Interleukin-2 polypeptide conjugate and method of using the same

Abstract

The present invention provides compositions and methods comprising interleukin-2 (IL-2) polypeptide conjugates. Also described are IL-2 conjugates for treating diseases or disorders including cancer.

Description

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 987,872, filed Mar. 11, 2020, the contents of which are hereby incorporated by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy was created on Mar. 3, 2021, is named AMBX_0232_00PCT_ST25.txt, and is 27,704 bytes in size. Technical Field

[0005] Embodiments of the present disclosure relate at least to the fields of immunotherapy, immuno-oncology, and cancer therapy. More specifically, the present disclosure relates to interleukin-2 (IL-2) conjugates and their use. Background Art

[0006] Cancer is one of the most important health conditions. In the United States, cancer has the second highest mortality rate after heart disease, accounting for one quarter of deaths. It is generally expected that the incidence of cancer will increase as the U.S. population ages, further exacerbating the impact of the condition. The current treatment regimens for cancer established in the 1970s and 1980s have not changed much. When used in most common advanced cancers, these treatments, including chemotherapy, radiation, and other modalities (including newer targeted therapies), show limited overall survival benefit because these therapies primarily target the tumor mass.

[0007] More specifically, to date, conventional cancer diagnosis and therapies have attempted to selectively detect and eradicate mainly rapidly growing neoplastic cells (i.e., cells that form tumor masses). Standard oncological treatment regimens are generally designed primarily to administer the highest dose of radiation or chemotherapeutic agents without excessive toxicity, i.e., what is commonly referred to as the "maximum tolerated dose" (MTD) or the "no-observed-adverse-effect level" (NOAEL). Many conventional cancer chemotherapies (such as alkylating agents like cyclophosphamide, antimetabolites like 5-fluorouracil, and plant alkaloids like vincristine) and conventional radiotherapy exert their toxic effects on cancer cells mainly by interfering with cellular mechanisms involved in cell growth and DNA replication. Chemotherapy regimens often also include administering combinations of chemotherapeutic agents in an attempt to enhance therapeutic efficacy. Despite the availability of a large number of different chemotherapeutic agents, these therapies have many drawbacks. For example, chemotherapeutic agents are notoriously toxic due to their non-specific side effects on both normal and malignant rapidly growing cells; for example, chemotherapeutic agents cause significant and often dangerous side effects, including myelosuppression, immunosuppression, and gastrointestinal discomfort, among others.

[0008] Cancer stem cells

[0009] Cancer stem cells comprise a unique subpopulation of tumors (usually around 0.1 - 10%), which have higher tumorigenicity relative to the remaining approximately 90% of the tumor (and tumor mass), grow relatively more slowly or quiescently, and are generally relatively more chemoresistant compared to the tumor mass. Given that conventional therapies and regimens are mostly designed to attack rapidly proliferating cells (i.e., those cancer cells that make up the tumor mass), cancer stem cells, which generally grow more slowly compared to rapidly growing tumor masses, may be relatively more resistant to conventional therapies and regimens. Cancer stem cells can exhibit other characteristics that make them relatively more chemoresistant, such as multidrug resistance and anti-apoptotic pathways. The above factors constitute the key reasons why standard oncological treatment regimens fail to ensure long-term benefits in the majority of patients with advanced cancer, i.e., fail to sufficiently target and eradicate cancer stem cells. In some cases, cancer stem cells are the generating cells of the tumor (i.e., it is the ancestor of the cancer cells that make up the tumor mass).

[0010] IL-2 has been used to treat several cancers, such as renal cell carcinoma and metastatic melanoma. Commercially available IL-2 is a recombinant protein that is non-glycosylated, has alanine-1 removed and cysteine-125 replaced with serine-125 (Whittington et al., 1993). Although IL-2 was the earliest FDA-approved cytokine in cancer treatment, IL-2 has been shown to exhibit severe side effects when used at high doses. This greatly limits its application in potential patients. The potential mechanism of said severe side effects has been attributed to the binding of IL-2 to one of its receptors, IL-2Rα. Generally, IL-2 can not only form a heterotrimeric complex with its receptors including IL-2Rα (or CD25), IL-2Rβ (or CD122) and IL-2Rγ (or CD132) (when all three receptors are present in tissues), but also form a heterodimeric complex with IL-2Rβ and IL-2Rγ. In a clinical context, when high-dose IL-2 is used, IL-2 begins to bind IL-2αβγ, which is the major receptor form in reg T reg cells. The inhibitory effect on T

[0011] The ability to incorporate non-genetically encoded amino acids into proteins allows the introduction of chemical functional groups that can provide valuable alternatives to naturally occurring functional groups such as the ε–NH2 of lysine, the thiol –SH of cysteine, the imino group of histidine, etc. Certain chemical functional groups are known to be inert to the functional groups present in the 20 common genetically encoded amino acids but react cleanly and efficiently to form stable bonds. For example, it is known in the art that azide and ethynyl groups undergo a Huisgen [3+2] cycloaddition reaction in aqueous conditions in the presence of a catalytic amount of copper. See, e.g., Tornoe et al., (2002) J. Org. Chem. 67:3057-3064; and Rostovtsev et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599. By introducing, for example, an azide moiety into a protein structure, one can incorporate a functional group that is chemically inert to amines, thiols, carboxylic acids, and hydroxyl groups present in the protein but reacts smoothly and efficiently with an ethynyl moiety to form a cycloaddition product. Importantly, in the absence of an ethynyl moiety, the azide remains chemically inert and non-reactive in the presence of other protein side chains and under physiological conditions.

[0012] Among other problems, the present invention is directed to solving problems related to the activity and production of IL-2 polypeptide conjugates and is also directed to producing IL-2 polypeptides having improved biological or pharmacological properties such as improved activity against tumors and / or improved conjugation and / or improved therapeutic half-life. The IL-2 polypeptides of the present invention target both Treg cells that are known to express the trimeric IL-2 receptor (α, β, and γ) and CD8 cells that predominantly express the β and γ dimers of the IL-2 receptor. The IL-2 polypeptides of the present invention reduce binding to the α receptor of Treg cells and promote biased binding to the β and γ dimers of CD8 cells, thereby providing improved therapeutic applications and improved prognosis for diseases or disorders in which IL-2 receptor α is highly expressed. Summary of the Invention

[0013] In certain embodiments, the present disclosure provides a modified IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and comprising: a non-naturally encoded amino acid incorporated at position 42; one or more amino acid substitutions at selected positions within SEQ ID NO: 2; and one or more PEG molecules; wherein the polypeptide is conjugated to the one or more PEG molecules via the non-naturally encoded amino acid incorporated into the polypeptide. In certain embodiments, the present disclosure provides a modified IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and comprising: a non-naturally encoded amino acid incorporated at position 45; one or more amino acid substitutions at selected positions within SEQ ID NO: 2; and one or more PEG molecules; wherein the polypeptide is conjugated to the one or more PEG molecules via the non-naturally encoded amino acid incorporated into the polypeptide. In certain embodiments, the modified IL-2 polypeptide comprises a non-naturally encoded amino acid incorporated at position 42 corresponding to the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the modified IL-2 polypeptide comprises a non-naturally encoded amino acid incorporated at position 45 of SEQ ID NO: 2. In certain embodiments, the present invention provides a modified IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and comprising: a non-naturally encoded amino acid incorporated at position 42; one or more PEG molecules; and optionally one or more amino acid substitutions at selected positions within SEQ ID NO: 2; wherein the polypeptide is conjugated to the one or more PEG molecules via the non-naturally encoded amino acid incorporated into the polypeptide. In certain embodiments, the present invention provides a modified IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and comprising: a non-naturally encoded amino acid incorporated at position 45; one or more PEG molecules; and optionally one or more amino acid substitutions at selected positions within SEQ ID NO: 2; wherein the polypeptide is conjugated to the one or more PEG molecules via the non-naturally encoded amino acid incorporated into the polypeptide. In certain embodiments, the modified IL-2 polypeptide of the present invention optionally comprises one or more amino acid substitutions at selected positions within SEQ ID NO: 2.

[0014] In certain embodiments, the modified IL-2 polypeptide comprises non-naturally encoded amino acids selected from p-acetylphenylalanine, p-nitrophenylalanine, p-sulfotyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-boronylcarbonylphenylalanine, o-boronylcarbonylphenylalanine, m-boronylcarbonylphenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-OMe phenylalanine, o-OMe phenylalanine, m-OMe phenylalanine, p-sulfophenylalanine, o-sulfophenylalanine, m-sulfophenylalanine, 5-nitroHis, 3-nitroTyr, 2-nitroTyr, nitro-substituted Leu, nitro-substituted His, nitro-substituted De, nitro-substituted Trp, 2-nitroTrp, 4-nitroTrp, 5-nitroTrp, 6-nitroTrp, 7-nitroTrp, 3-aminotyrosine, 2-aminotyrosine, o-sulfotyrosine, 2-sulfoxyphenylalanine, 3-sulfoxyphenylalanine, o-carboxyphenylalanine, m-carboxyphenylalanine, p-acetyl-L-phenylalanine, p-propargyl-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-dopa, fluorophenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphoserine, phosphotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propargyloxyphenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine, and p-azidomethyl-phenylalanine. In certain embodiments, the non-naturally encoded amino acid is p-acetylphenylalanine.

[0015] In certain embodiments, the modified IL-2 polypeptide comprises one or more amino acid substitutions at positions R38 and P65 of SEQ ID NO: 2. In certain embodiments, the modified IL-2 polypeptide comprises one or more amino acid substitutions at positions 38 and 65 of SEQ ID NO: 2. In certain embodiments, the modified IL-2 polypeptide comprises one or more amino acid substitutions at position 38 or 65 of SEQ ID NO: 2. In certain embodiments, the modified IL-2 polypeptide comprises one or more amino acid substitutions at position 38 of SEQ ID NO: 2. In certain embodiments, the modified IL-2 polypeptide comprises one or more amino acid substitutions at position 65 of SEQ ID NO: 2. In certain embodiments, the amino acid substitution at position 38 of SEQ ID NO: 2 is a substitution to alanine.

[0016] In certain embodiments, the modified IL-2 polypeptide comprises one or more PEG molecules, wherein the one or more PEG molecules are linear or branched or multi-armed. In certain embodiments, the one or more PEG molecules are linear. In certain embodiments, the one or more PEG molecules are branched. In certain embodiments, the one or more PEG molecules are multi-armed. In certain embodiments, the one or more PEG molecules have an average molecular weight of 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, and 50 kDa or greater. In certain embodiments, the one or more PEG molecules are 30 kDa. In certain embodiments, the one or more PEG molecules are 40 kDa. In certain embodiments, the one or more PEG molecules are linear 30 kDa PEG molecules. In certain embodiments, the one or more PEG molecules are branched 30 kDa PEG molecules. In certain embodiments, the one or more PEG molecules are linear 40 kDa PEG molecules. In certain embodiments, the one or more PEG molecules are branched 40 kDa PEG molecules. In certain embodiments, the modified IL-2 polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 2, which comprises site-specifically incorporated non-naturally encoded amino acids, one or more amino acid substitutions at selected positions within SEQ ID NO: 2, and one or more PEG molecules conjugated through the site-specifically incorporated non-naturally encoded amino acids. In certain embodiments, the modified IL-2 polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 2, which comprises site-specifically incorporated non-naturally encoded amino acids and one or more PEG molecules conjugated through the site-specifically incorporated non-naturally encoded amino acids. In certain embodiments, the modified IL-2 polypeptides comprising site-specifically incorporated non-naturally encoded amino acids are selected from SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23. In certain embodiments, the modified IL-2 polypeptide comprising site-specifically incorporated non-naturally encoded amino acids is SEQ ID NO: 9. In certain embodiments, the modified IL-2 polypeptide comprising site-specifically incorporated non-naturally encoded amino acids is SEQ ID NO: 10. In certain embodiments, the modified IL-2 polypeptide comprising site-specifically incorporated non-naturally encoded amino acids is SEQ ID NO: 11.In certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO: 12. In certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO: 13. In certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO: 14. In certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO: 15. In certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO: 16. In certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO: 17. In certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO: 18. In certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO: 19. In certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO: 20. In certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO: 21. In certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO: 22. In certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO: 23.

[0017] In certain embodiments, the invention relates to interleukin-2 (IL-2) polypeptides comprising one or more non-naturally encoded amino acids. In certain embodiments, the invention provides IL-2 polypeptide conjugates comprising one or more non-naturally encoded amino acids. In certain embodiments, the invention provides IL-2 polypeptide conjugates in which a water-soluble polymer such as PEG is conjugated to the IL-2 variant via one or more non-naturally encoded amino acids within the IL-2 variant. In certain embodiments, the invention provides IL-2 polypeptide conjugates having one or more non-naturally encoded amino acids and one or more natural amino acid substitutions. In certain embodiments, the invention provides IL-2 polypeptide conjugates having one or more non-naturally encoded amino acids, one or more natural amino acid substitutions, and one or more PEG molecules. The one or more naturally occurring amino acid substitutions can be selected from any of the 20 common amino acids, including but not limited to alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0018] In one embodiment, the PEG-IL-2 is mono-PEGylated. In one embodiment, the PEG-IL-2 is di-PEGylated. In one embodiment, the PEG-IL-2 has more than two (2) polyethylene glycol molecules attached to it. Another embodiment of the invention provides a method of modulating the activity of immune system cells using the PEG-IL-2 polypeptides of the invention.

[0019] In this or any embodiment of the invention, the PEG-IL-2 can comprise full-length, mature (lacking the signal peptide) human interleukin-2 linked to the PEG polymer. In this or any embodiment of the invention, the PEG-IL-2 can comprise full-length, mature (lacking the signal peptide) human interleukin-2 covalently linked to the PEG polymer or other bioactive molecule. In certain embodiments, the bioactive molecule is modified and, by way of non-limiting example, the bioactive molecule can include one or more non-naturally encoded amino acids.

[0020] In the PEG-IL2 conjugate, the PEG or other water-soluble polymer can be conjugated directly or via a linker to the IL-2 protein or the bioactive molecule. Suitable linkers include, for example, cleavable and non-cleavable linkers.

[0021] The present invention provides a method for treating cancer in a mammal by administering an effective amount of a PEG-IL-2 polypeptide, said mammal being, for example, a mammal including but not limited to those having one or more of the following conditions: solid tumors, hematological tumors, colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, glioblastoma, and leukemia. In certain embodiments, the cancer is small cell lung cancer, prostate cancer, gastric cancer, gastroenteropancreatic tumors, cervical cancer, esophageal cancer, colorectal cancer, cancers or tumors of epithelial origin, kidney cancer, brain cancer, pancreatic cancer, thyroid cancer, endometrial cancer, pancreatic cancer, head and neck cancer, or skin cancer. In certain embodiments, the cancer is characterized by a high level of Treg cells. In certain embodiments, the cancer is characterized by high expression of IL-2 receptor α. In certain embodiments, the present invention provides a method for treating cancer or a disorder or disease by administering to a subject an effective amount of a composition comprising the IL-2 polypeptide of the present invention. In certain embodiments, the present invention provides a method for treating a genetic disorder by administering to a patient an effective amount of the IL-2 composition of the present invention. In certain embodiments, the disorder or disease is characterized by high expression of IL-2 receptor α. In certain embodiments, the disorder or disease is characterized by a high level of Treg cells. In certain embodiments, the cancer, disorder or disease is treated by reducing, blocking or silencing the expression of IL-2 receptor α. In certain embodiments, the cancer, disorder or disease is treated by reducing the binding of IL-2 receptor α on the surface of Treg cells, resulting in a reduction in the proliferation of Treg cells in the cancer, disorder or disease to be treated.

[0022] As used herein, interleukin 2 or IL-2 is defined as a protein having the following properties: (a) having an amino acid sequence that is substantially identical to a known sequence of IL-2 (including IL-2 mutant proteins, mature IL-2 sequences (i.e., lacking the secretory leader sequence), and IL-2 as disclosed in SEQ ID NO: 1, 2, 3, 5, or 7 of the present application), and (b) having at least one biological activity common to native or wild-type IL-2. For the purposes of the present invention, both glycosylated (e.g., produced in eukaryotic cells such as yeast or CHO cells) and non-glycosylated (e.g., chemically synthesized or produced in Escherichia coli (E. coli)) IL-2 are equivalent and can be used interchangeably. Also included are other mutants and other analogs that retain the biological activity of IL-2, including viral IL-2.

[0023] The present invention provides IL-2 polypeptides conjugated to one or more water-soluble polymers via one or more non-naturally encoded amino acids incorporated into the polypeptide. The present invention provides IL-2 polypeptides conjugated to one or more water-soluble polymers, wherein the PEGylated IL-2 polypeptides are also linked to another drug or bioactive molecule, and wherein the IL-2 polypeptide comprises one or more non-naturally encoded amino acids conjugated to the one or more water-soluble polymers. The present invention also provides monomers and dimers of the IL-2 polypeptide. The present invention also provides trimers of the IL-2 polypeptide. The present invention provides multimers of the IL-2 polypeptide. The present invention also provides IL-2 dimers comprising one or more non-naturally encoded amino acids. The present invention provides IL-2 multimers comprising one or more non-naturally encoded amino acids. The present invention provides homogeneous IL-2 multimers comprising one or more non-naturally encoded amino acids, wherein each IL-2 polypeptide has the same amino acid sequence. The present invention provides heterogeneous IL-2 multimers, wherein at least one of the IL-2 polypeptides comprises at least one non-naturally encoded amino acid, and wherein any or each IL-2 polypeptide in the multimer can have a different amino acid sequence.

[0024] In certain embodiments, the IL-2 polypeptide comprises one or more post-translational modifications. In certain embodiments, the IL-2 polypeptide is linked to a linker, polymer, or bioactive molecule. In certain embodiments, the IL-2 monomer is homogeneous. In certain embodiments, the IL-2 dimer is homogeneous. In certain embodiments, the IL-2 multimer is conjugated to one water-soluble polymer. In certain embodiments, the IL-2 multimer is conjugated to two water-soluble polymers. In certain embodiments, the IL-2 multimer is conjugated to three water-soluble polymers. In certain embodiments, the IL-2 multimer is conjugated to more than three water-soluble polymers. In certain embodiments, the IL-2 polypeptide is linked to a linker long enough to permit dimer formation. In certain embodiments, the IL-2 polypeptide is linked to a linker long enough to permit trimer formation. In certain embodiments, the IL-2 polypeptide is linked to a linker long enough to permit multimer formation. In certain embodiments, the IL-2 polypeptide is linked to a bifunctional polymer, bifunctional linker, or at least one additional IL-2 polypeptide. In certain embodiments, the IL-2 polypeptide comprises one or more post-translational modifications. In certain embodiments, the IL-2 polypeptide is linked to a linker, polymer, or bioactive molecule.

[0025] In certain embodiments, the non-naturally encoded amino acid is linked to a water-soluble polymer. In certain embodiments, the water-soluble polymer comprises a polyethylene glycol (PEG) moiety. In certain embodiments, the non-naturally encoded amino acid is linked to the water-soluble polymer using a linker or is bonded to the water-soluble polymer. In certain embodiments, the polyethylene glycol molecule is a bifunctional polymer. In certain embodiments, the bifunctional polymer is linked to a second polypeptide. In certain embodiments, the second polypeptide is IL-2. In certain embodiments, the IL-2 or a variant thereof comprises at least two amino acids linked to a water-soluble polymer comprising a polyethylene glycol moiety. In certain embodiments, at least one amino acid is a non-naturally encoded amino acid.

[0026] In certain embodiments, the IL-2 or PEG-IL-2 of the present invention is linked to a therapeutic agent such as an immunomodulator. The immunomodulator can be any agent that exerts a therapeutic effect on immune cells and can be used as a therapeutic agent for conjugation to IL-2, PEG-IL-2, or an IL-2 variant. In certain embodiments, the IL-2 or PEG-IL-2 of the present invention is linked to a therapeutic agent such as a cytokine, chemotherapeutic agent, immunotherapeutic agent, hormonal agent, anti-tumor agent, immunostimulant, or a combination thereof.

[0027] In certain embodiments, a non-naturally encoded amino acid is incorporated into one or more of the following positions of IL-2 or a variant thereof: before position 1 (i.e., at the N-terminus), positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or added to the carboxyl terminus of the protein, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5, or 7). In certain embodiments, one or more bioactive molecules are directly conjugated to the IL-2 variant. In certain embodiments, the one or more bioactive molecules are conjugated to the one or more non-naturally encoded amino acids in the IL-2 polypeptide. In certain embodiments, the IL-2 variant of the invention is linked to a linker. In certain embodiments, the IL-2 variant linked to the linker further comprises a bioactive molecule. In certain embodiments of the invention, the linker is linked to a non-naturally encoded amino acid.

[0028] In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 3, 32, 35, 37, 38, 42, 43, 44, 45, 48, 49, 61, 62, 64, 65, 68, 72, 76, and 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NOs: 3, 5, or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: before positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, and 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NOs: 3, 5, or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 35, 37, 42, 45, 49, 61, or 65, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NOs: 3, 5, or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 42, 45, 61, and 65, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NOs: 3, 5, or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 45 and 65, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NOs: 3, 5, or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 3 in the IL-2 or a variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 32 in the IL-2 or a variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 35 in the IL-2 or a variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 37 in the IL-2 or a variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 38 in the IL-2 or a variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 41 in the IL-2 or a variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 42 in the IL-2 or a variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 43 in the IL-2 or a variant thereof of the present invention.In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 44 in the IL-2 or variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 45 in the IL-2 or variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 48 in the IL-2 or variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 49 in the IL-2 or variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 61 in the IL-2 or variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 62 in the IL-2 or variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 64 in the IL-2 or variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 65 in the IL-2 or variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 68 in the IL-2 or variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 72 in the IL-2 or variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 76 in the IL-2 or variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 107 in the IL-2 or variant thereof of the present invention.

[0029] In certain embodiments, one or more non-naturally encoded amino acids are incorporated at any position within one or more of the following regions corresponding to secondary structures or specific amino acids in IL-2 or its variants as described below: at sites of hydrophobic interaction; at or near sites that interact with IL-2 receptor subunits (including IL2Rα); within amino acid positions 3 or 35 to 45; within the first 107 N-terminal amino acids; within amino acid positions 61 - 72; each such position being a position of SEQ ID NO:2 or the corresponding amino acid position in SEQ ID NO:3, 5, or 7. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at one or more of the following positions in IL-2 or its variants: before position 1 of SEQ ID NO:2 (i.e., at the N-terminus), positions 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, and any combination thereof; or the corresponding amino acids in SEQ ID NO:3, 5, or 7. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at one or more of the following positions in IL-2 or its variants: positions 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133 of SEQ ID NO:2, or added to the carboxyl terminus of the protein, and any combination thereof; or the corresponding amino acids in SEQ ID NO:3, 5, or 7.

[0030] In certain embodiments, the non-naturally occurring amino acid(s) at one or more of these positions in IL-2 or its variant are linked to a drug or other bioactive molecule, and the positions include, but are not limited to, the following positions: before position 1 (i.e., at the N-terminus), positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or added to the carboxyl terminus of the protein, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5, or 7).

[0031] In certain embodiments, the non-naturally occurring amino acids at one or more of these positions in IL-2 or its variants are linked to a drug or other bioactive molecule, and the positions include, but are not limited to, positions at sites of hydrophobic interaction, at or near sites that interact with IL-2 receptor subunits (including IL2Rα), within amino acid positions 3 or 35 to 45, within the first 107 N-terminal amino acids, within amino acid positions 61-72; each such position is a position of SEQ ID NO:2 or the corresponding amino acid position in SEQ ID NO:3, 5, or 7. In certain embodiments, the non-naturally occurring amino acids at one or more of these positions in IL-2 or its variants are linked to a drug or other bioactive molecule, and the positions include, but are not limited to, the following positions: before position 1 of SEQ ID NO:2 (i.e., at the N-terminus), positions 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, and any combination thereof; or the corresponding amino acids in SEQ ID NO:3, 5, or 7. In certain embodiments, the non-naturally occurring amino acids at one or more of these positions in IL-2 or its variants are linked to a drug or other bioactive molecule, and the positions include, but are not limited to, the following positions in IL-2 or its variants: positions 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133 of SEQ ID NO:2, or added to the carboxyl terminus of the protein, and any combination thereof; or the corresponding amino acids in SEQ ID NO:3, 5, or 7.In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof and linked to a drug or other bioactive molecule: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, and 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5, or 7).

[0032] In certain embodiments, the non-naturally occurring amino acids at one or more of these positions in IL-2 or a variant thereof are linked to a linker, including but not limited to the following positions: before position 1 (i.e., at the N-terminus), positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or added to the carboxyl terminus of the protein, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5, or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof and linked to a linker: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, and 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5, or 7).

[0033] In certain embodiments, the non-naturally occurring amino acid(s) at one or more of these positions in IL-2 or its variant is / are linked to a linker, which is further linked to a water-soluble polymer or a bioactive molecule. The positions include, but are not limited to, the following positions: before position 1 (i.e., at the N-terminus), positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or added to the carboxyl terminus of the protein, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5, or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or its variant and linked to a linker, which is further linked to a water-soluble polymer or a bioactive molecule. The positions include, but are not limited to, the following positions: before positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, and 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5, or 7).

[0034] In certain embodiments, the non-naturally occurring amino acids at one or more of these positions in IL-2 or its variants are linked to a water-soluble polymer, and the positions include but are not limited to the following positions: before position 1 (i.e., at the N-terminus), positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or added to the carboxyl terminus of the protein, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NOs: 3, 5, or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or its variants and linked to a linker, which is further linked to a water-soluble polymer, and the positions include but are not limited to the following positions: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, and 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NOs: 3, 5, or 7). In certain embodiments, the present disclosure provides IL-2 polypeptides corresponding to SEQ ID Nos: 9-23 that contain site-specifically incorporated non-naturally encoded amino acids.

