Polynucleotides encoding apoa1-rnase fusion polypeptides

Abstract

Compositions and methods relating to ApoA-1 fusion polypeptides are disclosed. The fusion polypeptides include a first polypeptide segment corresponding to an ApoA-1 polypeptide or ApoA-1 mimetic, and may also include a dimerizing domain such as, e.g., an Fc region, which is typically linked carboxyl-terminal to the first polypeptide segment via a flexible linker. In some embodiments, the fusion polypeptide further includes a second polypeptide segment located carboxyl-terminal to the first polypeptide segment and which confers a second biological activity (e.g., an RNase, paraoxonase, platelet-activating factor acetylhydrolase, cholesterol ester transfer protein, lecithin-cholesterol acyltransferase, polypeptide that specifically binds to proprotein convertase subtilisin/kexin type 9, or polypeptide that specifically binds to amyloid beta). Also disclosed are dimeric proteins comprising first and second ApoA-1 fusion polypeptides as disclosed herein. The fusion polypeptides and dimeric proteins are useful in methods for therapy.

What is claimed is: 
     
         1 . A polynucleotide encoding a fusion polypeptide, wherein the fusion polypeptide comprises, from an amino-terminal position to a carboxyl-terminal position, ApoA1-L1-D-L2-P, wherein:
 ApoA1 is a first polypeptide segment comprising an amino acid sequence having at least 95% sequence identity with amino acid residues 19-267 or 25-267 of SEQ ID NO:2, wherein said first polypeptide segment has cholesterol efflux activity;   L1 is a first polypeptide linker comprising at least 5 amino acid residues;   D is an immunoglobulin Fc region;   L2 is a second polypeptide linker; and   P is an RNase having at least 95% sequence identity with amino acid residues 544-675 or 548-675 of SEQ ID NO:4.   
     
     
         2 . The polynucleotide of  claim 1 , wherein the first polypeptide segment has the amino acid sequence shown in residues 19-267 or 25-267 of SEQ ID NO:2. 
     
     
         3 . The polynucleotide of  claim 1 , wherein L1 comprises at least 16 amino acid residues. 
     
     
         4 . The polynucleotide of  claim 1 , wherein L1 consists of from 10 to 60 amino acid residues. 
     
     
         5 . The polynucleotide of  claim 1 , wherein L1 comprises two or more tandem repeats of the amino acid sequence of SEQ ID NO:15. 
     
     
         6 . The polynucleotide of  claim 1 , wherein the Fc region is a human Fc region. 
     
     
         7 . The polynucleotide of  claim 6 , wherein the human Fc region is a γ1 Fc variant comprising one or more amino acid substitutions relative to a wild-type human γ1 Fc region sequence. 
     
     
         8 . The polynucleotide of  claim 7 , wherein the Fc region is a human γ1 Fc variant in which residues C220, C226, and C229, according to EU numbering for human IgG heavy chain constant region, are each replaced by serine. 
     
     
         9 . The polynucleotide of  claim 8 , wherein residue P238, according to EU numbering for human IgG heavy chain constant region, is replaced by serine. 
     
     
         10 . The polynucleotide of  claim 9 , wherein residue P331, according to EU numbering for human IgG heavy chain constant region, is replaced by serine. 
     
     
         11 . The polynucleotide of  claim 1 , wherein L2 has the amino acid sequence shown in residues 526-543 of SEQ ID NO:28. 
     
     
         12 . The polynucleotide of  claim 1 , wherein the RNase has the amino acid sequence shown in residues 544-675 or 548-675 of SEQ ID NO:4. 
     
     
         13 . The polynucleotide of  claim 1 , wherein the encoded fusion polypeptide comprises an amino acid sequence having at least 95% sequence identity with
 (i) residues 19-675 or 25-675 of SEQ ID NO:4,   (ii) residues 19-675 or 25-675 of SEQ ID NO:14,   (iii) residues 19-671 or 25-671 of SEQ ID NO:58, or   (iv) residues 19-671 or 25-671 of SEQ ID NO:59.   
     
