US2010092458A1PendingUtilityA1

Regulation of apg8 phosphorylation and uses thereof

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Assignee: WU CHUNPriority: Jul 18, 2008Filed: Jul 20, 2009Published: Apr 15, 2010
Est. expiryJul 18, 2028(~2 yrs left)· nominal 20-yr term from priority
Inventors:Chun-Wei WuJohn A. MountzourisBingren HuChunli Liu
C07K 5/1016C07K 5/1013C07K 5/101C07K 5/1021C07K 5/1024C07K 16/44A61P 37/02C07K 5/1008C07K 5/1027A61P 43/00C07K 5/1019
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Claims

Abstract

The invention relates to proteins and peptides of the alpha isoform of light chain 3 (APG8) and antibodies to the APG8 protein, particularly antibodies that specifically bind to the alpha isoform of APG8 when either phosphorylated or not phosphorylated at serine-12 and antibodies that bind to the gamma isoform of APG8 when either phosphorylated or not phosphorylated at serine-9. The invention also relates to methods of producing these antibodies and use of these antibodies in the treatment of diseases related to autophagocytosis.

Claims

exact text as granted — not AI-modified
1 . An isolated APG8 peptide comprising an amino acid sequence selected from the group consisting of QRRX (SEQ ID NO:11), RRXF (SEQ ID NO:12), RXFA (SEQ ID NO:13), XFAD (SEQ ID NO:14), KIPX (SEQ ID NO:15), IPXV (SEQ ID NO:16), PXVR (SEQ ID NO:17), and XVRP (SEQ ID NO:18) wherein X is serine or phosphoserine, and with the proviso that the peptide is not a full-length APG8 protein. 
     
     
         2 . An isolated APG8 peptide of  claim 1  wherein the peptide does not comprise a sequence of SEQ ID NO:1, 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, or SEQ ID NO:9. 
     
