Human somatomed in carrier protein subunits and process for producing them; recombinant DNA molecules, hosts, processes and human somatomedin carrier protein-like polypeptides

Abstract

This invention relates to polypeptides that are human somatomedin carrier protein subunits and to processes for producing them. The carrier protein subunits bind to human somatomedin-like polypeptides, also known as insulin-like growth factors. The process involves preparation from a human serum fraction, Cohn IV-1, by a molecule of various chromatographic steps. This invention also relates to DNA molecules encoding human somatomedin carrier protein-like polypeptides, recombinant DNA molecules, hosts, processes for producing carrier protein-like polypeptides, human somatomedin carrier protein-like polypeptides produced using those molecules, hosts and processes. The invention relates to DNA molecules and their expression in appropriate hosts. The recombinant DNA molecules contain DNA molecules that code for polypeptides which have a biological activity of the human carrier protein or a human carrier protein subunit capable of binding somatomedins. The DNA molecules, recombinant DNA molecules, hosts, and processes of this invention may be used in the production of polypeptides useful in a variety of therapeutic, diagnostic, and other useful applications.

What is claimed is:  
     
         1 . A carrier protein subunit capable of binding somatomedin-like polypeptides.  
     
     
         2 . A protein subunit of    claim 1    having a molecular weight of about 30,000 daltons or less.  
     
     
         3 . A protein subunit of    claim 1    having a molecular weight of about 15,000 to and including about 30,000 daltons.  
     
     
         4 . A carrier protein subunit capable of binding somatomedins characterized by an N-terminal amino acid sequence of the formula: 
 Gly-Ala-Ser-Ser-Ala-Gly-Leu-Gly-Pro-Val-(1) (5) (10)    Val-Arg-R-Glu-Pro-R-Asp-Ala-Arg-Ala-(15) (20)    Leu-Ala-,    wherein R is cysteine or half-cystine, the 5-Ala may be Gly, and the 14-Glu may be Phe.    
     
     
         5 . A protein subunit of    claim 4    having a molecular weight of about 30,000 daltons or less.  
     
     
         6 . A protein subunit of    claim 4    having a molecular weight of about 15,000 to and including about 30,000 daltons.  
     
     
         7 . An essentially pure carrier protein subunit capable of binding somatomedin-like polypeptides.  
     
     
         8 . An essentially pure carrier protein subunit of    claim 7    being essentially free of other proteins, peptides, nucleotides, polysaccharides, lipids and salts.  
     
     
         9 . An essentially pure carrier protein subunit of    claim 7    having a molecular weight of about 30,000 daltons or less.  
     
     
         10 . An essentially pure carrier protein subunit of    claim 7    having a molecular weight of about 15,000 to and including 30,000 daltons.  
     
     
         11 . An essentially pure carrier protein subunit of    claim 7    characterized by an N-terminal amino acid sequence of the formula: 
 Gly-Ala-Ser-Ser-Ala-Gly-Leu-Gly-Pro-Val-(1) (5) (10)  
 Val-Arg-R-Glu-Pro-R-Asp-Ala-Arg-Ala-15) (20)  
 Leu-Ala-,  
 wherein R is cysteine or half-cystine, the 5-Ala may be Gly and the 14-Glu may be Phe.  
 
     
     
         12 . A therapeutic composition for inhibiting the growth of somatomedin-dependent cancers, for inhibiting the effect of somatomedin-C in acromegaly, for inhibiting the growth of retinal blood vessels and fibrous tissues in diabetic retinopathy, for inhibiting growth of tall children, for inhibiting the growth of keloid scars, or for inhibiting the growth of tissue in the orbit of the eyes in malignant exophthalmos, comprising an effective amount of at least one carrier protein subunit according to any one of claims  1  through  11 , or a pharmacologically-acceptable salt thereof, and a pharmacologically acceptable carrier.  
     
     
         13 . A method for inhibiting the growth of somatomedin-dependent cancers, for inhibiting the effect of somatomedin-C in acromegaly, for inhibiting the growth of retinal blood vessels and fibrous tissues in diabetic retinopathy, for inhibiting growth of tall children, for inhibiting the growth of keloid scars, or for inhibiting the growth of tissue in the orbit of the eyes in malignant exophthalmos, comprising administering an effective amount of a composition according to    claim 12   .  
     
