Truncated Guinea Pig L-Asparaginase Variants and Methods of Use

Abstract

Variants of cleaved and humanized guinea pig L-asparaginase are described, as are fusion proteins containing said L-asparaginase and the use of said L-asparaginase in the treatment of cancers such as acute lymphoblastic leukemia and acute myeloid leukemia.

Description

[Technical Field]

[0001] introduction This application claims the benefit of priority to U.S. Provisional Application No. 62 / 544,396, filed August 11, 2017, and U.S. Provisional Application No. 62 / 544,411, filed August 11, 2017, the contents of which are incorporated herein by reference in their entireties.

[0002] This invention was made with government support under Grant No. EB013685 awarded by the National Institutes of Health and Grant No. BX001919 awarded by the Department of Veterans Affairs. The government has certain rights in this invention.

[0003] background Certain cancers, such as acute lymphocytic leukemia (ALL), depend on scavenging of Asn from the blood, a factor most commonly attributed to the absence / low expression of asparagine synthetase in such cancers. Consequently, L-asparaginase has been identified as a key component in the treatment of these cancers. All commercially available L-asparaginases have dual activities. The primary activity, L-asparaginase, hydrolyzes the amino acid L-asparagine (Asn) to L-aspartic acid (Asp) and ammonia. The secondary activity, L-glutaminase, hydrolyzes L-glutamine (Gln) to L-glutamic acid (Glu) and ammonia. For example, for FDA-approved enzymes such as ELSPAR® (an enzyme obtained from Escherichia coli) and ERWINAZE® (an enzyme obtained from Erwinia chrysanthemi), L-glutaminase activity ranges from 2 to 10% of the primary L-asparaginase activity. While the importance of L-asparaginase activity in these drugs is accepted, there are conflicting reports regarding the importance of L-glutaminase activity in killing leukemia cells. Furthermore, L-glutaminase activity is associated with many of the clinical toxicities of L-asparaginase. In fact, the toxic side effects of L-asparaginase treatment severely limit the use of this anticancer drug.

[0004] Another disadvantage of using bacterial enzymes as therapeutic agents is their immunogenicity, which can pose a direct threat to patients due to hypersensitivity reactions, even anaphylactic shock. Furthermore, the antibodies generated can inactivate and remove the enzyme drug, thereby reducing or even eliminating its effectiveness. Methods such as linking E. coli enzymes with polyethylene glycol (PEGylation) or deimmunization by mutating residues 115, 118, 120, 123, 215, 219, 307, and 312 of the wild-type E. coli enzyme have been developed to reduce these severe side effects ( WO 2012 / 075173 A2 ). However, there is a need for the preparation of alternative L-asparaginases with reduced immunogenicity and reduced L-glutaminase activity.

[0005] Guinea pig L-asparaginase was purified from guinea pig serum and characterized (Zhang, et al. (1995) Comp. Biochem. Physiol. B Biochem. Mol. Biol. 112(4):607-12). The guinea pig enzyme, annotated as H0W0T5_CAVPO, exhibits antitumor activity and has a low K for asparagine. m and lacks L-glutaminase activity (Schalk, et al. (2014) J. Biol. Chem. 289:33175-33186). See also EP0726313B1 and US6,537,547.

[0006] Other approaches to treating cancer using L-asparaginase include co-administration of L-asparaginase with a TNF-related apoptosis-inducing ligand (TRAIL) agonist or a TRAIL receptor agonist, such as three soluble TRAIL domains and additional functional domains such as antibody fragments. See US2015 / 0337027, US2009 / 0131317, and WO2012 / 170640. In this regard, L-asparaginase has been shown to overcome resistance to both intrinsic apoptosis induced by the Bcl-2 / Bcl-xL inhibitor ABT263 and TRAIL-mediated extrinsic apoptosis in glioma cells that are resistant to L-asparaginase monotherapy (Karpel-Massler, et al. (2016) Oncotarget 7(23):33512-28). Summary of the Invention

[0007] SUMMARY OF THE INVENTION The present invention provides C-terminally truncated guinea pig L-asparaginase (GpA) variants that share at least 85% sequence identity with residues 1-359 of SEQ ID NO: 1. In some embodiments, the C-terminal truncation is at positions between 359 and 396 of SEQ ID NO: 1. In certain embodiments, the C-terminal truncation is at position 369 of SEQ ID NO: 1. In other embodiments, the truncated GpA variant further comprises at least one amino acid substitution relative to SEQ ID NO:1, e.g., at positions 7, 10, 23, 25, 48, 49, 52, 53, 54, 57, 58, 59, 60, 62, 92, 98, 101, 102, 106, 108, 121, 122, 134, 147, 193, 198, 217, 233, 236, 250, 257, 281, 301, 311, 340, 344, 360, 362, 363, 364, 365, 366, 367, or 368, or a combination thereof. In certain embodiments, the at least one amino acid substitution compared to SEQ ID NO:1 is H10R, Q23R, K25E, K48E, Q52R, Q54R, P57S, D58E, H59D, A60T, A62V, D91A, D92E, K98Q, E101K, Q108H, S121F, G122A, H134Q, R147H, K193R, C198A, C198S, C198V, D217E, N233S, H236Q, S250A, Q288E, R301Q, E344D, L360P, T362S, A363V, D364E, L365E, H366R, Q367R, or S368P, or a combination thereof. In still other embodiments, the at least one amino acid substitution compared to SEQ ID NO: 1 includes: (a) a cysteine ​​residue at position 49, 52, 225, 257, 281, or 340, or a combination thereof; or (b) a lysine residue at position 7, 53, 54, 57, 58, 98, 106, 233, 250, 257, 281, 311, or 340, or a combination thereof. Ideally, the variant has catalytic activity equal to or greater than wild-type GpA.Optionally, the truncated GpA variant may further comprise a histidine tag, a SUMO tag, an albumin binding domain, the three tandem soluble domains of TRAIL (e.g., residues 115-281 of human TRAIL), or a combination thereof.

[0008] Nucleic acid molecules, expression vectors, host cells and pharmaceutical compositions containing the truncated GpA variants or fusion proteins are also provided, as are methods of treating cancer by administering to a subject in need thereof an effective amount of the truncated GpA variants or fusion proteins, optionally in combination with a stable form of TRAIL. [Brief explanation of the drawings]

[0009] Brief description of the drawings [Figure 1] Figure 1 is a schematic diagram of the DNA shuffling process and clones obtained from C-terminal domain swapping. The hASNase 1 sequence is shown in open boxes, and gpASNase 1 is shown in shaded boxes. Clones isolated from selection (#63, 64, 65, and SA) or generated by C-terminal swapping (#63-hC, 64-hC, 65-hC, and SA-hC) are shuffles between the hASNase 1 (open boxes) and gpASNase 1 (filled boxes) sequences.

[0010] [Figure 2A] Figures 2A and 2B show amino acid sequence alignments of hASNase 1 (hASN), gpASNase 1 (GpA), and selected humanized clones. The underlined residues are those from GpA. [Figure 2B] Figures 2A and 2B show amino acid sequence alignments of hASNase 1 (hASN), gpASNase 1 (GpA), and selected humanized clones. The underlined residues are those from GpA. DETAILED DESCRIPTION OF THE INVENTION

[0011] Detailed Description of the Invention L-asparaginase is a chemotherapy drug used for the treatment of acute lymphocytic leukemia (ALL). The main prerequisite for clinical effectiveness of L-asparaginase is a micromolar Km for asparagine, which allows for complete depletion of this amino acid in the blood. The bacterial origin of currently approved L-asparaginases poses immunogenicity challenges that would be mitigated by human enzymes. However, all human L-asparaginases have a millimolar Km for asparagine. m Has low K m A guinea pig L-asparaginase (gpASNase1) was identified, which shares approximately 70% amino acid identity with human L-asparaginase 1 (hASNase1). Similar to the human enzyme, gpASNase1 contains two domains: an N-terminal domain of approximately 360 residues where L-asparaginase activity resides, and a C-terminal domain of approximately 200 residues of unknown function. To improve half-life, shorter but stable versions of GpA were sought. Consequently, GpA was truncated at the C-terminus to identify the shortest fragment of GpA that retained activity. This analysis indicated that the 359 amino acid N-terminal catalytic domain could be expressed with a SUMO tag and retained wild-type activity. However, removal of the SUMO tag destabilized the protein. Extending the truncation to residue 369 provided an enzyme that was both stable and active.

[0012] To reduce the immunogenicity of truncated GpA variants, two different approaches were used to humanize the enzyme: DNA shuffling and structure-based mutation of surface residues. Humanization of GpA resulted in approximately 80% sequence identity with hASNase1, wild-type GpA activity, and a low K for asparagine. mThis resulted in variants that share the same L-asparaginase activity and no detectable L-glutaminase activity. Introducing cysteine ​​or lysine residues and conjugating them to PEG maintained the truncated GpA variants and, in some instances, showed an increase in their L-asparaginase activity. Additionally, modifications such as the addition of a histidine tag, a SUMO tag, and / or an albumin-binding domain to the N- or C-terminus can increase circulation time in vivo, and fusion of a truncated GpA variant to the three tandem soluble domains of TRAIL can promote cell death by both providing the necessary signals for the intrinsic apoptotic cascade (L-asparaginase) and inducing the apoptotic cascade externally (TRAIL).

[0013] Consequently, the present invention provides truncated GpA variants and fusion proteins thereof for use in the treatment of cancers, such as lymphomas and leukemias, that depend on the presence of an external supply of Asn. The improved safety of the present L-asparaginase will benefit its long-term use in current patient populations (e.g., patients with pediatric ALL) and other patient populations (e.g., adult ALL, AML, and other cancers).

[0014] As known in the art, L-asparaginase (L-asparagine aminohydrolase, EC 3.5.1.1) is an amidase that hydrolyzes the amide bond at Asn to Asp and ammonia (Kumar & Verma (2012) Asian J. Biochem. Pharma Res. 3:197-205). Wild-type guinea pig (Cavia porcellus) L-asparaginase, referred to herein as "gpASNase1" or "GpA," is a 565 amino acid residue protein available under Uniprot accession number H0W0T5_CAVPO and SEQ ID NO: 1. GpA exhibits antitumor activity and has a low K for asparagine. mhASNase1 has a nucleotide sequence similar to that of wild-type GpA and lacks L-glutaminase activity. However, wild-type GpA shares only approximately 70% sequence identity with hASNase1. Consequently, the present invention provides, in some embodiments, truncated GpA that is humanized to reduce immunogenicity. In particular, the present invention provides truncated GpA variants that share at least 85% sequence identity with residues 1-359 of SEQ ID NO:1.

[0015] The "GpA variant" exhibits L-asparaginase activity and has a low K for Asn. m and lacks L-glutaminase activity. In comparison, "wild-type" GpA refers to the typical form of L-asparaginase when isolated from a naturally occurring source. The wild-type form is that most frequently observed in natural populations and is therefore arbitrarily referred to as the normal or wild-type form. Wild-type GpA has a 39s -1 The reaction rate (k cat ) and a K of 58 μM for Asn m (Schalk, et al. (2014) J. Biol. Chem. 289:33175-33186). The truncated GpA variants of the invention typically have a k of at least 75%, 80%, 85%, 90%, 95%, or 100% of the wild-type GpA enzyme. cat and a K for Asn lower than 250 μM, 200 μM, 150 μM, 100 μM, 80 μM, or 60 μM m Shows.

