# US 6,897,039 B2 · Apolipoprotein analogues > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 6,897,039 B2 - Document ID: US6897039B2 - Status: Granted - Kind: B2 - Application number: 09/987,107 - Filed: 2001-11-13 - Published: 2002-10-24 - Granted: 2005-05-24 - Earliest claimed: 2000-11-10 - Assignees: ProteoPharma ApS - Inventors: Jonas Graversen; Søren Moestrup - Web page: https://patentgrep.com/patent/US6897039B2 ## CPC - C07K 14/775: Apolipopeptides - A61K 38/47: acting on glycosyl compounds (3.2), e.g. cellulases, lactases - A61P 7/00: Drugs for disorders of the blood or the extracellular fluid - A61P 9/00: Drugs for disorders of the cardiovascular system - A61P 9/08: Vasodilators for multiple indications - A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis - A61P 39/02: Antidotes - C12N 15/62: DNA sequences coding for fusion proteins - C07K 2319/00: Fusion polypeptide - C07K 2319/02: containing a signal sequence - C07K 2319/21: containing a His-tag - C07K 2319/31: fusions, other than Fc, for prolonged plasma life, e.g. albumin - C07K 2319/50: containing protease site - C07K 2319/73: containing coiled-coiled motif (leucine zippers) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The invention relates to a pharmaceutical composition comprising an apolipoprotein construct, to an apolipoprotein construct, a nucleic acid sequence encoding the apolipoprotein construct, a vector comprising the nucleic acid sequence, a method for producing the apolipoprotein construct, and a method of treatment comprising administering the apolipoprotein construct. The presented data document that the constructs according to the invention are capable of binding lipids, are capable of binding cubilin, which is a strong Apo AI receptor, stronger than native Apo A-I and that the plasma half life of the constructs is at least tripled compared to native Apo A-I. Together these data document that the constructs according to the invention are strong candidates for treatment of cardiovascular diseases. ## Claims ### Claim 1 A pharmaceutical composition comprising an apolipoprotein protein construct having the general formula apo-A-X, where apo-A is an apolipoprotein component selected from the group consisting of apolipoprotein A-I, apolipoprotein A-II, apolipoprotein A-IV, and X is a teeranectin trimerising module. ### Claim 2 (depends on 1) The composition of claim 1, further comprising a spacer peptide between the apo-A component and the tetranectin trimerising module, wherein the spacer peptide comprises at least two amino acids. ### Claim 3 (depends on 2) The composition according to claim 2, wherein the spacer peptide is essentially non-immunogenic, and/or is not prone to proteolytic cleavage and/or does not comprise any cystein residues. ### Claim 4 (depends on 2) The composition according to claim 2, wherein the three-dimensional structure of the spacer linear. ### Claim 5 (depends on 2) The composition according to claim 2, wherein the spacer peptide comprises an amino acid sequence selected from the group consisting of GTKVHMK (SEQ ID NO:69) PGTSGQQPSVGQQ (SEQ ID NO:70), GTSGQ (residues 2-6 of SEQ ID NO:70), PKPSTPPGSS (SEQ ID NO:71), SGGTSGSTSGTGST (SEQ in NQ:72), AGSSTGSSTGPGSTT (SEQ ID NO:73) and GGSGGAP (SEQ ID NO:74). ### Claim 6 (depends on 1) The composition of claim 1, wherein the tetranectin trimerising module is linked by a covalent link to the N-terminal or the C-terminal amino acid of apo-A. ### Claim 7 (depends on 1) The composition of claim 1, wherein the tetranectin trimerising module is part of a stable trimeric complex with two other tetranectin trimerising modules. ### Claim 8 (depends on 7) The composition of claim 7, wherein the stable trimeric complex includes a coiled coil structure. ### Claim 9 (depends on 8) The composition of claim 8, wherein the coiled coil structure is a triple alpha helical coiled coil. ### Claim 10 (depends on 7) The composition of claim 7, wherein the stable trimeric complex comprises two tetranectin trimerising modules linked by a spacer moiety, which allows both of the two tetranectin trimerising modules to take part in the complex formation with a third tetranectin trimerising module not being part of the apolipoprotein protein construct. ### Claim 11 (depends on 1) The composition of claim 1, wherein the tetranectin trimerising module is selected from the group consisting of human tetranectin, murine tetranectin or C-type lectin of human, bovine or shark cartilage. ### Claim 12 (depends on 1) The composition of claim 