     
         14 . A composition comprising at least one carrier protein subunit according to any one of claims  1  through 11 substantially completed with at least one human somatomedin-like polypeptide.  
     
     
         15 . A method for treating osteoporosis in humans, for stimulating the growth of bone, for stimulating animal growth, for stimulating the healing of human and other animal wounds, and for stimulating the growth of patients with growth hormone deficiency, comprising administering an effective amount of a composition of    claim 14   .  
     
     
         16 . Monoclonal and polyclonal antibodies against a carrier protein subunit of    claim 1   .  
     
     
         17 . A method for measuring the level of free somatomedins in human fluids comprising separating somatomedins complexed with the carrier protein subunits of    claim 1    from unbound somatomedins.  
     
     
         18 . A process for producing a carrier protein subunit from human plasma Cohn fraction IV-1 comprising: 
 (a) chromatographing the portions of Cohn fraction IV-1 that are soluble in an aqueous solution of pH of about 4.5 to 7.5 on a sulfopropyl derivative of a cross-linked dextran adsorbent by sequentially eluting with aqueous solutions of increasing pH;    (b) chromatographing an acidic solution of pH less than about 4.0 of the fractions from step (a) that contain somatomedin binding activity on the same adsorbent as step (a) and collecting the pass-through fraction or chromatographing the fractions from step (a) on a phenyl derivative of agarose by adsorption from a neutral solution of about 10% ammonium sulfate and eluting with about 0.5 M sodium thiocyanate solution at about neutral pH;    (c) chromatographing the fraction from step (b) containing somatomedin binding activity by gel filtration and eluting with an acidic aqueous solution;    (d) chromatographing the fraction from step (c) containing somatomedin binding activity on a solid support cross-linked to substantially pure somatomedin-C by adsorbing at about neutral pH and eluting with an acidic aqueous solution; and    (e) chromatographing the fraction from step (d) containing somatomedin binding activity by reverse phase high performance liquid chromatography.    
     
     
         19 . A therapeutic composition for stimulating the healing of human or other animal wounds, comprising an effective amount of at least one carrier protein subunit according to any one of claims  1  through  11 , or a pharmacologically-acceptable salt thereof, and a pharmacologically effective carrier.  
     
     
         20 . A method for stimulating the healing of human or other animal wounds, comprising administering an effective amount of a composition of    claim 19   .  
     
     
         21 . A DNA molecule comprising a gene which codes for a carrier protein-like polypeptide.  
     
     
         22 . A DNA molecule of    claim 21   , wherein the gene is free of introns.  
     
     
         23 . A DNA molecule of    claim 21   , wherein the DNA molecule is essentially free of genes which code for any other polypeptide coded for by the human genome.  
     
     
         24 . A DNA molecule of    claim 21   , wherein the gene codes for a polypeptide having a molecular weight of about 40,000-50,000 daltons or less, if measured in a form accompanied by glycosylation.  
     
     
         25 . A DNA molecule of    claim 21    wherein the gene codes for a polypeptide displaying a somatomedin-C regulating activity of the carrier protein.  
     
     
         26 . A DNA molecule comprising a gene which codes for a carrier protein-like polypeptide that is a, carrier protein subunit capable of binding somatomedin-like polypeptides.  
     
     
         27 . A process for obtaining a DNA molecule, comprising preparing cDNA molecules from mRNA found in cells or tissues that produce the carrier protein, determining which of the cDNA molecules hybridize to one or more labelled polynucleotide probes based on the DNA sequence of FIG. 4, analyzing the cDNA molecules that hybridized, and obtaining a DNA molecule having a gene which codes for a carrier protein-like polypeptide.  
     
     
         28 . The process of    claim 27   , wherein a DNA molecule having the gene is obtained by ligating one or more cDNA molecules that hybridized with other cDNA molecules, synthetic DNA molecules, or recombinant DNA molecules.  
     
     
         29 . The process of    claim 27   , wherein the labelled polynucleotide probe has the DNA sequence shown in FIG. 2 a.    
     
     
         30 . A DNA molecule obtainable by the process of    claim 27   , and a DNA molecule which encodes a carrier protein-like polypeptide coded for by a DNA molecule obtainable by the process of    claim 27   .  
     
