Interleukin-2 polypeptide conjugates and methods of use thereof

Abstract

The present invention relates to interleukin-2 polypeptide conjugates and methods of use thereof. In particular, the present invention provides compositions and methods comprising an interleukin-2 (IL-2) polypeptide conjugate. Also described are IL-2 conjugates for the treatment of diseases or conditions, including cancer.

Description

[0001] This application is a divisional application of the application with international application date of March 11, 2021, international application number PCT / US2021 / 022011 entering the Chinese national phase on September 8, 2022, application number 202180020053.8, and invention name “Interleukin-2 polypeptide conjugates and methods of use thereof”.

[0002] Cross-references

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 987,872, filed March 11, 2020, the contents of which are incorporated herein by reference in their entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing submitted in ASCII format via EFS-Web and incorporated herein by reference in its entirety. The ASCII copy was created on March 3, 2021, is named AMBX_0232_00PCT_ST25.txt, and is 27,704 bytes in size. Technical Field

[0006] 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 uses. Background Art

[0007] Cancer is one of the most significant health conditions. In the United States, cancer is the second leading cause of death after heart disease, accounting for a quarter of all deaths. It is widely expected that the incidence of cancer will increase as the U.S. population ages, further exacerbating the impact of this condition. Current treatment options for cancer, established in the 1970s and 1980s, have not changed significantly. When used in the most common advanced cancers, these treatments, which include chemotherapy, radiation, and other modalities (including newer targeted therapies), have shown limited overall survival benefits because they primarily target the tumor mass.

[0008] More specifically, to date, conventional cancer diagnosis and therapy attempts to selectively detect and eradicate primarily rapidly growing neoplastic cells (i.e., cells that form tumor masses). Standard oncology treatment regimens are typically designed to administer the highest dose of radiation or chemotherapeutic agents without excessive toxicity, commonly referred to as the "maximum tolerated dose" (MTD) or "no observed adverse effect level" (NOAEL). Many conventional cancer chemotherapies (e.g., alkylating agents such as cyclophosphamide, antimetabolites such as 5-fluorouracil and plant alkaloids such as vincristine) and conventional radiotherapy exert their toxic effects on cancer cells primarily by interfering with the cellular mechanisms involved in cell growth and DNA replication. Chemotherapy regimens also typically include administering a combination of chemotherapeutic agents in an attempt to improve therapeutic efficacy. Although a large number of different chemotherapeutic agents are available, these therapies have many disadvantages. For example, chemotherapeutic agents are well known to be toxic due to their nonspecific side effects on rapidly growing cells, whether normal or malignant; for example, chemotherapeutic agents cause significant and often dangerous side effects, including bone marrow suppression, immunosuppression, and gastrointestinal discomfort.

[0009] cancer stem cells

[0010] Cancer stem cells comprise a unique subpopulation of tumors (usually around 0.1-10%) that is more tumorigenic, grows relatively slower or quiescent, and is generally more resistant to chemotherapy than the tumor mass, relative to the remaining 90% or so of the tumor (and tumor mass). Considering 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 that generally grow slowly compared to the rapidly growing tumor mass may be relatively more resistant to conventional therapies and regimens. Cancer stem cells may exhibit other characteristics that make them relatively more resistant to chemotherapy, such as multidrug resistance and anti-apoptotic pathways. The above factors constitute the key reason why standard tumor treatment regimens fail to ensure long-term benefits in most patients with advanced cancer, i.e., fail to adequately target and eradicate cancer stem cells. In some cases, cancer stem cells are the generative cells of the tumor (i.e., they are the ancestors of the cancer cells that make up the tumor mass).

