Targeted Therapeutic Proteins

Abstract

Targeted therapeutics that localize to a specific subcellular compartment such as the lysosome are provided. The targeted therapeutics include a therapeutic agent and a targeting moiety that binds a receptor on an exterior surface of the cell, permitting proper subcellular localization of the targeted therapeutic upon internalization of the receptor. Nucleic acids, cells, and methods relating to the practice of the invention are also provided.

We claim: 
     
         1 . A targeted therapeutic comprising:
 a therapeutic agent that is therapeutically active in a mammalian lysosome; and   means for binding an extracellular domain of human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner.   
     
     
         2 . The targeted therapeutic of  claim 1 , wherein the means for binding comprises retinoic acid or a derivative thereof. 
     
     
         3 . The targeted therapeutic of  claim 1 , wherein the means for binding comprises a protein having an amino acid sequence at least 70% identical to a domain of urokinase-type plasminogen activator receptor. 
     
     
         4 . The targeted therapeutic of  claim 1 , wherein the means for binding comprises IGF-II or an antibody variable domain. 
     
     
         5 . The targeted therapeutic of  claim 1 , wherein association of the therapeutic agent with the means for binding is destabilized by a pH change from about pH 7.4 to about pH 5.5. 
     
     
         6 . The targeted therapeutic of  claim 1 , wherein the means for binding binds to the extracellular domain with a submicromolar dissociation constant at about pH 7.4. 
     
     
         7 . The targeted therapeutic of  claim 6 , wherein the dissociation constant is less than 10 −8  M. 
     
     
         8 . The targeted therapeutic of  claim 6 , wherein the dissociation constant is less than 10 −9  M. 
     
     
         9 . The targeted therapeutic of  claim 6 , wherein the dissociation constant is less than 10 −10  M. 
     
     
         10 . The targeted therapeutic of  claim 6 , wherein the dissociation constant is between 10 −7  M and 10 −11  M. 
     
     
         11 . The targeted therapeutic of  claim 6 , wherein the means for binding binds to the extracellular domain with an dissociation constant at about pH 5.5 at least ten times the dissociation constant at about pH 74. 
     
     
         12 . The targeted therapeutic of  claim 11 , wherein the dissociation constant at about pH 5.5 is at least 10 −6  M. 
     
     
         13 . A targeted therapeutic comprising:
 a therapeutic agent that is therapeutically active in a mammalian lysosome; and   an unglycosylated lysosomal targeting domain that binds an extracellular domain of human cation-independent mannose-6-phosphate receptor.   
     
     
         14 . A targeted therapeutic comprising:
 a therapeutic agent that is therapeutically active in a human lysosome; and   a lysosomal targeting domain that binds an extracellular domain of human cation-independent mannose-6-phosphate receptor and   (i) does not bind a mutein in which amino acid 1572 of the human cation-independent mannose-6-phosphate receptor is changed from isoleucine to threonine; or   (ii) binds the mutein with a dissociation constant at least ten times the dissociation constant for binding the extracellular domain of human cation-independent mannose-6-phosphate receptor.   
     
     
         15 . A targeted therapeutic comprising:
 a therapeutic agent that is therapeutically active in a human lysosome; and   a lysosomal targeting domain that is capable of binding a receptor domain consisting essentially of repeats 10-15 of the human cation-independent mannose-6-phosphate receptor.   
     
     
         16 . The targeted therapeutic of  claim 15 , wherein the lysosomal targeting domain is capable of binding a receptor domain consisting essentially of repeats 10-13 of the human cation-independent mannose-6-phosphate receptor. 
     
     
         17 . The targeted therapeutic of  claim 16 , wherein the lysosomal targeting domain binds a receptor domain consisting essentially of repeats 11-12 of the human cation-independent mannose-6-phosphate receptor. 
     
     
         18 . The targeted therapeutic of  claim 17 , wherein the lysosomal targeting domain binds a receptor domain consisting essentially of repeat 11 of the human cation-independent mannose-6-phosphate receptor. 
     
     
         19 . The targeted therapeutic of  claim 18 , wherein the lysosomal targeting domain binds a receptor domain consisting essentially of amino acids 1508-1566 of the human cation-independent mannose-6-phosphate receptor. 
     
     
         20 . The targeted therapeutic of  claim 15 , wherein the lysosomal targeting domain binds the receptor domain with a submicromolar dissociation constant at pH 7.4. 
     
     
         21 . The targeted therapeutic of  claim 20 , wherein the dissociation constant is about 10 −7  M. 
     
     
         22 . The targeted therapeutic of  claim 20 , wherein the dissociation constant is less than about 10 −7  M. 
     
     
         23 . A targeted therapeutic comprising:
 a therapeutic agent that is therapeutically active in a human lysosome; and   a binding moiety sufficiently duplicative of human IGF-II such that the binding moiety binds the human cation-independent mannose-6-phosphate receptor.   
     
     
         24 . The targeted therapeutic of  claim 23 , wherein the binding moiety is an organic molecule having a three-dimensional shape representative of at least a portion of IGF-II. 
     
     
         25 . The targeted therapeutic of  claim 24 , wherein the portion of IGF-II comprises amino acids 48-55 of human IGF-II. 
     
     
         26 . The targeted therapeutic of  claim 24 , wherein the portion of IGF-II comprises at least three amino acids selected from the group consisting of amino acids 8, 48, 49, 50, 54, and 55 of human IGF-II. 
     
     
         27 . The targeted therapeutic of  claim 24 , wherein the organic molecule has a hydrophobic moiety at a position representative of amino acid 48 of human IGF-II and has a positive charge at about pH 7.4 at a position representative of amino acid 49 of human IGF-II. 
     
