Modified polypeptides and methods of making and uses thereof

Abstract

Provided herein are engineered polypeptides, including engineered uricase polypeptides, that compnse one or more non-standard ammo acids (NSAAs). Compositions, pharmaceutical compositions, kits, or reagents are also provided. Additionally, the methods for making the engineered polypeptides are provided. Also provided herein are methods treating a disease or condition using the engineered polypeptides.

Description

MODIFIED POLYPEPTIDES AND METHODS OF MAKING AND USES THEREOFCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 587,924 filed on October 4, 2023, and U.S. Provisional Patent Application No. 63 / 556,130 filed on February 21, 2024, each of which is incorporated by reference in its entirety.BACKGROUND

[0002] Glycosylation is one of the most critical posttranslational modifications for many therapeutic proteins. Eliminating glycosylation can impair circulating half-life and impact downstream protein interactions and functions. In addition, it has become increasingly clear that glycan composition at specific sites plays a critical role in tuning half-life and functionality of the protein. The glycans of the glycoproteins are directly involved in biological recognition processes including intracellular trafficking, cell adhesion, signaling, development, host-pathogen interactions, and immune responses.SUMMARY

[0003] Provided herein is a composition comprising an engineered uricase polypeptide with uricase activity, wherein the engineered uricase polypeptide comprises a non-standard amino acid (NSAA).

[0004] Also provided herein is a composition comprising an engineered uricase polypeptide with uricase activity, wherein the engineered uricase polypeptide comprises an amino acid linked to a glycan, and wherein the engineered uricase polypeptide comprises an amino acid sequence derived from a Sus scrofa uricase, a Papio uricase, aMus musculus uricase, an Aspergillus flavus uricase, an Arthrobacter globiformis uricase, or a Paeniglutamicibacter gangotriensis uricase. In some embodiments, the engineered uricase polypeptide comprises a non-standard amino acid (NSAA).

[0005] In some embodiments, the engineered uricase polypeptide comprises at least two NSAAs. In some embodiments, the engineered uricase polypeptide comprises at least three NSAAs. In some embodiments, each NSAA is the same NSAA.

[0006] In some embodiments, the NSAA comprises a sidechain comprising an azide, a ketone, an aldehyde, a selenol, an alkyne, an amine, a hydrazide, an aminooxy, a boronate, or a vicinal diol. In some embodiments, the NSAA comprises a sidechain comprising an aliphatic alkyne or a strained alkyne. In some embodiments, the NSAA comprises a sidechain comprising an azide. In some embodiments, the NSAA comprises para-azidophenylalanine (pAzF), para-azidomethyl-1- phenylalanine (pAMF), azido-lysine, azido-alanine, p-propargyloxy-l-phenylalanine, p- benzoylphenylalanine, p-acetylphenylalanine, p-propargyloxy-l-phenylalanine, propargylglycine, homopropargylglycine, selenocysteine, or para-isothiocyanate phenylalanine. In some embodiments, the NSAA comprises pAzF. In some embodiments, the NSAA comprises pAMF.

[0007] In some embodiments, the engineered uricase polypeptide comprises an amino acid sequence derived from a Sus scrofa uricase, a Papio uricase, a Mus musculus uricase, an Aspergillus flavus uricase, an Arthrobacter globiformis uricase, or a Paeniglutamicibacter gangotriensis uricase.

[0008] In some embodiments, the engineered uricase polypeptide comprises an amino acid sequence derived from an Aspergillus flavus uricase.

[0009] In some embodiments, the NSAA comprises a sugar moiety coupled thereto. In some embodiments, each NSAA comprises a sugar moiety coupled thereto. In some embodiments, the NSAA comprises a glycan coupled thereto. In some embodiments, each NSAA comprises a glycan coupled thereto. In some embodiments, the engineered uricase polypeptide comprises at least two glycans coupled thereto. In some embodiments, the engineered uricase polypeptide comprises at least three glycans coupled thereto. In some embodiments, the engineered uricase comprises at least two different glycans coupled thereto or at least three different glycans coupled thereto. In some embodiments, each of the glycans is the same glycan. In some embodiments, at least one NSAA comprises at least two or at least three glycans coupled thereto. In some embodiments, each glycan is independently a glycan comprising one or more of the sugar moieties listed in Table 4. In some embodiments, each glycan is independently a glycan comprising a sugar moiety selected from the group consisting of N-Acetylglucosamine (GlcNAc), N-Acetylgalactosamine (GalNAc), GlcNAc- al,6-fucose (Fuc), galactose (Gal), mannose (Man), sialic acid (Sia), Sia-a2,3-Gal, Sia-a2,6-Gal, Sia- a2,3-GlcNAc, Sia-a2,6-GlcNac, Sia-a2, 3 -GalNAc, Gal-01,3 -GalNAc, Sia-a2,3-Gal-01,3-GalNAc, GlcNAc-pi,4-GlcNAc, GlcNAc-pi,4-[Fuc-al,3]GlcNAc, GlcNAc-pi,4-[Fuc-al,3][Fuc- al,6]GlcNAc, N-Acetylneuraminic acid (Neu5Ac), Neu5Ac-a2,3Gal- i,4[Fuc-al,3]GlcNAc, Gal- pi,4[Fuc-al,3]GlcNAc, Gal-al,3-Gal- i,4-GlcNAc-P, Gal-al,3-Gal, and any combination thereof.

[0010] In some embodiments, the sugar moiety or the glycan is coupled to the NSAA via a linker. In some embodiments, the linker comprises an alkyne. In some embodiments, the linker comprises 4- pentynoic acid, propargylamine, 6-azidohexanoic acid, dibenzocyclooctyne (DBCO), 3-butyn-l-ol, ethynylbenzene, or a PEG-alkyne. In some embodiments, the linker comprises a tetrazine. In some embodiments, the linker comprises a linker with trans cyclooctene (TCO) reactivity. In some embodiments, the linker comprises dibenzocyclooctyne (DBCO).

[0011] In some embodiments, the engineered uricase polypeptide comprises an amino acid sequence with at least 50% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-13. In some embodiments, the engineered uricase polypeptide comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-13. In some embodiments, the engineered uricase polypeptide comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to at least 200, 225, 250, 275, 300, 301 or 302 contiguous amino acids of the amino acid sequence according to SEQ ID NO: 5, wherein the NSAA is located at a position selected from the group consisting of atleast one of the following positions: a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the engineered uricase polypeptide comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to at least 200, 225, 250, 275, 300, 301 or 302 contiguous amino acids of the amino acid sequence according to SEQ ID NO: 5, wherein the NSAA is located at a position selected from the group consisting of at least two of the following positions: a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the engineered uricase polypeptide comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to at least 200, 225, 250, 275, 300, 301 or 302 contiguous amino acids of the amino acid sequence according to SEQ ID NO: 5, wherein the NSAA is located at a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the engineered uricase polypeptide comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to at least 200, 225, 250, 275, 300, 301 or 302 contiguous amino acids of the amino acid sequence according to SEQ ID NO: 8 or 11. In some embodiments, the engineered uricase polypeptide comprises an amino acid sequence with 100% sequence identity to at least 200, 225, 250, 275, 300, 301 or 302 contiguous amino acids of the sequence according to SEQ ID NO: 8 or 11. In some embodiments, the engineered uricase polypeptide comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence according to SEQ ID NO: 8 or 11. In some embodiments, the engineered uricase polypeptide comprises an amino acid sequence according to SEQ ID NO: 8 or 11. In some embodiments, the engineered uricase polypeptide comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to at least 200, 225, 250, 275, 300, 301 or 302 contiguous amino acids of the amino acid sequence according to any one of SEQ ID NOs: 9, 10, 12 or 13. In some embodiments, the engineered uricase polypeptide comprises an amino acid sequence with 100% sequence identity to at least 200, 225, 250, 275, 300, 301 or 302 contiguous amino acids of the sequence according to any one of SEQ ID NOs: 9, 10, 12 or 13. In some embodiments, the engineered uricase polypeptide comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence according to any one of SEQ ID NOs: 9, 10, 12 or 13. In some embodiments, the engineered uricase polypeptide comprises an amino acid sequence according to any one of SEQ ID NOs: 9, 10, 12 or 13.

[0012] In some embodiments, the engineered uricase polypeptide is configured to form a multimer. In some embodiments, the engineered uricase polypeptide is present as a multimer. In some embodiments, the multimer is a tetramer. In some embodiments, the tetramer is a homotetramer comprising four molecules of the engineered uricase polypeptide.

[0013] In some embodiments, the engineered uricase polypeptide comprises an NSAA linked to a glycan, wherein the NSAA linked to a glycan has a structure according to Formula (IA): AA-L'-L2-L3-X (Formula (IA)) wherein: AA is the NSAA; AA-L1comprises, whereinis where L is coupled to the NSAA, wherein is coupled to L2; L2is a bond oris coupled to L1, wherein iscoupled to L3; L3is asparagine or, wherein " is coupled to L2; wherein is coupled to X; and X is at least one glycan.

[0014] In some embodiments, the glycan comprises GlcNAc,Man - GlcNAc— GlcNAc - GlcNAc - ManMan - GlcNAc ; orMan - GlcNAc — Gal— Sia— GlcNAc - GlcNAc - ManMan - GlcNAc Gal— Sia

[0015] In some embodiments, each of at least two glycan-linked NSAAs of the engineered uricase polypeptide has a structure according to Formula (IA). In some embodiments, each of at least three glycan-linked NSAAs of the engineered uricase polypeptide has a structure according to Formula (IA).

[0016] In some embodiments, the engineered uricase polypeptide comprises a sialic acid (“Sia”). In some embodiments, the engineered uricase polypeptide is configured to form a multimer or is present as multimer in the composition, wherein the multimer is a tetramer, and wherein the multimer comprises from 4 to 48 total sialic acids. In some embodiments, each monomer of the tetramer comprises 1 to 12 sialic acids. In some embodiments, each monomer of the tetramer comprises 6 sialic acids. In some embodiments, each monomer of the tetramer comprises 12 sialic acids.

[0017] In some embodiments, the engineered uricase polypeptide is a recombinant polypeptide. In some embodiments, the engineered uricase polypeptide is not a synthetic polypeptide. In some embodiments, the engineered uricase polypeptide is not PEGylated.

[0018] In some embodiments, the engineered uricase polypeptide further comprises a tag. In some embodiments, the tag comprises an affinity tag. In some embodiments, the affinity tag comprises a His-tag, a GST-tag, a Strep-tag, a SUMO-tag, a MBP-tag, or any combination thereof. In some embodiments, the affinity tag comprises a His-tag.

[0019] In some embodiments, the engineered uricase polypeptide comprises uricase activity when present in a serum. In some embodiments, the serum is human serum. In some embodiments, the uricase activity of the engineered uricase polypeptide is at least 50% of the uricase activity of a control uricase polypeptide, wherein the control uricase polypeptide lacks the NSAA having a sugar moiety coupled thereto. In some embodiments, the uricase activity of the engineered uricase polypeptide is higher than the uricase activity of the control uricase polypeptide. In some embodiments, the uricase activity of the engineered uricase polypeptide and the control polypeptide are measured as the consumption of uric acid over time when the engineered uricase polypeptide and the control polypeptide are incubated at 25°C or 37°C in the presence of uric acid. In some embodiments, the engineered uricase polypeptide is configured to elicit an anti-drug antibody (ADA) response following administration to a subject, wherein the ADA response elicited is lower than an ADA response elicited by a control uricase polypeptide, and wherein the control uricase polypeptide lacks the NSAA having a sugar moiety coupled thereto. In some embodiments, the subject does not elicit a substantial ADA response to the engineered uricase polypeptide. In some embodiments, the engineered uricase polypeptide is an engineered uricase polypeptide described herein, and wherein the control uricase polypeptide comprises an amino acid sequence with 100% sequence identity to at least 200, 225, 250, 275, 300, 301 or 302 contiguous amino acids of the amino acid sequence according to SEQ ID NO: 5.

[0020] Also provided herein is a polynucleic acid comprising a sequence encoding an engineered uricase polypeptide described herein, wherein the NSAA is encoded by a stop codon. Also provided herein is a polynucleic acid comprising a sequence encoding the engineered uricase polypeptide described herein, wherein the NSAA is encoded by a sense codon. In some embodiments, the NSAA is encoded by a codon selected from the group consisting of UAG, UAA, UGA, AGY, UUR, UGC, and AGR, wherein Y is U or C, and wherein R is A or G.

[0021] Also provided herein is a pharmaceutical composition comprising the engineered uricase polypeptide described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for intravenous injection, subcutaneous injection, intradermal injection, intrathecal administration, intracerebral administration, intracerebro ventricular injection, topical administration, inhalation or nasal administration. In some embodiments, thepharmaceutical composition is a liquid. In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent.

[0022] Also provided herein is a method of treating a subject with a condition or a disease comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. In some embodiments, the condition or disease is an inflammatory disease. In some embodiments, the condition or disease is an arthritis. In some embodiments, the condition or disease is gout. In some embodiments, the subject has hyperuricemia. In some embodiments, the condition or disease is hyperuricemia. In some embodiments, the therapeutically effective amount of the pharmaceutical composition is sufficient to decrease a level of uric acid of the subject. In some embodiments, the therapeutically effective amount of the pharmaceutical composition is sufficient to decrease a level of uric acid of the subject to about 6 mg / dL or less. In some embodiments, the subject is a human.

[0023] Also provided herein is a method of making the engineered uricase polypeptide described herein, comprising expressing the engineered uricase polypeptide in a cell or in vitro. In some embodiments, the engineered uricase polypeptide is expressed within the cell. In some embodiments, the cell is abacterial cell or an A. coli cell. In some embodiments, the cell comprises a recoded genome. In some embodiments, a particular codon at substantially all instances within a template genome is changed to an alternative codon in the recoded genome. In some embodiments, a cognate transfer RNA (tRNA) to the particular codon is not present in the recoded genome. In some embodiments, the cell comprises an orthogonal translation system comprising an aminoacyl tRNA synthetase (aaRS) and a cognate tRNA comprising an anticodon that can bind to the particular codon. In some embodiments, the cognate tRNA comprises a bacterial tRNA, a eukaryotic tRNA, a plant tRNA, an archaeal tRNA or a variant thereof. In some embodiments, the cognate tRNA is selected from the group consisting of Methanococcus jannaschii tyrosyl tRNA, Methanosarcina barkeri pyrrolysine tRNA, Methanosarcina mazei pyrrolysine tRNA, Metahnomethylophilus alvus pyrrolysine tRNA, Pyrococcus horokoshii prolyl tRNA, Pyrococcus horokoshii tyrosyl tRNA, and Archaeoglobus fulgidus prolyl tRNA. In some embodiments, a release factor to the particular codon is not present in the recoded genome. In some embodiments, the cell comprises a polynucleotide comprising a sequence encoding the engineered uricase polypeptide, wherein the sequence encoding the engineered uricase polypeptide comprises the particular codon. In some embodiments, the particular codon is reassigned to a non-standard amino acid. In some embodiments, the particular codon is UAG, UAA, UGA, AGY, UUR, UGC, AGR, or a combination thereof, wherein: AGY is selected from the group consisting of AGU, and AGC; UUR is selected from the group consisting of UUA, and UUG; and AGR is selected from the group consisting of AGA, and AGG.

[0024] In some embodiments, the method further comprises culturing the cell under conditions sufficient for the cell to express the engineered uricase polypeptide. In some embodiments, the methodcomprises enriching the engineered uricase polypeptide from the cell. In some embodiments, the method comprises conjugating a sugar moiety or a glycan to a sidechain of the NSAA of the engineered uricase polypeptide. In some embodiments, the conjugating is performed in vitro. In some embodiments, the conjugating comprises chemically conjugating. In some embodiments, the chemically conjugating comprises a click chemistry reaction. In some embodiments, the click chemistry reaction comprises a copper-catalyzed azide-alkyne cycloaddition.

[0025] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0026] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1A depicts a schematic of an exemplary workflow for identifying engineered uricase polypeptide variants.

[0028] FIG. IB depicts a schematic of biological activities of a nonlimiting exemplary engineered uricase polypeptide.

[0029] FIG. 1C depicts a schematic of nonlimiting exemplary engineered glycans.

[0030] FIG. 2A shows the enzymatic activity of a nonlimiting exemplary engineered uricase polypeptide in human serum compared to the control.

[0031] FIG. 2B shows anti-drug antibody (ADA) levels in mice elicited against wild-type uricase or a nonlimiting example of an engineered uricase polypeptide.

[0032] FIG. 3 depicts an exemplary schematic for testing the clinical efficacy of an engineered uricase polypeptide.

[0033] FIG. 4 shows a schematic of a nonlimiting exemplary method for producing a first nonlimiting exemplary glycan-linker conjugate.

[0034] FIG. 5 shows a schematic of a nonlimiting exemplary method for producing a second nonlimiting exemplary glycan-linker conjugate.

[0035] FIG. 6 shows a schematic of a nonlimiting exemplary method for producing a third nonlimiting exemplary glycan-linker conjugate.

[0036] FIG. 7 shows a schematic of a nonlimiting exemplary method for producing a fourth nonlimiting exemplary glycan-linker conjugate.

[0037] FIG. 8 depicts serum uric acid levels (sUA) in mg / dL in mice treated intravenously with wildtype uricase, pegloticase, or a nonlimiting example of an engineered uricase polypeptide (e.g., ProGly- uricase). Serum uric acid levels were measured on days 0, 1, 3, 8, 15, and 22.

[0038] FIG. 9 depicts serum uric acid levels (sUA) in mg / dL in mice treated subcutaneously with wild-type uricase, pegloticase, or a nonlimiting example of an engineered uricase polypeptide (e.g., ProGly-uricase). Serum uric acid levels were measured on days 0, 1, 3, 8, 15, and 22.

[0039] FIG. 10 shows IgG anti-drug antibody (ADA) levels in mice elicited against wild-type uricase or a nonlimiting example of an engineered uricase polypeptide (e.g., an engineered uricase polypeptide comprising four sialic acids per glycan).

[0040] FIG. 11 depicts IgM antibody levels in mice intravenously treated with wild-type uricase, an exemplary engineered uricase polypeptide comprising two sialic acids per glycan (ProGly-BiSia uricase), or an exemplary engineered uricase polypeptide comprising four sialic acids per glycan (ProGly-TetraSia uricase).

[0041] FIG. 12A shows the concentration of uricase (ug / mL) in serum samples from mice taken 0.5 hours after treatment with wild-type uricase, an exemplary engineered uricase polypeptide comprising two sialic acids per glycan (ProGly-BiSia uricase), or an exemplary engineered uricase polypeptide comprising four sialic acids per glycan (ProGly-TetraSia uricase).

[0042] FIG. 12B depicts the concentration of uricase (ug / mL) in serum samples from animals taken 6 hours after treatment with wild-type uricase, an exemplary engineered uricase polypeptide comprising two sialic acids per glycan (ProGly-BiSia uricase), or an exemplary engineered uricase polypeptide comprising four sialic acids per glycan (ProGly-TetraSia uricase).

[0043] FIG. 12C shows the concentration of uricase (ug / mL) in serum samples from animals taken 24 hours after treatment with wild-type uricase, an exemplary engineered uricase polypeptide comprising two sialic acids per glycan (ProGly-BiSia uricase), or an exemplary engineered uricase polypeptide comprising four sialic acids per glycan (ProGly-TetraSia uricase).DETAILED DESCRIPTION

[0044] Certain specific details of this description are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the present disclosure may be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring descriptions of the embodiments. Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in anopen, inclusive sense, that is, as “including, but not limited to.” Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure.

[0045] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. In this application, the use of the singular includes the plural unless specifically stated otherwise.

[0046] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%.

[0047] The term “about” or “approximately” can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i. e. , the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0048] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0049] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0050] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description areexemplary and explanatory only and are not restrictive of any subject matter claimed. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.Definitions

[0051] The term “effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological or therapeutic result.

[0052] "PEGylated” can refer to a molecule with one or more PEG (polyethylene glycol), poly (ethylene oxide) or poly (oxy ethylene)) molecules (e.g., a poly(ethylene glycol), poly (ethylene oxide) or poly (oxy ethylene) polymer or unit thereof) attached thereto. The terms "pegylated" or "pegylation" can refer to one or more PEG conjugated to a molecule (e.g., a polypeptide, an amino acid, an NSAA, a linker, etc.), or the act of conjugating one or more PEG to a molecule (e.g., a polypeptide, an amino acid, an NSAA, a linker, etc.), respectively. A pegylated polypeptide can refer to a polypeptide modified with one or more PEG. For example, a pegylated polypeptide can refer to a pegylated bovine adenosine deaminase (e.g., pegademase), a pegylated 1-asparaginase (e.g., pegaspargase), or a pegylated granulocyte colony stimulating factor (e.g., pegfilgrastim). Pegylated uricases can include, but are not limited to, pegsiticase and pegloticase.

[0053] The term “non-standard amino acid” or “NSAA” refers to any amino acid other than the 20 standard amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine).

[0054] The terms, “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. For example, the term can refer to both short chains, which also commonly are referred to in the art as peptides, for example, and to longer chains, which generally are referred to in the art as proteins. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, a polypeptide comprising an NSAA, or a combination thereof.

[0055] The term “sequence identity” or “percent identity” in the context of two or more nucleic acids or polypeptide sequences, generally refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a local or global comparison window, as measured using a sequence comparisonalgorithm. Suitable sequence comparison algorithms for polypeptide sequences include, e.g., BLASTP using parameters of a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using parameters of a wordlength (W) of 2, an expectation (E) of 1000000, and the PAM30 scoring matrix setting gap costs at 9 to open gaps and 1 to extend gaps for sequences of less than 30 residues (these are the default parameters for BLASTP in the BLAST suite available at https: / / blast.ncbi.nlm.nih.gov); CLUSTALW with parameters of; the Smith- Waterman homology search algorithm with parameters of a match of 2, a mismatch of -1, and a gap of -1; MUSCLE with default parameters; MAEFT with parameters retree of 2 and maxiterations of 1000; Novafold with default parameters; HMMER hmmalign with default parameters.Overview

[0056] Protein pharmaceuticals represent a rapid growing class of drug molecules and are being used clinically for various indications. Unfortunately, the clinical utility of many therapeutic proteins has been undermined by the potential development of unwanted immune responses against the therapeutic protein. The incidence of immunogenicity of several clinically approved therapeutic proteins has been well documented, and some have failed before even reaching clinical trials. The development of antidrug antibodies (AD As) can limit efficacy and negatively impact safety, hampering the clinical utility of the therapeutic protein.

[0057] Glycosylation is a critical posttranslational modification for many therapeutic proteins. Eliminating glycosylation can impair circulating half-life and impact downstream protein interactions and functions. In addition, glycan composition at specific sites can play a critical role in tuning half- life and functionality of a protein. Glycans of glycoproteins can be directly involved in biological recognition processes including intracellular trafficking, cell adhesion, signaling, development, hostpathogen interactions, and immune responses.

[0058] Glycan moieties can be used by the immune system to distinguish “self’ versus “non-sel ’ molecules. Glycolic differences in phylogeny can be used by the immune system to determine whether a molecule is foreign or pathogenic. When a therapeutic protein is recognized by the immune system as a non-self molecule, the immune system can mount an immune response against the therapeutic protein and render the therapeutic protein ineffective. For example, administration of therapeutic proteins may induce humoral immune responses, causing the formation of AD As. In turn, AD As may inactivate the therapeutic protein and cause a loss of targeting and / or an increased clearance of ADA- drug complexes and render the therapeutic protein ineffective. As many therapeutic proteins, including monoclonal antibodies, are glycosylated, and the presence and the composition of the sugar chains are critical for the stability and biological functions, understanding the structure, functions, and the controlof the glycosylation status is crucial for developing and producing homogeneous therapeutic proteins in an efficient manner.

[0059] Carbohydrates are attached to glycopeptides in several ways, including carbohydrates N- linked to asparagine and carbohydrates O-linked to serine and threonine. There is great diversity in the structures created by glycosylation. Such glycans are produced by the catalytic activity of hundreds of enzymes (glycosyltransferases) that are resident in the Golgi complex. Diversity exists at the level of the glycan structure and in positions of attachment of glycans to the protein backbones. Despite the high degree of potential diversity, it is clear that glycosylation is a highly regulated process that shows a high degree of conservation among multicellular organisms.

[0060] Glycosylation is not a template-driven process and two copies of the same protein produced from the same cell can carry different glycoforms with variations of glycan components and / or the sites of glycosylation. The structural complexity and heterogeneity of glycoproteins make it difficult to define a specific glycoform of glycoproteins and to manufacture homogeneous glycoproteins or glycoproteins with minimal batch-to -batch variations, complicating therapeutic applications of glycoproteins. Also, the heterogeneity of glycoproteins is one of the focuses of regulatory agencies, highlighting the significant regulatory and clinical value in developing a method to template glycosylation. Although various chemical and biological strategies have been developed for producing glycan-defmed homogeneous glycoproteins, these strategies do not allow to template glycosylation of multiple defined glycans at multiple amino acids.

[0061] The lack of expression systems that can be used to manufacture polypeptides with wild-type glycosylation patterns has limited the use of such polypeptides as therapeutic agents. It is known in the art that improperly or incompletely glycosylated peptides can be immunogenic, leading to neutralization of the peptide and / or the development of an allergic response. Other deficiencies of recombinantly produced glycopeptides include suboptimal potency and rapid clearance from the bloodstream.

[0062] One approach to solving the problems inherent in the production of glycosylated peptide therapeutics has been to modify the peptides in vitro after their expression. Post-expression in vitro modification of polypeptides has been used for both the modification of existing glycan structures and the attachment of glycosyl moieties to non-glycosylated amino acid residues. A comprehensive selection of recombinant eukaryotic glycosyltransferases has become available, making in vitro enzymatic synthesis of mammalian gly coconjugates with custom designed glycosylation patterns and glycosyl structures possible. See, for example, U.S. Pat. Nos. 5,876,980; 6,030,815; 5,728,554; 5,922,577; as well as WO / 9831826; US2003180835; and WO 03 / 031464.

[0063] There is a need in the art for methods that can generate therapeutic molecules without triggering an immune response against the molecule (such as ADAs). For example, a therapeutic molecule may be designed to not trigger the immune response against itself by having glycan moietiesthat will not trigger the immune response through immune tolerance and / or immune masking. Thus, there is a need in the art for methods that permit one to precisely select the positions within a protein sequence as sites for glycosylation, and to precisely determine the composition of glycans, and to produce batches of glycoproteins with homogenous compositions of glycans. There is a need in the art for methods that produce polypeptides with homogenous glycosylation patterns.

[0064] The compositions and methods described herein can be used to regulate of uric acid levels and thus provide therapeutic benefit. Uric acid is a product generated by purine metabolism in various animals including humans, via the oxidation of xanthine and hypoxanthine. Xanthine and hypoxanthine are produced during the breakdown of guanine and adenine nucleotides, respectively. Uric acid is relatively insoluble, and because humans lack the enzyme to break down uric acid, humans are susceptible to pathologic conditions and diseases associated with heightened levels of uric acid (hyperuricemia). Hyperuricemia facilitates the formation of uric acid crystals in serum, which can precipitate in joints, skin and kidneys, resulting in inflammation of the joints, Gout, renal failure, metabolic acidosis, and hyperkalemia. Various causes of hyperuricemia include, but are not limited to, excessive intake of purine and / or proteins, treatments with uricosuric drugs, congenital metabolic defects, and Lesch-Nyhan syndrome (Kelley, W. N. and Wortman, R. L. Textbook of Rheumatology, 5thedition, pp 1313-1351, 1997; which is incorporated by reference in its entirety). Hyperuricemia can also occur in patients that have had heart or kidney transplants and are being treated with immunosuppressive agents, and can lead to loss of kidney function in these patients and produce significant morbidity and mortality. Renal handling of uric acid can require complicated procedures such as glomerular filtration, reabsorption of filtered urate, tubular secretion, and finally postsecretory reabsorption. Hyperuricemia is also found in patients with malignant diseases, including hematologic malignancies, such as leukemias and lymphomas. CAR-T cell therapies, chemotherapy and radiation therapy can induce a life-threatening condition known as tumor lysis syndrome (Kalemkerian, G. P., Darwish, B., and Varterasian, M. L. Am J. Med. 103, 363-367, 1997; Lorigan, P. C., Woodings, P. L., Morgenstern, G. R., and Scarffe, J. H. Ann Oncol. 7, 631-636, 1996; Hande, K. R., and Garrow, G. C. Am. J. Med. 94, 133-139, 1993; each of which is incorporated by reference in its entirety). Tumor lysis syndrome is characterized by the rapid development of hyperuricemia, hyperkalemia, hyperphosphatemia, and acute renal failure. Acute renal failure is the result of the intrarenal precipitation of uric acid. Tumor lysis syndrome can be triggered by cell death induced by CAR-T cell therapies, chemotherapy or radiotherapy, resulting in the release of intracellular substances. However, occasionally cancer patients with a heavy tumor burden may exhibit hyperuricemia and other features of tumor lysis syndrome even in the absence of radiotherapy or chemotherapy because of the high turnover of malignant cells with subsequent catabolism of released purines into uric acid.

[0065] Therapy for the prevention and treatment of the acute renal failure associated with tumor lysis syndrome is a considerable challenge and is currently unsatisfactory for a number of reasons. Methodsof treating hyperuricemia include hydration, urinary alkalinization, osmotic diuresis and allopurinol therapy. However, the difficulty in the treatment of hyperuricemia lies in the potential for aggravating other consequences of tumor lysis syndrome. For example, alkalinization of urine to increase uric acid solubility facilitates precipitation of calcium phosphate. Allopurinol (4-hydroxypurinol), an analogue of xanthine, has long been the standard treatment for hyperuricemia and also for the prevention of tumor lysis syndrome. Allopurinol is converted to oxypurinol, which then binds to and inhibits xanthine oxidase, the enzyme that catalyzes the conversion of hypoxanthine and xanthine to uric acid. As a result, uric acid production is inhibited, and xanthine and hypoxanthine concentrations increase. However, allopurinol does not remove uric acid that is already present and deposited intrarenally as crystals. As a result, it is often several days (sometimes 10-14 days) from the initial treatment with allopurinol before a significant decrease in uric acid in serum can be observed. While the solubilities of xanthine and hypoxanthine are only slightly greater than that of uric acid, the purines are catabolized to xanthine, hypoxanthine, and uric acid during allopurinol treatment, rather than mostly to uric acid. The result is that the urinary concentration of uric acid decreases below its solubility and further formation of uric acid crystals is prevented. However, during excessive catabolism of purines, allopurinol therapy may lead to intrarenal precipitation of hypoxanthine and xanthine, with further aggravation of acute renal failure. This situation has been well documented in clinical practice. Furthermore, allopurinol therapy is also associated with significant toxicity and can cause death. Allopurinol can produce severe toxic effects, including cutaneous hypersensitivity reactions, leukopenia, and hepatomegaly. This drug has been also implicated in the induction of tubulointerstitial nephritis. In addition, allopurinol may cause adverse drug interactions, as has been shown for 6- mercaptopurine and adenine arabinoside, drugs often used to treat lymphoproliferative disease and leukemia (Lauter, C B, Bailey, E J, Lerner, AM. J Infect. Dis 1976; 134, 75-79). Finally, although allopurinol can block the formation of uric acid, it does little to solubilize the uric acid which is already present. All of these adverse effects sometimes make allopurinol ineffective for the treatment of acute hyperuricemia in tumor patients. Thus, allopurinol is not an ideal drug for the treatment of hyperuricemia.

