Variant Asparaginase Polypeptides for Medical Use
Provided are various embodiments relating to variant asparaginase polypeptides with enhanced stability, pharmacodynamics, and/or reduced immunogenicity. The variant asparaginase polypeptides may be used as therapeutics in mammals, including human, canines, felines, and equines.
1 . A variant Erwinia chrysanthemi ( E. chrysanthemi ) asparaginase polypeptide comprising at least one cysteine substitution.
2 .- 3 . (canceled)
4 . The variant E. chrysanthemi asparaginase polypeptide of claim 1 , wherein the at least one cysteine or the at least one PEG-conjugatable amino acid is surface-exposed.
5 .- 7 . (canceled)
8 . The variant E. chrysanthemi asparaginase polypeptide of claim 1 , wherein the variant polypeptide lacks a leader sequence.
9 .- 13 . (canceled)
14 . The variant E. chrysanthemi asparaginase polypeptide of claim 1 , comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical a wildtype asparaginase amino acid sequence, such as SEQ ID NO: 2.
15 .- 16 . (canceled)
17 . The variant E. chrysanthemi asparaginase polypeptide of claim 1 , wherein the at least one cysteine, at least one amino acid substitution, or the at least one PEG-conjugatable amino acid substitution is located at a position corresponding to a position selected from position 3, 4, 17, 26, 37, 38, 41, 42, 44, 45, 47, 48, 51, 53, 54, 55, 56, 59, 60, 68, 72, 79, 82, 83, 84, 85, 87, 110, 112, 123, 124, 125, 127, 180, 190, 191, 192, 198, 202, 205, 206, 208, 210, 212, 213, 215, 216, 219, 231, 239, 240, 241, 243, 257, 260, 261, 264, 265, 267, 268, 269, 270, 280, 286, 287, 288, 289, 312, 313, 315, 316, 317, 318, and 322 of SEQ ID NO: 2.
18 .- 19 . (canceled)
20 . The variant E. chrysanthemi asparaginase polypeptide of claim 1 comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO 8, or SEQ ID NO: 9.
21 . (canceled)
22 . A tetramer comprising the variant E. chrysanthemi asparaginase polypeptide of claim 1 .
23 .- 47 . (canceled)
48 . The variant asparaginase polypeptide of claim 1 , wherein the variant asparaginase polypeptide is modified.
49 .- 65 . (canceled)
66 . A pharmaceutical composition comprising the variant asparaginase polypeptide of claim 1 , and a pharmaceutically acceptable carrier.
67 .- 71 . (canceled)
72 . The variant E. chrysanthemi asparaginase polypeptide of claim 1 comprising the amino acid sequence of SEQ ID NO: 4.
73 . The variant E. chrysanthemi asparaginase polypeptide of claim 1 comprising the amino acid sequence of SEQ ID NO: 5.
74 . The variant E. chrysanthemi asparaginase polypeptide of claim 1 consisting essentially of the amino acid sequence of SEQ ID NO: 4.
75 . The variant E. chrysanthemi asparaginase polypeptide of claim 1 consisting essentially of the amino acid sequence of SEQ ID NO: 5.
76 . The variant E. chrysanthemi asparaginase polypeptide of claim 1 consisting of the amino acid sequence of SEQ ID NO: 4.
77 . The variant E. chrysanthemi asparaginase polypeptide of claim 1 consisting of the amino acid sequence of SEQ ID NO: 5.
78 . The variant asparaginase polypeptide of claim 48 , wherein the modified variant asparaginase polypeptide is sialyated.
79 . The variant asparaginase polypeptide of claim 48 , wherein the modified variant asparaginase polypeptide is pegylated.
80 . The variant E. chrysanthemi asparaginase polypeptide of claim 1 , wherein the at least one cysteine, at least one amino acid substitution, or the at least one PEG-conjugatable amino acid substitution is located at a position corresponding to position 41 of SEQ ID NO: 2.
81 . The variant E. chrysanthemi asparaginase polypeptide of claim 1 , wherein the at least one cysteine, at least one amino acid substitution, or the at least one PEG-conjugatable amino acid substitution is located at a position corresponding to position 72 of SEQ ID NO: 2.
82 . The variant E. chrysanthemi asparaginase polypeptide of claim 1 , wherein the at least one cysteine, at least one amino acid substitution, or the at least one PEG-conjugatable amino acid substitution is located at a position corresponding to position 265 of SEQ ID NO: 2.