Conditionally active polypeptides

Abstract

A non-naturally occurring polypeptide or isolated polypeptide having a ratio of at least 1.3 of an activity in an assay at a first pH in the presence of at least one species having a molecular weight of less than 900 a.m.u. and a pKa up to 4 pH units away from said first pH, to an activity in an assay at a second pH in the presence of the same at least one species. The species has a pKa between said first pH and said second pH and can be a small molecule. Also disclosed are pharmaceutical formulations including the polypeptide and uses thereof. Methods of producing conditionally active polypeptides are also disclosed.

What is claimed is: 
     
         1 . A non-naturally occurring polypeptide or isolated polypeptide having a ratio of at least 1.3 of an activity in an assay at a first pH in the presence of at least one species having a molecular weight of less than 900 a.m.u. and a pKa up to 0.5, 1, 2, 3 or 4 pH units away from said first pH, to an activity in an assay at a second pH in the presence of the same at least one species. 
     
     
         2 . A non-naturally occurring polypeptide or isolated polypeptide having a ratio of at least 1.3 of an activity in an assay at a first pH in the presence of at least one species having a molecular weight of less than 900 a.m.u., to an activity in an assay at a second pH in the presence of the same at least one species, and wherein said species has a pKa beween said first pH and said second pH. 
     
     
         3 . A non-naturally occurring polypeptide or isolated polypeptide having a ratio of at least 1.3 of an activity in an assay at a first pH in the presence of a species selected from histidine, histamine, hydrogenated adenosine diphosphate, hydrogenated adenosine triphosphate, citrate, bicarbonate, acetate, lactate, bisulfide, hydrogen sulfide, ammonium, dihydrogen phosphate and any combination thereof, to an activity in an assay at a second pH in the presence of the same species. 
     
     
         4 . The polypeptide of any one of  claims 1 - 3 , wherein the ratio of the activity in the assay at the first pH to the activty in the assay at the second pH is at least 1.5, or at least 1.7, or at least 2.0, or at least 3.0, or at least 4.0, or at least 6.0, or at least 8.0, or at least 10.0, or at least 20.0, or at least 40.0, or at least 60.0, or at least 100.0. 
     
     
         5 . The polypeptide of any one of  claims 1 - 4 , wherein the first pH is an acidic pH and the second pH is an alkaline pH or neutral pH. 
     
     
         6 . The polypeptide of any one of  claims 1 - 5 , wherein the second pH is a normal physiological pH that is within a normal range of the physiological condition at a site of administration of the polypeptide to a subject, or at a tissue or organ at a site of action of the polypeptide of a subject, and the first pH is an aberrant pH that deviates from the normal range of the physiological condition at the site of administration of the polypeptide, or at the tissue or organ at the site of action of the polypeptide. 
     
     
         7 . The polypeptide of  claim 6 , wherein the first pH is in a range of 5.5-7.2, or a range of 6.2-6.8. 
     
     
         8 . The polypeptide of any one of  claims 6 - 7 , wherein the second pH is in a range of 7.2-7.6. 
     
     
         9 . The polypeptide of  claim 6 , wherein the first pH is about 6.0 and the second pH is about 7.4. 
     
     
         10 . The polypeptide of any one of  claims 1 - 9 , wherein the polypeptide is a non-naturally occurring mutant polypeptide evolved from a parent polypeptide. 
     
     
         11 . The mutant polypeptide of  claim 10 , wherein the parent polypeptide is a wild-type polypeptide. 
     
     
         12 . The mutant polypeptide of  claim 10 , wherein the parent polypeptide is a non-naturally occurring polypeptide. 
     
     
         13 . The mutant polypeptide of any one of  claims 10 - 12 , wherein the mutant polypeptide contains at least one amino acid substitution in comparison with the parent polypeptide. 
     
     
         14 . The mutant polypeptide of any one of  claims 10 - 13 , wherein the mutant polypeptide has a higher proportion of charged amino acid residues than the parent polypeptide. 
     
     
         15 . The polypeptide or mutant polypeptide of any one of  claims 1 - 14 , wherein the polypeptide or mutant polypeptide is a protein or protein fragment. 
     
     
         16 . The polypeptide or mutant polypeptide of any one of  claims 1 - 14 , wherein the polypeptide or mutant polypeptide is selected from an antibody, a single chain antibody, and an antibody fragment and the activity is a binding activity to an antigen. 
     
     
         17 . The polypeptide or mutant polypeptide of  claim 16 , the polypeptide or mutant polypeptide is an Fc region of an antibody. 
     
     
         18 . The polypeptide or mutant polypeptide of any one of  claims 1 - 14 , wherein the polypeptide or mutant polypeptide is an enzyme and the activity is an enzymatic activity. 
     
     
         19 . The polypeptide or mutant polypeptide of any one of  claims 1 - 14 , wherein the polypeptide or mutant polypeptide is selected from a receptor, a regulatory protein, a soluble protein, a cytokine and a fragment of a receptor, a regulatory protein, a soluble protein or a cytokine. 
     
     
         20 . The polypeptide or mutant polypeptide of any one of  claims 1 - 18 , wherein the species is bicarbonate. 
     