[0066] Dialysis and continuous arteriovenous hemodialysis are additional methods that are used to remove uric acid in patients with hyperuricemia. However, these treatment methods are problematic in patients with malignancies because of the risk for severe bleeding caused by thrombocytopenia or the need for anticoagulation. Furthermore, because of the invasive nature of these treatments, there is an increased risk for fatal infections in patients who are in many instances immunocompromised due to their illness or therapy they have received.

[0067] In most mammals other than humans, uric acid is oxidized by uricase, an enzyme of the purine degradation pathway, to allantoin (conversion of uric acid+Ch into allantoin+CCh). Uricase, also known as urate oxidase, is composed of four uricase subunits, forming a homotetrameric enzyme. Theuricase subunits are arranged in a tetrameric structure, with the active sites located at the interfaces between the subunits. Because humans lack uricase, they do not produce allantoin.

[0068] Uricase can be used for treating hyperuricemia and tumor lysis syndrome (London, M., and Hudson, P. B. Science, 125, 937-938, 1957; Oberling, F. and Lang, J. M. Nouvelle Presse Med 3, 2026, 1974; Robert, A., Corberand, J. and Regnier, C. Rev. Med. Toulouse 12, 1093-1100, 1976; Masera, G., et. Al., J. Pediatrics 100, 152-155, 1982; Jankovic, M., et. Al. Am. J. Pediatr. Hematol. Onocol. 7, 202-204, 1985; Masera, G. and Jankovic, M. Ann. Oncol. 8, 407, 1996; Jones, D. P, Mahmoud, H. and Chesney, R. W. Pediatr. Nephrol. 9, 206-212, 1995; each of which is incorporated in its entirety). Treatment with uricase converts uric acid into the highly soluble allantoin. Furthermore, uricase, if adequately filtered into the urine, may even dissolve already precipitated uric acid crystals and improve renal function.

[0069] Annually, about 10 million patients are diagnosed with Gout in the U.S. Currently available therapeutics for Gout include pegylated uricases (such as pegloticase, rasburicase, or pegadricase) and non-pegylated uricases, both of which have a variety of drawbacks. For example, only -100,000 refractory patients are eligible for pegloticase. Furthermore, the successful penetration of pegloticase is only applicable to < 5% of the patients administered with the drug. Similar drawbacks can also occur with other non-pegylated uricase therapies. Currently available uricase therapy can trigger anti-drug antibodies (AD As) against the administered uricase, rendering the therapy ineffective. For example, anti-pegloticase antibodies develops in 92% of patients treated with pegloticase every 2 weeks versus 28% for placebo.

[0070] Additionally, ADAs have been detected to be elicited against the PEG molecule itself. Anti- PEG antibodies are detected in 42% of patients treated with pegloticase. High anti-pegloticase antibody titer is associated with a failure to maintain pegloticase-induced normalization of uric acid. The impact of anti-PEG antibodies on patients’ responses to other PEG-containing therapeutics is unknown. There is a higher incidence of infusion reactions in patients with high anti-drug antibody (for example, anti-pegloticase antibody) titer: 53% (16 of 30) of patients treated with pegloticase every 2 weeks had infusion reactions and 6% of patients treated with pegloticase every 2 weeks had undetectable or low antibody titers. Additionally, other anti-drug antibodies, such as anti-uricase antibodies, can be produced within the patients administered non-pegylated (or pegylated) uricase therapies. Currently available uricase therapeutics also have low response rates and are administered intravenously. Additionally, PEGylated uricase may require chemical synthesis, which reduces its applicability and scalability.

[0071] The methods and compositions described herein can overcome these drawbacks. The methods described herein can be used to produce engineered uricase polypeptides that avoid inducing an ADA immune response or elicit less of an ADA immune response than elicited against current therapies. The methods described herein can be used to produce engineered uricase polypeptides that are able toreduce uric acid levels, such as serum uric acid levels, to a greater extent than the level of uric acid reduced using current therapies, such as pegloticase. The compositions described herein, such as engineered uricase polypeptides, can contain a non-standard amino acid (NSAA), which provides a handle to attach a glycan, such as via click chemistry. The compositions described herein, such as engineered uricase polypeptides containing a glycan attached to an NSAA, can be used to treat subject with high uric acid levels and avoid inducing an ADA immune response or elicit less of an ADA immune response than elicited against current therapies. The glycan(s) of the compositions described herein can be used to mask the polypeptide from recognition by the host immune system and / or induce immune tolerance such that an ADA immune response is prevented or such that less of an ADA immune response is elicited than elicited using current therapies. There are over 700 known nonstandard amino acids any of which may be included in a polypeptide composition described herein. Examples of non-standard amino acids include, but are not limited to, P-alanine, D-alanine, 4- hydroxyproline, desmosine, D-glutamic acid, y-aminobutyric acid, P-cyanoalanine, norvaline, p- azido-l-phenylalanine (pAzF), p-acetophenylalanine (pAcF), p-azidomethyl-L-phenylalanine (pAMF), 4-Biphenylyl-L-alanine (BipA), azido-lysine (AzK), 4-(E)-butenyl-4(R)-methyl-N-methyl- L-threonine, N-methyl-L-leucine, selenocysteine, and statine. Another exemplary non-standard amino acid (NSAA) is selenocysteine (Sec). Examples of preferred NSAAs for incorporation into the engineered uricase polypeptides described herein are p-azido-l-phenylalanine (pAzF) and para- azidomethyl-1 -phenylalanine (pAMF).

[0072] In one aspect, provided herein is a polypeptide with uricase activity comprising a nonstandard amino acid (NSAA), such as an engineered uricase polypeptide. For example, an engineered uricase polypeptide containing an NSAA can be produced in a bacterial cell and purified therefrom. In some embodiments, the NSAA is conjugated to a linker. For example, an engineered uricase polypeptide containing an NSAA that was produced in a bacterial cell and purified therefrom can be reacted with a molecule containing a linker to link the linker to the NSAA. In some embodiments, the molecule containing a linker also comprises another chemical moiety, such as a glycan. For example, an engineered uricase polypeptide containing an NSAA that was produced in a bacterial cell and purified therefrom can be reacted with a molecule containing a linker and a glycan to link the linker and the glycan to the NSAA. In some embodiments, the molecule containing a linker can be reacted with another chemical moiety, such as a glycan, to link the other chemical moiety to the linker. For example, an engineered uricase polypeptide containing an NSAA that was produced in a bacterial cell and purified therefrom can first be reacted with a molecule containing a linker to form an engineered uricase polypeptide containing a linker linked to an NSAA; the linker linked to an NSAA of engineered uricase polypeptide can then be linked to another chemical moiety, such as a glycan. For example, an engineered uricase polypeptide containing an NSAA that was produced in a bacterial cell and purified therefrom can be reacted with a molecule containing a linker previously linked to another chemicalmoiety, such as a glycan, to form an engineered uricase polypeptide containing a linker linking an NSAA to the other chemical moiety, such as a glycan. In some embodiments, the linker is capable of reacting with an NSAA such that the linker forms a covalent bond with the NSAA. In some embodiments, the linker is capable of reacting with a glycan containing moiety such that the linker forms a covalent bond with the glycan. In some embodiments, the NSAA comprises an azide group. In some embodiments, the NSAA a 1,2, 3 -triazole is formed when the linker is linked to the NSAA. In some embodiments, the linker comprises dibenzocyclooctyne (DBCO).

[0073] In some embodiments, the one or more sugar moieties comprises a glycan. In some embodiments, the NSAA comprises or be conjugated to a glycan stub. In some embodiments, the glycan stub is a moiety to which one or more sugar moieties can be conjugated thereto. In some embodiments, a glycan stub can also comprise a glycan or sugar moiety.

[0074] Provided herein are polypeptides, such as engineered uricase polypeptides, that have minimal immunogenicity when administered to a host. The polypeptides, such as engineered uricase polypeptides, may have substantially homogenous glycan moieties which mitigate immunogenicity and / or mask the polypeptides from the immune system of a host. In some embodiments, the polypeptides have uricase activity. In some embodiments, the polypeptides, such as engineered uricase polypeptides, can be used for treating hyperuricemia or a disease or condition associated with hyperuricemia (e.g., gout). Also provided herein are compositions and kits comprising the polypeptides, such as engineered uricase polypeptides. Also provided herein are methods for using the polypeptide, such as engineered uricase polypeptides, for treating a disease or condition. In some embodiments, the disease or condition is associated with hyperuricemia (such as but not limited to gout). In some embodiments, the polypeptides are not pegylated. In some embodiments, the polypeptides, such as engineered uricase polypeptides, do not induce anti-drug antibodies (ADAs) when administered to a host, or trigger significantly less ADAs compared to currently available therapeutics when administered to a host. The compositions described herein can be administered intravenously but also via other routes, such as subcutaneously.Polypeptides

[0075] Provided herein is a polypeptide comprising a non-standard amino acid (NSAA). In some embodiments, a polypeptide comprising a NSAA is an engineered uricase polypeptide. In some embodiments, a polypeptide comprises an engineered polypeptide with uricase activity. In some embodiments, at least one amino acid of the polypeptide is replaced with the NSAA.

[0076] There are over 700 known non-standard amino acids any of which may be included in a polypeptide described herein. Non-limiting examples of NSAAs include L-Tryptazan, 5-Fluoro-L- tryptophan, L-Ethionine, L-Selenomethionine, selenocysteine, P-alanine, D-alanine, 4- hydroxyproline, desmosine, D-glutamic acid, y-aminobutyric acid, P-cyanoalanine, norvaline, 4-(E)- butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-leucine, statine, Trifluoro-L-methionine, L-Norleucine, L-Homopropargylglycine, (2S)-2-amino-5-(methylsulfanyl) pentanoic acid, (2S)-2- amino-6-(methylsulfanyl) hexanoic acid, cystine, Para-fluoro-L-phenylalanine, Para-iodo-L- phenylalanine, demosine, isodesmosine, Para-azido-L-phenylalanine, Para-acetyl-L-phenylalanine, Para-benzoyl-L-phenylalanine, Meta-fluoro-L-tyrosine, O-methyl-L-tyrosine, Para-propargyloxy-L- phenylalanine, hydroxyproline, gamma-carboxygluamate, phosphoserine, phosphothreonine, phosphotyrosine, N-acetyl lysine, methyl lysine, (2S)-2-aminooctanoic acid, (2S)-2-aminononanoic acid, (2S)-2-aminodecanoic acid, (2S)-2-aminohept-6-enoic acid, (2S)-2-aminooct-7-enoic acid, L- Homocysteine, (2S)-2-amino-5-sulfanylpentanoic acid, (2S)-2-amino-6-sulfanylhexanoic acid, L-S- (2 -nitrobenzyl) cysteine, L-S-ferrocenyl-cysteine, L-O-crotylserine, L-O-(pent-4-en-l-yl)serine, L-O- (4,5-dimethoxy-2-nitrobenzyl)serine, (2S)-2-amino-3-({[5-(dimethylamino)naphthalen-l- yl]sulfonyl}amino)propanoic acid, (2S)-3-[(6-acetyl-naphthalen-l-yl)amino]-2-aminopropanoic acid, pyrrolysine, N6-[(propargyloxy)carbonyl]-L-lysine, L-N6-acetyllysine, N6 -trifluoroacetyl-L-lysine, N6-{[l-(6-nitro-l,3-benzodioxol-5-yl)ethoxy]carbonyl}-L-lysine, N6-{[2-(3-methyl-3H-diaziren-3- yl)ethoxy] carbonyl }-L-ly sine, p-azidophenylalanine, and 2-aminoisobutyric acid.

[0077] In some embodiments, an engineered uricase polypeptide comprises an NSAA that is selenocysteine. In some embodiments, an engineered uricase polypeptide comprises an NSAA that is not selenocysteine. In some embodiments, an engineered uricase polypeptide comprises an NSAA that is pyrrolysine. In some embodiments, an engineered uricase polypeptide comprises an NSAA that is not pyrrolysine. In some embodiments, an engineered uricase polypeptide comprises an NSAA that is p-azidophenylalanine (pAzF). In some embodiments, an engineered uricase polypeptide comprises an NSAA that is p-acetophenylalanine (pAcF). In some embodiments, an engineered uricase polypeptide comprises an NSAA that is p-azidomethyl-L-phenylalanine (pAMF). In some embodiments, an engineered uricase polypeptide comprises an NSAA that is azido-lysine (AzK). In some embodiments, an engineered uricase polypeptide comprises an NSAA that is 2-aminoisobutyric acid. In some embodiments, an engineered uricase polypeptide comprises an NSAA that is cystine. In some embodiments, an engineered uricase polypeptide comprises an NSAA that is desmosine. In some embodiments, an engineered uricase polypeptide comprises an NSAA that is isodesmosine. In some embodiments, an engineered uricase polypeptide comprises an NSAA that is hydroxyproline. In some embodiments, an engineered uricase polypeptide comprises an NSAA that is gammacarboxyglutamate. In some embodiments, an engineered uricase polypeptide comprises an NSAA that is phosphoserine. In some embodiments, an engineered uricase polypeptide comprises an NSAA that is phosphothreonine. In some embodiments, an engineered uricase polypeptide comprises an NSAA that is phosphotyrosine. In some embodiments, an engineered uricase polypeptide comprises an NSAA that is N-acetyl lysine. In some embodiments, an engineered uricase polypeptide comprises an NSAA that is methyl -lysine.

[0078] Provided herein is a polypeptide, such as an engineered uricase polypeptide, comprising a nonstandard amino acid (NSAA). The NSAA can comprise any amino acid other than the 20 standard amino acids. In some embodiments, a polypeptide, such as an engineered uricase polypeptide, comprises an NSAA linked to a glycan, wherein the NSAA linked to a glycan has a structure of Formula (I):AA-L-XFormula (I) wherein AA is an NSAA, L comprises a linker, and X is absent or comprises a glycan.

[0079] In some embodiments, a polypeptide, such as an engineered uricase polypeptide, comprises an NSAA linked to a glycan, wherein the NSAA linked to a glycan has a structure of Formula (IA):AA- I -L2-L3-XFormula (IA) wherein AA is an NSAA, L1-!? is a linker, L3is a standard amino acid, and X is absent or a glycan (or any sugar moiety described herein).

[0080] In some embodiments, the NSAA is pAzF. In some embodiments, the NSAA is P-alanine, D- alanine, 4-hydroxyproline, desmosine, D-glutamic acid, y-aminobutyric acid, P-cyanoalanine, norvaline, p-azido-l-phenylalanine (pAzF), p-acetophenylalanine (pAcF), p-azidomethyl-L- phenylalanine (pAMF), 4-Biphenylyl-L-alanine (BipA), azido-lysine (AzK), 4-(E)-butenyl-4(R)- methyl-N-methyl-L-threonine, N-methyl-L-leucine, selenocysteine, statine, or selenocysteine (Sec). In some embodiments, the NSAA is selected from the group consisting of para-azidophenylalanine, azido-lysine, azido-alanine, para-azidomethyl-l-phenylalanine, p-propargyloxy-l-phenylalanine, p- benzoylphenylalanine, p-acetylphenylalanine, p-propargyloxy-l-phenylalanine, propargylglycine, homopropargylglycine, selenocysteine, p-azido-L-phenylalanine, and para-isothiocyanate phenylalanine. In some embodiments, the NSAA is p-azido-L-phenylalanine.

[0081] In some embodiments, L1-!? of Formula (IA) comprises an alkyne. In some embodiments, L1- L2-L3of Formula (IA) comprises an alkyne. In some embodiments, L'-L2or L1-L2-L3comprises 4- pentynoic acid, propargylamine, 6-azidohexanoic acid, dibenzocyclooctyne (DBCO), 3-butyn-l-ol, ethynylbenzene, and / or PEG-alkyne.

[0082] In some embodiments, L3of Formula (IA) comprises an amino acid. In some embodiments, L3-X of Formula (IA) comprises a structure according to Table 3. In some embodiments, L3-X of Formula (IA) comprises a structure of Formula (II): AA1-L4-X; wherein X is a glycan or a glycan stub, L4is a linker, and AA1is any amino acid (such as standard amino acid). In some embodiments, X of Formula (I), (IA), and / or (II) comprises a sugar moiety or a glycan. In some embodiments, X is a standard amino acid selected from the group consisting of glycine (Gly or G), alanine (Ala or A), valine (Vai or standard V), leucine (Leu or L), isoleucine (He or I), proline (Pro or P), phenylalanine (Phe or F), tryptophan (Trp or W), methionine (Met or M), serine (Ser or S), threonine (Thr or T),cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), glutamine (Gin or Q), aspartic acid (Asp or D), glutamic acid (Glu or E), lysine (Lys or K), arginine (Arg or R), and histidine (His or H).In some embodiments, L3is a linker. In some embodiments, L3is

[0083] In some embodiments, L3-X of Formula (IA) has the structure of Formula (II): AA1-L4-X; wherein X is a glycan, L4is a linker, and AA1is any amino acid.

[0084] In some embodiments, X is a glycan. In some embodiments, X is a glycan stub. In some embodiments, the glycan stub is a sugar moiety to which one or more sugar moieties can be conjugated.

[0085] In some embodiments, I -L2-L3-X of Formula (IA) can refer to a gly can-linker conjugate. In some embodiments, the gly can-linker conjugate comprises one or more sugar moieties and an alkyne- containing linker. In some embodiments, click chemistry is used to conjugate the glycan-linker conjugate to a NSAA. In some embodiments, click chemistry is used to conjugate the glycan-linker conjugate to a polypeptide comprising one or more NSAAs. In some embodiments, click chemistry is used to conjugate the glycan-linker conjugate to an engineered uricase polypeptide comprising one or more NSAAs. In some embodiments, Formula (IA) can refer to a glycan-linker-NSAA, wherein X is the glycan, L1-L2-L3-is the linker, and NSAA is an amino acid other than the 20 standard amino acids.

[0086] In some embodiments, the polypeptide, such as an engineered uricase polypeptide, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NSAAs. In some embodiments, the polypeptide, such as an engineered uricase polypeptide, comprises at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NSAAs. In some embodiments, the polypeptide, such as an engineered uricase polypeptide, comprises more than one NSAA, and the at least two NSAAs of the polypeptide are the same. In some embodiments, the polypeptide, such as an engineered uricase polypeptide, comprises more than one NSAA, and at least two NSAAs of the polypeptide are different NSAAs. In some embodiments, the polypeptide, such as an engineered uricase polypeptide, comprises one NSAA. In some embodiments, the polypeptide, such as an engineered uricase polypeptide, comprises two NSAAs. In some embodiments, the polypeptide, such as an engineered uricase polypeptide, comprises three NSAAs. In some embodiments, the polypeptide, such as an engineered uricase polypeptide, comprises four NSAAs. In some embodiments, the polypeptide, such as an engineered uricase polypeptide, comprises five NSAAs. In some embodiments, the polypeptide, such as an engineered uricase polypeptide, comprises six NSAAs. In some embodiments, the polypeptide, such as an engineered uricase polypeptide, comprises seven NSAAs. In some embodiments, the polypeptide, such as an engineered uricase polypeptide, comprises eight NSAAs. In some embodiments, the polypeptide, such as an engineered uricase polypeptide, comprises nine NSAAs. In some embodiments, the polypeptide, such as an engineered uricase polypeptide, comprises ten NSAAs. In some embodiments, the polypeptide, such as an engineered uricase polypeptide, comprises eleven NSAAs. In some embodiments, the polypeptide, such as an engineered uricase polypeptide, comprises twelve NSAAs. In some embodiments, thepolypeptide, such as an engineered uricase polypeptide, is a homotetramer, wherein each monomer the homotetramer comprises three NSAAs.

[0087] The polypeptides disclosed herein may further comprises one or more sugar moieties or glycans conjugated to one or more of the NSAAs directly or indirectly (such as through a linker). In some embodiments, the sugar moieties or glycans are heterogenous. Heterogeneous sugar moieties or glycans can refer to the presence of different types of sugar molecules. Heterogeneous glycans can differ in terms of their composition, length, branching patterns, and types of sugar moieties contained therein. In some embodiments, the one or more sugar moieties or glycans are homogenous. In some embodiments, the one or more sugar moieties or glycans are conjugated to an NSAA. In some embodiments, the one or more sugar moieties or glycans are conjugated to an NSAA directly. In some embodiments, the one or more sugar moieties or glycans are conjugated to an NSAA via a linker. In some embodiments, the presence of glycans can play a crucial role in mitigating immunogenicity. For example, glycosylation can influence immune system interactions by modulating the recognition and signaling processes involved in immune cell activation and regulation. In some embodiments, glycan structures can be involved in immune suppression and tolerance induction. For example, glycosylation can help in allowing the immune system to recognize the polypeptide comprising a NSAA disclosed herein as a self-molecule rather that a non-self-molecule. In some embodiments, the one or more sugar moieties comprise an immune system masking glycan.

[0088] Glycan masking can refer to a technique in which sugar chains (glycans) are added on the surface of therapeutic proteins to hide all or part of the therapeutic protein and thus evade the immune system. By incorporating glycosylation, this masking strategy enables the polypeptide disclosed herein to escape the immune system and prevents or reduces production and / or binding of ADAs compared to the polypeptide without glycosylation. In some embodiments, the polypeptide, such as an engineered uricase polypeptide, has substantially homogenous glycan moieties which mitigate immunogenicity and / or mask the polypeptide from recognition and / or clearance by the immune system. In some embodiments, the polypeptide, such as an engineered uricase polypeptide, has uricase activity. In some embodiments, the polypeptide, such as an engineered uricase polypeptide, is enzymatically active within human serum.

[0089] Provided herein are engineered polypeptides, such as engineered uricase polypeptides, that are less immunogenic than the non-engineered version of the polypeptide. Immune responses against therapeutic proteins that contribute to immunogenicity can arise from two different mechanisms: a classical immune response, or by breaking tolerance. In healthy individuals, the immune system maintains homeostasis between self and non-self-proteins by negative selection of self-reactive immune cells, and proteins that are recognized as foreign can initiate a classical immune response in patients. This response can be characterized by the formation of antibodies (e.g., ADAs) and is typically first seen within days to weeks after administration. These types of responses can be longlasting and very difficult to reverse once memory B-cells have been generated. Immunogenicity of therapeutic proteins can manifest as the development of AD As as well as hypersensitivity reactions. Hypersensitivity reactions can be distinguished into four types (Type I, II, III, and IV) depending on the mechanism of response and effector molecules generated. ADA responses against therapeutic proteins can be separated into two distinct classes; neutralizing and binding antibodies. Measurement of ADAs or hypersensitivity reactions can provide insight into the immunogenicity of therapeutic proteins. In some aspects, provided herein is a polypeptide comprising an NSAA linked to a glycan, such as an engineered uricase polypeptide comprising an NSAA linked to a glycan, with immunogenicity mitigating activity, such as the ability suppress or attenuate immune responses against the polypeptide. In some embodiments, a polypeptide comprising an NSAA linked to a glycan, such as an engineered uricase polypeptide comprising an NSAA linked to a glycan, can avoid eliciting and immune response when administered to a host. For example, the immunogenicity mitigating activity of a polypeptide can aid to hide or disguise the polypeptide as a self-molecule, preventing an immune response to the polypeptide. In some embodiments, the immunogenicity mitigating activity of the polypeptide disclosed herein can aid in maintaining immune homeostasis and preventing hypersensitivity reactions.

[0090] In some embodiments, a polypeptide comprising an NSAA linked to a glycan, such as an engineered uricase polypeptide comprising an NSAA linked to a glycan, comprises immunomodulatory activity, such as the ability to regulate or modify an immune system's response. Immunomodulatory activity can involve influencing the balance and function of immune cells, cytokines, and other components of the immune system. In some embodiments, a polypeptide comprising an NSAA linked to a glycan, such as an engineered uricase polypeptide comprising an NSAA linked to a glycan, can suppress an immune response. For example, a polypeptide comprising an NSAA linked to a glycan, such as an engineered uricase polypeptide comprising an NSAA linked to a glycan, may not be cleared or cleared to a lesser extent, by the immune system compared to a nonengineered version of the polypeptide that does not comprise a NSAA and / or that does not comprises a glycan.

[0091] In some embodiments, a polypeptide comprises oxidoreductase activity. In some embodiments, a polypeptide comprises a uricase, a functional fragment thereof, or a variant thereof. In some embodiments, the uricase comprises a NSAA. In some embodiments, the uricase comprising a NSAA comprises one or more sugar moieties or glycans linked to the NSAA. The modified or glycosylated uricases disclosed herein offers several advantages over unmodified uricases and pegloticase for treating diseases or conditions, such as gout. For example, the glycosylated uricases disclosed herein can have prolonged half-life, enhanced stability, and / or reduced immunogenicity when compared to pegloticase, a non-glycosylated uricase and / or a uricase that does not comprise a NSAA. In some embodiments, the glycosylated uricase has a higher molecular weight compared to anon-glycosylated uricase and / or a uricase that does not comprise a NSAA. The increased molecular weight of the glycosylated uricase can allow the glycosylated uricase to circulate in a host for a longer duration, thus prolonging the half-life of the glycosylated uricase when compared to a nonglycosylated uricase and / or a uricase that does not comprise a NSAA. In some embodiments, the glycosylated uricase has increased bioavailability and / or therapeutic effectiveness when compared to a non-glycosylated uricase and / or a uricase that does not comprise a NSAA. For example, extended half-life of the glycosylated uricase can allow for less frequent dosing compared to a non-glycosylated uricase and / or a uricase that does not comprise a NSAA. In some embodiments, the glycosylated uricase has increased stability when compared to a non-glycosylated uricase and / or a uricase that does not comprise a NSAA. For example, the glycosylation can help to protect the uricase from enzymatic degradation and prolong the functional lifespan of the glycosylated uricase when compared to a non- glycosylated uricase and / or a uricase that does not comprise a NSAA.

[0092] In some embodiments, the glycosylated uricase exhibits reduced immunogenicity compared to a non-glycosylated uricase and / or a uricase that does not comprise a NSAA. By conjugating glycans to the uricase, the immune system is less likely to recognize the uricase as foreign and mount an immune response against the uricase. In some embodiments, the glycosylated uricase reduces the risk of developing neutralizing antibodies when compared to a non-glycosylated uricase and / or a uricase that does not comprise a NSAA. For example, the glycosylation can potentially mitigate hypersensitivity reactions, infusion-related reactions, and other adverse events that may occur with non-glycosylated uricase therapy.

[0093] In some embodiments, the glycosylated uricase can have enhanced interaction with certain cell types when compared to a uricase without glycosylation and / or a NSAA. For example, by incorporating specific glycans, glycosylation can enhance the interaction and uptake of antigens by hepatic antigen-presenting cells (APCs), potentially influencing immune responses and promoting antigen presentation. Glycosylation can play a crucial role in targeting to APCs due to the unique characteristics of these cells and their microenvironment in the liver. For example, many hepatic APCs, such as liver-resident macrophages and liver dendritic cells, express a mannose receptor on their cell surface. ASGPR (asialoglycoprotein receptor) is a specific receptor expressed on the surface of hepatocytes and liver sinusoidal endothelium, which recognizes and binds to specific glycan structures, particularly those containing terminal galactose or N-acetylgalactosamine residues. In some embodiments, the glycosylated uricase can have enhanced interaction with hepatic APCs when compared to a uricase without glycosylation and / or a NSAA. In some embodiments, the glycosylated uricase comprises glycan structures which can bind to and / or be recognized by an APC mannose receptor. For example, the glycosylation can play a significant role in increasing the avidity of hepatocyte ASGPR interactions with the glycosylated uricase. In some case, the glycosylation can increase the avidity through enhanced affinity and / or cooperative binding to ASGPR. In someembodiments, the increased avidity can enhance the delivery and efficacy of the glycosylated uricase when compared to a uricase without glycosylation and / or a NSAA. In some embodiments, the avidity of glycosylated uricase and ASGPR can be assessed by assaying the uptake of the glycosylated uricase by the APC in APC monoculture in a glycan-dependent manner. Additionally, cytokine profiling of the tolerogenic response (for example, but not limited to, via TGF-beta).

[0094] In some embodiments, the glycosylated uricase can have enhanced interaction with peripheral dendritic cells (DCs) when compared to a uricase without glycosylation and / or a NSAA. Glycosylation can be utilized to facilitate the targeting of peripheral DCs by enhancing their recognition and uptake of gly can-modified molecules. For example, DCs express various C-type lectin receptors on their cell surface, which are involved in glycan recognition. These receptors, such as DC-SIGN, DEC-205, and others, specifically bind to certain glycan structures. By incorporating these specific glycans into the engineered uricase disclosed herein, glycosylation can exploit the recognition capabilities of C-type lectin receptors and enhance the targeting of peripheral DCs. In some embodiments, the glycosylated uricase comprises glycan structures which can bind to and / or be recognized by DC-specific C-type lectin receptors. In some embodiments, the glycosylated uricase comprises glycan structures which can bind to and / or be recognized by DC-specific I-type lectins (immunoglobulin-type lectins). For example, glycosylation can play a crucial role in mediating interactions with Siglecs (sialic acidbinding I-type lectins), a family of cell surface receptors that bind specifically to sialic acid-containing glycans. In some embodiments, the glycosylated uricase comprises glycan structures which can bind to and / or be recognized by DC-specific Siglecs.

[0095] For example, glycan 2 of FIG. 1C can have increased avidity to ASGPR and / or enhance antigen delivery relative to glycan 1 of FIG. 1C. Glycan 4 of FIG. 1C can have increased avidity to a siglec, enhanced antigen delivery, and / or increased potentiation of tolerizing siglec signaling, relative to glycan 3 of FIG. 1C. For example, a glycosylated uricase, as disclosed herein, comprising four sialic acids per glycan (e.g., TetraSialylated or TetraSia glycan) can elicit decreased IgG ADA levels in a subject compared to the glycosylated uricase comprising two sialic acids per glycan (e.g., BiSialylated or BiSia glycan), see FIG. 10 and FIG. 2B, respectively. A glycosylated uricase, as disclosed herein, comprising four sialic acids per glycan (e.g., ProGly-TetraSia uricase) can also elicit decreased IgM antibodies in a subject when compared to the glycosylated uricase comprising two sialic acids per glycan (e.g., ProGly-BiSia uricase), see FIG. 11. Additionally, a glycosylated uricase, as disclosed herein, comprising four sialic acids per glycan (e.g., ProGly-TetraSia uricase) can elicit increased circulating uricase levels in a subject compared to the glycosylated uricase comprising two sialic acids per glycan (e.g., ProGly-BiSia uricase), see FIGs. 12A-12C. Without wishing to be bound by theory, such increased biological activities can be due to the structure / complexity of the sugar moiety(ies) (and thus the molecule(s) that the sugar moiety(ies) can interact with).

