Epithelial cell specific growth factor keratinocyte growth factor (KGF)

Abstract

Discoveries are disclosed that show particular aspects of recombinant DNA technology can be used successfully to produce hitherto unknown human keratinocyte growth factor (KGF) protein free of other polypeptides. These proteins can be produced in various functional forms from spontaneously secreting cells or from DNA segments introduced into cells. These forms variously enable biochemical and functional studies of this novel protein as well as production of antibodies. Means are described for determining the level of expression of genes for the KGF protein, for example, by measuring mRNA levels in cells or by measuring antigen secreted in extracellular or body fluids.

1 . A human keratinocyte growth factor (KGF) having an apparent molecular weight of about 28 kDa as determined by migration in NaDodSO 4 /PAGE, and a specific activity of at least about 3.4×10 4  units per milligram of protein, where one unit of activity is defined as that amount which causes half of the maximal possible stimulation of DNA synthesis in BALB/MK keratinocyte cells under standard assay conditions.  
     
     
         2 . Human KGF according to  claim 1 , wherein said specific activity is at least about 3.2×10 5  units per milligram protein.  
     
     
         3 . A bioassay for KGF-like activity in a test sample which comprises the following steps: 
 i) growing keratinocytes in culture to confluence and maintaining said confluent culture in serum-free medium;    ii) adding a test sample to said confluent culture of keratinocytes; and    iii) determining the stimulation of DNA synthesis in said keratinocytes.    
     
     
         4 . A method of producing KGF from cultured cells comprising the following steps: 
 i) Culturing KGF-producing cells in culture medium under conditions such that KGF is produced;    ii) concentrating said culture medium so that a first concentrate is formed;    iii) contacting said concentrate with heparin under conditions such that KGF present in said first concentrate binds to the heparin whereby a heparin-KGF complex is formed;    iv) separating said heparin-KGF complex from said concentrate;    v) treating said heparin-KGF complex under conditions such that said KGF dissociates from the heparin so that a solution of free KGF is formed;    vi) concentrating said solution so that a second concentrate is formed;    vii) fractionating said second concentrate so that KGF is separated from the remaining components.    
     
     
         5 . A method of producing KGF from cultured cells, according to  claim 4 , wherein said KGF-producing cells are M426 human embryonic fibroblasts.  
     
     
         6 . A DNA segment encoding a human keratinocyte growth factor (KGF) protein.  
     
     
         7 . A DNA segment, according to  claim 6 , wherein said protein has the amino acid sequence defined in FIG. II- 1 .  
     
     
         8 . A DNA segment encoding a chimeric KGF-like protein which comprises within a single polypeptide molecule functional segments of human KGF and at least one other polypeptide of the fibroblast growth factor family.  
     
     
         9 . A recombinant DNA molecule comprising a DNA segment according to  claim 6  or  claim 8  and a vector.  
     
     
         10 . A culture of cells transformed with said recombinant DNA molecule according to  claim 9 .  
     
     
         11 . A method of producing a human KGF protein comprising culturing said cells according to  claim 10  in a culture medium under conditions such that said protein is produced and isolating said protein from said cells.  
     
     
         12 . A method of producing a human KGF protein comprising culturing said cells according to  claim 10  in a culture medium, wherein said protein is secreted from said cell, and isolating said protein from said medium.  
     
     
         13 . A human KGF or KGF-like protein having the amino acid sequence in FIG. II- 1 B.  
     
     
         14 . A human KGF or KGF-like protein, according to  claim 13 , which is not glycosylated.  
     
     
         15 . An antibody specific for a peptide having the amino acid sequence of human KGF or KGF-like protein, according to  claim 13 .  
     
     
         16 . The antibody according to  claim 15  which neutralizes the mitogenic activity of human KGF.  
     
     
         17 . A bioassay for expression of a gene encoding KGF, comprising the steps of: 
 i)isolating mRNA from tissues or cells; and    ii) annealing said RNA to a DNA probe encoding a human KGF;    iii) determining the amount of DNA:RNA hybrid containing said DNA probe.    
     
     
         18 . A bioassay for KGF antigen comprising the steps of: 
 i) extracting polypeptides from body fluids or tissue samples; and    ii) determining the level of human KGF antigen by reaction with an antibody specific for a peptide having the amino acid sequence of human KGF or KGF like protein, according to  claim 13 .    
     
     
         19 . A pharmaceutical composition for treatment of conditions requiring specific stimulation of epithelial cells, comprising KGF according to  claim 1  or  claim 13 , and an acceptable pharmaceutical carrier.  
     
     
         20 . A pharmaceutical composition for treatment of conditions requiring specific inhibition of stimulation of epithelial cells by KGF, comprising antibodies to KGF according to  claim 15 , and an acceptable pharmaceutical carrier.