U.S. patent application number 10/037218 was filed with the patent office on 2002-10-17 for novel neutrophil chemotactic factor cloned cdna and monoclonal antibodies thereto.
Invention is credited to Appella, Ettore, Leonard, Edward J., Matsushima, Kouji, Oppenheim, Joost, Showalter, Stephen D., Yoshimura, Teizo.
Application Number | 20020151706 10/037218 |
Document ID | / |
Family ID | 22614006 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020151706 |
Kind Code |
A1 |
Matsushima, Kouji ; et
al. |
October 17, 2002 |
Novel neutrophil chemotactic factor cloned cDNA and monoclonal
antibodies thereto
Abstract
An isolated, synthetic preparation of a novel
neutrophil-specific chemotactic factor (NCF), monoclonal antibodies
having specific binding affinity for NCF and a clone containing the
complete cDNA coding sequence for NCF are disclosed.
Inventors: |
Matsushima, Kouji;
(Frederick, MD) ; Yoshimura, Teizo; (Frederick,
MD) ; Leonard, Edward J.; (Chevy Chase, MD) ;
Oppenheim, Joost; (Bethesda, MD) ; Appella,
Ettore; (Chevy Chase, MD) ; Showalter, Stephen
D.; (Frederick, MD) |
Correspondence
Address: |
MORGAN & FINNEGAN, L.L.P.
345 Park Avenue
New York
NY
10154-0053
US
|
Family ID: |
22614006 |
Appl. No.: |
10/037218 |
Filed: |
November 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10037218 |
Nov 9, 2001 |
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08818631 |
Mar 14, 1997 |
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08818631 |
Mar 14, 1997 |
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07169033 |
Mar 16, 1988 |
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Current U.S.
Class: |
536/23.5 ;
435/326; 435/6.1; 435/6.18; 530/324; 530/350; 530/388.24 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 16/244 20130101; Y10S 930/12 20130101; Y10S 435/81 20130101;
C07K 14/5421 20130101; Y10S 514/885 20130101; A61P 29/00
20180101 |
Class at
Publication: |
536/23.5 ; 435/6;
435/326; 530/350; 530/388.24; 530/324 |
International
Class: |
C12Q 001/68; C07H
021/04; C07K 014/435; C07K 016/18; C12N 005/06 |
Claims
What is claimed is:
1. An isolated, synthetic neutrophil chemotactic polypeptide (NCF)
composed in whole or in part of the following amino acid sequence
represented by single letter code:
3 NH.sub.2-S-A-K-E-L-R-C-Q-C-I-K-T-Y-S-K-P-F-H-P-K-F-I-K
-E-L-R-V-I-E-S-G-P-H-C-A-N-T-E-I-I-V-K-L-S-D-G-R-
E-L-C-L-D-P-K-E-N-W-V-Q-R-V-V-E-K-F-L-K-R-A-E-N-S
2. A molecular clone containing the complete cDNA coding sequence
for the synthesis of the NCF of claim 1 when said cDNA is inserted
into genome of an eukaryotic or prokary tic expression vector.
3. The clone of claim 2 having identifying characteristics of ATCC
40412.
4. A hybridoma producing monoclonal antibodies having specific
binding affinity for NCF of claim 1.
5. The hybridoma of claim 4 having the identifying characteristics
of ATCC HB9647.
6. The anti-NCF monoclonal antibodies having specific binding
affinity for NCF of claim 1.
7 A kit for determining the level of NCF mRNA present in a sample,
comprising a container containing cDNA for NCF for preforming assay
to datermine the level of mRNA for NCF in a biological sample.
8. A kit for determining the level of NCF present in a sample,
comprising a container containing anti-NCF antibodies.
9. A method for detecting the level of mRNA for NCF in a sample,
comprising reacting said sample with cDNA and determining the
occurrence of hybridization by any conventional technique, the
presence of said cDNA-mRNA hybrids being indicative of the presence
of mRNA for NCF in said sample.
10. A method of treating inflammatory condition, comprising
administering to a host inflicted with an inflammatory condition,
anti-inflammatory effective amount of anti-NCF monoclonal
antibodies to alleviate clinico-pathological condition caused by or
related to NCF.
11. A pharmaceutical composition, comprising anti-inflammatory
effective amount of anti-NCF monoclonal antibodies and
pharmaceutically acceptable carrier.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Technical Field
[0002] The present invention relates to an isolated, synthetic
preparation of a novel neutrophil-specific chemotactic factor
(NCF), monoclonal antibodies having specific binding affinity for
NCF and a clone containing the complete coding sequence for
NCF.
[0003] 2. State of the Art
[0004] Activated monocytes/macrophages produce various mediators
that cause inflammation. Among them are chemotactic factors which
cause white blood cells to migrate into inflammatory sites where
these factors are released. Neutrophils, the dominant leukocytes
attracted by the chemotactic factors, are believed to play a
critical role in the inflammatory reactions. Such diseases as
rheumatoid arthritis, idiopathic pulmonary fibrosis and certain
pathological inflammatory changes in many other conditions are
believed to be caused by neutrophils and/or their products.
However, a specific pro-inflammatory mediator released by tissue
macrophages and other cells in response to inflammatory stimuli and
leading to neutrophil-rich leukocyte accumulation in host defense
and disease, has not heretofore been identified and isolated.
SUMMARY OF THE INVENTION
[0005] It is, therefore, an object of the present invention to
provide a biologically active novel synthetic polypeptide acting as
a neutrophil-specific chemotactic factor (NCF).
