Modified IL-2 polypeptides for the treatment of inflammatory and autoimmune diseases - Patents.com

Abstract

The present disclosure relates to modified IL-2 polypeptides, compositions comprising modified IL-2 polypeptides, methods of production thereof, and methods of using modified IL-2 polypeptides for the treatment of diseases, including autoimmune diseases. In one aspect, the disclosure relates to the treatment of autoimmune diseases using modified IL-2 polypeptides. In some embodiments, the disclosed IL-2 polypeptides exhibit enhanced binding to IL-2 receptor alpha and / or decreased binding to IL-2 receptor beta. In another aspect, the modified IL-2 polypeptides exhibit enhanced ability to activate regulatory T cells relative to T effector cells.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 219,995, filed July 9, 2021, and U.S. Provisional Patent Application No. 63 / 219,989, filed July 9, 2021, which applications are incorporated by reference herein in their entireties. [Background technology]

[0002] Interleukin-2 (IL-2) is a cytokine signaling molecule important in regulating the immune system. IL-2 is involved in helping the immune system differentiate between foreign and endogenous cell types, thereby preventing the immune system from attacking a subject's own cells. IL-2 achieves its activity through interactions with the IL-2 receptor (IL-2R) expressed by lymphocytes. Through these binding interactions, IL-2 mediates the expression of T effector (T eff ) cells, natural killer (NK) cells, and regulatory T cells (T reg ) subject population can be adjusted.

[0003] The ability of IL-2 to regulate the immune system is driven, at least in part, by its differential affinity for the IL-2Rα (CD25) and IL-2Rβ (CD122) subunits. Natural IL-2 acts on resting lymphocytes through an intermediate affinity receptor composed of the IL-2Rβ and IL-2Rγ subunits. Activated lymphocytes and T reg The cells further express the IL-2R α subunit, which combines with the β and γ subunits to form a high affinity receptor for IL-2. Upon acting on the high affinity αβγ receptor, IL-2 transduces T reg It is possible to enhance cell activation and proliferation, thus modulating the immune response of a subject.

[0004] For these reasons, IL-2 has been used alone and in combination with other therapies to treat various diseases involving the immune system.However, the therapeutic use of IL-2 has been limited by its toxicity, including life-threatening and sometimes fatal vascular leak syndrome, as well as its short half-life, which requires administration three times a day for 8 days.There is a need for improved IL-2 polypeptides with different selectivities for various IL-2 receptor subunits, such as enhanced binding of IL-2Rα to enhance therapeutic potential and minimize the risk of side effects of IL-2 therapy. Summary of the Invention

[0005] In one aspect, provided herein is a modified interleukin-2 (IL-2) polypeptide, comprising a modified IL-2 polypeptide, wherein the modified IL-2 polypeptide comprises up to seven naturally occurring amino acid substitutions, wherein the seven naturally occurring amino acid substitutions comprise amino acid substitutions at residues Y31, K35 and Q74, and wherein the residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO:1 as a reference sequence.

[0006] In another aspect, provided herein are modified IL-2 polypeptides, including modified IL-2 polypeptides that exhibit a binding affinity to the IL-2 receptor alpha subunit (IL-2Rα) that is between about 0.1 nM and about 100 nM, and that exhibit a binding affinity to the IL-2 receptor beta subunit (IL-2Rβ) that is at least about 1000 nM.

[0007] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0008] Incorporation by Reference 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. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over such conflicting material. [Brief description of the drawings]

[0009] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also referred to herein as "FIG" and "FIG").

[0010] [Figure 1] FIG. 1 shows the synthetic scheme used to synthesize modified IL-2 polypeptides as provided herein as linear depsipeptides. [Diagram 2] FIG. 1 shows a scheme for rearrangement and folding of linear depsipeptides to provide a folded modified IL-2 polypeptide as provided herein. [Diagram 3] FIG. 1 shows a scheme for producing the PEGylated modified IL-2 polypeptides provided herein. [Figure 4A] FIG. 1 shows the mean fluorescence intensity (MFI) of STAT5 phosphorylation in Teff cells by various concentrations of aldesleukin, composition A and composition A1. [Figure 4B] FIG. 1 shows the MFI of STAT5 phosphorylation in Treg cells by aldesleukin, composition A and composition A1 at various concentrations. [Figure 4C] FIG. 1 shows EC50 values ​​for STAT5 phosphorylation of various T cell subtypes by modified IL-2 polypeptides provided herein. [Diagram 5] FIG. 1 shows the binding affinity of Composition A1 and Aldesleukin to the IL-2Rα and IL-2Rβ subunits as determined by Biolayer Interferometry (BLI). [Figure 6] FIG. 1 shows the pharmacokinetics of Composition A1 administered subcutaneously to mice at 0.1 mg / kg or 0.3 mg / kg. [Figure 7] 1 shows the immunopharmacodynamic effects of Composition A1 or Aldesleukin on various lymphocyte populations at various time points after administration of the indicated doses. The top left graph shows Treg% pSTAT5 positive cells, the top middle graph shows Teff% pSTAT5 positive cells, the top right graph shows NK% pSTAT5 positive cells, the middle left graph shows Treg% Ki67 positive cells, the middle middle graph shows Teff% Ki67 positive cells, the middle right graph shows NK% Ki67 positive cells, the bottom left graph shows the fold change in Treg counts relative to baseline, the bottom middle graph shows the fold change in Teff counts relative to baseline, and the bottom right graph shows the fold change in NK counts relative to baseline. [Figure 8A] FIG. 1 shows the experimental design for evaluating the ability of Composition A1 to delay hypersensitivity to keyhole limpet hemocyanin in mice. [Figure 8B] Figure 1 shows the difference in ear thickness between the right ear (challenged with KLH) and the contralateral ear (injected with saline) reported in mm as a measure of swelling at 24, 48, 72 and 96 hours. A two-way ANOVA was performed revealing significant effects of time (F(4,216)=48.16, p<0.0001) and treatment (F(5,54)=13.74, p<0.0001), suggesting a change over time that was modulated by treatment with Composition A1. Data are reported as mean ± SEM (n=10 per experimental group). [Figure 8C]FIG. 1 illustrates the difference in ear thickness between the right ear (challenged with KLH) and the contralateral ear (injected with saline), reported as area under the curve (AUC) as a measure of overall swelling after challenge. A one-way ANOVA was performed revealing a significant effect of treatment (F(5,54)=12.59, p<0.0001), suggesting that this parameter was modulated by treatment. A Dunnett's test versus vehicle multiple comparisons showed that Composition A1 significantly reduced ear swelling in all regimens (**p<0.01, ****p<0.0001). Data are reported as mean±SEM (n=10 per experimental group). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present disclosure relates to modified interleukin-2 (IL-2) polypeptides useful as therapeutic agents. The modified IL-2 polypeptides provided herein can be used as treatments for a variety of diseases and disorders, including inflammatory or other autoimmune diseases. Such modified IL-2 polypeptides can exhibit binding properties for the IL-2 receptor (IL-2R) that differ from wild-type IL-2 (SEQ ID NO: 1) or aldesleukin (SEQ ID NO: 2). In one aspect, the modified IL-2 polypeptides described herein have increased affinity for the IL-2Rα complex. In some embodiments, the modified IL-2 polypeptides have unregulated affinity for the IL-2Rβγ complex. In some embodiments, the modified IL-2 polypeptides have reduced affinity for the IL-2Rβγ complex. In some embodiments, the modified IL-2 polypeptides provided herein can include amino acid substitutions that increase binding affinity for the IL-2Rα receptor subunit. In some embodiments, the modified IL-2 polypeptides provided herein include amino acid substitutions that reduce the affinity of the modified IL-2 polypeptide for the IL-2Rβ receptor subunit. In some embodiments, the modified IL-2 polypeptide induces fewer T effectors (Ts) when administered in vivo compared to wild-type IL-2 or aldesleukin. effIn some embodiments, the modified IL-2 polypeptides provided herein have a biological activity to induce regulatory T cells (T cells) when administered in vivo, as compared to wild-type IL-2 or aldesleukin. reg ) (e.g., EC 50 is 10 times or less, 100 times or less).

[0012] In some embodiments, the modified IL-2 polypeptides described herein contain modified amino acid residues. Such modifications can take the form of amino acid substitutions of the wild-type IL-2 polypeptide, such as the amino acid sequence of SEQ ID NO:1, addition or deletion of amino acids from the sequence of SEQ ID NO:1, or addition of moieties to amino acid residues. In some embodiments, the modified IL-2 polypeptides described herein include a deletion of the first amino acid from the sequence of SEQ ID NO:1. In some embodiments, the modified IL-2 polypeptides described herein include a C125S substitution, using the sequence of SEQ ID NO:1 as a reference sequence. In some embodiments, the modified IL-2 polypeptides described herein include a substitution at one or more residues selected from Y31, K35, Q74 and / or N88, with the residue position numbering of the modified IL-2 polypeptide based on SEQ ID NO:1 as the reference sequence. These substitutions may be combined with a C125S substitution and / or an N-terminal deletion, such as a deletion of the first amino acid from the sequence of SEQ ID NO:1. In some embodiments, the Y31 substitution is a Y31H substitution. In some embodiments, the K35 substitution is a K35R substitution. In some embodiments, the Q74 substitution is a Q74P substitution. In some embodiments, the N88 substitution is a N88D substitution. In some embodiments, the modified IL-2 polypeptide comprises a Y31H substitution, a K35R substitution, and a Q74P substitution. In some embodiments, the modified IL-2 polypeptide comprises a Y31H substitution, a K35R substitution, a Q74P substitution, and a N88D substitution. In some embodiments, the modified IL-2 polypeptide comprises a Y31H substitution, a K35R substitution, a Q74P substitution, and a N88D substitution. In some embodiments, the modified IL-2 polypeptide comprises a Y31H substitution, a K35S substitution, a Q74P substitution, and a C125S substitution. In some embodiments, the modified IL-2 polypeptide comprises a Y31H substitution, a K35S substitution, a Q74P substitution, a N88D substitution, and a C125S substitution.

[0013] In some embodiments, the modified IL-2 polypeptide is a synthetic polypeptide. In some embodiments, the modified IL-2 polypeptide is synthesized by alpha-keto acid-hydroxylamine (KAHA) amide forming ligation. In some embodiments, the modified IL-2 polypeptide includes unnatural amino acids, such as homoserine, that are used during the KAHA ligation reaction to join multiple polypeptide fragments to synthesize a full-length modified IL-2 polypeptide. In some embodiments, these are the only unnatural amino acids in the modified IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide includes norleucine (Nle) residue substitutions at one or more methionine residues present in wild-type IL-2 or aldesleukin. In some embodiments, the modified IL-2 polypeptide includes norleucine residues at positions 23, 39, and 46.

[0014] The modified IL-2 polypeptides described herein can include one or more non-standard amino acids (also referred to herein as "unnatural amino acids"). A "non-standard" amino acid can refer to an amino acid residue in D- or L-form that is not among the 20 standard amino acids commonly incorporated into naturally occurring proteins. In some embodiments, one or more amino acids of the modified IL-2 polypeptides are substituted with one or more non-standard amino acids. Non-standard amino acids include, but are not limited to, N-α-(9-fluorenylmethyloxycarbonyl)-L-azidolysine (Fmoc-L-Lys(N3)-OH), N-α-(9-fluorenylmethyloxycarbonyl)-L-biphenylalanine (Fmoc-L-Bip-OH), and N-α-(9-fluorenylmethyloxycarbonyl)-O-benzyl-L-tyrosine (Fmoc-L-Tyr(Bzl)-OH, or unprotected analogs thereof.

[0015] Additionally, polymers may be added to the modified IL-2 polypeptide. In some embodiments, the polymer is added to increase the half-life of the polypeptide. Such half-life extending polymers can be added to the N-terminus of the modified IL-2 polypeptide. The half-life extending polymer can be of any size, including up to about 6 kDa, up to about 30 kDa, or up to about 50 kDa. In some embodiments, the half-life extending polymer is a PEG polymer.

[0016] In some embodiments, the modified IL-2 polypeptide comprises one or more amino acid substitutions or deletions selected from Table 1.

[0017] [Table 1]

[0018] In some embodiments, the modified IL-2 polypeptides provided herein contain one or more amino acid substitutions selected from Table 2.

[0019] [Table 2]

[0020] In some embodiments, a modified IL-2 polypeptide provided herein comprises one or more polymers selected from Table 3. In some embodiments, the one or more polymers are covalently attached to the N-terminus of the modified IL-2 polypeptide.

[0021] [Table 3]

[0022] The modified IL-2 polypeptides described herein may also be chemically synthesized rather than expressed as recombinant polypeptides. The modified IL-2 polypeptides may be made by synthesizing one or more fragments of a full-length modified IL-2 polypeptide, ligating the fragments together, and folding the ligated full-length polypeptide. In some embodiments, the modified IL-2 polypeptide comprises Y31H, K35R, Q74P, and C125S substitutions, and optionally a PEG polymer covalently attached to the N-terminus of the modified IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide comprises Y31H, K35R, Q74P, N88D, and C125S substitutions, and optionally a PEG polymer covalently attached to the N-terminus of the modified IL-2 polypeptide.

[0023] In some embodiments, the modified IL-2 polypeptide, when administered to a subject, activates regulatory T cells (T reg In some embodiments, the modified IL-2 polypeptide, when administered to a subject, enhances T effector cell (T eff ) and / or natural killer (NK) cells, while sparing T reg In some embodiments, the modified IL-2 polypeptides, when administered to a subject, increase Treg cells without substantially increasing CD8+ T cells and NK cells.

[0024] The following description and examples illustrate the embodiments of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific embodiments described herein and may therefore vary. Those skilled in the art will recognize that there are numerous variations and modifications of the present disclosure that fall within the scope of the present disclosure.

[0025] Although various features of the present disclosure may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein for clarity in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment.

[0026] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0027] All terms are intended to be understood as those terms are understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0028] The following definitions supplement those in the art and are intended for this application and should not be applied to related or unrelated cases, such as co-owned patents or applications. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present disclosure, preferred materials and methods are described herein. Thus, the terminology used herein is intended only to describe specific embodiments and is not intended to be limiting.

[0029] I. Definition The terms used herein are for the purpose of describing particular instances only and are not intended to be limiting. In this application, the use of the singular includes the use of the plural unless specifically stated otherwise. As used herein, the singular forms "a," "an," and "the" are intended to include the plural unless the context clearly indicates otherwise.

[0030] In this application, the use of "or" means "and / or" unless otherwise specified. As used herein, the terms "and / or" and "any combination thereof" and their grammatical equivalents can be used interchangeably. These terms can convey that any combination is specifically envisioned. For illustrative purposes only, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" can mean "A individually, B individually, C individually, A and B, B and C, A and C, and A, B, and C." The term "or" can be used conjunctively or disjunctively, unless the context specifically dictates disjunctive use.

[0031] The term "about" or "approximately" can mean within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, as is customary in the art. Alternatively, "about" can mean within a range of up to 20%, up to 15%, 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 5-fold or within 2-fold order of magnitude of a value. When a particular value is described in the present application and claims, unless otherwise indicated, the term "about" should be assumed to mean within an acceptable error range of the particular value.

[0032] As used in this specification and claims, the terms "comprising" (and any of the comprising forms, such as "comprise" and "comprises"), "having" (and any of the having forms, such as "have" and "has"), "including" (and any of the including forms, such as "includes" and "include"), or "containing" (and any of the containing forms, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0033] Reference herein to "some embodiments," "one embodiment," "one embodiment," or "other embodiments" means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the disclosure, but not necessarily in all embodiments. To facilitate understanding of this disclosure, a number of terms and phrases are defined below.

[0034] Polymers that are "attached" or "covalently attached" to a residue of an IL-2 polypeptide are referred to herein. As used herein, "attached" or "covalently attached" means that the polymer is tethered to the indicated residue, and such tethering may include a linking group (i.e., a linker). Thus, when referring to a polymer that is "attached" or "covalently attached" to a residue, it is expressly intended that such linking groups are also encompassed.

[0035] Binding affinity refers to the strength of the binding interaction between a single molecule and its ligand / binding partner. A higher binding affinity refers to a stronger binding than a lower binding affinity. In some cases, binding affinity is measured by the dissociation constant (K D ) is measured by K D When comparing values, a binding interaction with a lower value has a higher binding affinity than a binding interaction with a higher value. For protein-ligand interactions, K D is calculated according to the following formula:

[0036]

number

[0037] As used herein, a specific amino acid sequence (e.g., a polypeptide sequence) that has a certain percent sequence identity to a reference sequence or refers to residues at positions corresponding to positions in a reference sequence. Sequence identity is measured by the protein-protein BLAST algorithm using the parameters of Matrix BLOSUM62, Gap Costs Existence: 11, Extension: 1, and Compositional Adjustments Conditional Compositional Score Matrix Adjustment. This alignment algorithm is also used to assess whether residues are at "corresponding" positions through analysis of the alignment of the two sequences being compared.

[0038] The term "pharmaceutical acceptable" refers to approved or approvable by a regulatory agency of the Federal or state government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, including humans.

[0039] A "pharmaceutical acceptable excipient, carrier, or diluent" refers to an excipient, carrier, or diluent that may be administered to a subject together with a drug, does not destroy its pharmacological activity, and is non-toxic when administered in a dosage sufficient to deliver a therapeutic amount of the drug.

[0040] A "pharmaceutically acceptable salt" suitable for this disclosure may be a salt of an acid or base that is generally considered in the art to be suitable for use in contact with the tissues of humans or animals without undue toxicity, irritation, allergic response, or other problems or complications. Such salts include inorganic and organic acid salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Particular pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, malic acid, glycolic acid, fumaric acid, sulfuric acid, sulfamic acid, sulfanilic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxyethylsulfonic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid, salicylic acid, glutamic acid, ascorbic acid, pamoic acid, succinic acid, fumaric acid, maleic acid, propionic acid, hydroxymaleic acid, hydroiodic acid, phenylacetic acid, alkanoic acids such as acetic acid, HOOC-(CH2)n-COOH, where n is 0 to 4. Similarly, pharma- ceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. One skilled in the art will recognize from this disclosure and knowledge in the art that additional pharma-ceutically acceptable salts include those listed by Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p.1418 (1985). In general, pharma-ceutically acceptable acid or base salts can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or free base form of these compounds with a stoichiometric amount of the appropriate base or acid in a suitable solvent.

[0041] Ranges provided herein are understood to be shorthand for all values ​​within that range, for example, a range of 1 to 50 is understood to include any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values ​​between the aforementioned integers, such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested subranges" extending from either end of the range are specifically contemplated. For example, nested subranges of the exemplary range of 1 to 50 could include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0042] The term "subject" refers to an animal that is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a human or a non-human mammal, such as a mammal including a non-human primate, cow, horse, dog, sheep, or cat.

[0043] Certain formulas and other examples provided herein depict triazole reaction products resulting from azide-alkyne cycloaddition reactions. Although such formulas generally depict only a single regioisomer of the resulting triazole formed in the reaction, the formulas are intended to encompass both resulting regioisomers. Thus, the formulas may depict only a single regioisomer (e.g.,

[0044] [ka] ) are shown, while other positional isomers (e.g.

