PEGYLATED KYNURENINASE ENZYMES AND USES THEREOF FOR THE TREATMENT OF CANCER
20210115416 ยท 2021-04-22
Inventors
Cpc classification
A61K9/0019
HUMAN NECESSITIES
A61K35/17
HUMAN NECESSITIES
A61K47/6803
HUMAN NECESSITIES
A61K47/10
HUMAN NECESSITIES
A61K47/60
HUMAN NECESSITIES
International classification
A61K35/17
HUMAN NECESSITIES
A61K47/10
HUMAN NECESSITIES
Abstract
Kynureninase enzymes covalently bound to polyethylene glycol are described. Aspects of the disclosure provide compositions and methods for improving the effective treatment of cancer by way of kynurenine depletion using such molecules.
Claims
1. A kynureninase homodimer covalently bound to one or more polyethylene glycol (PEG) molecules, wherein the ratio of PEG molecules to homodimer is from about 10:1 to about 40:1
2. The homodimer of claim 1, wherein the ratio of PEG molecules to homodimer is from about 20:1 to about 30:1, optionally wherein the ratio of PEG molecules to homodimer is about 20:1, 21:2, 22:1, 23:1, 24:1, or 25:1.
3. The homodimer of claim 1, wherein the ratio of PEG molecules to homodimer is about 25:1.
4. The homodimer of any one of claims 1-3, wherein the PEG molecule has a molecular weight from about 1 kDa to about 10 kDa.
5. The homodimer of claim 4, wherein the PEG molecule has molecular weight from about 3 kDa to about 7 kDa.
6. The homodimer of claim 5, wherein the PEG molecule has a molecular weight of about 5 kDa.
7. The homodimer of any one of claims 1-6, wherein the PEG molecule is linear.
8. The homodimer of any one of claims 1-6, wherein the PEG molecule is branched
9. The homodimer of any one of claims 1-8, wherein the homodimer is covalently bound to the PEG molecule by way of one or more lysine or cysteine residues.
10. The homodimer of claim 9, wherein the homodimer is covalently bound to the PEG molecule by way of one or more lysine residues.
11. The homodimer of any one of claims 1-10, wherein the homodimer comprises two polypeptide monomers each having an amino acid sequence that is at least 85% identical to SEQ ID NO: 1, and wherein each polypeptide monomer has one or more amino acid substitutions, relative to SEQ ID NO: 1, selected from the group consisting of L8P, K38E, Y47L, I48F, K50Q, I51M, S60N, K64N, D65G, E66K, N67D, N67P, D67S, A68F, A68T, A68V, F71L, F71M, L72N, K84E, E88N, E89K, E89S, E90Q, D92E, K93N, K93T, A95H, A95Q, K96N, I97H, 197L, I97V, A98G, A99G, A99I, A99R, A99S, A99T, A99V, Y100N, Y100S, Y100T, G101A, H102W, E103F, E103H, E103N, E103Q, E103R, E103V, E103W, V104D, V104E, V104F, V104H, V104K, V104L, V104R, G105A, G105H, G105S, G105T, E106D, K106D, K106E, K106H, K106N, R107P, R107S, P108R, I110A, I110F, I110L, I110M, I110T, T111 D, T111H, T111N, T111R, G112A, G112C, G112D, G112K, G112L, G112M, G112Q, G112R, G112S, G112T, G112Y, N127K, I131V, A132V, L133V, A136T, L137T, T138S, N140D, H142Q, Q14R, Y156H, K163T, D168E, H169R, Q175L, I183F, I183L, I183P, I183S, E184A, E184D, E184R, E184T, E184V, E185T, M187L, M189I, K191A, K191G, K191H, K191M, K191N, K191R, K191S, K191T, K191W, E197A, E197D, E197F, E197K, E197M, E197Q, E197S, E197T, E197V, I201C, I201E, I201F, I201H, I201L, I201S, I201T, I201V, H203K, L219M, L219W, F220L, V223I, F225Y, H230F, H230L, H230Y, N232S, Y246F, F249W, D250E, S274A, S274C, S274G, S274N, S274T, L278M, A280G, A280S, A280T, G281S, A282M, A282P, G284N, I285L, V303L, V303S, F306W, F306Y, S311N, K315E, D317E, D317K, I331C, I331L, I331N, I331S, I331T, I331V, N333T, P334N, P335T, L337T, L338A, L338Q, S341I, K373E, K373N, N375A, N375H, Y376C, Y376F, Y376L, K378G, K378P, K378Q, K378R, K380G, K380S, A382G, A382R, A382T, T383S, K384G, K384N, P386K, P386S, V387L, N389E, I405L, F407Y, S408D, S408N, N411R, D413S, D413V, Q416T, E419A, E419L, K420E, R421N, V424I, K427M, N429E, G432A, and A436T.
12. The homodimer of claim 11, wherein each polypeptide monomer has an amino acid sequence that is at least 90% identical to SEQ ID NO: 1.
13. The homodimer of claim 12, wherein each polypeptide monomer has an amino acid sequence that is at least 95% identical to SEQ ID NO: 1.
14. The homodimer of any one of claims 11-13, wherein each polypeptide monomer comprises a substitution at A282, F306 or F249 relative to SEQ ID NO: 1.
15. The homodimer of any one of claims 11-14, wherein each polypeptide monomer comprises a substitution at A99 relative to SEQ ID NO: 1.
16. The homodimer of any one of claims 11-15, wherein each polypeptide monomer comprises a substitution at G112 relative to SEQ ID NO: 1.
17. The homodimer of any one of claims 11-16, wherein each polypeptide monomer comprises a substitution at E103 relative to SEQ ID NO: 1.
18. The homodimer of any one of claims 11-17, wherein each polypeptide monomer comprises a substitution at V104 relative to SEQ ID NO: 1.
19. The homodimer of any one of claims 11-18, wherein each polypeptide monomer comprises a substitution at S408 relative to SEQ ID NO: 1.
20. The homodimer of any one of claims 11-19, wherein each polypeptide monomer comprises the F306W substitution.
21. The homodimer of any one of claims 11-20, wherein each polypeptide monomer comprises the L72N substitution.
22. The homodimer of any one of claims 11-21, wherein each polypeptide monomer comprises the H102W and N333T substitutions.
23. The homodimer of any one of claims 11-22, wherein each polypeptide monomer comprises the I183P substitution.
24. The homodimer of any one of claims 11-23, wherein each polypeptide monomer comprises the R107P substitution.
25. The homodimer of any one of claims 11-24, wherein each polypeptide monomer comprises the A436T substitution.
26. The homodimer of any one of claims 11-25, wherein each polypeptide monomer comprises at least one substitution selected from L72N, H102W, A282P, F306W, I331S and N333T relative to SEQ ID NO: 1.
27. The homodimer of claim 26, wherein each polypeptide monomer comprises at least two, three, four or five substitutions selected from L72N, H102W, A282P, F306W, I331S and N333T.
28. The homodimer of claim 27, wherein each polypeptide monomer comprises the substitutions L72N, H102W, A282P, F306W, I331S and N333T.
29. The homodimer of any one of claims 1-28, wherein the homodimer comprises two polypeptide monomers each having an amino acid sequence that is at least 85% identical to SEQ ID NO: 3.
30. The homodimer of claim 29, wherein each polypeptide monomer has an amino acid sequence that is at least 90% identical to SEQ ID NO: 3.
31. The homodimer of claim 30, wherein each polypeptide monomer has an amino acid sequence that is at least 95% identical to SEQ ID NO: 3.
32. The homodimer of claim 31, wherein each polypeptide monomer has the amino acid sequence of SEQ ID NO: 3.
33. The homodimer of any one of claims 1-28, wherein the homodimer comprises two polypeptide monomers each having an amino acid sequence that is at least 85% identical to SEQ ID NO: 2.
34. The homodimer of claim 33, wherein each polypeptide monomer has an amino acid sequence that is at least 90% identical to SEQ ID NO: 2.
35. The homodimer of claim 34, wherein each polypeptide monomer has an amino acid sequence that is at least 95% identical to SEQ ID NO: 2.
36. The homodimer of claim 35, wherein each polypeptide monomer has the amino acid sequence of SEQ ID NO: 2.
37. The homodimer of any of claims 1-36, wherein the homodimer has a catalytic activity (kcat/K.sub.M) of at least 8000M.sup.1 s.sup.1.
38. The homodimer of claim 37, wherein the homodimer has a catalytic activity (kcat/K.sub.M) of between about 8000M.sup.1 s.sup.1 and 40000 M.sup.1 s.sup.1; 10000M.sup.1 s.sup.1 and 40000 M.sup.1 s.sup.1; 20000M.sup.1 s.sup.1 and 40000 M.sup.1 s.sup.1; or 25000M.sup.1 s.sup.1 and 35000 M.sup.1 s.sup.1.
39. The homodimer of any one of claims 1-38, wherein the ratio of PEG molecules to homodimer is about 25:1, the PEG molecule has a molecular weight of about 5 kDa and is bound to the homodimer by way of an N-hydroxysuccinimide ester carbonate linking group.
40. A method of producing a PEGylated kynureninase homodimer, wherein the homodimer comprises two polypeptide monomers, the method comprising contacting the homodimer with a PEGylation agent, and wherein the molar input ratio of PEGylation agent to homodimer is from about 10:1 to about 50:1.
41. The method of claim 40, wherein the molar input ratio of PEGylation agent to homodimer is from about 20:1 to about 40:1, optionally wherein the molar input ratio of PEGylation agent to homodimer is from about 20:1 to about 30:1, optionally wherein the molar input ratio of PEGylation agent to homodimer is about 20:1.
42. The method of claim 40, wherein the molar input ratio of PEGylation agent to homodimer is from about 25:1 to about 35:1.
43. The method of claim 40, wherein the molar input ratio of PEGylation agent to homodimer is about 30:1.
44. The method of any one of claims 40-43, wherein the PEG molecule has a molecular weight from about 1 kDa to about 10 kDa.
45. The method of claim 44, wherein the PEG molecule has molecular weight from about 3 kDa to about 7 kDa.
46. The method of claim 45, wherein the PEG molecule has a molecular weight of about 5 kDa.
47. The method of any one of claims 40-46, wherein the PEG molecule is linear.
48. The method of any one of claims 40-46, wherein the PEG molecule is branched
49. The method of any one of claims 40-48, wherein the homodimer is covalently bound to the PEG molecule by way of one or more lysine or cysteine residues.
50. The method of claim 49, wherein the homodimer is covalently bound to the PEG molecule by way of one or more lysine residues.