1, wherein the tetranectin trimerising module comprises a sequence having at least 68% identity with the sequence of SEQ ID NO 12 and is capable of forming a stable trimeric complex with other tetranectin trimerising modules. ### Claim 13 (depends on 12) The composition of claim 12, wherein the cysteine residue 50 in SEQ ID NO 12 is substituted by a serine residue, a threonine residue, or a methionine residue. ### Claim 14 (depends on 1) The composition of claim 1, wherein the tetranectin trimerisation module has at least 68% sequence identity with the Trip A module (SEQ ID NO 13) and is capable of forming a stable trimeric complex with other tetranectin trimerising modules. ### Claim 15 (depends on 7) The composition of claim 7, wherein the stable trimeric complex has a half-life at least 2 times the half-life of native apolipoprotein A-I, A-II or A-IV. ### Claim 16 (depends on 7) The composition of claim 7, wherein said stable trimeric complex is capable of binding to a receptor or protein selected from the group consisting of cubilin, megalin, Scavenger receptor class B, type 1 (SR-B1), ATP-binding cassette 1 (ABC1), Lecithin:cholesterol acyltransferase (LCAT), Cholesteryl-ester transfer protein (CETP), and Phospolipid transfer protein (PLTP). ### Claim 17 (depends on 16) The composition according to claim 16, wherein the trimeric complex comprises an amino acid sequence having at least 70% sequence identity to one of the sequences SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 11. ### Claim 18 (depends on 1) The composition of claim 1, further comprising pharmaceutical acceptable excipients, adjuvants, or additives. ### Claim 19 An apolipoprotein protein construct having the general formula apo-A-X, where apo-A is an apolipoprotein component selected from the group consisting of apolipoprotein A-I, apolipoprotein A-II, and apolipoprotein A-IV, and X is a tetranectin trimerising module. ### Claim 20 (depends on 19) The construct of claim 19, further comprising a spacer peptide between the apo-A component and the tetranectin trimerising module, wherein the spacer peptide comprises at least two amino acids. ### Claim 21 (depends on 20) The construct according to claim 20, wherein the spacer peptide is essentially non-immunogenic, and/or is not prone to proteolytic cleavage and/or does not comprise any cysteine residues. ### Claim 22 (depends on 20) The construct according to claim 20, wherein the three-dimensional structure of the spacer peptide is linear. ### Claim 23 (depends on 20) The construct according to claim 20, wherein the spacer peptide comprises an amino acid sequence selected from the group consisting of GTKVHMK (SEQ ID NO:69), PGTSGQQPSVGQQ (SEQ ID NO:70), GTSGQ (residues 2-6 of SEQ ID NO:70), PKPSTPPGSS (SEQ ID NO:71), SGGTSGSTSGTGST (SEQ ID NO:72), AGSSTCSSTGPGSTT (SEQ ID NO:73) and GGSGGAP (SEQ ID NO:74). ### Claim 24 (depends on 19) The construct of claim 19, wherein the tetranectin trimerising module is part of a stable trimeric complex with two other tetranectin trimerising modules. ### Claim 25 (depends on 24) The construct of claim 24, wherein the stable complex comprises a coiled coil structure. ### Claim 26 (depends on 25) The construct of claim 25, wherein the coiled coil structure is a triple alpha helical coiled coil. ### Claim 27 (depends on 24) The construct of claim 24, wherein the stable trimeric complex comprises two tetranectin trimerising modules linked by a spacer moiety, which allows both of the two tetranectin trimerising modules to take part in the complex formation with a third tetranectin trimerising module not being part of the apolipoprotein protein construct. ### Claim 28 (depends on 24) The construct of claim 24, wherein the tetranectin trimerising module is selected from the group consisting of human tetranectin, murine tetranectin or C-type lectin of human, bovine or shark cartilage. ### Claim 29 (depends on 24) The construct of claim 24, wherein the tetranectin trimerising module comprises a sequence having at least 68% sequence identity with the sequence of SEQ ID NO 12 and is capable of forming a stable trimeric complex with other tetranectin trimerising modules. ### Claim 30 (depends on 29) The construct of claim 29, wherein the cysteine residue 50 in SEQ ID NO 12 is substituted by a serine residue, a threonine residue, or a methionine residue. ### Claim 31 (depends on 24) The construct of claim 24, wherein the tetranectin trimerising module has at least 68% sequence identity with the Trip A module (SEQ ID NO 13) and