IL107809A - Method and kit for the detection of mRNA encoding a neutrophil polypeptide - Google Patents

Description

107,809/2 ->3mi7T>J TUD3'7137 ΤΠρίϊΠ mRNA "7*A7 iDTjn JWW METHOD AND KIT FOR THE DETECTION OF mRNA ENCODING A NEUTROPHIL POLYPEPTIDE The present invention relates to a method and kit for the detection of a neutrophil polypeptide.

More particularly, the present invention relates to a kit for determining the amount of neutrophil chemotactic polypeptide (NCF) mRNA present in a sample, and to a method of detecting mRNA that encodes neutrophil chemotactic polypeptide (NCF) .

The present specification is divided from Israel Specification 89600, filed March 14, 1989.

In order that the invention may be better understood and appreciated, description from Israel Specification 89600 is included herein, it being understood that this is for background purposes only, the subject matter of Israel Specification 89600 being specifically disclaimed and not forming a part of the present invention.

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.

A number of researchers have investigated and reported on the purification and characterization of neutrophil activating or chemotactic factors derived from monocytes and lymphocytes. For example, Walz, et al., in "Purification and Amino Acid Sequencing of NAF, a Novel Neutrophil-Activating Factor Produced by Monocytes," Biochemical and Biophysical Research Communications , Vol. 149, No. 2, pp. 755-761 (1987), describe the amino acid sequencing of a monocyte-derived, neutrophil-activating factor (NAF) purified from a culture of human blood mononuclear cells.

Gregory, et al., in "Structure Determination of a Human Lymphocyte-Derived Neutrophil Activating Peptide (LYNAP) ," Biochemical and Biophysical Research Communications, Vol. 151, No. 2, pp. 883-890 (1988), describe the amino acid sequencing of a human lymphocyte-derived neutrophil activating factor with chemotactic and enzyme degranulating activity in peripheral human neutrophils.

Yoshimura, et al., describe a chemotactic factor for human neutrophils in "Neutrophil Chemotactic Factor Produced by Lipopolysaccharide (LPS) - Stimulated Human Mononuclear Leukocytes: Partial Characterization and Separation from Interleukin 1 (IL 1)," The Journal of Immunology, Vol. 139, No. 2, pp. 788-793 (1987).

However, prior to the present invention, monoclonal antibodies having specific binding affinity for NCF and hybridomas which produce anti-NCF monoclonal antibodies were not known.

In Israel specification 89600 there is described and claimed a hybridoma that produces monoclonal antibodies that have specific binding affinity for neutrophil chemotactic - 3 - 107,809/2 polypeptide (NCF) , wherein said NCF has the amino acid sequence : NH2-S-A- -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.

Said specification also describes and claims a kit for determining the amount of neutrophil chemotactic polypeptide (NCF) with the amino acid sequence depicted above which is present in a sample, comprising a container that contains anti-NCF monoclonal antibodies that are produced by the hybridoma defined above.

In addition, said specification claims a pharmaceutical composition comprising an anti-inflammatory effective concentration of anti-neutrophil chemotactic polypeptide (NCF) monoclonal antibodies that are produced by the hybridoma defined above, in a pharmaceutically acceptable carrier.

The present invention is directed to and provides a kit for use in determining the amount of neutrophil chemotactic polypeptide (NCF) mRNA present in a sample, comprising a container that contains DNA that encodes NCF with the amino acid sequence: NH2-S-A-K-E-L-R-C-Q-C-I-K-T-Y-S- -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 wherein said DNA is used to assess the amount of said mRNA in said sample.

The present invention also provides a method of detecting mRNA that encodes neutrophil chemotactic polypeptide (NCF) with the amino acid sequence: 107,809/2 NH2-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- -F-L-K-R-A-E-N-S comprising hybridizing the mRNA in a sample with DNA that encodes said NCF; and detecting the presence of DNA-RNA hybrids in said sample.

As background for the present invention, it is important to note that human monocytes are known to produce and secrete a potent neutrophil chemotactic and activating protein, now termed "neutrophil activating protein" (NAP-1) and more recently proposed to be named "Interleukin 8 (IL-8) [Gregory, et al., "Structure Determination of a Human Lymphocyte-Derived Neutrophil Activating Peptide ( LYNAP) , " Biochem. Blophys. Res. Commun., Vol. 151, pp. 883-890 (1988); J. Kuby, Immunology, pp. 249-258 (1992)]. IL-8 is a cytokine secreted primarily by monocytes and has a variety of effects on neutrophils. In the presence of IL-8, neutrophils adhere to vascular endothelial cells and emmigrate from the blood into the tissue compartment toward a concentration gradient of IL-8. IL-8 is such a potent chemotactic factor for neutrophils that nanogram quantities are activated.

The sequence of neutrophil activating protein is identical to that reported for melanoma growth stimulating activity (MGSA) , Richmond, et al., "Molecular Characterization and Chromosomal Mapping of Melanoma Growth Stimulatory Activity, a Factor Structurally Related to B-thromboglobulin," Embo J., Vol. 7, pp. 2025-2033 (1988). The neutrophil activity protein sequence was also reported to be identical as well as the gene product of oncogene (GRO) detected in tumor-derived human T24 cells [Anisowic, et al., "Constitutive Overexpression of a Growth Regulated - 5 - 107,809/2 Gene In Transformed Chinese Hamster and Human Cells," Proc. Natl. Acad. Sci. U.S.A. , Vol. 84, pp. 7188-7192 (1987)]. Note Table 1: TABLE 1 NH2 Terminal Sequence MGSA ASVATELRCQCLQTLQG lHPKNIQSVNVKSP GRO ASVATELRCQCLQTLQG lHPKNIQSVNVKSP NCF SAKELRCQCIKTYSKPFHPKFIKELRVIES — The invention will now be described in connection with certain preferred embodiments with reference to the following illustrative figures so that it may be more fully understood.