[0045] [ka] ) are also intended to be included.

[0046] The term "optional" or "optionally" means that the subsequently described event or circumstance may occur, but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0047] The term "moiety" refers to a specific segment or functional group of a molecule. A chemical moiety is often recognized as a chemical entity that is embedded in or appended to a molecule.

[0048] As used herein, "N-terminus with glutaric acid and 0.5 kDa azido PEG" refers to a modification of the N-terminal amine of an IL-2 polypeptide provided herein having the following structure:

[0049] [ka] Although described as having an azide functionality, it is contemplated that the azide can be replaced with an alternative conjugation handle in each case where the modified IL-2 polypeptide comprises an N-terminus with glutaric acid and a 0.5 kDa azide PEG.

[0050] "Composition A" refers to a modified IL-2 polypeptide of SEQ ID NO:3 comprising an N-terminus bearing glutaric acid and a 0.5 kDa azido PEG.

[0051] "Composition A1" refers to the reaction product formed between Composition A and DBCO containing PEG having a molecular weight of about 30 kDa.

[0052] "Composition B" refers to a modified IL-2 polypeptide of SEQ ID NO:4 comprising an N-terminus bearing glutaric acid and a 0.5 kDa azido PEG.

[0053] "Composition B1" refers to the reaction product formed between Composition B and DBCO containing PEG having a molecular weight of about 30 kDa.

[0054] "Composition C" refers to a modified IL-2 polypeptide of SEQ ID NO:5 comprising an N-terminus bearing glutaric acid and a 0.5 kDa azido PEG.

[0055] "Composition C1" refers to the reaction product formed between Composition B and DBCO containing PEG having a molecular weight of about 30 kDa.

[0056] "Composition D" refers to a modified IL-2 polypeptide of SEQ ID NO:6 comprising an N-terminus bearing glutaric acid and a 0.5 kDa azido PEG.

[0057] "Composition D1" refers to the reaction product formed between Composition D and DBCO containing PEG having a molecular weight of about 30 kDa.

[0058] "Composition E" refers to a modified IL-2 polypeptide of SEQ ID NO:7 comprising an N-terminus bearing glutaric acid and a 0.5 kDa azido PEG.

[0059] "Composition E1" refers to the reaction product formed between Composition E and DBCO containing PEG having a molecular weight of about 30 kDa.

[0060] "Composition F" refers to a modified IL-2 polypeptide of SEQ ID NO:8 comprising an N-terminus bearing glutaric acid and a 0.5 kDa azido PEG.

[0061] As used herein, "conjugation handle" refers to a reactive group capable of forming a bond upon contact with a complementary reactive group. In some instances, the conjugation handle preferably has no substantial reactivity with other molecules that do not contain the intended complementary reactive group. Non-limiting examples of conjugation handles, their respective complementary conjugation handles, and corresponding reaction products can be found in the table below. Although the table headings list specific reactive groups under the headings "conjugation handle" or "complementary conjugation handle," it is intended that any reference to a conjugation handle may instead encompass the complementary conjugation handle listed in the table (e.g., trans-cyclooctene may be a conjugation handle, in which case tetrazine is the complementary conjugation handle). In some instances, amine conjugation handles and amine-complementary conjugation handles are less preferred for use in biological systems due to the ubiquitous presence of amines in biological systems and the increased likelihood of off-target conjugation.

[0062] [Table 4]

[0063] As used herein, the term "number average molecular weight" (Mn) means the statistical average molecular weight of all individual units in a sample, and is represented by the formula (1):

[0064]

number

[0065] As used herein, the term “weight average molecular weight” (Mw) refers to a molecular weight of a compound represented by the formula (2):

[0066]

number

[0067] As used herein, "peak molecular weight" (Mp) means the molecular weight of the highest peak in a given analytical method (e.g., mass spectrometry, size exclusion chromatography, dynamic light scattering, analytical centrifugation, etc.).

[0068] II. Description In one embodiment, T eff T compared with cells reg Described herein are modified IL-2 polypeptides that are biased in favor of cellular activation. In one aspect, described herein are modified polypeptides, including modified interleukin-2 (IL-2) polypeptides, the modified IL-2 polypeptides comprising one or more amino acid substitutions. In some embodiments, the modified IL-2 polypeptide comprises at least one amino acid substitution at a residue selected from Y31, K35, Q74, and N88, and the residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the modified IL-2 polypeptide comprises an amino acid substitution at each of residues Y31, K35, and Q74, and the residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the modified IL-2 polypeptide comprises an amino acid substitution at Y31H, K35R, and Q74P. In some embodiments, the modified IL-2 polypeptide comprises an amino acid substitution at each of residues Y31, K35, Q74, and N88, and the residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the modified IL-2 polypeptide comprises the amino acid substitutions Y31H, K35R, Q74P, and N88D, in some embodiments, the modified IL-2 polypeptide does not comprise any additional substitutions that substantially affect the binding of the modified IL-2 polypeptide to the IL-2Rα receptor.

[0069] In another aspect, a modified polypeptide comprising a modified interleukin-2 (IL-2) polypeptide has a substantially lower T than the IL-2 polypeptide of SEQ ID NO:1 and / or SEQ ID NO:2. eff Described herein are modified IL-2 polypeptides that exhibit the ability to activate T cells. In some embodiments, the modified IL-2 polypeptides reg In some embodiments, the modified IL-2 polypeptide retains the ability to activate T cells compared to the IL-2 polypeptide of SEQ ID NO: 1 and / or SEQ ID NO: 2. reg In some embodiments, the modified IL-2 polypeptides have a dissociation constant (K) for IL-2Rα that is at least about 4-fold lower than the IL-2 polypeptides of SEQ ID NO:1 and / or SEQ ID NO:2. d In some embodiments, the modified IL-2 polypeptide has a dissociation constant (K) for IL-2Rα that is 2-fold to 10-fold lower than the IL-2 polypeptide of SEQ ID NO:1 and / or SEQ ID NO:2. d ) is shown.

[0070] binding affinity In one aspect, described herein are modified IL-2 polypeptides that exhibit greater affinity for the IL-2 receptor α subunit than the IL-2 polypeptides of SEQ ID NO: 1 and / or SEQ ID NO: 2. In some embodiments, the affinity for the IL-2 receptor subunit is measured by the dissociation constant (K d As used herein, the K d The phrase "dissociation constant" refers to the dissociation constant of the binding interaction between a modified IL-2 polypeptide and CD25.

[0071] In some embodiments, the K d In some embodiments, the K of the modified IL-2 polypeptide / IL-2 receptor α subunit is less than 10 nM. dIn some embodiments, the K of the modified IL-2 polypeptide / IL-2 receptor α subunit is less than 10 nM, less than 7.5 nM, less than 5 nM, less than 4 nM, or less than 3 nM. d In some embodiments, the K of the modified IL-2 polypeptide / IL-2 receptor α subunit is about 1 nM to 0.1 nM. d In some embodiments, the K of the modified IL-2 polypeptide / IL-2 receptor α subunit is about 10 nM to about 0.1 nM. d In some embodiments, the K of the modified IL-2 polypeptide / IL-2 receptor α subunit is about 10 nM to about 1 nM. d In some embodiments, the K of the modified IL-2 polypeptide / IL-2 receptor α subunit is about 7.5 nM to about 0.1 nM. d In some embodiments, the K of the modified IL-2 polypeptide / IL-2 receptor α subunit is about 7.5 nM to about 1 nM. d In some embodiments, the K of the modified IL-2 polypeptide / IL-2 receptor α subunit is about 5 nM to about 0.1 nM. d In some embodiments, K d is measured by surface plasmon resonance.

[0072] In some embodiments, the modified IL-2 polypeptide exhibits at least about 10%, 50%, 100%, 250% or 500% greater affinity for the IL-2 receptor alpha subunit than the IL-2 polypeptide of SEQ ID NO: 1 and / or SEQ ID NO: 2. In some embodiments, the modified IL-2 polypeptide exhibits up to about 500%, 750% or 1000% greater affinity for the IL-2 receptor alpha subunit than the IL-2 polypeptide of SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0073] In some embodiments, the modified IL-2 polypeptide exhibits about 1.5- to about 10-fold greater affinity for the IL-2 receptor α-subunit than the IL-2 polypeptide of SEQ ID NO:1 and / or SEQ ID NO:2.

[0074] In some embodiments, the modified IL-2 polypeptide exhibits substantially the same binding affinity for IL-2Rα as compared to the IL-2 polypeptide of SEQ ID NO: 1 and / or SEQ ID NO: 2. ... d K d Shows.

[0075] In some embodiments, the modified IL-2 polypeptides exhibit reduced affinity for the IL-2 receptor beta subunit (IL-2Rβ) compared to the IL-2 polypeptides of SEQ ID NO:1 and / or SEQ ID NO:2. In some embodiments, the modified IL-2 polypeptides exhibit at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or at least about 500-fold lower affinity for IL-2Rβ. In some embodiments, the modified IL-2 polypeptides exhibit at least about 100-fold lower affinity for IL-2Rβ. In some embodiments, the modified IL-2 polypeptides exhibit substantially no affinity for IL-2Rβ. In some embodiments, the affinity is measured using a dissociation constant K d (e.g., lower affinity correlates with higher dissociation constant).

[0076] In some embodiments, the modified IL-2 polypeptide exhibits a binding affinity for IL-2Rβ that is at least 500 nM, at least 1000 nM, at least 5000 nM, at least 10000 nM, at least 50000 nM, or at least 100000 nM. In some embodiments, the modified IL-2 polypeptide exhibits substantially no binding affinity for IL-2Rβ.

[0077] In some embodiments, the modified IL-2 polypeptides exhibit affinity for IL-2Rα that is at least about 30-fold greater, at least about 50-fold greater, at least about 75-fold greater, at least about 100-fold greater, at least about 500-fold greater, or at least about 1000-fold greater than IL-2Rβ. In some embodiments, the modified IL-2 polypeptides exhibit affinity for IL-2Rα that is at least about 100-fold greater than IL-2Rβ. In some embodiments, the modified IL-2 polypeptides exhibit affinity for IL-2Rα that is at least about 1000-fold greater than IL-2Rβ.

[0078] biological activity In some embodiments, the modified IL-2 polypeptides described herein are capable of inhibiting regulatory T cells (T reg In some embodiments, the modified IL-2 polypeptides described herein can be used to expand effector T cells (T eff ) proliferation is avoided.

[0079] In some embodiments, the modified IL-2 polypeptide has up to a moderately reduced T compared to the IL-2 polypeptide of SEQ ID NO:1 and / or SEQ ID NO:2. reg Half-maximal effective concentration (EC) for cell activation 50 In some embodiments, T reg Cell activation is measured by assessing changes in STAT5 phosphorylation in a population of T cells upon contact with a modified IL-2 polypeptide. In some embodiments, T reg The cells are CD4 + , CD25+ and FoxP3 + In some embodiments, the T reg The cells were stained with CD25 (CD25 Hi In some embodiments, the modified IL-2 polypeptide is identified by also exhibiting elevated expression of a T of up to about 100 nM, about 75 nM, about 50 nM, about 40 nM, about 35 nM, about 30 nM, or about 25 nM. reg EC for cell activation50 In some embodiments, the modified IL-2 polypeptide has a T of up to about 50 nM, about 40 nM, about 35 nM, about 30 nM, or about 25 nM, about 20 nM, about 15 nM, about 10 nM, or about 5 nM. reg EC for cell activation 50 In some embodiments, the modified IL-2 polypeptide has the following structure: reg EC up to approximately 100 nM for cell activation 50 In some embodiments, the modified IL-2 polypeptide has the following structure: reg EC up to approximately 50 nM for cell activation 50 In some embodiments, the modified IL-2 polypeptide has the following structure: reg EC up to approximately 25 nM for cell activation 50 In some embodiments, the modified IL-2 polypeptide has a T of about 0.1 nM to about 100 nM, about 1 nM to about 100 nM, about 0.1 nM to about 50 nM, about 1 nM to about 50 nM, about 0.1 nM to about 25 nM, about 1 nM to about 25 nM, about 0.1 nM to about 10 nM, or about 1 nM to about 10 nM. reg EC for cell activation 50 has.

[0080] In some embodiments, the modified IL-2 polypeptide has a T that is up to 2-fold, up to 5-fold, up to 10-fold, up to 20-fold, up to 50-fold, up to 100-fold, up to 200-fold, up to 500-fold, or up to 1000-fold greater than the IL-2 polypeptide of SEQ ID NO:1 and / or SEQ ID NO:2. reg EC for cell activation 50 In some embodiments, the modified IL-2 polypeptide has a T reg EC for cell activation 50 In some embodiments, the modified IL-2 polypeptide has up to 5-fold greater T reg EC for cell activation 50 In some embodiments, the modified IL-2 polypeptide has a T reg EC for cell activation 50In some embodiments, the modified IL-2 polypeptide has a T reg EC for cell activation 50 In some embodiments, the modified IL-2 polypeptide has a T reg EC for cell activation 50 In some embodiments, the modified IL-2 polypeptide has a T reg EC for cell activation 50 In some embodiments, the modified IL-2 polypeptide has a T reg EC for cell activation 50 In some embodiments, the modified IL-2 polypeptide has a T reg EC for cell activation 50 has.

[0081] In some embodiments, the modified IL-2 polypeptide has a substantially greater T than the IL-2 polypeptide of SEQ ID NO:1 and / or SEQ ID NO:2. eff Half-maximal effective concentration (EC 50 In some embodiments, T eff The cells are CD8 T eff Cells (e.g., CD8 + ), naive CD8 cells (e.g., CD8 + , CD45RA + ), or CD4 Con cells (e.g., CD4 + , FoxP3 - ), or any combination thereof. In some embodiments, T cell activation is measured by assessing a change in STAT5 phosphorylation in a population of T cells upon contact with a modified IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide has a T phosphorylation of at least about 10 nM, at least about 50 nM, at least about 100 nM, at least about 500 nM, at least about 1000 nM, at least about 2000 nM, at least about 3000 nM, at least about 4000 nM, or at least about 5000 nM. effEC for cell activation 50 In some embodiments, the modified IL-2 polypeptide has a T of at least about 100 nM. eff EC for cell activation 50 In some embodiments, the modified IL-2 polypeptide has a T of at least about 500 nM. eff EC for cell activation 50 In some embodiments, the modified IL-2 polypeptide has a T of at least about 1000 nM. eff EC for cell activation 50 In some embodiments, the modified IL-2 polypeptide has a T of at least about 5000 nM. eff EC for cell activation 50 In some embodiments, the modified IL-2 polypeptide has a T that is at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold greater than the IL-2 polypeptide of SEQ ID NO:1 and / or SEQ ID NO:2. eff EC for cell activation 50 In some embodiments, the modified IL-2 polypeptide has the following structure: eff EC for cell activation 50 In some embodiments, the modified IL-2 polypeptide has a T eff EC for cell activation 50 In some embodiments, the modified IL-2 polypeptide has a T eff EC for cell activation 50 In some embodiments, the modified IL-2 polypeptide has a T eff EC for cell activation 50 In some embodiments, the modified IL-2 polypeptide has a T eff EC for cell activation 50 is at least 1000 times larger.

[0082] In some embodiments, the modified IL-2 polypeptide is effCompared with cells, T reg In some embodiments, the T reg T for EC50 of cell type activation eff The ratio of EC50 for activation of the cell types is at least 10, at least 20, at least 50, at least 100, at least 150, or at least 200. reg T for EC50 of cell type activation eff The ratio of EC50 for activation of the cell type is at least 100. reg T for EC50 of cell type activation eff The ratio of EC50 for activation of the cell type is at least 200. reg T for EC50 of cell type activation eff The ratio of EC50 for activation of the cell type is at least 300. reg T for EC50 of cell type activation eff The ratio of EC50 for activation of the cell type is at least 500. reg T for EC50 of cell type activation eff The EC50 ratio for activation of the cell type is at least 1000.

[0083] In some embodiments, the level of activation is measured about 0.5 hours to about 1 hour after incubation with the modified IL-2 polypeptide (e.g., 0.5 hours to 1 hour before fixing the cells for in vitro experiments).

[0084] In some embodiments, the modified IL-2 polypeptides described herein comprise a covalently attached polymer for half-life extension. In some embodiments, the modified IL-2 polypeptides comprise a covalently attached polymer for plasma or serum half-life extension. In some embodiments, the plasma or serum half-life of the polymer-attached modified IL-2 polypeptide is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold longer than the plasma or serum half-life of the wild-type IL-2 polypeptide (SEQ ID NO: 1) or aldesleukin (SEQ ID NO: 2) without the polymer attached.

[0085] In some embodiments, the plasma or serum half-life of a modified IL-2 polypeptide described herein is at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times longer than the plasma or serum half-life of a modified IL-2 polypeptide that does not contain a half-life extending polymer.

[0086] site-specific modification In some embodiments, the modified IL-2 polypeptides described herein contain one or more modifications at one or more amino acid residues. In some embodiments, the residue position numbering of the modified IL-2 polypeptides is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the residue position numbering of the modified IL-2 polypeptides is based on wild-type human IL-2 polypeptide as a reference sequence.

[0087] Modifications to the polypeptides described herein include amino acid substitutions, addition of various functional groups, deletion of amino acids, addition of amino acids, or any other modification of the wild-type version of the protein or protein fragment. Functional groups that can be added to the polypeptide include polymers, linkers, alkyl groups, detectable molecules such as chromophores or fluorophores, reactive functional groups, or any combination thereof. In some embodiments, functional groups are added to individual amino acids of the polypeptide. In some embodiments, functional groups are added site-specifically to the polypeptide.

[0088] In one aspect, provided herein is a modified IL-2 polypeptide comprising one or more amino acid substitutions. In some embodiments, the amino acid substitution affects the binding properties of the modified IL-2 polypeptide to an IL-2 receptor subunit (e.g., α, β or γ subunit) or an IL-2 receptor complex (e.g., IL-2 receptor αβγ complex or βγ complex). In some embodiments, the amino acid substitution is at a position on the interface of the binding interaction between the modified IL-2 polypeptide and the IL-2 receptor subunit or IL-2 receptor complex. In some embodiments, the amino acid substitution causes an increase in affinity for the IL-2 receptor αβγ complex or α subunit. In some embodiments, the amino acid substitution causes a decrease in affinity for the IL-2 receptor βγ complex or β subunit.

[0089] In one aspect, provided herein are modified IL-2 polypeptides comprising modified IL-2 polypeptides comprising naturally occurring amino acid substitutions compared to WT IL-2 (SEQ ID NO:1). In some embodiments, the modified IL-2 polypeptide comprises up to seven naturally occurring amino acid substitutions. In some embodiments, the modified IL-2 polypeptide comprises up to six amino acid substitutions. In some embodiments, the modified IL-2 polypeptide comprises up to five amino acid substitutions. In some embodiments, the modified IL-2 polypeptide comprises up to four amino acid substitutions. In some embodiments, the modified IL-2 polypeptide comprises up to three amino acid substitutions. In some embodiments, the modified IL-2 polypeptide comprises 3-7, 3-6, 3-5, 3-4, 4-7, 4-6, 4-5, 5-7, 5-6, or 6-7 naturally occurring amino acid substitutions. In some embodiments, the modified IL-2 polypeptide comprises at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 amino acid substitutions.

