WO2023060230A2 - Engineered human thymidine phosphorylase and pharmacological preparations thereof - Google Patents

Abstract

A recombinant human thymidine phosphorylase (rHsTP) enzyme is described. The rHsTP includes one or more modifications relative to wild-type thymidine phosphorylase. The one or more modifications includes one or more chemical modifications, substitutions, insertions, deletions, and/or truncations. The rHsTP has a sequence similarity in a range of from 90% to 100% compared to SEQ ID NO:1.

Description

ENGINEERED HUMAN THYMIDINE PHOSPHORYLASE AND PHARMACOLOGICAL PREPARATIONS THEREOF

TECHNICAL FIELD

[0001] The present disclosure generally relates to compositions and formulations of recombinant Human thymidine phosphorylases (rHsTP).

BACKGROUND

[0002] Efficient recombinant production of proteins and more specifically of biologies plays a crucial role in the viability of a drug development process. Availability of large quantities of a protein of interest facilitates the activities associated with all the stages of the drug development trajectory including biochemical and biophysical characterization, downstream processing, manufacturing and formulation optimizations as well as pharmacological and toxicity assessments. Despite the immense progress in the field of molecular biology with the development of engineered E. coli strains and improved plasmids capable of expressing complex proteins of mammalian origins, the efficient expression of certain proteins can still be very challenging, which in turn, can have a profound impact on cost and time. Thus, achieving high expression levels of high-quality protein that retains its biological activity (binding and catalytic properties) represents an important early focus in the field of biologies. Similarly, the PEGylation of therapeutic proteins receives equal attention during the early phase of drug candidate selection and optimization. The conjugation of PEG to proteins has emerged as a powerful strategy to overcome significant limitations of protein pharmaceuticals such as limited in-vivo half-life and efficacy, thermodynamic and serum instability, immunogenicity.

[0003] Although alternative time-extension approaches have been recently developed (Despanie et al., 2016), (Haeckel et al., 2016), (Ding et al., 2014) and hold high promise, their clinical efficacy has not yet been demonstrated in case of large biomacromolecules such as proteins. In contrast, FDA has approved 24 PEGylated macromolecular drugs as of 2020 with 6 being enzymes (adenosine deaminase, asparaginase, uricase, phenylalanine ammonia lyase). Therefore, PEGylation still represents a very attractive approach for the enhancement of the pharmacological features of therapeutic proteins, and enzymes. However, the covalent attachment of PEG to the surface of enzymes (either targeting lysine or cysteine residues) often suffers by two main limitations: i) poor PEGylation efficiency due to the low number of surface exposed lysine residues, thereby resulting in a polydisperse population of species with distinct biochemical properties attributed to the different degrees of PEGylation and complicating the biochemical and biophysical characterization, and ii) conjugation of PEG can negatively impact the enzyme’s catalytic activity and thus, higher doses of a drug may be required to achieve a desirable pharmacological effect.

[0004] While the mature HsTP displays very promising catalytic activity against the toxic metabolites which drive the disease, its recombinant expression in E. coli was very poor, yielding ~ 5 mg/L of culture medium. Multiple attempts to improve the expression levels of the enzyme by scouting different conditions, E. coli strains and plasmids failed to benefit protein production. It is possible that protein expression in E. coli can be governed by several underlying mechanisms with the protein’s primary and secondary structure at the N’ -terminus and mRNA’s secondary structure being the most important factors (Wruck et al., 2017), (de Smit and van Duin, 1994).

SUMMARY

[0005] Embodiments of the present disclosure utilize modified human thymidine phosphorylase (“HsTP”) enzymes that have been engineered such that phosphorolysis (ie efficient degradation) of deoxythymidine (dThd) and deoxyuridine (dUrd) to 2-deoxyribose-l- phosphate provides a human therapy for MNGIE. Methods are described to produce proteins with deoxythymidine (dThd) and deoxyuridine (dUrd) catalytic activity that are soluble, stable, and can be used in vivo as well as in vitro. Some of the enzymes are based upon the native or wild-type amino acid sequence, whereas some are based on mutated sequences.

[0006] One aspect of the present disclosure is directed to a recombinant HsTP (“rHsTP”) enzyme. In some embodiments, the rHsTP enzyme comprises one or more modifications relative to wild-type HsTP. In some embodiments, the one or more modifications comprise one or more chemical modifications, substitutions, insertions, deletions, and/or truncations. In some embodiments, the rHsTP enzyme has a sequence similarity in a range of from 90% to 100% compared to SEQ ID NO:1.

[0007] In some embodiments, the rHsTP enzyme comprises a truncated rHsTP enzyme. In some embodiments, the truncated rHsTP enzyme comprises an amino acid sequence that is 90% to 100% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, ID NO: 6, SEQ ID NO: 11 or SEQ ID NO: 12.

[0008] In some embodiments, the rHsTP enzyme comprises an amino acid sequence that is identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, including any and all of the above in a range of from 90% to 100% similarity.

[0009] In some embodiments, the rHsTP enzyme comprises one or more amino acid residue mutations at K139, K275, R329, R342, R345, R358, and R453. In some embodiments, the rHsTP enzyme comprises one or more amino acid residue mutations K139R, K275R, R329K, R342K, R345K, R358K, and R453K.

[0010] In some embodiments, the rHsTP enzyme comprises one or more of an affinity tag, a linker peptide and a cleavage site. In some embodiments, the affinity tag comprises a HIS tag.

[0011] In some embodiments, the rHsTP enzyme comprises a catalytic efficiency (kcat/Kw) in a range of from 1 mM-ls-1 to 500 mM-ls-1. In some embodiments, the rHsTP enzyme comprises a catalytic efficiency (kcat/Kw) in a range of from 100 mM-ls-1 to 500 mM-ls-1. In some embodiments, the rHsTP enzyme comprises a catalytic efficiency (kcat/Kw) in a range of 450 mM-ls-1 to 750 mM-ls-1. In some embodiments, the rHsTP enzyme comprises a catalytic efficiency (kcat/Kw) in a range of 700 mM-ls-1 to 1,250 mM-ls-1. In some embodiments, the rHsTP enzyme comprises a catalytic efficiency (kcat/Kw) in a range of 1,200 mM-ls-1 to 2,500 mM-ls-1. In some embodiments, the rHsTP enzyme comprises a catalytic efficiency (kcat/Kw) in a range of 50 mM-ls-1 to 250 mM-ls-1. In some embodiments, the rHsTP enzyme comprises a catalytic efficiency (kcat/Kw) in a range of from in the range of 5 mM-ls-1 to 50 mM-ls-1. In some embodiments, the rHsTP enzyme comprises a catalytic efficiency (kcat/Kw) in a range of 0.1 mM-ls-1 to 2.5 mM-ls-1. In some embodiments, the rHsTP enzyme comprises a catalytic efficiency (kcat/Kw) in a range of 1,000 mM-ls-1 to 5,000 mM-ls-1. In some embodiments, the rHsTP enzyme comprises a catalytic efficiency (kcat/Kw) in a range of 200 mM-ls-1 to 3,000 mM-ls-1

[0012] In one or more embodiments, the calculated molecular weight of monomeric is about 49,955 Da. In one or more embodiments, rHsTP is a homodimer. In some embodiments, the rHsTP has an apparent molecular weight of 90,000 to 110,00 Daltons. [0013] In one or more embodiments, the average number of PEG is about 0.1 to about 10 moles of PEG/mole rHsTP monomer, such as about 0.5 to about 2.0 moles of PEG/mole rHsTP monomer. Exemplary amounts of PEG include about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.7, about 1.8, about 1.9 and about 2.0 moles of PEG/mole rHsTP monomer. In one or more embodiments, each PEG has an average molecular weight of about 1,000 to about 50,000 Daltons, such as about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 11,000, about 12,000, about 13,000, , about 14,000, about 15,000, about 16,000, about 17,000, about 18,000, about 19,000, about 20,000, about 21,000, about 22,000, about 23,000, about 24,000, about 25,000, about 26,000, about 27,000, about 28,000, about 29,000, about 30,000, about 31,000, about 32,000, about 33,000, about 34,000, about 35,000, about 36,000, about 37,000, about 38,000, about 39,000, about 40,000, about 41,000, about 42,000, about 43,000, about 44,000, about 45,000, about 46,000, about 47,000, about 48,000, about 49,000, or about 50,000 Daltons. In a particular embodiment, the average MW of the PEG is about 20,000 Daltons.

[0014] In some embodiments, the average molecular weight for rHsTP is approximately 100 kDa for the dimer.

[0015] In some embodiments, PEGylation reaction may be performed on the rHsTP. In one or more embodiments, amount of reactants, time, temperature and solution and reactant handling (such as mixing, addition rate, PEG handling) are important to produce consistent PEGylated product. In one embodiment, the PEGylation reaction on rHsTP is performed in a reaction buffer at pH 8.4. In one or more embodiments, the PEGylation reaction comprises reactant ratio, PEG (g) to rHsTP (g), is in the range of 4:1 to 1: 1. In one or more embodiments, the PEGylation reaction comprises reactant ratio, PEG (g) to Co-rhARGl (g), is about 5:1. In one or more embodiments, the PEGylation reaction is performed by misting PEG and rHsTP from about 5 minutes to about 300 minutes, about 10 minutes to about 300 minutes, about 20 minutes to about 300 minutes, about 30 minutes to about 300 minutes, about 5 minutes to about 280 minutes, about 10 minutes to about 280 minutes, about 20 minutes to about 280 minutes, about 30 minutes to about 280 minutes, about 5 minutes to about 260 minutes, about 10 minutes to about 260 minutes, about 20 minutes to about 260 minutes, about 30 minutes to about 260 minutes, about 5 minutes to about 240 minutes, about 10 minutes to about 240 minutes, about 20 minutes to about 240 minutes, about 30 minutes to about 240 minutes. In one or more embodiments, the PEGylation reaction is stopped by removing excess PEG and reducing pH of the reaction buffer. In some embodiments, the excess PEG is removed by filtration technique. In one or more embodiments, the pH is reduced by exchanging the reaction buffer with a storage buffer.

[0016] In one or more embodiments, the rHsTP is PEGylated at one or more lysine amino acid residues.

[0017] In one or more embodiments of the rHsTP is PEGylated in the range of about 15% to about 60%. In one or more embodiments of the rHsTP is PEGylated in the range of about 35% to about 80%. In one or more embodiments of the rHsTP is PEGylated in the range of about 20% to about 85%. In one or more embodiments of the rHsTP is PEGylated in the range of about 10% to about 60%. In one or more embodiments of the rHsTP is PEGylated in the range of about 10% to about 60%. In one or more embodiments the rHsTP is PEGylated in the range of about 40% to about 90%. In one or more embodiments of the rHsTP is PEGylated in the range of about 30% to about 95%. In one or more embodiments of the rHsTP is PEGylated in the range of about 30% to about 98%. In one or more embodiments of the rHsTP is PEGylated in the range of about 15% to about 65%. In one or more embodiments of the rHsTP PEGylated in the range of about 25% to about 70%. In one or more embodiments the rHsTP is PEGylated in the range of about 25% to about 85%. In one or more embodiments the rHsTP is PEGylated in the range of about 15% to about 65%. In one or more embodiments the rHsTP is PEGylated in the range of about 20% to about 75%. In one or more embodiments the rHsTP is PEGylated in the range of 0% to about 30%. In one or more embodiments the rHsTP is PEGylated in the range of 0% to about 35%. In one or more embodiments the rHsTP is PEGylated in the range of 0% to about 45%. In one or more embodiments the rHsTP is PEGylated in the range of 0% to about 45%.

