DESCRIPTION
Immunoassay Method for Human CXCL1 Protein
TECHNICAL FIELD
The present invention relates to a method for measuring a
human CXCL1 protein (hereinafter, referred to as human CXCL1).
More specifically, the present invention relates to an immunoassay
method that is a method for measuring human CXCL1 using a
combination of monoclonal antibodies or fragments thereof each
specifically binding to a partial sequence region at any one of three
specific positions on the amino acid sequence composing human
CXCL1. Also, the present invention relates to monoclonal antibodies
or fragments thereof specifically binding to human CXCL1, which can
be used for the above method for measuring a human CXCL1 protein.
BACKGROUND ART
Human CXCL1 is a kind of chemokine belonging to the CXC
family. In blood, this protein is present in blood platelets and is
known to be over-expressed upon inflammation reactions in a manner
similar to that of other members of the CXC family.
In recent years, it has been reported that human CXCL1
functions as a tumor-related factor. Therefore, it is expected that
human CXCL1 can serve as a urothelial cancer marker through
quantitative detection of human CXCL1 in urine (Hiroaki Kawanishi et
al, 2008, Clinical Cancer Research, vol. 14, No. 9, 2579-2587; and
WO2007/026895). It has been further reported that the amount of
human CXCL1 fluctuates at the gene level and at the protein level in
the tissue or blood of patients with other malignant tumors such as
large-bowel cancer, ovary cancer, or malignant melanoma (Gong Yang
et al, 2006, PNAS, vol. 103, No. 44, 16472-16477; Yu Wen et al, 2006,
Clinical Cancer Research, vol. 12, No. 20, 5951-5959; and Jing Luan
et al, 1997, Journal of Leukocyte Biology, vol. 62, No. 5, 588-597).
The human CXCL1 potentially enables extremely early and/or early
detection of such cancers, in addition to urothelial cancer.
However, measurement methods using an existing enzyme
immunoassay have insufficient sensitivity for detection of the above
cancers. For example, WO2007/026895 discloses a kit for measuring
human CXCL1 produced by R&D SYSTEMS, Inc. (hereinafter, R&D
SYSTEMS), wherein, although the detection limit concentration is
around 20 pg/mL, the concentration of human CXCL1 in the urine of a
healthy subject does not reach the detection limit. Hence, a method
for measuring human CXCL1 with higher sensitivity has been desired.
DISCLOSURE OF THE INVENTION
In an embodiment of the present invention, an object of the
present invention is thus to provide an immunoassay method for
detecting human CXCL1 with higher sensitivity. More specifically,
an object of the present invention is to realize a method for detecting
human CXCL1 with higher sensitivity by an immunoassay using a
combination of antibodies or antigen recognition regions each
specifically binding to a sequence at any one of 3 specific positions in
the amino acid sequence composing human CXCL1.
Also, in another embodiment of the present invention, an
object of the present invention is to provide monoclonal antibodies or
fragments thereof specifically recognizing human CXCL1. More
specifically, another object of the present invention is to provide
monoclonal antibodies or fragments thereof that can be used for the
above method for detecting human CXCL1 with higher sensitivity,
comprise new amino acid sequences specifically recognizing human
CXCL1, and have affinity for human CXCL1 higher than that of
conventional antibodies.
Means to Solve the Problems
As a result of intensive studies to achieve the above objects,
the present inventors have discovered that human CXCL1 can be
detected with higher sensitivity than conventional methods by
immunological determination using a combination of monoclonal
antibodies or antigen recognition portions specifically binding to
partial sequence regions at 3 specific positions in the amino acid
sequence composing human CXCL1. Also, the present inventors have
succeeded in preparation of monoclonal antibodies comprising new
amino acid sequences specifically binding to partial sequences at the 3
positions on the above human CXCL1 and hybridomas producing such
antibodies. Moreover, the present inventors have isolated genes
encoding the amino acid sequences composing the antibodies and
determined the nucleotide sequences. This has made it possible to
prepare recombinant antibodies and recombinant fragments thereof.
The present invention has been achieved based on the above findings
in order to provide the following (1) to (15).
(1) An immunoassay method for a human CXCL1 protein, by which
human CXCL1 or a fragment thereof in a sample is measured using
two or more types of anti-human CXCL1 monoclonal antibodies or
fragments thereof, wherein:
the two or more types of anti-human CXCL1 monoclonal antibodies or
fragments thereof specifically recognize any one of the sequence
regions of the amino acid sequences shown in SEQ ID NOS: 1-3 that
are partial sequences of the amino acid sequence composing the human
CXCL1 protein; and the two or more types of anti-human CXCL1
monoclonal antibodies or fragments thereof specifically recognize
sequence regions that differ from each other.
