U.S. patent application number 13/148233 was filed with the patent office on 2011-12-01 for remedy for chronic inflammation and antibody to be used therein.
This patent application is currently assigned to Tokyo Universtiy of Science Educational Foundation Administrative Organization. Invention is credited to Fumio Fukai.
Application Number | 20110294985 13/148233 |
Document ID | / |
Family ID | 42542164 |
Filed Date | 2011-12-01 |
United States Patent
Application |
20110294985 |
Kind Code |
A1 |
Fukai; Fumio |
December 1, 2011 |
Remedy for Chronic Inflammation and Antibody to be Used Therein
Abstract
The invention provides a remedy for chronic inflammation and an
anti-TNIIIA2 antibody to be used therein. The remedy includes an
antibody recognizing TNIIIA2, that is a peptide derived from a
partial sequence A2 of a human tenascin-C fibronectin III-like
repetitive sequence and having the amino acid sequence
RSTDLPGLKAATHYTITIRGVC (SEQ ID NO: 1).
Inventors: |
Fukai; Fumio; (Tokyo,
JP) |
Assignee: |
Tokyo Universtiy of Science
Educational Foundation Administrative Organization
Tokyo
JP
|
Family ID: |
42542164 |
Appl. No.: |
13/148233 |
Filed: |
February 4, 2010 |
PCT Filed: |
February 4, 2010 |
PCT NO: |
PCT/JP2010/051639 |
371 Date: |
August 5, 2011 |
Current U.S.
Class: |
530/387.9 |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 16/18 20130101; C07K 2317/76 20130101; A61P 29/00 20180101;
C12N 5/0634 20130101 |
Class at
Publication: |
530/387.9 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 6, 2009 |
JP |
2009-025707 |
Claims
1. An antibody recognizing a peptide derived from a partial
sequence A2 of a human Tenascin-C fibronectin III-like repetitive
sequence.
2. The antibody of claim 1 that recognizes a peptide having the
amino acid sequence of SEQ ID NO: 2.
3. An immune cell infiltration inhibitor comprising the antibody of
claim 1.
4. A cell death inducer for inflammatory cells, the cell death
inducer comprising the antibody of claim 1.
5. A chronic inflammation remedy comprising the antibody of claim
1.
6. The antibody of claim 1 that recognizes a peptide having the
amino acid sequence of SEQ ID NO: 1.
7. The antibody of claim 1 that recognizes an antigen, the antigen
being a fusion protein containing a hapten, the hapten having the
amino acid sequence of SEQ ID NO: 3.
8. An immune cell infiltration inhibitor comprising the antibody of
claim 2.
9. A cell death inducer for inflammatory cells, the cell death
inducer comprising the antibody of claim 2.
10. A chronic inflammation remedy comprising the antibody of claim
2.
11. An immune cell infiltration inhibitor comprising the antibody
of claim 6.
12. A cell death inducer for inflammatory cells, the cell death
inducer comprising the antibody of claim 6.
13. A chronic inflammation remedy comprising the antibody of claim
6.
14. An immune cell infiltration inhibitor comprising the antibody
of claim 7.
15. A cell death inducer for inflammatory cells, the cell death
inducer comprising the antibody of claim 7.
16. A chronic inflammation remedy comprising the antibody of claim
7.
Description
TECHNICAL FIELD
[0001] The present invention relates to a remedy for chronic
inflammation and a novel antibody to be used therein.
BACKGROUND ART
[0002] The expression of tenascin-C is rarely observed in healthy
cells, except for the immune system. Expression of tenascin-C is
induced under pathological conditions such as inflammation and
tumor growth.
[0003] As a method for detecting tenascin-C, some anti-tenascin-C
antibodies have been known (for example, see JP-A Nos. 2004-217546
and 2002-234900). An arthritis diagnosis method using tenascin-C as
a marker has been also known (for example, see
JP-A-2004-138489).
