PEGylated kynureninase enzymes and their use for the treatment of cancer

Abstract

Kynureninase enzymes covalently linked to polyethylene glycol are described. Aspects of the present disclosure provide compositions and methods for improving the effective treatment of cancer by kynurenine ablation using such molecules.

Description

[Technical Field]

[0001] The present disclosure relates to compositions and methods of synthesis of PEGylated kynureninase enzymes and their use for the treatment of cancer.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on October 16, 2020, has the name 51175-014WO2_Sequence_Listing_10_16_20_ST25, and is 12,461 bytes in size. [Background technology]

[0003] Kynurenine is a metabolite of the amino acid tryptophan, produced by the action of either indoleamine 2,3 dioxygenase (IDO) or tryptophan 2,3 dioxygenase (TDO). Many tumor cells regulate the expression of IDO and / or TDO to increase local kynurenine concentrations, which in turn inhibit the function of tumor-infiltrating T cells that would otherwise attack tumors. Kynurenine concentrations can be removed by the enzyme kynureninase, which catalyzes the degradation of kynurenine to anthranilic acid. To achieve long-term removal of kynurenine so that T cells can effectively identify and attack tumor cells, there is a continuing need to develop therapeutic compounds with enhanced catalytic activity for kynurenine degradation and enhanced stability against inactivation in serum. Summary of the Invention

[0004] The present disclosure provides kynureninase enzymes covalently linked to polyethylene glycol (PEG) molecules. PEG molecules covalently linked to kynureninase can be used, for example, to achieve greater serum stability and improved catalytic efficiency for kynurenine degradation. A large series of kynureninase enzyme variants with improved catalytic efficiency, along with a variety of PEG molecules with different linking groups, can be used in conjunction with the compositions and methods described herein. The molecules described herein can be administered to patients to treat cancers expressing indoleamine 2,3 dioxygenase (IDO) and / or tryptophan 2,3 dioxygenase (TDO), which have elevated local kynurenine levels, thereby preventing targeting and attack by T cells.

[0005] In a first aspect, the present disclosure provides a kynureninase homodimer covalently linked to one or more polyethylene glycol (PEG) molecules, wherein the ratio of PEG molecules to homodimer is about 10:1 to about 40:1, e.g., about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, or 40:1.

[0006] In some embodiments, the ratio of PEG molecules to homodimers is about 11:1 to about 39:1, e.g., about 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, or 39:1. In some embodiments, the ratio of PEG molecules to homodimers is about 12:1 to about 38:1, e.g., about 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, or 38:1. In some embodiments, the ratio of PEG molecules to homodimers is about 13:1 to 37:1, e.g., about 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, or 37:1. In some embodiments, the ratio of PEG molecules to homodimers is about 14:1 to 36:1, e.g., about 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, or 36:1. In some embodiments, the ratio of PEG molecules to homodimers is about 15:1 to 35:1, e.g., about 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, or 35:1.In some embodiments, the ratio of PEG molecules to homodimer is about 16:1 to 34:1, e.g., about 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, or 34:1. In some embodiments, the ratio of PEG molecules to homodimer is about 17:1 to 33:1, e.g., about 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, or 33:1. In some embodiments, the ratio of PEG molecules to homodimer is about 18:1 to 32:1, e.g., about 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, or 32:1. In some embodiments, the ratio of PEG molecules to homodimer is about 19:1 to 31:1, e.g., about 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, or 31:1. In some embodiments, the ratio of PEG molecules to homodimers is about 20:1 to 30:1, e.g., about 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, or 30:1. In some embodiments, the ratio of PEG molecules to homodimers is about 21:1 to 29:1, e.g., about 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, or 29:1. In some embodiments, the ratio of PEG molecules to homodimers is about 22:1 to 28:1, e.g., about 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, or 28:1. In some embodiments, the ratio of PEG molecules to homodimers is about 23:1 to 27:1, e.g., about 23:1, 24:1, 25:1, 26:1, or 27:1. In some embodiments, the ratio of PEG molecules to homodimers is about 24:1 to 26:1, e.g., about 24:1, 25:1, or 26:1. In some embodiments, the ratio of PEG molecules to homodimers is about 25:1.

[0007] In some embodiments, the ratio of PEG molecules to homodimers is about 10:1. In some embodiments, the ratio of PEG molecules to homodimers is about 11:1. In some embodiments, the ratio of PEG molecules to homodimers is about 12:1. In some embodiments, the ratio of PEG molecules to homodimers is about 13:1. In some embodiments, the ratio of PEG molecules to homodimers is about 14:1. In some embodiments, the ratio of PEG molecules to homodimers is about 15:1. In some embodiments, the ratio of PEG molecules to homodimers is about 16:1. In some embodiments, the ratio of PEG molecules to homodimers is about 17:1. In some embodiments, the ratio of PEG molecules to homodimers is about 18:1. In some embodiments, the ratio of PEG molecules to homodimers is about 19:1. In some embodiments, the ratio of PEG molecules to homodimers is about 20:1. In some embodiments, the ratio of PEG molecules to homodimers is about 21:1. In some embodiments, the ratio of PEG molecules to homodimers is about 22:1. In some embodiments, the ratio of PEG molecules to homodimers is about 23:1. In some embodiments, the ratio of PEG molecules to homodimers is about 24:1. In some embodiments, the ratio of PEG molecules to homodimers is about 25:1. In some embodiments, the ratio of PEG molecules to homodimers is about 26:1. In some embodiments, the ratio of PEG molecules to homodimers is about 27:1. In some embodiments, the ratio of PEG molecules to homodimers is about 28:1. In some embodiments, the ratio of PEG molecules to homodimers is about 29:1. In some embodiments, the ratio of PEG molecules to homodimers is about 30:1. In some embodiments, the ratio of PEG molecules to homodimers is about 31:1. In some embodiments, the ratio of PEG molecules to homodimers is about 32:1. In some embodiments, the ratio of PEG molecules to homodimers is about 33:1. In some embodiments, the ratio of PEG molecules to homodimers is about 34:1. In some embodiments, the ratio of PEG molecules to homodimers is about 35:1. In some embodiments, the ratio of PEG molecules to homodimers is about 36:1.In some embodiments, the ratio of PEG molecules to homodimers is about 37:1. In some embodiments, the ratio of PEG molecules to homodimers is about 38:1. In some embodiments, the ratio of PEG molecules to homodimers is about 39:1. In some embodiments, the ratio of PEG molecules to homodimers is about 40:1.

[0008] In some embodiments, the ratio of PEG molecules to homodimers is about 13:1 to about 33:1, e.g., about 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, or 33:1.

[0009] In some embodiments, the ratio of PEG molecules to homodimers is about 18:1 to about 33:1, e.g., about 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, or 33:1.

[0010] In some embodiments, the ratio of PEG molecules to homodimers is about 18:1 to about 28:1, e.g., about 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, or 28:1.

[0011] In some embodiments, the ratio of PEG molecules to homodimers is about 18:1 to about 23:1, e.g., about 18:1, 19:1, 20:1, 21:1, 22:1, or 23:1.

[0012] In some embodiments, the ratio of PEG molecules to homodimers is about 23:1 to about 33:1, e.g., about 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, or 33:1.

[0013] In some embodiments, the ratio of PEG molecules to homodimers is about 23:1 to about 28:1, such as about 23:1, 24:1, 25:1, 26:1, 27:1, or 28:1.

[0014] In some embodiments, the PEG molecule has a molecular weight of about 1 kDa to about 10 kDa, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kDa. In some embodiments, the PEG molecule has a molecular weight of about 2 kDa to about 9 kDa, e.g., about 2, 3, 4, 5, 6, 7, 8, or 9 kDa. In some embodiments, the PEG molecule has a molecular weight of about 3 kDa to about 8 kDa, e.g., about 3, 4, 5, 6, 7, or 8 kDa. In some embodiments, the PEG molecule has a molecular weight of about 4 kDa to about 7 kDa, e.g., about 4, 5, 6, or 7 kDa. In some embodiments, the PEG molecule has a molecular weight of about 5 kDa to about 6 kDa, e.g., about 5 or 6 kDa. In some embodiments, the PEG molecule has a molecular weight of about 5 kDa. In some embodiments, the PEG molecule is linear. In some embodiments, the PEG molecule is branched.

[0015] In some embodiments, the homodimer is covalently attached to the PEG molecule through one or more lysine or cysteine ​​residues, e.g., through one or more lysine residues via a linking group, e.g., a linking group formed by reaction of a lysine amine with a succinimide group, an aldehyde group, an amide group, a carbamate group, an ester group, an epoxy group, a carboxyl group, a hydroxyl group, or a combination thereof. In some embodiments, the linking group is a succinimide group, e.g., succinimidyl succinate, succinimidyl propionate, succinimidyl carboxymethylate, succinimidyl carbonate, succinimidyl succinamide, N-hydroxysuccinimide, or a combination thereof, e.g., N-hydroxysuccinimide carbonate. In some embodiments, the linking group is monofunctional.

[0016] In some embodiments, the homodimer comprises two polypeptide monomers each having an amino acid sequence that is at least 85% identical to SEQ ID NO:1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:1). Each polypeptide monomer has the following structure compared to SEQ ID NO: 1: 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, V104 H, V104K, V104L, V104R, G105A, G105H, G105S, G105T, E106D, K106D, K106E, K106H, K106N, R107P, R107S, P108R, I110A, I110F, I1 10L, 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, I183 P, I183S, E184A, E184D, E184R, E184T, E184V, E185T, M187L, M189I, K191A, K191G, K191H, K191M, K191N, K191R, K191S, K191T, K1 91W, 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, F24 9W, D250E, S274A, S274C, S274G, S274N, S274T, L278M, A280G, A280S, A280T, G281S, A 282M, A282P, G284N, I285L, V303L, V303S, F306W, F306Y, S311N, K315E, D317E, D317 K, I331C, I331L, I331N, I331S, I331T, I331V, N333T, P334N, P335T, L337T, L338A, L3 38Q, S341I, K373E, K373N, N375A, N375H, Y376C, Y376F, Y376L, K378G, K378P, K378Q , K378R, K380G, K380S, A382G, A382R, A382T, T383S, K384G, K384N, P386K, P386S, V38 7L, N389E, I405L, F407Y, S408D, S408N, N411R, D413S, D413V, Q416T, E419A, E419L, K420E, R421N, V424I, K427M, N429E, G432A, and A436T. In some embodiments, the amino acid sequence is at least 90% identical to SEQ ID NO: 1 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1), or at least 95% identical to SEQ ID NO: 1 (e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1).

[0017] In some embodiments, each polypeptide monomer comprises a substitution at A282 (e.g., A282M or A282P), F306 (e.g., F306W or F306Y), and / or F249, e.g., F249W, compared to SEQ ID NO:1. In some embodiments, each polypeptide monomer comprises a substitution at A99 compared to SEQ ID NO:1. In some embodiments, each polypeptide monomer comprises a substitution at G112 compared to SEQ ID NO:1. In some embodiments, each polypeptide monomer comprises a substitution at E103 compared to SEQ ID NO:1. In some embodiments, each polypeptide monomer comprises a substitution at V104 compared to SEQ ID NO:1. In some embodiments, each polypeptide monomer comprises a substitution at S408 compared to SEQ ID NO:1.

[0018] In some embodiments, each polypeptide monomer comprises a F306W substitution. In some embodiments, each polypeptide monomer comprises a L72N substitution. In some embodiments, each polypeptide monomer comprises a H102W and a N333T substitution. In some embodiments, each polypeptide monomer comprises a I183P substitution. In some embodiments, each polypeptide monomer comprises a R107P substitution. In some embodiments, each polypeptide monomer comprises an A436T substitution. In some embodiments, each polypeptide monomer comprises at least one substitution selected from L72N, H102W, A282P, F306W, I331S, and N333T relative to SEQ ID NO: 1. In some embodiments, each polypeptide monomer comprises at least two, three, four, or five substitutions selected from L72N, H102W, A282P, F306W, I331S, and N333T. In some embodiments, each polypeptide monomer comprises the following substitutions relative to SEQ ID NO:1: L72N, H102W, A282P, F306W, I331S, and N333T. In some embodiments, each polypeptide monomer comprises the following substitutions relative to SEQ ID NO:1: L72N, A99R, H102W, E103R, V104H, R107P, G112Y, I183P, A282P, F306W, I331S, N333T, S408N, and A436T.

[0019] In some embodiments, each polypeptide monomer has an amino acid sequence that is at least 85% identical to SEQ ID NO:3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:3). In some embodiments, each polypeptide monomer has an amino acid sequence that is at least 90% identical to SEQ ID NO:3 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:3). In some embodiments, each polypeptide monomer has an amino acid sequence that is at least 95% identical to SEQ ID NO:3 (e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:3). In some embodiments, each polypeptide monomer has the amino acid sequence of SEQ ID NO:3. In some embodiments, each polypeptide monomer has one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 3. For example, each polypeptide monomer can have 50 or fewer conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 3. In some embodiments, each polypeptide monomer may have 25 or fewer conservative amino acid substitutions (e.g., 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 conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 3. In some embodiments, each polypeptide monomer may have 10 or fewer conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 3. In some embodiments, each polypeptide monomer may have an amino acid sequence that differs from SEQ ID NO: 3 only by conservative amino acid substitutions.

[0020] In some embodiments, each polypeptide monomer has an amino acid sequence that is at least 85% identical to SEQ ID NO:2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:2). In some embodiments, each polypeptide monomer has an amino acid sequence that is at least 90% identical to SEQ ID NO:2 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:2). In some embodiments, each polypeptide monomer has an amino acid sequence that is at least 95% identical to SEQ ID NO:2 (e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:2). In some embodiments, each polypeptide monomer has the amino acid sequence of SEQ ID NO:2. Each polypeptide monomer can have one or more conservative amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 2. For example, each polypeptide monomer can have 50 or fewer conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 2. In some embodiments, each polypeptide monomer may have 25 or fewer conservative amino acid substitutions (e.g., 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 conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 2. In some embodiments, each polypeptide monomer may have 10 or fewer conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 2. In some embodiments, each polypeptide monomer may have an amino acid sequence that differs from SEQ ID NO: 2 only by conservative amino acids.

[0021] In some embodiments, the homodimer is at least 8000 M -1 s -1 Catalytic activity (kcat / K M In some embodiments, the homodimer has a -1 s -1 ~40,000M -1 s -1 ;10000M -1 s -1 ~40,000M -1 s -1 ;20000M -1 s -1 ~40000M-1s -1 ;or 25000M -1 s -1 ~35000M -1 s -1 Catalytic activity (kcat / K M )

[0022] In some embodiments, the ratio of PEG molecules to homodimers is about 25:1, and the PEG molecules have a molecular weight of about 5 kDa and are attached to the homodimers by N-hydroxysuccinimide ester carbonate linking groups.

[0023] In another aspect, the disclosure provides a method for increasing the circulating half-life of a kynureninase polypeptide, comprising contacting the kynureninase polypeptide with a PEGylation agent, wherein the kynureninase polypeptide forms a homodimer, and the molar input ratio of the PEGylation agent to the kynureninase homodimer is between about 10:1 and about 50:1, e.g., about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, , 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1 or 50:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is from about 11:1 to about 49:1, e.g., 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, or 49:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is from about 12:1 to about 48:1, e.g., 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, or 48:1.In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is from about 13:1 to about 47:1, e.g., 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, or 47:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is from about 14:1 to about 46:1, e.g., 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, or 46:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 15:1 to about 45:1, e.g., 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, or 45:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is from about 16:1 to about 44:1, e.g., 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, or 44:1.In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 17:1 to about 43:1, e.g., 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, or 43:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 18:1 to about 42:1, e.g., 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, or 42:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 19:1 to about 41:1, e.g., 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1 or 41:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 20:1 to about 40:1, e.g., 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, or 40:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 21:1 to about 39:1, e.g., 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, or 39:1.In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 22:1 to about 38:1, e.g., 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, or 38:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 23:1 to about 37:1, e.g., 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, or 37:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 24:1 to about 36:1, e.g., 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, or 36:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 25:1 to about 35:1, e.g., 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, or 35:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 26:1 to about 34:1, e.g., 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, or 34:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 27:1 to about 33:1, e.g., 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, or 33:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 28:1 to about 32:1, e.g., 28:1, 29:1, 30:1, 31:1, or 32:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 29:1 to about 31:1, e.g., 29:1, 30:1, or 31:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 30:1.

