Method of producing 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 production of 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, comprising the steps of (i) culturing a transformed host cell under conditions promoting the expression of said apolipoprotein protein construct, and (ii) obtaining and recovering said apolipoprotein protein construct.  
     
     
         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 cysteine residues.  
     
     
         4 . A method according to  claim 2 , wherein the three-dimensional structure of the spacer peptide 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 1 , wherein the tetranectin trimerising module is part of a stable trimeric complex with two other tetranectin trimerising modules.  
     
     
         7 . A method according to  claim 6 , wherein the stable complex comprises a coiled coil structure.  
     
     
         8 . A method according to  claim 7 , wherein the coiled coil structure is a triple alpha helical coiled coil.  
     
     
         9 . A method according to  claim 6 , 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.  
     
     
         10 . A method according to  claim 6 , 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.  
     
     
         11 . A method according to  claim 6 , 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.  
     
     
         12 . A method according to  claim 11 , wherein the cysteine residue 50 in SEQ ID NO 12 is substituted by a serine residue, a threonine residue, or a methionine residue.  
     
     
         13 . A method according to  claim 6 , 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.  
     
     
         14 . A method according to  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.  
     
     
         15 . A method according to  claim 6 , 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 6 , 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 6 , 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 6 , 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 1 , wherein the apolipoprotein A-I is human apolipoprotein A-I.  
     
     
         20 . A method according to  claim 1 , wherein the apolipoprotein A-I is a fragment of human apolipoprotein A-I, where said fragment substantially retains the lipid binding function of human apolipoprotein A-I.  
     
     
         21 . A method according to  claim 20 , wherein the fragment of human apolipoprotein A-I comprises at least the amino acids 100-186 of human apolipoprotein A-I.  
     
     
         22 . A method according to  claim 20 , 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).  
     
     
         23 . A method according to  claim 20 , wherein the fragment of human apolipoprotein A-I is amino acids no 68-267 from human apolipoprotein A-I.  
     
     
         24 . A method according to  claim 6 , 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).  
     
     
         25 . A method according to  claim 24 , 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.  
     
     
         26 . A method according to  claim 1 , comprising further processing of the apolipoprotein protein construct.