[0096] Glycosylation can play a significant role in increasing the avidity of DCs interactions with the glycosylated uricase. In some embodiments, the glycosylation can increase the avidity through enhanced affinity and / or cooperative binding to Siglecs. In some embodiments, the glycosylation can potentiate tolerizing Siglec signaling. In some embodiments, the increased avidity can enhance the delivery and efficacy of the glycosylated uricase when compared to a uricase without glycosylation and / or a NSAA. In some embodiments, treatment of a subject in need thereof with a glycosylated uricase comprising four sialic acids per glycan (e.g., ProGly-TetraSia uricase) or two sialic acids per glycan (e.g., ProGly-BiSia uricase) can elicit decreased IgG ADA levels in the subject compared to IgG ADA levels in a subject treated with a wild-type uricase, see for example FIG. 10 and FIG. 2B, respectively and Example 4. In some embodiments, treatment of a subject in need thereof with a glycosylated uricase comprising four sialic acids per glycan (e.g., ProGly- TetraSia uricase) or two sialic acids per glycan (e.g., ProGly-BiSia uricase) can elicit decreased IgM antibodies levels in a subject compared to IgM antibodies levels in a subject treated with a wild-type uricase, see for example FIG. 11 and Example 4. In some embodiments, treatment of a subject in need thereof with a glycosylated uricase comprising four sialic acids per glycan (e.g., ProGly-TetraSia uricase) or two sialic acids per glycan (e.g., ProGly-BiSia uricase) can elicit increased circulating uricase levels in a subject compared to circulating uricase levels in a subject treated with a wild-type uricase, see for example FIGs. 12A-12C and Example 4.

[0097] In some embodiments, the polypeptide comprises a uricase derived from a fungus, an animal, or a bacteria. In some embodiments, the polypeptide comprises a uricase derived from a mammal. In some embodiments, the polypeptide comprises a uricase derived from any one of Sus scrofa, Papio, Mus musculus, Candida utilis, Aspergillus flavus, Arthrobacter globiformis, Paeniglutamicibacter gangotriensis, a functional fragment or variant thereof, or any combination thereof. In some embodiments, the polypeptide comprises a uricase from Sus scrofa. In some embodiments, the polypeptide comprises a uricase derived fromPapzo. In some embodiments, the polypeptide comprises a uricase from Mus musculus. In some embodiments, the polypeptide comprises a uricase derived from Candida utilis. In some embodiments, the polypeptide comprises a uricase derived from an Aspergillus fungus. In some embodiments, the polypeptide comprises a uricase derived from Aspergillus flavus. In some embodiments, the polypeptide comprises a uricase derived from Arthrobacter globiformis. In some embodiments, the polypeptide comprises a uricase derived from Paeniglutamicibacter gangotriensis .

[0098] In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to a uricase from any one of Sus scrofa, Papio, Mus musculus, Candida utilis, Aspergillus flavus, Arthrobacter globiformis, or Paeniglutamicibactergangotriensis . In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to a uricase from any one of Sus scrofa, Papio, Mus musculus, Candida utilis, Aspergillus flavus, Arthrobacter globiformis, or Paeniglutamicibacter gangotriensis . In some embodiments, the polypeptide comprises less than 100% sequence identity to a uricase from any one of Sus scrofa, Papio, Mus musculus, Candida utilis, Aspergillus flavus, Arthrobacter globiformis, or Paeniglutamicibacter gangotriensis. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to a uricase from any one of Sus scrofa, Papio, Mus musculus, Candida utilis, Aspergillus flavus, Arthrobacter globiformis, or Paeniglutamicibacter gangotriensis. In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to a uricase from any one of Sus scrofa, Papio, Mus musculus, Candida utilis, Aspergillus flavus, Arthrobacter globiformis, or Paeniglutamicibacter gangotriensis.

[0099] In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-13. In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 5. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to any one of SEQ ID NOs: 1-13. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NOs: 5. In some embodiments, the polypeptide comprises less than 100% sequence identity to any one of SEQ ID NOs: 1-13. In someembodiments, the polypeptide comprises less than 100% sequence identity to SEQ ID NO: 5. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to any one of SEQ ID NOs: 1-13. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 5. In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to any one of SEQ ID NOs: 1-13. In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 5.

[0100] In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.7% or more sequence identity to any one of SEQ ID NOs: 1-7. In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.7% or more sequence identity to SEQ ID NO: 5. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to any one of SEQ ID NOs: 1-7. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 5. In some embodiments, the polypeptide comprises less than 100% sequence identity to SEQ ID NO: 5. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2,at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to any one of SEQ ID NOs: 1-7. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 5. In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to any one of SEQ ID NOs: 1-7. In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 5.

[0101] In some embodiments, the polypeptide is not pegylated. In some embodiments, the polypeptide does not comprise a PEG moiety. In some embodiments, the polypeptide does not comprise pegloticase, rasburicase, or pegadricase; or a peptide with 100% identity to any of pegloticase, rasburicase, or pegadricase. Pegloticase, rasburicase, and pegadricase are described in (Schlesinger N, Yasothan U, Kirkpatrick P. Pegloticase. Nat Rev Drug Discov. 2011 Jan;10(l): 17-8; Ueng S. Rasburicase (Elitek): a novel agent for tumor lysis syndrome. Proc (Bayl Univ Med Cent). 2005 Jul;18(3):275-9; Schlesinger N, Padnick-Silver L, LaMoreaux B. Enhancing the Response Rate to Recombinant Uricases in Patients with Gout. BioDrugs. 2022 Mar;36(2):95-103), each of which is incorporated by reference in its entirety. TABLE 1 below lists the sequences of SEQ ID NOs: 1-7.TABLE 1: A summary of exemplary polypeptide sequences

[0102] In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity to SEQ ID NO: 1. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 1. In some embodiments, the polypeptide comprises less than 100% sequence identity to SEQ ID NO: 1. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 1 In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, atmost 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 1 In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 2. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 2. In some embodiments, the polypeptide comprises less than 100% sequence identity to SEQ ID NO: 2. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 2 In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 2 In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 3. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 3. In some embodiments, the polypeptide comprises less than 100% sequence identity to SEQ ID NO: 3. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 3 In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative toSEQ ID NO: 3 In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 4. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 4. In some embodiments, the polypeptide comprises less than 100% sequence identity to SEQ ID NO: 4. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 4 In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 4 In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 5. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 5. In some embodiments, the polypeptide comprises less than 100% sequence identity to SEQ ID NO: 5. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 5 In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 5 In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 6. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 6. In some embodiments, the polypeptide comprises less than 100% sequence identity to SEQ ID NO: 6. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 6 In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 6 In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 7. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 7. In some embodiments, the polypeptide comprises less than 100% sequence identity to SEQ ID NO: 7. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 7. In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 7

[0103] In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or more sequence identity to any one of SEQ ID NOs: 8-10. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to any one of SEQ ID NOs: 8-10. In some embodiments, the polypeptide comprises 100% sequence identity to any one of SEQ ID NOs: 8-10. In some embodiments, the polypeptide comprises less than 100% sequence identity to any one of SEQ ID NOs: 8-10. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to any one of SEQ ID NOs: 8-10. In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to any one of SEQ ID NOs: 8-10.

[0104] In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to any one of SEQ ID NOs: 11-13. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to any one of SEQ ID NOs: 11-13. In some embodiments, the polypeptide comprises 100% sequence identity to any one of SEQ ID NOs: 11-13. In some embodiments, the polypeptide comprises less than 100% sequence identity to any one of SEQ ID NOs: 11-13. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to any one of SEQ ID NOs: 11-13. In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, suchas a substitution of an NSAA for a standard amino acid, or mutations relative to any one of SEQ ID NOs: 11-13. TABLE 2 below lists the sequences of SEQ ID NOs: 8-13.TABLE 2: A summary of exemplary polypeptide sequences comprising an NSAA

[0105] In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 8. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 8. In some embodiments, the polypeptide comprises 100% sequence identity to SEQ ID NO: 8. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 8. In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 8. In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 9. In some embodiments, the polypeptidecomprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 9 In some embodiments, the polypeptide comprises less than 100% sequence identity to SEQ ID NO: 9 In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 9. In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 9. In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 10. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 10. In some embodiments, the polypeptide comprises less than 100% sequence identity to SEQ ID NO: 10 In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 10. In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 10. In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 11. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%,at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 11. In some embodiments, the polypeptide comprises 100% sequence identity to SEQ ID NO: 11. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 11. In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 11. In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 12. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 12. In some embodiments, the polypeptide comprises 100% sequence identity to SEQ ID NO: 12. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 12. In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 12. In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 13. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 13. In some embodiments, the polypeptide comprises 100% sequence identity to SEQ IDNO: 13. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 13. In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 13.

[0106] In some embodiments, provided herein is an engineered uricase polypeptide with uricase activity, wherein the engineered uricase polypeptide is derived from an Aspergillus uricase and wherein the engineered uricase polypeptide comprises a non-standard amino acid (NSAA). In some embodiments, the polypeptide is derived from an Aspergillus flavus uricase. In some embodiments, the polypeptide is derived from an Aspergillus flavus uricase and the NSAA is located at a position selected from the group consisting of: a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide is derived from an Aspergillus flavus uricase and the NSAA is located at a position selected from the group consisting of at least two of the following positions: a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and aposition corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide is derived from an Aspergillus flavus uricase and the NSAA is located at a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and aposition corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the NSAA comprises para-azidophenylalanine (pAzF). In some embodiments, the polypeptide is derived from an Aspergillus flavus uricase, the NSAA comprises pAzF, and the pAzF is located at a position selected from the group consisting of: a position corresponding to position 139 of SEQ ID NO: 5, aposition corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide is derived from an Aspergillus flavus uricase the NSAA comprises pAzF, and the pAzF is located at a position selected from the group consisting of at least two of the following positions: a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide is derived from an Aspergillus flavus uricase, the NSAA comprises pAzF, and the pAzF is located at aposition corresponding to position 139 of SEQ ID NO: 5, aposition corresponding to position 175 of SEQ ID NO: 5, and aposition corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the NSAA comprises para-azidomethylphenylalanine (pAMF). Insome embodiments, the polypeptide is derived from an Aspergillus flavus uricase, the NSAA comprises pAMF, and the pAMF is located at a position selected from the group consisting of: a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide is derived from an Aspergillus flavus uricase the NSAA comprises pAMF, and the pAMF is located at a position selected from the group consisting of at least two of the following positions: a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and aposition corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide is derived from an Aspergillus flavus uricase, the NSAA comprises pAMF, and the pAMF is located at a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5.

[0107] In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 9, wherein the polypeptide comprises an NSAA located at a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 9, wherein the polypeptide comprises a pAzF located at a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 9, wherein the polypeptide comprises a pAMF located at a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 9, wherein the polypeptide comprises an NSAA located at a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQID NO: 5. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 9, wherein the polypeptide comprises a pAzF located at a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide comprises at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% sequence identity to SEQ ID NO: 9, wherein the polypeptide comprises a pAMF located at a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide comprises less than 100% sequence identity to SEQ ID NO: 9, wherein the polypeptide comprises an NSAA located at a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide comprises less than 100% sequence identity to SEQ ID NO: 9, wherein the polypeptide comprises a pAzF located at a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide comprises less than 100% sequence identity to SEQ ID NO: 9, wherein the polypeptide comprises a pAMF located at a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 9, wherein the polypeptide comprises an NSAA located at a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide comprises a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to SEQ ID NO: 9, wherein the polypeptide comprises a pAzF located at a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide comprises a sequence that has at least1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions or mutations relative to SEQ ID NO: 9, wherein the polypeptide comprises a pAMF located at a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to SEQ ID NO: 9, wherein the polypeptide comprises an NSAA located at a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions or mutations relative to SEQ ID NO: 9, wherein the polypeptide comprises a pAzF located at a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide comprises a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most24, or at most 25 amino acid substitutions or mutations relative to SEQ ID NO: 9, wherein the polypeptide comprises a pAMF located at a position corresponding to position 139 of SEQ ID NO: 5, a position corresponding to position 175 of SEQ ID NO: 5, and a position corresponding to position 218 of SEQ ID NO: 5. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 9, and three NSAAs. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 9, and three NSAAs, wherein each of the three NSAAs comprises pAzF. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 9, and three NSAAs. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 9, and three NSAAs, wherein each of the three NSAAs comprises pAMF.

[0108] In some embodiments, the polypeptide comprises an N-terminal variant. In some embodiments, the N-terminal variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid additions between the C-terminus of M (i.e., amino acid position 1 relative to any one of SEQ ID NOs: 8-10) and the N-terminus of the next amino acid in the sequence (e.g., the N-terminus of the Sat amino acid position 2 relative to any one of SEQ ID NOs: 8-10). For example, the N-terminal variant can comprise an amino acid addition of HHHHHH (SEQ ID NO: 14), wherein HHHHHH is located between the C-terminus of M (amino acid position 1 relative to any one of SEQ ID NOs: 8- 10) and the N-terminus of S (amino acid position 2 relative to any one of SEQ ID NOs: 8-10). In some embodiments, the N-terminal variant can comprise an amino acid addition of HHHHHHGSLEVLFQGP (SEQ ID NO: 15), wherein HHHHHHGSLEVLFQGP is located between the C-terminus of M (amino acid position 1 relative to any one of SEQ ID NOs: 8-10) and the N- terminus of S (amino acid position 2 relative to any one of SEQ ID NOs: 8-10).

[0109] In some embodiments, a polypeptide comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acids is attached to the N-terminus of any one of SEQ ID NOs: 11-13). In some embodiments, the sequence attached to the N-terminus of any one of SEQ ID NOs: 11-13 comprises the sequence: MHHHHHH (SEQ ID NO: 16). In some embodiments, the sequence attached to the N-terminus of any one of SEQ ID NOs: 11-13 comprises the sequence: MHHHHHHGSLEVLFQGP (SEQ ID NO: 17).

[0110] In some embodiments, the polypeptide comprises a tag. In some embodiments, the polypeptide comprises an affinity tag. In some embodiments, the polypeptide comprises at least one affinity tag. In some embodiments, the affinity tag of a polypeptide is a C-terminal affinity tag. In some embodiments, the affinity tag of a polypeptide is an N-terminal affinity tag. In some embodiments, the polypeptide comprises more than one affinity tag. In some embodiments, the polypeptide comprises at least two affinity tags. In some embodiments, the polypeptide comprises a first affinity tag and a second affinity tag. In some embodiments, the first affinity tag of a polypeptide is an N-terminal affinity tag and the second affinity tag of a polypeptide is a C-terminal affinity tag. In some embodiments, the first affinity tag of a polypeptide is a first N-terminal affinity tag and the second affinity tag of a polypeptide is a second N-terminal affinity tag. In some embodiments, the first affinity tag of a polypeptide is a first C-terminal affinity tag and the second affinity tag of a polypeptide is a second C-terminal affinity tag. In some embodiments, the first affinity tag and the second affinity tag are the same affinity tags. In some embodiments, the first affinity tag and the second affinity tag are not the same affinity tags. In some embodiments, the first affinity tag and the second affinity tag are different affinity tags.[OHl] Non-limiting examples of the affinity tag comprise a poly-histidine (His) tag, poly-histidine- glycine tag, poly-arginine tag, poly-aspartate tag, poly-cysteine tag, poly-phenylalanine, c-myc tag, Herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene 10 protein peptide tag, streptavidin tag, streptavidin binding peptide (SPB) tag, Strep-tag, Strep- tag II, albumin-binding protein (ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag), calmodulin binding peptide (CBP) tag, chloramphenicol acetyl transferase (CAT) tag, choline- binding domain (CBD) tag, chitin binding domain (CBD) tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP), glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSV tag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag, NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag, Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity eXact tag, Protein C tag, SI -tag, S-tag, biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinity purification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, or a combination thereof.

[0112] In some embodiments, the affinity tag is selected from the group consisting of a His-tag, a GST-tag, a Strep-tag, a SUMO-tag, and an MBP-tag. In some embodiments, the polypeptide comprises a His-tag. In some embodiments, the His-tag is a 6x-His tag. In some embodiments, the His-tag is located near the N-terminal of the polypeptide. In some embodiments, the His-tag is located at position 2 of the N-terminal, after the M amino acid residue. In some embodiments, the His-tag is located at position 2 of the N-terminal, according to the formula M-His-tag-Xn, wherein Xnrepresents the remaining amino acid residues in the polypeptide. In some embodiments, the polypeptide comprises a GST-tag. In some embodiments, the polypeptide comprises a Strep-tag. In some embodiments, the polypeptide comprises a SUMO tag. In some embodiments, the polypeptide comprises an MBP-tag.

[0113] The polypeptide described herein may have an enzymatic activity. In some embodiments, the polypeptide described herein may be an enzyme. In some embodiments, the polypeptide may have an oxidoreductase activity. In some embodiments, the polypeptide may be an oxidoreductase.

[0114] In some embodiments, an oxidoreductase can catalyze a transfer of an electron from an electron donor (or a reductant) to an electro acceptor (or an oxidant). An oxidoreductase may be an enzyme of EC (Enzyme Commission) group 1 (EC 1). In some case, the polypeptide may be an enzyme or have the enzymatic activity of enzyme group EC 1.7 (enzymes acting on other nitrogenous compounds as electron donors). In some embodiments, the polypeptide may be an enzyme or have the enzymatic activity of enzyme group EC 1.7.3 (enzymes uses oxygen as electron acceptors). In some embodiments, the polypeptide may be an enzyme or have the enzymatic activity of enzyme group EC 1.7.3.3 (factor-independent urate hydroxylase). In some embodiments, the polypeptide may be an enzyme or have the enzymatic activity of enzyme group uricase (urate oxidase or urate hydroxylase). In some embodiments, uricase can catalyze: (1) uric acid + O2 + H2O5-hydroxyisourate + H2O2; (2) 5-hydroxyisourate + H2O allantoin + CO2; or (3) both (1) and (2).

[0115] The enzymatic activity of the enzyme can be measured by KM (the Michaelis-Menten constant); kcat (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 specificity constant (kcat / KM),' or Vmax(the maximal rate of an enzymatic reaction). The assay used to measure KM (Michaelis constant) can be a kinetic assay. For example, the kinetic assay can determine the substrate concentration at which the enzyme achieves half of its maximum velocity (VmK). The assay used to measure kcat (catalytic constant or turnover number) can be a turnover assay or a time-course assay. This assay determines the rate at which the enzyme converts substrate into product per active site, providing a measure of the enzyme's catalytic efficiency. For example, in a turnover assay, the enzyme reaction is allowed to proceed for a specific period of time, and the amount of product formed is measured. The reaction is typically performed under conditions where the substrate concentration is much higher than the KM value, ensuring that the enzyme is operating at its maximum velocity (FmiK). The enzyme reaction, kinetic assay, or the substrates / products being measured in the assays can comprise those described herein, such as those of the uricase enzymatic reaction. In some embodiments, the assays are carried out in a serum. The serum can comprise a mammalian serum. In some embodiments, the serum is a human serum. For example, the turnover assay may be measured by the method described in Example 3.

[0116] The polypeptide described herein may have & KM of at least 1 x 10’3mM, 2 x 10’3mM, 3 x 10’3mM, 4 x 10’3mM, 5 x 10’3mM, 6 x 10’3mM, 7 x 10’3mM, 8 x 10’3mM, 9 x 10’3mM, 1 x 10’2mM, 2 x 10’2mM, 3 x 10’2mM, 4 x 10’2mM, 5 x 10’2mM, 6 x 10’2mM, 7 x 10’2mM, 8 x 10’2mM, 9 x 10’2mM, 1 x 10’1mM, 2 x 10’1mM, 3 x 10’1mM, 4 x 10’1mM, 5 x 10’1mM, 6 x 10’1mM, 7 x 10’1mM, 8 x 10’1mM, 9 x 10’1mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, or 90 mM or more. The polypeptide described herein may have a KM of at most 1 x 1 O'3mM, 2 x 1 O'3mM, 3 x 1 O'3mM, 4 x 1 O'3mM, 5 x 10’3mM, 6 x 10’3mM, 7 x 10’3mM, 8 x 10’3mM, 9 x 10’3mM, 1 x 10’2mM, 2 x 10’2mM, 3 x 10’2mM, 4 x 10’2mM, 5 x 10’2mM, 6 x 10’2mM, 7 x 10’2mM, 8 x 10’2mM, 9 x 10’2mM, 1 x 10-1mM, 2 x 10’1mM, 3 x 10’1mM, 4 x 10’1mM, 5 x 10’1mM, 6 x 10’1mM, 7 x 10’1mM, 8 x 10’1mM, 9 x 10’1mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, or 90 mM. The polypeptide described herein may have akcat / KM of at least 1 x 102M’1s’1, 2 x 102M’1s’1, 3 x 102M’1s’1, 4 x 102M’1s’1, 5 x 102M’1s’1, 6 x 102M’1s’ \ 7 x 102M’1s’1, 8 x 102M’1s’1, 9 x 102M’1s’1, 1 x 103M’1s’1, 2 x 103M’1s’1, 3 x 103M’1s’1, 4 x 103M’1s’1, 5 x 103M S’1, 6 x 103M’1S’1, 7 x 103M’1s’1, 8 x 103M’1s’1, 9 x 103M’1s’1, 1 x I O4M’' s’1, 2 x I O4M’' S’1, 3 x 104M’1S’1, 4 x 104M’1s’1, 5 x 104M’1s’1, 6 x 104M’1s’1, 7 x 104M’1s’1, 8 x 104M’1s’1, 9 x 104M’1S’1, 1 x 105M’1s’1, 2 x 105M’1s’1, 3 x 105M’1s’1, 4 x 105M’1s’1, 5 x I 05M’1s’1, 6 x 105M’1s’1, 7 x 105M’1s’1, 8 x 105M’1s’1, 9 x 105M’1s’1, 1 x 106M’1s’1, 2 x I O6M’' s’1, 3 x I O6M’' s’1, 4 x I O6M’' S’1, 5 x I O6M’' S’1, 6 X 106M’1s’1, 7 x 106M’1s’1, 8 x I O6M’' s’1, 9 x I O6M’' s’1, 1 x lO’M’1s’1, 2 X 107M’1S’1, 3 X lO’M’1s’1, 4 x lO’M’1s’1, 5 x lO’M’1s’1, 6 x 1O7M s’1, 7x 1O7M s’1, 8 x 107M’1s’1, 9 x 1O7M S’1, 1 x 108M’1s’1, 2 x 108M’1s’1, 3 x 108M’1s’1, 4 x 108M’1s’1, 5 x 108M’1s’1, 6 x 108M’1S’1, 7X 108M’1S’1, 8X 108M’1S’1, 9X 108M’1S’1, 1 x IO9M’' s’1, 2x 109M’1s’1, 3 x 109M’1s’1, 4 x 109M’1S’1, 5 x 109M’1S’1, 6 x 109M’1s’1, 7 x 109M’1s’1, 8 x 109M’1s’1, 9x 109M’1s’1or more. The polypeptide described herein may have a kcat / K of at most 1 x 102M’1s’1, 2 x 102M’1s’1, 3 x 102M’1s’1, 4 x 102M’1s’1, 5 x 102M’1s’1, 6 x 102M’1s’1, 7 x 102M’1s’1, 8 x 102M’1s’1, 9 x 102M’1s’1, 1 x IO3M’' S’1, 2X 103M’1S’1, 3X 103M’1S’1, 4X 103M’1S’1, 5 x 103M’1s’1, 6x 103M’1s’1, 7x 103M’1s’1, 8 x 103M’1S’1, 9X 103M’1S’1, 1 x IO4M’' s’1, 2x 104M’1s’1, 3 x 104M’1s’1, 4x 104M’1s’1, 5 x 104M’1s’1, 6 x 1O4M S’1, 7 x 104M’1S’1, 8 x 104M’1s’1, 9 x 104M’1s’1, 1 x IO5M’1s’1, 2 x IO5M’1s’1, 3 x IO5M’1s’1, 4 x IO5M’1s’1, 5 x IO5M’1s’1, 6 x IO5M’1s’1, 7 x IO5M’1s’1, 8 x IO5M’1s’1, 9 x IO5M’1s’1, 1 x IO6M’' S’1, 2X 106M’1S’1, 3 x IO6M’' s’1, 4x 106M’1s’1, 5 x IO6M’' s’1, 6x IO6M’' s’1, 7x 106M’1s’1, 8 x 106M’1s’1, 9 x 106M’1s’1, 1 x lO’M’1s’1, 2 x lO’M’1s’1, 3 x lO’M’1s’1, 4 x lO’M’1s’1, 5 x lO’M’1s’1, 6 x lO’M’1s’1, 7x 1O7M s’1, 8 x lO’M’1s’1, 9 x lO’M’1s’1, 1 x 108M’1s’1, 2 x 108M’1s’1, 3 x 108M’1S’1, 4 x 108M’1S’1, 5 x 108M’1s’1, 6 x 108M’1s’1, 7 x 108M’1s’1, 8 x 108M’1s’1, 9 x 108M’1s’1, 1 x 1O9M S’1, 2 x 109M’1S’1, 3 x 109M’1s’1, 4 x 109M’1s’1, 5 x 109M’1s’1, 6 x 109M’1s’1, 7 x 109M’1s’1, 8 x 109M’1s’1, 9 x 109M’1s’1. The polypeptide described herein may have a kcat of at least1 x 10’2s’1, 2 x 10’2s’1, 3 x 10’2s’1, 4 x 10’2s’1, 5 x 10’2s’1, 6 x 10’2s’1, 7 x 10’2s’1, 8 x 10’2s’1, 9 x 10’2s’1, 1 x IO’1s’1, 2 x IO’1s’1, 3 x IO’1s’1, 4 x IO’1s’1, 5 x IO’1s’1, 6 x IO’1s’1, 7 x IO’1s’1, 8 x IO’1s’1, 9 x IO’1s’1, 1 x 100 s’1, 2 s’1, 3 s’1, 4 s’1, 5 s’1, 6 s’1, 7 s’1, 8 s’1, 9 s’1, 10 s’1, 20 s’1, 30 s’1, 40 s’1, 50 s’1, 60 s’1, 70 s’1, 80 s’1, 90 s’1, 100 s’1, 200 s’1, 300 s’1, 400 s’1, 500 s’1, 600 s’1, 700 s’1, 800 s’1, 900 s’1or more. The polypeptide described herein may have a kcat of at most 1 x 10’2s’1, 2 x 10’2s’1, 3 x 10’2s’1, 4x10’2s’1, 5 x 10’2s’1, 6 x 10’2s’1, 7 x 10’2s’1, 8 x 10’2s’1, 9 x 10’2s’1, 1 x IO’1s’1, 2 x IO’1s’1, 3 x IO’1s’1, 4mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, or 90 mM.

[0117] A polypeptide with at least one NSAA and / or at least one sugar or glycan moiety coupled to the NSAA, such as an engineered uricase polypeptide, may have a KM that is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold higher than that of a control polypeptide or corresponding polypeptide lacking the NSAA and / or the at least one sugar or glycan moiety. In some embodiments, a corresponding polypeptide comprises a polypeptide having the same or substantially the same (having about 80%, 85%, 90%, 95%, 99% or more sequence identity) polypeptide sequence as the sequence of the polypeptide with at least one NSAA and / or at least one sugar or glycan moiety coupled to the NSAA except the corresponding polypeptide lacks: (1) the NSAA; (2) the at least one sugar moiety coupled to the NSAA; or (3) a combination thereof. In some embodiments, a control polypeptide comprises a polypeptide having the same or substantially the same (having about 80%, 85%, 90%, 95%, 99% or more sequence identity) polypeptide sequence as the sequence of the polypeptide with at least one NSAA and / or at least one sugar or glycan moiety coupled to the NSAA, but is pegylated. The control polypeptide may be pegylated at the NSAA. The control polypeptide may have 100% sequence identity to any one of the sequences of Table 1. The control polypeptide may be a non-engineered, wildtype uricase polypeptide. In some embodiments, a control polypeptide comprises pegloticase, rasburicase, or pegadricase. A polypeptide with at least one NSAA or at least one sugar moiety coupled to the NSAA, such as an engineered uricase polypeptide, may have a KM that is at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 100%, at most 150%, at most 2-fold, at most 3 -fold, at most 4-fold, or at most 5- fold higher than that of a control polypeptide or corresponding polypeptide. A polypeptide with at least one NSAA or at least one sugar moiety coupled to the NSAA, such as an engineered uricase polypeptide, may have a kcat / KM that is at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 100%, at most 150%, at most 2-fold, at most 3-fold, at most 4-fold, at most 5-fold or more of that of a control polypeptide or corresponding polypeptide. A polypeptide with at least one NSAA or at least one sugar moiety coupled to the NSAA, such as an engineered uricase polypeptide, may have a Vmaxthat is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold or more of that of a control polypeptide or corresponding polypeptide. A polypeptide with at least one NSAA or at least one sugar moiety coupled to the NSAA, such as an engineered uricase polypeptide, may have a Vmaxthat is at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 100%, at most 150%, at most 2- fold, at most 3-fold, at most 4-fold, at most 5-fold or more of that of a control polypeptide or corresponding polypeptide. A polypeptide with at least one NSAA or at least one sugar moiety coupled to the NSAA, such as an engineered uricase polypeptide, may have a kcat that is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%,at least 2-fold, at least 3 -fold, at least 4-fold, at least 5 -fold or more of that of a control polypeptide or corresponding polypeptide. A polypeptide with at least one NSAA or at least one sugar moiety coupled to the NSAA, such as an engineered uricase polypeptide, may have a feat that is at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 100%, at most 150%, at most 2-fold, at most 3-fold, at most 4-fold, at most 5-fold or more of that of a control polypeptide or corresponding polypeptide.

[0118] Subsequent to being administered to a subject(s), a polypeptide with at least one NSAA or at least one sugar moiety coupled to the NSAA, such as an engineered uricase polypeptide, may elicit an anti-drug response (ADA) in the subject that is lower than the ADA elicited in the subject by a control or corresponding polypeptide. For example, subsequent to being administered to a subject(s), a polypeptide with at least one NSAA or at least one sugar moiety coupled to the NSAA, such as an engineered uricase polypeptide, may elicit an anti-drug response (ADA) in the subject that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 500-fold or at least 100-fold less than the ADA elicited in the subject by a control or corresponding polypeptide. For example, subsequent to being administered to a subject(s), a polypeptide with at least one NSAA or at least one sugar moiety coupled to the NSAA, such as an engineered uricase polypeptide, may elicit an anti-drug response (ADA) in the subject that is at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99% of the ADA elicited in the subject by a control or corresponding polypeptide. The ADA response can comprise production of or increasing the level of an antibody that can target or bind the polypeptide or a portion thereof. The ADA response can be measured by the amount or titer of the antibody elicited by a polypeptide administered to the body against the polypeptide. For example, the ADA response may be measured by the method described in Example 4.

[0119] Subsequent to being administered to a subject(s), a polypeptide with at least one NSAA or at least one sugar moiety coupled to the NSAA may reduce the amount or level of uric acid within the subject(s) by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10- fold, at least 100-fold or more, relative to a control amount or level of uric acid. The control amount or level of uric acid can comprise: (1) the amount or level of uric acid of a subject prior to administration of the polypeptide; (2) the amount or level of uric acid of a subject not administered the polypeptide; or (3) the amount or level of uric acid of a subject subsequent to administration of acontrol polypeptide described herein. For example, subsequent to being administered to a subject(s), an engineered uricase polypeptide with at least one NSAA or at least one sugar moiety coupled to the NSAA reduces the amount or level of uric acid within the subject(s) by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 100-fold or more, relative to that reduced by a control polypeptide described herein.