[0035] In certain embodiments, the IL-2 or its variant comprises a substitution, addition, or deletion that modulates the affinity of the IL-2 for the IL-2 receptor subunit or its variant. In certain embodiments, the IL-2 or its variant comprises a substitution, addition, or deletion that modulates the affinity of the IL-2 or its variant for the IL-2 receptor or a binding partner (including but not limited to a protein, polypeptide, lipid, fatty acid, small molecule, or nucleic acid). In certain embodiments, the IL-2 or its variant comprises a substitution, addition, or deletion that modulates the stability of the IL-2 compared to the stability of the corresponding IL-2 without the substitution, addition, or deletion. Stability and / or solubility can be measured using a variety of different assays known to those of ordinary skill in the art. These assays include but are not limited to SE-HPLC and RP-HPLC. In certain embodiments, the IL-2 comprises a substitution, addition, or deletion that modulates the immunogenicity of the IL-2 compared to the immunogenicity of the corresponding IL-2 without the substitution, addition, or deletion. In certain embodiments, the IL-2 comprises a substitution, addition, or deletion that modulates the serum half-life or circulation time of the IL-2 compared to the serum half-life or circulation time of the corresponding IL-2 without the substitution, addition, or deletion.

[0036] In certain embodiments, the IL-2 or its variant comprises a substitution, addition, or deletion that increases the water solubility of the IL-2 compared to the water solubility of the corresponding IL-2 or its variant without the substitution, addition, or deletion. In certain embodiments, the IL-2 or its variant comprises a substitution, addition, or deletion that increases the solubility of the IL-2 or its variant produced in a host cell compared to the solubility of the corresponding IL-2 or its variant without the substitution, addition, or deletion. In certain embodiments, the IL-2 or its variant comprises a substitution, addition, or deletion that increases the expression of the IL-2 in a host cell or increases in vitro synthesis compared to the expression or synthesis of the corresponding IL-2 or its variant without the substitution, addition, or deletion. The IL-2 or its variant comprising such a substitution retains agonist activity or retains or increases the expression level in a host cell. In certain embodiments, the IL-2 or its variant comprises a substitution, addition, or deletion that increases the protease resistance of the IL-2 or its variant compared to the protease resistance of the corresponding IL-2 or its variant without the substitution, addition, or deletion. In certain embodiments, the IL-2 or its variant comprises a substitution, addition, or deletion that modulates the signal transduction activity of the IL-2 receptor compared to the activity of the IL-2 receptor after interaction with the corresponding IL-2 or its variant without the substitution, addition, or deletion. In certain embodiments, the IL-2 or its variant comprises a substitution, addition, or deletion that modulates its binding to another molecule such as a receptor compared to the binding of the corresponding IL-2 without the substitution, addition, or deletion.

[0037] In certain embodiments, the present invention provides methods of treating a proliferative disorder, cancer, tumor, or pre-cancerous condition such as dysplasia using PEG-IL-2 and at least one additional therapeutic or diagnostic agent. The additional therapeutic agent can be, for example, a cytokine or cytokine antagonist such as IL-12, interferon-α, or an anti-epidermal growth factor receptor antibody, doxorubicin, epirubicin, an anti-folate agent such as methotrexate or fluorouracil, irinotecan, cyclophosphamide, radiotherapy, hormonal or anti-hormonal therapy such as androgen, estrogen, anti-estrogen antibody, flutamide, or diethylstilbestrol, surgery, tamoxifen, ifosfamide, dibromodulcitol, an alkylating agent such as melphalan or cisplatin, etoposide, vinorelbine, vinblastine, vindesine, glucocorticoid, histamine receptor antagonist, angiogenesis inhibitor, radiation, radiation sensitizer, anthracycline, vinca alkaloid, taxane such as paclitaxel and docetaxel, a cell cycle inhibitor such as a cyclin-dependent kinase inhibitor, checkpoint inhibitor, immunomodulatory drug, immunostimulatory drug, a monoclonal antibody against another tumor antigen, a complex of a monoclonal antibody with a bioactive molecule, a T cell adjuvant, a bone marrow graft, or an antigen presenting cell such as dendritic cell therapy. The vaccine can be provided as, for example, a soluble protein or a nucleic acid encoding the protein (see, e.g., Le et al., supra; Greco and Zellefsky eds. (2000) Radiotherapy of Prostate Cancer, Harwood Academic, Amsterdam; Shapiro and Recht (2001) New Engl. J. Med. 344:1997-2008; Hortobagyi (1998) New Engl. J. Med. 339:974-984; Catalona (1994) New Engl. J. Med. 331:996-1004; Naylor and Hadden (2003) Int. Immunopharmacol. 3:1205-1215; The Int. Adjuvant Lung Cancer Trial Collaborative Group (2004) New Engl. J. Med. 350:351-360; Slamon et al., (2001) New Engl. J. Med. 344:783-792; Kudelka et al., (1998) New Engl. J. Med. 338:991-992; van Netten et al., (1996) New Engl. J. Med. 334:920-921).

[0038] Methods for treating extramedullary hematopoiesis (EMH) in cancer are also provided. EMH has been described (see, e.g., Rao et al., (2003) Leuk. Lymphoma 44:715-718; Lane et al., (2002) J. Cutan. Pathol. 29:608-612).

[0039] In certain embodiments, the PEG-IL-2 or variant thereof comprises a substitution, addition, or deletion that modulates binding to its receptor or receptor subunit as compared to the binding activity of the corresponding IL-2 or variant thereof without the substitution, addition, or deletion. In certain embodiments, the IL-2 or variant thereof comprises a substitution, addition, or deletion that inhibits its activity related to binding to the receptor or receptor subunit as compared to the binding activity of the corresponding IL-2 or variant thereof without the substitution, addition, or deletion.

[0040] In certain embodiments, the IL-2 or variant thereof comprises a substitution, addition, or deletion that increases the compatibility of the IL-2 or variant thereof with a pharmaceutical preservative (e.g., m-cresol, phenol, benzyl alcohol) as compared to the compatibility of the corresponding wild-type IL-2 without the substitution, addition, or deletion. This increased compatibility enables the preparation of a well-preserved pharmaceutical formulation that maintains the physicochemical properties and biological activity of the protein during storage.

[0041] In certain embodiments, one or more non-natural amino acids are used to generate one or more engineered linkages. The intramolecular linkages can be generated in a number of ways, including but not limited to the reaction between two amino acids in the protein under suitable conditions (one or both amino acids can be non-natural amino acids), the reaction with two amino acids (each amino acid can be naturally encoded or non-naturally encoded), with a linker, polymer, or other molecule under suitable conditions, etc.

[0042] In certain embodiments, one or more amino acid substitutions in the IL-2 or its variant can be made using one or more naturally occurring or non-naturally occurring amino acids. In certain embodiments, the amino acid substitutions in the IL-2 or its variant can be made using naturally occurring or non-naturally occurring amino acids, provided that at least one substitution is made using a non-naturally encoded amino acid. In certain embodiments, one or more amino acid substitutions in the IL-2 or its variant can be made using one or more naturally occurring amino acids, and additionally at least one substitution is made using a non-naturally encoded amino acid. In certain embodiments, the amino acid substitutions in the IL-2 or its variant can be made using any naturally occurring amino acid, and at least one substitution is made using a non-naturally encoded amino acid. In certain embodiments, one or more natural amino acids can be substituted at one or more of the following positions in the IL-2 or its variant: before position 1 (i.e., at the N-terminus), positions 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, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5, or 7). In certain embodiments, one or more natural amino acid substitutions can be at one or more of the following positions in the IL-2 or its variant: positions 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or added to the carboxyl terminus of the protein, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitutions in the IL-2 or its variant can be made using at least one naturally occurring amino acid, and at least one substitution is made using a non-naturally encoded amino acid.In certain embodiments, the amino acid substitutions in the IL-2 or its variant can use at least two naturally occurring amino acids, and at least one substitution is made using a non-naturally encoded amino acid. In certain embodiments, the one or more naturally occurring or encoded amino acids can be any of the 20 common amino acids, including but not limited to alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In certain embodiments, the at least one naturally occurring amino acid substitution can be at the following positions of IL-2 or its variant: positions 38 and / or 46 and / or 65 or any combination thereof. In certain embodiments, the naturally occurring amino acid substitution can be at position 38 of IL-2 or its variant. In certain embodiments, the naturally occurring amino acid substitution at position 38 of the IL-2 or its variant can be selected from any of the 20 common naturally occurring amino acids, including but not limited to alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In certain embodiments, the naturally occurring amino acid substitution at position 38 of the IL-2 or its variant can be an alanine substitution. In certain embodiments, the naturally occurring amino acid substitution can be at position 46 of IL-2 or its variant. In certain embodiments, the naturally occurring amino acid substitution at position 46 of the IL-2 or its variant can be selected from any of the 20 common naturally occurring amino acids, including but not limited to alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In certain embodiments, the naturally occurring amino acid substitution at position 46 of the IL-2 or its variant can be a leucine or isoleucine substitution. In certain embodiments, the naturally occurring amino acid substitution can be at position 65 of IL-2 or its variant. In certain embodiments, the naturally occurring amino acid substitution at position 65 of the IL-2 or its variant can be selected from any of the 20 common naturally occurring amino acids, including but not limited to alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In certain embodiments, the naturally occurring amino acid substitution at position 65 of the IL-2 or its variant can be an arginine substitution.In certain embodiments, the amino acid substitutions in the IL-2 or its variant can be naturally occurring amino acid substitutions at positions 38, 46, or 65, and at least one substitution is made using non-naturally encoded amino acids incorporated into one or more of the following positions of the IL-2 or its variant: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, and 107, and any combination thereof (corresponding amino acid positions in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7). In certain embodiments, the amino acid substitution in the IL-2 or its variant can be a naturally occurring amino acid substitution at position 38, and at least one substitution is made using non-naturally encoded amino acids incorporated into one or more of the following positions of the IL-2 or its variant: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, and 107, and any combination thereof (corresponding amino acid positions in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7). In certain embodiments, the amino acid substitution in the IL-2 or its variant can be a naturally occurring amino acid substitution at position 46, and at least one substitution is made using non-naturally encoded amino acids incorporated into one or more of the following positions of the IL-2 or its variant: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, and 107, and any combination thereof (corresponding amino acid positions in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7). In certain embodiments, the amino acid substitution in the IL-2 or its variant can be a naturally occurring amino acid substitution at position 65, and at least one substitution is made using non-naturally encoded amino acids incorporated into one or more of the following positions of the IL-2 or its variant: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, and 107, and any combination thereof (corresponding amino acid positions in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7). In certain embodiments, the amino acid substitutions in the IL-2 or its variant can be naturally occurring amino acid substitutions at positions 38 and / or 46 and / or 65, and at least one substitution is made using non-naturally encoded amino acids incorporated into one or more of the following positions of the IL-2 or its variant: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, and 107, and any combination thereof (corresponding amino acid positions in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7).In certain embodiments, the amino acid substitutions in the IL-2 or its variant can be naturally occurring amino acid substitutions at position 38 and non-naturally encoded amino acids incorporated at position 42 in the IL-2 or its variant (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitutions in the IL-2 or its variant can be naturally occurring amino acid substitutions at positions 38 and 46 and non-naturally encoded amino acids incorporated at position 42 in the IL-2 or its variant (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitutions in the IL-2 or its variant can be naturally occurring amino acid substitutions at positions 38 and 65 and non-naturally encoded amino acids incorporated at position 42 in the IL-2 or its variant (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitutions in the IL-2 or its variant can be naturally occurring amino acid substitutions at positions 38, 46, and 65 and non-naturally encoded amino acids incorporated at position 42 in the IL-2 or its variant (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitutions in the IL-2 or its variant can be naturally occurring amino acid substitutions at position 38 and non-naturally encoded amino acids incorporated at position 45 in the IL-2 or its variant (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitutions in the IL-2 or its variant can be naturally occurring amino acid substitutions at positions 38 and 46 and non-naturally encoded amino acids incorporated at position 45 in the IL-2 or its variant (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitutions in the IL-2 or its variant can be naturally occurring amino acid substitutions at positions 38 and 65 and non-naturally encoded amino acids incorporated at position 45 in the IL-2 or its variant (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitutions in the IL-2 or its variant can be naturally occurring amino acid substitutions at positions 38, 46, and 65 and non-naturally encoded amino acids incorporated at position 45 in the IL-2 or its variant (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5, or 7).In certain embodiments, the amino acid substitutions in the IL-2 or its variant can be naturally occurring amino acid substitutions at position 38 and non-naturally encoded amino acids incorporated at position 65 in the IL-2 or its variant (the corresponding amino acid positions in SEQ ID NO: 2, or SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitutions in the IL-2 or its variant can be naturally occurring amino acid substitutions at positions 38 and 46 and non-naturally encoded amino acids incorporated at position 65 in the IL-2 or its variant (the corresponding amino acid positions in SEQ ID NO: 2, or SEQ ID NO: 3, 5, or 7).

[0043] In certain embodiments, the non-naturally encoded amino acid comprises a carbonyl group, an acetyl group, an aminooxy group, a hydrazino group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group.

[0044] In certain embodiments, the non-naturally encoded amino acid comprises a carbonyl group. In certain embodiments, the non-naturally encoded amino acid has the following structure:

[0045]

[0046] where n is 0 - 10; R1 is an alkyl group, an aryl group, a substituted alkyl group, or a substituted aryl group; R2 is H, an alkyl group, an aryl group, a substituted alkyl group, or a substituted aryl group; R3 is H, an amino acid, a polypeptide, or an amino-terminal modifying group, and R4 is H, an amino acid, a polypeptide, or a carboxyl-terminal modifying group.

[0047] In certain embodiments, the non-naturally encoded amino acid comprises an aminooxy group. In certain embodiments, the non-naturally encoded amino acid comprises a hydrazide group. In certain embodiments, the non-naturally encoded amino acid comprises a hydrazino group. In certain embodiments, the non-naturally encoded amino acid residue comprises a semicarbazide group.

[0048] In certain embodiments, the non-naturally encoded amino acid residue comprises an azide group. In certain embodiments, the non-naturally encoded amino acid has the following structure:

[0049]

[0050] where n is 0 - 10; R1 is an alkyl group, an aryl group, a substituted alkyl group, a substituted aryl group, or absent; X is O, N, S, or absent; m is 0 - 10; R2 is H, an amino acid, a polypeptide, or an amino-terminal modifying group, and R3 is H, an amino acid, a polypeptide, or a carboxyl-terminal modifying group.

[0051] In certain embodiments, the non-naturally encoded amino acid comprises an alkyne group. In certain embodiments, the non-naturally encoded amino acid has the following structure:

[0052]

[0053] Wherein n is from 0 to 10; R1 is alkyl, aryl, substituted alkyl or substituted aryl; X is O, N, S or absent; m is from 0 to 10, R2 is H, an amino acid, a polypeptide or an amino-terminal modifying group, and R3 is H, an amino acid, a polypeptide or a carboxyl-terminal modifying group.

[0054] In certain embodiments, the polypeptide is an IL-2 agonist, partial agonist, antagonist, partial antagonist or inverse agonist. In certain embodiments, the IL-2 agonist, partial agonist, antagonist, partial antagonist or inverse agonist comprises a non-naturally encoded amino acid linked to a water-soluble polymer. In certain embodiments, the water-soluble polymer comprises a polyethylene glycol moiety. In certain embodiments, the IL-2 agonist, partial agonist, antagonist, partial antagonist or inverse agonist comprises a non-naturally encoded amino acid and one or more post-translational modifications, linkers, polymers or bioactive molecules.

[0055] The present invention also provides an isolated nucleic acid comprising a polynucleotide encoding a polypeptide of SEQ ID NO: 1, 2, 3, 5 or 7, and the present invention provides an isolated nucleic acid comprising a polynucleotide that hybridizes under stringent conditions to a polynucleotide encoding a polypeptide of SEQ ID NO: 1, 2, 3, 5 or 7. The present invention also provides an isolated nucleic acid comprising a polynucleotide encoding a polypeptide shown as SEQ ID NO: 1, 2, 3, 5 or 7, wherein the polynucleotide comprises at least one selector codon. The present invention also provides an isolated nucleic acid comprising a polynucleotide encoding a polypeptide shown as SEQ ID NO: 1, 2, 3, 5 or 7 and having one or more non-naturally encoded amino acids. It will be apparent to those of ordinary skill in the art that many different polynucleotides can encode any polypeptide of the present invention.

[0056] In certain embodiments, the selector codon is selected from the group consisting of amber codon, ochre codon, opal codon, unique codon, rare codon, pentacodon and quadruplet codon.

[0057] The present invention also provides a method for making IL-2 or a variant thereof conjugated to a bioactive molecule. In certain embodiments, the method comprises contacting an isolated IL-2 or a variant thereof comprising a non-naturally encoded amino acid with a bioactive molecule comprising a moiety reactive with the non-naturally encoded amino acid. In certain embodiments, the non-naturally encoded amino acid incorporated into the IL-2 or a variant thereof is reactive with a bioactive molecule that is not reactive with any of the 20 commonly used amino acids. In certain embodiments, the non-naturally encoded amino acid incorporated into the IL-2 is reactive with a linker, polymer, or bioactive molecule that is not reactive with any of the 20 commonly used amino acids conjugated to a bioactive molecule.

[0058] In certain embodiments, the IL-2 or a variant thereof conjugated to a water-soluble polymer or bioactive molecule is made by reacting an IL-2 or a variant thereof comprising a carbonyl-containing amino acid with a water-soluble polymer or bioactive molecule comprising an aminooxy, hydrazine, hydrazide, or semicarbazide moiety. In certain embodiments, the aminooxy, hydrazine, hydrazide, or semicarbazide moiety is linked to the bioactive molecule by an amide bond. In certain embodiments, the aminooxy, hydrazine, hydrazide, or semicarbazide moiety is linked to the water-soluble polymer or bioactive molecule by a carbamate bond.

[0059] The present invention also provides a method for making an IL-2 conjugate conjugated to a water-soluble polymer. In certain embodiments, the method comprises contacting an isolated IL-2-bioactive molecule conjugate comprising a non-naturally encoded amino acid with a water-soluble polymer comprising a moiety reactive with the non-naturally encoded amino acid. In certain embodiments, the non-naturally encoded amino acid incorporated into the IL-2 conjugate is reactive with a water-soluble polymer that is not reactive with any of the 20 commonly used amino acids. In certain embodiments, the non-naturally encoded amino acid incorporated into the IL-2 conjugate is reactive with a linker, polymer, or bioactive molecule that is not reactive with any of the 20 commonly used amino acids.

[0060] The present invention also provides a method for manufacturing IL-2 or a variant thereof linked to a water-soluble polymer. In certain embodiments, the method comprises contacting an isolated IL-2 or a variant thereof comprising a non-naturally encoded amino acid with a water-soluble polymer comprising a moiety reactive with the non-naturally encoded amino acid. In certain embodiments, the non-naturally encoded amino acid incorporated into the IL-2 or a variant thereof is reactive with a water-soluble polymer that is not reactive with any of the 20 common amino acids. In certain embodiments, the non-naturally encoded amino acid incorporated into the IL-2 is reactive with a linker, polymer, or bioactive molecule that is not reactive with any of the 20 common amino acids.

[0061] In certain embodiments, the IL-2 or a variant thereof linked to a water-soluble polymer is manufactured by reacting an IL-2 or a variant thereof comprising a carbonyl-containing amino acid with a polyethylene glycol molecule comprising an aminooxy, hydrazine, hydrazide, or semicarbazide group. In certain embodiments, the aminooxy, hydrazine, hydrazide, or semicarbazide group is linked to the polyethylene glycol molecule via an amide bond. In certain embodiments, the aminooxy, hydrazine, hydrazide, or semicarbazide group is linked to the polyethylene glycol molecule via a carbamate bond.

[0062] In certain embodiments, the IL-2 or a variant thereof linked to a water-soluble polymer is manufactured by reacting a polyethylene glycol molecule comprising a carbonyl group with a polypeptide comprising a non-naturally encoded amino acid comprising an aminooxy, hydrazine, hydrazide, or semicarbazide group.

[0063] In certain embodiments, the IL-2 or a variant thereof linked to a water-soluble polymer is manufactured by reacting an IL-2 comprising an alkynyl-containing amino acid with a polyethylene glycol molecule comprising an azide moiety. In certain embodiments, the azide or alkynyl group is linked to the polyethylene glycol molecule via an amide bond.

[0064] In certain embodiments, the IL-2 or a variant thereof linked to a water-soluble polymer is manufactured by reacting an IL-2 or a variant thereof comprising an azide-containing amino acid with a polyethylene glycol molecule comprising an alkynyl moiety. In certain embodiments, the azide or alkynyl group is linked to the polyethylene glycol molecule via an amide bond.

[0065] In certain embodiments, the polyethylene glycol molecule has a molecular weight between about 0.1 kDa and about 100 kDa. In certain embodiments, the polyethylene glycol molecule has a molecular weight between 0.1 kDa and 50 kDa. In certain embodiments, the polyethylene glycol has a molecular weight between 1 kDa and 50 kDa, between 1 kDa and 25 kDa, between 2 kDa and 22 kDa, between 5 kDa and 20 kDa, between 5 kDa and 30 kDa, or between 5 kDa and 40 kDa. For example, the molecular weight of the polyethylene glycol polymer can be about 5 kDa or about 10 kDa or about 20 kDa or about 30 kDa or about 40 kDa. For example, the molecular weight of the polyethylene glycol polymer can be 5 kDa, 10 kDa, 20 kDa, 30 kDa, or 40 kDa. In certain embodiments, the polyethylene glycol molecule is 20K 2-branched PEG. In certain embodiments, the polyethylene glycol molecule is 40K 2-branched PEG. In certain embodiments, the polyethylene glycol molecule is 30K branched PEG. In certain embodiments, the polyethylene glycol molecule is 40K or greater branched PEG. In certain embodiments, the polyethylene glycol molecule is linear 5K PEG. In certain embodiments, the polyethylene glycol molecule is linear 10K PEG. In certain embodiments, the polyethylene glycol molecule is linear 15K PEG. In certain embodiments, the polyethylene glycol molecule is linear 20K PEG. In certain embodiments, the polyethylene glycol molecule is linear 25K PEG. In certain embodiments, the polyethylene glycol molecule is linear 30K PEG. In certain embodiments, the polyethylene glycol molecule is linear 35K PEG. In certain embodiments, the polyethylene glycol molecule is linear 40K PEG. In certain embodiments, the polyethylene glycol molecule is linear 45K PEG. In certain embodiments, the polyethylene glycol molecule is linear 50K PEG. In certain embodiments, the polyethylene glycol molecule is linear 60K PEG. In certain embodiments, the molecular weight of the polyethylene glycol polymer is the average molecular weight. In certain embodiments, the average molecular weight is the number average molecular weight (Mn). The average molecular weight can be determined or measured using GPC or SEC, SDS / PAGE analysis, RP-HPLC, mass spectrometry, or capillary electrophoresis. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, or 107, and any combination thereof (corresponding amino acid positions in SEQ ID NO: 2, or SEQ ID NO: 3, 5, or 7), and the IL-2 or a variant thereof is linked to a linear 20K, 30K, 40K, 50K, or 60K polyethylene glycol molecule.In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 35, 37, 42, 45, 49, 61 or 65, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5 or 7), and the IL-2 or variant thereof is linked to a linear 20K or 30K or 40K or 50K or 60K polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into the polypeptide at position 65 in IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5 or 7), and the IL-2 or variant thereof is linked to a linear 20K or 30K or 40K or 50K or 60K polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into the polypeptide at position 61 in IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5 or 7), and the IL-2 or variant thereof is linked to a linear 20K or 30K or 40K or 50K or 60K polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into the polypeptide at position 49 in IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5 or 7), and the IL-2 or variant thereof is linked to a linear 20K or 30K or 40K or 50K or 60K polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into the polypeptide at position 45 in IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5 or 7), and the IL-2 or variant thereof is linked to a linear 20K or 30K or 40K or 50K or 60K polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into the polypeptide at position 42 in IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5 or 7), and the IL-2 or variant thereof is linked to a linear 20K or 30K or 40K or 50K or 60K polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into the polypeptide at position 37 in IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5 or 7), and the IL-2 or variant thereof is linked to a linear 20K or 30K or 40K or 50K or 60K polyethylene glycol molecule.In certain embodiments, a non-naturally encoded amino acid is incorporated into a polypeptide at position 35 of IL-2 or a variant thereof (the corresponding amino acid position in SEQ ID NO: 2, or SEQ ID NO: 3, 5, or 7), and the IL-2 or variant thereof is linked to a linear 20K or 30K or 40K or 50K or 60K polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, or 107, and any combination thereof (the corresponding amino acid positions in SEQ ID NO: 2, or SEQ ID NO: 3, 5, or 7), and the IL-2 or variant thereof is linked to a linear 20K polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 35, 37, 42, 45, 49, 61, or 65, and any combination thereof (the corresponding amino acid positions in SEQ ID NO: 2, or SEQ ID NO: 3, 5, or 7), and the IL-2 or variant thereof is linked to a linear 20K polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, or 107, and any combination thereof (the corresponding amino acid positions in SEQ ID NO: 2, or SEQ ID NO: 3, 5, or 7), and the IL-2 or variant thereof is linked to a linear 30K polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 35, 37, 42, 45, 49, 61, or 65, and any combination thereof (the corresponding amino acid positions in SEQ ID NO: 2, or SEQ ID NO: 3, 5, or 7), and the IL-2 or variant thereof is linked to a linear 30K polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, or 107, and any combination thereof (the corresponding amino acid positions in SEQ ID NO: 2, or SEQ ID NO: 3, 5, or 7), and the IL-2 or variant thereof is linked to a linear 40K polyethylene glycol molecule.In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 35, 37, 42, 45, 49, 61 or 65, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5 or 7), and the IL-2 or a variant thereof is linked to a linear 40K polyethylene glycol molecule.

