U.S. patent application number 13/523791 was filed with the patent office on 2013-03-14 for method and reagent for diagnosis and/or evaluation of progression of graft-versus-host disease.
This patent application is currently assigned to Immuno-Biological Laboratories Co., Ltd. and Sapporo Medical University. The applicant listed for this patent is Tsukasa HORI, Kohzoh Imai, Yasuo Kokai, Yasuyoshi Naishiro, Hiroyuki Tsutsumi. Invention is credited to Tsukasa HORI, Kohzoh Imai, Yasuo Kokai, Yasuyoshi Naishiro, Hiroyuki Tsutsumi.
Application Number | 20130065779 13/523791 |
Document ID | / |
Family ID | 40185370 |
Filed Date | 2013-03-14 |
United States Patent
Application |
20130065779 |
Kind Code |
A1 |
HORI; Tsukasa ; et
al. |
March 14, 2013 |
METHOD AND REAGENT FOR DIAGNOSIS AND/OR EVALUATION OF PROGRESSION
OF GRAFT-VERSUS-HOST DISEASE
Abstract
Disclosed is a method of diagnosing graft-versus-host disease,
comprising measuring the level of CCL8 protein in a sample obtained
from a subject as an indicator for the diagnosis or course of
graft-versus-host disease. Also a diagnostic reagent for
graft-versus-host disease comprising an anti-CCL8 antibody is
disclosed. The method of the present invention enables diagnosis of
the onset of graft-versus-host disease and monitoring of the
progress, in particular, differential diagnosis between
graft-versus-host disease and an infectious disease. The present
invention also provides a method for treating graft-versus-host
disease utilizing the anti-CCL8 antibody.
Inventors: |
HORI; Tsukasa; (Hokkaido,
JP) ; Kokai; Yasuo; (Hokkaido, JP) ; Naishiro;
Yasuyoshi; (Hokkaido, JP) ; Tsutsumi; Hiroyuki;
(Hokkaido, JP) ; Imai; Kohzoh; (Hokkaido,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HORI; Tsukasa
Kokai; Yasuo
Naishiro; Yasuyoshi
Tsutsumi; Hiroyuki
Imai; Kohzoh |
Hokkaido
Hokkaido
Hokkaido
Hokkaido
Hokkaido |
|
JP
JP
JP
JP
JP |
|
|
Assignee: |
Immuno-Biological Laboratories Co.,
Ltd. and Sapporo Medical University
|
Family ID: |
40185370 |
Appl. No.: |
13/523791 |
Filed: |
June 14, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12666209 |
Mar 15, 2010 |
|
|
|
PCT/JP2008/001625 |
Jun 23, 2008 |
|
|
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13523791 |
|
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Current U.S.
Class: |
506/9 ; 435/23;
435/7.92; 436/501; 506/18 |
Current CPC
Class: |
G01N 2800/245 20130101;
A61P 37/06 20180101; G01N 2333/523 20130101; G01N 33/6863 20130101;
G01N 2030/8831 20130101; A61K 2039/505 20130101; C07K 16/24
20130101 |
Class at
Publication: |
506/9 ; 436/501;
435/7.92; 435/23; 506/18 |
International
Class: |
G01N 33/566 20060101
G01N033/566; C12Q 1/37 20060101 C12Q001/37; C40B 40/10 20060101
C40B040/10; G01N 27/26 20060101 G01N027/26; G01N 30/02 20060101
G01N030/02; C40B 30/04 20060101 C40B030/04; G01N 27/62 20060101
G01N027/62 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 22, 2007 |
JP |
2007-165547 |
Claims
1. A method for diagnosing graft-versus-host disease, comprising:
measuring the level of CCL8 protein in a first sample obtained from
a human subject or animal subject at a first time point; measuring
the level of CCL8 protein in a second sample obtained from said
human subject or animal subject at a second, later time point;
comparing the levels of CCL8 protein to thereby diagnose the
development of graft-versus-host disease; wherein increased level
of CCL8 protein in the second sample indicates the development of
graft-versus-host disease.
2. The method according to claim 1, wherein the diagnosis of
graft-versus-host disease is made before clinical manifestations of
graft-versus-host disease.
3. The method according to claim 1, wherein the level of CCL8
protein is measured using an anti-CCL8 antibody.
4. The method according to claim 1, wherein the level of CCL8
protein is measured using a method selected from mass spectrometry,
high-performance liquid chromatography, and two-dimensional
electrophoresis.
5-8. (canceled)
9. A method for evaluating therapeutic efficacy of
graft-versus-host disease, comprising treating a human subject or
animal subject in need of therapy for graft-versus-host disease;
measuring the level of CCL8 protein in a first sample obtained from
said human subject or animal subject at a first time point before
or after the therapy has begun; measuring the level of CCL8 protein
in a second sample obtained from said human subject or animal
subject at a second, later time point after the therapy has begun;
comparing the levels of CCL8 protein to thereby evaluate the
therapeutic efficacy of the treatment for graft-versus-host
disease; wherein increased level of CCL8 protein in the second
sample indicates the therapy is not effective for treatment of
graft-versus-host disease, and wherein decreased level of CCL8
protein in the second sample indicates the therapy is effective for
treatment of graft-versus-host disease.
10. The method according to claim 9, wherein the level of CCL8
protein is measured using an anti-CCL8 antibody.
11. The method according to claim 9, wherein the level of CCL8
protein is measured using a method selected from mass spectrometry,
high-performance liquid chromatography, and two-dimensional
electrophoresis.
12. The method according to claim 2, wherein the level of CCL8
protein is measured using an anti-CCL8 antibody.
13. The method according to claim 2, wherein the level of CCL8
protein is measured using a method selected from mass spectrometry,
high-performance liquid chromatography, and two-dimensional
electrophoresis.
14. A method of diagnosing graft-versus-host disease, comprising:
identifying a human subject who is treated with hematopoietic stem
cell transplantation; measuring the level of CCL8 protein in a
first sample obtained from the human subject at a first time point
and in a second sample obtained from the human subject at a second,
later time point, by: obtaining an anti-CCL8 antibody reagent and
using the anti-CCL8 antibody reagent to measure the level of CCL8
protein, or using a method selected from mass spectrometry,
high-performance liquid chromatography, and two-dimensional
electrophoresis to measure the level of CCL8 protein; and comparing
the levels of CCL8 protein so obtained wherein increased level of
CCL8 protein in the second sample indicates the development of
graft-versus-host disease.
15. A method of evaluating the development of clinical
manifestations of graft-versus-host disease, comprising:
identifying a human subject who is treated with hematopoietic stem
cell transplantation; measuring the level of CCL8 protein in a
first sample obtained from the human subject at a first time point
and in a second sample obtained from the human subject at a second,
later time point, by: obtaining an anti-CCL8 antibody reagent and
using the anti-CCL8 antibody reagent to measure the level of CCL8
protein, or using a method selected from mass spectrometry,
high-performance liquid chromatography, and two-dimensional
electrophoresis to measure the level of CCL8 protein; and comparing
the levels of CCL8 protein so obtained wherein increased level of
CCL8 protein in the second sample indicates the development of
clinical manifestations of graft-versus-host disease.
16. A method of evaluating the progression of clinical
manifestations of graft-versus-host disease, comprising:
identifying a human subject who has graft-versus-host disease;
measuring the level of CCL8 protein in a first sample obtained from
the human subject at a first time point and in a second sample
obtained from the human subject at a second, later time point, by:
obtaining an anti-CCL8 antibody reagent and using the anti-CCL8
antibody reagent to measure the level of CCL8 protein, or using a
method selected from mass spectrometry, high-performance liquid
chromatography, and two-dimensional electrophoresis to measure the
level of CCL8 protein; and comparing the levels of CCL8 protein so
obtained wherein increased level of CCL8 protein in the second
sample indicates the progression of clinical manifestations of
graft-versus-host disease.