[0004] A polypeptide which forms tenascin-C includes an epidermal
growth factor-like domain, a fibronectin (FN) III-like domain and a
fibrinogen-like domain. Among them, it has been known that the FN
III-like domain includes a continuation between 8 basic types of
repetitive sequences (1 to 8) and 9 types of repetitive sequences
to be spliced (A1, A2, A3, A4, B, AD2, AD1, C and D), which are
inserted between the fifth and the sixth sequences of the 8 basic
types thereof. These repetitive sequence sites to be spliced are
those easily cut by matrix metalloproteinases (MMP), and a peptide
produced by a cut in tenascin-C by MMP are thought as having
various functions.
[0005] On the other hand, it has been known that, in chronic
inflammation, immune cells including monocytes such as macrophages
and lymphocytes infiltrate from the blood vessels into the vascular
endothelium and penetrate through the vascular basement membrane to
gather at an inflammatory site. Such an immune cell infiltration at
the inflammatory site is regulated by the interaction between
immune cells, vascular endothelial cells and extracellular matrixes
via adhesion molecules, integrins, on the immune cell membrane. It
has been known that the integrins exist in two conformations, the
active form and the inactive form and only the active form of
integrins can adhere to extracellular matrixes.
[0006] A peptide derived from the A2 domain of the FNIII-like
domain of tenascin-C (hereinafter may be referred to as "TNIIIA2")
has been known as a peptide causing the activation of the integrins
(for example, see J. Biol. Chem., Vol. 282, pp. 34929-34937
(2007)).
[0007] However, no details have been given as to the roles of
tenascin-C and the peptide derived therefrom in inflammatory sites,
and in particular, there has been obtained no clear finding
regarding their relationship with chronic inflammation. Further,
there are cases that the effects of conventionally-known
anti-tenascin-C antibodies against inflammation are inconsistent
depending on the site.
SUMMARY
[0008] An object of the present invention is to provide a novel
remedy effective against chronic inflammation.
[0009] A first aspect of the present invention provides an
anti-TNIIIA2 antibody recognizing a peptide derived from a partial
sequence A2 of a human tenascin-C fibronectin III-like repetitive
sequence. The antibody preferably recognizes a peptide having the
amino acid sequence of SEQ ID NO: 1.
[0010] A second aspect of the present invention provides an immune
cell vascular infiltration inhibitor including the antibody.
[0011] A third aspect of the present invention provides a cell
death inducer for inflammatory cells, including the antibody.
[0012] A fourth aspect of the present invention provides a chronic
inflammation remedy including the antibody.
[0013] The present invention can provide the novel remedy against
chronic inflammation.
BRIEF DESCRIPTION OF DRAWINGS
[0014] FIG. 1 is a view showing the inhibition effects of
anti-human TNIIIA2 antibodies against cell adhesion induced by a
peptide TNIIIA2, according to Example 1 of the invention.
DESCRIPTION OF EMBODIMENTS
[0015] An antibody according to the invention is an anti-TNIIIA2
antibody recognizing a peptide derived from a partial sequence A2
of a human tenascin-C fibronectin III-like repetitive sequence.
[0016] The antibody recognizes the peptide (TNIIIA2) derived from
the partial sequence A2 present in the human tenascin-C fibronectin
III-like repetitive sequence. The antibody selectively binds to the
TNIIIA2, thereby inhibiting immune cell infiltration that can be
induced by the TNIIIA2.
[0017] The present inventor found that the peptide TNIIIA2 derived
from human tenascin-C is associated with the infiltration of immune
cells and a cell death-inducing mechanism for inflammatory cells in
inflammation sites. The invention is based on this finding.
[0018] In the present description, the term "step" encompasses not
only independent steps but a case in which even if a step cannot be
clearly distinguished from any other step, as long as an effect
expected from the step is achieved.
[0019] Additionally, in the present description, the numerical
range indicated by using "to" refers to a range including
respective values presented before and after "to" as a minimum and
a maximum, respectively.
[0020] Hereinafter, the invention will be described.
[0021] The anti-TNIIIA2 antibody of the invention is not
specifically limited as long as the antibody can recognize the
peptide (TNIIIA2) derived from the partial sequence A2 of the human
tenascin-C FNIII-like domain. The anti-TNIIIA2 antibody may be a
monoclonal antibody or a polyclonal antibody.
[0022] The TNIIIA2 is a peptide composed of 22 amino acids:
RSTDLPGLKAATHYTITIRGVC (SEQ ID NO: 1) (See J. Biol. Chem., Vol.
