1d05 pcsk9 antagonists

Abstract

Antagonists of human proprotein convertase subtilisin-kexin type 9 (“PCSK9”) are disclosed. The disclosed antagonists are effective in the inhibition of PCSK9 function and, accordingly, present desirable antagonists for use in the treatment of conditions associated with PCSK9 activity. The present invention also discloses nucleic acid encoding said antagonists, vectors, host cells, and compositions comprising the antagonists. Methods of making PCSK9-specific antagonists as well as methods of using the antagonists for inhibiting or antagonizing PCSK9 function are also disclosed and form important additional aspects of the present disclosure.

What is claimed is: 
     
         1 . An isolated PCSK9-specific antagonist which comprises:
 (a) a heavy chain variable region comprising a CDR3 domain comprising SEQ ID NO: 17 or an equivalent thereof, said equivalent characterized as having one or more conservative amino acid substitutions in the CDR3 domain; and/or   (b) a light chain variable region comprising a CDR3 domain comprising SEQ ID NO: 7 or an equivalent thereof, said equivalent characterized as having one or more conservative amino acid substitutions in the CDR3 domain;   wherein said PCSK9-specific antagonist antagonizes PCSK9's inhibition of cellular LDL uptake.   
     
     
         2 . The PCSK9-specific antagonist of  claim 1  wherein the CDR3 domain(s) are in a human germline region in the CDR3 region thereof. 
     
     
         3 . The PCSK9-specific antagonist of  claim 1  that binds to human PCSK9 with an equilibrium dissociation constant (K D ) of less than 1200 nM. 
     
     
         4 . The PCSK9-specific antagonist of  claim 1  that binds to human PCSK9 with a K D  of less than 500 nM. 
     
     
         5 . The PCSK9-specific antagonist of  claim 1  that binds to human PCSK9 with a K D  of less than 100 nM. 
     
     
         6 . The PCSK9-specific antagonist of  claim 1  that binds to human PCSK9 with a K D  of less than 5 nM. 
     
     
         7 . The PCSK9-specific antagonist of  claim 1  that antagonizes PCSK9's inhibition of cellular LDL uptake at an IC 50  of less than 500 nM. 
     
     
         8 . The PCSK9-specific antagonist of  claim 1  that antagonizes PCSK9's inhibition of cellular LDL uptake at an IC 50  of less than 200 nM. 
     
     
         9 . The PCSK9-specific antagonist of  claim 1  that antagonizes PCSK9's inhibition of cellular LDL uptake at an IC 50  of less than 100 nM. 
     
     
         10 . The PCSK9-specific antagonist of  claim 1  that antagonizes PCSK9's inhibition of cellular uptake by at least 20%. 
     
     
         11 . The PCSK9-specific antagonist of  claim 1  which is an antibody molecule. 
     
     
         12 . The PCSK9-specific antagonist of  claim 1  which further comprises:
 (a) a heavy chain variable CDR1 sequence comprising SEQ ID NO: 13; 
 (b) a heavy chain variable CDR2 sequence comprising SEQ ID NO: 15; 
 (c) a light chain variable CDR1 sequence comprising SEQ ID NO: 3; and/or 
 (d) a light chain variable CDR2 sequence comprising SEQ ID NO: 5. 
 
     
     
         13 . The PCSK9-specific antagonist of  claim 12  wherein the CDR1, CDR2 and/or CDR3 domain(s) are in a human germline region in the respective CDR1, CDR2 and/or CDR3 regions thereof. 
     
     
         14 . The PCSK9-specific antagonist of  claim 1  comprising:
 (a) a heavy chain variable region comprising a CDR3 domain comprising SEQ ID NO: 17; 
 (b) a light chain variable region comprising a CDR3 domain comprising SEQ ID NO: 7; 
 (c) a heavy chain variable CDR1 sequence comprising SEQ ID NO: 13; 
 (d) a light chain variable CDR1 sequence comprising SEQ ID NO: 3; 
 (e) a heavy chain variable CDR2 sequence comprising SEQ ID NO: 15; and 
 (f) a light chain variable CDR2 sequence comprising SEQ ID NO: 5. 
 
     
     
         15 . The PCSK9-specific antagonist of  claim 14  wherein the CDR1, CDR2 and/or CDR3 domain(s) are in a human germline variable region in the respective CDR1, CDR2 and/or CDR3 regions thereof. 
     
     
         16 . The PCSK9-specific antagonist of  claim 12  which comprises a heavy chain variable region comprising SEQ ID NO: 11 and/or a light chain variable region comprising SEQ ID NO: 27. 
     
     
         17 . The PCSK9-specific antagonist of  claim 12  which comprises a heavy chain having constant sequence comprising: SEQ ID NO: 24. 
     
     
         18 . The PCSK9-specific antagonist of  claim 16  which comprises a heavy chain having constant sequence comprising: SEQ ID NO: 24. 
     
     
         19 . A PCSK9-specific antagonist which comprises:
 (a) a light chain comprising SEQ ID NO: 1; and   (b) a heavy chain comprising SEQ ID NO: 11;   wherein said PCSK9-specific antagonist is an antibody molecule that antagonizes PCSK9's inhibition of cellular LDL uptake.   
     