17. A method of diagnosing graft-versus-host disease made before
clinical manifestations of graft-versus-host disease, comprising:
identifying a human subject who is treated with hematopoietic stem
cell transplantation but is not yet showing any clinical
manifestations of graft-versus-host disease; measuring the level of
CCL8 protein in a first sample obtained from the human subject at a
first time point and in a second sample obtained from the human
subject at a second, later time point, by: obtaining an anti-CCL8
antibody reagent and using the anti-CCL8 antibody reagent to
measure the level of CCL8 protein, or using a method selected from
mass spectrometry, high-performance liquid chromatography, and
two-dimensional electrophoresis to measure the level of CCL8
protein; and comparing the levels of CCL8 protein so obtained
wherein increased level of CCL8 protein in the second sample
indicates the development of graft-versus-host disease irrespective
of the presence of clinical manifestations of graft-versus-host
disease.
18. A method of initiating treatment of graft-versus-host disease
conducted before clinical manifestations of graft-versus-host
disease, comprising: identifying a human subject who is treated
with hematopoietic stem cell transplantation but is not yet showing
any clinical manifestations of graft-versus-host disease; measuring
the level of CCL8 protein in a first sample obtained from the human
subject at a first time point and in a second sample obtained from
the human subject at a second, later time point, by: obtaining an
anti-CCL8 antibody reagent and using the anti-CCL8 antibody reagent
to measure the level of CCL8 protein, or using a method selected
from mass spectrometry, high-performance liquid chromatography, and
two-dimensional electrophoresis to measure the level of CCL8
protein; and comparing the levels of CCL8 protein so obtained
wherein increased level of CCL8 protein in the second sample
indicates the development of graft-versus-host disease irrespective
of the presence of clinical manifestations of graft-versus-host
disease; and beginning treatment for graft-versus-host disease
based on the increased levels in the second sample.
19. A method of diagnosing graft-versus-host disease, comprising:
identifying a human subject who is treated with hematopoietic stem
cell transplantation; and measuring the level of CCL8 protein in a
sample obtained from the human subject, by: obtaining an anti-CCL8
antibody reagent and using the anti-CCL8 antibody reagent to
measure the level of CCL8 protein, or using a method selected from
mass spectrometry, high-performance liquid chromatography, and
two-dimensional electrophoresis to measure the level of CCL8
protein; wherein the level of CCL8 protein of at least 52.0 pg/ml
indicates the development of graft-versus-host disease.
20. A kit for diagnosis of graft-versus-host disease, comprising an
antibody reagent for measuring the level of a CCL8 protein,
reagents for use with the antibody reagent, and means for detecting
the level of the CCL8 protein.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation application of U.S. application Ser.
No. 12/666,209, whose 35 U.S.C. .sctn.371(c) date is Mar. 15, 2010,
which is a national stage of International Application No.
PCT/JP2008/001625, filed Jun. 23, 2008, all of which claim priority
to Japanese Patent Application No. 2007-165547, filed Jun. 22,
2007, and the entire disclosures of all of which are incorporated
herein by reference.
TECHNICAL FIELD
[0002] 1. Technical Field
[0003] The present invention relates to a method and reagent for
diagnosis of graft-versus-host disease, as well as a method and a
pharmaceutical composition for treating graft-versus-host
disease.
[0004] 2. Background Art
[0005] Hematopoietic stem cell transplantation (HSCT) is a therapy
where hematopoietic stem cells from another individual is
transplanted into a patient to restore hematopoiesis and immune
function, and it has been established as a mode of therapy in a
broad range of blood, tumor, metabolism, and immune diseases.
Post-transplantation immunosuppression therapy has made
considerable progress in the last 20 years, but graft-versus-host
disease (GVHD) continues to be a major, life-threatening
post-transplantation complication. Despite preventive therapy using
immunosuppressants, GVHD occurs in 30 to 80% of HSCT recipients
(patients). Therefore, early diagnosis of GVHD, early initiation of
therapy, and objective monitoring of therapeutic efficacy are
needed. Moreover, current therapeutic methods do not always result
in a cure, and the development of new therapeutic methods is
needed. For information about the incidence, diagnosis, and
treatment of GVHD, see the report by Sullivan et al. (Sullivan K M.
Graft vs. host disease. In: Blume K G, Forman S J, Appelbaum F R,
eds. Thomas' Hematopoietic Cell Transplantation. 3rd ed. Malden,
Mass.: Blackwell Publishing; 2004:635-664).
[0006] At present, however, the diagnosis of GVHD is mainly carried
out based on clinical findings such as skin rash,
hyperbilirubinemia, diarrhea, etc., and no determinative biomarker
exists that can distinguish GVHD from other similar complications
(veinous occlusion, viral reactivation, a treatment regimen-related
toxicity, and the like). Therefore, an invasive method such as
liver biopsy is required for a differential diagnosis of GVHD.
However, as biopsy is an invasive and subjective diagnostic method,
it is desired to develop a new method that can facilitate early,
accurate, and objective quantitative diagnosis of GVHD without
reliance on biopsy, leading to suitable therapy for GVHD and
improvement of the outcome of HSCT.
[0007] Recent advances in proteomics have provided several methods
for investigating global protein expression in biological fluids
and identifying a new biomarker for the disease or pathological
conditions. One such method is surface-enhanced laser
desorption/ionization time-of-flight mass spectrometry (SELDI-TOF
MS). This is a high-throughput, highly sensitive proteomic approach
for isolating proteins from a body fluid with a complex composition
such as plasma to generate a comparative protein profile. Petricoin
et al. (Petricoin E F, Ardekani A M, Hitt B A, et al. Use of
proteomic patterns in serum to identify ovarian cancer. Lancet.
2002; 359:572-577) have reported a biomarker for ovarian cancer
based on proteomic analysis using SELDI. In SELDI, proteins
obtained from a biological sample are allowed to selectively bind
to chemically modified affinity surfaces on a ProteinChip
(Ciphergen Biosystems, Fremont, Calif.), and then nonspecifically
bound impurities are washed away. Next, the captured proteins are
analyzed by TOF-MS to obtain a spectrum of the molecular mass of
each protein (m/z) and relative concentration (intensity). Through
recent studies this type of technologies have been successfully
applied to the diagnosis of cancer and other diseases.
[0008] Recent reports describe proteomic analysis of body fluids
from GVHD patients. In tests using human clinical samples, however,
artifacts related to genetic background and the environment are
unavoidable, and this has confounded the discovery of a new
biomarker. This is particularly true for post-HSCT patients, who
have a wide variety of pre-existing diseases, and undergo diverse
conditioning regimens and GVHD prophylaxis. There have been no
reports of the discovery of a useful marker based on biochemical
methods including proteomic analysis. For example, Kaiser et al.
(Kaiser T, Kamal H, Rank A, et al. Proteomics applied to the
clinical follow-up of patients after allogeneic hematopoietic stem
cell transplantation. Blood. 2004; 104:340-349) report on their
investigation of GVHD markers by proteomic analysis using urine as
a sample. Two proteins (a leukotriene, i.e., an inflammation
mediator, and serum albumin, i.e., the most frequent protein in
serum) were identified, but no GVHD-specific protein was found.
[0009] The reference documents cited in the present description are
listed below. The contents of these publications are hereby
incorporated by reference in its entirety. However, none of these
documents is admitted to be prior art of the present invention.
[0010] Non-patent document 1: Sullivan K M. Graft vs. host disease.
In: Blume K G, Forman S J, Appelbaum F R, eds. Thomas'
Hematopoietic Cell Transplantation. 3rd ed. Malden, Mass.:
Blackwell Publishing; 2004:635-664
[0011] Non-patent document 2: Petricoin E F, Ardekani A M, Hitt B
A, et al. Use of proteomic patterns in serum to identify ovarian
cancer. Lancet. 2002; 359:572-577
[0012] Non-patent document 3: Kaiser T, Kamal H, Rank A, et al.
