Compositions of adenosine deaminase-2 (ada2), variants thereof and methods of using same

Abstract

Provided are variant adenosine deaminase 2 (ADA2) proteins, conjugates thereof and compositions containing the proteins and/or conjugates. Also provided are methods and uses of the ADA2 proteins or conjugates for treating diseases and conditions, such as a tumor or cancer, and in particular any disease or condition associated with elevated adenosine or other associated marker.

1 . A variant adenosine deaminase 2 protein (ADA2) protein or a catalytically active portion thereof, comprising one or more modifications in the sequence of amino acids of an unmodified ADA2 polypeptide or a catalytically active portion thereof,
 the unmodified ADA2 protein comprises the sequence of amino acids set forth in SEQ ID NO:5 or a catalytically active portion thereof, or a sequence of amino acids that has at least 95% sequence identity to the sequence of amino acids set forth in SEQ ID NO:5 or a catalytically active portion thereof,   the amino acid modification(s) are selected from among amino acid replacement(s), deletion(s) and insertion(s);   the variant ADA2 protein comprises one or more amino acid replacements at an amino acid position corresponding to amino acid residue 11, 13, 20, 22, 26, 86, 109, 118, 119, 124, 133, 139, 179, 183, 191, 217, 219, 221, 224, 258, 262, 264, 266, 267, 277, 283, 296, 309, 317, 321, 352, 366, 371, 372, 373, 374, 403, 404, 405, 406, 441, 444, 452, 461, 469 or 470, with reference to amino acid positions set forth in SEQ ID NO:5;   the variant ADA2 protein that has at least 85% sequence identity to the sequence of amino acids set forth in SEQ ID NO:5 or to a corresponding catalytically active portion thereof,   the catalytically active portion comprises the PRB domain or a deletion of all or a portion of the PRB domain, and optionally comprises a linker in place of all or a portion of PRB domain;   the variant ADA2 protein, when in dimer form, exhibits one or more properties selected from among increased adenosine deaminase activity, reduced heparin binding, longer serum half-life, altered pH optimum, increased thermal stability, altered receptor binding, and hyperglycosylation compared to the corresponding dimer form of the unmodified ADA2 protein of SEQ ID NO:5 or dimer form of the corresponding catalytically activity portion thereof; and   the variant ADA2 protein or catalytically active portion thereof, when in dimer form, exhibits adenosine deaminase activity to convert adenosine to inosine.   
     
     
         2 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein:
 the variant ADA2 protein or catalytically active portion thereof has at least 95% sequence identity with the unmodified ADA2 protein of any of SEQ ID NOs: 326-330, and 380-383 or with a catalytically active portion of ADA2 protein of any of SEQ ID NOs: 326-330, and 380-383; and   the catalytically active portion includes all of the PRB, or deletion of all or a portion of the PRB.   
     
     
         3 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein the variant ADA2 protein or catalytically active portion thereof has at least 95% sequence identity with the unmodified ADA2 protein of SEQ ID NO: 5. 
     
     
         4 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein the variant ADA2 protein or catalytically active portion thereof exhibits altered heparin binding. 
     
     
         5 . The variant ADA2 protein or catalytically active portion thereof of  claim 4 , wherein the variant ADA2 protein or catalytically active portion thereof comprises a replacement at one or more of residues 11, 13, 20, 26, 217, 258, 277, 283, 309, 317, 321, 352, 366, 371, 372, 441, 444, 452, 461, 469 and 470, with reference to amino acid positions set forth in SEQ ID NO:5. 
     
     
         6 . The variant ADA2 protein or catalytically active portion thereof of  claim 5 , wherein the amino acid replacement is selected from among one or more of K11A, K11D, K11E, K13A, K13D, K13E, K371A, K371D, K371E, K372A, K372D, K372E, K452A, K452D, K452E, R20A, R20D, R20E, R366A, R366D, R366E, K11A/R20A, K11A/R20A/K371A, R20A/K371A, and K11A/K371A, with reference to amino acid positions set forth in SEQ ID NO:5. 
     
     
         7 . The variant ADA2 protein or catalytically active portion thereof of  claim 6 , wherein the amino acid replacement is selected from among one or more of R20E, K371D, K371E, K372D, K372E, K452D, K452E, and R366E, with reference to amino acid positions set forth in SEQ ID NO:5. 
     
     
         8 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein the variant ADA2 protein or catalytically active portion thereof exhibits altered catalytic efficiency (k cat /K m ) for adenosine or altered adenosine deaminase activity. 
     