     
         31 . A DNA molecule selected from the group consisting of the DNA molecules LCP 0.70, LCP 0.77, LCP 2.3, LCP 2.5, FCP 1.8, and FCP 2.5, DNA molecules which hybridize to any of the DNA molecules LCP 0.70, LCP 0.77, LCP 2.3, LCP 2.5, FCP 1.8 and FCP 2.5 and which code for a carrier protein-like polypeptide, and DNA molecules which code for a polypeptide coded for by any of the foregoing DNA molecules.  
     
     
         32 . A DNA molecule comprising a gene which codes for a polypeptide having the sequence of amino acids −1 to 290 of FIG. 4.  
     
     
         33 . A DNA molecule comprising a gene which codes for a polypeptide having the sequence of amino acids 1 to 290 of FIG. 4.  
     
     
         34 . A DNA molecule comprising a gene which codes for a polypeptide having the sequence of amino acids 27 to 290 of FIG. 4.  
     
     
         35 . A DNA molecule comprising a gene which codes for a polypeptide having the sequence of amino acids 27 to 290 of FIG. 4 and having a methionine residue preceding amino acid 27.  
     
     
         36 . A DNA molecule comprising a gene which codes for a polypeptide having the sequence of amino acids 27 to 290 and having a sequence of amino acid residues preceding amino acid 27 that constitute a secretion, signal or other precursor sequence recognized by a host.  
     
     
         37 . A recombinant DNA molecule comprising a DNA molecule according to any one of    claims 21    to    26    and    30    to  36  operatively linked to an expression control sequence.  
     
     
         38 . A host transformed with at least one recombinant DNA molecule according to    claim 37   .  
     
     
         39 . The transformed host according to    claim 38   , selected from the group consisting of strains of  E. coli , Pseudomonas,  Bacillus subtilis, Bacillus stearothermophilus , other bacteria, yeasts, fungi, animal, insect or plant hosts, and human tissue cells.  
     
     
         40 . A method for producing a carrier protein-like polypeptide, comprising transforming an appropriate host with a recombinant DNA molecule according to    claim 37   , and culturing said host to make said polypeptide.  
     
     
         41 . The method of    claim 40   , comprising the additional step of collecting the polypeptide.  
     
     
         42 . A method for producing a carrier protein-like polypeptide, comprising culturing a host according to    claim 38    to make said polypeptide.  
     
     
         43 . A vector comprising a recombinant DNA molecule according to    claim 37   .  
     
     
         44 . A host transformed with at least one vector of    claim 43   .  
     
     
         45 . A transformed host of    claim 44    selected from the group consisting of strains of  E. coli , Pseudomonas.  Bacillus subtilis, Bacillus stearothermophilus , other bacteria, yeast, other fungi, mouse or other animal, insect or plant hosts and human tissue cells.  
     
     
         46 . A method for producing a carrier protein-like polypeptide, comprising transforming an appropriate host with a vector according to    claim 43   , and culturing said host to make said polypeptide.  
     
     
         47 . The method of    claim 46   , comprising the additional step of collecting the polypeptide.  
     
     
         48 . A method for producing a carrier protein-like polypeptide, comprising culturing a host transformed by a vector according to claims  43  to make said polypeptide.  
     
     
         49 . An essentially pure carrier protein-like polypeptide other than a carrier protein subunit capable of binding somatomedin-like polypeptides.  
     
     
         50 . An essentially pure polypeptide of    claim 49    being essentially free of substances naturally present in human serum.  
     
     
         51 . An essentially pure polypeptide of    claim 49    being a mature carrier protein-like polypeptide.  
     
     
         52 . A carrier protein-like polypeptide lacking the natural glycosylation of the carrier protein.  
     
     
         53 . A polypeptide of    claim 52    being essentially free of substances naturally present in human serum.  
     
     
         54 . An essentially pure polypeptide having the sequence of amino acids −1 to 290 of FIG. 4.  
     
     
         55 . An essentially pure polypeptide having the sequence of amino acids 1 to 290 of FIG. 4.  
     
     
         56 . An essentially pure polypeptide having the sequence of amino acids 27 to 290 of FIG. 4 and such a polypeptide having a methionine residue preceding amino acid 27.  
     