51. The method of any one of claims 40-50, wherein each polypeptide monomer has an amino acid sequence that is at least 85% identical to SEQ ID NO: 1, and wherein each polypeptide monomer has one or more amino acid substitutions, relative to SEQ ID NO: 1, selected from the group consisting of L8P, K38E, Y47L, I48F, K50Q, I51M, S60N, K64N, D65G, E66K, N67D, N67P, D67S, A68F, A68T, A68V, F71L, F71M, L72N, K84E, E88N, E89K, E89S, E90Q, D92E, K93N, K93T, A95H, A95Q, K96N, I97H, 197L, I97V, A98G, A99G, A99I, A99R, A99S, A99T, A99V, Y100N, Y100S, Y100T, G101A, H102W, E103F, E103H, E103N, E103Q, E103R, E103V, E103W, V104D, V104E, V104F, V104H, V104K, V104L, V104R, G105A, G105H, G105S, G105T, E106D, K106D, K106E, K106H, K106N, R107P, R107S, P108R, I110A, I110F, I110L, I110M, I110T, T111D, T111H, T111N, T111R, G112A, G112C, G112D, G112K, G112L, G112M, G112Q, G112R, G112S, G112T, G112Y, N127K, I131V, A132V, L133V, A136T, L137T, T138S, N140D, H142Q, Q14R, Y156H, K163T, D168E, H169R, Q175L, I183F, I183L, I183P, I183S, E184A, E184D, E184R, E184T, E184V, E185T, M187L, M189I, K191A, K191G, K191H, K191M, K191N, K191R, K191S, K191T, K191W, E197A, E197D, E197F, E197K, E197M, E197Q, E197S, E197T, E197V, I201C, I201E, I201F, I201H, I201L, I201S, I201T, I201V, H203K, L219M, L219W, F220L, V223I, F225Y, H230F, H230L, H230Y, N232S, Y246F, F249W, D250E, S274A, S274C, S274G, S274N, S274T, L278M, A280G, A280S, A280T, G281S, A282M, A282P, G284N, I285L, V303L, V303S, F306W, F306Y, S311N, K315E, D317E, D317K, I331C, I331L, I331N, I331S, I331T, I331V, N333T, P334N, P335T, L337T, L338A, L338Q, S341I, K373E, K373N, N375A, N375H, Y376C, Y376F, Y376L, K378G, K378P, K378Q, K378R, K380G, K380S, A382G, A382R, A382T, T383S, K384G, K384N, P386K, P386S, V387L, N389E, I405L, F407Y, S408D, S408N, N411R, D413S, D413V, Q416T, E419A, E419L, K420E, R421N, V424I, K427M, N429E, G432A, and A436T.
52. The method of claim 51, wherein each polypeptide monomer has an amino acid sequence that is at least 90% identical to SEQ ID NO: 1.
53. The method of claim 52, wherein each polypeptide monomer has an amino acid sequence that is at least 95% identical to SEQ ID NO: 1.
54. The method of any one of claims 40-53, wherein each polypeptide monomer comprises a substitution at A282, F306 or F249.
55. The method of any one of claims 40-54, wherein each polypeptide monomer comprises a substitution at A99 relative to SEQ ID NO: 1.
56. The method of any one of claims 40-55, wherein each polypeptide monomer comprises a substitution at G112 relative to SEQ ID NO: 1.
57. The method of any one of claims 40-56, wherein each polypeptide monomer comprises a substitution at E103 relative to SEQ ID NO: 1.
58. The method of any one of claims 40-57, wherein each polypeptide monomer comprises a substitution at V104 relative to SEQ ID NO: 1.
59. The method of any one of claims 40-58, wherein each polypeptide monomer comprises a substitution at S408 relative to SEQ ID NO: 1.
60. The method of any one of claims 51-59, wherein each polypeptide monomer comprises the F306W substitution.
61. The method of any one of claims 51-60, wherein each polypeptide monomer comprises the L72N substitution.
62. The method of any one of claims 51-61, wherein each polypeptide monomer comprises the H102W and N333T substitutions.
63. The method of any one of claims 51-62, wherein each polypeptide monomer comprises the I183P substitution.
64. The method of any one of claims 51-63, wherein each polypeptide monomer comprises the R107P substitution.
65. The method of any one of claims 51-64, wherein each polypeptide monomer comprises the A436T substitution.
66. The method of any one of claims 40-65, wherein each polypeptide monomer comprises at least one substitution selected from L72N, H102W, A282P, F306W, I331S and N333T relative to SEQ ID NO: 1.
67. The method of claim 66, wherein each polypeptide monomer comprises at least two, three, four or five substitutions selected from L72N, H102W, A282P, F306W, I331S and N333T relative to SEQ ID NO: 1.
68. The method of claim 67, wherein each polypeptide monomer comprises the substitutions L72N, H102W, A282P, F306W, I331S and N333T relative to SEQ ID NO: 1.
69. The method of any one of claims 40-68, wherein each polypeptide monomer has an amino acid sequence that is at least 85% identical to SEQ ID NO: 3.
70. The method of claim 69, wherein each polypeptide monomer has an amino acid sequence that is at least 90% identical to SEQ ID NO: 3.
71. The method of claim 70, wherein each polypeptide monomer has an amino acid sequence that is at least 95% identical to SEQ ID NO: 3.
72. The method of claim 71, wherein each polypeptide monomer has the amino acid sequence of SEQ ID NO: 3.
73. The method of any one of claims 40-68, wherein each polypeptide monomer has an amino acid sequence that is at least 85% identical to SEQ ID NO: 2.
74. The method of claim 73, wherein each polypeptide monomer has an amino acid sequence that is at least 90% identical to SEQ ID NO: 2.
75. The method of claim 74, wherein each polypeptide monomer has an amino acid sequence that is at least 95% identical to SEQ ID NO: 2.
76. The method of claim 75, wherein each polypeptide monomer has the amino acid sequence of SEQ ID NO: 2.
77. The method of any of claims 40-76, wherein the homodimer has a catalytic activity (kcat/K.sub.M) of at least 8000M.sup.1 s.sup.1.
78. The method of claim 77, wherein the homodimer has a catalytic activity (kcat/K.sub.M) of between about 8000M.sup.1 s.sup.1 and 40000 M.sup.1 s.sup.1; 10000M.sup.1 s.sup.1 and 40000 M.sup.1 s.sup.1; 20000M.sup.1 s.sup.1 and 40000 M.sup.1 s.sup.1; or 25000M.sup.1 s.sup.1 and 35000 M.sup.1 s.sup.1.
79. The method of claim 78, wherein the ratio of PEG molecules to homodimer is about 25:1, the PEG molecule has a molecular weight of about 5 kDa and is bound to the homodimer by way of an N-hydroxysuccinimide ester carbonate linking group.
80. The method of any of claims 40-79, wherein the homodimer is contacted with the PEGylation agent in an aqueous buffer.
81. The method of claim 80, wherein the aqueous buffer comprises disodium phosphate.
82. The method of claim 80, wherein the aqueous buffer comprises sodium acetate.
83. The method of any one of claims 80-82, wherein the aqueous buffer ranges in pH from about 7.5 to about 9.5.
84. The method of any one claims 80-83, wherein the aqueous buffer ranges in ionic strength from about 250 mM to about 350 mM.
85. The method of any one of claims 40-84, wherein size exclusion chromatography is used to isolate the homodimer.
86. A kynureninase homodimer produced by the method of any one of claims 40-85.
87. A pharmaceutical formulation comprising the kynureninase homodimer of any one of claims 1-39 and 86 in a pharmaceutically acceptable carrier.
88. A method of treating a subject having a tumor comprising administering to a subject an effective amount of the formulation of claim 87 or the enzyme of any one of claims 1-39 and 86.
89. The method of claim 88, wherein the subject has been identified as having an IDO1, IDO2, or TDO expressing tumor.
90. The method of claim 88 or 89, wherein the tumor is a solid tumor.
91. The method of claim 88 or 89, wherein the tumor is a hematological tumor.
92. The method of any of one of claims 88-91, wherein the subject is a human patient.
93. The method of any one of claims 88-92, wherein the formulation is administered intratumorally, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, orally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, or via a lavage.
94. The method of any one of claims 88-93, further comprising administering a second anti-cancer therapy.
95. The method of claim 94, wherein the second anticancer therapy is radiation therapy, surgical therapy, an immunotherapy or a second anticancer compound.
96. The method of claim 95, wherein the second anticancer compound is an immune checkpoint inhibitor.
97. The method of claim 95, wherein the second anticancer compound comprises an anti-PDI, anti-CTLA-4, or anti-PD-LI antibody.
98. The method of claim 95, wherein the second anticancer compound is an antibody.
99. The method of claim 95, wherein the second anticancer compound is an antibody-drug conjugate.
100. The method of claim 95, wherein the immunotherapy comprises administering immune effector cells or an immunogenic composition.
101. The method of claim 100, wherein the immunogenic composition comprises cancer cell antigens.
102. The method of claim 100, wherein the immune effector cells comprise NK-cells or T-cells.
103. The method of claim 100, wherein the immune effector cells comprise CAR T-cells.
104. A method of providing a T-cell response in a human subject having a tumor, the method comprising administering to the subject a kynureninase homodimer in accordance with the method of any one of claims 88-103.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0081] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0082]
[0083] All molar input ratios shown in
[0084]
[0085] All molar input ratios shown in
DETAILED DESCRIPTION
[0086] The present disclosure provides kynureninase proteins that are covalently bound to polyethylene glycol (PEG), as well as methods of synthesizing such proteins and methods of using the same for the treatment of patients having cancer, such as those that have tumors. In particular, the disclosure provides PEGylated kynureninase proteins characterized by ratios of PEG to kynureninase that effectuate enhanced pharmacokinetic and/or pharmacodynamic properties, such as improved tumor accumulation, prolonged half-life, and greater enzymatic active as compared to kynureninase proteins that are not covalently bound to PEG. Without being limited by mechanism, the kynureninase proteins described herein may exhibit increased resistance to deactivation in serum, which allows for the kynureninase to maintain activity for kynurenine degradation for an increased number of hours. Thus, PEGylated kynureninase enzymes characterized by the PEG:kynureninase ratios described herein may exhibit substantially improved therapeutic properties that make them ideal for therapeutic disease intervention, such as the treatment of cancer. In some embodiments, the kynureninase proteins of the disclosure are used to treat cancer patients, such as those with tumors that express indolamine 2,3 dioxygenase (IDO) or tryptophan 2,3 dioxygenase (TDO).
[0087] The sections that follow describe the ratios of PEG:kynureninase polypeptide that result in the advantageous therapeutic properties described above, as well as processes that can be used to produce such proteins and to use the same for treating patients.
Kynureninase Proteins
[0088] Some embodiments of the PEGylated kynureninase enzymes concern modified proteins and polypeptides. Particular embodiments concern a modified protein or polypeptide that exhibits at least one functional activity that is comparable to the unmodified version, preferably, the kynurenine degrading activity. In further embodiments, the protein or polypeptide may be further modified to increase serum stability.
