) ( (51) International Patent Classification: (74) Agent: SCHNEPP, Amanda, S.J.; Parker Highlander A61B 17/225 (2006.01) A61K 38/38 (2006.01) PLLC, 1120 S. Capital of Taxas Highway, Bldg. One, Suite A 61K 31/70 76 2006.01) A 61K 38/51 (2006.01) 200, Austin, TX 78701 (US). A61K 38/16 (2006.01) A61K 45/06 (2006.01) (81) Designated States (unless otherwise indicated, for every (21) International Application Number: kind of national protection av ailable) . AE, AG, AL, AM, PCT/US20 19/058021 AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, (22) International Filing Date: DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, 25 October 2019 (25. 10.2019) HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, (25) Filing Language: English KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (26) Publication Language: English OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (30) Priority Data: SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, 62/75 1,197 26 October 2018 (26. 10.2018) US TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicant: BOARD OF REGENTS, THE UNIVERSI¬ (84) Designated States (unless otherwise indicated, for every TY OF TEXAS SYSTEM [US/US]; 210 West 7th Street, kind of regional protection available) . ARIPO (BW, GH, Austin, TX 78701 (US). GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (72) Inventors: STONE, Everett; 7503 Hart Lane, Austin, TX TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, 7873 1 (US). LU, Wei-Cheng; 7600 Wood Hollow Dr., EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, #202, Austin, TX 7873 1 (US). KARAMITROS, Christos; MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, 581 1Mesa Dr., Apt. 614, Austin, TX 7873 1 (US). (54) Title: ENGINEERED PRIMATE CYSTINE/CYSTEINE DEGRADING ENZYMES FOR THERAPEUTIC USES © (57) Abstract: Methods and compositions related to the engineering of a protein with L-cyst(e)ine degrading enzyme activity are © described. For example, disclosed are modified cystathionine-y-lyases comprising one or more amino acid substitutions and capable of degrading L-cyst(e)ine. Furthermore, compositions and methods are provided for the treatment of cystinuria using the disclosed modified enzymes or nucleic acids encoding said enzymes. & [Continued on next page] ||| ||||| ||||| ||||| |||| 11| ||| ||||| ||||| ||||| ||||| ||||| |||| limn nil nil nil Published: with international search report (Art. 21(3)) before the expiration of the time limit for amending the claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) with sequence listing part of description (Rule 5.2(a)) DESCRIPTION ENGINEERED PRIMATE CYSTINE/CYSTEINE DEGRADING ENZYMES FOR THERAPEUTIC USES REFERENCE TO RELATED APPLICATIONS [0001] The present application claims the priority benefit of United States provisional application number 62/751,197, filed October 26, 2018, the entire contents of which is incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under Grant No. R01 CA1 89623 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO A SEQUENCE LISTING [0003] The instant application contains a Sequence Listing, which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on October 22, 2019, is named UTFBPl2l2WO_ST25.txt and is 365 kilobytes in size. PARTIES TO JOINT RESEARCH AGREEMENT [0004] The inventions disclosed and claimed herein were developed within the scope of a Joint Research Agreement between Aeglea BioTherapeutics, Inc. and The Board of Regents of The University of Texas System. BACKGROUND 1. Field [0005] Disclosed are recombinantly engineered primate enzyme variants having high cysteine/cysteine degrading activity suitable for human therapy. Compositions and methods for the treatment of cystinuria with enzymes that deplete both L-cystine and L-cysteine are also provided. 2. Description of Related Art [0006] Cystinuria is a hereditary disorder caused by mutations in the SLC3A1 and SLC7A9 genes encoding the kidney proximal tubule’s cystine and dibasic amino acid transporter that leads to abnormal excretion of cystine (the disulfide form of the amino acid cysteine) and the formation of cystine crystals/stones in the urinary tract. There are few therapeutics available to patients suffering from the hereditary disorder cystinuria wherein a defective kidney transporter is unable to re-uptake cystine during renal filtration. Cystine, the disulfide form of the amino acid L-cysteine, is highly insoluble and in cystinuria patients reaches high concentrations in the urinary tract resulting in the formation of cystine crystals and stones. Existing therapies that