Apolipoprotein analogues

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.

1 . A method for the prevention and/or treatment of a disease or condition comprising administering to an individual in need thereof, 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, and apolipoprotein A-IV, and X is a tetranectin trimerising module. 
     
     
         2 . A method according to  claim 1 , wherein the apolipoprotein protein construct further comprises a spacer peptide between the apo-A component and the tetranectin trimerising module, wherein the spacer peptide comprises at least two amino acids. 
     
     
         3 . A method 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. 
     
     
         4 . A method according to  claim 2 , wherein the three-dimensional structure of the spacer is linear. 
     
     
         5 . A method 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 ID NO: 72), AGSSTGSSTGPGSTT (SEQ ID NO: 73) and GGSGGAP (SEQ ID NO: 74). 
     
     
         6 . A method according to  claim 2 , wherein the tetranectin trimerising module is linked by a covalent link to the N-terminal or the C-terminal amino acid of apo-A. 
     
     
         7 . A method according to  claim 1 , wherein the tetranectin trimerising module is part of a stable trimeric complex with two other tetranectin trimerising modules. 
     
     
         8 . A method according to  claim 7 , wherein the stable trimeric complex includes a coiled coil structure. 
     
     
         9 . A method according to  claim 8 , wherein the coiled coil structure is a triple alpha helical coiled coil. 
     
     
         10 . A method according to  claim 9 , 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 fusion protein construct. 
     
     
         11 . A method according to  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. 
     
     
         12 . A method according to  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. 
     
     
         13 . A method according to  claim 12 , wherein the cystein residue 50 in SEQ ID NO 12 is substituted by a serine residue, a threonine residue, or a methionine residue. 
     
     
         14 . A method according to  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. 
     
     
         15 . A method according to  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. 
     
     
         16 . A method according to  claim 7 , 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. 
     
     
         17 . A method according to  claim 7 , 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. 
     
     
         18 . A method according to  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. 
     
     
         19 . A method according to  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). 
     
     
         20 . A method according to  claim 19 , 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 C, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 11. 
     
     
         21 . A method according to  claim 7 , wherein the composition further comprises pharmaceutical acceptable excipients, adjuvants, or additives. 
     
     
         22 . A method according to  claim 1 , wherein the apolipoprotein A-I is human apolipoprotein A-I. 
     
     
         23 . A method according to  claim 1 , wherein the apolipoprotein A-I is a fragment of human apolipoprotein A-I, wherein said fragment substantially retains the lipid binding function of human apolipoprotein A-I. 
     
     
         24 . A method according to  claim 23 , wherein the fragment of human apolipoprotein A-I comprises at least the amino acids 100-186 of human apolipoprotein A-I. 
     
     
         25 . A method according to  claim 23 , 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). 
     
     
         26 . A method according to  claim 23 , wherein the fragment of human apolipoprotein A-I is amino acids 68-267 from human apolipoprotein A-I. 
     
     
         27 . A method according to  claim 1 , wherein the individual has a condition related to a disorder selected from cholesterol disorders, phospholipid disorders, triacylglyceride disorders, LDL disorders and HDL disorders. 
     
     
         28 . A method according to  claim 1 , wherein the disease is an arteriosclerotic disease. 
     
     
         29 . A method according to  claim 1 , wherein the disease is a cardiovascular disease. 
     
     
         30 . A method according to  claim 1 , wherein the disease is angina pectoris. 
     
     
         31 . A method according to  claim 1 , wherein the disease is myocardial infarction. 
     
     
         32 . A method according to  claim 1 , wherein the disease is plaque angina pectoris. 
     
     
         33 . A method according to  claim 1 , wherein the disease is unstable angina pectoris. 
     
     
         34 . A method according to  claim 1 , wherein the disease is arterial stenosis. 
     
     
         35 . A method according to  claim 1 , wherein the disease is claudicatio. 
     
     
         36 . A method according to  claim 1 , wherein the disease is carotis stenosis. 
     
     
         37 . A method according to  claim 1 , wherein the disease is cerebral arterial stenosis. 
     
     
         38 . A method according to  claim 1 , wherein the condition is caused by endotoxins. 
     
     
         39 . A method according to  claim 1 , wherein the pharmaceutical composition is administered intravenously, intraarterially, intramusculary, transdermally, pulmonary, subcutaneously, intradermally, intratechally, through the buccal-, anal-, vaginal-, conjunctival-, or intranasal tissue, by inoculation into tissue, by an implant, or orally. 
     
     
         40 . A method according to  claim 7 , wherein the trimeric complex is administered in an amount of at least 50 mg per week. 
     
     
         41 . A method according to  claim 7 , wherein the trimeric complex is administered in an amount of at least 100 mg per week. 
     
     
         42 . A method according to  claim 7 , wherein the trimeric complex is administered in an amount of at least 250 mg per week. 
     
     
         43 . A method according to  claim 7 , wherein the trimeric complex is administered in an amount of at least 500 mg per week. 
     
     
         44 . A method according to  claim 7 , wherein the trimeric complex is administered in an amount of at least 750 mg per week. 
     
     
         45 . A method according to  claim 7 , wherein the trimeric complex is administered in an amount of at least 1000 mg per week. 
     
     
         46 . A method according to  claim 7 , wherein the trimeric complex is administered in an amount of at least 1250 mg per week. 
     
     
         47 . A method according to  claim 7 , wherein the trimeric complex is administered in an amount of at least 1500 mg per week. 
     
     
         48 . A method according to  claim 7 , wherein the trimeric complex is administered in an amount of at least 2000 mg per week. 
     
     
         49 . A method according to  claim 7 , wherein the trimeric complex is administered in an amount of at least 2500 mg per week. 
     
     
         50 . A method according to  claim 7 , wherein the trimeric complex is administered in an amount of at least 5000 mg per week. 
     
     
         51 . A method according to  claim 1 , comprising administration during at least 1 day. 
     
     
         52 . A method according to  claim 7 , wherein the trimeric complex is administered in an amount of at least 10 mg/kg body weight. 
     
     
         53 . A method according to  claim 7 , wherein the trimeric complex is administered in an amount of at least 50 mg/kg body weight. 
     
     
         54 . A method according to  claim 7 , wherein the trimeric complex is administered in an amount of at least 100 mg/kg body weight. 
     
     
         55 . A method according to  claim 7 , wherein the trimeric complex is administered in an amount of at least 200 mg/kg body weight. 
     
     
         56 . A method according to  claim 7 , wherein the trimeric complex is administered in an amount of at least 500 mg/kg body weight. 
     
     
         57 . A method according to  claim 7 , wherein the trimeric complex is administered in an amount of at least 1000 mg/kg body weight. 
     
     
         58 . A method according to  claim 1 , comprising administering a dose of said pharmaceutical composition once a week. 
     
     
         59 . A method according to  claim 1 , comprising administering a dose of said pharmaceutical composition once every second week, once every third week, or once every fourth week. 
     
     
         60 . A method for the prevention and/or treatment of a disease or condition, said method comprising transfecting a cell population with a nucleic acid sequence encoding an apolipoprotein protein construct as defined in  claim 1 , and subsequently transferring said cell population to an individual in need thereof. 
     
     
         61 . A method according to  claim 60 , wherein the cell population comprises macrophages. 
     
     
         62 . A method according to  claim 60 ; wherein the cell population comprises liver cells.