(2) The immunoassay method for a human CXCL1 protein according to
(1), by which a human CXCL1 protein or a fragment thereof in a
sample is measured using an anti-human CXCL1 monoclonal antibody
or a fragment thereof that specifically recognizes the amino acid
sequence region shown in SEQ ID NO: 3.
(3) The immunoassay method for a human CXCL1 protein according to
(1) or (2), by which a human CXCL1 protein or a fragment thereof in a
sample is measured using an anti-human CXCL1 monoclonal antibody
or a fragment thereof that specifically recognizes the amino acid
sequence region shown in SEQ ID NO: 1 and an anti-human CXCL1
monoclonal antibody or a fragment thereof that specifically recognizes
the amino acid sequence region shown in SEQ ID NO: 3.
(4) The immunoassay method for a human CXCL1 protein according to
(1) or (2), by which a human CXCL1 protein or a fragment thereof in a
sample is measured using an anti-human CXCL1 monoclonal antibody
or a fragment thereof that specifically recognizes the amino acid
sequence region shown in SEQ ID NO: 2 and an anti-human CXCL1
monoclonal antibody or a fragment thereof that specifically recognizes
the amino acid sequence region shown in SEQ ID NO: 3.
(5) The immunoassay method for a human CXCL1 protein according to
any one of (1) to (4), wherein the sample is a tissue collected after an
operation, blood, serum, blood plasma, urine, spinal fluid, saliva,
lymph fluid, lacrimal fluid, or seminal fluid.
(6) An anti-human CXCL1 monoclonal antibody or a fragment thereof,
which specifically recognizes the partial sequence shown in SEQ ID
NO: 3 in the amino acid sequence composing the human CXCL1
protein, wherein:
in its light chains, CDR1 comprises the amino acid sequence
shown in SEQ ID NO: 4, CDR2 comprises the amino acid sequence
shown in SEQ ID NO: 5, and CDR3 comprises the amino acid sequence
shown in SEQ ID NO: 6;
in its heavy chains, CDR1 comprises the amino acid sequence
shown in SEQ ID NO: 7, CDR2 comprises the amino acid sequence
shown in SEQ ID NO: 8, and CDR3 comprises the amino acid sequence
shown in SEQ ID NO: 9.
(7) The anti-human CXCL1 monoclonal antibody or a fragment thereof
according to (6), containing the amino acid sequence shown in SEQ ID
NO: 10 in a light-chain variable region and the amino acid sequence
shown in SEQ ID NO: 11 in a heavy-chain variable region.
(8) An anti-human CXCL1 monoclonal antibody or a fragment thereof,
which specifically recognizes the partial sequence shown in SEQ ID
NO: 1 in the amino acid sequence composing a human CXCL1 protein,
wherein:
in its light chains, CDR1 comprises the amino acid sequence
shown in SEQ ID NO: 12, CDR2 comprises the amino acid sequence
shown in SEQ ID NO: 13, and CDR3 comprises the amino acid
sequence shown in SEQ ID NO: 14; and
in its heavy chains, CDR1 comprises the amino acid sequence
shown in SEQ ID NO: 15, CDR2 comprises the amino acid sequence
shown in SEQ ID NO: 16, and CDR3 comprises the amino acid
sequence shown in SEQ ID NO: 17.
(9) The anti-human CXCL1 monoclonal antibody or a fragment thereof
according to (8), containing the amino acid sequence shown in SEQ ID
NO: 18 in a light-chain variable region and the amino acid sequence
shown in SEQ ID NO: 19 in a heavy-chain variable region.
(10) An anti-human CXCL1 monoclonal antibody or a fragment
thereof, which that specifically recognizes the partial sequence shown
in SEQ ID NO: 3 in the amino acid sequence composing a human
CXCL1 protein, wherein:
in its light chains, CDR1 comprises the amino acid sequence
shown in SEQ ID NO: 20, CDR2 comprises the amino acid sequence
shown in SEQ ID NO: 21, and CDR3 comprises the amino acid
sequence shown in SEQ ID NO: 22; and
in its heavy chains, CDR1 comprises the amino acid
sequence shown in SEQ ID NO: 23, CDR2 comprises the amino acid
sequence shown in SEQ ID NO: 24, and CDR3 comprises the amino
acid sequence shown in SEQ ID NO: 25.
(11) The anti-human CXCL1 monoclonal antibody or a fragment
thereof according to (10), containing the amino acid sequence shown
in SEQ ID NO: 26 in a light-chain variable region and the amino acid
sequence shown in SEQ ID NO: 27 in a heavy-chain variable region.