     
         21 . The polypeptide or mutant polypeptide of any one of  claims 1 - 19 , wherein the species has a pKa greater than 6.2. 
     
     
         22 . The polypeptide of any one of  claims 1 - 21 , wherein the polypeptide has two functional domains and the activity is an activity of one of the two functional domains. 
     
     
         23 . The polypeptide of  claim 22 , wherein both of the two functional domains have a pH-dependent activity. 
     
     
         24 . The polypeptide of any one of  claims 22 - 23 , wherein the polypeptide is a bispecific antibody. 
     
     
         25 . A pharmaceutical composition comprising an effective amount of the polypeptide of any one of  claims 1 - 24  and a pharmaceutically acceptable carrier. 
     
     
         26 . Use of the polypeptide of any one of  claims 1 - 24  for treatment of solid tumors, inflamed joints, or brain diseases or disorders. 
     
     
         27 . A method of treatment of solid tumors, inflamed joints, or brain diseases or disorders comprising a step of administering a polypeptide as claimed in any one of  claims 1 - 24 . 
     
     
         28 . The method of  claim 27 , wherein the polypeptide is administered as part of a chimeric antigen receptor for T-cells comprising the polypeptide. 
     
     
         29 . The method of  claim 27 , wherein the polypeptide is administered linked to a nanoparticle. 
     
     
         30 . The method of  claim 27 , wherein the polypeptide is administered as an antibody-drug conjugate comprising the polypeptide. 
     
     
         31 . A chimeric antigen receptor for T-cells comprising the polypeptide of any one of  claims 1 - 24 . 
     
     
         32 . The polypeptide of any one of  claims 1 - 24  linked to a nanoparticle. 
     
     
         33 . An antibody-drug conjugate comprising the polypeptide of any one of  claims 1 - 24 . 
     
     
         33 . A bispecific antibody comprising at least one binding domain that contains the polypeptide of any one of  claims 1 - 24 . 
     
     
         34 . A chimeric protein containing two different binding or catalytic domains, wherein one or both of the domains comprise the polypeptide of any one of  claims 1 - 24 . 
     
     
         35 . An oncolytic virus comprising the polypeptide of any one of  claims 1 - 24 . 
     
     
         36 . The polypeptide of any one of  claims 1 - 24 , wherein the polypeptide is conjugated to a nucleic acid. 
     
     
         37 . The polypeptide of  claim 36 , wherein the nucleic acid is conjugated non-covalently or covalently and the nucleic acid is a nucleic acid comprising at least one non-natural nucleotide. 
     
     
         38 . The polypeptide of any one of  claims 1 - 2 , wherein the at least one species interacts with the polypeptide through a hydrophilic interaction, a hydrophobic interaction, or a covalent interaction. 
     
     
         39 . The polypeptide of any one of  claims 1 - 24 , comprising two or more species. 
     
     
         40 . A method of producing a conditionally active polypeptide from a parent polypeptide, comprising steps of:
 (i) evolving the parent polypeptide by mutating at least one region outside of its active site to produce one or more mutant polypeptides;   (ii) subjecting the one or more polypeptides and the parent polypeptide to a first assay under a normal physiological condition to measure the activity of the active site under the normal physiological condition and a second assay under an aberrant condition to measure the activity of the active site under the aberrant condition, wherein the normal physiological condition and the aberrant condition are the same condition but having different values;   (iii) selecting the conditionally active polypeptide from the one or more mutant polypeptides which exhibits both (a) a decrease in an activity compared to the same activity of the parent polypeptide in the first assay, and (b) an increase in the activity compared to the same activity of the parent polypeptide in the second assay.   
     
     
         41 . The method of  claim 40 , wherein the physiological condition and aberrant condition are selected from pH, temperature, pH, osmotic pressure, osmolality, oxidative stress, and electrolyte concentration. 
     
     
         42 . The method of any one of  claims 40 - 41 , wherein the at least one region outside of the active site is a region adjacent to the active site. 
     
     
         43 . The method of any one of  claims 40 - 41 , wherein the at least one region outside of the active site is a region remote from the active site. 
     
     
         44 . The method of any one of  claims 40 - 43 , wherein the parent polypeptide is a heavy chain or light chain of an antibody and the active site is a complementarity determining region of the parent polypeptide. 
     
     
         45 . The method of  claim 44 , wherein the at least one region outside of the active site is an Fc region of the parent polypeptide. 
     
     
         46 . The method of  claim 45 , wherein the evolving step comprises replacing the Fc region with a variable region of a separate antibody to form a bispecific antibody. 
     
     
         47 . The method of  claim 45 , wherein the evolving step comprises replacing the Fc region with a variable region of a separate antibody to form a single chain antibody. 
     
     
         48 . The method of  claim 44 , wherein the at least one region outside of the active site is a framework region of the parent polypeptide. 
     
     
         49 . The method of any one of  claims 40 - 47 , where the at least one region outside of the active site is a plurality of regions and the evolving step comprises evolving the plurality of regions sequentially. 
     
     
         50 . The method of any one of  claims 40 - 48 , wherein the evolving step comprising substitution, insertion, deletion, or combinations thereof.