[0016] At least the GpA variant is truncated. "Truncated GpA" refers to GpA from which all or part of the approximately 206 C-terminal amino acid residues have been removed. In some embodiments, the truncated GpA protein retains the 359 amino acid residue catalytic domain at the N-terminus. Ideally, full-length GpA (SEQ ID NO: 1) is truncated at the C-terminus between residues 359 and 396. In particular, full-length GpA (SEQ ID NO: 1) is truncated at the C-terminus at positions 359, 367, 369, 374, 384, 392, or 396. In some embodiments, the truncated GpA variant is truncated at the C-terminus at position 369. Exemplary truncated GpA variants are provided in SEQ ID NO: 3 (GpA359), SEQ ID NO: 4 (GpA367), SEQ ID NO: 5 (GpA369), SEQ ID NO: 6 (GpA374), SEQ ID NO: 7 (GpA384), SEQ ID NO: 8 (GpA392), and SEQ ID NO: 9 (GpA396). In some embodiments, the C-terminally truncated GpA variant comprises or consists of SEQ ID NO: 3. In certain embodiments, the C-terminally truncated GpA variant comprises or consists of SEQ ID NO: 5.

[0017] According to another aspect of the present invention, GpA variants are truncated and contain at least one amino acid substitution or modification (e.g., pegylation or protein fusion) that increases stability, increases sequence identity with hASNase 1, and / or increases the in vivo circulation time of GpA compared to the wild-type GpA enzyme. As demonstrated herein, amino acid residues located on the surface of GpA are mutated to either humanize GpA and / or provide suitable sites for pegylation. Furthermore, fusion of GpA to a protein tag or trimeric TRAIL provides stability and / or enhanced tumor cell killing activity.

[0018] Consequently, in some aspects, a GpA variant has at least one amino acid substitution compared to wild-type GpA (SEQ ID NO: 1). In certain embodiments, the substituted amino acid residue is a surface residue. The term "surface residue" refers to a residue located on the surface of a protein. In contrast, a buried residue is a residue that is not located on the surface of a protein. Surface residues often contain hydrophilic side chains. Operationally, surface residues can be computationally identified from a structural model of a protein as residues that contact a sphere of hydration that rolls over the surface of the molecular structure. Surface residues can also be experimentally identified through deuterium exchange studies or the use of accessibility of various labeling reagents, such as, for example, hydrophilic alkylating agents. In certain embodiments, the amino acid substitution is not at one of the active site residues, for example, Thr19, Ser85, Ser86, Thr116, Asp117, Ala142, Lys188, Asn272, and Tyr308.

[0019] Surface residues of GpA that can be substituted to generate GpA variants of the invention include, but are not limited to, positions 7, 10, 23, 25, 40, 48, 49, 52, 53, 54, 57, 58, 59, 60, 62, 92, 98, 101, 106, 108, 121, 122, 131, 132, 134, 147, 193, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 27 , 217, 221, 222, 223, 224, 225, 226, 233, 236, 250, 253, 257, 261, 281, 282, 283, 284, 301, 311, 340, 344, 345, 347, 352, 358, 359, 360, 362, 363, 364, 365, 366, 367, or 368 amino acid residues. In some embodiments, the GpA variant comprises at least one amino acid substitution. In other embodiments, the GpA variant comprises about 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-7, or 1-4 amino acid substitutions. In certain embodiments, the GpA variant comprises an amino acid substitution at one or more of positions 7, 10, 23, 25, 48, 49, 52, 53, 54, 57, 58, 59, 60, 62, 92, 98, 101, 106, 108, 121, 122, 134, 147, 193, 198, 217, 233, 236, 250, 257, 281, 301, 311, 340, 344, 360, 362, 363, 364, 365, 366, 367, and 368 of SEQ ID NO:1.

[0020] As indicated herein, truncated GpA369 shares approximately 72% sequence identity with the N-terminal 371 amino acid residues of hASNase 1. Ideally, at least one amino acid substitution in GpA generates a GpA variant with increased amino acid sequence identity with hASNase 1 compared to wild-type GpA. Consequently, one aspect of the present invention provides for humanization of GpA. According to this aspect of the present invention, the truncated GpA variant preferably has at least 70%, 72%, 74%, 76%, 78%, 80%, 82%, or 84% amino acid sequence identity with the N-terminal 371 amino acid residues of hASNase 1. Furthermore, the truncated GpA variant shares at least 85%, 87%, 89%, 91%, 93%, 95%, 97%, or 99% sequence identity with wild-type truncated GpA.

[0021] Preferably, humanization of truncated GpA is achieved by replacing one or more surface residues of wild-type GpA with the corresponding surface residues of hASNase 1. In particular, humanization of truncated GpA is achieved by replacing wild-type GpA residues H10, Q23, K25, K48, Q52, Q54, D91, D92, K98, E101, Q108, S121, G122, H134, R147, K193, D217, N233, H236, S250, Q288, R301, E344, L360, T362, A363, L365, H366, Q367, or S368, or any combination thereof, with the corresponding surface residues of hASNase 1. Specifically, humanization of truncated GpA is achieved by making one or more of the following amino acid substitutions relative to wild-type GpA: H10R, Q23R, K25E, K48E, Q52R, Q54R, D91A, D92E, K98Q, E101K, Q108H, S121F, G122A, H134Q, R147H, K193R, D217E, N233S, H236Q, S250A, Q288E, R301Q, E344D, L360P, T362S, A363V, L365E, H366R, Q367R and / or S368P. Exemplary truncated and humanized GpA variants are provided in SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:86.

[0022] PEGylation of L-asparaginase has been shown to increase its in vivo circulation time. The term "PEGylated" or "PEGylation" refers to the association of L-asparaginase with polyethylene glycol (PEG). "PEG" or "polyethylene glycol" refers to either water-soluble poly(ethylene glycol) or poly(ethylene oxide). PEG is thus represented by the structure (CH2CHO) n, where n is an integer from 2 to about 1000. Commonly used PEGs are end-capped PEGs, where one end of the PEG is end-capped with a relatively inert group such as alkoxy, while the other end is a hydroxyl group which may be further modified by a linker moiety. In one embodiment, the capping group is methoxy, and the corresponding end-capped PEG is designated mPEG. Thus, mPEG is CHO(CHCHO) n where n is an integer from 2 to about 1000. In another embodiment, the capping group is hydroxyl, and the corresponding end-capped PEG is a hydroxyPEG. "PEG" followed by a number (not a subscript) refers to a PEG moiety having a molecular weight approximately equal to the number multiplied by 1,000. Thus, "PEG40" or "PEG40K" is a PEG moiety having a molecular weight of approximately 40 kDa. Examples of methods that may be used to determine the molecular weight of PEG include, but are not limited to, mass spectrometry, such as TOF-MS. PEG may be provided, for example, by NOF Corporation, Tokyo, Japan; Creative PEG-works, Winston Salem, NC; and Nanocs, Boston, MA.

[0023] In one embodiment, the PEG moiety may be attached by nucleophilic substitution (acylation) on the N-terminal alpha-amino group or on the lysine residue(s) at the gamma position, for example, using an OSu-activated ester. In another embodiment, the PEG moiety may be attached by reductive alkylation at the amino group present in the GpA protein using a PEG-aldehyde reagent and a reducing agent such as sodium cyanoborohydride. In another embodiment, the PEG moiety may be attached to the side chain of an unpaired cysteine ​​residue in a Michael addition reaction, for example, using a PEG maleimide reagent. Other PEGylation methods include, but are not limited to, bridging PEGylation, transglutaminase PEGylation, glycoPEGylation, PEGylation using genetic engineering, and releasable linker PEGylation. For reviews of PEGylation methods, see Pasut & Veronese (2012) J. Contr. Rel. 161:461-472; and Roberts, et al. (2012) Adv. Drug Del. Rev. 64:116-127. In one embodiment, the PEG moiety is attached to the side chain(s) of a lysine or cysteine ​​residue(s).

[0024] "Linker" refers to the chemical moiety that connects the -HN- group of GpA protein and the -O- group of the PEG moiety. In a preferred embodiment, the linker does not have any adverse effect on the activity of GpA. The linker is typically a carboxylic acid derivative, where the carboxylic acid functionality is used to attach to the GpA protein via an amide bond. Examples of linkers include, but are not limited to, an acetic acid moiety having a linking motif: CHCO, a propionic acid moiety having a linking motif: CHCHCO or CHCHCO, a butyric acid moiety having a linking motif: CHCHCHCO or CHCHCHCO, a CO group, N-(aminocarbonyl)succinimide derivatives (e.g., N-(N-propylpropanamido)succinimide, N-(N-propylhexanamido)succinimide, and N-(N-ethylpropanamido)succinimide, etc.), pentanoic acid ((CH)CO), α-methylbutanoic acid (CHCHCH(CH)CO), succinic acid (CO(CH)CO), glutaric acid (CO(CH)CO), succinamide derivatives (e.g., (CH)NHCO(CH)CO, etc.), glutaramide derivatives (e.g., (CH)NHCO(CH)CO, and (CH)NHCO(CH)CO, etc.). Exemplary PEG molecules for use in the present invention include, but are not limited to, methoxyPEG succinimidyl carbonate (mPEG-SC), mPEG-succinimidyl carboxymethyl ester (mPEG-SCM), and mPEG-succinamidyl succinimidyl ester (mPEG-SAS).

[0025] PEGylation methods that can be used to PEGylate the L-asparaginase of the present invention are provided, for example, in US Pat. Nos. 4,179,337, 5,766,897, 2002 / 0065397, and 2009 / 0054590. Ideally, PEGylated GpA variants are produced by reacting PEG with truncated GpA variants at a PEG:GpA ratio of about 20:1 to 100:1, more specifically 20:1 to 50:1, or most specifically 20:1, depending on the size of the PEG. In some aspects, the GpA variant has at least one cysteine ​​residue, the PEG is maleimide PEG, the reaction is carried out in the absence of glycine or aspartic acid, and the reaction product is treated with beta-mercaptoethanol. In another aspect, the GpA variant has at least one lysine residue, the PEG is mPEG-SC, mPEG-SCM or mPEG-SAS, and the reaction is carried out in the absence of DTT and glycerol.

