser 1 5
989PRTMus musculus 98Ser Gln Ser Thr His Phe Pro Phe Thr 1 5
9933DNAMus musculus 99ggcttctcca tcaccagtgg ttattactgg acc
3310048DNAMus musculus 100tacataggct acgatggtag caatgactcc
aacccatctc tcaaaaat 4810127DNAMus musculus 101gcgatgttgc ggcgaggatt
tgactac 2710230DNAMus musculus 102agtgccagct caagtttaag ttacatgcac
3010321DNAMus musculus 103gacacatcca aactggcttc t 2110421DNAMus
musculus 104catcggcgga gtagttacac g 2110511PRTMus musculus 105Gly
Phe Ser Ile Thr Ser Gly Tyr Tyr Trp Thr 1 5 10 10616PRTMus musculus
106Tyr Ile Gly Tyr Asp Gly Ser Asn Asp Ser Asn Pro Ser Leu Lys Asn
1 5 10 15 1079PRTMus musculus 107Ala Met Leu Arg Arg Gly Phe Asp
Tyr 1 5 10810PRTMus musculus 108Ser Ala Ser Ser Ser Leu Ser Tyr Met
His 1 5 10 1097PRTMus musculus 109Asp Thr Ser Lys Leu Ala Ser 1 5
1107PRTMus musculus 110His Arg Arg Ser Ser Tyr Thr 1 5 1116PRTMus
musculus 111Ser Gly Tyr Tyr Trp Thr 1 5 112455DNAMus musculus
112aagcttgccg ccaccatgga atggagctgg gtgttcctgt tctttctgtc
cgtgaccaca 60ggcgtgcatt ctcaggtcaa gctgcagcag tctggacctg agctggtaag
gcctgggact 120tcagtgaagg tgtcctgcac ggcttctgga gacgccttca
caaattactt aatagagtgg 180gtaaaacaga ggcctggaca gggccttgag
tggattggac tgatttatcc tgatagtggt 240tacattaact acaatgagaa
cttcaagggc aaggcaacac tgactgcaga cagatcctcc 300agcactgcct
acatgcagct cagcagcctg acatctgagg actctgcggt ctatttctgt
360gcaagaagat ttgcttacta cggtagtggc tactactttg actactgggg
ccaaggcacc 420actctcacag tctcctcagc ctccaccaag ggccc
455113417DNAMus musculus 113aagcttgccg ccaccatgtc tgtgcctacc
caggtgctgg gactgctgct gctgtggctg 60acagacgccc gctgtgatgt tgtgatgacc
caaactccac tctccctgcc tgtcagtctt 120ggagatcaag cctccatctc
ttgcagatct agtcagagcc ttctaaaaac taatggaaac 180acctatttac
attggtacct gcagaagcca ggccagtctc caaaactcct aatcttcaaa
240gtttccaacc gattttctgg ggtcccggac aggttcagtg gcagtggatc
agggacagac 300ttcacactca agatcagcag agtggaggct gaggatctgg
gagtttattt ctgctctcaa 360agtacacatt ttccattcac gttcggcacg
gggacaaaat tggaaataaa acgtacg 417114151PRTMus musculus 114Lys Leu
Ala Ala Thr Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu 1 5 10 15
Ser Val Thr Thr Gly Val His Ser Gln Val Lys Leu Gln Gln Ser Gly 20
25 30 Pro Glu Leu Val Arg Pro Gly Thr Ser Val Lys Val Ser Cys Thr
Ala 35 40 45 Ser Gly Asp Ala Phe Thr Asn Tyr Leu Ile Glu Trp Val
Lys Gln Arg 50 55 60 Pro Gly Gln Gly Leu Glu Trp Ile Gly Leu Ile
Tyr Pro Asp Ser Gly 65 70 75 80 Tyr Ile Asn Tyr Asn Glu Asn Phe Lys
Gly Lys Ala Thr Leu Thr Ala 85 90 95 Asp Arg Ser Ser Ser Thr Ala
Tyr Met Gln Leu Ser Ser Leu Thr Ser 100 105 110 Glu Asp Ser Ala Val
Tyr Phe Cys Ala Arg Arg Phe Ala Tyr Tyr Gly 115 120 125 Ser Gly Tyr
Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val 130 135 140 Ser
Ser Ala Ser Thr Lys Gly 145 150 115139PRTMus musculus 115Lys Leu
Ala Ala Thr Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu 1 5 10 15
Leu Leu Trp Leu Thr Asp Ala Arg Cys Asp Val Val Met Thr Gln Thr 20
25 30 Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser
Cys 35 40 45 Arg Ser Ser Gln Ser Leu Leu Lys Thr Asn Gly Asn Thr
Tyr Leu His 50 55 60 Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys
Leu Leu Ile Phe Lys 65 70 75 80 Val Ser Asn Arg Phe Ser Gly Val Pro
Asp Arg Phe Ser Gly Ser Gly 85 90 95 Ser Gly Thr Asp Phe Thr Leu
Lys Ile Ser Arg Val Glu Ala Glu Asp 100 105 110 Leu Gly Val Tyr Phe
Cys Ser Gln Ser Thr His Phe Pro Phe Thr Phe 115 120 125 Gly Thr Gly
Thr Lys Leu Glu Ile Lys Arg Thr 130 135 116455DNAMus musculus
116aagcttgccg ccaccatgga atggagctgg gtgttcctgt tctttctgtc
cgtgaccaca 60ggcgtgcatt ctcaggtcca actgcagcag tctggagctg agctggtaag
gcctgggact 120tcagtgaagg tgtcctgcaa ggcttctgga tacggcttca
ttaattactt aatagagtgg 180ataaaacaga ggcctggaca gggccttgag
tggattggac tgattaatcc tggaagtgat 240tatactaact acaatgagaa
cttcaagggc aaggcaacac tgactgcaga caagtcctcc 300agcactgcct
acatgcacct cagcagcctg acatctgagg actctgcggt ctatttctgt
360gcaagaagat ttggttacta cggtagcggc aactactttg actactgggg
ccaaggcacc 420actctcacag tctcctcagc ctccaccaag ggccc
455117417DNAMus musculus 117aagcttgccg ccaccatgtc tgtgcctacc
caggtgctgg gactgctgct gctgtggctg 60acagacgccc gctgtgatgt tgtgatgacc
caaactccac tctccctgcc tgtcagtctt 120ggagatcaag cctccatctc
ttgcacatct ggtcagagcc ttgtccacat taatggaaac 180acctatttac
attggtacct gcagaagcca ggccagtctc caaagctcct catctacaaa
240gtttccaacc tattttctgg ggtcccagac aggttcagtg gcagtggatc
agggacagat 300ttcacactca agatcagcag agtggaggct gaggatctgg
gagtttattt ctgctctcaa 360agtacacatt ttccattcac gttcggcacg
gggacaaaat tggaaataaa acgtacg 417118151PRTMus musculus 118Lys Leu
Ala Ala Thr Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu 1 5 10 15
Ser Val Thr Thr Gly Val His Ser Gln Val Gln Leu Gln Gln Ser Gly 20
25 30 Ala Glu Leu Val Arg Pro Gly Thr Ser Val Lys Val Ser Cys Lys
Ala 35 40 45 Ser Gly Tyr Gly Phe Ile Asn Tyr Leu Ile Glu Trp Ile
Lys Gln Arg 50 55 60 Pro Gly Gln Gly Leu Glu Trp Ile Gly Leu Ile
Asn Pro Gly Ser Asp 65 70 75 80 Tyr Thr Asn Tyr Asn Glu Asn Phe Lys
Gly Lys Ala Thr Leu Thr Ala 85 90 95 Asp Lys Ser Ser Ser Thr Ala
Tyr Met His Leu Ser Ser Leu Thr Ser 100 105 110 Glu Asp Ser Ala Val
Tyr Phe Cys Ala Arg Arg Phe Gly Tyr Tyr Gly 115 120 125 Ser Gly Asn
Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val 130 135 140 Ser
Ser Ala Ser Thr Lys Gly 145 150 119139PRTMus musculus 119Lys Leu
Ala Ala Thr Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu 1 5 10 15
Leu Leu Trp Leu Thr Asp Ala Arg Cys Asp Val Val Met Thr Gln Thr 20
25 30 Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser
Cys 35 40 45 Thr Ser Gly Gln Ser Leu Val His Ile Asn Gly Asn Thr
Tyr Leu His 50 55 60 Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys
Leu Leu Ile Tyr Lys 65 70 75 80 Val Ser Asn Leu Phe Ser Gly Val Pro
Asp Arg Phe Ser Gly Ser Gly 85 90 95 Ser Gly Thr Asp Phe Thr Leu
Lys Ile Ser Arg Val Glu Ala Glu Asp 100 105 110 Leu Gly Val Tyr Phe
Cys Ser Gln Ser Thr His Phe Pro Phe Thr Phe 115 120 125 Gly Thr Gly
Thr Lys Leu Glu Ile Lys Arg Thr 130 135 120455DNAMus musculus
120aagcttgccg
ccaccatgga atggagctgg gtgttcctgt tctttctgtc cgtgaccaca 60ggcgtgcatt
ctcaggtcca gctgcagcag tctggagctg agctggtcag gcctgggact
120tcagtgaagg tgtcctgcaa ggcttctgga gacgccttca ctaattactt
gatcgagtgg 180gtaaagcaga ggcctggaca gggccttgag tggattggac
tgattattcc tggaactggt 240tatactaact acaatgagaa cttcaagggc
aaggcaacac tgactgcaga caaatcctcc 300agcactgcct acatgcagct
cagcagcctg acatctgagg actctgcggt ctatttctgt 360gcaagaagat
ttggttacta cggtagtagc aactactttg actactgggg ccaaggcacc
420actctcacag tctcctcagc ctccaccaag ggccc 455121417DNAMus musculus
121aagcttgccg ccaccatgtc tgtgcctacc caggtgctgg gactgctgct
gctgtggctg 60acagacgccc gctgtgatgt tgtgatgacc caaactccac tctccctgcc
tgtcagtctt 120ggagatcaag cctccatctc ttgcagatct agtcagagcc
ttgtacacag taatggaaac 180acctatttac attggtacct gcagaagcca
ggccagtctc caaagctcct gatctacaaa 240gtttccaacc gattttctgg
ggtcccagac aggttcagtg gcagtggacc agggacagat 300ttcacactca
agatcagcag agtggaggct gaggatctgg gaatttattt ctgctctcaa
360agtacacatt ttccattcac tttcggcacg gggacaaaat tggaaataaa acgtacg
417122151PRTMus musculus 122Lys Leu Ala Ala Thr Met Glu Trp Ser Trp
Val Phe Leu Phe Phe Leu 1 5 10 15 Ser Val Thr Thr Gly Val His Ser
Gln Val Gln Leu Gln Gln Ser Gly 20 25 30 Ala Glu Leu Val Arg Pro
Gly Thr Ser Val Lys Val Ser Cys Lys Ala 35 40 45 Ser Gly Asp Ala
Phe Thr Asn Tyr Leu Ile Glu Trp Val Lys Gln Arg 50 55 60 Pro Gly
Gln Gly Leu Glu Trp Ile Gly Leu Ile Ile Pro Gly Thr Gly 65 70 75 80
Tyr Thr Asn Tyr Asn Glu Asn Phe Lys Gly Lys Ala Thr Leu Thr Ala 85
90 95 Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr
Ser 100 105 110 Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Arg Phe Gly
Tyr Tyr Gly 115 120 125 Ser Ser Asn Tyr Phe Asp Tyr Trp Gly Gln Gly
Thr Thr Leu Thr Val 130 135 140 Ser Ser Ala Ser Thr Lys Gly 145 150
123139PRTMus musculus 123Lys Leu Ala Ala Thr Met Ser Val Pro Thr
Gln Val Leu Gly Leu Leu 1 5 10 15 Leu Leu Trp Leu Thr Asp Ala Arg
Cys Asp Val Val Met Thr Gln Thr 20 25 30 Pro Leu Ser Leu Pro Val
Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys 35 40 45 Arg Ser Ser Gln
Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 50 55 60 Trp Tyr
Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys 65 70 75 80
Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly 85
90 95 Pro Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu
Asp 100 105 110 Leu Gly Ile Tyr Phe Cys Ser Gln Ser Thr His Phe Pro
Phe Thr Phe 115 120 125 Gly Thr Gly Thr Lys Leu Glu Ile Lys Arg Thr
130 135 124455DNAMus musculus 124aagcttgccg ccaccatgga atggagctgg
gtgttcctgt tctttctgtc cgtgaccaca 60ggcgtgcatt ctcaggtcca gctgcagcag
tctggagctg agctggtcag gcctgggact 120tcagtgaagt tgtcctgcaa
ggcttctgga gacgccttca ctaattactt gatcgagtgg 180gtaaagcaga
ggcctggaca gggccttgag tggattggac tgattattcc tggaactggt
240tatactaact acaatgagaa cttcaagggc aaggcaacac tgactgcaga
caagtcctcc 300agcactgcct acatgcagct cagcagcctg acatctgagg
actctgcggt ctatttctgt 360gcaagaagat ttggttacta cggtagtggc
tactactttg actactgggg ccaaggcacc 420actctcacag tctcctcagc
ctccaccaag ggccc 455125417DNAMus musculus 125aagcttgccg ccaccatgtc
tgtgcctacc caggtgctgg gactgctgct gctgtggctg 60acagacgccc gctgtgatgt
tgtgatgacc caaactccac tctccctgcc tgtcagtctt 120ggagatcaag
cctccatctc ttgcagatct agtcagagcc ttgtacacag taatggaaac
180acctatttac attggtacct gcagaagcca ggccagtctc caaagctcct
gatctacaaa 240gtttccaacc gattttctgg ggtcccagac aggttcagtg
gcagtggacc agggacagat 300ttcacactca agatcagcag agtggaggct
gaggatctgg gagtttattt ctgctctcaa 360agtacacatt ttccattcac
gttcggcacg ggcacaaaat tggaaataaa acgtacg 417126151PRTMus musculus
126Lys Leu Ala Ala Thr Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu
1 5 10 15 Ser Val Thr Thr Gly Val His Ser Gln Val Gln Leu Gln Gln
Ser Gly 20 25 30 Ala Glu Leu Val Arg Pro Gly Thr Ser Val Lys Leu
Ser Cys Lys Ala 35 40 45 Ser Gly Asp Ala Phe Thr Asn Tyr Leu Ile
Glu Trp Val Lys Gln Arg 50 55 60 Pro Gly Gln Gly Leu Glu Trp Ile
Gly Leu Ile Ile Pro Gly Thr Gly 65 70 75 80 Tyr Thr Asn Tyr Asn Glu
Asn Phe Lys Gly Lys Ala Thr Leu Thr Ala 85 90 95 Asp Lys Ser Ser
Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser 100 105 110 Glu Asp
Ser Ala Val Tyr Phe Cys Ala Arg Arg Phe Gly Tyr Tyr Gly 115 120 125
Ser Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val 130