     
         9 . The variant ADA2 protein or catalytically active portion thereof of  claim 8 , wherein the variant ADA2 protein or catalytically active portion thereof comprises a replacement at one or more of residues 86, 179, 219, 221, 262, 264, 266, 267, and 296, with reference to amino acid positions set forth in SEQ ID NO:5. 
     
     
         10 . The variant ADA2 protein or catalytically active portion thereof of  claim 9 , wherein the amino acid replacement is selected from among one or more of H264A; H264Q; H264N; H264G; R219K; R219Q; R219N; R219A; L221A; L221V; L221G; E179D; E179A; E179S; E179T; E179V; E179G; S262A; S262V; S262M; S262N; D86A; D86C; D86E; D86F; D86G; D86H; D86I; D86K; D86L; D86M; D86N; D86P; D86Q; D86R; D86S; D86T; D86V; D86W; D86Y; E179C; E179F; E179H; E179I; E179K; E179L; E179M; E179N; E179P; E179Q; E179R; E179W; E179Y; R219C; R219D; R219E; R219F; R219G; R219H; R219I; R219L; R219M; R219P; R219S; R219T; R219V; R219W; R219Y; L221C; L221D; L221E; L221F; L221H; L221I; L221K; L221M; L221N; L221P; L221Q; L221R; L221S; L221T; L221W; L221Y; S262C; S262D; S262E; S262F; S262G; S262H; S262I; S262K; S262L; S262P; S262Q; S262R; S262T; S262W; S262Y; H264C; H264D; H264E; H264F; H264I; H264K; H264L; H264M; H264P; H264R; H264S; H264T; H264V; H264W; H264Y; S266A; S266C; S266D; S266E; S266F; S266G; S266H; S266I; S266K; S266L; S266M; S266N; S266P; S266Q; S266R; S266T; S266V; S266W; S266Y; K267A; K267C; K267D; K267E; K267F; K267G; K267H; K267I; K267L; K267M; K267N; K267P; K267Q; K267R; K267S; K267T; K267V; K267W; K267Y; V296A; V296C; V296D; V296E; V296F; V296G; V296H; V296I; V296K; V296L; V296M; V296N; V296P; V296Q; V296R; V296S; V296T; V296W; and V296Y, with reference to amino acid positions set forth in SEQ ID NO:5. 
     
     
         11 . The variant ADA2 protein or catalytically active portion thereof of  claim 9 , wherein the amino acid replacement is selected from among one or more of H264Q, H264G, R219K, R219Q, R219N, L221A, L221V, L221G, S262M, and S262N, with reference to amino acid positions set forth in SEQ ID NO:5. 
     
     
         12 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein the variant ADA2 protein or catalytically active portion thereof has reduced binding to a receptor. 
     
     
         13 . The variant ADA2 protein or catalytically active portion thereof of  claim 12 , wherein the variant ADA2 protein or catalytically active portion thereof comprises a replacement or deletion in the putative receptor binding (PRB) domain. 
     
     
         14 . The variant ADA2 protein or catalytically active portion thereof of  claim 13 , wherein the variant ADA2 protein or catalytically active portion thereof comprises a replacement at one or more of residues 109, 118, 119, 124, 133, 139, 183, 191, and 224, with reference to amino acid positions set forth in SEQ ID NO:5. 
     
     
         15 . The variant ADA2 protein or catalytically active portion thereof of  claim 14 , wherein the variant ADA2 protein or catalytically active portion thereof comprises a replacement selected from one or more of F109S, F109A, R118D, R118A, F119S, F119K, P124A, P124S, W133S, W133T, Y139A, Y139T, F183K, Y191S, Y191D, Y191D/Y224R, Y224R, and Y224N, with reference to amino acid positions set forth in SEQ ID NO:5. 
     
     
         16 . The variant ADA2 protein or catalytically active portion thereof of  claim 13 , wherein the variant ADA2 protein or catalytically active portion thereof comprises a deletion of residues V99-Q144, or C105-T147, or N98-N156, or C105-E148, with reference to amino acid positions set forth in SEQ ID NO:5, or a portion thereof that eliminates binding to a receptor. 
     
     
         17 . The variant ADA2 protein or catalytically active portion thereof of  claim 16 , wherein the variant ADA2 protein or catalytically active portion thereof includes a linker comprising at least 4 amino acid residues in place of deleted residues. 
     
     
         18 . The variant ADA2 protein or catalytically active portion thereof of  claim 17 , wherein the linker comprises Gly and Ser residues or Gly residues. 
     