     
         57 . The polypeptide of claims  54 ,  55  or  56 , wherein one or more amino acid residues have been added, deleted or substituted and the polypeptide constitutes a carrier protein-like polypeptide.  
     
     
         58 . A polypeptide selected from the group consisting of a polypeptide having the sequence of amino acids −1 to 290 of FIG. 4, and polypeptides having a portion of that sequence and constituting a carrier protein-like polypeptide.  
     
     
         59 . A therapeutic composition comprising an effective amount for inhibiting the effect of somatomedin-C in acromegaly, for inhibiting the growth of retinal blood vessels and fibrous tissues in diabetic retinopathy, for inhibiting growth of tall children, for inhibiting the growth of keloid scars, for inhibiting the growth of tissue in the orbit of the eyes in malignant exophthalmos or for stimulating the healing of human or animal wounds, of at least one polypeptide selected from the group consisting of a polypeptide according to any one of claims  49  to 58, or a pharmacologically-acceptable salt thereof, and a pharmacologically acceptable carrier.  
     
     
         60 . A method for inhibiting the growth of somatomedin-dependent cancers, for inhibiting the effect of somatomedin-C in acromegaly, for inhibiting the growth of retinal blood vessels and fibrous tissues in diabetic retinopathy, for inhibiting growth of tall children, for inhibiting the growth of keloid scars, for inhibiting the growth of tissue in the orbit of the eyes in malignant exophthalmos or for stimulating the healing of human or animal wounds, comprising administering an effective amount of a composition according to    claim 59   .  
     
     
         61 . A composition comprising at least one polypeptide selected from the group consisting of a polypeptide according to any one of    claims 49    to    58    substantially complexed with at least one somatomedin-like polypeptide.  
     
     
         62 . A therapeutic composition comprising an effective amount for treating osteoporosis in humans, for stimulating the growth of bone, for stimulating animal growth, for stimulating the healing of human and animal wounds, for stimulating the growth of patients with growth hormone deficiency, of a composition of    claim 61   .  
     
     
         63 . A method for treating osteoporosis in humans, for stimulating the growth of bone, for stimulating animal growth, for stimulating the healing of human and animal wounds, for stimulating the growth of patients with growth hormone deficiency, comprising administering an effective amount of a composition according to    claim 62   .  
     
     
         64 . An oligonucleotide probe having all or a portion of the DNA sequence of any one of the DNA molecules LCP 2.3, LCPO 0.70, LCP 0.77, LCP 2.5, FCP 1.8 and FCP 2.5, which selectively hybridizes to a DNA molecule encoding a carrier protein-like polypeptide.  
     
     
         65 . A recombinant DNA molecule according to    claim 37    further comprising a DNA molecule having a gene which codes for a somatomedin-like polypeptide operatively linked to an expression control sequence.  
     
     
         66 . A host transformed with at least one recombinant DNA molecule of    claim 65   .  
     
     
         67 . A vector comprising a recombinant DNA molecule according to    claim 37    and a DNA molecule which codes for a somatomedin-like polypeptide operatively linked to an expression control sequence.  
     
     
         68 . A host transformed with a vector in    claim 67   .  
     
     
         69 . A method for producing a composition according to    claim 61    comprising transforming an appropriate host with a vector according to    claim 67   , and culturing said host to make said polypeptides.  
     
     
         70 . A method of    claim 69   , comprising the additional step of collecting the polypeptide.  
     
     
         71 . A method for producing a composition according to    claim 61   , comprising culturing a host transformed with a vector according to    claim 67   .  
     
     
         72 . A method for producing a composition of    claim 61   , comprising transforming an appropriate host with at least one recombinant DNA molecule of    claim 37   , co-transforming such host with at least one recombinant DNA molecule comprising a gene which codes for a somatomedin-like polypeptide operatively linked to an expression control sequence, and culturing such host to produce such polypeptides.  
     
     
         73 . A host transformed with a recombinant DNA molecule of    claim 37    and with a recombinant DNA molecule comprising a gene which codes for a somatomedin-like polypeptide operatively linked to an expression control sequence.  
     
     
         74 . Monoclonal and polyclonal antibodies against a polypeptide of    claim 49   .  
     
     
         75 . A method for measuring the level of free somatomedins in human fluids comprising separating somatomedins completed with a polypeptide of    claim 49    from unbound somatomedins.