(12) An anti-human CXCL1 monoclonal antibody or a fragment
thereof, which is a monoclonal antibody or a fragment thereof that
specifically recognizes the partial sequence shown in SEQ ID NO: 2
in the amino acid sequence composing a human CXCL1 protein,
wherein:
in its light chains, CDR1 comprises the amino acid sequence
shown in SEQ ID NO: 28, CDR2 comprises the amino acid sequence
shown in SEQ ID NO: 29, and CDR3 comprises the amino acid
sequence shown in SEQ ID NO: 30; and
in its heavy chains, CDR1 comprises the amino acid sequence
shown in SEQ ID NO: 31, CDR2 comprises the amino acid sequence
shown in SEQ ID NO: 32, and CDR3 comprises the amino acid
sequence shown in SEQ ID NO: 33.
(13) The anti-human CXCL1 monoclonal antibody or a fragment
thereof according to (12), containing the amino acid sequence shown
in SEQ ID NO: 34 in a light-chain variable region and the amino acid
sequence shown in SEQ ID NO: 35 in a heavy-chain variable region.
(14) An anti-human CXCL1 monoclonal antibody or a
fragment thereof, which is a monoclonal antibody or a fragment
thereof that specifically recognizes the partial sequence shown in SEQ
ID NO: 3 in the amino acid sequence composing a human CXCL1
protein, wherein:
in its light chains, CDR1 comprises the amino acid sequence
shown in SEQ ID NO: 36, CDR2 comprises the amino acid sequence
shown in SEQ ID NO: 37, and CDR3 comprises the amino acid
sequence shown in SEQ ID NO: 38; and
in its heavy chains, CDR1 comprises the amino acid
sequence shown in SEQ ID NO: 39, CDR2 comprises the amino acid
sequence shown in SEQ ID NO: 40, and CDR3 comprises the amino
acid sequence shown in SEQ ID NO: 41.
(15) The anti-human CXCL1 monoclonal antibody or a fragment
thereof according to (14), containing the amino acid sequence shown
in SEQ ID NO: 42 in a light-chain variable region and the amino acid
sequence shown in SEQ ID NO: 43 in a heavy-chain variable region.
Effect of the Invention
According to the present invention, the concentration of
human CXCL1 can be measured with higher sensitivity than
conventional methods. Also, a high-affinity anti-human CXCL1
monoclonal antibody or a fragment thereof specifically recognizing
human CXCL1 can be provided.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows immunoassay according to a conventional
method using a sandwich ELISA method using a monoclonal antibody
that specifically recognizes any one of the amino acid sequences
shown in SEQ ID NOS: 1-3 and a commercial kit.
Figure 2 shows sandwich ELISA method using as a labeled
antibody a monoclonal antibody that specifically recognizes the amino
acid sequence shown in SEQ ID NO: 3.
Figure 3 shows sandwich ELISA method for detection of
human CXCL1 in urine using a monoclonal antibody that specifically
recognizes the amino acid sequence shown in SEQ ID NO: 1 or 2 and a
monoclonal antibody that specifically recognizes the amino acid
sequence shown in SEQ ID NO: 3.
Figure 4 is a graph showing the results of detecting human
CXCL1 in a buffer by immobilizing a commercial antibody that
specifically recognizes the amino acid sequence shown in SEQ ID NO:
2 and performing a sandwich ELISA method using the biotin-labeled
antibody of the present invention recognizing the amino acid sequence
shown in SEQ ID NO: 3 or a commercial biotin-labeled anti-human
CXCL1 polyclonal antibody.
Figure 5 is a graph showing the results of detecting human
CXCL1 in a buffer by immobilizing the 5 types of antibody (IgGl-1,
IgGl-3, IgGl-10, IgGl-14, and IgG2b-l) of the present invention and
a commercial antibody and then carrying out a sandwich ELISA
method using a biotin-labeled anti-human CXCL1 polyclonal antibody.
Figure 6 is a graph showing the results of detecting human
CXCL1 in blood plasma by immobilizing the 5 types of antibody of
the present invention and a commercial antibody and then carrying out
a sandwich ELISA method using a biotin-labeled anti-human CXCL1
polyclonal antibody.
Figure 7 is a graph showing the results of detecting human
CXCL1 in urine by immobilizing the 5 types of antibody of the
present invention and a commercial antibody and then carrying out a
sandwich ELISA method using a biotin-labeled anti-human CXCL1
polyclonal antibody.
Figure 8 is graph (1) showing the bladder cancer cell
invasion-suppressing capability of the 5 types of antibody of the
present invention and a commercial antibody.