[0026] As disclosed herein, L-asparaginase can be pegylated by site-specific pegylation at 1 to 6 cysteine ​​residues or 10 to 30 lysine residues introduced into the amino acid sequence of L-asparaginase by amino acid substitution. See Examples 4 and 5. In particular, truncated GpA or humanized truncated GpA can be pegylated by introducing: (a) cysteine ​​residues at positions 49, 52, 225, 257, 281, and / or 340; or (b) lysine residues at positions 7, 53, 54, 57, 58, 98, 106, 233, 250, 257, 281, 311, and / or 340. In addition to introducing cysteine ​​or lysine residues at specific locations, endogenous cysteine ​​or lysine residues can be mutated (i.e., replaced with another residue) to more evenly distribute pegylation sites along the protein length. In this regard, certain embodiments include replacing residue Cys198 with Ala, Val, or Ser, and / or replacing residue Lys223 with Asp. In one embodiment, Cys198 is replaced with Ala, and the L-asparaginase derivative is pegylated at Cys79. In other embodiments, Cys198 is replaced with Ala, and one or more of the amino acids at positions 49, 225, and 340 are replaced with cysteine. Exemplary truncated GpA variants suitable for pegylation are SEQ ID NO: 51 (GpA369-C198A), SEQ ID NO: 52 (GpA369-C198S), SEQ ID NO: 53 (GpA369-C198V), SEQ ID NO: 54 (GpA369-C198A+K225C), SEQ ID NO: 55 (GpA369-C198A+K225C+E340C), SEQ ID NO: 56 (GpA369-C198A), SEQ ID NO: 57 (GpA369-C198A+K225C+E340C), SEQ ID NO: 58 (GpA369-C198A+K225C+E340C), SEQ ID NO: 59 (GpA369-C198A+K225C+E340C), SEQ ID NO: 60 (GpA369-C198A+K225C+E340C), SEQ ID NO: 61 (GpA369-C198A+K225C+E340C), SEQ ID NO: 62 (GpA369-C198A+K225C+E340C), SEQ ID NO: 63 (GpA369-C198A+K225C+E340C), SEQ ID NO: 64 (GpA369-C198A+K225C+E340C), SEQ ID NO: 65 (GpA369-C198A+K225C+E340C), SEQ ID NO: 66 (GpA369-C198A+K225C+E340C), SEQ 8A+K225C+E340C+E49C), SEQ ID NO:57 (GpA369-C198A+K225C+E340C+E49C+Q257C), SEQ ID NO:58 (GpA369(hum)-Group1+2+3-C198A), SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:86.

[0027] To increase the in vivo circulation time of truncated GpA, the present invention also provides variants, particularly truncated GpA variants having a histidine tag (His), a SUMO tag (SUMO), an albumin-binding domain (ABD), or a combination thereof. The truncated GpA variant may contain a histidine tag, a SUMO tag, and / or an albumin-binding domain at its C-terminus, N-terminus, and / or an internal location in the GpA sequence. Ideally, when the His tag, SUMO tag, or ABD is inserted into the GpA sequence (i.e., not at the N- or C-terminus), the insertion is in one or more flexible loops of the GpA protein. Illustrative truncated GpA variants include SUMO-asparaginase-ABD, His-SUMO-ABD-asparaginase, His-SUMO-asparaginase-ABD, His-SUMO-asparaginase, His-ABD-asparaginase, ABD-asparaginase, ABD-SUMO-asparaginase, and asparaginase. 1-225 -ABD-asparaginase 226-369 , asparaginase 1-340 -ABD-asparaginase 341-369 The nucleic acid encoding His, SUMO, or ABD can be inserted in frame either 5' or 3' to the nucleic acid encoding the truncated GpA variant, thereby creating a fusion protein.

[0028] Ideally, the inclusion of one or more of a histidine tag, a SUMO tag, an albumin-binding domain, or a combination thereof significantly increases the in vivo circulation time of the L-asparaginase. Alternatively, the variant has a longer t than wild-type L-asparaginase administered at an equivalent protein dose. 1 / 2 As used herein, the term "t 1 / 2" or "half-life" refers to the time required for the concentration of a truncated GpA variant or its fusion protein to decrease by half in vitro or in vivo (e.g., after injection in a mammal). Given that the effectiveness of L-asparaginase is related to the in vivo half-life of the drug, the truncated GpA variants of the present invention are particularly useful in treating cancers such as leukemia and lymphoma.

[0029] As used herein, a "histidine tag" refers to an amino acid motif composed of at least one histidine (His) residue, preferably six His residues. A histidine tag includes polyhistidines of up to six (hexahistidine tag, 6xHis tag, or His6 tag), seven, eight, nine, ten, or twenty histidine residues.

[0030] "SUMO tag" refers to the fusion of a SUMO (small ubiquitin-like modifier) ​​protein with a protein of interest to enhance the solubility / stability of the protein of interest. The inclusion of a SUMO tag can be achieved using known expression systems such as the CHAMPION pET SUMO expression system (Invitrogen), the EXPRESSO T7 SUMO cloning and expression system (Lucigen), or the pET His6 SUMO TEV LIC cloning vector (Addgene). In addition to SUMO tags, it is contemplated that other Ubl proteins, including, but not limited to, Ub, Rub1, Hub1, ISG15, Ubi-L (MNSF), FAT10, Apg12, Apg8, and Urm1, can be used (Larsen & Wang (2002) J. Proteome Res. 1(5):411-9). See also US Pat. No. 7,655,413, incorporated herein by reference in its entirety. Exemplary SUMO tags are set forth in SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:70.

[0031] "Albumin binding domain" or "ABD" refers to a polypeptide that binds to albumin in vivo or in vitro and enhances the serum half-life and biodistribution of a therapeutic agent. Albumin can be derived from any animal species, such as humans, monkeys, or rodents. Albumin binding domains are described, for example, in US 6,267,964, WO 1991 / 19741, WO 2005 / 097202, WO 2001 / 45746, WO 2013 / 043071, and US 2004 / 0001827. Furthermore, US 9,156,887 discloses non-natural albumin binding domains that can be used in the present invention. In some embodiments, the albumin binding domain has the amino acid sequence set forth in SEQ ID NO:71, SEQ ID NO:72, or SEQ ID NO:73.

[0032] The enhanced cytotoxic activity of the truncated GpA variants disclosed herein can be achieved by conjugating or fusing GpA to the three tandem soluble domains of TRAIL, or by co-administering GpA with a stable form of TRAIL. The resulting fusion protein exhibits a significantly increased IC compared to truncated GpA variants lacking the three tandem soluble domains of TRAIL. 50 The truncated GpA variants fused to the three tandem soluble domains of TRAIL may provide at least a 20-fold, 25-fold, 30-fold, 35-fold, or 40-fold reduction in the level of L-asparaginase. Consequently, truncated GpA variants fused to the three tandem soluble domains of TRAIL are particularly useful in treating cancer, particularly cancers that are insensitive to L-asparaginase.

[0033] "Fusion protein" refers to a chimeric protein containing proteins or protein fragments (e.g., GpA variants) operably linked in a non-natural manner. According to the fusion protein of the present invention, three tandem soluble domains of TRAIL (TRAIL トリマー) is fused in-frame with a truncated GpA variant. Ideally, a glycine or serine residue is inserted between each TRAIL repeat to facilitate folding of the TRAIL trimer (see, for example, SEQ ID NO:87 and SEQ ID NO:88). Optionally, the TRAIL trimer and asparaginase components can be separated by a linker as set forth in SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, or SEQ ID NO:84. The fusion protein comprises a GpA trimer at the C-terminus of GpA (GpA-TRAIL), where the GpA component can be any of the variants previously described. トリマー ; for example, SEQ ID NO: 89 and SEQ ID NO: 90) or N-terminus (TRAIL トリマー When further used in combination with a tag or modification, the fusion protein may comprise a TRAIL trimer fused to SUMO-TRAIL-GpA; e.g., SEQ ID NO: 91 and SEQ ID NO: 92. トリマー -GpA-ABD, His-SUMO-ABD-TRAIL トリマー -GpA, His-SUMO-TRAIL トリマー -GpA-ABD, His-SUMO-GpA-TRAIL トリマー , His-ABD-TRAIL トリマー -GpA, ABD-GpA-TRAIL トリマー , ABD-SUMO-TRAIL トリマー -GpA. In addition to being inserted at the N- or C-terminus, the ABD peptide can be added to one or more flexible loops of a GpA variant. In one example, the flexible loop spans residues 215-228. In another example, the flexible loop spans residues 337-342 of GpA. See, for example, SEQ ID NO: 94 and SEQ ID NO: 95. TRAIL, particularly TRAIL トリマー The nucleic acid encoding the L-asparaginase can be inserted in frame either 5' or 3' of the nucleic acid encoding the L-asparaginase, thereby creating a fusion protein.

[0034] Preferably, the soluble domain of TRAIL is derived from mammalian, particularly human, TRAIL, including allelic variants and / or derivatives thereof. The soluble domain comprises the extracellular portion of TRAIL, including the receptor-binding domain without the membrane-localization domain. Like other proteins of the TNF superfamily, TRAIL is membrane-anchored via a 15-30 amino acid N-terminal portion, the so-called stalk region. The stalk region contributes to trimer formation and provides a certain distance to the cell membrane. However, the stalk region is not part of the receptor-binding domain (RBD). Consequently, the soluble TRAIL domain preferably comprises the receptor-binding domain of TRAIL lacking any amino acids from the stalk region (see US2015 / 0337027).

[0035] The soluble TRAIL domain may be derived from human TRAIL. Preferably, the soluble TRAIL domain is derived from human TRAIL, particularly starting from amino acid residues 115-122, and includes amino acid residues 115-281, 120-281, 121-281, or 122-281 of human TRAIL. In certain embodiments, the soluble domain of each TRAIL トリマー is composed of residues 115-281 of human TRAIL. Residues 115-281 of human TRAIL are set forth herein in SEQ ID NO: 80. To promote correct folding, glycine or serine residues may be inserted between each TRAIL repeat. See, for example, SEQ ID NO: 87 and SEQ ID NO: 88.

[0036] All derivatives and variants of death receptor-binding TRAIL domains are contemplated and can be made by altering their amino acid sequences by substitution, addition, and / or deletion / truncations, or by introducing chemical modifications that result in functionally equivalent polypeptides. It will be understood by those skilled in the art that certain amino acids in the sequence of any polypeptide can be substituted for other amino acids without adversely affecting the activity of the polypeptide.

[0037] The TRAIL domain disclosed herein comprises one or more amino acid substitutions in the disclosed sequence.Those skilled in the art can use well-known techniques to determine suitable sequence variants of the peptides shown herein.In some embodiments, those skilled in the art can identify suitable areas of the molecule that can be changed without destroying activity by targeting regions that are not considered important for activity.In other embodiments, those skilled in the art can identify residues and portions of the molecule that are conserved between similar polypeptides.In further embodiments, even amino acid residues that are important for biological activity or structure can be subjected to conservative amino acid substitutions without destroying their biological activity or adversely affecting the peptide structure.