[0018] PEG-Protein Molar ratio is an attribute indicative of the extent of PEGylation. In one or more embodiments, about 0.1 to about 2.0 moles of PEG has PEGaylated one mole of rHsTP. Exemplary range of molar ratio for PEG:rHsTP include 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4: 1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1: 1, 2.25:1, 2.5:1, 3.0:1 and 4.0:1. In some embodiments, the molar ratio of PEG:rHsTP is in the range of about 0.5 moles/mole to about 4.0 moles/mole. [0019] The free PEG is measured to demonstrate PEG clearance and stability. In some embodiments, free PEG concentration (pg) in PEGylated rHsTP (mL) is less than or equal to 500 pg/mL, less than or equal to 400 pg/mL, less than or equal to 300 pg/mL, less than or equal to 200 pg/mL, less than or equal to 100 pg/mL, and less than or equal to 50 pg/mL.

[0020] rHsTP catalyzes thymidine into 2-deoxyribose 1 -phosphate and thymine. The PEGylated drug substance, rHsTP-PEG, catalyzes the same reaction. The assay to assess enzyme activity measures the conversion of thymidine into 2-deoxyribose 1 -phosphate and thymine during a fixed reaction time. The amount of conversion of product is converted to a reaction rate and fit to the Michaelis-Menten equation to determine Km and kcat.

[0021] Vmax is the maximum reaction rate achieved at saturating substrate concentration; Km is the Michaelis-Menten binding constant to measure the substrate concentration yielding a velocity at the half of Vmax. The enzymatic turnover number, kcat is calculated by Vmax/[E].

[0022] In one or more embodiments, the protein (e.g. rHsTP or rHsTP-PEG) displays a kcat/KM greater than 25 mM-1 s-1 at pH 7.4. In a particular embodiment, the protein displays a kcat/KM in the range of about 250 mM-1 s-1 to about 5,000 mM-1 s-1 at pH 7.4. In another embodiment, the protein displays a kcat/KM in the range of about 40 mM-1 s-1 to about 250 mM-1 s-1 at pH 7.4 at 37° C. In a particular embodiment, the present invention contemplates a protein comprising an amino acid sequence of human rHsTP in a pharmaceutically acceptable carrier, wherein said protein exhibits a kcat/KM greater than 25 mM-1 s-1 at 37° C., pH 7.4. Exemplary kcat/KM values include about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 650, about 70, about 80, about 90, about 100, about 110, about 120, about 150, about 200, about 250, about 300, about 350, about 400, about 450 and about 500 mM-1 s-1 at pH 7.4 at 37° C, or any range in between these values.

[0023] The specific activity is an indication of potency of the protein (e.g. Co- rHsTP or rHsTP -PEG). In one or more embodiments, the specific activity of rHsTP-PEG is in the range of about 20 U/mg to about 1000 U/mg. Exemplary ranges of the specific activity include about 50 U/mg to about 1000 U/mg, about 100 U/mg to about 1000 U/mg, about 200 U/mg to about 1000 U/mg, about 250 U/mg to about 900 U/mg, about 300 U/mg to about 900 U/mg, about 400 U/mg to about 900 U/mg, about 200 U/mg to about 800 U/mg, about 300 U/mg to about 800 U/mg, about 400 U/mg to about 800 U/mg, about 200 U/mg to about 700 U/mg, about 300 U/mg to about 700 U/mg, and about 400 U/mg to about 700 U/mg. [0024] In one or more embodiments, the rHsTP, or rHsTP-PEG can have at least 98%, 98.5%, 99% or 99.5% identity to SEQ ID NO: 1. In one or more embodiments, rHsTP, or rHsTP-PEG can have at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more deletions, substitutions and/or insertions to the amino acid sequence described by SEQ ID NO: 1. Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/). In one or more embodiments, the rHsTP, or rHsTP-PEG has at least one amino acid substitution.

[0025] In some embodiments, the rHsTP enzyme comprises PEGylated rHsTP enzyme. In some embodiments, the PEGylated rHsTP enzyme comprises 1 to 20 PEGylated amino acid residues per rHsTP monomer. In some embodiments, the rHsTP enzyme is PEGylated by a branched PEG or a linear PEG. In some embodiments, the PEGylated rHsTP enzyme has non- site-specific PEGylation. In some embodiments, the PEGylation chemistry preferentially PEGylates lysine residues. In some embodiments, the PEGylation chemistry preferentially PEGylates arginine residues. In some embodiments, the PEGylated rHsTP enzyme has a sitespecific PEGylation. In some embodiments, the specific site for PEGylation is chosen based on factors comprising one or more of accessibility of the amino acid at the surface of the molecule, non-essential role of the amino acid in structural function, mutations which cause rHsTP in humans, proximity to disease causing mutations, proximity to the active site residues, proximity to the dimer-dimer interface, and a degree of amino acid conservation across different species. In some embodiments, the site- specific PEGylation is cysteine directed.

[0026] Another aspect of the disclosure is directed to a method of preventing or treating MNGIE. In some embodiments, the method comprises administering a pharmaceutical composition or formulation comprising the rHsTP enzyme to a subject in need thereof, wherein the rHsTP enzyme is according to any of the embodiments described herein. In some embodiments, the subject comprises human, mice, rat, rabbit or monkey. In some embodiments, the subject is a human. In some embodiments, the pharmaceutical composition or formulation comprises buffers containing any of citrate, phosphate, and acetate. In some embodiments, the pharmaceutical composition or formulation contains additional stabilizing additives including but not limited to NaCl, sulfate, arginine, sucrose, dextrose, sorbitol, and/or glycine. In some embodiments, liquid formulations combine the rHsTP enzyme with compounds to enhance a stable active medication following storage. These include solubilizers, stabilizers, buffers, tonicity modifiers, bulking agents, viscosity enhancer s/reducers, surfactants, chelating agents, and adjuvants. In some embodiments, the stabilizing additive comprises NaCl. Stability during the freeze/thaw cycle is an important characteristic to facilitate batch purification in a stepwise manner because the drug substance can be pooled and stored until a subsequent purification or filling step is performed.

[0027] In some embodiments, the invention relates to erythrocytes containing rHsTP. Some embodiments further relate to a suspension of erythrocytes containing rHsTP in a pharmaceutically acceptable solution (for example a solution containing NaCl and one or more ingredients selected from glucose, dextrose, adenine and mannitol; e.g. SAG-mannitol or ADsol). Said solution can provide preservation of the erythrocytes, and it can include a preservative such as L-carnitine. Said suspension can be packaged ready for use or for dilution before use. The final hematocrit value of the ready-to-use product (after dilution before use, if necessary) is preferably between 40 and 70%. It can be administered intravenously, preferably by perfusion.

[0028] A suspension of rHsTP-containing erythrocytes or any administrable formulation containing the erythrocytes according to the invention constitutes in itself a medicinal product or a pharmaceutical composition covered by the invention. Said medicinal product or composition can notably be intended for the various applications. It can be packaged for example as a flexible bag for perfusion, or in some other form for administration by injection.

[0029] In some embodiments, the pharmaceutical composition is administered intravenously, intradermally, intraarterially, intraperitoneally, intramuscularly, subcutaneously, by infusion, by continuous infusion, via a catheter, or in lipid composition. In some embodiments, the method further comprises combining administration of rHsTP with an additional therapy.

[0030] Another aspect of the disclosure is directed to a method of determining enzymic activity of the rHsTP enzyme, wherein the rHsTP enzyme is according to any of the embodiments described herein. In some embodiments, the method comprises incubating the rHsTP enzyme with different concentrations of substrate, spectroscopically measuring absorbance, and determining the Km and Kcat by plotting Michaelis-Menten curve. Another aspect of the disclosure is directed to a method of determining serum stability of the rHsTP enzyme, wherein the rHsTP enzyme is according to any of the embodiments described herein. In some embodiments, the method comprises reacting the rHsTP enzyme with substrate in serum for a predetermined time, quenching the reaction and determining the enzyme activity over time in serum.

[0031] Another aspect of the disclosure is directed to a method of making a rHsTP enzyme, wherein the rHsTP enzyme is according to any of the embodiments described herein. In some embodiments, the method comprises expressing the rHsTP in E. coli cells, lysing the cells using sonication in lysis buffer consisting of 50 mM Na2HPC>4, 300 mM NaCl, 10 mM Imidazole, ImM PMSF protease inhibitors, IpL of Img/mL DNase I for each mL of cell suspension, pH 8. After sonication, the lysate containing rHsTP can be clarified using centrifuged at 12,000xg for 1 hour. In some embodiments, the method further comprises applying the lysate to a NiNTA resin, washing the resin with 50 mM Na2HPO4, 300 mM NaCl, 20 mM Imidazole (pH 8), then eluting the purified rHsTP with 50 mM Na2HPC>4, 300 mM NaCl, 300 mM Imidazole, pH 8). In some embodiments, the rHsTP can be purified using anion exchange (capture) chromatography (for example Q-sepharose). In some embodiments, the rHsTP is applied to a Q-FF resin that is equilibrated with 20 mM Tris-Cl, 20 mM NaCl, pH 7.5, the column washed, then rHsTP eluted with a 50-500 mM NaCl gradient. Purified rHsTP can be used immediately or mixed with 15% (v/v) final glycerol concentration and stored at - 80 °C for future use.