Proteomics applied to the clinical follow-up of patients after
allogeneic hematopoietic stem cell transplantation. Blood. 2004;
104:340-349
DISCLOSURE OF THE INVENTION
[0013] An object of the present invention is to provide a method
and reagent for diagnosis of GVHD, and a method and a
pharmaceutical composition for treating GVHD.
[0014] The present inventors investigated a broad range of proteins
that are expressed differently in a GVHD model animal and a control
animal, and discovered that the expression level of CCL8 is
significantly higher in GVHD. Additionally, they discovered that
there is a correlation between the expression level of CCL8 and the
manifestation of clinical signs and course of GVHD, to achieve the
present invention.
[0015] The present invention provides a method for testing GVHD
comprising measuring the level of CCL8 protein in a sample obtained
from a human subject or animal subject as an indicator for
diagnosis or course of GVHD. Preferably, the diagnosis of GVHD is
made before the manifestation of clinical signs.
[0016] In the method of the present invention, preferably the level
of CCL8 protein is measured using an anti-CCL8 antibody. Also
preferably the level of CCL8 protein is measured using a method
selected from the group consisting of mass spectrometry (MS),
high-performance liquid chromatography (HPLC), and two-dimensional
electrophoresis.
[0017] The present invention also provides a diagnostic reagent for
GVHD comprising an anti-CCL8 antibody.
[0018] The present invention also provides a method for selecting a
candidate substance for a therapeutic agent for GVHD. This method
comprises the steps of: administering the test substance to a GVHD
model animal; measuring the level of CCL8 protein in a sample
obtained from the model animal; and selecting the test substance as
a candidate substance for a therapeutic agent for GVHD if the CCL8
protein expression level is lower than the level without
administration of the test substance.
[0019] The present invention also provides a pharmaceutical
composition for treating GVHD comprising an anti-CCL8 antibody as
an active ingredient. The present invention also provides a method
for treating GVHD comprising administering an anti-CCL8 antibody to
a subject suffering from graft-versus-host disease.
[0020] In accordance with the present invention, the development
and course of GVHD is diagnosed in a highly reliable manner,
leading to objective (rather than subjective as in the conventional
method), quantitative, and more accurate diagnosis of GVHD.
Furthermore, the present invention allows treatment of GVHD
especially in patients resistant to existing therapeutic
methods.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 shows the time course of clinical signs and pathology
scores in a GVHD model mouse;
[0022] FIGS. 2A-B show a typical SELDI spectrum from a sample
obtained from a GVHD model mouse (A) and the average normalized
intensity of the 8972 Da peak in the sample at each time point
(B);
[0023] FIGS. 3A-B show a representative SELDI spectrum from a
sample obtained from a model mouse receiving cyclosporin-A (CsA)
(A), and the average normalized intensity of the 8972 Da peak in
the sample at each time point (B);
[0024] FIGS. 4A-B show a representative SELDI spectrum from a
sample obtained from an syngeneic transplant model mouse (A), and
the average normalized intensity of the 8972 Da peak in the sample
at each time point (B);
[0025] FIGS. 5A-C show the SELDI spectrum of the HPLC fraction
containing the 8972 Da protein (A)and the results of
two-dimensional electrophoresis (B and C);
[0026] FIG. 6 shows a representative MS/MS spectrum of the peptide
derived from the 8972 Da protein and the amino acid sequence
identified;
[0027] FIG. 7 shows the detection of the CCL8 protein by
immunoassay;
[0028] FIG. 8 shows the time course of the CCL8 protein level in a
human clinical sample;
[0029] FIG. 9 shows the time course of the CCL8 protein level in a
human clinical sample;
[0030] FIG. 10 shows the detection of the CCL8 protein in a human
clinical sample;
[0031] FIG. 11 shows the detection of the CCL8 protein in a
plurality of human clinical samples;
[0032] FIG. 12 shows the time course of the CCL8 protein level in a
human clinical sample;
[0033] FIG. 13 shows the time course of the CCL8 protein level in a
human clinical sample;
[0034] FIG. 14 shows the time course of the CCL8 protein level in a
mouse receiving TLR ligand and in a GVHD model mouse;
[0035] FIG. 15 shows the time course of the clinical signs and in
the level of CCL8 protein in a GVHD model mouse;
[0036] FIGS. 16a-b show histochemical staining of a GVHD model
mouse receiving an anti-CCL8 antibody (a) or a normal rabbit
antibody (control) (b); and
[0037] FIG. 17 shows the pathological evaluation of therapeutic
efficacy of the anti-CCL8 antibody in a GVHD model mouse.
PREFERRED EMBODIMENTS OF THE INVENTION
[0038] The present invention features a method for diagnosing GVHD
and monitoring the course of GVHD by measuring the expression level
of CCL8 in a test sample such as blood from a subject. As
specifically illustrated in the following examples, among patients
that have undergone bone marrow transplantation or umbilical cord
blood transplantation, those who develop GVHD shows significantly
higher expression level of CCL8, and a correlation was found
between the manifestation of clinical signs of GVHD and the
expression level of CCL8.
[0039] CCL8 is a basic, heparin-binding secretory protein belonging
to the chemokine family, also called MCP-2 (GenBank Accession No.
NP.sub.--005614). This protein is produced in monocytes,
fibroblasts, epithelial cells, etc., and is known to bind to
receptors CCR2, CCR3, CCR5 and CCR11. It has been shown that CCL8
targets CD4-positive T cells, CD8-positive T cells, monocytes, NK
cells, eosinophils, basophils, and the like. It is believed that
GVHD develops through 3 steps subsequent to hematopoietic stem cell
transplantation (HSCT). The first step is conditioning, which
includes exposure to radiation for preparing the host (patient) for
HSCT; the second step is the activation, proliferation, and
differentiation of transplanted T cells; and the third step is the
appearance of cell-mediated or inflammatory effectors (for the
mechanisms, see the following two reviews: Reddy P. Pathophysiology
of acute graft-versus-host disease. Hematol Oncol. 2003;
21:149-161., Ferrara J L, Reddy P. Pathophysiology of
graft-versus-host disease. Semin Hematol. 2006; 43:3-10). In the
second step, antigen presentation by dendrocytes is necessary for
activation of transplanted T cell. Chemokines including CCL8 play
an important role in the differentiation, proliferation, and
activation of these dendrocytes (for the importance of chemokines
in the second step, see: Wysocki C A, Panoskaltsis-Mortari A,
Blazar B R, Serody J S. Leukocyte migration and graft-versus-host
disease. Blood. 2005; 105:4191-4199). However, the role of CCL8 in
the body and its significance in regulation of the immune system
are not fully understood. Therefore, the mechanism underlying the
correlation between the expression of CCL8 and GVHD that was
discovered in the present invention is still unknown.
[0040] A test sample to be measured for the level of CCL8 protein
includes, for example, body fluid, blood, serum, and plasma from a
human subject or animal subject. Tissue and cells collected from a
subject may also be used as a test sample.
[0041] The amount of CCL8 protein in a test sample obtained from a
subject can be measured by using an anti-CCL8 antibody in
immunological assay methods well known in the art. The anti-CCL8
antibody may be a polyclonal antibody or a monoclonal antibody.
Various types of anti-CCL8 polyclonal antibodies and monoclonal
antibodies are commercially available, and any of these antibodies
can be used in the present invention.
[0042] Alternatively, an antibody can be prepared by any of the
methods well known in the art. A polyclonal antibody that binds to
CCL8 can be obtained by a well-known method in the art that
involves immunizing an animal using CCL8 or a peptide fragment
thereof as the sensitizing antigen, isolating antiserum containing
the antibody from the immunized animal, and verifying the presence
of an antibody with the desired binding specificity by an ELISA
assay, Western blot analysis, radioimmunoassay and the like.