Figure 9 is a graph (2) showing bladder cancer cell
invasion-suppressing capability of the 4 types of antibody of the
present invention and a commercial antibody.
MODE OF CARRYING OUT THE INVENTION
Embodiments of the invention will be hereafter described in
detail.
1. Anti-human CXCL1 monoclonal antibody and a fragment thereof
The term "human CXCL1" as used herein refers to a protein
or a natural mutant thereof comprising the amino acid sequence
according to Genbank NM_001511. The term "natural mutant" refers
to a mutant existing in the nature. Examples of such a mutant
include a mutant comprising an amino acid sequence having a
deletion, a substitution, an addition, or an insertion of one or several
amino acids in the aforementioned amino acid sequence of human
CXCL1 and a mutant having 95% or more, preferably 98% or more,
and more preferably 99% or more amino acid sequence identity with
the aforementioned amino acid sequence of human CXCL1. Here, the
term "identity" refers to the percentage (%) of the total number of
amino acid residues of the amino acid sequence in question that are
identical to amino acid residues of the amino acid sequence of human
CXCL1 when the two amino acid sequences are aligned such that the
highest possible degree of agreement between them is achieved. In
this case, sequence alignment can be carried out by introducing or not
introducing gaps, and the number of gaps introduced is included when
the percentage is calculated. Also, the term "several" refers to an
integer between 2 and 10, such as between 2 and 7, 2 and 5, 2 and 4,
and 2 and 3. Specific examples of a natural mutant include mutants
based on polymorphism such as SNP (single nucleotide polymorphism)
and splicing mutants. The above substitution is preferably a
conservative amino acid substitution. If the substitution is a
conservative amino acid substitution, a mutant resulting from the
conservative amino acid substitution may have a structure or
properties substantially equivalent to those of human CXCL1 having
the above amino acid sequence. As conservative amino acids,
nonpolar amino acids (glycine, alanine, phenylalanine, valine, leucine,
isoleucine, methionine, proline, and tryptophan) and polar amino acids
(amino acids other than nonpolar amino acids), charged amino acids
(acidic amino acids (aspartic acid and glutamic acid) and basic amino
acids (arginine, histidine, and lysine)) and non-charged amino acids
(amino acids other than charged amino acids), aromatic amino acids
(phenylalanine, tryptophan, and tyrosine), branched amino acids
(leucine, isoleucine, and valine), and aliphatic amino acids (glycine,
alanine, leucine, isoleucine, and valine), are known, for example.
The term "monoclonal antibody" as used herein refers to a
polypeptide containing an immunoglobulin- or its fragment-derived
framework region (FR) and a complementarity determining region
(CDR) and being capable of specifically binding to and recognizing an
antigen. Therefore, the term "anti-human CXCL1 monoclonal
antibody" in the present invention refers to a polypeptide capable of
specifically binding to human CXCL1 or a fragment thereof and
recognizing the human CXCL1 or a fragment thereof. The term
"specifically binding" refers to binding to only a target antigen
(human CXCL1 or a fragment thereof in the present invention).
A typical immunoglobulin molecule consists of a tetramer in
which two sets, each consisting of two polypeptide chains referred to
as a heavy chain and a light chain, are connected to each other via
disulfide bond. A heavy chain comprises a heavy chain variable
region (V ) on the N-terminus and a heavy chain constant region (C )
H H
on the C-terminus. A light chain comprises a light chain variable
region (V ) on the N-terminus and a light chain constant region (C )
on the C-terminus. Of these regions, V and V are particularly
H L
important since they are involved in the binding specificity of the
antibody. V and V each comprises about 110 amino acid residues,
H L
wherein three complementarity determining regions (CDR1, CDR2,
and CDR3) directly involved in binding specificity with an antigen
and four framework regions (FR1, FR2, FR3, and FR4) functioning as
framework structures for variable regions are present. A
complementary determining region is known to form conformation
complementary to an antigen molecule and determine the specificity of
the relevant antibody (E. A. Kabat et al., 1991, Sequences of proteins
of immunological interest, Vol. 1, eds. 5, NIH publication). Whereas
amino acid sequences of constant regions remain almost unchanged
among antibodies of the same species, amino acid sequences of
complementary determining regions are highly variable among
antibodies. Hence, complementary strand determining regions are
also referred to as hypervariable regions. In a variable region, such
complementarity determining regions (CDRs) and framework regions
are arranged in the direction from an amino acid terminus to a carboxy
terminus in order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
V and V form a dimer with each other so as to form an antigen
L H 6
binding site within an immunoglobulin molecule. Regarding
immunoglobulin, IgG, IgM, IgA, IgE, and IgD classes are known.
