Novel binding proteins for pcsk9

Abstract

The present disclosure relates to novel lipocalin muteins which bind to PCSK9. The disclosure also provides corresponding nucleic acid molecules encoding lipocalin muteins and methods for producing lipocalin muteins as well as their encoding nucleic acid molecules.

1 . A method for lowering plasma level of low density lipoprotein cholesterol (LDL-C), comprising administering to a human subject in need thereof a composition comprising an effective amount of a mutein of human tear lipocalin, wherein the mutein comprises:
 (a) a mutated amino acid residue at any one or more of the sequence positions 26-34, 56-58, 80, 83, 104-106 and 108 of the linear polypeptide sequence of the mature human tear lipocalin, wherein the mutein comprises at least one of the following amino acid substitutions in comparison to mature human tear lipocalin: (i) Arg 26→Ser, Phe, Trp, His or Thr and (ii) Glu 34→Asn, Thr, Arg or Gly, and   (b) a mutated amino acid residue at any one or more of the sequence positions 61, 101, 111, 114 and 153 of the linear polypeptide sequence of the mature human tear lipocalin,   and wherein the mutein specifically binds to proprotein convertase subtilisin/kexin type 9 (PCSK9).   
     
     
         2 . The method according to  claim 1 , wherein the mutein reduces binding of PCSK9 to low density lipoprotein receptors (LDL-R). 
     
     
         3 . The method according to  claim 1 , wherein the mutein enhances recycling of LDL-R to plasma membrane and intake of LDL-C by LDL-R. 
     
     
         4 . The method according to  claim 1 , wherein the mutein binds to human PCSK9 or to a fragment thereof with a dissociation constant (K D ) selected from the group consisting of a dissociation constant (K D ) equal to or less than 10 nM, with a dissociation constant (K D ) equal to or less than 1 nM, with a dissociation constant (K D ) equal to or less than 0.1 nM and with a dissociation constant (K D ) equal to or less than 1 pM. 
     
     
         5 . The method according to  claim 1 , wherein the mutein comprises one of the following sets of amino acid substitutions in comparison to mature human tear lipocalin:
 (a) at least one of the following amino acid substitutions in comparison to mature human tear lipocalin: Arg 26→Ser, Phe, Trp, His or Thr,   (b) at least one of the following amino acid substitutions in comparison to mature human tear lipocalin: Glu 34→Asn, Thr, Arg or Gly; Leu 56→Met, Ser, Gln, Phe, His or Asn,   (c) at least one of the following amino acid substitutions in comparison to mature human tear lipocalin: Ser 58→Lys, Ala, Arg, Trp or Pro,   (d) at least one of the following amino acid substitutions in comparison to mature human tear lipocalin: Met 31→Ala, Gly, His, Pro, Ser Aps, Glu or Gln,   (e) at least one of the following amino acid substitutions in comparison to mature human tear lipocalin: Leu 33→Tyr, Trp, Tyr, Phe, Pro or Ala,   (f) at least one of the following amino acid substitutions in comparison to mature human tear lipocalin: Ser 61→Trp or Phe; Asp 80→Ser, Met, Pro, Ile, Gln, Tyr, Ser, Val or Thr,   (h) at least one of the following amino acid substitutions in comparison to mature human tear lipocalin: Glu 104→Leu, Pro, Ser, Ala, Asn, Thr, Lys or Asp,   (i) at least one of the following amino acid substitutions in comparison to mature human tear lipocalin: His 106→Pro, Gln, Gly, Arg, Val, Thr, Asn or Leu,   (j) at least one of the following amino acid substitutions in comparison to mature human tear lipocalin: Lys 108→Gln, Ala, Trp, Tyr, Arg, Asp, Asn, Ser, Glu or Thr,   (l) at least one of the following amino acid substitutions in comparison to mature human tear lipocalin: Glu 27→Arg, Ser, Gln, Thr, Phe, Lys, Ala or Arg,   (m) at least one of the following amino acid substitutions in comparison to mature human tear lipocalin: Pro 29→Gly, Asp, Asn, Ile, Leu or Met,   (o) at least one of the following amino acid substitutions in comparison to mature human tear lipocalin: Asn 32→Ile, Leu, Tyr, Met or Trp.   (p) at least one of the following amino acid substitutions in comparison to mature human tear lipocalin: Leu 105→Cys, Tyr, Trp, Glu, Arg, Ser, His, Ala, Val, Asp, Pro, Gly or Lys,   (q) at least one of the following amino acid substitutions in comparison to mature human tear lipocalin: Phe 28→Cys, Arg, Lys, Trp, Asp, Gly, His, Leu or Asn,   (r) at least one of the following amino acid substitutions in comparison to mature human tear lipocalin: Glu 30→Arg, Asp, Thr, Ser, Gly, Ala or Asn,   (s) at least one of the following amino acid substitutions in comparison to mature human tear lipocalin: Ile 57→Tyr, Trp, His, Gln, Thr or Arg, or   (t) at least one of the following amino acid substitutions in comparison to mature human tear lipocalin: Lys 83→Arg, Ser, Gln, Thr or Glu.   
     