[0120] In some embodiments, subsequent to an engineered uricase polypeptide with at least one NSAA or at least one sugar moiety coupled to the NSAA being administered to a subject(s), serum uric acid level of the subject is reduced to a concentration of at most 2 mg / dL, at most 2.1 mg / dL, at most 2.2 mg / dL, at most 2.3 mg / dL, at most 2.4 mg / dL, at most 2.5 mg / dL, at most 2.6 mg / dL, at most 2.7 mg / dL, at most 2.8 mg / dL, at most 2.9 mg / dL, at most 3 mg / dL, at most 3.1 mg / dL, at most 3.2 mg / dL, at most 3.3 mg / dL, at most 3.4 mg / dL, at most 3.5 mg / dL, at most 3.6 mg / dL, at most 3.7 mg / dL, at most 3.8 mg / dL, at most 3.9 mg / dL, at most 4 mg / dL, at most 4. 1 mg / dL, at most 4.2 mg / dL, at most 4.3 mg / dL, at most 4.4 mg / dL, at most 4.5 mg / dL, at most 4.6 mg / dL, at most 4.7 mg / dL, at most 4.8 mg / dL, at most 4.9 mg / dL, at most 5 mg / dL, at most 5.1 mg / dL, at most 5.2 mg / dL, at most5.3 mg / dL, at most 5.4 mg / dL, at most 5.5 mg / dL, at most 5.6 mg / dL, at most 5.7 mg / dL, at most 5.8 mg / dL, at most 5.9 mg / dL, at most 6 mg / dL, at most 6. 1 mg / dL, at most 6.2 mg / dL, at most 6.3 mg / dL, at most 6.4 mg / dL, at most 6.5 mg / dL, at most 6.6 mg / dL, at most 6.7 mg / dL, at most 6.8 mg / dL, at most 6.9 mg / dL, at most 7 mg / dL, at most 7.1 mg / dL, at most 7.2 mg / dL, at most 7.3 mg / dL, at most7.4 mg / dL, at most 7.5 mg / dL, at most 7.6 mg / dL, at most 7.7 mg / dL, at most 7.8 mg / dL, at most 7.9 mg / dL, or at most 8.0 mg / dL.

[0121] In some embodiments, an engineered uricase polypeptide with at least one NSAA or at least one sugar moiety coupled to the NSAA is not pegylated. In some embodiments, an engineered uricase polypeptide with at least one NSAA or at least one sugar moiety coupled to the NSAA is not pegylated at the NSAA. In some embodiments, an engineered uricase polypeptide with at least one NSAA or at least one sugar moiety coupled to the NSAA is pegylated at a site that is not the NSAA. In some embodiments, the engineered uricase polypeptide is PEGylated at an amino acid other than lysine. In some embodiments, the engineered uricase polypeptide is PEGylated at the NSAA. In some embodiments, the engineered uricase polypeptide is PEGylated, wherein each PEG chain consists of less than 200, less than 175, less than 150, less than 125, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, or less than 20 ethylene glycol units. In some embodiments, the engineered uricase polypeptide is PEGylated, wherein each PEG chain consists of 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 ethylene glycol units. In some embodiments, the engineered uricase polypeptide is PEGylated, wherein each PEG chain consists of 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 ethylene glycol units, or no greater than 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 ethylene glycol units. In some embodiments, the engineered uricase polypeptide is PEGylated, wherein each PEG chain consists of 10 ethylene glycol units. In some embodiments, the engineered uricase polypeptide is PEGylated, wherein each PEG chain is a branched PEG chain. In some embodiments, the engineered uricase polypeptide is PEGylated, wherein each PEG chain is a branched PEG chain comprising 2 or more PEG molecules attached to a central core. In some embodiments, the linker comprises one or more PEG units (see for example, Glycan4of FIG. 1C). In some embodiments, the linker consists of less than 200, less than 175, less than 150, less than 125, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, or less than 20 ethylene glycol units. In some embodiments, the linker consists of 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 ethylene glycol units. In some embodiments, the linker consists of 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 ethylene glycol units, or no greater than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 ethylene glycol units. In some embodiments, the linker consists of 10 ethylene glycol units. In some embodiments, the linker comprises a branched PEG chain. In some embodiments, the linker comprises a branched PEG chain comprising 2 or more PEG molecules attached to a central core.

[0122] Also provided herein is a polynucleotide comprising a sequence that encodes a polypeptide disclosed herein, such as an engineered uricase polypeptide.

[0123] In some embodiments, the polynucleotide comprises a sequence that encodes a polypeptide having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.7% or more sequence identity to any one of SEQ ID NOs: 1-13. For example, a polynucleotide can comprise a sequence that encodes a polypeptide having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.7% sequence identity to SEQ ID NO: 5, but less than 100% sequence identity to SEQ ID NO: 5 For example, a polynucleotide can comprise a sequence that encodes a polypeptide having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.7% sequence identity or 100% sequence identity to SEQ ID NO: 8 or 11, or any one of SEQ ID Nos: 9, 10, 12 or13. In some embodiments, the polynucleotide comprises a sequence that encodes a polypeptide having at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, or at most 99.7% sequence identity to any one of SEQ ID NOs: 1-13. In some embodiments, the polynucleotide comprises asequence that encodes a polypeptide having 100% sequence identity to any one of SEQ ID NOs: 8- 13. In some embodiments, the polynucleotide comprises a sequence that encodes a polypeptide having a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to any one of SEQ ID NOs: 1-7. In some embodiments, the polynucleotide comprises a sequence that encodes a polypeptide having a sequence that has at most 1, at most 2, at most 3, at most 4, at most 5, at most 10, at most 11, at most 12, at most13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 amino acid substitutions, such as a substitution of an NSAA for a standard amino acid, or mutations relative to any one of SEQ ID NOs: 1-7.Non-Standard Amino Acid and Sugar Moiety (ies) Conjugation

[0124] The use of non-standard amino acids in proteins offers the possibility of polypeptides having greatly expanded functionality that could be exploited for wide range of applications. In one example, by incorporation of a NSAA (such as selenocysteine) into a polypeptide, enzymes having enhanced levels of stability or activity can be engineered. In another example, by incorporation of a NSAA (such as pAzF) into a polypeptide, specific and / or homogeneous glycoforms of the polypeptide can be engineered, for example, by chemically or enzymatically linking one or more glycans to a NSAA of the polypeptide.

[0125] Studies detailed herein and described elsewhere have demonstrated that by using orthogonal aminoacyl-tRNA synthetase (aaRS) / tRNA pairs in bacterial cells, NSAAs (such as pAzF) can be incorporated into polypeptides during translation to produce NSAA containing polypeptides. However, although bacterial cells are capable of producing eukaryotic proteins, bacterial cells cannot produce eukaryotic glycoproteins. The compositions and methods described herein allow for the production of eukaryotic glycoproteins by producing eukaryotic proteins with NSAAs in bacterial cells and, after isolating the eukaryotic proteins with NSAAs from the bacterial cells, linking glycans to the NSAAs of the isolated eukaryotic proteins.Glycan-coupled or Glycan-Containing Non-Standard Amino Acid

[0126] The present disclosure provides an engineered uricase polypeptide comprising a non-standard amino acid (NSAA). The NSAA can be conjugated to one or more sugar moieties or glycans. The NSAA can be conjugated to one or more sugar moieties or glycans chemically or enzymatically. When sugar moieties are chemically conjugated, it can involve the use of reactive chemical groups to attach the sugar to the target biomolecule. This method can allow for precise control over the site and type of linkage between the sugar moiety and the molecule. In some embodiments, chemical conjugation offers control over the site and type of linkage. Enzymatic conjugation can involve the action of specific enzymes known as glycosyltransferases. These enzymes can catalyze the transfer of sugarmolecules to specific target sites on biomolecules. Enzymatic conjugation is highly specific and can generate complex and diverse glycan structures that closely resemble those found in nature. In some case, enzymatic conjugation can allow for the creation of biologically relevant glycosylation patterns. In some embodiments, enzymatic conjugation can allow for the production of complex and diverse glycan structures. The NSAA can be coupled to one or more sugar moieties chemically or enzymatically. In some embodiments, at least one amino acid of the polypeptide is replaced with the NSAA.

[0127] In one aspect, provided herein is a polypeptide comprising (1) a sequence of a uricase or a fragment thereof and (2) a non-standard amino acid (NSAA), wherein the NSAA is conjugated to a 1,2, 3 -triazole linker. In some embodiments, the sequence of the uricase or the fragment thereof comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1-7. In some embodiments, the NSAA is conjugated to one or more sugar moieties. In some embodiments, the NSAA is not conjugated to one or more sugar moieties. In some embodiments, the NSAA is paraazidophenylalanine (pAzF). In some embodiments, the NSAA linked to a glycan comprises a structure of Formula (I):AA-L-XFormula (I) wherein AA is a NSAA, AA-L comprises a 1,2, 3 -triazole, and X is absent or comprises a glycan.

[0128] In some embodiments, the polypeptide comprises a structure of Formula (IA): AA-L '-lA-L3- X. In some embodiments, L'-L2is an alkyne-containing linker. In some embodiments, L3comprises an amino acid, such as a standard amino acid. In some embodiments, X is a glycan (or any sugar moiety as described herein).

[0129] In some embodiments, the non-standard amino acid is P-alanine, D-alanine, 4-hydroxyproline, desmosine, D-glutamic acid, y-aminobutyric acid, P-cyanoalanine, norvaline, p-azido-l-phenylalanine (pAzF), p-acetophenylalanine (pAcF), p-azidomethyl-L-phenylalanine (pAMF), 4-Biphenylyl-L- alanine (BipA), azido-lysine (AzK), 4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L- leucine, selenocysteine, statine, and / or selenocysteine (Sec or U). In some embodiments, the NSAA is selected from the group consisting of para-azidophenylalanine, azido-lysine, azido-alanine, p- propargyloxy-l-phenylalanine, p-benzoylphenylalanine, p-acetylphenylalanine, p-propargyloxy-1- phenylalanine, propargylglycine, homopropargylglycine, selenocysteine, p-azido-L-phenylalanine, and para-isothiocyanate phenylalanine.

[0130] In some embodiments, L'-L2of Formula (IA) comprises an alkyne-containing linker. In some embodiments, the linker is an alkyne-containing linker. In some embodiments, the linker comprises 4- pentynoic acid, propargylamine, 6-azidohexanoic acid, dibenzocyclooctyne (DBCO), 3-butyn-l-ol, ethynylbenzene, and / or PEG-alkyne.

[0131] In some embodiments, AA-L1of Formula (IA) comprises, whereinN| / N >" is coupled to the NS AA of Formula (IA), wherein • is coupled to L2.

[0132] In some embodiments, L2of Formula (IA) comprises a bond. In some embodiments, L2ofcoupled to L1, wherein is coupled to L3.

[0133] In some embodiments, L3of Formula (IA) comprises a standard amino acid. In some embodiments, L3-X of Formula (IA) comprises a structure according to Table 3. In some embodiments, L3-X of Formula (IA) comprises a structure of Formula (II): AAJ-L4-X; wherein X is absent or a glycan, L4is a linker, and AA1is any amino acid. In some embodiments, AA1comprises glycine (Gly or G), alanine (Ala or A), valine (Vai or standard V), leucine (Leu or L), isoleucine (He or I), proline (Pro or P), phenylalanine (Phe or F), tryptophan (Trp or W), methionine (Met or M), serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), glutamine (Gin or Q), aspartic acid (Asp or D), glutamic acid (Glu or E), lysine (Lys or K), arginine (Arg or R), or histidine (His or H).

[0134] In some embodiments, L3of Formula (IA) comprises, iscoupled to L2; wherein is coupled to X.

[0135] In some embodiments, X of Formula (I) and / or Formula (IA) comprises a glycan. In some embodiments, the X is a sugar moiety to which one or more sugar moieties can be conjugated. In some embodiments, a glycan stub can refer to a glycan.

[0136] In some embodiments, X of Formula (I) and / or Formula (IA) comprises GlcNAc,Man - GlcNAcMa,n- GlcNAc— Gal-Sia / _ / — GlcNAc - GlcNAc Man § GlcNAc GlcNAc ManMan - GlcNAc ; or Man - GlcNAc Gal— Sia

[0137] In some embodiments, the glycan stub comprises one or more sugar moieties. In some embodiments, the glycan stub comprises a moiety that one or more sugar moieties is configured thereto. In some embodiments, the glycans are sourced from natural sources. In some embodiments, natural glycoproteins are treated with pronase to degrade the protein and retain the glycan linked to an amino acid. In some embodiments, the glycans are biantennary glycans. In some embodiments, the biantennary glycans are linked to Asn. In some embodiments, L3of Formula (IA) can be Asn.

[0138] In some embodiments, the L3-X of Formula (IA) comprises a structure of Formula structure of Formula (II): AAJ-L4-X. In some embodiments, the X of Formula (II) is a glycan. In some embodiments, the L4of Formula (II) is a linker. In some embodiments, the AA1of Formula (II) is any standard amino acid.

[0139] In some embodiments, AA of Formula ( wherein the carbohydrate moiety is linked to the AA by RA. In some embodiments,-CH2-, -CH2CH2-, -CH2CH2CH2- , -CH2CH2CH2CH2-, -(C=O)-, -(C=O)CH2-, -CH2(C=O)-, -CH2CH2(C=O)-, -(C=O)CH2CH2-, - CH2(C=O)CH2-, -(C=O)CH2(C=O)CH2-, -CH2(C=O)CH2(C=O)-, -CH2S-, -CH2CH2S-, - CH2CH2SCH2-, -CH2CH2CH2S-, -CH2CH2CH2CH2S-, -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, - CH2CH2 CH2CH2NH-, -OCH2-, -OCH2CH2-, -OCH2CH2CH2-, -OCH2CH2CH2CH2-, -CH2O-, - CH2CH2O-, -CH2CH2CH2O-, -CH2CH2CH2CH2O-, -OCH2O-, -OCH2CH2O-, -OCH2CH2OCH2-, - CH(CH3)O-, -CH2(C=O)NH-, -CH2CH2(C=O)NH-, -CH2CH2CH2NH(C(NH2))NH-, -CH2(C=O)O-, - CH2CH2(C=O)O-, -CH(CH3)NH-, -CH(CH3)CH2-, -CH(CH3)CH2CH2-, or -CH2CH(CH3)CH2-.

[0140] In some embodiments, L4of Formula (II) is an alpha linkage (a) or a beta linkage (0).

[0141] In some embodiments, the X of Formula (I), Formula (IA) and / or Formula (II) is selected from the group consisting of N-Acetylglucosamine (GlcNAc), N-Acetylgalactosamine (GalNAc), GlcNAc-al,6-fucose (Fuc), galactose (Gal), sialic acid (Sia), Sia-a2,3-Gal, Sia-a2,6-Gal, Sia-a2,3- GlcNAc, Sia-a2,6-GlcNac, Sia-a2, 3 -GalNAc, Gal-01,3 -GalNAc, Sia-a2,3-Gal-01,3-GalNAc, GlcNAc-01,4-GlcNAc, GlcNAc-01,4-[Fuc-al,3]GlcNAc, GlcNAc-01,4-[Fuc-al,3][Fuc- al,6]GlcNAc, N-Acetylneuraminic acid (Neu5Ac), Neu5Ac-a2,3Gal-01,4[Fuc-al,3]GlcNAc, Gal- 01, 4[Fuc-al,3]GlcNAc, Gal-al,3-Gal-01,4-GlcNAc-0, and Gal-al,3-Gal.

[0142] In some embodiments, the acetyl group (-NHAc) off C2 of GlcNAc can be functionalized with a chemical group to facilitate a specific chemical reaction to a glycan or sugar moieties described herein with a complimentary chemical group. In some embodiments, the acetyl group (-NHAc) off C2 of GlcNAc may be replaced with an azide, a ketone, an aldehyde, a selenol, an aliphatic alkyne, a strained alkyne, an amine, a hydrazide, an aminooxy, a boronate, or a vicinal diol. In some embodiments, one or more sugar moieties can be added to the glycan, including a side chain pair selected from the group consisting of an azido / aliphatic alkyne, an azido / strained alkyne, aketone / amine, an aldehyde / amine, a ketone / hydrazide, a ketone / aminooxy, an aldehyde / hydrazide, an aldehyde / aminooxy, and a boronate / vicinal diol.

[0143] In some embodiments, the hydroxyl group (-OH) off C4 of GlcNAc can be functionalized with a chemical group to facilitate a specific chemical reaction to a glycan or sugar moieties described herein with a complimentary chemical group. In some embodiments, the hydroxyl group (-OH) off C4 of GlcNAc may be replaced with an azide, a ketone, an aldehyde, a selenol, an aliphatic alkyne, a strained alkyne, an amine, a hydrazide, an aminooxy, a boronate, or a vicinal diol. In some embodiments, one or more sugar moieties can be added to the glycan, including a side chain pair selected from the group consisting of an azido / aliphatic alkyne, an azido / strained alkyne, a ketone / amine, an aldehyde / amine, a ketone / hydrazide, a ketone / aminooxy, an aldehyde / hydrazide, an aldehyde / aminooxy, and a boronate / vicinal diol.

[0144] In some embodiments, the -OH of the C4 of GlcNAc can be replaced with an azide to react with an alkyne-functionalized glycan. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with an alkyne to react with an azide-functionalized glycan. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with a ketone to react with an amine-functionalized glycan to form an imine. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with an amine to react with a ketone-functionalized glycan to form an imine. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with an aldehyde to react with an amine-functionalized glycan to form an imine. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with an amine to react with an aldehyde-functionalized glycan to form an imine. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with an aldehyde to react with a cyanide-functionalized glycan to form a cyanohydrin. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with a cyanide to react with an aldehyde-functionalized glycan to form a cyanohydrin. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with a ketone to react with an alcohol-functionalized glycan. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with an aldehyde to react with an alcohol-functionalized glycan. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with a selenol group to react with a selenol-functionalized glycan. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with a ketone to react with a hydrazide-functionalized glycan. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with a hydrazide to react with a ketone-functionalized glycan. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with a ketone to react with an aminooxy-functionalized glycan. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with an aminooxy to react with a ketone-functionalized glycan. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with an aldehyde to react with a hydrazide-functionalized glycan. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with a hydrazide to react with an aldehyde-functionalized glycan. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with an aldehyde to react with an aminooxy-functionalized glycan.In some embodiments, the -OH of the C4 of GlcNAc can be replaced with an aminooxy to react with an aldehyde-functionalized glycan. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with a boronate to react with a vicinal diol-functionalized glycan. In some embodiments, the -OH of the C4 of GlcNAc can be replaced with a vicinal diol to react with a boronate-functionalized glycan.

[0145] In some embodiments, GlcNAc has a structure of Formula (HI):Formula (III) wherein R is a hydroxyl group, an azide, a ketone, an aldehyde, a sei enol, an aliphatic alkyne, a strained alkyne, an amine, a hydrazide, an aminooxy, a boronate, or a vicinal diol.

[0146] In some embodiments, the hydroxyl group (-OH) off C3, C4, or C6 of GalNAc can be functionalized with a chemical group to facilitate a specific chemical reaction to a glycan or sugar moieties described herein with a complimentary chemical group. In some embodiments, the hydroxyl group (-OH) off C3, C4, or C6 of GalNAc can be functionalized with a chemical group to increase the affinity for ASGPR. In some embodiments, the hydroxyl group (-OH) off the C3, C4, or C6 of GalNAc may be replaced with an azide, a ketone, an aldehyde, a selenol, an aliphatic alkyne, a strained alkyne, an amine, a hydrazide, an aminooxy, a boronate, or a vicinal diol. In some embodiments, a side chain pair can be added to the glycan, including a side chain pair selected from the group consisting of an azido / aliphatic alkyne, an azido / strained alkyne, a ketone / amine, an aldehyde / amine, a ketone / hydrazide, a ketone / aminooxy, an aldehyde / hydrazide, an aldehyde / aminooxy, and a boronate / vicinal diol.

[0147] In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with an azide to react with an alkyne-functionalized glycan. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with an alkyne to react with an azide-functionalized glycan. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with a ketone to react with an amine-functionalized glycan to form an imine. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with an amine to react with a ketone-functionalized glycan to form an imine. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with an aldehyde to react with an amine-functionalized glycan to form an imine. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with an amine to react with an aldehyde-functionalized glycan to form an imine. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with an aldehyde to react with a cyanide-functionalized glycan to form a cyanohydrin. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with a cyanide to react with an aldehyde-functionalizedglycan to form a cyanohydrin. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with a ketone to react with an alcohol-functionalized glycan. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with an aldehyde to react with an alcohol-functionalized glycan. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with a sei enol group to react with a selenol-functionalized glycan. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with a ketone to react with a hydrazide-functionalized glycan. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with a hydrazide to react with a ketone-functionalized glycan. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with a ketone to react with an aminooxy-functionalized glycan. In some embodiments, the - OH of C3, C4, or C6 of GalNAc can be replaced with an aminooxy to react with a ketone- functionalized glycan. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with an aldehyde to react with a hydrazide-functionalized glycan. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with a hydrazide to react with an aldehyde-functionalized glycan. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with an aldehyde to react with an aminooxy-functionalized glycan. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with an aminooxy to react with an aldehyde-functionalized glycan. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with a boronate to react with a vicinal diol-functionalized glycan. In some embodiments, the -OH of C3, C4, or C6 of GalNAc can be replaced with a vicinal diol to react with a boronate-functionalized glycan.

[0148] In some embodiments, GalNAc has a structure of Formula (IV):Formula (IV) wherein R is a hydroxyl group, an azide, a ketone, an aldehyde, a sei enol, an aliphatic alkyne, a strained alkyne, an amine, a hydrazide, an aminooxy, a boronate, or a vicinal diol.

[0149] In some embodiments, GalNAc has a structure of Formula (V):Formula (V)wherein Ri and R2 are each a hydroxyl group, an azide, a ketone, an aldehyde, a selenol, an aliphatic alkyne, a strained alkyne, an amine, a hydrazide, an aminooxy, a boronate, or a vicinal diol.

[0150] In some embodiments, the linker or L of Formula (V) comprises a bond or R4, wherein R4 is - O-, -S-, -Se-, -N-, -S-S-, -Se-Se-, -CH2-, -CH=CH-, -C=C-, -CH2CH2-, -(C=O)-, -(C=O)NH-, - NH(C=O)-, -(C=O)CH2-, -CH2(C=O)-, -CH2S-, -CH2NH-, -NHCH2-, -OCH2-, -CH(CH3)O-, -CH2O- , -OCH2O-, -CH(CH3)O-, -CH2(C=O)NH-, -CH2(C=O)O-, -CH(CH3)NH-, or -CH(CH3)CH2-.

[0151] In some embodiments, L3-X of Formula (IA) comprises a structure of Formula (II): AA1-! / - X; wherein X is a glycan, L4is a linker, and AA1is any amino acid (such as standard amino acid or an NSAA). In some embodiments, the L3-X is selected from the group consisting of structures listed in Table 3. In some embodiments, AA1-L4-X has a structure selected from the group consisting of structures listed in Table 3. In some embodiments, the NSAA is selected from the group consisting of: GlcNAc-0-Asn, Fuc-al, 6-GlcNAc-P-Asn, GalNAc-P-Asn, Sia-a2,3-GalNAc-P-Asn, Sia-a2,6- GalNAc-P-Asn, Sia-a2,3-GlcNAc-P-Asn, Sia-a2,6-GlcNAc-P-Asn, Sia-a-Asn, GlcNAc-P-Ser, Fuc- al,6-GlcNAc-P-Ser, GalNAc-a-Ser, Sia-a2,3-GalNAc-a-Ser, Sia-a2,6-GalNAc-a-Ser, Sia-a2,3- GlcNAc-a-Ser, Sia-a2,6-GlcNAc-a-Ser, Sia-a-Ser, GlcNAc-a-Thr, Fuc-al, 6-GlcNAc-a-Thr, GalNAc-a-Thr, Sia-a2,3-GalNAc-a-Thr, Sia-a2,6-GalNAc-a-Thr, Sia-a2,3-GlcNAc-a-Thr, Sia-a2,6- GlcNAc-a-Thr, Sia-a-Thr, GalNAc-a-Thr, Gal-pi,3-GalNAc-a-Ser, Gal-pi,3-GalNAc-a-Thr, Sia- a2,3-Gal-pi,3-GalNAc-a-Ser, Sia-a2,3-Gal-pi,3-GalNAc-a-Thr, GlcNAc-pi,4-GlcNAc-P-Asn, GlcNAc-pi,4-GlcNAc-P-Ser, GlcNAc-P 1,4-GlcNAc-P-Thr, GlcNAc-pi,4-[Fuc-al,3]GlcNAc-P- Asn, GlcNAc-pi,4-[Fuc-al,3]GlcNAc-P-Ser, GlcNAc-pi,4-[Fuc-al,3]GlcNAc-P-Thr, GlcNAc- pi, 4-[Fuc-al, 3] [Fuc-al, 6]GlcNAc-P-Asn, GlcNAc-pi,4-[Fuc-al, 3] [Fuc-al, 6]GlcNAc-P-Ser,GlcNAc-P l,4-[Fuc-al, 3] [Fuc-al, 6]GlcNAc-P-Thr, Neu5Ac-a2,3Gal-pi,4[Fuc-al,3]GlcNAc-P-Asn, Neu5Ac-a2,3Gal-pi,4[Fuc-al,3]GlcNAc-P-Ser, Neu5Ac-a2,3Gal-pi,4[Fuc-al,3]GlcNAc-P-Thr, Gal-pi,4[Fuc-al,3]GlcNAc-P-Asn, Gal-pi,4[Fuc-al,3]GlcNAc-P-Ser, Gal-pi,4[Fuc-al,3]GlcNAc- P-Thr, Gal-al,3-Gal-pi,4-GlcNAc-P-Asn, Gal-al,3-Gal-pi,4-GlcNAc-P-Ser, Gal-al,3-Gal-pi,4- GlcNAc-P-Thr, Gal-al,3-Gal-P-Asn, Gal-al,3-Gal-P-Ser, Gal-al,3-Gal-P-Thr, GlcNAc-a-Cys, Fuc- al,6-GlcNAc-a-Cys, GalNAc-a-Cys, Sia-a2,3-GalNAc-a-Cys, Sia-a2,6-GalNAc-a-Cys, Sia-a2,3- GlcNAc-a-Cys, Sia-a2,6-GlcNAc-a-Cys, Sia-a-Cys, GalNAc-a-Cys, Sia-a2,3-GalNAc-a-Cys, Gal- pi,3-GalNAc-a-Cys, Sia-a2,3-Gal-pi,3-GalNAc-a-Cys, GlcNAc-pi,4-GlcNAc-P-Cys, GlcNAc- P 1 ,4-[Fuc-al ,3 ]GlcNAc-P-Cys, GlcNAc-P 1 ,4-[Fuc-al ,3 ] [Fuc-al ,6]GlcNAc-P-Cys, Neu5Ac- a2,3Gal-pi,4[Fuc-al,3]GlcNAc-P-Cys, Gal-pi,4[Fuc-al,3]GlcNAc-P-Cys, Gal-al,3-Gal-pi,4- GlcNAc-P-Cys, Gal-al,3-Gal-P-Cys, GlcNAc-a-Sec, Fuc-al, 6-GlcNAc-a-Sec, GalNAc-a-Sec, Sia- a2,3-GalNAc-a-Sec, Sia-a2,6-GalNAc-a-Sec, Sia-a2,3-GlcNAc-a-Sec, Sia-a2,6-GlcNAc-a-Sec, Sia- a-Sec, GalNAc-a-Sec, Sia-a2,3-GalNAc-a-Sec, Gal-pi,3-GalNAc-a-Sec, Sia-a2,3-Gal-pi,3- GalNAc-a-Sec, GlcNAc-P 1,4-GlcNAc-P-Sec, GlcNAc-P l,4-[Fuc-al,3]GlcNAc-P-Sec, GlcNAc-pi,4-[Fuc-al,3][Fuc-al,6]GlcNAc-P-Sec, Neu5Ac-a2,3Gal-pi,4[Fuc-al,3]GlcNAc-P-Sec, Gal- pi,4[Fuc-al,3]GlcNAc-P-Sec, Gal-al,3-Gal-pi,4-GlcNAc-P-Sec, and Gal-al,3-Gal-P-Sec.

[0152] In some embodiments, a glycan comprises one or more of N-Acetylglucosamine (GlcNAc), N-Acetylgalactosamine (GalNAc), GlcNAc-al,6-fucose (Fuc), galactose (Gal), sialic acid (Sia), Sia- a2,3-Gal, Sia-a2,6-Gal, Sia-a2, 3 -GlcNAc, Sia-a2,6-GlcNac, Sia-a2, 3 -GalNAc, Gal-P 1,3 -GalNAc, Sia-a2, 3 -Gal-P 1,3 -GalNAc, GlcNAc-pi,4-GlcNAc, GlcNAc-pi,4-[Fuc-al,3]GlcNAc, GlcNAc-pi,4- [Fuc-al,3][Fuc-al,6]GlcNAc, N-Acetylneuraminic acid (Neu5Ac), Neu5Ac-a2,3Gal-pi,4[Fuc- al,3]GlcNAc, Gal-pi,4[Fuc-al,3]GlcNAc, Gal-al,3-Gal-pi,4-GlcNAc-P, and Gal-al,3-Gal.

[0153] In some embodiments, the AAJ-L4-X is Sia-a2,3-GalNAc-P-Asn with a structure of Formula (VI):Formula (VI) wherein R2= H, R3= NHCOCH3 or NHCOCH2OH, R4= H, R5= H, and R6= H or COCH3.Table 3. Exemplary amino acids coupled to sugar moiety(ies)R1= OH or linkageR2, R4, R5, R6= H or AcR3= NHAc, NHGc, OH

[0154] In some embodiments, at least one amino acid of the engineered uricase polypeptide is replaced with an NSAA. In some embodiments, the at least one amino acid is a naturally occurring amino acid. In some embodiments, the at least one amino acid is a standard amino acid, such as glycine (Gly or G), alanine (Ala or A), valine (Vai or V), leucine (Leu or L), isoleucine (He or I), proline (Pro or P), phenylalanine (Phe or F), tryptophan (Trp or W), methionine (Met or M), serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), glutamine (Gin or Q), aspartic acid (Asp or D), glutamic acid (Glu or E), lysine (Lys or K), arginine (Arg or R), or histidine (His or H). In some embodiments, at least one Asn, Ser, Thr, Trp, or Cys is replaced with the NSAA. In some embodiments, the polypeptide further comprises one or more sugar moieties o glycans.

[0155] In some embodiments, an NSAA without a gly can is incorporated into the engineered uricase polypeptide, such as during translation of the polypeptide. In some embodiments, the NSAA comprises a sidechain which can react with a functional group of a glycan (or glycan-containing molecule) to link the glycan to the NSAA. Non-limiting examples of chemical groups of the sidechain of an NSAA which can react with a functional group of a glycan (or glycan-containing molecule) to link the glycan to the NSAA include, but are not limited to, an azide, a ketone, an aldehyde, a selenol, an alkyne, an aliphatic alkyne, a strained alkyne, an amine, an alcohol, a cyanide, a hydrazide, an aminooxy, a boronate, and a vicinal diol. Examples of NSAAs with a sidechain which can react with a functional group of a glycan (or glycan-containing molecule) to link the glycan to the NSAA include, but are not limited to, para-azidophenylalanine, azido-lysine, azido-alanine, para-azidomethyl-1- phenylalanine, p-benzoylphenylalanine, p-acetylphenylalanine, p-propargyloxy-l-phenylalanine, propargylglycine, homopropargylglycine, selenocysteine, and para-isothiocyanate phenylalanine.