[0032] In some embodiments, the method further comprises exchanging buffer of the purified rHsTP enzyme to a PEGylation buffer, incubating the purified rHsTP enzyme with a PEGylating agent for a predetermined time at a predetermined temperature. In some embodiments, the PEGylation buffer is exchanged with a storage buffer. In some embodiments, the PEGylation buffer comprises 100 mM Na2HPO4, pH 8.5. In some embodiments the PEGylation buffer comprises 50 mM Na2HPO4, 50 mM NaCl, pH 8.0 and the final enzyme concentration is 200 pM. In some embodiments, the storage buffer comprises 50 mM Na2HPO4, 50 mM NaCl, pH 7.0, with 15% glycerol. In some embodiments, the PEGylating agent comprises polyethylene glycol (PEG) and a conjugating agent. In some embodiments, the PEG comprises by a branched PEG or a linear PEG. In some embodiments, the PEG comprises a molecular weight in a range of from 2,000 kDa to 20,000 kDa. In some embodiments, the conjugating agent comprises Methoxy Succinimidyl Carboxymethyl Ester. In some embodiments, the conjugation reaction contains methoxy-5-kDa-PEG-succinimidyl- succinate. In some embodiments, the conjugating agent comprises Methoxy Maleimide. In some embodiments, the purified rHsTP enzyme is incubated with the PEGylating agent at a molar ratio (proteimPEG ratio) in a range of from 1:10 to 1:50. In some embodiments, the predetermined temperature is in a range of from 4 °C to 37 °C. In some embodiments, the predetermined time is in a range of from 15 minutes to 24 hours.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Fig. 1(a) shows the primary amino acid sequence of native human Thymidine Phosphorylase (HsTP) (SEQ ID NO: 1)

[0034] Fig. 1(b) shows the primary amino acid sequence of human Thymidine Phosphorylase construct HsTP¹⁹⁹ containing an N-terminal truncation and an N-terminal polyhistidine tag. (SEQ ID NO: 2)

[0035] Fig. 1(c) shows the primary amino acid sequence of human Thymidine Phosphorylase construct HsTP²⁰⁰ containing an N-terminal polyhistidine tag. The sequence of this TP is also known as isoform 2. (SEQ ID NOG)

[0036] Fig. 1(d) shows the primary amino acid sequence of human Thymidine Phosphorylase construct HsTP²¹⁶ containing an N-terminal truncation and a C-terminal polyhistidine tag. (SEQ ID NO:4)

[0037] Fig. 1(e) shows the primary amino acid sequence of human Thymidine Phosphorylase construct HsTP²¹⁷ containing an N-terminal truncation and a C-terminal polyhistidine tag. (SEQ ID NOG)

[0038] Fig. 1(f) shows the primary amino acid sequence of human Thymidine Phosphorylase construct HsTP²¹⁸ containing an N-terminal truncation and a C-terminal polyhistidine tag. (SEQ ID NOG)

[0039] Fig. 1(g) shows the primary amino acid sequence of E.coli Thymidine Phosphorylase construct HsTP²⁰¹ containing an N-terminal polyhistidine tag. (SEQ ID NO:7) [0040] Fig. 1(h) shows the primary amino acid sequence of human Thymidine Phosphorylase HsTP²¹⁵ containing a C-terminal polyhistidine tag (SEQ ID NOG)

[0041] Fig. l(i) shows the primary amino acid sequence of human Thymidine Phosphorylase HsTP²¹⁷ containing an N-terminal deletion and a C-terminal polyhistidine tag. (SEQ ID NO: 9) [0042] Fig. l(j) shows the primary amino acid sequence of human Thymidine Phosphorylase HsTP²¹⁸ containing an N-terminal deletion and a C-terminal polyhistidine tag. (SEQ ID NO: 10)

[0043] Fig. l(k) shows the primary amino acid sequence of human Thymidine Phosphorylase construct HsTP²⁴⁰ containing K139R-K275R-R342K-R345K-R358K mutations as well as a C-terminal polyhistidine tag. (SEQ ID NO: 11)

[0044] Fig. 1(1) shows the primary amino acid sequence of human Thymidine Phosphorylase construct HsTP²⁴¹ containing K139R-K275R-R329K-R342K-R345K-R358K- R453K and a C-terminal polyhistidine tag. (SEQ ID NO: 12)

[0045] Fig. 2. The crystal structures and the constructs of native HsTP and EcTP. (A) Overlaid crystal structures of HsTP (PDB: 2WK6) and EcTP (PDB entry 4LHM) shown in cyan and orange color respectively. Dashed arrow indicates the N’ -terminus of each enzyme. (B) Zoomed, overlaid a-helices located at the N’ -terminus of HsTP (cyan) and EcTP (orange). Q35 residue shown in red sticks is the first amino acid residue that is structurally-resolved in case of HsTP, with F2 and L3 (shown in orange sticks) occupying the respective structurally- equivalent positions of EcTP. (C) N’ -terminal domain of HsTP. Unstructured region without electron density is shown as circles, where each circle represents the respective amino acid residue. Pro-peptide corresponds to the first ten residues and are shown in yellow. Residues Al l, S19 and K34 are shown in orange, green and dark pink and represent the positions at which the HsTP constructs were truncated (constructs were truncated at the residue preceding Al l, S19 and K34). (D) Cartoon representation of all the HsTP and EcTP constructs tested in the present study. The position of His6-tag as well as the truncation positions are also shown. Starting methionine and a glycine residue (result of the cloning process as described) are preceding the starting position (Al l, S19, K34, F2) of all constructs shown in this panel.

[0046] Fig. 3. SDS-PAGE analysis of HsTP constructs. Lane 1: pET28a-HsTP199- whole cell lysate; Lane 2: pET28a-HsTP199-soluble fraction; lane 3: pJC20-HsTP199-whole cell lysate; lane 4: pJC20-HsTP199-soluble fraction; lane 5: pET28a-HsTP215-whole cell lysate; lane 6: pET28a-HsTP215-soluble fraction; lane 7: pET28a-HsTP216-whole cell lysate; lane 8: pET28a-HsTP216-soluble fraction; lane 9: pET28a-HsTP217-whole cell lysate; lane 10: pET28a-HsTP217-soluble fraction; lane 11: pET28a-HsTP218-whole cell lysate; lane 12: pET28a-HsTP218-soluble fraction. Red square in lanes 11 and 12 shows the overexpression of pET28a-HsTP218 which is predominantly detected in the insoluble fraction. Expression of all constructs was induced with 0.5 mM IPTG in TB medium (at OD600- 1) followed by overnight incubation at 30 °C. For more details about the expression conditions, see Methods section.

[0047] Fig. 4. Two-step purification of HsTP218. Lane 1 shows HsTP218 eluted from Ni²⁺ agarose beads and lanes 2, 3 and 4 display representative elutions from the Q-Sepharose anion exchange chromatography step at 100, 125 and 150 mM NaCl respectively. ~10 pg of protein was loaded in each lane.

[0048] Fig. 5. mPEG5kDa-conjugation reaction with HsTP¹⁹⁹. (A) Scheme of the conjugation reaction of HsTP¹⁹⁹ with mPEG5kDa. The N-hydroxysuccinimide ester (NHS ester) reactive group of mPEG5kDa reacts with primary amines (lysine side group and N’- terminus amino-group) leading to the final conjugation adduct with methoxy-PEG and the simultaneous liberation of NHS. (B) Lane 1: Purified HsTP¹⁹⁹ (2-step purification including IMAC and anion exchange as described in Methods section) used for PEGylation; lanes 2 and 3 show mPEG5kDa-conjugated HsTP218 at 1:20 and 1:30 protein :mPEG5kDa ratio respectively. Each lane contains ~15 pg of protein.

[0049] Fig. 6. Crystal structure of HsTP homodimer (PDB: 2WK6). (A) Subunits A and B are shown in cyan and green respectively. All lysine residues of subunit A are shown as sticks. Buried lysine residues characterized by very low probability for PEGylation are colored as salmon red whereas four, more surface exposed lysine residues (K43, K139, K253, K275) with higher likelihood for conjugation are shown in red. (B) Surface representation of HsTP’s crystal structure. Monomers and lysine residues are colored as in panel A. K43, K139, K253 and K275 are labeled and their exposed molecular surface is shown in red dots.

[0050] Fig. 7. Spatial distribution of arginine residues mapped on the crystal structure of HsTP (PDB: 2WK6). (A) All arginine residues from subunit A (cyan color) are shown in magenta sticks. (B) Positions of arginine residues R329, R342, R345, R358 and R453 which satisfied our selection criteria and were substituted with lysine. (C) Surface representation of HsTP (at 20% transparency) highlighting the exposed molecular surface of the five arginine residues (magenta dots) shown in previous panel B.

[0051] Fig. 8. PEGylation efficiency assessment by SDS-PAGE of HsTP²¹⁸, HsTP²⁴⁰ and HsTP²⁴¹. Lanes 1-2-3, 4-5-6 and 7-8-9 show the HsTP²¹⁸, HsTP²⁴⁰ and HsTP²⁴¹ prior to and after mPEG5kDa-conjugation at 1:10 and 1:20 proteimPEG molar ratio respectively. Approximately 15 pg of protein was loaded in each lane. [0052] Fig. 9. SDS-PAGE of the pET28a-HsTP¹⁹⁹ expression tests in BL21(DE3) and C41(DE3). Lanes 1-2: BL21(DE3) whole-cell lysate and soluble fractions respectively of pET28a-HsTP¹⁹⁹ at 22 °C; lanes 3-4: BL21(DE3) whole-cell lysate and soluble fractions respectively of pET28a-HsTP¹⁹⁹ at 32 °C; lanes 5-6: C41(DE3) whole-cell lysate and soluble fractions respectively of pET28a-HsTP¹⁹⁹ at 22 °C and lanes 7-8: C41(DE3) whole-cell lysate and soluble fractions respectively of pET28a-HsTP¹⁹⁹ at 32 °C. Approximately 15 pg of protein was loaded in each lane.

[0053] Fig. 10. Immunoblotting analysis of the pET28a-HsTP¹⁹⁹ expression tests in BL21(DE3) and C41(DE3). The order of the samples is the same as in Figure 9 above. Lanes 1-2: BL21(DE3) whole-cell lysate and soluble fractions respectively of pET28a-HsTP¹⁹⁹ at 22 °C; lanes 3-4: BL21(DE3) whole-cell lysate and soluble fractions respectively of pET28a- HsTP¹⁹⁹ at 32 °C; lanes 5-6: C41(DE3) whole-cell lysate and soluble fractions respectively of pET28a-HsTP¹⁹⁹ at 22 °C and lanes 7-8: C41(DE3) whole-cell lysate and soluble fractions respectively of pET28a-HsTP¹⁹⁹ at 32 °C.

[0054] Fig. 11. Recombinant expression comparison of pET28a-HsTP¹⁹⁹ and pJC20- HsTP¹⁹⁹ in BL21(DE3). Lanes 1-2-3-4: pJC20-HsTP¹⁹⁹ whole-cell lysate, soluble fraction, flow-through from IMAC and IMAC elution respectively; lanes 5-6-7-8: pET28a-HsTP¹⁹⁹ whole-cell lysate, soluble fraction, flow-through from IMAC and IMAC elution respectively. Each lane contains ~10 pg of protein.

[0055] Fig. 12. Recombinant expression assessment of HsTP²¹⁸ in BL21(DE3) at 16 and 22 °C. Lanes 1 -2-3-4: pET28a-HsTP²¹⁸ whole-cell lysate, soluble fraction, flow-through from IMAC and IMAC elution respectively at 16 °C; lanes 5-6-7-8: pET28a-HsTP²¹⁸ wholecell lysate, soluble fraction, flow-through from IMAC and IMAC elution respectively at 22 °C.

[0056] Fig. 13. Analytical size-exclusion chromatography analysis of HsTP²¹⁸. (A) standard gel filtration marker (Bio-Rad catalog number 151-1901). (B) Size-exclusion chromatography profile of HsTP218 eluting at 38.94 minutes.