282, pp. 34929-34937 (2007)). The anti-TNIIIA2 antibody of the
invention is the antibody that recognizes the TNIIIA2, in which at
least a part of the amino acid sequence of the TNIIIA2 shown in SEQ
ID NO: 1 is an epitope. The epitope recognized by the anti-TNIIIA2
can be any as long as it is a partial peptide including an amino
acid sequence: YTITIRGV (SEQ ID NO: 2).
[0023] The peptide to be used to produce the antibody that
recognizes the TNIIIA2, namely the peptide (a target peptide)
recognized by the anti-TNIIIA2 antibody can be any peptide as long
as the peptide includes the sequence of SEQ ID NO: 2, and in
embodiments it may be a peptide having the entire length (SEQ ID
NO: 1). From the viewpoint of improving antigenicity and stability,
a linker function or one or more compounds having a linker function
(for example, an amino acid) may be added to the peptide having the
amino acid sequence of SEQ ID NO: 2. Examples of such an additional
amino acid include amino acids capable of adding an amino acid to
be bound to a carrier protein to the peptide having the amino acid
sequence of SEQ ID NO: 2, such as cysteine, acidic amino acids or
basic amino acids. From the viewpoint of antibody production
efficiency, it may be more preferable that the target peptide has
an amino acid sequence: CATHTITIRGV (SEQ ID NO: 3).
[0024] The antibody recognizing the TNIIIA2 can be prepared using
the target peptide by a usually-conducted method.
[0025] For example, when the antibody is a polyclonal antibody, it
may be obtained in the following manner. Any of the amino acid
sequences of SEQ ID NOs: 1 to 3 or a mixture thereof is used as the
target peptide. The target peptide is used to immunize a small
animal such as a rabbit to obtain serum. The antibody is then
prepared by purification thereof from the obtained serum by using a
known antibody-purifying means, such as ammonium sulfate
precipitation, protein A, protein G columns, DEAE ion-exchange
chromatography, or affinity columns prepared by coupling the
specific peptide.
[0026] Alternatively, when the antibody is a monoclonal antibody,
it may be obtained in the following manner. Any small animal such
as a mouse is immunized with the target peptide. A spleen is taken
out of the mouse and crushed to isolate cells. The spleen cells are
fused with mouse myeloma cells by using a reagent such as
polyethylene glycol to form fused cells (hybridomas). Clones which
produce antibodies binding to the target peptide are selected from
hybridomas. Next, the selected hybridomas are transplanted in the
peritoneal cavity of a mouse to collect ascitic fluid from the
mouse. The obtained monoclonal antibody is purified by, for
example, ammonium sulfate precipitation, protein A, protein G
columns, DEAE ion-exchange chromatography or affinity columns
prepared by coupling the specific peptide, so as to prepare the
antibody.
[0027] The target peptide may be used in immunization in a form of
a fusion protein in which the by target peptide is fused with a
known carrier protein in view of improving antigenicity. Any known
molecule used for this purpose can be used as such a carrier
protein without any specific restriction, and examples of the
carrier protein include KLH, GST and BSA.
[0028] The immune cells the infiltration of which is inhibited by
the anti-TNIIIA2 antibody are not specifically restricted as long
as they are immune cells the infiltration of which is observed in
inflammatory sites. Examples of such immune cells include
neutrophils, eosinophils, basophils, monocytes, lymphocytes
(including plasma cells) and combinations of two or more kinds
thereof.
[0029] Not being restricted by any specific theory, it is expected
that the anti-TNIIIA2 antibody can inhibit the adhesion of immune
cells to an extracellular matrix (for example, fibronectin or the
like) by hampering the activation of integrins expressed on the
immune cell surface induced by tenascin-C or the like. Therefore,
the use of the anti-TNIIIA2 antibody may inhibit occurrence of
immune cell infiltration across the vascular endothelium.
[0030] In addition, at an inflammatory site in chronic
inflammation, activated resident immune cells or immune cell
infiltrate (these cells are sometimes generically called
"inflammatory cells") are known to exist for a longer period of
time than usual. The anti-TNIIIA2 antibody can induce the cell
death of such inflammatory cells, thereby inhibiting their
long-term existence.