[0038] The three soluble domains of TRAIL are preferably linked to each other via a peptide bond consisting of 1 to 8 amino acid residues. トリマー is preferably linked to L-asparaginase via a peptide linking group consisting of 1 to 20 amino acid residues. The term "peptide linking group" or "linker" is meant to refer to a peptide moiety that acts as a molecular bridge operatively linking two different molecules together. Desirably, the linker of the present invention is composed of glycine or serine, or a combination thereof. In a specific embodiment, each soluble domain of TRAIL トリマー are preferably linked to each other by a single glycine or a single serine residue. トリマー contains a single serine residue between the three soluble TRAIL domains (SEQ ID NO: 88). トリマー With respect to the peptide bond located between the TRAIL and the L-asparaginase, it is desirable that this linker be a flexible linker. The flexible linker preferably has a length of 1 to 20 amino acid residues, in particular a length of 6, 9, 12, 15, or 18 amino acid residues. The flexible linker is preferably a glycine / serine linker, i.e., a peptide linker composed mainly of glycine and serine amino acids. In a particular embodiment, TRAIL トリマーThe linker between the enzyme and L-asparaginase is (GGGS) n linker (SEQ ID NO: 81), where n is 1 to 4, or, for example, GGGS (GGGGS) n (SEQ ID NO: 82), wherein n is 1 to 4, and permutations thereof. トリマー and L-asparaginase has the amino acid sequence set forth in SEQ ID NO: 83. トリマー and L-asparaginase has the amino acid sequence set forth in SEQ ID NO: 84. トリマー Examples of -GpA variant fusion proteins include a single glycine or serine residue between the three soluble TRAIL domains, トリマー N-terminal TRAIL containing a glycine / serine linker between トリマー See, for example, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92.

[0039] A stable form of TRAIL is intended to refer to a form of TRAIL that promotes trimerization. In particular, to promote trimerization of TRAIL, the small trimerization domain FOLDON sequence (GYIPEAPRDGQAYVRKDGEWVLLSTFL; SEQ ID NO: 85) has been shown to maintain the stability and biological activity of TRAIL for at least 48 hours at 37°C (Kouno, et al. (2013) J. Invest. Dermatol. 133(9):2212-2220). Consequently, the FOLDON peptide was inserted between the His-SUMO tag and the N-terminus of TRAIL to produce a His-SUMO-FOLDON-TRAIL fusion protein. The fusion protein was expressed, and a yield of >10 mg / L of fusion protein was obtained in bacterial culture. The protein was highly stable, indicating that the inclusion of FOLDON results in a stable form of TRAIL. Consequently, stable forms of TRAIL can be co-administered and / or combined in pharmaceutical compositions with truncated GpA variants to enhance the cytotoxic activity of GpA.

[0040] The truncated GpA variants and fusion proteins disclosed herein can be easily prepared by conventional recombinant protein technology, in which recombinant host cells are transformed or transduced with an expression construct or vector containing a nucleic acid molecule encoding the variant or fusion protein, and the recombinant host cells are grown under suitable conditions to provide expression of the variant or fusion protein, which may then be isolated and optionally purified. Consequently, the present invention also provides nucleic acid molecules encoding the truncated GpA variants or fusion proteins thereof, as well as expression cassettes and / or expression vectors containing them. Ideally, the expression cassettes and expression vectors contain the necessary regulatory sequences (e.g., promoters, terminators, etc.) to promote expression in the desired host cell. Host cells containing nucleic acid molecules encoding the truncated GpA variants or fusion proteins are also within the scope of the present invention. Host cells can include eukaryotic cells (e.g., mammalian, fungal, or yeast cells) or prokaryotic cells (e.g., E. coli).

[0041] Once produced and isolated / purified, the truncated GpA variant and / or fusion protein of the present invention can be used as is, or can be formulated into a pharmaceutical composition containing pharmaceutically acceptable excipients.The pharmaceutical compositions provided herein can be specially formulated for intravenous administration or intravenous injection in solid or liquid form.The optimal pharmaceutical composition can be determined by those skilled in the art, depending on, for example, the intended administration route, delivery form, and desired dosage.See, for example, Remington's Pharmaceutical Sciences (19th edition, 1995).

[0042] The truncated GpA variants and / or fusion proteins can be incorporated into conventional systemic administration forms, such as injectable formulations, which may also contain necessary physiologically acceptable carrier materials, excipients, lubricants, buffers, surfactants, antibacterial agents, bulking agents (such as mannitol), antioxidants (ascorbic acid or sodium bisulfite), and the like.

[0043] The primary carrier or excipient in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, suitable carriers or excipients can be water for injection, saline, or artificial cerebrospinal fluid, optionally supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. The pharmaceutical composition can contain Tris buffer at about pH 7.0-8.5 or acetate buffer at about pH 4.0-5.5, and can further contain sorbitol or a suitable substitute therefor. The pharmaceutical compositions of the present invention can be prepared for storage by mixing the selected composition having the desired purity with any formulating agent in the form of a lyophilized cake or aqueous solution (Remington's Pharmaceutical Sciences, Id.). Additionally, the truncated GpA variants or fusion proteins of the present invention can be formulated as lyophilizates using appropriate excipients such as sucrose or glycine.

[0044] The administration routes of the truncated GpA variants, fusion proteins, or pharmaceutical compositions of the present invention include intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional injection; sustained-release systems, or implanted devices. The compositions can be administered by bolus injection or continuously by infusion or implanted device. The compositions can also be administered locally via implantation of a membrane, sponge, or another suitable material into which the desired molecule is absorbed or encapsulated. When an implanted device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, sustained-release bolus, or continuous administration.

[0045] The compositions of the present invention can be delivered parenterally. When parenteral administration is intended, the therapeutic composition for use in the present invention can be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired compound identified by the screening method of the present invention in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the compound identified by the screening method of the present invention is formulated as a sterile, isotonic solution, properly preserved. Preparation can involve formulating the desired molecule with agents such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, which can provide controlled or sustained release of the product and can then be delivered via depot injection. Formulation with hyaluronic acid has the effect of promoting sustained duration in the circulation. The desired molecule can be introduced using an implantable drug delivery device.

[0046] Compositions can also be formulated for inhalation.In these embodiments, truncated GpA variants or fusion proteins are formulated as dry powders for inhalation, or inhalation solutions can also be formulated with propellants for aerosol delivery, such as by nebulization.Pulmonary administration is further described in WO1994 / 020069, which describes the pulmonary delivery of chemically modified proteins.

[0047] The compositions of the present invention can be delivered through the gastrointestinal tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the knowledge of those skilled in the art. The truncated GpA variants or fusion proteins of the present invention administered in this manner can be formulated with or without carriers commonly used in the formulation of solid dosage forms such as tablets and capsules. Capsules can be designed to release the active portion of the formulation at a point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to promote absorption of the peptides of the present invention disclosed herein. Diluents, flavorings, low-melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be used.

[0048] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of microbial action can be ensured by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars and sodium chloride. Prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0049] The truncated GpA variants and / or fusion proteins of the present invention are particularly useful for treating diseases or conditions treatable by asparagine depletion. Consequently, the present invention also provides a method for treating diseases, particularly cancer, by administering an effective amount of truncated GpA variants or fusion proteins to a subject in need of treatment. As used herein, "effective amount" refers to an amount of active ingredient sufficient to achieve the intended purpose: (a) reducing the severity of the disorder; (b) limiting or preventing the onset of symptoms characteristic of the disorder(s) being treated; (c) inhibiting the worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing the recurrence of the disorder(s) in patients who previously had the disorder; (e) limiting or preventing the recurrence of symptoms in patients who previously showed signs of the disorder(s); (f) reducing mortality after the onset of the disease or disorder; (g) cure; and (h) preventing disease. The effective amount in each individual case can be determined empirically by those skilled in the art according to methods established in the art. As used in the context of the present invention, "administering" includes in vivo administration to an individual, as well as in vitro or ex vivo direct administration to cells or tissues. An effective amount of a truncated GpA variant or fusion protein is generally one that can induce apoptosis and reduce circulating L-asparagine in the target cancer cells or tumor. A clinician can titrate the dosage or administration route to obtain optimal therapeutic effect.

[0050] In some embodiments, the truncated GpA variant or fusion protein is useful in the treatment or manufacture of pharmaceuticals for use in the treatment of cancers such as acute lymphocytic leukemia (ALL) in both adults and children, as well as other conditions in which asparagine depletion is expected to have a beneficial effect. Such conditions include, but are not limited to, malignant tumors or cancers, including, but not limited to, hematological malignancies, non-Hodgkin's lymphoma, NK lymphoma, pancreatic cancer, ovarian cancer, Hodgkin's disease, acute myeloid leukemia, acute myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulum cell sarcoma, and melanosarcoma. Representative non-malignant hematological diseases that respond to asparagine depletion include immune system-mediated hematological diseases (e.g., infectious diseases such as those caused by HIV infection (i.e., AIDS)). Non-hematological diseases associated with asparagine dependency include autoimmune diseases (e.g., rheumatoid arthritis, SLE, autoimmune, collagen-related vascular diseases, AIDS, etc.). Other autoimmune diseases include osteoarthritis, Isaacs syndrome, psoriasis, insulin-dependent diabetes mellitus, multiple sclerosis, sclerosing panencephalitis, systemic lupus erythematosus, rheumatic fever, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), primary biliary cirrhosis, chronic active hepatitis, glomerulonephritis, myasthenia gravis, pemphigus vulgaris, and Graves' disease. In certain embodiments, the truncated GpA variant or fusion protein is used in the treatment of non-Hodgkin's lymphoma, leukemia, acute lymphocytic leukemia, acute myeloid leukemia, B-cell lymphoma, Burkitt's lymphoma, chronic myelogenous leukemia, chronic lymphocytic leukemia, and hairy cell leukemia.

[0051] Cells suspected of causing disease can be tested for asparagine dependency in any suitable in vitro or in vivo assay (e.g., an in vitro assay in which the growth medium lacks asparagine). Thus, in one aspect, the present invention relates to a method for treating a treatable disease in a patient, comprising administering to the patient an effective amount of a truncated GpA variant or fusion protein of the present invention. In a specific embodiment, the disease is ALL. In a specific embodiment, the truncated GpA variant or fusion protein used in the treatment of a treatable disease by asparagine depletion comprises a truncated GpA variant derived from guinea pigs.

[0052] The truncated GpA variant or fusion protein can be administered on a schedule ranging from about three times a week to about once a month, typically once a week or once every other week, as a single agent (e.g., monotherapy) or as part of a combination of chemotherapy drugs, including but not limited to glucocorticoids, corticosteroids, anticancer compounds, or other drugs (including, but not limited to, methotrexate, dexamethasone, prednisone, prednisolone, vincristine, cyclophosphamide, and anthracyclines). For example, a patient with ALL will be administered the truncated GpA variant or fusion protein of the present invention as a component of multi-drug chemotherapy during three chemotherapy phases, including induction, intensification or boost, and maintenance. In certain instances, the truncated GpA variant or fusion protein is not administered together with an asparagine synthetase inhibitor (see, for example, WO 2007 / 103290). In another specific example, the truncated GpA variant or fusion protein is not administered with an asparagine synthetase inhibitor, but is administered with other chemotherapeutic agents. The truncated GpA variant or fusion protein can be administered before, after, or simultaneously with other compounds as part of a multi-drug chemotherapy regimen.