     
         19 . The variant ADA2 protein or catalytically active portion thereof of  claim 17 , wherein the linker is:
 (GGGGS) n , and n is 1-5; or   (Gly)n, where n=2 to 20.   
     
     
         20 . The variant ADA2 protein or catalytically active portion thereof of  claim 19 , wherein the variant ADA2 protein or catalytically active portion thereof comprises a replacement, deletion, and/or insertion selected from among K371D/V99-Q144del→(GGGGS) 1 , K371D/V99-Q144del→(GGGGS) 2 , K371D/V99-Q144del→(GGGGS) 3 , K371D/C105-T147del→(GGGGS) 1 , K371D/C105-T147del→(GGGGS) 2 , K371D/C105-T147del→(GGGGS) 3 , C105-T147del→(G)n, where n is 1-15, N98-N156del, C105-E148del, and C105-T147del, with reference to amino acid positions set forth in SEQ ID NO:5. 
     
     
         21 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein the variant ADA2 protein or catalytically active portion thereof comprises replacements that add one or more non-native glycosylation sites. 
     
     
         22 . The variant ADA2 protein or catalytically active portion thereof of  claim 21 , wherein the variant ADA2 protein or catalytically active portion thereof is hyperglycosylated and comprises the replacements selected from among R125N/P126A, S127N/K129S, P126N/E128T, R112N/1114T, 1134N/L135C/L136T, I134N/L135S/L136T, R142N/Q144S, P111N/G113S, and E137N/Y 139 T, with reference to amino acid positions set forth in SEQ ID NO:5. 
     
     
         23 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein the variant ADA2 protein or catalytically active portion thereof comprises one or more of a replacement, deletion, and insertion in two or more of: the putative receptor binding (PRB) domain to reduce binding to a receptor; the heparin binding domain to decrease heparin binding; and the active site to increase catalytic activity in an active dimer form of the variant ADA2 protein, compared to a dimer of the unmodified ADA2 protein. 
     
     
         24 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein the variant ADA2 protein or catalytically active portion thereof is PEGylated. 
     
     
         25 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein:
 the variant ADA2 is a multimer, comprising a plurality of variant ADA2 proteins or catalytically active portions thereof, and   the variant ADA2 protein or catalytically active portion thereof are the same or different.   
     
     
         26 . The variant ADA2 protein or catalytically active portion thereof of  claim 25 , wherein the variant ADA2 is a dimer, comprising a variant ADA2 protein or catalytically active portion thereof. 
     
     
         27 . The variant ADA2 protein or catalytically active portion thereof of  claim 26 , wherein the variant ADA2 dimer or catalytically active portion thereof is a homodimer comprising two variant ADA2 proteins or catalytically active portions thereof that are the same. 
     
     
         28 . The variant ADA2 protein or catalytically active portion thereof of  claim 26 , wherein the variant ADA2 dimer or catalytically active portion thereof is a heterodimer comprising two variant ADA2 proteins or catalytically active portions thereof that are different from each other. 
     
     
         29 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein the variant ADA2 protein or catalytically active portion, is linked by chemical or physical interaction directly or indirectly via a linker to a half-life extending moiety. 
     
     
         30 . The variant ADA2 protein or catalytically active portion thereof of  claim 29 , wherein:
 the half-life extending moiety is a PEG; and   the ADA2 protein or catalytically active portion thereof is PEGylated.   
     
     
         31 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein the ADA2 protein is PEGylated. 
     
     
         32 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein the ADA2 protein comprises an ADA2 dimer. 
     
     
         33 . The variant ADA2 protein or catalytically active portion thereof of  claim 32 , wherein the dimer is a homodimer. 
     
     
         34 . The variant ADA2 protein or catalytically active portion thereof of  claim 33 , wherein the dimer is PEGylated. 
     
     
         35 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein the ADA2 protein is modified by deletion of all or part of the putative receptor binding (PRB), whereby the ADA2 protein and dimer thereof do not bind to a growth factor receptor. 
     
     
         36 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein the ADA2 protein is modified, whereby heparin binding is reduced and catalytic activity is increased. 
     
     
         37 . The variant ADA2 protein or catalytically active portion thereof of  claim 35 , wherein the ADA2 protein is modified, whereby heparin binding is reduced and catalytic activity is increased. 
     
     
         38 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein the unmodified ADA2 protein comprises the sequence of amino acids set forth in any of SEQ ID NOs: 5, 326-330, and 380-383 or is a catalytically active portion thereof, wherein the catalytically active portion comprises the PRB domain or a deletion of all or a portion of the PRB domain. 
     