[0011] 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 has replaced the residue cysteine-125 with serine-125 (Whittington et al., 1993). Although IL-2 is the earliest FDA-approved cytokine in cancer treatment, IL-2 has been shown to exhibit severe side effects when used in high doses. This has greatly limited its use in potential patients. The underlying mechanism of the severe side effects has been attributed to the binding of IL-2 to one of its receptors, IL-2Rα. Normally, IL-2 can form heterotrimeric complexes not only with its receptors including IL-2Rα (or CD25), IL-2Rβ (or CD122) and IL-2Rγ (or CD132) (when all three receptors are present in the tissue), but also with IL-2Rβ and IL-2Rγ. In a clinical setting, when high doses of IL-2 are used, IL-2 begins to bind to IL-2αβγ, which is a T cell receptor. reg The main receptor form in cells. reg The inhibitory effect on cells causes an unwanted effect of using IL-2 in cancer immunotherapy. In order to reduce the side effects of IL-2, many methods have been used in the art. For example, a form of IL-2 manufactured by Nektar uses 6 PEGylated lysines to mask the IL2Rα binding region on the surface of IL-2 (Charych et al., 2016). This PEGylated IL-2 form has an extended half-life, contains a mixture of single and multi-PEGylated forms, contains a very large amount of PEG, and shows improved side effects. However, results from activity studies show that the effective PEGylated IL-2 form in this heterogeneous 6-PEGylated IL-2 mixture is the only monoPEGylated form. Therefore, there is a need for more effective PEGylated IL-2 with a homogeneous, well-defined product composition that modulates the side effects of IL-2.

[0012] 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 sulfhydryl-SH of cysteine, the imino group of histidine, and the like. 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 acetylene groups undergo a Huisgen [3+2] cycloaddition reaction under aqueous conditions in the presence of a catalytic amount of copper. 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. By introducing, for example, an azide moiety into a protein structure, one can incorporate a functional group that is chemically inert toward amines, sulfhydryls, carboxylic acids, and hydroxyl groups present in proteins, but reacts smoothly and efficiently with the acetylene moiety to form cycloaddition products. Importantly, in the absence of the acetylene moiety, the azide group remains chemically inert and unreactive in the presence of other protein side chains and under physiological conditions.

[0013] The present invention is directed to solving problems associated with the activity and production of IL-2 polypeptide conjugates, among other problems, and is also directed to producing IL-2 polypeptides with improved biological or pharmacological properties, such as increased activity against tumors and / or improved binding and / or improved therapeutic half-life. The IL-2 polypeptides of the present invention target both Treg cells, which are known to express trimeric IL-2 receptors (α, β, and γ), and CD8 cells, which primarily 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 preferential binding to the β and γ dimers of CD8 cells, thereby providing improved therapeutic applications and improved prognosis for diseases or conditions in which the IL-2 receptor α is highly expressed. Summary of the Invention

[0014] 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 coupled 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 coupled 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 of the amino acid sequence corresponding to 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 coupled 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 coupled 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.

[0015] In certain embodiments, the modified IL-2 polypeptide comprises a non-naturally encoded amino acid selected from the group consisting of p-acetylphenylalanine, p-nitrophenylalanine, p-sulfotyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-boronophenylalanine, o-boronophenylalanine, m-boronophenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-OM e-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-aminotyramine Acid, 2-aminotyrosine, o-sulfotyrosine, 2-sulfoxyphenylalanine, 3-sulfophenylalanine, 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, In some embodiments, the non-naturally encoded amino acid is p-acetylphenylalanine.

[0016] 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 positions 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 with alanine.