[0066] In certain embodiments, the polyethylene glycol molecule is a branched polymer. In certain embodiments, each branch of the polyethylene glycol branched polymer has a molecular weight between 1 kDa and 100 kDa or between 1 kDa and 50 kDa. In certain embodiments, each branch of the polyethylene glycol branched polymer has a molecular weight between 1 kDa and 25 kDa or between 2 kDa and 22 kDa or between 5 kDa and 20 kDa or between 5 kDa and 30 kDa or between 5 kDa and 40 kDa or between 5 kDa and 50 kDa or between 5 kDa and 60 kDa. For example, the molecular weight of each branch of the polyethylene glycol branched polymer can be about 5 kDa or about 10 kDa or about 20 kDa or about 30 kDa or about 40 kDa or about 50 kDa or about 60 kDa or greater. For example, the molecular weight of each branch of the polyethylene glycol branched polymer can be 5 kDa or 10 kDa or 15 kDa or 20 kDa or 25 kDa or 30 kDa or 35 kDa or 40 kDa or 45 kDa or 50 kDa or 55 kDa or 60 kDa or greater. In certain embodiments, the polyethylene glycol molecule is 20K 2-branched PEG. In certain embodiments, the polyethylene glycol molecule is 20K 4-branched PEG. In certain embodiments, the polyethylene glycol molecule is 40K 2-branched PEG. In certain embodiments, the molecular weight of the polyethylene glycol polymer is the average molecular weight. In certain embodiments, the average molecular weight is the number-average molecular weight (Mn). The average molecular weight can be determined or measured using GPC or SEC, SDS / PAGE analysis, RP-HPLC, mass spectrometry, or capillary electrophoresis. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, or 107, and any combination thereof (the corresponding amino acid positions in SEQ ID NO: 2, or SEQ ID NO: 3, 5, or 7), and the IL-2 or a variant thereof is linked to a branched 20K polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 35, 37, 42, 45, 49, 61, or 65, and any combination thereof (the corresponding amino acid positions in SEQ ID NO: 2, or SEQ ID NO: 3, 5, or 7), and the IL-2 or a variant thereof is linked to a branched 20K polyethylene glycol molecule.In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, or 107, and any combination thereof (the corresponding amino acid positions in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is conjugated to a branched 30K polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 35, 37, 42, 45, 49, 61, or 65, and any combination thereof (the corresponding amino acid positions in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is conjugated to a branched 30K polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, or 107, and any combination thereof (the corresponding amino acid positions in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is conjugated to a branched 40K polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 35, 37, 42, 45, 49, 61, or 65, and any combination thereof (the corresponding amino acid positions in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is conjugated to a branched 40K polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, or 107, and any combination thereof (the corresponding amino acid positions in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is conjugated to a 20K 2-branched or 40K 2-branched polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 35, 37, 42, 45, 49, 61, or 65, and any combination thereof (the corresponding amino acid positions in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is conjugated to a 20K 2-branched or 40K 2-branched polyethylene glycol molecule.In certain embodiments, a non-naturally encoded amino acid is incorporated into position 65 of IL-2 or a variant thereof (the corresponding amino acid position in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is linked to a 20K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 61 of IL-2 or a variant thereof (the corresponding amino acid position in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is linked to a 20K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 49 of IL-2 or a variant thereof (the corresponding amino acid position in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is linked to a 20K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 45 of IL-2 or a variant thereof (the corresponding amino acid position in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is linked to a 20K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 42 of IL-2 or a variant thereof (the corresponding amino acid position in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is linked to a 20K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 37 of IL-2 or a variant thereof (the corresponding amino acid position in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is linked to a 20K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 35 of IL-2 or a variant thereof (the corresponding amino acid position in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is linked to a 20K 2-branched polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions of IL-2 or a variant thereof: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, or 107, and any combination thereof (the corresponding amino acid positions in SEQ ID NO:2, or SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is linked to a 20K 4-branched polyethylene glycol molecule.In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 35, 37, 42, 45, 49, 61 or 65, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5 or 7), and the IL-2 or variant thereof is linked to a 20K 4-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 65 in IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5 or 7), and the IL-2 or variant thereof is linked to a 20K 4-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 61 in IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5 or 7), and the IL-2 or variant thereof is linked to a 20K 4-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 49 in IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5 or 7), and the IL-2 or variant thereof is linked to a 20K 4-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 45 in IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5 or 7), and the IL-2 or variant thereof is linked to a 20K 4-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 42 in IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5 or 7), and the IL-2 or variant thereof is linked to a 20K 4-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 37 in IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5 or 7), and the IL-2 or variant thereof is linked to a 20K 4-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 35 in IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5 or 7), and the IL-2 or variant thereof is linked to a 20K 4-branched polyethylene glycol molecule.In certain embodiments, a non-naturally encoded amino acid is incorporated into position 65 of IL-2 or a variant thereof (the corresponding amino acid position in SEQ ID NO: 2, or SEQ ID NO: 3, 5, or 7), and the IL-2 or variant thereof is conjugated to a 40K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 61 of IL-2 or a variant thereof (the corresponding amino acid position in SEQ ID NO: 2, or SEQ ID NO: 3, 5, or 7), and the IL-2 or variant thereof is conjugated to a 40K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 49 of IL-2 or a variant thereof (the corresponding amino acid position in SEQ ID NO: 2, or SEQ ID NO: 3, 5, or 7), and the IL-2 or variant thereof is conjugated to a 40K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 45 of IL-2 or a variant thereof (the corresponding amino acid position in SEQ ID NO: 2, or SEQ ID NO: 3, 5, or 7), and the IL-2 or variant thereof is conjugated to a 40K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 42 of IL-2 or a variant thereof (the corresponding amino acid position in SEQ ID NO: 2, or SEQ ID NO: 3, 5, or 7), and the IL-2 or variant thereof is conjugated to a 40K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 37 of IL-2 or a variant thereof (the corresponding amino acid position in SEQ ID NO: 2, or SEQ ID NO: 3, 5, or 7), and the IL-2 or variant thereof is conjugated to a 40K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 35 of IL-2 or a variant thereof (the corresponding amino acid position in SEQ ID NO: 2, or SEQ ID NO: 3, 5, or 7), and the IL-2 or variant thereof is conjugated to a 40K 2-branched polyethylene glycol molecule.

[0067] In certain embodiments, the water-soluble polymer linked to IL-2 or a variant thereof comprises a polyalkylene glycol moiety. In certain embodiments, the non-naturally encoded amino acid residue incorporated into IL-2 comprises a carbonyl group, an aminoxy group, a hydrazide group, a hydrazine, a semicarbazide group, an azide group or an alkyne group. In certain embodiments, the non-naturally encoded amino acid residue incorporated into IL-2 or a variant thereof comprises a carbonyl moiety, and the water-soluble polymer comprises an aminoxy, hydrazide, hydrazine or semicarbazide moiety. In certain embodiments, the non-naturally encoded amino acid residue incorporated into IL-2 or a variant thereof comprises an alkyne moiety, and the water-soluble polymer comprises an azide moiety. In certain embodiments, the non-naturally encoded amino acid residue incorporated into IL-2 or a variant thereof comprises an azide moiety, and the water-soluble polymer comprises an alkyne moiety.

[0068] The present invention also provides a composition comprising IL-2 or a variant thereof containing a non-naturally encoded amino acid and a pharmaceutically acceptable carrier. In certain embodiments, the non-naturally encoded amino acid is linked to a water-soluble polymer.

[0069] The present invention also provides a cell comprising a polynucleotide encoding IL-2 or an IL-2 variant comprising a selector codon. In certain embodiments, the cell comprises an orthogonal RNA synthetase and / or an orthogonal tRNA for replacing a non-naturally encoded amino acid into the IL-2.

[0070] The present invention also provides a cell comprising a polynucleotide encoding IL-2 or a variant thereof comprising a selector codon. In certain embodiments, the cell comprises an orthogonal RNA synthetase and / or an orthogonal tRNA for replacing a non-naturally encoded amino acid into the IL-2 or a variant thereof.

[0071] In certain embodiments, the present invention provides a method for modulating receptor interaction of the IL-2 polypeptide of the present invention. In certain embodiments, the present invention provides a method for inhibiting or reducing the interaction of the PEGylated IL-2 with the IL2Rα subunit of the trimeric IL-2 receptor using the PEGylated IL-2 polypeptide of the present invention.

[0072] The present invention also provides a method for manufacturing PEG-IL-2, IL-2 or any variant thereof containing a non-naturally encoded amino acid. In certain embodiments, the method comprises culturing a cell comprising one or more polynucleotides encoding IL-2, an orthogonal RNA synthetase and / or an orthogonal tRNA under conditions allowing the expression of the IL-2 or a variant thereof; and purifying the IL-2 or a variant thereof from the cell and / or the culture medium.

[0073] The present invention also provides methods for increasing the therapeutic half-life, serum half-life or circulation time of IL-2 or its variants. In certain embodiments, the half-life (t 1 / 2 ) or circulation time of IL-2 or an IL-2 variant or a PEGylated IL-2 conjugate or a glycosylated IL-2 conjugate is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, 96, 120, 240 hours or longer. The present invention also provides methods for modulating the immunogenicity of IL-2 or its variants. In certain embodiments, the method includes replacing any one or more amino acids in a naturally occurring IL-2 or its variant with non-naturally encoded amino acids and / or conjugating the IL-2 or its variant to a linker, polymer, water-soluble polymer or bioactive molecule. In one embodiment of the present invention, the linker is long enough to allow flexibility and allow dimer formation. In one embodiment of the present invention, the linker has a length of at least 3 amino acids or 18 atoms to allow dimer formation.

[0074] The present invention also provides methods for treating a patient in need thereof with an effective amount of a PEG-IL-2 conjugate or a variant thereof of the present invention. In certain embodiments, the method includes administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising a PEG-IL-2 or a variant thereof containing non-naturally encoded amino acids and a pharmaceutically acceptable carrier. In certain embodiments, the method includes administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising a PEG-IL-2 or a variant thereof containing non-naturally encoded amino acids and natural amino acid substitutions and a pharmaceutically acceptable carrier. In certain embodiments, the non-naturally encoded amino acids are conjugated to a water-soluble polymer. In certain embodiments, the PEG-IL-2 or a variant thereof is glycosylated. In certain embodiments, the PEG-IL-2 or a variant thereof is not glycosylated.

[0075] The present invention also provides a method of treating a patient in need thereof with an effective amount of the IL-2 or IL-2 variant molecule of the present invention. In certain embodiments, the method comprises administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising an IL-2 or IL-2 variant molecule containing a non-naturally encoded amino acid and a pharmaceutically acceptable carrier. In certain embodiments, the method comprises administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising an IL-2 or a variant thereof containing one or more non-naturally encoded amino acids and one or more naturally occurring amino acid substitutions and a pharmaceutically acceptable carrier. In certain embodiments, the non-naturally encoded amino acid is linked to a water-soluble polymer. In certain embodiments, the naturally occurring amino acid is linked to a water-soluble polymer. In certain embodiments, the IL-2 is glycosylated. In certain embodiments, the IL-2 is not glycosylated. In certain embodiments, the patient in need of treatment has a cancer, disorder or disease characterized by high expression of IL-2 receptor α, but is not limited thereto. In certain embodiments, the present invention provides a method of treating a cancer or disorder or disease by administering to a subject a therapeutically effective amount of the IL-2 composition of the present invention. In certain embodiments, the present invention provides a method of treating a genetic disorder by administering to a patient a therapeutically effective amount of the IL-2 composition of the present invention. The IL-2 polypeptide of the present invention is used to treat a disease or disorder in cells having high expression of IL-2 receptor α. In certain embodiments, the cancer, disorder or disease is treated by reducing, blocking or silencing IL-2 receptor α expression. The IL-2 polypeptide or variant of the present invention is used to manufacture a medicament for treating a cancer, disease or disorder associated with high expression of IL-2 receptor α. The IL-2 polypeptide or variant of the present invention is used to manufacture a medicament for treating a cancer. The IL-2 polypeptide or variant of the present invention is used to manufacture a medicament for treating a genetic disorder.

[0076] The present invention also provides an IL-2 comprising the sequences shown in SEQ ID NO: 1, 2, 3, 5 or 7 or any other IL-2 sequence, provided that at least one amino acid is replaced by a non-naturally encoded amino acid. In certain embodiments, the present invention provides novel IL-2 polypeptides corresponding to SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23, and at least one amino acid is replaced by a non-naturally encoded amino acid. In certain embodiments, the present invention provides novel IL-2 polypeptides comprising SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23, and site-specifically incorporating non-naturally encoded amino acids. In certain embodiments, the non-naturally encoded amino acid is linked to a water-soluble polymer. In certain embodiments, the water-soluble polymer comprises a polyethylene glycol moiety. In certain embodiments, the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group or an alkyne group.

[0077] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a PEG-IL-2 or a native variant thereof comprising the sequence shown in SEQ ID NO: 1, 2, 3, 5 or 7 or any other IL-2 sequence, provided that at least one amino acid is replaced by a non-naturally encoded amino acid. The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an IL-2 or a native variant thereof comprising the sequence shown in SEQ ID NO: 1, 2, 3, 5 or 7. In certain embodiments, the non-naturally encoded amino acid comprises a sugar moiety. In certain embodiments, the water-soluble polymer is linked to the IL-2 or its native variant through a sugar moiety. In certain embodiments, a linker, a polymer or a bioactive molecule is linked to the IL-2 or its native variant through a sugar moiety.

[0078] The present invention also provides an IL-2 or a native variant thereof comprising a water-soluble polymer covalently linked to the IL-2 at a single amino acid. In certain embodiments, the water-soluble polymer comprises a polyethylene glycol moiety. In certain embodiments, the amino acid covalently linked to the water-soluble polymer is a non-naturally encoded amino acid present in the polypeptide.

[0079] The present invention provides an IL-2 or a variant thereof comprising at least one linker, polymer, or bioactive molecule, wherein the linker, polymer, or bioactive molecule is attached to the polypeptide through a functional group of a non-naturally encoded amino acid incorporated into the polypeptide via ribosome. In certain embodiments, the IL-2 or its variant is mono-PEGylated. The present invention also provides an IL-2 or a variant thereof comprising a linker, polymer, or bioactive molecule attached to one or more non-naturally encoded amino acids, wherein the non-naturally encoded amino acids are incorporated into the polypeptide via ribosome at preselected sites.

[0080] Within the scope of the present invention are the leader or signal sequences of IL-2 or its variants linked to the IL-2 coding region and heterologous signal sequences linked to the IL-2 coding region. The selected heterologous leader or signal sequence should be a sequence that is recognized and processed by the host cell secretion system for secretion and is possibly cleaved by the host cell signal peptidase. The method of treating a disease or disorder with the IL-2 of the present invention implies treating with IL-2 or its variant with or without a signal or leader peptide.

[0081] In another embodiment, the conjugation of the IL-2 or its variant comprising one or more non-naturally occurring amino acids with another molecule (including but not limited to PEG) provides substantially purified IL-2 due to the unique chemical reaction used for conjugating with the non-natural amino acids. The conjugation of the IL-2 or its variant comprising one or more non-naturally encoded amino acids with another molecule such as PEG can be carried out using other purification techniques before or after the conjugation step to provide substantially pure IL-2 or its variant.

[0082] In certain embodiments, the present invention provides a modified IL-2 polypeptide for use in preparing a medicament. In certain embodiments, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of IL-2 and a pharmaceutically acceptable carrier or excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 A model depicting a view of the IL-2 polypeptide, wherein potential receptor interaction sites are labeled with the structure of IL-2Rα and its interface with IL-2.

[0084] Figure 2 A plasmid map of an expression vector for expressing IL-2 in Escherichia coli is depicted.

[0085] Figures 3A - 3B A Western blot analysis of the expression of IL-2 protein in Escherichia coli is depicted ( Figure 3A ) and the titer of IL-2 variants in Escherichia coli ( Figure 3B ).

[0086] Figures 4A - 4B Depicts the binding kinetics sensorgram and model fitting lines and calculated measurements of wild-type IL-2 with CD25( Figure 4A ), and the plasmid map of the expression vector for expressing IL-2 in mammalian cells( Figure 4B ).

[0087] Figure 5 Shows the design of the UPF1 genomic DNA sequence and CRISPR gRNA sites.

[0088] Figure 6 Depicts the sequence verification of the UPF1 knockout cell line.

[0089] Figures 7A - 7B Depicts the transient expression of various different IL-2 variants in mammalian cells( Figure 7A ) and Western blot analysis of wild-type IL-2 and IL-2 variants produced in mammalian cells( Figure 7B ).

[0090] Figure 8 Depicts the CTLL-2 proliferation assay of the F42 variant of IL-2.

[0091] Figure 9 Shows the screening of IL-2 variants by CTLL-2 proliferation assay.

[0092] Figures 10A - 10C Depicts the binding kinetics sensorgram of wild-type IL-2 and F42 variant( Figure 10A ), the binding kinetics sensorgram of K35 and Y45 variants( Figure 10B ), and the binding kinetics sensorgram of T37 and P65 variants( Figure 10C ).

[0093] Figure 11 Shows an illustrative diagram of the IL-2 receptor dimerization assay.

[0094] Figure 12 Shows an illustrative diagram of the ex vivo pSTAT5 assay.

[0095] Figure 13 Depicts the clonal growth and long-term proliferation of CTLL-2 cells in the presence of glycosylated or non-glycosylated IL-2.

[0096] Figure 14 Shows a comparison of the titers before and after the production of the stable pools of the corresponding wild-type IL-2 or its selected variants.

[0097] Figures 15A - 15CDepicts the titer in mammalian cells expressing the F42-R38A variant ( Figure 15A ), the CTLL-2 binding assay of the F42-R38A variant ( Figure 15B ), and the binding kinetics sensorgram of the F42-R38A variant ( Figure 15C ).

[0098] Figure 16 Depicts the plot of the mean plasma concentration over time for the Y45-PEG20K-BR2 and F42-R38A-PEG20K-BR2 variants.

[0099] Figures 17A - 17D Depicts the binding kinetics sensorgrams of IL-2 wild type (WT; Figure 17A ) with F42-R38A-P65R-PEG20K-BR2 ( Figure 17B ), IL2-Y45-M46L-PEG20K-BR2 ( Figure 17C ), and IL2-Y45-M46I-PEG20K-BR2 ( Figure 17D ) variants.

[0100] Figure 18 Shows the CTLL-2 cell proliferation assay of the PEGylated IL-2 variants.

[0101] Figure 19 Depicts the curve of the mean plasma concentration over time for the PEGylated IL-2 variants.

[0102] Figures 20A - 20B Depicts the activity of the PEGylated IL-2 variants on tumor volume ( Figure 20A ) and body weight ( Figure 20B ).

[0103] Figures 21A - 21B Depicts the effects of the IL-2 variants F42-R38A-P65R-PEG30K-L, F42-R38A-P65R-PEG40K-BR2, Y45-PEG30K-L, and Y45-PEG40K-BR2 at 2 mg / kg ( Figure 21A ) and 5 - 8 mg / kg ( Figure 21B ) on B16F10 tumor growth inhibition in C57BL / 6 mice.

[0104] Figure 22 Depicts the final tumor volume in BALB / c mice bearing B16F10 tumors.

[0105] Figures 23A - 23C Depicts the PEGylated IL-2 variant F42-R38A-P65R-PEG30K-L ( Figure 23A) and Y45-PEG30K-L( Figure 23B ) on CT26 tumor growth inhibition and mouse body weight( Figure 23C ) effects.

[0106] Figure 24 Depicts the final tumor volume in BALB / c mice bearing CT26 tumors.

[0107] Figures 25A - 25C Depicts the effects of the PEGylated IL-2 variants F42-R38A-P65R-PEG30K-L and Y45-PEG30K-L on CD8+ cells( Figure 25A ) and CD4+ cells( Figure 25B ) in the blood of mice bearing CT26 tumors and the CD8+ / CD4+ ratio( Figure 25C ) effects.

[0108] Figure 26 Depicts the melting temperature of wild-type IL-2 analyzed by DSF.

[0109] Definition

[0110] It should be understood that the present invention is not limited to the specific methods, protocols, cell lines, constructs, and reagents described herein, and that they can vary themselves. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present invention, the scope of which is limited only by the appended claims.

[0111] When used herein and in the appended claims, the singular forms without a specific quantity include plural referents unless the context clearly indicates otherwise. Thus, for example, references to "IL-2", "PEG-IL-2", "PEG-IL-2 conjugate", and various different capitalized, hyphenated, and non-hyphenated forms refer to one or more such proteins and include their equivalents known to those of ordinary skill in the art, and so on.

[0112] 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 to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials will now be described.

[0113] All publications and patents mentioned herein are incorporated herein by reference for the purpose of describing and disclosing, for example, the constructs and methods described in the publications that can be used in conjunction with the presently described invention. The publications discussed herein are provided solely because of their publication prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or any other reason.

[0114] The term "substantially purified" refers to an IL-2 or variant thereof that is substantially or essentially free of components that are normally associated with or interact with the protein in its natural environment, i.e., in the native cell or, in the case of recombinantly produced IL-2, in the host cell. IL-2 that is substantially free of cellular material includes protein preparations having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating protein. When the IL-2 or variant thereof is recombinantly produced by a host cell, the protein can be present in an amount of about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cell. When the IL-2 or variant thereof is recombinantly produced by a host cell, the protein can be present in the culture medium in an amount of about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L or less. Thus, "substantially purified" IL-2 produced by the methods of the present invention can have a purity level of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, particularly at least about 75%, 80%, 85%, more particularly at least about 90%, at least about 95%, at least about 99% or higher, as determined by suitable methods such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0115] "Recombinant host cell" or "host cell" refers to a cell that comprises an exogenous polynucleotide, regardless of the method used for insertion, e.g., direct uptake, transduction, f-mating, or other methods known in the art for generating recombinant host cells. The exogenous polynucleotide can be maintained as a non-integrated vector, such as a plasmid, or can be integrated into the host genome.

[0116] As used herein, the term "culture medium" includes any culture medium, solution, solid, semi-solid or rigid support that can support or contain any host cell, including bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, Escherichia coli or Pseudomonas host cells, and cell inclusions. Thus, the term can encompass the culture medium in which the host cells have grown, such as the culture medium into which IL-2 has been secreted, including the culture medium before or after the proliferation step. The term can also encompass a buffer or reagent containing host cell lysates, such as in the case where IL-2 is produced intracellularly and the host cells are lysed or disrupted to release IL-2.

[0117] When used herein in the context of protein refolding, a "reducing agent" is defined as any compound or material that maintains sulfhydryl groups in a reduced state and reduces intra- or intermolecular disulfide bonds. Suitable reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2-aminoethanethiol), and reduced glutathione. It will be apparent to those of ordinary skill in the art that a wide variety of reducing agents are suitable for use in the methods and compositions of the present invention.

[0118] When used herein in the context of protein refolding, an "oxidizing agent" is defined as any compound or material that is capable of removing electrons from an oxidized compound. Suitable oxidizing agents include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythritol, and oxygen. It will be apparent to those of ordinary skill in the art that a wide variety of oxidizing agents are suitable for use in the methods of the present invention.

[0119] As used herein, "denaturant" is defined as any compound or material that causes reversible unfolding of a protein. The strength of a denaturant is determined by both the nature and concentration of the particular denaturant. Suitable denaturants can be chaotropes, detergents, organic solvents, water-miscible solvents, phospholipids, or combinations of two or more such reagents. Suitable chaotropes include, but are not limited to, urea, guanidine, and sodium thiocyanate. Useful detergents can include, but are not limited to, strong detergents such as sodium dodecyl sulfate or polyoxyethylene ethers (e.g., Tween or Triton detergents), Sarkosyl, mild nonionic detergents (e.g., digitonin), mild cationic detergents such as N->2,3-(dioleoyloxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents (e.g., sodium cholate or sodium deoxycholate), or zwitterionic detergents, including but not limited to sulfobetaine (Zwittergent), 3-(3-cholamidopropyl)dimethylammonium-1-propane sulfate (CHAPS), and 3-(3-cholamidopropyl)dimethylammonium-2-hydroxy-1-propane sulfonate (CHAPSO). Water-miscible organic solvents such as acetonitrile, lower alkanols (especially C2-C4 alkanols such as ethanol or isopropanol), or lower alkylene glycols (especially C2-C4 alkylene glycols such as ethylene glycol) can be used as denaturants. Phospholipids useful in the present invention can be naturally occurring phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol, or synthetic phospholipid derivatives or variants such as dihexanoyl phosphatidylcholine or diheptanoyl phosphatidylcholine.

[0120] As used herein, "refolding" describes any process, reaction, or method of converting a polypeptide containing disulfide bonds from an incorrectly folded or unfolded state to a native or correctly folded conformation for the disulfide bonds.

[0121] As used herein, "co-folding" specifically refers to a refolding process, reaction, or method that uses at least two polypeptides that interact with each other and causes an unfolded or incorrectly folded polypeptide to be converted into a native, correctly folded polypeptide.

[0122] As used herein, "Interleukin-2", "IL-2", and their hyphenated and non-hyphenated forms include polypeptides and proteins having at least one biological activity of IL-2, as well as IL-2 analogs, IL-2 mutant proteins, IL-2 variants, IL-2 subtypes, IL-2 mimetics, IL-2 fragments, chimeric IL-2 proteins, fusion proteins, oligomers and polymers, homologs, variants of glycosylation patterns, variants, spliced variants, and mutant proteins, regardless of their biological activity and regardless of the method of synthesis and manufacture, which methods include, but are not limited to, recombinant (produced from cDNA, genomic DNA, synthetic DNA, or other forms of nucleic acid), in vitro, in vivo, by microinjection of nucleic acid molecules, synthesis, transgenic, and gene activation methods. The terms "IL-2", "IL-2 variant", and "IL-2 polypeptide" encompass IL-2s that contain one or more amino acid substitutions, additions, or deletions.

[0123] The sequence of IL-2 lacking a leader sequence and having no methionine at the N-terminus is shown as SEQ ID NO: 2 herein. The sequence of IL-2 having no leader sequence and having methionine at the N-terminus is shown as SEQ ID NO: 3, 5, or 7. In certain embodiments, the IL-2 or its variant of the present invention is substantially identical to SEQ ID NO: 2, 3, 5, or 7 or any other sequence of IL-2. Nucleic acid molecules encoding IL-2 (including mutant IL-2 and other variants), as well as methods for expressing and purifying these polypeptides, are well known in the art.

[0124] The term "IL-2" also includes pharmaceutically acceptable salts and prodrugs of naturally occurring IL-2 and prodrugs of said salts, polymorphs, hydrates, solvates, bioactive fragments, bioactive variants, and stereoisomers, as well as agonist, mimetic, and antagonist variants of naturally occurring IL-2 and polypeptide fusions thereof.

[0125] Various different references disclose modifications of polypeptides by polymer conjugation or glycosylation. The term "IL-2" includes polypeptides conjugated to polymers such as PEG and may contain one or more additional derivatizations of cysteine, lysine, or other residues. Additionally, the IL-2 may contain a linker or polymer, where the amino acid to which the linker or polymer is conjugated may be a non-natural amino acid according to the present invention or may be conjugated to a naturally encoded amino acid using techniques known in the art, such as conjugation to lysine or cysteine.