     
         28 . The targeted therapeutic of  claim 23 , wherein the binding moiety comprises a polypeptide comprising the amino acid sequence of IGF-I or of a mutein of IGF-I in which
 (i) amino acids 55 and 56 are changed,   (ii) amino acids 1-4 are deleted or changed, or   (iii) amino acids 55 and 56 are changed and amino acids 1-4 are deleted or changed.   
     
     
         29 . The targeted therapeutic of  claim 23 , wherein the binding moiety comprises a polypeptide comprising an amino acid sequence at least 60% identical to human IGF-II. 
     
     
         30 . The targeted therapeutic of  claim 29 , wherein the amino acid sequence comprises, at positions corresponding to positions 54 and 55 of human IGF-11, amino acids each of which are uncharged or negatively charged at pH 7.4. 
     
     
         31 . The targeted therapeutic of  claim 23  wherein the binding moiety comprises a polypeptide having antiparallel alpha-helices separated by not more than five amino acids. 
     
     
         32 . A targeted therapeutic comprising:
 a therapeutic agent that is therapeutically active in a human lysosome; and   a polypeptide comprising the amino acid sequence phenylalanine-arginine-serine.   
     
     
         33 . A targeted therapeutic comprising:
 a therapeutic agent that is therapeutically active in a human lysosome; and   a polypeptide comprising an amino acid sequence at least 75% homologous to amino acids 48-55 of human IGF-II.   
     
     
         34 . A targeted therapeutic comprising:
 a therapeutic agent that is therapeutically active in a human lysosome;   amino acids 8-28 of human IGF-II; and   amino acids 41-61 of human IGF-II.   
     
     
         35 . The targeted therapeutic of  claim 34 , wherein amino acids 8-28 and 41-61 are present in a single polypeptide. 
     
     
         36 . A targeted therapeutic comprising:
 a therapeutic agent that is therapeutically active in a human lysosome;   amino acids 41-61 of human IGF-II; and   a mutein of amino acids 8-28 of human IGF-II, the mutein differing from human IGF-II at a position selected from the group consisting of amino acid 9, amino acid 19, amino acid 26, and amino acid 27.   
     
     
         37 . A therapeutic fusion protein comprising:
 a therapeutic domain and   a subcellular targeting domain that binds to an extracellular domain of a receptor on an exterior surface of a cell and, upon internalization of the receptor, permits localization of the therapeutic domain to a subcellular compartment where the therapeutic domain is therapeutically active.   
     
     
         38 . The therapeutic fusion protein of  claim 37 , wherein the subcellular compartment is selected from the group consisting of a lysosome, an endosome, endoplasmic reticulum, and golgi complex. 
     
     
         39 . The therapeutic fusion protein of  claim 38 , wherein the subcellular compartment is a lysosome. 
     
     
         40 . The therapeutic fusion protein of  claim 37 , wherein the receptor undergoes continuous endocytosis. 
     
     
         41 . The therapeutic fusion protein of  claim 37 , wherein the therapeutic domain has a therapeutic enzymatic activity. 
     
     
         42 . The therapeutic fusion protein of  claim 41 , wherein a cellular or subcellular deficiency in the enzymatic activity is associated with a human disease. 
     
     
         43 . The therapeutic fusion protein of  claim 42 , wherein the human disease is a lysosomal storage disease. 
     
     
         44 . A nucleic acid encoding the therapeutic fusion protein of  claim 37 . 
     
     
         45 . A cell comprising the nucleic acid of  claim 44 . 
     
     
         46 . A method of producing a therapeutic fusion protein, the method comprising the step of providing to the cell of  claim 45  conditions permitting expression of the therapeutic fusion protein. 
     
     
         47 . The method of  claim 46 , comprising culturing the cell in vitro. 
     
     
         48 . The method of  claim 46 , comprising maintaining the cell inside a mammalian body. 
     
     
         49 . The method of  claim 46 , further comprising harvesting the therapeutic fusion protein. 
     
     
         50 . A method of treating a patient, the method comprising administering to the patient the therapeutic fusion protein of  claim 37 . 
     
     
         51 . A method of treating a patient, the method comprising administering to the patient the nucleic acid of  claim 44 . 
     
     
         52 . A method of treating a patient, the method comprising administering to the patient the cell of  claim 45 . 
     
     
         53 . A method of treating a patient, the method comprising:
 (i) synthesizing a targeted therapeutic comprising a therapeutic agent that is therapeutically active in a mammalian lysosome and a targeting moiety that binds human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner; and   (ii) administering the targeted therapeutic to the patient.   
     
     
         54 . The method of  claim 53 , further comprising, prior to step (i),
 identifying a targeting moiety that binds human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner.   
     
     
         55 . The method of  claim 54 , wherein the targeting moiety is identified by screening a nucleic acid or peptide library. 
     
     
         56 . A method of producing a targeted therapeutic, the method comprising the steps of:
 (a) providing a molecular model defining a three-dimensional shape representative of at least a portion of human IGF-II;   (b) identifying a candidate IGF-II analog having a three-dimensional shape corresponding to the three-dimensional shape representative of at least a portion of human IGF-II; and   (c) producing a therapeutic agent directly or indirectly bound to the candidate IGF-II analog, wherein the therapeutic agent is therapeutically active in a mammalian lysosome.   
     
     
         57 . The method of  claim 56 , further comprising determining whether the compound produced in step c binds to the human cation-independent mannose-6-phosphate receptor.