[0043] A monoclonal antibody that binds to CCL8 can be obtained in
accordance with a well-known method in the art that involves
immunizing an animal using CCL8 or a peptide fragment thereof as
the sensitizing antigen, collecting the resulting immune cells and
fusing them with myeloma cells, selecting a hybridoma producing the
antibody, and culturing the hybridoma.
[0044] The anti-CCL8 monoclonal antibody used in the present
invention encompasses not only antibodies produced by hybridomas,
but also recombinant antibodies produced by transformants
transfected with an expression vector carrying the antibody gene. A
recombinant antibody can be produced by cloning cDNA encoding a
monoclonal antibody that binds to CCL8 from an antibody-producing
hybridoma, inserting the cDNA into an expression vector,
transforming an animal cell or a plant cell with the vector, and
culturing the transformant. Alternatively, the gene encoding the
anti-CCL8 antibody may be introduced into a transgenic animal to
obtain the anti-CCL8 antibody produced in the transgenic
animal.
[0045] For use in treating a human patient, it is preferable for
the anti-CCL8 antibody of the present invention to be a human
chimeric antibody or humanized antibody. A human chimeric antibody
is an antibody constructed from an antibody heavy-chain variable
region and light-chain variable region of an nonhuman animal, and a
heavy-chain constant region and light-chain constant region of a
human antibody. A humanized antibody is constructed from an
antibody complementarity determining region (CDR) originating in a
nonhuman animal and a framework region (FR) and C region
originating in a human antibody. Human chimeric antibodies and
humanized antibodies will have reduced antigenicity in the human
body, and therefore are useful as an active ingredient of the
pharmaceutical composition of the present invention. Conventional
gene recombination techniques for obtaining human chimeric
antibodies and humanized antibodies, and the methods for evaluating
the binding activity of these antibodies are well known in the art.
Alternatively, an anti-CCL8 human antibody can be obtained by
introducing CCL8 into a transgenic animal having a complete
repertoire of human antibody genes.
[0046] In addition, an antibody fragment may also be used in the
present invention. An antibody fragment refers to a peptide that
lacks a part of the entire anti-CCL8 antibody but still has CCL8
binding capability. Examples of antibody fragments include Fab,
Fab', F(ab')2, Fv, and the like. The antibody fragment can be
obtained by enzymatic treatment of the antibody to produce
fragments. The antibody fragment of the present invention also
includes antibody fragments and dimers thereof constructed by
joining a VL chain and VH chain of an anti-CCL8 antibody with a
linker. Examples include an ScFv, diabody, sc(Fv).sub.2, and the
like.
[0047] The CCL8 protein in a test sample obtained from a human
subject or animal subject is assayed by an immunological method
using an antibody obtained in this manner. The assay may be
qualitative or quantitative. An immunoassay for the expression of
CCL8 in a sample obtained from a subject can be carried out using a
radioimmunoassay, ELISA, immunoprecipitation, immunoagglutination,
Western blotting, and the like.
[0048] As a typical example, sandwich ELISA can be carried out in
the following manner. Peripheral blood is collected from the
subject and plasma is prepared, added to a plate or chip where the
anti-CCL8 antibody is immobilized, and incubated for a suitable
period of time. After the plate or chip is washed to remove unbound
components, another anti-CCL8 antibody is added. The antibody can
be detectably labeled with an enzyme, fluorescent dye,
chemoluminescent substance, biotin, radioactive compound, and the
like. After incubation for a suitable period of time, the plate or
chip is washed, and the label is detected by fluorescence,
luminescence, radioactivity, and the like. Optionally, after the
anti-CCL8 antibody is bound to the protein, a secondary antibody
(for example, goat anti-mouse antibody) may be added in order to
amplify the signal. The secondary antibody can be detectably
labeled with an enzyme, fluorescent dye, chemoluminescent
substance, biotin, radioactive compound, and the like. The amount
of CCL8 protein in plasma obtained from the subject can be measured
in this manner.
[0049] In another aspect, the CCL8 protein can be detected using a
detection method involving an agglutination reaction. In this
method CCL8 can be detected using a carrier, for example latex
particles carrying the anti-CCL8 antibody. When the latex particles
carrying the anti-CCL8 antibody are mixed with the test sample and
incubated for a predetermined period of time, the particles will
agglutinate if CCL8 is contained in the sample. The CCL8 in the
sample can be detected by observing the extent of the agglutination
with the naked eye or by quantifying using a spectrophotometer.
[0050] In still another aspect, the CCL8 protein can be detected
using a biosensor utilizing the surface plasmon resonance
phenomenon. A biosensor based on the surface plasmon resonance
phenomenon enables monitoring protein-protein interactions as a
surface plasmon resonance signal. For example, binding between the
CCL8 protein and the anti-CCL8 antibody can be detected using a
biosensor such as BIAcore (Pharmacia). More specifically, the test
sample is brought into contact with a sensor chip having the
anti-CCL8 antibody and the CCL8 protein bound to the anti-CCL8
antibody can be detected as a change in the resonance signal.
[0051] In another embodiment, the test sample is partially purified
(enriched) using a metal chelating agent and an affinity support
such as heparin, and the CCL8 protein can be detected and
quantified by MS, as shown in Example 2. Furthermore, the CCL8
protein can be detected and quantified by HPLC as shown in Example
3, or it can be detected and quantified by two-dimensional
electrophoresis and silver staining.
[0052] The present invention also provides a diagnostic reagent for
GVHD comprising an anti-CCL8 antibody. The diagnostic reagent for
GVHD of the present invention can be provided in the form of a test
kit. The test kit contains a reagent for detecting CCL8, e.g., an
anti-CCL8 antibody, as the active ingredient. The kit may also
contain suitable reagents necessary for the assay such as a buffer
solution, diluent, reaction stopping solution, washing solution,
control sample, and the like.
[0053] In the present invention, the level of CCL8 protein measured
in this manner may be used as an indicator for diagnosis of GVHD.
According to the present invention, GVHD can be diagnosed
objectively by using the CCL8 protein as a marker rather than
depending on observations such as visual examination and the amount
of diarrhea, and thus the development and course of GVHD can be
monitored. The diagnostic method of the present invention is
useful, for example, for diagnosis before the manifestation of GVHD
(early diagnosis), definitive diagnosis of the development, scoring
of severity, monitoring the course of the disease, evaluating
therapeutic efficacy and prognosis. In particular, as shown in FIG.
14, the expression level of CCL8 does not increase via a pathway
mediated by TLR (Toll-like receptor), which allows differential
diagnosis between GVHD and a bacterial or viral infection using the
CCL8 protein as a marker.
[0054] As shown in the following examples, the CCL8 expression
level in patients is significantly higher after the development of
GVHD than before the development. Example 8 demonstrates that in a
bone marrow transplant model mouse, the amount of CCL8 expression
began to increase two days before clinical signs of GVHD were
recognized. In addition, as shown in FIGS. 8 and 9, the amount of
CCL8 expression starts to increase before clinical signs of GVHD is
observed in human patients as well, indicating that the method of
the present invention enables early diagnosis of GVHD. Furthermore,
the method of the present invention is useful for evaluating
therapeutic efficacy. As shown in FIGS. 8 and 9, as the clinical
signs of GVHD improved through therapy, a decrease in the CCL8
expression level was observed. In treatment-resistant GVHD,
conventional methylprednisolone therapy is not effective, and more
aggressive treatment is required. Because such treatment is often
accompanied by adverse side effects, it is beneficial to adjust the
dose and control the therapy while suitably monitoring therapeutic
efficacy by the method of the present invention.
[0055] Furthermore, as shown in FIG. 11, the CCL8 expression level
correlates with the severity and prognosis of GVHD, thus the method
of the present invention will enable earlier identification of
patient with severe conditions and implementation of suitable
proper treatments.