     
         6 . The method according to  claim 1 , wherein the mutein comprises one of the following sets of amino acid substitutions in comparison to mature human tear lipocalin:
 (a) Arg 26→Phe; Asn 32→Ile; Glu 34→Thr; Leu 56→Met; Ser 58→Ala and Lys 83→Ser,   (b) Arg 26→Trp; Asn 32→Leu; Glu 34→Thr; Leu 56→Ser and Ser 58→Ala,   (c) Arg 26→His; Asn 32→Tyr; Glu 34→Thr; Leu 56→Ser; Ser 58→Arg and Lys 83→Gln;   (d) Arg 26→Phe; Asn 32→Met; Glu 34→Thr; Leu 56→Gln; Ser 58→Ala and Lys 83→Thr;   (e) Asn 32→Trp; Glu 34→Arg; Leu 56→Asn; Ser 58→Trp and Lys 83→Ser,   (f) Arg 26→Phe; Asn 32→Leu; Glu 34→Thr; Leu 56→Phe; Ser 58→Ala and Lys 83→Arg,   (g) Arg 26→Thr; Asn 32→Trp; Glu 34→Asn; Leu 56→His; Ser 58→Pro and Lys 83→Ser,   (h) Asn 32→Trp; Glu 34→Asn; Leu 56→Phe; Ser 58→Arg and Lys 83→Glu,   (i) Arg 26→Trp; Asn 32→Leu; Glu 34→Thr; Leu 56→Met; Ser 58→Ala and Lys 83→Ser, or   (j) Asn 32→Trp; Glu 34→Gly; Leu 56→Gln; Ser 58→Ala and Lys 83→Gln.   
     
     
         7 . The method according to  claim 1 , wherein the mutein comprises one of the following sets of amino acid substitutions in comparison to mature human tear lipocalin:
 (a) Glu 27→Ser; Phe 28→Arg; Pro 29→Gly; Glu 30→Asp; Met 31→Ala; Leu 33→Trp; Ile 57→Tyr; Asp 80→Met; Glu 104→Pro; Leu 105→Tyr; His 106→Gln; Lys 108→Ala,   (b) Glu 27→Gln; Phe 28→Cys; Pro 29→Asp; Glu 30→Thr; Met 31→Gly; Leu 33→Trp; Ile 57→Tyr; Leu 105→Cys; His 106→Gly; Lys 108→Trp,   (c) Glu 27→Glu; Phe 28→Trp; Pro 29→Asn; Glu 30→Gly; Met 31→His; Leu 33→Tyr; Ile 57→Tyr; Asp 80→Pro; Glu 104→Ser; Leu 105→Trp; His 106→Pro; Lys 108→Tyr,   (d) Glu 27→Thr; Phe28→Asp; Pro 29→Asn; Glu 30→Ser; Met 31→Pro; Leu 33→Phe; Ile 57→Tyr; Asp 80→Ile; Glu 104→Ala; Leu 105→Glu; His 106→Arg; Lys 108→Arg,   (e) Glu 27→Phe; Phe 28→Lys; Pro 29→Ile; Glu 30→Ala; Met 31→Ser; Leu 33→Pro; Ile 57→Trp; Asp 80→Gln; Glu 104→Asn; Leu 105→Arg; His 106→Gln; Lys 108→Asp,   (f) Glu 27→Lys; Phe 28→Gly; Pro 29→Pro; Glu 30→Thr; Met 31→Pro; Leu 33→Trp; Ile 57→His; Asp 80→Tyr; Glu 104→Ala; Leu 105→Ser; His 106→Val; Lys 108→Asn,   (g) Glu 27→Glu; Phe 28→His; Pro 29→Leu; Glu 30→Ala; Met 31→Asp; Leu 33→Ala; Ile 57→Gln; Asp 80→Ile; Glu 104→Ala; Leu 105→Tyr; His 106→Pro; Lys 108→Ser,   (h) Glu 27→Ala; Phe 28→Asp; Pro 29→Met; Glu 30→Gly; Met 31→Asp; Leu 33→Pro; Ile 57→Thr; Asp 80→Thr; Glu 104→Thr; His 106→Thr; Lys 108→Arg,   (i) Glu 27→Arg; Phe 28→Leu; Pro 29→Asp; Glu 30→Asn; Met 31→Glu; Leu 33→Trp; Ile 57→Tyr; Asp 80→Gln; Glu 104→Pro; Leu 105→Arg; His 106→Asn; Lys 108→Ala,   (j) Glu 27→Lys; Phe 28→Asn; Pro 29→Met; Glu 30→Gly; Met 31→Gln; Leu 33→Pro; Ile 57→Arg; Asp 80→Ile; Glu 104→Asp; Leu 105→Arg; His 106→Leu; Lys 108→Thr, or   (k) Glu 27→Ser; Phe 28→Arg; Pro 29→Gly; Glu 30→Asp; Met 31→Ala; Leu 33→Trp; Ile 57→Tyr; Asp 80→Met; Glu 104→Pro; Leu 105→Gly; His 106→Gln; Lys 108→Ala.   
     