[0053] In a particular embodiment, the method involves administering a truncated GpA variant or fusion protein of the invention in an amount of about 1 U / kg to about 1000 U / kg. In a more particular embodiment, the truncated GpA variant or fusion protein is administered in an amount selected from the group consisting of about 20, 50, 60, 70, 100, 200, 300, 400, 500, and 600 U / kg. In another particular embodiment, the truncated GpA variant or fusion protein is administered in an amount of about 1000 IU / kg. 2 ~About 20000IU / m 2 Doses ranging from 1000 IU / m 2 , 2000 IU / m 2 , 3000IU / m 2 , 4000 IU / m 2 , 5000IU / m 2 , 6000 IU / m 2 , 7000 IU / m 2 , 8000IU / m 2 , 9000IU / m 2 , 10,000 IU / m 2 , 11,000 IU / m 2 , 12000IU / m 2 , 13,000 IU / m 2 , 14000IU / m 2 , 15,000 IU / m 2 , 16000IU / m 2 , 17,000 IU / m 2 , 18,000 IU / m 2 , 19,000 IU / m 2 , or 20,000 IU / m 2 In another specific embodiment, the truncated GpA variant or fusion protein is administered at a dose that depletes Asn to undetectable levels in a single dose for a period of about 3 days to about 10 days (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 days). In another embodiment, the method involves administering a truncated GpA variant or fusion protein of the invention that has a longer in vivo circulatory half-life after a single dose compared to wild-type L-asparaginase.

[0054] The following non-limiting examples are provided to further illustrate the present invention.

[0055] Example 1: Generation of an active, C-terminally truncated GpA Mammalian L-asparaginases, such as human L-asparaginase (hASNase 1; UNIPROT entry Q86U10; SEQ ID NO: 2) and guinea pig L-asparaginase (gpASNase 1 or GpA; UNIPROT entry H0W0T5; SEQ ID NO: 1), contain two domains: an N-terminal domain of approximately 360 residues in which L-asparaginase activity resides, and a C-terminal domain of approximately 200 residues of unknown function. In comparison, L-asparaginases from the clinically relevant bacteria Escherichia coli and Erwinia chrysanthemi are approximately 350 amino acid residues long and do not contain such C-terminal domains.

[0056] One of the challenges of injectable biologics for treatment is their short half-life, which results in poor bioavailability. Elimination occurs primarily through proteolysis, renal filtration, or neutralization by the immune system. Larger molecules with foreign sequences have a greater chance of being detected by the immune system and are rapidly cleared from the system. Identifying a shorter, more stable version of GpA was expected to provide better treatment, especially in situations where the C-terminal domain of the full-length protein is of unknown function.

[0057] To assess the function of the C-terminal residues of GpA, C-terminally truncated GpA variants were constructed and analyzed for stability and overall L-asparaginase activity. Sequence alignment and crystal structure analysis of hASNase1, GpA, E. coli L-asparaginase, and Erwinia L-asparaginase enzymes indicated the catalytic domain ends at residue 359. A construct consisting of residues 1-359 of GpA (SEQ ID NO: 3) was recombinantly expressed, but the purified C-terminally truncated enzyme was unstable. Therefore, additional C-terminally truncated GpA constructs fused to a SUMO tag were prepared (Table 1). [Table 1]

[0058] Recombinant overexpression of these C-terminally truncated GpA constructs indicated that all proteins were fully overexpressed and purified to >95% purity (as measured by SDS-PAGE). While GpA367 rapidly precipitated within minutes after SUMO tag cleavage, the longer variants exhibited superior stability and activity comparable to that of the full-length protein. Notably, the longer constructs possessing an extra Cys388 residue tended to form unspecific disulfide bonds, altering the tetrameric form required for L-asparaginase activity. While C-terminally truncated GpA consisting of at least residues 1-369 yields a stable GpA enzyme with L-asparaginase activity, as shown herein, the stability of C-terminally truncated GpA consisting of residues 1-359 or 1-367 can be stabilized by fusion with heterologous peptides / proteins (e.g., SUMO tag or albumin) or chemical modification (e.g., PEGylation).

[0059] Example 2: Humanized GpA variants generated by directed evolution Overview. Directed evolution, or the process of mimicking natural evolutionary processes in the laboratory, is widely used to improve enzyme properties. See, for example, Dalby (2011) Curr. Opin. Struct. Biol. 21:473-480; Goldsmith & Tawfik (2012) Curr. Opin. Struct. Biol. 22:406-412; Labrou (2010) Curr. Protein Pept. Sci. 11:91-100; Wang & Zhao (2012) Bioresour. Technol. 115:117-125. Given advances in library generation, screening techniques, and primarily a better understanding of the mechanisms of natural protein evolution, significant improvements in evolved catalytic activity (compared to the starting point and absolute k cat / K m values) were obtained (Fasan, et al. (2008) J. Mol. Biol. 383:1069-80; Bar-Even, et al. (2011) Biochemistry (Mosc.) 50:4402-10).

[0060] To overcome immunogenicity, a human-like L-asparaginase with kinetic properties similar to those of type II E. coli L-asparaginase was generated via a directed evolution approach. DNA family shuffling was used to generate a human-like L-asparaginase with high sequence identity to hASNase1 but the low K of gpASNase1. m Several clones with specific properties were identified.

[0061] The chromosomal gene deletions were performed using the λ-red recombinase system (Datsenko & Wanner (2000) Proc. Natl. Acad. Sci. USA 97:6640-6645). The tyrosine aminotransferase gene tyrB, the aspartate aminotransferase gene aspC, and the L-asparaginase genes ansA, ansB, and iaaA were transfected into E. coli BW25113F. -, DE(araD-araB)567, lacZ4787(del)::rrnB-3, LAM-, rph-1, DE(rhaD-rhaB)568, and hsdR514 were deleted from the chromosome, resulting in E. coli BW5Δ. Briefly, primer pairs were used to amplify the replaced gene with a kanamycin resistance cassette from the appropriate keio strain, according to known methods (Datsenko & Wanner (2000) Proc. Natl. Acad. Sci. USA 97:6640-6645). The linear PCR product was then used to replace the entire open reading frame of the target gene on the chromosome of BW25113. Colonies containing the correct gene deletion were transformed with the FLP recombinase plasmid pCP20 to remove the kanamycin resistance marker, and pCP20 was then cured from the resulting strain. The E. coli BW2Δ strain was obtained following the same process and after deletion of the ansA and ansB genes.

[0062] Cloning of hASNase1 and gpASNase1 in BW2Δ and BW5Δ. The gene encoding the codon-optimized sequence of hASNase1 was amplified using primers NdeI-hA_F1 and hA-BamHI_R573, and the gene encoding the codon-optimized sequence of gpASNase1 was amplified using primers NdeI-gpA_F1 and gpA-BamHI_R565 (Table 2). After BamHI / NdeI digestion, the PCR product was inserted into the pBAD vector. The resulting vectors were subsequently used to transform BW5Δ and BW2Δ strains, resulting in BW5Δ pBAD_hASNase1, BW5Δ pBAD_gpASNase1, BW2Δ pBAD_hASNase1, and BW2Δ pBAD_gpASNase1 strains. BW5Δ and BW2Δ were also transformed with the empty pBAD vector to serve as controls. [Table 2-1] [Table 2-2]

[0063] Culture medium and growth experiments. M9 complete medium was made with M9 minimal salts (Sigma) supplemented with 0.4% glycerol, 2 μM thiamine, 1 mM MgSO, 0.1 mM CaCl, and 100 μg / mL ampicillin. For complete M9 plates, 15 g / L of agar was added. For experiments using the BW2Δ strain, M9 medium was made without NHCl. When necessary, L-asparaginase was added to M9 medium at various concentrations. 0.02% arabinose was added to M9 medium to induce expression of hASNase1 and gpASNase1 cloned into the pBAD vector. For growth experiments on complete M9 agar plates, strains were first grown overnight in LB at 37°C, spun down, and washed in M9 medium. Appropriate dilutions of this suspension were then spread onto M9 plates to obtain equal numbers of colonies for each strain. Plates were incubated at 37°C for 48-96 hours.

[0064] DNA shuffling. DNA shuffling was performed as described (Meyer, et al. (2014) Curr. Protoc. Mol. Biol., Ausubel (ed) 105:Unit-15.12) with minor adjustments. Briefly, an equimolar mixture of hASNase1 and GpA genes was digested with 0.5 U of DNase (NEB) for 2 minutes and 30 seconds. Fragments between 100 and 200 bp were extracted using a QIAQUICK gel extract kit (Qiagen), reassembled by PCR, and then amplified using primers specific for either the GpA gene or the hASNase1 gene. The resulting shuffled fragments were cloned into the pBAD vector using the Megawhop method (Miyazaki (2003) Methods Mol. Biol. 231:23-28). Briefly, PCR of the entire plasmid was performed using 100–300 ng of the shuffled fragment as a megaprimer and either pBAD_hASNase1 or pBAD_gpASNase1 as a template. After digestion of the template with DpnI, 20–40 ng of the newly synthesized plasmid containing the shuffled sequence was transformed into electrocompetent BW5Δ cells. After pulsing, the cells were resuspended in 1 ml of SOC medium and incubated at 37°C for 1 h with shaking. The cells were then spun down at 4000 x g for 4 min and gently resuspended in 200 μL of M9 medium. 100 μL of the mixture was plated onto M9 plates supplemented with 0.2 mM Asn or 2 mM Asn. After 4 days of incubation at 37°C, colonies from the 2 mM Asn plates were isolated and pooled in 200 μL of fresh M9 medium. Two serial dilutions were performed and used to plate onto fresh 0.2 mM AsnM9 plates. After 4 days of incubation at 37°C and an additional 3 days at room temperature, clones capable of growing on 0.2 mM Asn plates were isolated and streaked onto LB plates.

[0065] C-terminal domain swapping.N -g C To assemble the clone, the N-terminal domain of hASNase1 (hASNase1) was used. N The sequence corresponding to the C-terminal domain of gpASNase1 (gC, residues 360-565) was amplified using primers hA354-361_F and gpA-BamHI_R565. N -h C To assemble the clone, the N-terminal domain of gpASNase1 (g N The sequence corresponding to the C-terminal domain of hASNase1 (hASNase1, residues 1-359) was amplified using primers NdeI-gpA_F1 and hA362-369_R. C The sequence corresponding to the fragment hA_BamHI_R573 (residues 362-573) was amplified using primers gpA352-359_F and hA_BamHI_R573. The primers are listed in Table 2. The chimera was then amplified using the appropriate primers to amplify the fragment hA_BamHI_R573. N and g C or g N and h C and then subsequently cloned into pBAD and pET vectors.

[0066] Cloning and Expression of Selected Clones. Isolated clones were cultured; plasmids were extracted and sequenced. The corresponding genes were introduced into a pET vector (modified pET14b containing a His-SUMO tag using the same primers used to clone into the pBAD vector) to allow expression of His-tagged proteins in C41(DE3) cells. Cultivation was carried out in 1 L of 2YT medium supplemented with 100 μg / mL ampicillin. Expression was induced with 0.1 mM IPTG, and cells were grown overnight at 18°C. Cells were harvested, lysed, and purified as previously described for wild-type gpASNase1 (Schalk, et al. (2014) J. Biol. Chem. 289:33175-86). The protein was eluted in a buffer of 25 mM Tris-HCl, 200 mM KCl, 500 mM imidazole, pH 7.5, and dialyzed against the same buffer without imidazole but containing 1 mM DTT. Expression and purification of the E. coli ansB enzyme have been previously described (Schalk, et al. (2014) J. Biol. Chem. 289:33175-86).