[0056] In addition, the method of the present application is useful
for the development and improvement of therapeutic modes for GVHD.
As shown in FIG. 11, patients with treatment-resistant GVHD can be
identified by measuring the level of CCL8 expression. There is no
established mode of therapy for such patients. A suitable
therapeutic mode may be developed by studying the efficacy of
combination therapy with multiple agents with monitoring the CCL8
level in the patients.
[0057] Furthermore, because at present no magic drug for GVHD is
available, the present invention is also useful in screening for
candidate substances that would be a powerful drug for the
treatment of GVHD based on the CCL8 expression level as an
indicator. Screening is carried out by administering a test
substance to a GVHD model animal, and measuring the amount of CCL8
protein in a test sample obtained from the model animal. For
example, an anti-CCL8 antibody can be used as a test substance.
Also the test substance can be obtained from a library such as a
library of various synthetic or naturally occurring compounds, a
combinatorial library, oligonucleotide library, peptide library,
and the like. The test substance may also include an extract of a
natural substance or partially refined product originating in
bacteria, fungi, algae, plants, animals and the like. If the level
of CCL8 protein is lower in animals receiving the test substance,
the test substance can be selected as a candidate substance for a
therapeutic agent for GVHD. In other words, the diagnostic method
of the present invention provides a platform for the development of
new modes of therapy for GVHD.
[0058] In another aspect, the present invention provides a
pharmaceutical composition for treating GVHD comprising an
anti-CCL8 antibody as the active ingredient, as well as a method
for the treatment of GVHD comprising administrating an anti-CCL8
antibody. As shown in Example 9 below, when an anti-CCL8 antibody
was administered to a GVHD model animal and evaluating pathological
conditions, a decrease in inflammatory cell infiltration into the
dermoepidermal junction in the skin and amelioration of damage to
hair follicles and sebaceous glands were observed, demonstrating
that administration of the anti-CCL8 antibody is effective in the
treatment of GVHD.
[0059] The pharmaceutical composition of the present invention may
be formulated by a method known in the art. For example, the
composition may be formulated by suitably combining with a
pharmaceutically acceptable carrier or excipient, such as sterile
water and physiological saline, vegetable oil, emulsifier,
suspending agent, surfactant, stabilizer, flavoring, filler,
vehicle, preservative, binder, and the like, and compounding them
into the form of a unit dose required for generally recognized
pharmaceutical manufacturing.
[0060] For oral administration, the compound of the present
invention can be formulated into a tablet, pill, dragee, capsule,
liquid, gel, syrup, slurry, suspension, and the like by admixing
with a pharmaceutically acceptable carrier well known in the
art.
[0061] For parenteral administration, the compound of the present
invention can be formulated into an injectable, an aerosol spray,
product for dermal administration and the like using a
pharmaceutically acceptable vehicle well known in the art.
[0062] The pharmaceutical composition of the present invention can
be administered to a patient by oral or parenteral administration.
Parenteral administration is preferred. Examples of the route of
administration include intravenous injection, intramuscular
injection, intraperitoneal injection, subcutaneous injection,
intrarectal administration, transnasal administration,
transpulmonary administration, transcutaneous administration and
the like. The dosage can be selected from a range of 0.0001 mg to
1000 mg/kg of body weight for a single dose. The route of
administration and dose can be selected as needed by the attending
physician in consideration of the age of the patient, severity of
symptoms, concomitant drugs, and the like.
[0063] The content of all patents and reference documents
specifically cited in the present description are hereby
incorporated by reference in its entirety.
[0064] The present invention is described in greater detail below
through the following examples, but is by no means limited by the
examples.
EXAMPLE 1
[0065] Bone Marrow Transplantation (BMT)
[0066] Acute GVHD was induced in mice via allogeneic bone marrow
transplantation. BALB/c (H-2.sup.d) mice were used as the
recipients, C57BL/6 (H-2.sup.b) mice were used as the allogeneic
BMT donors, and BALB/c (H-2.sup.d) mice were used as the syngeneic
BMT donors. All mice were 7 to 12 weeks old (Sankyo Labo Service
Corporation, Japan).
[0067] On the day of BMT, the donor mice were sacrificed by
cervical dislocation. Donor bone marrow cells were collected by
flushing the shaft of the femur and tibia. The cells were placed in
modified Eagle's medium containing 2% fetal calf serum/1%
penicillin-streptomycin, and prepared as a single cell suspension.
The cells were rinsed with RPMI 1640 medium and resuspended in that
same medium. The bone marrow cell inoculum was prepared to contain
2.times.10.sup.7 bone marrow cells/200 .mu.L for allogeneic BMT and
1.times.10.sup.6 bone marrow cells/100 .mu.L for syngeneic BMT.
[0068] The recipient BALB/c mice were raised on acidic water for at
least 7 days before BMT to prevent sepsis after a lethal dose of
radiation. The recipient mice were given a total of 8.5 Gy of total
body irradiation at a rate of 0.34 Gy/min, and within 3 hours after
irradiation the donor bone marrow cells were injected intravenously
via the caudal vein.
[0069] Monitoring of GVHD
[0070] The recipient mice were observed every day for clinical
signs of GVHD, i.e., weight loss, hunched posture, skin erythema,
alopecia, and diarrhea. In the allogeneic BMT mice, clinical signs
of acute GVHD, e.g., diarrhea and ruffled fur within 7 days
post-transplantation, and skin erythema and alopecia within 21
days. Some animal deaths were found at days 14 and 21
post-transplantation.
[0071] Histopathologic Analysis
[0072] The recipient mice were sacrificed on post-transplantation
days 7, 14, 21, and 28. The skin, liver, and small intestine were
removed and fixed in 10% buffered formalin. The fixed tissue was
embedded in paraffin, sections were prepared and stained with
hematoxylin/eosin, and then observations were made under an optical
microscope. Histologic changes thought to correspond to GVHD in
typical organs were as follows: skin (mononuclear infiltration into
the dermoepidermal junction, and damage to hair follicles or
sebaceous glands); liver (periportal mononuclear infiltration and
hepatocellular necrosis); and small intestine (apoptosis of crypt
cells and dilatation or flattening of the villi). For each of these
changes in the organs the scoring system assigned a score of zero
for negative findings and a score of 1 for positive findings (the
maximum possible score for each mouse was 6). FIG. 1 shows the
average pathology score at each time point together with the
observed clinical signs. These results were typical throughout 5
independent studies. The recipient mice have pathological signs of
GVHD at all post-transplantation time points, and the pathology
score was highest on day 14. The pathology score matched the
clinical findings for GVHD at each time point.
[0073] Plasma Samples
[0074] Blood samples were taken before BMT and on days 7, 14, 21,
and 28 after BMT. The blood samples were collected using a
heparin-coated capillary tube from the caudal veins of living mice,
and centrifuged at 10,000 rpm for 5 min within 30 min to prepare
plasma. The plasma samples were stored at -80.degree. C. until
assay.
EXAMPLE 2
[0075] SELDI Protein Chip Array Analysis
[0076] To 10 .mu.L of each plasma sample was added 20 .mu.L of a
solution comprising 9 mol/L urea and 10 g/L CHAPS in Tris-HCl (pH
7.4). The mixture was mixed for 15 min at 4.degree. C. with a
vortex mixer, and was then diluted to 1:40 in Tris-HCl. Eight-spot
immobilized metal affinity capture arrays (IMAC-30) were activated
with 50 mmol/L CuSo.sub.4. Diluted samples (50 .mu.L) were applied
to each spot of the protein chip array, and incubated for 1 h on a
shaker. After washed with the same Tris-HCl, the chip was gently
rinsed with water, and 0.5 .mu.L of saturated sinapinic acid (SPA)
was applied twice to each spot, and then air-dried. The mass/charge
(m/z) spectra of the proteins bound to the chelated metal were
measured using a Ciphergen Protein Biology System II Time-Of-Flight
mass spectrometer (PBS II, Ciphergen Biosystems, Inc.) The data
were calculated by averaging 65 laser shots obtained at a laser
intensity of 200 and a detector sensitivity of 8.