[0057] Fig. 14. Surface-exposed arginine residues of HsTP that were mutated to lysine. (A) Crystal structure of HsTP showing in magenta sticks the arginine residues that were substituted with lysine along with their level of conservation as assessed by amino acid sequence alignment of eukaryotic and prokaryotic thymidine phosphorylase enzymes. Subunits A and B are colored in cyan and green respectively. The bound substrate 5-iodouracil (5IUR) is also shown as green sticks in the active site of each subunit. (B) Calculated solvent- accessible surface area (SASA) in A for each of the arginine residues that were selected for lysine substitution as well as for the two lysine residues that were mutated to arginine. Values were calculated with PyMol.

[0058] Fig. 15. mPEG5kDa-conjugation of EcTP and the impact on its catalytic activity against dThd. (A) Lysine residues shown as red sticks mapped on EcTP’s subunit A (PDB entry 4LHM). (B) HsTP¹⁹⁹ and EcTP prior and post-PEGylation. Lanes 1-2-3: HsTP¹⁹⁹ after 2-step purification (IMAC and Q-column anion exchange), PEGylated HsTP¹⁹⁹ at 1:10 molar ratio of proteimPEG, PEGylated HsTP¹⁹⁹ at 1:30 molar ratio of proteimPEG; lanes 4-5- 6: EcTP after single IMAC purification step, PEGylated EcTP at 1 :10 molar ratio of proteimPEG, PEGylated EcTP at 1:30 molar ratio of proteimPEG. (C) Steady-state kinetic parameters of EcTP against dThd prior and post-PEGylation. Assays were performed in assay buffer consisting of 20 mM Hepes, 50 mM KH2PO4, pH 7.5 at the indicated temperature.

DETAILED DESCRIPTION

[0059] Human thymidine phosphorylase catalyzes the reversible phosphorolysis of deoxythymidine (dThd) and deoxyuridine (dUrd) to 2-deoxyribose-l -phosphate, is encoded by the TYMP gene, and forms a homodimer. PEGylation of thymidine phosphorylase extends the circulating half-life significantly. Although specific reference is made herein to thymidine phosphorylase, the methods, formulations and uses described herein can also be applied to variants of the native human enzyme that retain enzymatic activity.

[0060] Definitions

[0061] As used in the specification, “a” or “an” may mean one or more. As used in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.

[0062] As used here, the term “about” is understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. Generally, about encompasses a range of values that are plus/minus 10% of a referenced value.

[0063] The terms “enzyme” and “protein” and “polypeptide” refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably.

[0064] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0065] The term “expression vector” refers to any type of genetic construct comprising a nucleic acid coding for an RNA capable of being transcribed and translated. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions.

[0066] The term “fusion protein” refers to a chimeric protein containing proteins or protein fragments operably linked in a non-native way.

[0067] The term “gene” refers to a DNA sequence that comprises control and coding sequences necessary to produce a polypeptide or precursor thereof. The polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence so as the desired enzymatic activity is retained.

[0068] As used herein, the term “half-life” (Vi-life) refers to the time taken for the activity of an enzyme to reduce by half, either in vitro or in vivo. Methods to measure “halflife” include determining the catalytic activity of the enzyme as a function of time by any assay that detects the production of any substrates resulting from conversion of, for example, deoxythymidine (dThd) such as the detection of the reaction product(s) following conversion of deoxythymidine (dThd) to 2-deoxyribose-l -phosphate. An alternative expression of half-life is the time that required for the concentration of a polypeptide to reduce by half in vitro (i.e., as measured in the cell culture media) or in vivo (i.e., as measured in serum), for example, after injection in a mammal. Methods to measure “half-life” include the use of antibodies specific for rHsTP or PEG used in an ELISA format such that the physical amount of protein measured as a function of time.

[0069] The term “HIS tagged” or “polyhistidine tag” or “His6” refers to consecutive histidine residues introduced into a protein sequence. The HIS tag includes constructs with six consecutive histidine residues which facilitates purification of the resulting tagged protein via affinity with a metal ion immobilized on a solid matrix. The metal ion containing matrix can be referred to as Immobilized Metal Affinity Chromatography. The term “N-terminal HIS tagged” refers to a protein wherein the histidine residues are introduced at, or near, the N-terminus of the protein. “HIS tagged HsTP” refers to an rHsTP protein (or its equivalent DNA construct) which as the histidine residues incorporated into the HsTP enzyme.

[0070] The term “KM” refers to the Michaelis-Menten constant for an enzyme and is defined as the concentration of the specific substrate at which a given enzyme yields one -half its maximum velocity in an enzyme catalyzed reaction. The term “k_ca ” refers to the turnover number or the number of substrate molecules each enzyme site converts to product per unit time, and in which the enzyme is working at maximum efficiency. The term “k_catlKM” as used herein is the specificity constant, which is a measure of how efficiently an enzyme converts a substrate into product.

[0071] The term “linker” refers to peptide that is spliced between the polypeptides connected in tandem. Production of fusion proteins that recover the functional activities of the parent proteins may be facilitated by connecting genes with a bridging DNA segment encoding the peptide linker. The linker would be of sufficient length to allow proper folding of the resulting fusion protein.

[0072] Mitochondrial neurogastrointestinal encephalopathy syndrome (MNGIE) is caused by a deficiency in the thymidine phosphorylase enzyme. Alternative names for MNGIE include Myoneurogastrointestinal encephalopathy syndrome, MNGIE syndrome, Oculogastrointestinal muscular dystrophy; OGIMD; Polyneuropathy, ophthalmoplegia, leukoencephalopathy, and intestinal pseudo-obstruction; POLIP; and Thymidine phosphorylase deficiency.

[0073] The term “native” refers to the typical or wild-type form of a gene, a gene product (such as RNA, a protein, or an enzyme), or a characteristic of that gene or gene product when isolated from a naturally occurring source. In contrast, the term “modified,” “variant,” “mutein,” or “mutant” refers to a gene or gene product that displays modification in sequence and functional properties (i.e., altered characteristics) when compared to the native gene or gene product, wherein the modified gene or gene product is genetically engineered and not naturally present or occurring. [0074] The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” may mean at least a second or more.

[0075] The term “PEGylated” refers to conjugation with polyethylene glycol (PEG). PEG can be coupled (e.g., covalently linked) to active agents through a variety of chemistries including through the hydroxy groups at the end of the PEG chain. PEG can be coupled (e.g., covalently linked) to active agents through the hydroxy groups at the end of the PEG chain via chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids have been explored as novel biomaterial that would retain the biocompatibility of PEG, but that would have the added advantage of numerous attachment points per molecule (thus providing greater drug loading), and that can be synthetically designed to suit a variety of applications.

[0076] The term “recombinant” refers to any of a variety of techniques, and products generated from using those techniques, for separating and recombining segments of nucleic acids, DNA, or genes, often employing a gene of interest from a donor organism (for example a mammal or human), and combining with a different DNA such as a plasmid or viral DNA for transplantation into a host organism, wherein this process causes the production of a desired substance or gene product such as a protein for harvesting or for benefit of the host organism itself.

[0077] As used herein, the term “rHsTP” refers to the human Thymidine Phosphorylase enzymes or variants thereof. The rHsTP may be produced using any variety of techniques including but not limited to recombinant technology.

[0078] As used herein, “SASA” refers to the solvent-accessible surface area of a molecule. This includes the surface area of a biomolecule that is accessible to a solvent. Measurement of SASA is typically described in units of square Angstroms (A ).

[0079] The term “Thymidine Phosphorylase” (TP) refers to the enzyme that catalyzes the conversion of thymidine and 2'-deoxyuridine to their respective bases and 2-a-d- deoxyribose-1 -phosphate. The Enzyme Commission number for Thymidine phosphorylase is EC 2.4.2.4. Synonyms for Thymidine phosphorylase include TP, EC 2.4.2.4, Gliostatin, Platelet-derived endothelial cell growth factor, PD-ECGF, and TdRPase. [0080] The terms "treatment" and "treating" refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject to obtain a therapeutic benefit of a disease or health-related condition. For example, treatment includes administration of a therapeutically effective amount of a modified HsTP enzyme.

[0081] The term “vector” is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be “exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques.

[0082] Thymidine phosphorylases (E.C. 2.4.2.4.) are enzymes that play a pivotal role in the salvage pathway of pyrimidine nucleoside metabolism in both lower and higher organisms (Desgranges et al., 1981) (Okuyama et al., 1996) (Pugmire and Ealick, 2002). They catalyze the reversible phosphorolysis of deoxythymidine (dThd) and deoxyuridine (dUrd) to 2-deoxyribose-l -phosphate and to their respective bases with the latter being fluxed towards nucleotide synthesis.

[0083] Humans have two cytosolic pyrimidine nucleoside phosphorylases, namely thymidine phosphorylase (HsTP; encoded by TYMP gene) and uridine phosphorylase (HsUP; encoded by UPP1 and UPP2 genes) that have totally distinct structural and biochemical features (Johansson, 2003) (Norman et al., 2004). HsTP does not show any significant amino acid sequence similarity against any of the HsUP enzymes whereas HsUPl and HsUP2 share 66% sequence identity between them. HsTP is a highly specific N’-ribosyl phosphorylase displaying k_cat/KM values against dThd and dUrd in the range of 10⁵ M’¹ s’¹ and 10⁴ M’¹ s’¹ respectively (Deves et al., 2014) (Schwartz et al., 2010) , whereas UP enzymes exhibit higher specificity against uridine (Urd) (k_cat/KM ~10⁵ M’¹ s’¹) and to a lesser extend towards dUrd and dThd (k_cat/KM ~10³ M’¹ s’¹) (Liu et al., 1998) (Renck et al., 2010). Thymidine phosphorylases from lower organisms like Bacillus stearothermophilus (BsTP) and Escherichia coli (EcTP) exhibit similar fold and belong to the same family (family II) of nucleoside phosphorylases with HsTP while their amino acid sequence identity is in the range of -40%. Particularly, EcTP is highly active against dThd showing a k_cat/KM ~10⁶ M ¹ s ¹ (Panova et al., 2008) (Gbaj et al., 2006).

[0084] HsTP can have substantial implication in tumor cell growth (Matsushita et al., 1999) (Moghaddam et al., 1995). The platelet-derived endothelial cell growth factor (PD- ECGF) is identical to HsTP and is involved in angiogenic and endothelial cell chemotactic activities (Sumizawa et al., 1993). Overexpression of HsTP and elevated enzyme activity can be detected and measured in plasma of cancer patients that may be associated with invasive and metastatic profiles of various cancer types including bladder, colorectal, ovarian and pancreatic tumors (Pauly et al., 1977) (Takebayashi et al., 1996a) (Takebayashi et al., 1996b) (Reynolds et al., 1994). HsTP can degrade the anti-viral drug trifluorothymidine (TFT) as well as other fluoropyrimidines and chemotherapeutic agents (Patterson et al., 1995). HsTP’s kinetic properties and the elucidation of its catalytic mechanism (Schwartz et al., 2010), (Birck and Schramm, 2004), (Oh and el Kouni, 2018) may help developing potent inhibitors (de Moura Sperotto et al., 2019), (Perez-Perez et al., 2005). The inhibitor 5-chloro-6-[l-(2- iminopyrrolidinyl) methyl] uracil hydrochloride (TPI) is a potent HsTP inhibitors (Ki ~ 20 nM) and can cause considerable reduction in tumor growth in mice (Matsushita et al., 1999).