[0067] Kinetic assay. The catalytic activity of clones was determined using a spectroscopic NADH-dependent enzyme-coupled assay (Fernandez, et al. (2013) Int. J. Clin. Exp. Med. 6:478-487; Hejazi, et al. (2002) Biochem. J. 364:129-136), which measures the production of L-aspartate (Asp) through the 1:1 oxidation of reduced NADH. The conversion of NADH to NAD was measured spectrophotometrically as the decrease in absorbance at 340 nm at 37 °C. All measurements were performed in triplicate in a buffer containing 100 mM Tris at pH 7.5, 0.4 mM α-ketoglutarate, and 0.4 mM NADH with 50 nM (hASNase 1, hN-gC); 10 nM (gpASNase 1, gN-hC, 63-hC, 64-hC, 65-hC, SA-hC), or 3 nM (ansB) enzyme. Glutamate oxaloacetic transaminase (Sigma) and malate dehydrogenase (Sigma) were used as helper enzymes for the coupled enzyme reactions; 5 and 1 unit were used, respectively. Data were fitted to the Michaelis-Menten equation using SigmaPlot (Systat Software Inc.). Due to the cooperative nature of hASNase 1, this enzyme was analyzed using the Hill equation.

[0068] Cell culture. The LOUCY (Ben-Bassat, et al. (1990) Cancer Genet. Cytogenet. 49(2):241-8) and SUP-B15 (ATCC CRL-1929) cell lines have been described in the art. All cell lines were analyzed by short tandem repeat (STR) and confirmed to be 100% consistent with the corresponding STR profile data from the Global Bioresource Center ATCC. All cell lines were confirmed to be mycoplasma-free. The LOUCY and SUP-B15 lines were cultured in a humid atmosphere (5% CO2, 37°C) using RPMI 1640 medium supplemented with 10% FBS (Hyclone) and 1x penicillin-streptomycin solution (Invitrogen). L-glutamine was added directly to the cell culture to a final concentration of 2 mM. Aliquots of 90 μL of the cell suspension (5 × 10 per mL) were cultured in the presence of 10 μL of either DPBS (Dulbecco's Phosphate Buffered Saline, Mediatech) or various L-asparaginases. 5 L-asparaginase (1000 cells) was cultured in triplicate in round-bottom 96-well microtiter plates to final concentrations ranging from 0.00001 to 0.1 IU / mL. After incubating the plates at 37°C in humidified air containing 5% CO2 for 4 days, Alamar Blue (Invitrogen) was added to a final concentration of 10% (v / v), and the plates were incubated for an additional 2 hours, after which the fluorescent signal was read. Leukemia cell viability was calculated as the percentage of fluorescent counts in the presence of L-asparaginase relative to the fluorescent counts in the DPBS control.

[0069] Development of Selection Systems. To create a selection system for L-asparaginase activity, a bacterial strain whose growth depended on one of the products of the enzymatic reaction was required. Since L-asparaginase catalyzes the hydrolysis of Asn to Asp and ammonia, two selection systems were developed and tested: one using a bacterial strain that depended on the reaction product ammonia as the sole nitrogen source, and the other using a strain auxotrophic for Asp.

[0070] BW2Δ strain: Dependence of L-asparaginase reaction on nitrogen source. All organisms require a nitrogen source. For growth of E. coli in minimal media such as M9, the nitrogen source is usually obtained in the form of NH4Cl salt. However, it was demonstrated that the E. coli BW strain used in this study can grow using 2 mM Asn in the medium in the absence of NH4Cl. This indicates that E. coli can use Asn as a nitrogen source through the activity of its endogenous L-asparaginase. To create an NH4Cl-dependent BW E. coli strain produced by exogenous L-asparaginase activity, two endogenous L-asparaginase genes (ansA and ansB) were removed from the E. coli BW parent strain, which is then designated BW2Δ. The extremely high K for Asn was m In particular, the third endogenous L-asparaginase (iiiA) was not removed, due to its value in the BW2Δ strain. Indeed, under the experimental conditions, growth of the BW2Δ strain was greatly reduced even with 2 mM Asn in the medium.

[0071] BW5Δ strain: Dependence on the L-asparaginase reaction for aspartate. E. coli can produce Asp by hydrolyzing Asn (L-asparaginase reaction) or by an aminotransferase reaction. In fact, the parent BW strain grows well in M9 medium alone, and supplementation with Asp is not affected. To create an E. coli strain auxotrophic for Asp, all three endogenous L-asparaginase genes (ansA, ansB, and iiiA) as well as two related aminotransferase genes (aspC and tyrB) were deleted. The strain with these five genes deleted is referred to as the BW5Δ strain. To confirm that the BW5Δ strain had acquired Asp auxotrophy, growth was tested in M9 minimal medium with and without Asp supplementation. We observed that BW5Δ could not grow in medium not supplemented with Asp. However, growth was observed under Asp-supplemented conditions.

[0072] Use of BW2Δ and BW5Δ strains for the selection of clones expressing L-asparaginase. Whether these bacterial strains can be used as a selection system for L-asparaginase activity and, first, their K m To investigate whether a low K would allow discrimination between L-asparaginases based on their properties, m L-asparaginase with high K m The growth of strains BW2Δ and BW5Δ expressing either L-asparaginase with a K for Asn in the millimolar range was analyzed. The protein target to be evolved, human L-asparaginase type I (hASNase1), has a K for Asn in the millimolar range. m (Karamitros & Konrad (2014) J. Biol. Chem. 289:12962-75; Schalk, et al. (2014) J. Biol. Chem. 289:33175-86), and therefore have a high K mGuinea pig L-asparaginase I (gpASNase1) was used as the L-asparaginase of choice. The K for Asn is in the micromolar range. m (Schalk, et al. (2014) J. Biol. Chem. 289:33175-86) and therefore have a low K m In order to have well-controlled protein expression, both of the genes encoding the L-asparaginases were cloned into the pBAD vector.

[0073] In the first screening system, based on the L-asparaginase reaction supplying a single source of nitrogen, BW2Δ was transfected with pBAD (control vector), pBAD_hASNase1 (high K m enzyme) or pBAD_gpASNase1 (low K m The transformed cells were grown in M9 medium lacking NH4Cl but supplemented with increasing concentrations of Asn. Low Asn concentrations resulted in low K M It was expected that the growth of the BW2Δ strains carrying the pBAD_gpASNase1 plasmid, encoding the enzyme, would be preferentially promoted. The results show that the growth of both pBAD_hASNase1 and pBAD_gpASNase1 BW2Δ strains was indeed dependent on the Asn concentration (i.e., enhanced Asn concentration resulted in better growth). However, there was no significant difference in growth between pBAD_hASNase1 and pBAD_gpASNase1 BW2Δ strains at each Asn concentration, e.g., at 0.2 mM Asn, a low K m BW2Δ bacteria expressing the gpASNase1 enzyme exhibited a high K m In summary, this screening system demonstrated that the K of these Asn m It has been found that it is not suitable to distinguish between L-asparaginases that differ in their values.

[0074] In a second screening system, based on Asp auxotrophy, BW5Δ was similarly transformed with pBAD, pBAD_hASNase1, or pBAD_gpASNase1. Transformed cells were grown in complete M9 medium supplemented with increasing concentrations of Asn. Growth of BW5Δ in pBAD_hASNase1 and pBAD_gpASNase1 was found to be dependent on the concentration of Asn. Interestingly, the guinea pig enzyme was found to be dependent on the substrate (K m = 50 μM), and the human enzyme is only partially saturated (K m At 2 mM Asn, representing a concentration of Asn (K = 3,500 μM), the BW5Δ strains transformed with hASNase1 and gpASNase1 show similar growth. In other words, at this relatively high Asn concentration, the BW5Δ strain exhibits a low K m Those bacteria expressing L-asparaginase and high K m In contrast, only BW5Δ carrying pBAD_gpASNase1 is able to grow at lower concentrations of the substrate Asn. Indeed, the K m Although the K m At a higher Asn concentration of 0.2 mM, colonies of pBAD_gpASNase1-expressing BW5Δ developed, while pBAD_hASNase1-expressing BW5Δ failed to grow. Notably, this difference in growth was found to be independent of the expression level of the enzyme, as no change in growth was observed when arabinose (the inducer for protein expression) was used in the range of 0.0002% to 0.2%. Taken together, the results suggest that the difference in growth observed between hASNase1-expressing BW5Δ and gpASNase1-expressing BW5Δ on minimal medium plates is due to the K values ​​of the respective expressed L-asparaginases. m This suggests that the data directly reflects the

[0075] The conclusion from this first set of experiments is that a screening based on the nitrogen-feeding L-asparaginase reaction (using the BW2Δ strain) can m Enzymes and Low K m While the enzymes do not discriminate well between the two, a screen based on the L-asparaginase reaction (using the BW5Δ strain) supplying the amino acid Asp does indeed distinguish between enzymes with different affinities for Asn—low K m Therefore, only bacteria expressing L-asparaginase at low K m All further selection steps aimed at finding human L-asparaginase variants acquiring the nucleotide sequence were carried out using the BW5Δ E. coli strain.

[0076] DNA family shuffling. DNA family shuffling is an alternative method for generating genetic diversity. The protein sequences of hASNase 1 and gpASNase 1 contain 573 and 565 amino acids, respectively, and are 69.8% identical at the amino acid level (170 amino acid differences). When synthetic codon-optimized versions of both hASNase 1 (SEQ ID NO: 35) and gpASNase 1 (SEQ ID NO: 36) are used, the genes encoding hASNase 1 and gpASNase 1 show 75% identity at the DNA level. As mentioned herein, hASNase 1 has a K of 3.5 mM for Asn. m whereas the K of the guinea pig enzyme m The K was determined to be 50 μM (Table 3). The DNA shuffling method was performed using guinea pig low K m The resulting chimeric library was used to transform the BW5Δ strain. mThe presence of mutants possessing the α-asn-binding domain was discovered using the selection protocol described above. Four clones (#63, #64, #65, and #SA) were isolated from M9 plates at an Asn concentration of 0.2 mM. Sequence analysis of these clones revealed a shuffling pattern with recombination events occurring primarily in the N-terminal (i.e., catalytic) domain; one of the selected clones (#SA) harbored a mutation that introduced a premature stop codon (STOP) beyond the catalytic domain (Figure 1).

[0077] Swapping of the C-terminal domain to minimize immunogenicity to GpA, making it as identical as possible to human L-asparaginase, but with the low K of the guinea pig / E. coli type II enzyme. m Shuffling experiments suggested that a chimera with a guinea pig L-asparaginase domain followed by a human C-terminal domain would still retain the favorable kinetic properties of gpASNase1, but with increased sequence identity to the human enzyme.