[0090] In some embodiments, the modified IL-2 polypeptides provided herein comprise a natural amino acid substitution at at least one of Y31, K35, Q74, and N88D, where the residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as the reference sequence. In some embodiments, the modified IL-2 polypeptide comprises at least two natural amino acid substitutions at Y31, K35, Q74, and N88. In some embodiments, the modified IL-2 polypeptide comprises at least three natural amino acid substitutions at Y31, K35, Q74, and N88. In some embodiments, the modified IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide comprises a natural amino acid substitution at each of Y31, K35, Q74, and N88. In some embodiments, the modified IL-2 polypeptide comprises the amino acid substitutions Y31H, K35R, Q74P, and N88D. In some embodiments, the modified IL-2 polypeptide further comprises an optional C125 substitution (e.g., C125S or C125A). In some embodiments, the modified IL-2 polypeptide further comprises an optional A1 deletion or substitution of residue A1. In some embodiments, the modified IL-2 polypeptide further comprises an optional A1 deletion.

[0091] In some embodiments, a modified IL-2 polypeptide provided herein comprises a Y31 substitution, wherein the residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO:1 as a reference sequence. In some embodiments, the Y31 substitution is for an aromatic amino acid. In some embodiments, the Y31 substitution is for a basic amino acid. In some embodiments, the basic amino acid is weakly basic. In some embodiments, the Y31 substitution is selected from Y31F, Y31H, Y31W, Y31R, and Y31K. In some embodiments, the Y31 substitution is Y31H.

[0092] In some embodiments, a modified IL-2 polypeptide provided herein comprises a K35 substitution, wherein the residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the K35 substitution is for a basic amino acid. In some embodiments, the K35 substitution is for a positively charged amino acid. In some embodiments, the K35 substitution is K35R, K35E, K35D, or K35Q. In some embodiments, the K35 substitution is K35R.

[0093] In some embodiments, a modified IL-2 polypeptide provided herein comprises a Q74 substitution, wherein the residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO:1 as the reference sequence. In some embodiments, the Q74 substitution is a cyclic amino acid. In some embodiments, the cyclic amino acid comprises a cyclic group covalently attached to the alpha carbon and a nitrogen attached to the alpha carbon. In some embodiments, the Q74 substitution is Q74P.

[0094] In some embodiments, a modified IL-2 polypeptide provided herein comprises an N88 substitution, wherein the residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the N88 substitution is a charged amino acid residue. In some embodiments, the N88 substitution is a negatively charged amino acid residue. In some embodiments, the N88 substitution is N88D or N88E. In some embodiments, the N88 substitution is N88D or N88E. In some embodiments, the N88 substitution is N88D.

[0095] In some embodiments, the modified IL-2 polypeptide comprises a C125 substitution, where the residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as the reference sequence. In some embodiments, the C125 substitution stabilizes the modified IL-2 polypeptide. In some embodiments, the C125 substitution does not substantially change the activity of the modified IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide comprises a C125S substitution. In some embodiments, the modified IL-2 polypeptide comprises a C125A substitution.

[0096] In some embodiments, the modified IL-2 polypeptide comprises a modification at residue A1, and the residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as the reference sequence. In some embodiments, the modification is an A1 deletion.

[0097] In some embodiments, the modified IL-2 polypeptide comprises an additional amino acid substitution. In some embodiments, the modified IL-2 polypeptide comprises an additional amino acid substitution that affects binding to the IL-2 receptor α subunit or the αβγ complex. In some embodiments, the modified IL-2 polypeptide comprises an additional amino acid substitution that affects binding to the IL-2 receptor β subunit or the βγ complex. In some embodiments, the modified IL-2 polypeptide comprises at least one additional amino acid substitution selected from Table 1. In some embodiments, the modified IL-2 polypeptide comprises at least one amino acid substitution at residues E15, N29, N30, T37, K48, V69, N71, N88, I89, or I92. In some embodiments, the modified IL-2 polypeptide comprises at least one amino acid substitution at residues E15, N29, N30, T37, K48, V69, N71, I89, or I92. In some embodiments, the modified IL-2 polypeptide comprises one, two, three or four naturally occurring amino acid substitutions at residues selected from E15, N29, N30, T37, K48, V69, N71, N88, I89 or I92, and in some embodiments, the modified IL-2 polypeptide comprises one, two, three or four naturally occurring amino acid substitutions at residues selected from E15, N29, N30, T37, K48, V69, N71, I89 or I92. In some embodiments, the modified IL-2 polypeptide comprises one naturally occurring amino acid substitution at a residue selected from E15, N29, N30, T37, K48, V69, N71, N88, I89 or I92. In some embodiments, the modified IL-2 polypeptide comprises two. In some embodiments, the modified IL-2 polypeptide comprises up to two naturally occurring amino acid substitutions at residues selected from E15, N29, N30, T37, K48, V69, N71, N88, I89, or I92. In some embodiments, the modified IL-2 polypeptide comprises up to three naturally occurring amino acid substitutions at residues selected from E15, N29, N30, T37, K48, V69, N71, N88, I89, or I92. In some embodiments, the additional amino acid substitution comprises E15A, E15G, or E15S. In some embodiments, the additional amino acid substitution comprises N29S.In some embodiments, the additional amino acid substitution comprises N30S. In some embodiments, the additional amino acid substitution comprises T37A or T37R. In some embodiments, the additional amino acid substitution comprises K48E. In some embodiments, the additional amino acid substitution comprises V69A. In some embodiments, the additional amino acid substitution comprises N71R. In some embodiments, the additional amino acid substitution comprises N88A, N88D, N88E, N88F, N88G, N88H, N88I, N88M, N88Q, N88R, N88S, N88T, N88V or N88W. In some embodiments, the additional amino acid substitution comprises N88D. In some embodiments, the additional amino acid substitution comprises I89V. In some embodiments, the additional amino acid substitution comprises I92K or I92R.

[0098] In some embodiments, the modified IL-2 polypeptides provided herein include substitutions at Y31, K35, Q74, and optionally C125S. In some embodiments, the modified IL-2 polypeptides do not include any additional substitutions that substantially affect binding to the IL-2 receptor α subunit or the αβγ complex. In some embodiments, the modified IL-2 polypeptides do not include additional amino acid substitutions that affect binding to the IL-2 receptor β subunit or the βγ complex. In some embodiments, the modified IL-2 polypeptides do not include additional naturally occurring amino acid substitutions selected from the positions identified in Table 1. In some embodiments, the modified IL-2 polypeptides do not include additional amino acid substitutions selected from Table 1. In some embodiments, the modified IL-2 polypeptides do not include additional naturally occurring amino acid substitutions at residues E15, N29, N30, T37, K48, V69, N71, N88, I89, or I92. In some embodiments, the modified IL-2 polypeptide does not comprise an additional amino acid substitution at residues E15, N29, N30, T37, K48, V69, N71, N88, I89, or I92. In some embodiments, the modified IL-2 polypeptide does not have a V69 substitution. In some embodiments, the modified IL-2 polypeptide does not have a V69A substitution. In some embodiments, the modified IL-2 polypeptide does not have a K48 substitution. In some embodiments, the modified IL-2 polypeptide does not have a K48E substitution. In some embodiments, the modified IL-2 polypeptide does not comprise a substitution at V69 or K48. In some embodiments, the modified IL-2 polypeptide does not comprise a substitution at either V69 or K48. In some embodiments, the modified IL-2 polypeptide does not comprise a V69A or K48E substitution. In some embodiments, the modified IL-2 polypeptide does not comprise either a V69A or K48E substitution.

[0099] In some embodiments, the modified IL-2 polypeptides provided herein include substitutions at Y31, K35, Q74, N88, and optionally C125S. In some embodiments, the modified IL-2 polypeptides do not include any additional substitutions that substantially affect binding to the IL-2 receptor α subunit or the αβγ complex. In some embodiments, the modified IL-2 polypeptides do not include additional amino acid substitutions that affect binding to the IL-2 receptor β subunit or the βγ complex. In some embodiments, the modified IL-2 polypeptides do not include additional naturally occurring amino acid substitutions selected from the positions identified in Table 1. In some embodiments, the modified IL-2 polypeptides do not include additional amino acid substitutions selected from Table 1. In some embodiments, the modified IL-2 polypeptides do not include additional naturally occurring amino acid substitutions at residues E15, N29, N30, T37, K48, V69, N71, I89, or I92. In some embodiments, the modified IL-2 polypeptide does not comprise an additional amino acid substitution at residues E15, N29, N30, T37, K48, V69, N71, I89, or I92. In some embodiments, the modified IL-2 polypeptide does not have a V69 substitution. In some embodiments, the modified IL-2 polypeptide does not have a V69A substitution. In some embodiments, the modified IL-2 polypeptide does not have a K48 substitution. In some embodiments, the modified IL-2 polypeptide does not have a K48E substitution. In some embodiments, the modified IL-2 polypeptide does not comprise a substitution at V69 or K48. In some embodiments, the modified IL-2 polypeptide does not comprise a substitution at either V69 or K48. In some embodiments, the modified IL-2 polypeptide does not comprise a V69A or K48E substitution. In some embodiments, the modified IL-2 polypeptide does not comprise either a V69A or K48E substitution.

[0100] In some embodiments, the modified IL-2 polypeptides provided herein comprise an N-terminal deletion. In some embodiments, the N-terminal deletion is of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acids. In some embodiments, the N-terminal deletion is of at least one amino acid. In some embodiments, the N-terminal deletion is of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, the N-terminal deletion is from 1 to 15 amino acids. In some embodiments, the N-terminal deletion is a deletion of a single amino acid (e.g., an A1 deletion of SEQ ID NO:1).

[0101] The modified IL-2 polypeptides as described herein can include one or more non-natural amino acids. An "unnatural" amino acid can refer to an amino acid residue in D- or L-form that is not among the 20 standard amino acids commonly incorporated into naturally occurring proteins. In some embodiments, one or more amino acids of the modified IL-2 polypeptides are substituted with one or more non-natural amino acids. Non-natural amino acids include, but are not limited to, L-azidolysine and L-biphenylalanine.

[0102] Exemplary unnatural amino acids also include homoserine, norleucine, p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, tri-O-acetyl-GlcNAcp-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, , p-benzoyl-L-phenylalanine, p-boronophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, an analog of the amino acid tyrosine, an analog of the amino acid glutamine, an analog of the amino acid phenylalanine, an analog of the amino acid serine, an analog of the amino acid threonine, a β-amino acid, a cyclic amino acid other than proline or histidine, an aromatic amino acid other than phenylalanine, tyrosine or tryptophan, or a combination thereof. In some embodiments, the unnatural amino acid is selected from a β-amino acid, a homoamino acid, and a cyclic amino acid. In some embodiments, the unnatural amino acid is selected from a β-alanine, a β-amino acid, a piperidinic acid, an aminocaproic acid, an aminoheptanoic acid, an aminopimelic acid, a desmosine, a diaminopimelic acid, a N-amino acid, a cyclic amino ... α -Ethylglycine, N α -Ethylasparagine, isodesmosine, allo-isoleucine, N α -Methylglycine, N α -Methylisoleucine, N α -Methylvaline, γ-carboxyglutamate, N α -Acetylserine, N α -formylmethionine, 3-methylhistidine, and / or other similar amino acids.

[0103] In some embodiments, the unnatural amino acid substitutions provided herein can be incorporated into an IL-2 polypeptide in addition to any combination of the natural amino acid substitutions provided herein, unless otherwise indicated. For example, when a modified IL-2 polypeptide is described that includes natural amino acid substitutions of, for example, Y31H, K35R, and Q74P, it is expressly contemplated that the modified IL-2 polypeptide can also include unnatural amino acid substitutions (e.g., Hse41, Hse71, Hse104, Nle23, Nle39, and Nle46). As another example, when a modified IL-2 polypeptide provided herein is described as having Y31H, K35R, Q74P, and N88D natural amino acid substitutions, the modified IL-2 polypeptide can further include unnatural amino acid substitutions (e.g., Hse41, Hse71, Hse104, Nle23, Nle39, and Nle46). In particular, any combination of the naturally occurring amino acid substitutions present in the recombinant modified IL-2 polypeptides provided herein can also be incorporated into a synthetic version of the modified IL-2 polypeptide (e.g., a corresponding modified IL-2 polypeptide that contains, for example, Hse41, Hse71, Hse104, Nle23, Nle39, and Nle46).

[0104] In one aspect, disclosed herein are modified IL-2 polypeptides comprising one or more unnatural amino acid substitutions. In some embodiments, the modified IL-2 polypeptide comprises at least two unnatural amino acid substitutions. In some embodiments, the modified IL-2 polypeptide comprises at least one amino acid substitution at a residue selected from Y31, K35, Q74 and N88, and the residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the modified IL-2 polypeptide comprises a homoserine (Hse) residue located at any one of residues 36-45. In some embodiments, the modified IL-2 polypeptide comprises an Hse residue located at any one of residues 61-81. In some embodiments, the modified IL-2 polypeptide comprises an Hse residue located at any one of residues 94-114. In some embodiments, the modified IL-2 polypeptide comprises one, two, three or more Hse residues. In some embodiments, the modified IL-2 polypeptide comprises Hse41, Hse71, Hse104, or a combination thereof. In some embodiments, the modified IL-2 polypeptide comprises Hse41, Hse71, and Hse104. In some embodiments, the modified IL-2 polypeptide comprises at least two amino acid substitutions, wherein the at least two amino acid substitutions are selected from: (a) a homoserine (Hse) residue located at any one of residues 36-45; (b) a homoserine residue located at any one of residues 61-81; and (c) a homoserine residue located at any one of residues 94-114. In some embodiments, the modified IL-2 polypeptide comprises Hse41 and Hse71. In some embodiments, the modified IL-2 polypeptide comprises Hse41 and Hse104. In some embodiments, the modified IL-2 polypeptide comprises Hse71 and Hse104. In some embodiments, the modified IL-2 polypeptide comprises Hse41. In some embodiments, the modified IL-2 polypeptide comprises Hse71. In some embodiments, the modified IL-2 polypeptide comprises Hse104. In some embodiments, the modified IL-2 polypeptide comprises one, two, three or more norleucine (Nle) residues.In some embodiments, the modified IL-2 polypeptide comprises an Nle residue located at any one of residues 18-28. In some embodiments, the modified IL-2 polypeptide comprises one or more Nle residues located at any one of residues 34-50. In some embodiments, the modified IL-2 polypeptide comprises an Nle residue located at any one of residues 20-60. In some embodiments, the modified IL-2 polypeptide comprises three Nle substitutions. In some embodiments, the modified IL-2 polypeptide comprises Nle23, Nle39, and Nle46. In some embodiments, the modified IL-2 polypeptide comprises SEQ ID NO:3. In some embodiments, the modified IL-2 polypeptide comprises SEQ ID NO:3 with an A1 deletion.

[0105] In some embodiments, the modified IL-2 polypeptides provided herein comprise an amino acid sequence of any one of SEQ ID NOs: 3-43 provided in Table 7. In some embodiments, the modified IL-2 polypeptides comprise an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 3-43. In some embodiments, the modified IL-2 polypeptides comprise an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 3-43, where each residue in the reference amino acid sequence that is substituted relative to SEQ ID NO: 1 is retained. In some embodiments, the modified IL-2 polypeptides comprise an amino acid sequence of SEQ ID NO: 3. In some embodiments, the modified IL-2 polypeptides comprise an amino acid sequence at least 85% identical to the sequence of SEQ ID NO: 3. In some embodiments, the modified IL-2 polypeptides comprise an amino acid sequence at least 85%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 3, where each residue substituted in SEQ ID NO: 3 relative to SEQ ID NO: 1 is retained. In some embodiments, the modified IL-2 polypeptides comprise an amino acid sequence of SEQ ID NO: 4. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO:4, where each residue substituted in SEQ ID NO:4 relative to SEQ ID NO:1 is retained.

[0106] In some embodiments, the modified IL-2 polypeptides described herein comprise at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 3. In some embodiments, the modified IL-2 polypeptides described herein comprise at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 4. In some embodiments, sequence identity is measured by the protein-protein BLAST algorithm using the parameters of Matrix BLOSUM62, Gap Costs Existence:11, Extension:1, and Compositional Adjustments Conditional Compositional Score Matrix Adjustment.

[0107] In some embodiments, the modified IL-2 polypeptides described herein comprise at least 3, at least 4, at least 5, at least 6, at least 7, or at least 9 amino acid substitutions. In some embodiments, the modified IL-2 polypeptides comprise 3-9 amino acid substitutions. In some embodiments, the modified IL-2 polypeptides comprise 3 or 4 amino acid substitutions, 3-5 amino acid substitutions, 3-6 amino acid substitutions, 3-7 amino acid substitutions, 3-9 amino acid substitutions, 4 or 5 amino acid substitutions, 4-6 amino acid substitutions, 4-7 amino acid substitutions, 4-9 amino acid substitutions, 5 or 6 amino acid substitutions, 5-7 amino acid substitutions, 5-9 amino acid substitutions, 6 or 7 amino acid substitutions, 6-9 amino acid substitutions, or 7-9 amino acid substitutions. In some embodiments, the modified IL-2 polypeptides comprise 3 amino acid substitutions, 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions. In some embodiments, the modified IL-2 polypeptide comprises up to 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions. In some embodiments, one or more amino acid substitutions are selected from Table 1. In some embodiments, one or more amino acid substitutions are selected from Table 2.

[0108] In some embodiments, the modified IL-2 polypeptide comprises a substitution of a modified native amino acid residue that can be used for attachment of additional functional groups that can be used to facilitate conjugation reactions or attachment of various payloads to the modified IL-2 polypeptide (e.g., polymers). The substitution can be to a native amino acid that is more suitable for attachment of additional functional groups (e.g., aspartic acid / asparagine cysteine, glutamic acid / glutamine, lysine, serine, threonine, or tyrosine), derivatives of modified versions of any native amino acid, or any non-natural amino acid (e.g., amino acids containing a desired reactive group, e.g., CLICK chemistry reagents such as azides, alkynes, etc.). Non-limiting examples of modified native amino acid residues include the modified lysine, glutamic acid, aspartic acid, and cysteine ​​provided below.

[0109] [ka] where each n is an integer from 1 to 30. Other examples of natural amino acids that may be similarly modified include those that have a heteroatom that can readily form a bond with a group suitable for linking a polymeric group to the amino acid (e.g., tyrosine, serine, threonine). These non-limiting examples of modified amino acid residues can be used at any position where it is desirable to add an additional functional group (e.g., a polymer or additional polypeptide) to the modified IL-2 polypeptide.