[0031] A chronic inflammation remedy according to the invention
includes at least the anti-TNIIIA2 antibody.
[0032] Inclusion of the anti-TNIIIA2 antibody may allow for the
inhibition of immune cell infiltration into an inflammatory site
and may also allow for the induction of cell death of the
inflammatory cells to inhibit the longer existence of the
inflammatory cells, whereby chronic inflammation can be
treated.
[0033] Subjects of treatment with the chronic inflammation remedy
may be preferably mammals, and more preferably humans.
[0034] The chronic inflammation remedy according to the invention
may be administered orally or parenterally, and systemically or
locally. Examples of the selectable administration method include
intravenous injection such as drip infusion, intramuscular
injection, intraperitoneal injection, subcutaneous injection,
suppository, enema, oral enteric tablet and the like. The
administration method can be selected as appropriate, depending on
patients' age and condition of disease. In addition, the amount of
administration can also be selected as appropriate, depending on
patients' age, condition of disease and the like.
[0035] The chronic inflammation remedy may further include a
carrier and/or an additive that are pharmaceutically acceptable in
accordance with the administration route. Examples of such a
carrier and an additive include water, pharmaceutically acceptable
organic solvents, collagen, polyvinyl alcohol, polyvinyl
pyrrolidone, carboxyvinyl polymer, carboxymethyl cellulose sodium,
sodium polyacrylate, sodium alginate, water-soluble dextran,
carboxymethyl starch sodium, pectin, methyl cellulose, ethyl
cellulose, xanthan gum, gum arabic, casein, gelatin, agar,
diglycerin, propylene glycol, polyethylene glycol, Vaseline,
paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA),
mannitol, sorbitol, lactose and surfactants acceptable as
pharmaceutical additives. The additives to be used may be selected
as appropriate from the above examples in accordance with the
dosage form, but not limited thereto.
[0036] An immune cell infiltration inhibitor according to the
invention includes at least the anti-TNIIIA2 antibody.
[0037] Inclusion of the anti-TNIIIA2 antibody may allow for the
inhibition of infiltration of immune cells into the vascular
endothelium and effects such as chronic inflammation inhibition or
the like can be exhibited.
[0038] The immune cell infiltration inhibitor may further include
any other component if needed. The carriers and additives
pharmaceutically acceptable in the chronic inflammation remedy may
be similarly used as such other components.
[0039] A cell death inducer for inflammatory cells according to the
invention includes at least the anti-TNIIIA2 antibody. Inclusion of
the anti-TNIIIA2 antibody may allow for the induction of cell death
of inflammatory cells at an inflammatory site, thereby inhibiting
their long-term existence and to exhibits effects such as
alleviation of symptoms due to chronic inflammation.
[0040] The cell death inducer for inflammatory cells may further
include any other component if needed. The carriers and additives
pharmaceutically acceptable in the chronic inflammation remedy may
be similarly used as such other components.
[0041] The invention further includes a method for treating or
inhibiting chronic inflammation, the method including
administration of the remedy including the anti-TNIIIA2 antibody to
patients who have or may develop chronic inflammation. Herein, any
improvement in symptoms is included in the scope of the "treating
chronic inflammation", and thus alleviation of symptoms and
inhibition of advances in severity are also included therein.
Symptoms of chronic inflammation or symptoms occurring due to
inflammation can be inhibited, reduced or alleviated thereby.
[0042] The amount of administration in patients may be different
depending on the dosage form of a remedy to be applied, patients'
sex, age, symptoms and the like or combinations thereof. In
general, administration can be done by intravenous injection,
intramuscular injection, intraperitoneal injection, subcutaneous
injection, suppository, enema and oral enteric tablet, and
preferably by intravenous injection.
[0043] The method of administration to patients are different
depending on the dosage form of a remedy to be applied, patients'
sex, age, symptoms and the like or combinations thereof, and, in
general, examples of the administration method can include
intravenous injection, intramuscular injection, intraperitoneal
injection, subcutaneous injection, suppository, enema and oral
enteric tablet. From these administration methods, an appropriate
method may be selected depending on the condition of the patient.