[0156] Examples of pairings of NSAA sidechains / functional groups of glycans (or glycan-containing molecules) include, but are not limited to, an azido / aliphatic alkyne, an azido / strained alkyne, a ketone / amine, a ketone / alcohol, a ketone / cyanide, an aldehyde / amine, an aldehyde / alcohol, an aldehyde / cyanide, a ketone / hydrazide, a ketone / aminooxy, an aldehyde / hydrazide, an aldehyde / aminooxy, a selenol / selenol, and a boronate / vicinal diol.

[0157] In some embodiments, an azide of the NSAA can react with an alkyne-functionalized glycan to link the glycan to the NSAA. In some embodiments, an alkyne of the NSAA can react with an azide- functionalized glycan to link the glycan to the NSAA. In some embodiments, a ketone of the NSAA can react with an amine-functionalized glycan to link the glycan to the NSAA. In some embodiments, an amine of the NSAA can react with a ketone-functionalized glycan to link the glycan to the NSAA. In some embodiments, a ketone of the NSAA can react with an alcohol-functionalized glycan to link the glycan to the NSAA. In some embodiments, an alcohol of the NSAA can react with a ketone- functionalized glycan to link the glycan to the NSAA. In some embodiments, a ketone of the NSAA can react with a cyanide-functionalized glycan to link the glycan to the NSAA. In some embodiments, a cyanide of the NSAA can react with a ketone-functionalized glycan to link the glycan to the NSAA. In some embodiments, an aldehyde of the NSAA can react with an amine-functionalized glycan to link the glycan to the NSAA. In some embodiments, an amine of the NSAA can react with an aldehyde-functionalized glycan to link the glycan to the NSAA. In some embodiments, an aldehyde of the NSAA can react with an alcohol-functionalized glycan to link the glycan to the NSAA. In some embodiments, an alcohol of the NSAA can react with an aldehyde-functionalized glycan to link the glycan to the NSAA. In some embodiments, an aldehyde of the NSAA can react with a cyanide- functionalized glycan to link the glycan to the NSAA. In some embodiments, a cyanide of the NSAA can react with an aldehyde-functionalized glycan to link the glycan to the NSAA. In some embodiments, a ketone of the NSAA can react with a hydrazide-functionalized glycan to link the glycan to the NSAA. In some embodiments, a hydrazide of the NSAA can react with a ketone- functionalized glycan to link the glycan to the NSAA. In some embodiments, a ketone of the NSAA can react with an aminooxy-functionalized glycan to link the glycan to the NSAA. In some embodiments, an aminooxy of the NSAA can react with a ketone-functionalized glycan to link the glycan to the NSAA. In some embodiments, an aldehyde of the NSAA can react with a hydrazide- functionalized glycan to link the glycan to the NSAA. In some embodiments, a hydrazide of the NSAA can react with an aldehyde-functionalized glycan to link the glycan to the NSAA. In some embodiments, an aldehyde of the NSAA can react with an aminooxy-functionalized glycan to link the glycan to the NSAA. In some embodiments, an aminooxy of the NSAA can react with an aldehyde- functionalized glycan to link the glycan to the NSAA. In some embodiments, a strained alkene of the NSAA can react with a tetrazine functionalized glycan to link the glycan to the NSAA. In some embodiments, a tetrazine of the NSAA can react with a strained alkene functionalized glycan to link the glycan to the NSAA. In some embodiments, a selenol of the NSAA can react with a selenol- functionalized glycan to link the glycan to the NSAA. In some embodiments, a boronate of the NSAA can react with a vicinal diol-functionalized glycan to link the glycan to the NSAA. In some embodiments, a vicinal diol of the NSAA can react with a boronate-functionalized glycan to link theglycan to the NSAA. In some embodiments, the polypeptide further comprises one or more sugar moieties conjugated to the sidechain.

[0158] In some embodiments, an azide of a NSAA can react with an alkyne-functionalized glycan to link the glycan to the NSAA. In some embodiments, an alkyne of a NSAA can react with an azide- functionalized glycan to link the glycan to the NSAA. In some embodiments, a ketone of a NSAA can react with an amine-functionalized glycan to link the glycan to the NSAA. In some embodiments, an amine of a NSAA can react with a ketone-functionalized glycan to link the glycan to the NSAA. In some embodiments, a ketone of a NSAA can react with an alcohol-functionalized glycan to link the glycan to the NSAA. In some embodiments, an alcohol of a NSAA can react with a ketone- functionalized glycan to link the glycan to the NSAA. In some embodiments, a ketone of a NSAA can react with a cyanide-functionalized glycan to link the glycan to the NSAA. In some embodiments, a cyanide of a NSAA can react with a ketone-functionalized glycan to link the glycan to the NSAA. In some embodiments, an aldehyde of a NSAA can react with an amine-functionalized glycan to link the glycan to the NSAA. In some embodiments, an amine of a NSAA can react with an aldehyde- functionalized glycan to link the glycan to the NSAA. In some embodiments, an aldehyde of a NSAA can react with an alcohol-functionalized glycan to link the glycan to the NSAA. In some embodiments, an alcohol of a NSAA can react with an aldehyde-functionalized glycan to link the glycan to the NSAA. In some embodiments, an aldehyde of a NSAA can react with a cyanide-functionalized glycan to link the glycan to the NSAA. In some embodiments, a cyanide of a NSAA can react with an aldehyde-functionalized glycan to link the glycan to the NSAA. In some embodiments, a ketone of a NSAA can react with a hydrazide-functionalized glycan to link the glycan to the NSAA. In some embodiments, a hydrazide of a NSAA can react with a ketone-functionalized glycan to link the glycan to the NSAA. In some embodiments, a ketone of a NSAA can react with an aminooxy-functionalized glycan to link the glycan to the NSAA. In some embodiments, an aminooxy of a NSAA can react with a ketone-functionalized glycan to link the glycan to the NSAA. In some embodiments, an aldehyde of a NSAA can react with a hydrazide-functionalized glycan to link the glycan to the NSAA. In some embodiments, a hydrazide of a NSAA can react with an aldehyde-functionalized glycan to link the glycan to the NSAA. In some embodiments, an aldehyde of a NSAA can react with an aminooxy- functionalized glycan to link the glycan to the NSAA. In some embodiments, an aminooxy of a NSAA can react with an aldehyde-functionalized glycan to link the glycan to the NSAA. In some embodiments, a sei enol of a NSAA can react with a selenol-functionalized glycan to link the glycan to the NSAA. In some embodiments, a boronate of a NSAA can react with a vicinal diol-functionalized glycan to link the glycan to the NSAA. In some embodiments, a vicinal diol of a NSAA can react with a boronate-functionalized glycan to link the glycan to the NSAA. In some embodiments, the polypeptide further comprises one or more sugar moieties conjugated to the sidechain.

[0159] In some embodiments, the engineered uricase polypeptide comprises one or more sugar moieties conjugated to the NSAA. In some embodiments, the engineered uricase polypeptide comprises one or more sugar moieties linked to the NSAA. In some embodiments, the engineered uricase polypeptide further comprises one or more sugar moieties conjugated to the sidechain of the NSAA. In some embodiments, the one or more sugar moieties comprises branched glycans. A glycan branch can refer to a substructure or side chain that extends from the main linear chain of a glycan. In some cases glycan branching involves the attachment of additional sugar units to specific positions in the glycan structure, creating a branching point. The branch can consist of one or more sugar moieties, and it can further extend with additional branches or modifications. In some embodiments, the branched glycans comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more glycan branches. In some embodiments, the branched glycans comprises at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 glycan branches.

[0160] In some embodiments, the one or more sugar moieties comprises a glycosidic bond. In some embodiments, a glycosidic bond comprises an alpha-glycosidic bond or a beta glycosidic bond. In some embodiments, an alpha-glycosidic bond comprises the bond in which both carbons of the bond have the same stereochemistry. In some embodiments, an alpha-glycosidic bond comprises alpha- 1,3 glycosidic bond, alpha- 1,4 glycosidic bond, alpha- 1, 6 glycosidic bond, or alpha-2, 6 glycosidic bond. In some embodiments, a beta-glycosidic bond comprises the bond in which the two carbons of the bond have different stereochemistry. In some embodiments, a beta-glycosidic bond comprises beta-1, 2 glycosidic bond, or beta-1, 4 glycosidic bond. In some embodiments, a glycosidic bond comprises C-, N-,O-, or S-glycosidic bonds.

[0161] In some embodiments, a glycan comprises an N-glycan and / or an O-glycan. N-glycans can be divided into three types according to the sugar moiety structures: high-mannose type, hybrid type, and complex type. In some embodiments, a sugar moiety of an N-glycan comprises mannose, glucose, N- acetylglucosamines, galactose, sialic acid, or any combination thereof.

[0162] Methods described herein can determine the identity and quantity of the multitude of glycan types. For example, an N-glycan may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 200, 300, 400, 500, 1000 or more sugar moieties. For example, an N-glycan may comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, or 1000 sugar moieties. In some embodiments, an N-glycan is branched. In some embodiments, an N- glycan is linear. In some embodiments, an N-glycan comprises high-mmannose types (two N- acetylglucosamines with various mannoses), complex oligosaccharide type (any number of saccharides including more than two N-acetylglucosamines), or hybrid oligosaccharide (with a mannose on one side of the branch and a N-acetylglucosamine on another side of the branch).

[0163] In some embodiments, a sugar moiety of an O-glycan comprises N-acetyl-galactosamine (GalNAc), galactose (GAL), N-acetyl-glucosamine, sialic acid (Sia), N-acetylneuraminic acid, fucose, or a combination thereof. In some embodiments, an O-glycan comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 1000 or more sugar moieties. In some embodiments, an O-glycan comprises at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 200, 300, 400, 500, or 1000 sugar moieties. An O-glycan can comprise various Cores. Core 1 may be generated by the addition of galactose. Core 2 may be generated by the addition of N-acetyl- glucosamine to the N-acetyl-galactosamine of Core 1. Core 3 may be generated by the addition of a single N-acetyl-glucosamine to the original N-acetyl-galactosamine. Core 4 may be generated by the addition of a second N-acetyl-glucosamine to Core. Cores 3, 4, and 6 are P-GIcN Acylated on C3- hydroxyl (C3-OH) and / or C6-OH of the initiating GalNAc. Cores 5, 7, and 8 contain a-linked extensions (al -3 GalNAc, al-6GalNAc, and al -3 Gal, respectively). O-glycan may also comprise other cores not comprising Cores 1-8. In some embodiments, an O-glycan is branched. In some embodiments, an O-glycan is linear.

[0164] In some embodiments, the one or more sugar moieties linked to the engineered uricase polypeptides described herein comprise one or more sugars selected from the group consisting of sugars listed in Table 4 or combinations thereof.Table 4. Exemplary sugars (KEGG*)* Kyoto Encyclopedia of Genes and Genomes# Symbol Nomenclature for Glycans

[0165] In some embodiments, the glycan linked to an engineered polypeptide described herein comprises a galactosylated, an N-acetylglucosylated, and / or a sialylated version of MansGlcNAc2. In some embodiments, the glycan the glycan linked to an engineered polypeptide described herein comprises MamGlcNAc^ Man4GlcNAc2, MansGlcNAc2, MangGlcNAc2, Man?GlcNAc2, MansGlcNAc2, MangGlcNAc2, Gal, Sia, or SiaGal. In some embodiments, the glycan the glycan linked to an engineered polypeptide described herein comprises a sialylated version of GalNac or a sialylated version of GlcNAcGalNAc. In some embodiments, the glycan the glycan linked to an engineered polypeptide described herein comprises a sialic acid attached in the a2,3 or a2,6 conformation to underlying sugars, or is comprised of polysialic acid ([Siaa2,8]n, where n is between 2 and 150).

[0166] In some embodiments, the glycan the glycan linked to an engineered polypeptide described herein comprises fucosylated GlcNAc.

[0167] In some embodiments, the engineered polypeptide is a recombinant polypeptide. In some embodiments, the engineered polypeptide is not a synthetic polypeptide.

[0168] Also provided herein is a population of engineered polypeptide molecules. In some embodiments, the population of engineered polypeptide molecules comprises polypeptide molecules with homogeneous sugar moieties. In some aspects, each polypeptide molecule of the population of engineered polypeptide molecules comprises one or more NSAAs as described herein. In one aspect, provided herein is a polypeptide comprising a non-standard amino acid (NSAA). The NSAA can comprise any amino acid other than the 20 standard amino acids. In some embodiments, the NSAA linked to a glycan comprises a structure a structure of Formula (I): AA-L-X, wherein AA is an amino acid, L comprises a linker, and X is absent, or comprises a glycan or a glycan stub. In some embodiments, Formula (I) comprises a structure of Formula (IA): AA-L1-L2-L3-X. In some embodiments, the NSAA linked to a glycan comprises a structure of Formula (IA): AA-LJ-L2-L3-X. In some embodiments, L'-L2is an alkyne-containing linker. In some embodiments, L3comprises a standard amino acid. In some embodiments, X is a glycan (or any sugar moiety as described herein).

[0169] In some embodiments, the non-standard amino acid is P-alanine, D-alanine, 4-hydroxyproline, desmosine, D-glutamic acid, y-aminobutyric acid, P-cyanoalanine, norvaline, p-azido-l-phenylalanine (pAzF), p-acetophenylalanine (pAcF), p-azidomethyl-L-phenylalanine (pAMF), 4-Biphenylyl-L- alanine (BipA), azido-lysine (AzK), 4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L- leucine, selenocysteine, statine, and / or selenocysteine (Sec or U). In some embodiments, the NSAA is selected from the group consisting of para-azidophenylalanine, azido-lysine, azido-alanine, p- propargyloxy-l-phenylalanine, p-benzoylphenylalanine, p-acetylphenylalanine, p-propargyloxy-1- phenylalanine, propargylglycine, homopropargylglycine, selenocysteine, p-azido-L-phenylalanine, and para-isothiocyanate phenylalanine.

[0170] In some embodiments, L'-L2of Formula (IA) comprises an alkyne-containing linker. In some embodiments, the linker can be an alkyne-containing linker. In some embodiments, the linker comprises 4-pentynoic acid, propargylamine, 6-azidohexanoic acid, dibenzocyclooctyne (DBCO), 3- butyn-l-ol, ethynylbenzene, and / or PEG-alkyne. In some embodiments, AA-L or AA-L1comprises a 1,2, 3 -tri azole.In some embodiments, L3of Formula (IA) comprises a standard amino acid. In some embodiments, L3-X of Formula (IA) comprises a structure according to Table 3. In some embodiments, L3-X of Formula (IA) comprises a structure of Formula (II): AA1-L4-X; wherein X is a glycan, L4is a linker, and AA1is any amino acid (such as standard amino acid). In some embodiments, X comprises a glycan stub. In some embodiments, the standard amino acid comprises glycine (Gly or G), alanine (Ala or A), valine (Vai or standard V), leucine (Leu or L), isoleucine (He or I), proline (Pro or P), phenylalanine (Phe or F), tryptophan (Trp or W), methionine (Met or M), serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), glutamine (Gin or Q), aspartic acid (Asp or D), glutamic acid (Glu or E), lysine (Lys or K), arginine (Arg or R), or histidine(His or H). In some embodiments, L can be a linker. In some embodiments, L isH

[0171] In some embodiments, X of Formula (I) and / or Formula (IA) comprises a glycan. In some embodiments, the glycan can be a glycan stub. In some embodiments, X is a glycan stub.

[0172] In some embodiments, I -L2-L3-X of Formula (IA) comprises one or more sugar moieties and an alkyne-containing linker. In some embodiments, click chemistry is used to conjugate the gly canlinker conjugate to a NSAA. In some embodiments, click chemistry is used to conjugate the glycan- linker conjugate to a polypeptide comprising one or more NSAAs. In some embodiments, click chemistry is used to conjugate the glycan-linker conjugate to a uricase comprising one or more NSAAs.

[0173] In some embodiments, the population of engineered uricase polypeptide molecules comprises polypeptide molecules with homogeneous glycosylation or a uniform glycosylation pattern. In some embodiments, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of the polypeptide molecules of the population have homogeneous glycosylation or a uniform glycosylation pattern. In some embodiments, at least 80% of the polypeptide molecules of the population have homogeneous glycosylation or a uniform glycosylation pattern. In some embodiments, at least 85% of the polypeptide molecules of the population have homogeneous glycosylation or a uniform glycosylation pattern. In some embodiments, at least 90% of the polypeptide molecules of the population have homogeneous glycosylation or a uniform glycosylation pattern. In some embodiments, at least 95% of the polypeptide molecules of the population have homogeneous glycosylation or a uniform glycosylation pattern.

[0174] In some embodiments, the population of engineered uricase polypeptide molecules comprises at least 10; 50; 100; 500; 1,000; 5,000; 10,000; 50,000; 100,000; 500,000; 1,000,000 or more polypeptide molecules. In some embodiments, the population of the polypeptide molecules comprises from 10 to 5,000,000 or more polypeptide molecules. In some embodiments, the population of the polypeptide molecules comprises from 10 to 50, from 10 to 100, from 10 to 500, from 10 to 1,000, from 10 to 5,000, from 10 to 10,000, from 10 to 50,000, from 10 to 100,000, from 10 to 500,000, from 10 to 1,000,000, from 10 to 5,000,000, from 50 to 100, from 50 to 500, from 50 to 1,000, from 50 to 5,000, from 50 to 10,000, from 50 to 50,000, from 50 to 100,000, from 50 to 500,000, from 50 to 1,000,000, from 50 to 5,000,000, from 100 to 500, from 100 to 1,000, from 100 to 5,000, from 100 to 10,000, from 100 to 50,000, from 100 to 100,000, from 100 to 500,000, from 100 to 1,000,000, from 100 to 5,000,000, from 500 to 1,000, from 500 to 5,000, from 500 to 10,000, from 500 to 50,000, from 500 to 100,000, from 500 to 500,000, from 500 to 1,000,000, from 500 to 5,000,000, from 1,000 to 5,000, from 1,000 to 10,000, from 1,000 to 50,000, from 1,000 to 100,000, from 1,000 to 500,000, from 1,000 to 1,000,000, from 1,000 to 5,000,000, from 5,000 to 10,000, from 5,000 to 50,000, from 5,000 to 100,000, from 5,000 to 500,000, from 5,000 to 1,000,000, from 5,000 to 5,000,000, from10,000 to 50,000, from 10,000 to 100,000, from 10,000 to 500,000, from 10,000 to 1,000,000, from 10,000 to 5,000,000, from 50,000 to 100,000, from 50,000 to 500,000, from 50,000 to 1,000,000, from 50,000 to 5,000,000, from 100,000 to 500,000, from 100,000 to 1,000,000, from 100,000 to 5,000,000, from 500,000 to 1,000,000, from 500,000 to 5,000,000, or from 1,000,000 to 5,000,000 polypeptide molecules. In some embodiments, the population of the polypeptide molecules comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000, at least 90,000, at least 100,000, at least 200,000, at least 300,000, at least 400,000, at least 500,000, at least 600,000, at least 700,000, at least 800,000, at least 900,000, at least 1,000,000, at least 2,000,000, at least 3,000,000, at least 4,000,000, or at least 5,000,000 or more polypeptide molecules. In some embodiments, the population of the polypeptide molecules comprises at least IxlO6, 2xl06, 3xl06, 4x106, 5xl06, 6xl06, 7xl06, 8xl06, 9xl06, 1x107, 2xl07, 3xl07, 4xl07, 5xl07, 6xl07, 7xl07, 8xl07, 9xl07, IxlO8, 2xl08, 3xl08, 4xl08, 5xl08, 6xl08, 7xl08, 8xl08, 9xl08, IxlO9, 2xl09, 3xl09, 4x109, 5xl09, 6xl09, 7xl09, 8xl09, 9xl09, IxlO10, 2xlO10, 3xl010, 4xlO10, 5xl010, 6xlO10, 7xlO10, 8xl010, 9xlO10, IxlO11, 2xlOn, 3xl0n, 4xlOn, SxlO11, 6xlOn, 7xlOn, SxlO11, 9xlOn, IxlO12, 2xl012, 3xl012, 4xl012, 5xl012, 6xl012, 7xl012, 8xl012, or 9xl012or more polypeptide molecules. In some embodiments, the population of the polypeptide molecules comprises at most 50, at most 60, at most 70, at most 80, at most 90, at most 100, at most 150, at most 200, at most 250, at most 300, at most 350, at most 400, at most 450, at most 500, at most 600, at most 700, at most 800, at most 900, at most 1,000, at most 2,000, at most 3,000, at most 4,000, at most 5,000, at most 6,000, at most 7,000, at most 8,000, at most 9,000, at most 10,000, at most 20,000, at most 30,000, at most 40,000, at most 50,000, at most 60,000, at most 70,000, at most 80,000, at most 90,000, at most 100,000, at most 200,000, at most 300,000, at most 400,000, at most 500,000, at most 600,000, at most 700,000, at most 800,000, at most 900,000, at most 1,000,000, at most 2,000,000, at most 3,000,000, at most 4,000,000, or at most 5,000,000 polypeptide molecules. In some embodiments, the population of polypeptide molecules comprises at most IxlO6, 2xl06, 3xl06, 4xl06, 5xl06, 6xl06, 7xl06, 8xl06, 9xl06, IxlO7, 2xl07, 3xl07, 4xl07, 5xl07, 6xl07, 7xl07, 8xl07, 9xl07, IxlO8, 2xl08, 3xl08, 4xl08, 5xl08, 6xl08, 7x108, 8x108, 9x108, IxlO9, 2x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109, 9x109, IxlO10, 2x1010, 3xlO10, 4xlO10, 5xlO10, 6xlO10, 7xlO10, 8xlO10, 9xlO10, IxlO11, 2xlOn, 3xlOn, 4xlOn, SxlO11, 6xlOn, 7xlOn, 8xlOn, 9xlOn, IxlO12, 2xl012, 3xl012, 4xl012, 5xl012, 6xl012, 7xl012, 8xl012, or 9xl012polypeptide molecules.Method of Making Polypeptides

[0175] Also provided herein are methods of making the engineered polypeptides described herein, such as engineered uricase polypeptides comprising an NSAA engineered uricase polypeptides comprising an NSAA linked to a glycan. In some embodiments, a polypeptide described herein can be produced by expressing a polypeptide encoded by an exogenous polynucleotide sequence in a host cell in the presence of an NSAA or precursor thereof. For example, an engineered uricase polypeptide described herein can be produced by expressing a uricase polypeptide encoded by an exogenous polynucleotide sequence in a host cell in the presence of an NSAA or precursor thereof. In some embodiments, the polypeptide comprising a NSAA is encoded by a polynucleotide sequence comprising a codon that has been reassigned to the NSAA and that has been substantially or completely removed from a host genome. In some embodiments, the method of making the polypeptide comprising the NSAA comprises translating the polypeptide from an exogenous RNA sequence present in a host cell containing an orthogonal AARS / tRNA pair that effectively charges the specific NSAA onto the tRNA with an anti-codon complementary to the reassigned codon (e.g., amber codon) in the presence of an NSAA or precursor thereof. In some embodiments, the orthogonal AARS / tRNA pair can be provided on a plasmid or into the genome of a genomically recoded organism with a genome in which a codon has been removed and reassigned to a NSAA.

[0176] In some embodiments, the method further comprises purifying a polypeptide comprising an NSAA produced according to these methods from the host cell. In some embodiments, the method further comprises conjugating (such as conjugation through click chemistry) the polypeptide comprising the NSAA to a glycan or glycan-linker conjugate as described herein.

[0177] As described above, an orthogonal aaRS / tRNA pair can be used to bioorthogonally incorporate NSAAs into proteins. Over 100 NSAAs with diverse chemistries have been synthesized and co- translationally incorporated into proteins using evolved orthogonal aminoacyl -tRNA synthetase (aaRSs) / tRNA pairs. Vector-based over-expression systems may be used to outcompete natural codon function with its reassigned function. If one completely abolishes natural UAG translation function, far lower aaRS / tRNA function may be sufficient to achieve efficient NSAA incorporation. Genomically recoded organism (GRO)-based NSAA incorporation can use either vector- and / or genome-based aaRS / tRNA pairs. In some embodiments, the NSAA is supplied to the genomically recoded organism by supplementing the growth media with the NSAA or a precursor thereof. In some embodiments, the AARS is expressed in the genomically recoded organism by inducing expression of the AARS. In some embodiments, the AARS is expressed in the genomically recoded organism by constitutive expression of the AARS. Genome-based aaRS / tRNA pairs have been used to reduce the mis-incorporation of standard amino acids in the absence of available NSAAs. A genomically recoded organism has been made in which the UAG codon has been completely removed from the genomeand reassigned to a NSAA. Thus, a polypeptide containing an NSAA, such as an NSAA with glycan stub, can be produced in the genomically recoded organism.

[0178] The engineered polypeptides described herein can be made in a host cell, or in vitro, in cell- free synthetic systems. Host cells may be any that can be robustly recoded. These can be bacterial cells that have well developed genetic systems, of which E. coli is exemplary. Other bacterial species can also be used. In some embodiments, cell-free systems for producing the proteins may be coupled transcription / translation systems or only translation systems. Notably, in some embodiments, biological syntheses are utilized rather than chemical synthesis.

[0179] Recoded cells with polynucleotide sequences encoding the engineered polypeptides described herein may be cultured by any suitable means known in the art. In some embodiments, the culturing may be batch or continuous, in shaker flasks or in fermenters or immobilized on solid surfaces, such as small particles contained in larger vessels. In some embodiments, the culture medium is supplemented with the NSAA or a precursor thereof.

[0180] In some embodiments, the culture medium may be supplemented with a source of GlcNAc-0- Asn, Fuc-al, 6-GlcNAc-P-Asn, GalNAc-P-Asn, Sia-a2,3-GalNAc-P-Asn, Sia-a2,6-GalNAc-P-Asn, Sia-a2,3-GlcNAc-P-Asn, Sia-a2,6-GlcNAc-P-Asn, Sia-a-Asn, GlcNAc-P-Ser, Fuc-al, 6-GlcNAc-P- Ser, GalNAc-a-Ser, Sia-a2,3-GalNAc-a-Ser, Sia-a2,6-GalNAc-a-Ser, Sia-a2,3-GlcNAc-a-Ser, Sia- a2,6-GlcNAc-a-Ser, Sia-a-Ser, GlcNAc-a-Thr, Fuc-al, 6-GlcNAc-a-Thr, GalNAc-a-Thr, Sia-a2,3- GalNAc-a-Thr, Sia-a2,6-GalNAc-a-Thr, Sia-a2,3-GlcNAc-a-Thr, Sia-a2,6-GlcNAc-a-Thr, Sia-a- Thr, GalNAc-a-Thr, Gal-pi,3-GalNAc-a-Ser, Gal-pi,3-GalNAc-a-Thr, Sia-a2,3-Gal-pi,3-GalNAc- a-Ser, Sia-a2,3-Gal-pi,3-GalNAc-a-Thr, GlcNAc-pi,4-GlcNAc-P-Asn, GlcNAc-P 1,4-GlcNAc-P- Ser, GlcNAc-pi,4-GlcNAc-P-Thr, GlcNAc-pi,4-[Fuc-al,3]GlcNAc-P-Asn, GlcNAc-pi,4-[Fuc- al,3]GlcNAc-P-Ser, GlcNAc-pi,4-[Fuc-al,3]GlcNAc-P-Thr, GlcNAc-pi,4-[Fuc-al, 3] [Fuc- al ,6]GlcNAc-P-Asn, GlcNAc-P 1 ,4-[Fuc-al ,3 ] [Fuc-al ,6]GlcNAc-P-Ser, GlcNAc-P 1 ,4-[Fuc- al ,3 ] [Fuc-al ,6]GlcNAc-P-Thr, Neu5 Ac-a2,3 Gal-P 1 ,4[Fuc-al ,3 ]GlcNAc-P-Asn, Neu5 Ac-a2,3 Gal- pi,4[Fuc-al,3]GlcNAc-P-Ser, Neu5Ac-a2,3Gal-pi,4[Fuc-al,3]GlcNAc-P-Thr, Gal-P l,4[Fuc- al,3]GlcNAc-P-Asn, Gal-pi,4[Fuc-al,3]GlcNAc-P-Ser, Gal-pi,4[Fuc-al,3]GlcNAc-P-Thr, Gal- al,3-Gal-pi,4-GlcNAc-P-Asn, Gal-al,3-Gal-pi,4-GlcNAc-P-Ser, Gal-al,3-Gal-pi,4-GlcNAc-P- Thr, Gal-al,3-Gal-P-Asn, Gal-al,3-Gal-P-Ser, Gal-al,3-Gal-P-Thr, GlcNAc-a-Cys, Fuc-al, 6- GlcNAc-a-Cys, GalNAc-a-Cys, Sia-a2,3-GalNAc-a-Cys, Sia-a2,6-GalNAc-a-Cys, Sia-a2,3- GlcNAc-a-Cys, Sia-a2,6-GlcNAc-a-Cys, Sia-a-Cys, GalNAc-a-Cys, Sia-a2,3-GalNAc-a-Cys, Gal- pi,3-GalNAc-a-Cys, Sia-a2,3-Gal-pi,3-GalNAc-a-Cys, GlcNAc-P 1,4-GlcNAc-P-Cys, GlcNAc- P 1 ,4-[Fuc-al ,3 ]GlcNAc-P-Cys, GlcNAc-P 1 ,4-[Fuc-al ,3 ] [Fuc-al ,6]GlcNAc-P-Cys, Neu5Ac- a2,3Gal-pi,4[Fuc-al,3]GlcNAc-P-Cys, Gal-pi,4[Fuc-al,3]GlcNAc-P-Cys, Gal-al,3-Gal-pi,4- GlcNAc-P-Cys, Gal-al,3-Gal-P-Cys, GlcNAc-a-Sec, Fuc-al, 6-GlcNAc-a-Sec, GalNAc-a-Sec, Sia- a2,3-GalNAc-a-Sec, Sia-a2,6-GalNAc-a-Sec, Sia-a2,3-GlcNAc-a-Sec, Sia-a2,6-GlcNAc-a-Sec, Sia-a-Sec, GalNAc-a-Sec, Sia-a2,3-GalNAc-a-Sec, Gal-01, 3 -GalNAc-a-Sec, Sia-a2,3-Gal-01,3- GalNAc-a-Sec, GlcNAc-01,4-GlcNAc-0-Sec, GlcNAc-01,4-[Fuc-al,3]GlcNAc-0-Sec, GlcNAc- 01,4-[Fuc-al,3][Fuc-al,6]GlcNAc-0-Sec, Neu5Ac-a2,3Gal-01,4[Fuc-al,3]GlcNAc-0-Sec, Gal- 01,4[Fuc-al,3]GlcNAc-0-Sec, Gal-al,3-Gal-01,4-GlcNAc-0-Sec, or Gal-al,3-Gal-0-Sec. As is known in the art, production of the desired protein variant may be under the control of an inducer or a repressor. Any such systems which are known in the art may be selected for convenience of construction and protein production.