The antibody of the present invention may be of any class and is
preferably IgG.
An antibody useful in the present invention may be derived
from every animal source including birds and mammals. Examples of
such the animal or bird source include mice, rats, guinea pigs, rabbits,
goats, donkeys, sheep, camels, horses, chickens, and humans. Also,
"monoclonal antibody" in the present invention may be chemically
synthesized or synthesized using a recombinant DNA method. For
example, recombinant antibodies such as chimeric antibodies and
humanized antibodies are also encompassed in the present invention.
The term "chimeric antibody" refers to an antibody prepared
by substituting a constant region of an antibody with a constant region
of another antibody. An example of such an antibody is an antibody
prepared by substituting a constant region of an anti-human CXCL1
mouse monoclonal antibody with a constant region of a human
antibody. A more specific example of such an antibody is an
antibody in which V comprises any one of the amino acid sequence
shown in SEQ ID NO: 10, 18, 26, 34, or 42 of V in an anti-human
CXCL1 mouse monoclonal antibody, C comprises the amino acid
sequence of C in an arbitrary human antibody, and/or V comprises
L H
the amino acid sequence shown in SEQ ID NO: 11, 19, 27, 35, or 43 of
V in an anti-human CXCL1 mouse monoclonal antibody, and C
H J' H
comprises the amino acid sequence of C in an arbitrary human
antibody.
The term "humanized antibody" refers to a mosaic antibody
prepared by artificially combining an antibody (generally a non-human
antibody such as a mouse antibody)-derived CDR group with FR and a
constant region of a human antibody. An example of such an
antibody is an antibody prepared by combining each CDR of an
anti-human CXCL1 mouse monoclonal antibody with each FR and a
constant region of an arbitrary human antibody. CDR groups in a
variable region are mainly responsible for the antigen-binding
specificity of an antibody. Hence, when a recombinant antibody
having binding properties similar to those of an antibody as described
above is prepared, obtainment of the entire amino acid sequence of the
antibody is not required. Specifically, by using an existing
recombinant DNA technique, a mosaic antibody is prepared by
substituting a DNA sequence encoding each CDR derived from an
antibody with a DNA sequence encoding the corresponding CDR
derived from a human antibody and then expressed. Thus, a
recombinant antibody that mimics the characteristics of the antibody
can be obtained. Such a technique is referred to as a CDR-grafted
antibody (Nature, 1986, Vol. 321, 522). In addition, when the
anti-human CXCL1 antibody or a fragment thereof of the present
invention is used for detecting human CXCL1 or a fragment thereof,
the antibody is not always required to be a humanized antibody. By
using this grafting antibody technique, FR and a constant region may
be derived from an antibody of an arbitrary non-human animal.
Furthermore, an "antibody" in the present invention may also
be a multiple specific antibody. The term "multiple specific
antibody" refers to a multivalent antibody that is specifically an
antibody having a plurality of antigen binding sites within one
molecule in which these antigen binding sites bind to different
epitopes. An example of such a multivalent antibody is a bispecific
antibody having two antigen binding sites such as IgG, wherein the
two antigen binding sites bind to different epitopes. In the present
invention, such a multiple specific antibody is preferable, in which
antigen binding sites are capable of binding to different epitopes
existing on human CXCL1. These antibodies can be obtained by
artificially altering IgG or the like by a known method using a
recombinant DNA technique.
The term "fragment thereof" in "monoclonal antibody or a
fragment thereof" as used herein refers to a partial region of the
antibody and specifically refers to a polypeptide chain or a complex
thereof having activity substantially equivalent to the antigen-specific
binding activity of the antibody. Examples of such a fragment
include an antibody portion containing at least one of the above
antigen binding sites and specifically, a polypeptide chain or a
complex thereof having at least one V and at least one V . Specific
L H
examples of such a polypeptide chain or a complex thereof include
many sufficiently characterized antibody fragments and the like
generated via cleavage of immunoglobulin with various peptidases.
More specific examples of such antibody fragments include Fab,
F(ab' ) , and Fab' . Fab is a fragment generated by cleaving an IgG
molecule with papain, by which cleavage is carried out at a position
closer to the N-terminal side than the disulfide linkage of a hinge
part. Fab is composed of a polypeptide comprising V and C 1 which
H H
is adjacent to V among the 3 domains (C 1, C 2, and C 3) composing
H H H H
C and a light chain. F(ab' ) is a dimer of Fab' , which is generated
H 2
by cleaving an IgG molecule with pepsin at a position closer to the
C-terminal side than the disulfide linkage of the hinge part. Fab'
has a structure substantially equivalent to that of Fab, although the H
chain is somewhat longer than that of Fab since it contains the hinge
part (Fundamental Immunology, Paul ed., 3d ed., 1993). Fab' can be
obtained by reducing F(ab' ) under mild conditions and then cleaving
the disulfide linkage in the hinge region. All of these antibody
fragments contain antigen binding sites, so that they are capable of
specifically binding to antigens (that is, human CXCL1 or a fragment
thereof in the present invention).