     
         3 . The isolated APG8 peptide of  claim 1 , which comprises an amino acid sequence selected from the group consisting of KQRRX (SEQ ID NO:19), QRRXF (SEQ ID NO:20), RRXFA (SEQ ID NO:21), RXFAD (SEQ ID NO:22), XFADR (SEQ ID NO:23), FKQRRX (SEQ ID NO:24), KQRRXF (SEQ ID NO:25), QRRXFA (SEQ ID NO:26), RRXFAD (SEQ ID NO:27), RXFADR (SEQ ID NO:28), PFKQRRX (SEQ ID NO:29), FKQRRXF (SEQ ID NO:30), KQRRXFA (SEQ ID NO:31), QRRXFAD (SEQ ID NO:32), RRXFADR (SEQ ID NO:33), RPFKQRRX (SEQ ID NO:34), PFKQRRXF (SEQ ID NO:35), FKQRRXFA (SEQ ID NO:36), KQRRXFAD (SEQ ID NO:37), QRRXFADR (SEQ ID NO:38), RPFKQRRX (SEQ ID NO:34), RPFKQRRXF (SEQ ID NO:39), PFKQRRXFA (SEQ ID NO:40), FKQRRXFAD (SEQ ID NO:41), KQRRXFADR (SEQ ID NO:42), SDRPFKQRRX (SEQ ID NO:43), RPFKQRRXF (SEQ ID NO:39), RPFKQRRXFA (SEQ ID NO:44), PFKQRRXFAD (SEQ ID NO:45), FKQRRXFADR (SEQ ID NO:46), RPFKQRRXFA (SEQ ID NO:44), RPFKQRRXFAD (SEQ ID NO:47), PFKQRRXFADR (SEQ ID NO:48), MPSDRPFKQRRX (SEQ ID NO:49), PSDRPFKQRRXF (SEQ ID NO:50), SDRPFKQRRXFA (SEQ ID NO:51), RPFKQRRXFAD (SEQ ID NO:47), RPFKQRRXFADR (SEQ ID NO:52), MPSDRPFKQRRXF (SEQ ID NO:53), PSDRPFKQRRXFA (SEQ ID NO:54), SDRPFKQRRXFAD (SEQ ID NO:55), RPFKQRRXFADR (SEQ ID NO:52), MPSDRPFKQRRXFA (SEQ ID NO:56), PSDRPFKQRRXFAD (SEQ ID NO:57), SDRPFKQRRXFADR (SEQ ID NO:58), MPSDRPFKQRRXFAD (SEQ ID NO:59), PSDRPFKQRRXFADR (SEQ ID NO:60), MPSDRPFKQRRXFADR (SEQ ID NO:61), QKIPX (SEQ ID NO:62), KIPXV (SEQ ID NO:63), IPXVR (SEQ ID NO:64), PXVRP (SEQ ID NO:65), XVRPF (SEQ ID NO:66), PQKIPX (SEQ ID NO:67), QKIPXV (SEQ ID NO:68), KIPXVR (SEQ ID NO:69), IPXVRP (SEQ ID NO:70), PXVRPF (SEQ ID NO:71), XVRPFK (SEQ ID NO:72), IPQKIPX (SEQ ID NO:73), PQKIPXV (SEQ ID NO:74), QKIPXVR (SEQ ID NO:75), KIPXVRP (SEQ ID NO:76), IPXVRPF (SEQ ID NO:77), PXVRPFK (SEQ ID NO:78), XVRPFKQ (SEQ ID NO:79), KIPQKIPX (SEQ ID NO:80), IPQKIPXV (SEQ ID NO:81), PQKIPXVR (SEQ ID NO:82), QKIPXVRP (SEQ ID NO:83), KIPXVRPF (SEQ ID NO:84), IPXVRPFK (SEQ ID NO:85), PXVRPFKQ (SEQ ID NO:86), QKIPQKIPX (SEQ ID NO:87), KIPQKIPXV (SEQ ID NO:88), IPQKIPXVR (SEQ ID NO:89), PQKIPXVRP (SEQ ID NO:90), QKIPXVRPF (SEQ ID NO:91), KIPXVRPFK (SEQ ID NO:92), IPXVRPFKQ (SEQ ID NO:93), PXVRPFKQR (SEQ ID NO:94), PQKIPQKIPX (SEQ ID NO:95), QKIPQKIPXV (SEQ ID NO:96), KIPQKIPXVR (SEQ ID NO:97), IPQKIPXVRP (SEQ ID NO:98), PQKIPXVRPF (SEQ ID NO:99), QKIPXVRPFK (SEQ ID NO:100), KIPXVRPFKQ (SEQ ID NO:101), IPXVRPFKQR (SEQ ID NO:102), PXVRPFKQRK (SEQ ID NO:103), XVRPFKQRKS (SEQ ID NO:104), PPQKIPQKIPX (SEQ ID NO:105), PQKIPQKIPXV (SEQ ID NO:106), QKIPQKIPXVR (SEQ ID NO:107), KIPQKIPXVRP (SEQ ID NO:108), IPQKIPXVRPF (SEQ ID NO:109), PQKIPXVRPFK (SEQ ID NO:110), QKIPXVRPFKQ (SEQ ID NO:111), KIPXVRPFKQR (SEQ ID NO:112), IPXVRPFKQRK (SEQ ID NO:113), PXVRPFKQRKS (SEQ ID NO:114), XVRPFKQRKSL (SEQ ID NO:115), PPPQKIPQKIPX (SEQ ID NO:116), PPQKIPQKIPXV (SEQ ID NO:117), PQKIPQKIPXVR (SEQ ID NO:118), QKIPQKIPXVRP (SEQ ID NO:119), KIPQKIPXVRPF (SEQ ID NO:120), IPQKIPXVRPFK (SEQ ID NO:121), PQKIPXVRPFKQ (SEQ ID NO:122), QKIPXVRPFKQR (SEQ ID NO:123), KIPXVRPFKQRK (SEQ ID NO:124), IPXVRPFKQRKS (SEQ ID NO:125), PXVRPFKQRKSL (SEQ ID NO:126), XVRPFKQRKSLA (SEQ ID NO:127), MPPPQKIPQKIPX (SEQ ID NO:128), PPPQKIPQKIPXV (SEQ ID NO:129), PPQKIPQKIPXVR (SEQ ID NO:130), PQKIPQKIPXVRP (SEQ ID NO:131), QKIPQKIPXVRPF (SEQ ID NO:132), KIPQKIPXVRPFK (SEQ ID NO:133), IPQKIPXVRPFKQ (SEQ ID NO:134), PQKIPXVRPFKQR (SEQ ID NO:135), QKIPXVRPFKQRK (SEQ ID NO:136), KIPXVRPFKQRKS (SEQ ID NO:137), IPXVRPFKQRKSL (SEQ ID NO:138), PXVRPFKQRKSLA (SEQ ID NO:139), XVRPFKQRSKLAI (SEQ ID NO:140), MPPPQKIPQKIPXVR (SEQ ID NO:141), PPPQKIPQKIPXVRP (SEQ ID NO:142), PPQKIPQKIPXVRPF (SEQ ID NO:143), PQKIPQKIPXVRPFK (SEQ ID NO:144), QKIPQKIPXVRPFKQ (SEQ ID NO:145), KIPQKIPXVRPFKQR (SEQ ID NO:146), IPQKIPXVRPFKQRK (SEQ ID NO:147), PQKIPXVRPFKQRKS (SEQ ID NO:148), QKIPXVRPFKQRKSL (SEQ ID NO:149), KIPXVRPFKQRKSLA (SEQ ID NO:150), IPXVRPFKQRKSLAI (SEQ ID NO:151), and PXVRPFKQRKSLAIR (SEQ ID NO:152). 
     