[0024] In another aspect, the disclosure provides a method for producing a PEGylated kynureninase homodimer, wherein the homodimer comprises two polypeptide monomers, the method comprising contacting the homodimer with a PEGylation agent, wherein the molar input ratio of the PEGylation agent to the kynureninase homodimer is between about 10:1 and about 50:1, e.g., about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78 :1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1 or 50:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 11:1 to about 49:1, e.g., about 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, or 49:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 12:1 to about 48:1, e.g., about 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, or 48:1.In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 13:1 to about 47:1, e.g., about 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, or 47:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 14:1 to about 46:1, e.g., about 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, or 46:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 15:1 to about 45:1, e.g., about 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, or 45:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 16:1 to about 44:1, e.g., about 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, or 44:1.In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 17:1 to about 43:1, e.g., about 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, or 43:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 18:1 to about 42:1, e.g., about 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, or 42:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 19:1 to about 41:1, e.g., about 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1 or 41:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 20:1 to about 40:1, e.g., about 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, or 40:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 21:1 to about 39:1, e.g., about 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, or 39:1.In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 22:1 to about 38:1, e.g., about 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, or 38:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 23:1 to about 37:1, e.g., about 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, or 37:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 24:1 to about 36:1, e.g., about 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, or 36:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 25:1 to about 35:1, e.g., about 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, or 35:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 26:1 to about 34:1, e.g., about 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, or 34:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 27:1 to about 33:1, e.g., about 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, or 33:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 28:1 to about 32:1, e.g., about 28:1, 29:1, 30:1, 31:1, or 32:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 29:1 to about 31:1, e.g., about 29:1, 30:1, or 31:1. In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 30:1.

[0025] In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 20:1 to about 30:1, e.g., about 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, or 30:1.

[0026] In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 10:1 to about 30:1, e.g., about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, or 30:1.

[0027] In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 11:1 to about 29:1, e.g., about 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, or 29:1.

[0028] In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 12:1 to about 28:1, e.g., about 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, or 28:1.

[0029] In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 13:1 to about 27:1, e.g., about 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, or 27:1.

[0030] In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 14:1 to about 26:1, e.g., about 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, or 26:1.

[0031] In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 15:1 to about 25:1, e.g., about 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, or 25:1.

[0032] In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 16:1 to about 24:1, e.g., about 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, or 24:1.

[0033] In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 17:1 to about 23:1, for example, about 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, or 23:1.

[0034] In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 18:1 to about 22:1, for example, about 18:1, 19:1, 20:1, 21:1, or 22:1.

[0035] In some embodiments, the molar input ratio of PEGylating agent to kynureninase homodimer is about 19:1 to about 21:1, for example, about 19:1, 20:1, or 21:1.

[0036] In some embodiments, the molar input ratio of PEGylation agent to kynureninase homodimer is about 20:1.

[0037] In some embodiments of any of the above aspects of the present disclosure, the PEG molecule has a molecular weight of about 1 kDa to about 10 kDa, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kDa. In some embodiments, the PEG molecule has a molecular weight of about 2 kDa to about 9 kDa, e.g., about 2, 3, 4, 5, 6, 7, 8, or 9 kDa. In some embodiments, the PEG molecule has a molecular weight of about 3 kDa to about 8 kDa, e.g., about 3, 4, 5, 6, 7, or 8 kDa. In some embodiments, the PEG molecule has a molecular weight of about 4 kDa to about 7 kDa, e.g., about 4, 5, 6, or 7 kDa. In some embodiments, the PEG molecule has a molecular weight of about 5 kDa to about 6 kDa, e.g., about 5 or 6 kDa. In some embodiments, the PEG molecule has a molecular weight of about 5 kDa.

[0038] In some embodiments, the PEGylating agent is a lysine-reactive PEGylating agent, e.g., a PEGylated agent in which the PEG molecule is attached to a reactive group containing an N-hydroxysuccinimide (NHS) moiety, among other lysine-reactive moieties described herein or known in the art.

[0039] In some embodiments, the PEGylating agent is a cysteine-reactive PEGylating agent, e.g., a PEG molecule attached to a reactive group containing an iodoacetamide moiety or a maleimide moiety, among other cysteine-reactive moieties described herein or known in the art.

[0040] In some embodiments, the homodimer is covalently attached to the PEG molecule through one or more lysine or cysteine ​​residues, e.g., through one or more lysine residues, by a linking group formed from the reaction of a lysine amine with a succinimide group, an aldehyde group, an amide group, a carbamate group, an ester group, an epoxy group, a carboxyl group, a hydroxyl group, or combinations thereof. In some embodiments, the linking group is a succinimide group, e.g., succinimidyl succinate, succinimidyl propionate, succinimidyl carboxymethylate, succinimidyl carbonate, succinimidyl succinamide, N-hydroxysuccinimide, or combinations thereof, e.g., N-hydroxysuccinimide carbonate. In some embodiments, the linking group is monofunctional.

[0041] In some embodiments, each polypeptide monomer has an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:1, and each polypeptide monomer has at least one of the following amino acid sequences as compared to SEQ ID NO:1: L8P, K38E, Y47L, I48F, K50Q, I51M, S60N, K64N, D65G, E66K, N67D, N67P, D67S, A68F, A68T, A68V, F71L, F71M, L72N, K84E, E88N, E89K, E89S, E 90Q, D92E, K93N, K93T, A95H, A95Q, K96N, I97H, I97L, I97V, A98G, A99G, A99 I, A99R, A99S, A99T, A99V, Y100N, Y100S, Y100T, G101A, H102W, E103F, E103H , E103N, E103Q, E103R, E103V, E103W, V104D, V104E, V104F, V104H, V104K, V 104L, V104R, G105A, G105H, G105S, G105T, E106D, K106D, K106E, K106H, K106 N, R107P, R107S, P108R, I110A, I110F, I110L, I110M, I110T, T111D, T111H, T111N, T111R, G112A, G112C, G112D, G112K, G112L, G112M, G112Q, G112R, G1 12S, G112T, G112Y, N127K, I131V, A132V, L133V, A136T, L137T, T138S, N140 D, H142Q, Q14R, Y156H, K163T, D168E, H169R, Q175L, I183F, I183L, I183P, I1 83S, E184A, E184D, E184R, E184T, E184V, E185T, M187L, M189I, K191A, K191 G, K191H, K191M, K191N, K191R, K191S, K191T, K191W, E197A, E197D, E197F, E 197K, E197M, E197Q, E197S, E197T, E197V, I201C, I201E, I201F, 1201H, I20 1L, I201S, I201T, I201V, H203K, L219M, L219W, F220L, V223I, F225Y, H230F,H230L, H230Y, N232S, Y246F, F249W, D250E, S274A, S274C, S274G, S274N, S274 T, L278M, A280G, A280S, A280T, G281S, A282M, A282P, G284N, I285L, V303L, V3 03S, 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, each polypeptide monomer has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, each polypeptide monomer has an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0042] In some embodiments, each polypeptide monomer comprises a substitution at A282 (e.g., A282M or A282P), F306 (e.g., F306W or F306Y), or F249, e.g., F249W, relative to SEQ ID NO:1. In some embodiments, each polypeptide monomer comprises a substitution at A99 relative to SEQ ID NO:1. In some embodiments, each polypeptide monomer comprises a substitution at G112 relative to SEQ ID NO:1. In some embodiments, each polypeptide monomer comprises a substitution at E103 relative to SEQ ID NO:1. In some embodiments, each polypeptide monomer comprises a substitution at V104 relative to SEQ ID NO:1. In some embodiments, each polypeptide monomer comprises a substitution at S408 relative to SEQ ID NO:1. In some embodiments, each polypeptide monomer comprises a F306W substitution. In some embodiments, each polypeptide monomer comprises a L72N substitution. In some embodiments, each polypeptide monomer comprises a H102W and a N333T substitution. In some embodiments, each polypeptide monomer comprises a I183P substitution. In some embodiments, each polypeptide monomer comprises a R107P substitution. In some embodiments, each polypeptide monomer comprises a A436T substitution. In some embodiments, each polypeptide monomer comprises at least one substitution selected from L72N, H102W, A282P, F306W, I331S, and N333T relative to SEQ ID NO:1. In some embodiments, 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. In some embodiments, each polypeptide monomer comprises the substitutions L72N, H102W, A282P, F306W, I331S, and N333T relative to SEQ ID NO:1.

[0043] In some embodiments, each polypeptide monomer has an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3. In some embodiments, each polypeptide monomer has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3. In some embodiments, each polypeptide monomer has an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3. In some embodiments, each polypeptide monomer has the amino acid sequence of SEQ ID NO: 3.

[0044] In some embodiments, each polypeptide monomer has an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2. In some embodiments, each polypeptide monomer has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2. In some embodiments, each polypeptide monomer has an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2. In some embodiments, each polypeptide monomer has the amino acid sequence of SEQ ID NO: 2.

[0045] In some embodiments, the homodimer is at least 8000 M -1 s -1 Catalytic activity (kcat / K M In some embodiments, the homodimer has a -1 s -1 ~40,000M -1 s -1 ;10000M -1 s -1 ~40,000M -1 s -1 ;20000M -1 s -1~40,000M -1 s -1 ;or 25000M -1 s -1 ~35000M -1 s -1 Catalytic activity (kcat / K M )

[0046] In some embodiments of any of the above aspects of the disclosure, the ratio of PEG molecules to homodimers is about 25:1, and the PEG molecules have a molecular weight of about 5 kDa and are attached to the homodimers by N-hydroxysuccinimide ester carbonate linking groups.

[0047] In some embodiments, the homodimer is contacted with the PEGylation agent in an aqueous buffer, such as sodium diphosphate or sodium acetate. In some embodiments, the pH of the aqueous buffer is in the range of about 7.5 to about 9.5, e.g., 7.5, 7.6, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, or 9.5. In some embodiments, the ionic strength of the aqueous buffer is in the range of about 250 mM to about 350 mM. In some embodiments, the homodimer is isolated using size exclusion chromatography.

[0048] In some embodiments of any of the above aspects or embodiments of the present disclosure, the kynureninase homodimer may contain one or more of the amino acid substitutions disclosed in WO2015 / 031771, WO2016 / 033488, or WO2017 / 151860, each of which is incorporated by reference in its entirety.

[0049] The modified polypeptides described above and herein can be characterized by having a certain percentage of identity compared to an unmodified polypeptide (e.g., a naturally occurring polypeptide, such as the human kynureninase of SEQ ID NO: 1) or any polypeptide sequence disclosed herein. For example, an unmodified polypeptide can contain at least or up to about 150, 200, 250, 300, 350, 400, 450, or 465 residues of a naturally occurring kynureninase (or any range derivable therein). The percentage identity between a modified and unmodified polypeptide, or between any two sequences being compared, can be about, up to, or at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any range derivable therein). It is also contemplated that the above-mentioned percentage identities can be with respect to a particular modified region of the polypeptide as compared to an unmodified region of the polypeptide. For example, a polypeptide may contain a modified or mutant substrate recognition site of a kynureninase that can be characterized based on the identity of the amino acid sequence of the modified or mutant substrate recognition site of a kynureninase to that of an unmodified or mutant kynureninase from the same species or across species. For example, a modified or mutant human polypeptide characterized as having at least 90% identity to an unmodified kynureninase means that at least 90% of the amino acids in the modified or mutant human polypeptide are identical to those in the unmodified polypeptide.

[0050] Such unmodified polypeptides can be naturally occurring kynureninases, particularly human isoforms or other primate isoforms. For example, naturally occurring human kynureninases can have the sequence of SEQ ID NO: 1. Non-limiting examples of other naturally occurring primate kynureninases include Pongo abelii kynureninases (Genbank ID: XP_009235962.1, GI: 686708656), Macaca fascicularis kynureninases (Genbank ID: EHH54849.1, GI: 355750522), and Pan troglodytes kynureninases (Genbank ID: XP_003309314.1, GI: 332814521). Exemplary naturally occurring polypeptides include sequences that are about, at most, or at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any range derivable therein) to SEQ ID NO: 1. For example, a naturally occurring polypeptide can include at least or at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 465 (or any range derivable therein) residues of the sequence of SEQ ID NO: 1.

[0051] In another aspect, the disclosure includes a pharmaceutical formulation comprising a kynureninase homodimer having any one of the above compositions in a pharmaceutically acceptable carrier, hi some embodiments, the pharmaceutical formulation comprises a kynureninase homodimer having any one of the above compositions in a pharmaceutically acceptable carrier.

[0052] In another aspect, the disclosure features a method of treating a subject (e.g., a mammalian subject, e.g., a human subject) having a tumor, the method including administering to the subject an effective amount of an enzyme having any one of the formulations described herein or compositions described herein. In some embodiments, the method includes administering to the subject an effective amount of an enzyme having any one of the formulations described herein or compositions described herein. In some embodiments, the subject is confirmed to have an IDO1-, IDO2-, or TDO-expressing tumor, e.g., a solid tumor or a hematopoietic tumor. In some embodiments, the subject is a human subject.

[0053] In some embodiments, the formulation is administered intratumorally, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrathoracically, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctivally, intravesically, transmucosally, intrapericardially, intraumbilically, orally, by aspiration, by injection, by infusion, by continuous infusion, by local perfusion directly into target cells, by catheter, or by lavage.