The effective dose of the antibody in treatment may vary depending
on the level of symptom and the condition of the patient. In
embodiments, it may be in a range of from approximately 0.1 mg/kg
weight to approximately 50 mg/kg weight, but not limited thereto.
In addition, the frequency of administration may be set, for
example, in a range of twice per daily to once per week, but not
limited thereto.
EXAMPLES
[0044] Hereinafter, the invention will be described by way of
Examples, but the invention is not limited to these Examples.
Unless specifically stated otherwise, "%" is based on mass.
Example 1
<Production of Anti-Human TNIIIA2 Antibody>
[0045] A peptide having the amino acid sequence of SEQ ID NO: 3 was
synthesized by a usual method. The synthesized peptide as hapten
was fused with KLH, and the obtained fusion peptide was used as an
antigenic peptide. Immunization was conducted in accordance with a
usual method using rabbits. The mixture including a target antibody
obtained was purified by a usual method.
[0046] In the manner as above, three kinds (No. 1 to No. 3) of
polyclonal antibodies (anti-human TNIIIA2 antibodies) against the
human TNIIIA2 were obtained.
[0047] <Evaluation of Antibody Activity>
[0048] Antibody activity of the anti-human TNIIIA2 polyclonal
antibodies obtained above were evaluated as follows by using, as
indicators, their inhibition effects against cell adhesion induced
by the human TNIIIA2. The results are shown in Table 1.
[0049] Using RPMI 1640 (20% FBS added) as a cell culture medium,
KOP 2.16 cells (a mouse bone marrow-derived vascular endothelial
cell line) were seeded in each well of a 96-hole plate at a density
of 5.times.10.sup.4 cells/200 .mu.L/well to be cultured at
37.degree. C. and 5% CO.sub.2 for 4 hours. After 100 .mu.L of the
culture supernatant was removed and 100 .mu.L of 20% TCA solution
was added, the cells were allowed to stand at 4.degree. C.
overnight or at room temperature for 1 hour to be solid-phased. The
plate was washed 5 times with PBS, once with RPMI 1640 (20% FBS
added) and once with PBS in this order to produce a 96-hole plate
with each well coated with KOP 2.16 cells.
[0050] Into each well of the 96-hole plate coated with KOP 2.16
cells obtained above was added RPMI 1640 (20% FBS added) containing
12.5 .mu.g/mL of the human TNIIIA2. The anti-human TNIIIA2
antibodies (No. 1 to No. 3) obtained above were added such that
their concentrations were 1.5 .mu.g/mL, 3 .mu.g/mL and 6 .mu.g/mL,
respectively, and then, K562 cells (a leukemia cell line) were
seeded in each well at the density of 1.5.times.10.sup.4 cells/well
to be cultured at 37.degree. C. and 5% CO.sub.2 for 1 hour.
[0051] Following the addition of formalin, the cells were allowed
to stand at room temperature for 1 hour to be immobilized. Then,
the plate was washed three times with PBS and stained with Lily
Mayer's Hematoxylin. The stained cells were observed with an
optical microscope to count adherent cells in a predetermined
visual field.
[0052] FIG. 1 indicates that the normal rabbit IgG does not inhibit
cell adhesion induced by the human TNIIIA2, whereas the anti-human
TNIIIA2 antibodies dose-dependently inhibit cell adhesion. In
addition, all of the anti-human TNIIIA2 antibodies of No. 1 to No.
3 are shown to exhibit similar degrees of antibody activity.
Further, the anti-human TNIIIA2 antibodies did not exhibit
inhibition effect against cell adhesion induced by magnesium
ion.
[0053] The above results show that the anti-human TNIIIA2 antibody
of the invention may specifically bind to the human TNIIIA2 to
inhibit its function.
[0054] [Example 2]
<Evaluation 1 of Infiltration Inhibiting Properties>
[0055] The inhibition of infiltration of Jurkat cells (a T
cell-based cell line) into the vascular endothelium by the
anti-human TNIIIA2 antibody was evaluated as follows.
[0056] Into each well of a 96-hole plate, human tenascin-C at a
concentration of 5 .mu.g/mL was added, and the cells were incubated
at 37.degree. C. for 1 hour. The plate was blocked by adding BSA at
a concentration of 2.5 mg/mL and incubating at 37.degree. C. for 1
hour. The plate was washed three times with PBS to obtain an assay
well plate with each well coated with human-tenascin C.