The above "fragment thereof" in the present invention may be
chemically synthesized or synthesized using a recombinant DNA
method. An example of such a fragment is an antibody fragment
newly synthesized using a recombinant DNA method. Specific
examples of such a fragment include, but are not limited to, a
monomeric polypeptide molecule prepared by artificially linking one
or more V and one or more V of the antibody of the present
L H
invention via a linker peptide or the like having an appropriate length
and sequence and a multimeric polypeptide thereof. Examples of
such a polypeptide include single chain Fv (scFv: single chain
fragment of variable region) (see Pierce catalog and Handbook,
1994-1995, Pierce Chemical co., Rockford, IL) and synthetic
antibodies such as a diabody, a triabody, and a tetrabody. In an
immunoglobulin molecule, V and V are generally separately located
L H
on different polypeptide chains (a light chain and a heavy chain).
Single chain Fv is a synthetic antibody fragment that has a structure
in which these variable regions are linked with a flexible linker
havine a sufficient leneth and the linked regions are cnntained in a
single polypeptide chain. Within single chain Fv, both variable
regions can be self-assembled to form a single functional antigen
binding site. Single chain Fv can be obtained by incorporating a
recombinant DNA encoding the single chain Fv into a phage genome
using a known technique and then causing the expression of the DNA.
A diabody is a molecule having a structure based on the dimeric
structure of single chain Fv (Holliger et al., 1993, Proc. Natl. Acad.
Sci. U.S.A., 90: 6444-6448). For example, when the length of the
above linker is shorter than about 12 amino acid residues, two
variable sites within single chain Fv cannot undergo self assembly.
However, the two variable sites are caused to form a diabody and
specifically two single chain Fvs are caused to interact with each
other, enabling the assembling of V of one Fv chain and V of the
L H
other Fv chain. Hence, two functional antigen binding sites can be
formed (Marvin et al., 2005, Acta Pharmacol. Sin., 26: 649-658).
Moreover, a cysteine residue is added to the C-terminus of single
chain Fv, so that disulfide bond of the two Fv chains can be formed
and thereby formation of a stable diabody become possible (Olafsen et
al, 2004, Prot. Engr. Des. Sel., 17: 21-27). As described above, a
diabody is a divalent antibody fragment. However, each antigen
binding site is not required to bind to the same epitope and may have
bispecificity such that the antigen binding sites recognize and
specifically bind to different epitopes. For example, one antigen
binding site may comprise V that contains CDRs comprising the
amino acid sequences shown in SEQ ID NOS: 36, 37 and 38
(corresponding to CDR1, CDR2 and CDR3, respectively), and V that
H
contains CDRs comprising the amino acid sequences shown in SEQ
ID NOS: 39, 40 and 41 (corresponding to CDR1, CDR2 and CDR3,
respectively). The other antigen binding site may comprise V that
contains CDRs comprising the amino acid sequences shown in SEQ
ID NOS: 28, 29 and 30 (corresponding to CDR1, CDR2 and CDR3,
respectively) and V that contains CDRs comprising the amino acid
H
sequences shown in SEQ ID NOS: 31, 32 and 33 (corresponding to
CDR1, CDR2 and CDR3 respectively). A triabody and a tetrabody
have a trimeric structure and a tetrameric structure, respectively,
based on a single chain Fv structure in a manner similar to a diabody.
A triabody and a tetrabody are a trivalent antibody fragment and a
quadrivalent antibody fragment, respectively, or may be multiple
specific antibodies.
Furthermore, examples of the above "fragment thereof"
include antibody fragments that are identified using phage display
libraries (e.g., see McCafferty et al., 1990, Nature, Vol. 348, 522-554)
and have antigen-binding capacity. In addition, also see Kuby, J.,
Immunology, 3rd ed., 1998, W. H. Freeman & Co., New York, for
example.
The present invention provides an antibody or a fragment
thereof having amino acid sequences composing variable regions
having desirable activity to bind to human CXCL1 and CDRs thereof.
Specifically, the present invention provides an antibody or a fragment
thereof containing an immunoglobulin variable region comprising the
amino acid sequence shown in any one of SEQ ID NOS: 4-43.