     
         14 . The polynucleotide of  claim 13 , wherein the encoded fusion polypeptide comprises an amino acid sequence having at least 98% sequence identity with
 (i) residues 19-675 or 25-675 of SEQ ID NO:4,   (ii) residues 19-675 or 25-675 of SEQ ID NO:14,   (iii) residues 19-671 or 25-671 of SEQ ID NO:58, or   (iv) residues 19-671 or 25-671 of SEQ ID NO:59.   
     
     
         15 . The polynucleotide of  claim 13 , wherein the encoded fusion polypeptide comprises the amino acid sequence shown in
 (i) residues 19-675 or 25-675 of SEQ ID NO:4,   (ii) residues 19-675 or 25-675 of SEQ ID NO:14,   (iii) residues 19-671 or 25-671 of SEQ ID NO:58, or   (iv) residues 19-671 or 25-671 of SEQ ID NO:59.   
     
     
         16 . A method of making a fusion polypeptide, the method comprising:
 (a) culturing a mammalian cell into which has been introduced an expression vector comprising the following operably linked elements:
 (i) a transcription promoter; 
 (ii) a DNA segment encoding a fusion polypeptide, wherein the fusion polypeptide comprises, from an amino-terminal position to a carboxyl-terminal position, ApoA1-L1-D-L2-P, wherein:
 ApoA1 is a first polypeptide segment comprising an amino acid sequence having at least 95% sequence identity with amino acid residues 19-267 or 25-267 of SEQ ID NO:2, wherein said first polypeptide segment has cholesterol efflux activity; 
 L1 is a first polypeptide linker comprising at least 5 amino acid residues; 
 D is an immunoglobulin Fc region; 
 L2 is a second polypeptide linker; and 
 P is an RNase having at least 95% sequence identity with amino acid residues 544-675 or 548-675 of SEQ ID NO:4; and 
 
 (iii) a transcription terminator, 
   wherein the cell expresses the DNA segment and the encoded fusion polypeptide is produced; and
 (b) recovering the fusion polypeptide. 
   
     
     
         17 . The method of  claim 16 , wherein the human Fc region is a γ1 Fc variant comprising one or more amino acid substitutions relative to a wild-type human γ1 Fc region sequence. 
     
     
         18 . The method of  claim 16 , wherein the RNase has the amino acid sequence shown in residues 544-675 or 548-675 of SEQ ID NO:4. 
     
     
         19 . The method of  claim 16 , wherein the encoded fusion polypeptide comprises an amino acid sequence having at least 95% sequence identity with
 (i) residues 19-675 or 25-675 of SEQ ID NO:4,   (ii) residues 19-675 or 25-675 of SEQ ID NO:14,   (iii) residues 19-671 or 25-671 of SEQ ID NO:58, or   (iv) residues 19-671 or 25-671 of SEQ ID NO:59.   
     
     
         20 . The method of  claim 19 , wherein the encoded fusion polypeptide comprises an amino acid sequence having at least 98% sequence identity with
 (i) residues 19-675 or 25-675 of SEQ ID NO:4,   (ii) residues 19-675 or 25-675 of SEQ ID NO:14,   (iii) residues 19-671 or 25-671 of SEQ ID NO:58, or   (iv) residues 19-671 or 25-671 of SEQ ID NO:59.   
     
     
         21 . The method of  claim 19 , wherein the encoded fusion polypeptide comprises the amino acid sequence shown in
 (i) residues 19-675 or 25-675 of SEQ ID NO:4,   (ii) residues 19-675 or 25-675 of SEQ ID NO:14,   (iii) residues 19-671 or 25-671 of SEQ ID NO:58, or   (iv) residues 19-671 or 25-671 of SEQ ID NO:59.   
     
     
         22 . The method of  claim 16 , wherein the encoded fusion polypeptide is produced in the cell and recovered as a dimeric protein.