[0006] It is a further object of the present invention to provide a
molecular clone containing the complete coding sequence for the
synthesis of NCF by either prokaryotic or eukaryotic expression
vectors.
[0007] It is a still further object of the present invention to
provide monoclonal antibodies having specific binding affinity for
NCF of the present invention.
[0008] It is another object of the present invention to provide a
kit comprising a container containing the cDNA for NCF
quantitation, detection or localization of NCF mRNA in a body
sample.
[0009] It is yet another object of the present invention to provide
a kit comprising a container containing anti-NCF antibodies having
specific binding affinity for NCF for quantitation, detection or
localization of NCF in a body sample.
[0010] Other objects and advantages will become evident from the
following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] These and other objects, features and many of the attendant
advantages of the invention will be better understood upon a
reading of the followings detailed description when considered in
connection with the accompanying drawings wherein:
[0012] FIG. 1 demonstrates translation of cDNA into NCF protein in
reticulocyte lysate system; and
[0013] FIG. 2 shows:
[0014] (a) Northern blot analysis of mRNA induction in
lipopolysaccharide (LPS) stimulated peripheral blood monomuclear
cells (PBMC);
[0015] (b) The time course of the accumulation of neutrophil
chemotactic activity in culture media of PBMC after stimulation
with LPS;
[0016] (c) Induction of NCF mRNA in PBMC by IL 1 or TNF, but not by
IL 2, gamma-IFN, and alpha-IFN;
[0017] (d) HPLC gel filtration analysis of IL 1 and TNF induced
neutrophil chemotactic activity.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The above and various other objects and advantages of the
present invention are achieved by a homogeneously pure, isolated,
synthetic neutrophil chemotactic protein, designated herein NCF,
composed in the whole or in part only of the following amino acid
sequence (single letter code):
1 NH.sub.2-S-A-K-E-L-R-C-Q-C-I-K-T-Y-S-K-P-F-H-P-K-F-I-K
-E-L-R-V-I-E-S-G-P-H-C-A-N-T-E-I-I-V-K-L-S-D-G-R-
E-L-C-L-D-P-K-E-N-W-V-Q-R-V-V-E-K-F-L-K-R-A-E-N-S
[0019] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned hereunder are incorporated herein by
reference. Unless mentioned otherwise, the techniques employed
herein are standard methodologies well known to one of ordinary
skill in the art.
[0020] Chemical synthesis of the NCF of the present invention
composed of the 72 amino acid residues as shown above, is achieved
by commercially available polypeptide synthesizers. Alternatively,
the NCF of the present invention is synthesized by standard
techniques employing an expression vector containing in its genome
the cloned complete coding sequence of NCF. Anti-NCF monoclonal
antibodies of the present invention are prepared by standard
hybridoma technology and utilized for purification and assaying
purposes following standard immunological methodologies well known
in the art.
[0021] High performance liquid chromatography (HPLC), in situ
hybridization assays, Northern blotting analysis and the like are
typical examples of the standard conventional techniques well known
to one of ordinary skill in the art, which can be employed for
isolation, localization, differentiation, detection, or measurement
of the mRNA for NCF in biological samples.
[0022] It should be noted that the fact that chemically synthesized
polypeptide of the present invention at 10 factor, is shown by the
results presented in Table 1.
2TABLE 1 Chemotactic response of human neutrophils to chemically
synthesized NCF. Concentration of Percentage of assay NCF,
nanomolar neutrophils that migrated 1000 23 100 34 10 32 1 5 0.1 1
Hanks medium 0.3 10-.sup.7M fMet-Leu-Phe 40 1. It is typical for
chemoattractant dose-response curves to show an optimum, with a
decreased response at concentrations above the optimum. 2.
fMet-Leu-Phe is a commonly used reference chemoattractant.
[0023] It may be pointed out that various stimuli cause the release
or secretion of more than one chemoattractant. Without the cDNA of
the present invention, it is clear, of course, that the presence,
specificially of the mRNA for NCF as an involved factor in a
particular clinico-pathological situation, could not be
definitively identified and diagnosed. cDNA of the present
invention due to its binding affinity for mRNA for NCF, for the
first time makes it possible to analyze body samples such as joint
fluid, sputum, alveolar lavage fluid, tissue samples and the like
to detect the presence or absence of mRNA for NCF. Of course, the
antibodies can also be utilized for diagnostic purposes to detect
the NCF and to neutralize the NCF for alleviating any disease or
anomalous conditions in which the presence of NCF is found to be a
causative factor.
[0024] A pharmaceutical composition for use in treating
inflammatory condition comprises and anti-inflammatory effective
amount of the anti-NCF monoclonal antibodies in pharmaceutically
acceptable carrier, such as physiological saline, sterile non-toxic
buffer and the like.
[0025] A deposit of cDNA for NCF and of the hybridoma for anti-NCF
monoclonal antibodies have been made at the ATCC, Rockville, Md. on
Jan. 12, 1988 and Feb. 17, 1988, respectively, under the accession
numbers 40412 and HB9647, respectively. The deposits shall be
viably maintained, replacing if they became non-viable, for a
period of 30 years from the date of the deposit, or for 5 years
from the last data of request for a sample of the deposit,
whichever is longer, and made available to the public without
restriction in accordance with the provisions of the law. The
Commissioner of Patents and Trademarks, upon request, shall have
access to the deposits.
[0026] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
Sequence CWU 1
1
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