[0085] HsTP may contribute to rare metabolic diseases. Mutations in the HsTP gene (TYMP) which may result in partial or complete loss of enzymatic activity, cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) which is an autosomal recessive disease (Slama et al., 2005). HsTP deficiency may lead to elevated systemic levels of dThd and dUrd and patients with MNGIE may suffer by neurological and gastrointestinal symptoms (Hirano, 1993), (Teitelbaum et al., 2002). The underlying mechanism for all those symptoms and manifestations may be associated with a disorder known as mitochondrial DNA (mtDNA) depletion syndrome (Pacitti et al., 2018), (El-Hattab and Scaglia, 2013). That is, build-up of dThd and dUrd may lead to an imbalance of nucleotide levels which, in turn, negatively affects the mitochondrial DNA replication that relies on the cytoplasmic pool of nucleotides for proper function. Ultimately, this nucleotide imbalance may lead to mtDNA instability which may be responsible for the neurological and gastrointestinal manifestations of MNGIE. Current management of MNGIE has been primarily supportive with the main goal being to ease the disease manifestations. Thus, MNGIE therapy represents an unmet medical need that requires further attention for the development of effective treatments.

[0086] Cloning of HsTP and EcTP genes [0087] Amino acid sequences for wild-type HsTP and EcTP were codon-optimized for E. coli and gene blocks with the resultant sequences were used as a template for polymerase chain reaction (PCR) amplification (See Table 1). Primers were designed to amplify the HsTP and EcTP gene fragments as follows: the 5’ primer introduced a His6-tag at the N-terminus and an Ncol restriction site; the 3’ primer introduced an EcoRI restriction site. The amplicons were digested with Ncol and EcoRI, gel purified, ligated into pET28a (Novagen) with T4 DNA ligase and transformed into MCI 061 cells (Lucigen). Primers used to generate the variants in this study are provided in Table 1 below. Finally, inserts were sequence-verified by Sanger sequencing. [0088] Table 1: 5’ and 3’ primers used to generate codon-optimized genes for expression in E. coli

[0089] Expression and purification of HsTP and EcTP constructs

[0090] Expression

[0091] Protein synthesis can be initiated by either methionine in eukaryotes or formylmethionine in prokaryotes, mitochondria, and chloroplasts (Wingfield, 2017). The N- terminal methionine can be co-translationally cleaved by the enzyme methionine aminopeptidase (MAP). In eukaryotes, methionine can be removed either by cleavage of N- terminal signal peptide used for secretion etc., or by MAP. In prokaryotes, formylmethionine can be first removed by formylmethionine deformylase resulting in N-terminal methionine which is then processed by MAP (Wingfield, 2017).

[0092] When recombinant proteins are expressed, especially in E. coli, the N-terminal methionine can be retained regardless, possibly due to saturation of MAP, depletion of required cofactors, or other factors. It is also possible for recombinantly expressed proteins to have ragged N-termini resulting from additional processing beyond N-methionine by other proteases. Thus, regardless of which expression host is used, the N-terminal methionine can be retained, excised, or additional processing by other proteases further truncates the N-terminus of the enzyme. These further truncations are within the scope of the HsTP constructs disclosed herein. It is also within the scope of this disclosure that these additional truncations are engineered into the constructs for expression in a host cell.

[0093] HsTP and EcTP constructs either in BL21(DE3) or C41(DE3) cells were cultured in terrific broth media supplemented with kanamycin at 37 °C until OD at 600 nm reached between and 0.8 and 1 units when standard shake flasks were used, or OD of 5 when ultra-yield flasks (Thompson) were used. Then, for HsTP¹⁹⁹ and EcTP, 0.5 mM IPTG was added, temperature was reduced to 30°C, and expression allowed to proceed for 20-22 hours. Expression optimized variants, including HsTP²¹⁸, were induced with 0.1 mM IPTG and expressed at 16 °C for 40 hours.

[0094] Purification

[0095] Cells were pelleted by centrifugation at 4°C and resuspended in lysis buffer consisting of 50 m Na2HPO4, 300 mM NaCl, 10 mM Imidazole, ImM PMSF protease inhibitors, IpL of Img/mL DNase I for each mL of cell suspension, pH 8. Resuspended samples were kept on ice and lysed by sonication. After sonication, the samples were centrifuged at 12,000xg for 1 hour at 4°C, the supernatant decanted, filtered through 0.2 pm filter and the pellet discarded.

[0096] Immobilized metal affinity chromatography (IMAC)

[0097] Lysate was mixed with Ni-NTA resin (Qiagen) (Ni-NTA resin volume was in the range of 4-8 mL slurry mixture depending on the expected protein amount in the cell lysate) which had been previously equilibrated with 20 bed volumes of lysis buffer (50 mM Na2HPO4, 300 mM NaCl, 10 mM Imidazole, pH 8). The lysate -resin mixture was incubated at 4°C for 2 hours and subsequently was applied to a polypropylene column. The column was washed with 20 bed volumes of washing buffer (50 mM Na2HPO4, 300 mM NaCl, 20 mM Imidazole, pH 8) followed by elution with 3 bed volumes of elution buffer (50 mM Na2HPO4, 300 mM NaCl, 300 mM Imidazole, pH 8). The eluted protein was buffer exchanged against 20 mM Tris-Cl, 20 mM NaCl, pH 7.5 using 10 kDa MWCO protein concentrator tubes (Amicon) and was subjected to ion-exchange chromatography described below.

[0098] Q-Sepharose purification

[0099] Following buffer exchange, the IMAC-eluted protein was applied by gravity flow to a polypropylene column loaded with 2 mL Q-FF resin which had been previously equilibrated with 20 bed volumes of binding & washing buffer (20 mM Tris-Cl, 20 mM NaCl, pH 7.5). The column was washed with 20 bed volumes of binding & washing buffer, then eluted with a 50-500 mM NaCl gradient. Purity of the protein elutions was assessed by SDS- PAGE, and the fractions with the highest concentration and purity were pooled. HsTP was observed to elute between 100-200 mM NaCl. Purified HsTP was aliquoted, mixed with 15% (v/v) final glycerol concentration, flash-frozen with liquid nitrogen and stored at -80 °C for future use.

[00100] Table 2. Summary of the HsTP and EcTP constructs and their expression yields.

Expression yield refers to the soluble protein that was recovered after employing the standard 2-step purification scheme described.

[00101] Western blotting

[00102] Immunoblotting for the detection of expressed HsTP was carried out according to standard protocols. Briefly, protein concentration was calculated using a NanoDrop (One microvolume UV-Vis spectrophotometer from Thermo Scientific) by recording the A280. For crude extracts A280 of 1 was assumed to be equivalent to 1 mg/mL whereas for HsTP and EcTP the respective molar extinction coefficient values (aHsTP=23490 M- 1cm- land aEcTP=24410 M^-1cm^_1) were used for the conversion of absorbance to molar concentration. The typical protein amount loaded per well was in the range of 10-15 pg. The protein samples were analyzed by SDS-PAGE and subsequently, the separated proteins were transferred onto a nitrocellulose membrane in transfer buffer (25 mM Tris-Cl, 190 mM glycine, 0.1% SDS) at a fixed current of 10 mA overnight at 4 °C. Membranes were blocked with 5% skim milk dissolved in TBST buffer for 2 hours at room temperature, followed by incubation with monoclonal anti-His6 antibodies (Sigma- Aldrich, SAB2702218). Incubation with secondary goat anti-mouse HRP-linked antibodies (ThermoFisher Scientific, G-21040) was carried out for 1 hour at room temperature. Upon immunoblotting, bands were detected using an enhanced chemiluminescence (ECL) kit (SuperSignal West Pico PLUS from ThermoFisher Scientific, 34580).

[00103] Steady-state kinetic analysis of HsTP and EcTP [00104] Steady-state kinetic characterization of HsTP and EcTP against dThd and dUrd was performed by continuously monitoring the decrease in absorbance of dThd and dUrd upon depyrimidination at 290 nm and 282 nm respectively. Enzyme concentration in the range of 10-20 nM were used for all the steady-state kinetic measurements. Reactions took place in assay buffer consisting of 20 mM Hepes, 50 mM KH2PO4, pH 7.5 in a final volume of 1 mL placed in UV cuvettes with 1 cm ¹. Reaction progress was typically monitored for 2 minutes using a Jasco V750 spectrophotometer with temperature-controlled cuvette holder and the absorbance was converted to concentration using the extinction coefficient of dThd and dUrd (Aa290 = lOOOM-lcm ¹ and Aa282 = 1370M ¹cm ¹)(Krenitsky et al., 1976). The obtained v/[E] (initial velocity/total enzyme concentration) values from the linear region of the reaction progress curves with <10% of substrate conversion were plotted against the respective substrate concentrations and the steady-state kinetic parameters k_cat and kcat/Kw were calculated by nonlinear regression using the Michaelis-Menten model (equation 1 below) analyzed by the SoftZymics software (Igor Pro, Wavemetrics).

[00105] v=(k_cat x [S])/(KM+S) (equation 1)

[00106] Analytical size-exclusion chromatography of HsTP.

[00107] The purified protein samples were diluted in PBS buffer to a final concentration of approximately 2 mg/mL. 2 pL of diluted samples were injected on a size exclusion column (TSKgel G4000SWxl, 7.8x300 mm, 8 p particle size, from Tosoh). Isocratic elution was performed at a flow rate of 0.3 mL/min, using a Thermo ultimate HPLC system. The mobile phase contains 90% of phosphate buffer (50 mM sodium phosphate, 200 mM sodium chloride, adjusted to pH 7.2) and 10% ethanol. The column eluent was monitored by UV detection at 214 nm. The BioRad gel filtration standard was used as molecular weight markers.

[00108] Table 3. Steady-state kinetic parameters of HsTP¹⁹⁹, HsTP²¹⁸ and EcTP against dThd and dUrd.

Assays were performed in assay buffer consisting of 20 mM Hepes, 50 mM KH2PO4, pH 7.5 at the indicated temperature.

[00109] Phylogenetic analysis of thymidine phosphorylase enzymes

[00110] Phylogenetic analysis was performed by using the program Geneious prime. The full-length amino acid sequences of HsTP and EcTP deposited in the Uniprot database (P19971 and P07650) were used as query sequences to perform massive BLAST alignments and thus, determine conserved residues. Geneious is directly connected to NCBI database and searches for homologous sequences to the query entry. For restricting the search exclusively for either eukaryotic or prokaryotic thymidine phosphorylase enzymes, the commands “Eukaryotes [organism]” or “Prokaryotes [organism]” were used in the “entrez query” option respectively. The maximum number of thymidine phosphorylase hits to be searched for from the NCBI database was set to 1000.