135 140 Ser Ser Ala Ser Thr Lys Gly 145 150 127139PRTMus musculus
127Lys Leu Ala Ala Thr Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu
1 5 10 15 Leu Leu Trp Leu Thr Asp Ala Arg Cys Asp Val Val Met Thr
Gln Thr 20 25 30 Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala
Ser Ile Ser Cys 35 40 45 Arg Ser Ser Gln Ser Leu Val His Ser Asn
Gly Asn Thr Tyr Leu His 50 55 60 Trp Tyr Leu Gln Lys Pro Gly Gln
Ser Pro Lys Leu Leu Ile Tyr Lys 65 70 75 80 Val Ser Asn Arg Phe Ser
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly 85 90 95 Pro Gly Thr Asp
Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp 100 105 110 Leu Gly
Val Tyr Phe Cys Ser Gln Ser Thr His Phe Pro Phe Thr Phe 115 120 125
Gly Thr Gly Thr Lys Leu Glu Ile Lys Arg Thr 130 135 128443DNAMus
musculus 128aagcttgccg ccaccatgga atggagctgg gtgttcctgt tctttctgtc
cgtgaccaca 60ggcgtgcatt ctgatataca gcttcaggag tcaggacctg gcctcgtgaa
accttctcag 120tctctgtctc tcacctgctc tgtcactggc ttctccatca
ccagtggtta ttactggacc 180tggatccggc agtttccagg aaacaaactg
gagtgggtgg cctacatagg ctacgatggt 240agcaatgact ccaacccatc
tctcaaaaat cgaatctcca tcacccgtga cacatctaag 300aaccagtttt
tcctgaagtt gaattctgtg actactgagg acacagccac atattactgt
360gcaagagcga tgttgcggcg aggatttgac tactggggcc aaggcaccac
tctcacagtc 420tcctcagcct ccaccaaggg ccc 443129393DNAMus musculus
129aagcttgccg ccaccatgtc tgtgcctacc caggtgctgg gactgctgct
gctgtggctg 60acagacgccc gctgtcaaat tgttctcacc cagtctccag caatcatgtc
tgcatctcca 120ggggagaagg tcaccatgac ctgcagtgcc agctcaagtt
taagttacat gcactggtac 180cagcagaagc caggcacctc ccccaaaaga
tggatttatg acacatccaa actggcttct 240ggagtccctg ctcgcttcag
tggcagtggg tctgggacct cttattctct cacaatcagc 300agcatggagg
ctgaagatgc tgccacttat tactgccatc ggcggagtag ttacacgttc
360ggagggggga ccaagctgga aataaaacgt acg 393130147PRTMus musculus
130Lys Leu Ala Ala Thr Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu
1 5 10 15 Ser Val Thr Thr Gly Val His Ser Asp Ile Gln Leu Gln Glu
Ser Gly 20 25 30 Pro Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu
Thr Cys Ser Val 35 40 45 Thr Gly Phe Ser Ile Thr Ser Gly Tyr Tyr
Trp Thr Trp Ile Arg Gln 50 55 60 Phe Pro Gly Asn Lys Leu Glu Trp
Val Ala Tyr Ile Gly Tyr Asp Gly 65 70 75 80 Ser Asn Asp Ser Asn Pro
Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg 85 90 95 Asp Thr Ser Lys
Asn Gln Phe Phe Leu Lys Leu Asn Ser Val Thr Thr 100 105 110 Glu Asp
Thr Ala Thr Tyr Tyr Cys Ala Arg Ala Met Leu Arg Arg Gly 115 120 125
Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser 130
135 140 Thr Lys Gly 145 131131PRTMus musculus 131Lys Leu Ala Ala
Thr Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu 1 5 10 15 Leu Leu
Trp Leu Thr Asp Ala Arg Cys Gln Ile Val Leu Thr Gln Ser 20 25 30
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys 35
40 45 Ser Ala Ser Ser Ser Leu Ser Tyr Met His Trp Tyr Gln Gln Lys
Pro 50 55 60 Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys
Leu Ala Ser 65 70 75 80 Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser
Gly Thr Ser Tyr Ser 85 90 95 Leu Thr Ile Ser Ser Met Glu Ala Glu
Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 His Arg Arg Ser Ser Tyr Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125 Lys Arg Thr 130
132122PRTMus musculus 132Gln Val Lys Leu Gln Gln Ser Gly Pro Glu
Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Thr Ala
Ser Gly Asp Ala Phe Thr Asn Tyr 20 25 30 Leu Ile Glu Trp Val Lys
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Tyr
Pro Asp Ser Gly Tyr Ile Asn Tyr Asn Glu Asn Phe 50 55 60 Lys Gly
Lys Ala Thr Leu Thr Ala Asp Arg Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85
90 95 Ala Arg Arg Phe Ala Tyr Tyr Gly Ser Gly Tyr Tyr Phe Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120
133112PRTMus musculus 133Asp Val Val Met Thr Gln Thr Pro Leu Ser
Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Leu Lys Thr 20 25 30 Asn Gly Asn Thr Tyr Leu
His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu
Ile Phe Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85
90 95 Thr His Phe Pro Phe Thr Phe Gly Thr Gly Thr Lys Leu Glu Ile
Lys 100 105 110 134122PRTMus musculus 134Gln Val Gln Leu Gln Gln
Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Gly Phe Ile Asn Tyr 20 25 30 Leu Ile
Glu Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45
Gly Leu Ile Asn Pro Gly Ser Asp Tyr Thr Asn Tyr Asn Glu Asn Phe 50
55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala
Tyr 65 70 75 80 Met His Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Gly Tyr Tyr Gly Ser Gly Asn
Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Leu Thr Val Ser
Ser 115 120 135112PRTMus musculus 135Asp Val Val Met Thr Gln Thr
Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile
Ser Cys Thr Ser Gly Gln Ser Leu Val His Ile 20 25 30 Asn Gly Asn
Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro
Lys Leu Leu Ile Tyr Lys Val Ser Asn Leu Phe Ser Gly Val Pro 50 55
60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser
Gln Ser 85 90 95 Thr His Phe Pro Phe Thr Phe Gly Thr Gly Thr Lys
Leu Glu Ile Lys 100 105 110 136122PRTMus musculus 136Gln Val Gln
Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Asp Ala Phe Thr Asn Tyr 20 25
30 Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 Gly Leu Ile Ile Pro Gly Thr Gly Tyr Thr Asn Tyr Asn Glu
Asn Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Gly Tyr Tyr Gly
Ser Ser Asn Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Leu
Thr Val Ser Ser 115 120 137112PRTMus musculus 137Asp Val Val Met
Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Pro Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr
Phe Cys Ser Gln Ser 85 90 95 Thr His Phe Pro Phe Thr Phe Gly Thr
Gly Thr Lys Leu Glu Ile Lys 100 105 110 138122PRTMus musculus
138Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr
1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Asp Ala Phe Thr
Asn Tyr 20 25 30 Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45 Gly Leu Ile Ile Pro Gly Thr Gly Tyr Thr
Asn Tyr Asn Glu Asn Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala
Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu
Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Phe
Gly Tyr Tyr Gly Ser Gly Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln
Gly Thr Thr Leu Thr Val Ser Ser 115 120 139112PRTMus musculus
139Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val
His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys
Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn
Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Pro
Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu
Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Phe Pro
Phe Thr Phe Gly Thr Gly Thr Lys Leu Glu Ile Lys 100 105 110
140118PRTMus musculus 140Asp Ile Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser Val
Thr Gly Phe Ser Ile Thr Ser Gly 20 25
30 Tyr Tyr Trp Thr Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp
35 40 45 Val Ala Tyr Ile Gly Tyr Asp Gly Ser Asn Asp Ser Asn Pro
Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys
Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Thr Glu Asp
Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Ala Met Leu Arg Arg Gly
Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser
115 141104PRTMus musculus 141Gln Ile Val Leu Thr Gln Ser Pro Ala
Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys
Ser Ala Ser Ser Ser Leu Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln
Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser
Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly
Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70
75 80 Asp Ala Ala Thr Tyr Tyr Cys His Arg Arg Ser Ser Tyr Thr Phe
Gly 85 90 95 Gly Gly Thr Lys Leu Glu Ile Lys 100
142366DNAArtificialhumanized antibody variant 142gaggtgcagc
tggtgcagag cggagccgag gtgaagaagc ccggcgagag cctgaagatc 60agctgccagg
ccttcggcta cggcttcatc aactacctga tcgagtggat ccggcagatg
120cccggccagg gcctggaatg gatcggcgca atcaaccccg gcagcgacta
caccaactac 180aacgagaact tcaagggcca ggccaccctg agcgccgaca
agagcagcag caccgcctac 240ctgcagtgga gcagcctgaa ggccagcgac
accgccatgt acttttgcgc caggcggttc 300ggctactacg gcagcggcaa
ctacttcgac tactggggcc agggcaccat ggtgaccgtg 360agcagc
366143122PRTArtificialhumanized antibody variant 143Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu
Lys Ile Ser Cys Gln Ala Phe Gly Tyr Gly Phe Ile Asn Tyr 20 25 30
Leu Ile Glu Trp Ile Arg Gln Met Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Ala Ile Asn Pro Gly Ser Asp Tyr Thr Asn Tyr Asn Glu Asn
Phe 50 55 60 Lys Gly Gln Ala Thr Leu Ser Ala Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Gly Tyr Tyr Gly Ser
Gly Asn Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 144336DNAArtificialhumanized antibody variant
144gacgtggtga tgacccagac ccccctgagc ctgcccgtga ccccaggcga
acccgccagc 60atcagctgta gaagctccca gtccctgctg aaaaccaacg gcaacaccta
tctgcactgg 120tatctgcaga agcccggcca gagccccaag ctgctgatct
acaaggtgtc caaccggttc 180agcggcgtgc ccgacagatt cagcggcagc
ggctccggca ccgacttcac cctgaagatc 240agccgggtgg aggccgagga
cgtgggcgtg tacttctgca gccagtccac ccacttccct 300ttcaccttcg
gccagggcac aaagctggaa atcaag 336145112PRTArtificialhumanized
antibody variant 145Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu Leu Lys Thr 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr
Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg
Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95
Thr His Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105 110 146336DNAArtificialhumanized antibody variant 146gacgtggtga
tgacccagac ccccctgagc ctgcccgtga ccccaggcga acccgccagc 60atcagctgta
gaagctccca gtccctgctg aaaaccaacg gcaacaccta tctgcactgg
120tatctgcaga agcccggcca gagcccccag ctgctgatct acaaggtgtc
caaccggttc 180agcggcgtgc ccgacagatt cagcggcagc ggctccggca
ccgacttcac cctgaagatc 240agccgggtgg aggccgagga cgtgggcgtg
tactactgca gccagtccac ccacttccct 300ttcaccttcg gccagggcac
caagctggaa atcaag 336147112PRTArtificialhumanized antibody variant
147Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu
Lys Thr 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys
Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn
Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu
Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Phe Pro
Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110
148336DNAArtificialhumanized antibody variant 148gacgtggtga
tgacccagac ccccctgagc ctgcccgtga ccccaggcga acccgccagc 60atcagctgta
gaagctccca gagcctgctg aaaaccaacg gcaacaccta tctgcactgg
120tatctgcaga agcccggcca gagccccaag ctgctgattt tcaaggtgtc
caaccggttc 180agcggcgtgc ccgacagatt cagcggcagc ggctccggca
ccgacttcac cctgaagatc 240agccgggtgg aggccgagga cgtgggcgtg
tacttctgca gccagtccac ccacttccct 300ttcaccttcg gccagggcac
aaagctggaa atcaag 336149112PRTArtificialhumanized antibody variant
149Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu
Lys Thr 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys
Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Phe Lys Val Ser Asn
Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu
Asp Val Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Phe Pro
Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110
150366DNAArtificialhumanized antibody variant 150gaggtgcagc
tggtgcagag cggagccgaa gtgaagaagc ccggcgagag cctgaagatc 60agctgccagg
ccttcggcta cggcttcatc aactacctga tcgagtggat ccggcagatg
120cccggacagg gcctggaatg gatcggcctg atctaccccg acagcggcta
catcaattac 180aacgagaact tcaagggcca ggccaccctg agcgccgaca
agagcagcag caccgcctat 240ctgcagtgga gcagcctgaa ggccagcgac
accgccatgt acttttgcgc caggcggttc 300gcctactacg gcagcggcta
ctacttcgac tactggggcc agggcacaat ggtgaccgtg 360tctagc
366151122PRTArtificialhumanized antibody variant 151Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu
Lys Ile Ser Cys Gln Ala Phe Gly Tyr Gly Phe Ile Asn Tyr 20 25 30
Leu Ile Glu Trp Ile Arg Gln Met Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Leu Ile Tyr Pro Asp Ser Gly Tyr Ile Asn Tyr Asn Glu Asn
Phe 50 55 60 Lys Gly Gln Ala Thr Leu Ser Ala Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Ala Tyr Tyr Gly Ser
Gly Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 152366DNAArtificialhumanized antibody variant
152gaggtgcagc tggtgcagag cggcgctgaa gtgaagaagc ccggcgagag
cctgaagatc 60agctgccagg ccttcggcta cgccttcacc aactacctga tcgagtgggt
gcgccagatg 120cccggacagg gcctggaatg gatgggcctg atctaccccg
acagcggcta catcaactac 180aacgagaact tcaagggcca ggtgaccatc
agcgccgaca agagcagcag caccgcctat 240ctgcagtgga gcagcctgaa
ggccagcgac accgccatgt acttttgcgc caggcggttc 300gcctactacg
gcagcggcta ctacttcgac tactggggcc agggcacaat ggtgaccgtg 360tccagc
366153122PRTArtificialhumanized antibody variant 153Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu
Lys Ile Ser Cys Gln Ala Phe Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Met Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Leu Ile Tyr Pro Asp Ser Gly Tyr Ile Asn Tyr Asn Glu Asn
Phe 50 55 60 Lys Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Ala Tyr Tyr Gly Ser
Gly Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 154366DNAArtificialhumanized antibody variant
154gaggtgcagc tggtgcagag cggcgctgaa gtgaagaagc ccggcgagag
cctgaagatc 60agctgccagg ccttcggcta cgccttcacc aactacctga tcgagtgggt
gcgccagatg 120cccggacagg gcctggaatg gatcggcctg atctaccccg
acagcggcta catcaactac 180aacgagaact tcaagggcca ggccaccctg
agcgccgaca agagcagcag caccgcctat 240ctgcagtgga gcagcctgaa
ggccagcgac accgccatgt acttttgcgc caggcggttc 300gcctactacg
gcagcggcta ctacttcgac tactggggcc agggcacaat ggtgaccgtg 360tccagc
366155122PRTArtificialhumanized antibody variant 155Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu
Lys Ile Ser Cys Gln Ala Phe Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Met Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Leu Ile Tyr Pro Asp Ser Gly Tyr Ile Asn Tyr Asn Glu Asn
Phe 50 55 60 Lys Gly Gln Ala Thr Leu Ser Ala Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Ala Tyr Tyr Gly Ser
Gly Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 156366DNAArtificialhumanized antibody variant
156gaggtgcagc tggtgcagag cggagccgaa gtgaagaagc ccggcgagag
cctgaagatc 60agctgccagg ccttcggcga cgccttcacc aactacctga tcgagtgggt
gcgccagatg 120cccggacagg gcctggaatg gatgggcctg atctaccccg
acagcggcta catcaactac 180aacgagaact tcaagggcca ggtgaccatc
agcgccgaca gaagcagcag caccgcctat 240ctgcagtgga gcagcctgaa
ggccagcgac accgccatgt acttttgcgc caggcggttc 300gcctactacg
gcagcggcta ctacttcgac tactggggcc agggcacaat ggtgaccgtg 360tccagc
366157122PRTArtificialhumanized antibody variant 157Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu
Lys Ile Ser Cys Gln Ala Phe Gly Asp Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Met Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Leu Ile Tyr Pro Asp Ser Gly Tyr Ile Asn Tyr Asn Glu Asn
Phe 50 55 60 Lys Gly Gln Val Thr Ile Ser Ala Asp Arg Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Ala Tyr Tyr Gly Ser
Gly Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 158366DNAArtificialhumanized antibody variant
158gaggtgcagc tggtgcagag cggagccgaa gtgaagaagc ccggcgagag
cctgaagatc 60agctgccagg ccttcggcga cgccttcacc aactacctga tcgagtgggt
gcgccagatg 120cccggacagg gcctggaatg gatcggcctg atctaccccg
acagcggcta catcaactac 180aacgagaact tcaagggcca ggccaccctg
agcgccgaca gaagcagcag caccgcctat 240ctgcagtgga gcagcctgaa
