Variants of C-type natriuretic peptide
View Patent ↗The present disclosure provides variants of C-type natriuretic peptide (CNP), pharmaceutical compositions comprising CNP variants, and methods of making CNP variants. The CNP variants are useful as therapeutic agents for the treatment of diseases responsive to CNP, including but not limited to bone-related disorders, such as skeletal dysplasias (e.g., achondroplasia), and vascular smooth muscle disorders (e.g., restenosis and arteriosclerosis).
1. A variant of C-type natriuretic peptide (CNP) selected from the group consisting of:
(SEQ ID NO: 179)
GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS
GLGC (Gly-CNP53);
(SEQ ID NO: 185)
PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS
GLGC (Pro-CNP53);
(SEQ ID NO: 190)
MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS
GLGC (Met-CNP53)
(SEQ ID NO: 180)
DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNS
GLGC [CNP-53(M48N)].
2. A pharmaceutical composition comprising a CNP variant of claim 1 15, and a pharmaceutically acceptable excipient, carrier or diluent.
3. The composition of claim 2 , which is a lyophilized formulation prepared from a formulation that comprises a citric acid/citrate buffer or an acetic acid/acetate buffer having a pH from about 4 to about 6.
4. The composition of claim 3 , wherein the lyophilized formulation is prepared from a formulation that further comprises (a) an isotonicity-adjusting agent or a bulking agent or (b) an antioxidant.
5. A method of treating a bone-related disorder or skeletal dysplasia, comprising administering a CNP variant to a subject in need thereof, wherein the CNP variant is a CNP variant according to claim 1 15, and wherein the bone-related disorder or skeletal dysplasia is selected from the group consisting of achondroplasia, hypochondroplasia, short stature, dwarfism and homozygous achondroplasia.
6. A method for recombinant production of a CNP variant, comprising culturing in a medium a host cell comprising a first polynucleotide encoding a CNP variant polypeptide linked to a second polynucleotide encoding a cleavable peptide or protein under conditions that result in expression of a fusion polypeptide encoded by the polynucleotides, wherein the fusion polypeptide comprises the CNP variant polypeptide directly linked to the cleavable peptide or protein or indirectly linked thereto via a linker, wherein the CNP variant is selected from the group consisting of:
(SEQ ID NO: 179)
GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS
GLGC (Gly-CNP53);
(SEQ ID NO: 185)
PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS
GLGC (Pro-CNP53);
(SEQ ID NO: 190)
MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS
GLGC (Met-CNP53);
(SEQ ID NO: 180)
DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNS
GLGC [CNP-53(M48N)].
7. The method of claim 6 16, wherein the cleavable peptide or protein is selected from the group consisting of histidine tags, human transcription factor TAF12, TAF12 fragments, TAF12 histone fold domain, mutants of TAF12 and fragments thereof, TAF12(C/A), TAF12(D/E), TAF12(4D/4E), TAF12(6D/6E), TAF12(10D/10E), TAF12(C/A & D/E), TAF12(C/A & 4D/4E), TAF12(C/A & 6D/6E), TAF12(C/A & 10D/10E), ketosteroid isomerase, maltose-binding protein, β-galactosidase, glutathione-S-transferase, thioredoxin, chitin-binding domain, BMP-2, BMP-2 mutants, BMP-2(C/A), and mutants and fragments thereof.
8. The method of claim 6 16, wherein the host cell is bacterial.
9. The method of claim 6 16, wherein the fusion polypeptide is expressed as a soluble protein or as an inclusion body.
10. The method of claim 6 16, further comprising isolating the expressed fusion polypeptide from the host cell or culture medium.
11. The method of claim 10 , further comprising contacting the isolated fusion polypeptide with a cleaving agent selected from the group consisting of formic acid, cyanogen bromide (CNBr), hydroxylamine, protein self cleavage, Factor Xa, enterokinase, ProTEV, and SUMO protease.
12. A CNP variant produced according to the method of claim 6 , wherein the CNP variant is selected from the group consisting of:
(SEQ ID NO: 179)
GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS
GLGC (Gly-CNP53));
(SEQ ID NO: 185)
PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS
GLGC (Pro-CNP53));
(SEQ ID NO: 190)
MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS
GLGC (Met-CNP53));
(SEQ ID NO: 180)
DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSG
LGC [CNP-53(M48N)]).
13. A method of increasing long bone growth, comprising administering a CNP variant according to claim 1 15 to a subject in need thereof, where the administration increases long bone growth.
14. A CNP variant according to claim 1 15 useful for increasing long bone growth or treating achondroplasia, hypochondroplasia, short stature, dwarfism, or homozygous achondroplasia in a subject in need thereof.
15. A variant of C-type natriuretic peptide (CNP) selected from the group consisting of:
GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSM
SGLGC (SEQ ID NO: 179) (Gly-CNP53);
PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSM
SGLGC (SEQ ID NO: 185) (Pro-CNP53);
MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSM
SGLGC (SEQ ID NO: 190) (Met-CNP53);
DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNS
GLGC (SEQ ID NO: 180) [CNP-53(M48N)];
a CNP53 variant comprising a K4R mutation in the CNP22 portion of the peptide;
a CNP53 variant comprising a furin cleavage site mutation;
PEG-GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDR
IGSMSGLGC (SEQ ID NO: 179) (PEG-Gly-CNP53);
PEG-PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDR
IGSMSGLGC (SEQ ID NO: 185) (PEG-Pro-CNP53);
PEG-MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDR
IGSMSGLGC (SEQ ID NO: 190) (PEG-Met-CNP53); and
PEG-DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRI
GSNSGLGC (SEQ ID NO: 180) [PEG-CNP-53(M48N)].
16. A method for recombinant production of a CNP variant, comprising culturing in a medium a host cell comprising a first polynucleotide encoding a CNP variant polypeptide linked to a second polynucleotide encoding a cleavable peptide or protein under conditions that result in expression of a fusion polypeptide encoded by the polynucleotides, wherein the fusion polypeptide comprises the CNP variant polypeptide directly linked to the cleavable peptide or protein or indirectly linked thereto via a linker, wherein the CNP variant is selected from the group consisting of:
GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSM
SGLGC (SEQ ID NO: 179) (Gly-CNP53);
PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSM
SGLGC (SEQ ID NO: 185) (Pro-CNP53);
MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSM
SGLGC (SEQ ID NO: 190) (Met-CNP53);
DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNS
GLGC (SEQ ID NO: 180) [CNP-53(M48N)];
a CNP53 variant comprising a K4R mutation in the CNP22 portion of the peptide; and
a CNP53 variant comprising a furin cleavage site mutation.
17. A CNP variant produced according to the method of claim 16, wherein the CNP variant is selected from the group consisting of:
GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSM
SGLGC (SEQ ID NO: 179) (Gly-CNP53);
PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSM
SGLGC (SEQ ID NO: 185) (Pro-CNP53);
MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSM
SGLGC (SEQ ID NO: 190) (Met-CNP53);
DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNS
GLGC (SEQ ID NO: 180) [CNP-53(M48N)];
a CNP53 variant comprising a K4R mutation in the CNP22 portion of the peptide; and
a CNP53 variant comprising a furin cleavage site mutation.