[00111] Translation initiation rate in-silico analysis

[00112] Translation initiation rates (TIR) were calculated using the online webserver De Novo DNA at the following URL: https://salislab.net/software/. The mRNA of each construct starting from the transcriptional start site and ending at the transcriptional terminator sequence was subjected to analysis based on the ribosome binding site (RBS) predict-mode algorithm using E. coli as the host organism for recombinant expression. The RBS calculator in the predict-mode identifies all the starting codons in the mRNA sequence and calculates a translation initiation rate (TIR) in arbitrary units (a.u. ; the larger the value, the higher the TIR) for each of them according to its statistical thermodynamics algorithm as described elsewhere (Salis et al., 2009). The TIR calculations are accompanied by ribosome binding Gibbs free energy calculations that represent the quantification of the interaction between the ribosome and the mRNA that, in turn, affects the TIR. The reported AGtotai is defined as follows: AGtotai = AGmRNA-rRNA + AGspacing + AGstart - AGstandby - AGmRNA, where AGmRNA-rRNA is the energy released when the last nine nucleotides of the E. coli 16S rRNA hybridizes and co-folds to the mRNA sub-sequence. AGstart is the energy released when the start codon hybridizes to the initiating tRNA anticodon loop. AG_spacing is the free energy cost caused by a non-optimal physical distance between the 16S rRNA binding site and the start codon. AGmRNA is the work required to unfold the mRNA sub-sequence when it folds to its most stable secondary structure and AGstandby is the work required to unfold any secondary structures sequestering the standby site after the 30S complex assembly.

[00113] Site-directed mutagenesis and generation of HsTP variants

[00114] Modified HsTP enzymes may possess deletions and/or substitutions of amino acids; thus, an enzyme with a deletion, an enzyme with a substitution, and an enzyme with a deletion and a substitution are modified HsTP enzymes. These modified HsTP enzymes may further include insertions or added amino acids, such as with fusion proteins or proteins with linkers, for example. A modified deleted HsTP enzyme that lacks one or more residues of the native enzyme may possess the specificity and/or activity of the native enzyme. A modified HsTP enzyme may also have reduced immunogenicity or antigenicity.

[00115] HsTP enzyme variants may contain the exchange of one amino acid for another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide, particularly its effector functions and/or bioavailability. Substitutions may or may not be conservative. A conservative change includes one amino acid replaced with one of similar size and charge. Conservative substitutions include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

[00116] Amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.

[00117] For the generation of the PEGylation variants HsTP²⁴⁰ and HsTP²⁴¹ site-directed mutagenesis was performed following the overlap extension PCR methodology (Nelson and Fitch, 2011). Briefly, this method comprises three successive amplification steps and involves four primers as follows: two external ones that cover the 5’- and 3’- ends of the parental sequence and two additional ones which carry the desired mutations to be incorporated in the final sequence. Two independent PCR reactions (PCR1: forward external primer covering the 5’ combined with the reverse carrying the mutations and PCR2: reverse external at the 3 ’ combined with forward carrying the mutations) were performed to amplify two fragments which overlap at the regions flanking the mismatches. The two amplified fragments were agarose gel-purified and in a final third step, they were combined in equal molar quantities with the initial external primers and were subjected to the last PCR reaction resulting in the final amplicon that carries the desired point mutations. The PCR amplicons along with pET28a plasmid were digested overnight at 37 °C with NcoI/EcoRI and ultimately were cloned using T4 DNA ligase. The incorporated mutations were sequence-verified by Sanger sequencing.

[00118] In some embodiments, recombinant HsTP enzymes are used to treat MNGIE. In some embodiments, the recombinant HsTP enzymes are PEGylated. In some embodiments, truncated recombinant HsTP enzymes are used to treat MNGIE. Accordingly, in some embodiments, several truncation variants are generated to determine whether the variants would be suitable for use in the treatment of MNGIE. In some embodiments, the truncated HsTP enzyme comprises truncations of 10, 18, or 33 amino acids that had N-terminal truncations of ten amino acids (A 10), eighteen amino acids (A 18), or thirty-three amino acids (A 33) respectively. In some embodiments, the N-terminal methionine has been excised.

[00119] An equivalent to an HsTP disclosed herein may be a biologically functional equivalent of the native HsTP, or may include amino acid sequences that have about 90% or more sequence identity to any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, or even between about 91 % and about 99% of amino acids (including 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) that are identical to, or have conservative substitution of the amino acids of, a modified HsTP enzyme disclosed herein. An equivalent will retain some biological activity of the enzyme such that a measurable biological activity parameter (e.g., phosphorolysis of deoxythymidine (dThd) or deoxyuridine (dUrd)) can be observed in vitro or in vivo.

[00120] Native HsTP is poorly expressed in E. coli [00121] Use of the synthetic, codon-optimized gene for optimal expression in E. coli failed to yield large quantities of HsTP (Figure 3). Recombinant expression of human proteins in E. coli can be very challenging because multiple factors need to be optimized at the molecular level such as mRNA stability and structure (Simonetti et al., 2009), (de Smit and van Duin, 1994), translation initiation rates (Espah Borujeni et al., 2014) and structural features of the protein (particularly at the N’ -terminus) (Wruck et al., 2017) can play a pivotal role in achieving high levels of protein expression.

[00122] The crystal structure of HsTP (PDB entry 2WK6) (Mitsiki et al., 2009) was compared to the structure of the bacterial ortholog EcTP (PDB entry 4LHM) (Timofeev et al., 2014). The two enzymes belong to the same family II of nucleoside phosphorylases, they are relatively close structural homologs (Ca-RMSD -5.2A) (Figure 1A) and they share 38.4 % amino acid sequence identity. None of the available HsTP crystal structures deposited in protein data bank (PDB) (2WK6, 1UOU, 2J0F) showed a well-resolved secondary structure of the N’-terminal 10-residue pro-peptide. In fact, a longer sequence consisting of the first thirty- three residues of HsTP is structurally missing, possibly due to its large intrinsic flexibility which can result in low electron density as has been shown in other studies (Palamini et al., 2016). The structurally-visible residue of HsTP is Q35 which is located on the top of an uncapped a-helix (Figure 2A, 2B), whereas EcTP has a leucine (L3) at the respective structurally equivalent position and is lacking an unstructured domain at its N’ -terminus (Figure 2B). Amino acid sequence alignments of eukaryotic and prokaryotic nucleoside phosphorylases belonging to family II revealed that, while eukaryotic species harbor a N’- terminus sequence of variable length which is very poorly conserved (part of which corresponds to the pro-peptide), the prokaryotic orthologs totally lack such a sequence.

[00123] HsTP and EcTP have distinct N’-terminal domains and recombinant expression profiles

[00124] An EcTP construct was designed and generated in a similar manner with the HsTP (i.e. codon optimized gene was cloned in pET28a with a His6-tag at the N’ -terminus) (Figure 2D) using the predicted amino acid sequence encoded by the deoA gene from the K-12 strain as has been deposited in the EcoGene 3.0 database (Zhou and Rudd, 2013). Prior to the experimental expression assessment of EcTP, analysis of the translation initiation rate (TIR) for both constructs (HsTP¹⁹⁹ & EcTP) using the algorithm developed by Salis lab (Salis et al., 2009), (Salis, 2011) yielded identical TIR values (11643 arbitrary units). This algorithm uses a statistical-thermodynamics-derived model to calculate the free energy of different interactions between the mRNA and the ribosome and the sum of all those binding free energies (as AGtotai). This determines how likely it is for the ribosome to bind to an mRNA molecule and initiate translation (the higher the translation rate, the more likely for the ribosome to initiate translation). Particularly, calculations have indicated that the secondary structure of the mRNA may play a determining role in those interactions and, in turn, in the translation rate (Espah Borujeni et al., 2017). The modelled mRNA structures of HsTP¹⁹⁹ and EcTP including the 5’- UTR and the first 10 codons of each construct were predicted to be identical as both were cloned into the same plasmid (identical ribosome binding sites) harboring a His6-tag at the N’- terminus along with the same linkers.

[00125] Thus, based on the TIR analysis very similar expression levels of HsTP¹⁹⁹ and EcTP were anticipated. However, surprisingly, expression of EcTP in BL21(DE3) under the same conditions as HsTP¹⁹⁹ yielded ~ 100-120 mg of protein per L of culture medium (entire amount in the soluble fraction) which is a ~ 28-fold larger quantity relative to the 3-5 mg/L for the HsTP¹⁹⁹ (Table 2). These findings led us to hypothesize that the primary and secondary structure of HsTP’s N’ -terminal domain could possibly impede efficient protein expression. Therefore, N’ -terminal truncations of HsTP were made to improve recombinant protein expression of HsTP.

[00126] Example 2 shows that N’ -terminal truncation of HsTP significantly improved its expression in E. coli. Primary and secondary protein structure can have a profound effect on the translation, as well as protein solubility & toxicity. For example, if the N-terminal region contains many prolines or positively charged amino acids, it can slow down ribosome translocation & elongation (lower protein synthesis & higher ribosome sequestration) (Wruck et al., 2017), (Charneski and Hurst, 2013), (Lu and Deutsch, 2014). Close inspection of the HsTP¹⁹⁹ construct revealed that among the first five residues at the N’ -terminus, three correspond to proline residues i.e. M1-A2-P3-P4-A5-P6 (Figure 2C, A11-P12-P13-A14-P15 based on the full-length numbering). This could negatively affect co-translational folding and in turn, explain the presence of a considerable amount of HsTP in the insoluble fraction. In addition, the N’-terminal highly flexible and unstructured polypeptide fragment of HsTP199 spanning the range A11-K34 could further impede co-translational folding (Waudby et al., 2019), (Ciryam et al., 2013). [00127] TIR analysis of the HsTP218 (truncated construct) revealed that this construct is characterized by the highest translation rate (18179 arbitrary units) among all the constructs tested in the present study. This high value of TIR is indicative of a high propensity for translation initiation as a result of the strong interactions between the HsTP²¹⁸ mRNA’s secondary structure and the respective ribosomal binding sites essential for proper translation. Although the interactions between the mRNA and ribosomal RNA (that are governed and mediated by the mRNA’s structure) affect TIR in a totally different mechanism relative to the protein’s primary and secondary structure at the N’ -terminus, HsTP²¹⁸ expression may overall have been benefited by both mechanisms. That is, from one side, the first ten codons of the new HsTP²¹⁸ truncated construct (His6-tag present at the C’ -terminus) altered the secondary structure of its mRNA in a way that enhanced its binding to the ribosome and consequently the translation as evidenced by the higher TIR relative to the initial construct HsTP¹⁹⁹. On the other hand, the lack of multiple successive proline residues in the new N’ -terminus facilitated the co-translational folding and improved soluble expression. An analysis of 200 highly expressed endogenous E. coli proteins showed that the most abundant amino acid residue at the 3rd position of the polypeptide chain is lysine (Bivona et al., 2010), which is in agreement with the HSTP²¹⁸’S sequence (M-G-K3-Q4— , Gly2 is a scar residue as a result of the cloning process using Ncol site). HsTP²¹⁶ and HsTP²¹⁷ constructs appeared to have very similar TIRs, which is in-line with their low expression levels, but HsTP²¹⁶ showed ~4-fold lower soluble protein, possibly attributed to the successive proline residues at its N’ -terminus that could stall translation (Starosta et al., 2014), (Peil et al., 2013), (Huter et al., 2017).