[0110] In some embodiments, the modified IL-2 polypeptide comprises a modification of a terminal residue (e.g., the N-terminal residue or the C-terminal residue) that comprises a polymer. In some embodiments, the modification to the terminal residue comprises attachment of a conjugation handle to the terminal residue of the modified IL-2 polypeptide. In some embodiments, the conjugation handle is attached to the modified IL-2 polypeptide via the N-terminal amino group or the C-terminal carboxyl group of the modified IL-2 polypeptide. In some embodiments, the conjugation handle is attached to the modified IL-2 polypeptide via the N-terminal amino group of the modified IL-2 polypeptide. In some embodiments, the conjugation handle is attached to the N-terminal amino group of the modified IL-2 polypeptide via a glutarylamino-PEG linker. In some embodiments, the conjugation handle is attached to the N-terminal amino group of the modified IL-2 polypeptide via a glutarylamino-PEG linker having the structure

[0111] [ka] and wherein each n is independently an integer from 1 to 30 (e.g., 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, or 30) and X is a conjugation handle (e.g., an azide or other conjugation handle provided herein, such as a DBCO group). In some embodiments, the modified IL-2 polypeptide comprises an adduct as described above, but the conjugation handle X is replaced with the reaction product of a conjugation handle that links the modified IL-2 polypeptide to an additional moiety (e.g., a larger polymer or additional polypeptide) and a complementary conjugation handle (e.g., a 1,2,3-triazole). In some embodiments, the N-terminal amino group of the modified IL-2 polypeptide comprises an adduct having the structure:

[0112] [ka]

[0113] In some embodiments, the modified IL-2 polypeptide is linked to an additional polypeptide. In some embodiments, the modified IL-2 polypeptide and the additional polypeptide form a fusion polypeptide. In some embodiments, the modified IL-2 polypeptide and the additional polypeptide are conjugated together. In some embodiments, the additional polypeptide comprises an antibody or a binding fragment thereof. In some embodiments, the antibody comprises a humanized antibody, a murine antibody, a chimeric antibody, a bispecific antibody, any fragment thereof, or any combination thereof. In some embodiments, the antibody is a monoclonal antibody or any fragment thereof (e.g., an antigen-binding fragment).

[0114] In some embodiments, the modified IL-2 polypeptide is not conjugated to an additional polypeptide. In some embodiments, the modified IL-2 polypeptide is not conjugated to an antibody. In some embodiments, the modified IL-2 polypeptide is not conjugated to an anti-TNFα antibody.

[0115] polymer In some embodiments, the modified IL-2 polypeptides described herein comprise one or more polymers covalently attached thereto. In some embodiments, the modified IL-2 polypeptides described comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polymers covalently attached to the modified IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptides described comprise a polymer covalently attached to the N-terminus of the IL-2 polypeptide. The polymers provided herein may be attached directly to a residue of the modified IL-2 polypeptide or may be attached via a small linking group (e.g., attached by reaction with a conjugation handle incorporated into the modified IL-2 polypeptide).

[0116] The polymers provided herein can be attached to any desired residue of an IL-2 polypeptide. In some embodiments, the polymer is preferably attached to a residue that does not affect the binding of the IL-2 polypeptide to an IL-2 receptor or a specific IL-2 receptor subunit (e.g., the IL-2 receptor α subunit). In some embodiments, the polymer is attached to or near the N-terminus of the modified IL-2 polypeptide. In some embodiments, the polymer is attached to the N-terminus of the modified IL-2 polypeptide. In some embodiments, the N-terminus is residue A1 of the IL-2 polypeptide, and the numbering of the residue positions is based on SEQ ID NO: 1 as the reference sequence. In some embodiments, the N-terminus is residue P2 of the IL-2 polypeptide, and the numbering of the residue positions is based on SEQ ID NO: 1 as the reference sequence (e.g., the modified IL-2 polypeptide includes a deletion of residue A1 from the sequence). In some embodiments, the polymer is attached to a residue position that blocks or reduces the binding of the modified IL-2 polypeptide to an IL-2 receptor β subunit. Such residue positions are provided in U.S. Patent Application Publication No. 20200231644, which is incorporated by reference as if set forth in its entirety, and include, for example, residue positions K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95, and Q126.

[0117] In some embodiments, the polymer comprises a water soluble polymer. In some embodiments, the water soluble polymer comprises a poly(alkylene oxide), a polysaccharide, poly(vinylpyrrolidone), poly(vinyl alcohol), polyoxazoline, poly(acryloylmorpholine), or a combination thereof. In some embodiments, the water soluble polymer is a poly(alkylene oxide). In some embodiments, the water soluble polymer is a polysaccharide. In some embodiments, the water soluble polymer is a poly(ethylene oxide).

[0118] In some embodiments, the modified IL-2 polypeptide described herein comprises a polymer covalently attached to the N-terminus of the IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide comprises a second polymer covalently attached thereto. In some embodiments, the modified IL-2 polypeptide comprises a second and a third polymer covalently attached thereto.

[0119] In some embodiments, the attached polymer has a weight average molecular weight of about 6,000 daltons to about 50,000 daltons. In some embodiments, the polymer has a weight average molecular weight of about 6,000 daltons to about 10,000 daltons, about 6,000 daltons to about 30,000 daltons, about 6,000 daltons to about 50,000 daltons, about 10,000 daltons to about 30,000 daltons, about 10,000 daltons to about 50,000 daltons, or about 30,000 daltons to about 50,000 daltons. In some embodiments, the polymer has a weight average molecular weight of about 6,000 daltons, about 10,000 daltons, about 30,000 daltons, or about 50,000 daltons. In some embodiments, the polymer has a weight average molecular weight of at least about 6,000 daltons, about 10,000 daltons, or about 30,000 daltons, hi some embodiments, the polymer has a weight average molecular weight of at most about 10,000 daltons, about 30,000 daltons, or about 50,000 daltons.

[0120] In some embodiments, the conjugated polymer, such as the polymer attached to the N-terminus, has a weight average molecular weight of about 120 daltons to about 1,000 daltons. In some embodiments, the polymer has a weight average molecular weight of about 120 daltons to about 250 daltons, about 120 daltons to about 300 daltons, about 120 daltons to about 400 daltons, about 120 daltons to about 500 daltons, about 120 daltons to about 1,000 daltons, about 250 daltons to about 300 daltons, about 250 daltons to about 400 daltons, about 250 daltons to about 500 daltons, about 250 daltons to about 1,000 daltons, about 300 daltons to about 400 daltons, about 300 daltons to about 500 daltons, about 300 daltons to about 1,000 daltons, about 400 daltons to about 500 daltons, about 400 daltons to about 1,000 daltons, or about 500 daltons to about 1,000 daltons. In some embodiments, the polymer has a weight average molecular weight of about 120 daltons, about 250 daltons, about 300 daltons, about 400 daltons, about 500 daltons, or about 1,000 daltons. In some embodiments, the polymer has a weight average molecular weight of at least about 120 daltons, about 250 daltons, about 300 daltons, about 400 daltons, or about 500 daltons. In some embodiments, the polymer has a weight average molecular weight of at most about 250 daltons, about 300 daltons, about 400 daltons, about 500 daltons, or about 1,000 daltons.

[0121] In some embodiments, the attached polymer comprises a water soluble polymer. In some embodiments, the water soluble polymer comprises a poly(alkylene oxide), a polysaccharide, a poly(vinylpyrrolidone), a poly(vinyl alcohol), a polyoxazoline, a poly(acryloylmorpholine), or a combination thereof. In some embodiments, the water soluble polymer is a poly(alkylene oxide), such as polyethylene glycol (e.g., polyethylene oxide). In some embodiments, the water soluble polymer is polyethylene glycol. In some embodiments, the water soluble polymer comprises a modified poly(alkylene oxide).

[0122] In some embodiments, the modified poly(alkylene oxide) comprises one or more linker groups. In some embodiments, the one or more linker groups comprise bifunctional linkers, such as amide, ester, ether, thioether, carbonyl, etc. In some embodiments, the one or more linker groups comprise amide linker groups. In some embodiments, the modified poly(alkylene oxide) comprises one or more spacer groups. In some embodiments, the spacer group comprises a substituted or unsubstituted C1-C6 alkylene group. In some embodiments, the spacer group comprises -CH2-, -CH2CH2-, or -CH2CH2CH2-. In some embodiments, the linker group is the product of a biorthogonal reaction (e.g., a biocompatible and selective reaction). In some embodiments, the bioorthogonal reaction is a metal-mediated process such as Cu(I)-catalyzed or "copper-free" alkyne-azide triazole formation reaction, Staudinger ligation, inverse electron-on-demand Diels-Alder (IEDDA) reaction, "photo-click" chemistry, or olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling. In some embodiments, the polymer is attached to the IL-2 polypeptide via click chemistry.

[0123] In some embodiments, the modified IL-2 polypeptides provided herein comprise reactive groups that facilitate conjugation of the modified IL-2 polypeptides to derivatized molecules or moieties, such as antibodies and polymers (e.g., further larger polymers). In some embodiments, the reactive group comprises one or more of active esters derived from carboxylic acids, mixed anhydrides, acyl halides, acyl azides, alkyl halides, N-maleimides, imino esters, isocyanates, and isothiocyanates. In some embodiments, the reactive group comprises an azide. In some embodiments, the reactive group comprises an alkyne.

[0124] In some embodiments, the polymer comprises a conjugation handle that can be used to further attach additional moieties to the modified IL-2 polypeptide (e.g., the addition of additional polypeptides such as antibodies). Any suitable reactive group that can react with a complementary reactive group attached to another moiety can be used as a conjugation handle.

[0125] In some embodiments, the polymer comprises a conjugation handle or a reaction product of a conjugation handle with a complementary conjugation handle. In some embodiments, the reaction product of the conjugation handle and the complementary conjugation handle results from a KAT ligation (reaction of potassium acyltrifluoroborate with hydroxylamine), a Staudinger ligation (reaction of an azide with a phosphine), a tetrazine cycloaddition (reaction of tetrazine with trans-cyclooctene), or a Huisgen cycloaddition (reaction of an alkyne with an azide). In some embodiments, the polymer comprises a reaction product of a conjugation handle and a complementary conjugation handle used to attach the polymer to the modified IL-2 polypeptide. In some embodiments, the polymer comprises an azide moiety. In some embodiments, the polymer comprises an alkyne moiety. In some embodiments, the polymer comprises an azide moiety, an alkyne moiety, or the reaction product of an azide-alkyne cycloaddition reaction. In some embodiments, the reaction product of the azide-alkyne cycloaddition reaction is a 1,2,3-triazole.

[0126] In some embodiments, the polymer is attached to the modified IL-2 polypeptide by use of a bifunctional linker. In some embodiments, the bifunctional linker reacts with a reactive group of an amino acid residue on the modified IL-2 polypeptide (e.g., a cysteine ​​sulfhydryl) to form a covalent bond. In some embodiments, in a second step, a second reactive group of the bifunctional linker (e.g., a conjugation handle such as an azide or alkyne) is used to attach a second moiety, such as a polymer.

[0127] In some embodiments, the polymer has formula (X):

[0128] [ka] The structure of In the formula, L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 and L 9 each independently represents -O-, -NR L -, -(C1-C6 alkylene)NR L -, -NR L (C1-C6 alkylene)-, -N(R L )2 + -, -(C1-C6 alkylene)N(R L )2 + -, -N(R L )2 + -(C1-C6 alkylene)-, -OP(=O)(OR L )O-, -S-, -(C1-C6 alkylene)S-, -S(C1-C6 alkylene)-, -S(=O)-, -S(=O)2-, -C(=O)-, -(C1-C6 alkylene)C(=O)-, -C(=O)(C1-C6 alkylene)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NR L -, -C(=O)NR L (C1-C6 alkylene)-, -(C1-C6 alkylene)C(=O)NR L -, -NR L C(=O)-, -(C1-C6 alkylene)NR L C(=O)-, -NR L C(=O)(C1-C6 alkylene)-, -OC(=O)NR L -, -NR L C(=O)O-, -NR L C(=O)NR L -, -NR L C(=S)NR L-, -CR L =N-, -N=CR L , -NR L S(=O)2-, -S(=O)2NR L -, -C(=O)NR L S(=O)2-, -S(=O)2NR L C(=O)-, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 heteroalkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene, substituted or unsubstituted C6-C 20 Arylene, substituted or unsubstituted C2-C 20 Heteroarylene, -(CH2-CH2-O) qa -, -(O-CH2-CH2) qb -, -(CH2-CH(CH3)-O) qc -, -(O-CH(CH3)-CH2) qd -, the reaction product of a conjugation handle and a complementary conjugation handle, or absent, (C1-C6 alkylene); Each R L are independently hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C7 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; Each of qa, qb, qc, and qd independently represents an integer of 1 to 100; During the ceremony, each

[0129] [ka] includes a linker that includes the structure, which is the point of attachment of the modified IL-2 polypeptide or polymer to the polymer portion.

[0130] In some embodiments, the polymer has formula (X'):

[0131] [ka] The structure of In the formula, each L' is independently -O-, -NR L -, -(C1-C6 alkylene)NR L -, -NR L (C1-C6 alkylene)-, -N(R L )2 + -, -(C1-C6 alkylene)N(R L )2 + -, -N(R L )2 + -(C1-C6 alkylene)-, -OP(=O)(OR L )O-, -S-, -(C1-C6 alkylene)S-, -S(C1-C6 alkylene)-, -S(=O)-, -S(=O)2-, -C(=O)-, -(C1-C6 alkylene)C(=O)-, -C(=O)(C1-C6 alkylene)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NR L -, -C(=O)NR L (C1-C6 alkylene)-, -(C1-C6 alkylene)C(=O)NR L -, -NR L C(=O)-, -(C1-C6 alkylene)NR L C(=O)-, -NR L C(=O)(C1-C6 alkylene)-, -OC(=O)NR L -, -NR L C(=O)O-, -NR L C(=O)NR L -, -NR L C(=S)NR L -, -CR L =N-, -N=CR L , -NR L S(=O)2-, -S(=O)2NR L -, -C(=O)NR L S(=O)2-, -S(=O)2NR LC(=O)-, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 heteroalkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene, substituted or unsubstituted C6-C 20 Arylene, substituted or unsubstituted C2-C 20 Heteroarylene, -(CH2-CH2-O) qa -, -(O-CH2-CH2) qb -, -(CH2-CH(CH3)-O) qc -, -(O-CH(CH3)-CH2) qd -, the reaction product of a conjugation handle and a complementary conjugation handle, or absent, (C1-C6 alkylene); Each R L are independently hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C7 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; Each of qa, qb, qc, and qd independently represents an integer of 1 to 100; g is an integer from 1 to 100; During the ceremony, each

[0132] [ka] is the point of attachment of the modified IL-2 polypeptide or polymer to the polymer portion.

[0133] In some embodiments, the modified IL-2 polypeptides provided herein comprise a polymer comprising a linker selected from Table 4. In Table 4, each

[0134] [ka] is the point of attachment to either the modified IL-2 polypeptide (eg, an amino group on the modified IL-2 polypeptide) or to the polymer portion of the polymer.

[0135] [Table 5]

[0136] In some embodiments, the water soluble polymer comprises 1 to 10 polyethylene glycol chains. In some embodiments, the water soluble polymer comprises 1 to 10 polyethylene glycol chains. In some embodiments, the first water soluble polymer comprises 1 to 2 polyethylene glycol chains, 1 to 4 polyethylene glycol chains, 1 to 6 polyethylene glycol chains, 1 to 10 polyethylene glycol chains, 2 to 4 polyethylene glycol chains, 2 to 6 polyethylene glycol chains, 2 to 10 polyethylene glycol chains, 4 to 6 polyethylene glycol chains, 4 to 10 polyethylene glycol chains, or 6 to 10 polyethylene glycol chains. In some embodiments, the water soluble polymer comprises 1 polyethylene glycol chain, 2 polyethylene glycol chains, 4 polyethylene glycol chains, 6 polyethylene glycol chains, or 10 polyethylene glycol chains. In some embodiments, the water soluble polymer comprises at least one polyethylene glycol chain, two polyethylene glycol chains, four polyethylene glycol chains, or six polyethylene glycol chains. In some embodiments, the first water soluble polymer comprises at most two polyethylene glycol chains, four polyethylene glycol chains, six polyethylene glycol chains, or ten polyethylene glycol chains. In some embodiments, the water soluble polymer comprises four polyethylene glycol chains. In some embodiments, the water soluble polymer comprises a compound of formula (I):

[0137] [ka] wherein each m is independently an integer from 4 to 30. In some embodiments, at least one polyethylene glycol chain of the water-soluble polymer comprises a structure of formula (II):

[0138] [ka] wherein each m is independently an integer from 4 to 30 and each n is independently an integer from 1 to 10. In some embodiments, each polyethylene glycol chain of the water soluble polymer comprises a structure of formula (II). In some embodiments of formula (II), m is 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, or 40. In some embodiments of formula (II), n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0139] In some embodiments, each of the polyethylene glycol chains independently contains from about 5 to about 300, from about 10 to about 200, from about 20 to about 100, or from about 25 to about 50 ethylene glycol units. In some embodiments, each of the polyethylene glycol chains independently contains from 5 ethylene glycol units to 300 ethylene glycol units. In some embodiments, each of the polyethylene glycol chains is independently selected from 5 ethylene glycol units to 10 ethylene glycol units, 5 ethylene glycol units to 20 ethylene glycol units, 5 ethylene glycol units to 25 ethylene glycol units, 5 ethylene glycol units to 50 ethylene glycol units, 5 ethylene glycol units to 100 ethylene glycol units, 5 ethylene glycol units to 200 ethylene glycol units, 5 ethylene glycol units to 300 ethylene glycol units, 10 ethylene glycol units to 20 ethylene glycol units, 10 ethylene glycol units to 25 ethylene glycol units, 10 ethylene glycol units to 50 ethylene glycol units, 10 ethylene glycol units to 100 ethylene glycol units, 10 ethylene glycol units to 200 ethylene glycol units, 10 ethylene glycol units to 300 ethylene glycol units, 20 ethylene glycol units to 25 ethylene glycol units, 25 ethylene glycol units to 30 ethylene glycol units, 26 ethylene glycol units to 30 ethylene glycol units, 27 ethylene glycol units to 30 ethylene glycol units, 28 ethylene glycol units to 40 ethylene glycol units, 29 ethylene glycol units to 50 ethylene glycol units, 30 ethylene glycol units to 40 ethylene glycol units, 31 ethylene glycol units to 45 ethylene glycol units, 32 ethylene glycol units to 40 ethylene glycol units, 33 ethylene glycol units to 45 ethylene glycol units, 34 ethylene glycol units to 40 ethylene glycol units, 35 ethylene glycol units to 50 ethylene glycol units, 36 ethylene glycol units to 50 ethylene glycol units, 37 ethylene glycol units to 50 ethylene glycol units, 38 ethylene glycol units to 50 ethylene glycol units, 39 ethylene glycol units to 60 ethylene glycol units, 38 ethylene glycol units to 60 ethylene glycol units, 39 ethylene glycol units to 70 ethylene glycol units, recall units, 20 ethylene glycol units to 50 ethylene glycol units, 20 ethylene glycol units to 100 ethylene glycol units, 20 ethylene glycol units to 200 ethylene glycol units, 20 ethylene glycol units to 300 ethylene glycol units, 25 ethylene glycol units to 50 ethylene glycol units, 25 ethylene glycol units to 100 ethylene glycol units, 25 ethylene glycol units to 200 ethylene glycol units, 25 ethylene glycol units to 300 ethylene glycol units, 50 ethylene glycol units to 100 ethylene glycol units, 50 ethylene glycol units to 200 ethylene glycol units, 50 ethylene glycol units to 300 ethylene glycol units, 100 ethylene glycol units to 200 ethylene glycol units, 100 ethylene glycol units to 300 ethylene glycol units, or 200 ethylene glycol units to 300 ethylene glycol units.In some embodiments, each of the polyethylene glycol chains independently comprises 5 ethylene glycol units, 10 ethylene glycol units, 20 ethylene glycol units, 25 ethylene glycol units, 50 ethylene glycol units, 100 ethylene glycol units, 200 ethylene glycol units, or 300 ethylene glycol units. In some embodiments, each of the polyethylene glycol chains independently comprises at least 5 ethylene glycol units, 10 ethylene glycol units, 20 ethylene glycol units, 25 ethylene glycol units, 50 ethylene glycol units, 100 ethylene glycol units, or 200 ethylene glycol units. In some embodiments, each of the polyethylene glycol chains independently comprises at most 10 ethylene glycol units, 20 ethylene glycol units, 25 ethylene glycol units, 50 ethylene glycol units, 100 ethylene glycol units, 200 ethylene glycol units, or 300 ethylene glycol units.