[0126] The term "IL-2 polypeptide" also includes glycosylated IL-2, such as but not limited to polypeptides glycosylated at any amino acid position, N-linked or O-linked glycosylated forms of said polypeptides. Variants containing single nucleotide changes are also regarded as bioactive variants of the IL-2 polypeptide. In addition, splice variants are also included.

[0127] The term "IL-2" also includes IL-2 heterodimers, homodimers, heteromultimers or homotrimers of any one or more IL-2s or any other polypeptides, proteins, carbohydrates, polymers, small molecules, linkers, ligands or any other type of bioactive molecule linked by chemical means or expressed as a fusion protein, as well as polypeptide analogs containing, for example, specific deletions or other modifications but still maintaining biological activity.

[0128] As used herein, whether conjugated to a bioactive molecule, conjugated to polyethylene glycol or in an unconjugated form, "interleukin-2" or "IL-2" is a protein that comprises two subunits non-covalently associated to form a homodimer. As used herein, "interleukin-2" and "IL-2" can refer to human or murine IL-2, which is also referred to as "hIL-2" or "mIL-2".

[0129] The terms "PEGylated IL-2", "PEGylated IL-2" or "PEG-IL-2" are IL-2 molecules having one or more polyethylene glycol molecules covalently attached through a linker to one or more amino acid residues of the IL-2 protein such that the attachment is stable. The terms "mono-PEGylated IL-2" and "mono-PEG-IL-2" mean that one polyethylene glycol molecule is covalently attached through a linker to a single amino acid residue of one subunit of the IL-2 dimer. The average molecular weight of the PEG moiety is preferably between about 5,000 and about 50,000 daltons. The method or site of PEG attachment to IL-2 is not critical, but preferably the PEGylation does not change or only minimally changes the activity of the bioactive molecule. Preferably, the increase in half-life exceeds any decrease in biological activity.

[0130] All references to amino acid positions in IL-2 described herein are based on the positions in SEQ ID NO:2, unless otherwise indicated (i.e., when stating that the comparison is based on SEQ ID NO:3, 5, or 7 or other IL-2). Those skilled in the art will recognize that the amino acid positions corresponding to the positions in SEQ ID NO:2 in any other IL-2, such as SEQ ID NO:3, 5, or 7, can be readily identified. Those skilled in the art will recognize that the amino acid positions corresponding to the positions in SEQ ID NO:2, 3, 5, or 7 or any other IL-2 sequence in any other IL-2 molecule, such as an IL-2 fusion, variant, fragment, etc., can be readily identified. For example, sequence alignment programs such as BLAST can be used to align and identify the specific positions in the protein corresponding to the positions in SEQ ID NO:2, 3, 5, or 7 or other IL-2 sequences. Substitutions, deletions, or additions of amino acids described herein with reference to SEQ ID NO:2, 3, 5, or 7 or other IL-2 sequences are intended to also refer to substitutions, deletions, or additions in the corresponding positions in IL-2 fusions, variants, fragments, etc. described herein or known in the art, and are expressly covered by the present invention.

[0131] IL-2 (IL2): Any form of IL-2 known in the art can be used in the compositions described herein. For experimental work, the murine form of IL-2 is particularly useful. Those skilled in the art will recognize that certain amino acid residues in IL2 can be altered without affecting its activity, and these modified forms of IL2 can also be conjugated to a carrier and used in the methods described herein.

[0132] The term "interleukin-2" or "IL-2" encompasses IL-2 containing one or more amino acid substitutions, additions, or deletions. The IL-2 of the present invention can contain modifications of one or more natural amino acids in combination with one or more non-natural amino acid modifications. Exemplary substitutions in a wide variety of amino acid positions in naturally occurring IL-2 polypeptides have been described, including but not limited to substitutions that modulate drug stability, modulate one or more biological activities of the IL-2 polypeptide such as but not limited to enhancing agonist activity, enhancing polypeptide solubility, reducing protease sensitivity, converting the polypeptide into an antagonist, etc., and are encompassed by the term "IL-2 polypeptide". In certain embodiments, the IL-2 antagonist contains a non-naturally encoded amino acid linked to a water-soluble polymer, which is present in the receptor binding region of the IL-2 molecule.

[0133] In certain embodiments, the IL-2 or variant thereof further comprises an addition, substitution, or deletion that modulates the biological activity of the IL-2 or variant polypeptide. In certain embodiments, the IL-2 or variant further comprises an addition, substitution, or deletion that modulates one or more known and experimentally verified properties of IL-2, such as the treatment or alleviation of one or more symptoms of cancer. The addition, substitution, or deletion can modulate one or more properties or activities of the IL-2 or variant. For example, the addition, substitution, or deletion can modulate the affinity for the IL-2 receptor or one or more subunits of the receptor, modulate the circulating half-life, modulate the therapeutic half-life, modulate the stability of the polypeptide, modulate cleavage by proteases, modulate the dose, modulate the release or bioavailability, facilitate purification, or improve or alter a particular route of administration. Similarly, the IL-2 or variant can comprise a protease cleavage sequence, a reactive group, an antibody binding domain (including but not limited to FLAG or poly-His), or other affinity-based sequences (including but not limited to FLAG, poly-His, GST, etc.) or a conjugated molecule (including but not limited to biotin), which improve the detection (including but not limited to GFP), purification, or other properties of the polypeptide.

[0134] The term "IL-2 polypeptide" also encompasses conjugated homodimers, heterodimers, homopolymers, or heteropolymers, including but not limited to those directly conjugated to the same or different non-naturally encoded amino acid side chains, conjugated to a naturally encoded amino acid side chain, or indirectly conjugated via a linker. Exemplary linkers include but are not limited to small organic compounds, water-soluble polymers of various different lengths such as polyethylene glycol or dextran, or polypeptides of various different lengths.

[0135] As used herein, the terms "conjugate of the invention", "IL-2-bioactive molecule conjugate", or "PEG-IL-2" refer to interleukin-2 or a portion, analogue, or derivative thereof that binds to the interleukin-2 receptor or a subunit thereof and is conjugated to a bioactive molecule, a portion thereof, or an analogue thereof. Unless otherwise specified, the terms "compound of the invention" and "composition of the invention" are used as alternative terms for the term "conjugate of the invention".

[0136] As used herein, the term "cytotoxic agent" can be any agent that has a therapeutic effect on cancer cells or activated immune cells and can be used as a therapeutic agent in combination with IL-2, PEG-IL-2 or an IL-2 variant (see, e.g., WO 2004 / 010957, "Drug Conjugates and Their Use for Treating Cancer, An Autoimmune Disease or an Infectious Disease"). Classes of cytotoxic or immunosuppressive agents for use in the present invention include, for example, antimicrotubule agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, monoplatins, diplatins and trinuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemosensitizers, esperamicins, etoposides, fluoropyrimidines, ionophores, lexitropsins, nitrosoureas, platinum alcohols, preformed compounds, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, etc.

[0137] Individual cytotoxic or immunosuppressive agents include, for example, androgens, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytarabine glycoside, cytochalasin B, dacarbazine, dactinomycin (formerly called actinomycin), daunorubicin, dacarbazine, docetaxel, doxorubicin, estrogen, 5-fluorodeoxyuridine, 5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel, plicamycin, procarbazine, streptozotocin, teniposide, 6-thioguanine, thiotepa, topotecan, vinblastine, vincristine, vinorelbine, VP-16 and VM-26.

[0138] In certain exemplary embodiments, the therapeutic agent is a cytotoxic agent. Suitable cytotoxic agents include, for example, dolastatins (e.g., auristatin E, AFP, MMAF, MMAE), DNA minor groove binders (e.g., enediynes and lexitropsins), calicheamicins, taxanes (e.g., paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin, lysocine, cyano-morpholino-doxorubicin, echinosporin, combretastatin, distamycin, epothilones A and B, estramustine, cryptophycins, zimadotin, maytansinoids, discodermolide, acanthopanaxsaponin, and mitoxantrone.

[0139] "Unnaturally encoded amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. Other terms that may be used synonymously with the term "unnaturally encoded amino acid" are "non-natural amino acid", "non-naturally occurring amino acid", and their various hyphenated and non-hyphenated forms. The term "unnaturally encoded amino acid" also includes, but is not limited to, amino acids that are produced by modification of a naturally encoded amino acid (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine), but that are not themselves naturally incorporated into the growing polypeptide chain by the translation complex. Examples of such non-naturally occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

[0140] "Amino-terminal modifying group" refers to any molecule that can be attached to the amino terminus of a polypeptide. Similarly, "carboxyl-terminal modifying group" refers to any molecule that can be attached to the carboxyl terminus of a polypeptide. Terminal modifying groups include, but are not limited to, various water-soluble polymers, peptides or proteins such as serum albumin or other components that increase the serum half-life of the peptide.

[0141] The terms "functional group", "active moiety", "activating group", "leaving group", "reactive site", "chemically reactive group", and "chemically reactive moiety" are used in the art and herein to refer to distinct, definable portions or units of a molecule. The terms are somewhat synonymous in the chemical art and are used herein to denote portions of a molecule that perform certain functions or activities and are reactive with other molecules.

[0142] The terms “linkage”, “link” or “connector” are used herein to refer to a group or bond that is typically formed as a result of a chemical reaction and is typically a covalent bond. A hydrolysis-stable bond means that the bond is substantially stable in water and does not react with water for an extended period, and possibly indefinitely, at useful pH values, including but not limited to physiological conditions. A hydrolysis-labile or degradable bond means that the bond is degradable in water or an aqueous solution, including for example blood. An enzyme-labile or degradable bond means that the bond can be degraded by one or more enzymes. As is understood in the art, PEG and related polymers can include degradable bonds in the polymer backbone or in the linker groups between the polymer backbone and one or more terminal functional groups of the polymer molecule. For example, an ester bond formed by the reaction of a PEG carboxylic acid or an activated PEG carboxylic acid with an alcohol group on a bioactive agent is typically hydrolyzed under physiological conditions to release the agent. Other hydrolytically degradable bonds include but are not limited to carbonate bonds, imine bonds obtained from the reaction of an amine with an aldehyde, phosphate ester bonds formed by the reaction of an alcohol with a phosphate group, hydrazone bonds as a byproduct of the reaction of a hydrazide with an aldehyde, acetal bonds as the reaction product of an aldehyde with an alcohol, orthoester bonds as the reaction product of formic acid with an alcohol, peptide bonds formed from an amino group at the terminus of a polymer, including but not limited to PEG, and a carboxyl group of a peptide, and oligonucleotide bonds formed from a phosphoramidite group at the terminus of a polymer, including but not limited to a polymer, and the 5′-hydroxyl of an oligonucleotide.

[0143] As used herein, the terms "bioactive molecule", "bioactive component", or "bioactive agent" mean any substance that can affect any physical or biochemical property of a biological system, pathway, molecule, or interaction associated with an organism, including but not limited to viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans. Specifically, as used herein, bioactive molecules include but are not limited to any substance intended for use in diagnosing, curing, alleviating, treating, or preventing a disease in humans or other animals or otherwise enhancing the physical or mental health of humans or animals. Examples of bioactive molecules include but are not limited to peptides, proteins, enzymes, small molecule drugs, vaccines, immunogens, addictive drugs, non-addictive drugs, saccharides, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxoids, bioactive molecules, prokaryotic and eukaryotic cells, viruses, polysaccharides, nucleic acids and portions thereof derived from or originating from viruses, bacteria, insects, animals, or any other cell or cell type, liposomes, microparticles, and micelles. Classes of bioactive agents suitable for use in the present invention include but are not limited to drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, bile acid resins, niacin and / or statins, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroid agents, bioactive molecules of microbial origin, etc. Bioactive agents also include amide compounds such as those described in Patent Application Publication No. 20080221112 of Yamamori et al., which are administered before, after, and / or co-administered with the IL-2 polypeptide of the present invention.

[0144] "Bifunctional polymer" refers to a polymer that contains two discrete functional groups capable of specifically reacting with other components (including but not limited to amino acid side chains) to form covalent or non-covalent bonds. A bifunctional linker having one functional group reactive with a group on a specific bioactive component and another group reactive with a group on a second biological component can be used to form a conjugate comprising the first bioactive component, the bifunctional linker, and the second bioactive component. Many procedures and linker molecules for attaching various different compounds to peptides are known. See, for example, European Patent Application No. 188,256, U.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784, 4,680,338, and 4,569,789, which are incorporated herein by reference. "Multifunctional polymer" refers to a polymer that contains two or more discrete functional groups capable of specifically reacting with other components (including but not limited to amino acid side chains) to form covalent or non-covalent bonds. The bifunctional polymer or multifunctional polymer can have any desired length or molecular weight and can be selected to provide a specific desired spacing or conformation between one or more molecules attached to IL-2 and its receptor or IL-2.

[0145] When substituents are described by their conventional chemical formulas written from left to right, they equally cover chemically identical substituents obtained by writing the structure from right to left. For example, the structure -CH2O- is equivalent to the structure -OCH2-.

[0146] The term "substituent" includes but is not limited to "non-interfering substituents". "Non-interfering substituents" are groups that produce stable compounds. Suitable non-interfering substituents or residues include but are not limited to halogens, C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, C1-C 10 alkoxy, C1-C 12 aralkyl, C1-C 12 alkaryl, C3-C 12 cycloalkyl, C3-C 12 cycloalkenyl, phenyl, substituted phenyl, toluoyl, xylene, biphenyl, C2-C 12 alkoxyalkyl, C2-C 12 alkoxyaryl, C7-C 12 aryloxyalkyl, C7-C 12 oxyaryl, C1-C6 alkylsulfinyl, C1-C 10 alkylsulfonyl, --(CH2) m --O--(C1-C 10(alkyl) (where m is from 1 to 8), aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic group, substituted heterocyclic group, nitroalkyl, --NO2, --CN, --NRC(O)--(C1-C 10 alkyl), --C(O)--(C1-C 10 alkyl), C2-C 10 alkylthioalkyl, --C(O)O--(C1-C 10 alkyl), --OH, --SO2, ═S, --COOH, --NR2, carbonyl, --C(O)--(C1-C 10 alkyl)-CF3, --C(O)—CF3, --C(O)NR2, --(C1-C 10 (aryl)-S--(C6-C 10 aryl), --C(O)--(C1-C 10 (aryl), --(CH2) m --O--(--(CH2) m --O--(C1-C 10 alkyl) (where each m is from 1 to 8), --C(O)NR2, --C(S)NR2, --SO2NR2, --NRC(O)NR2, --NRC(S)NR2, its salts, etc. When used herein, each R is H, alkyl or substituted alkyl, aryl or substituted aryl, aralkyl or alkaryl.

[0147] The term "halogen" includes fluorine, chlorine, iodine and bromine.

[0148] Unless otherwise stated, the term "alkyl" by itself or as part of another substituent means a straight-chain, branched-chain or cyclic hydrocarbon group or a combination thereof, which may be fully saturated, mono- or poly-unsaturated, and may include divalent and polyvalent residues, having the specified number of carbon atoms (i.e., C1-C 10 means 1 to 10 carbon atoms). Examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologues and isomers such as n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl groups are groups having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl and higher homologues and isomers. Unless otherwise specified, the term "alkyl" also means derivatives of alkyl as defined in more detail below, such as "heteroalkyl". Alkyl limited to hydrocarbon groups is called "homoalkyl".

[0149] The term "alkylene" by itself or as part of another substituent means a divalent residue derived from an alkane, such as, but not limited to, the structures –CH2CH2– and –CH2CH2CH2CH2–, and also includes groups described hereinbelow as "heteroalkylene". Generally, an alkyl (or alkylene) has 1 to 24 carbon atoms, with groups having 10 or fewer carbon atoms being specific embodiments of the methods and compositions described herein. "Lower alkyl" or "lower alkylene" is a shorter-chain alkyl or alkylene, typically having 8 or fewer carbon atoms.

[0150] The terms "alkoxy", "alkylamino", and "alkylthio" (or thioalkoxy) are used in their conventional meanings and refer to an alkyl group attached to the remainder of the molecule through an oxygen atom, an amino group, or a sulfur atom, respectively.

[0151] Unless otherwise stated, the term "heteroalkyl" by itself or in combination with another term means a stable straight-chain, branched-chain, or cyclic hydrocarbon group or combination thereof consisting of the stated number of carbon atoms and at least one heteroatom selected from O, N, Si, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatoms may be optionally quaternized. The heteroatoms O, N, S, and Si may be placed at any internal position of the heteroalkyl or at the position where the alkyl is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and –CH=CH-N(CH3)-CH3. Up to two heteroatoms may be consecutive, such as -CH2-NH-OCH3 and –CH2-O-Si(CH3)3. Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent residue derived from a heteroalkyl, such as, but not limited to, -CH2-CH2-S-CH2-CH2- and –CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene, the same or different heteroatoms may also occupy either or both chain termini (including, but not limited to, alkyleneoxy, alkylenedioxy, alkylamino, alkylenediamino, aminooxyalkylene, etc.). In addition, for alkylene and heteroalkylene linking groups, the direction in which the structural formula of the linking group is written does not imply the orientation of the linking group. For example, the formula –C(O)2R'- represents both –C(O)2R'- and –R'C(O)2-.

[0152] Unless otherwise stated, the terms "cycloalkyl" and "heterocycloalkyl", alone or in combination with other terms, respectively denote the cyclic forms of "alkyl" and "heteroalkyl". Thus, cycloalkyl or heterocycloalkyl includes saturated, partially unsaturated, and fully unsaturated cyclic linkages. In addition, for heterocycloalkyl, the heteroatom can occupy the position where the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1–(1,2,5,6-tetrahydropyridyl), 1-piperidyl, 2-piperidyl, 3-piperidyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1–piperazinyl, 2-piperazinyl, and the like. In addition, the terms encompass bicyclic and tricyclic ring structures. Similarly, the term "heterocycloalkylene" alone or as part of another substituent means a divalent residue derived from heterocycloalkyl, and the term "cycloalkylene" alone or as part of another substituent means a divalent residue derived from cycloalkyl.

[0153] As used herein, the term "water-soluble polymer" refers to any polymer that is soluble in an aqueous solvent. The attachment of a water-soluble polymer to IL-2 can result in changes including, but not limited to, increased or modulated serum half-life or increased or modulated therapeutic half-life relative to the unmodified form, modulated immunogenicity, modulated physical association characteristics such as aggregation and multimer formation, altered receptor binding, altered binding to one or more binding partners, and altered receptor dimerization or multimerization. The water-soluble polymer may or may not have its own biological activity and can be used as a linker to attach IL-2 to other substances, including but not limited to one or more IL-2s or one or more bioactive molecules. Suitable polymers include, but are not limited to, polyethylene glycol, polyethylene glycol propionaldehyde, its mono C1-C10 alkoxy or aryloxy derivatives (described in U.S. Patent No. 5,252,714, which is incorporated herein by reference), monomethoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran derivatives (including dextran sulfate), polypropylene glycol, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives (including but not limited to methylcellulose and carboxymethylcellulose), starch and starch derivatives, polypeptides, polyalkylene glycols and their derivatives, copolymers of polyalkylene glycols and their derivatives, polyvinyl ethyl ether, and α-β-poly(2-hydroxyethyl)-DL-asparagine, and the like, or mixtures thereof. Examples of these water-soluble polymers include, but are not limited to, polyethylene glycol and serum albumin.

[0154] As used herein, the term "polyalkylene glycol" refers to polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof. The term "polyalkylene glycol" encompasses both linear and branched polymers and has an average molecular weight between 0.1 kDa and 100 kDa. Other exemplary embodiments are listed, for example, in the catalogs of commercial suppliers such as the catalog "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001) of Shearwater Corporation.

[0155] Unless otherwise stated, the term "aryl" means a polyunsaturated aromatic hydrocarbon substituent which may be a single ring or multiple rings (including but not limited to 1 to 3 rings) fused together or covalently linked. The term "heteroaryl" means an aryl (or ring) containing 1 to 4 heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atoms are optionally quaternized. The heteroaryl may be attached to the remainder of the molecule through the heteroatom. Non-limiting examples of aryl and heteroaryl include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2- oxazolyl, 4- oxazolyl, 2-phenyl-4- oxazolyl, 5- oxazolyl, 3-isothia oxazolyl, 4-isothia oxazolyl, 5-isothia oxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. For each of the above-mentioned aryl and heteroaryl ring systems, the substituents are selected from the acceptable substituents described below.

[0156] Briefly, the term "aryl", when used in combination with other terms (including but not limited to aryloxy, arylthioxy, aralkyl), includes both aryl and heteroaryl rings as defined above. Thus, the term "aralkyl" is meant to include those residues in which an aryl group is attached to an alkyl group (including but not limited to benzyl, phenethyl, pyridylmethyl, etc.), said alkyl group including those alkyl groups in which a carbon atom (including but not limited to methylene) has been replaced by, for example, an oxygen atom (including but not limited to phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, etc.).

[0157] Each of the above terms (including but not limited to "alkyl", "heteroalkyl", "aryl", and "heteroaryl") is meant to include both substituted and unsubstituted forms of the indicated residues. Exemplary substituents for each type of residue are provided below.

[0158] Substituents for alkyl and heteroalkyl residues (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl and heterocycloalkenyl) can be one or more of a variety of different groups selected from, but not limited to, the following: -OR’, =O, =NR’, =N-OR’, -NR’R”, -SR’, -halogen, -SiR’R”R”’, -OC(O)R’, -C(O)R’, -CO2R’, -CONR’R”, -OC(O)NR’R”, -NR”C(O)R’, -NR’-C(O)NR”R”’, -NR”C(O)2R’, -NR-C(NR’R”R’”) = NR””, -NR-C(NR’R”) = NR’”’, -S(O)R’, -S(O)2R’, -S(O)2NR’R”, -NRSO2R’, -CN and –NO2, the number of which is in the range of 0 to (2m’ + 1), where m’ is the total number of carbon atoms in such a residue. R’, R”, R”’ and R”” each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (including but not limited to aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy or aralkyl. When the compounds of the present invention include more than one R group, for example, each said R group is independently selected, and when there are more than one R’, R”, R’” and R”” groups, each of these groups is also so. When R’ and R” are attached to the same nitrogen atom, they can combine with the nitrogen atom to form a 5-, 6- or 7-membered ring. For example, -NR’R” means including but not limited to 1-pyrrolidinyl and 4-morpholinyl. From the discussion of the above substituents, those skilled in the art will understand that the term “alkyl” means including groups containing carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (including but not limited to -CF3 and –CH2CF3) and acyl (including but not limited to -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0159] Similar to the substituents described for alkyl residues, the substituents used for aryl and heteroaryl are variable and are selected from, but not limited to: halogen, -OR’, =O, =NR’, =N-OR’, -NR’R”, -SR’, -halogen, -SiR’R”R”’, -OC(O)R’, -C(O)R’, -CO2R’, -CONR’R”, -OC(O)NR’R”, -NR”C(O)R’, -NR’-C(O)NR”R”’, -NR”C(O)2R’, -NR-C(NR’R”R’”) = NR””, -NR-C(NR’R”) = NR’”’, -S(O)R’, -S(O)2R’, -S(O)2NR’R”, -NRSO2R’, -CN and –NO2, -R’, -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy and fluoro(C1-C4)alkyl, the number of which is in the range of 0 to the total number of open valences on the aromatic ring system; and wherein R’, R”, R”’ and R”” are independently selected from hydrogen, alkyl, heteroalkyl, aryl and heteroaryl. When the compounds of the present invention include more than one R group, for example, each of said R groups is independently selected, and when there are more than one R’, R”, R’” and R”” groups, each of these groups is also so.

[0160] As used herein, the term “modulated serum half-life” means a positive or negative change in the circulating half-life of modified IL-2 relative to its unmodified form. Serum half-life is measured by obtaining blood samples at various time points after IL-2 administration and determining the concentration of the molecule in each sample. The correlation of serum concentration with time allows calculation of the serum half-life. An increased serum half-life desirably has at least about two-fold, although smaller increases may also be useful, for example, in cases where it can achieve a satisfactory dosing regimen or avoid toxic effects. In certain embodiments, the increase is at least about three-fold, at least about five-fold or at least about ten-fold.

[0161] As used herein, the term “modulated therapeutic half-life” means a positive or negative change in the half-life of a therapeutically effective amount of IL-2 relative to its unmodified form. Therapeutic half-life is measured by measuring the pharmacokinetic and / or pharmacodynamic properties of the molecule at various time points after administration. An increased therapeutic half-life desirably enables a particularly beneficial dosing regimen, a particularly beneficial total dose or avoidance of adverse effects. In certain embodiments, the increased therapeutic half-life is caused by an increase in potency, an increase or decrease in the binding of the modified molecule to its target, an increase or decrease in the degradation of the molecule by an enzyme such as a protease, or an increase or decrease in another parameter or mechanism of action of the unmodified molecule or an increase or decrease in the receptor-mediated clearance of the molecule.

[0162] The term "isolated", when used in reference to a nucleic acid or protein, means that the nucleic acid or protein is at least free of some of the cellular components that are associated with it in its natural state, or that the nucleic acid or protein has been concentrated to a level higher than the concentration at which it is produced in vivo or in vitro. It may be in a homogeneous state. An isolated substance may be in a dry or semi-dry state, or in solution (including but not limited to an aqueous solution). It may be a component of a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier and / or excipient. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the major species present in a preparation is substantially purified. Specifically, an isolated gene is separated from open reading frames that flank the gene and encode proteins other than the gene of interest. The term "purified" means that the nucleic acid or protein gives essentially one band on an electrophoretic gel. Specifically, this may mean that the nucleic acid or protein is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or more pure.