[0089] Determination of activity may be achieved using assays familiar to those of skill in the art, particularly with respect to the protein's activity and may include for comparison purposes, the use of native and/or recombinant versions of either the modified or unmodified protein or polypeptide.
[0090] In some embodiments, a modified polypeptide, such as a modified kynureninase, may be identified based on its increase in kynurenine. For example, substrate recognition sites of the unmodified polypeptide may be identified. This identification may be based on structural analysis or homology analysis. A population of mutants involving modifications of such substrate recognition sites may be generated. In a further embodiment, mutants with increased kynurenine degrading activity may be selected from the mutant population. Selection of desired mutants may include methods, such as detection of byproducts or products from kynurenine degradation.
[0091] Modified proteins may possess deletions and/or substitutions of amino acids; thus, a protein with a deletion, a protein with a substitution, and a protein with a deletion and a substitution are modified proteins. In some embodiments, these modified proteins may further include insertions or added amino acids, such as with fusion proteins or proteins with linkers. A modified deleted protein lacks one or more residues of the native protein, but may possess the specificity and/or activity of the native protein. A modified deleted protein may also have reduced immunogenicity or antigenicity. An example of a modified deleted protein is one that has an amino acid residue deleted from at least one antigenic region that is, a region of the protein determined to be antigenic in a particular organism, such as the type of organism that may be administered the modified protein.
[0092] Substitution or replacement variants typically contain the exchange of one amino acid for another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide, particularly its effector functions and/or bioavailability. Substitutions may or may not be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
[0093] In addition to a deletion or substitution, a modified protein may possess an insertion of residues, which typically involves the addition of at least one residue in the polypeptide. This may include the insertion of a targeting peptide or polypeptide or simply a single residue. Terminal additions, called fusion proteins, are discussed below.
[0094] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5 or 3 sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5 or 3 portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
[0095] In some embodiments, a kynureninase according to the embodiments comprises and amino acid sequence at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the kynureninase of SEQ ID NOs: 1, 2 or 3. In still further embodiments, a kynureninase is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the kynureninase of SEQ ID NOs: 1, 2 or 3 and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 of the amino acid substitutions selected from the group consisting of L8P, K38E, Y47L, I48F, K50Q, I51M, S60N, K64N, D65G, E66K, N67D, N67P, D67S, A68F, A68T, A68V, F71L, F71M, L72N, K84E, E88N, E89K, E89S, E90Q, D92E, K93N, K93T, A95H, A95Q, K96N, I97H, I97L, I97V, A98G, A99G, A99I, A99R, A99S, A99T, A99V, Y100N, Y100S, Y100T, G101A, H102W, E103F, E103H, E103N, E103Q, E103R, E103V, E103W, V104D, V104E, V104F, V104H, V104K, V104L, V104R, G105A, G105H, G105S, G105T, E106D, K106D, K106E, K106H, K106N, R107P, R107S, P108R, I110A, I110F, I110L, I110M, I110T, T111D, T111H, T111N, T111R, G112A, G112C, G112D, G112K, G112L, G112M, G112Q, G112R, G112S, G112T, G112Y, N127K, I131V, A132V, L133V, A136T, L137T, T138S, N140D, H142Q, Q14R, Y156H, K163T, D168E, H169R, Q175L, I183F, I183L, I183P, I183S, E184A, E184D, E184R, E184T, E184V, E185T, M187L, M189I, K191A, K191G, K191H, K191M, K191N, K191R, K191S, K191T, K191W, E197A, E197D, E197F, E197K, E197M, E197Q, E197S, E197T, E197V, I201C, I201E, I201F, I201H, I201L, I201S, I201T, I201V, H203K, L219M, L219W, F220L, V223I, F225Y, H230F, H230L, H230Y, N232S, Y246F, F249W, D250E, S274A, S274C, S274G, S274N, S274T, L278M, A280G, A280S, A280T, G281S, A282M, A282P, G284N, I285L, V303L, V303S, F306W, F306Y, S311N, K315E, D317E, D317K, I331C, I331L, I331N, I331S, I331T, I331V, N333T, P334N, P335T, L337T, L338A, L338Q, S341I, K373E, K373N, N375A, N375H, Y376C, Y376F, Y376L, K378G, K378P, K378Q, K378R, K380G, K380S, A382G, A382R, A382T, T383S, K384G, K384N, P386K, P386S, V387L, N389E, I405L, F407Y, S408D, S408N, N411R, D413S, D413V, Q416T, E419A, E419L, K420E, R421N, V424I, K427M, N429E, G432A and A436T. In some embodiments, a kynureninase is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one of the kynureninases provided in Table 2. In some embodiments, the kynureninase is selected from one of those provided in Table 2.
[0096] Table 2, below, sets forth various kynureninase enzymes that may be used in conjunction with the compositions and methods of the present disclosure. Although not listed explicitly in Table 2, Protein (1) herein refers to wild-type human kynureninase, the amino acid sequence of which is set forth in SEQ ID NO: 1.
TABLE-US-00002 Wild-typehumankynureninase: SEQIDNO:1 MEPSSLELPADTVQRIAAELKCHPTDERVALHLDEEDKLRHFRECFYIPK IQDLPPVDLSLVNKDENAIYFLGNSLGLQPKMVKTYLEEELDKWAKIAAY GHEVGKRPWITGDESIVGLMKDIVGANEKEIALMNALTVNLHLLMLSFFK PTPKRYKILLEAKAFPSDHYAIESQLQLHGLNIEESMRMIKPREGEETLR IEDILEVIEKEGDSIAVILFSGVHFYTGQHFNIPAITKAGQAKGCYVGFD LAHAVGNVELYLHDWGVDFACWCSYKYLNAGAGGIAGAFIHEKHAHTIKP ALVGWFGHELSTRFKMDNKLQLIPGVCGFRISNPPILLVCSLHASLEIFK QATMKALRKKSVLLTGYLEYLIKHNYGKDKAATKKPVVNIITPSHVEERG CQLTITFSVPNKDVFQELEKRGVVCDKRNPNGIRVAPVPLYNSFHDVYKF TNLLTSILDSAETKN
TABLE-US-00003 TABLE2 Exemp1arykynureninaseenzymesofthedisclosure Protein ListofMutations 2 N67D/L72N/E103Q/M189I/F225Y/S274G/I331V/I405L/S408N 3 A99I/G112A/F306Y/I331N/I405L/S408N/A436T 4 Q14R/A99I/G112A/M189I/H230Y/F306Y/I331N/I405L/S408N/A436T 5 N67D/L72N/E103Q/A99I/G112A/M189I/F225Y/S274G/I331V/F306Y/K380S/A382R/ K384N/P386K/I405L/S408N/A436T 6 N67D/L72N/A99I/H102W/E103F/V104E/K106E/R107S/I110M/A136T/M189I/F225Y/ S274G/I331C/N333T/S341I/I405L/S408N 7 N67D/L72N/A99I/H102W/E103F/V104E/K106E/R107S/I110M/I131V/L133V/A136T/ T138S/M189I/F225Y/G274T/L278M/G281S/A282P/F306W/K315E/D317E/I331C/N333T/ S341I/I405L/S408N 8 N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/A132V/ A136T/M189I/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/ I405L/S408N 9 A99I/G112A/G284N/I285L/F306Y/I331N/I405L/S408N/A436T 10 A99I/G112A/K191N/F306Y/I331N/I405L/S408N/A436T 11 L8P/A99I/G112A/Q175L/F306Y/I331N/K373E/K378R/I405L/S408N/A436T 12 A99I/G112A/F306Y/I331N/K380S/A382R/K384N/P386K/I405L/S408N/A436T 13 A99I/G112A/F306Y/I331N/N375H/Y376L/K378P/I405L/S408N/A436T 14 N67D/A68T/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110L/T111H/G112Y/ A136T/M189I/F225Y/S274G/F306W/I331C/N333T/S341I/I405L/S408N 15 N67D/L72N/A99I/H102W/E103F/V104E/K106E/R107S/I110M/A132V/A136T/M189I/ F225Y/G274S/A280S/G281S/A282P/F306W/I331C/N333T/S341I/I405L/S408N 16 N67D/F71L/L72N/A99I/H102W/E103FN104E/E106D/R107S/M110I/G112Y/A136T/ M189I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/I405L/S408N 17 D67N/A68V/L72N/A99I/H102W/E103F/V104E/K106E/R107S/M110I/T111H/G112Y/ A136T/M189I/F225Y/G274T/A280S/G281S/A282P/F306W/K315E/I331C/N333T/ S341I/I405L/S408N 18 D67N/L72N/A99I/H102W/E103F/V104E/K106E/R107S/I110M/T111H/G112Y/A136T/ M189I/F225Y/G274S/A280S/G281S/A282P/F306W/K315E/I331C/N333T/S341I/ I405L/S408N 19 N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/A132V/ A136T/M189I/V223I/F225Y/S274G/A280S/G281S/A282P/V303L/F306W/S311N/ K315E/I331C/N333T/S341I/I405L/S408N 20 D67S/L72N/A99I/H102W/E103F/V104E/K106E/R107S/I110M/A132V/L133V/A136T/ M189I/F225Y/G274A/A280S/A282P/F306W/I331C/N333T/S341I/I405L/S408N 21 D67N/L72N/A99I/H102W/E103F/V104E/K106E/R107S/I110M/T111H/G112Y/A136T/ M189I/F225Y/G274N/L278M/A282P/F306W/K315E/I331C/N333T/S341I/I405L/S408N 22 D67N/A68V/L72N/A99I/H102W/E103F/V104E/K106E/R107S/M110I/A136T/M189I/ F225Y/G274T/G281S/A282P/F306W/I331C/N333T/S341I/I405L/S408N 23 N67D/F71L/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ A136T/M189I/V223I/F225Y/G274S/A280S/G281S/A282P/F306W/K315E/I331C/ N333T/S341I/I405L/S408N 24 D67N/L72N/A99I/H102W/E103F/V104E/K106E/R107S/I110M/T111H/G112Y/A136T/ M189I/F225Y/G274S/A280S/G281S/A282P/F306W/D317E/I331C/N333T/S341I/ I405L/S408N 25 N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/A132V/ A136T/V223I/M189I/F225Y/S274G/A280S/G281S/A282P/V303S/F306W/K315E/ D317K/I331C/N333T/S341I/I405L/S408N 26 N67D/A68T/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ A136T/M189I/F225Y/G274N/A280S/A282P/F306W/I331C/N333T/S341I/I405L/S408N 27 L72N/A99V/Y100N/H102W/E103F/V104E/K106D/R107S/T111H/G112Y/L133V/A136T/ F225Y/S274N/A280S/G281S/A282P/F306W/I331C/N333T 28 L72N/A99V/Y100N/H102W/E103F/V104E/K106D/R107S/T111N/G112Y/I131V/L133V/ A136T/F225Y/S274T/A280S/G281S/A282P/F306W/I331C/N333T 29 L72N/A99V/Y100N/H102W/E103F/V104E/K106D/R107S/I110M/T111N/G112Y/I131V/ L133V/A136T/F225Y/S274N/A280S/G281S/A282P/F306W/I331C/N333T 30 N67D/F71L/L72N/A99V/Y100N/H102W/E103F/V104E/K106D/R107S/I110M/T111H/ G112Y/A136T/F225Y/S274G/A280S/G281S/A282P/F306W/I331C/N333T 31 L72N/E90Q/A99V/Y100N/H102W/E103F/V104E/K106D/R107S/I110M/T111D/G112Y/ A136T/F225Y/S274N/A280S/G281S/A282M/F306W/I331C/N333T 32 Y100T/H102W/E103F/V104E/K106D/R107S/T111H/G112Y/A136T/M189I/F225Y/A280G/ A282P/F306W/I331C/N333T 33 A99S/Y100S/G101A/H102W/E103F/V104E/K106D/R107S/I110M/T111H/G112Y/A136T/ F225Y/A280T/A282P/F306W/I331C/N333T/K373N 34 K191S-E197T-I201H 35 K191R-E197S-I201T 36 K191S-E197T-I201T 37 I183P-E184A 38 K191R-E197F-I201T 39 K191R-I201E 40 K191W-E197V-I201E 41 K191H-E197V-I201E 42 E197M-I201L 43 K191G-E197V-I201H 44 K191T-E1970-I201E 45 K191H-E197V-I201E 46 N67D/A68T/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ A136T/M189I/F225Y/G274N/A280S/A282P/F306W/I331C/N333T/S341I/K373N/ I405L/S408N 47 L72N/E90Q/A99V/Y100N/H102W/E103F/V104E/K106D/R107S/I110M/T111D/G112Y/ A136T/F225Y/S274N/A280S/G281S/A282M/F306W/I331C/N333T/K373N 48 N67D/A68T/L72N/E88N/E89K/D92E/K93T/A95Q/K96N/I97H/A98G/A99I/H102W/E103F/ V104E/E106D/R107S/M110L/T111H/G112Y/A136T/M189I/F225Y/A280S/A282P/F306W/ I331L/N333T/P335T/L338A/S341I/I405L/S408N/E419A/K420E/R421N/V424I 49 Y47L/I48F/K50Q/del51-62/K64N/D65G/E66K/N67P/A68F/L72N/E88N/E89S/E90Q/ K93N/K96N/I97L/A99I/H102W/E103F/V104E/E106D/R107S/M110L/T111H/G112Y/ A136T/M189I/F225Y/A280G/A282P/F306W/I331C/N333T/S341I/I405L/S408N 50 Q14R/K38E/I51M/N67D/A68T/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110L/ T111H/G112Y/N127K/A136T/M189I/K191N/E197T/F225Y/H230F/S274G/F306W/ I331C/N333T/S341I/I405L/S408N 51 S60N/N67D/L72N/K84E/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/ G112Y/A132V/A136T/Y156H/K163T/M1891/V223I/F225Y/S274G/A280S/G281S/A282P/ I331C/N333T/S341I/I405L/S408N 52 N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/A132V /A136T/M189I/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/L338Q/ S341I/I405L/S408N 53 N67D/L72N/A99I/Y100N/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y /A132V/A136T/M189I/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/ S341I/I405L/S408N 54 N67D/L72N/I97V/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ A132V/A136T/N140D/M189I/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/ S341I/I405L/S408N 55 N67D/A68T/L72N/E90Q/K93N/A95H/K96N/I97L/A99I/H102W/E103F/V104E/E106D/ R107S/M110L/T111H/G112Y/A136T/M189I/F225Y/A280S/A282P/F306W/I331C/ N333T/S341I/I405L/S408N 56 N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/A132V/ A136T/M189I/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/ I405L/S408N 57 N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/A132V/ A136T/D168E/M189I/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/N333T/S341I/ I405L/S408N 58 N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/A132V /A136T/M189I/V223I/F225Y/D250E/S274G/A280S/G281S/A282P/I331C/N333T/ S341I/I405L/S408N 59 N67D/A68T/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ A136T/D168E/M189I/F225Y/G274N/A280S/A282P/F306W/I331C/N333T/S341I/ I405L/S408N 60 N67D/A68T/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/ A136T/M189I/F225Y/D250E/G274N/A280S/A282P/F306W/I331C/N333T/S341I/ I405L/S408N 61 L72N/A99I/H102W/G112Y/A282P/F306W/I331S/N333T 62 L72N/A99I/H102W/G112Y/A282P/F306W/I331S/N333T/P334N 63 L72N/A99I/H102W/E103F/E106D/M110I/T111H/G112Y/A132V/A136T/M189I/A282P/ I331C/N333T/S341I/S408N 64 N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/T111H/G112Y/A136T/M189I/ A280S/A282P/F306W/I331C/N333T/I405L/S408N 65 N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/A132V/ A136T/M189I/K191S/E197T/I201H/V223I/F225Y/S274G/A280S/G281S/A282P/ I331C/N333T/S341I/I405L/S408N 66 N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/A132V/ A136T/M189I/K191R/E197F/I201T/V223I/F225Y/S274G/A280S/G281S/A282P/ I331C/N333T/S341I/I405L/S408N 67 N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/A132V/ A136T/M189I/K191SW/E197V/I201E/V223I/F225Y/S274G/A280S/G281S/A282P/ I331C/N333T/S341I/I405L/S408N 68 N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/A132V/ A136T/M189I/E197M/I201L/V223I/F225Y/S274G/A280S/G281S/A282P/I331C/ N333T/S341I/I405L/S408N 69 N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/A132V/ A136T/M189I/K191H/E197V/I201E/V223I/F225Y/S274G/A280S/G281S/A282P/ I331C/N333T/S341I/I405L/S408N 70 N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/A132V/ A136T/M189I/K191R/E197S/I201T/V223I/F225Y/S274G/A280S/G281S/A282P/ I331C/N333T/S341I/I405L/S408N 71 N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/A132V/ A136T/M189I/K191G/E197V/I201H/V223I/F225Y/S274G/A280S/G281S/A282P/ I331C/N333T/S341I/I405L/S408N 72 N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/A132V/ A136T/M189I/K191S/E197T/I201T/V223I/F225Y/S274G/A280S/G281S/A282P/ I331C/N333T/S341I/I405L/S408N 73 N67D/L72N/A99I/H102W/E103F/V104E/E106D/R107S/M110I/T111H/G112Y/A132V/ A136T/M189I/K191G/E197A/I201E/V223I/F225Y/S274G/A280S/G281S/A282P/ I331C/N333T/S341I/I405L/S408N 74 L72N/A99S/H102W/E103F/V104K/K106H/R107S/G112Y/A282P/F306W/I331C/N333T 75 N67D/L72N/A99I/H102W/K106D/S274G/A280S/G281S/A282P/F306W/I331S/N333T 76 N67D/A68T/L72N/A99I/H102W/E103F/V104E/K106D/R107S/I110L/T111H/G112Y/ A136T/M189I/F225Y/S274N/A280S/A282P/F306W/I331C/N333T/S341I/I405L/S408N 77 L72N/A99I/H102W/E103V/V104E/K106D/R107S/T111H/G112Y/A282P/F306W/I331S/ N333T 78 L72N/A99V/H102W/E103F/V104E/K106N/T111N/A280S/A282P/F306W/I331S/N333T 79 L72N/A99I/H102W/G112Y/A282P/F306W/I331S/N333T/T383S/D413V 80 L72N/A99I/Y100N/H102W/G112Y/H169R/M187L/Y246F/A282P/F306W/I331S/N333T/ A382T 81 L72N/A99I/H102W/V104R/G105H/R107P/G112Y/A282P/F306W/I331S/N333T 82 L72N/H102W/E103N/K106D/R107P/T111R/G112Y/A282P/F306W/I331T/N333T 83 L72N/A99I/H102W/G112Y/A282P/F306W/I331S/N333T/Y376F/K3780 84 L72N/A99I/H102W/G112Y/K191S/E197D/I201E/A282P/F306W/I331S/N333T 85 L72N/A99I/H102W/G112Y/K191A/I201V/A282P/F306W/I331S/N333T 86 L72N/A99I/H102W/G112Y/I183P/E184A/M187L/M189I/K191N/E197T/I201T/A282P/ F306W/I331S/N333T 87 F71M/L72N/E103F/V104H/G105T/I183P/E184A/M187L/M189I/K191N/E197T/I201T/ A2805/A282P/F306Y 88 L72N/H102W/G112Y/A282P/F306W/I331S/N333T 89 L72N/G101A/H102W/G112Y/E184A/A282P/F306W/I331S/N333T 90 L72N/A99I/H102W/G112Y/I183P/E184A/A282P/F306W/I331S/N333T 91 L72N/A99I/H102W/G112Y/I183L/M189I/K191S/I201T/A282P/F306W/I331S/N333T 92 L72N/A99I/H102W/G112Y/M187L/M189I/K191R/I201T/A282P/F306W/I331S/N333T 93 L72N/A99V/E103Q/H102W/G112Y/I183P/E184A/E197A/I201T/A282P/F306W/I331S/ N333T 94 L72N/A99I/H102W/E103Q/V104D/G105S/G112Y/I183P/E184A/A282P/F306W/I331S/ N333T 95 L72N/A99I/H102W/G112Y/I183P/A282P/F306W/I331S/N333T 96 L72N/A99V/H102W/E103Q/V104H/G112Y/I183P/M189I/K191S/A282P/F306W/I331S/ N333T 97 L72N/H102W/R107P/G112Y/I183P/E184A/E197A/A282P/F306W/I331S/N333T 98 L72N/A99T/H102W/G112Y/I183P/E184A/I201T/A282P/F306W/I331S/N333T 99 L72N/A99I/H102W/G112Y/A136T/L137T/F225Y/A282P/F306W/I331S/N333T/F407Y 100 L72N/A99I/H102W/G112Y/A282P/F306W/I331S/N333T/I405L/S408D/A436T 101 L72N/A99I/H102W/G112Y/I183P/E184A/M187L/M189I/K191N/A282P/F306W/I331S/ N333T 102 L72N/A99V/H102W/E103Q/G112Y/I183P/E184A/E197A/I201T/A282P/F306W/I331S/ N333T 103 L72N/A99V/H102W/E103Q/V104H/G105A/R107P/G112Y/M187L/K191S/I201T/A282P/ F306W/I331S/N333T 104 L72N/H102W/V104D/G105A/G112Y/I183F/E184T/M189I/K191S/I201T/A282P/F306W/ I331S/N333T 105 L72N/A99V/H102W/V104H/G105A/G112Y/M187L/M189I/K191S/I201T/A282P/F306W/ I331S/N333T 106 L72N/A99I/H102W/V104D/G105S/G112Y/I183P/M187L/M189I/I201T/A282P/F306W/ I331S/N333T 107 L72N/A99V/H102W/E103Q/V104D/G112Y/I183P/E184A/M189I/K191R/I201T/A280S/ A282P/F306W/I331S/N333T 108 L72N/A99I/H102W/E103Q/V104H/G112Y/I183L/M189I/A282P/F306W/I331S/N333T 109 L72N/A99I/H102W/G112Y/I183S/E183A/M189I/K191N/I201T/A282P/F306W/I331S/ N333T 110 L72N/A99I/H102W/G112Y/A136T/L137T/A282P/F306W/I331S/N333T 111 L72N/A99I/H102W/G112Y/A136T/L137T/F220L/F225Y/H230L/A282P/F306W/I331S/ N333T 112 L72N/A99I/H102W/G112Y/L137T/F220L/H203K/A282P/F306W/I331S/N333T 113 L72N/A99I/H102W/G112Y/A136T/L137T/F225Y/A282P/F306W/I331S/N333T/F407Y 114 L72N/A99I/H102W/R107P/G112Y/I183L/E184A/A282P/F306W/I331S/N333T 115 L72N/A99V/H102W/E103Q/V104H/G105S/R107P/G112Y/M187L/K191R/I201T/A282P/ F306W/I331S/N333T 116 L72N/A99I/H102W/G112Y/I183L/E184R/M187L/M189I/K191N/E197K/I201T/A282P/ F306W/I331S/N333T 117 L72N/A99I/H102W/G112Y/E184D/M187L/M189I/K191R/E197A/I201T/A282P/F306W/ I331S/N333T 118 L72N/A99I/H102W/G112Y/I183S/E184V/M187L/M189I/K191R/A282P/F306W/I331S/ N333T 119 L72N/A99V/H102W/V104D/G112Y/E184A/M187L/M189I/K191R/E197A/A282P/F306W/ I331S/N333T 120 L72N/A99V/H102W/V104H/G105A/G112Y/M189I/K191S/I201T/A282P/F306W/I331S/ N333T 121 L72N/A99I/H102W/E103Q/V104D/G105S/G112Y/I183P/M189I/K191S/I201T/A280S/ A282P/F306W/I331S/N333T 122 L72N/A99V/H102W/E103Q/R107P/G112Y/I183P/E184A/I201T/A282P/F306W/I331S/ N333T 123 L72N/A99T/H102W/E103F/R107P/G112Y/I183P/E184N1201T/A282P/S724Q/F306W/ I331S/N333T 124 L72N/A99V/H102W/R107P/G112Y/A282P/F306W/I331S/N333T 125 L72N/A99V/H102W/E103Q/V104H/G105A/R107P/G112Y/M187L/K191S/I201T/A282P/ F306W/I331S/N333T/I405L/S408D 126 L72N/A99V/H102W/E103Q/V104D/G112Q/I183P/E184A/M189I/K191R/I201T/A280S/ A282P/F306W/I331S/N333T 127 L72N/A99V/H102W/E103Q/V104H/R107P/G112Y/I183P/A282P/F306W/I331S/N333T/ S408N/A436T 128 L72N/A99V/H102W/E103Q/V104H/G105A/R107P/G112L/M187L/K191S/I201T/F220L/ F225Y/A282P/F306W/I331S/N333T 129 L72N/A99V/H102W/E103Q/V104H/G105A/R107P/G112L/A136T/M187L/K191S/I201T/ H230L/A282P/F306W/I331S/N333T 130 L72N/A99V/H102W/E103Q/V104H/G105A/R107P/G112Q/M187L/K191S/I201T/F220L/ F225Y/A282P/F306W/I331S/N333T 131 L72N/A99V/H102W/E103Q/V104D/G112R/I183P/E184A/M189I/K191R/I201T/A280S/ A282P/F306W/I331S/N333T 132 L72N/A99V/H102W/V104H/G105A/M189I/K191S/I201T/A282P/F306W/I331S/N333T 133 L72N/A99V/H102W/E103Q/V104D/R107P/G112Q/I201E/A282P/F306W/I331S/N333T 134 L72N/A99V/H102W/E103Q/V104D/G112L/M187L/I201T/A282P/F306W/I331S/N333T 135 L72N/A99V/H102W/E103Q/V104L/G112D/K191S/A282P/F306W/I331S/N333T/S408N 136 L72N/A99I/H102W/E103Q/V104L/G112Y/A136T/K191S/A282P/F306W/I331S/N333T/ S408N/A436T 137 L72N/A99V/H102W/E103Q/V104L/R107P/G112Y/A136T/I183P/K191S/I201T/A282P/ F306W/I331S/N333T 138 L72N/A99V/H102W/E103Q/V104H/R107P/G112Y/H142Q/K191S/I201T/A282P/F306W/ I331S/N333T 139 L72N/A99I/H102W/E103Q/V104H/G112D/K191S/I201T/F225Y/A282P/F306W/I331S/ N333T/S408N 140 L72N/A99V/H102W/E103Q/V104H/G105A/R107P/G112Y/M187L/K191S/I201T/A282P/ F306W/I331S/N333T/S408D 141 L72N/A99G/H102W/E103H/V104H/R107P/G112Y/I183P/A282P/F306W/I331S/N333T/ S408N/A436T 142 L72N/A99R/H102W/E103R/V104H/R107P/G112Y/I183P/A282P/F306W/I331S/N333T/ (SEQIDNO:3) S408N/A436T 143 L72N/A99G/H102W/E103W/V04H/R107P/G112Y/I183P/A282P/F306W/I331S/N333T/ 5408N/A436T 144 L72N/A99V/H102W/E103Q/V104H/R107P/I183P/A282P/F306W/I331S/N333T/S408N/ A436T 145 L72N/A99V/H102W/E103Q/V104H/R107P/G112Y/N140D/I183P/A282P/F306W/I331S/ N333T/S408N/A436T 146 L72N/A99V/H102W/E103Q/V104H/R107P/I110F/G112Y/I183P/A282P/F306W/I331S/ N333T/S408N/A436T 147 L72N/A99V/H102W/E103Q/V104H/R107P/G112L/I183P/A282P/F306W/I331S/N333T/ S408N/A436T 148 L72N/A99V/H102W/E103Q/V104H/R107P/G112K/I183P/A282P/F306W/I331S/N333T/ S408N/A436T 149 L72N/A99V/H102W/E103Q/V104H/R107P/T111R/G112R/I183P/A282P/F306W/I331S/ N333T/S408N/A436T 150 L72N/A99V/H102W/E103Q/V104H/R107P/G112Y/I183P/L219M/A282P/F306W/I331S/ N333T/S408N/A436T 151 L72N/A99V/H102W/E103Q/V104H/G105A/R107P/T111N/Y112A/I183P/A282P/F306W/ I331S/N333T/S408N/A436T 152 L72N/A99V/H102W/E103Q/V104H/R107P/G112Y/I183P/E184A/M189I/K191R/I201T/ L219W/A282P/F306W/I331S/N333T/S408N/A436T 153 L72N/A99V/H102W/E103Q/V104H/R107P/G112Y/D168E/I183P/A282P/F306W/I331S/ N333T/S408N/A436T 154 L72N/A99V/H102W/E103Q/V104H/R107P/G112Y/I183P/E184A/M189I/K191R/I201T/ F249W/A282P/F306W/I331S/N333T/S408N/A436T 155 L72N/A99V/H102W/E103Q/V104H/R107P/G112Y/I183P/F249W/A282P/F306W/I331S/ N333T/S408N/A436T 156 Identica1AAsequenceto(126) 157 L72N/A99R/H102W/E103R/V104H/R107P/G112Y/I183P/F249W/A282P/F306W/I331S/ (SEQIDNO:2) N333T/5408N/A436T 158 L72N/A99G/H102W/E103/VN104H/R107P/G112Y/I183P/F249W/A282P/F306W/I331S/ N333T/S408N/A436T 159 L72N/A99G/H102W/E103W/V104H/R107P/Y112K/I183P/F249W/A282P/F306W/I331S/ N333T/S408N/A436T 160 L72N/A99G/H102W/E103W/V104F/R107P/G112Y/I183P/A282P/F306W/I331S/N333T/ S408N/A436T 161 L72N/A99G/H102W/E103/V104H/R107P/G112Y/I183P/A282P/F306W/I331S/N333T/ S408N/A436TANDArginineInsertionbetweenP107andP108 162 L72N/A99G/H102W/E103W/V104H/R107P/P108R/G112Y/I183P/A282P/F306W/I331S/ N333T/S408N/A436T-ANDLeucineinsertionbetweenP107andR108 163 L72N/A99G/H102W/E103W/V104H/R107P/Y112K/I183P/A282P/F306W/I331S/N333T/ S408N/A436T 164 L72N/A99G/H102W/E103W/V104H/R107P/G112Y/I183P/K191M/A282P/F306W/I331S/ N333T/S408N/A436T 165 L72N/A99G/H102W/E103W/V104H/R107P/G112Y/I183P/M189I/I201S/N232S/A282P/ F306W/I331S/N333T/S408N/A436T 166 L72N/A99G/H102W/E103W/V104H/R107P/G112Y/I183P/E184A/M189I/K191R/I201F/ A282P/F306W/I331S/N333T/S408N/A436T 167 F71M/L72N/E103F/V104H/G105T/I110T/G112T/I183P/E184A/M187L/M189I/K191N/ E197T/I201T/A280S/A282P/F306Y 168 I183P/E184A/M187L/M189I/K191S/E197T/I201T/N375A/Y376Q/K378G/K380G/A382G/ K384G/P3865/N411R/D413S/Q416T/E419L/K427M/N429E/G432A/A436T 169 A99I/G1125/F306Y/I405L/5408N/A436T/I183P/E185T/M189I/K191N/N375H/Y376L/ K378P/K3805/A382R/K384N/P386KN387L/N389E 170 L72N/A99R/H102W/E103R/V104D/G112M/I183P/A282P/F306W/I331S/N333T/5408N/ A436T 171 L72N/A99G/H102W/E103W/V104H/R107P/G112M/I183P/I201F/F249W/A282P/F306W/ I331S/N333T/5408N/A436T 172 L72N/A99G/H102W/E103W/V104H/R107P/G112Y/I183P/M189I/I201C/F249W/A282P/ F306W/I331S/N333T/S408N/A436T 173 L72N/A99R/H102W/E103R/V104H/R107P/G112Y/I183P/A282P/F306W/I331S/N333T/ L337T/S408N/A436T 174 L72N/A99R/H102W/E103R/V104H/R107P/I110A/G112Y/I183P/A280G/A282P/F306W/ I331S/N333T/L337T/S408N/A436T
Conjugates
[0097] Compositions and methods of the present disclosure involve modified kynureninases, such as by forming conjugates with heterologous peptide segments or polymers, such as polyethylene glycol (PEG). The kynureninase is linked to PEG to increase the hydrodynamic radius of the enzyme and hence increase the serum persistence. In some embodiments, the disclosed polypeptide may be conjugated to any targeting agent, such as a ligand having the ability to specifically and stably bind to an external receptor or binding site on a tumor cell (U.S. Patent Publ. 2009/0304666).