[0140] In some embodiments, each of the polyethylene glycol chains is independently linear or branched. In some embodiments, each of the polyethylene glycol chains is linear polyethylene glycol. In some embodiments, each of the polyethylene glycol chains is branched polyethylene glycol. For example, in some embodiments, each of the first and second polymers comprises a linear polyethylene glycol chain.

[0141] In some embodiments, each of the polyethylene glycol chains is independently end-capped with a hydroxy, alkyl, alkoxy, amido, or amino group. In some embodiments, each of the polyethylene glycol chains is independently end-capped with an amino group. In some embodiments, each of the polyethylene glycol chains is independently end-capped with an amide group. In some embodiments, each of the polyethylene glycol chains is independently end-capped with an alkoxy group. In some embodiments, each of the polyethylene glycol chains is independently end-capped with an alkyl group. In some embodiments, each of the polyethylene glycol chains is independently end-capped with a hydroxy group. In some embodiments, one or more polyethylene glycol chains are independently of the structure

[0142] [ka] wherein n is an integer from 4 to 30. In some embodiments, one or more polyethylene glycol chains independently have the structure

[0143] [ka] wherein m is an integer of 4 to 30.

[0144] In some embodiments, the modified IL-2 polypeptide comprises multiple polymers covalently attached thereto. In some embodiments, each polymer comprises a water soluble polymer. In some embodiments, the water soluble polymer comprises a poly(alkylene oxide), a polysaccharide, poly(vinylpyrrolidone), poly(vinyl alcohol), polyoxazoline, poly(acryloylmorpholine), or a combination thereof. In some embodiments, each water soluble polymer is a poly(alkylene oxide). In some embodiments, each water soluble polymer is a polyethylene glycol.

[0145] In some embodiments, the modified IL-2 polypeptide comprises 1 to 10 covalently attached water soluble polymers. In some embodiments, the modified IL-2 polypeptide comprises 1 to 10 covalently attached water soluble polymers. In some embodiments, the modified IL-2 polypeptide comprises 1 or 2 covalently attached water soluble polymers, 1 to 3 covalently attached water soluble polymers, 1 to 4 covalently attached water soluble polymers, 1 to 6 covalently attached water soluble polymers, 1 to 8 covalently attached water soluble polymers, 1 to 10 covalently attached water soluble polymers, 2 or 3 covalently attached water soluble polymers, 2 to 4 covalently attached water soluble polymers, 2 to 6 covalently attached water soluble polymers, 2 to 8 covalently attached water soluble polymers, 2 to 10 covalently attached water soluble polymers, 3 or 4 covalently attached water soluble polymers, 3 to 6 covalently attached water soluble polymers, 3 to 8 covalently attached water soluble polymers, 3 to 10 covalently attached water soluble polymers, 4 to 6 covalently attached water soluble polymers, 4 to 8 covalently attached water soluble polymers, 4 to 10 covalently attached water soluble polymers, 6 to 8 covalently attached water soluble polymers, 6 to 10 covalently attached water soluble polymers, or 8 to 10 covalently attached water soluble polymers.

[0146] In some embodiments, the water soluble polymer that can be attached to the modified IL-2 polypeptide has the structure of Formula (A):

[0147] [ka] Includes.

[0148] In some embodiments, the water soluble polymer that can be attached to the modified IL-2 polypeptide has the structure of Formula (B):

[0149] [ka] Includes.

[0150] In some embodiments, the water soluble polymer that can be attached to the modified IL-2 polypeptide has the structure of Formula (C):

[0151] [ka] Includes.

[0152] In some embodiments, the water soluble polymer that can be attached to the modified IL-2 polypeptide has the structure of formula (D):

[0153] [ka] Includes.

[0154] In some embodiments, the water soluble polymer that can be attached to the modified IL-2 polypeptide comprises the structure of formula (E):

[0155] [ka]

[0156] In some embodiments, the water soluble polymer attached to the modified IL-2 polypeptide comprises one or more linkers and / or spacers. In some embodiments, the one or more linkers comprise one or more amide groups. In some embodiments, the one or more linkers comprise one or more lysine groups. In some embodiments, the water soluble polymer attached to the modified IL-2 polypeptide comprises a structure of Formula (I), Formula (II), Formula (III), or a combination thereof. In some embodiments, the water soluble polymer attached to the modified IL-2 polypeptide comprises a structure of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), or a combination thereof. In some embodiments, the water soluble polymer attached to the modified IL-2 polypeptide comprises the structure:

[0157] [ka]

[0158] In some embodiments, the water soluble polymer attached to the N-terminus comprises one or more linkers and / or spacers. In some embodiments, the one or more linkers comprise one or more amide groups. In some embodiments, the one or more linkers comprise one or more lysine groups. In some embodiments, the water soluble polymer attached to the N-terminus comprises a structure of Formula (I), Formula (II), Formula (III), or a combination thereof. In some embodiments, the water soluble polymer attached to the N-terminus comprises a structure of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), or a combination thereof. In some embodiments, the attached water soluble polymer comprises the following structure:

[0159] [ka]

[0160] In some embodiments, the polymer is synthesized from a suitable precursor material. In some embodiments, the polymer is synthesized from a precursor material of Structure 5, Structure 6, Structure 7, or Structure 8, where Structure 5 is:

[0161] [ka] and Structure 6 is

[0162] [ka] and Structure 7 is

[0163] [ka] and Also, structure 8 is

[0164] [ka] It is.

[0165] III. Composition Pharmaceutical Formulations In one aspect, described herein is a pharmaceutical formulation comprising a modified IL-2 polypeptide described herein and a pharma- ceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical formulation comprises more than one modified IL-2 polypeptide. In some embodiments, the pharmaceutical formulation further comprises one or more excipients selected from a carbohydrate, an inorganic salt, an antioxidant, a surfactant, or a buffering agent.

[0166] In some embodiments, the pharmaceutical formulation further comprises a carbohydrate, hi certain embodiments, the carbohydrate is selected from the group consisting of fructose, maltose, galactose, glucose, D-mannose, sorbose, lactose, sucrose, trehalose, cellobiosraffinose, melezitose, maltodextrin, dextran, starch, mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myo-inositol, cyclodextrin, and combinations thereof.

[0167] In some embodiments, the pharmaceutical formulation comprises an inorganic salt, hi certain embodiments, the inorganic salt is selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium sulfate, or a combination thereof.

[0168] In certain embodiments, the pharmaceutical formulation comprises an antioxidant, hi certain embodiments, the antioxidant is selected from the group consisting of ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, potassium metabisulfite, propyl gallate, sodium metabisulfite, sodium thiosulfate, vitamin E, 3,4-dihydroxybenzoic acid, and combinations thereof.

[0169] In certain embodiments, the pharmaceutical formulation comprises a surfactant, hi certain embodiments, the surfactant is selected from the group consisting of polysorbates, sorbitan esters, lipids, phospholipids, phosphatidylethanolamines, fatty acids, fatty acid esters, steroids, EDTA, zinc, and combinations thereof.

[0170] In certain embodiments, the pharmaceutical formulation comprises a buffering agent, hi certain embodiments, the buffering agent is selected from the group consisting of citric acid, sodium phosphate, potassium phosphate, acetic acid, ethanolamine, histidine, an amino acid, tartaric acid, succinic acid, fumaric acid, lactic acid, Tris, HEPES, or a combination thereof.

[0171] In some embodiments, the pharmaceutical formulation is prepared for parenteral or enteral administration. In some embodiments, the pharmaceutical formulation is formulated for intravenous or subcutaneous administration. In some embodiments, the pharmaceutical formulation is in lyophilized form.

[0172] In one aspect, described herein are liquid or lyophilized formulations comprising the modified IL-2 polypeptides described. In some embodiments, the modified IL-2 polypeptides are lyophilized powders. In some embodiments, the lyophilized powders are resuspended in a buffer solution. In some embodiments, the buffer solution comprises a buffering agent, a sugar, a salt, a surfactant, or any combination thereof. In some embodiments, the buffer solution comprises a phosphate salt. In some embodiments, the phosphate salt is sodium Na2HPO4. In some embodiments, the salt is sodium chloride. In some embodiments, the buffer solution comprises phosphate buffered saline. In some embodiments, the buffer solution comprises mannitol. In some embodiments, the lyophilized powders are suspended in a solution comprising 10 mM Na2HPO4 buffer pH 7.5, 0.022% SDS, and 50 mg / mL mannitol.

[0173] Dosage form The modified IL-2 polypeptides described herein may be in a variety of dosage forms. In some embodiments, the modified IL-2 polypeptide is administered as a lyophilized powder. In some embodiments, the modified IL-2 polypeptide is administered as a suspension. In some embodiments, the modified IL-2 polypeptide is administered as a solution. In some embodiments, the modified IL-2 polypeptide is administered as an injectable solution. In some embodiments, the modified IL-2 polypeptide is administered as an IV solution.

[0174] IV. Treatment Method In one aspect, described herein is a method of treating an autoimmune disease or disorder in a subject in need of such treatment, comprising administering to the subject an effective amount of a modified IL-2 polypeptide or pharmaceutical composition as described herein. In one aspect, described herein is a method of treating an inflammatory disease or disorder in a subject in need of such treatment, comprising administering to the subject an effective amount of a modified IL-2 polypeptide or pharmaceutical composition as described herein. In some embodiments, the autoimmune disease is a T cell mediated autoimmune disease.In some embodiments, the inflammatory disease or disorder is inflammation (e.g., cartilage inflammation), autoimmune disease, atopic disease, paraneoplastic autoimmune disease, arthritis, rheumatoid arthritis (e.g., active), juvenile arthritis, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile psoriatic arthritis, psoriatic arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis , ankylosing spondylitis, juvenile ankylosing spondylitis, juvenile enterocolitis, reactive arthritis, juvenile reactive arthritis, Reiter's syndrome, juvenile Reiter's syndrome, juvenile dermatomyositis, juvenile scleroderma, juvenile vasculitis, enterocolitis, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myositis, polyarteritis, dermatomyositis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, rheumatic myalgia, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythema psoriasis, dermatitis, atopic dermatitis, dermatitis herpetiformis, Behçet's disease, alopecia, alopecia areata, alopecia totalis, atherosclerosis, lupus, Still's disease, myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, asthma, COPD, rhinosinusitis, rhinosinusitis with polyps, esophageal eosinophilic hepatitis, eosinophilic hepatitis, Guillain-Barre disease, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, graft-versus-host disease, steroid-refractory chronic graft-versus-host disease, transplant rejection (e.g., kidney, lung, heart, skin, etc.), kidney damage, Hepatitis C induced vasculitis, spontaneous abortion, vitiligo, focal segmental glomerulosclerosis (FSGS), minimal change disease, membranous nephropathy, ANCA-associated glomerulonephropathy, membranoproliferative glomerulonephritis, IgA nephropathy, lupus nephritis, or a combination thereof.

[0175] In some embodiments, the inflammatory disease or disorder is a neuroinflammatory disorder. In some embodiments, the neuroinflammatory disorder is neuromyelitis optica spectrum disorder, multiple sclerosis, anti-myelin oligodendrocyte glycoprotein antibody disorder, autoimmune dermatitis, transverse myelitis, optic neuritis, or neurosarcoidosis. In some embodiments, the disease or disorder is amyotrophic lateral sclerosis.

[0176] V. Manufacturing method In one aspect, described herein is a method of making a modified IL-2 polypeptide. In another aspect, described herein is a method of making a modified IL-2 polypeptide, comprising synthesizing two or more fragments of a modified IL-2 polypeptide and ligating the fragments. In another aspect, described herein is a method of making a modified IL-2 polypeptide, comprising: a. synthesizing two or more fragments of a modified IL-2 polypeptide; b. ligating the fragments; and c. folding the ligated fragments. Examples of methods of synthesizing IL-2 polypeptides can be found, for example, at least in PCT Publication No. WO 2021140416, U.S. Patent Publication No. 20190023760, and Asahina et al., Angew. Chem. Int. Ed. 2015, 54, 8226-8230, each of which is incorporated herein by reference as if set forth in its entirety.

[0177] In some embodiments, two or more fragments of a modified IL-2 polypeptide are chemically synthesized. In some embodiments, two or more fragments of a modified IL-2 polypeptide are synthesized by solid phase peptide synthesis. In some embodiments, two or more fragments of a modified IL-2 polypeptide are synthesized on an automated peptide synthesizer.

[0178] In some embodiments, the modified IL-2 polypeptide is ligated from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptide fragments. In some embodiments, the modified peptide is ligated from two peptide fragments. In some embodiments, the modified IL-2 polypeptide is ligated from three peptide fragments. In some embodiments, the modified IL-2 polypeptide is ligated from four peptide fragments. In some embodiments, the modified IL-2 polypeptide is ligated from 2 to 10 peptide fragments.

[0179] In some embodiments, two or more fragments of the modified IL-2 polypeptide are ligated together. In some embodiments, three or more fragments of the modified IL-2 polypeptide are ligated in a sequential manner. In some embodiments, three or more fragments of the modified IL-2 polypeptide are ligated in a one-pot reaction.

[0180] In some embodiments, the ligated fragments are folded. In some embodiments, folding comprises forming one or more disulfide bonds in the modified IL-2 polypeptide. In some embodiments, the ligated fragments are subjected to a folding step. In some embodiments, the ligated fragments are folded using methods known in the art. In some embodiments, the ligated or folded polypeptide is further modified by attaching one or more polymers thereto. In some embodiments, the ligated or folded polypeptide is further modified by PEGylation.

[0181] In some embodiments, the modified IL-2 polypeptide is synthetic.

[0182] In some embodiments, the modified IL-2 polypeptide is recombinant. In one aspect, a host cell comprising a modified IL-2 polypeptide is described herein. In some embodiments, the host cell is a prokaryotic or eukaryotic cell. In some embodiments, the host cell is a mammalian cell, an avian cell, and an insect cell. In some embodiments, the host cell is a CHO cell, a COS cell, or a yeast cell.

[0183] In one aspect, described herein is a method of producing a modified IL-2 polypeptide, comprising expressing the modified IL-2 polypeptide in a host cell. In some embodiments, the host cell is a prokaryotic or eukaryotic cell. In some embodiments, the host cell is a mammalian cell, an avian cell, or an insect cell. In some embodiments, the host cell is a CHO cell, a COS cell, or a yeast cell. VI. SEQ ID NO:

[0184] [Table 6-1]

[0185] [Table 6-2]

[0186] [Table 6-3]

[0187] [Table 6-4]

[0188] [Table 6-5]

[0189] [Table 6-6]

[0190] [Table 6-7]

[0191] [Table 6-8]

[0192] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made therein without departing from the spirit and scope of the present disclosure as defined in the appended claims.

[0193] The present disclosure is further illustrated in the following examples, which are provided for purposes of illustration only and are not intended to limit the disclosure in any way. EXAMPLES

[0194] Example 1: Synthesis of modified IL-2 polypeptides - general procedure Preparation of IL-2 linear protein (representative protocol) General Strategy: The modified IL-2 polypeptides described herein, for example, modified IL-2 polypeptides having the amino acid sequence of SEQ ID NO:3, or any of SEQ ID NOs:3-8 or 40-43, or synthetic versions of any one of SEQ ID NOs:9-39, or modified IL-2 polypeptides described elsewhere herein, can be synthesized by ligating individual peptide segments prepared by solid phase peptide synthesis (SPPS). Individual peptides were synthesized on an automated peptide synthesizer using the methods described below.

[0195] Materials and solvents: Fmoc-amino acids with side chain protecting groups suitable for Fmoc-SPPS, resins polyethylene glycol derivatives and reagents used for peptide functionalization were commercially available and used without further purification. HPLC grade CH3CN was used for analytical and preparative RP-HPLC purification.

[0196] Loading of amine-based resins with protected keto acid derivatives (segments 1-3): 5 g of Rink-amide MBHA or ChemMatrix resin (1.8 mmol scale) was swollen in DMF for 30 min. Fmoc deprotection was performed by treating the resin twice with 20% piperidine in DMF (v / v) for 10 min at room temperature, followed by several washes with DMF. Fmoc-AA protected α-keto acid (1.8 mmol, 1.00 equiv) was dissolved in 20 mL of DMF and preactivated with HATU (650 mg, 1.71 mmol, 0.95 equiv) and DIPEA (396 μL, 3.6 mmol, 2.00 equiv). The reaction mixture was added to the swollen resin. The reaction was allowed to proceed with gentle stirring at room temperature for 6 h. The resin was rinsed thoroughly with DMF. The resin was capped of unreacted amines by adding a solution of acetic anhydride (1.17 mL) and DIPEA (2.34 mL) in DMF (20 mL). The reaction was allowed to proceed at room temperature for 15 min under gentle agitation. The resin was rinsed thoroughly with DCM followed by diethyl ether and dried. The loading of the resin was determined to be 0.25 mmol / g by UV quantification of dibenzofulvene.

[0197] [Table 7]

[0198] Loading of Wang resin (segment 4) with Fmoc-Thr(tBu)-OH: Preloading of Fmoc-Thr-OH was performed on Wang resin. 4 g of resin (loading: 0.56 mmol / g, 2.24 mmol scale) was swollen in DMF for 15 min. The resin was treated with 20% (v / v) piperidine in DMF for 20 min at room temperature. The resin was washed several times with DMF. Fmoc-Thr(tBu)-OH (638 mg, 1.68 mmol, 0.75 equiv) and HATU (638 mg, 1.68 mmol, 0.75 equiv) were dissolved in DMF (12 mL). Preactivation was performed by addition of DIPEA (585 μL, 3.36 mmol, 1.5 equiv) for 3 min at room temperature. The reaction mixture was added to the swollen resin. The reaction was allowed to proceed overnight at room temperature with gentle stirring. The resin was rinsed thoroughly with DMF. Capping of unreacted amines on the resin was initiated by adding a solution of acetic anhydride (1.27 mL) and DIPEA (2.34 mL) in DMF (12 mL). The reaction was allowed to proceed for 15 min at room temperature under gentle agitation. The resin was rinsed thoroughly with DCM and dried. The loading of the resin was measured (0.34 mmol / g).