[0054] In some embodiments, the method also includes administering a second anti-cancer therapy to the subject. The second anti-cancer therapy can be surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy. In some embodiments, the second anti-cancer therapy can be immune checkpoint inhibitor therapy. For example, the kynureninase homodimer can be mixed with, conjugated to, administered together with, or administered separately from an immunotherapeutic agent, for example, to treat cancer or an infectious disease, such as a cancer or infectious disease described herein. Exemplary immunotherapeutic agents useful in connection with the compositions and methods described herein include, but are not limited to, anti-CTLA-4 agents, anti-PD-1 agents, anti-PD-L1 agents, anti-PD-L2 agents, anti-TNF-α cross-linking agents, anti-TRAIL cross-linking agents, anti-CD27 agents, anti-CD30 agents, anti-CD40 agents, anti-4-1BB agents, anti-GITR agents, anti-OX40 agents, anti-TRAILR1 agents, anti-TRAILR2 agents, and anti-TWEAKR agents, as well as agents directed against immune targets listed, for example, in Table 1 of Mahoney et al., Cancer Immunotherapy, 14:561-584 (2015), the disclosure of which is incorporated herein by reference in its entirety. For example, the immunotherapeutic agent can be an anti-CTLA-4 antibody or antigen-binding fragment thereof, such as ipilimumab and tremelimumab. The immunotherapeutic agent may be an anti-PD-1 antibody or an antigen-binding fragment thereof, such as nivolumab, pembrolizumab, avelumab, durvalumab, and atezolizumab. The immunotherapeutic agent may be an anti-PD-L1 antibody or an antigen-binding fragment thereof, such as atezolizumab or avelumab. As other examples, the immune target 4-1BB ligand may be targeted by an anti-4-1BB ligand antibody; the immune target OX40L may be targeted by an anti-OX40L antibody; the immune target GITR may be targeted by an anti-GITR antibody; the immune target CD27 may be targeted by an anti-CD27 antibody; the immune target TL1A may be targeted by an anti-TL1A antibody; the immune target CD40L or CD40 may be targeted by an anti-CD40L antibody; the immune target LIGHT may be targeted by an anti-LIGHT antibody; the immune target BTLA may be targeted by an anti-BTLA antibody; the immune target LAG3 may be targeted by an anti-LAG3 antibody;The immune target TIM3 may be targeted by an anti-TIM3 antibody; the immune target Singlecs may be targeted by an anti-Singlecs antibody; the immune target ICOS ligand may be targeted by an anti-ICOS ligand antibody; the immune target B7-H3 may be targeted by an anti-B7-H3 antibody; the immune target B7-H4 may be targeted by an anti-B7-H4 antibody; the immune target VISTA may be targeted by an anti-VISTA antibody; and the immune target TMIGD2 may be targeted by an anti-TMIGD2 antibody. immune targeting BTNL2 can be targeted by an anti-BTNL2 antibody; immune targeting CD48 can be targeted by an anti-CD48 antibody; immune targeting KIR can be targeted by an anti-KIR antibody; immune targeting LIR can be targeted by an anti-LIR antibody; immune targeting ILT can be targeted by an anti-ILT antibody; immune targeting NKG2D can be targeted by an anti-NKG2D antibody; immune targeting NKG2A can be targeted by an anti-NKG2A antibody; Targeting MICA can be targeted by an anti-MICA antibody; immune targeting MICB can be targeted by an anti-MICB antibody; immune targeting CD244 can be targeted by an anti-CD244 antibody; immune targeting CSF1R can be targeted by an anti-CSF1R antibody; immune targeting IDO can be targeted by an anti-IDO antibody; immune targeting TGFβ can be targeted by an anti-TGFβ antibody; immune targeting CD39 can be targeted by an anti-CD39 antibody; immune targeting CD73 can be targeted by an anti-CD73 antibody; immune targeting CXCR4 can be targeted by an anti-CXCR4 antibody; immune targeting CXCL12 can be targeted by an anti-CXCL12 antibody; immune targeting SIRPA can be targeted by an anti-SIRPA antibody; immune targeting CD47 can be targeted by an anti-CD47 antibody; immune targeting VEGF can be targeted by an anti-VEGF antibody; and immune targeting neuropilin can be targeted by an anti-neuropilin antibody (e.g., Mahoney, (See Table 1 in [End Page 110] et al.).

[0055] Immunotherapeutic agents that may be used in connection with the compositions and methods described herein include, for example, anti-TWEAK agents, anti-cell surface lymphocyte protein agents, anti-BRAF agents, anti-MEK agents, anti-CD33 agents, anti-CD20 agents, anti-HLA-DR agents, anti-HLA class I agents, anti-CD52 agents, anti-A33 agents, anti-GD3 agents, anti-PSMA agents, anti-Ceacan1 agents, anti-Galedin9 agents, anti-HVEM agents, anti-VISTA agents, and anti-B7 agents. Examples of such agents include H4 agents, anti-HHLA2 agents, anti-CD155 agents, anti-CD80 agents, anti-BTLA agents, anti-CD160 agents, anti-CD28 agents, anti-CD226 agents, anti-CEACAM1 agents, anti-TIM3 agents, anti-TIGIT agents, anti-CD96 agents, anti-CD70 agents, anti-CD27 agents, anti-LIGHT agents, anti-CD137 agents, anti-DR4 agents, anti-CR5 agents, anti-TNFRS agents, anti-TNFR1 agents, anti-FAS agents, anti-CD95 agents, anti-TRAIL agents, anti-DR6 agents, anti-EDAR agents, anti-NGFR agents, anti-OPG agents, anti-RANKL agents, anti-LTβ receptor agents, anti-BCMA agents, anti-TACI agents, anti-BAFFR agents, anti-EDAR2 agents, anti-TROY agents, and anti-RELT agents. For example, the immunotherapeutic agent may be an anti-TWEAK antibody or antigen-binding fragment thereof, an anti-cell surface lymphocyte protein antibody or antigen-binding fragment thereof, an anti-BRAF antibody or antigen-binding fragment thereof, an anti-MEK antibody or antigen-binding fragment thereof, an anti-CD33 antibody or antigen-binding fragment thereof, an anti-CD20 antibody or antigen-binding fragment thereof, an anti-HLA-DR antibody or antigen-binding fragment thereof, an anti-HLA class I antibody or antigen-binding fragment thereof, an anti-CD52 antibody or antigen-binding fragment thereof, an anti-A33 antibody or antigen-binding fragment thereof, an anti-GD3 antibody or antigen-binding fragment thereof, an anti-PSMA antibody or antigen-binding fragment thereof, an anti-Ceacan1 antibody or antigen-binding fragment thereof, an anti-Galedin9 antibody or antigen-binding fragment thereof, an anti-HVEM antibody or antigen-binding fragment thereof, an anti-VISTA antibody or antigen-binding fragment thereof, or an anti-B7 antibody.H4 antibody or its antigen-binding fragment, anti-HHLA2 antibody or its antigen-binding fragment, anti-CD155 antibody or its antigen-binding fragment, anti-CD80 antibody or its antigen-binding fragment, anti-BTLA antibody or its antigen-binding fragment, anti-CD160 antibody or its antigen-binding fragment, anti-CD28 antibody or its antigen-binding fragment, anti-CD226 antibody or its antigen-binding fragment, anti-CEACAM1 antibody or its antigen-binding fragment, anti-TIM3 antibody or its antigen-binding fragment, anti-TIGIT antibody or its antigen-binding fragment, anti-CD96 antibody or its antigen-binding fragment, anti-CD70 antibody or its antigen-binding fragment, anti-CD27 antibody or its antigen-binding fragment, anti-LIGHT antibody or its antigen-binding fragment, anti-CD137 antibody or its antigen-binding fragment, anti-DR4 antibody or its antigen-binding fragment, anti-CR5 antibody or its antigen-binding fragment The antibody may be an antigen-binding fragment thereof, an anti-TNFRS antibody or an antigen-binding fragment thereof, an anti-TNFR1 antibody or an antigen-binding fragment thereof, an anti-FAS antibody or an antigen-binding fragment thereof, an anti-CD95 antibody or an antigen-binding fragment thereof, an anti-TRAIL antibody or an antigen-binding fragment thereof, an anti-DR6 antibody or an antigen-binding fragment thereof, an anti-EDAR antibody or an antigen-binding fragment thereof, an anti-NGFR antibody or an antigen-binding fragment thereof, an anti-OPG antibody or an antigen-binding fragment thereof, an anti-RANKL antibody or an antigen-binding fragment thereof, an anti-LTβ receptor antibody or an antigen-binding fragment thereof, an anti-BCMA antibody or an antigen-binding fragment thereof, an anti-TACI antibody or an antigen-binding fragment thereof, an anti-BAFFR antibody or an antigen-binding fragment thereof, an anti-EDAR2 antibody or an antigen-binding fragment thereof, an anti-TROY antibody or an antigen-binding fragment thereof, or an anti-RELT antibody or an antigen-binding fragment thereof.

[0056] In some embodiments, the second anticancer therapy is immunotherapy, e.g., administering immune effector cells or an immunogenic composition. For example, the immunogenic composition comprises a cancer cell antigen and, optionally, an adjuvant. In some embodiments, the immune effector cells may comprise NK cells, T cells (e.g., CAR T cells), or NK / T cells. In some embodiments, T cells comprising a chimeric antigen receptor (CAR) and a kynureninase homodimer of the embodiments are contemplated for use in treating a subject with cancer. In some embodiments, cells may be transfected with DNA encoding the CAR and kynureninase, and optionally a transposase.

[0057] In some embodiments, the present disclosure provides a method for producing a T cell response in a human subject having a tumor, the method comprising administering to the subject a kynureninase homodimer according to any one of the above-described embodiments of the treatment method.

[0058] definition As used herein, the term "about" refers to a value within 10% above or below the stated value. For example, the phrase "about 5,000 Da" refers to a value between 4,500 and 5,500 Da, inclusive.

[0059] As used herein, the terms "administering," "administration," and the like refer to providing a therapeutic agent (e.g., a formulation) directly to a patient by any effective route, which agent (e.g., a formulation) is administered in conjunction with one or more proteins described herein. Exemplary routes of administration are described herein and include systemic routes, such as intravenous injection, and routes of administration directly into a patient's central nervous system, such as by intracerebroventricular injection, intrathecal injection, and stereotactic injection, among others.

[0060] As used herein, the term "biologically functional equivalent" is well-recognized in the art and is further defined in detail herein. Thus, it includes sequences in which about 70% to about 80%, or about 81% to about 90%, or even about 91% to about 99% of the amino acids are identical or functionally equivalent to the amino acids of a reference polypeptide, provided that the biological activity of the protein is maintained. In some embodiments, a modified protein may be biologically functionally equivalent to its native counterpart.

[0061] As used herein in the context of a protein of interest, the term "catalytic activity" refers to a biological function associated with the wild-type form of the protein. For example, in the context of an enzyme, the term "catalytic activity" refers to the ability of a protein to effect substrate turnover in a manner that yields the product of a corresponding chemical reaction. The activity level of an enzyme can be detected and quantified, for example, using substrate turnover assays known in the art. As another example, in the context of a membrane-bound receptor, the term "activity" can refer to signal transduction initiated by the receptor, for example, upon binding to its cognate ligand.

[0062] The term "chimeric antigen receptor (CAR)," as used herein, can refer to, for example, an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor, and can encompass engineered receptors that graft artificial specificity onto specific immune effector cells. CARs can be employed to confer the specificity of a monoclonal antibody to T cells, thereby enabling the generation of large numbers of specific T cells, for example, for use in adoptive cell therapy. In specific embodiments, a CAR directs the specificity of a cell to, for example, a tumor-associated antigen. In some embodiments, a CAR comprises an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor-associated antigen-binding region. In certain embodiments, a CAR comprises a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a CD3-zeta transmembrane and endodomain (e.g., as described in U.S. Pat. No. 7,109,304, incorporated herein by reference in its entirety). The specificity of other CAR designs can be derived from the receptor's ligand (e.g., a peptide) or from a pattern-recognition receptor, such as dectin. In certain embodiments, malignant B cells can be targeted by redirecting T cell specificity using a CAR specific for the B lineage molecule CD19. In some cases, the spacing of the antigen recognition domain can be modified to reduce activation-induced cell death. In some cases, the CAR contains domains for additional costimulatory signaling, such as CD3-zeta, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some cases, molecules including costimulatory molecules, reporter genes for imaging (e.g., positron emission tomography), gene products that conditionally eliminate T cells when a prodrug is administered, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors may be co-expressed with the CAR.

[0063] As used herein, the term "homodimer" refers to a protein (e.g., a kynureninase) comprising two polypeptide chains that are identical to each other with respect to the order, number, and types of amino acid residues. Thus, the term "kynureninase homodimer" refers to a kynureninase comprising two monomeric kynureninase polypeptide chains that are identical to each other with respect to the order, number, and types of amino acid residues.

[0064] As used herein, the terms "conservative mutation," "conservative substitution," "conservative amino acid substitution," and the like refer to the replacement of one or more amino acids with one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric bulk. These properties for each of the 20 naturally occurring amino acids are summarized in Table 1 below. [Table 1]

[0065] As used herein, the terms "contacted" and "exposed," when applied to a cell, are used herein to refer to the process of delivering a therapeutic construct and a chemotherapeutic or radiotherapeutic agent to or placing them in the immediate vicinity of a target cell, e.g., delivering both agents to the cell in a combined amount effective to kill the cell or prevent it from dividing, to achieve cell killing.

[0066] As used herein, the terms "effective amount," "therapeutically effective amount," and the like, when used in reference to a therapeutic composition, e.g., a vector construct, viral vector, or cell described herein, refer to an amount sufficient to produce a beneficial or desired result, e.g., a clinical outcome, when administered to a subject, including a mammal, e.g., a human. For example, in the context of treating cancer as described herein, these terms refer to an amount of a composition sufficient to produce a therapeutic benefit compared to the response obtained without administration of the composition, vector construct, viral vector, or cell. The amount of a given composition described herein that corresponds to such an amount can vary depending on various factors, such as the agent or pharmaceutical formulation given, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, weight), or the host being treated. An "effective amount," "therapeutically effective amount," and the like, of a composition, e.g., a vector construct, viral vector, or cell of the present disclosure, also includes an amount that produces a beneficial or desired result in a subject relative to a control.

[0067] As used herein, the term "fusion protein" refers to a chimeric protein containing proteins or protein fragments operably linked in a manner not found in nature.

[0068] As used herein, the term "half-life" (1 / 2 life) refers to the time that would be required for the concentration of a polypeptide to fall by half in vitro or in vivo, for example after injection into a mammal.

[0069] As used herein, "K M The term "k" refers to the Michaelis-Menten constant for an enzyme and is defined as the concentration of a particular substrate at which a given enzyme produces half its maximum rate in an enzyme-catalyzed reaction. cat The term "k" refers to the turnover number, or the number of substrate molecules that each enzyme site converts to product per unit time when the enzyme is working at maximum efficiency. cat / K MThe term "specificity constant" is a measure of how efficiently an enzyme converts a substrate into a product.

[0070] As used herein, the term "kynureninase" refers to an enzyme responsible for the catalytic degradation of kynurenine to anthranilic acid. The term "kynureninase" refers to variants of the wild-type kynureninase enzyme and nucleic acids encoding same, for example, variants having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity or greater) to the amino acid sequence of the wild-type kynureninase enzyme (e.g., SEQ ID NO: 1). "Kynureninase" also refers to a protein or polynucleotide having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity or greater) to the nucleic acid sequence of a wild-type kynureninase gene, provided that the encoded kynureninase analog retains the therapeutic function of the wild-type kynureninase. "Kynureninase" can also refer to a kynureninase protein in which the native signal peptide is present. Alternatively, "kynureninase" can refer to a kynureninase protein (e.g., a mature protein) from which the native signal peptide has been removed. Kynureninase can also refer to the catalytic domain of a kynureninase or a variant having at least 70% sequence identity to such a domain (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% or greater identity). In addition, the term "kynureninase" also includes variants in which the polypeptide is operably linked to another agent, eg, another polypeptide, a half-life modifying agent, eg, a PEGylated agent, or a therapeutic agent.As used herein, "kynureninase" can refer to the enzyme or the gene encoding this protein, depending on the context, as will be understood by those of skill in the art.