[0181] In some embodiments, the tRNA of an orthogonal aaRS / tRNA pair used for incorporating an NSAA into a polypeptide is an engineered tRNA. In some embodiments, a tRNA / tRNA synthetase pair is engineered based on an anticodon-agnostic tRNA. In some embodiments, a tRNA / tRNA synthetase is engineered based on a polyspecific tRNA. In some embodiments, a tRNA / tRNA synthetase pair is engineered to utilize an anticodon corresponding to a codon that is unassigned in a genomically recoded organism (GRO) such as genomically recoded E. coli cell line (e.g. , an amber stop codon or a redundant rare codon for a standard amino acid). In some embodiments, a seryl- tRNA / seryl-tRNA synthetase pair, a leucyl-tRNA / leucyl-tRNA synthetase pair, an arginyl- tRNA / arginyl-tRNA synthetase pair, or a cysteinyl-tRNA / cysteinyl-tRNA synthetase pair may be used. In some embodiments, a tRNA / tRNA synthetase pair can be from any species such as bacteria (e.g., E. coll), archaea, plants, and eukaryotes (e.g., human).

[0182] In some embodiments, the method comprises expressing an amino acid sequence of the polypeptide. In some embodiments, expressing comprises expressing in a cell or in vitro. In some embodiments, the cell is abacterial cell. In some embodiments, the cell is a genomically recoded cell. In some embodiments, the cell comprises an unassigned codon recognized by a stabilizing nonstandard amino acid tRNA comprising an anticodon corresponding to the unassigned codon. In some embodiments, the polypeptide comprising a NSAA is encoded by a polynucleotide sequence comprising a codon that has been reassigned to the NSAA and that has been substantially or completely removed from a host genome. In some embodiments, the method comprises culturing the cell under conditions in which the amino acid sequence of an engineered uricase polypeptide is expressed. In some embodiments, the codon reassigned to a NSAA is UAG, UAA, or UGA, or where multiple different NSAAs are intended, any combination thereof. In some embodiments, the codon reassigned to a NSAA is UAG, UAA, UGA, AGY, UUR, UGC, AGR, or a combination thereof. In some embodiments, AGY is selected from the group consisting of AGU, and AGC. In some embodiments, UUR is selected from the group consisting of UUA, and UUG In some embodiments, AGR is selected from the group consisting of AGA, and AGG.

[0183] In some embodiments, the cell comprises a reassigned codon recognized by a tRNA charged with an NSAA. In some embodiments, the tRNA charged with an NSAA comprises an anticodon corresponding to the reassigned codon. In some embodiments, the tRNA charged with an NSAAcomprises a bacterial tRNA or variant thereof. In some embodiments, the tRNA charged with an NSAA comprises a eukaryotic tRNA or variant thereof. In some embodiments, the tRNA charged with an NSAA comprises a plant tRNA or variant thereof. In some embodiments, the tRNA charged with an NSAA comprises an archaeal tRNA or variant thereof. In some embodiments, the archaeal tRNA or variant thereof is selected from the group consisting of Methanococcus jannaschii tyrosyl tRNA, Methanosarcina barkeri pyrrolysine tRNA, Methanosarcina mazei pyrrolysine tRNA, Methanomethylophilus alvus pyrrolysine tRNA, Pyrococcus horokoshii prolyl tRNA, Pyrococcus horokoshii tyrosyl tRNA, Archaeoglobus fulgidus prolyl tRNA and any variant thereof.

[0184] In some embodiments, the cell comprises an aminoacyl-tRNA synthetase (aaRS) capable of attaching an NSAA to a tRNA. In some embodiments, the aaRS comprises abacterial tRNA synthetase or variant thereof. In some embodiments, the aaRS comprises a eukaryotic tRNA synthetase or variant thereof. In some embodiments, the aaRS comprises a plant tRNA synthetase or variant thereof. In some embodiments, the aaRS comprises an archaeal tRNA synthetase or variant thereof. In some embodiments, the archaeal aaRS or variant thereof is selected from the group consisting of Methanococcus jannaschii tyrosyl tRNA synthetase, Methanosarcina barkeri pyrrolysine tRNA synthetase, Methanosarcina mazei pyrrolysine tRNA synthetase, Methanomethylophilus alvus pyrrolysine tRNA, Pyrococcus horokoshii prolyl tRNA synthetase, Pyrococcus horokoshii tyrosyl tRNA synthetase, Archaeoglobus fulgidus prolyl tRNA synthetase, and any variant thereof.

[0185] Incorporation of NSAAs into a polypeptide involves the aminoacylation of a tRNA by an aminoacyl tRNA synthetase (AARS), which aminoacylates the tRNA with the desired NSAA instead of a standard amino acid. In some embodiments, the method of making a polypeptide comprising a NSAA uses an orthogonal AARS / tRNA pair that effectively charges the specific NSAA onto the tRNA with a reassigned codon (e.g., amber codon). For incorporating NSAAs into a polypeptide in vivo, an ideal orthogonal AARS / tRNA pair does not cross-react with the endogenous amino acids, AARSs, or tRNAs of a host cell but can be recognized by the host cell’s ribosome. In some embodiments, the NSAA is biphenylalanine / 4-Biphenylyl-L-alanine / L-4,4'-biphenylalanine (BipA). In some embodiments, the NSAA is p-azidophenylalanine / para-azido-phenylalanine / p-azido-1- phenylalanine (pAzF). In some embodiments, the NSAA is p-azidomethyl-l-phenylalanine (pAMF). In some embodiments, the tRNA is a tRNA-Tyr. In some embodiments, the tRNA is an engineered cognate tRNA. In some embodiments, the engineered AARS is a BipA-AARS (BipA-RS). In some embodiments, the engineered AARS is a pAzF-AARS (pAzF-RS).

[0186] Methods disclosed herein may include collecting the polypeptide comprising the NSAA described herein. In some embodiments, the collecting comprises lysing a cell. A cell may be lysed via high pressure, osmotic shock or pressure, low temperature, sonication, homogenization, or a combination thereof. The resulting solution, or lysate of the cell, may be collected and subjected to downstream analysis or processing such as purification. In some embodiments, the collecting thepolypeptide comprises purifying the polypeptide. In some embodiments, purifying does not comprise lysing the cell. In some embodiments, the polypeptide comprising a NSAA may be secreted. The secretion of the polypeptide may allow the polypeptide to be collected in the media. The secretion of the polypeptide may allow the polypeptide to be collected without lysis of the cell.

[0187] The polypeptide comprising a NSAA (glycosylated or not) may be purified, or otherwise concentrated or isolated such a solution contains predominantly the polypeptide comprising a NSAA. In some embodiments, the purification is performed on the soluble fraction of the cell lysate. In some embodiments, the soluble fraction of the cell lysate is obtained by the centrifugation of cell lysate and collecting the supernatant. In some embodiments, the purification is performed on the insoluble fraction of the cell lysate. In some embodiments, the insoluble fraction of the cell lysate is obtained by the centrifugation of cell lysate and collecting the pellet formed. In some embodiments, the insoluble fraction comprises inclusion bodies that include the polypeptide comprising a NSAA. In some embodiments, the purifying comprises isolating the polypeptide is purified from the inclusion bodies. In some embodiments, purifying comprises isolating from inclusion bodies. Isolating the polypeptide from the inclusion bodies may comprise the use of denaturants and chaotropes, such as guanidine or urea, to solubilize the inclusion bodies. In some embodiments, the purifying uses an agent that specifically binds the polypeptide. For example, the agent may be protein with a binding affinity to the polypeptide. In some embodiment, the agent that specifically binds the polypeptide is immobilized on a solid support such as a bead and is used to capture the polypeptide.

[0188] In some embodiments, purifying comprises using a chromatography. Chromatography may be performed by using the physical characteristics of the polypeptide, such as charge, shape, or polarity, to separate the polypeptide from other protein. In some embodiments, the chromatography comprises ion exchange chromatography, size exclusion chromatography, reverse phase chromatography, affinity chromatography, or a combination thereof. Multiple forms of chromatography may be used sequentially to increase the purity of the polypeptide. Chromatography may be performed by introducing the cell lysate or polypeptide containing solution into a column containing resin or other solid supports. The resin may interact different with different proteins and polypeptides resulting in a separation of the proteins based on physical characteristics. For example, the resin may contain nickel and may interact with a His tag on the polypeptide. In some embodiments, the chromatography is selected from the group consisting of an ion exchange chromatography, a size exclusion chromatography, a reverse phase chromatography, and an affinity chromatography. In some embodiments, purifying comprises using an agent that specifically binds the polypeptide. In some embodiments, the tag or affinity tag of the polypeptide as described herein can be used for the purification of the polypeptide as described herein.

[0189] The methods as disclosed elsewhere herein may yield a particular polypeptide yield. In some embodiments, the methods as disclosed elsewhere herein produce polypeptide at an amount of at least0.1 mgs (milligrams) per liter (L) of media. In some embodiments, the methods produce polypeptide at an amount of at least 1 mg per liter of media. In some embodiments, the methods produce polypeptide at an amount of at least 5 mgs per liter of media. In some embodiments, the methods produce polypeptide at an amount of at least 10 mgs per liter of media. In some embodiments, the methods produce polypeptide at an amount of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more mgs per liter of media.

[0190] Also provided herein is a cell comprising any polypeptide described herein and / or a polynucleotide encoding the polypeptide described herein, a NSAA tRNA described herein, a NSAA tRNA synthetase described herein, and optionally one or more sugar moieties described herein. In some embodiments, the one or more sugar moieties comprise one or more sugars selected from the group consisting of sugars listed in Table 4 or combinations thereof.Click-chemistry

[0191] In some embodiments, conjugating comprises chemically conjugating. Many chemical approaches have been developed to crosslink carbohydrate and protein, all of which can be used to conjugate a glycan to an NSAA of a polypeptide described herein. For example, the Staudinger ligation employs a substituted phosphite to react with the azide-modified protein to form the carbohydrate- protein conjugation via the amide bond formation (C. Grandjean, A. Boutonnier, C. Guerreiro, J. M. Fournier, L. A. Mulard, J Org Chem 2005, 70, 7123-7132). For example, oxime conjugation introduces an aminooxy group on the protein to react with oligosaccharide containing aldehyde or keto group (J. Kubler-Kielb, V. Pozsgay, J Org Chem 2005, 70, 6987-6990). For example, Michael addition often uses thiol group addition to maleimide to form a stable thioester linkage (T. Masuko, A. Minami, N. Iwasaki, T. Majima, S. Nishimura, Y. C. Lee, Biomacromolecules 2005, 6, 880-884). For example, the method of copper (I)-catalyzed cycloaddition of azide to alkyens (click chemistry) provides efficient glycoconjugation (a) H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew Chem Int Ed Engl 2001, 40, 2004-2021; b) S. Hotha, S. Kashyap, J Org Chem 2006, 71, 364-367).

[0192] In some embodiments, chemically conjugating comprises a click chemistry reaction. Click chemistry comprises a group of biocompatible small molecule reactions commonly used in bioconjugation that are stereospecific, high yielding, wide in scope, and simple to perform. The primary click reaction utilizes copper-catalyzed coupling of a terminal alkyne with a terminal azide to exclusively form the 1,2, 3 -triazole unit. These reactions create only byproducts that can be removed without chromatography, and can be conducted in easily removable or benign solvents. Examples of the click chemistry reaction includes, but are not limited to, Huisgen azi de-alkyne 1,3 -dipolar cycloaddition, copper-catalyzed azide-alkyne cycloaddition, ruthenium-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, strain-promoted alkyne-nitronecycloaddition, and reactions of strained alkenes such as alkene and azide [3+2] cycloaddition, alkene and tetrazine inverse-demand Diels-Alder, and alkene and tetrazole photoclick reaction. In some embodiments, the click chemistry reaction comprises a copper-catalyzed azide-alkyne cycloaddition. The mechanism of the copper-catalyzed azide-alkyne cycloaddition is described in Himo, et al. J. Am. Chem. Soc. 2005. 127: 210-216.

[0193] In some embodiments, a polypeptide with an NSAA conjugated to a glycan comprises a triazole ring. In some embodiments, the triazole ring of a polypeptide with an NSAA conjugated to a glycan is formed by reacting a sialic acid derivative-conjugated binding polypeptide comprising an NSAA with terminal azide moiety with a glycan comprising an alkyne or bound to a moiety comprising an alkyne using click chemistry. In some embodiments, the triazole ring of a polypeptide with an NSAA conjugated to a glycan is formed by reacting a sialic acid derivative-conjugated binding polypeptide comprising an NSAA comprising an alkyne or bound to a moiety comprising an alkyne with a glycan comprising a terminal azide moiety using click chemistry. In some embodiments, the click chemistry reaction to form the polypeptide with an NSAA conjugated to a glycan occurs at ambient temperatures. In some embodiments, the click chemistry reaction to form a polypeptide with an NSAA conjugated to a glycan occurs in the presence of a metal catalyst, for example, a copper(I)- catalyzed azide-alkyne cycloaddition. In some embodiments, the click chemistry reaction to form a polypeptide with an NSAA conjugated to a glycan is performed in the absence of copper.

[0194] In some embodiments, I -L2-L3-X of Formula (IA) can refer to a glycan-linker. In some embodiments, the glycan-linker conjugate comprises one or more sugar moieties and an alkyne- containing linker. In some embodiments, click chemistry is used to conjugate the glycan-linker conjugate to a NSAA. In some embodiments, click chemistry is used to conjugate the glycan-linker conjugate to a polypeptide comprising one or more NSAAs. In some embodiments, click chemistry is used to conjugate the glycan-linker conjugate to a uricase comprising one or more NSAAs. In some embodiments, Formula (IA) can refer to a glycan-linker-NSAA, wherein X is the glycan, I -L2-L3-is the linker, and NSAA is an amino acid other than the 20 standard amino acids. A glycan-linker conjugate can refer to a molecular entity that incorporates glycan moieties, or sugar moieties, into a linker molecule. In some case, the glycan-linker conjugate is designed to connect or bridge two different entities, such as the polypeptide disclosed herein and one or more sugar moieties. In some embodiments, the glycan-linker conjugate provides the benefits of glycosylation, as described above, to the polypeptide disclosed herein. The glycan component in the glycan-linker conjugate can add glycosylation functionality, offering advantages in terms of solubility, stability, bioactivity, and targeting. A glycan-linker conjugate can be synthesized through various methods, including chemical synthesis or enzymatic approaches. In some embodiments, the glycan-linker conjugate is synthesized as described in Example 6 of the present disclosure. The specific design and structure of the glycan- linker conjugate can vary depending on the intended application and desired properties. For example,the glycan-linker conjugate can have various structures as depicted in FIG. 1C, and FIGs. 4-7. In some embodiments, the linker incorporates a glycan moiety, which can be derived from natural sources or can be chemically synthesized. In some embodiments, the glycan, glycan moieties, or sugar moieties, can be attached to the linker molecule through specific chemical or enzymatic conjugation methods. In some embodiments, the glycan-linker conjugate is attached to a NSAA through click chemistry. In some embodiments, the glycan-linker conjugate is attached to a NSAA, which is incorporated into the polypeptide disclosed herein, through click chemistry. In some embodiments, the glycan-linker conjugate is attached to a NSAA, which is incorporated into a uricase disclosed herein, through click chemistry. In some embodiments, the glycan-linker conjugate is attached to a NSAA through click chemistry to form the glycosylated uricase disclosed herein.

[0195] In some embodiments, the glycan-linker conjugate contains a spacer or linking group that connects the glycan component to the target entity, wherein the target entity can be a NSAA, a polypeptide comprising a NSAA, and / or a uricase comprising a NSAA. The spacer can be a simple linear molecule or a more complex structure. The spacer can provide flexibility and appropriate distance between the glycan and the target. Glycan-linkers can be designed to be compatible with biological systems. For example, glycan-linkers can have minimal toxicity, low immunogenicity, and / or the ability to undergo biodegradation or elimination from the body. Glycan-linkers can be designed to suit specific needs, allowing for customization of the glycan structure, linker length, and other chemical or biological properties. In some embodiments, this flexibility enables fine-tuning of the glycan-linker conjugate for various applications, such as drug delivery. In some embodiments, the linker can participate in click chemistry reactions. In some embodiments, the linker can be an alkyne- containing linker. In some embodiments, the linker comprises 4-pentynoic acid, propargylamine, 6- azidohexanoic acid, dibenzocyclooctyne (DBCO), 3-butyn-l-ol, ethynylbenzene, and / or PEG-alkyne. In some embodiments, click chemistry can be used to click on glycan groups to a NSAA using an alkyne-containing linker. In some embodiments, the glycan-linker conjugate comprises one or more sugar moieties and an alkyne-containing linker. In some embodiments, click chemistry is used to click on the glycan-linker conjugate to a NSAA. In some embodiments, click chemistry is used to click on the glycan-linker conjugate to a polypeptide comprising a NSAA. In some embodiments, click chemistry is used to click on the glycan-linker conjugate to a uricase comprising a NSAA.Polypeptide stabilizing moieties

[0196] In some case, the engineered uricase polypeptides may also comprise an additional stabilizing moiety that increase the half-life of the polypeptides in the bloodstream of a subject or in other in vivo applications. The polypeptide may be glycosylated such that a sugar moiety is attached to the polypeptide. The polypeptide may be acylated.

[0197] The addition of a sugar moiety or acyl moiety may work to prolong circulation in the blood by multiple distinct mechanisms. The addition of the stabilizing moiety may first work by slowing therelease from the subcutaneous injection site into the bloodstream. The addition of the stabilizing moiety may work by increasing the hydrodynamic radius to reduce renal clearance from the bloodstream. The addition of the stabilizing moiety may work by binding the abundant circulating protein Human Serum Albumin (HSA). Upon receptor-mediated endocytosis, HSA may engage the neonatal receptor recycling system that returns HSA and HSA-associated proteins back into the bloodstream rather than trafficking them to lysosomal degradation. By binding transiently to HSA, the stabilizing moiety conjugated polypeptide may obtain a larger hydrodynamic radius to slow renal clearance and may gain access to the same neonatal receptor recycling system.

[0198] The acylation may comprise the addition of fatty acids. For example, the acylation may comprise the addition of palmitic acid. The acylation may comprise the addition of myristic acid. The fatty acid may be a member selected from the group consisting of butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, oleic acid, elaidic acid, eicosenoic acid, eruic acid, nervonic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, gamma-linolenic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoeic acid. The fatty acid may be of a particular length. For example, the fatty acid may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, or more carbons. The fatty acid may comprise linear chains. For example a linear chain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, or more carbons. The fatty acid may be branched. For example, the fatty acid may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, or more branches, wherein each branch may comprise an additional carbon or chain of carbon. The fatty acid may be saturated or unsaturated. The fatty acid may comprise a double bond. The fatty acid may comprise a triple bond. The fatty acid may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, or more unsaturations. The acylation of polypeptides with a particular fatty acids may be advantageous over another fatty acid based on the ability to increase the half-life or activity of the polypeptide.

[0199] The glycosylation may comprise the addition of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 200, 500 or more sugar moieties. The glycosylation may comprise the addition of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 200, or 500 sugar moieties. The sugar moieties may be of a certain size. For example, the sugar moieties may comprise at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8,4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5,11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4,13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3,15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2,17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1,19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21,21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8,22.9, 23, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7,24.8, 24.9, 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, 26, 26.1, 26.2, 26.3, 26.4, 26.5, 26.6,26.7, 26.8, 26.9, 27, 27.1, 27.2, 27.3, 27.4, 27.5, 27.6, 27.7, 27.8, 27.9, 28, 28.1, 28.2, 28.3, 28.4, 28.5,28.6, 28.7, 28.8, 28.9, 29, 29.1, 29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 29.8, 29.9, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 200, 500 or more kiloDaltons (kDa). The sugar moieties may comprise at most 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4,4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2,11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1,13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15,15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8,16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7,18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6,20.7, 20.8, 20.9, 21, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5,22.6, 22.7, 22.8, 22.9, 23, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2, 24.3, 24.4,24.5, 24.6, 24.7, 24.8, 24.9, 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, 26, 26.1, 26.2, 26.3,26.4, 26.5, 26.6, 26.7, 26.8, 26.9, 27, 27.1, 27.2, 27.3, 27.4, 27.5, 27.6, 27.7, 27.8, 27.9, 28, 28.1, 28.2,28.3, 28.4, 28.5, 28.6, 28.7, 28.8, 28.9, 29, 29.1, 29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 29.8, 29.9, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 200, or 500 kiloDaltons (kDa).

[0200] The sugar moieties may also be functionalized, for example, with n-succinimidyl or p- nitrophenyl carbonate to facilitate conjugation to the polypeptide. The glycosylation of polypeptides with a particular size and length of sugar moieties may be advantageous over another size and length based on the ability to increase the half-life or activity of the polypeptide. In some embodiments the sugar moieties are selected based on glycosylation characteristics described herein. For example, theone or more sugar moieties can mitigate immunogenicity, mask immunogenicity, increase the halflife or activity of the polypeptide, and / or decrease the ADA response against the polypeptide. In some embodiments the glycan structure (e.g., number of branches, types of bonds, specific sugar moieties) is selected based on glycosylation characteristics described herein. For example, the glycan structure can mitigate immunogenicity, mask immunogenicity, increase the half-life or activity of the polypeptide, and / or decrease the ADA response against the polypeptide.

[0201] In some embodiments, therapeutic, diagnostic, biosensing, and prophylactic biologicals with NSAAs may be made and used to allow templating glycosylation and production of homogeneous glycoforms. In some embodiments, this technique and modification may be useful for any protein biological, including but not limited to hormones, antibodies, cytokines, growth factors, structural proteins, vaccines, cancer therapeutics, and the like.

[0202] Although particular exemplary proteins are shown with a particular sequence in the sequence listing, which forms part of this disclosure, these sequences need not be used and reproduced with total fidelity. As is known in the art, slight modifications to proteins including allelic variations and polymorphisms may occur in parts of proteins which are not detrimental to their use and function. In some embodiments, useful proteins according to the present disclosure may have the precise sequences which are shown in the sequence listing or they may be slightly different. In some embodiments, for example, useful proteins may be at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85%, at least 84%, at least 83%, at least 82%, 81%, or at least 80% identical. The proteins that can be made have a wide variety of applications and purposes. In some embodiments, they can be therapeutic proteins, prophylactic proteins (such as vaccines), enzymatic proteins, endocrine proteins, signaling proteins, scaffolding proteins, diagnostic proteins, analytic proteins, etc. In some embodiments, the diselenide bonds will generally make the proteins more stable and less susceptible to reduction. In some embodiments, incorporation of one or more NSAAs into the proteins will allow templating glycosylation and addition of one or more sugar moieties on specific positions with particular patterns.Glycosylation

[0203] Template-driven glycosylation permits production therapeutic proteins bearing specific glycans at one or more positions of interest, including, but not limited to, new classes of biologies for applications in immunomodulation (e.g., immune activation, immune tolerance), autoimmune disease (e.g., immune tolerance) and immuno-oncology (e.g., immune activation). The approach employs modular NSAAs to which a variety of immunoactivating and immunogenicity mitigating glycans can be conjugated in a site-specific manner, thereby providing biomimicry of immunomodulatory approaches utilized by a variety of cells including, but not limited to, healthy cells and cancer cells, and cellular pathogens.

[0204] Glycosylation may allow a biomolecule (such as a protein or peptide) to acquire a physiochemical property not found in the biomolecule not glycosylated. For example, various sugar moieties can have various physiochemical properties. When a protein or peptide is glycosylated, the protein or peptide can then acquire physiochemical properties of the sugar moieties, wherein the protein or peptide not glycosylated may not have the same physiochemical properties. Additionally, various distinct proteins or peptides, when glycosylated, can acquire the physiochemical properties of the sugar moieties. The proteins can be useful in enhancing the response towards specific antigens. For example, such proteins with an NSAA conjugated to an oligosaccharide can be used to target cells via binding to receptors, such as mannose-binding receptors, thereby increasing the immunogenicity of various vaccine constituents.

[0205] In some embodiments, a method comprises a method to template glycosylation by incorporating one or more NSAAs into the proteins, which allows addition of one or more sugar moieties on specific positions with particular patterns. Template-driven glycosylation to produce structurally well-defined, homogeneous glycoproteins can utilize a variety of glycosylation methods including, but not limited to, total chemical synthesis, “tag and modify” approaches for site-selective protein-glycan conjugation, chemoenzymatic synthesis using enzymes for key modification and ligation, and glycosylation engineering by manipulating the biosynthetic pathways in different host expression systems.

[0206] In some embodiments, the method of making the polypeptide comprising an NSAA described herein or the population of the polypeptide molecules comprising the NSAA described herein further comprises conjugating one or more sugar moieties to the NSAA or the sidechain of the NSAA of the purified polypeptide or the population of the polypeptide molecules. In some embodiments, conjugating is performed in vitro. In some embodiments, the purified polypeptide or the population of the polypeptide molecules may have glycans conjugated to the NSAA or the sidechain of the NSAA before purification from the expression system host cells (e.g., bacteria cells, yeast cells, etc.). These glycans may be heterogeneous and are cleaved before in vitro glycosylation.

[0207] In some embodiments, a method comprises producing a polypeptide with an NSAA, such as a polypeptide as described herein, having a desired glycosylation state comprising the steps of expressing the polypeptide with an NSAA, and attaching (e.g., enzymatically or chemically) a sugar to the polypeptide with a NSAA. In some embodiments, the method comprises expressing the polypeptide with an NSAA in a host cell, such as a genetically recoded organism. In some embodiments, expressing the polypeptide with an NSAA in a host cell comprises transfecting or transducing a polynucleotide encoding the polypeptide into the host cell. In some embodiments, the method comprises expressing the polypeptide with an NSAA using an in vitro translation system. In some embodiments, the method comprises purifying, enriching or isolating the expressed polypeptide with an NSAA. In some embodiments, the method comprises removing one or more sugars from theexpressed polypeptide with an NSAA. In some embodiments, the method comprises enzymatically attaching a sugar to the polypeptide with an NSAA. In some embodiments, the method comprises chemically attaching a sugar to the polypeptide with an NSAA. In some embodiments, the sugar is a chemically synthesized sugar. In some embodiments, the sugar comprises an oxazoline ring. In some embodiments, the enzyme is an endoglycosidase. In some embodiments, the enzymatic attachment comprises a transglycosylation.

[0208] In some embodiments, the sugar removed is an asparagine linked sugar. In some embodiments, the sugar removed is a serine linked sugar. In some embodiments, the enzymatic attachment is to N- acetylglucosamine. In some embodiments, the method results in substantially pure polypeptide with an NSAA. In some embodiments, the attachment of the sugar results in a non-natural carbohydrate structure. In some embodiments, the polypeptide with an NSAA comprises a glycosylation state capable of modulating a biological activity. In some embodiments, the biological activity is (i) a reduced binding affinity for an Fcgamma Receptor or (ii) reduced antibody-dependent cell-mediated cytotoxicity. The polypeptide can be any polypeptide. In various embodiments, the polypeptide is a therapeutic polypeptide. In one example, the polypeptide is a recombinant polypeptide. In one example, the polypeptide is a synthetic polypeptide. The polypeptide can be a glycopeptide and can have any number of amino acids. In one embodiment, the polypeptide of the present disclosure has a molecular weight of about 5 kDa to about 500 kDa. In another embodiment, the polypeptide has a molecular weight of about 10 kDa to about 400 kDa, about 10 kDa to about 350 kDa, about 10 kDa to about 300 kDa, about 10 kDa to about 250 kDa, about 10 kDa to about 200 kDa, or about 10 kDa to about 150 kDa. In another embodiment, the polypeptide has a molecular weight of about 10 kDa to about 100 kDa. In yet another embodiment, the polypeptide has a molecular weight of about 10 kDa to about 50 kDa. In a further embodiment, the polypeptide has a molecular weight of about 10 kDa to about 25 kDa. In one embodiment, a polypeptide described herein can have a molecular weight of at least 1 kilodalton (kDa), at least 2 kDa, at least 3 kDa, at least 4 kDa, at least 5 kDa, at least 6 kDa, at least 7 kDa, at least 8 kDa, at least 9 kDa, at least 10 kDa, at least 11 kDa, at least 12 kDa, at least 13 kDa, at least 14 kDa, at least 15 kDa, at least 16 kDa, at least 17 kDa, at least 18 kDa, at least 19 kDa, at least 20 kDa, at least 21 kDa, at least 22 kDa, at least 23 kDa, at least 24 kDa, at least 25 kDa, at least 26 kDa, at least 27 kDa, at least 28 kDa, at least 29 kDa, at least 30 kDa, at least 31 kDa, at least 32 kDa, at least 33 kDa, at least 34 kDa, at least 35 kDa, at least 36 kDa, at least 37 kDa, at least 38 kDa, at least 39 kDa, at least 40 kDa, at least 41 kDa, at least 42 kDa, at least 43 kDa, at least 44 kDa, at least 45 kDa, at least 46 kDa, at least 47 kDa, at least 48 kDa, at least 49 kDa, at least 50 kDa, at least 51 kDa, at least 52 kDa, at least 53 kDa, at least 54 kDa, at least 55 kDa, at least 56 kDa, at least 57 kDa, at least 58 kDa, at least 59 kDa, at least 60 kDa, at least 61 kDa, at least 62 kDa, at least 63 kDa, at least 64 kDa, at least 65 kDa, at least 66 kDa, at least 67 kDa, at least 68 kDa, at least 69 kDa, at least 70 kDa, at least 71 kDa, at least 72 kDa, at least 73 kDa, at least 74 kDa, at least 75 kDa, atleast 76 kDa, at least 77 kDa, at least 78 kDa, at least 79 kDa, at least 80 kDa, at least 81 kDa, at least 82 kDa, at least 83 kDa, at least 84 kDa, at least 85 kDa, at least 86 kDa, at least 87 kDa, at least 88 kDa, at least 89 kDa, at least 90 kDa, at least 91 kDa, at least 92 kDa, at least 93 kDa, at least 94 kDa, at least 95 kDa, at least 96 kDa, at least 97 kDa, at least 98 kDa, at least 99 kDa, at least 100 kDa, at least 105 kDa, at least 110 kDa, at least 115 kDa, at least 120 kDa, at least 125 kDa, at least 130 kDa, at least 135 kDa, at least 140 kDa, at least 145 kDa, at least 150 kDa, at least 160 kDa, at least 170 kDa, at least 180 kDa, at least 190 kDa, at least 200 kDa, at least 210 kDa, at least 220 kDa, at least 230 kDa, at least 240 kDa, at least 250 kDa, at least 300 kDa, at least 350 kDa, at least 400 kDa, at least 450 kDa, at least 500 kDa, at least 550 kDa, at least 600 kDa, at least 650 kDa, at least 700 kDa, at least 750 kDa, at least 800 kDa, at least 850 kDa, at least 900 kDa, at least 950 kDa, at least 1000 kDa or more. In one embodiment, a polypeptide described herein can have a molecular weight of at most 1 kilodalton (kDa), at most 2 kDa, at most 3 kDa, at most 4 kDa, at most 5 kDa, at most 6 kDa, at most 7 kDa, at most 8 kDa, at most 9 kDa, at most 10 kDa, at most 11 kDa, at most 12 kDa, at most 13 kDa, at most 14 kDa, at most 15 kDa, at most 16 kDa, at most 17 kDa, at most 18 kDa, at most 19 kDa, at most 20 kDa, at most 21 kDa, at most 22 kDa, at most 23 kDa, at most 24 kDa, at most 25 kDa, at most 26 kDa, at most 27 kDa, at most 28 kDa, at most 29 kDa, at most 30 kDa, at most 31 kDa, at most 32 kDa, at most 33 kDa, at most 34 kDa, at most 35 kDa, at most 36 kDa, at most 37 kDa, at most 38 kDa, at most 39 kDa, at most 40 kDa, at most 41 kDa, at most 42 kDa, at most 43 kDa, at most 44 kDa, at most 45 kDa, at most 46 kDa, at most 47 kDa, at most 48 kDa, at most 49 kDa, at most 50 kDa, at most 51 kDa, at most 52 kDa, at most 53 kDa, at most 54 kDa, at most 55 kDa, at most 56 kDa, at most 57 kDa, at most 58 kDa, at most 59 kDa, at most 60 kDa, at most 61 kDa, at most 62 kDa, at most 63 kDa, at most 64 kDa, at most 65 kDa, at most 66 kDa, at most 67 kDa, at most 68 kDa, at most 69 kDa, at most 70 kDa, at most 71 kDa, at most 72 kDa, at most 73 kDa, at most 74 kDa, at most 75 kDa, at most 76 kDa, at most 77 kDa, at most 78 kDa, at most 79 kDa, at most 80 kDa, at most 81 kDa, at most 82 kDa, at most 83 kDa, at most 84 kDa, at most 85 kDa, at most 86 kDa, at most 87 kDa, at most 88 kDa, at most 89 kDa, at most 90 kDa, at most 91 kDa, at most 92 kDa, at most 93 kDa, at most 94 kDa, at most 95 kDa, at most 96 kDa, at most 97 kDa, at most 98 kDa, at most 99 kDa, at most 100 kDa, at most 105 kDa, at most 110 kDa, at most 115 kDa, at most 120 kDa, at most 125 kDa, at most 130 kDa, at most 135 kDa, at most 140 kDa, at most 145 kDa, at most 150 kDa, at most 160 kDa, at most 170 kDa, at most 180 kDa, at most 190 kDa, at most 200 kDa, at most 210 kDa, at most 220 kDa, at most 230 kDa, at most 240 kDa, at most 250 kDa, at most 300 kDa, at most 350 kDa, at most 400 kDa, at most 450 kDa, at most 500 kDa, at most 550 kDa, at most 600 kDa, at most 650 kDa, at most 700 kDa, at most 750 kDa, at most 800 kDa, at most 850 kDa, at most 900 kDa, at most 950 kDa, or at most 1000 kDa.