In the antibody or a fragment thereof of the present
invention, CDR1, CDR2 and CDR3 can comprise the amino acid
sequences shown in SEQ ID NOS: 4, 5 and 6, respectively, in its light
chains, and CDR1, CDR2 and CDR3 can comprise the amino acid
sequences shown in SEQ ID NOS: 7, 8 and 9, respectively, in its heavy
chains.
Also, in the antibody or a fragment thereof of the present
invention, V and V can comprise the amino acid sequences shown in
L H
SEQ ID NOS: 10 and 11, respectively.
In the antibody or a fragment thereof of the present
invention, CDR1, CDR2, and CDR3 can comprise the amino acid
sequences shown in SEQ ID NOS: 12, 13, and 14, respectively, in its
light chains and CDR1, CDR2, and CDR3 can comprise the amino acid
sequences shown in SEQ ID NOS: 15, 16, and 17, respectively, in its
heavy chains.
Also, in the antibody or a fragment thereof of the present
invention, V and V can comprise the amino acid sequences shown in
L H
SEQ ID NOS: 18 and 19, respectively.
In the antibody or a fragment thereof of the present
invention, CDR1, CDR2 and CDR3 can comprise the amino acid
sequences shown in SEQ ID NOS: 20, 21 and 22, respectively, in its
light chains, and CDR1, CDR2 and CDR3 can comprise the amino acid
sequences shown in SEQ ID NOS: 23, 24 and 25, respectively, in its
heavy chains.
Also, in the antibody or a fragment thereof of the present
invention, V and V can comprise the amino acid sequences shown in
L H
SEQ ID NOS: 26 and 27, respectively.
In the antibody or a fragment thereof of the present
invention, CDR1, CDR2 and CDR3 can comprise the amino acid
sequences shown in SEQ ID NOS: 28, 29 and 30, respectively, in its
light chains, and CDR1, CDR2 and CDR3 can comprise the amino acid
sequences shown in SEQ ID NOS: 31, 32 and 33, respectively, in its
heavy chains.
Also, in the antibody or a fragment thereof of the present
invention, V and V can comprise the amino acid sequences shown in
L H
SEQ ID NOS: 34 and 35, respectively.
In the antibody or a fragment thereof of the present
invention, CDR1, CDR2 and CDR3 can comprise the amino acid
sequences shown in SEQ ID NOS: 36, 37 and 38, respectively, in its
light chains, and CDR1, CDR2 and CDR3 can comprise the amino acid
sequences shown in SEQ ID NOS: 39, 40 and 41, respectively, in its
heavy chains.
Also, in the antibody or a fragment thereof of the present
invention, V and V can comprise the amino acid sequences shown in
L H
SEQ ID NOS: 42 and 43, respectively.
The antibody or a fragment thereof of the present invention
can be modified. The term "modified or modification" used herein
refers to both functional modification required for the antibody or a
fragment thereof of the present invention to have activity of
specifically binding to human CXCL1 (e.g., glycosylation) and
modification for labeling required for detection of the antibody or a
fragment thereof of the present invention. Examples of the above
antibody labels include fluorescent dyes (FITC, rhodamine, Texas red,
Cy3, and Cy5), fluorescent proteins (e.g., PE, APC, and GFP),
enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and
glucose oxidase), and biotin or (strept)avidin. Also, glycosylation of
the antibody of the present invention may be altered for adjusting the
affinity of an antibody for a target antigen. Such alteration can be
achieved by, for example, changing one or more glycosylation sites
within the antibody sequence. More specifically, for example, one or
more amino acid substitutions are introduced into an amino acid
sequence composing one or more glycosylation sites within FR so as
to remove the glycosylation sites, so that deglycosylation can be
achieved at the sites. Such deglycosylation is effective for
increasing the affinity of an antibody for an antigen (US Patent No.
5714350 and US Patent No. 6350861).
Monoclonal antibodies or fragments thereof to be used in the
measurement method of the present invention is preferably verified
before use concerning their cross-reactivity with other antigens
(proteins or fragments thereof), so as to ensure their activity of
specifically binding to human CXCL1 or a fragment thereof.