reduce circulating cystine levels partially prevent urinary tract stone formation but have significant adverse effects that limit their use. Therapies are needed to reduce and prevent cystine stone formation in the kidney and bladder. SUMMARY [0007] The present invention concerns the engineering of primate cystathionine- gamma-lyase (“CGL”) enzymes such that both L-cystine and L-cysteine (referred to herein as “L-cyst(e)ine”) can be efficiently degraded from serum, and providing the modified CGL enzymes in a formulation suitable for human therapy. To develop an enzyme displaying low K M and high catalytic activity, hat, as compared to the native enzyme, the native enzyme was engineered by modifying selected amino acids, which modifications result in an enzyme having dramatically improved enzymatic properties. As such, modified CGL enzymes, as described herein, overcome a major deficiency in the art by providing novel enzymes that comprise human or primate polypeptide sequences having improved L-cyst(e)ine-degrading catalytic activity. As this enzyme is comprised of a human sequence, it is not likely to induce adverse immunological responses. As such, these modified enzymes may be suitable for human therapy and have low immunogenicity. [0008] Methods are disclosed of utilizing an engineered human cystathionine-gamma- lyase (CGL) enzyme that efficiently converts cystine to cysteine-persulfide, which subsequently decays to free cysteine and L S, such that it is a suitable therapy for treating cystinuria patients by preventing cystine accumulation and formation of stones in the kidney and urinary tract. In addition, the engineered human CGL enzyme can convert cysteine to pyruvate, ammonia, and hydrogen sulfide, in effect reducing the amount of cysteine that can oxidize to form cystine. As cystine is a non-essential amino acid, which is normally produced by most cells, no toxicities have been found to be induced by long-term cystine depletion in animal models. The ability of a cystine-degrading therapeutic to non-toxically ablate the total levels of circulating cystine indicate that it would be a superior therapeutic regimen for preventing cystine stone formation than existing therapeutic regimens. [0009] Provided herein are modified CGL enzymes, having L-cyst(e)ine degrading activity, that are derived from primate CGL enzymes. A modified CGL enzyme may be derived from a human CGL enzyme (SEQ ID NO: 1), a Pongo abelii CGL enzyme (Genbank ID: NP_00 1124635.1; SEQ ID NO: 2), a Macaca fascicularis CGL enzyme (Genbank ID: AAW71993.1; SEQ ID NO: 3), a Pan troglodytes CGL enzyme (Genbank ID: XP_5l3486.2; SEQ ID NO: 4), or a Pan paniscus CGL enzyme (Genbank ID: XP_003830652.l; SEQ ID NO: 5). The native CGL enzyme may be modified by one or more other modifications, such as chemical modifications, substitutions, insertions, deletions, and/or truncations. [0010] A modified CGL enzyme may be derived from a native, primate CGL enzyme by modifying by one, two, three, four or more substitutions at amino acid position(s) 51, 55, 59, 91, 163, 189, 193, 200, 234, 311, 336, 339, and/or 353 of SEQ ID NOs: 1-5. In these examples, the first methionine of each sequence corresponds to amino acid position 1, and each amino acid is numbered sequentially therefrom. The substitutions at amino acid positions 5 1 may be tryptophan (W), 55 may be glutamic acid (E), 59 may be threonine (T) or isoleucine (I), 9 1 may be methionine (M) or serine (S), 163 may be arginine (R), 189 may be serine (S), 193 may be glycine (G) or alanine (A), 200 may be proline (P) or histidine (H), 234 may be lysine (K), 311 may be glycine (G), 336 may be aspartic acid (D), 339 may be valine (V), and/or 353 may be serine (S). [0011] A modified CGL enzyme may have an amino acid sequence according to SEQ ID NOs: 6-95. A modified CGL enzyme may be capable of degrading L-cyst(e)ine under physiological conditions. The modified CGL enzyme may have a catalytic efficiency for L- cyst(e)ine k lK of at least or about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 104, 105, 106 s 'M 1 or any range derivable therein. An exemplary CGL enzyme may have a catalytic efficiency of >l0 4 s^M 1 for L-cystineand >l0 3 s 1 M 1 for L-cysteine. [0012] The substitutions may be a combination of P193A, T311G, E339V, and I353S of human CGL (for example, the modified polypeptide having the amino acid sequence of SEQ ID NO: 6 or a fragment or homolog thereof). For example, an equivalent substitution of 1353 in SEQ ID NO: 1 for SEQ ID NO: 2 would modify a valine and not isoleucine as in SEQ ID NO: 1.