[0071] Kynureninase can exist as dimers of individual "kynureninase monomers," e.g., homodimers composed of two kynureninase monomers, each having the same amino acid sequence. As used herein, the term "kynureninase monomer" refers to an individual polypeptide that forms a dimeric kynureninase enzyme when non-covalently associated with another such polypeptide. A kynureninase monomer of the present disclosure can have an amino acid sequence that is, for example, at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 herein. In some embodiments, the kynureninase monomers of the disclosure contain one or more amino acid substitutions compared to SEQ ID NO:1, e.g., L8P, K38E, Y47L, I48F, K50Q, I51M, S60N, K64N, D65G, E66K, N67D, N67P, D67S, A68F, A68T, A68V, F71L, F71M, L72N, K84E, E88N, E89K, E89S, E 90Q, D92E, K93N, K93T, A95H, A95Q, K96N, I97H, I97L, I97V, A98G, A99G, A99I, A99R, A99S, A99T, A99V, Y 100N, Y100S, Y100T, G101A, H102W, E103F, E103H, E103N, E103Q, E103R, E103V, E103W, V104D, V104E, V1 04F, V104H, V104K, V104L, V104R, G105A, G105H, G105S, G105T, E106D, K106D, K106E, K106H, K106N, R1 07P, R107S, P108R, I110A, I110F, I110L, I110M, I110T, T111D, T111H, T111N, T111R, G112A, G112C, G11 2D, G112K, G112L, G112M, G112Q, G112R, G112S, G112T, G112Y, N127K, I131V, A132V, L133V, A136T, L137 T, 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, K19 1W, E197A, E197D, E197F, E197K, E197M, E197Q, E197S, E197T, E197V, I201C, I201E, I201F, 1201H, I201L, I201S, I 201T, I201V, H203K, L219M, L219W, F220L, V223I, F225Y, H230F, H230L, H230Y, N232S, Y246F, F249W, D250E, S274 A, S274C, S274G, S274N, S274T, L278M, A280G, A280S, A280T, G281S, A282M, A282P, G284N, I285L, V303L, V303S, F3 06W, 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, K38 1, A282G, 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. Kynureninase monomers of the disclosure can have, for example, substitutions at A282 (e.g., A282M or A282P), F306 (e.g., F306W or F306Y), or F249, e.g., F249W, compared to SEQ ID NO: 1. In some embodiments, a kynureninase monomer of the disclosure comprises a substitution at A99 compared to SEQ ID NO: 1. In some embodiments, a kynureninase monomer of the disclosure comprises a substitution at G112 compared to SEQ ID NO: 1. In some embodiments, a kynureninase monomer of the disclosure comprisesIt comprises a substitution at E103 compared to SEQ ID NO:1. In some embodiments, a kynureninase monomer of the disclosure comprises a substitution at V104 compared to SEQ ID NO:1. In some embodiments, a kynureninase monomer of the disclosure comprises a substitution at S408 compared to SEQ ID NO:1. In some embodiments, a kynureninase monomer of the disclosure comprises a F306W substitution. In some embodiments, a kynureninase monomer of the disclosure comprises a L72N substitution. In some embodiments, a kynureninase monomer of the disclosure comprises a H102W and N333T substitutions. In some embodiments, a kynureninase monomer of the disclosure comprises a I183P substitution. In some embodiments, a kynureninase monomer of the disclosure comprises a R107P substitution. In some embodiments, a kynureninase monomer of the disclosure comprises an A436T substitution. In some embodiments, a kynureninase monomer of the disclosure comprises at least one substitution selected from L72N, H102W, A282P, F306W, I331S, and N333T compared to SEQ ID NO: 1. In some embodiments, a kynureninase monomer of the disclosure comprises at least two, three, four, or five substitutions selected from L72N, H102W, A282P, F306W, I331S, and N333T compared to SEQ ID NO: 1. In some embodiments, a kynureninase monomer of the disclosure comprises the following substitutions compared to SEQ ID NO: 1: L72N, H102W, A282P, F306W, I331S, and N333T.

[0072] As used herein, the term "linking group" refers to a compound or portion thereof that acts as a molecular bridge operably linking two different molecules, with one portion of the linking group operably linked to a first molecule and another portion of the linking group operably linked to a second molecule.

[0073] As used herein, the term "modified protein" or "modified polypeptide" includes, for example, proteins or polypeptides that have additional advantages over the unmodified protein or polypeptide, such as kynurenine degrading activity or thermodynamic stability.

[0074] As used herein, the term "monomer" refers to a single polypeptide chain that is the simplest basic subunit of a protein's further quaternary structure.

[0075] As used herein, the term "PEGylation agent" refers to a polyethylene glycol (PEG) molecule that can be attached (e.g., covalently linked) to, for example, an active agent via the hydroxy groups at the ends of the PEG chains by means of a variety of chemical methods to proteins. In some embodiments, the PEG itself limits the number of active agents to at most two per PEG molecule. A different approach can use copolymers of PEG and amino acids, which retain the biocompatibility of PEG but have the added advantage of multiple attachment points per molecule, thus resulting in higher drug loading.

[0076] As used herein, "percent sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in the reference polynucleotide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be accomplished in a variety of ways within the skill of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences to be compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A to, with, or against a given nucleic acid or amino acid sequence B (which may alternatively be expressed as a given nucleic acid or amino acid sequence A having a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence B) is calculated as follows: Multiply by 100 (fraction X / Y) where X is the number of nucleotides or amino acids that a sequence alignment program (e.g., BLAST) evaluates as an identical match in its alignment of A and B, and Y is the total number of nucleic acids in B. It will be recognized that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0077] As used herein, the term "pharmaceutically acceptable" refers to compounds, substances, compositions and / or dosage forms that are suitable for contact with the tissues of a subject, e.g., a mammal (e.g., a human) without undue toxicity, irritation, allergic response and other problematic complications commensurate with a reasonable benefit / risk ratio.

[0078] As used herein, the terms "protein" and "polypeptide" refer to compounds comprising amino acids joined by peptide bonds and are used interchangeably.

[0079] As used herein, "subject" and "patient" refer to either a human or non-human, such as a primate, mammal, or vertebrate. In certain embodiments, the subject is a human.

[0080] As used herein, "treatment" and "treating" refer to an approach for obtaining beneficial or desired results, e.g., clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or symptoms, whether detectable or undetectable; reduction in the severity of the disease or condition; a stabilized (i.e., not worsening) state of the disease, disorder, or condition; preventing the spread of the disease or condition; slowing or slowing the disease or condition; improvement or palliative care of the disease or condition; and remission (partial or complete). "Ameliorating" or "palliating" a disease or condition means a decrease in the severity and / or undesirable clinical signs of the disease, disorder, or condition, and / or a slowing or prolongation of the time course of progression compared to the severity or time course without treatment. "Treatment" can also mean prolonging survival compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, those susceptible to or at risk of developing the condition or disorder, and those in whom the condition or disorder is to be prevented.

[0081] As used herein, the term "unit dose," when used in reference to a therapeutic composition, refers to physically discrete units suitable as unitary dosages for subjects, each unit containing a predetermined quantity of active material calculated to produce a desired therapeutic effect, together with the required diluent, i.e., carrier or vehicle.

[0082] 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. [Brief explanation of the drawings]

[0083] [Figure 1A] Production of PEGylated kynureninase conjugates. Number of conjugated PEG molecules per kynureninase monomer obtained at PEG:kynureninase homodimer input ratios of 20:1 to 60:1. All molar input ratios shown in Figures 1A-1F are PEG to kynureninase homodimer input ratios. [Figure 1B] Production of PEGylated kynureninase conjugates. Number of conjugated PEG molecules per kynureninase homodimer obtained at PEG:kynureninase homodimer input ratios ranging from 20:1 to 60:1. All molar input ratios shown in Figures 1A-1F are PEG to kynureninase homodimer input ratios. [Figure 1C] Production of PEGylated kynureninase conjugates. Efficiency of the conjugation reaction between PEG molecules and kynureninase at input ratios of 20:1 to 60:1 PEG:kynureninase homodimer. All molar input ratios shown in Figures 1A-1F are the input ratios of PEG to kynureninase homodimer. [Figure 1D] Production of PEGylated kynureninase conjugates. SEC-MALS HPLC of the PEGylated kynureninase homodimer product obtained at a 20:1 input ratio of PEG to kynureninase homodimer. All molar input ratios shown in Figures 1A-1F are the input ratio of PEG to kynureninase homodimer. [Figure 1E]Production of PEGylated kynureninase conjugates. SEC-MALS HPLC of the PEGylated kynureninase homodimer product obtained at a 30:1 input ratio of PEG to kynureninase homodimer. All molar input ratios shown in Figures 1A-1F are the input ratio of PEG to kynureninase homodimer. [Figure 1F] Production of PEGylated kynureninase conjugates. SEC-MALS HPLC of the PEGylated kynureninase homodimer product obtained at a 60:1 input ratio of PEG to kynureninase homodimer. All molar input ratios shown in Figures 1A-1F are the input ratio of PEG to kynureninase homodimer. [Figure 2A] Comparison of the biological activity of PEGylated kynureninase enzymes. SEC-HPLC profiles of kynureninase at different feed ratios (shown as PEG:kynureninase homodimer). Peaks, from left to right, correspond to a 100:1 molar feed ratio of PEG:kynureninase homodimer, an 80:1 molar feed ratio of PEG:kynureninase homodimer, a 40:1 molar feed ratio of PEG:kynureninase homodimer, a 20:1 molar feed ratio of PEG:kynureninase homodimer, a 10:1 molar feed ratio of PEG:kynureninase homodimer, and unPEGylated kynureninase homodimer. All molar feed ratios shown in Figures 2A-2C are the PEG to kynureninase homodimer feed ratios. [Figure 2B] Comparison of the biological activity of PEGylated kynureninase enzymes. Mouse plasma PD 72 hours after a single iv dose of PEGylated kynureninase. Values ​​on the x-axis correspond to the molar input ratio of PEG:kynureninase homodimer. All molar input ratios shown in Figures 2A-2C are the input ratio of PEG to kynureninase homodimer. [Figure 2C]Comparison of the biological activity of PEGylated kynureninase enzymes. CT26 tumor accumulation of PEGylated kynureninase quantified by anti-PEG Western blot. The x-axis values ​​represent the time points at which tumor accumulation was assessed after administration of kynureninase to BALB / c mice as described in Example 3. Four vertical bars are shown for each time point shown in Figure 2C, each representing the relative tumor accumulation of kynureninase formed at a particular input ratio of PEG to kynureninase homodimer. From left to right, the vertical bars at each time point correspond to kynureninase homodimers formed at molar input ratios of 10:1, 20:1, 40:1, and 100:1, respectively. All molar input ratios shown in Figures 2A-2C are the input ratio of PEG to kynureninase homodimer. DETAILED DESCRIPTION OF THE INVENTION

[0084] The present disclosure provides kynureninase proteins covalently linked to polyethylene glycol (PEG), as well as methods for synthesizing such proteins and using them to treat patients with cancer, e.g., tumor-bearing patients. In particular, the present disclosure provides PEGylated kynureninase proteins characterized by a PEG to kynureninase ratio that results in improved pharmacokinetic and / or pharmacodynamic properties, e.g., improved tumor accumulation, extended half-life, and greater enzymatic activity, compared to kynureninase proteins that are not covalently linked to PEG. Without being limited by mechanism, the kynureninase proteins described herein may exhibit improved resistance to inactivation in serum, which allows the kynureninase to maintain kynurenine-degrading activity for a longer period of time. Thus, PEGylated kynureninase enzymes characterized by the PEG:kynureninase ratios described herein may exhibit substantially improved therapeutic attributes that make them ideal for therapeutic disease intervention, e.g., the treatment of cancer. In some embodiments, kynureninase proteins of the disclosure are used to treat cancer patients, for example, patients with tumors that express indoleamine 2,3 dioxygenase (IDO) or tryptophan 2,3 dioxygenase (TDO).

[0085] The following sections describe ratios of PEG:kynureninase polypeptide that result in the beneficial therapeutic attributes noted above, as well as processes that can be used to produce such proteins and use them to treat patients.

[0086] Kynureninase protein Some embodiments of PEGylated kynureninase enzymes relate to modified proteins and polypeptides. Certain embodiments relate to modified proteins or polypeptides that exhibit at least one functional activity, preferably kynurenine degrading activity, to the same extent as the unmodified form. In further embodiments, the proteins or polypeptides may be further modified to improve serum stability.

[0087] Determining activity can be accomplished using assays well known to those skilled in the art, particularly for protein activity, and can include the use of native and / or recombinant forms of either modified or unmodified proteins or polypeptides for comparison purposes.

[0088] In some embodiments, a modified polypeptide, e.g., a modified kynureninase, can be identified based on its increased kynurenine. For example, the substrate recognition site of the unmodified polypeptide can be identified. This identification can be based on structural or homology analysis. A population of mutants with such modified substrate recognition sites can be generated. In further embodiments, mutants with increased kynurenine degradation activity can be selected from the mutant population. Selection of the desired mutant can include methods such as detecting by-products or products of kynurenine degradation.

[0089] Modified proteins can have amino acid deletions and / or substitutions; thus, proteins with deletions, proteins with substitutions, and proteins with deletions and substitutions are modified proteins. In some embodiments, these modified proteins can further include inserted or added amino acids, as in the case of fusion proteins or proteins with linkers. Modified deletion proteins lack one or more residues of the native protein but can retain the specificity and / or activity of the native protein. Modified deletion proteins can also have reduced immunogenicity or antigenicity. One example of a modified deletion protein is one in which amino acid residues have been deleted from at least one antigenic region, i.e., from a region of the protein determined to be antigenic in a particular organism, e.g., one species of organism to which the modified protein may be administered.

[0090] Substitution or substitution variants typically involve the exchange of one amino acid for another at one or more sites in the protein, and can 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, i.e., replacing one amino acid with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, 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.

[0091] In addition to deletions or substitutions, modified proteins can have residue insertions, which typically involve the addition of at least one residue in the polypeptide. This can include the insertion of a targeting peptide or polypeptide, or simply a single residue. Terminal additions, called fusion proteins, are discussed below.

[0092] It will also be understood that amino acid and nucleic acid sequences can include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and still be essentially as set forth in one of the sequences disclosed herein, so long as the sequences meet the criteria set forth above, including maintenance of biological protein activity related to protein expression. The addition of terminal sequences applies in particular to nucleic acid sequences, which can include, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region, or can include various internal sequences, i.e., introns, that are known to occur within genes.

[0093] In some embodiments, a kynureninase according to the embodiments comprises an amino acid sequence that 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 NO: 1, 2 or 3. In further embodiments, the 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 NO: 1, 2 or 3 and is 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, Y1 00S, Y100T, G101A, H102W, E103F, E103H, E103N, E103Q, E103R, E103V, E103W, V104D, V104E, V104F, V104H, V104K, V104L, V104R, G10 5A, G105H, G105S, G105T, E106D, K106D, K106E, K106H, K106N, R107P, R107S, P108R, I110A, I110F, I110L, I110M, I110T, T111D, T11 1H, T111N, T111R, G112A, G112C, G112D, G112K, G112L, G112M, G112Q, G112R, G112S, G112T, G112Y, N127K, I131V, A132V, L133V, A136 T, 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, L2 19W, F220L, V223I, F225Y, H230F, H230L, H230Y, N232S, Y246F, F249W, D250E, S274A, S274C, S274G, S274 N, 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, L33 8A, 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, S4 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 amino acid substitutions selected from the group consisting of: O8N, N411R, D413S, D413V, Q416T, E419A, E419L, K420E, R421N, V424I, K427M, N429E, G432A and A436T. In some embodiments, the 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 shown in Table 2. In some embodiments, the kynureninase is selected from one of those shown in Table 2.

[0094] Table 2 below lists various kynureninase enzymes that may be used with the compositions and methods of the present disclosure. Although not explicitly listed 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.