[0163] The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in either single-stranded or double-stranded form. Unless otherwise specifically restricted, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically restricted, the term also refers to oligonucleotide analogues, including PNA (peptide nucleic acid), analogues of DNA used in antisense technology (phosphorothioates, phosphoroamidates, etc.). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to degenerate codon substitutions) and complementary sequences as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions may be achieved by generating sequences in which one or more selected (or all) codons' third positions are replaced with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0164] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description of a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The term applies to both naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the term encompasses amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0165] The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrrolysine and selenocysteine. Amino acid analogs are compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a compound that is bound to a hydrogen, a carboxyl group, an amino group, and the α-carbon of an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. These analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as the naturally occurring amino acid. References to amino acids include, for example, naturally occurring protein L-amino acids, D-amino acids, chemically modified amino acids such as amino acid variants and derivatives, naturally occurring non-protein amino acids such as β-alanine, ornithine, etc., and chemically synthesized compounds that have properties known in the art to be characteristic of amino acids. Examples of non-naturally occurring amino acids include, but are not limited to, α-methyl amino acids (e.g., α-methyl alanine), D-amino acids, histidine-like amino acids (e.g., 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine, and α-methyl-histidine), amino acids having an additional methylene in the side chain ("homo" amino acids), and amino acids in which the carboxylic acid functional group in the side chain is replaced by a sulfonic acid group (e.g., sulfoalanine). The incorporation of non-natural amino acids (including synthetic non-natural amino acids, substituted amino acids, or one or more D-amino acids) into the proteins of the present invention can be advantageous in a number of different ways. Peptides containing D-amino acids, etc., exhibit increased stability in vitro or in vivo compared to their L-amino acid counterparts. Thus, the construction of peptides, etc., incorporating D-amino acids can be particularly useful when higher intracellular stability is desired or required. More specifically, D-peptides, etc., are resistant to endogenous peptidases and proteases, and thus provide increased bioavailability and extended in vivo lifetimes of the molecules when these properties are desirable. In addition, D-peptides, etc., cannot be efficiently processed for presentation to T helper cells restricted by class II major histocompatibility complex, and thus are less likely to induce a humoral immune response in a whole organism.

[0166] Amino acids can be referred to herein by their well-known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Similarly, nucleotides can be referred to by their generally accepted single-letter codes.

[0167] "Conservative modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservative modified variants" refers to nucleic acids encoding the same or substantially the same amino acid sequence or, where the nucleic acid does not encode an amino acid sequence, to sequences which are substantially the same. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. These nucleic acid variations are "silent variations" and represent one species of conservative modified variation. Each nucleic acid sequence encoding a polypeptide herein also describes every possible silent variation of the nucleic acid. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except the AUG which is ordinarily the only codon for methionine and the TGG which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Thus, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.

[0168] With respect to amino acid sequences, one of ordinary skill in the art will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alter, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservative modified variants" where the alteration results in the deletion of an amino acid, the addition of an amino acid, or the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are known to one of ordinary skill in the art. These conservative modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[0169] Conservative substitution tables providing functionally similar amino acids are known to one of ordinary skill in the art. The following eight groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M); (see, e.g., Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co., Second Edition (December 1993)).

[0170] In the case of two or more nucleic acid or polypeptide sequences, the terms "identical" or percent "identity" refer to two or more sequences or subsequences that are the same. Measured by using one of the following sequence comparison algorithms (or other algorithms available to those of ordinary skill in the art) or by manual alignment and visual inspection, when the sequences are compared and aligned in a comparison window or specified region to obtain maximum correspondence, if they have a certain percentage of identical amino acid residues or nucleotides (i.e., about 60% identity, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% identity in the specified region), then the sequences are "substantially identical". This definition also applies to the complementary sequences of the test sequences. The identity can exist in a region of at least about 50 amino acids or nucleotides or in a region of 75 - 100 amino acids or nucleotides, or across the entire sequence of the polynucleotide or polypeptide if not otherwise specified. Polynucleotides encoding the polypeptides of the present invention (including homologs from species other than humans) can be obtained by a method comprising the steps of screening a library using a labeled probe having the polynucleotide sequence of the present invention or a fragment thereof under stringent hybridization conditions and isolating the full-length cDNA and genomic clones containing the polynucleotide sequence. Such hybridization techniques are well known to those of ordinary skill in the art.

[0171] The phrase "selectively (or specifically) hybridizes to" means that when a particular nucleotide sequence is present in a complex mixture (including but not limited to total cellular or library DNA or RNA), a molecule will bind only to that particular sequence, form a duplex or hybridize under stringent hybridization conditions.

[0172] As is known in the art, the phrase "stringent hybridization conditions" refers to the hybridization of sequences of DNA, RNA, PNA or other nucleic acid mimetics or combinations thereof under conditions of low ionic strength and high temperature. Typically, under stringent conditions, a probe will hybridize to its target sequence in a complex mixture of nucleic acids (including but not limited to total cellular or library DNA or RNA), but not to other sequences in the complex mixture. Stringent conditions are sequence-dependent and vary in different circumstances. Longer sequences hybridize specifically at higher temperatures.

[0173] As used herein, the term "eukaryote" refers to an organism belonging to the domain Eukarya, such as animals (including but not limited to mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to monocots, dicots, algae, etc.), fungi, yeast, flagellates, microsporidia, protists, etc.

[0174] As used herein, the term "non-eukaryote" refers to an organism that is not eukaryotic. For example, a non-eukaryotic organism can belong to the eubacteria (including but not limited to Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.) domain or the archaea (including but not limited to Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.) domain.

[0175] As used herein, the term "subject" refers to an animal that is the target of treatment, observation, or experiment, which in certain embodiments is a mammal and in other embodiments is a human. The animal can be a companion animal (such as a dog, cat, etc.), a farm animal (such as a cow, sheep, pig, horse, etc.), or a laboratory animal (such as a rat, mouse, guinea pig, etc.).

[0176] As used herein, the term "effective amount" refers to an amount of the administered modified unnatural amino acid polypeptide that alleviates to some extent one or more symptoms of the disease, disorder, or condition to be treated. Compositions containing the modified unnatural amino acid polypeptides described herein can be administered for prophylactic, enhancing, and / or therapeutic treatment.

[0177] The term "enhance" means to increase or prolong the desired effect in either aspect of potency or duration. Thus, for enhancing the effect of a therapeutic agent, the term "enhance" refers to the ability to increase or prolong the effect of another therapeutic agent on the system in either aspect of potency or duration. As used herein, an "enhancing effective amount" refers to an amount sufficient to enhance the effect of another therapeutic agent in the desired system. When used in a patient, the amount effective for this use depends on the severity and course of the disease, disorder, or condition, previous treatment, the health status of the patient and the response to the drug, and the judgment of the treating physician.

[0178] As used herein, the term "modified" refers to any change made to a given polypeptide, such as a change in the length, amino acid sequence, chemical structure, co-translational modification or post-translational modification of the polypeptide. The term in the form "(modified)" means that the polypeptide in question is optionally modified, that is, the polypeptide in question may or may not be modified.

[0179] The term "post-translational modification" refers to any modification that occurs on an amino acid after it has been incorporated into a polypeptide chain. Merely by way of example, the term encompasses co-translational in vivo modification, co-translational in vitro modification (e.g., in a cell-free translation system), post-translational in vivo modification and post-translational in vitro modification.

[0180] In a prophylactic application, a composition containing the IL-2 is administered to a patient who is susceptible or otherwise at risk of a particular disease, disorder or condition. Such an amount is defined as a "prophylactically effective amount". In such use, the exact amount also depends on the health status, weight, etc. of the patient. Determining such a prophylactically effective amount by routine experimentation (e.g., dose escalation clinical trials) is considered to be well within the scope of those skilled in the art.

[0181] In a therapeutic application, a composition containing the modified unnatural amino acid polypeptide is administered to a patient who already has the disease, disorder or condition in an amount sufficient to cure or at least partially arrest the symptoms of the disease, disorder or condition. Such an amount is defined as a "therapeutically effective amount" and will depend on the severity and course of the disease, disorder or condition, previous treatment, the health status of the patient and the response to the drug, as well as the judgment of the treating physician. Determining such a therapeutically effective amount by routine experimentation (e.g., dose escalation clinical trials) is considered to be well within the scope of those skilled in the art.

[0182] The term "treatment" is used to refer to either prophylactic and / or therapeutic treatment.

[0183] The unnatural amino acid polypeptides presented herein may include isotopically labeled compounds having one or more atoms replaced by atoms having an atomic weight or mass number different from the atomic weight or mass number commonly found in nature. Examples of isotopes that may be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 35 S, 18 F, 36Cl. Certain isotopically labeled compounds described herein, such as those incorporating a radioactive isotope such as 3 H and 14 C, may be useful in the determination of the tissue distribution of drugs and / or substances. In addition, replacement with an isotope such as deuterium, i.e., 2 H, can result in certain therapeutic advantages arising from higher metabolic stability, such as an increased in vivo half-life or a reduced dosage requirement.

[0184] All isomers, including but not limited to diastereoisomers, enantiomers, and mixtures thereof, are considered to be part of the compositions described herein. In additional or alternative embodiments, the non-naturally encoded amino acid polypeptide is metabolized after administration to a subject in need to produce metabolites, which are then used to produce the desired effect, including the desired therapeutic effect. In other or alternative embodiments, the active metabolite of the non-naturally encoded amino acid polypeptide.

[0185] In certain instances, the non-naturally encoded amino acid polypeptide may exist as a tautomer. In addition, the non-naturally encoded amino acid polypeptides described herein may exist in unsolvated forms as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, etc. The solvated forms are also considered to be disclosed herein. One of ordinary skill in the art will recognize that certain compounds herein may exist in several tautomeric forms. All such tautomeric forms are considered to be part of the compositions described herein.

[0186] Unless otherwise indicated, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology within the skill of the art are used.

[0187] Detailed Description

[0188] I. Introduction

[0189] The present invention provides an IL-2 molecule comprising at least one unnatural amino acid. In certain embodiments of the present invention, the IL-2 having at least one unnatural amino acid comprises at least one post-translational modification. In one embodiment, the at least one post-translational modification comprises attaching a molecule comprising a second reactive group to at least one unnatural amino acid comprising a first reactive group using chemical methods known to those of ordinary skill in the art suitable for specific reactive groups, and the molecule includes but is not limited to markers, dyes, polymers, water-soluble polymers, derivatives of polyethylene glycol, photo-crosslinking agents, radionuclides, cytotoxic compounds, drugs, affinity labels, photoaffinity labels, reactive compounds, resins, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelators, cofactors, fatty acids, saccharides, polynucleotides, DNA, RNA, antisense polynucleotides, sugars, water-soluble dendritic polymers, cyclodextrins, inhibitory ribonucleic acids, biomaterials, nanoparticles, spin labels, fluorophores, metal-containing moieties, radioactivity-containing moieties, new functional groups, groups that interact covalently or non-covalently with other molecules, photocaging moieties, components excitable by actinic radiation, photoisomerizable components, biotin, derivatives of biotin, biotin analogs, components doped with heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox-active agents, aminothio acids, toxic components, isotope-labeled components, biophysical probes, phosphorescent groups, chemiluminescent groups, electron-dense groups, magnetic groups, intercalating groups, chromophores, energy transfer reagents, bioactive agents, detectable labels, small molecules, quantum dots, nanoemitters, radioactive nucleotides, radioactive emitters, neutron capture agents, or any combination of the above molecules or any other desired compounds or substances. For example, the first reactive group is an alkynyl moiety (including but not limited to p-propargyloxyphenylalanine in the unnatural amino acid, where the propargyl group is sometimes also referred to as an ethynyl moiety) and the second reactive group is an azide moiety, and [3+2] cycloaddition chemistry is used. In another example, the first reactive group is an azide moiety (including but not limited to p-azido-L-phenylalanine (pAZ) in the unnatural amino acid) and the second reactive group is an alkynyl moiety. In certain embodiments of the modified IL-2 of the present invention, at least one unnatural amino acid comprising at least one post-translational modification (including but not limited to unnatural amino acids containing a keto functional group) is used, and the at least one post-translational modification comprises a sugar moiety. In certain embodiments, the post-translational modification is produced in vivo in eukaryotic cells or in non-eukaryotic cells. A linker, polymer, water-soluble polymer, or other molecule can attach the molecule to the polypeptide. In another embodiment, the linker attached to IL-2 is long enough to allow dimer formation.The molecule can also be directly linked to the polypeptide.

[0190] In certain embodiments, the IL-2 protein comprises at least one post-translational modification made in vivo by a host cell, wherein the post-translational modification is not typically made by another host cell type. In certain embodiments, the protein comprises at least one post-translational modification made in vivo by a eukaryotic cell, wherein the post-translational modification is not typically made by a non-eukaryotic cell. Examples of post-translational modifications include, but are not limited to, glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitic acid addition, phosphorylation, glycolipid linkage modification, and the like.

[0191] In certain embodiments, the IL-2 comprises one or more non-naturally encoded amino acids for glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitic acid addition, phosphorylation, or glycolipid linkage modification of the polypeptide. In certain embodiments, the IL-2 comprises one or more non-naturally encoded amino acids for glycosylation of the polypeptide. In certain embodiments, the IL-2 comprises one or more naturally encoded amino acids for glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitic acid addition, phosphorylation, or glycolipid linkage modification of the polypeptide. In certain embodiments, the IL-2 comprises one or more naturally encoded amino acids for glycosylation of the polypeptide.

[0192] In certain embodiments, the IL-2 comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation of the polypeptide. In certain embodiments, the IL-2 comprises one or more deletions that enhance glycosylation of the polypeptide. In certain embodiments, the IL-2 comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation at different amino acids in the polypeptide. In certain embodiments, the IL-2 comprises one or more deletions that enhance glycosylation at different amino acids in the polypeptide. In certain embodiments, the IL-2 comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation at non-naturally encoded amino acids in the polypeptide. In certain embodiments, the IL-2 comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation at naturally encoded amino acids in the polypeptide. In certain embodiments, the IL-2 comprises one or more naturally encoded amino acid additions and / or substitutions that enhance glycosylation at different amino acids in the polypeptide. In certain embodiments, the IL-2 comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation at naturally encoded amino acids in the polypeptide. In certain embodiments, the IL-2 comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation at non-naturally encoded amino acids in the polypeptide.

[0193] In one embodiment, the post-translational modification includes attaching an oligosaccharide (including but not limited to cases where the oligosaccharide comprises (GlcNAc-Man)2-Man-GlcNAc-GlcNAc, etc.) to asparagine via a GlcNAc-asparagine linkage. In another embodiment, the post-translational modification includes attaching an oligosaccharide (including but not limited to Gal-GalNAc, Gal-GlcNAc, etc.) to serine or threonine via a GalNAc-serine, GalNAc-threonine, GlcNAc-serine, or GlcNAc-threonine linkage. In certain embodiments, the protein or polypeptide of the present invention may comprise a secretion or localization sequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, etc. Examples of secretion signal sequences include but are not limited to prokaryotic secretion signal sequences, eukaryotic secretion signal sequences, eukaryotic secretion signal sequences optimized at the 5'-end for bacterial expression, novel secretion signal sequences, pectate lyase secretion signal sequences, Omp A secretion signal sequences, and phage secretion signal sequences. Examples of secretion signal sequences include but are not limited to STII (prokaryote), Fd GIII and M13 (phage), Bgl2 (yeast), and the signal sequence bla derived from a transposon. Any such sequence can be modified to provide the desired result for the polypeptide, including but not limited to replacing one signal sequence with a different signal sequence, replacing a leader sequence with a different leader sequence, etc.

[0194] The protein or polypeptide of interest may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more non-natural amino acids. The non-natural amino acids may be the same or different. For example, in the protein, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different sites that contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different non-natural amino acids. In certain embodiments, at least one but less than all of the specific amino acids present in the native form of the protein are replaced with non-natural amino acids.

[0195] The present invention provides methods and compositions based on IL-2 comprising at least one non-naturally encoded amino acid. The introduction of at least one non-naturally encoded amino acid into IL-2 allows the use of conjugation chemistries involving specific chemical reactions that involve, but are not limited to, reacting with one or more non-naturally encoded amino acids while not reacting with the common 20 amino acids. In certain embodiments, the IL-2 comprising the non-naturally encoded amino acid is linked to a water-soluble polymer such as polyethylene glycol (PEG) via the side chain of the non-naturally encoded amino acid. The present invention provides an efficient method for selectively modifying a protein with a PEG derivative, the method comprising the selective incorporation of a non-genetically encoded amino acid (including but not limited to an amino acid containing a functional group or substituent not present in the 20 naturally incorporated amino acids (including but not limited to keto, azide or acetylene moieties)) in response to a selector codon into the protein, followed by modification of those amino acids with a suitable reactive PEG derivative. Once incorporated, the amino acid side chains can then be modified using chemical methods known to those of ordinary skill in the art that are suitable for the specific functional groups or substituents present in the non-naturally encoded amino acid. A wide variety of known chemical methods are suitable for use in the present invention for incorporating a water-soluble polymer into the protein. These methods include but are not limited to the use of Huisgen [3+2] cycloaddition reactions using, for example, acetylene or azide derivatives (see, for example, Padwa, A., Comprehensive Organic Synthesis, Vol. 4, (1991) Ed. Trost, B.M., Pergamon, Oxford, p. 1069-1109; and Huisgen, R., 1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, New York, p. 1-176).

[0196] Since the Huisgen [3+2] cycloaddition method involves cycloaddition rather than nucleophilic substitution reactions, proteins can be modified with extremely high selectivity. By adding a catalytic amount of a Cu(I) salt to the reaction mixture, the reaction can proceed with excellent regioselectivity (1,4>1,5) at room temperature under aqueous conditions. See, for example, Tornoe et al., (2002) J. Org. Chem. 67:3057-3064; and Rostovtsev et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599; and WO 03 / 101972. Molecules that can be added to the proteins of the present invention by [3+2] cycloaddition include, in fact, any molecule having a suitable functional group or substituent, including but not limited to azide or acetylene derivatives. These molecules can be added to unnatural amino acids having an acetylene group, including but not limited to p-propargyloxyphenylalanine, or unnatural amino acids having an azide group, including but not limited to p-azido-phenylalanine.

[0197] The 5-membered ring resulting from the Huisgen [3+2] cycloaddition is generally irreversible in a reducing environment and is stable against hydrolysis in an aqueous environment for a long time. Therefore, the physical and chemical properties of a wide variety of substances can be modified with the active PEG derivatives of the present invention under demanding aqueous conditions. Even more importantly, since the azide and acetylene moieties are specific to each other (and do not react with any of the 20 commonly used genetically encoded amino acids, for example), proteins can be modified with extremely high selectivity at one or more specific sites.

[0198] The present invention also provides water-soluble and hydrolysis-stable PEG derivatives and related hydrophilic polymers having one or more acetylene or azide moieties. The PEG polymer derivatives containing an acetylene moiety are highly selective for coupling with an azide moiety that has been selectively introduced into a protein in response to a selector codon. Similarly, the PEG polymer derivatives containing an azide moiety are highly selective for coupling with an acetylene moiety that has been selectively introduced into a protein in response to a selector codon.

[0199] More specifically, the azide moiety includes but is not limited to alkyl azides, aryl azides, and derivatives of these azides. The derivatives of the alkyl and aryl azides can include other substituents as long as the acetylene-specific reactivity is maintained. The acetylene moiety includes alkyl and aryl acetylene compounds and derivatives of each. The derivatives of the alkyl and aryl acetylene compounds can include other substituents as long as the azide-specific reactivity is maintained.

[0200] The present invention provides conjugates of a substance having a wide variety of types of functional groups, substituents, or moieties with other substances, said other substances including but not limited to labels, dyes, polymers, water-soluble polymers, derivatives of polyethylene glycol, photo-crosslinking agents, radionuclides, cytotoxic compounds, drugs, affinity labels, photoaffinity labels, reactive compounds, resins, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, metal chelators, cofactors, fatty acids, saccharides, polynucleotides, DNA, RNA, antisense polynucleotides, sugars, water-soluble dendrimers, cyclodextrins, inhibitory ribonucleic acids, biomaterials, nanoparticles, spin labels, fluorophores, metal-containing moieties, radioactivity-containing moieties, novel functional groups, groups that interact covalently or non-covalently with other molecules, photocaging moieties, components excitable by actinic radiation, photoisomerizable components, biotin, derivatives of biotin, biotin analogs, components doped with heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox active agents, aminothiocarboxylic acids, toxic components, isotope-labeled components, biophysical probes, phosphorescent groups, chemiluminescent groups, electron-dense groups, magnetic groups, intercalating groups, chromophores, energy transfer reagents, bioactive agents, detectable labels, small molecules, quantum dots, nanoemitters, radiolabeled nucleotides, radioemitters, neutron capture agents, or any combination of the foregoing or any other desired compound or substance. The present invention also includes conjugates of a substance having an azide or alkyne moiety with a PEG polymer derivative having a corresponding alkyne or azide moiety. For example, a PEG polymer containing an azide moiety can be conjugated to a bioactive molecule at the position in the protein containing a non-genetically encoded amino acid having an alkyne functional group. The linkages for conjugating the PEG to the bioactive molecule include but are not limited to Huisgen [3+2] cycloaddition products.

[0201] It has been well established in the art that PEG can be used to modify the surface of biomaterials (see, e.g., U.S. Patent 6,610,281; Mehvar, R., J. Pharm Sci., 3(1):125-136 (2000), which are incorporated herein by reference). The present invention also includes biomaterials having one or more reactive azide or alkyne sites on the surface and to which one or more azide- or alkyne-containing polymers of the present invention are conjugated via Huisgen [3+2] cycloaddition linkages. Biomaterials and other substances can also be conjugated to the azide- or alkyne-activated polymer derivatives via linkages other than azide or alkyne linkages, such as linkages containing carboxylic acid, amine, alcohol, or thiol moieties, leaving the azide or alkyne moieties available for subsequent reactions.

[0202] The present invention includes a method for synthesizing a polymer containing azide and acetylene of the present invention. In the case of the azide-containing PEG derivative, the azide can be directly bonded to the carbon atom of the polymer. Alternatively, the azide-containing PEG derivative can be prepared by attaching a linking reagent having an azide moiety to one end of a conventionally activated polymer, such that the resulting polymer has the azide moiety at its end. In the case of the acetylene-containing PEG derivative, the acetylene can be directly bonded to the carbon atom of the polymer. Alternatively, the acetylene-containing PEG derivative can be prepared by attaching a linking reagent having an acetylene moiety to one end of a conventionally activated polymer, such that the resulting polymer has the acetylene moiety at its end.

[0203] More specifically, in the case of the azide-containing PEG derivative, a water-soluble polymer having at least one active hydroxyl moiety undergoes a reaction to produce a substituted polymer having a more reactive moiety, such as a mesylate, tribenzoate, tosylate, or halogen leaving group, thereon. The preparation and use of PEG derivatives containing sulfonyl halides, halogen atoms, and other leaving groups are known to those of ordinary skill in the art. The resulting substituted polymer then undergoes a reaction to replace the azide moiety at the end of the polymer with the more reactive moiety. Alternatively, a water-soluble polymer having at least one active nucleophilic or electrophilic moiety undergoes a reaction with a linking reagent having an azide at one end in order to form a covalent bond between the PEG polymer and the linking reagent, and the azide moiety is located at the end of the polymer. Nucleophilic and electrophilic moieties, including amines, thiols, hydrazides, hydrazines, alcohols, carboxylic esters, aldehydes, ketones, thioesters, etc., are known to those of ordinary skill in the art.

[0204] More specifically, in the case of the acetylene-containing PEG derivative, a water-soluble polymer having at least one active hydroxyl moiety undergoes a reaction to displace a halogen or other activated leaving group from a precursor containing an acetylene moiety. Alternatively, a water-soluble polymer having at least one active nucleophilic or electrophilic moiety undergoes a reaction with a linking reagent having an acetylene at one end in order to form a covalent bond between the PEG polymer and the linking reagent, and the acetylene moiety is located at the end of the polymer. The use of halogen moieties, activated leaving groups, nucleophilic and electrophilic moieties in the context of organic synthesis and in the preparation and use of PEG derivatives is clear to those skilled in the art.

[0205] The present invention also provides a method for selectively modifying a protein to add other substances to the modified protein, said other substances including but not limited to water-soluble polymers such as PEG and PEG derivatives containing azide or alkyne moieties. The azide- and alkyne-containing PEG derivatives can be used to modify properties of surfaces and molecules where biocompatibility, stability, solubility, and lack of immunogenicity are important, and at the same time provide a more selective means of attaching the PEG derivatives to proteins than previously known in the art.

[0206] II. General recombinant nucleic acid methods for the present invention

[0207] In numerous embodiments of the present invention, nucleic acids encoding IL-2 of interest will be isolated, cloned, and generally altered using recombinant methods. These embodiments are used during, including but not limited to, protein expression or the generation of variants, derivatives, expression cassettes, or other sequences derived from IL-2. In certain embodiments, the sequences encoding the polypeptides of the present invention are operably linked to a heterologous promoter.

[0208] The amino acid sequence of the mature human IL-2 protein is shown in Table 1 below.

[0209] Table 1 - IL-2 Protein and DNA Sequences

[0210]

[0211]

[0212]

[0213]

[0214]

[0215] Nucleotide sequences encoding IL-2 containing non-naturally encoded amino acids can be synthesized on the basis of the amino acid sequences of parent polypeptides including but not limited to the amino acid sequences shown in SEQ ID NO: 1, 2, 3, 5, or 7, and then the nucleotide sequences are altered to effect introduction (i.e., incorporation or replacement) or removal (i.e., deletion or replacement) of the relevant amino acid residues. The nucleotide sequences can be conveniently modified by site-directed mutagenesis according to conventional methods. Alternatively, the nucleotide sequences can be prepared by chemical synthesis, including but not limited to by using an oligonucleotide synthesizer, where the oligonucleotides are designed on the basis of the amino acid sequence of the desired polypeptide and preferably those codons that are favorable in the host cell in which the recombinant polypeptide is to be produced are selected. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and assembled by PCR, ligation, or ligase chain reaction. See, e.g., Barany et al., Proc. Natl. Acad. Sci. 88:189-193 (1991); U.S. Patent 6,521,427, which is incorporated herein by reference.

[0216] The DNA sequence of the synthetic human IL-2 gene cloned into the pKG0269 expression plasmid is shown as SEQ ID NO: 4 in Table 1 above. This DNA sequence has been optimized for E. coli codons.

[0217] The present invention utilizes conventional techniques in the field of recombinant genetics. Fundamental texts disclosing the general methods used in the present invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd ed., 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994).