ggccagcgac accgccatgt acttttgcgc caggcggttc 300gcctactacg
gcagcggcta ctacttcgac tactggggcc agggcacaat ggtgaccgtg 360tccagc
366159122PRTArtificialhumanized antibody variant 159Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu
Lys Ile Ser Cys Gln Ala Phe Gly Asp Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Met Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Leu Ile Tyr Pro Asp Ser Gly Tyr Ile Asn Tyr Asn Glu Asn
Phe 50 55 60 Lys Gly Gln Ala Thr Leu Ser Ala Asp Arg Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Ala Tyr Tyr Gly Ser
Gly Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 160112PRTArtificialhumanized antibody variant
160Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Thr Ser Gly Gln Ser Leu Val
His Ile 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys
Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn
Leu Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu
Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Phe Pro
Phe Thr Phe Gly Thr Gly Thr Lys Leu Glu Ile Lys 100 105 110
161112PRTArtificialhumanized antibody variant 161Asp Ile Val Met
Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Thr Ser Gly Gln Ser Leu Val His Ile 20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Leu Phe Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Ser Gln Ser 85 90 95 Thr His Phe Pro Phe Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 110
162112PRTArtificialhumanized antibody variant 162Asp Val Val Met
Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Thr Ser Gly Gln Ser Leu Val His Ile 20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Leu Phe Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Phe Cys Ser Gln Ser 85 90
95 Thr His Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110 163112PRTArtificialhumanized antibody variant 163Asp
Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10
15 Glu Pro Ala Ser Ile Ser Cys Thr Ser Gly Gln Ser Leu Val His Ile
20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Leu Phe
Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Phe Pro Phe Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110
164112PRTArtificialhumanized antibody variant 164Asp Val Val Met
Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Thr Ser Gly Gln Ser Leu Val His Ile 20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Leu Phe Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Phe Cys Ser Gln Ser 85 90 95 Thr His Phe Pro Phe Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 110
165112PRTArtificialhumanized antibody variant 165Asp Val Val Met
Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Thr Ser Gly Gln Ser Leu Val His Ile 20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Leu Phe Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Ser Gln Ser 85 90 95 Thr His Phe Pro Phe Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 110
166112PRTArtificialhumanized antibody variant 166Asp Val Val Met
Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Thr Ser Gly Gln Ser Leu Val His Ile 20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Leu Phe Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Ser Gln Ser 85 90 95 Thr His Phe Pro Phe Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 110
167112PRTArtificialhumanized antibody variant 167Asp Val Val Met
Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Lys Thr 20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Phe Cys Ser Gln Ser 85 90 95 Thr His Phe Pro Phe Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 110
168112PRTArtificialhumanized antibody variant 168Asp Val Val Met
Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Lys Thr 20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Ser Gln Ser 85 90 95 Thr His Phe Pro Phe Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 110
169112PRTArtificialhumanized antibody variant 169Asp Val Val Met
Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Lys Thr 20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Lys Leu Leu Ile Phe Lys Val Ser Asn Arg Phe Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Phe Cys Ser Gln Ser 85 90 95 Thr His Phe Pro Phe Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 110
170122PRTArtificialhumanized antibody variant 170Gln Val Gln Leu
Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Gly Phe Ile Asn Tyr 20 25 30
Leu Ile Glu Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Leu Ile Asn Pro Gly Ser Asp Tyr Thr Asn Tyr Asn Glu Asn
Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Met His Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Gly Tyr Tyr Gly Ser
Gly Asn Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Leu Thr
Val Ser Ser 115 120 171122PRTArtificialhumanized antibody variant
171Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15 Ser Leu Lys Ile Ser Cys Gln Ser Phe Gly Tyr Ile Phe Ile
Asn Tyr 20 25 30 Leu Ile Glu Trp Val Arg Gln Met Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45 Gly Leu Ile Asn Pro Gly Ser Asp Tyr Thr
Asn Tyr Asn Glu Asn Phe 50 55 60 Lys Gly Gln Val Thr Ile Ser Ala
Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu
Lys Ala Ser Asp Thr Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe
Gly Tyr Tyr Gly Ser Gly Asn Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln
Gly Thr Met Val Thr Val Ser Ser 115 120
172122PRTArtificialhumanized antibody variant 172Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu
Lys Ile Ser Cys Gln Ala Phe Gly Tyr Gly Phe Ile Asn Tyr 20 25 30
Leu Ile Glu Trp Ile Arg Gln Met Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Leu Ile Asn Pro Gly Ser Asp Tyr Thr Asn Tyr Asn Glu Asn
Phe 50 55 60 Lys Gly Gln Ala Thr Leu Ser Ala Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Gly Tyr Tyr Gly Ser
Gly Asn Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 173122PRTArtificialhumanized antibody variant
173Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15 Ser Leu Lys Ile Ser Cys Gln Ser Phe Gly Tyr Gly Phe Ile
Asn Tyr 20 25 30 Leu Ile Glu Trp Ile Arg Gln Met Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Gly Ser Asp Tyr Thr
Asn Tyr Asn Glu Asn Phe 50 55 60 Lys Gly Gln Ala Thr Leu Ser Ala
Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu
Lys Ala Ser Asp Thr Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe
Gly Tyr Tyr Gly Ser Gly Asn Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln
Gly Thr Met Val Thr Val Ser Ser 115 120
174122PRTArtificialhumanized antibody variant 174Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu
Lys Ile Ser Cys Gln Ala Phe Gly Tyr Ile Phe Ile Asn Tyr 20 25 30
Leu Ile Glu Trp Ile Arg Gln Met Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Leu Ile Asn Pro Gly Ser Asp Tyr Thr Asn Tyr Asn Glu Asn
Phe 50 55 60 Lys Gly Gln Ala Thr Leu Ser Ala Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Gly Tyr Tyr Gly Ser
Gly Asn Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 175122PRTArtificialhumanized antibody variant
175Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15 Ser Leu Lys Ile Ser Cys Gln Ala Phe Gly Tyr Gly Phe Ile
Asn Tyr 20 25 30 Leu Ile Glu Trp Val Arg Gln Met Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Gly Ser Asp Tyr Thr
Asn Tyr Asn Glu Asn Phe 50 55 60 Lys Gly Gln Ala Thr Leu Ser Ala
Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu
Lys Ala Ser Asp Thr Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe
Gly Tyr Tyr Gly Ser Gly Asn Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln
Gly Thr Met Val Thr Val Ser Ser 115 120
176122PRTArtificialhumanized antibody variant 176Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu
Lys Ile Ser Cys Gln Ala Phe Gly Tyr Gly Phe Ile Asn Tyr 20 25 30
Leu Ile Glu Trp Ile Arg Gln Met Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Leu Ile Asn Pro Gly Ser Asp Tyr Thr Asn Tyr Asn Glu Asn
Phe 50 55 60 Lys Gly Gln Ala Thr Leu Ser Ala Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Gly Tyr Tyr Gly Ser
Gly Asn Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 177122PRTArtificialhumanized antibody variant
177Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15 Ser Leu Lys Ile Ser Cys Gln Ala Phe Gly Tyr Gly Phe Ile
Asn Tyr 20 25 30 Leu Ile Glu Trp Ile Arg Gln Met Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Gly Ser Asp Tyr Thr
Asn Tyr Asn Glu Asn Phe 50 55 60 Lys Gly Gln Val Thr Leu Ser Ala
Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu
Lys Ala Ser Asp Thr Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe
Gly Tyr Tyr Gly Ser Gly Asn Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln
Gly Thr Met Val Thr Val Ser Ser 115 120
178122PRTArtificialhumanized antibody variant 178Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu
Lys Ile Ser Cys Gln Ala Phe Gly Tyr Gly Phe Ile Asn Tyr 20 25 30
Leu Ile Glu Trp Ile Arg Gln Met Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Leu Ile Asn Pro Gly Ser Asp Tyr Thr Asn Tyr Asn Glu Asn
Phe 50 55 60 Lys Gly Gln Ala Thr Ile Ser Ala Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Gly Tyr Tyr Gly Ser
Gly Asn Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 179122PRTArtificialhumanized antibody variant
179Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15 Ser Leu Lys Ile Ser Cys Gln Ala Phe Gly Tyr Gly Phe Ile
Asn Tyr 20 25 30 Leu Ile Glu Trp Val Arg Gln Met Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Gly Ser Asp Tyr Thr
Asn Tyr Asn Glu Asn Phe 50 55 60 Lys Gly Gln Val Thr Ile Ser Ala
Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu
Lys Ala Ser Asp Thr Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe
Gly Tyr Tyr Gly Ser Gly Asn Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln
Gly Thr Met Val Thr Val Ser Ser 115 120
180122PRTArtificialhumanized antibody variant 180Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu
Lys Ile Ser Cys Gln Ser Phe Gly Tyr Gly Phe Ile Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Met Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Leu Ile Asn Pro Gly Ser Asp Tyr Thr Asn Tyr Asn Glu Asn
Phe 50 55 60 Lys Gly Gln Ala Thr Leu Ser Ala Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Gly Tyr Tyr Gly Ser