[0095] SEQ ID NO:1 - Wild-type human kynureninase: MEPSSLELPADTVQRIAAELKCHPTDERVALHLDEEDKLRHFRECFYIPKIQDLPPVDLSLVNKDENAIYFLGNSLGLQPKMVKTYLEEELDKWAKIAAYGHEVGKRPWITGDESI VGLMKDIVGANEKEIALMNALTVNLHLLMLSFFKPTPKRYKILLEAKAFPSDHYAIESQLQLHGLNIEESMRMIKPREGEETLRIEDILEVIEKEGDSIAVILFSGVHFYTGQHFN IPAITKAGQAKGCYVGFDLAHAVGNVELYLHDWGVDFACWCSYKYLNAGAGGIAGAFIHEKHAHTIKPALVGWFGHELSTRFKMDNKLQLIPGVCGFRISNPPILLVCSLHASLEI FKQATMKALRKKSVLLTGYLEYLIKHNYGKDKAATKKPVVNIITPSHVEERGCQLTITFSVPNKDVFQELEKRGVVCDKRNPNGIRVAPVPLYNSFHDVYKFTNLLTSILDSAETKN [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10]

[0096] combination The compositions and methods of the present disclosure include modified kynureninase, for example, by conjugation with a heterologous peptide segment or polymer, such as polyethylene glycol (PEG). The kynureninase is linked to PEG to increase the hydrodynamic radius of the enzyme, thereby improving serum persistence. In some embodiments, the polypeptides of the present disclosure can be conjugated to any targeting agent, such as a ligand capable of specifically and stably binding to an external receptor or binding site on tumor cells (U.S. Patent Publication No. 2009 / 0304666).

[0097] PEGylation In some embodiments of the present disclosure, methods and compositions related to the PEGylation of kynureninase are disclosed, for example, kynureninase is PEGylated according to the methods disclosed herein.

[0098] PEGylation is the process of covalently attaching poly(ethylene glycol) polymer chains to another molecule, usually a drug or therapeutic protein. PEGylation is traditionally accomplished by incubation of a reactive derivative of PEG with the target macromolecule. Covalent attachment of PEG to a drug or therapeutic protein can mask the drug from the host's immune system (reducing immunogenicity and antigenicity) or increase the drug's hydrodynamic size (size in solution), which extends its circulation time by reducing renal clearance. PEGylation can also impart water solubility to hydrophobic drugs and proteins. The first step in PEGylation is the appropriate functionalization of one or both termini of the PEG polymer. PEGs activated at each terminus with the same reactive moiety are known as "homobifunctional," while PEG derivatives with different functional groups present are termed "heterobifunctional" or "heterofunctional." Chemically active or activated derivatives of the PEG polymer are prepared to conjugate PEG to the desired molecule.

[0099] The selection of a suitable functional group for a PEG derivative is based on the type of available reactive group on the molecule to be linked to PEG. In proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. N-terminal amino groups and C-terminal carboxylic acids can also be used. The technique used to form first-generation PEG derivatives generally involves reacting PEG polymers with groups reactive with hydroxyl groups, typically anhydrides, acid chlorides, chloroformates, and carbonates. Second-generation PEGylation chemistry makes more efficient functional groups available for conjugation, such as aldehydes, esters, and amides.

[0100] As PEGylation applications become more advanced and sophisticated, the need for heterobifunctional PEGs for conjugation has increased. These heterobifunctional PEGs are very useful for linking two entities when a hydrophilic, flexible, and biocompatible spacer is required. Preferred terminal groups for heterobifunctional PEGs are maleimide, vinyl sulfone, pyridyl disulfide, amine, carboxylic acid, and NHS ester.

[0101] The most common modifiers or linkers are based on methoxy PEG (mPEG) molecules. Their activity depends on the addition of protein-modifying groups to the alcohol termini. In some cases, polyethylene glycol (PEG diol) is used as a precursor molecule. Both termini of the diol are then modified to create hetero- or homodimeric PEG-linked molecules.

[0102] Proteins are typically PEGylated at nucleophilic sites, such as unprotonated thiols (cysteinyl residues) or amino groups. Examples of cysteinyl-specific (i.e., cystine-reactive) modifying reagents include PEG-maleimide, PEG-iodoacetate, PEG-thiol, and PEG-vinylsulfone. All four exhibit strong cysteinyl specificity under mild conditions and at neutral to slightly alkaline pH.

[0103] Amine-specific (i.e., lysine-reactive) modifiers include PEG NHS ester, PEG tresylate, PEG aldehyde, PEG isothiocyanate, and several others. All react under mild conditions and are highly specific for amino groups. PEG NHS ester is considered one of the more reactive agents, but its high reactivity can make PEGylation reactions difficult to control on a large scale. PEG aldehyde forms an imine with the amino group, which is then reduced to a secondary amine by sodium cyanoborohydride. Unlike sodium borohydride, sodium cyanoborohydride does not reduce disulfide bonds. However, this chemical is highly toxic and must be handled with care, especially at lower pHs where it becomes volatile.

[0104] Site-specific PEGylation can be challenging because most proteins contain multiple lysine residues. Because these reagents react with unprotonated amino groups, PEGylation can be directed to amino groups with low pK by performing the reaction at a lower pH. Generally, the pK of alpha amino groups is 1–2 pH units lower than that of the epsilon amino groups of lysine residues. High selectivity for the N-terminus can frequently be achieved by PEGylating molecules at pH 7 or below. However, this is only feasible if the N-terminal portion of the protein is not required for biological activity. Nevertheless, the pharmacokinetic benefits of PEGylation often outweigh the significant loss of in vitro bioactivity, resulting in products with much greater in vivo bioactivity despite the PEGylation chemistry.

[0105] 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 possibly the exclusion of oxygen (oxygen can cause intermolecular disulfide formation with the protein, which reduces the yield of the PEGylated product). For amine-specific modifications, the same factors (apart from oxygen) should be considered, although pH may be even more critical, especially when targeting the N-terminal amino group.

[0106] In both amine- and thiol-specific modifications, reaction conditions can affect protein stability. This can limit temperature, protein concentration, and pH. In addition, the reactivity of the PEG linker must be known before initiating the PEGylation reaction. For example, if the PEGylation agent is only 70% active, the amount of PEG used must ensure that only active PEG molecules are included in the stoichiometry of the protein-PEG reaction.

[0107] Linkers for chemical conjugation Various linkers can be used to covalently link the reactive residues of a kynureninase homodimer and the PEG molecule to form, for example, the conjugates described herein. Exemplary linkers include those that can be cleaved, for example, 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 for purposes of describing linkers suitable for chemical coupling). Examples of linkers useful in synthesizing the conjugates described herein include, among others, those containing electrophiles suitable for reaction with nucleophilic substituents present in antibodies, antigen-binding fragments, proteins, peptides, and small molecules, such as amine and thiol moieties, such as Michael acceptors (e.g., maleimides), active esters, electron-deficient carbonyl compounds, and aldehydes. For example, linkers suitable for synthesis of therapeutic agent conjugates include, but are not limited to, alkyl, cycloalkyl, and heterocycloalkyl linkers, such as open-chain ethyl, propyl, butyl, hexyl, heptyl, octyl, nonyl, or decyl chains, cyclohexyl, cyclopentyl, cyclobutyl, cyclopropyl, piperidinyl, morpholino, 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 in kynureninase), aryl, or heteroaryl linkers, such as benzyl, naphthyl, or pyridyl groups that contain two halomethyl groups that can be displaced by reactive nucleophilic atoms present in 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, as described, for example, in Liu et al., 18:690-697, 1979, the disclosure of which is incorporated herein by reference for linkers for chemical conjugation. Additional linkers include the non-cleavable maleimidocaproyl linker, as described in Doronina et al., Bioconjugate Chem. 17:14-24, 2006, the disclosure of which is incorporated herein by reference for linkers for chemical conjugation.

[0108] Additional linkers that can connect one component of a conjugate to another as described herein include linkers that are covalently attached at one end to one component of the conjugate (e.g., kynureninase) and contain a chemical moiety at the other end formed by a coupling reaction between a reactive substituent present on the linker and a reactive substituent present in the other component of the conjugate (e.g., a PEG molecule described herein). Exemplary reactive substituents that can be present in the components of the conjugate include the hydroxyl moieties of serine, threonine, and tyrosine residues; the amino moieties of lysine residues; the carboxy moieties of aspartic acid and glutamic acid residues; and the 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 with the conjugates described herein include, but are not limited to, linkers containing a chemical moiety formed by a coupling reaction, as depicted in Table 3 below. The curved lines represent the points of attachment to each component of the conjugate.

[0109] Linkers that can be used to conjugate PEG molecules to enzymes include those that are covalently attached to the enzyme at one end of the linker and contain a chemical moiety at the other end formed by a coupling reaction between a reactive substituent present on the linker and a reactive substituent present in one of the side chains of an amino acid residue present as part of the enzyme. Reactive substituents that can be present in kynureninase enzymes include the hydroxyl moieties of serine, threonine, and tyrosine residues, the amino moieties of lysine residues, the carboxyl moieties of aspartic acid and glutamic acid residues, and the 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 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0110] Recombinant expression of kynureninase in host cells The present disclosure includes compositions and methods for recombinantly expressing kynureninase proteins from host cells, such as prokaryotic or eukaryotic producer cells. Exemplary methods that can be used to effect expression of kynureninase proteins in host cells are described in more detail in the following sections.

[0111] Polynucleotides encoding kynureninase One platform that can be used to express kynureninase proteins from host cells, such as mammalian cells, is by stable integration of one or more genes encoding kynureninase into the host cell genome (e.g., integration into the nuclear genome of mammalian cells). These genes are polynucleotides that encode the primary amino acid sequence of the corresponding protein. To introduce such foreign genes into mammalian cells, these genes can be incorporated into vectors. Vectors can be introduced into cells by a variety of methods, including transformation, transfection, direct uptake, particle bombardment, and by encapsulating the vector in liposomes. Examples of suitable methods for 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.

[0112] The gene encoding the therapeutic protein of the present disclosure can also be introduced into mammalian cells by directing the vector containing the gene encoding such drug to cell membrane phospholipids.For example, by linking the vector molecule to the VSV-G protein, which is a viral protein that has affinity for all cell membrane phospholipids, the vector can be directed to the phospholipids on the extracellular surface of the cell membrane.Such constructs can be produced using methods well known to those skilled in the art.

[0113] Recognition and binding of a polynucleotide encoding one or more therapeutic proteins of the present disclosure by mammalian RNA polymerase is important for gene expression. Therefore, sequence elements may be included within the polynucleotide that exhibit high affinity for transcription factors that recruit RNA polymerase and promote assembly of a transcription complex at the transcription initiation site. Such sequence elements include, for example, mammalian promoters, sequences that can be recognized and bound by specific transcription initiation factors and ultimately RNA polymerase. Examples of mammalian promoters are described in Smith et al., Mol. Sys. Biol., 3:73, published online, the disclosure of which is incorporated herein by reference.

[0114] Once a polynucleotide encoding one or more therapeutic proteins has been integrated into the nuclear DNA of a mammalian cell, transcription of the 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 transcription factors and / or RNA polymerase to the mammalian promoter, thereby controlling gene expression. The chemical reagent may serve to facilitate the binding of RNA polymerase and / or transcription factors to the mammalian promoter, for example, by removing a promoter-bound repressor protein. Alternatively, the chemical reagent may serve to enhance the affinity of the mammalian promoter for RNA polymerase and / or transcription factors, such that the rate of transcription of genes located downstream of the promoter increases in the presence of the chemical reagent. Examples of chemical reagents that enhance polynucleotide transcription by the above mechanisms are tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, CA) and can be administered to mammalian cells according to established protocols to promote gene expression.

[0115] Another DNA sequence element that can be included in a polynucleotide for use in the compositions and methods described herein is an enhancer sequence. Enhancers represent another class of regulatory elements that induce conformational changes in a polynucleotide containing a gene of interest, causing the DNA to adopt a three-dimensional orientation that favors the binding of transcription factors and RNA polymerase at the transcription start site. Thus, polynucleotides for use in the compositions and methods described herein include those that encode one or more therapeutic proteins and also contain mammalian enhancer sequences. Many enhancer sequences derived from mammalian genes are currently known, including enhancers from genes encoding mammalian globin, elastase, albumin, alpha-fetoprotein, and insulin. Enhancers for use in the compositions and methods described herein also include those derived from the genetic material of viruses capable of infecting eukaryotic cells. Examples include 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).

[0116] fusion proteins Some embodiments of the present disclosure relate to fusion proteins. These molecules can have a native or modified kynureninase linked at the N- or C-terminus to a heterologous domain. For example, fusions can also use leader sequences from other species to enable recombinant expression of the 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 immunoreactive domain, such as an antibody epitope, which is preferably cleavable to facilitate purification of the fusion protein. Affinity tags include, but are not limited to, polyhistidine, chitin-binding protein (CBP), maltose-binding protein (MBP), and glutathione-S-transferase (GST).

[0117] In certain embodiments, kynureninase may be linked to a peptide that increases its half-life in vivo, such as an XTEN polypeptide (Schellenberger et al., 2009), an IgG Fc domain, albumin, or an albumin-binding peptide.

[0118] Methods for generating fusion proteins are well known to those skilled in the art. Such proteins can be produced, for example, by de novo synthesis from an intact fusion protein or by joining DNA sequences encoding heterologous domains and then expressing the intact fusion protein. Production of fusion proteins that restore the functional activity of the parent protein can be facilitated by linking the gene to a bridging DNA segment encoding a peptide linker joined between the tandemly linked polypeptides. The linker will be of sufficient length to allow proper folding of the resulting fusion protein.

[0119] Enzymatic Kynurenine Degradation for Therapeutic Use In some embodiments, the kynureninases of the present disclosure can be used to treat diseases, including cancers, that are sensitive to kynurenine removal by enzymes that remove kynurenine, thereby inhibiting tumor-mediated immune tolerance and instead mediating a tumor-eliminating pro-inflammatory response. In some embodiments, kynureninases are contemplated for use in treating tumors that express IDO1, IDO2, and / or TDO.

[0120] Some embodiments of the present disclosure provide modified kynureninases for treating diseases, such as tumors. Specifically, the modified kynureninases may have a human polypeptide sequence and therefore may prevent allergic reactions in human patients, allow repeated dosing, and improve therapeutic efficacy.

[0121] Tumors for which the therapeutic methods of the present invention are useful include any malignant cell type, such as those found in solid tumors or hematopoietic tumors. Exemplary solid tumors may include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, pharynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematopoietic tumors include tumors of the bone marrow, T- or B-cell malignancies, leukemia, lymphoma, blastoma, myeloma, and the like. Further examples of cancers that may be treated using the methods provided herein include carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), cancer of the peritoneum, hepatocellular carcinoma, gastric or gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colorectal cancer, colon cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentigo melanoma, nodular melanoma, and the like. and B-cell lymphomas (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 mass 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.

[0122] The cancer may be of the following histological types, specifically but not limited to: malignant neoplasm; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; calcifying epithelioma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; bile duct carcinoma; hepatocellular carcinoma; mixed hepatocellular and bile duct carcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; familial polyposis coli adenocarcinoma; solid carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; Eosinophilic adenocarcinoma; Basophilic carcinoma; Clear cell adenocarcinoma; Granular cell carcinoma; Follicular adenocarcinoma; Papillary and follicular adenocarcinoma; Non-encapsulating sclerosing carcinoma; Adrenal cortical carcinoma; Endometrioid carcinoma; Cutaneous adnexal carcinoma; Apocrine adenocarcinoma; Sebaceous gland carcinoma; Ceruminous adenocarcinoma; Mucoepidermoid carcinoma; Cystadenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous adenocarcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma of the breast; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; Acinic cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Malignant thymoma; Malignant ovarian stromal tumor; Malignant theca cell tumor; Malignant granulosa cell tumor; Malignant androblastoma; Certo Lymphocyte carcinoma; Malignant Leydig cell tumor; Malignant lipid cell tumor; Malignant paraganglioma; Malignant extramammary paraganglioma; Pheochromocytoma; Glomus angiosarcoma; Malignant melanoma; Amelanotic melanoma; Superficial spreading melanoma; Malignant melanoma in giant pigmented nevus; Epithelioid cell melanoma, malignant blue nevus; Sarcoma; Fibrosarcoma; Malignant fibrous histiocytoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Malignant mixed tumor; Mixed Müllerian tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Malignant mesenchymoma; Malignant Brenner tumor; Malignant phyllodes tumor; Synovial sarcoma tumor; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; parosteal osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal; cerebellar sarcoma;Ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's; paragranulomatous; malignant small lymphocytic lymphoma; malignant diffuse large cell lymphoma; malignant follicular lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphocytic 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.