[0078] As a result, two chimeras were generated; N -g C One is called g, which contains a human N-terminal domain fused to a guinea pig C-terminal domain, and the other is called g N -h C The first chimera, designated guinea pig chimera 1, contains a guinea pig N-terminal domain fused to a human C-terminal domain (Figure 1, Table 3). In vitro kinetic characterization of these chimeras has shown that N -g C showed similar kinetic properties to hASNase1, and g N -h C We verified the prediction that gpASNase1 exhibits similar kinetic properties to gpASNase1 (Table 4).

[0079] This result indicates that the C-terminal domain of GpA does not affect the catalytic activity of L-asparaginase, and most importantly, K mThe goal was to identify a clone with the kinetic properties of gpASNase1 but with the highest sequence homology to hASNase1. Four clones, #63, #64, #65, and #SA, were engineered by replacing their shuffled C-terminal domains with the exact sequence of the human C-terminal domain (Figure 1). The engineered clone, #63 N -h C , 64 N -h C , 65 N -h C and SA N -h C showed 85.7%, 91.1%, 87.1% and 91.6% identity with the wild-type hASNase1 sequence, respectively (Table 3 and Figures 2A-2B). [Table 3]

[0080] Catalytic properties of variants. 63 N -h C , 64 N -h C , 65 N -h C and SA N -h C To determine the detailed kinetic properties of hASNase1, the genes encoding these enzymes were subcloned into the pET14b expression vector and expressed in C41 E. coli cells. Purified clones were tested for their L-asparaginase activity (Table 4). For comparison with L-asparaginases approved for cancer therapy, the E. coli L-asparaginase ansB was also included. Four clones selected by directed evolution and carrying the C-terminal domain of hASNase1 showed high sequence identity (>85%) with hASNase1 but exhibited K values ​​in the micromolar range. m We observed that clone 63 exhibited similar kinetic properties to gpASNase1. N -h C (85.7% identity with the sequence of hASNase1) had the lowest K at 47 μMm Clone SA N -h C had the highest sequence identity with hASNase1 (91.6% identity), but this clone had a somewhat higher K of Asn at 165 μM. m To relate this to blood Asn concentration, the observed Asn hydrolysis rate (k obs@50μM ) were compared (Ollenschlaeger (1988) Eur. J. Clin. Invest. 18:512-6). obs@50μM is 41±0.3 seconds -1 and that of wild-type gpASNase1 was found to be 20 sec -1 Importantly, the k for the humanized clones obs@50μM The values ​​were also within this range, i.e. clone 63 N -h C , 64 N -h C , 65 N -h C and SA N -h C , 17 seconds for each -1 , 10sec -1 , 17sec -1 and 6sec -1 It was. [Table 4]

[0081] Cell culture evaluation of humanized L-asparaginase clones. To determine whether the GpA enzyme exhibits anti-ALL activity, human T-ALL LOUCY and B-ALL SUP-B15 cell lines were exposed to increasing concentrations of gpASNase 1. Results of this analysis showed that gpASNase 1 exhibited IC50 activity of 0.00015 IU / ml and 0.00036 IU / ml for the LOUCY and SUP-B15 cell lines, respectively. 50 (Table 5). In particular, these IC 50 The values ​​are comparable to those of the E. coli type II enzyme.

[0082] The hASNase1 and gpASNase1 chimeras were also evaluated for their anti-ALL efficacy. N -h C、 63 N -hC and 65 N -hC, these clones have the lowest Asn K m (35, 47, and 74 μM, respectively), and were therefore chosen for these experiments because they have an activity almost similar to that of gpASNase 1 (50 μM). N -hC highest K m Due to the high IC value, this clone has the highest IC compared to other enzymes. 50 had low IL-1 levels but were still highly effective in killing both T-ALL and B-ALL cells (IC in the range of mIU / mL) 50 (Table 5). Clone g N -h C and 63 N -h C demonstrated similar cell killing potency compared to gpASNase1. Clone 63 N -h C This is particularly noteworthy since this clone increased the percent identity to hASNase 1 from 69.8% to 85.7% as present in gpASNase 1. [Table 5]

[0083] In summary, this analysis demonstrates the required low K m Two of the identified clones, 63N-hC and 65N-hC, shared 85.7% and 87.1% amino acid sequence identity with hA-FL, respectively, but had similar K values ​​to full-length GpA. mThese clones possess 100- to 140-fold enhanced catalytic efficiency compared to full-length hASNase 1. Notably, these highly human-like L-asparaginases maintain their in vitro ALL-killing potential.

[0084] Example 3: Humanized GpA variants produced via a structure-based approach As an alternative to DNA shuffling and domain swapping, a structure-based approach was taken to humanize the GpA enzyme and reduce its immunogenicity. For this approach, the truncated GpA369 variant (SEQ ID NO: 5) was modified. The crystal structure of GpA was examined, and predictions were made regarding which residues on the surface of the enzyme could be mutated to the corresponding amino acids in hASNase 1, and their presence would not be detrimental to the activity or stability of the enzyme. The candidate surface residues were divided into three groups based on the possibility that they may affect the activity or stability of GpA (Group 1, no effect; Group 2, likely no effect; and Group 3, possible effect) (Table 6). [Table 6]

[0085] GpA variants were prepared containing all Group 1 mutations, combinations of Group 1 and 2 mutations, and combinations of Group 1, 2, and 3 mutations (Table 7). The GpA variants were recombinantly expressed and found to retain 100% of wild-type activity and stability.

[0086] In vivo stability data suggested potential cleavage of one of the surface-exposed loops present in the GpA enzyme. In particular, the data indicated that loop 1 (loop1), as present in hASNase 1, may be involved in the cleavage of the GpA enzyme. hum , residues 57-62: SEDTLV (SEQ ID NO: 43)) and a sequence similar to that present in GpA (loop1gp These results suggest a difference in stability between proteins with the mutations from Groups 1, 2, and 3 (residues 57-62: PDHALA (SEQ ID NO: 44)). Consequently, GpA variants containing combinations of mutations from Groups 1, 2, and 3 were further analyzed using loop 1. hum The Loop1GpA variant was mutated to include the following sequence (Table 7): Furthermore, the Loop1GpA variant was recombinantly expressed and found to retain 100% of the wild-type activity and stability. [Table 7]

[0087] Example 4: Mutation of surface cysteine ​​residues for PEGylation PEGylation is known to increase in vivo circulation time. PEG molecules can be easily attached to cysteine ​​residues of proteins of interest using maleimide-based PEGylation chemistry. GpA369 and humanized GpA369 variants contain five unique cysteine ​​residues: Cys79, Cys173, Cys198, Cys296, and Cys299. Based on crystal structure analysis, Cys173 and Cys296 are predicted to be buried and therefore unaffected by PEGylation. Cys299 is less accessible than Cys198 and is thought to contribute to tetramer stabilization. Attempts to replace Cys299 with either Ala or Ser to avoid PEGylation at this site resulted in unstable proteins. Conversely, mutation of the cysteine ​​residue at position 198 to alanine (GpA369-C198A, SEQ ID NO: 51), serine (GpA369-C198S, SEQ ID NO: 52), or valine (GpA369-C198V, SEQ ID NO: 53) resulted in proteins of comparable stability and activity compared to the reference GpA369 enzyme. Furthermore, maleimide-PEGylation of the GpA369-C198A, GpA369-C198S, and GpA369-C198V proteins, which now contain only a single reactive surface cysteine ​​(Cys79), resulted in homogeneous products.

[0088] The degree of protection of biologics by PEGylation depends on the structure and complexity of the PEG agent, but in many cases, single-site PEGylation is insufficient to cover the entire macromolecule. Therefore, additional GpA variants containing one to five surface cysteine ​​residues were generated. In this regard, the structure of GpA was analyzed to identify regions of residues that could be mutated to cysteines for multi-site PEGylated products. Useful regions must be on the surface, away from the oligomerization interface (because GpA is a tetramer, PEGylation of residues near the interface could be detrimental to enzyme activity), and contain outward-facing residues. Regions of GpA that meet these criteria are listed in Table 8. [Table 8]

[0089] Among the possible residues that could be mutated (Table 8), E49 from region 1, K225 from region 2, Q257 from region 3, and E340 from region 4 were identified as good candidates for mutation to cysteine. Variants of GpA369-C198 and GpA369(hum)-Group1+2+3-C198 containing one or more of the K225C, E340C, E49C, and Q257C mutations were generated (Table 9). [Table 9]

[0090] Each of the variants listed in Table 9 was recombinantly expressed, purified, and PEGylated with one or more of mPEG-10K, mPEG-20K, and mPEG-40K (Table 10). As determined by SDS-PAGE analysis, the size of the PEGylated enzyme increased with the number of active cysteines. For example, using maleimide-PEG10K, GpA369(hum)-C198A migrated at a higher molecular weight after PEGylation (due to the additional 10 KDa provided by the PEG), and GpA369(hum)-C198A+R52C migrated at an even higher apparent molecular weight because this variant was able to react with two PEG molecules (due to the additional 20 KDa total provided by the two 10 K molecules attached to the enzyme). Notably, the GpA369-C198A-K225C-E340C-E49C-Q257C mutant was prepared, expressed, purified, and PEGylated with mPEG-10K. However, PEGylation of this variant with mPEG-10K resulted in multiple products. The chaperone 60KDa was present in a 1:1 ratio with the protein, suggesting that the GpA369-C198A-K225C-E340C-E49C-Q257C mutant was not fully folded. [Table 10-1] [Table 10-2]

[0091] To further assess the PEGylation of the variants, various PEG:protein ratios were used. For this analysis, 2 mg / mL of GpA369-C198A was used along with 2x, 10x, and 20x excess m-PEG10K linear. Furthermore, analyses were performed to determine the effects of adding 5 mM beta-mercaptoethanol after the PEGylation reaction was complete and including additives such as 10 mM or 100 mM glycine or 10 mM aspartic acid during the PEGylation reaction. This analysis indicated that a 20:1 molar ratio of maleimide PEG:protein was required to ensure complete PEGylation of the GpA369 variants. Furthermore, the addition of beta-mercaptoethanol was found to provide a homogeneous product, while glycine or aspartic acid should be omitted from the PEGylation reaction.

[0092] In addition to linear maleimide-PEG10K, GpA369 variants were PEGylated using linear maleimide-PEG20K, linear maleimide-PEG40K, biantennary maleimide-PEG20K, four-arm maleimide-PEG10K, and Y-shaped maleimide-PEG40K. PEGylation with each of these different types of PEG was observed. Notably, the PEGylated variants showed a significant increase in L-asparaginase activity compared to the naked GpA369 variant (Table 10).

[0093] Example 5: Mutation of surface lysine residues for PEGylation Many conventional biologics are PEGylated on lysine residues, where the epsilon-amino group of the lysine side chain reacts with a PEG molecule. Lysine is a common amino acid present on the surface of proteins. Therefore, PEGylation using this strategy often results in a less uniform product with a variable number of PEG molecules attached to the protein.