[0077] Statistical Analysis of SELDI-TOF MS
[0078] All spectra were compiled and the data was preliminary
analyzed using Ciphergen Protein Chip Software 3.2.0. For the
plasma samples, 50 samples from the GVHD group
(post-transplantation days 7, 14, 21, and 28) and 28 samples from
the control group (before BMT) were used for a total of 78 samples.
A total of 169 peaks that appeared to differ were detected in the
range of m/z=2 k to 200 k. When peaks with a change in intensity of
5-fold or greater and a level of significance of p<0.05 were
extracted by making a comparison between the two groups using
Biomarker Pattern's Software, in the GVHD group there were 10 peaks
that were higher and 9 peaks that were lower than in the
control.
[0079] Protein Profiling by SELDI-TOF MS
[0080] The 169 peaks in the above mass range of 2.0 to 200 kDa was
further analyzed using the peak intensity values. A classification
tree was developed with Biomarker Pattern's Software (BPS,
Ciphergen Biosystems, Inc.) using all 169 peaks by a cross
variation procedure. Simply put, in a classification tree the data
are split into two nodes using one rule at a time in the form of a
question. In this study the splitting decisions were made based on
the normalized intensity level of peaks or clusters identified from
the SELDI protein expression profile. In other words, each peak or
cluster identified from the SELDI profile becomes a variable in the
classification process. The splitting process was continued until
terminal nodes were reached and further splitting yielded no gain
in data classification.
[0081] A plurality of classification trees were generated using
this process, and the best performing tree was selected based on
classification tree analysis. As a result, a peak at 8972 Da was
selected as one peak that was significantly higher in the GVHD
group than in the control group. The 8972 Da peak provided
differentiation of the GVHD group and the control group at 100%
both in terms of sensitivity and specificity.
[0082] FIG. 2 shows an example of the 8972 Da peak. FIG. 2A shows
representative SELDI spectra from a normal control sample (pre-BMT,
pre-transplantation) and GVHD sample (post-transplantation days 7,
14, 21, and 28) obtained from the same individual at a range from
6000 to 10,000 Da. The part surrounded by the line shows a peak
with an average mass of 8972 Da. This peak is overexpressed in GVHD
plasma compared with normal plasma, and the peak intensity on day 7
and beyond was significantly higher than in the control. Both the
tissue score and peak intensity decreased on day 28. FIG. 2B shows
the average normalized intensity values of the 8972 Da peak in
samples at various time points (n=9 at each point). The average
expression of the peak in the GVHD sample was significantly higher
than the average expression in the control sample (pre-BMT,
pre-transplantation).
[0083] CsA Treatment Model
[0084] Cyclosporin-A (CsA) (Novartis Pharma), a therapeutic drug
for GVHD, was diluted to 1.67 mg/mL in a 0.9% NaCl solution. CsA
was administered intraperitoneally at a dose of 20 mg/kg daily from
post-transplantation day 8 through day 13. FIG. 3 shows the change
in the 8972 Da peak in the same individuals treated with CsA, and
the average normalized intensity value of the 8972 Da peak in the
sample at each time point (n=4 at each point). In the GVHD mice
treated with CsA, the 8972 Da peak intensity was high on day 7, and
then dropped after the administration of CsA.
[0085] Syngeneic Transplantation Model
[0086] In an syngeneic transplantation model using BALB/c mice with
a bone marrow graft transplanted from a BALB/c mouse, GVHD was not
induced, and no significant difference in average peak expression
was found between pre-transplantation and post-transplantation
samples (FIGS. 4A and 4B).
EXAMPLE 3
[0087] Separation of Proteins
[0088] The three most abundant proteins in the plasma (albumin,
IgG, transferrin) were removed from the pooled plasma sample by
immunodepletion chromatography (Multiple Affinity Removal Column
MS-3, 4.6 mm ID.times.50 mm; Agilent). A 5-fold dilution of 50
.mu.L of plasma in Buffer A (Agilent) was prepared and injected
into the immunodepletion column. The flow-through fractions were
collected and further separated by high-performance liquid
chromatography (HPLC). The separation column used in HPLC was an
Inertsil.TM. Ph column (5 .mu.m, 4.6 mm ID.times.150 mm; GL
Sciences, Japan). The elution gradient profile was as follows: (1)
Elution solvent A: 2% ACN/0.1% TFA, solvent B: 80% ACN/0.1% TFA;
(2) Linear gradient: 0 to 100% for solvent B for 50 min; flow rate
1.0 mL/min.
[0089] The fractions were collected at 30 sec intervals, 2 .mu.L of
each fraction was applied to an Au chip (Cipheragen), processed
with an SPA matrix, and the composition of each fraction was
monitored by SELDI-TOF MS. The HPLC fraction from 31.0 to 31.5 min
(approximately 38% acetonitrile) was collected based on SELDI-TOF
MS monitoring. A large amount of the 8972 Da protein was contained
in the GVHD sample, but almost none was present in the control
sample (FIG. 5A). This fraction was lyophilized and dissolved in
200 .mu.L of solubilization buffer (7 M urea, 2 M thiourea, 50 mM
DTT, 2% ampholine, 3% CHAPS, 1% Triton X-100).
[0090] Next, the sample concentrated in that manner was separated
with two-dimensional electrophoresis. After sonication the sample
solutions were loaded onto IPG gel strips (pH 3-11, NL, 11 cm long,
Amersham Bioscience), and the strips were rehydrated for 10 hours
at 30 V. The first-dimensional separation by isoelectric focusing
(IEF) was performed at 20.degree. C. for a total of 12 kV/hr using
the IPGphor system (Amersham Bioscience). After IEF, the IPG strips
were equilibrated for 15 min in 50 mM Tris-HCl (pH 8.8) containing
6 M urea, 2% SDS, 30% glycerol, 0.002% bromophenol blue, and 1%
dithiothreitol. Next, the strips were equilibrated for 15 min in
the same buffer except 2.5% iodoacetamide replaced the
dithiothreitol. For the second-dimensional separation, SDS-PAGE was
performed using a polyacrylamide gel with an 8 to 20% gradient at a
constant current of 40 mA/gel. After two-dimensional
electrophoresis, the proteins were made visible by silver nitrate
staining. By comparing the images of the two gels from the GVHD and
control samples, a spot located at 6,500 Da to 12,300 Da was
identified that was highly expressed in the GVHD sample (FIGS. 5B
and 5C).
[0091] Protein Identification
[0092] The spot of the 8972 Da candidate protein was digested in
the gel. In short, the gel spot was cut out and washed with 100%
ACN and 100 mM NH.sub.4HCO.sub.3, vacuum dried, and incubated at
37.degree. C. for 16 hours in 5 .mu.L of trypsin solution (12.5
ng/.mu.L in 50 mM NH.sub.4HCO.sub.3 and 5 mM CaCl.sub.2). The
resulting peptides were extracted once in 20 .mu.L of 20 mM
NH.sub.4HCO.sub.3 and three times in 20 .mu.L of 5% formic acid in
50% ACN. The collected extracts were vacuum dried to approximately
40 .mu.L, and analyzed by nanoflow HPLC-ESI-MS/MS. For HPLC a DiNa
system (KYA Technology) was used, and the trypsin-digested samples
were separated on a HIQsil.TM. C18 column (75 .mu.m ID.times.50 mm;
KYA Technology). The separation conditions were as follows: Elution
solvent A: 0.1% formic acid, solvent B: 0.1% formic acid in 70%
ACN; gradient: 0 to 100% for solvent B for 40 min; flow rate: 200
nL/min. The properties of the separated peptides were determined
using a QSTAR XL Q-TOF mass spectrometer (Applied Biosystems). A
search of the NCBI protein database was performed with MASCOT
software (Matrix Science Inc.) using the obtained mass spectral
data.