[0218] The present invention also relates to eukaryotic host cells, non-eukaryotic host cells, and organisms for in vivo incorporation of non-naturally occurring amino acids by an orthogonal tRNA / RS pair. The host cells are genetically engineered (including but not limited to transformation, transduction, or transfection) with the polynucleotides of the present invention or constructs comprising the polynucleotides of the present invention, including but not limited to the vectors of the present invention, which can be, for example, cloning vectors or expression vectors.

[0219] Several well-known methods for introducing target nucleic acids into cells are available, any of which can be used in the present invention. These methods include: fusion of recipient cells with bacterial protoplasts containing the DNA, electroporation, particle bombardment, and infection with viral vectors (discussed further below), etc. Bacterial cells can be used to amplify the number of plasmids containing the DNA constructs of the present invention. The bacteria are grown to the logarithmic phase, and the plasmids within the bacteria can be isolated by various different methods known in the art (see, for example, Sambrook). In addition, kits for purifying plasmids from bacteria are commercially available (see, for example, EasyPrep TM , FlexiPrep TM ; Stratagene's StrataClean TM ; and Qiagen's QIAprep TM)。Then the isolated and purified plasmid is further manipulated to generate other plasmids for transfection of cells or incorporation into related vectors for infection of organisms. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters that can be used to regulate the expression of the specific target nucleic acid. The vector optionally contains a universal expression cassette that contains at least one independent terminator sequence, sequences that permit replication of the expression cassette in eukaryotes or prokaryotes or both (including but not limited to shuttle vectors), and selectable markers for both prokaryotic and eukaryotic systems. The vector is suitable for replication and integration in prokaryotes, eukaryotes, or both. See Gillam & Smith, Gene 8:81 (1979); Roberts et al., Nature, 328:731 (1987); Schneider, E. et al., Protein Expr. Purif. 6(1):10 - 14 (1995); Ausubel, Sambrook, Berger (all supra). Catalogs of bacteria and bacteriophages available for cloning are provided, for example, by the ATCC, such as the 《ATCC Catalogue of Bacteria and Bacteriophage》(1992), edited by Gherna et al. and published by the ATCC. Other basic procedures for sequencing, cloning, and other aspects of molecular biology, as well as the underlying theoretical considerations, can also be found in Watson et al., (1992), 《Recombinant DNA》, Second Edition, Scientific American Books, NY. In addition, substantially any nucleic acid (and indeed any labeled nucleic acid, whether standard or non - standard) can be custom - ordered or obtained as a standard product from any of a variety of different commercial sources, such as Midland Certified Reagent Company (Midland, TX, available at the World Wide Web URL mcrc.com), The Great American Gene Company (Ramona, CA, available at the World Wide Web URL genco.com), ExpressGen Inc. (Chicago, IL, available at the World Wide Web URL expressgen.com), Operon Technologies Inc. (Alameda, CA), and many other sources.

[0220] Selector codon

[0221] The selector codons of the present invention expand the genetic codon framework of the protein biosynthesis machinery. For example, selector codons include, but are not limited to, unique three-base codons, nonsense codons such as stop codons (including, but not limited to, amber codon (UAG), ochre codon or opal codon (UGA)), unnatural codons, four- or more-base codons, rare codons, etc. It will be apparent to those of ordinary skill in the art that the number of selector codons that can be introduced into a desired gene or polynucleotide has a wide range, including, but not limited to, one or more, two or more, three or more, 4, 5, 6, 7, 8, 9, 10 or more in a single polynucleotide encoding at least a portion of the IL-2.

[0222] In one embodiment, the method includes using a selector codon that functions as a stop codon for in vivo incorporation of one or more unnatural amino acids. For example, an O-tRNA is produced that recognizes a stop codon (including, but not limited to, UAG) and is aminoacylated with the desired unnatural amino acid by an O-RS. This O-tRNA is not recognized by the naturally occurring host aminoacyl-tRNA synthetase. Conventional site-directed mutagenesis can be used to introduce the stop codon (including, but not limited to, TAG) into the site of interest in the polypeptide of interest. See, for example, Sayers, J.R. et al., (1988), 5'-3' Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis, Nucleic Acids Res, 16:791-802. When the O-RS, O-tRNA, and nucleic acid encoding the polypeptide of interest are combined in vivo, the unnatural amino acid is incorporated in response to the UAG codon to give a polypeptide containing the unnatural amino acid at the designated position.

[0223] In vivo incorporation of unnatural amino acids can be carried out without significantly perturbing eukaryotic host cells. For example, since the suppression efficiency of the UAG codon depends on the competition between the O-tRNA (including, but not limited to, amber suppressor tRNA) and the eukaryotic release factor (including, but not limited to, eRF) (which binds to the stop codon and initiates the release of the growing peptide from the ribosome), the suppression efficiency can be regulated by, including, but not limited to, increasing the expression level of the O-tRNA and / or suppressor tRNA.

[0224] Non-natural amino acids can also be encoded by rare codons. For example, it has been demonstrated that when the concentration of arginine is reduced in an in vitro protein synthesis reaction, the rare arginine codon AGG is efficiently inserted as Ala by a synthetic tRNA acylated with alanine. See, e.g., Ma et al., Biochemistry, 32:7939 (1993). In this case, the synthetic tRNA competes with the naturally occurring tRNAArg that is present as a minor species in E. coli. Some organisms do not use all of the triplet codons. The unassigned codon AGA in Micrococcus luteus has been used for the insertion of amino acids in an in vitro transcription / translation extract. See, e.g., Kowal and Oliver, Nucl. Acid. Res., 25:4685 (1997). Components of the present invention can be produced to use these rare codons in vivo.

[0225] The selector codons also include extended codons, including but not limited to four or more base codons such as four, five, six or more base codons. Examples of four-base codons include but are not limited to AGGA, CUAG, UAGA, CCCU, etc. Examples of five-base codons include but are not limited to AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC, etc. Features of the present invention include the use of extended codons based on frameshift suppression. The four or more base codons can insert one or more non-natural amino acids into the same protein. For example, in the presence of a mutant O-tRNA with an anticodon loop, such as an anticodon loop having at least 8-10 nt (including but not limited to a special frameshift suppressor tRNA), the four or more base codons are read as a single amino acid. In other embodiments, the anticodon loop can decode at least a four-base codon, at least a five-base codon, or at least a six-base codon or more base codons. Since there are 256 possible four-base codons, four or more base codons can be used to encode multiple non-natural amino acids in the same cell. See Anderson et al., Exploring the Limits of Coden and Anticodon Size, Chemistry and Biology, 9:237-244, (2002); Magliery, Expanding the Genetic Code: Selection of Efficient Suppressors of Four-base Codens and Identification of “Shifty” Four-base Codens with a Library Approach in Escherichia coli, J. Mol. Biol. 307:755-769 (2001).

[0226] For example, four-base codons have been used to incorporate unnatural amino acids into proteins in in vitro biosynthetic methods. See, for example, Ma et al., Biochemistry, 32:7939, (1993); and Hohsaka et al., J. Am. Chem. Soc., 121:34 (1999). CGGG and AGGU have been used to incorporate 2-naphthylalanine and an NBD derivative of lysine into streptavidin in vitro using two chemically acylated frameshift suppressor tRNAs. See, for example, Hohsaka et al., J. Am. Chem. Soc., 121:12194 (1999). In in vivo studies, Moore et al. examined the ability of tRNA Leu derivatives with NCUA anticodons to suppress UAGN codons (where N can be U, A, G, or C) and found that the quadruplet UAGA could be decoded by tRNA Leu with a UCUA anticodon with an efficiency of 13 to 26%, and rarely decoded in the 0 or -1 frames. See Moore et al., J. Mol. Biol., 298:195 (2000). In one embodiment, extended codons based on rare or nonsense codons can be used in the present invention, which can reduce missense readthrough and frameshift suppression at other unwanted sites.

[0227] For a given system, the selector codon can also include one of the natural three-base codons, where the endogenous system does not use (or rarely uses) the natural base codon. For example, this includes systems that lack a tRNA that recognizes the natural three-base codon and / or where the three-base codon is a rare codon.

[0228] The selector codon optionally includes unnatural base pairs. These unnatural base pairs further expand the existing genetic alphabet. An additional base pair increases the number of triplet codons from 64 to 125. The properties of the third base pair include stable and selective base pairing, efficient enzymatic incorporation into DNA by a polymerase with high fidelity, and efficient continued primer extension after the synthesis of the nascent unnatural base pair. Descriptions of unnatural base pairs that can be engineered for use in methods and compositions include, for example, Hirao et al., An unnatural base pair for incorporating amino acid analogues into protein, Nature Biotechnology, 20:177-182, (2002). See also Wu, Y. et al., J. Am. Chem. Soc. 124:14626-14630 (2002). Other relevant publications are listed below.

[0229] For in vivo use, the unnatural nucleosides are membrane-permeable and phosphorylated to form the corresponding triphosphates. In addition, the added genetic information is stable and not destroyed by the enzymes of the cell. Previous attempts by Benner and others have utilized hydrogen bonding patterns different from those in the classical Watson-Crick pairs, the most notable example being the iso-C:iso-G pair. See, for example, Switzer et al., J. Am. Chem. Soc., 111:8322 (1989); and Piccirilli et al., Nature, 343:33 (1990); Kool, Curr. Opin. Chem. Biol., 4:602 (2000). These bases generally mispair to some extent with the natural bases and cannot be enzymatically replicated. Kool and co-workers have demonstrated that hydrophobic stacking interactions between bases can replace hydrogen bonding to drive base pair formation. See Kool, Curr. Opin. Chem. Biol., 4:602 (2000); and Guckian and Kool, Angew. Chem. Int. Ed. Engl., 36, 2825 (1998). In attempts to develop unnatural base pairs that meet all of the above requirements, Schultz, Romesberg and co-workers have systematically synthesized and studied a series of unnatural hydrophobic bases. The PICS:PICS self-pair was found to be more stable than natural base pairs and could be efficiently incorporated into DNA by the Klenow fragment (KF) of Escherichia coli DNA polymerase I. See, for example, McMinn et al., J. Am. Chem. Soc., 121:11585-6 (1999); and Ogawa et al., J. Am. Chem. Soc., 122:3274 (2000). The 3MN:3MN self-pair could be synthesized by KF with sufficient efficiency and selectivity for biological function. See, for example, Ogawa et al., J. Am. Chem. Soc., 122:8803 (2000). However, both bases act as chain terminators for further replication. Mutant DNA polymerases have recently been evolved that can be used to replicate the PICS self-pair. In addition, the 7AI self-pair can be replicated. See, for example, Tae et al., J. Am. Chem. Soc., 123:7439 (2001). The new metal base pair Dipic:Py has also been developed, which forms a stable pair upon binding of Cu(II). See Meggers et al., J. Am. Chem. Soc., 122:10714 (2000). Since the extended codons and unnatural codons are inherently orthogonal to the natural codons, the methods of the invention can utilize this property to generate orthogonal tRNAs for them.

[0230] Translation bypass systems can also be used to incorporate unnatural amino acids into desired polypeptides. In a translation bypass system, a large sequence is incorporated into a gene but not translated into protein. The sequence contains structures that act as cues to induce ribosomes to skip the sequence and resume translation downstream of the insertion.

[0231] In certain embodiments, in the methods and / or compositions of the invention, a protein or polypeptide of interest (or a portion thereof) is encoded by a nucleic acid. Generally, the nucleic acid comprises at least one selector codon, at least two selector codons, at least three selector codons, at least four selector codons, at least five selector codons, at least six selector codons, at least seven selector codons, at least eight selector codons, at least nine selector codons, ten or more selector codons.

[0232] The gene encoding the protein or polypeptide of interest can be mutagenized using methods known to those of ordinary skill in the art and described herein to contain, for example, one or more selector codons for incorporation of unnatural amino acids. For example, the nucleic acid for the protein of interest is mutagenized to contain one or more selector codons for incorporation of one or more unnatural amino acids. The invention includes any such variant of any protein, including but not limited to mutant forms, e.g., including at least one unnatural amino acid. Similarly, the invention also includes the corresponding nucleic acids, i.e., any nucleic acid having one or more selector codons encoding one or more unnatural amino acids.

[0233] A nucleic acid molecule encoding a protein of interest such as IL-2 can be readily mutated to introduce cysteine at any desired position in the polypeptide. Cysteine is widely used to introduce reactive molecules, water-soluble polymers, proteins, or other molecules of a wide variety of types onto the protein of interest. Methods suitable for incorporating cysteine into the desired position in a polypeptide are known to those of ordinary skill in the art, e.g., as described in U.S. Patent No. 6,608,183, which is incorporated herein by reference, and standard mutagenesis techniques.

[0234] III. Unnaturally Encoded Amino Acids

[0235] A wide variety of non-naturally encoded amino acids are applicable to the present invention. Any number of non-naturally encoded amino acids can be introduced into IL-2. Generally, the introduced non-naturally encoded amino acids are substantially chemically inert to the 20 common genetically encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). In certain embodiments, the non-naturally encoded amino acids include side chain functional groups that react efficiently and selectively with functional groups not present in the 20 common amino acids (including but not limited to azide, ketone, aldehyde, and aminooxy) to form conjugates. For example, IL-2 comprising a non-naturally encoded amino acid containing an azide functional group can react with a polymer (including but not limited to polyethylene glycol or a second polypeptide containing an alkyne moiety) to form a stable conjugate because of the selective reaction of the azide and alkyne functional groups to form a Huisgen [3+2] cycloaddition product.

[0236] The general structure of an α-amino acid is shown below (Formula I):

[0237] I

[0238]

[0239] Unnatural encoded amino acids generally have any structure with the structural formula listed above, where the R group is any substituent other than those used in the 20 natural amino acids and can be suitably used in the present invention. Since the unnatural encoded amino acids of the present invention generally differ from natural amino acids only in the structure of the side chain, the unnatural encoded amino acids form amide bonds with other amino acids (including but not limited to natural or unnatural encoded amino acids) in the same manner as they form in naturally occurring polypeptides. However, the unnatural encoded amino acids have side chain groups different from those of the natural amino acids. For example, R optionally includes alkyl-, aryl-, acyl-, keto-, azido-, hydroxy-, hydrazine, cyano-, halo-, acylhydrazide, alkenyl, alkynyl, ether, thiol, seleno-, sulfonyl-, borate ester, boronic acid group, phosphoryl, phosphonyl, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino, etc. or any combination thereof. Other unnatural amino acids of interest that may be suitable for the present invention include but are not limited to amino acids containing photoactivatable crosslinkers, spin-labeled amino acids, fluorescent amino acids, metal-binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with new functional groups, amino acids that covalently or non-covalently interact with other molecules, photocaged and / or photoisomerizable amino acids, amino acids containing biotin or biotin analogs, glycosylated amino acids such as sugar-substituted serine, other sugar-modified amino acids, keto-containing amino acids, amino acids containing polyethylene glycol or polyether, heavy atom-substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids with extended side chains compared to natural amino acids (including but not limited to polyethers or long-chain hydrocarbons, including but not limited to long-chain hydrocarbons having greater than about 5 or greater than about 10 carbons), carbon-linked sugar-containing amino acids, redox-active amino acids, amino acids containing aminothioacids, and amino acids containing one or more toxic moieties.

[0240] Exemplary non-naturally encoded amino acids that may be suitable for use in the present invention and may be used to react with water-soluble polymers include, but are not limited to, non-naturally encoded amino acids having carbonyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide, and alkyne reactive groups. In certain embodiments, the non-naturally encoded amino acids comprise a sugar moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine. Examples of such amino acids also include instances where the natural N- or O-linkage between the amino acid and the sugar is replaced by a covalent linkage not common in nature, including but not limited to alkenes, oximes, thioethers, amides, etc. Examples of such amino acids also include sugars not common in naturally occurring proteins, such as 2-deoxyglucose, 2-deoxygalactose, etc.

[0241] Many of the non-naturally encoded amino acids provided herein are commercially available, for example, from Sigma-Aldrich (St. Louis, MO, USA), Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), or Peptech (Burlington, MA, USA). Those non-naturally encoded amino acids that are not commercially available are optionally synthesized as provided herein or using standard methods known to those of ordinary skill in the art. For organic synthesis techniques, see, for example, Organic Chemistry, 2nd ed., by Fessendon and Fessendon, Willard Grant Press, Boston Mass (1982); Advanced Organic Chemistry, 3rd ed., by March, Wiley and Sons, New York (1985); and Advanced Organic Chemistry, 3rd ed., parts A and B, by Carey and Sundberg, Plenum Press, New York (1990). Also see U.S. Patent Nos. 7,045,337 and 7,083,970, which are incorporated herein by reference. In addition to non-natural amino acids having new side chains, non-natural amino acids that may be suitable for use in the present invention optionally also comprise modified backbone structures, including but not limited to those shown by the structures of Formulas II and III:

[0242] II

[0243]

[0244] III

[0245]

[0246] Wherein Z generally contains OH, NH2, SH, NH-R′ or S-R′; X and Y may be the same or different and generally contain S or O, and the optionally same or different R and R' are generally selected from the same list of constituents as the R groups described above for the unnatural amino acids of formula I and hydrogen. For example, the unnatural amino acids of the present invention optionally contain substitutions in the amino or carboxyl groups, as shown for formulas II and III. This type of unnatural amino acid includes, but is not limited to, α-hydroxy acids, α-thio acids, α-aminothiocarboxylic acid esters, including but not limited to those compounds having side chains corresponding to the common 20 natural amino acids or unnatural side chains. In addition, substitutions at the α-carbon optionally include, but are not limited to, L, D or α,α-disubstituted amino acids such as D-glutamic acid, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, etc. Other structural alternatives include cyclic amino acids such as proline analogs and 3-, 4-, 6-, 7-, 8- and 9-membered ring proline analogs, β and γ amino acids such as substituted β-alanine and γ-aminobutyric acid.

[0247] Many unnatural amino acids are based on natural amino acids such as tyrosine, glutamine, phenylalanine, etc. and are suitable for the present invention. Tyrosine analogs include, but are not limited to, para-substituted tyrosine, ortho-substituted tyrosine and meta-substituted tyrosine, wherein the substituted tyrosine contains, including but not limited to, keto groups (including but not limited to acetyl), benzoyl, amino, hydrazine, hydroxylamine, thiol, carboxyl, isopropyl, methyl, C6-C 20Straight-chain or branched-chain hydrocarbons, saturated or unsaturated hydrocarbons, O-methyl, polyether group, nitro, alkynyl, etc. In addition, poly-substituted aryl rings are also considered. Glutamine analogs applicable to the present invention include, but are not limited to, α-hydroxy derivatives, γ-substituted derivatives, cyclic derivatives, and amide-substituted glutamine derivatives. Examples of phenylalanine analogs applicable to the present invention include, but are not limited to, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine, wherein the substituents include, but are not limited to, hydroxyl, methoxy, methyl, allyl, aldehyde, azide, iodo, bromo, keto (including but not limited to acetyl), benzoyl, alkynyl, etc. Specific examples of unnatural amino acids applicable to the present invention include, but are not limited to, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-dopa, fluorophenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphoserine, phosphotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, and p-propynyloxy-phenylalanine, etc. Examples of the structures of various different unnatural amino acids applicable to the present invention are provided in, for example, WO 2002 / 085923 entitled "In vivo incorporation of unnatural amino acids". For other methionine analogs, also see Kiick et al., Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation, PNAS 99:19-24 (2002), which is incorporated herein by reference. International Application No. PCT / US06 / 47822 entitled "Compositions Containing, Methods Involving, and Uses of Non-natural Amino Acids and Polypeptides", which is incorporated herein by reference, describes the reductive alkylation and reductive amination of aromatic amine moieties (including but not limited to p-amino-phenylalanine).

[0248] In another embodiment of the present invention, the IL-2 polypeptide having one or more non-naturally encoded amino acids is covalently modified. Selective chemical reactions orthogonal to the diverse functional groups of biological systems are considered important tools in chemical biology. As relatively latecomers to the synthetic chemistry family, these bioorthogonal reactions have inspired new strategies for compound library synthesis, protein engineering, functional proteomics, and cell surface chemical remodeling. Azides have played an important role as unique chemical handles for bioconjugation. The Staudinger ligation has been used with phosphines to label azidosugars metabolically introduced into cellular glycoconjugates. The Staudinger ligation can be carried out in live animals and is physiologically innocuous; however, the Staudinger reaction is not without disadvantages. The required phosphines are susceptible to air oxidation, and optimizations for increased water solubility and improved reaction rates have proven synthetically challenging.

[0249] Azides have an alternative bioorthogonal reactivity pattern: the [3+2] cycloaddition with alkynes described by Huisgen. In its classical form, this reaction has limited applicability in biological systems due to the elevated temperature (or pressure) required for reasonable reaction rates. Sharpless and co-workers overcame this obstacle by developing a copper(I)-catalyzed form known as "click chemistry", which proceeds readily at physiological temperature and in a richly functionalized biological environment. This discovery has enabled the selective modification of virus particles, nucleic acids, and proteins from complex tissue lysates. Unfortunately, the obligatory copper catalyst is toxic to both bacteria and mammalian cells, thus precluding applications in which cells must remain viable. Catalyst-free Huisgen cycloadditions of alkynes activated by electron-withdrawing substituents have been reported to occur at ambient temperature. However, these compounds undergo Michael reactions with biological nucleophiles.

[0250] In one embodiment, there is provided a composition of IL-2 comprising a non-natural amino acid (such as p-(propargyloxy)-phenylalanine). Also provided are various different compositions comprising p-(propargyloxy)-phenylalanine, including but not limited to proteins and / or cells. In one aspect, the composition comprising the p-(propargyloxy)-phenylalanine non-natural amino acid further comprises an orthogonal tRNA. The non-natural amino acid can be bonded (including but not limited to covalently) to the orthogonal tRNA, including but not limited to covalently bonding to the orthogonal tRNA through an amino-acyl bond, covalently bonding to the 3'OH or 2'OH of the terminal ribose of the orthogonal tRNA, etc.

[0251] Chemical moieties that can be incorporated into proteins via non-natural amino acids provide various different advantages and operations to the proteins. For example, the unique reactivity of the keto functional group allows for the selective modification of proteins either in vitro or in vivo with any one of a large number of reagents containing hydrazine or hydroxylamine. For example, heavy atom non-natural amino acids may be useful for phasing X-ray structure data. The site-specific introduction of heavy atoms using non-natural amino acids also provides selectivity and flexibility in choosing positions for the heavy atoms. Photo-reactive non-natural amino acids (including but not limited to amino acids having benzophenone and aryl azide (including but not limited to phenyl azide) side chains) allow for efficient in vivo and in vitro photo-crosslinking of proteins, for example. Examples of photo-reactive non-natural amino acids include but are not limited to para-azido-phenylalanine and para-benzoyl-phenylalanine. Proteins having photo-reactive non-natural amino acids can thus be crosslinked at will by excitation of the photo-reactive groups, providing temporal control. In one example, the methyl group of a non-natural amino acid can be replaced with, including but not limited to, an isotope-labeled methyl group to serve as a probe for local structure and dynamics when using, including but not limited to, nuclear magnetic resonance and vibrational spectroscopy. Alkyne or azide functional groups allow for the selective modification of proteins with molecules, for example, by [3+2] cycloaddition reactions.

[0252] Non-natural amino acids incorporated at the amino terminus of a polypeptide can be composed of an R group that is any substituent other than those used among the 20 natural amino acids and a second reactive group that is different from the NH2 group normally present in α-amino acids (see formula I). Similar non-natural amino acids can be incorporated at the carboxyl terminus, which has a second reactive group different from the COOH group normally present in α-amino acids (see formula I).

[0253] The non-natural amino acids of the present invention can be selected or designed to provide other properties not available among the 20 natural amino acids. For example, non-natural amino acids can be optionally designed or selected to modify, for example, the biological properties of the proteins into which they are incorporated. For example, by including non-natural amino acids in a protein, the following properties can be optionally modified: toxicity, biodistribution, solubility, stability such as thermal, hydrolytic, oxidative stability, resistance to enzymatic degradation, etc., ease of purification and processing, structural properties, spectral properties, chemical and / or photochemical properties, catalytic activity, redox potential, half-life, ability to react with other molecules such as covalently or non-covalently, etc.

[0254] In certain embodiments, the present invention provides an IL-2 linked to a water-soluble polymer such as PEG by an oxime bond. Many types of non-naturally encoded amino acids are suitable for forming an oxime bond. They include, but are not limited to, non-naturally encoded amino acids containing a carbonyl, a dicarbonyl, or a hydroxylamine group. These amino acids are described in U.S. Patent Publication Nos. 2006 / 0194256, 2006 / 0217532, and 2006 / 0217289, and in WO 2006 / 069246 entitled "Compositions Containing, Methods Involving, and Uses of Non-natural Amino Acids and Polypeptides", which are hereby incorporated by reference in their entirety. Non-naturally encoded amino acids are also described in U.S. Patent No. 7,083,970 and U.S. Patent No. 7,045,337, which are hereby incorporated by reference in their entirety.

[0255] Certain embodiments of the present invention utilize an IL-2 polypeptide in which the p-acetylphenylalanine amino acid is substituted at one or more positions. The synthesis of p-acetyl-(+ / -)-phenylalanine and m-acetyl-(+ / -)-phenylalanine is described in Zhang, Z. et al., Biochemistry 42:6735-6746 (2003), which is incorporated by reference. Other carbonyl- or dicarbonyl-containing amino acids can be prepared similarly by those of ordinary skill in the art. In addition, non-limiting exemplary syntheses of the non-natural amino acids included herein are presented in FIGS. 4, 24-34, and 36-39 of U.S. Patent No. 7,083,970, which are hereby incorporated by reference in their entirety.