[0057] Next, into each well of the assay well plate, KOP 2.16 (the
mouse bone marrow-derived vascular endothelial cell line) suspended
in a DMEM (+) culture medium was seeded so as to be the condition
of 5.0.times.10.sup.4 cells/well and cultured at 37.degree. C. and
5% CO.sub.2 for 3 hours. Thereby, a single layer composed of the
KOP 2.16 cells was formed on the wells coated with human
tenascin-C.
[0058] A normal rabbit IgG or the anti-human tenascin-C antibody
was added to a suspension of Jurkat cells such that its
concentration was 20 .mu.g/mL. The resulting cells were gently
seeded on the single layer composed of the KOP 2.16 cells at a
density of .1.0.times.10.sup.4 cells/well and cultured at
37.degree. C. and 5% CO.sub.2 for 3 hours.
[0059] After adding formalin and allowing to stand at room
temperature for more 1 hour to be immobilized, the cells were
washed three times with PBS (-) and stained with Lily Mayer's
Hematoxylin. Under an optical microscope (at 200-fold
magnification), the numbers of infiltration in predetermined four
fields were counted concerning each three wells and an arithmetic
average value thereof was determined as an infiltration count.
[0060] The infiltration count in a case without adding the human
tenascin-C were similarly calculated and was used as a background
to subtract from the infiltration count of the human
tenascin-C-added sample, whereby obtained infiltration counts is
shown in Table 1. Further, the ratio of a difference between the
infiltration count of a sample using the normal rabbit IgG and that
of the sample using the anti-human TNIIIA2 antibody to the
infiltration count of the sample using the normal rabbit IgG was
calculated as an infiltration inhibition rate (%).
[0061] In addition, the inhibition of THP-1 infiltration into the
vascular endothelium by the anti-human TNIIIA2 antibody was
evaluated in the same manner as above, except that THP-1 (a
monocytes-like cell line) was used instead of the Jurkat cells. The
results are shown in Table 1.
TABLE-US-00001 TABLE 1 Jurkat THP-1 Normal rabbit IgG 27 41
Anti-human TNIIIA2 antibody 7 1 Infiltration inhibition rate (%)
74% 98%
[0062] Table 1 indicates that the anti-human TNIIIA2 antibody can
effectively inhibit the infiltration of 1-lymphocyte cells or
monocytes-like cells into the vascular endothelial layer induced by
human tenascin-C.
Example 3
<Evaluation 2 of Infiltration Inhibiting Properties>
[0063] The infiltration inhibition effect of the anti-human TnIIIA2
antibody obtained in Example 1 was compared with those of another
antibody 4F10TT and a normal rabbit IgG, against human tenascin-C
as follows.
[0064] The 4F10TT is a rabbit IgG antibody that recognizes a
peptide sequence of the epithelium growth factor-like domain of
human tenascin-C. The 4F10TT used here was obtained from
Immuno-Biological Laboratories Co., Ltd (Tsunoda T. et al., Am J
Pathol 162: 1857-1867, 2003; Sato A. et al., J Am Coll Cardiol 47:
2319-2325, 2006).
[0065] The count of Jurkat cells which infiltrated into the single
layer of the KOP 2.16 cells was measured in the same manner as in
Example 2 except that the above antibodies were respectively
used.
[0066] The infiltration count in a case without adding any antibody
was similarly counted. The ratio of the infiltration count of each
case of the addition of the normal mouse IgG (normal IgG), the
addition of the 4F10TT, and the addition of the anti-human TNIIIA2
antibody to the infiltration count obtained without adding any
antibody were calculated. The results are shown in Table 2 ("Coated
TNC").
[0067] Additionally, the cell infiltration inhibition effect of
each antibody was evaluated in the same manner as above, except
that, instead of the assay plate with each well coated with human
tenascin-C, an uncoated 96-hole plate was prepared and human
tenascin-C was added at a concentration of 2 .mu.g/mL to bring the
total volume per well to 100 .mu.L. The results are shown in Table
2 ("Soluble TNC").