     
         4 . The isolated APG8 peptide of  claim 1 , which comprises an amino acid sequence selected from the group consisting of MPSDRPFKQRRXFADR (SEQ ID NO:61) and MPPPQKIPXVRPFKQRKSLAIR (SEQ ID NO:153). 
     
     
         5 . The isolated APG8 peptide of  claim 1 , wherein X is serine. 
     
     
         6 . The isolated APG8 peptide of  claim 1 , wherein X is phosphoserine. 
     
     
         7 . A method for producing an antibody to an APG8 polypeptide, which method comprises:
 (a) introducing an isolated APG8 peptide of  claim 1  to a mammal in an amount sufficient to produce an antibody to said APG8 peptide; and   (b) recovering said antibody from said mammal.   
     
     
         8 . The method of  claim 7 , wherein the X in the isolated APG8 peptide is serine and the method is used to produce an antibody to a nonphosphorylated APG8 polypeptide. 
     
     
         9 . The method of  claim 7 , wherein the X in the isolated APG8 peptide is phosphoserine and the method is used to produce an antibody to a phosphorylated APG8 polypeptide. 
     
     
         10 . The method of  claim 9 , which further comprises a step of removing an antibody that binds to an isolated APG8 peptide of embodiment 1 wherein X is serine. 
     
     
         11 . The method of  claim 7 , wherein the isolated APG8 peptide is conjugated to a carrier to enhance the peptide's immunogenicity. 
     
     
         12 . The method of  claim 7 , wherein the isolated APG8 peptide is comprised in an immunogen of embodiment 10 or a MAP of embodiment 12. 
     
     
         13 . An antibody to an APG8 polypeptide produced by the method of  claim 7 . 
     
     
         14 . An isolated antibody that specifically binds to an epitope that comprises the amino acid residue X in the amino acid sequence MPSDRPFKQRRXFADR (SEQ ID NO:61), wherein X is serine or phosphoserine. 
     