     
         20 . A PCSK9-specific antagonist of  claim 19  wherein SEQ ID NO: 11 is followed in sequence by an amino acid sequence selected from the group consisting of: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24. 
     
     
         21 . An isolated PCSK9-specific antagonist which comprises:
 (a) a light chain comprising SEQ ID NO: 26; and   (b) a heavy chain comprising SEQ ID NO: 25;   wherein said PCSK9-specific antagonist is an antibody molecule that antagonizes PCSK9's inhibition of cellular LDL uptake.   
     
     
         22 . An isolated PCSK9-specific antagonist that:
 (a) inhibits the binding of a 1D05 Fab to PCSK9 by at least 50%; said 1D05 Fab characterized as comprising a light chain comprising SEQ ID NO: 1 and an Fd chain comprising amino acids 1-233 of SEQ ID NO: 9; and   (b) antagonizes (i) PCSK9 binding to the LDL receptor and/or (ii) PCSK9 internalization into cells.   
     
     
         23 . A PCSK9-specific antagonist that:
 (a) inhibits the binding of a 1D05 IgG to PCSK9 by at least 50%; said 1D05 IgG characterized as comprising (i) a light chain comprising SEQ ID NO: 1 and (ii) a heavy chain comprising SEQ ID NO: 11; and   (b) antagonizes (i) PCSK9 binding to the LDL receptor and/or (ii) PCSK9 internalization into cells.   
     
     
         24 . A PCSK9-specific antagonist of  claim 23  wherein SEQ ID NO: 11 is followed in sequence by an amino acid sequence selected from the group consisting of: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24. 
     
     
         25 . An isolated PCSK9-specific antagonist which comprises:
 (a) heavy chain variable region CDR3 sequence of SEQ ID NO: 45;   (b) heavy chain variable region CDR3 sequence of SEQ ID NO: 45; heavy chain CDR1 sequence of SEQ ID NO: 43 and heavy chain CDR2 sequence of SEQ ID NO: 44;   (c) light chain variable region CDR3 sequence of SEQ ID NO: 48;   (d) light chain variable region CDR3 sequence of SEQ ID NO: 48; light chain CDR1 sequence of SEQ ID NO: 46 and light chain CDR2 sequence of SEQ ID NO: 47;   (e) both (a) and (c);   (f) both (b) and (d);   (g) heavy and/or light chain variable regions comprising SEQ ID NOs: 50 and 49, respectively;   (h) a heavy chain variable region comprising any one of SEQ ID NOs: 51-56 and optionally a light chain variable region comprising SEQ ID NO: 27; or   (i) a light chain variable region comprising any one of SEQ ID NOs: 57-60 and optionally a heavy chain variable region comprising SEQ ID NO: 11;   wherein said PCSK9-specific antagonist is an antibody molecule that antagonizes PCSK9's inhibition of cellular LDL uptake.   
     
     
         26 . A composition comprising the PCSK9-specific antagonist of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         27 . A composition in accordance with  claim 26  which comprises:
 (a) about 50 mg/mL to about 200 mg/mL of the PCSK9-specific antagonist; 
 (b) a polyhydroxy hydrocarbon (including but not limited to sorbitol, mannitol, glycerol and dulcitol) and/or a disaccharide (including but not limited to sucrose, lactose, maltose and trehalose); the total of said polyhydroxy hydrocarbon and/or disaccharide being about 1% to about 6% w/v of the formulation; 
 (c) about 5 mM to about 200 mM of histidine, imidazole, phosphate or acetic acid; 
 (d) about 5 mM to about 200 mM of arginine, proline, phenylalanine, alanine, glycine, lysine, glutamic acid, aspartic acid or methionine; 
 (e) about 0.01 M to about 0.1 M of hydrochloric acid (“HCl”) in an amount sufficient to achieve a pH in the range of about 5.5 to about 7.5; and 
 (f) a liquid carrier including but not limited to sterile water, petroleum, animal oil, vegetable oil, mineral oil, synthetic oil, physiological saline solution, dextrose or other saccharide solution or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol; 
 wherein said pharmaceutical composition has a pH in the range of about 5.5 to about 7.5; and wherein said pharmaceutical composition optionally comprises about 0.01% to about 1% w/v of the formulation of a non-ionic surfactant (including but not limited to Polysorbate-80 (Tween 80™), Polysorbate-60 (Tween 60™), Polysorbate-40 (Tween 40™), and Polysorbate-20 (Tween 20™), polyoxyethylene alkyl ethers, including but not limited to Brij 58™, Brij35™, as well as others such as Triton X-100™, Triton X-114™, NP40™, Span 85 and the Pluronic series of non-ionic surfactants (e.g., Pluronic 121)). 
 