[0017] In certain embodiments, the modified IL-2 polypeptide comprises one or more PEG molecules, wherein the one or more PEG molecules are linear, 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, an average molecular weight of 10 kDa, an average molecular weight of 15 kDa, an average molecular weight of 20 kDa, an average molecular weight of 25 kDa, an average molecular weight of 30 kDa, an average molecular weight of 35 kDa, an average molecular weight of 40 kDa, an average molecular weight of 45 kDa, or an average molecular weight of 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, comprising a site-specifically incorporated non-naturally encoded amino acid, one or more amino acid substitutions at selected positions within SEQ ID NO: 2, and one or more PEG molecules coupled via the site-specifically incorporated non-naturally encoded amino acid. In certain embodiments, the modified IL-2 polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 2, comprising a site-specifically incorporated non-naturally encoded amino acid, and one or more PEG molecules coupled via the site-specifically incorporated non-naturally encoded amino acid. In certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is selected from the group consisting of 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 a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO: 9. In certain embodiments, the modified IL-2 polypeptide comprising a site-specific incorporation of a non-naturally encoded amino acid is SEQ ID NO: 10. In certain embodiments, the modified IL-2 polypeptide comprising a site-specific incorporation of a non-naturally encoded amino acid is SEQ ID NO: 11.In certain embodiments, the modified IL-2 polypeptide comprising a site-specific incorporation of a non-naturally encoded amino acid is SEQ ID NO: 12. In certain embodiments, the modified IL-2 polypeptide comprising a site-specific incorporation of a non-naturally encoded amino acid is SEQ ID NO: 13. In certain embodiments, the modified IL-2 polypeptide comprising a site-specific incorporation of a non-naturally encoded amino acid is SEQ ID NO: 14. In certain embodiments, the modified IL-2 polypeptide comprising a site-specific incorporation of a non-naturally encoded amino acid is SEQ ID NO: 15. In certain embodiments, the modified IL-2 polypeptide comprising a site-specific incorporation of a non-naturally encoded amino acid is SEQ ID NO: 16. In certain embodiments, the modified IL-2 polypeptide comprising a site-specific incorporation of a non-naturally encoded amino acid is SEQ ID NO: 17. In certain embodiments, the modified IL-2 polypeptide comprising a site-specific incorporation of a non-naturally encoded amino acid is SEQ ID NO: 18. In certain embodiments, the modified IL-2 polypeptide comprising a site-specific incorporation of a non-naturally encoded amino acid is SEQ ID NO: 19. In certain embodiments, the modified IL-2 polypeptide comprising a site-specific incorporation of a non-naturally encoded amino acid is SEQ ID NO: 20. In certain embodiments, the modified IL-2 polypeptide comprising a site-specific incorporation of a non-naturally encoded amino acid is SEQ ID NO: 21. In certain embodiments, the modified IL-2 polypeptide comprising a site-specific incorporation of a non-naturally encoded amino acid is SEQ ID NO: 22. In certain embodiments, the modified IL-2 polypeptide comprising a site-specific incorporation of a non-naturally encoded amino acid is SEQ ID NO: 23.

[0018] In certain embodiments, the present invention relates to interleukin-2 (IL-2) polypeptides comprising one or more non-naturally encoded amino acids. In certain embodiments, the present invention provides IL-2 polypeptide conjugates comprising one or more non-naturally encoded amino acids. In certain embodiments, the present 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 present 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 present invention provides IL-2 polypeptide conjugates having one or more non-naturally encoded amino acids and 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.

[0019] In one embodiment, the PEG-IL-2 is monoPEGylated. In one embodiment, the PEG-IL-2 is diPEGylated. In one embodiment, the PEG-IL-2 has more than two (2) polyethylene glycol molecules attached to it. Another embodiment of the present invention provides a method of using the PEG-IL-2 polypeptides of the present invention to modulate the activity of immune system cells.

[0020] In this or any embodiment of the invention, the PEG-IL-2 may comprise full-length, mature (lacking a signal peptide) human interleukin-2 linked to a PEG polymer. In this or any embodiment of the invention, the PEG-IL-2 may comprise full-length, mature (lacking a signal peptide) human interleukin-2 covalently linked to a PEG polymer or other biologically active molecule. In certain embodiments, the biologically active molecule is modified, and as a non-limiting example, the biologically active molecule may include one or more non-naturally encoded amino acids.

[0021] In PEG-IL2 conjugates, the PEG or other water-soluble polymer can be coupled to the IL-2 protein or the biologically active molecule directly or through a linker. Suitable linkers include, for example, cleavable and non-cleavable linkers.

[0022] The present invention provides a method for treating cancer in a mammal, such as, but not limited to, a mammal having one or more of the following conditions, by administering an effective amount of a PEG-IL-2 polypeptide. The mammal includes, but is not limited to, a mammal 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 tumor, cervical cancer, esophageal cancer, colorectal cancer, an epithelial cancer or tumor, 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 high levels of Treg cells. In certain embodiments, the cancer is characterized by high expression of IL-2 receptor alpha. In certain embodiments, the present invention provides a method for treating cancer, a condition, or a disease by administering to a subject an effective amount of a composition comprising an IL-2 polypeptide of the present invention. In certain embodiments, the present invention provides a method for treating a genetic disease by administering to a patient an effective amount of an IL-2 composition of the present invention. In certain embodiments, the condition or disease is characterized by high expression of IL-2 receptor alpha. In certain embodiments, the condition or disease is characterized by elevated levels of Treg cells. In certain embodiments, the cancer, condition, or disease is treated by reducing, blocking, or silencing the expression of IL-2 receptor alpha. In certain embodiments, the cancer, condition, or disease is treated by reducing the binding of IL-2 receptor alpha on the surface of Treg cells, resulting in a decrease in Treg cell proliferation in the cancer, condition, or disease being treated.