PEGylation
[0098] In some embodiments of the disclosure, methods and compositions related to PEGylation of kynureninase are disclosed. For example, the kynureninase is PEGylated in accordance with the methods disclosed herein.
[0099] PEGylation is the process of covalent attachment of poly(ethylene glycol) polymer chains to another molecule, normally a drug or therapeutic protein. PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target macromolecule. The covalent attachment of PEG to a drug or therapeutic protein can mask the agent from the host's immune system (reduced immunogenicity and antigenicity) or increase the hydrodynamic size (size in solution) of the agent, which prolongs its circulatory time by reducing renal clearance. PEGylation can also provide water solubility to hydrophobic drugs and proteins. The first step of the PEGylation is the suitable functionalization of the PEG polymer at one or both terminals. PEGs that are activated at each terminus with the same reactive moiety are known as homobifunctional, whereas if the functional groups present are different, then the PEG derivative is referred as heterobifunctional or heterofunctional. The chemically active or activated derivatives of the PEG polymer are prepared to attach the PEG to the desired molecule.
[0100] The choice of the suitable functional group for the PEG derivative is based on the type of available reactive group on the molecule that will be coupled to the PEG. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. The N-terminal amino group and the C-terminal carboxylic acid can also be used. The techniques used to form first generation PEG derivatives are generally reacting the PEG polymer with a group that is reactive with hydroxyl groups, typically anhydrides, acid chlorides, chloroformates, and carbonates. In the second generation PEGylation chemistry more efficient functional groups, such as aldehyde, esters, amides, etc., are made available for conjugation.
[0101] As applications of PEGylation have become more and more advanced and sophisticated, there has been an increase in need for heterobifunctional PEGs for conjugation. These heterobifunctional PEGs are very useful in linking two entities, where a hydrophilic, flexible, and biocompatible spacer is needed. Preferred end groups for heterobifunctional PEGs are maleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acids, and NHS esters.
[0102] The most common modification agents, or linkers, are based on methoxy PEG (mPEG) molecules. Their activity depends on adding a protein-modifying group to the alcohol end. In some instances, polyethylene glycol (PEG diol) is used as the precursor molecule. The diol is subsequently modified at both ends in order to make a hetero- or homo-dimeric PEG-linked molecule.
[0103] Proteins are generally PEGylated at nucleophilic sites, such as unprotonated thiols (cysteinyl residues) or amino groups. Examples of cysteinyl-specific (i.e., cystine-reactive) modification reagents include PEG maleimide, PEG iodoacetate, PEG thiols, and PEG vinylsulfone. All four are strongly cysteinyl-specific under mild conditions and neutral to slightly alkaline pH.
[0104] Amine-specific (i.e., lysine-reactive) modification agents include PEG NHS ester, PEG tresylate, PEG aldehyde, PEG isothiocyanate, and several others. All react under mild conditions and are very specific for amino groups. The PEG NHS ester is probably one of the more reactive agents; however, its high reactivity can make the PEGylation reaction difficult to control on a large scale. PEG aldehyde forms an imine with the amino group, which is then reduced to a secondary amine with sodium cyanoborohydride. Unlike sodium borohydride, sodium cyanoborohydride will not reduce disulfide bonds. However, this chemical is highly toxic and must be handled cautiously, particularly at lower pH where it becomes volatile.
[0105] Due to the multiple lysine residues on most proteins, site-specific PEGylation can be a challenge. Because these reagents react with unprotonated amino groups, it is possible to direct the PEGylation to lower-pK amino groups by performing the reaction at a lower pH. Generally, the pK of the alpha-amino group is 1-2 pH units lower than the epsilon-amino group of lysine residues. By PEGylating the molecule at pH 7 or below, high selectivity for the N-terminus frequently can be attained. However, this is only feasible if the N-terminal portion of the protein is not required for biological activity. Still, the pharmacokinetic benefits from PEGylation frequently outweigh a significant loss of in vitro bioactivity, resulting in a product with much greater in vivo bioactivity regardless of PEGylation chemistry.
[0106] There are several parameters to consider when developing a PEGylation procedure. For thiol-specific PEGylation reactions, parameters to consider include: protein concentration, PEG-to-protein ratio (on a molar basis), temperature, pH, reaction time, and in some instances, the exclusion of oxygen. (Oxygen can contribute to intermolecular disulfide formation by the protein, which will reduce the yield of the PEGylated product.) The same factors should be considered (with the exception of oxygen) for amine-specific modification except that pH may be even more critical, particularly when targeting the N-terminal amino group.
[0107] For both amine- and thiol-specific modifications, the reaction conditions may affect the stability of the protein. This may limit the temperature, protein concentration, and pH. In addition, the reactivity of the PEG linker should be known before starting the PEGylation reaction. For example, if the PEGylation agent is only 70 percent active, the amount of PEG used should ensure that only active PEG molecules are counted in the protein-to-PEG reaction stoichiometry.
Linkers for Chemical Conjugation
[0108] A variety of linkers can be used to covalently couple reactive residues within a kynureninase homodimer and a PEG molecule to one another, for instance, so as to form a conjugate as described herein. Exemplary linkers include those that may be cleaved, for instance, by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, for example, Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012, the disclosure of which is incorporated herein by reference as it pertains to linkers suitable for chemical coupling). Examples of linkers useful for the synthesis of conjugates described herein include those that contain electrophiles, such as Michael acceptors (e.g., maleimides), activated esters, electron-deficient carbonyl compounds, and aldehydes, among others, suitable for reaction with nucleophilic substituents present within antibodies, antigen-binding fragments, proteins, peptides, and small molecules, such as amine and thiol moieties. For instance, linkers suitable for the synthesis of therapeutic conjugates include, without limitation, alkyl, cycloalkyl, and heterocycloalkyl linkers, such as open-chain ethyl, propyl, butyl, hexyl, heptyl, octyl, nonyl, or decyl chains, cyclohexyl groups, cyclopentyl groups, cyclobutyl groups, cyclopropyl groups, piperidinyl groups, morpholino groups, or others containing two reactive moieties (e.g., halogen atoms, aldehyde groups, ester groups, acyl chloride groups, acyl anhydride groups, tosyl groups, mesyl groups, or brosyl groups, among others, that can be displaced by reactive nucleophilic atoms present within a kynureninase), aryl or heteroaryl linkers, such as benzyl, napthyl, or pyridyl groups containing two halomethyl groups that can be displaced by reactive nucleophilic atoms present within a kynureninase. Exemplary linkers include succinimidyl 4-(N-maleimidomethyl)-cyclohexane-L-carboxylate (SMCC), N-succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), sulfo-MBS, and succinimidyl iodoacetate, among others described, for instance, Liu et al., 18:690-697, 1979, the disclosure of which is incorporated herein by reference as it pertains to linkers for chemical conjugation. Additional linkers include the non-cleavable maleimidocaproyl linkers, which are described by Doronina et al., Bioconjugate Chem. 17:14-24, 2006, the disclosure of which is incorporated herein by reference as it pertains to linkers for chemical conjugation.
[0109] Additional linkers through which one component of a conjugate may be bound to another as described herein include linkers that are covalently bound to one component of the conjugate (e.g., a kynureninase) on one end of the linker and, on the other end of the linker, contain a chemical moiety formed from a coupling reaction between a reactive substituent present on the linker and a reactive substituent present within the other component of the conjugate (e.g., a PEG molecule described herein). Exemplary reactive substituents that may be present within a component of the conjugate include, without limitation, hydroxyl moieties of serine, threonine, and tyrosine residues; amino moieties of lysine residues; carboxyl moieties of aspartic acid and glutamic acid residues; and thiol moieties of cysteine residues, as well as propargyl, azido, haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of non-naturally occurring amino acids. Linkers useful in conjunction with the conjugates described herein include, without limitation, linkers containing chemical moieties formed by coupling reactions as depicted in Table 3 below. Curved lines designate points of attachment to each component of the conjugate
[0110] Linkers that can be used to conjugate a PEG molecule to an enzyme include those that are covalently bound to the enzyme on one end of the linker and, on the other end of the linker, contain a chemical moiety formed from a coupling reaction between a reactive substituent present on the linker and a reactive substituent present within one of the side chains of the amino acid residues present as part of the enzyme. Reactive substituents that may be present within the kynureninase enzyme include, without limitation, hydroxyl moieties of serine, threonine, and tyrosine residues; amino moieties of lysine residues; carboxyl moieties of aspartic acid and glutamic acid residues; and thiol moieties of cysteine residues, as well as propargyl, azido, haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of non-naturally occurring amino acids.
TABLE-US-00004 TABLE 3 Exemplary chemical moieties formed by coupling reactions in the formation of kynureninase homodimer conjugates Exemplary Coupling Reactions Chemical Moiety Formed by Coupling Reactions [3 + 2] Cycloaddition
Recombinant Expression of Kynureninases in Host Cells
[0111] The present disclosure includes compositions and methods for expressing kynureninase proteins recombinantly from a host cell, such as a prokaryotic or eukaryotic producer cell. Exemplary methods that can be used for effectuating the expressing a kynureninase protein in a host cell are described in further detail in the sections that follow.
Polynucleotides Encoding Kynureninases
[0112] One platform that can be used to express a kynureninase protein from a host cell, such as a mammalian cell, is by way of the stable incorporation of one or more genes encoding the kynureninase into the host cell genome (e.g., by integration into the nuclear genome of a mammalian cell). These genes are polynucleotides that encode the primary amino acid sequence of the corresponding protein. In order to introduce such exogenous genes into a mammalian cell, these genes can be incorporated into a vector. Vectors can be introduced into a cell by a variety of methods, including transformation, transfection, direct uptake, projectile bombardment, and by encapsulation of the vector in a liposome. Examples of suitable methods of transfecting or transforming cells are calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake. Such methods are described in more detail, for example, in Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition (Cold Spring Harbor University Press, New York (2014)); and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York (2015)), the disclosures of each of which are incorporated herein by reference.