[0256] Amino acids having an electrophilic reactive group allow the use of a variety of different reactions, especially for linking molecules by nucleophilic addition reactions. Such electrophilic reactive groups include a carbonyl (including a ketone group and a dicarbonyl), a carbonyl-like group (which has reactivity similar to that of a carbonyl (including a ketone group and a dicarbonyl) and is structurally similar to a carbonyl), a masked carbonyl (which can be easily converted into a carbonyl (including a ketone group and a dicarbonyl)), or a protected carbonyl (which has reactivity similar to that of a carbonyl (including a ketone group and a dicarbonyl) after deprotection). These amino acids include amino acids having a structure of formula (IV):

[0257]

[0258] Wherein:

[0259] A is optional and, when present, is a lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene or substituted aralkylene;

[0260] B is optional and, when present, is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2 or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) k N(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)2-N=N- and -C(R’)2-N(R’)-N(R’)- linkers, where each R’ is independently H, alkyl or substituted alkyl;

[0261] J is

[0262] R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

[0263] Each R” is independently H, alkyl, substituted alkyl or a protecting group, or when more than one R” group is present, two R”s optionally form a heterocycloalkyl;

[0264] R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

[0265] R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

[0266] Each R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form cycloalkyl or heterocycloalkyl;

[0267] Or the –A-B-J-R group together forms a bicyclic or tricyclic cycloalkyl or heterocycloalkyl containing at least one carbonyl group, said carbonyl group including a dicarbonyl group, a protected carbonyl group (including a protected dicarbonyl group), or a masked carbonyl group (including a masked dicarbonyl group);

[0268] Or the –J-R group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl containing at least one carbonyl group, said carbonyl group including a dicarbonyl group, a protected carbonyl group (including a protected dicarbonyl group), or a masked carbonyl group (including a masked dicarbonyl group);

[0269] Provided that when A is phenylene and each R3 is H, B is present; and when A is –(CH2)4- and each R3 is H, B is not –NHC(O)(CH2CH2)-; and when A and B are absent and each R3 is H, R is not methyl.

[0270] In addition, it includes amino acids having the structure of formula (V):

[0271]

[0272] Wherein:

[0273] A is optional and, when present, is lower alkylene, substituted lower alkylene, lower subcycloalkylene, substituted lower subcycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower subheteroalkylene, substituted subheteroalkylene, lower subheterocycloalkylene, substituted lower subheterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkyl, or substituted aralkyl;

[0274] B is optional and, when present, is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower subheteroalkylene, substituted lower subheteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2, or 3), -S(O) k(Alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) k Linkers of -N(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)2-N=N- and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl or substituted alkyl;

[0275] R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

[0276] R1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

[0277] R2 is optional and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;

[0278] Provided that when A is phenylene, B is present; and when A is –(CH2)4-, B is not –NHC(O)(CH2CH2)-; and when A and B are absent, R is not methyl.

[0279] In addition, it includes amino acids having the structure of formula (VI):

[0280]

[0281] Wherein:

[0282] B is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2 or 3), -S(O) k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) k linkers of -N(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)2-N=N- and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl or substituted alkyl;

[0283] R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

[0284] R1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

[0285] R2 is optional and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;

[0286] each R a is independently selected from H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O) k R’ (where k is 1, 2 or 3), -C(O)N(R’)2, -OR’ and -S(O) k R’, where each R’ is independently H, alkyl or substituted alkyl.

[0287] In addition, the following amino acids are included:

[0288]

[0289] wherein these compounds are optionally protected at the amino group, protected at the carboxyl group or are salts thereof. In addition, any of the following unnatural amino acids can be incorporated into unnatural amino acid polypeptides.

[0290] In addition, the following amino acids having the structure of formula (VII) are included:

[0291]

[0292] wherein

[0293] B is optional and, when present, is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2 or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) k N(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)2-N=N- and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl or substituted alkyl;

[0294] R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

[0295] R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

[0296] R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;

[0297] each R a is independently selected from H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O) k R’(where k is 1, 2 or 3), -C(O)N(R’)2, -OR’ and -S(O) k R’, where each R’ is independently H, alkyl or substituted alkyl; and n is from 0 to 8;

[0298] provided that when A is –(CH2)4-, B is not –NHC(O)(CH2CH2)-.

[0299] In addition, the following amino acids are included:

[0300]

[0301] Wherein these compounds are optionally protected at the amino group, optionally protected at the carboxyl group, optionally protected at both the amino and carboxyl groups, or are salts thereof. In addition, these unnatural amino acids and any of the following unnatural amino acids can be incorporated into unnatural amino acid polypeptides.

[0302] In addition, the following amino acids having the structure of formula (VIII) are included:

[0303]

[0304] Wherein A is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkyl or substituted aralkyl;

[0305] B is optional and when present is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2 or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) kLinkers of -N(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)2-N=N- and -C(R’)2-N(R’)-N(R’)-, wherein each R’ is independently H, alkyl or substituted alkyl;

[0306] R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

[0307] R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide.

[0308] In addition, it includes amino acids having the following structure of formula (IX):

[0309]

[0310] B is optional and, when present, is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2 or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) k Linkers of -N(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)2-N=N- and -C(R’)2-N(R’)-N(R’)-, wherein each R’ is independently H, alkyl or substituted alkyl;

[0311] R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

[0312] R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0313] R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

[0314] wherein each R a is independently selected from H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O) k R’ (where k is 1, 2, or 3), -C(O)N(R’)2, -OR’, and -S(O) k R’, where each R’ is independently H, alkyl, or substituted alkyl.

[0315] In addition, it includes the following amino acids:

[0316]

[0317] wherein these compounds are optionally protected at the amino group, optionally protected at the carboxyl group, optionally protected at both the amino and carboxyl groups, or are salts thereof. In addition, these non-natural amino acids and any of the following non-natural amino acids can be incorporated into non-natural amino acid polypeptides.

[0318] In addition, it includes the following amino acids having the structure of formula (X):

[0319]

[0320] wherein B is optional and, when present, is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) kLinkers of -N(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)2-N=N- and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl or substituted alkyl;

[0321] R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

[0322] R1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

[0323] R2 is optional and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;

[0324] Each R a is independently selected from H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O) k R’ (where k is 1, 2 or 3), -C(O)N(R’)2, -OR’ and -S(O) k R’, where each R’ is independently H, alkyl or substituted alkyl; and n is from 0 to 8.

[0325] In addition, the following amino acids are included:

[0326]

[0327] wherein these compounds are optionally protected at the amino group, optionally protected at the carboxyl group, optionally protected at both the amino and carboxyl groups, or are salts thereof. In addition, these unnatural amino acids and any of the following unnatural amino acids can be incorporated into unnatural amino acid polypeptides.

[0328] In addition to the monocarbonyl structure, the unnatural amino acids described herein can include groups such as dicarbonyl, dicarbonyl-like, masked dicarbonyl and protected dicarbonyl.

[0329] For example, amino acids having the structure of formula (XI) below are included:

[0330]

[0331] where A is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkyl or substituted aralkyl;

[0332] B is optional and, when present, is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2 or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) k linkers of -N(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)2-N=N- and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl or substituted alkyl;

[0333] R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

[0334] R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

[0335] R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide.

[0336] In addition, it includes amino acids having the following structure of formula (XII):

[0337]

[0338] B is optional and, when present, is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k-(where k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) k linkers of -N(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl;

[0339] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0340] R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0341] R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

[0342] where each R a is independently selected from H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O) k R’ (where k is 1, 2, or 3), -C(O)N(R’)2, -OR’, and -S(O) k R’, where each R’ is independently H, alkyl, or substituted alkyl.

[0343] In addition, it includes the following amino acids:

[0344]

[0345] where these compounds are optionally protected at the amino group, optionally protected at the carboxyl group, optionally protected at both the amino and carboxyl groups, or are salts thereof. In addition, these unnatural amino acids and any of the following unnatural amino acids can be incorporated into unnatural amino acid polypeptides.

[0346] In addition, it includes the following amino acids having the structure of formula (XIII):

[0347]

[0348] wherein B is optional and when present is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2 or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) k N(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)2-N=N- and -C(R’)2-N(R’)-N(R’)- linkers, where each R’ is independently H, alkyl or substituted alkyl;

[0349] R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; R1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and R2 is optional and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; each R a is independently selected from H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O) k R’(where k is 1, 2 or 3), -C(O)N(R’)2, -OR’ and -S(O) k R’, where each R’ is independently H, alkyl or substituted alkyl; and n is from 0 to 8.

[0350] In addition, it includes the following amino acids:

[0351]

[0352] Among these compounds, the amino group is optionally protected, the carboxyl group is optionally protected, the amino group is optionally protected and the carboxyl group is protected, or it is a salt thereof. In addition, these unnatural amino acids and any of the following unnatural amino acids can be incorporated into unnatural amino acid polypeptides.

[0353] In addition, it includes amino acids having the structure of formula (XIV) below:

[0354]

[0355] Wherein:

[0356] A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene or substituted aralkylene;

[0357] R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and R2 is optional, and when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; X1 is C, S or S(O); and L is alkylene, substituted alkylene, N(R’)(alkylene) or N(R’)(substituted alkylene), wherein R’ is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.

[0358] In addition, it includes amino acids having the structure of formula (XIV-A) below:

[0359]

[0360] Wherein:

[0361] A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene or substituted aralkylene;

[0362] R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;

[0363] L is alkylene, substituted alkylene, N(R’)(alkylene) or N(R’)(substituted alkylene), where R’ is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.

[0364] In addition, it includes amino acids having the structure of formula (XIV-B) below:

[0365]

[0366] where:

[0367] A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

[0368] R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; L is alkylene, substituted alkylene, N(R’)(alkylene) or N(R’)(substituted alkylene), where R’ is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.

[0369] In addition, it includes amino acids having the structure of formula (XV) below:

[0370]

[0371] where:

[0372] A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

[0373] R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; X1 is C, S, or S(O); and n is 0, 1, 2, 3, 4, or 5; and each CR 8 R 9 On each R of the 8 and R 9 groups are independently selected from H, an alkoxy group, an alkylamine, a halogen, an alkyl group, an aryl group, or any R 8 and R 9 may together form ═O or a cycloalkyl group, or any adjacent R 8 groups may together form a cycloalkyl group.

[0374] In addition, it includes amino acids having the structure of formula (XV-A) below:

[0375]

[0376] Wherein:

[0377] A is optional and, when present, is a lower alkylene group, a substituted lower alkylene group, a lower cycloalkylene group, a substituted lower cycloalkylene group, a lower alkenylene group, a substituted lower alkenylene group, an alkynylene group, a lower heteroalkylene group, a substituted heteroalkylene group, a lower heterocycloalkylene group, a substituted lower heterocycloalkylene group, an arylene group, a substituted arylene group, a heteroarylene group, a substituted heteroarylene group, an alkarylene group, a substituted alkarylene group, an aralkyl group, or a substituted aralkyl group;

[0378] R is H, an alkyl group, a substituted alkyl group, a cycloalkyl group, or a substituted cycloalkyl group; R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

[0379] n is 0, 1, 2, 3, 4, or 5; and each CR 8 R 9 On each R of the 8 and R 9 groups are independently selected from H, an alkoxy group, an alkylamine, a halogen, an alkyl group, an aryl group, or any R 8 and R 9 may together form ═O or a cycloalkyl group, or any adjacent R 8 groups may together form a cycloalkyl group.

[0380] In addition, it includes amino acids having the structure of formula (XV-B) below:

[0381]

[0382] Wherein:

[0383] A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkyl or substituted aralkyl;

[0384] R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; n is 0, 1, 2, 3, 4 or 5; and each CR 8 R 9 On each R of the group 8 and R 9 Are independently selected from H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 and R 9 May together form ═O or cycloalkyl, or any adjacent R 8 Groups may together form cycloalkyl.

[0385] In addition, it includes the amino acids having the structure of formula (XVI) as follows:

[0386]

[0387] Wherein:

[0388] A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkyl or substituted aralkyl; R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

[0389] R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

[0390] R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; X1 is C, S, or S(O); and L is an alkylene, a substituted alkylene, N(R’)(alkylene), or N(R’)(substituted alkylene), where R’ is H, an alkyl, a substituted alkyl, a cycloalkyl, or a substituted cycloalkyl.

[0391] In addition, it includes the following amino acids having the structure of formula (XVI-A):

[0392]

[0393] where:

[0394] A is optional and, when present, is a lower alkylene, a substituted lower alkylene, a lower cycloalkylene, a substituted lower cycloalkylene, a lower alkenylene, a substituted lower alkenylene, an alkynylene, a lower heteroalkylene, a substituted heteroalkylene, a lower heterocycloalkylene, a substituted lower heterocycloalkylene, an arylene, a substituted arylene, a heteroarylene, a substituted heteroarylene, an alkarylene, a substituted alkarylene, an aralkyl, or a substituted aralkyl; R is H, an alkyl, a substituted alkyl, a cycloalkyl, or a substituted cycloalkyl;

[0395] R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; L is an alkylene, a substituted alkylene, N(R’)(alkylene), or N(R’)(substituted alkylene), where R’ is H, an alkyl, a substituted alkyl, a cycloalkyl, or a substituted cycloalkyl.

[0396] In addition, it includes the following amino acids having the structure of formula (XVI-B):

[0397]

[0398] where:

[0399] A is optional and, when present, is a lower alkylene, a substituted lower alkylene, a lower cycloalkylene, a substituted lower cycloalkylene, a lower alkenylene, a substituted lower alkenylene, an alkynylene, a lower heteroalkylene, a substituted heteroalkylene, a lower heterocycloalkylene, a substituted lower heterocycloalkylene, an arylene, a substituted arylene, a heteroarylene, a substituted heteroarylene, an alkarylene, a substituted alkarylene, an aralkyl, or a substituted aralkyl; R is H, an alkyl, a substituted alkyl, a cycloalkyl, or a substituted cycloalkyl;

[0400] R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; L is an alkylene, a substituted alkylene, N(R’)(alkylene) or N(R’)(substituted alkylene), where R’ is H, an alkyl, a substituted alkyl, a cycloalkyl or a substituted cycloalkyl.

[0401] In addition, it includes amino acids having the structure of formula (XVII):

[0402]

[0403] Wherein:

[0404] A is optional and, when present, is a lower alkylene, a substituted lower alkylene, a lower sub-cycloalkyl, a substituted lower sub-cycloalkyl, a lower alkenylene, a substituted lower alkenylene, an alkynylene, a lower heteroalkylene, a substituted heteroalkylene, a lower heterocycloalkyl, a substituted lower heterocycloalkyl, an arylene, a substituted arylene, a heteroarylene, a substituted heteroarylene, an alkarylene, a substituted alkarylene, an aralkylene or a substituted aralkylene;

[0405] M is -C(R3)-,

[0406] where (a) indicates bonding to the A group and (b) indicates bonding to the corresponding carbonyl, R3 and R4 are independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl, or R3 and R4 or two R3 groups or two R4 groups optionally form a cycloalkyl or a heterocycloalkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; T3 is a bond, C(R)(R), O or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide.

[0407] In addition, it includes amino acids having the structure of formula (XVIII):

[0408]

[0409] Wherein:

[0410] M is -C(R3)-,

[0411] (a) indicates bonding to group A, and (b) indicates bonding to the corresponding carbonyl. R3 and R4 are independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl, or R3 and R4 or two R3 groups or two R4 groups optionally form cycloalkyl or heterocycloalkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; T3 is a bond, C(R)(R), O or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; R1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and R2 is optional and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; each R a is independently selected from H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O) k R’ (where k is 1, 2 or 3), -C(O)N(R’)2, -OR’ and -S(O) k R’, where each R’ is independently H, alkyl or substituted alkyl.

[0412] In addition, it includes amino acids having the structure of formula (XIX):

[0413]

[0414] wherein:

[0415] R is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; and

[0416] T3 is O or S.

[0417] In addition, it includes amino acids having the structure of formula (XX):

[0418]

[0419] wherein:

[0420] R is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.

[0421] In addition, it includes the following amino acids having the structure of formula (XXI):

[0422]

[0423] In certain embodiments, polypeptides containing unnatural amino acids are chemically modified to generate reactive carbonyl or dicarbonyl functional groups. For example, an aldehyde functional group that can be used in a coupling reaction can be generated from a functional group having an adjacent amino and hydroxyl group. For example, in the case where the bioactive molecule is a polypeptide, N-terminal serine or threonine (which may be present normally or may be exposed by chemical or enzymatic digestion) can be used to generate an aldehyde functional group using periodate under mild oxidative cleavage conditions. See, e.g., Gaertner et al., Bioconjug. Chem. 3:262-268 (1992); Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertner et al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known in the art are limited to amino acids at the N-terminus of a peptide or protein.

[0424] In the present invention, unnatural amino acids bearing adjacent hydroxyl and amino groups can be incorporated into polypeptides as "masked" aldehyde functional groups. For example, 5-hydroxylysine bears a hydroxyl group adjacent to the ε amino group. Reaction conditions for generating aldehydes generally include adding a molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other sites within the polypeptide. The pH of the oxidation reaction is typically about 7.0. A typical reaction involves adding about 1.5 molar equivalents of sodium metaperiodate to a buffered solution of the polypeptide, followed by incubation in the dark for about 10 minutes. See, e.g., U.S. Patent No. 6,423,685.

[0425] Carbonyl or dicarbonyl functional groups can react selectively with reagents containing hydroxylamine under mild conditions in an aqueous solution to form the corresponding oxime bonds that are stable under physiological conditions. See, e.g., Jencks, W.P., J. Am. Chem. Soc. 81, 475-481 (1959); Shao, J. and Tam, J.P., J. Am. Chem. Soc. 117:3893-3899 (1995). In addition, the unique reactivity of carbonyls or dicarbonyls allows for selective modification in the presence of other amino acid side chains. See, e.g., Cornish, V.W. et al., J. Am. Chem. Soc. 118:8150-8151 (1996); Geoghegan, K.F. & Stroh, J.G., Bioconjug. Chem. 3:138-146 (1992); Mahal, L.K. et al., Science 276:1125-1128 (1997).

[0426] A. Carbonyl Reactive Groups

[0427] Amino acids having a carbonyl-reactive group allow for a variety of different reactions to attach molecules (including but not limited to PEG or other water-soluble molecules), particularly via nucleophilic addition or aldol condensation reactions.

[0428] Exemplary carbonyl-containing amino acids can be represented as follows:

[0429]

[0430] where n is 0 - 10; R1 is alkyl, aryl, substituted alkyl or substituted aryl; R2 is H, alkyl, aryl, substituted alkyl and substituted aryl; and R3 is H, an amino acid, a polypeptide or an amino-terminal modifying group, and R4 is H, an amino acid, a polypeptide or a carboxyl-terminal modifying group. In certain embodiments, n is 1, R1 is phenyl and R2 is a simple alkyl (i.e., methyl, ethyl or propyl), and the keto moiety is in the para position relative to the alkyl side chain. In certain embodiments, n is 1, R1 is phenyl and R2 is a simple alkyl (i.e., methyl, ethyl or propyl), and the keto moiety is in the meta position relative to the alkyl side chain.

[0431] The synthesis of p-acetyl-(+ / -)-phenylalanine and m-acetyl-(+ / -)-phenylalanine is described in Zhang, Z. et al., Biochemistry 42:6735 - 6746 (2003), which is incorporated herein by reference. Other carbonyl-containing amino acids can be prepared similarly by those of ordinary skill in the art.

[0432] In certain embodiments, polypeptides containing non-naturally encoded amino acids are chemically modified to generate reactive carbonyl functional groups. For example, an aldehyde functional group for coupling reactions can be generated from a functional group having adjacent amino and hydroxyl groups. For example, in the case where the bioactive molecule is a polypeptide, N-terminal serine or threonine (which may be present normally or may be exposed by chemical or enzymatic digestion) can be used to generate an aldehyde functional group using periodate under mild oxidative cleavage conditions. See, e.g., Gaertner et al., Bioconjug. Chem. 3:262 - 268 (1992); Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138 - 146 (1992); Gaertner et al., J. Biol. Chem. 269:7224 - 7230 (1994). However, methods known in the art are limited to amino acids at the N-terminus of the peptide or protein.

[0433] In the present invention, non-naturally encoded amino acids bearing adjacent hydroxyl and amino groups can be incorporated into polypeptides as "masked" aldehyde functional groups. For example, 5-hydroxylysine bears a hydroxyl group adjacent to the ε-amino group. Reaction conditions for generating the aldehyde typically include adding a molar excess of sodium periodate under mild conditions to avoid oxidation at other sites within the polypeptide. The pH of the oxidation reaction is typically about 7.0. A typical reaction involves adding about 1.5 molar equivalents of sodium periodate to a buffered solution of the polypeptide, followed by incubation in the dark for about 10 minutes. See, e.g., U.S. Patent No. 6,423,685, which is incorporated herein by reference.

[0434] The carbonyl functional group can react selectively with reagents containing hydrazine, hydrazide, hydroxylamine, or semicarbazide under mild conditions in aqueous solution to form the corresponding hydrazone, oxime, or semicarbazone bonds, which are stable under physiological conditions. See, e.g., Jencks, W.P., J. Am. Chem. Soc. 81, 475 - 481 (1959); Shao, J. and Tam, J.P., J. Am. Chem. Soc. 117:3893 - 3899 (1995). In addition, the unique reactivity of the carbonyl allows for selective modification in the presence of other amino acid side chains. See, e.g., Cornish, V.W. et al., J. Am. Chem. Soc. 118:8150 - 8151 (1996); Geoghegan, K.F. & Stroh, J.G., Bioconjug. Chem. 3:138 - 146 (1992); Mahal, L.K. et al., Science 276:1125 - 1128 (1997).

[0435] B. Hydrazine, Hydrazide, or Semicarbazide Reactive Groups

[0436] Non-naturally encoded amino acids containing nucleophilic groups such as hydrazine, hydrazide, or semicarbazide allow for reaction with a variety of different electrophilic groups to form conjugates (including but not limited to conjugates with PEG or other water-soluble polymers).

[0437] Exemplary hydrazine-, hydrazide-, or semicarbazide-containing amino acids can be represented as follows:

[0438]

[0439] where n is 0 - 10; R1 is alkyl, aryl, substituted alkyl, or substituted aryl or absent; X is O, N, or S or absent; R2 is H, amino acid, polypeptide, or amino-terminal modifying group, and R3 is H, amino acid, polypeptide, or carboxyl-terminal modifying group.

[0440] In certain embodiments, n is 4, R1 is absent, and X is N. In certain embodiments, n is 2, R1 is absent, and X is absent. In certain embodiments, n is 1, R1 is phenyl, X is O, and the oxygen atom is para to the aliphatic group on the aryl ring.

[0441] Amino acids containing hydrazide, hydrazine, and semicarbazide can be obtained from commercial sources. For example, L-glutamic acid-γ-hydrazide can be obtained from Sigma Chemical (St. Louis, MO). Other non-commercially available amino acids can be prepared by those of ordinary skill in the art. See, for example, U.S. Patent No. 6,281,211, which is incorporated herein by reference.

[0442] Polypeptides containing non-naturally encoded amino acids with hydrazide, hydrazine, or semicarbazide functional groups can react efficiently and selectively with molecules containing aldehyde or other functional groups with similar chemical reactivity. See, for example, Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995). The unique reactivity of hydrazide, hydrazine, and semicarbazide functional groups makes them significantly more reactive towards aldehydes, ketones, and other electrophilic groups compared to the nucleophilic groups present on the 20 common amino acids, including but not limited to the hydroxyl groups of serine or threonine or the amino groups at lysine and the N-terminus.

[0443] C. Aminooxy-containing amino acids

[0444] Non-naturally encoded amino acids containing an aminooxy (also known as hydroxylamine) group allow for reaction with a variety of different electrophilic groups to form conjugates (including but not limited to conjugates with PEG or other water-soluble polymers). Similar to hydrazine, hydrazide, and semicarbazide, the high nucleophilicity of aminooxy allows it to react efficiently and selectively with a variety of different molecules containing aldehyde or other functional groups with similar chemical reactivity. See, for example, Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34:727-736 (2001). Although the reaction of hydrazine groups results in the corresponding hydrazone, the reaction of aminooxy with a carbonyl group such as a ketone typically gives an oxime.

[0445] Exemplary amino acids containing aminooxy can be represented as follows:

[0446]

[0447] Wherein n is from 0 to 10; R1 is alkyl, aryl, substituted alkyl or substituted aryl or absent; X is O, N, S or absent; m is from 0 to 10; Y = C(O) or absent; R2 is H, an amino acid, a polypeptide or an amino-terminal modifying group, and R3 is H, an amino acid, a polypeptide or a carboxyl-terminal modifying group. In certain embodiments, n is 1, R1 is phenyl, X is O, m is 1, and Y is present. In certain embodiments, n is 2, R1 and X are absent, m is 0, and Y is absent.

[0448] Aminooxy-containing amino acids can be prepared from readily available amino acid precursors (homoserine, serine and threonine). See, for example, M. Carrasco and R. Brown, J. Org. Chem. 68:8853-8858 (2003). Certain aminooxy-containing amino acids such as L-2-amino-4-(aminooxy)butyric acid have been isolated from natural sources (Rosenthal, G., Life Sci. 60:1635-1641 (1997)). Other aminooxy-containing amino acids can be prepared by those of ordinary skill in the art.

[0449] D. Azide and alkyne reactive groups

[0450] The unique reactivity of azide and alkyne functional groups makes them extremely useful for the selective modification of polypeptides and other biomolecules. Organic azides, especially aliphatic azides and alkynes, are generally stable to common reactive chemical conditions. Specifically, both azide and alkyne functional groups are inert to the side chains (i.e., R groups) of the 20 common amino acids present in naturally occurring polypeptides. However, when in proximity, the "spring-loaded" nature of azide and alkyne groups is revealed, and they react selectively and efficiently via the Huisgen [3+2] cycloaddition reaction to produce the corresponding triazoles. See, for example, Chin J. et al., Science 301:964-7 (2003); Wang, Q. et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Chin, J. W. et al., J. Am. Chem. Soc. 124:9026-9027 (2002).

[0451] Since the Huisgen cycloaddition reaction involves a selective cycloaddition reaction (see, for example, Padwa, A., Comprehensive Organic Synthesis, Vol. 4, Trost, B. M. ed. (1991), p. 1069-1109; Huisgen, R., 1,3-Dipolar Cycloaddition Chemistry IPOLAR CYCLOADDITION C HEMISTRY), Padwa, A. Ed., (1984), p. 1 - 176), rather than nucleophilic substitution, so the incorporation of non - natural - coded amino acids with azido - and alkyne - containing side chains allows the resulting polypeptides to be selectively modified at the positions of the non - natural - coded amino acids. The cycloaddition reactions involving azido - or alkyne - containing IL - 2 can be carried out in situ at room temperature and in aqueous conditions by adding a catalytic amount of Cu(II) (including but not limited to in the form of catalytic amounts of CuSO4) in the presence of a reducing agent for reducing Cu(II) to Cu(I). See, for example, Wang, Q. et al., J. Am. Chem. Soc. 125, 3192 - 3193 (2003); Tornoe, C. W. et al., J. Org. Chem. 67:3057 - 3064 (2002); Rostovtsev et al., Angew. Chem. Int. Ed. 41:2596 - 2599 (2002). Exemplary reducing agents include but are not limited to ascorbate, metallic copper, quinine, hydroquinone, vitamin K, glutathione, cysteine, Fe 2+ , Co 2+ and the applied potential.