[0209] Exemplary polypeptides can include EC 1.1 enzymes (enzymes acting on the CH-OH group of donors (alcohol oxi doreductases such as methanol dehydrogenase)), EC 1.2 enzymes (enzymesacting on the aldehyde or oxo group of donors), EC 1.3 enzymes (enzymes acting on the CH-CH group of donors (CH-CH oxidoreductases)), EC 1.4 enzymes (enzymes acting on the CH-NH2 group of donors (Amino acid oxidoreductases, Monoamine oxidase)), EC 1.5 enzymes (enzymes acting on CH- NH group of donors), EC 1.6 enzymes (enzymes acting on NADH or NADPH), EC 1.7 enzymes (enzymes acting on other nitrogenous compounds as donors), EC 1.8 enzymes (enzymes acting on a sulfur group of donors), EC 1.9 enzymes (enzymes acting on a heme group of donors), EC 1.10 enzymes (enzymes acting on diphenols and related substances as donors), EC 1.11 enzymes (enzymes acting on peroxide as an acceptor (peroxidases)), EC 1.12 enzymes (enzymes acting on hydrogen as donors), EC 1.13 enzymes (enzymes acting on single donors with incorporation of molecular oxygen (oxygenases)), EC 1.14 enzymes (enzymes acting on paired donors with incorporation of molecular oxygen), EC 1.15 enzymes (enzymes acting on superoxide radicals as acceptors), EC 1.16 enzymes (oxidoreductases that oxidize metal ions), EC 1.17 enzymes (enzymes acting on CH or CH2 groups), EC 1.18 enzymes (enzymes acting on iron-sulfur proteins as donors), EC 1.19 enzymes (enzymes acting on reduced flavodoxin as a donor), EC 1.20 enzymes (enzymes acting on phosphorus or arsenic in donors), EC 1.21 enzymes (enzymes acting on X-H and Y-H to form an X-Y bond), or a combination thereof.

[0210] Other exemplary polypeptides include enzymes, such as glucocerebrosidase, alphagalactosidase, acid-alpha-glucosidase (acid maltase), iduronidases, such as alpha-L-iduronidase, thyroid peroxidase (TPO), beta-glucosidase, arylsulfatase, asparaginase, alpha-glucoceramidase (e.g., imiglucerase), sphingomyelinase, butyrylcholinesterase, urokinase and alpha-galactosidase A.

[0211] In some embodiments, the polypeptide with an NSAA has a substantially pure glycosylation state. In some embodiments, the glycosylation state comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more sugars. A glycosylated polypeptide disclosed herein can also be referred to as a glycoprotein or a glycopeptide. A glycoprotein or glycopeptide may comprise a protein or peptide that is glycosylated. Glycosylation of a protein or peptide may comprise attaching or coupling a sugar moiety (saccharide moiety) to the protein or peptide (forming the glycoprotein or glycopeptide). The sugar or saccharide moiety may comprise any of those sugars or saccharides described herein.

[0212] Also included herein are proteins carrying N- or O-linked, or both, oligosaccharide, such as oligomannose, structures conjugated to an NSAA of a polypeptide. Glycosylation may comprise N- linked glycosylation, O-linked glycosylation, C-linked glycosylation, S-linked glycosylation, glycation, or a combination thereof.

[0213] N-linked glycans are characterized by their five sugar moieties, composed of two N- Acetylglucosamine (GlcNAc) followed by three mannose (Man) units resulting in two available antennae for further glycosylation. N-linked glycosylation may comprise the attachment of sugar moieties (such as oligosaccharides) to a nitrogen atom (such as N4 of asparagine residues) of the protein / peptide. N-linked glycosylation may comprise the covalent attachment of the amide group onan asparagine (N) amino acid to a glycoform. The sequon of the peptide backbone may comprise the motif of NXS / T, where X is not a proline. N-linked glycans are characterized by their five sugar moieties, composed of two N-Acetylglucosamine (GlcNAc) followed by three mannose (Man) units resulting in two available antennae for further glycosylation. N-glycosylation can occur on secreted or membrane bound proteins / peptides. In eukaryotic cells, N-glycosylation can be initiated as a co- translational event in the endoplasmic reticulum, wherein preassembled blocks of 14 sugar moieties (such as 2 N-acetylglucosamines, 9 mannoses and 3 glucoses) are first added to the nascent peptide. After cleavage of 3 glucose and 1 mannose residues, the protein can then be transferred to the Golgi apparatus where the glycans lose a variable number of mannose residues and acquire a more complex structure (the process is referred to as terminal glycosylation). Mature N-glycans may comprise 3 types: high mannose (those that have escaped terminal glycosylation), hybrid complex (with different combinations of mannose, N-acetylglucosamine, N-acetylgalactosamine, fucose and sialic acid residues). The consensus sequence for N-glycosylation can comprise Asn-Xaa-Ser / Thr (where Xaa is not Pro; note that Thr is more common than Ser) or Asn-X-Cys.

[0214] O-linked glycans are the attachment of the hydroxyl functional group on serine (S) or threonine (T) amino acids to a glycoform with no specific sequence motif. O-linked glycans may comprise the attachment of the hydroxyl functional group on serine (S) or threonine (T) amino acids to a glycoform with no specific sequence motif. O-linked glycans may comprise various different core structures. Core 1 structure, usually more commonly found, may comprise Gal01-3 GalNAc. Other less commonly found structures may comprise: Core 2 may comprise GlcNAcpi-6(Gaipi-3)GalNAca, Core 3 is GlcNAcpi-3GalNAca; Core 4 may comprise GlcNAcpi-6(GlcNAcpi-3)GalNAca; Core 5 may comprise GalNAcal-3GalNAca; Core 6 may comprise GlcNAcpi-6GalNAca’ Core 7 may comprise GalNAcal-6GalNAca; Core 8 may comprise Galal-3GalNAca.3 Additionally saccharides may attach to any of these cores. O-linked glycosylation of secreted and membrane bound proteins may comprise a post-translational event that takes place in the cis-Golgi compartment after N- glycosylation and folding of the protein. O-linked glycosylation can comprise the attachment of glycans to serine and threonine, or to hydroxyproline and hydroxylysine. O-linked glycans can be involved in protein localization and trafficking, protein solubility, antigenicity and cell-cell interactions. O-linked glycans can be built up in a stepwise fashion with sugars added incrementally. O-glycosylation in secreted and membrane-bound mammalian proteins / peptides can comprise the addition of reducing terminal N-acetylgalactosamine (GalNAc, a mucin-type glycan). The reducing terminal GalNAc residue can be further extended with galactose (Gal), N-acetylglucosamine (GlcNAc) or GlcNAc and Gal resulting in 8 common core structures, which can be decorated with the addition of up to three sialic acid residues. Some cytoplasmic and nuclear proteins / peptides can comprise a simple O-linked glycans in which a single N-acetylglucosamine residue is linked to a serine or a threonine. This modification may be found in plants and filamentous fungi. This type of O-linkedglycosylation can be involved in the modulation of the biological activity of intracellular proteins / peptides. In some embodiments, the same residue may be subject to competing phosphorylation and O-linked glycosylation.

[0215] C-linked glycosylation can comprise the covalent attachment of a mannose residue to a tryptophan residue within an extracellular protein. Two recognition signals for C-mannosylation may comprise: W-X-X-W (in which the first or both tryptophan residues become mannosylated) or W-S / T- X-C.

[0216] S-linked glycosylation can comprise the attachment of oligosaccharides to the sulfur atom of the cysteine.

[0217] In some embodiments, the methods comprise native chemical ligation of preassembled peptides and glycopeptide fragments, chemoselective ligation between tagged proteins and activated glycans, endoglycosidase-catalyzed convergent glycosylation for N-gly coprotein synthesis and glycan remodeling, direct enzymatic glycosylation of intact polypeptides and proteins, or posttranslational enzymatic glycan modification of glycoproteins or combinations thereof. Details of these methods are described in Li & Wang, Chem Rev. 2018. 118(17): 8359-8413; Krasnova & Wong, Annu. Rev. Biochem. 2016. 85: 599-630; Zou, et al. J Am Chem Soc. 2011. 133(46): 18975-18991; Du, et al. Glycobiology 2009. 19(12): 1382-1401; Duong, et al. 2010. J. Exp. Med. 207(1): 173-187; Yang, et al. ACS Chem Biol. 2017. 12(6): 1665-1673; Kim, et al. MABS 2017. 9(4): 704-714; Perdicchio, et al., PNAS 2016. 113(12): 3329-3334; Zhang, et al. Drug Discovery Today 2016. 21(5): 740-765; Seeberger & Overkleeft, Chemical Synthesis of Glycans and Glycoconjugates. 2017. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology 3rd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2015-2017. Chapter 53., each of which is hereby incorporated by reference in its entirety.

[0218] In some embodiments, conjugating comprises enzymatically conjugating. Specifically modified, homogeneous peptide therapeutics can be produced in vitro through the use of enzymes. Unlike non-specific methods for attaching a modifying group (e.g., a synthetic polymer) to a peptide, enzyme-based syntheses have the advantages of regioselectivity and stereoselectivity. In some embodiments, enzymatically conjugating comprises using a glycoside hydrolase or a glycosidase. Any glycoside hydrolase or glycosidase known in the art can be utilized (e.g. , see Carbohydrate- Active Enzyme database (CAZy)). In some embodiments, enzymatically conjugating comprises using a glycosynthase. There are a number of glyco synthases that have been used in vitro to catalyze the addition of complex or complete N-gly cans on amino acid residues (e.g., Asp) of proteins. Complex or complete N-glycans can be provided as a sugar oxazoline where the N-acetyl group of the base GlcNAc is bound to the nearby hydroxyl from Cl of GlcNAc after the final deprotection step during glycan synthesis. Reaction conditions can vary based on the enzyme used (e.g., typical PBS at room temperature) and are known to those of ordinary skill in the art. Non-limiting examples ofglycosynthases include Arthrobacter protophormiae endoglycosidase A (Endo-A), Mucor hiemalis Endo-M, Streptococcus pneumoniae Endo-D, Streptococcus pyogenes Endo-S, Streptococcus pyogenes Endo-S2, and Elizabethkingia miricola Endo-F3. In some embodiments, the glycosynthase comprises a mutant of Arthrobacter protophormiae endoglycosidase A (Endo-A), Mucor hiemalis Endo-M, Streptococcus pneumoniae Endo-D, Streptococcus pyogenes Endo-S, Streptococcus pyogenes Endo-S2, or Elizabethkingia miricola Endo-F3. In some embodiments, the glycosynthase comprises a derivative or a mutant of Arthrobacter protophormiae endoglycosidase A (Endo-A), Mucor hiemalis Endo-M, Streptococcus pneumoniae Endo-D, Streptococcus pyogenes Endo-S, Streptococcus pyogenes Endo-S2, o Elizabethkingia miricola Endo-F3.

[0219] In some embodiments, enzymatically conjugating comprises using a glycosidase.

[0220] In some embodiments, enzymatically conjugating comprises using a glycosyltransferase (e.g., a sialyltransferase, an oligosaccharyltransferase, N-acetylglucosaminyltransferase).

[0221] In some embodiments, enzymatically conjugating comprises using a sialytransferase. Sialyltransferases are enzymes that transfer a sialic acid to nascent oligosaccharide. Each sialyltransferase is specific for a particular sugar nucleotide donor substrate. Sialyltransferases add sialic acid to the terminal galactose in glycolipids (gangliosides), or N- or O-linked glycans of glycoproteins. There are about twenty different sialyltransferases which can be distinguished on the basis of the acceptor substrate on which they act and on the type of sugar linkage they form. Exemplary sialyl-transferases belong to the EC 2.4.99 class of enzymes. Non limiting examples are ST3Gal-l (specific for O-glycans), and ST3 Gal-111 (specific for N-gly cans), and ST6GalNAc-l. It is thus possible to engineer the structure of the conjugated protein according to the present disclosure by e.g. selection of a specific sialyltransferase and / or engineering of protein with a particular glycosylation pattern or particular glycan liked NSAA.

[0222] Another exemplary method of forming a covalent conjugate between a polypeptide and a modifying group (e.g. , a polymeric modifying group) involves intracellular glycosylation within a host cell, in which the polypeptide is expressed. The method takes advantage of endogenous and / or coexpressed oligosaccharyl transferases. The method includes contacting the polypeptide, which includes an N-linked glycosylation sequence (e.g. a polypeptide of the disclosure, and a glycosyl donor species in the presence of an intracellular enzyme (e.g., an oligosaccharyltransferase) under conditions sufficient for the enzyme to transfer a glycosyl moiety from the glycosyl donor species onto an asparagine residue of the N-linked glycosylation sequence. In one example, the glycosyl donor species is added to the cell culture medium, internalized by the host cell and used as a substrate by an intracellular (endogenous or co-expressed) oligosaccharyltransferase.

[0223] In some embodiments, a glycosylation sequence comprising an NSAA is a substrate for an enzyme (e.g., an oligosaccharyltransferase, such as PglB). In some embodiments, the enzyme catalyzes the transfer of a glycosyl moiety from a glycosyl donor species (e.g., a lipid-pyrophosphate-linked glycosyl moiety) to a glyco-stub NSAA residue. Exemplary glycosyl moieties that can be conjugated to the NSAA of a glycosylation sequence include GlcNAc, GlcNH, bacillosamine, 6- hydroybacillosamine, GalNAc, GalNH, GlcNAc-GlcNAc, GlcNAc-GlcNH, GlcNAc-Gal, GlcNAc-GlcNAc-Gal-Sia, GlcNAc-Gal-Sia, GlcNAc-GlcNAc-Man, and GlcNAc-GlcNAc- Man(Man)2.

[0224] In some embodiments, the polypeptide with an NSAA is an engineered uricase polypeptide (such as SEQ ID NO. 9). In some embodiments, the engineered uricase polypeptide is glycosylated. In some embodiments, the engineered uricase polypeptide comprises at least one NSAA. In some embodiments, the at least one NSAA comprises at least two or at least three glycans coupled thereto. In some embodiments, each glycan (the at least two or at least three glycans) is independently a glycan comprising one or more of the sugar moieties listed in Table 4. In some embodiments, the engineered uricase polypeptide comprises N-acetylgalactosamine (GalNAc). In some embodiments, the engineered uricase polypeptide comprises at least one GalNAc, at least two GalNAc, at least three GalNAc, at least four GalNAc, at least five GalNAc, at least six GalNAc, at least seven GalNAc, at least eight GalNAc, at least nine GalNAc, at least ten GalNAc, at least eleven GalNAc, at least twelve GalNAc, or more GalNAc. In some embodiments, the engineered uricase polypeptide comprises N- Acetylglucosamine (GlcNAc). In some embodiments, the engineered uricase polypeptide comprises at least one GlcNAc, at least two GlcNAc, at least three GlcNAc, at least four GlcNAc, at least five GlcNAc, at least six GlcNAc, at least seven GlcNAc, at least eight GlcNAc, at least nine GlcNAc, at least ten GlcNAc, at least eleven GlcNAc, at least twelve GlcNAc, or more GlcNAc. In some embodiments, the engineered uricase polypeptide comprises at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or more GlcNAc. In some embodiments, the engineered uricase polypeptide comprises galactosylated, mannosylated, N-acetylglucosylated, and / or sialylated versions of GlcNAc2. In some embodiments, the engineered uricase polypeptide comprises mannose (Man). In some embodiments, the engineered uricase polypeptide comprises at least one Man, at least two Man, at least three Man, at least four Man, at least five Man, at least six Man, at least seven Man, at least eight Man, at least nine Man, at least ten Man, at least eleven Man, at least twelve Man, or more Man. In some embodiments, the engineered uricase polypeptide comprises at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more Man. In some embodiments, the engineered uricase polypeptide comprises galactosylated, N-acetylglucosylated, and / or sialylated versions of MansGlcNAc2. In some embodiments, the engineered uricase polypeptide comprises Man3GlcNAc2, Man4GlcNAc2, MansGlcNAc2, MangGlcNAc2, Man?GlcNAc2, MansGlcNAc2, Man9GlcNAc2, Gal, Sia, or SiaGal. In some embodiments, the engineered uricase polypeptide comprises MamGlcNAc?. MamGlcNAc-i, MamGlcNAcs, MamGlcNAcg, MamGlcNAc?, MamGlcNAcs, MamGlcNAcy, Gal, Sia, or SiaGal. In some embodiments, the engineered uricase polypeptide comprises galactosylated, N-acetylglucosylated, and / or sialylated versions of GlcNAc2Man3GlcNAc2 (see for example FIG. 1C).In some embodiments, the engineered uricase polypeptide comprises at least one, at least two, at least three, at least four, at least five, or at least six galactosylated, N-acetylglucosylated, and / or sialylated versions of GlcNAc2Man3GlcNAc2. In some embodiments, the engineered uricase polypeptide comprises galactose (Gal). In some embodiments, the engineered uricase polypeptide comprises at least one galactose, at least two galactose, at least three galactose, at least four galactose, at least five galactose, at least six galactose, at least seven galactose, at least eight galactose, at least nine galactose, at least ten galactose, at least eleven galactose, at least twelve galactose, or more galactose. In some embodiments, the engineered uricase polypeptide comprises galactosylated versions of GlcNAc2Man3GlcNAc2. In some embodiments, the engineered uricase polypeptide comprises at least one, at least two, at least three, at least four, at least five, or at least six galactosylated versions of GlcNAc2Man3GlcNAc2. In some embodiments, the engineered uricase polypeptide comprises GlcNAc2Man3GlcNAc2Gab. In some embodiments, the engineered uricase polypeptide comprises at least one, at least two, at least three, at least four, at least five, or at least six GlcNAc2Man3GlcNAc2Gab (see for example FIG. 1C). In some embodiments, the engineered uricase polypeptide comprises a sialic acid (“Sia”). In some embodiments, the engineered uricase polypeptide comprises at least one Sia, at least two Sia, at least three Sia, at least four Sia, at least five Sia, at least six Sia, at least seven Sia, at least eight Sia, at least nine Sia, at least ten Sia, at least eleven Sia, at least twelve Sia, or more Sia. In some embodiments, the engineered uricase polypeptide comprises sialylated versions of GlcNAc2Man3GlcNAc2. In some embodiments, each glycan of the engineered uricase polypeptide comprises at least one Sia, at least two Sia, at least three Sia, at least four Sia, at least five Sia, at least six Sia, at least seven Sia, at least eight Sia, at least nine Sia, at least ten Sia, at least eleven Sia, at least twelve Sia, or more Sia. In some embodiments, each glycan of the engineered uricase polypeptide comprises 2 Sia (i.e., BiSialylated or BiSia). In some embodiments, each glycan of the engineered uricase polypeptide comprises 4 Sia (i.e., TetraSialylated or TetraSia). In some embodiments, the engineered uricase polypeptide comprises at least one, at least two, or at least three sialylated versions of GlcNAc2Man3GlcNAc2. In some embodiments, the engineered uricase polypeptide comprises at least one, at least two, or at least three sialylated versions of GlcNAc2Man3GlcNAc2Gab. In some embodiments, the engineered uricase polypeptide comprises GlcNAc2Man3GlcNAc2GabSia2. In some embodiments, the engineered uricase polypeptide comprises at least one, at least two, at least three, at least four, at least five, or at least six GlcNAc2Man3GlcNAc2GabSia2 (see for example FIG. 1C). In some embodiments, the engineered uricase polypeptide comprises at least a portion of a uric acid binding active site. In some embodiments, an engineered uricase polypeptide is configured to form a multimer. In some embodiments, the engineered uricase polypeptide is present as multimer. In some embodiments, the multimer is a tetramer. In some embodiments, the tetramer is a homotetramer comprising four molecules of the engineered uricase polypeptide. In some embodiments, the tetramer comprises anengineered uricase with uricase activity. In some embodiments, a monomeric engineered uricase comprises an engineered uricase with uricase activity. For example, a monomeric engineered uricase can comprise four engineered uricase polypeptide sequences linked together (e.g., amino acid linker) to form a monomeric engineered uricase with uricase activity.

[0225] In some embodiments, the engineered uricase polypeptide comprises a sialic acid (“Sia”). In some embodiments, the engineered uricase polypeptide is configured to form a multimer or is present as multimer in the composition, wherein the multimer is a tetramer, and wherein the multimer comprises from 4 to 48 total sialic acids. In some embodiments, each monomer of the tetramer comprises 1 to 12 sialic acids. In some embodiments, each monomer of the tetramer comprises 6 sialic acids. In some embodiments, each monomer of the tetramer comprises 12 sialic acids . In some embodiments, the tetramer (or the monomeric engineered uricase comprising four engineered uricase polypeptide sequences) comprises at least 4 Sia, at least 5 Sia, at least 6 Sia, at least 7 Sia, at least 8 Sia, at least 9 Sia, at least 10 Sia, at least 11 Sia, at least 12 Sia, at least 13 Sia, at least 14 Sia, at least 15 Sia, at least 16 Sia, at least 17 Sia, at least 18 Sia, at least 19 Sia, at least 20 Sia, at least 21 Sia, at least 22 Sia, at least 23 Sia, at least 24 Sia, at least 25 Sia, at least 26 Sia, at least 27 Sia, at least 28 Sia, at least 29 Sia, at least 30 Sia, at least 31 Sia, at least 32 Sia, at least 33 Sia, at least 34 Sia, at least 35 Sia, at least 36 Sia, at least 37 Sia, at least 38 Sia, at least 39 Sia, at least 40 Sia, at least 41 Sia, at least 42 Sia, at least 43 Sia, at least 44 Sia, at least 45 Sia, at least 46 Sia, at least 47 Sia, or at least 48 Sia.

[0226] In some embodiments, the tetramer (or the monomeric engineered uricase comprising four engineered uricase polypeptides) comprises at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 GlcNAc2Man3GlcNAc2Gal2Sia2 (see for example FIG. 1C). In some embodiments, the tetramer (or the monomeric engineered uricase comprising four engineered uricase polypeptides) comprises at least 12 GlcNAc2Man3GlcNAc2Gal2Sia2 (see for example FIG. 1C). In some embodiments, the tetramer (or the monomeric engineered uricase comprising four engineered uricase polypeptides) comprises at least 24 GlcNAc2Man3GlcNAc2Gal2Sia2 (see for example FIG. 1C). In some embodiments, the engineered uricase polypeptide comprises sialylated versions of GalNac, or GlcNAcGalNAc. In some embodiments, the engineered uricase polypeptide comprises sialic acid that is attached in the a2,3 or a2,6 conformation to underlying sugars, or is comprised of poly sialic acid ([Siaa2,8]n, where n is between 2 and 150). In some embodiments, the engineered uricase polypeptide comprises galactose (Gal) that is attached in the [31,4 conformation to underlying sugars, such as a GlcNAc. In some embodiments, the engineered uricase polypeptide comprises GlcNAc that is attached in the [31,2 conformation to underlying sugars, such as a mannose. In some embodiments, the engineered uricase polypeptide comprises mannose (Man) that is attached in the al, 3 or pi, 4 conformationto underlying sugars, such as a GlcNAc. In some embodiments, the glycan may be fucosylated at the base GlcNAc.

[0227] The present disclosure provides polypeptide conjugates, in which a modified or non-modified sugar moiety is attached to an NSAA sequence. In a representative embodiment, the method is a cell- free in vitro method, wherein the polypeptide containing an NSAA is contacted (e.g., in a reaction vessel) with a glycosyl donor species (e.g., a lipid-pyrophosphate-linked glycosyl moiety, such as a undecaprenyl-pyrophosphate-linked glycosyl moiety) in the presence of a carbohydrate conjugating enzyme, such as an oligosaccharyl transferase, for which the glycosyl donor species is a substrate. The glycosyl moiety in this glycosyl donor species is optionally derivatized with a modifying group, such as a water-soluble polymeric modifying group. The enzyme transfers the modified or non-modified glycosyl moiety onto the polypeptide containing an NSAA thereby creating the polypeptide conjugate.

[0228] In another representative method, the above-described enzymatic reaction occurs within a host-cell, in which the polypeptide is expressed. The carbohydrate conjugating enzyme, such as an oligosaccharyl transferase, may be endogenously present in the host-cell or may be over-expressed in the host cell. Intracellular glycosylation according to this method offers a variety of advantages over cell-free in vitro glycosylation. For example, there is no need for purification of the polypeptide from cell-culture before glycosylation. In addition, advantage may be taken of other endogenous or coexpressed enzymes, which can be utilized for further modification of the initially formed glycosylated polypeptide.Pharmaceutical Compositions

[0229] Also provided herein are pharmaceutical compositions comprising any polypeptide comprising the NSAA conjugated to one or more sugar moieties described herein. The present disclosure also relates to a pharmaceutical composition comprising the population of polypeptide molecules with homogeneous sugar moieties described herein. In some aspects, provided herein is a pharmaceutical composition comprising: (i) any polypeptide described herein or the population of polypeptide molecules described herein; and (ii) a pharmaceutically acceptable carrier, excipient, or additive. The pharmaceutical compositions described herein can provide homogenous protein therapeutics for disease and conditions for which homogeneity of glycosylation of the therapeutic protein(s) is crucial for treatment.

[0230] In some embodiments, the pharmaceutical composition of the present disclosure may contain pharmaceutically acceptable carriers, excipients, or additives depending on the route of administration. In some embodiments, carriers, excipients, or additives may include, but are not limited to, water (e.g.ssterile water), saline, buffered saline, dextrose, glycerol, ethanol, sterile isotonic aqueous buffer, binding agent, a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, carboxymethylcellulose sodium, polyacrylic sodium, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methyl cellulose, ethylcellulose, xanthan gum, gum Arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, a pharmaceutically acceptable surfactant and the like, and combinations thereof. In some embodiments, the at least one pharmaceutically acceptable carrier, excipient, or additive comprises saline or sterile water. Additives used are chosen from, but not limited to, the above or combinations thereof depending on the dosage form.

[0231] In some embodiments, the pharmaceutical composition is formulated for oral administration, intravenous injection, intradermal injection, subcutaneous injection, intrathecal administration, intracerebral administration, intracerebroventricular injection, topical administration, inhalation, or nasal administration. In some embodiments, the pharmaceutical composition is formulated for transdermal administration (which may include a penetration enhancement agent), buccal administration, or suppository administration. In some embodiments, the pharmaceutical composition of the present disclosure may be administered, either orally or parenterally, systemically or locally. For example, intravenous injection such as drip infusion, intramuscular injection, intrapleural injection, intraperitoneal injection, subcutaneous injection, suppositories, intestinal lavage, oral enteric coated tablets, and the like can be selected, and the method of administration may be chosen, as appropriate, depending on the age and the condition of the subject.

[0232] In some embodiments, the pharmaceutical composition is a tablet, a pill, a capsule, a liquid, an inhalant, a nasal spray solution, a suppository, a suspension, a gel, a colloid, a dispersion, a suspension, a solution, an emulsion, an ointment, a lotion, an eye drop, or an ear drop.

[0233] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a drug, a compound, a small molecule, a macrocycle, an antibody or fragment thereof, a polynucleotide, a peptide, a payload, or a protein. In some embodiments, the therapeutic agent is synthetic or biosynthetic. In some embodiments, the additional therapeutic agent reduces expression of an efflux transporter or inhibits an efflux transporter. In some embodiments, the additional therapeutic agent increases expression of an influx transporter or promotes activity of an influx transporter.

[0234] The herein described pharmaceutical compositions may be formulated into different forms; for example, they may be formulated into a solid form (e.g., powders, tablets, capsule, creams, films, granules, paste, and gels) or a liquid form (e.g. , suspension, solution, and syrup). In embodiments, the pharmaceutical composition is formulated into a tablet, a capsule, a powder, which tablet, capsule or powder may or may not have sustained release characteristics.

[0235] In some embodiments, the pharmaceutical composition is prepared by a method comprising (a) spray-drying a liquid composition comprising one or more polypeptides and the at least one pharmaceutically acceptable carrier, excipient, or additive, and (b) collecting the spray-dried product of step (a) as a dispersible powder containing one or more polypeptides, functional fragment thereof,or variant thereof. In embodiments, the liquid composition comprises a carrier such as saline, water, or alcohol. In embodiments, the liquid composition comprises sterile water. In some embodiments, the liquid composition comprises sodium chloride. In some embodiments, the liquid composition comprises a sugar.

[0236] In some embodiments, the liquid composition has a concentration of the polypeptide of about from 0.1 mg / ml to 400 mg / ml, 1 mg / ml to 300 mg / ml, 1 mg / ml to 200 mg / ml, 1 mg / ml to 100 mg / ml, 1 mg / ml to 80 mg / ml, 1 mg / ml to 50 mg / ml, 1 mg / ml to 25 mg / ml, 5 mg / ml to 100 mg / ml, 5 mg / ml to 50 mg / ml, or 5 mg / ml to 25 mg / ml. In some embodiments, the liquid composition has a concentration of the polypeptide of about from 1 mg / ml to 80 mg / ml. In some embodiments, the polypeptide is present in the dispersible powder in an amount of about 1% to 100% by weight. In some embodiments, the polypeptide is present in the dispersible powder in an amount of about at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% by weight. In some embodiments, the polypeptide is present in the dispersible powder in an amount of about at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% by weight. In some embodiments, the dispersible powder comprises a sugar in an amount of about from 10% to 90% by weight. In some embodiments, the dispersible powder comprises sodium chloride in an amount of about from 10% to 90% by weight.