     
         15 . The isolated antibody of  claim 14 , wherein the epitope is comprised of an amino acid sequence selected from the group consisting of KQRRX (SEQ ID NO:19), QRRXF (SEQ ID NO:20), RRXFA (SEQ ID NO:21), RXFAD (SEQ ID NO:22), XFADR (SEQ ID NO:23), FKQRRX (SEQ ID NO:24), KQRRXF (SEQ ID NO:25), QRRXFA (SEQ ID NO:26), RRXFAD (SEQ ID NO:27), RXFADR (SEQ ID NO:28), PFKQRRX (SEQ ID NO:29), FKQRRXF (SEQ ID NO:30), KQRRXFA (SEQ ID NO:31), QRRXFAD (SEQ ID NO:32), RRXFADR (SEQ ID NO:33), RPFKQRRX (SEQ ID NO:34), PFKQRRXF (SEQ ID NO:35), FKQRRXFA (SEQ ID NO:36), KQRRXFAD (SEQ ID NO:37), QRRXFADR (SEQ ID NO:38), RPFKQRRX (SEQ ID NO:34), RPFKQRRXF (SEQ ID NO:39), PFKQRRXFA (SEQ ID NO:40), FKQRRXFAD (SEQ ID NO:41), KQRRXFADR (SEQ ID NO:42), SDRPFKQRRX (SEQ ID NO:43), RPFKQRRXF (SEQ ID NO:39), RPFKQRRXFA (SEQ ID NO:44), PFKQRRXFAD (SEQ ID NO:45), FKQRRXFADR (SEQ ID NO:46), RPFKQRRXFA (SEQ ID NO:44), RPFKQRRXFAD (SEQ ID NO:47), PFKQRRXFADR (SEQ ID NO:48), MPSDRPFKQRRX (SEQ ID NO:49), PSDRPFKQRRXF (SEQ ID NO:50), SDRPFKQRRXFA (SEQ ID NO:51), RPFKQRRXFAD (SEQ ID NO:47), RPFKQRRXFADR (SEQ ID NO:52), MPSDRPFKQRRXF (SEQ ID NO:53), PSDRPFKQRRXFA (SEQ ID NO:54), SDRPFKQRRXFAD (SEQ ID NO:55), RPFKQRRXFADR (SEQ ID NO:52), MPSDRPFKQRRXFA (SEQ ID NO:56), PSDRPFKQRRXFAD (SEQ ID NO:57), SDRPFKQRRXFADR (SEQ ID NO:58), MPSDRPFKQRRXFAD (SEQ ID NO:59), PSDRPFKQRRXFADR (SEQ ID NO:60), and MPSDRPFKQRRXFADR (SEQ ID NO:61). 
     
     
         16 . The isolated antibody of  claim 14 , wherein the epitope is comprised of an amino acid sequence selected from the group consisting of QRRX (SEQ ID NO:11), RRXF (SEQ ID NO:12), RXFA (SEQ ID NO:13), XFAD (SEQ ID NO:14), KIPX (SEQ ID NO:15), IPXV (SEQ ID NO:16), PXVR (SEQ ID NO:17), and XVRP (SEQ ID NO:18). 
     
     
         17 . An isolated antibody that specifically binds to an epitope that comprises the amino acid residue X in the amino acid sequence MPPPQKIPXVRPFKQRKSLAIR (SEQ ID NO:153), wherein X is serine or phosphoserine. 
     