[0093] The result of the search showed that a partial amino acid
sequence of the 8972 Da protein matched the sequence of a CCL8
precursor. FIG. 6 shows a typical MS/MS spectrum of a peptide
separated from the 8972 Da protein. This peptide was identified by
nano LC-MS/MS as CCL8 peptide 68-79 (QGMSLCVDPTQK). Similarly, 11
peptides were identified that matched the theoretical mass. When
these peptide sequences were combined, 52% of the amino acid
sequence of the CCL8 precursor was covered (underlines). [0094] 1
MKIYAVLLCL LLIAVPVSPE KLTGPDKAPV TCCFHVLKLK IPLRVLKSYE [0095] 51
RINNIQCPME AVVFQTKQGM SLCVDPTQKW VSEYMEILDQ KSQILQP (SEQ ID NO:
1)
[0096] The predicted mass of the CCL8 precursor is 11,017 Da, and
the predicted pI is 8.64. The CCL8 precursor contains a signal
peptide of 19 amino acids followed by a mature CCL8 sequence of 78
amino acid residues. The predicted mass of the mature CCL8 is 8972
Da, and the predicted pI is 8.45. These numbers are consistent with
the data obtained by SELDI-TOF MS and two-dimensional
electrophoresis (2D-PAGE).
EXAMPLE 4
[0097] Verification of CCL8 Expression by Immunoassay
[0098] The fact that the 8972 Da marker is CCL8 was verified by
SELDI immunoassay using a specific anti-mouse CCL8 rabbit antibody.
To each spot on a PS20 (preactivated surface) ProteinChip
(Ciphergen Biosystems) was added 0.1 .mu.g of anti-mouse CCL8
antibody, and the chip was incubated for 2 hours at room
temperature in a humidified chamber. After the residual active
sites were blocked for 30 min with 5 .mu.L of 1M ethanolamine (pH
8.0), the spots were washed three times with 0.5% Triton X-100 in
PBS and twice with PBS. The plasma sample was diluted 1:75 in PBS,
applied to the antibody-immobilized spots on the PS20 chip, and
incubated for 2 hours with gentle mixing at room temperature using
a bioprocessor. Each spot was washed twice with 0.5% Triton X-100
in PBS and twice with PBS. After a brief wash with 5 mM HEPES, SPA
matrix was added, and MS analysis was performed using a PBS II
ProteinChip reader. CCL8 was detected in the GVHD plasma sample,
but was only barely detected in the control sample (FIG. 7).
EXAMPLE 5
[0099] CCL8 Expression in Human Clinical Samples
[0100] Plasma obtained from patients that had undergone bone marrow
transplantation was diluted 1:25 in PBS and used for human clinical
samples. As in Example 4, a PS20 ProteinChip was used together with
an anti-human CCL8 antibody to investigate the expression of CCL8
in human patients by SELDI immunoassay.
[0101] Patient 1 (FIG. 8)
[0102] Five-year-old male
[0103] Diagnosis: Fanconi anemia
[0104] Treatment: Umbilical cord blood stem cell transplantation
from unrelated donor (CBSCT (UR))
[0105] Prophylaxis: CsA+MMF
[0106] Course: GVHD first developed on post-transplantation day 13
with a skin rash, and methylprednisolone (mPSL) therapy was started
the same day. CCL8 was detected on post-transplantation day 10
before the clinical manifestation of GVHD. The level of CCL8
temporarily decreased with this treatment. However, the amount of
CCL8 expression rose once again together with the recurrence of
GVHD. The patient had responded to treatment at first, but
ultimately died due to treatment-resistant GVHD. The amount of CCL8
expression correlated with the development of GVHD and therapeutic
efficacy. The level of CCL8 no longer fell after resistance to
therapy developed.
[0107] Patient 2 (FIG. 9)
[0108] Ten-year-old male
[0109] Diagnosis: Chronic myelogenous leukemia (CML)
[0110] Treatment: Bone marrow transplantation from unrelated donor
(BMT (UR))
[0111] Prophylaxis: FK+MTX
[0112] Course: GVHD first developed on post-transplantation day 19
with a skin rash, and methylprednisolone (mPSL) therapy was started
the same day. On that day the level of CCL8 showed a clear
increase. The GVHD progressed temporarily from stage 2 to stage 3,
but improved with therapy, and a high level of CCL8 expression was
not found thereafter.
[0113] Patient 3 (FIG. 10)
[0114] Three-year-old female
[0115] Diagnosis: Acute lymphoblastic leukemia (ALL)
[0116] Treatment: Bone marrow transplantation from matched sibling
(BMT (matched-sib.))
[0117] Prophylaxis: MTX
[0118] Course: GVHD did not develop. No elevation of CCL8
expression was found at any time throughout the course.
[0119] Patient 4 (FIG. 12)
[0120] Eleven-year-old female
[0121] Diagnosis: Acute lymphoblastic leukemia (ALL)
[0122] Treatment: Bone marrow transplantation from matched sibling
donor (MSD-BMT)
[0123] Prophylaxis: Short-term MTX
[0124] Course: GVHD developed on post-transplantation day 11. The
level of CCL8 expression showed an increase. On the same day
cyclosporin-A was administered by continuous intravenous infusion
(C.I.V.). On day 14 the symptoms of GVHD had improved, and the
level of CCL8 expression had fallen.
[0125] Patient 5 (FIG. 13)
[0126] A 19-year-old female with severe aplastic anemia underwent
bone marrow stem cell transplantation from a parent without a
matching HLA-1 antigen. The patient underwent pre-transplantation
conditioning consisting of 150 mg/m.sup.2 fludarabine (Flu), 120
mg/kg cyclophosphamide (CY), and 24 mg/kg anti-thymocyte globulin
(ATG), and prophylaxis for GVHD with tacrolimus. On day 10, the
patient developed a high fever, and on day 16 an atypical skin rash
appeared on the limbs and trunk, but without diarrhea. On day 20
the level of CCL8 showed a clear increase. On day 23 a skin rash
biopsy was performed, and methylprednisolone (mPSL) therapy was
started. On day 31 the symptoms of GVHD had improved, and the level
of CCL8 expression had fallen.
EXAMPLE 6
[0127] CCL8 Plasma Concentration in GVHD Patients and Normal
Individuals
[0128] The amount of CCL8 was quantified in the plasma of normal
individuals, and among post-HSCT patients, in the plasma of those
who developed GVHD and those who did not. FIG. 11 shows the
results. The numerical units are pg/mL. In patients that had
undergone HSCT, the CCL8 plasma concentration in those who did not
develop GVHD was 6.92 to 48.0 pg/mL, and the mean was 23.3 pg/mL.
The CCL8 plasma concentration in those who did develop GVHD was
52.0 to 333.6 pg/mL, and the mean was 133.3 pg/mL, which was
clearly a higher concentration. Furthermore, in the two
treatment-resistant GVHD cases the concentrations were 333.6 pg/mL
and 290.4 pg/mL, which were extremely high concentrations. These
two patients were resistant to GVHD therapy and died. In normal
individuals the CCL8 plasma concentration was 0 to 32.6 pg/mL and
the mean was 18.9 pg/mL, which was an extremely low value. From
these findings it was learned that the amount of CCL8 in the plasm
of patients who developed GVHD was significantly higher than in
normal individuals, and in treatment-resistant GVHD patients, it
was dramatically higher.
[0129] The above results clearly indicate that there is a strong
correlation between the amount of CCL8 expression and the
development and course of GVHD in human patients.
EXAMPLE 7
[0130] Infection and Differential Diagnosis
[0131] The level of CCL8 expression was measured in mice
administered a TLR ligand.