[0123] Kynureninase, as used herein, can be used as an antitumor agent in various ways to remove kynurenine and / or kynurenine-derived metabolites from tumor tissue or the circulatory system of a mammal with cancer, or to remove kynurenine when such removal is deemed desirable. Removal can be performed in vivo in the circulatory system of a mammal, in vitro when removal of kynurenine and / or kynurenine-derived metabolites is desired in tissue culture or other biological media, or in ex vivo procedures when biological fluids, cells, or tissues are manipulated outside the body and then returned to the patient mammal's body. Removal of kynurenine from the circulatory system, culture medium, biological fluid, or cells is performed to reduce the amount of kynurenine that can reach the material being treated, and therefore involves contacting the material to be removed with a kynurenine-removing dose of kynureninase under kynurenine-removing conditions that degrade ambient kynurenine in the material being contacted.

[0124] The target of elimination need not necessarily be the cells themselves, but may be a source of nutrition for the cells. Thus, in in vivo applications, treating tumor cells involves contacting a nutrient medium for a population of tumor cells with kynureninase. In this embodiment, the medium may be blood, lymph, cerebrospinal fluid, or similar bodily fluid from which kynurenine removal is desired.

[0125] The efficiency of kynurenine and / or kynurenine-derived metabolite removal can vary widely depending on the application and is typically determined by the amount of kynurenine present in the material, the desired removal rate, and the material's resistance to exposure to kynureninase. Kynurenine and kynurenine metabolite levels in a material, and thus the rate of kynurenine and kynurenine metabolite removal from the material, can be readily monitored and assessed by a variety of chemical and biochemical methods well known in the art. Exemplary kynurenine removal doses are further described herein and can range, for example, from 0.001 to 1,000 units (U) of kynureninase, depending on the subject being treated and the severity of the disease.

[0126] Kynurenine removal conditions are buffer and temperature conditions compatible with the biological activity of kynureninase, including mild temperature, salt, and pH conditions compatible with the enzyme, such as physiological conditions. Exemplary conditions include about 4-40°C, an ionic strength equivalent to about 0.05-0.2 M NaCl, and a pH of about 5-9, including physiological conditions.

[0127] In some embodiments, the present disclosure contemplates a method of using kynureninase as an anti-tumor agent, thus comprising contacting a population of tumor cells with a therapeutically effective amount of kynureninase for a period of time sufficient to inhibit tumor cell growth.

[0128] A therapeutically effective amount of kynureninase is a predetermined amount calculated to achieve the desired effect, i.e., to remove kynurenine in tumor tissue or the patient's circulatory system, thereby mediating a tumor-eliminating pro-inflammatory response. Thus, the dosage range for administration of the kynureninase of the present disclosure should be large enough to produce the desired effect of reducing symptoms of tumor cell division and cell circulation. The dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndrome, pulmonary edema, congestive heart failure, neurological effects, etc. Generally, dosage will vary depending on the patient's age, symptoms, sex, and extent of disease, and can be determined by one skilled in the art. Dosage can be adjusted by an individual physician in the event of any complications.

[0129] Kynureninase can be administered parenterally by injection or by slow infusion over time. Kynureninase can be administered intravenously, intraperitoneally, orally, intramuscularly, subcutaneously, intracavity, transdermally, dermally, delivered by peristaltic means, injected directly into tissue containing tumor cells, or by a pump connected to a catheter that may contain an electric potential biosensor for kynurenine.

[0130] Therapeutic compositions containing kynureninase are conventionally administered intravenously, for example, by injection of a unit dose.

[0131] The composition is administered in a therapeutically effective amount and in a manner compatible with the dosage formulation. The amount to be administered depends on the subject being treated, the subject's body's ability to utilize the active ingredient, and the degree of therapeutic effect desired. The precise amount of active ingredient required to be administered depends on the judgment of the practitioner and is peculiar to each individual. However, suitable dosage ranges for systemic use are disclosed herein and will depend on the route of administration. Suitable dosing regimens for initial and booster administrations are also contemplated, typically an initial administration followed by subsequent injections or other administrations at intervals of one or more hours. Exemplary multiple administrations are described herein and are particularly suitable for maintaining continuously high serum and tissue levels of kynureninase and conversely low serum and tissue levels of kynurenine. Alternatively, continuous intravenous infusion sufficient to maintain blood concentrations within the range specified for in vivo treatment is contemplated.

[0132] Proteins and Peptides In some embodiments, the present disclosure relates to novel compositions comprising at least one protein or peptide, such as kynureninase, which may be included in a fusion protein or conjugated to an agent as described above.

[0133] As used herein, protein or peptide generally refers to, but is not limited to, proteins of more than about 200 amino acids, up to the full-length sequence translated from a gene; polypeptides of more than about 100 amino acids; and / or peptides of about 3 to about 100 amino acids. For convenience, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein.

[0134] As used herein, "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimetic known in the art. In some embodiments, the residues of a protein or peptide are consecutive, and no non-amino acids interrupt the sequence of amino acid residues. In some embodiments, the sequence may contain one or more non-amino acid moieties. In certain embodiments, the sequence of residues of a protein or peptide may be interrupted by one or more non-amino acid moieties.

[0135] Thus, the term "protein or peptide" encompasses amino acid sequences that contain at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid.

[0136] Proteins or peptides can be made by any technique known to those skilled in the art, including expressing proteins, polypeptides, or peptides using standard molecular biology techniques, isolating proteins or peptides from natural sources, or chemically synthesizing proteins or peptides. Nucleotide sequences corresponding to various genes, as well as protein, polypeptide, and peptide sequences, have been previously disclosed and can be found in computerized databases known to those skilled in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information (available on the World Wide Web at ncbi.nlm.nih.gov / ). The coding regions of known genes can be amplified and / or expressed using techniques disclosed herein or known to those skilled in the art. Alternatively, various commercial preparations of proteins, polypeptides, and peptides are known to those skilled in the art.

[0137] Pharmaceutical Composition 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 cancer. It can be administered intravenously, intrathecally, and / or intraperitoneally. It can be administered alone or in combination with an antiproliferative agent. In one embodiment, it is administered to reduce the cancer tumor burden in a patient before surgery or other procedures. Alternatively, it is administered after surgery to ensure that any remaining cancer (e.g., cancer not removed by surgery) does not survive.

[0138] It is not intended that the present disclosure be limited by the particular nature of the therapeutic agent formulation. For example, such compositions may be provided as a formulation together with physiologically tolerable liquid, gel, or solid carriers, diluents, and excipients. These therapeutic agent formulations may be administered to mammals in the same manner as other therapeutic agents for veterinary use, such as for domestic animals, and for clinical use in humans. In general, the dosage required for therapeutic effectiveness will vary depending on the type of use and mode of administration, as well as the individual requirements of the individual subject.

[0139] Such compositions are typically prepared as injections, either as liquid solutions or suspensions. Suitable diluents and excipients include, for example, water, saline, dextrose, glycerol, and the like, and combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, stabilizers, or pH buffering agents.

[0140] Where clinical use is contemplated, pharmaceutical compositions containing proteins, antibodies, and drugs may need to be prepared in a form appropriate for the intended use. Generally, pharmaceutical compositions may contain an effective amount of one or more kynureninase or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when appropriately administered to an animal, e.g., a human. The preparation of pharmaceutical compositions containing at least one kynureninase isolated by the methods disclosed herein, or additional active ingredients, will be known to those skilled in the art in light of this disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference. Furthermore, it will be understood that for administration to animals (e.g., humans), preparations must meet the standards of sterility, pyrogenicity, general safety, and purity required by the FDA's Office of Biological Products Standards.

[0141] As will be appreciated by those skilled in the art, as used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonicity agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and similar materials, and combinations thereof (see, e.g., 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.

[0142] Some embodiments of the present disclosure may include different types of carriers depending on whether they are administered in solid, liquid, or aerosol form, and whether they need to be sterile for administration such as injection. The compositions may be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, transmucosally, orally, topically, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by local perfusion directly into target cells, by catheter, by lavage, in lipid compositions (e.g., liposomes), or by other methods known to those skilled in the art or any combination of the above (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference).

[0143] The modified polypeptide can be formulated into the composition in free base, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts, such as those formed with the free amino groups of the proteinaceous composition, or those formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid or mandelic acid. Salts formed with free carboxyl groups can also be obtained from inorganic bases such as sodium, potassium, ammonium, calcium or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine or procaine. After formulation, the solution is administered in a manner compatible with the dosage formulation and in an amount that is therapeutically effective. The formulation can be easily administered in a variety of dosage forms, for example, formulated for parenteral administration, such as injection solutions or aerosols for pulmonary delivery, or formulated for ingestion administration, such as drug-releasing capsules.

[0144] Furthermore, according to some embodiments of the present disclosure, compositions suitable for administration may be provided in a pharmaceutically acceptable carrier, with or without an inert diluent. Carriers must be assimilable and include liquids, semi-solids (i.e., pastes), or solid carriers. Except for any conventional vehicle, agent, diluent, or carrier having an adverse effect on the recipient or on the therapeutic effectiveness of the composition contained therein, its use in administrable compositions for use in practicing the methods is appropriate. Examples of carriers or diluents include fats, oils, water, saline, lipids, liposomes, resins, binders, fillers, and the like, or combinations thereof. The compositions may also contain various antioxidants to retard oxidation of one or more components. In addition, the prevention of microbial activity can be achieved by preservatives, such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0145] According to some embodiments of the present disclosure, the composition is combined with the carrier in any convenient and practical manner, i.e., by dissolving, suspending, emulsifying, mixing, encapsulating, absorbing, etc. Such procedures are common to those skilled in the art.

[0146] In a specific embodiment of the present disclosure, the composition is combined or thoroughly mixed with a semi-solid or solid carrier. Mixing can be performed by any convenient method, such as grinding. Stabilizers can also be added during the mixing process to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

[0147] In further embodiments, the present disclosure may relate to the use of pharmaceutical lipid vehicle compositions comprising kynureninase, one or more lipids, and an aqueous solvent. As used herein, the term "lipid" is defined to include any of a wide variety of substances characterized by being insoluble in water and extractable with an organic solvent. This broad class of compounds is well known to those skilled in the art, and the term "lipid," as used herein, is not limited to any particular structure. Examples include compounds containing long-chain aliphatic hydrocarbons and their derivatives. Lipids can be naturally occurring or synthetic (i.e., designed or manufactured by humans). However, lipids are typically biological substances. Biological lipids are well known in the art and include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids with ether- and ester-linked fatty acids, polymeric lipids, and combinations thereof. Of course, compounds that would be understood by those skilled in the art as lipids, other than those specifically described herein, are also encompassed by the compositions and methods.

[0148] Those skilled in the art will be familiar with various techniques that can be employed to disperse a composition in a lipid vehicle. For example, the kynureninase or its fusion protein may be dispersed in a solution containing a lipid, dissolved in a lipid, emulsified in a lipid, mixed with a lipid, bound to a lipid, covalently bound to a lipid, contained in a lipid as a suspension, encapsulated or complexed in a micelle or liposome, or associated with a lipid or lipid structure by any means known to those skilled in the art. Dispersion may or may not result in the formation of liposomes.

[0149] The actual dosage of a composition administered to an animal patient can be determined by physical and physiological factors, such as body weight, severity of symptoms, the type of disease being treated, previous or concurrent therapeutic interventions, any idiopathic illness in the patient, and the route of administration. Depending on the dosage and route of administration, the preferred dosage and / or frequency of administration of an effective amount can vary depending on the subject's response. In any event, the practitioner responsible for administration will determine the concentration of active ingredient(s) in the composition and the appropriate dose(s) for the individual subject.

[0150] In some embodiments, pharmaceutical compositions may contain, for example, at least about 0.1% of the active compound. In some embodiments, the active compound may comprise, for example, from about 2% to about 75%, or from about 25% to about 60%, of the weight of the unit, and any range derivable therein. Of course, the amount of active compound(s) in each therapeutically useful composition may be arranged in such a way that a suitable dosage will be obtained in any given unit dose of compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be taken into account by those skilled in the art of preparing such pharmaceutical formulations, and thus various dosages and treatment regimens may be desirable.

[0151] In other non-limiting examples, dosages can also include from about 1 microgram / kg / body weight, from about 5 micrograms / kg / body weight, from about 10 micrograms / kg / body weight, from about 50 micrograms / kg / body weight, from about 100 micrograms / kg / body weight, from about 200 micrograms / kg / body weight, from about 350 micrograms / kg / body weight, from about 500 micrograms / kg / body weight, from about 1 milligram / kg / body weight, from about 5 milligrams / kg / body weight, from about 10 milligrams / kg / body weight, from about 50 milligrams / kg / body weight, from about 100 milligrams / kg / body weight, from about 200 milligrams / kg / body weight, from about 350 milligrams / kg / body weight, from about 500 milligrams / kg / body weight, to about 1000 milligrams / kg / body weight or more per administration, or any range derivable therein. Non-limiting examples of ranges derivable from the numbers recited herein include about 5 milligrams / kg / body weight to about 100 milligrams / kg / body weight, about 5 micrograms / kg / body weight to about 500 milligrams / kg / body weight, etc., based on the above numbers.

[0152] Combination treatment In some embodiments, the compositions and methods of the present invention involve the administration of kynureninase in combination with a second or additional therapy. Such therapy may be applied in the treatment of any disease associated with kynurenine dependency. For example, the disease may be cancer.

[0153] The methods and compositions, including combination therapies, enhance the therapeutic or protective effect and / or augment the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. The therapeutic and prophylactic methods and compositions can be provided in combined amounts effective to produce the desired effect, such as killing cancer cells and / or inhibiting cellular hyperproliferation. This process can involve administering kynureninase and a second therapy. The second therapy may or may not have a direct cytotoxic effect. For example, the second therapy can be an agent that upregulates the immune system without a direct cytotoxic effect. This can be by exposing the tissue, tumor, or cells to one or more compositions or pharmaceutical formulations containing one or more of the agents (e.g., kynureninase or an anti-cancer agent), or by exposing the tissue, tumor, and / or cells to two or more separate compositions or formulations, each of which provides 1) kynureninase, 2) an anti-cancer agent, or 3) both kynureninase and an anti-cancer agent. It is also contemplated that such combination therapy may be used in conjunction with chemotherapy, radiation therapy, surgery, or immunotherapy.

[0154] Kynureninase can be administered before, during, after, or in various combinations with the anti-cancer therapy. Administration can occur at intervals ranging from concurrently to minutes, days, or weeks. In embodiments in which kynureninase is provided to a patient separately from the anti-cancer drug, it will typically be desirable to ensure that not too much time elapses between the times of delivery, so that the two compounds can still exert their beneficially combined effects on the patient. In such cases, it is contemplated that the kynureninase and anti-cancer therapy may be provided to a patient within about 12 to 24 or 72 hours of each other, more particularly within about 6 to 12 hours of each other. In some situations, it may be desirable to allow days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7, or 8) to elapse between each administration, significantly extending the period for treatment.

