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.

Description:
This application is a division of Ser. No. 08/818,631 filed Mar. 14, 1997, which is a division of Ser. No. 07/169,033 filed Mar. 16, 1988 now U.S. Pat. No. 5,652,338. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Technical Field 
     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. 
     2. State of the Art 
     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 
     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). 
     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. 
     It is a still further object of the present invention to provide monoclonal antibodies having specific binding affinity for NCF of the present invention. 
     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. 
     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. 
     Other objects and advantages will become evident from the following detailed description of the invention. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     These and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein: 
     FIG. 1 demonstrates translation of cDNA into NCF protein in reticulocyte lysate system; and 
     FIG. 2 shows: 
     (a) Northern blot analysis of mRNA induction in lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cells (PBMC); 
     (b) The time course of the accumulation of neutrophil chemotactic activity in culture media of PBMC after stimulation with LPS; 
     (c) Induction of NCF mRNA in PBMC by IL 1 or TNF, but not by IL 2, gamma-IFN, and alpha-IFN; 
     (d) HPLC gel filtration analysis of IL 1 and TNF induced neutrophil chemotactic activity. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     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): 
     NH 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-K-F-L-K-R-A-E-N-S (SEQ ID NO:2) 
     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. 
     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 analyses 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. 
     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 in Table 1. 
     
       
         
               
             
               
               
             
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Chemotactic response of human neutrophils to chemically 
               
               
                 synthesized NCF. 
               
             
          
           
               
                   
                 Percentage of assay neutrophils 
               
               
                 Concentration of NCF, nanomolar 
                 that migrated 
               
               
                   
               
             
          
           
               
                 1000 
                 23 
               
               
                 100 
                 34 
               
               
                 10 
                 32 
               
               
                 1 
                 5 
               
               
                 0.1 
                 1 
               
               
                 Hanks medium 
                 0.3 
               
               
                 10 −7 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 chemoattractant.  
               
             
          
         
       
     
     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 clinicopathological 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. 
     A pharmaceutical composition for use in treating inflammatory condition comprises an 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. 
     A deposit of cDNA for NCF and of the hybridoma for anti-NCF monoclonal antibodies have been made at the ATCC, 10801 University Boulevard, Manassas, Va. 20110-209 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 date of request for a sample of the deposit, which is longer, and made available to the public without restriction in accordance with the provisions of the law. The Commissioner for 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 scope of the appended claims. 
     
       
         
           
             2 
           
           
             1 
             1560 
             DNA 
             homo sapiens 
           
            1
ctccataagg cacaaacttt cagagacagc agagcacaca agcttctagg acaagagcca     60
ggaagaaacc accggaagga accatctcac tgtgtgtaaa catgacttcc aagctggccg    120
tggctctctt ggcagccttc ctgatttctg cagctctgtg tgaaggtgca gttttgccaa    180
ggagtgctaa agaacttaga tgtcagtgca taaagacata ctccaaacct ttccacccca    240
aatttatcaa agaactgaga gtgattgaga gtggaccaca ctgcgccaac acagaaatta    300
ttgtaaagct ttctgatgga agagagctct gtctggaccc caaggaaaac tgggtgcaga    360
gggttgtgga gaagtttttg aagagggctg agaattcata aaaaaattca ttctctgtgg    420
tatccaagaa tcagtgaaga tgccagtgaa acttcaagca aatctacttc aacacttcat    480
gtattgtgtg ggtctgttgt agggttgcca gatgcaatac aagattcctg gttaaatttg    540
aatttcagta aacaatgaat agtttttcat tgtaccatga aatatccaga acatacttat    600
atgtaaagta ttatttattt gaatctacaa aaaacaacaa ataattttta aatataagga    660
ttttcctaga tattgcacgg gagaatatac aaatagcaaa attgggccaa gggccaagag    720
aatatccgaa ctttaatttc aggaattgaa tgggtttgct agaatgtgat atttgaagca    780
tcacataaaa atgatgggac aataaatttt gccataaagt caaatttagc tggaaatcct    840
ggattttttt ctgttaaatc tggcaaccct agtctgctag ccaggatcca caagtccttg    900
ttccactgtg ccttggtttc tcctttattt ctaagtggaa aaagtattag ccaccatctt    960
acctcacagt gatgttgtga ggacatgtgg aagcacttta agttttttca tcataacata   1020
aattattttc aagtgtaact tattaaccta tttattattt atgtatttat ttaagcatca   1080
aatatttgtg caagaatttg gaaaaataga agatgaatca ttgattgaat agttataaag   1140
atgttatagt aaatttattt tattttagat attaaatgat gttttattag ataaatttca   1200
atcagggttt ttagattaaa caaacaaaca attgggtacc cagttaaatt ttcatttcag   1260
atatacaaca aataattttt tagtataagt acattattgt ttatctgaaa ttttaattga   1320
actaacaatc ctagtttgat actcccagtc ttgtcattgc cagctgtgtt ggtagtgctg   1380
tgttgaatta cggaataatg agttagaact attaaaacag ccaaaactcc acagtcaata   1440
ttagtaattt cttgctggtt gaaacttgtt tattatgtac aaatagattc ttataatatt   1500
atttaaatga ctgcattttt aaatacaagg ctttatattt ttaactttaa aaaaaaccgg   1560
 
           
             2 
             72 
             PRT 
             homo sapiens 
           
            2
Ser Ala Lys Glu Leu Arg Cys Gln Cys Ile Lys Thr Tyr Ser Lys Pro
1               5                   10                  15
Phe His Pro Lys Phe Ile Lys Glu Leu Arg Val Ile Glu Ser Gly Pro
            20                  25                  30
His Cys Ala Asn Thr Glu Ile Ile Val Lys Leu Ser Asp Gly Arg Glu
        35                  40                  45
Leu Cys Leu Asp Pro Lys Glu Asn Trp Val Gln Arg Val Val Glu Lys
    50                  55                  60
Phe Leu Lys Arg Ala Glu Asn Ser
65                  70