[0023] As used herein, interleukin-2 or IL-2 is defined as a protein having the following properties: (a) an amino acid sequence that is substantially identical to the known sequence of IL-2 (including IL-2 muteins, mature IL-2 sequences (i.e., lacking a secretory leader sequence), and IL-2 disclosed in SEQ ID NOs: 1, 2, 3, 5, or 7 of the present application), and (b) at least one biological activity shared by native or wild-type IL-2. For the purposes of this 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 E. coli) IL-2 are equivalent and can be used interchangeably. Other mutants and other analogs that retain the biological activity of IL-2 are also included, including viral IL-2.

[0024] 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 polypeptide is also linked to another drug or biologically active 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 IL-2 polypeptides. The present invention also provides trimers of IL-2 polypeptides. The present invention provides multimers of IL-2 polypeptides. 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, wherein any or each IL-2 polypeptide in the multimer may have a different amino acid sequence.

[0025] 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, a polymer, or a 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 allow dimer formation. In certain embodiments, the IL-2 polypeptide is linked to a linker long enough to allow trimer formation. In certain embodiments, the IL-2 polypeptide is linked to a linker long enough to allow multimer formation. In certain embodiments, the IL-2 polypeptide is linked to a bifunctional polymer, a 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, a polymer, or a biologically active molecule.

[0026] In certain embodiments, the non-naturally encoded amino acid is connected to a water-soluble polymer. In certain embodiments, the water-soluble polymer comprises a polyethylene glycol (PEG) component. In certain embodiments, the non-naturally encoded amino acid is connected to the water-soluble polymer using a connector 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 connected to a second polypeptide. In certain embodiments, the second polypeptide is IL-2. In certain embodiments, the IL-2 or its variant comprises at least two amino acids connected to a water-soluble polymer comprising a polyethylene glycol component. In certain embodiments, at least one amino acid is a non-naturally encoded amino acid.

[0027] In certain embodiments, IL-2 or PEG-IL-2 of the present invention are connected to therapeutic agents such as immunomodulators. The immunomodulator can be any agent that has a therapeutic effect on immune cells, which can be used as a therapeutic agent for coupling to IL-2, PEG-IL-2 or IL-2 variants. In certain embodiments, IL-2 or PEG-IL-2 of the present invention are connected to therapeutic agents such as cytokines, chemotherapeutic agents, immunotherapeutic agents, hormone agents, antitumor agents, immunostimulants or combinations thereof.

[0028] 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 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 8, 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 biologically active molecules are directly conjugated to the IL-2 variant. In certain embodiments, the one or more biologically active 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 present invention is linked to a linker. In certain embodiments, the IL-2 variant linked to the linker further comprises a biologically active molecule. In certain embodiments of the present invention, the linker is linked to a non-naturally encoded amino acid.

[0029] In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following 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 NO: 3, 5, or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following positions: before position 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). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following 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). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following positions: 42, 45, 61, and 65, 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 non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following positions: 45 and 65, 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 non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof of the present invention at position 3. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 of the present invention or its variants at position 32. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 of the present invention or its variants at position 35. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 of the present invention or its variants at position 37. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 of the present invention or its variants at position 38. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 of the present invention or its variants at position 41. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 of the present invention or its variants at position 42. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 of the present invention or its variants at position 43.In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 or variants thereof of the present invention at position 44. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 or variants thereof of the present invention at position 45. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 or variants thereof of the present invention at position 48. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 or variants thereof of the present invention at position 49. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 or variants thereof of the present invention at position 61. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 or variants thereof of the present invention at position 62. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 or variants thereof of the present invention at position 64. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 or variants thereof of the present invention at position 65. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 or variants thereof of the present invention at position 68. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 of the invention or its variants at position 72. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 of the invention or its variants at position 76. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 of the invention or its variants at position 107.

