Polynucleotides encoding APOA1-PON1 fusion polypeptides
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.
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 97% 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 Fe region;
L2 is a second polypeptide linker; and
P is a paraoxonase having at least 98% sequence identity with amino acid residues 16-355 of SEQ ID NO:12, amino acid residues 16-355 of SEQ ID NO:42, or amino acid residues 16-355 of SEQ ID NO:44,
wherein the encoded fusion polypeptide comprises an amino acid sequence having at least 99% sequence identity with (i) residues 19-883 or 25-883 of SEQ ID NO:28, (ii) residues 19-873 or 25-873 of SEQ ID NO:38, (iii) residues 19-883 or 25-883 of SEQ ID NO:46, or (iv) residues 19-883 or 25-883 of SEQ ID NO:48.
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 31 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 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.
7. The polynucleotide of claim 6 , wherein residue P238, according to EU numbering for human IgG heavy chain constant region, is replaced by serine.
8. The polynucleotide of claim 7 , wherein residue P331, according to EU numbering for human IgG heavy chain constant region, is replaced by serine.
9. The polynucleotide of claim 1 , wherein L2 has the amino acid sequence shown in residues 526-543 of SEQ ID NO:28.
10. The polynucleotide of claim 1 , wherein the paraoxonase has the amino acid sequence shown in residues 16-355 of SEQ ID NO:12, residues 16-355 of SEQ ID NO:42, or residues 16-355 of SEQ ID NO:44.
11. The polynucleotide of claim 1 , wherein the encoded fusion polypeptide comprises the amino acid sequence shown in
(i) residues 19-883 or 25-883 of SEQ ID NO:28,
(ii) residues 19-873 or 25-873 of SEQ ID NO:38,
(iii) residues 19-883 or 25-883 of SEQ ID NO:46, or
(iv) residues 19-883 or 25-883 of SEQ ID NO:48.
12. A method of making a fusion polypeptide, the method comprising:
(1) culturing a mammalian cell into which has been introduced an expression vector comprising the following operably linked elements:
(a) a transcription promoter;
(b) 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 97% 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 Fe region;
L2 is a second polypeptide linker; and
P is a paraoxonase having at least 98% sequence identity with amino acid residues 16-355 of SEQ ID NO:12, amino acid residues 16-355 of SEQ ID NO:42, or amino acid residues 16-355 of SEQ ID NO:44; wherein the encoded fusion polypeptide comprises an amino acid sequence having at least 99% sequence identity with (i) residues 19-883 or 25-883 of SEQ ID NO:28, (ii) residues 19-873 or 25-873 of SEQ ID NO:38, (iii) residues 19-883 or 25-883 of SEQ ID NO:46, or (iv) residues 19-883 or 25-883 of SEQ ID NO:48; and
(c) a transcription terminator, wherein the cell expresses the DNA segment and the encoded fusion polypeptide is produced; and
(2) recovering the fusion polypeptide.
13. The method of claim 12 , 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.
14. The method of claim 12 , wherein the paraoxonase has the amino acid sequence shown in residues 16-355 of SEQ ID NO:12, residues 16-355 of SEQ ID NO:42, or residues 16-355 of SEQ ID NO:44.
15. The method of claim 12 , wherein the encoded fusion polypeptide comprises the amino acid sequence shown in
(i) residues 19-883 or 25-883 of SEQ ID NO:28,
(ii) residues 19-873 or 25-873 of SEQ ID NO:38,
(iii) residues 19-883 or 25-883 of SEQ ID NO:46, or
(iv) residues 19-883 or 25-883 of SEQ ID NO:48.
16. The method of claim 12 , wherein the encoded fusion polypeptide is produced in the cell and recovered as a dimeric protein.