     
         8 . The method according to  claim 1 , wherein the mutein comprises the following combination of amino acid substitutions:
 (a) Arg 26→Phe; Glu 27→Ser; Phe 28→Arg; Pro 29→Gly; Glu 30→Asp; Met 31→Ala; Asn 32→Ile; Leu 33→Trp; Glu 34→Thr; Leu 56→Met; Ile 57→Tyr; Ser 58→Ala; Lys 83→Ser; Glu 104→Pro and Lys 108→Thr, and   (b) optionally further comprises one or more of the following amino acid substitutions:   Thr 43→Ile or Ala; Glu 45→Gly; Asn 48→Gly; Glu 63→Gly; Ala 66→Vla; Glu 69→Vla; Lys 70→Arg; Ala 79→Thr, Met or Vla; Asp 80→Met or Ser; Gly 82→Ser; His 84→Gln; Vla 85→Gly; Tyr 87→Ser; Ile 88→Thr or Leu; His 92→Pro; Leu 105→His, Gly or Tyr; and His 106→Gln or Arg   in comparison to mature human tear lipocalin.   
     
     
         9 . The method according to  claim 1 , wherein the mutein comprises the following combination of amino acid substitutions:
 (a) Glu 27→Phe; Phe 28→Lys; Pro 29→Ile; Asn 32→Trp; Leu 33→Pro; Glu 34→Arg; Leu 56→Asn; Ile 57→Trp; His 106→Gln and Lys 108→Glu, and   (b) optionally further comprises one or more of the following amino acid substitutions:   Glu 43→Gly or Ala; Glu 45→Gly; Ser 58→Trp or Arg; Glu 63→Asp; Glu 69→Gly; Lys 70→Arg; Asp 80→Gln, Val or Thr; Gly 82→Asp; Lys 83→Ser or Arg; Ala 86→Glu or Ser; Phe 99→Leu; Glu 102→Lys or Val; Glu 104→Asn or Lys; and Pro 106→Thr   in comparison to mature human tear lipocalin.   
     
     
         10 . The method according to  claim 1 , wherein the mutein comprises an amino acid substitution of a native amino acid by a cysteine residue at positions 28 or 105 with respect to the amino acid sequence of mature human tear lipocalin. 
     
     
         11 . The method according to  claim 1 , wherein the mutein has at least 85% or 95% identity to the sequence of mature human tear lipocalin. 
     
     
         12 . The method according to  claim 1 , wherein the mutein has an amino acid sequence as set forth in any one of SEQ ID NOs: 3-28, 62-71 and 82. 
     
     
         13 . The method according to  claim 12 , wherein the mutein has the amino acid sequence as set forth in SEQ ID NO: 23, the amino acid sequence as set forth in SEQ ID NO: 13, the amino acid sequence as set forth in SEQ ID NO: 20, or the amino acid sequence as set forth in SEQ ID NO: 22. 
     
     
         14 . The method according to  claim 1 , wherein the mutein comprises four loops of one of SEQ ID NOs: 3-28, 62-71 and 82 which together form a binding pocket for PCSK9. 
     
     
         15 . The method according to  claim 14 , wherein the mutein comprises four loops of SEQ ID NO: 23 which together form a binding pocket for PCSK9, four loops of SEQ ID NO: 13 which together form a binding pocket for PCSK9, four loops of SEQ ID NO: 20 which together form a binding pocket for PCSK9, or four loops of SEQ ID NO: 22 which together form a binding pocket for PCSK9. 
     
     
         16 . The method according to  claim 1 , wherein the mutein is conjugated to a moiety that extends the serum half-life of the mutein. 
     
     
         17 . The method according to  claim 16 , wherein the mutein is conjugated to a polyalkylene glycol molecule. 
     
     
         18 . The method according to  claim 17 , wherein the conjugated mutein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 30-32. 
     
     
         19 . The method according to  claim 16 , wherein the mutein is conjugated to an albumin binding protein. 
     
     
         20 . The method according to  claim 19 , wherein the conjugated mutein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 83-84.