[0155] In some embodiments, a course of treatment will last from 1 to 90 days or more (such ranges including the day of intervention). It is contemplated that one agent may be given on any or any combination of days between days 1 and 90 (such ranges including the day of intervention) and another agent may be given on any or any combination of days between days 1 and 90 (such ranges including the day of intervention). One or more administrations of agent(s) may be given to a patient within a single day (24-hour period). Furthermore, it is contemplated that a course of treatment will be followed by a period during which no anti-cancer treatment is administered. This period may last from 1 to 7 days and / or from 1 to 5 weeks and / or from 1 to 12 months or more (such ranges including the day of intervention), depending on the patient's condition, e.g., prognosis, strength, health, etc. It is anticipated that treatment cycles will be repeated as necessary.

[0156] Various combinations can be employed. In the example below, kynureninase is "A" and anti-cancer therapy is "B": A / B / AB / A / BB / B / AA / A / BA / B / B B / A / AA / B / B / BB / A / B / BB / B / B / A B / B / A / BA / A / B / BA / B / A / BA / B / B / A B / B / A / AB / A / B / AB / A / A / BA / A / A / B B / A / A / AA / B / A / AA / A / B / A

[0157] Administration of any compound or therapy of the present embodiments to a patient will be according to standard protocols for the administration of such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there will be a step to follow up and assess toxicity resulting from the combination therapy. [Example]

[0158] The following examples are presented to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used, made, and evaluated, and are intended merely as illustrative of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure.

[0159] Example 1. Generation of PEGylated Kynureninase Homodimers An exemplary method for producing PEGylated kynureninase homodimers for use in the compositions and methods described herein is by chemical conjugation. Kynureninase can be dissolved in an aqueous buffer containing 100 mM disodium phosphate at pH 8.5, to which a solid PEGylation agent, such as methoxy-PEG-CHCOO-N-hydroxysuccinimide (NHS) having a molecular weight of 5 kDa, can be added at a loading ratio of PEG molecules to kynureninase homodimer, e.g., 20:1, 30:1, 40:1, 50:1, or 60:1. The reaction mixture can be placed at about 22°C for about 30 minutes.

[0160] A further exemplary method for producing PEGylated kynureninase homodimers may use kynureninase homodimers in an aqueous buffer containing 50 mM sodium acetate, 230 mM NaCl, and 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-CHCOO-NHS with a molecular weight of 5 kDa, may be dissolved in 3 mM HCl to a final concentration of approximately 0.2 g of PEG per milliliter of HCl. This PEG solution may be added to the kynureninase solution in a controlled manner to achieve a final ratio of PEG to kynureninase homodimer of 30:1 in the reaction mixture. The reaction may be carried out at approximately 22°C for 30 minutes.

[0161] The PEGylated homodimers can then be analyzed using size-exclusion chromatography with multi-angle light scattering on a TSKgel G3000 SWxl column running a 25 mM NaHPO and 150 mM NaCl buffer at a flow rate of 0.6 mL / min for 25 min at 25°C, resulting in chromatograms for each individual PEG:kynureninase input ratio (Figures 1D-1F).

[0162] The number of PEG molecules per resulting kynureninase monomer and homodimer was calculated from the molecular weight of PEGylated kynureninase measured at various input ratios of PEG:kynureninase homodimer (Figure 1A-1B), and the efficiency of the PEGylation reaction at each input ratio of PEG:kynureninase homodimer was also determined (Figure 1C).

[0163] Example 2. Administration of PEGylated kynureninase homodimer to patients with malignant tumors According to the methods disclosed herein, a physician skilled in the art can treat a patient, e.g., a human patient, to reduce or alleviate the symptoms of an IDO1-, IDO2-, or TDO-expressing tumor. To this end, a physician skilled in the art can administer a PEGylated kynureninase homodimer to a human patient. The homodimer can be administered to a patient, for example, systemically (e.g., intravenously), by local perfusion directly into target cells, by catheter, by lavage, in a lipid composition (e.g., liposomes), or by any other method known to those skilled in the art of treating tumors, or any combination of the above. The homodimer may also be administered to a patient by multiple routes of administration, e.g., intravenously and intracerebroventricularly. An exemplary kynurenine-cleaving dose can be in the range of 0.001 to 1,000 units (U) of kynureninase.

[0164] The patient may be administered one or more anti-cancer therapies (e.g., radiation therapy, surgery, immunotherapy) in combination with the PEGylated kynureninase homodimer to eliminate tumor cells in the patient. Cell removal methods well known in the art, such as radiation, can be used alone or in combination with one or more anti-cancer or anti-hyperproliferative therapies to eliminate tumor cells in the patient. These agents and / or treatments may eliminate at least 5% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 99% or more) of tumor cells as assessed by PET imaging techniques known in the art. The homodimer may be administered to the patient, 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 therapies.

[0165] The homodimer can be administered to a patient in an amount sufficient to treat tumors expressing IDO1, IDO2, or TDO2. Standard tests can also be performed by a physician before and after treatment to assess changes in tumor size. Patients can be evaluated, for example, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more after administration of the homodimer, depending on the route of administration used for treatment. A decrease in tumor size or a decrease in local kynureninase concentration at the site of the tumor after administration of the PEGylated kynureninase homodimer indicates successful treatment in treating tumors expressing IDO1, IDO2, or TDO2.

[0166] Example 3. Optimization of kynureninase homodimer PEGylation density for in vivo delivery In this example, purified kynureninase having the amino acid sequence of SEQ ID NO:2 was conjugated to 5,000 dalton PEG using α-[3-(3-maleimido-1-oxopropyl)amino]propyl-ω-methoxyPEG reagent (CAS#883993-35-9) at pH 8.5 for 30 minutes at room temperature. The conjugation reaction was carried out at kynureninase concentrations ranging from 5.5 to 8 mg / ml, with various molar equivalents of the 5 kDa PEG reagent added directly (100:1, 80:1, 40:1, 20:1, and 10:1 PEGylation agent to kynureninase homodimer). After conjugation, the final product was diafiltered against 1x phosphate-buffered saline at pH 7.4 through a 30 kDa molecular weight cutoff spin filter to remove any remaining free PEG. The samples were analyzed by isocratic size exclusion chromatography (SEC) using a Superdex 200 10 / 300 column (Fig. 2A).

[0167] To test the PEGylated kynureninase homodimer, female BALB / c mice (6-8 weeks old) were inoculated subcutaneously into the right flank with 0.1 ml of PBS containing CT-26 tumor cells (1 × 10 ). Animals were randomized and intravenously administered vehicle (PBS pH 7.4) or 10 mg / kg of differentially PEGylated kynureninase having the amino acid sequence of SEQ ID NO: 2 until the mean tumor volume reached approximately 200 mm . 3 The study began when the kynurenine level reached 100%. Mice were exsanguinated at 6, 24, 48, 72, and 120 hours post-dose (n = 4 mice / time point). Plasma samples were immediately quenched with acid containing internal standard (IS) solution and processed for LC / MS analysis of kynurenine (Figure 2B). Tumor samples were collected from all groups and lysed in RIPA buffer. 20 μg of tumor lysate was loaded onto an SDS-PAGE gel for Western blot analysis with anti-PEG antibody (Abcam #abS 1257; 1:2000 dilution) and anti-β-actin antibody (Cell Signaling Technology #CST-4967). Chemiluminescent signals were detected, and the intensity of individual bands was quantified using Alphaview SA densitometry software (Figure 2C).

[0168] Other embodiments Various modifications and variations of the disclosed subject matter will be apparent to those skilled in the art without departing from the scope and spirit of the present disclosure. Although the disclosed subject matter has been described in connection with specific embodiments, it should be understood that the disclosed subject matter 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 disclosed subject matter. (Addendum) The technical ideas that can be understood from the above-described embodiment and modified examples will be described. [Item 1] A kynureninase homodimer covalently linked 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. [Item 2] 2. The homodimer of item 1, wherein the ratio of PEG molecules to homodimer is about 20:1 to about 30:1, and optionally the ratio of PEG molecules to homodimer is about 20:1, 21:2, 22:1, 23:1, 24:1, or 25:1. [Item 3] 2. The homodimer according to item 1, wherein the ratio of PEG molecules to homodimer is about 25:1. [Item 4] 4. The homodimer according to any one of items 1 to 3, wherein the PEG molecule has a molecular weight of about 1 kDa to about 10 kDa. [Item 5] 5. The homodimer according to item 4, wherein the PEG molecule has a molecular weight of about 3 kDa to about 7 kDa. [Item 6] 6. The homodimer of item 5, wherein the PEG molecule has a molecular weight of about 5 kDa. [Item 7] 7. The homodimer according to any one of items 1 to 6, wherein the PEG molecule is linear. [Item 8] 7. The homodimer according to any one of items 1 to 6, wherein the PEG molecule is branched. [Item 9] 9. The homodimer according to any one of items 1 to 8, wherein the homodimer is covalently attached to the PEG molecule by one or more lysine or cysteine ​​residues. [Item 10] 10. The homodimer of item 9, wherein the homodimer is covalently attached to the PEG molecule by one or more lysine residues. [Item 11] The homodimer comprises two polypeptide monomers each having an amino acid sequence at least 85% identical to SEQ ID NO:1, and each polypeptide monomer has, compared to SEQ ID NO:1: 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、1201H、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, A382 11. The homodimer according to any one of items 1 to 10, having one or more amino acid substitutions selected from the group consisting of R, 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. [Item 12] 12. The homodimer of item 11, wherein each polypeptide monomer has an amino acid sequence that is at least 90% identical to SEQ ID NO:1. [Item 13] 13. The homodimer of item 12, wherein each polypeptide monomer has an amino acid sequence that is at least 95% identical to SEQ ID NO:1. [Item 14] 14. The homodimer according to any one of items 11 to 13, wherein each polypeptide monomer comprises a substitution at A282, F306, or F249 compared to SEQ ID NO: 1. [Item 15] 15. The homodimer according to any one of items 11 to 14, wherein each polypeptide monomer comprises a substitution at A99 compared to SEQ ID NO:1. [Item 16] 16. The homodimer according to any one of items 11 to 15, wherein each polypeptide monomer comprises a substitution at G112 compared to SEQ ID NO: 1. [Item 17] 17. The homodimer according to any one of items 11 to 16, wherein each polypeptide monomer comprises a substitution at E103 compared to SEQ ID NO: 1. [Item 18] 18. The homodimer according to any one of items 11 to 17, wherein each polypeptide monomer comprises a substitution at V104 compared to SEQ ID NO: 1. [Item 19] 19. The homodimer according to any one of items 11 to 18, wherein each polypeptide monomer comprises a substitution at S408 compared to SEQ ID NO: 1. [Item 20] 20. The homodimer according to any one of items 11 to 19, wherein each polypeptide monomer comprises the F306W substitution. [Item 21] 21. The homodimer according to any one of items 11 to 20, wherein each polypeptide monomer comprises the L72N substitution. [Item 22] 22. The homodimer according to any one of items 11 to 21, wherein each polypeptide monomer comprises the H102W and N333T substitutions. [Item 23] 23. The homodimer according to any one of items 11 to 22, wherein each polypeptide monomer comprises the I183P substitution. [Item 24] 24. The homodimer according to any one of items 11 to 23, wherein each polypeptide monomer comprises the R107P substitution. [Item 25] 25. The homodimer according to any one of items 11 to 24, wherein each polypeptide monomer comprises the A436T substitution. [Item 26] 26. The homodimer according to any one of items 11 to 25, wherein each polypeptide monomer comprises at least one substitution selected from L72N, H102W, A282P, F306W, I331S and N333T compared to SEQ ID NO: 1. [Item 27] 27. The homodimer according to item 26, wherein each polypeptide monomer comprises at least two, three, four or five substitutions selected from L72N, H102W, A282P, F306W, I331S and N333T. [Item 28] 28. The homodimer according to item 27, wherein each polypeptide monomer comprises the substitutions L72N, H102W, A282P, F306W, I331S and N333T. [Item 29] 29. The homodimer according to any one of items 1 to 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. [Item 30] 30. The homodimer of item 29, wherein each polypeptide monomer has an amino acid sequence that is at least 90% identical to SEQ ID NO:3. [Item 31] 31. The homodimer of item 30, wherein each polypeptide monomer has an amino acid sequence that is at least 95% identical to SEQ ID NO:3. [Item 32] 32. The homodimer of item 31, wherein each polypeptide monomer has the amino acid sequence of SEQ ID NO:3. [Item 33] 29. The homodimer according to any one of items 1 to 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. [Item 34] 34. The homodimer of item 33, wherein each polypeptide monomer has an amino acid sequence that is at least 90% identical to SEQ ID NO:2. [Item 35] 35. The homodimer of item 34, wherein each polypeptide monomer has an amino acid sequence that is at least 95% identical to SEQ ID NO:2. [Item 36] 36. The homodimer of item 35, wherein each polypeptide monomer has the amino acid sequence of SEQ ID NO:2. [Item 37] The homodimer is at least 8000M -1 s -1 Catalytic activity (kcat / K M 37. The homodimer according to any one of items 1 to 36, having the following structure: [Item 38] The homodimer has a molecular weight of about 8000M -1 s -1 ~40000M -1 s -1 ;10000M -1 s -1 ~40000M -1 s -1 ;20000M -1 s -1 ~40000M -1 s -1 ;or 25000M -1 s -1 ~35000M -1 s -1 Catalytic activity (kcat / K M 38. The homodimer according to item 37, having [Item 39] the ratio of PEG molecules to homodimers is about 25:1; 39. The homodimer according to any one of items 1 to 38, wherein the PEG molecule has a molecular weight of about 5 kDa and is attached to the homodimer by an N-hydroxysuccinimide ester carbonate linking group. [Item 40] 1. A method for producing a PEGylated kynureninase homodimer, comprising: the homodimer comprises two polypeptide monomers; the method comprises contacting the homodimer with a PEGylation agent; The above method, wherein the molar input ratio of the PEGylating agent to the homodimer is about 10:1 to about 50:1. [Item 41] Item 41. The method according to Item 40, wherein the molar input ratio of PEGylation agent to homodimer is about 20:1 to about 40:1, optionally the molar input ratio of PEGylation agent to homodimer is about 20:1 to about 30:1, optionally the molar input ratio of PEGylation agent to homodimer is about 20:1. [Item 42] Item 41. The method according to item 40, wherein the molar input ratio of the PEGylating agent to the homodimer is about 25:1 to about 35:1. [Item 43] 41. The method of claim 40, wherein the molar input ratio of PEGylating agent to homodimer is about 30:1. [Item 44] 44. The method according to any one of items 40 to 43, wherein the PEG molecule has a molecular weight of about 1 kDa to about 10 kDa. [Item 45] Item 45. The method of item 44, wherein the PEG molecule has a molecular weight of about 3 kDa to about 7 kDa. [Item 46] 46. ​​The method of claim 45, wherein the PEG molecule has a molecular weight of about 5 kDa. [Item 47] 47. The method according to any one of items 40 to 46, wherein the PEG molecule is linear. [Item 48] 47. The method according to any one of items 40 to 46, wherein the PEG molecule is branched. [Item 49] 49. The method of any one of items 40 to 48, wherein the homodimer is covalently attached to the PEG molecule by one or more lysine or cysteine ​​residues. [Item 50] 50. The method of claim 49, wherein the homodimer is covalently attached to the PEG molecule by one or more lysine residues. [Item 51] Each polypeptide monomer has an amino acid sequence that is at least 85% identical to SEQ ID NO:1, and each polypeptide monomer has, compared to SEQ ID NO:1: 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、1201H、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, L338 A, L338Q, S341I, K373E, K373N, N375A, N375H, Y376C, Y376F, Y376L, K378G, K378P, K378Q, K378R, K380G, K380S, A382G, A38 51. The method according to any one of items 40 to 50, wherein the nucleotide sequence has one or more amino acid substitutions selected from the group consisting of 2R, 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. [Item 52] 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. [Item 53] 53. The method of item 52, wherein each polypeptide monomer has an amino acid sequence that is at least 95% identical to SEQ ID NO:1. [Item 54] 54. The method of any one of items 40 to 53, wherein each polypeptide monomer comprises a substitution at A282, F306 or F249. [Item 55] 55. The method according to any one of items 40 to 54, wherein each polypeptide monomer comprises a substitution at A99 compared to SEQ ID NO:1. [Item 56] 56. The method of any one of items 40 to 55, wherein each polypeptide monomer comprises a substitution at G112 compared to SEQ ID NO: 1. [Item 57] 57. The method of any one of items 40 to 56, wherein each polypeptide monomer comprises a substitution at E103 compared to SEQ ID NO: 1. [Item 58] 58. The method of any one of items 40 to 57, wherein each polypeptide monomer comprises a substitution at V104 compared to SEQ ID NO: 1. [Item 59] 59. The method of any one of items 40 to 58, wherein each polypeptide monomer comprises a substitution at S408 compared to SEQ ID NO: 1. [Item 60] 60. The method of any one of items 51 to 59, wherein each polypeptide monomer comprises the F306W substitution. [Item 61] 61. The method of any one of items 51 to 60, wherein each polypeptide monomer comprises the L72N substitution. [Item 62] 62. The method of any one of items 51 to 61, wherein each polypeptide monomer comprises the H102W and N333T substitutions. [Item 63] 63. The method of any one of items 51 to 62, wherein each polypeptide monomer comprises the I183P substitution. [Item 64] 64. The method of any one of items 51 to 63, wherein each polypeptide monomer comprises the R107P substitution. [Item 65] 65. The method of any one of items 51 to 64, wherein each polypeptide monomer comprises the A436T substitution. [Item 66] 66. The method according to any one of items 40 to 65, wherein each polypeptide monomer comprises at least one substitution selected from L72N, H102W, A282P, F306W, I331S and N333T compared to SEQ ID NO: 1. [Item 67] 67. The method of item 66, wherein each polypeptide monomer comprises at least two, three, four, or five substitutions selected from L72N, H102W, A282P, F306W, I331S, and N333T compared to SEQ ID NO: 1. [Item 68] 68. The method of item 67, wherein each polypeptide monomer comprises the substitutions L72N, H102W, A282P, F306W, I331S, and N333T compared to SEQ ID NO: 1. [Item 69] 69. The method of any one of items 40 to 68, wherein each polypeptide monomer has an amino acid sequence that is at least 85% identical to SEQ ID NO:3. [Item 70] 70. The method of item 69, wherein each polypeptide monomer has an amino acid sequence that is at least 90% identical to SEQ ID NO:3. [Item 71] 71. The method of item 70, wherein each polypeptide monomer has an amino acid sequence that is at least 95% identical to SEQ ID NO:3. [Item 72] 72. The method of item 71, wherein each polypeptide monomer has the amino acid sequence of SEQ ID NO:3. [Item 73] 69. The method of any one of items 40 to 68, wherein each polypeptide monomer has an amino acid sequence that is at least 85% identical to SEQ ID NO:2. [Item 74] 74. The method of item 73, wherein each polypeptide monomer has an amino acid sequence that is at least 90% identical to SEQ ID NO:2. [Item 75] 75. The method of item 74, wherein each polypeptide monomer has an amino acid sequence that is at least 95% identical to SEQ ID NO:2. [Item 76] 76. The method of item 75, wherein each polypeptide monomer has the amino acid sequence of SEQ ID NO:2. [Item 77] The homodimer is at least 8000M -1 s -1 Catalytic activity (kcat / K M 77. The method according to any one of Items 40 to 76, wherein [Item 78] The homodimer has a molecular weight of about 8000M -1 s -1 ~40000M -1 s -1 ;10000M -1 s -1 ~40000M -1 s -1 ;20000M -1 s -1 ~40000M -1 s -1 ;or 25000M -1 s -1 ~35000M -1 s -1 Catalytic activity (kcat / K M Item 78. The method according to Item 77, wherein [Item 79] the ratio of PEG molecules to homodimers is about 25:1; 79. The method of claim 78, wherein the PEG molecule has a molecular weight of about 5 kDa and is attached to the homodimer by an N-hydroxysuccinimide ester carbonate linking group. [Item 80] 80. The method of any of items 40 to 79, wherein the homodimer is contacted with the PEGylation agent in an aqueous buffer. [Item 81] 81. The method of claim 80, wherein the aqueous buffer comprises disodium phosphate. [Item 82] 81. The method of claim 80, wherein the aqueous buffer comprises sodium acetate. [Item 83] 83. The method according to any one of items 80 to 82, wherein the pH of the aqueous buffer solution is in the range of about 7.5 to about 9.5. [Item 84] 84. The method according to any one of items 80 to 83, wherein the ionic strength of the aqueous buffer is in the range of about 250 mM to about 350 mM. [Item 85] 85. The method according to any one of items 40 to 84, wherein the homodimer is isolated using size exclusion chromatography. [Item 86] A kynureninase homodimer produced by the method according to any one of items 40 to 85. [Item 87] A pharmaceutical formulation comprising the kynureninase homodimer according to any one of items 1 to 39 and 86 in a pharmaceutically acceptable carrier. [Item 88] 1. A method of treating a subject having a tumor, comprising: The method comprises administering an effective amount of the formulation according to Item 87 or the enzyme according to any one of Items 1 to 39 and 86 to a subject. [Item 89] 89. The method of claim 88, wherein the subject is confirmed to have an IDO1, IDO2, or TDO-expressing tumor. [Item 90] 89. The method of claim 88 or 89, wherein the tumor is a solid tumor. [Item 91] 89. The method of claim 88, wherein the tumor is a hematopoietic tumor. [Item 92] 92. The method of any one of items 88 to 91, wherein the subject is a human patient. [Item 93] 93. The method of any one of items 88-92, wherein the formulation is administered intratumorally, intravenously, intracutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrathoracically, intratracheally, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, transmucosally, intrapericardially, intraumbilically, orally, by aspiration, by injection, by infusion, by continuous infusion, by local perfusion directly into target cells, by catheter, or by lavage. [Item 94] 94. The method of any one of items 88 to 93, further comprising administering a second anticancer therapy. [Item 95] 95. The method of item 94, wherein the second anti-cancer therapy is radiation therapy, surgical therapy, immunotherapy, or a second anti-cancer compound. [Item 96] 96. The method of item 95, wherein the second anticancer compound is an immune checkpoint inhibitor. [Item 97] Item 96. The method of item 95, wherein the second anti-cancer compound comprises an anti-PDI, anti-CTLA-4, or anti-PD-LI antibody. [Item 98] 96. The method of item 95, wherein the second anti-cancer compound is an antibody. [Item 99] 96. The method of item 95, wherein the second anticancer compound is an antibody-drug conjugate. [Item 100] 96. The method of claim 95, wherein the immunotherapy comprises administering immune effector cells or an immunogenic composition. [Item 101] 101. The method of claim 100, wherein the immunogenic composition comprises a cancer cell antigen. [Item 102] 101. The method of claim 100, wherein the immune effector cells comprise NK cells or T cells. [Item 103] 101. The method of claim 100, wherein the immune effector cells comprise CAR T cells. [Item 104] 1. A method for generating a T cell response in a human subject having a tumor, comprising: 104. The method comprising administering to the subject a kynureninase homodimer according to the method of any one of items 88 to 103.