With specific reference now to the figures in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this context, it is to be noted that only subject matter embraced in the scope of the claims appended hereto is intended to be included in the scope of the present invention, while subject matter of Israel Specification 89600, although described and exemplified to provide background and better understanding of the invention, is not intended for inclusion as part of the present invention.

While the invention will now be described in connection with certain preferred embodiments in the following examples so that aspects thereof may be more fully understood and appreciated, it is not intended to limit the invention to these particular embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included within the scope of the invention as defined by the appended claims, while subject matter of Israel Specification 89600, although described and exemplified to provide background and better understanding of the invention, is not intended for inclusion as part of the present invention. Thus, the following examples which include preferred embodiments will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of formulation procedures as well as of the principles and conceptual aspects of the invention.

In the drawings: Fig. 1 demonstrates translation of cDNA into NCF protein in reticulocyte lysate system; Fig. 2a shows a Northern blot analysis of mRNA induction in lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cells (PBMC); Fig. 2b illustrates the time course of the accumulation of neutrophil chemotactic activity in culture media of PBMC after stimulation with LPS; Fig. 2c depicts the induction of NCF mRNA in PBMC by IL-1 or TNF, but not by IL-2, gamma-IFN, or alpha-IFN; and Fig. 2d shows HPLC gel filtration analysis of IL-1 and TNF-induced neutrophil chemotactic activity.

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 application 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 herein 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.

EXAMPLES 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.

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. - 8 - 107,809/2 cDNA cloning of monocyte-derived NCF and Northern blotting analysis are described by Matsushima, et al., J. Exp. Med.. Vol. 167, pp. 1883-1893 (1988), the teachings of which are also incorporated herein by reference.

The process for detecting the level of mRNA includes reacting the cDNA and determining the occurrence of hybridization by conventional techniques, such as in situ hybridization. The assay requires a single-stranded DNA fragment that is complementary in sequence to the mRNA. The hybridization permits the detection of mRNA that is complementary to a single-stranded molecule of known sequence, called a probe. The detection of mRNA hybridization is done with nitrocellulose filters. Hybridization can be done after gel electrophoresis. The mRNA is transferred by blotting from the gel to a sheet of membrane filter material, and the probe is then added to the filter. When the RNA is in the gel and DNA is the probe, the process is called Northern blotting. The presence of cDNA-mRNA hybrids is indicative of the presence of mRNA for NCF in the sample. A kit for determining the amount of mRNA in body fluid or tissue samples is provided in accordance with the present invention.

It should be noted that the fact that chemically synthesized polypeptide of the present invention at 10 nanomolar concentrations acts as a neutrophil-attracting factor, is shown by the results presented in Table 2: - 9 - 107,809/2 TABLE 2 Chemotactic Response of Human Neutrophils to Chemically-Synthesized NCP Concentration of NCF, Percentage of Assay Nanomolar Neutrophils that Migrated 1000 23 100 34 32 1 5 0.1 1 Hanks medium 0.3 lO-^M 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 chemoatt actant.

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, specifically of the mRNA for NCF as an involved factor in a particular clinico-pathological situation, could not be definitively identified and diagnosed. cDNA used in the present invention, due to its binding affinity for mRN 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 cna also be utilized for diagnostic purposes to detect the NCF and to neutralize the NCF for alleviating any disease or anomolous conditions in which the presence of NCF is found to be a causative factor.

A pharmaceutical composition for use in treating inflammatory conditions comprises an anti-inflammatory effective amount of the anti-NCF monoclonal antibodies in a pharmaceutically acceptable carrier, such as physiological saline, sterile, non-toxic buffer, and the like.

Deposits of cDNA for NCF and of the hybridoma for anti-NCF monoclonal antibodies have been made at the ATCC, Rockville, Maryland, on January 12, 1988, and February 17, 1988, respectively, under the accession numbers 40412 and HB9647, respectively. The deposits shall be viably maintained and replaced if they become non-viable, for a period of 30 years from the date of the deposit, or for 5 years from the last date 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 U.S. Commissioner of Patents and Trademarks, upon request, shall have access to the deposits.

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 the scope of the appended claims.

Claims (2)

- 11 - 107,809/2 WHAT IS CLAIMED IS:

1. A kit for use In determining the amount of neutrophil chemotactlc polypeptide (NCF) mRNA present In a sample, comprising a container that contains DNA that encodes NCF with the amino acid sequence: NH2-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 wherein said DNA Is used to assess the amount of said mRNA In said sample, substantially as described In the specification.

2. A method of detecting mRNA that encodes neutrophil chemotactlc polypeptide (NCF) with the amino acid sequence: NH2-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 comprising hybridizing the mRNA in a sample with DNA that encodes said NCF; and detecting the presence of DNA-RNA hybrids in said sample, substantially as described in the specification. for the Applicant: WOLFF, BREGMAN AND GOLLER