     
     
         28 . The composition of  claim 27  which comprises:
 (a) about 50 mg/mL to about 200 mg/mL of the PCSK9-specific antagonist; 
 (b) about 1% to about 6% w/v of mannitol, trehalose or sucrose; 
 (c) about 10 mM to about 150 mM of histidine; 
 (d) about 10 mM to about 150 mM of arginine or proline; 
 (e) about 0.003 M to about 0.005 M of hydrochloric acid (“HCl”) in an amount sufficient to achieve a pH in the range of about 5.8 to about 6.5; and 
 (f) a liquid carrier including but not limited to sterile water; petroleum, animal oil, vegetable oil, mineral oil, synthetic oil, physiological saline solution dextrose, or other saccharide solution or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol; 
 wherein said pharmaceutical composition has a pH in the range of about 5.8 to about 6.5; and wherein said pharmaceutical composition optionally comprises about 0.01% to about 1% w/v of Polysorbate-80 (Tween 80™) or Polysorbate-20 (Tween 20™). 
 
     
     
         29 . The composition of  claim 28  which comprises:
 (a) about 50 mg/mL to about 200 mg/mL of the PCSK9-specific antagonist; 
 (b) about 2% to about 6% w/v of sucrose; 
 (c) about 25 mM to about 100 mM of histidine; 
 (d) about 25 mM to about 100 mM of arginine; 
 (e) about 0.0040 M to about 0.0045 M of hydrochloric acid (“HCl”) in an amount sufficient to achieve a pH in the range of about 6; and 
 (f) sterile water; 
 wherein said pharmaceutical composition has a pH in the range of about 6; and 
 wherein said pharmaceutical composition optionally comprises about 0.01% to about 1% w/v of Polysorbate-80 (Tween 80™) or Polysorbate-20 (Tween 20™). 
 
     
     
         30 . The composition of  claim 29  which comprises:
 (a) about 50 mg/mL to about 200 mg/mL of the PCSK9-specific antagonist; 
 (b) sucrose, histidine and arginine in one of the following amounts: (i) about 3% w/v sucrose, about 50 mM histidine and about 50 mM arginine; or (ii) about 6% w/v sucrose, about 100 mM histidine and about 100 mM arginine; 
 (c) about 0.0040 M to about 0.0045 M of hydrochloric acid (“HCl”) in an amount sufficient to achieve a pH in the range of about 6; and 
 (d) sterile water; 
 wherein said pharmaceutical composition has a pH in the range of about 6; and wherein said pharmaceutical composition optionally comprises about 0.01% to about 1% w/v of Polysorbate-80 (Tween 80™) or Polysorbate-20 (Tween 20™). 
 
     
     
         31 . A method for antagonizing PCSK9 function which comprises administering a PCSK9-specific antagonist of  claim 1 . 
     
     
         32 . Use of a PCSK9-specific antagonist of  claim 1  in the manufacture of a medicament for ameliorating a disorder, condition or disease caused and/or exacerbated by PCSK9 function. 
     
     
         33 . Isolated nucleic acid encoding a PCSK9-specific antagonist of  claim 1 . 
     
     
         34 . Isolated nucleic acid which encodes a PCSK9-specific antagonist of  claim 1 ; wherein the CDR3 domain of the heavy chain variable region is encoded by a nucleotide sequence comprising SEQ ID NO: 18; and/or wherein the CDR3 domain of the light chain variable region is encoded by a nucleotide sequence comprising SEQ ID NO: 8. 
     
     
         35 . The isolated nucleic acid of  claim 34  which further comprises:
 (a) CDR1 and/or CDR2 domains in the heavy chain variable region that are encoded, respectively, by a nucleotide sequence comprising SEQ ID NO: 14 and SEQ ID NO: 16; and/or 
 (b) CDR1 and/or CDR2 domains in the light chain variable region that are encoded, respectively, by a nucleotide sequence comprising SEQ ID NO: 4 and SEQ ID NO: 6. 
 
     
     
         36 . Isolated nucleic acid which encodes a PCSK9-specific antagonist of  claim 1 ; wherein said PCSK9-specific antagonist comprises:
 (a) a heavy chain variable region encoded by a nucleotide sequence comprising SEQ ID NO: 12; and/or   (b) a light chain variable region encoded by a nucleotide sequence comprising SEQ ID NO: 28.   
     
     
         37 . Isolated nucleic acid which encodes a PCSK9-specific antagonist of  claim 1 ; wherein said PCSK9-specific antagonist comprises:
 (a) a heavy chain region encoded by a nucleotide sequence comprising SEQ ID NO: 29; and/or   (b) a light chain region encoded by a nucleotide sequence comprising SEQ ID NO: 30.   
     
     
         38 . A vector comprising nucleic acid of  claim 33 . 
     
     
         39 . An isolated host cell or population of host cells in vitro or in situ comprising nucleic acid of  claim 33 . 
     
     
         40 . A method for producing a PCSK9-specific antagonist which comprises:
 (a) culturing the cell(s) of  claim 39  under conditions appropriate for production of the PCSK9-specific antagonist; and   (b) isolating the PCSK9-specific antagonist produced.   
     
     
         41 . An isolated host cell or population of host cells in vitro or in situ comprising a PCSK9-specific antagonist of  claim 1 .