[0199] Solid-phase peptide synthesis (SPPS): Peptide segments were synthesized on an automated peptide synthesizer using Fmoc-SPPS chemistry. The following Fmoc-amino acids with side-chain protecting groups were used: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH. H, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Nle-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc or Boc-Opr-OH (Opr=5-(S)-oxaproline). When necessary, Fmoc-pseudoproline dipeptide was incorporated into the synthesis. Fmoc deprotection was performed with 20% piperidine in DMF (2 × 8 min) or 25% piperidine in DMF containing 0.1 M Cl-HOBt (2 × 8 min) or 20% piperidine in DMF containing 0.1 M Cl-HOBt (2 × 8 min) and monitored by UV at 304 nm with a feedback loop to ensure complete Fmoc removal. Coupling was performed at room temperature or at 50 °C with Fmoc-amino acids (3.0-5.0 equiv. relative to resin substitution), HCTU or HATU (2.9-4.9 equiv.) as coupling reagents, and DIPEA or NMM (6-10 equiv.) in DMF. After 3 min preactivation, the solution containing the reagents was added to the resin and allowed to react for 30 min or 2 h depending on the amino acid. In some cases, double bonds were required. In some cases, the resin was treated with 20% acetic anhydride in DMF to cap unreacted free amines.

[0200] Resin cleavage of peptides and side chain deprotection: Upon completion of peptide synthesis, the peptides were cleaved from the resin using a cleavage cocktail at room temperature for 2 hours. The resin was filtered off and the filtrate was concentrated, treated with cold diethyl ether, triturated and centrifuged. The ether layer was carefully decanted and the residue was resuspended in diethyl ether, triturated and centrifuged. The ether wash was repeated twice. The resulting crude peptides were dried under vacuum and stored at -20°C. An aliquot of the resulting solid was solubilized in 1:1 CH3CN / H2O containing 0.1% TFA (v / v) and analyzed by analytical RP-HPLC using a C18 column (4.6 x 150 mm) at 60°C. The molecular weight of the products was identified using MALDI-TOF or LC-MS.

[0201] Ligation and photodeprotection of IL-2 segments 1 and 2: IL-2 Seg1 (1.2 equiv.) and IL-2 Seg2 (1 equiv.) were dissolved in DMSO:H2O (9:1, v / v) containing 0.1 M oxalic acid (20 mM peptide concentration) and reacted at 60 °C for 22 h. The ligation vial was protected from light by wrapping in aluminum foil. The progress of the KAHA ligation was monitored by HPLC using a C18 column (4.6 × 150 mm) at 60 °C with a gradient of 5 to 95% CH3CN in 7 min using CH3CN / H2O containing 0.1% TFA as the mobile phase. After completion of the ligation, the mixture was diluted with CH3CN / H2O (1:1) containing 0.1% TFA and irradiated at 365 nm wavelength for 1 h. Completion of the photolysis reaction was confirmed by injecting a sample into the HPLC using the method described above. The solution was then purified by preparative HPLC.

[0202] Ligation and Fmoc deprotection of IL-2 segments 3 and 4: IL2-Seg3 (1.2 equiv.) and IL2-Seg4 (1 equiv.) were dissolved in DMSO / H2O (9.8:0.2) containing 0.1 M oxalic acid (15 mM) and reacted at 60° C. for 20 h. The progress of the KAHA ligation was monitored by HPLC using a C18 column (4.6×150 mm) at 60° C. with a gradient of 30 to 70% CH3CN in 7 min using CH3CN / H2O containing 0.1% TFA as the mobile phase. After completion of the ligation, the reaction mixture was diluted with DMSO (6 mL), 5% diethylamine (300 μL) was added, and the reaction mixture was shaken at room temperature for 7 min. To prepare the sample for purification, it was diluted with DMSO (4 mL) containing TFA (300 μL).

[0203] Final ligation: IL2-Seg12 (1.2 equiv.) and IL2-Seg34 (1 equiv.) were dissolved in DMSO / H2O (9:1) or (9.8:0.2) containing 0.1 M oxalic acid (15 mM peptide concentration) and the ligation was allowed to proceed for 24 h at 60 °C. The progress of the KAHA ligation was monitored by analytical HPLC using a C18 column (4.6 × 250 mm) and CH3CN / H2O containing 0.1% TFA as the mobile phase at 60 °C with a gradient of 30 to 95% CH3CN in 14 min. After completion of the ligation, the reaction mixture was diluted with DMSO, followed by further dilution with a mixture of (1:1) CH3CN:H2O containing 0.1% TFA (7 mL). Samples were purified by injection into a preparative HPLC.

[0204] Acm deprotection: IL2 linear protein with 2xAcm was dissolved in AcOH / H2O (1:1) (0.25 mM protein concentration) and AgOAc (1% m / v) was added to the solution. The mixture was protected from light and shaken at 50°C for 2.5 h. After completion of the reaction as confirmed by HPLC, the sample was diluted with CH3CN:H2O (1:1) containing 0.1% TFA and purified by preparative HPLC.

[0205] Peptide purification: Peptide segments, ligated peptides and linear proteins were purified by RP-HPLC. Different gradients were applied for different peptides. The mobile phases were MilliQ-HO with 0.1% TFA (v / v) (buffer A) and HPLC grade CHCN with 0.1% TFA (v / v) (buffer B). Preparative HPLC was performed on a (50×250 mm) or C18 column (50×250 mm) at a flow rate of 40 mL / min at 40° C. or 60° C.

[0206] Peptide characterization: Peptide segments, ligated peptides and linear proteins were analyzed by RP-HPLC. The mobile phases were MilliQ-H2O with 0.1% TFA (v / v) (buffer A) and HPLC grade CH3CN with 0.1% TFA (v / v) (buffer B). Analytical HPLC was performed on a C4 column (3.6 μm, 150 × 4.6 mm) at room temperature or a C18 column (3.6 μm, 150 × 4.6 mm) at 60 °C with a flow rate of 1 mL / min. Peptides and proteins were characterized by high-resolution Fourier transform mass spectrometry (FTMS) using a SolariX (9.4 T magnet) spectrometer (Bruker, Billerica, USA) equipped with a dual ESI / MALDI-FTICR source, using 4-hydroxy-α-cyanocinnamic acid (HCCA) as matrix.

[0207] Example 2: Composition A and synthesis of the A1 variant of IL-2 (SEQ ID NO:3) Synthesis of IL-2(1-39)-Leu-α-keto acid of composition A (segment 1A)

[0208] [ka] Peptide synthesis: IL2(1-39)-Leu-α-keto acid segment 1A (see residues 1-40 of SEQ ID NO:3) was synthesized on a 0.2 mmol scale on Rink-Amide MBHA resin preloaded with Fmoc-Leu-protected-α-keto acid (0.8 g) with a substitution capacity of about 0.25 mmol / g. Automated Fmoc-SPPS of segment 1A was performed according to the general procedure "Solid Phase Peptide Synthesis (SPPS)". The insertion of the conjugation handle was performed as follows: The initial manual coupling reaction was performed at room temperature for 30 min by adding glutaric anhydride (CAS RN108-55-4, 114.10 mg, 5 eq.) and DIPEA (242 μL, 7 eq.) in DMF to the resin. Coupling with commercially available O-(2-aminoethyl)-O'-(2-azidoethyl)nonaethylene glycol (compound 2,421 mg, equiv.) in DMF was then carried out at room temperature for 3 h by adding DMF containing DIPEA (276 μL, 8 equiv.) and HATU (300 mg, 3.95 equiv.) to the resin. The resin was washed with DCM and dried under vacuum. The mass of the peptidyl resin after drying was 1.6 g. The crude peptide was precipitated according to the procedure in "Resin cleavage and side chain deprotection of peptide" using a cocktail of 95:2.5:2.5 TFA / DODT / H2O v / v / v (10 mL / g resin) for 2.0 h at room temperature.

[0209] [ka]

[0210] Purification: C18 column (5 μm, 50 × 250 mm), flow rate 40 mL / min at 60 °C, gradient: 30 to 80% B in 25 min. Fractions containing purified product were pooled and lyophilized to give segment 1A as a white solid with 97% purity. The isolated yield based on resin loading was 260 mg (25%). HRMS (ESI): C 228 H 394 N 64 O 72 , average isotope calculated value 5182.9193 Da [M+H] + , Measured value: 5182.9111Da [M+H] +.

[0211] Synthesis of Opr-IL2(42-69) photoprotected Leu-α-keto acid of composition A (segment 2A)

[0212] [ka] Peptide synthesis: Opr-IL2(42-69)-Leu-photoprotected-α-keto acid segment 2A (see 41-70 in SEQ ID NO:3) was synthesized on a 0.2 mmol scale on Rink-Amide MBHA resin preloaded with Fmoc-Leu-photoprotected-α-keto acid (0.8 g) at a substitution capacity of approximately 0.25 mmol / g. Automated Fmoc-SPPS of segment 2A was performed according to the general procedure "Solid-phase peptide synthesis (SPPS)". The resin was washed with DCM and dried under vacuum. The mass of the peptidyl resin after drying was 1.8 g. The crude peptide was precipitated according to the procedure "Resin cleavage and side chain deprotection of peptide" using a cocktail of 95:2.5:2.5 TFA / DODT / H2O v / v / v (15 mL / g resin) for 2.0 h at room temperature.

[0213] Purification: C18 column (5 μm, 50 × 250 mm), flow rate 40 mL / min at 60 °C, gradient: 10 to 60% B in 30 min. Fractions containing purified product were pooled and lyophilized to give segment 2A as a white solid with 97% purity. The isolated yield based on resin loading was 203 mg (20%). HRMS (ESI): C 184 H 286 N 40 O 52 S, calculated average isotope: 3922.0742 Da [M+H] +, observed: 3922.0680 Da [M+H] + .

[0214] Synthesis of Fmoc-OprIL2(72-102)-Phe-α-keto acid of composition A (segment 3A)

[0215] [ka] Peptide synthesis: Fmoc-OprIL2(72-102)-Phe-α-keto acid segment 3A (see residues 71-103 of SEQ ID NO:3) was synthesized on a 0.2 mmol scale on Rink-Amide ChemMatrix resin preloaded with Fmoc-Phe-photoprotected α-keto acid (0.8 g) with a substitution capacity of about 0.286 mmol / g. Automated Fmoc-SPPS of segment 3A was performed according to the general procedure "Solid-Phase Peptide Synthesis (SPPS)". The resin was washed with DCM and dried under vacuum. The mass of the peptidyl resin after drying was 2.17 g. The crude peptide was precipitated according to the procedure "Resin cleavage and side chain deprotection of peptide" using a cocktail of 95:2.5:2.5 TFA / DODT / H2O v / v / v (10 mL / g resin) for 2.0 h at room temperature.

[0216] Purification: C18 column (5 μm, 50 × 250 mm), flow rate 40 mL / min at 40 °C, two-step gradient: 10 to 30% B in 10 min, followed by 30 to 80% B in 30 min. Fractions containing purified product were pooled and lyophilized to give segment 3A as a white solid with 98% purity. The isolated yield based on resin loading was 200 mg (17.6%). HRMS (ESI): C 184 H 283 N 45 O 53 , average isotope calculated value 3973.0891 Da [M+H] + , Measured value: 3973.0995Da [M+H] + .

[0217] Synthesis of IL-2Opr-IL2(105-133) of Composition A (Segment 4A)

[0218] [ka] Peptide synthesis: Opr-IL2(105-133) segment 4A (see residues 104-133 of SEQ ID NO:3) was synthesized on a 0.1 mmol scale on Wang resin preloaded with Fmoc-Thr-OH (0.294 g) with a substitution capacity of approximately 0.34 mmol / g. Automated Fmoc-SPPS of segment 4A was performed according to the general procedure "Solid-phase peptide synthesis (SPPS)". The resin was washed with DCM and dried under vacuum. The mass of the peptidyl resin after drying was 725 mg. The crude peptide was precipitated according to the procedure "Resin cleavage and side chain deprotection of peptide" using a cocktail of 92.5:2.5:2.5:2.5 TFA / TIPS / DODT / H2O v / v / v / v / v (10 mL / g resin) for 2 h at room temperature.

[0219] Purification: C18 column (5 μm, 50×250 mm), flow rate 40 mL / min at 40° C., gradient: 10 to 50% B in 40 min. Fractions containing purified product were pooled and lyophilized to give segment 4A as a white solid with 90.8% purity. The isolated yield based on resin loading was 40 mg (8%). HRMS (ESI): C 158 H 241 N 37 O 52 S, average isotope calculated value 1175.2449 Da [M+H] +3 , Measured value: 1175.2440 [M+H] +3 .

[0220] KAHA Ligation to Prepare IL2-Seg12 of Composition A (Segment 12A)

[0221] [ka] Ligation and photodeprotection: Segment 12A was obtained following the general procedure “Ligation and photodeprotection of IL-2 segments 1 and 2” by dissolving 34 mg (6.56 μmol, 1.1 equiv.) of segment 1A and 19 mg (4.9 μmol, 1.0 equiv.) of segment 2A in 241 μL of 9.5:0.5 v / v DMSO / HO solution containing 0.1 M oxalic acid.

[0222] Purification: C18 column (5 μm, 50 × 250 mm), flow rate 40 mL / min at 40 °C, two-step gradient: 10 to 40% B in 5 min, followed by 40 to 70% B in 30 min. Fractions containing purified product were pooled and lyophilized to give segment 12A as a white solid with 98% purity. The isolated yield was 55% (25.5 mg). HRMS (ESI): C 400 H 667 N 103 O 119 S, calculated average isotope: 8855.2806Da [M+H] + , Measured value: 8855.9008Da [M+H] + .

[0223] KAHA Ligation to Prepare IL2-Seg34 of Composition A (Segment 34A)

[0224] [ka] Ligation: Following the general procedure "Ligation of IL-2 segments 3 and 4 and Fmoc deprotection", 69 mg (17.5 μmol, 1.1 equiv.) of segment 3A and 59 mg (16.6 μmol, 1.0 equiv.) of segment 4A were dissolved in 1100 μL of 9.9:0.1 DMSO / HO v / v containing 0.1 M oxalic acid to give segment 34A.

[0225] Purification: C18 column (5 μm, 50×250 mm), flow rate 40 mL / min at 40° C., gradient: 20 to 60% B in 40 min. Fractions containing purified product were pooled and lyophilized to give segment 34A as a white solid with 95% purity. The isolated yield was 33% (42.3 mg). HRMS (ESI): C 326 H 514 N 82 O 101 S, average isotope calculated value 7229.7437 Da [M+H] + , Measured value: 7229.7618Da [M+H] + .

[0226] Final KAHA ligation to prepare IL2 linear protein of composition A (segment 1234A)

[0227] [ka] Ligation: Segment 1234A was obtained following the general procedure "Final Ligation" in which 45 mg (5.1 μmol, 1.2 eq.) of segment 12A and 31 mg (16.6 μmol, 1.0 eq.) of segment 34A were dissolved in 220 μL of 9.5:0.5 DMSO / H2O v / v containing 0.1 M oxalic acid.

[0228] Purification: C18 column (5 μm, 50 × 250 mm), flow rate 40 mL / min at 40 °C, gradient: 30 to 80% B in 30 min. Fractions containing purified product were pooled and lyophilized to give Acm protected segment 1234A as a white solid with 95% purity. The isolated yield was 26% (18 mg).

[0229] Acm deprotection: Deprotection of cysteine ​​residues was carried out following the general procedure "Acm deprotection" using 18 mg of Acm protected segment 1234A as starting material.

[0230] Purification: C18 column (5 μm, 20 × 250 mm), flow rate 10 mL / min at 40 °C, two-step gradient: 10 to 30% B in 5 min, followed by 30 to 95% B in 20 min. Fractions containing purified product were pooled and lyophilized to give segment 1234A as a white solid with 97% purity. The isolated yield was 17% (11.6 mg). HRMS (ESI): C 719 H 1171 N 183 O 216 S2, calculated average isotope: 15898.5963Da [M+H] + , Measured value: 15898.6118Da [M+H] + .

[0231] Folding of IL-2 linear protein of composition A Reconstitution of linear protein: IL2-Seg1234-A linear protein (11.7 mg, 0.736 μmol) was dissolved in 6 M aqueous Gu·HCl containing 0.1 M Tris and 30 mM reduced glutathione (15 μM protein concentration), and the mixture was gently shaken at 50 °C for 2 h.

[0232] Folding of linear reconstituted protein (Method 1): After completion of the reconstitution reaction, the sample was cooled to room temperature and diluted with 0.1 M Tris and 1.5 mM oxidized glutathione, pH 8.0 (5 μM protein concentration). Folding was allowed to proceed for 20 h at room temperature. The sample was then acidified to pH 3 with TFA and purified by preparative HPLC using a C4 column (20×250 mm) at a flow rate of 10.0 mL / min, using CH3CN / H2O with 0.1% TFA (v / v) as the mobile phase with a two-step gradient from 5 to 40 to 95% acetonitrile with 0.1% TFA in 60 min at room temperature. Fractions containing the product were pooled and lyophilized to obtain the pure folded protein composition A as a white powder with a purity of 98% (2.2 mg, 19% yield for folding and purification steps). The purity and identity of the pure protein were confirmed by analytical RP-HPLC, MALDI-TOF and analytical size exclusion. HRMS(ESI):C 719 H 1169 N 183 O 216 S2, calculated average isotope: 15896.5806Da [M+H] + , Measured value: 15896.6322Da [M+H] +

[0233] Synthesis of IL-2 Protein Composition A1 IL-2 Composition A-fold protein (39.76 mg, 1 eq.) was first dissolved in 8 mL of 10 mM sodium acetate buffer, 8.4% sucrose, 0.02% polysorbate 80 pH 5.0. 22 mL of 50 mM sodium acetate buffer pH 5.0 and 30 kDa DCO-polyethylene glycol polymer (392.05 mg, 5 eq.) were added to the solution and the reaction was mixed gently for 17 hours at 25° C. The reaction mixture was loaded onto a HiTrap Capto S ImpAct column (5 mL) in 10 mL portions and purified with a 20 CV gradient from 50 mM sodium acetate buffer pH 5.0 to 50 mM sodium acetate buffer containing 1 M NaCl at a flow rate of 2.5 mL / min. Fractions containing IL-2 Composition A1 PEGylated protein were pooled together and dialyzed against 10 mM sodium acetate buffer, 8.4% sucrose, 0.02% polysorbate 80 (pH 5.0) to yield 12.26 mg protein fraction IL-2 Composition A1 PEGylated protein as measured by BCA (31% yield for PEGylation and purification steps). Purity and identity of the pure PEGylated protein were confirmed by analytical RP-HPLC, MALDI-TOF and analytical size exclusion. Example 3: Composition B and synthesis of the B1 variant of IL-2 In this variant, except for segment 3, all other segments are the same as those used in composition A.

[0234] Synthesis of Fmoc-OprIL2(72-102)-Phe-α-keto acid of composition B (segment 3B) Fmoc-OprIL2(72-102)-Phe-α-keto acid segment 3B (see residues 71-103 of SEQ ID NO:4) was synthesized by automated Fmoc-SPPS synthesis similar to the procedure described for the synthesis of segment 3A to give segment 3B as a white solid in >98% purity. The isolated yield based on resin loading was 18% (200 mg). HRMS (ESI): C 184 H 284 N 46 O 52 , average isotope calculated value 3972.1051 Da [M+H] +, Measured value: 3972.1054 Da [M+H] + .