Regarding the antibody or a fragment thereof of the present invention,
examples of antigens for which cross-reactivity should be confirmed
include proteins belonging to the CXC family, and particularly, a
human CXCL2 protein and a human CXCL3 protein structurally
analogous to human CXCL1. Also, in addition to the above proteins,
other proteins having common partial structures with human CXCL1
are more preferably confirmed in advance for their cross-reactivity
with antibodies or fragments thereof to be used in the measurement
method of the present invention. For confirmation of
cross-reactivity, an ELISA method using human CXCL1 as an antigen
can be employed. At a site of reaction of an antibody to be tested for
its reaction specificity, that is, an anti-human CXCL1 monoclonal
antibody and a fragment thereof, with human CXCL1, another antigen
protein to be confirmed for its cross-reactivity is also caused to
co-exist at the reaction site. By observing the competition of them,
cross-reactivity can be confirmed. Such a method for confirming
cross-reactivity using the principle of competitive inhibition does not
require preparation of reaction systems for all antigens, so that
screening can be rapidly performed.
2. Method for preparing monoclonal antibody and hybridoma
The anti-human CXCL1 monoclonal antibody of the present
invention or a hybridoma producing the antibody can be prepared by
the following method, but is not limited thereto. The antibody and
the hybridoma can also be prepared by any other methods known in the
art.
A. Method for preparing anti-human CXCL1 monoclonal antibody
An anti-human CXCL1 monoclonal antibody specifically
binding to the partial amino acid sequence region shown in any one of
SEQ ID NOS: 1-3 in the amino acid sequence composing human
CXCL1 is prepared by:
a method comprising preparing a monoclonal antibody using the
full-length human CXCL1 as an immunogen and then screening for an
antibody specifically binding to the partial amino acid sequence
region of any one of SEQ ID NOS: 1-3; or
a method comprising preparing in advance a monoclonal antibody
using the partial sequence of human CXCL1, which is shown in SEQ
ID NO: 1, 2, or 3, as an immunogen.
Al. Preparation of human CXCL1
First, human CXCL1 to be used as an immunogen (antigen) is
prepared. Human CXCL1 may be any human CXCL1 such as natural
human CXCL1, recombinant human CXCL1, or human CXCL1 prepared
via chemical synthesis of a full-length or a partial amino acid
sequence such as peptide synthesis.
Natural human CXCL1 can be collected from human samples
including a human body fluid such as blood or urine or from
supernatants of cultured human cells by the known protein separation •
purification techniques such as gel chromatography, ion exchange
chromatography, and affinity chromatography.
Recombinant human CXCL1 can be collected by the known
protein separation and/or purification techniques from cells after
introduction of DNA encoding the protein into microorganisms, insect
cells, or animal cells followed by expression of the DNA.
Synthetic human CXCL1 can be synthesized by techniques
known in the technical field such as a solid phase peptide synthesis
method, for example, using public human CXCL amino acid sequence
information. Incidentally, the cDNA sequence of human CXCL1 is
disclosed at GenBank under Accession No. X12510. A carrier protein
such as KLH (keyhole limpet hemocyanin), OVA (ovalbumin), or BSA
(bovine serum albumin) may be ligated to the synthetic human CXCL1
and then the resultant may be used.
Furthermore, when the partial sequence of human CXCL1
shown in any one of SEQ ID NOS: 1-3 is used as an immunogen, the
immunogen may be a natural, recombinant, or chemically synthesized
immunogen similarly to a case of immunizing with a full-length
sequence.
For example, when a partial sequence of natural human
CXCL1 is used as an immunogen, first, purified human CXCL1 is
treated with appropriate protease such as trypsin and then peaks are
isolated and fractionated using a reverse phase column. The amino
acid sequence of a peptide contained in each fractionated peak is
determined using a mass spectrometer. The partial sequence shown
in SEQ ID NO: 1, 2, or 3 or a peak that is a portion of the partial
sequence can be used as an immunogen.
Also, when a partial amino acid sequence of recombinant
human CXCL1 is used as an immunogen, partial amino acid sequences
shown in SEQ ID NOS: 1-3 in the above DNA sequence encoding
human CXCL1 or a DNA sequence portion encoding a portion of the
partial amino acid sequences is inserted into an expression vector in a
manner similar to a case of preparing full-length human CXCL1 and
then the vector is introduced into various cells. Hence, recombinant
human CXCL1 comprising partial amino acid sequences shown in SEQ
ID NOS: 1-3 or a portion thereof can be obtained.
Preparation of the partial amino acid sequences of
recombinant human CXCL1, which are shown in SEQ ID NOS: 1-3
(hereinafter, referred to as partial human CXCL1 sequence(s)), is as
described in detail herein.
(a) Preparation of polynucleotide encoding recombinant partial human
CXCL1 sequence
A method for preparing the polynucleotide is described in
detail in Example 1 below, so that it is not mentioned herein.
As a vector to be used for expression of a partial human
CXCL1 sequence, a phage or a plasmid that is capable of
autonomously replicating in host microorganisms can be used. For
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