[0452] In some cases, when a Huisgen [3 + 2] cycloaddition reaction between azide and alkyne is desired, the IL - 2 contains a non - natural - coded amino acid containing an alkyne moiety and a water - soluble polymer containing an azido moiety to be attached to the amino acid. Alternatively, the reverse reaction can also be carried out (i.e., using the azido moiety on the amino acid and the alkyne moiety present on the water - soluble polymer).

[0453] The azido functional group can also selectively react with a water - soluble polymer suitably functionalized with an aryl ester and an arylphosphine moiety to form an amide bond. The arylphosphine group reduces the azide in situ, and the resulting amine then reacts efficiently with the neighboring ester bond to produce the corresponding amide. See, for example, E. Saxon and C. Bertozzi, Science 287, 2007 - 2010 (2000). The azido - containing amino acid can be an alkyl azide (including but not limited to 2 - amino - 6 - azido - 1 - hexanoic acid) or an aryl azide (p - azido - phenylalanine).

[0454] Exemplary water - soluble polymers containing aryl esters and phosphine moieties can be represented as follows:

[0455]

[0456] Wherein X can be O, N, S or absent, Ph is phenyl, W is a water-soluble polymer, and R can be H, alkyl, aryl, substituted alkyl and substituted aryl. Exemplary R groups include, but are not limited to, -CH2, -C(CH3)3, -OR’, -NR’R”, -SR’, -halogen, -C(O)R’, -CONR’R”, -S(O)2R’, -S(O)2NR’R”, -CN and –NO2. R’, R”, R”’ and R”” each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (including, but not limited to, aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy or aralkyl. When the compounds of the present invention include more than one R group, for example, each of said R groups is independently selected, and the same is true for each of these groups when there are more than one R’, R”, R’” and R”” groups. When R’ and R” are attached to the same nitrogen atom, they can combine with said nitrogen atom to form a 5-, 6- or 7-membered ring. For example, -NR’R” means including, but not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will understand that the term “alkyl” means including groups containing carbon atoms bonded to groups other than hydrogen groups such as haloalkyl (including, but not limited to, -CF3 and –CH2CF3) and acyl (including, but not limited to, -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0457] The azide functional group can also selectively react with a water-soluble polymer containing a thioester and suitably functionalized with an arylphosphine moiety to produce an amide bond. The arylphosphine group reduces the azide in situ, and the resulting amine then reacts efficiently with the thioester bond to produce the corresponding amide. Exemplary water-soluble polymers containing a thioester and a phosphine moiety can be represented as follows:

[0458]

[0459] Wherein n is 1-10; X can be O, N, S or absent, Ph is phenyl, and W is a water-soluble polymer.

[0460] Exemplary alkynyl amino acids can be represented as follows:

[0461]

[0462] wherein n is from 0 to 10; R1 is alkyl, aryl, substituted alkyl or substituted aryl or absent; X is O, N, S or absent; m is from 0 to 10, R2 is H, an amino acid, a polypeptide or an amino-terminal modifying group, and R3 is H, an amino acid, a polypeptide or a carboxyl-terminal modifying group. In certain embodiments, n is 1, R1 is phenyl, X is absent, m is 0 and the acetylene moiety is in the para position relative to the alkyl side chain. In certain embodiments, n is 1, R1 is phenyl, X is O, m is 1 and the propargyloxy group is in the para position relative to the alkyl side chain (i.e., O-propargyl-tyrosine). In certain embodiments, n is 1, R1 and X are absent and m is 0 (i.e., propargylglycine).

[0463] Alkynyl-containing amino acids are commercially available. For example, propargylglycine can be purchased from Peptech (Burlington, MA). Alternatively, alkynyl-containing amino acids can be prepared according to standard methods. For example, p-propargyloxyphenylalanine can be synthesized as described, for example, in Deiters, A. et al., J. Am. Chem. Soc. 125:11782-11783 (2003), and 4-alkynyl-L-phenylalanine can be synthesized as described in Kayser, B. et al., Tetrahedron 53(7):2475-2484 (1997). Other alkynyl-containing amino acids can be prepared by those of ordinary skill in the art.

[0464] Exemplary azido-containing amino acids can be represented as follows:

[0465]

[0466] wherein n is from 0 to 10; R1 is alkyl, aryl, substituted alkyl, substituted aryl or absent; X is O, N, S or absent; m is from 0 to 10; R2 is H, an amino acid, a polypeptide or an amino-terminal modifying group, and R3 is H, an amino acid, a polypeptide or a carboxyl-terminal modifying group. In certain embodiments, n is 1, R1 is phenyl, X is absent, m is 0 and the azide moiety is in the para position of the alkyl side chain. In certain embodiments, n is from 0 to 4 and R1 and X are absent, and m = 0. In certain embodiments, n is 1, R1 is phenyl, X is O, m is 2 and the β-azidoethoxy moiety is in the para position relative to the alkyl side chain.

[0467] Azide-containing amino acids can be obtained from commercial sources. For example, 4-azidophenylalanine can be obtained from Chem-Impex International, Inc. (Wood Dale, IL). For those azide-containing amino acids that are not commercially available, the azide group can be relatively easily prepared using standard methods known to those of ordinary skill in the art, including but not limited to displacement by a suitable leaving group (including but not limited to halides, mesylates, tosylates) or by opening of a suitably protected lactone. See, for example, March's Advanced Organic Chemistry (Third Edition, 1985, Wiley and Sons, New York).

[0468] E. Aminothiol reactive groups

[0469] The unique reactivity of β-substituted aminothiol functional groups makes them extremely useful for the selective modification of aldehyde-containing polypeptides and other biomolecules through the formation of thiazolidines. See, for example, J. Shao and J. Tam, J. Am. Chem. Soc., 117(14) 3893-3899 (1995). In certain embodiments, β-substituted aminothiol amino acids can be incorporated into an IL-2 polypeptide and then reacted with a water-soluble polymer containing an aldehyde functional group. In certain embodiments, a water-soluble polymer, a drug conjugate, or other payload can be conjugated to an IL-2 containing a β-substituted aminothiol amino acid through the formation of a thiazolidine.

[0470] F. Additional reactive groups

[0471] Additional reactive groups and non-naturally encoded amino acids (including, but not limited to, para-aminophenylalanine) that can be incorporated into the IL-2 polypeptides of the invention are described in the following patent applications, which are hereby incorporated by reference in their entirety: U.S. Patent Publication No. 2006 / 0194256, U.S. Patent Publication No. 2006 / 0217532, U.S. Patent Publication No. 2006 / 0217289, U.S. Provisional Patent No. 60 / 755,338; U.S. Provisional Patent No. 60 / 755,711; U.S. Provisional Patent No. 60 / 755,018; International Patent Application No. PCT / US06 / 49397; WO 2006 / 069246; U.S. Provisional Patent No. 60 / 743,041; U.S. Provisional Patent No. 60 / 743,040; International Patent Application No. PCT / US06 / 47822; U.S. Provisional Patent No. 60 / 882,819; U.S. Provisional Patent No. 60 / 882,500; and U.S. Provisional Patent No. 60 / 870,594. These applications also discuss reactive groups that may be present on PEG or other polymers for conjugation, including, but not limited to, hydroxylamine (aminoxy) groups.

[0472] Polypeptide with unnatural amino acid

[0473] The incorporation of non-natural amino acids can be carried out for various different purposes, including but not limited to modulating the interaction of a protein with its receptor or one or more subunits of its receptor, tailoring changes in protein structure and / or function, altering size, acidity, nucleophilicity, hydrogen bond formation, hydrophobicity, accessibility of protease target sites, targeting components (including but not limited to for protein arrays), adding bioactive molecules, attaching polymers, attaching radionuclides, modulating serum half-life, modulating tissue penetration (e.g., tumors), modulating active transport, modulating tissue, cell or organ specificity or distribution, modulating immunogenicity, modulating protease resistance, etc. Proteins incorporating non-natural amino acids can have enhanced or even entirely new catalytic or biophysical properties. For example, by including non-natural amino acids within a protein, the following properties can optionally be modified: receptor binding, toxicity, biodistribution, structural properties, spectral properties, chemical and / or photochemical properties, catalytic ability, half-life (including but not limited to serum half-life), the ability to react with other molecules (including but not limited to covalent or non-covalent reactions), etc. Compositions containing proteins incorporating at least one non-natural amino acid can be used for, including but not limited to, new therapeutic agents, diagnostic agents, catalytic enzymes, industrial enzymes, binding proteins (including but not limited to antibodies), and including but not limited to the study of protein structure and function. See, e.g., Dougherty, Unnatural Amino Acids as Probes of Protein Structure and Function, Current Opinion in Chemical Biology, 4:645-652 (2000).

[0474] In one aspect of the present invention, the composition comprises at least one protein having at least one non-natural amino acid including but not limited to at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten or more non-natural amino acids. The non-natural amino acids may be the same or different, including but not limited to that there may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different sites in the protein, which contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different non-natural amino acids. In another aspect, the composition comprises a protein in which at least one but less than all of the specific amino acids present are replaced by non-natural amino acids. For a given protein having more than one non-natural amino acid, the non-natural amino acids may be identical or different (including but not limited to that the protein may comprise two or more different types of non-natural amino acids, or may comprise two identical non-natural amino acids). For a given protein having more than two non-natural amino acids, the non-natural amino acids may be the same, different or a combination of multiple identical types of non-natural amino acids with at least one different non-natural amino acid.

[0475] A protein or polypeptide of interest having at least one non-natural amino acid is a feature of the present invention. The present invention also includes polypeptides or proteins having at least one non-natural amino acid produced using the compositions and methods of the present invention. Excipients (including but not limited to pharmaceutically acceptable excipients) may also be present with the protein.

[0476] By producing a protein or polypeptide of interest having at least one non-natural amino acid in a eukaryotic cell, the protein or polypeptide will generally include eukaryotic post-translational modifications. In certain embodiments, the protein comprises at least one non-natural amino acid and at least one post-translational modification made in vivo by the eukaryotic cell, wherein the post-translational modification is not made by a prokaryotic cell. For example, the post-translational modification includes but is not limited to acetylation, acylation, lipid modification, palmitoylation, palmitic acid addition, phosphorylation, glycolipid linkage modification, glycosylation, etc.

[0477] One advantage of non-natural amino acids is that they provide additional chemical moieties that can be used to attach other molecules. These modifications can be made in eukaryotic or prokaryotic cells in vivo or in vitro. Thus, in certain embodiments, the post-translational modification is achieved by the non-natural amino acid. For example, the post-translational modification can be achieved by a nucleophilic-electrophilic reaction. Most current reactions for the selective modification of proteins involve the formation of a covalent bond between a nucleophilic and an electrophilic reaction partner, including but not limited to the reaction of an α-halo ketone with a histidine or cysteine side chain. In these cases, the selectivity is determined by the number and accessibility of the nucleophilic residues in the protein. In the proteins of the present invention, other more selective reactions can be used in vitro and in vivo, such as the reaction of a non-natural keto-amino acid with a hydrazide or an aminooxy compound. See, e.g., Cornish et al., J. Am. Chem. Soc., 118:8150-8151 (1996); Mahal et al., Science, 276:1125-1128 (1997); Wang et al., Science 292:498-500 (2001); Chin et al., J. Am. Chem. Soc. 124:9026-9027 (2002); Chin et al., Proc. Natl. Acad. Sci., 99:11020-11024 (2002); Wang et al., Proc. Natl. Acad. Sci., 100:56-61 (2003); Zhang et al., Biochemistry, 42:6735-6746 (2003); and Chin et al., Science, 301:964-7 (2003), all of which are incorporated herein by reference. This allows for the selective labeling of virtually any protein with a large number of reagents, including fluorophores, cross-linkers, sugar derivatives, and cytotoxic molecules. See U.S. Patent No. 6,927,042, entitled "Glycoprotein synthesis," which is incorporated herein by reference. Post-translational modifications, including but not limited to those achieved by azido amino acids, can be made by the Staudinger ligation (including but not limited to the use of triarylphosphine reagents). See, e.g., Kiick et al., Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation, PNAS 99:19-24 (2002).

[0478] IV. In Vivo Production of IL-2 Comprising Non-Naturally Encoded Amino Acids

[0479] The IL-2 polypeptide of the present invention can be produced in vivo using modified tRNA and tRNA synthetase to add or replace amino acids not encoded in a naturally occurring system.

[0480] Methods for generating tRNA and tRNA synthetase using amino acids not encoded in a naturally occurring system are described, for example, in U.S. Patent Nos. 7,045,337 and 7,083,970, which are incorporated herein by reference. These methods involve generating a translation machinery that functions independently of the endogenous synthetase and tRNA of the translation system (and is thus sometimes referred to as "orthogonal"). Generally, the translation system comprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl-tRNA synthetase (O-RS). Generally, the O-RS preferentially aminoacylates the O-tRNA with at least one non-naturally occurring amino acid in the translation system, and the O-tRNA recognizes at least one selector codon that is not recognized by other tRNAs in the system. Thus, in response to the encoded selector codon, the translation system inserts the non-naturally encoded amino acid into the protein produced in the system, thereby "replacing" the amino acid into the position in the encoded polypeptide.

[0481] A wide variety of orthogonal tRNAs and aminoacyl-tRNA synthetases for inserting specific synthetic amino acids into polypeptides have been described in the art and are generally suitable for use in the present invention. For example, keto-specific O-tRNA / aminoacyl-tRNA synthetases are described in Wang, L. et al., Proc. Natl. Acad. Sci. USA 100:56-61 (2003) and Zhang, Z. et al., Biochem. 42(22):6735-6746 (2003). Exemplary O-RSs or portions thereof are encoded by polynucleotide sequences and include the amino acid sequences disclosed in U.S. Patent Nos. 7,045,337 and 7,083,970, each of which is incorporated herein by reference. The corresponding O-tRNA molecules used with the O-RS are also described in U.S. Patent Nos. 7,045,337 and 7,083,970, which are incorporated herein by reference. Additional examples of O-tRNA / aminoacyl-tRNA synthetase pairs are described in WO 2005 / 007870, WO 2005 / 007624, and WO 2005 / 019415.

[0482] Examples of azide-specific O-tRNA / aminoacyl-tRNA synthetase systems are described in Chin, J.W. et al., J. Am. Chem. Soc. 124:9026-9027 (2002). Exemplary O-RS sequences for azido-L-Phe include, but are not limited to, the nucleotide sequences SEQ ID NOs: 14-16 and 29-32 and the amino acid sequences SEQ ID NOs: 46-48 and 61-64 disclosed in U.S. Patent No. 7,083,970, which is incorporated herein by reference. Exemplary O-tRNA sequences suitable for use in the present invention include, but are not limited to, the nucleotide sequences SEQ ID NOs: 1-3 disclosed in U.S. Patent No. 7,083,970, which is incorporated herein by reference. Other examples of O-tRNA / aminoacyl-tRNA synthetase pairs specific for particular non-naturally encoded amino acids are described in U.S. Patent No. 7,045,337, which is incorporated herein by reference. O-RS and O-tRNA for incorporation of both keto- and azido-containing amino acids in Saccharomyces cerevisiae are described in Chin, J.W. et al., Science 301:964-967 (2003).

[0483] Several other orthogonal pairs have been reported. Glutaminyl (see, e.g., Liu, D.R. and Schultz, P.G. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:4780-4785), asparaginyl (see, e.g., Pastrnak, M. et al., (2000) Helv. Chim. Acta 83:2277-2286), and tyrosyl (see, e.g., Ohno, S. et al., (1998) J. Biochem. (Tokyo, Jpn.) 124:1065-1068; and Kowal, A.K. et al., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2268-2273) systems derived from Saccharomyces cerevisiae tRNA and synthetases have been described for potential incorporation of unnatural amino acids in Escherichia coli. Glutaminyl (see, e.g., Kowal, A.K. et al., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2268-2273) and tyrosyl (see, e.g., Edwards, H. and Schimmel, P. (1990) Mol. Cell. Biol. 10:1633-1641) synthetase systems derived from E. coli have been described for use in S. cerevisiae. The E. coli tyrosyl system has been used for in vivo incorporation of 3-iodo-L-tyrosine in mammalian cells. See Sakamoto, K. et al., (2002) Nucleic Acids Res. 30:4692-4699.

[0484] The use of an O-tRNA / aminoacyl-tRNA synthetase involves the selection of a specific codon (selector codon) encoding an unnaturally encoded amino acid. Although any codon can be used, it is generally desirable to select a codon that is rare or never used in the cell in which the O-tRNA / aminoacyl-tRNA synthetase is expressed. For example, exemplary codons include nonsense codons such as stop codons (amber, ochre, and opal), four or more base codons, and other rare or unused natural three-base codons.

[0485] A specific selector codon can be introduced into a suitable position in the IL-2 coding sequence using mutagenesis methods known in the art (including but not limited to site-directed mutagenesis, cassette mutagenesis, restriction selection mutagenesis, etc.).

[0486] V. Position of the unnatural amino acid in IL-2

[0487] The present invention contemplates incorporating one or more non-naturally occurring amino acids into IL-2. One or more non-naturally occurring amino acids can be incorporated at specific positions without disrupting the activity of the polypeptide. This can be achieved by making "conservative" substitutions, including but not limited to replacing hydrophobic amino acids with hydrophobic amino acids, replacing large amino acids with large amino acids, replacing hydrophilic amino acids with hydrophilic amino acids, and / or inserting the non-naturally occurring amino acids into positions that are not required for activity.

[0488] A variety of different biochemical and structural methods can be used to select the desired sites for replacement of non-naturally encoded amino acids within the IL-2. It will be apparent to one of ordinary skill in the art that any position of the polypeptide chain is suitable for selection to incorporate non-naturally encoded amino acids, and the selection can be based on rational design or by random selection, for any or no particular desired purpose. The selection of the desired sites can be used to generate IL-2 molecules having any desired property or activity, including but not limited to modulating receptor binding or binding to one or more subunits of its receptor, agonists, superagonists, inverse agonists, antagonists, receptor binding modulators, receptor activity modulators, dimer or multimer formation, not changing the activity or properties relative to the native molecule, or manipulating any physical or chemical property of the polypeptide such as solubility, aggregation or stability. For example, the positions required for the biological activity of IL-2 in the polypeptide can be identified using methods known in the art such as point mutation analysis, alanine scanning, saturation mutagenesis and bioactivity screening or homology scanning methods. Other methods can be used to identify residues for modification of IL-2, including but not limited to sequence profiling (Bowie and Eisenberg, Science 253(5016):164-70,(1991)), rotamer library selection (Dahiyat and Mayo, Protein Sci 5(5):895-903(1996); Dahiyat and Mayo, Science 278(5335):82-7(1997); Desjarlais and Handel, ProteinScience 4:2006-2018(1995); Harbury et al., PNAS USA 92(18):8408-8412(1995); Kono et al., Proteins:Structure,Function and Genetics 19:244-255(1994); Hellinga and Richards, PNAS USA 91:5803-5807(1994)) and residue pairing potential (Jones, Protein Science 3:567-574(1994)) and using Protein Design Rational design of the technology (see U.S. Patent Nos. 6,188,965, 6,269,312, 6,403,312, WO98 / 47089, which are incorporated herein by reference). Residues other than those identified as critical for biological activity by alanine or homologous scanning mutagenesis may be good candidates for replacement with non-naturally encoded amino acids, depending on the desired activity sought for the polypeptide. Alternatively, sites identified as critical for biological activity may also be good candidates for replacement with non-naturally encoded amino acids, again depending on the desired activity sought for the polypeptide. Another alternative is to simply make a series of replacements with non-naturally encoded amino acids at each position in the polypeptide chain and observe the effect on the activity of the polypeptide. It will be apparent to those of ordinary skill in the art that any means, technique, or method for selecting positions for replacement with non-natural amino acids in any polypeptide is suitable for use in the present invention.

[0489] The structure and activity of IL-2 polypeptide mutants containing deletions can also be examined to determine regions of the protein that may be tolerant to replacement with non-naturally encoded amino acids. In a similar manner, protease digestion and monoclonal antibodies can be used to identify regions of IL-2 responsible for binding to the IL-2 receptor. Once residues that are likely to be intolerant to replacement with non-naturally encoded amino acids have been eliminated, the effect of the proposed replacements at each remaining position can be examined. Thus, those of ordinary skill in the art can readily identify amino acid positions that can be replaced with non-naturally encoded amino acids.

[0490] Those of ordinary skill in the art recognize that this analysis of IL-2 can determine which amino acid residues are surface-exposed compared to amino acid residues buried within the tertiary structure of the protein. Accordingly, an embodiment of the present invention is to replace amino acids that are surface-exposed residues with non-naturally encoded amino acids.

[0491] In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in LI-2: before position 1 (i.e., at the N-terminus), positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or added to the carboxyl terminus of the protein, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5, or 7).

[0492] In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: before positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, and 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5, or 7).

[0493] In certain embodiments, one or more non-naturally encoded amino acids are incorporated at any position in one or more of the following regions corresponding to a secondary structure or specific amino acids in IL-2 or a variant thereof as described below: at sites of hydrophobic interaction; at or near sites that interact with IL-2 receptor subunits (including IL2Rα); within amino acid positions 3 or 35 to 45; within the first 107 N-terminal amino acids; within amino acid positions 61 - 72; each such position being the position in SEQ ID NO:2 or the corresponding amino acid position in SEQ ID NO:3, 5, or 7. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at one or more of the following positions in IL-2 or a variant thereof: before position 1 of SEQ ID NO:2 (i.e., at the N-terminus), positions 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, and any combination thereof; or the corresponding amino acids in SEQ ID NO:3, 5, or 7. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at one or more of the following positions in IL-2 or a variant thereof: positions 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133 of SEQ ID NO:2, or added to the carboxyl terminus of the protein, and any combination thereof; or the corresponding amino acids in SEQ ID NO:3, 5, or 7.

[0494] In certain embodiments, the IL-2 polypeptide is an agonist, and non-naturally occurring amino acids in one or more of these regions are conjugated to a water-soluble polymer, including but not limited to: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, and 107. In certain embodiments, the IL-2 polypeptide is an agonist, and non-naturally occurring amino acids in one or more of these regions are conjugated to a water-soluble polymer, including but not limited to: near positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, and 107.

[0495] A wide variety of non-naturally encoded amino acids can be substituted or incorporated at a given position in IL-2. Generally, the specific non-naturally encoded amino acid to be incorporated is selected on the basis of: examination of the three-dimensional crystal structure of the IL-2 polypeptide or other IL-2 family members with their receptors, the preference for conservative substitutions (i.e., aryl-based non-naturally encoded amino acids such as p-acetylphenylalanine or O-propargyltyrosine substituting for Phe, Tyr, or Trp), and the particular coupling chemistry one wishes to introduce into the IL-2 (e.g., introduction of 4-azidophenylalanine if one wishes to effect a Huisgen [3+2] cycloaddition with a water-soluble polymer bearing an alkyne moiety or an amide bond formation with a water-soluble polymer bearing an aryl ester and thus incorporating a phosphine moiety).

[0496] In one embodiment, the method further includes: incorporating the unnatural amino acid into the protein, wherein the unnatural amino acid comprises a first reactive group; and contacting the protein with a molecule comprising a second reactive group (the molecule includes but is not limited to a label, a dye, a polymer, a water-soluble polymer, a derivative of polyethylene glycol, a photo-crosslinker, a radionuclide, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound, a resin, a second protein or polypeptide or polypeptide analogue, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a saccharide, a polynucleotide, DNA, RNA, an antisense polynucleotide, a sugar, a water-soluble dendrimer, a cyclodextrin, an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactivity-containing moiety, a new functional group, a group that covalently or non-covalently interacts with other molecules, a photocaging moiety, a photoirradiation-excitable moiety, a photoisomerizable moiety, biotin, a derivative of biotin, a biotin analogue, a moiety doped with heavy atoms, a chemically cleavable group, a photocleavable group, an extended side chain, a carbon-linked sugar, a redox-active agent, an aminothio acid, a toxic moiety, an isotope-labeled moiety...

Claims

1. A modified IL-2 polypeptide, which comprises the amino acid sequence of SEQ ID NO: 2 and comprises: a non-naturally encoded amino acid incorporated at position 42, wherein the non-naturally encoded amino acid is p-acetylphenylalanine; an amino acid substitution at position 38 of SEQ ID NO: 2, and the amino acid substitution at position 38 is alanine; and one or more PEG molecules; wherein the polypeptide is coupled to the one or more PEG molecules through the non-naturally encoded amino acid incorporated into the polypeptide.

2. The modified IL-2 polypeptide according to claim 1, wherein the modified IL-2 polypeptide further comprises an amino acid substitution at position 65 of SEQ ID NO: 2, and the amino acid substitution at position 65 is arginine.

3. The modified IL-2 polypeptide according to claim 1, wherein the one or more PEG molecules are linear or branched.

4. The modified IL-2 polypeptide according to claim 1, wherein the one or more PEG molecules have an average molecular weight of 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa or 50 kDa.

5. The modified IL-2 polypeptide according to claim 1, wherein the average molecular weight of the one or more PEG molecules is 30 kDa.

6. The modified IL-2 polypeptide according to claim 1, wherein the average molecular weight of the one or more PEG molecules is 40 kDa.

7. Use of the modified IL-2 polypeptide according to any one of claims 1-6 in the preparation of a medicament for treating cancer in a subject.

8. The use according to claim 7, wherein the cancer is ovarian cancer, colon cancer, kidney cancer or melanoma.

9. The use according to claim 7 or 8, wherein the modified IL-2 polypeptide is further used in combination with other therapeutic agents.

10. The use according to claim 9, wherein the other therapeutic agent is a chemotherapeutic agent, a hormonal agent, an anti-tumor agent, an immune stimulant, an immunomodulator, an immunotherapeutic agent or a combination thereof.

11. A pharmaceutical composition, which comprises a therapeutically effective amount of the modified IL-2 according to any one of claims 1-6 and a pharmaceutically acceptable carrier or excipient.

Images