[00128] PEGylation optimization of HsTP

[00129] Lysine Based PEGylation.

[00130] PEG can be used in the field of biologies as a time extension strategy aiming at the improvement of the thermodynamic stability (Santos et al., 2019), (Moreno-Perez et al., 2016), immunogenicity (Gefen et al., 2013) and the pharmacokinetic (PK) properties of therapeutic proteins (Harris et al., 2001), (Hamidi et al., 2006).

[00131] Initial efforts to conjugate HsTP¹⁹⁹ with methoxy-5-kDa-PEG-succinimidyl- succinate (mPEG5kDa) targeting primary amines on the enzyme’s surface (primarily lysines and N’ -terminus) resulted in an apparent heterogeneous mixture of three distinct PEGylated enzyme species (Figure 5A, 5B) indicative of an inefficient PEGylation reaction. Moreover, upon PEGylation, HsTP¹⁹⁹ showed a 2.8- and 2.2-fold decrease of its k_cat and k_cat/KMdThd respectively (Table 4) suggesting that chemical modification with mPEG5kDa of one or more lysine residues, negatively affects the enzyme’s catalytic activity.

[00132] Rational Surface Engineering.

[00133] To address the low PEGylation efficiency of HsTP as well as the negative impact on catalytic activity, we employed a rational surface engineering approach where arginine residues, located at surface-exposed positions suitable for PEGylation, were identified and were mutated to lysine. Conversely, lysine residues whose modification with mPEG5kDa could hamper the catalytic activity were engineered-out and mutated to arginine. Enzymology and enzyme-PEGylation criteria were applied to the engineering efforts. Unlike in case of lysine, HsTP has thirty-two arginine residues widely distributed around its surface (Figure 7A). Five residues were identified that satisfied all criteria (R329, R342, R345, R358, R453) and are shown in Figure 7B and 7C. These arginine residues i) are not highly conserved among thymidine phosphorylases that belong to family II (Figure 14A), ii) they have very large solvent-accessible surface area (SASA) with the only exception being R342 which barely met our SASA criterion (>75 A , Figure 14B), iii) they are not located on loops whose dynamics may be altered upon PEGylation and, in turn, negatively impact catalytic activity (rigidification issue (Rodriguez-Martinez et al., 2009)), iv) do not form salt bridges with aspartate or glutamate residues as this could alter the pKa of the newly introduced lysine residue, requiring the PEGylation reaction to be performed at highly alkaline pH and v) they are not located nearby the active site and thus, they do not prevent the substrate molecules from diffusing into it. Furthermore, analysis of the four lysine residues (K43, K139, K253, K275) based on our “engineer-out” criteria led us to mutate K139 and K275 to arginine and thus, prevent their PEGylation. Both residues are located on flexible loops (flexibility was assessed by the B- factor values of those domains) which connect 2nd-shell domains nearby the active site (Figure 6A). Previous studies have shown that PEGylation of enzymes correlates with reduced structural dynamics and this in turn, may impair catalytic activity which can be directly dependent on conformational dynamics (Rodriguez-Martinez et al., 2008), (Hsieh and Lin, 2015), (Morgenstern et al., 2017), (Santos et al., 2019). PEGylation of EcTP whose 8/22 lysine residues are located on surface-exposed flexible loops led to even greater reduction of its kcat relative to HsTP (400 s ¹ vs 90 s ¹ for un-PEGylated and PEGylated EcTP species respectively) (Figure 15). [00134] The results disclosed show experimental validation of a surface engineering strategy that enabled PEGylation improvement of HsTP at two different levels. That is, through rationally-designed Arg-to-Lys substitutions significantly high PEGylation efficiency was achieved and improved homogeneity using proteimPEG molar ratio at 1:20. This is advantageous on multiple levels: i) additional extensive ion exchange purification steps are not required to resolve and isolate the different enzyme PEG-mers which are obtained in case of partial and incomplete PEGylation ii) the more homogeneous PEGylated enzyme species allow for a more accurate biochemical assessment and iii) low proteimPEG molar ratios can lower the cost of manufacturing. On a second level, by mutating Lys-to-Arg residues at specific locations, the catalytic activity of HsTP was maintained upon PEGylation.

[00135] Table 4. dThd steady-state kinetic parameters of HsTP¹⁹⁹, HsTP²¹⁸, HsTP²⁴⁰ and HsTP²⁴¹ prior and post-PEGylation.

Conjugation reactions took place at either 1:10, 1:20 or 1:30 proteimPEG molar ratio as described in the Methods section.

[00136] Enzymatic Degradation for Therapy

[00137] Diagnosis of Mitochondrial neurogastrointestinal encephalopathy (MNGIE) syndrome can be confirmed by detection of TYMP gene variations in the patient or by the increased levels of thymidine and deoxyuridine in blood. Patients may experience a multitude of clinical symptoms including abdominal distention, abdominal pain, atrophic muscularis propria, cachexia, dysphagia.

[00138] Direct evidence of MNGIE syndrome can be provided by one of the following: (1) A blood test showing an increase in plasma thymidine concentration (greater than 3 pmol/L) and an increase in plasma deoxyuridine concentration (greater than 5 pmol/L). This is sufficient to make the diagnosis of MNGIE disease; (2) Thymidine phosphorylase enzyme activity in leukocytes (white blood cells) less than 10% of the control mean.

[00139] The present invention provides methods of using engineered, therapeutic enzymes that degrade deoxythymidine (dThd) and/or deoxyuridine (dUrd) to treat diseases, such as MNGIE.

[00140] Before describing several exemplary embodiments of the disclosure, it is to be understood that the disclosure is not limited to the details of construction or process steps set forth in the following description. The disclosure is capable of other embodiments and of being practiced or being carried out in various ways.

[00141] EXAMPLES

[00142] Example 1 - HsTP is poorly expressed in E. coli

[00143] Successful recombinant expression of HsTP in E. coli has been previously described by other groups with soluble expression levels reported in the range of ~ 3 mg/L of culture (Schwartz et al., 2010). Research databases such as NCBI and Uniprot suggests that human thymidine phosphorylase harbors a N’-terminal 10-residue long pro-peptide (Uniprot entry P19971) which is cleaved during a post-translational modification and processing step, thereby yielding the final mature polypeptide chain.

[00144] While the biological role of this pro-peptide as well as its impact on the enzyme’s biochemical features remain largely unclear, all prior studies focusing on the biochemical characterization of HsTP used the putatively mature version of the protein with the first ten residues at the N’-terminal site being truncated from the sequence (Schwartz et al., 2010), (Deves et al., 2014). A codon-optimized HsTP gene carrying a His6-tag at the N’- terminus was constructed, devoid of the first ten residues corresponding to the pro-peptide sequence, then cloned into pET28-a plasmid (construct termed HsTP¹⁹⁹, Figure ID). This construct was expressed in E. coli BL21(DE3). The levels of the finally recovered active enzyme (after one IMAC purification step with minimal protein loss in the flow through and washing fractions) were in the range of 3-5 mg/L of TB culture medium (Table 2) while a considerable amount of the produced protein was detected in the insoluble fraction (-50% more than the soluble) as inclusion bodies (Figures 9 & 10).

[00145] Multiple attempts to increase the expression titers (both soluble and total amount) by i) switching to C41(DE3) E. coli expression strain which has been shown to benefit soluble expression levels (Kwon et al., 2015) (Figures 9 & 10), ii) altering expression conditions (e.g. IPTG concentrations, temperature, OD600 at the induction, culture media) (Rosano and Ceccarelli, 2014), (Gutierrez-Gonzalez et al., 2019), and iii) testing a high-copy- number plasmid (pJC-20) (Clos and Brandau, 1994) (Figure 11) failed to improve expression levels of HsTP199. Taken together, these data showed that achieving high levels of HsTP bacterial expression required more extensive experimentation.

[00146] Example 2 - N’-terminal truncation of HsTP significantly improves its expression in E. coli

[00147] Truncated HsTP constructs were generated (Figure 2D) within the first 33 residues (Figure 2C) which are not visible in its crystal structure (Figures 2A, 2B). Among the four new constructs tested, one (HsTP218) yielded large amounts of expressed protein, yet the protein was almost exclusively observed in the insoluble fraction when expressed at 30 °C (Figure 3). Expression of HsTP²¹⁸ under low temperatures (16 °C and 22 °C) and IPTG conditions (Table 2, Figure 12) allowed the protein to fold properly and achieve soluble expression levels similar to those of EcTP (up to 70 mg/L, Table 2). HsTP²¹⁸ construct is missing the entire unstructured N’-terminal region comprising the first thirty-three amino acid residues of full-length HsTP. That is, downstream of the first K34 residue of HsTP²¹⁸ the enzyme’s secondary structure forms an a-helix similar to EcTP (Figure 2B). The steady-state kinetic analysis of purified HsTP²¹⁸ (Figure 4) and HsTP¹⁹⁹ (mature form of HsTP) against dThd and dUrd showed very similar kinetic properties against dThd but a 2-fold increase in kcat/KvidUrd in favor of HsTP²¹⁸ (Table 3). In addition, analytical size exclusion chromatography (aSEC) confirmed the oligomeric state of HsTP²¹⁸ exhibiting a dimer conformation (Figure 13), which agrees with the active form of the enzyme as suggested by its crystal structure and previous studies (Norman et al., 2004).

[00148] Example 3 - PEGylation optimization of HsTP

[00149] Lysine (Non-Site-Specific) Based PEGylation [00150] Conjugation reactions with methoxy-5-kDa-PEG-succinimidyl-succinate (mPEG5kDa, NOF corporation) were performed in 100 mM Na2HPO4, pH 8.5 using purified HsTP¹⁹⁹ and EcTP. The final enzyme concentration in the reaction was ~ 115 pM and depending on the proteimPEG ratio, the respective concentration of PEG was adjusted accordingly: 1150 pM, 2300 pM and 3450 pM for 1:10, 1:20 and 1:30 molar ratio respectively. The lyophilized PEG was weighed and added directly into a 2 mL tube containing either purified HsTP¹⁹⁹ or EcTP at a volume of 1 mL. Immediately after the addition of the PEG, the tube was vortexed continuously for 30 seconds, followed by incubation at room temperature for 30 minutes under rotating conditions. Subsequently, the mixture was buffer exchanged to remove excess of unreactive PEG using protein concentrators with a 50-kDa MWCO (Thermo Scientific). HsTP PEGylation variants and HsTP²¹⁸ were conjugated following the same process except for the buffer which was 50 mM Na2HPO4, 50 mM NaCl, pH 8.0 and the final enzyme concentration was 200 pM. Following the final buffer exchange step, PEGylation efficiency was assessed by SDS-PAGE and steady-state kinetics was performed using dThd as substrate. PEGylated enzymes were buffer exchanged against 50 mM Na2HPO4, 50 mM NaCl, pH 7.0, mixed with 15% final concentration of glycerol, flash-frozen in liquid nitrogen and stored at -80 °C.