     
         39 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein:
 replacements that confer increased adenosine deaminase activity are selected from among K11E, R20E, R219K, R219Q, R219N, L221A, L221V, L221G, S262M, S262N, H264Q, H264G, R366D, R366E, K371D, K371E, K372A, K372D, K372E, K452D, K452E, K11A/R20A, and R20A/K371A, with reference to amino acid positions set forth in SEQ ID NO:5;   replacements that confer longer serum half-life are selected from among R20E and K371D, with reference to amino acid positions set forth in SEQ ID NO:5;   replacements that confer altered pH optimum are selected from among R20E, R219Q, S262N, K371D, K371E, R219Q/S262N, S262N/K371D, S262N/K371E, R20E/R219Q, R20E/S262N, R20E/S262N/K371D, R20E/S262N/K371E, R219Q/K371D, R219Q/K371E, R20E/R219Q/K371E, R20E/R219Q/K371D, R219Q/S262N/K371E, R219Q/S262N/K371D, R20E/R219Q/S262N, R20E/R219Q/S262N/K371E, and R20E/R219Q/S262N/K371D, with reference to amino acid positions set forth in SEQ ID NO:5;   the replacement that confers increased thermal stability is K371E, with reference to amino acid positions set forth in SEQ ID NO:5;   replacements that confer altered receptor binding are selected from among F119S, F119K, Y224R, Y224N, Y191S, Y191D, F183K, Y191D/Y224R, F109S, F109A, R118D, R118A, Y139T, Y139A, W133S, W133T, P124A, and P124S, with reference to amino acid positions set forth in SEQ ID NO:5; and   amino acid modifications that result in one or more additional glycosylation sites are selected from among --→N1/--→A2/--→S3, R20N/V22S, K371N/D373S, K372N/I374S, T403N/H405S, G404N/P406S, R125N/P126A, S127N/K129S, P126N/E128T, R112N/I114T, I134N/L135C/L136T, I134N/L135S/L, 136T, R142N/Q144S, E137N/Y139T, and P111N/G113 S, with reference to amino acid positions set forth in SEQ ID NO:5.   
     
     
         40 . The variant ADA2 protein or catalytically active portion thereof of  claim 39  that lacks all or a portion of the PRB domain. 
     
     
         41 . A variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein:
 the unmodified ADA2 protein is selected from among an ADA2 protein that comprises the sequence of amino acids set forth in any one of SEQ ID NOs: 326-330, and 380-383, or is a catalytically active portion thereof that contains the ADA domain, and optionally, lacks all or a portion of the PRB domain, and optionally contains a linker in place thereof,   the PRB domain consists of all or a portion of the residues corresponding to residues 98-156 of SEQ ID NO:5;   the ADA domain corresponds to residues 77-473 of the mature protein set forth in SEQ ID NO:5.   
     
     
         42 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein:
 the ADA protein, or variant ADA2 protein, or catalytically active portion thereof comprises a deletion of all or a portion of the PRB domain; and   the PRB domain consists of residues corresponding to residues 98-156 of SEQ ID NO:5.   
     
     
         43 . The variant ADA2 protein or catalytically active portion thereof of  claim 42 , wherein the ADA2 protein, variant ADA2 protein, or catalytically active portion thereof comprises a linker in place of the deleted PRB domain or in place of a portion of the deleted PRB domain. 
     
     
         44 . The variant ADA2 protein or catalytically active portion thereof of  claim 43 , wherein the linker is (GGGGS) n , and n is 1-5; or is (Gly)n, where n=2 to 20. 
     
     
         45 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein:
 the ADA2 protein, variant ADA2 protein, or catalytically active portion comprises the catalytic domain of the ADA2;   the catalytic domain consists of residues corresponding to residues 77-473 of the sequence of amino acids set forth in SEQ ID NO:5;   the ADA2 protein, variant ADA2 protein, or catalytically active portion comprises a deletion of all or a portion of the PRB domain; and   the PRB domain consists of residues corresponding to residues 98-156 of SEQ ID NO:5.   
     
     
         46 . The variant ADA2 protein or catalytically active portion thereof of  claim 1 , wherein the ADA2 protein, variant ADA2 protein, or catalytically active form thereof comprises a linker in place of all or a portion of the deleted PRB domain or the deleted portion thereof. 
     
     
         47 . The variant ADA2 protein or catalytically active portion thereof of  claim 46 , wherein the linker is (GGGGS) n , and n is 1-5; or is (Gly)n, where n=2 to 20.