[0169] Other embodiments are within the scope of the following claims.

Claims

1. A kynureninase homodimer covalently linked to one or more polyethylene glycol (PEG) molecules, wherein the ratio of PEG molecules to homodimer is from about 15:1 to about 35:

1.

2. 2. The homodimer of claim 1, wherein the ratio of PEG molecules to homodimer is from about 20:1 to about 30:

1.

3. 2. The homodimer of claim 1, wherein the ratio of PEG molecules to homodimer is about 25:

1.

4. 2. The homodimer of claim 1, wherein each of said one or more PEG molecules independently has a molecular weight of about 1 kDa to about 10 kDa.

5. 5. The homodimer of claim 4, wherein each of said one or more PEG molecules independently has a molecular weight of about 3 kDa to about 7 kDa.

6. 2. The homodimer of claim 1, wherein said homodimer is covalently attached to said PEG molecule by one or more lysine or cysteine ​​residues.

7. The homodimer comprises two polypeptide monomers each having an amino acid sequence at least 90% identical to SEQ ID NO:1, and each polypeptide monomer has, compared to SEQ ID NO:1: 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、1201H、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, P335 T, L337T, L338A, L338Q, S341I, K373E, K373N, N375A, N375H, Y376C, Y376F, Y376L, K378G, K378P, K378Q, K37 8R, 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, The homodimer according to any one of claims 1 to 6, having one or more amino acid substitutions selected from the group consisting of:

8. 8. The homodimer of claim 7, wherein each polypeptide monomer comprises at least one substitution selected from L72N, H102W, A282P, F306W, I331S and N333T compared to SEQ ID NO:

1.

9. 9. The homodimer of claim 8, wherein each polypeptide monomer comprises at least two, three, four, or five substitutions selected from L72N, H102W, A282P, F306W, I331S, and N333T.

10. 10. The homodimer of claim 9, wherein each polypeptide monomer comprises the substitutions L72N, H102W, A282P, F306W, I331S and N333T.

11. 7. The homodimer of any one of claims 1 to 6, wherein the homodimer comprises two polypeptide monomers each having an amino acid sequence that is at least 90% identical to SEQ ID NO:

3.

12. 7. The homodimer of any one of claims 1 to 6, wherein the homodimer comprises two polypeptide monomers each having an amino acid sequence that is at least 90% identical to SEQ ID NO:

2.

13. The homodimer is at least 8000 M －１ s －１ Catalytic activity (kcat / K Ｍ The homodimer according to any one of claims 1 to 6, having

14. The homodimer has a molecular weight of about 8000 M －１ s －１ ~40,000m －１ s －１ ;10000M －１ s －１ ~40,000m －１ s －１ ;20000M －１ s －１ ~40,000m －１ s －１ or 25,000M －１ s －１ ~35,000M －１ s －１ Catalytic activity (kcat / K Ｍ 14. The homodimer of claim 13, having:

15. the ratio of PEG molecules to homodimers is about 25:1; 7. The homodimer of any one of claims 1 to 6, wherein the PEG molecule has a molecular weight of about 5 kDa and is attached to the homodimer by an N-hydroxysuccinimide ester carbonate linking group.

16. 1. A method for producing a PEGylated kynureninase homodimer, comprising: the homodimer comprises two polypeptide monomers; the method comprises contacting the homodimer with a PEGylation agent; the molar input ratio of PEGylating agent to homodimer is from about 15:1 to about 35:1; The method.

17. 17. The method of claim 16, wherein the molar input ratio of PEGylating agent to homodimer is from about 20:1 to about 35:

1.

18. 17. The method of claim 16, wherein the molar input ratio of PEGylating agent to homodimer is from about 25:1 to about 35:

1.

19. 17. The method of claim 16, wherein the molar input ratio of PEGylating agent to homodimer is about 30:

1.

20. A method described in any one of claims 16 to 19, wherein the PEG molecule has a molecular weight of about 1 kDa to about 10 kDa.

21. 21. The method of claim 20, wherein the PEG molecule has a molecular weight of about 3 kDa to about 7 kDa.

22. 20. The method of any one of claims 16 to 19, wherein the homodimer is covalently attached to a PEG molecule by one or more lysine or cysteine ​​residues.

23. Each polypeptide monomer has an amino acid sequence that is at least 90% identical to SEQ ID NO:1, and each polypeptide monomer has, compared to SEQ ID NO:1: 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、1201H、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, P335 T, L337T, L338A, L338Q, S341I, K373E, K373N, N375A, N375H, Y376C, Y376F, Y376L, K378G, K378P, K378Q, K37 8R, 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, The method of any one of claims 16 to 19, wherein the amino acid sequence has one or more amino acid substitutions selected from the group consisting of:

24. 20. The method of any one of claims 16 to 19, wherein each polypeptide monomer comprises at least one substitution selected from L72N, H102W, A282P, F306W, I331S and N333T compared to SEQ ID NO:

1.

25. 25. The method of claim 24, wherein each polypeptide monomer comprises at least two, three, four, or five substitutions selected from L72N, H102W, A282P, F306W, I331S, and N333T compared to SEQ ID NO:

1.

26. 26. The method of claim 25, wherein each polypeptide monomer comprises the substitutions L72N, H102W, A282P, F306W, I331S, and N333T compared to SEQ ID NO:

1.

27. 20. The method of any one of claims 16 to 19, wherein each polypeptide monomer has an amino acid sequence that is at least 90% identical to SEQ ID NO:

3.

28. 20. The method of any one of claims 16 to 19, wherein each polypeptide monomer has an amino acid sequence that is at least 90% identical to SEQ ID NO:

2.

29. The homodimer is at least 8000 M －１ s －１ Catalytic activity (kcat / K Ｍ 20. The method according to any one of claims 16 to 19, wherein

30. The homodimer has a molecular weight of about 8000 M －１ s －１ ~40,000m －１ s －１ ;10000M －１ s －１ ~40,000m －１ s －１ ;20000M －１ s －１ ~40,000m －１ s －１ or 25,000M －１ s －１ ~35,000M －１ s －１ Catalytic activity (kcat / K Ｍ 30. The method of claim 29, wherein

31. the ratio of PEG molecules to homodimers is about 25:1; 31. The method of claim 30, wherein the PEG molecule has a molecular weight of about 5 kDa and is attached to the homodimer by an N-hydroxysuccinimide ester carbonate linking group.

32. 20. The method of any one of claims 16 to 19, wherein the homodimer is contacted with the pegylation agent in an aqueous buffer.

33. 33. The method of claim 32, wherein the pH of the aqueous buffer ranges from about 7.5 to about 9.

5.

34. 33. The method of claim 32, wherein the ionic strength of the aqueous buffer ranges from about 250 mM to about 350 mM.

35. 33. The method of claim 16 or claim 32, wherein the homodimer is isolated using size exclusion chromatography.

36. 10. A pharmaceutical formulation comprising the kynureninase homodimer of claim 1 in a pharmaceutically acceptable carrier.

37. 37. The pharmaceutical preparation of claim 36 for use in a method for treating a subject having a tumor.

38. 38. The pharmaceutical preparation of claim 37, wherein the subject is confirmed to have an IDO1, IDO2, or TDO-expressing tumor.

39. 38. The pharmaceutical formulation of claim 37, wherein the subject is a human patient.

40. 38. The pharmaceutical formulation of claim 37, wherein the formulation is administered intratumorally, intravenously, intracutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrathoracically, intratracheally, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, transmucosally, intrapericardially, intraumbilically, orally, by aspiration, by injection, by infusion, by continuous infusion, by local perfusion directly into target cells, by catheter, or by lavage.

41. 38. The pharmaceutical preparation of claim 37, further comprising administering a second anti-cancer therapy.

42. 42. The pharmaceutical preparation of claim 41, wherein the second anti-cancer therapy is radiation therapy, surgery, immunotherapy, or a second anti-cancer compound.

43. 43. The pharmaceutical preparation of claim 42, wherein the immunotherapy comprises administering immune effector cells or an immunogenic composition.

44. 38. The pharmaceutical preparation of claim 37 for use in a method of generating a T cell response in a human subject bearing a tumor.

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