[0113] Genes encoding therapeutic proteins of the disclosure can also be introduced into mammalian cells by targeting a vector containing a gene encoding such an agent to cell membrane phospholipids. For example, vectors can be targeted to the phospholipids on the extracellular surface of the cell membrane by linking the vector molecule to a VSV-G protein, a viral protein with affinity for all cell membrane phospholipids. Such, a construct can be produced using methods well known to those of skill in the field.
[0114] Recognition and binding of the polynucleotide encoding one or more therapeutic proteins of the disclosure by mammalian RNA polymerase is important for gene expression. As such, one may include sequence elements within the polynucleotide that exhibit a high affinity for transcription factors that recruit RNA polymerase and promote the assembly of the transcription complex at the transcription initiation site. Such sequence elements include, e.g., a mammalian promoter, the sequence of which can be recognized and bound by specific transcription initiation factors and ultimately RNA polymerase. Examples of mammalian promoters have been described in Smith et al., Mol. Sys. Biol., 3:73, online publication, the disclosure of which is incorporated herein by reference.
[0115] Once a polynucleotide encoding one or more therapeutic proteins has been incorporated into the nuclear DNA of a mammalian cell, the transcription of this polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing the mammalian cell to an external chemical reagent, such as an agent that modulates the binding of a transcription factor and/or RNA polymerase to the mammalian promoter and thus regulates gene expression. The chemical reagent can serve to facilitate the binding of RNA polymerase and/or transcription factors to the mammalian promoter, e.g., by removing a repressor protein that has bound the promoter. Alternatively, the chemical reagent can serve to enhance the affinity of the mammalian promoter for RNA polymerase and/or transcription factors such that the rate of transcription of the gene located downstream of the promoter is increased in the presence of the chemical reagent. Examples of chemical reagents that potentiate polynucleotide transcription by the above mechanisms are tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, Calif.) and can be administered to a mammalian cell in order to promote gene expression according to established protocols.
[0116] Other DNA sequence elements that may be included in polynucleotides for use in the compositions and methods described herein are enhancer sequences. Enhancers represent another class of regulatory elements that induce a conformational change in the polynucleotide containing the gene of interest such that the DNA adopts a three-dimensional orientation that is favorable for binding of transcription factors and RNA polymerase at the transcription initiation site. Thus, polynucleotides for use in the compositions and methods described herein include those that encode one or more therapeutic proteins and additionally include a mammalian enhancer sequence. Many enhancer sequences are now known from mammalian genes, and examples are enhancers from the genes that encode mammalian globin, elastase, albumin, -fetoprotein, and insulin. Enhancers for use in the compositions and methods described herein also include those that are derived from the genetic material of a virus capable of infecting a eukaryotic cell. Examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Additional enhancer sequences that induce activation of eukaryotic gene transcription are disclosed in Yaniv et al., Nature 297:17 (1982).
Fusion Proteins
[0117] Some embodiments of the present disclosure concern fusion proteins. These molecules may have a native or modified kynureninase linked at the N- or C-terminus to a heterologous domain. For example, fusions may also employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. Another useful fusion includes the addition of a protein affinity tag, such as a serum albumin affinity tag or six histidine residues, or an immunologically active domain, such as an antibody epitope, preferably cleavable, to facilitate purification of the fusion protein. Non-limiting affinity tags include polyhistidine, chitin binding protein (CBP), maltose binding protein (MBP), and glutathione-S-transferase (GST).
[0118] In a particular embodiment, the kynureninase may be linked to a peptide that increases the in vivo half-life, such as an XTEN polypeptide (Schellenberger et al., 2009), IgG Fc domain, albumin, or albumin binding peptide.
[0119] Methods of generating fusion proteins are well known to those of skill in the art. Such proteins can be produced, for example, by de novo synthesis of the complete fusion protein, or by attachment of the DNA sequence encoding the heterologous domain, followed by expression of the intact fusion protein. Production of fusion proteins that recover the functional activities of the parent proteins may be facilitated by connecting genes with a bridging DNA segment encoding a peptide linker that is spliced between the polypeptides connected in tandem. The linker would be of sufficient length to allow proper folding of the resulting fusion protein.
Enzymatic Kynurenine Degradation for Therapy
[0120] In some embodiments, the kynureninases of the disclosure may be used for the treatment of diseases, including cancers that are sensitive to kynurenine depletion, with enzymes that deplete kynurenine, to prevent tumor-mediated tolerogenic effects and instead mediate tumor-ablating pro-inflammatory responses. In some embodiments, kynureninases are contemplated for use in treating tumors expressing IDO1, IDO2, and/or TDO.
[0121] Some embodiments of the present disclosure provide a modified kynureninase for treating diseases, such as tumors. Particularly, the modified kynureninases may have human polypeptide sequences and thus may prevent allergic reactions in human patients, allow repeated dosing, and increase the therapeutic efficacy.
[0122] Tumors for which the present treatment methods are useful include any malignant cell type, such as those found in a solid tumor or a hematological tumor. Exemplary solid tumors can include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, melanoma, superficial spreading melanoma, lentigo malignant melanoma, acral lentiginous melanomas, nodular melanomas, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairy cell leukemia, multiple myeloma, acute myeloid leukemia (AML) and chronic myeloblastic leukemia.
[0123] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
[0124] The kynureninase may be used herein as an antitumor agent in a variety of modalities for depleting kynurenine and/or kynurenine-derived metabolites from tumor tissue, or the circulation of a mammal with cancer, or for depletion of kynurenine where its depletion is considered desirable. Depletion can be conducted in vivo in the circulation of a mammal, in vitro in cases where kynurenine and/or kynurenine-derived metabolites depletion in tissue culture or other biological mediums is desired, and in ex vivo procedures where biological fluids, cells, or tissues are manipulated outside the body and subsequently returned to the body of the patient mammal. Depletion of kynurenine from circulation, culture media, biological fluids, or cells is conducted to reduce the amount of kynurenine accessible to the material being treated, and therefore comprises contacting the material to be depleted with a kynurenine-depleting amount of the kynureninase under kynurenine-depleting conditions as to degrade the ambient kynurenine in the material being contacted.
[0125] The depletion may be directed to the nutrient source for the cells, and not necessarily the cells themselves. Therefore, in an in vivo application, treating a tumor cell includes contacting the nutrient medium for a population of tumor cells with the kynureninase. In this embodiment, the medium may be blood, lymphatic fluid, spinal fluid and the like bodily fluid where kynurenine depletion is desired.
[0126] Kynurenine- and/or kynurenine-derived metabolites depletion efficiency can vary widely depending upon the application, and typically depends upon the amount of kynurenine present in the material, the desired rate of depletion, and the tolerance of the material for exposure to kynureninase. Kynurenine and kynurenine metabolite levels in a material, and therefore rates of kynurenine and kynurenine metabolite depletion from the material, can readily be monitored by a variety of chemical and biochemical methods well known in the art. Exemplary kynurenine-depleting amounts are described further herein, and can range, for example, from 0.001 to 1,000 units (U) of kynureninase, depending, for example, on the subject being treated and the severity of the disease.
[0127] Kynurenine-depleting conditions are buffer and temperature conditions compatible with the biological activity of a kynureninase, and include moderate temperature, salt, and pH conditions compatible with the enzyme, for example, physiological conditions. Exemplary conditions include about 4-40 C., ionic strength equivalent to about 0.05 to 0.2 M NaCl, and a pH of about 5 to 9, while physiological conditions are included.
[0128] In some embodiments, the disclosure contemplates methods of using a kynureninase as an antitumor agent, and therefore comprises contacting a population of tumor cells with a therapeutically effective amount of kynureninase for a time period sufficient to inhibit tumor cell growth.
[0129] A therapeutically effective amount of a kynureninase is a predetermined amount calculated to achieve the desired effect, i.e., to deplete kynurenine in the tumor tissue or in a patients circulation, and thereby mediate a tumor-ablating pro-inflammatory response. Thus, the dosage ranges for the administration of kynureninase of the disclosure are those large enough to produce the desired effect in which the symptoms of tumor cell division and cell cycling are reduced. The dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, neurological effects, and the like. Generally, the dosage will vary with age of, condition of, sex of, and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication.
[0130] The kynureninase can be administered parenterally by injection or by gradual infusion over time. The kynureninase can be administered intravenously, intraperitoneally, orally, intramuscularly, subcutaneously, intracavity, transdermally, dermally, can be delivered by peristaltic means, can be injected directly into the tissue containing the tumor cells, or can be administered by a pump connected to a catheter that may contain a potential biosensor for kynurenine.
[0131] The therapeutic compositions containing kynureninase are conventionally administered intravenously, as by injection of a unit dose, for example.
[0132] The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for initial administration and booster shots are also contemplated and are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Exemplary multiple administrations are described herein and are particularly preferred to maintain continuously high serum and tissue levels of kynureninase and conversely low serum and tissue levels of kynurenine. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.
Proteins and Peptides
[0133] In some embodiments, the present disclosure concerns novel compositions comprising at least one protein or peptide, such as a kynureninase. These peptides may be comprised in a fusion protein or conjugated to an agent as described supra.
[0134] As used herein, a protein or peptide generally refers, but is not limited to, a protein of greater than about 200 amino acids, up to a full-length sequence translated from a gene; a polypeptide of greater than about 100 amino acids; and/or a peptide of from about 3 to about 100 amino acids. For convenience, the terms protein, polypeptide, and peptide are used interchangeably herein.
[0135] As used herein, an amino acid residue refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimic known in the art. In some embodiments, the residues of the protein or peptide are sequential, without any non-amino acids interrupting the sequence of amino acid residues. In some embodiments, the sequence may comprise one or more non-amino acid moieties. In particular embodiments, the sequence of residues of the protein or peptide may be interrupted by one or more non-amino acid moieties.
[0136] Accordingly, the term protein or peptide encompasses amino acid sequences comprising at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid.
[0137] Proteins or peptides may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides, or peptides through standard molecular biological techniques, the isolation of proteins or peptides from natural sources, or the chemical synthesis of proteins or peptides. The nucleotide and protein, polypeptide, and peptide sequences corresponding to various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (available on the world wide web at ncbi.nlm.nih.gov/). The coding regions for known genes may be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art. Alternatively, various commercial preparations of proteins, polypeptides, and peptides are known to those of skill in the art.
Pharmaceutical Compositions
[0138] It is contemplated that the novel kynureninase can be administered systemically or locally to inhibit tumor cell growth and, most preferably, to kill cancer cells in cancer patients with locally advanced or metastatic cancers. They can be administered intravenously, intrathecally, and/or intraperitoneally. They can be administered alone or in combination with anti-proliferative drugs. In one embodiment, they are administered to reduce the cancer load in the patient prior to surgery or other procedures. Alternatively, they can be administered after surgery to ensure that any remaining cancer (e.g., cancer that the surgery failed to eliminate) does not survive.