[0132] Male BALB/c (H-2.sup.d) mice were purchased from Sankyo Labo
Service Corporation (Tokyo, Japan). The mice were 8 to 10 weeks old
at the start of testing. Unless otherwise indicated, all reagents
were purchased from SIGMA/ALDRICH (Tokyo, Japan).
[0133] Wild type BALB/c mice were given an intraperitoneal
injection of 5 .mu.g lipopolysaccharide (LPS) (Escherichia coli), 5
.mu.g of poly(I:C) (GE Healthcare Bio-sciences, Tokyo, Japan), and
20 mg of D-GalN, 100 mg of peptide glycan (PGN (Staphylococcus
aureus) (Invitrogen, CA, USA), 20 mg of Zymosan-A (Invitrogen, CA,
USA), and 20 nmol Cpg-ODN (Invitrogen, CA, USA) in 500 .mu.L of
PBS. The control mice were given an intraperitoneal injection of
500 .mu.L of PBS. Four hours after the injection, a plasma sample
was collected. The doses of LPS, poly(I:C), PGN, Zymosan-A, and
CpG-ODN were determined by preliminary experiments.
[0134] Four hours after the injection, the mouse blood was sampled
using a heparin-coated syringe, and centrifugal separation was
performed on the blood for 7 min at 5000 rpm within 30 min of
sampling to obtain plasma. The divided plasma samples were stored
at -80.degree. C. until assay. Blood samples were collected from
human volunteers and patients, plasma samples were prepared, and
those divided samples were stored at -80.degree. C. until
assay.
[0135] The human CCL8 was measured by enzyme-linked immunosorbent
assay (ELISA). An ELISA kit for human CCL8 was purchased from
RayBiotech (Norcross, Ga.), and the manufacturer's protocol was
followed. The plates were read with a plate reader at 450 nm
(Multiskan JX, Thermo Labsystems, Helsinki, Finland).
[0136] The results were expressed as mean.+-.S.E. A statistical
analysis for significance was performed using either a two-tailed
or one-tailed t-test. The level of significance was set at
p<0.05. A Bonferroni correction for multiple comparisons was
used. The results shown are representative data for a series of
tests.
[0137] As shown in FIG. 14, it is clearly demonstrated that the
level of CCL8 does not increase by the administration of TLR
ligand. This finding shows that the level of CCL8 expression does
not increase by bacterial or viral infection, and indicates that
differential diagnosis between GVHD and infection may be effected
by using CCL8 expression as an indicator.
EXAMPLE 8
[0138] Diagnosis before Manifestation of GVHD
[0139] As in Example 1 syngeneic BMT was performed in mice, blood
was collected on post-transplantation days 1, 3, 5, and 7, and
heparin-plasma samples were obtained. The concentration of CCL8 in
the plasma was quantified using ELISA for mouse CCL8.
[0140] Change in weight from syngeneic BMT post-transplantation day
0 to 7 and weight loss, hunched posture, coat, skin, and diarrhea
on day 7 were evaluated on a 3-step scale of 0, 1, or 2 points. A
score of 0 was assigned to a weight loss of <10%; score of 1 to
a weight loss of 10% to <25%; and score of 2 to a weight loss of
.gtoreq.25%. For hunched posture, a score of 0 was assigned to a
normal posture; score of 1 to a slightly hunched posture; and score
of 2 to a very hunched posture. For coat, a score of 0 was assigned
to normal; a score of 1 to slightly ruffled fur; and a score of 2
to whole body ruffled fur with almost no grooming. For skin, a
score of 0 was assigned to normal skin; a score of 1 to visible
sclerosis on the tail and legs; and a score of 2 to mice with
patchy alopecia. For diarrhea, a score of 0 was assigned to normal;
a score of 1 to slight diarrhea; and a score of 2 to full-blown
diarrhea. The clinical GVHD score represents the total number of
points for each criterion, and the maximum number of points is
10.
[0141] FIG. 15 shows the time-course changes in CCL8 plasma
concentration. The CCL8 plasma concentration increased soon after
bone marrow transplantation and was markedly higher after day 5. In
contrast, until day 6 no clinical manifestations of GVHD were
observed. The clinical evaluation of GVHD was approximately 2.5
points on day 7, which represents early-stage GVHD. Thereafter, the
GVHD signs progressed in all mice starting on day 7, and the score
on day 28 was 6.3.
[0142] Based on these findings it is believed that the quantitation
of the CCL8 protein in the blood is useful for the pre-clinical or
early stage diagnosis of GVHD in mice.
EXAMPLE 9
[0143] Treatment of GVHD with Anti-CCL8 Antibody
[0144] An anti-mouse CCL8 rabbit antibody was prepared by
administering a synthesized CCL8 peptide to a rabbit, and purifying
the anti-CCL8 IgG fraction from the resulting antiserum using an
affinity column. An IgG fraction from the serum of a normal rabbit
was used as a normal rabbit antibody control.
[0145] Bone marrow transplantation was performed on mice as in
Example 1. Anti-mouse CCL8 rabbit antibody or normal rabbit
antibody was administered in a dose of 100 .mu.g to the recipient
mouse via the caudal vein for 3 consecutive days counting from the
day before allogeneic BMT. Three mice each were treated with
anti-mouse CCL8 antibody (treatment group) and normal rabbit
antibody (control group). On post-BMT day 14 the mice were
sacrificed by cervical dislocation. The skin, liver and small
intestine were removed and fixed in 10% buffered formalin. The
fixed tissue was embedded in paraffin, sections were prepared and
stained with hematoxylin/eosin, and then observations were made
under an optical microscope to look for pathological signs
considered indicative of GVHD. The scoring method was as follows:
skin (infiltration of monocytes into the dermoepidermal junction,
and damage to follicles or sebaceous glands); liver (periportal
mononuclear infiltration and hepatocellular necrosis); and small
intestine (apoptosis of crypt cells and dilatation or flattening of
villi). Findings were given an interpretation of positive (+),
intermediate (+/-), or negative (-) on a 3-step scale.
[0146] A typical image of stained skin tissue is shown in FIG. 16,
with (a) showing the administration of the anti-mouse CCL8 antibody
and (b) showing administration of the normal rabbit antibody
(control). Damage (arrowhead) to the sebaceous glands and
lymphocyte infiltration (arrow) into the dermoepidermal junction,
which are signs of GVHD in skin, can be seen in the group
administered normal rabbit antibody, but these are not found in the
group administered the anti-mouse CCL8 antibody.
[0147] FIG. 17 shows the results using the above scoring system. A
decrease in inflammatory cell infiltration into the dermoepidermal
junction and amelioration of damage to hair follicles and sebaceous
glands in the skin were seen only in the mice treated with the
anti-CCL8 antibody. This finding indicates that treatment with an
anti-CCL8 antibody is effective in the treatment of GVHD.
INDUSTRIAL APPLICABILITY
[0148] The present invention is useful for the diagnosis, course
monitoring, and treatment of GVHD.
Sequence CWU 1
1
2197PRThomo sapiens 1Met Lys Ile Tyr Ala Val Leu Leu Cys Leu Leu
Leu Ile Ala Val Pro1 5 10 15Val Ser Pro Glu Lys Leu Thr Gly Pro Asp
Lys Ala Pro Val Thr Cys 20 25 30Cys Phe His Val Leu Lys Leu Lys Ile
Pro Leu Arg Val Leu Lys Ser 35 40 45Tyr Glu Arg Ile Asn Asn Ile Gln
Cys Pro Met Glu Ala Val Val Phe 50 55 60Gln Thr Lys Gln Gly Met Ser
Leu Cys Val Asp Pro Thr Gln Lys Trp65 70 75 80Val Ser Glu Tyr Met
Glu Ile Leu Asp Gln Lys Ser Gln Ile Leu Gln 85 90 95Pro212PRThomo
sapiens 2Gln Gly Met Ser Leu Cys Val Asp Pro Thr Gln Lys1 5 10
* * * * *