Polynucleotides encoding APOA-1 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, wherein:
ApoA1 is a first polypeptide segment comprising the amino acid sequence shown in 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 consisting of 16 to 36 amino acid residues; and
D is an immunoglobulin Fc region,
wherein the fusion polypeptide has increased cholesterol efflux activity as compared to the ApoA1-L1-D fusion polypeptide in which L1 is a two amino acid linker or is absent, and
wherein the fusion polypeptide comprises the amino acid sequence shown in
(i) residues 19-525, 19-524, 25-525, or 25-524 of SEQ ID NO:2,
(ii) residues 19-525, 19-524, 25-525, or 25-524 of SEQ ID NO:13,
(iii) residues 19-515, 19-514, 25-515, or 25-514 of SEQ ID NO:22,
(iv) residues 19-520, 19-519, 25-520, or 25-519 of SEQ ID NO:26, or
(v) residues 19-535, 19-534, 25-535, or 25-534 of SEQ ID NO:24.
2 . The polynucleotide of claim 1 , wherein the encoded fusion polypeptide comprises the amino acid sequence shown in residues 19-525, 19-524, 25-525, or 25-524 of SEQ ID NO:2.
3 . The polynucleotide of claim 1 , wherein the encoded fusion polypeptide comprises the amino acid sequence shown in residues 19-525, 19-524, 25-525, or 25-524 of SEQ ID NO:13.
4 . The polynucleotide of claim 1 , wherein the encoded fusion polypeptide comprises the amino acid sequence shown in residues 19-515, 19-514, 25-515, or 25-514 of SEQ ID NO:22.
5 . The polynucleotide of claim 1 , wherein the encoded fusion polypeptide comprises the amino acid sequence shown in residues 19-520, 19-519, 25-520, or 25-519 of SEQ ID NO:26.
6 . The polynucleotide of claim 1 , wherein the encoded fusion polypeptide comprises the amino acid sequence shown in residues 19-535, 19-534, 25-535, or 25-534 of SEQ ID NO:24.
7 . The polynucleotide of claim 1 , wherein the encoded fusion polypeptide further comprises a second polypeptide segment located carboxyl-terminal to the immunoglobulin Fc region, wherein the second polypeptide segment is an RNase,
wherein the fusion polypeptide comprises, from an amino-terminal position to a carboxyl-terminal position, ApoA1-L1-D-L2-P, wherein ApoA1, L1, and D are as defined in claim 1 , L2 is a second polypeptide linker, wherein L2 is optionally present, and P is the RNase.
8 . The polynucleotide of claim 7 , wherein the RNase has at least 95% sequence identity with amino acid residues 544-675 or 548-675 of SEQ ID NO:4.
9 . The polynucleotide of claim 8 , wherein the RNase has the amino acid sequence shown in residues 544-675 or 548-675 of SEQ ID NO:4.
10 . The polynucleotide of claim 7 , 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.
11 . The polynucleotide of claim 10 , wherein the encoded fusion polypeptide comprises the amino acid sequence shown in residues 19-675 or 25-675 of SEQ ID NO:4.
12 . The polynucleotide of claim 10 , wherein the encoded fusion polypeptide comprises the amino acid sequence shown in residues 19-675 or 25-675 of SEQ ID NO: 14.
13 . The polynucleotide of claim 10 , wherein the encoded fusion polypeptide comprises the amino acid sequence shown in 19-671 or 25-671 of SEQ ID NO:58.
14 . The polynucleotide of claim 10 , wherein the encoded fusion polypeptide comprises the amino acid sequence shown in residues 19-671 or 25-671 of SEQ ID NO:59.
15 . 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, wherein:
ApoA1 is a first polypeptide segment comprising the amino acid sequence shown in 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 consisting of 16 to 36 amino acid residues; and
D is an immunoglobulin Fc region,
wherein the fusion polypeptide has increased cholesterol efflux activity as compared to the ApoA1-L1-D fusion polypeptide in which L1 is a two amino acid linker or is absent, and
wherein the fusion polypeptide comprises the amino acid sequence shown in
(A) residues 19-525, 19-524, 25-525, or 25-524 of SEQ ID NO:2,
(B) residues 19-525, 19-524, 25-525, or 25-524 of SEQ ID NO:13,
(C) residues 19-515, 19-514, 25-515, or 25-514 of SEQ ID NO:22,
(D) residues 19-520, 19-519, 25-520, or 25-519 of SEQ ID NO:26, or
(E) residues 19-535, 19-534, 25-535, or 25-534 of SEQ ID NO:24; 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.
16 . The method of claim 15 , wherein the encoded fusion polypeptide further comprises a second polypeptide segment located carboxyl-terminal to the immunoglobulin Fc region, wherein the second polypeptide segment is an RNase,
wherein the fusion polypeptide comprises, from an amino-terminal position to a carboxyl-terminal position, ApoA1-L1-D-L2-P, wherein ApoA1, L1, and D are as defined in claim 15 , L2 is a second polypeptide linker, wherein L2 is optionally present, and P is the RNase.
17 . The method of claim 16 , wherein the RNase has at least 95% sequence identity with amino acid residues 544-675 or 548-675 of SEQ ID NO:4.
18 . The method of claim 17 , 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 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.
20 . The method of claim 15 , wherein the encoded fusion polypeptide is produced in the cell and recovered as a dimeric protein.