[0139] It is not intended that the present disclosure be limited by the particular nature of the therapeutic preparation. For example, such compositions can be provided in formulations together with physiologically tolerable liquid, gel, or solid carriers, diluents, and excipients. These therapeutic preparations can be administered to mammals for veterinary use, such as with domestic animals, and clinical use in humans in a manner similar to other therapeutic agents. In general, the dosage required for therapeutic efficacy will vary according to the type of use and mode of administration, as well as the particularized requirements of individual subjects.
[0140] Such compositions are typically prepared as liquid solutions or suspensions, as injectables. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired, the compositions may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, stabilizing agents, or pH buffering agents.
[0141] Where clinical applications are contemplated, it may be necessary to prepare pharmaceutical compositions comprising proteins, antibodies, and drugs in a form appropriate for the intended application. Generally, pharmaceutical compositions may comprise an effective amount of one or more kynureninase or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases pharmaceutical or pharmacologically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one kynureninase isolated by the method disclosed herein, or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards.
[0142] As used herein, pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.
[0143] Some embodiments of the present disclosure may comprise different types of carriers depending on whether it is to be administered in solid, liquid, or aerosol form, and whether it needs to be sterile for the route of administration, such as injection. The compositions can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, mucosally, orally, topically, locally, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, via a lavage, in lipid compositions (e.g., liposomes), or by other methods or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference).
[0144] The modified polypeptides may be formulated into a composition in a free base, neutral, or salt form. Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine, or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as formulated for parenteral administrations, such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations, such as drug release capsules and the like.
[0145] Further in accordance with some embodiments of the present disclosure, the composition suitable for administration may be provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent, or carrier is detrimental to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in administrable composition for use in practicing the methods is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers, and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives, such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
[0146] In accordance with some embodiments of the present disclosure, the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption, and the like. Such procedures are routine for those skilled in the art.
[0147] In a specific embodiment of the present disclosure, the composition is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner, such as grinding. Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in a composition include buffers, amino acids, such as glycine and lysine, carbohydrates, such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
[0148] In further embodiments, the present disclosure may concern the use of a pharmaceutical lipid vehicle composition that includes kynureninases, one or more lipids, and an aqueous solvent. As used herein, the term lipid will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds is well known to those of skill in the art, and as the term lipid is used herein, it is not limited to any particular structure. Examples include compounds that contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether- and ester-linked fatty acids, polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods.
[0149] One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the kynureninase or a fusion protein thereof may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.
[0150] The actual dosage amount of a composition administered to an animal patient can be determined by physical and physiological factors, such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient, and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
[0151] In some embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In some embodiments, an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors, such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations, will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
[0152] In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 milligram/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 milligram/kg/body weight to about 100 milligram/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.
Combination Treatments
[0153] In some embodiments, the compositions and methods of the present embodiments involve administration of a kynureninase in combination with a second or additional therapy. Such therapy can be applied in the treatment of any disease that is associated with kynurenine dependency. For example, the disease may be cancer.
[0154] The methods and compositions, including combination therapies, enhance the therapeutic or protective effect, and/or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect, such as the killing of a cancer cell and/or the inhibition of cellular hyperproliferation. This process may involve administering a kynureninase and a second therapy. The second therapy may or may not have a direct cytotoxic effect. For example, the second therapy may be an agent that upregulates the immune system without having a direct cytotoxic effect. A tissue, tumor, or cell can be exposed to one or more compositions or pharmacological formulation(s) comprising one or more of the agents (e.g., a kynureninase or an anti-cancer agent), or by exposing the tissue, tumor, and/or cell with two or more distinct compositions or formulations, wherein one composition provides 1) a kynureninase, 2) an anti-cancer agent, or 3) both a kynureninase and an anti-cancer agent. Also, it is contemplated that such a combination therapy can be used in conjunction with chemotherapy, radiotherapy, surgical therapy, or immunotherapy.
[0155] A kynureninase may be administered before, during, after, or in various combinations relative to an anti-cancer treatment. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where the kynureninase is provided to a patient separately from an anti-cancer agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one may provide a patient with the kynureninase and the anti-cancer therapy within about 12 to 24 or 72 h of each other and, more particularly, within about 6-12 h of each other. In some situations, it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.
[0156] In some embodiments, a course of treatment will last 1-90 days or more (this such range includes intervening days). It is contemplated that one agent may be given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof, and another agent is given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no anti-cancer treatment is administered. This time period may last 1-7 days, and/or 1-5 weeks, and/or 1-12 months or more (this such range includes intervening days), depending on the condition of the patient, such as their prognosis, strength, health, etc. It is expected that the treatment cycles would be repeated as necessary.
[0157] Various combinations may be employed. For the example below a kynureninase is A and an anti-cancer therapy is B:
[0158] A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
[0159] B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A
[0160] Administration of any compound or therapy of the present embodiments to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy.
EXAMPLES
[0161] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure.
Example 1. Generation of PEGylated Kynureninase Homodimer
[0162] An exemplary method of generating PEGylated kynureninase homodimer for use in the compositions and methods described herein is by way of chemical conjugation. Kynureninase can be dissolved in an aqueous buffer with a pH of 8.5, comprising 100 mM disodium phosphate, to which the solid PEGylation agent, such as Methoxy-PEG-CH.sub.2COON-hydroxysuccinimide (NHS) with a molecular weight of 5 kDa, can be added with an input ratio of, for example, 20:1, 30:1. 40:1, 50:1 or 60:1 PEG molecules to kynureninase homodimer. This reaction mixture can be left at about 22 C. for about 30 minutes.
[0163] Additional exemplary methods for generating PEGylated kynureninase homodimer can involve a kynureninase homodimer in an aqueous buffer, comprising 50 mM sodium acetate, 230 mM NaCl, 0.1 mM pyridoxal phosphate, adjusted to pH 8.6 using 0.6 M sodium borate, pH 9. A PEGylation agent, such as Methoxy-PEG-CH.sub.2COONHS with a molecular weight of 5 kDa, can be dissolved in 3 mM HCl such that a final concentration of about 0.2 g PEG per milliliter of HCl is achieved. This PEG solution can be added to the kynureninase solution in a controlled manner such that a final ratio of 30:1 PEG to kynureninase homodimer is achieved in the reaction mixture. The reaction can occur at about 22 C. for 30 minutes.
[0164] The PEGylated homodimer can then be analyzed using size exclusion chromatography with multi-angle light-scatter using a TSKgel G3000 SWxl column with a flow rate of 0.6 mL/min over 25 min in a 25 mM NaH.sub.2PO.sub.4, 150 mM NaCl buffer at 25 C. resulting in chromatograms for each respective input PEG:kynureninase ratio (
[0165] The resulting number of PEG molecules per kynureninase monomer and homodimer were calculated from the measured molecular weight of PEGylated kynureninase for various input ratios of PEG:kynureninase homodimer (
Example 2. Administration of a PEGylated Kynureninase Homodimer to a Patient Suffering from a Malignant Tumor
[0166] According to the methods disclosed herein, a physician of skill in the art can treat a patient, such as a human patient, so as to reduce or alleviate symptoms of an IDO1, IDO2, or TDO expressing tumor. To this end, a physician of skill in the art can administer to the human patient the PEGylated kynureninase homodimer. The homodimer may be administered to the patient, for example, systemically (e.g., intravenously), by localized perfusion bathing target cells directly, via a catheter, via a lavage, in lipid compositions (e.g., liposomes), or by other methods or any combination of the forgoing as would be known to one of ordinary skill in the art to treat the tumor. The homodimer can also be administered to the patient by multiple routes of administration, for example, intravenously and intracerebroventricularly. Exemplary kynurenine-depleting amounts can range from 0.001 to 1,000 units (U) of kynureninase.
[0167] In combination with the PEGylated kynureninase homodimer, one or more anti-cancer therapies may be administered to the patient (e.g. radiation therapy, surgical therapy, an immunotherapy) to ablate the patient's tumor cells. Methods of cell ablation well known in the art, such as irradiation, may be used alone or in combination with one or more anti-cancer or anti-hyperproliferative therapies to ablate the patients tumor cells. These agents and/or treatments may ablate tumor cells by at least 5% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 99%, or more), as assessed by PET imaging techniques known in the art. The homodimer can be administered to the patient from, for example, 12 hours to 1 month (e.g., 12 hours, 24 hours, 72 hours, 1 week, 2 weeks, 3 weeks, 4, weeks) or more before or after treatment with a second or more anti-cancer therapy.
[0168] The homodimer can be administered to the patient in an amount sufficient to treat a tumor expressing IDO1, IDO2 or TDO2. A standard examination can also be performed by the physician before and after treatment to evaluate changes in tumor size. The patient may be evaluated, for example, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or more following administration of the homodimer depending on the route of administration used for treatment. A finding of reduced tumor size or reduced local kynureninase concentrations at the site of the tumor following administration of the PEGylated kynureninase homodimer provides an indication that the treatment has successfully treated the tumor expressing IDO1, IDO2 or TDO2.
Example 3. Optimization of Kynureninase Homodimer PEGylation Density for In Vivo Delivery
[0169] In this example, purified kynureninase having the amino acid sequence of SEQ ID NO: 2 was conjugated with 5,000-dalton PEG using -[3-(3-maleimido-1-oxopropyl)amino]propyl-w-methoxy PEG reagent (CAS #883993-35-9) at pH 8.5 for 30 minutes at room temperature. The conjugation reaction was performed with kynureninase concentrations of 5.5-8 mg/ml and direct addition of various molar equivalents of the 5 kDa PEG reagent (100:1, 80:1, 40:1, 20:1, and 10:1 PEGylation agent to kynureninase homodimer). Following conjugation, the final products were diafiltered into 1 phosphate buffered saline, pH 7.4, with 30 kDa molecular weight cutoff spin filters to remove any residual free PEG. Samples were analyzed by an isocratic size exclusion chromatography (SEC) method using a Superdex 200 10/300 column (
[0170] For testing PEGylated kynureninase homodimers, each female BALB/c mouse (6-8 weeks) was inoculated subcutaneously at the right flank with CT-26 tumor cells (1105) in 0.1 ml of PBS for tumor development. The animals were randomized and dosed iv with vehicle (PBS pH7.4), or 10 mg/kg differentially pegylated kynureninase having the amino acid sequence of SEQ ID NO: 2 was started when the average tumor volume reached approximately 200 mm.sup.3. At 6, 24, 48, 72 & 120 hours after dosing, mice were terminally bled (n=4 mice/time point) and the plasma samples were immediately quenched with acid containing Internal Standard (IS) solution and processed for LC/MS analysis of Kynurenine (
Other Embodiments
[0171] Various modifications and variations of the described disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the art are intended to be within the scope of the disclosure.
[0172] Other embodiments are in the claims.