TABLE-US-00002 TABLE 2 Antibody Coated TNC Soluble TNC -- 1 1
Normal IgG 1.05 1.05 4F10TT 1.04 1.07 Anti-TNIIIA2 0.74 0.84
[0068] The results of Table 2 indicate that the cell infiltration
inhibition effects of the antibodies recognizing the human
tenascin-C-derived peptide differ from each other, and the
anti-TNIIIA2 antibody shows a remarkable cell infiltration
inhibition effect as compared to the 4F10TT.
Example 3
<Evaluation of Long-Term Existence Inhibiting Properties>
[0069] The effect of the anti-human TNIIIA2 antibody on changes in
viable macrophage cell counts over time in the presence of human
tenascin-C was evaluated as follows.
[0070] THP-1 cells were cultured in a complete medium, to which PMA
was added at a concentration of 10 ng/mL, at 37.degree. C. and 5%
CO.sub.2 for 24 hours to be induced to differentiate into
macrophages (hereinafter, the differentiated THP-1 is referred to
as "PMA-THP-1").
[0071] Into each well of a 96-hole plate, fibronectin (FN) was
added at a concentration of 5 .mu.g/mL, and the plate was incubated
at 37.degree. C. for 1 hour. The plate was blocked by adding BSA at
a concentration of 2.5 mg/mL and incubating at 37.degree. C. for 1
hour, and washed three times with PBS to obtain a plate with
FN-coated each well.
[0072] Into the FN-coated wells, an RPMI 1640 (serum-free) culture
medium, to which human tenascin-C was added such that its final
concentration was 2 .mu.g/mL, was added, and a normal rabbit IgG,
the 4F10TT antibody or the anti-human TNIIIA2 antibody was further
added to bring its final concentration to 50 .mu.g/mL. Herein,
PMA-THP-1 cells were seeded to bring the density to
2.times.10.sup.4 cells/well to 2.5.times.10.sup.4 cells/well and
cultured at 37.degree. C. and 5% CO.sub.2 for 24 hours.
[0073] Using a cell counting kit (manufactured by DOUJIN Co.) based
on the WST method, cell counts were measured to calculate the ratio
(viable cell count ratio) of post-culture viable cell counts (day
2) to viable cell counts upon seeding (day 0). The results are
shown in Table 3.
TABLE-US-00003 TABLE 3 Antibody Cell count ratio Day 0 1 Day 2 --
1.52 1 Normal IgG 1.48 0.97 4F10TT 1.57 1.03 Anti-TNIIIA2 0.93
0.61
[0074] Table 3 shows the increase of the macrophages in the
presence of human tenascin-C. On the contrary, it is shown that,
when the anti-human TNIIIA2 antibody is added, increase in the
macrophages was inhibited (the ratio of the cell count thereof to
that of no antibody-addition sample is 0.61), and the viable cell
count decreased (the ratio of the cell count thereof to that
measured upon seeding is 0.93). In other words, it is found that
the anti-human TNIIIA2 antibody can inhibit long-term existence of
the macrophages in the presence of human tenascin-C.
[0075] It was observed that the decrease in the viable cell count
by the anti-human TNIIIA2 antibody was due to cell death.
[0076] It is understood from the above that the anti-human TNIIA2
antibody of the invention may inhibit immune cell infiltration into
the vascular endothelium and may induce the cell death of
inflammatory cells in the presence of human tenascin-C.
[0077] The disclosure of Japanese Patent Application No.
2009-025707 filed on Feb. 6, 2009 is incorporated herein by
reference in its entirety.
[0078] All documents, patent applications and technical standards
described in the present description are incorporated herein by
reference to the same extent as if each individual document, patent
application and technical standard were specifically and
individually indicated to be incorporated by reference.
Sequence CWU 1
1
3122PRTHomo sapiens 1Arg Ser Thr Asp Leu Pro Gly Leu Lys Ala Ala
Thr His Tyr Thr Ile1 5 10 15Thr Ile Arg Gly Val Cys 2028PRTHomo
sapiens 2Tyr Thr Ile Thr Ile Arg Gly Val1
5312PRTArtificialsynthesised epitope 3Cys Ala Thr His Tyr Thr Ile
Thr Ile Arg Gly Val1 5 10
* * * * *