[0094] To increase uniformity, lysine residues in GpA that are in close proximity to other lysine residues were replaced with residues that would not react with PEG. In addition, residues that could adversely affect the structural integrity, and therefore the enzymatic activity, of tetrameric L-asparaginase were replaced with surface lysine residues to fully and evenly protect the entire surface of the enzyme.

[0095] The truncated GpA variants listed in Table 11 with mutations using this strategy were considered useful for use in the present invention. [Table 11]

[0096] To demonstrate activity, stability, and PEGylation via lysine residues, the GpA369(hum)-Group 1+2+3-C198A-R54K+A91K+K223D+S311K variant was generated, recombinantly expressed, purified, and PEGylated using various PEG:protein ratios. This analysis indicated that a molar ratio of amine PEG to protein greater than 20:1 was required to ensure complete PEGylation of the GpA369 variant. It was also observed that additives such as DTT and glycerol should be omitted during the PEGylation reaction. Various sizes and linker types were tested, including methoxy PEG succinimidyl carbonate 10K (mPEG-SC-10K), mPEG-succinimidyl carboxymethyl ester 5K (mPEG-SCM-5K), and mPEG-succinimidyl succinimidyl ester 5K (mPEG-SAS-5K). Notably, none of the PEGylated products showed a loss in L-asparaginase activity compared to the naked version (Table 12). [Table 12]

[0097] Example 6: Tags for increasing in vivo half-life The effectiveness of L-asparaginase is related to the drug's in vivo half-life; the longer the half-life, the longer the enzyme can work to hydrolyze blood asparagine. Consequently, to increase the in vivo half-life of the L-asparaginase variants disclosed herein, a tag can be fused to the N-terminus of L-asparaginase. Such tags can include a histidine tag, a yeast SUMO tag; a human SUMO tag; a His6-human SUMO tag, where the SUMO tag can be one of the four homologous SUMO domains (SUMO-1, SUMO-2, SUMO-3, or SUMO-4) present in humans; and an albumin-binding peptide tag. Each tag can increase the circulation time of the L-asparaginase enzyme. In addition, a combination of tags can be used. In particular, SA21 and SUMO tags can be combined to obtain a variant with an even longer half-life.

[0098] Histidine tag. DNA sequences specifying a series of 6 to 9 histidine residues are frequently used in vectors for the production of recombinant proteins. The result is the expression of a recombinant protein with a 6xHis or poly-His tag fused to its N- or C-terminus.

[0099] Expressed His-tagged proteins can be purified and easily detected, thereby providing a means to specifically purify or detect recombinant proteins without protein-specific antibodies or probes. Kits are commercially available for His-tagged proteins.

[0100] SUMO Modification. SUMO as an N-terminal fusion partner has been found to enhance the production of functional proteins in prokaryotic and eukaryotic expression systems due to significantly improved protein stability and solubility.

[0101] Following expression and purification of the fusion protein, the SUMO tag can be cleaved in vitro by a specific (SUMO) protease via endopeptidase activity to generate the desired N-terminus of the released protein partner. SUMO tag expression systems are commercially available. In some embodiments, the SUMO tag is a yeast SUMO tag (e.g., Smt3 (SEQ ID NO: 66)). In other embodiments, the SUMO tag is a human SUMO tag (e.g., SUMO-1 (SEQ ID NO: 67), SUMO-2 (SEQ ID NO: 68), SUMO-3 (SEQ ID NO: 69), or SUMO-4 (SEQ ID NO: 70)).

[0102] His-SUMO modification. Combining a histidine (e.g., 1x-6xHis) tag with SUMO modification provides efficient purification, increased expression and solubility, and increased half-life of L-asparaginase. Expression systems for providing His-SUMO modification to proteins of interest are commercially available. See, for example, the CHAMPION pET SUMO protein expression system (Invitrogen).

[0103] Albumin-binding domain. A series of peptides that bind to serum albumin from multiple species have been identified using phage display (Dennis, at al. (2002) J. Biol. Chem. 277(38):35035-35043; US2016 / 0185874; and US2004 / 0001827). One of these peptides, designated SA21, was found to have an extended serum half-life. Exemplary albumin-binding peptides include, but are not limited to, SA20 (QRLIEDICLPRWGCLWEDDF; SEQ ID NO: 71), SA21 (RLIEDICLPRWGCLWEDD; SEQ ID NO: 72), and SA31 (RLIEDICLPRWGCLW; SEQ ID NO: 73). Fusing such domains to the L-asparaginase enzyme disclosed herein is expected to improve pharmacokinetics through non-covalent association with albumin. See Dennis, et al. (2002) J. Biol. Chem. 277(38):35035-35043; US2016 / 0185874; and US2004 / 0001827.

[0104] Exemplary truncated GpA variants with various tags are provided in Table 13. [Table 13-1] [Table 13-2]

[0105] Example 7: TRAIL-GpA fusion protein TRAIL (TNF-associated apoptosis containing ligand) is a protein that induces cell death by apoptosis. Consequently, TRAIL-asparaginase fusion proteins are created to combine the activities of these two proteins. Using this fusion protein, the L-asparaginase component signals cells to undergo apoptosis, and the TRAIL component induces cell death. Illustratively, three tandem TRAIL soluble domains (TRAILトリマー ) was truncated and humanized, GpA369(hum)-Group1+2+3 (SEQ ID NO: 47) or GpA369(hum)-Group1+2+3-loop1 hum (SEQ ID NO: 50) or TRAIL トリマー - GpA or GpA-TRAIL トリマー A fusion protein is produced.

[0106] The efficacy of the fusion protein is assessed by culturing human acute myeloid leukemia MV4;11 cells in the presence of final concentrations of the fusion protein ranging from 0.0001 to 2.5 IU / ml. After incubating the plates for 4 days at 37°C in a humidified atmosphere containing 5% CO2, Alamar Blue (Invitrogen) is added to a final concentration of 10% v / v, and the plates are incubated for an additional 4 hours, following which the fluorescent signal is read. The viability of leukemia cells is calculated as the percentage of fluorescent counts in the presence of L-asparaginase versus that in the DPBS control. This analysis will demonstrate that the active fusion protein has significantly better killing activity against the MV4;11 cell line compared to truncated, humanized guinea pig L-asparaginase alone.

[0107] The in vivo efficacy of the fusion protein in killing leukemia cells was evaluated in 4-10 6-week-old non-obese diabetic / severe combined immunodeficiency gamma (NSG) mice (The Jackson Laboratory) at 5x10 6 The fusion protein is assessed by injecting 150 μL of DPBS containing luciferase-positive MV4;11 cells. At specific time points, bioluminescence is measured using an IVIS Lumina II imaging system (PerkinElmer). After engraftment (day 0), mice are treated with 15 IU / mouse of the fusion protein via intraperitoneal injection daily for one week. Bioluminescence signals are measured on days 0 and 7. The results of this analysis will demonstrate a significant killing effect of MV4;11 cells by the fusion protein.

[0108] The efficacy of the fusion proteins against solid cancers, such as pancreatic and ovarian cancer, will also be determined. Pancreatic cancer cell lines, such as Panc-1 and MiaPaca2, and ovarian cancer cell lines, such as OVCAR3 and OVCAR4, will be treated with final concentrations ranging from 0.0001 to 2.5 IU / ml of the fusion proteins or GpA alone, or with TRAIL at the corresponding concentrations used for the fusion proteins. トリマー After incubating the plates for 4 days at 37°C in humidified air containing 5% CO2, Alamar Blue (Invitrogen) is added to a final concentration of 10% v / v, and the plates are incubated for an additional 4 hours, following which the fluorescent signal is read. Cancer cell viability is calculated as the percentage of fluorescent counts in the presence of the fusion protein versus that in the DPBS control. This analysis demonstrates that active fusion proteins inhibit the proliferation of TRAIL トリマー Or it may be shown to have significantly better killing activity against cancer cell lines compared to GpA alone.

Claims

1. A C-terminally truncated guinea pig L-asparaginase (GpA) variant sharing at least 90% sequence identity with residues 1-359 of SEQ ID NO:1; wherein the variant further comprises at least one amino acid substitution compared to SEQ ID NO:1; At least one amino acid substitution compared to SEQ ID NO: 1 below: (i) comprising Q23R, K25E, K48E, Q52R, Q54R, K98Q, E101K, S121F, G122A, H134Q, N233S, Q288E, E344D, L360P, T362S, A363V, D364E, L365E, H366R, Q367R, and S368P; or below: together with the substitutions defined in (i) above, (ii) H10R, D91A, D92E, Q108H, H236Q, and S250A; (iii) R147H, K193R, D217E, and R301Q; (iv) P57S, D58E, H59D, A60T, and A62V; and / or (v) C198A, Including; and At least one amino acid substitution compared to SEQ ID NO: 1 is one of the following: (a) a cysteine ​​residue at position 49, 52, 225, 257, 281, or 340, or a combination thereof; or (b) the variant, comprising a lysine residue at position 7, 53, 54, 58, 98, 106, 233, 257, 281, 311, or 340, or a combination thereof.

2. 2. The truncated GpA variant of claim 1, wherein the C-terminal truncation is between positions 359 and 396 of SEQ ID NO:

1.

3. 3. The truncated GpA variant of claim 1 or 2, wherein the C-terminal truncation is at position 369 of SEQ ID NO:

1.

4. The truncated GpA variant of any one of claims 1 to 3, wherein the variant has L-asparaginase activity equal to or greater than that of wild-type GpA.

5. A GpA variant consisting of the amino acid sequence of SEQ ID NO: 45, 46, 47, 48, 49, 50, 51, 54, 55, 56, 58, 59, 60, 61, 62, 64, 65, or 86.

6. The truncated GpA variant of claim 1 or 5, further comprising a histidine tag, a SUMO tag, an albumin binding domain, or a combination thereof.

7. 10. The truncated GpA variant of claim 1 or 5, further comprising three tandem soluble domains of TRAIL.

8. 8. The truncated GpA variant of claim 7, wherein the soluble domain of TRAIL comprises residues 115 to 281 of human TRAIL.

9. A nucleic acid molecule encoding the truncated GpA variant of claim 1 or 5.

10. An expression vector comprising the nucleic acid molecule of claim 9.

11. A host cell comprising the nucleic acid molecule of claim 9.

12. A pharmaceutical composition comprising a truncated GpA variant according to claim 1 or 5 and a pharmaceutically acceptable excipient.

13. 13. The pharmaceutical composition of claim 12, further comprising a stable form of TRAIL.

14. 14. The pharmaceutical composition of claim 13, wherein the stable form of TRAIL comprises the FOLDON sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 85).

15. The pharmaceutical composition according to any one of claims 12 to 14 for treating cancer.

16. 16. The pharmaceutical composition of claim 15, wherein the cancer is selected from non-Hodgkin's lymphoma, leukemia, acute lymphocytic leukemia, acute myeloid leukemia, B-cell lymphoma, Burkitt's lymphoma, chronic myelogenous leukemia, chronic lymphocytic leukemia, and hairy cell leukemia.

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