[0237] In some embodiments, the pharmaceutical composition is prepared by a method further comprising combining one or more additional therapeutics with one or more polypeptides. For example, in some embodiments, the additional therapeutic may be combined with the dispersible powder that contains one or more polypeptides. For another example, in some embodiments, the additional therapeutic may be combined with one or more polypeptides to produce the liquid composition. For yet another example, in some embodiments, the additional therapeutic may be added into the liquid composition that comprises one or more polypeptides.

[0238] In some embodiments, the pharmaceutical composition is in a liquid form such as suspension, solution, and syrup, which liquid form may or may not have a sustained release feature.

[0239] In some embodiments, the pharmaceutical composition is a dosage form and comprises 0.1 to 50 mL, 0.1 to 20 mL, 0.1 to 10 mL, 0.5 to 20 mL, 0.5 to 10 mL, 0.5 to 5 mL, 0.5 to 4 mL, 0.5 to 3 mL, 0.5 to 2 mL, 0.5 to 1.5 mL, 0.5 to 1 mL, 0.75 to 5 mL, 0.75 to 4 mL, 0.75 to 3 mL, 0.75 to 2.5 mL, 0.75 to 2 mL, 0.75 to 1.5 mL, 0.75 to 1.25 mL, or 0.75 to 1 mL. In some embodiments, the pharmaceutical composition is a dosage form and comprises at least 0.1 mL, 0.25 mL, 0.5 mL, 0.75 mL, 1 mL, 1.25 mL, 1.5 mL, 1.75 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, or 20 mL. In some embodiments, the pharmaceutical composition is a dosage form and comprises at most 0.5 mL, 0.75 mL, 1 mL, 1.25 mL, 1.5 mL, 1.75 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 20 mL, or 50 mL.

[0240] In some embodiments, the pharmaceutical composition is a dosage form and the polypeptide is present in the dosage form at an amount of about 0.1 mg to 20 mg, 0.1 mg to 10 mg, 0.1 mg to 5mg, 0.5 mg to 4 mg, 0.5 mg to 3 mg, 0.5 mg to 2.5 mg, 0.5 mg to 2 mg, 0.5 mg to 1.5 mg, 0.75 mg to 1.5 mg, 0.75 mg to 1.25 mg, or 1.5 mg to 2.5 mg. In some embodiments, the pharmaceutical composition is a dosage form and the polypeptide is present in the dosage form at an amount of at least 0.1 mg, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, or 20 mg. In some embodiments, the pharmaceutical composition is a dosage form and the polypeptide is present in the dosage form at an amount of at most 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, or 50 mg.

[0241] In some embodiments, a weight ratio of the polypeptide and the additional therapeutic present in the pharmaceutical composition for an administration is at least 1:1, 2: 1, 3:1, 4:1, 5: 1, 6:1, 10: 1, 25: 1, 50:1, 100: 1, 200:1, 300:1, 400:1, 500:1, 600:1, 800:1, 6000: 1, or 60000:1. In some embodiments, a weight ratio of the polypeptide and the additional therapeutic present in the pharmaceutical composition for an administration is at most 1: 1, 2:1, 3:1, 4:1, 5: 1, 6:1, 10:1, 25:1, 50: 1, 100: 1, 200: 1, 300: 1, 400:1, 500:1, 600:1, 800:1, 6000: 1, or 60000:1. In some embodiments, a weight ratio of the polypeptide and the additional therapeutic present in the pharmaceutical composition for an administration is about from 200:1 to 1000:1, 400: 1 to 800:1, or 500:1 to 700: 1.

[0242] In some embodiments, a weight ratio of the additional therapeutic and the polypeptide present in the pharmaceutical composition for an administration is at least 1:1, 2: 1, 3:1, 4:1, 5: 1, 6:1, 10: 1, 25: 1, 50:1, 100: 1, 200:1, 300:1, 400:1, 500:1, 600:1, 800:1, 6000: 1, or 60000:1. In some embodiments, a weight ratio of the additional therapeutic and the polypeptide present in the pharmaceutical composition for an administration is at most 1: 1, 2:1, 3:1, 4:1, 5: 1, 6:1, 10:1, 25:1, 50: 1, 100: 1, 200: 1, 300: 1, 400:1, 500:1, 600:1, 800:1, 6000: 1, or 60000:1. In some embodiments, a weight ratio of the additional therapeutic and the polypeptide present in the pharmaceutical composition for an administration is about from 200:1 to 1000:1, 400: 1 to 800:1, or 500:1 to 700: 1.Methods of Treatment

[0243] The present disclosure discloses a method of treating a subject with a condition or a disease. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein. The present disclosure discloses a method of treating a subject with a condition or a disease associated with hyperuricemia, such has gout.

[0244] In some embodiments, the pharmaceutical composition mitigates immunogenicity in the subject. In some embodiments, the pharmaceutical composition is less immunogenic than a pharmaceutical composition comprising a corresponding polypeptide without the NSAA and / or without glycosylation. In some embodiments, the pharmaceutical composition is less immunogenic than a pharmaceutical composition comprising a corresponding polypeptide without the one or more sugar moieties conjugated to the NSAA.

[0245] The compositions disclosed herein can mitigate immunogenicity against the polypeptide described herein. In some aspects, provided herein is a method of mitigating immunogenicity againstthe polypeptide in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein. In some embodiments, the polypeptide comprises one or more sugar moieties comprising an immunogenicity mitigating glycan. In some embodiments, the immunogenicity mitigating glycan comprises galactosylated, N-acetylglucosylated, and / or sialylated versions of MansGlcNAc2. In some embodiments, the immunogenicity mitigating glycan comprises MamGlcNAc^. Man4GlcNAc2, MansGlcNAc2, MangGlcNAc2, Man?GlcNAc2, MansGlcNAc2, MangGlcNAc2, Gal, Sia, or SiaGal. In some embodiments, the immunogenicity mitigating glycan comprises sialylated versions of GalNac, or GlcNAcGalNAc. In some embodiments, the immunogenicity mitigating glycan comprises sialic acid that is attached in the a2,3 or a2,6 conformation to underlying sugars, or is comprised of poly sialic acid ([Siaa2,8]n, where n is between 2 and 150). In some embodiments, the glycan may be fucosylated at the base GlcNAc.

[0246] The compositions and methods disclosed herein can mask immunogenicity of a therapeutic agent. In some aspects, provided herein is a method of masking immunogenicity of a therapeutic agent in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein. In some embodiments, the polypeptide comprises one or more sugar moieties comprising an immunogenicity mitigating glycan. In some embodiments, the immunogenicity mitigating glycan comprises galactosylated, N-acetylglucosylated, and / or sialylated versions of MansGlcNAc2. In some embodiments, the immunogenicity mitigating glycan comprises MansGlcNAc2, Man4GlcNAc2, MansGlcNAc2, MangGlcNAc2, Man?GlcNAc2, MansGlcNAc2, MangGlcNAc2, Gal, Sia, or SiaGal. In some embodiments, the immunogenicity mitigating glycan comprises sialylated versions of GalNac, or GlcNAcGalNAc. In some embodiments, the immunogenicity mitigating glycan comprises sialic acid that is attached in the a2,3 or a2,6 conformation to underlying sugars, or is comprised of poly sialic acid ([Siaa2,8]n, where n is between 2 and 150). In some embodiments, the glycan may be fucosylated at the base GlcNAc.

[0247] In some embodiments, the immunogenicity rate of the pharmaceutical composition disclosed herein is measured by detecting AD As. In some embodiments, AD As are detected using a Bridging assay. In some embodiments, ADAs are detected using a Ligand Binding assay. In some embodiments, AD As are detected using a protein specific assay. In some embodiments, ADAs are detected using a drug tolerant assay. In some embodiments, ADAs are detected using an ELISA assay. In some embodiments, the method described herein mitigates the immunogenicity in a subject. The modified or glycosylated polypeptide disclosed herein offers several advantages over an unmodified polypeptide as a therapeutic protein. For example, the glycosylated polypeptide disclosed herein can have prolonged half-life, enhanced stability, and / or reduced immunogenicity when compared to a nonglycosylated polypeptide and / or a polypeptide that does not comprise a NSAA. In some embodiments, the glycosylated polypeptide is of a higher molecular weight compared to a non-glycosylated polypeptide and / or a polypeptide that does not comprise a NSAA. The increased molecular weight ofthe glycosylated polypeptide can allow the glycosylated polypeptide to circulate in a subject for a longer duration, thus prolonging the half-life of the glycosylated polypeptide when compared to a nonglycosylated polypeptide and / or a polypeptide that does not comprise a NSAA. In some embodiments, the glycosylated polypeptide has increased bioavailability and / or therapeutic effectiveness when compared to a non-glycosylated polypeptide and / or a polypeptide that does not comprise a NSAA. For example, extended half-life of the glycosylated polypeptide can allow for less frequent dosing compared to a non-glycosylated polypeptide and / or a polypeptide that does not comprise a NSAA. In some embodiments, the glycosylated polypeptide has increased stability when compared to a non- glycosylated polypeptide and / or a polypeptide that does not comprise a NSAA. For example, the glycosylation can help to protect the polypeptide from enzymatic degradation and prolong the functional lifespan of the glycosylated polypeptide when compared to a non-glycosylated polypeptide and / or a polypeptide that does not comprise a NSAA.

[0248] In some embodiments, the pharmaceutical composition described herein is less immunogenic than a pharmaceutical composition comprising a corresponding polypeptide without the sugar moiety or NSAA. In some embodiments, the pharmaceutical composition is less immunogenic than a pharmaceutical composition comprising a corresponding polypeptide without the one or more sugar moieties conjugated to the NSAA.

[0249] In some embodiments, the pharmaceutical composition is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 100-fold, at least 1000-fold, at least 10000-fold, at least 100000-fold, or at least 1000000-fold less immunogenic than a pharmaceutical composition comprising a corresponding polypeptide without the one or more sugar moieties conjugated to the NSAA. The immunogenicity of a protein refers to its ability to trigger an immune response in the body, leading to the production of antibodies or activation of immune cells. Some of the promising protein therapeutics fail to advance to the clinic due to immunogenicity, activation of off-target receptors or dangerous immune reactions. By incorporating the NSAAs (e.g., NSAA with a sidechain that can react with a functionalized glycan) described herein to therapeutic proteins, the surface of immunogenic therapeutics can be decorated with immunogenicity mitigating or immunosilent glycans (e.g., sialic acid, mannose, etc.).

[0250] In some embodiments, the pharmaceutical composition is at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most 9%, at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 100%, at most 150%, at most 2-fold, at most 3-fold, at most 4-fold, at most 5-fold, at most 6- fold, at most 7-fold, at most 8-fold, at most 9-fold, at most 10-fold, at most 100-fold, at most 1000-fold, at most lOOOO-fold, at most lOOOOO-fold, or at most lOOOOOO-fold less immunogenic than a pharmaceutical composition comprising a corresponding polypeptide without the one or more sugar moieties conjugated to the NSAA.Immunogenicity mitigating glycan

[0251] In some embodiments, the polypeptide comprises one or more sugar moieties comprising an immunogenicity mitigating glycan. Glycans can comprise simple or complex sugar structures that are found on the surface of cells, proteins, and other molecules in the body. In some embodiments, the immunogenicity mitigating glycan can refer to a type of carbohydrate molecule that has the ability to reduce or attenuate the immune response. In some embodiments, the immunogenicity mitigating glycan can refer to a type of carbohydrate molecule that has the ability to be immune-silent. An immunogenicity mitigating glycan can refer to a type of carbohydrate molecule that has the ability to mask or shield the polypeptide disclosed herein. For example, the glycosylated polypeptides disclosed herein can be involved in modulating the immune system's response, specifically by inducing a state of immunotolerance or suppressing immune reactions. In some embodiments, the immunogenicity mitigating glycan can play a role in immune recognition and modulation. In some embodiments, the immunogenicity mitigating glycans can increase the recognition of the polypeptide disclosed herein as a self-molecule, when compared to the polypeptide without glycosylation. For example, immunogenicity mitigating glycans may hide all or part of the polypeptide disclosed herein, thus evading the immune system. In some embodiments, the immunogenicity mitigating glycans can help to abate unwanted immune responses against the polypeptide disclosed herein, thus increasing the efficacy.Treating a subject

[0252] The compositions disclosed herein can be used in a method of treating a subject with a condition or a disease associated with hyperuricemia. The compositions disclosed herein can be used in a method of treating a subject with gout.

[0253] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a variety of mammalian species utilized in research for various purposes, including medical, biological, and veterinary investigations. In some embodiments, the mammal is a mouse, a rat, a guinea pig, a rabbit, a hamster, a ferret, a cat, a pig, a dog, or a non-human primate. In some embodiments, the nonhuman primate can be rhesus macaques, marmosets, and / or chimpanzees. In some embodiments, the subject is a human. The subject may have gout. The subject may be suspected of having gout. The subjected may have a risk of developing gout. Symptoms of gout can comprise pains in the joints, ankle, foot, knee, or toe of the subject. The joints of the subject may have lumps, stiffness, or swelling.

[0254] In some embodiments, the subject may have hyperuricemia or elevated serum uric acid level (or symptoms of hyperuricemia, such as but not limited to having warm, red, swollen, painful, inflamed, or throbbing joints). In some embodiments, hyperuricemia may comprise a serum uric acidlevel that is at least 6.8 milligrams per deciliter (mg / dL). A ...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A composition comprising an engineered uricase polypeptide with uricase activity, wherein the engineered uricase polypeptide comprises a non-standard amino acid (NSAA).

2. A composition comprising an engineered uricase polypeptide with uricase activity, wherein the engineered uricase polypeptide comprises an amino acid linked to a glycan, and wherein the engineered uricase polypeptide comprises an amino acid sequence derived from a Sus scrofa uricase, a Papio uricase, a Mus musculus uricase, an Aspergillus flavus uricase, an Arthrobacter globiformis uricase, or a Paeniglutamicibacter gangotriensis uricase.

3. The composition of claim 2, wherein the engineered uricase polypeptide comprises a nonstandard amino acid (NSAA).

4. The composition of any one of claims 1-3, wherein the engineered uricase polypeptide comprises at least two NSAAs.

5. The composition of any one of claims 1-3, wherein the engineered uricase polypeptide comprises at least three NSAAs.

6. The composition of claim 4 or 5, wherein each NSAA is the same NSAA.

7. The composition of any one of claims 1-6, wherein the NSAA comprises a sidechain comprising an azide, a ketone, an aldehyde, a selenol, an alkyne, an amine, a hydrazide, an aminooxy, a boronate, or a vicinal diol.

8. The composition of claim 7, wherein the NSAA comprises a sidechain comprising an aliphatic alkyne or a strained alkyne.

9. The composition of claim 7, wherein the NSAA comprises a sidechain comprising an azide.

10. The composition of any one of claims 1-9, wherein the NSAA comprises paraazidophenylalanine (pAzF), azido-lysine, azido-alanine, para-azidomethyl-l-phenylalanine (pAMF), p-propargyloxy-l-phenylalanine, p-benzoylphenylalanine, p-acetylphenylalanine, p- propargyloxy-l-phenylalanine, propargylglycine, homopropargylglycine, selenocysteine, or para-isothiocyanate phenylalanine.

11. The engineered uricase polypeptide of any one of claims 1-7, 9 or 10, wherein the NSAA comprises pAzF.

12. The composition of any one of claims 1-7, 9 or 10, wherein the NSAA comprises pAMF.

13. The composition of any one of claims 1-12, wherein the engineered uricase polypeptide comprises an amino acid sequence derived from a Sus scrofa uricase, Papio uricase, Mus musculus uricase, an Aspergillus flavus uricase, an Arthrobacter globiformis uricase, or a Paeniglutamicibacter gangotriensis uricase.

14. The composition of claim 13, wherein the engineered uricase polypeptide comprises an amino acid sequence derived from an Aspergillus flavus uricase.

15. The composition of any one of claims 1-14, wherein the NSAA comprises a sugar moiety coupled thereto.

16. The composition of claim 15, wherein each NSAA comprises a sugar moiety coupled thereto.

17. The composition of any one of claims 1-15, wherein the NSAA comprises a glycan coupled thereto.

18. The composition of claim 17, wherein each NSAA comprises a glycan coupled thereto.

19. The composition of any one of claims 1-18, wherein the engineered uricase polypeptide comprises at least two glycans coupled thereto.

20. The composition of claims 19, wherein the engineered uricase polypeptide comprises at least three glycans coupled thereto.

21. The composition of claim 19 or 20, wherein the engineered uricase comprises at least two different glycans coupled thereto or at least three different glycans coupled thereto.

22. The composition of claim 19 or 20, wherein each of the glycans is the same glycan.

23. The composition of any one of claims 1-22, wherein at least one NSAA comprises at least two or at least three glycans coupled thereto.

24. The composition of any one of claims 17-23, wherein each glycan is independently a glycan comprising one or more of the sugar moieties listed in Table 4.

25. The composition of any one of claims 17-24, wherein each glycan is independently a glycan comprising a sugar moiety selected from the group consisting of N-Acetylglucosamine (GlcNAc), N-Acetylgalactosamine (GalNAc), GlcNAc-al,6-fucose (Fuc), galactose (Gal), mannose (Man), sialic acid (Sia), Sia-a2,3-Gal, Sia-a2,6-Gal, Sia-a2, 3 -GlcNAc, Sia-a2,6- GlcNac, Sia-a2, 3 -GalNAc, Gal-P 1,3 -GalNAc, Sia-a2,3-Gal-pi,3-GalNAc, GlcNAc-pi,4- GlcNAc, GlcNAc-pi,4-[Fuc-al,3]GlcNAc, GlcNAc-pi,4-[Fuc-al,3][Fuc-al,6]GlcNAc, N- Acetylneuraminic acid (Neu5Ac), Neu5Ac-a2,3Gal-pi,4[Fuc-al,3]GlcNAc, Gal-pi,4[Fuc- al,3]GlcNAc, Gal-al,3-Gal-pi,4-GlcNAc-P, Gal-al,3-Gal, and any combination thereof.

26. The composition of any one of claims 15-25, wherein the sugar moiety or the glycan is coupled to the NSAA via a linker.

27. The composition of claim 26, wherein the linker comprises an alkyne.

28. The composition of claim 26, wherein the linker comprises 4-pentynoic acid, propargylamine, 6-azidohexanoic acid, dibenzocyclooctyne (DBCO), 3-butyn-l-ol, ethynylbenzene, or aPEG- alkyne.

29. The composition of claim 26, wherein the linker comprises a tetrazine.

30. The composition of claim 26, wherein the linker comprises a linker with trans-cyclooctene (TCO) reactivity.

31. The composition of claim 26, wherein the linker comprises dibenzocyclooctyne (DBCO).

32. The composition of any one of claims 1-31, wherein the engineered uricase polypeptide comprises an amino acid sequence with at least 50% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-13.

33. The composition of any one of claims 1-32, wherein the engineered uricase polypeptide comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-13.

34. The composition of any one of claims 1-33, wherein the engineered uricase polypeptide comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to at least 200, 225, 250, 275, 300, 301 or 302 contiguous amino acids of the amino acid sequence according to SEQ ID NO: 5, wherein the NSAA is located at a position selected from the group consisting of at least one of the following positions:(a) a position corresponding to position 139 of SEQ ID NO: 5,(b) a position corresponding to position 175 of SEQ ID NO: 5, and(c) a position corresponding to position 218 of SEQ ID NO: 5.

35. The composition of claim 34, wherein the engineered uricase polypeptide comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to at least 200, 225, 250, 275, 300, 301 or 302 contiguous amino acids of the amino acid sequence according to SEQ ID NO: 5, wherein the NSAA is located at a position selected from the group consisting of at least two of the following positions:(a) a position corresponding to position 139 of SEQ ID NO: 5,(b) a position corresponding to position 175 of SEQ ID NO: 5, and(c) a position corresponding to position 218 of SEQ ID NO: 5.

36. The composition of claim 34, wherein the engineered uricase polypeptide comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to at least 200, 225, 250, 275, 300, 301 or 302 contiguous amino acids of the amino acid sequence according to SEQ ID NO: 5, wherein the NSAA is located at(a) a position corresponding to position 139 of SEQ ID NO: 5,(b) a position corresponding to position 175 of SEQ ID NO: 5, and(c) a position corresponding to position 218 of SEQ ID NO: 5.

37. The composition of any one of claims 1-33, wherein the engineered uricase polypeptide comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to at least 200, 225, 250, 275, 300, 301 or 302 contiguous amino acids of the amino acid sequence according to SEQ ID NO: 8 or 11.

38. The composition of claim 37, wherein the engineered uricase polypeptide comprises an amino acid sequence with 100% sequence identity to at least 200, 225, 250, 275, 300, 301 or 302 contiguous amino acids of the sequence according to SEQ ID NO: 8 or 11.

39. The composition of claim 37 or 38, wherein the engineered uricase polypeptide comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence according to SEQ ID NO: 8 or 11.

40. The composition of any one of claims 37-39, wherein the engineered uricase polypeptide comprises an amino acid sequence according to SEQ ID NO: 8 or 11.

41. The composition of any one of claims 1-33, wherein the engineered uricase polypeptide comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to at least 200, 225, 250, 275, 300, 301 or 302 contiguous amino acids of the amino acid sequence according to any one of SEQ ID NOs: 9, 10, 12 or 13.

42. The composition of claim 41, wherein the engineered uricase polypeptide comprises an amino acid sequence with 100% sequence identity to at least 200, 225, 250, 275, 300, 301 or 302 contiguous amino acids of the sequence according to any one of SEQ ID Nos: 9, 10, 12 or 13.

43. The composition of claim 41 or 42, wherein the engineered uricase polypeptide comprises an amino acid sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence according to any one of SEQ ID Nos: 9, 10, 12 or 13.

44. The composition of any one of claims 41-43, wherein the engineered uricase polypeptide comprises an amino acid sequence according to any one of SEQ ID Nos: 9, 10, 12 or 13.

45. The composition of any one of claims 1-44, wherein the engineered uricase polypeptide is configured to form a multimer.

46. The composition of any one of claims 1-44, wherein the engineered uricase polypeptide is present as a multimer.

47. The composition of claim 45 or 46, wherein the multimer is a tetramer.

48. The composition of claim 47, wherein the tetramer is a homotetramer comprising four molecules of the engineered uricase polypeptide.

49. The composition of any one of claims 1-48, wherein the engineered uricase polypeptide comprises an NSAA linked to a glycan, wherein the NSAA linked to a glycan has a structure according to Formula (IA):AA-LJ-L2-L3-XFormula (IA) wherein:A A is the NSAA;AA-L1comprises, wherein1is where L1is coupled to the NSAA, wherein coupled to L2;L2is a bondwhereiniscoupled to L1, wherein is coupled to L3;L3is asparagine or, whereinis coupled to L2; wherein is coupled to X; andX is at least one glycan.

50. The composition of claim 49,Man - GIcNAcMan wherein the glycan comprises GIcNAc, Man GIcNAc ; or SiaMan - GIcNAc - Gal— Sia51. The composition of claim 49 or 50, wherein each of at least two glycan-linked NSAAs of the engineered uricase polypeptide has a structure according to Formula (IA).

52. The composition of claim 51, wherein each of at least three glycan-linked NSAAs of the engineered uricase polypeptide has a structure according to Formula (IA).

53. The composition of any one of claims 1-52, wherein the engineered uricase polypeptide comprises a sialic acid (“Sia”).

54. The composition of claim 53, wherein the engineered uricase polypeptide is configured to form a multimer or is present as multimer in the composition, wherein the multimer is a tetramer, and wherein the multimer comprises from 4 to 48 total sialic acids.

55. The composition of claim 54, wherein each monomer of the tetramer comprises 1 to 12 sialic acids.

56. The composition of claim 55, wherein each monomer of the tetramer comprises 6 sialic acids.

57. The composition of claim 55, wherein each monomer of the tetramer comprises 12 sialic acids.

58. The composition of any one of claims 1-57, wherein the engineered uricase polypeptide is a recombinant polypeptide.

59. The composition of any one of claims 1-57, wherein the engineered uricase polypeptide is not a synthetic polypeptide.

60. The composition of any one of claims 1-59, wherein the engineered uricase polypeptide is not PEGylated.

61. The composition of any one of claims 1 -60, wherein the engineered uricase polypeptide further comprises a tag.

62. The composition of claim 61, wherein the tag comprises an affinity tag.

63. The composition of claim 62, wherein the affinity tag comprises a His-tag, a GST-tag, a Strep- tag, a SUMO-tag, a MBP-tag, or any combination thereof.

64. The composition of claim 63, wherein the affinity tag comprises a His-tag.

65. The composition of any one of claims 1-64, wherein the engineered uricase polypeptide comprises uricase activity when present in a serum.

66. The composition of claim 65, wherein the serum is human serum.

67. The composition of any one of claims 1-66, wherein the uricase activity of the engineered uricase polypeptide is at least 50% of the uricase activity of a control uricase polypeptide, wherein the control uricase polypeptide lacks the NSAA having a sugar moiety coupled thereto.

68. The composition of claim 67, wherein the uricase activity of the engineered uricase polypeptide is higher than the uricase activity of the control uricase polypeptide.

69. The composition of claim 67 or 68, wherein the uricase activity of the engineered uricase polypeptide and the control polypeptide are measured as the consumption of uric acid over time when the engineered uricase polypeptide and the control polypeptide are incubated at 25°C or 37°C in the presence of uric acid.

70. The composition of any one of claims 1-69, wherein the engineered uricase polypeptide is configured to elicit an anti-drug antibody (ADA) response following administration to a subject, wherein the ADA response elicited is lower than an ADA response elicited by a control uricase polypeptide, and wherein the control uricase polypeptide lacks the NSAA having a sugar moiety coupled thereto.

71. The composition of claim 70, wherein the subject does not elicit a substantial ADA response to the engineered uricase polypeptide.

72. The composition of any one of claims 67-71, wherein the composition is a composition according to any one of claims 34-64, and wherein the control uricase polypeptide comprises an amino acid sequence with 100% sequence identity to at least 200, 225, 250, 275, 300, 301 or 302 contiguous amino acids of the amino acid sequence according to SEQ ID NO: 5.

73. A polynucleic acid comprising a sequence encoding the composition of any one of claims 1-72, wherein the NSAA is encoded by a stop codon.

74. A polynucleic acid comprising a sequence encoding the composition of any one of claims 1-73, wherein the NSAA is encoded by a sense codon.

75. The polynucleic acid of claim 73 or 74 wherein the NSAA is encoded by a codon selected from the group consisting of UAG, UAA, UGA, AGY, UUR, UGC, and AGR, wherein Y is U or C, and wherein R is A or G.

76. A pharmaceutical composition comprising the composition of any one of claims 1-75 and a pharmaceutically acceptable excipient.

77. The pharmaceutical composition of claim 76, wherein the pharmaceutical composition is formulated for intravenous injection, subcutaneous injection, intradermal injection, intrathecal administration, intracerebral administration, intracerebroventricular injection, topical administration, inhalation or nasal administration.

78. The pharmaceutical composition of claim 76 or 77, wherein the pharmaceutical composition is a liquid.

79. The pharmaceutical composition of any one of claims 76-78, wherein the pharmaceutical composition further comprises an additional therapeutic agent.

80. A method of treating a subject with a condition or a disease comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 76-79.

81. The method of claim 80, wherein the condition or disease is an inflammatory disease.

82. The method of claim 80, wherein the condition or disease is an arthritis.

83. The method of claim 80, wherein the condition or disease is gout.

84. The method of any one of claims 80-83, wherein the subject has hyperuricemia.

85. The method of claim 80, wherein the condition or disease is hyperuricemia.

86. The method of claim 80, wherein the therapeutically effective amount of the pharmaceutical composition is sufficient to decrease a level of uric acid of the subject.

87. The method of claim 80, wherein the therapeutically effective amount of the pharmaceutical composition is sufficient to decrease a level of uric acid of the subject to about 6 mg / dL or less.

88. The method of any one of claims 80-87, wherein the subject is a human.

89. A method of making the composition of any one of claims 1-72, comprising expressing the engineered uricase polypeptide in a cell or in vitro.

90. The method of claim 89, wherein the engineered uricase polypeptide is expressed within the cell.

91. The method of claim 90, wherein the cell is a bacterial cell or an E. coli cell.

92. The method of any one of claims 89-91, wherein the cell comprises a recoded genome.

93. The method of claim 92, wherein a particular codon at substantially all instances within a template genome is changed to an alternative codon in the recoded genome.

94. The method of claim 93, wherein a cognate transfer RNA (tRNA) to the particular codon is not present in the recoded genome.

95. The method of claim 93 or 94, wherein the cell comprises an orthogonal translation system comprising an aminoacyl tRNA synthetase (aaRS) and a cognate tRNA comprising an anticodon that can bind to the particular codon.

96. The method of claim 95, wherein the cognate tRNA comprises a bacterial tRNA, a eukaryotic tRNA, a plant tRNA, an archaeal tRNA or a variant thereof.

97. The method of claim 96, wherein the cognate tRNA is selected from the group consisting of Methanococcus jannaschii tyrosyl tRNA, Methanosarcina barkeri pyrrolysine tRNA, Methanomethylophilus alvus pyrrolysine tRNA, Methanosarcina mazei pyrrolysine tRNA, Pyrococcus horokoshii prolyl tRNA, Pyrococcus horokoshii tyrosyl tRNA, and Archaeoglobus fulgidus prolyl tRNA.

98. The method of any one of claims 94-97, wherein a release factor to the particular codon is not present in the recoded genome.

99. The method of any one of claims 93-98, wherein the cell comprises a polynucleotide comprising a sequence encoding the engineered uricase polypeptide, wherein the sequence encoding the engineered uricase polypeptide comprises the particular codon.

100. The method of any one of claims 93-99, wherein the particular codon is reassigned to a nonstandard amino acid.

101. The method of any one of claims 93-100, wherein the particular codon is UAG, UAA, UGA, AGY, UUR, UGC, AGR, or a combination thereof, wherein:AGY is selected from the group consisting of AGU, and AGC;UUR is selected from the group consisting of UUA, and UUG; andAGR is selected from the group consisting of AGA, and AGG.

102. The method of any one of claims 89-101, wherein the method further comprises culturing the cell under conditions sufficient for the cell to express the engineered uricase polypeptide.

103. The method of any one of claims 89-102, wherein the method comprises enriching the engineered uricase polypeptide from the cell.

104. The method of any one of claims 89-103, wherein the method comprises conjugating a sugar moiety or a glycan to a sidechain of the NSAA of the engineered uricase polypeptide.

105. The method of claim 104, wherein the conjugating is performed in vitro.

106. The method of claim 104 or 105, wherein the conjugating comprises chemically conjugating.

107. The method of claim 106, wherein the chemically conjugating comprises a click chemistry reaction.

108. The method of claim 107, wherein the click chemistry reaction comprises a copper-catalyzed azide-alkyne cycloaddition.

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