[0235] Synthesis of IL2-Seg34 of Composition B by KAHA Ligation Ligation: Following the general procedure "Ligation of IL-2 Segments 3 and 4 and Fmoc deprotection", 37 mg (9.3 μmol, 1.2 equiv.) of Segment 3B and 27 mg (7.8 μmol, 1.0 equiv.) of Segment 4A were dissolved in 517 μL of 9.8:0.2 DMSO / HO v / v containing 0.1 M oxalic acid to give Segment 34B.

[0236] Purification: C18 column (5 μm, 50 × 250 mm), flow rate 40 mL / min at 40 °C, two-step gradient: 10 to 40% %B in 5 min, followed by 40 to 80% %B in 35 min. Fractions containing purified product were pooled and lyophilized to give segment 34B as a white solid with >99% purity. The isolated yield was 22% (12.6 mg). HRMS (ESI): C 326 H 515 N 83 O 100 S, average isotope calculated value 7228.7597 Da [M+H] + , Measured value: 7228.7738Da [M+H] + .

[0237] Final KAHA ligation to prepare IL2 linear protein of composition B (segment 1234B) Ligation: Segment 1234B was obtained following the general procedure "Final Ligation" in which 34 mg (3.8 μmol, 1.2 eq.) of segment 12A and 23 mg (3.2 μmol, 1.0 eq.) of segment 34B were dissolved in 214 μL of 9.5:0.5 DMSO / HO v / v containing 0.1 M oxalic acid.

[0238] Purification: C18 column (5 μm, 50×250 mm), flow rate 40 mL / min at 40° C., two-step gradient: 10 to 40%% B in 5 min, followed by 40 to 80%% B in 35 min. Fractions containing purified product were pooled and lyophilized to give Acm protected segment 1234B as a white solid in 96% purity. The isolated yield was 52% (26.8 mg). HRMS (ESI): C 725 H 1182 N 186 O 217 S2, average isotope calculated value 16039.6865 Da [M+H] + , Measured value: 16039.6389Da [M+H] + .

[0239] Acm deprotection: Deprotection of cysteine ​​residues was carried out following the general procedure "Acm deprotection" using 26.8 mg of Acm protected segment 1234B as starting material.

[0240] Purification: C18 column (5 μm, 20 × 250 mm), flow rate 10 mL / min at 40 °C, two-step gradient: 10 to 30% B in 5 min, followed by 30 to 95% B in 30 min. Fractions containing purified product were pooled and lyophilized to give segment 1234B as a white solid with 97% purity. The isolated yield was 55% (14.5 mg). HRMS (ESI): C 719 H 1172 N 184 O 215 S2, calculated average isotope: 15897.6117Da [M+H] + , Measured value: 15897.6195Da [M+H] + .

[0241] Folding of IL-2 linear protein of composition B Reconstitution of linear protein: IL2-Seg1234-B linear protein (14.5 mg, 0.913 μmol) was dissolved in aqueous 6 M Gu·HCl containing 0.1 M Tris and 30 mM reduced glutathione (61 mL, 15 μM protein concentration), and the mixture was gently shaken at 50 °C for 2 h.

[0242] Folding of the linear reconstituted protein: After completion of the reconstitution reaction, the sample was cooled to room temperature and diluted with 0.1 M Tris and 1.5 mM oxidized glutathione, pH 8.0 (122 mL, 5 μM protein concentration). Folding was allowed to proceed for 20 h at room temperature. The sample was then acidified to pH 3 with TFA and purified by preparative HPLC on a C4 column (20×250 mm) kept at room temperature with a two-step gradient from 10 to 30% B in 5 min, followed by 30 to 95% B in 30 min, at a flow rate of 10.0 mL / min. Fractions containing the product were pooled and lyophilized to give pure folded IL2-Seg1234-B protein as a white powder with a purity of >98%. (3.9 mg, 27% yield for folding and purification steps). HRMS (ESI): C 719 H 1170 N 184 O 215 S2, calculated average isotope: 15895.5966Da [M+H] + , Measured value: 15895.5669Da [M+H] + .

[0243] Synthesis of IL-2 Protein Composition B1 To a solution of IL2-Seg1234-B (2 mg, 0.126 μmol, 1 equiv.) in 1:1 CH3CN:H2O (50 mM protein concentration) was added 30 kDa DBCO-polyethylene glycol polymer (11.7 mg, 0.403 μmol, 3.2 equiv.) and the reaction was mixed gently for 20 h at 25 °C. The reaction mixture was diluted with 1:1 CH3CN / H2O + 0.1% TFA and purified by preparative HPLC using a C4 column (20 × 250 mm) with a two-step gradient from 10 to 30% B in 5 min, followed by 30 to 95% B in 30 min, flow rate: 10.0 mL / min. Fractions containing PEGylated IL2-Seg1234-B1 protein were pooled together and lyophilized to yield 2.5 mg of IL2-Seg1234-B1 PEGylated protein as a white powder with 98% purity. (29% yield for the PEGylation and purification steps). The purity and identity of the pure PEGylated protein was confirmed by analytical RP-HPLC, MALDI-TOF, SEC-HPLC and SDS-page. Example 4: Composition C and synthesis of the C1 variant of IL-2 In this variant, except for segment 2, all other segments are the same as those used in composition A.

[0244] Synthesis of IL-2 Fmoc-OprIL2(42-69)-Leu-α-keto acid of composition C (segment 2C) Opr-IL2(42-69)-Leu-photoprotected a-keto acid segment 2C (see residues 41-70 of SEQ ID NO:5) was synthesized by automated Fmoc-SPPS synthesis similar to the procedure described for the synthesis of segment 2A to give segment 2C as a white solid in 94% purity. The isolated yield based on resin loading was 19.7% (153.7 mg). MALDI-TOF was used to confirm that the desired product mass was obtained.

[0245] Synthesis of IL2-Seg12 of Composition C by KAHA Ligation (Segment 12C) Ligation and photodeprotection: Segment 12C was obtained following the general procedure “Ligation and photodeprotection of IL-2 segments 1 and 2” by dissolving 90 mg of segment 1A (17.4 μmol, 1.1 equiv.) and 56 mg (14.5 μmol, 1.0 equiv.) of segment 2C in 1157 μL of 9.5:0.5 v / v DMSO / HO solution containing 0.1 M oxalic acid.

[0246] Purification: C18 column (5 μm, 50 × 250 mm), flow rate 40 mL / min at 40 °C, two-step gradient: 10 to 40% B in 5 min, followed by 40 to 70% B in 30 min. Fractions containing purified product were pooled and lyophilized to give segment 12C as a white solid with >99% purity. The isolated yield was 36% (46.7 mg).

[0247] Final KAHA ligation to prepare IL2 linear protein (segment 1234C) of composition C Ligation: Segment 1234C was obtained following the general procedure "Final Ligation" in which 46.7 mg (5.24 μmol, 1.2 eq.) of segment 12C and 32 mg (4.41 μmol, 1.0 eq.) of segment 34A were dissolved in 683 μL of 9.5:0.5 DMSO / HO v / v containing 0.1 M oxalic acid.

[0248] Purification: C18 column (5 μm, 50×250 mm), flow rate 40 mL / min at 60° C., gradient: 30 to 80% %B in 30 min. Fractions containing purified product were pooled and lyophilized to give Acm protected segment 1234C as a white solid. The isolated yield was 34% (24.5 mg).

[0249] Acm deprotection: Deprotection of cysteine ​​residues was carried out according to the general procedure "Acm deprotection" using 24.5 mg of Acm protected segment 1234C as starting material.

[0250] Purification: C18 column (5 μm, 20 × 250 mm), flow rate 10 mL / min at 40 °C, two-step gradient: 10 to 30% B in 5 min, followed by 30 to 95% B in 30 min. Fractions containing purified product were pooled and lyophilized to give segment 1234C as a white solid with 94% purity. The isolated yield was 69% (16.6 mg). HRMS (ESI): C 717 H 1167 N 183 O 216 S2, calculated average isotope: 15870.5650 Da [M+H] + , Measured value: 15870.6232 Da [M+H] + .

[0251] Folding of IL-2 linear protein of composition C Reconstitution of linear protein: IL2-Seg1234-C linear protein (16.6 mg, 1.04 μmol) was dissolved in aqueous 6 M Gu·HCl containing 0.1 M Tris and 30 mM reduced glutathione (69 mL, 15 μM protein concentration), and the mixture was gently shaken at 50 °C for 2 h.

[0252] Folding of the linear reconstituted protein: After completion of the translocation reaction, the sample was cooled to room temperature and diluted with 0.1 M Tris and 1.5 mM oxidized glutathione, pH 8.0 (140 mL, 5 μM protein concentration). Folding was allowed to proceed for 20 h at room temperature. The sample was then acidified to pH 3 with TFA and purified by preparative HPLC on a C4 column (20×250 mm) kept at room temperature with a two-step gradient from 10 to 30% B in 5 min, followed by 30 to 95% B in 30 min, at a flow rate of 10.0 mL / min. Fractions containing the product were pooled and lyophilized to give pure folded IL2-Seg1234-C protein as a white powder with a purity of >99% (3.4 mg, 20.5% yield for folding and purification steps). HRMS (ESI): C 717 H 1165 N 183 O 216 S2, calculated average isotope: 15868.5493Da [M+H] + , Measured value: 15868.5979Da [M+H] + .

[0253] Synthesis of IL-2 Protein Composition C1 To a solution of IL2-Seg1234-C (2 mg, 1 equiv.) in 1:1 CH3CN:H2O (50 μM protein concentration) was added 30 kDa DBCO-polyethylene glycol polymer (11.0 mg, 3 equiv.) and the reaction was mixed gently for 20 h at 25 °C. The reaction mixture was diluted with 1:1 CH3CN / H2O + 0.1% TFA and purified by preparative HPLC using a C4 column (20 × 250 mm) with a two-step gradient from 10 to 30% B in 5 min, followed by 30 to 95% B in 30 min, at a flow rate of 10.0 mL / min. Fractions containing PEGylated IL2-Seg1234-C1 protein were pooled together and lyophilized to give 1.4 mg of IL2-Seg1234-C1 PEGylated protein as a white powder with a purity of >99%. (25% yield for PEGylation and purification steps). The purity and identity of the pure PEGylated protein was confirmed by analytical RP-HPLC and MALDI-TOF. Example 5: Synthesis of Composition D and D1 Variants of IL-2 In this variation, except for segment 1, all other segments are the same as those used in composition B.

[0254] Synthesis of IL-2(1-39)-Leu-α-keto acid of composition D (segment 1D) IL2(1-39)-Leu-α-keto acid segment 1D (see residues 1-40 of SEQ ID NO:6) was synthesized by automated Fmoc-SPPS synthesis similar to the procedure described for the synthesis of segment 1A to give segment 1D as a white solid in 98% purity. The isolated yield based on resin loading was 20% (209 mg). MALDI-TOF was used to confirm that the desired product mass was obtained.

[0255] Synthesis of IL2-Seg12 of Composition D by KAHA Ligation (Segment 12D) Ligation and photodeprotection: Segment 12D was obtained following the general procedure “Ligation and photodeprotection of IL-2 segments 1 and 2” by dissolving 60 mg (11.7 μmol, 1.1 equiv.) of segment 1D and 38 mg (9.7 μmol, 1.0 equiv.) of segment 2A in 780 μL of 9.5:0.5 v / v DMSO / HO solution containing 0.1 M oxalic acid.

[0256] Purification: C18 column (5 μm, 50 × 250 mm), flow rate 40 mL / min at 40 °C, two-step gradient: 10 to 40% B in 5 min, followed by 40 to 70% B in 30 min. Fractions containing the purified product were pooled and lyophilized to give segment 12D as a white solid. The isolated yield was 54% (46.2 mg). HRMS (ESI): C 397 H 661 N 103 O 119 S, m / z calculated: 8812.8698 Da [M+H] + , Measured value: 8812.8833Da [M+H] + .

[0257] Final KAHA ligation to prepare IL2 linear protein of composition D (segment 1234D) Ligation: Segment 1234D was obtained following the general procedure "Final Ligation" where 35.2 mg (5.24 μmol, 1.2 eq.) of segment 12D and 26 mg (3.62 μmol, 1.0 eq.) of segment 34B were dissolved in 270 μL of 9.5:0.5 DMSO / HO v / v containing 0.1 M oxalic acid.

[0258] Purification: C18 column (5 μm, 50×250 mm), flow rate 40 mL / min at 60° C., gradient: 30 to 80% %B in 30 min. Fractions containing purified product were pooled and lyophilized to give Acm protected segment 1234D as a white solid. The isolated yield was 26% (15 mg).

[0259] Acm deprotection: Deprotection of cysteine ​​residues was carried out following the general procedure "Acm deprotection" using 15 mg (0.95 μmol) of Acm protected segment 1234D as starting material.

[0260] Purification: C18 column (5 μm, 20 × 250 mm), flow rate 10 mL / min at 40 °C, two-step gradient: 10 to 30% B in 5 min, followed by 30 to 95% B in 30 min. Fractions containing purified product were pooled and lyophilized to give segment 1234D as a white solid with 94% purity. The isolated yield was 80% (12 mg). HRMS (ESI): C 716 H 1166 N 184 O 215 S2, calculated average isotope: 15855.5653Da [M+H] + , Measured value: 15855.5300Da [M+H] + .

[0261] Folding of IL-2 linear protein of composition D Reconstitution of linear protein: IL2-Seg1234-D linear protein (9 mg, 0.568 μmol) was dissolved in aqueous 6 M Gu·HCl containing 0.1 M Tris and 30 mM reduced glutathione (38 mL, 15 μM protein concentration), and the mixture was gently shaken at 50 °C for 2 h.

[0262] Folding of the linear reconstituted protein: After completion of the translocation reaction, the sample was cooled to room temperature and diluted with 0.1 M Tris and 1.5 mM oxidized glutathione, pH 8.0 (76 mL, 5 μM protein concentration). Folding was allowed to proceed at room temperature for 44 h. The sample was then acidified to pH 3 with TFA and purified by preparative HPLC using a Shiseido ProteonAviC4 column (20×250 mm) with a two-step gradient from 10 to 30% B in 5 min, followed by 30 to 95% B in 30 min, at a flow rate of 10.0 mL / min and kept at room temperature. Fractions containing the product were pooled and lyophilized to give folded IL2-Seg1234-D as a white solid with a purity of >98% (0.3 mg, 3% yield for folding and purification steps).

[0263] Synthesis of IL-2 Protein Composition D1 To a solution of IL2-Seg1234-D (0.3 mg, 1 equiv.) in 1:1 CH3CN:H2O (50 μM protein concentration) was added 30 kDa DBCO-polyethylene glycol polymer (1.8 mg, 3.2 equiv.) and the reaction was mixed gently for 20 h at 25 °C. The reaction mixture was diluted with 1:1 CH3CN / H2O + 0.1% TFA and purified by preparative HPLC using a C4 column (20 × 250 mm) with a two-step gradient from 10 to 30% B in 5 min, followed by 30 to 95% B in 30 min, at a flow rate of 10.0 mL / min. Fractions containing PEGylated IL2-Seg1234-D1 protein were pooled together and lyophilized to give 0.1 mg of IL2-Seg1234-D1 PEGylated protein as a white powder with a purity of >98%. (11% yield for PEGylation and purification steps).

[0264] The purity and identity of the pure PEGylated protein was confirmed by analytical RP-HPLC and MALDI-TOF. Example 6: Synthesis of Composition E and E1 Variants of IL-2 In this variation, except for segment 1, all other segments are the same as those used in composition B.

[0265] Synthesis of IL-2(1-39)-Leu-α-keto acid of composition E (segment 1E) IL2(1-39)-Leu-α-keto acid segment 1E (see residues 1-40 of SEQ ID NO:7) was synthesized by automated Fmoc-SPPS synthesis similar to the procedure described for the synthesis of segment 1A to give segment 1E as a white solid in 97% purity. The isolated yield based on resin loading was 17% (180 mg). HRMS (ESI): C 225 H 388 N 64 O 72 , average isotope calculated value 5140.87247 Da [M+H] + , Measured value: 5141.8699Da [M+H] + .

[0266] Synthesis of IL2-Seg12 of Composition E by KAHA Ligation (Segment 12E) Ligation and photodeprotection: Segment 12E was obtained following the general procedure “Ligation and photodeprotection of IL-2 segments 1 and 2” by dissolving 60 mg (11.7 μmol, 1.1 equiv.) of segment 1E and 38 mg (9.7 μmol, 1.0 equiv.) of segment 2A in 780 μL of 9.5:0.5 v / v DMSO / HO solution containing 0.1 M oxalic acid.

[0267] Purification: C18 column (5 μm, 50 × 250 mm), flow rate 40 mL / min at 40 °C, two-step gradient: 10 to 40% B in 5 min, followed by 40 to 70% B in 30 min. Fractions containing the purified product were pooled and lyophilized to give segment 12E as a white solid. The isolated yield was 55% (33.4 mg). HRMS (ESI): C 397 H661 N 103 O 119 S, m / z calculated: 8812.8698 Da [M+H] + , Measured value: 8812.8833Da [M+H] + .

[0268] Final KAHA ligation to prepare IL2 linear protein of composition E (segment 1234E) Ligation: Segment 1234E was obtained following the general procedure "Final Ligation" in which 17.5 mg (1.98 μmol, 1.2 eq.) of segment 12E and 12 mg (1.66 μmol, 1.0 eq.) of segment 34B were dissolved in 256 μL of 9.5:0.5 DMSO / H2O v / v containing 0.1 M oxalic acid.

[0269] Purification: C18 column (5 μm, 50×250 mm), flow rate 40 mL / min at 60° C., gradient: 30 to 80% %B in 30 min. Fractions containing purified product were pooled and lyophilized to give Acm protected segment 1234E as a white solid. The isolated yield was 22% (6.6 mg). MALDI-TOF was used to confirm that the desired product mass was obtained.

[0270] Acm deprotection: Deprotection of cysteine ​​residues was carried out following the general procedure "Acm deprotection" using 6.6 mg (0.37 μmol) of Acm protected segment 1234E as starting material.

[0271] Purification: C18 column (5 μm, 20 × 250 mm), flow rate 10 mL / min at 40 °C, two-step gradient: 10 to 30% B in 5 min, followed by 30 to 95% B in 30 min. Fractions containing purified product were pooled and lyophilized to give segment 1234E as a white solid with 94% purity. The isolated yield was 98% (5.8 mg). HRMS (ESI): C 716 H 1166 N 184 O 215 S2, calculated average isotope: 15855.5653Da [M+H] +, Measured value: 15855.5479Da [M+H] + .

[0272] Folding of IL-2 linear protein of composition E Reconstitution of linear protein: IL2-Seg1234-E linear protein (5.8 mg, 0.366 μmol) was dissolved in aqueous 6 M Gu·HCl containing 0.1 M Tris and 30 mM reduced glutathione (24 mL, 15 μM protein concentration), and the mixture was gently shaken at 50 °C for 2 h.