is capable of forming a stable trimeric complex with other tetranectin trimerising modules. ### Claim 32 (depends on 7) The composition of claim 7, wherein the stable trimeric cowplex has a half-life at least 3 times the half-life of native apolipoprotein A-I, A-II or A-IV. ### Claim 33 (depends on 7) The composition of claim 7, wherein the stable trimeric complex has a half-life at least 4 time the half-life of native apolipoprotein A-I, A-II or A-IV. ### Claim 34 (depends on 7) The composition of claim 7, wherein the stable trimeric complex has a half-life at least 10 times the half-life of native apolipoprotein A-I, A-II or A-IV. ### Claim 35 (depends on 1) The composition of claim 1, wherein the apolipoprotein A-I is human apolipoprotein A-I. ### Claim 36 (depends on 1) The composition of claim 1, wherein apo-A is a fragment of human apolipoprotein A-I, where said fragment substantially retains the lipid binding function of human apolipoprotein A-I. ### Claim 37 (depends on 36) The composition of claim 36, wherein the fragment of human apolipoprotein A-I comprises at least the amino acids 100-186 of human apolipoprotein A-I. ### Claim 38 (depends on 36) The composition of claim 36, wherein the fragment of human apolipoprotein A-I comprises at least the amino acids 25-267 of human apolipoprotein A-I (SEQ ID NO 1). ### Claim 39 (depends on 36) The composition of claim 36, wherein the fragment of human apolipoprotein A-I is amino acids 68-267 from human apolipoprotein A-I. ### Claim 40 (depends on 19) The construct according to claim 19, wherein the tetranectin trimerising module is linked by a covalent link to the N-terminal or the C-terminal amino acid of apo-A. ### Claim 41 (depends on 24) The construct of claim 24, wherein the stable trimeric complex has a half-life at least 2 times the half-life of native apolipoprotein A-I, A-II or A-IV. ### Claim 42 (depends on 24) The construct of claim 24, wherein the stable trimeric complex has a half-life at least 3 times the half-life of native apolipoprotein A-I, A-II or A-IV. ### Claim 43 (depends on 24) The construct of claim 24, wherein the stable trimeric complex has a half-life at least 4 times the half-life of native apolipoprotein A-I, A-II or A-IV. ### Claim 44 (depends on 24) The construct of claim 24, wherein the stable trimeric complex has a half-life at least 10 times the half-life of native apolipoprotein A-I, A-II or A-IV. ### Claim 45 (depends on 19) The construct of claim 19, wherein apo-A is human apolipoprotein A-I. ### Claim 46 (depends on 24) The construct of claim 24, wherein apo-A is a fragment of human apolipoprotein A-I where said fragment substantially retains the lipid binding function of human apolipoprotein A-I. ### Claim 47 (depends on 46) The construct of claim 46, wherein the fragment of human apolipoprotein A-I comprises at least the amino acids 100-186 of human apolipoprotein A-I. ### Claim 48 (depends on 46) The construct of claim 46, wherein the fragment of human apolipoprotein A-I comprises at least the amino acids 25-267 of human apolipoprotein A-I (SEQ ID NO 1). ### Claim 49 (depends on 46) The construct of claim 46, wherein the fragment of human apolipoprotein A-I is amino acids 68-267 from human apolipoprotein A-I. ### Claim 50 (depends on 24) The construct of claim 24, wherein said stable trimeric complex is capable of binding to a receptor or protein selected from the group consisting of cubilin, megalin, Scavenger receptor class B, type 1 (SR-B1), ATP-binding cassette 1 (ABC1), Lecithin:cholesterol acyltransferase (LCAT), Cholesteryl-ester transfer protein (CETP), and Phospolipid transfer protein (PLTP). ### Claim 51 (depends on 50) The construct according to claim 50, wherein the trimeric complex comprises an amino acid sequence having at least 70% sequence identity to at least one of the sequences SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 11. ### Claim 52 A trimeric complex consisting essentially of three apolipoprotein protein constructs, each construct having the general formula apo-A-X, where apo-A is an apolipoprotein component selected from the group consisting of apolipoprotein A-I, apolipoprotein A-II, and apolipoprotein A-IV, and X is a tetranectin trimerising module. ### Claim 53 (depends on 36) The composition of claim 36, wherein said fragment retains all of the lipid binding function of human apolipoprotein A-I. ### Claim 54 (depends on 46) The construct of claim 46, wherein said fragment retains all of the lipid binding function of human apolipoprotein A-I.