[00151] Initial efforts to conjugate HsTP¹⁹⁹ with methoxy-5-kDa-PEG-succinimidyl- succinate (mPEG5kDa) targeting primary amines on the enzyme’s surface (primarily lysines and N’ -terminus) resulted in an apparent heterogeneous mixture of three distinct PEGylated enzyme species (Figure 5A, 5B) indicative of an inefficient PEGylation reaction. Indeed, HsTP contains eleven lysine residues per monomer (Figure 5A), however analysis of its crystal structure revealed that only four of them (K43, K139, K253, K275) are adequately surface- exposed and thus, accessible to react with mPEG5kDa (Figure 5B). In fact, as shown in Figure 5B, perhaps the only lysine residue that can react with mPEG5kDa with high certainty and efficiency due to its high solvent accessibility is K253 whereas K43, K139 and K275 are less exposed, thereby yielding a mixture of PEGylated species. Indeed, based on the PEGylation pattern shown in Figure 4, up to five sites appear to be modified with mPEG5kDa as this would result in a ~ 25 kDa increase in the molecular weight (MW) yielding a total of ~ 75 kDa per monomer (upper band in the gel). It appears reasonable to assume that those five sites consist of the four lysine residues mentioned above in addition to the N’ -terminus site of the molecule. Moreover, upon PEGylation, HsTP¹⁹⁹ showed a 2.8- and 2.2-fold decrease of its k_cat and kcat/Kw Thd respectively (Table 4) suggesting that chemical modification with mPEG5kDa of one or more lysine residues, negatively affects the enzyme’s catalytic activity. [00152] Rational Surface Engineering

[00153] To improve the very modest PEGylation efficiency and negative impact on catalytic activity a rational surface engineering approach was used to engineer modified HsTPs.

[00154] Two HsTP variants were generated (HsTP²⁴⁰ and HsTP²⁴¹) using as template the expression-optimized HsTP²¹⁸ enzyme. The PEGylation efficiency was assessed as well as their steady-state kinetics against dThd prior and post PEG conjugation. HsTP²⁴¹ harbors all residue substitutions identified above (K139R-K275R-R329K-R342K-R345K-R358K-R453K) whereas R329K and R453K were not incorporated on the second variant HsTP²⁴⁰ to investigate whether high PEGylation homogeneity and efficiency could be achieved with less substitutions.

[00155] Both HsTP²⁴⁰ and HsTP²⁴¹ variants showed a significantly improved PEGylation homogeneity relative to their parental HsTP²¹⁸ with HsTP²⁴¹. Figure 7 shows only one visible band around 100 kDa when 1:20 (proteimPEG) molar ratio conjugation reaction conditions were used. In contrast, HsTP²¹⁸ showed an almost identical profile for both PEGylation treatments tested (1:10 and 1:20 molar ratio) which consists of five distinct bands on the SDS-PAGE (Figure 8) suggesting lower conjugation efficiency.

[00156] Importantly, the steady-state kinetic characterization of HsTP²⁴¹ revealed that the PEGylated enzyme retained k_cat/KMdThd at the same levels as prior to PEGylation while PEGylation of HsTPiis caused a 2-fold decrease similar to HSTP199 (Table 4). Upon PEGylation, the second variant which is missing the two R329K and R453K mutations exhibited a decreased kcat/KMdThd. However, this decrease was due to an effect on KM rather than on k_cat which remained almost unaltered relative to the unPEGylated species (Table 4).

[00157] Taken together, the data demonstrate that by rational selection of suitable arginine and lysine residues for mutagenesis, there is significant improvement in the PEGylation efficiency and consistency while catalytic activity is preserved post-PEGylation.

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[00246] Reference throughout this specification to “one embodiment,” “certain embodiments,” “various embodiments,” “one or more embodiments” or "an embodiment" means that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of the phrases such as “in one or more embodiments,” “in certain embodiments,” “in various embodiments,” “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments.

[00247] Although the disclosure herein provided a description with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the present disclosure without departing from the spirit and scope thereof. Thus, it is intended that the present disclosure include modifications and variations that are within the scope of the appended claims and their equivalents.

Claims

47 What is claimed is:

1. A recombinant human thymidine phosphorylase (rHsTP) enzyme comprising one or more modifications relative to wild-type thymidine phosphorylase, wherein the one or more modifications comprise one or more chemical modifications, substitutions, insertions, deletions, and/or truncations, and wherein the rHsTP has a sequence similarity in a range of from 90% to 100% compared to SEQ ID NO:1.

2. The rHsTP enzyme of claim 1 comprising a truncated rHsTP enzyme.

3. The rHsTP enzyme of claim 2, wherein the truncated rHsTP enzyme comprises an amino acid sequence that is 90% to 100% identical to any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, or SEQ ID NO: 12.

4. The rHsTP enzyme of claim 1, wherein the modified rHsTP comprises an amino acid sequence that is 90% to 100% identical to any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

5. The rHsTP enzyme of any one of claims 1 to 4, wherein the rHsTP enzyme comprises one or more amino acid residue mutations at K139, K275, R329, R342, R345, R358, and R453.

6. The rHsTP enzyme of claim 5, wherein the rHsTP enzyme comprises one or more amino acid residue mutations K139R, K275R, R329K, R342K, R345K, R358K, and R453K.

7. The rHsTP enzyme of any one of claims 1 to 6, wherein the rHsTP enzyme comprises one or more of an affinity tag, a linker peptide and a cleavage site.

8. The rHsTP enzyme of claim 7, wherein the affinity tag comprises HIS tag.

9. The rHsTP enzyme of any one of claims 1 to 8 comprising a catalytic efficiency (kcat/KM) in a range of from 10 mM-ls-1 to 500 mM-ls-1.

10. The rHsTP enzyme of any one of claims 1 to 9, wherein the rHsTP enzyme is PEGylated.

11. The rHsTP enzyme of claim 10, wherein the PEGylated rHsTP enzyme comprises 1 to 20 PEGylated amino acid residues per rHsTP monomer.

12. The rHsTP enzyme of claim 10 or 11, wherein the rHsTP enzyme is PEGylated by a branched PEG or a linear PEG. 48 The rHsTP enzyme of any one of claims 10 to 12, wherein the PEGylated rHsTP enzyme has non- site- specific PEGylation. The rHsTP enzyme of claim 13, wherein the non-specific PEGylation is lysine directed. The rHsTP enzyme of any one of claims 10 to 12, wherein the PEGylated rHsTP enzyme has a site-specific PEGylation. The rHsTP enzyme of claim 15, wherein the specific site for PEGylation is chosen based on factors comprising one or more of accessibility of the amino acid at the surface of the molecule, non-essential role of the amino acid in structural function, mutations which cause rHsTP in humans, proximity to disease causing mutations, proximity to the active site residues, proximity to the dimer-dimer interface, and a degree of amino acid conservation across different species. The rHsTP enzyme of claim 15 or 16, wherein the site-specific PEGylation is directed towards cysteine or arginine. A method of preventing or treating mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), the method comprising administering a pharmaceutical composition comprising rHsTP enzyme of any one of claims 1 to 17 to a subject in need thereof. The method of claim 18, wherein the subject comprises a human, mouse, rat, rabbit or monkey. The method of claim 18, wherein the subject is a mouse. The method of claim 18, wherein the subject is a human. The method of any one of claim 18 to 21, wherein the pharmaceutical composition comprises 50 mM Na2HPO4, 50 mM NaCl, pH 7.0 The method of any one of claims 18 to 22, wherein the pharmaceutical composition is administered intravenously, intradermally, intraarterially, intraperitoneally, intramuscularly, subcutaneously, by infusion, by continuous infusion, via a catheter, or in lipid composition. The method of any one of claims 18 to 23, wherein the method further comprises another therapy for the treatment of MNGIE. The method of any one of claims 18 to 24, wherein the method further comprises managing MNGIE with dietary restrictions. 49 A method of determining the enzymatic activity of the recombinant human thymidine phosphorylase (rHsTP) enzyme of any one of claims 1 to 17, the method comprising: incubating the rHsTP enzyme with different concentrations of substrate, spectroscopically measuring the degradation of substrate and determining the Km by plotting Michaelis-Menten curve A method of determining the enzymatic activity of the recombinant human thymidine phosphorylase (rHsTP) enzyme of any one of claims 1 to 17, the method comprising: incubating the rHsTP enzyme with different concentrations of substrate, spectroscopically measuring the degradation of substrate and determining the Kcat by plotting Michaelis-Menten curve A method of determining serum stability of the recombinant human thymidine phosphorylase (rHsTP) enzyme of any one of claims 1 to 17, the method comprising: reacting the rHsTP enzyme with substrate in serum for a predetermined time; quenching the reaction; and determining the enzyme activity over time in serum. A method of manufacturing the recombinant human thymidine phosphorylase (rHsTP) enzyme of any one of claims 1 to 17, the method comprising: expressing the rHsTP enzyme in E. coli cells; lysing the cells using sonication in a lysis buffer comprising 50 mM Na2HPO4, 300 mM NaCl, 10 mM Imidazole, pH 8; optionally binding the rHsTP to a NiNTA resin, then eluting the purified rHsTP with 50 mM Na2HPO4, 300 mM NaCl, 300 mM Imidazole, pH 8. The method of claim 29 further comprising: exchanging the buffer of the purified rHsTP enzyme to a PEGylation buffer, the PEGylation buffer comprising 100 mM Na2HPO4, pH 8.5; incubating the purified rHsTP enzyme with a PEGylating agent for a predetermined time and a predetermined temperature. The method of claim 30, wherein the PEGylating agent comprises polyethylene glycol (PEG) and a conjugating agent. The method of claim 31, wherein the PEG comprises a branched PEG or a linear PEG. The method of claim 31 or 32, wherein the PEG comprises a molecular weight in a range of from 2,000 kDa to 20,000 kDa. The method of any one of claims 31 to 33, wherein the conjugating agent comprises Methoxy Succinimidyl Carboxymethyl Ester. 50 The method of any one of claims 31 to 34, wherein the conjugating agent comprises Methoxy Maleimide. The method of any one of claims 30 to 35, wherein the purified rHsTP enzyme is incubated with the PEGylating agent at a molar ratio in a range of enzyme to PEGylating agent of 1 : 10 to 1:50. The method of any one of claims 30 to 36, wherein the predetermined temperature is in a range of from 4 °C to 37 °C. The method of any one of claims 30 to 37, wherein the predetermined time is in a range of from 30 minutes to 24 hours.