[0273] Folding of the linear reconstituted protein: After completion of the translocation reaction, the sample was cooled to room temperature and diluted with 0.1 M Tris and 1.5 mM oxidized glutathione, pH 8.0 (48 mL, 5 μM protein concentration). Folding was allowed to proceed for 20 h at room temperature. The sample was then acidified to pH 3 with TFA and purified by preparative HPLC on a C4 column (20×250 mm) kept at room temperature with a two-step gradient from 10 to 30% B in 5 min, followed by 30 to 95% B in 30 min, at a flow rate of 10.0 mL / min. Fractions containing the product were pooled and lyophilized to give folded IL2-Seg1234-E as a white solid with a purity of >98% (0.6 mg, 10% yield for folding and purification steps).

[0274] Synthesis of IL-2 Protein Composition E1 To a solution of IL2-Seg1234-E (0.6 mg, 1 equiv.) in 1:1 CH3CN:H2O (50 μM protein concentration) was added 30 kDa DBCO-mPEG (3 mg, 2.7 equiv.) and the reaction was gently mixed for 20 h at 25 °C. The reaction mixture was diluted with 1:1 CH3CN / H2O + 0.1% TFA and purified by preparative HPLC using a Shiseido Proteonavi C4 column (20 × 250 mm) with a two-step gradient from 10 to 30% B in 5 min, followed by 30 to 95% B in 30 min, at a flow rate of 10.0 mL / min. Fractions containing PEGylated IL2-Seg1234-E1 protein were pooled together and lyophilized to give 0.2 mg of IL2-Seg1234-E1 PEGylated protein as a white powder with a purity of >98%. (12% yield for PEGylation and purification steps). The purity and identity of the pure PEGylated protein was confirmed by analytical RP-HPLC and MALDI-TOF. Example 7: Synthesis of Composition F In this variation, except for segment 2, all other segments are the same as those used in composition B.

[0275] Synthesis of Opr-IL2(42-69)-Leu-photoprotected α-keto acid of composition F (segment 2F) Opr-IL2(42-69)-Leu-photoprotected α-keto acid segment 2F (see residues 1-40 of SEQ ID NO:8) was synthesized by automated Fmoc-SPPS synthesis similar to the procedure described for the synthesis of segment 2A to give segment 2F as a white solid in 99% purity. The isolated yield based on resin loading was 35% (269 mg). HRMS (ESI): C 181 H 280 N 40 O 52 S, m / z calculated: 3880.0273 Da [M+H] + , Measured value: 3880.0207Da [M+H] + .

[0276] Synthesis of IL2-Seg12 of Composition F by KAHA Ligation (Segment 12F) Ligation and photodeprotection: Segment 12F was obtained following the general procedure "Ligation and photodeprotection of IL-2 segments 1 and 2" where 34 mg (6.6 μmol, 1.1 equiv.) of segment 1A and 19 mg (4.9 μmol, 1.0 equiv.) of segment 2F were dissolved in 385 μL of 9.5:0.5 v / v DMSO / HO solution containing 0.1 M oxalic acid. Purification: C18 column (5 μm, 50 × 250 mm), flow rate 40 mL / min at 40 °C, two-step gradient: 10 to 40% B in 5 min, followed by 40 to 70% B in 30 min. Fractions containing purified product were pooled and lyophilized to give segment 12F as a white solid with 96% purity. The isolated yield was 59% (25.5 mg). MALDI-TOF was used to confirm that the desired product mass was obtained.

[0277] Final KAHA ligation for preparation of IL2 linear protein of composition F (segment 1234F) Ligation: Segment 1234F was obtained following the general procedure "Final Ligation" in which 25.5 mg (2.89 μmol, 1.2 eq.) of segment 12F and 17.5 mg (2.42 μmol, 1.0 eq.) of segment 34B were dissolved in 373 μL of 9.5:0.5 DMSO / HO v / v containing 0.1 M oxalic acid.

[0278] Purification: C18 column (5 μm, 50×250 mm), flow rate 40 mL / min at 60° C., gradient: 30 to 80% %B in 30 min. Fractions containing purified product were pooled and lyophilized to give Acm protected segment 1234F as a white solid. The isolated yield was 31% (12 mg). MALDI-TOF was used to confirm that the desired product mass was obtained.

[0279] Acm deprotection: Deprotection of cysteine ​​residues was carried out following the general procedure "Acm deprotection" using 12 mg (0.75 μmol) of Acm protected segment 1234F as starting material.

[0280] Purification: C18 column (5 μm, 20 × 250 mm), flow rate 10 mL / min at 40 °C, two-step gradient: 10 to 30% B in 5 min, followed by 30 to 95% B in 30 min. Fractions containing purified product were pooled and lyophilized to give segment 1234F as a white solid with 94% purity. The isolated yield was 73% (8.7 mg). HRMS (ESI): C 716 H 1166 N 184 O 215 S2, calculated average isotope: 15855.5653Da [M+H] + , Measured value: 15855.6061Da [M+H] + .

[0281] Folding of IL-2 linear protein of composition F Reconstitution of linear protein: IL2-Seg1234-F linear protein (8.7 mg, 0.549 μmol) was dissolved in aqueous 6 M Gu·HCl containing 0.1 M Tris and 30 mM reduced glutathione (37 mL, 15 μM protein concentration), and the mixture was gently shaken at 50 °C for 2 h.

[0282] Folding of the linear reconstituted protein: After completion of the translocation reaction, the sample was cooled to room temperature and diluted with 0.1 M Tris and 1.5 mM oxidized glutathione, pH 8.0 (80 mL, 5 μM protein concentration). Folding was allowed to proceed for 44 h at room temperature. The sample was then acidified to pH 3 with TFA and purified by preparative HPLC on a C4 column (20×250 mm) kept at room temperature with a two-step gradient from 10 to 30% B in 5 min, followed by 30 to 95% B in 30 min, at a flow rate of 10.0 mL / min. Fractions containing the product were pooled and lyophilized to obtain pure folded IL2-Seg1234-F (0.2 mg, 2% yield for folding and purification steps). The purity and identity of the pure folded protein were confirmed by analytical RP-HPLC and MALDI-TOF.

[0283] Example 8: Selective activation of STAT5 by unconjugated and PEGylated IL-2 variants Engagement of IL-2R results in phosphorylation of signal transduction and activator of transcription 5 (STAT5), which can be used as a readout to assess selectivity for T cell subsets. Primary panT cells were obtained from buffy coats of healthy donors by peripheral blood mononuclear cell (PBMC) purification using Ficoll gradient centrifugation, followed by negative isolation with magnetic beads, then cryopreserved until further use. panT cells were thawed and incubated overnight in T cell medium (RPMI 10% FCS, 1% glutamine, 1% NEAA, 25 μM βMeoH, 1% sodium pyruvate), followed by two washes with PBS. Cells were resuspended in PBS and distributed at 200,000 cells per well, followed by incubation with aldesleukin, unconjugated IL-2 polypeptide (composition A), PEGylated IL-2 polypeptide (composition A1) or another indicated variant provided herein for 40 minutes at 37° C. / 5% CO2. After incubation, cells were fixed and permeabilized using a transcription factor phosphobuffer kit, followed by surface and intracellular immunostaining for CD4, CD8, CD25, FoxP3 and pSTAT5, allowing identification of cell subsets and measurement of STAT5 phosphorylation levels. FACS measurements were performed using either a NovoCyte or Quanteon Flow Cytometer from Acea. Tregs were classified as CD4+CD25+FoxP3+ cells and Teffs as CD8+ T cells. The EC50 results of the STAT5 phosphorylation assay from the indicated variants in various immune cell types are shown in Table 6 below.

[0284] [Table 8-1]

[0285] [Table 8-2]

[0286] Both unconjugated (composition A) and PEGylated IL-2 polypeptides (composition A1) selectively activated STAT5 in Tregs (Figure 4B) and had little effect on STAT5 activation in Teff cells (Figure 4A). On the other hand, aldesleukin nonselectively activated STAT5 in both Treg and Teff cells. The potency of STAT5 activation for PEGylated IL-2 polypeptides (composition A1) was lower than that for unconjugated IL-2 polypeptides (composition A), suggesting that PEGylation results in a slight decrease in activity.

[0287] Furthermore, both unconjugated and PEGylated IL-2 polypeptides activated STAT5 in Tregs from mice and cynomolgus monkeys with potency comparable to that on human Tregs, thus demonstrating cross-reactivity to mouse and cynomolgus monkey IL-2R (Table 7).

[0288] [Table 9]

[0289] Example 9: Binding affinity of PEGylated IL-2 polypeptides (composition A1) to the α and β subunits of the IL-2 receptor The binding affinity of PEGylated IL-2 polypeptide (composition A1) and SEQ ID NO: 2 (aldesleukin) to IL-2R α and β subunits was measured using Biolayer Interferometry (BLI) technology. Biotinylated IL-R2α and β (R&D, catalog AVI10305-050 and AVI10459-050) were loaded separately onto a streptavidin biosensor SAX2 and the sensor was immersed in 1× Octet® Kinetic Buffer to set the baseline. The sensor was incubated in the analyte solution for 300 s and then incubated in Kinetic Buffer for 600 s to measure dissociation. Kd values ​​were calculated using Octet® Analysis studio. Composition A1 had a higher affinity for the α subunit than aldesleukin (2.37 nM vs. 12.23 nM, respectively), but binding to the β subunit was abolished (Figure 5). Thus, composition A1 represents an α-enhanced β-dead IL-2 polypeptide.

[0290] Example 10: Pharmacokinetic / pharmacodynamic study in mice for Composition A1 Single-dose pharmacokinetic / pharmacodynamic (PK / PD) studies were conducted in C57BL / 6 mice receiving five daily subcutaneous (sc) injections of 0.3 mg / kg protein equivalent aldesleukin or a single sc injection of 0.1 or 0.3 mg / kg PEGylated IL-2 polypeptide composition A1. Blood was sampled at various time points in K2EDTA, plasma was generated by centrifugation and stored at -80°C until freshly subjected to PK analysis and cell pellets were stained for flow cytometry analysis.

[0291] The cell pellet was treated with 1x Lyse / Fix buffer (BD Bioscience, 558050) for 10 min. After washing, the cells were stained with anti-CD3, anti-CD335 and anti-CD25 antibodies for 30 min at 4°C. The cells were then permeabilized with cold BD Perm Buffer III and stained with antibodies against Ki67, Siglec-F, CD4, CD8, FoxP3, CD62L, CD44 or pSTAT5. FACS (fluorescence-activated cell sorting) measurements were performed on a BD Fortessa X-20 flow cytometer. For each cell subset, the percentage of pSTAT5 positive cells, as well as the percentage of Ki67 positive cells, cell number and cell frequency were determined.

[0292] The concentration of Composition A1 in plasma was determined using a qualified human IL-2 LegendPlex bead assay (Biolegend, #740717, #740368, #740758). The PK data were subjected to non-compartmental PK analysis by using Phoenix WinNonlin software version 6.3. The linear / logarithmic trapezoidal rule was applied in obtaining the PK parameters. The PK profile of the modified IL-2 polypeptide (Figure 6) peaked at 6 hours and the concentration declined with a long half-life of 26-30 hours upon PEGylation. The PK parameters (Table 8) showed a dose-proportional increase in exposure. This PK profile is superior to the wild-type polypeptide, which has a reported half-life in mice of within minutes.

[0293] [Table 10]

[0294] The immune PD profiles of aldesleukin and PEGylated IL-2 polypeptide composition A1 were evaluated simultaneously in the same study and monitored for 14 days. A single dose treatment with PEGylated IL-2 polypeptide resulted in strong and sustained STAT5 phosphorylation in the Treg population (CD3+, CD4+, CD25Hi, FoxP3+) with no or minimal effect on CD8+ Teff cells (CD3+, CD8+, CD4-) and NK cells (CD3-, CD49b+) (Figure 7). In contrast, five doses of treatment with aldesleukin resulted in a more modest STAT5 phosphorylation profile in Treg cells and no effect on CD8+ Teff and NK cells (Figure 7). Activation of the IL-2 receptor signaling pathway in Treg cells upon treatment with composition A1 translated into a significant and sustained upregulation of the proliferation marker Ki67, which was not observed in CD8+ Teff and NK cells. In contrast, five doses of treatment with aldesleukin resulted in limited Ki67 upregulation in Tregs (Figure 7). The increase in the proliferation activity of the Treg subpopulation upon treatment with Composition A1 resulted in a highly significant increase in Treg cell numbers compared to baseline, superior to that observed with the five-day dose of aldesleukin. Cell proliferation was selective for the Treg subpopulation, with CD8+ Teff and NK cells remaining unchanged to a similar extent (Figure 7).

[0295] Example 11: Composition A1 inhibits keyhole limpet hemocyanin-induced delayed-type hypersensitivity Delayed type hypersensitivity (DTH) represents a local T effector recall response to a previously encountered antigen. Here, mice were first sensitized to keyhole limpet hemocyanin (KLH) by subcutaneous immunization with KLH, then rechallenged several days later by intradermal injection of the same antigen into the ear, resulting in local tissue inflammation and swelling. Adult Balb / c mice were randomly assigned to experimental groups (n=10 / group) and allowed to acclimate for one week. On day 0, animals were administered an emulsion of 100 μg KLH in complete Freund's adjuvant (CFA) by sc injection between the shoulder blades. Composition A1 was administered at 0.3 mg / kg subcutaneously on either days 0, 0 and 3, 0, 3 and 5, or 0, 3, 5 and 8 (see FIG. 8A). Vehicle was administered subcutaneously on days 0, 3, 5 and 8. After baseline measurements of left and right ear thickness using a digital caliper, on day 7, all animals were challenged with an intradermal injection of 10 μg KLH in 0.9% sodium chloride in the right ear. The contralateral (left) ear was administered an equal amount of 0.9% sodium chloride. Ear thickness was measured at 24, 48, 72, and 96 hours using a digital caliper. In the vehicle-treated group, ear inflammation peaked 48 hours after antigen challenge and then slowly resolved (FIG. 8A). A single dose of Composition A1 strongly suppressed ear inflammation at all time points compared to vehicle. Multiple doses resulted in an earlier and shallower peak of inflammation 24 hours after antigen challenge, followed by rapid resolution back to near baseline. In each instance of Composition A1 administration, the difference in ear swelling, as measured by area under the curve (AUC), was significantly smaller than vehicle control (see FIG. 8B and FIG. 8C). Thus, Composition A1 potently suppresses antigen-driven tissue inflammation.

Claims

**Claim 1** A modified interleukin-2 (IL-2) polypeptide, comprising the modified IL-2 polypeptide, wherein the modified IL-2 polypeptide comprises up to 7 natural amino acid substitutions, the 7 natural amino acid substitutions comprising amino acid substitutions at residues Y31, K35 and Q74, and the residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as a reference sequence, a modified IL-2 polypeptide. **Claim 2** The modified IL-2 polypeptide according to claim 1, wherein the modified IL-2 polypeptide comprises 3, 4, 5 or 6 natural amino acid substitutions relative to the sequence shown in SEQ ID NO:

1. **Claim 3** The modified IL-2 polypeptide according to claim 1, wherein the modified IL-2 polypeptide comprises each of Hse41, Hse71 and Hse104. **Claim 4** The modified IL-2 polypeptide according to claim 3, wherein the modified IL-2 polypeptide comprises Nle substitutions at residue 23, residue 39 and residue 46. **Claim 5** The modified IL-2 polypeptide according to claim 1, wherein the modified IL-2 polypeptide comprises at least one amino acid substitution selected from Y31H, K35R, Q74P and N88D. **Claim 6** The modified IL-2 polypeptide according to claim 1, wherein the modified IL-2 polypeptide comprises two, three or four of the Y31H, K35R, Q74P and N88D amino acid substitutions. **Claim 7** The modified IL-2 polypeptide according to claim 1, wherein the modified IL-2 polypeptide comprises Y31H, K35R and Q74P amino acid substitutions. **Claim 8** The modified IL-2 polypeptide according to claim 1, wherein the modified IL-2 polypeptide comprises an amino acid substitution at residue N88. **Claim 9** The modified IL-2 polypeptide according to claim 8, wherein the amino acid substitution at residue N88 is an N88D substitution. **Claim 10** The modified IL-2 polypeptide according to claim 1, further comprising a C125S substitution. **Claim 11** The modified IL-2 polypeptide according to claim 1, wherein the modified IL-2 polypeptide comprises a V69A substitution. **Claim 12** The modified IL-2 polypeptide according to claim 1, wherein the modified IL-2 polypeptide is synthetic. **Claim 13**: The modified IL-2 polypeptide according to claim 1, wherein the modified IL-2 polypeptide exhibits binding affinity for IL-2Rβ that is at least about 1000 nM, at least about 2000 nM, at least about 3000 nM, at least about 5000 nM, or at least about 10000 nM. **Claim 14**: The modified IL-2 polypeptide according to claim 1, wherein the modified IL-2 polypeptide exhibits binding affinity for IL-2Rα that is at most about 100 nM, at most about 75 nM, at most about 50 nM, at most about 40 nM, at most about 30 nM, at most about 20 nM, at most about 10 nM, or at most about 5 nM. **Claim 15**: The modified IL-2 polypeptide according to claim 1, wherein the modified IL-2 polypeptide exhibits binding affinity for IL-2Rα that is from about 0.1 nM to about 100 nM, from about 0.1 nM to about 50 nM, from about 0.1 nM to about 20 nM, from about 1 nM to about 100 nM, from about 1 nM to about 50 nM, or from about 1 nM to about 20 nM. **Claim 16**: The modified IL-2 polypeptide according to claim 1, wherein the modified IL-2 polypeptide has an EC50 for activation of Treg cells that is at most about 100 nM, at most about 75 nM, at most about 50 nM, at most about 40 nM, at most about 35 nM, at most about 30 nM, or at most about 25 nM. **Claim 17**: The modified IL-2 polypeptide according to claim 1, wherein the modified IL-2 polypeptide has an EC50 for activation of Teff cells that is at least about 1000 nM. **Claim 18**: A modified IL-2 polypeptide comprising an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 3 - 43, wherein each residue in the reference amino acid sequence substituted relative to SEQ ID NO: 1 is retained. **Claim 19**: The modified IL-2 polypeptide according to claim 18, wherein the modified IL-2 polypeptide comprises a sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO:

42. **Claim 20**: The modified IL-2 polypeptide according to claim 18, wherein the modified IL-2 polypeptide comprises a sequence having at least about 95%, 98%, 99% or 100% sequence identity with the sequence shown in SEQ ID NO:

3. **Claim 21**: The modified IL-2 polypeptide according to claim 18, wherein the modified IL-2 polypeptide comprises the sequence shown in SEQ ID NO:

3. **Claim 22**: a) a modified IL-2 polypeptide according to any one of claims 1 to 21, and b) a pharmaceutically acceptable carrier or excipient A pharmaceutical composition comprising. **Claim 23**: Use of a modified IL-2 polypeptide according to any one of claims 1 to 21 in the manufacture of a medicament for treating an inflammatory disease or disorder in a subject in need thereof, in a pharmaceutically effective amount. **Claim 24**: Use of a pharmaceutical composition according to claim 22 in the manufacture of a medicament for treating an inflammatory disease or disorder in a subject in need thereof, in a pharmaceutically effective amount.

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