     
         18 . The antibody of  claim 17 , wherein the epitope is comprised in an amino acid sequence selected from the group consisting of QKIPX (SEQ ID NO:62), KIPXV (SEQ ID NO:63), IPXVR (SEQ ID NO:64), PXVRP (SEQ ID NO:65), XVRPF (SEQ ID NO:66), PQKIPX (SEQ ID NO:67), QKIPXV (SEQ ID NO:68), KIPXVR (SEQ ID NO:69), IPXVRP (SEQ ID NO:70), PXVRPF (SEQ ID NO:71), XVRPFK (SEQ ID NO:72), IPQKIPX (SEQ ID NO:73), PQKIPXV (SEQ ID NO:74), QKIPXVR (SEQ ID NO:75), KIPXVRP (SEQ ID NO:76), IPXVRPF (SEQ ID NO:77), PXVRPFK (SEQ ID NO:78), XVRPFKQ (SEQ ID NO:79), KIPQKIPX (SEQ ID NO:80), IPQKIPXV (SEQ ID NO:81), PQKIPXVR (SEQ ID NO:82), QKIPXVRP (SEQ ID NO:83), KIPXVRPF (SEQ ID NO:84), IPXVRPFK (SEQ ID NO:85), PXVRPFKQ (SEQ ID NO:86), QKIPQKIPX (SEQ ID NO:87), KIPQKIPXV (SEQ ID NO:88), IPQKIPXVR (SEQ ID NO:89), PQKIPXVRP (SEQ ID NO:90), QKIPXVRPF (SEQ ID NO:91), KIPXVRPFK (SEQ ID NO:92), IPXVRPFKQ (SEQ ID NO:93), PXVRPFKQR (SEQ ID NO:94), PQKIPQKIPX (SEQ ID NO:95), QKIPQKIPXV (SEQ ID NO:96), KIPQKIPXVR (SEQ ID NO:97), IPQKIPXVRP (SEQ ID NO:98), PQKIPXVRPF (SEQ ID NO:99), QKIPXVRPFK (SEQ ID NO:100), KIPXVRPFKQ (SEQ ID NO:101), IPXVRPFKQR (SEQ ID NO:102), PXVRPFKQRK (SEQ ID NO:103), XVRPFKQRKS (SEQ ID NO:104), PPQKIPQKIPX (SEQ ID NO:105), PQKIPQKIPXV (SEQ ID NO:106), QKIPQKIPXVR (SEQ ID NO:107), KIPQKIPXVRP (SEQ ID NO:108), IPQKIPXVRPF (SEQ ID NO:109), PQKIPXVRPFK (SEQ ID NO:110), QKIPXVRPFKQ (SEQ ID NO:111), KIPXVRPFKQR (SEQ ID NO:112), IPXVRPFKQRK (SEQ ID NO:113), PXVRPFKQRKS (SEQ ID NO:114), XVRPFKQRKSL (SEQ ID NO:115), PPPQKIPQKIPX (SEQ ID NO:116), PPQKIPQKIPXV (SEQ ID NO:117), PQKIPQKIPXVR (SEQ ID NO:118), QKIPQKIPXVRP (SEQ ID NO:119), KIPQKIPXVRPF (SEQ ID NO:120), IPQKIPXVRPFK (SEQ ID NO:121), PQKIPXVRPFKQ (SEQ ID NO:122), QKIPXVRPFKQR (SEQ ID NO:123), KIPXVRPFKQRK (SEQ ID NO:124), IPXVRPFKQRKS (SEQ ID NO:125), PXVRPFKQRKSL (SEQ ID NO:126), XVRPFKQRKSLA (SEQ ID NO:127), MPPPQKIPQKIPX (SEQ ID NO:128), PPPQKIPQKIPXV (SEQ ID NO:129), PPQKIPQKIPXVR (SEQ ID NO:130), PQKIPQKIPXVRP (SEQ ID NO:131), QKIPQKIPXVRPF (SEQ ID NO:132), KIPQKIPXVRPFK (SEQ ID NO:133), IPQKIPXVRPFKQ (SEQ ID NO:134), PQKIPXVRPFKQR (SEQ ID NO:135), QKIPXVRPFKQRK (SEQ ID NO:136), KIPXVRPFKQRKS (SEQ ID NO:137), IPXVRPFKQRKSL (SEQ ID NO:138), PXVRPFKQRKSLA (SEQ ID NO:139), XVRPFKQRSKLAI (SEQ ID NO:140), MPPPQKIPQKIPXVR (SEQ ID NO:141), PPPQKIPQKIPXVRP (SEQ ID NO:142), PPQKIPQKIPXVRPF (SEQ ID NO:143), PQKIPQKIPXVRPFK (SEQ ID NO:144), QKIPQKIPXVRPFKQ (SEQ ID NO:145), KIPQKIPXVRPFKQR (SEQ ID NO:146), IPQKIPXVRPFKQRK (SEQ ID NO:147), PQKIPXVRPFKQRKS (SEQ ID NO:148), QKIPXVRPFKQRKSL (SEQ ID NO:149), KIPXVRPFKQRKSLA (SEQ ID NO:150), IPXVRPFKQRKSLAI (SEQ ID NO:151), and PXVRPFKQRKSLAIR (SEQ ID NO:152). 
     
     
         19 . The antibody of  claim 17 , wherein the epitope is comprised in an amino acid sequence selected from the group consisting of QRRX (SEQ ID NO:11), RRXF (SEQ ID NO:12), RXFA (SEQ ID NO:13), XFAD (SEQ ID NO:14), KIPX (SEQ ID NO:15), IPXV (SEQ ID NO:16), PXVR (SEQ ID NO:17), and XVRP (SEQ ID NO:18). 
     
     
         20 . The antibody of  claim 17 , wherein the isolated antibody specifically binds to the epitope that comprises the amino acid residue X in the amino acid sequence MPPPQKIPXVRPFKQRKSLAIR (SEQ ID NO:153), wherein X is serine. 
     
     
         21 . The antibody of  claim 17 , wherein the isolated antibody specifically binds to the epitope that comprises the amino acid residue X in the amino acid sequence MPPPQKIPXVRPFKQRKSLAIR (SEQ ID NO:153), wherein X is phosphoserine.

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