Gly Asn Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 181122PRTArtificialhumanized antibody variant
181Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15 Ser Leu Lys Ile Ser Cys Gln Ser Phe Gly Tyr Gly Phe Ile
Asn Tyr 20 25
30 Leu Ile Glu Trp Val Arg Gln Met Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 Gly Leu Ile Asn Pro Gly Ser Asp Tyr Thr Asn Tyr Asn Glu
Asn Phe 50 55 60 Lys Gly Gln Ala Thr Leu Ser Ala Asp Lys Ser Ser
Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp
Thr Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Gly Tyr Tyr Gly
Ser Gly Asn Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Met Val
Thr Val Ser Ser 115 120 182122PRTArtificialhumanized antibody
variant 182Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Gln Ala Phe Gly Tyr Ile
Phe Ile Asn Tyr 20 25 30 Leu Ile Glu Trp Val Arg Gln Met Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Gly Ser Asp
Tyr Thr Asn Tyr Asn Glu Asn Phe 50 55 60 Lys Gly Gln Ala Thr Leu
Ser Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser
Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Phe Cys 85 90 95 Ala Arg
Arg Phe Gly Tyr Tyr Gly Ser Gly Asn Tyr Phe Asp Tyr Trp 100 105 110
Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120
183122PRTArtificialhumanized antibody variant 183Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu
Lys Ile Ser Cys Gln Ala Phe Gly Tyr Gly Phe Ile Asn Tyr 20 25 30
Leu Ile Glu Trp Ile Arg Gln Met Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Leu Ile Asn Pro Gly Ser Asp Tyr Thr Asn Tyr Asn Glu Asn
Phe 50 55 60 Lys Gly Gln Ala Thr Leu Ser Ala Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Gly Tyr Tyr Gly Ser
Gly Asn Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 184122PRTArtificialhumanized antibody variant
184Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15 Ser Leu Lys Ile Ser Cys Gln Ala Phe Gly Tyr Gly Phe Ile
Asn Tyr 20 25 30 Leu Ile Glu Trp Ile Arg Gln Met Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45 Gly Ala Ile Asn Pro Gly Ser Asp Tyr Thr
Asn Tyr Asn Glu Asn Phe 50 55 60 Lys Gly Gln Ala Thr Leu Ser Ala
Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu
Lys Ala Ser Asp Thr Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe
Gly Tyr Tyr Gly Ser Gly Asn Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln
Gly Thr Met Val Thr Val Ser Ser 115 120
185122PRTArtificialhumanized antibody variant 185Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu
Lys Ile Ser Cys Gln Ala Phe Gly Tyr Gly Phe Ile Asn Tyr 20 25 30
Leu Ile Glu Trp Ile Arg Gln Met Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Leu Ile Tyr Pro Asp Ser Gly Tyr Ile Asn Tyr Asn Glu Asn
Phe 50 55 60 Lys Gly Gln Ala Thr Leu Ser Ala Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Ala Tyr Tyr Gly Ser
Gly Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 186122PRTArtificialhumanized antibody variant
186Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15 Ser Leu Lys Ile Ser Cys Gln Ala Phe Gly Tyr Ala Phe Thr
Asn Tyr 20 25 30 Leu Ile Glu Trp Val Arg Gln Met Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45 Gly Leu Ile Tyr Pro Asp Ser Gly Tyr Ile
Asn Tyr Asn Glu Asn Phe 50 55 60 Lys Gly Gln Val Thr Ile Ser Ala
Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu
Lys Ala Ser Asp Thr Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe
Ala Tyr Tyr Gly Ser Gly Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln
Gly Thr Met Val Thr Val Ser Ser 115 120
187122PRTArtificialhumanized antibody variant 187Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu
Lys Ile Ser Cys Gln Ala Phe Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Met Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Leu Ile Tyr Pro Asp Ser Gly Tyr Ile Asn Tyr Asn Glu Asn
Phe 50 55 60 Lys Gly Gln Ala Thr Leu Ser Ala Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Ala Tyr Tyr Gly Ser
Gly Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 188122PRTArtificialhumanized antibody variant
188Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15 Ser Leu Lys Ile Ser Cys Gln Ala Phe Gly Asp Ala Phe Thr
Asn Tyr 20 25 30 Leu Ile Glu Trp Val Arg Gln Met Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45 Gly Leu Ile Tyr Pro Asp Ser Gly Tyr Ile
Asn Tyr Asn Glu Asn Phe 50 55 60 Lys Gly Gln Val Thr Ile Ser Ala
Asp Arg Ser Ser Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu
Lys Ala Ser Asp Thr Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe
Ala Tyr Tyr Gly Ser Gly Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln
Gly Thr Met Val Thr Val Ser Ser 115 120
189122PRTArtificialhumanized antibody variant 189Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu
Lys Ile Ser Cys Gln Ala Phe Gly Asp Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Met Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Leu Ile Tyr Pro Asp Ser Gly Tyr Ile Asn Tyr Asn Glu Asn
Phe 50 55 60 Lys Gly Gln Ala Thr Leu Ser Ala Asp Arg Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Phe Ala Tyr Tyr Gly Ser
Gly Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Met Val Thr
Val Ser Ser 115 120
* * * * *