[0030] In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at any position in one or more of the following regions corresponding to the secondary structure or specific amino acids as described below: at a site of hydrophobic interaction; at or near a site of interaction with an IL-2 receptor subunit (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 of which is a position in SEQ ID NO: 2 or the corresponding amino acid positions in SEQ ID NO: 3, 5, or 7. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of 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, and any combination thereof of 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 IL-2 or a variant thereof at one or more of 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, and any combination thereof of SEQ ID NO: 2 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 3, 5 or 7; or the corresponding amino acids in SEQ ID NO: 3, 5 or 7.

[0031] 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 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 6, 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 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).

[0032] In certain embodiments, the non-naturally occurring amino acid at one or more of these positions in IL-2 or a variant thereof is linked to a drug or other biologically active molecule, including but not limited to, at a site of hydrophobic interaction, at or near a site of interaction with an IL-2 receptor subunit (including IL2Rα), within amino acid positions 3 or 35 to 45, within the first 107 N-terminal amino acids, and within amino acid positions 61-72; each of which is a position in 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 acid at one or more of these positions in IL-2 or a variant thereof is linked to a drug or other biologically active molecule, 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, and any combination thereof of SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5, or 7. In certain embodiments, the non-naturally occurring amino acid at one or more of these positions in IL-2 or a variant thereof is linked to a drug or other biologically active molecule, including but not limited to the following positions in IL-2 or a variant thereof: 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 3, 5 or 7; 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 IL-2 or a variant thereof and linked to a drug or other biologically active molecule at one or more of the following 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 at one or more of these positions in IL-2 or a variant thereof is linked to a linker, including but not limited to the following positions: before position 1 (i.e., at the N-terminus), 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 5, 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 IL-2 or a variant thereof and linked to a linker at one or more of the following 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 acid at one or more of these positions in IL-2 or a variant thereof is linked to a linker which is further linked to a water soluble polymer or a biologically active molecule, including but not limited to the following positions: before position 1 (i.e., at the N-terminus), 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 or SEQ ID NO: 3, 5, or 7. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following positions and linked to a linker, which is further linked to a water soluble polymer or a biologically active molecule, including but not limited to positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, and before position 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5, or 7).

[0035] 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 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 IL-2 or a variant thereof at one or more of the following positions and linked to a linker, which is further linked 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, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5, or 7). In certain embodiments, the present disclosure provides IL-2 polypeptides corresponding to SEQ ID Nos: 9-23 comprising site-specifically incorporated non-naturally encoded amino acids.

[0036] In certain embodiments, the IL-2 or its variant comprises a substitution, addition or deletion that modulates the affinity of the IL-2 for an 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 an IL-2 receptor or a binding partner (including but not limited to proteins, polypeptides, lipids, fatty acids, small molecules or nucleic acids). 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 substitution, addition or deletion. Stability and / or solubility can be measured using a large number 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 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 as compared to the serum half-life or circulation time of the corresponding IL-2 without the substitution, addition, or deletion.

[0037] In certain embodiments, the IL-2 or variant thereof 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 variant thereof without the substitution, addition, or deletion. In certain embodiments, the IL-2 or variant thereof comprises a substitution, addition, or deletion that increases the solubility of the IL-2 or variant thereof produced in a host cell compared to the solubility 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 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 variant thereof without the substitution, addition, or deletion. The IL-2 or variant thereof comprising such a substitution retains agonist activity or retains or increases expression levels in a host cell. In certain embodiments, the IL-2 or variant thereof comprises a substitution, addition, or deletion that increases the protease resistance of the IL-2 or variant thereof compared to the protease resistance 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 modulates the signal transduction activity of the IL-2 receptor compared to the activity of the IL-2 receptor upon interaction with 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 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.

[0038] In certain embodiments, the present invention provides methods of treating a proliferative disorder, cancer, tumor, or precancerous 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 antifolate, such as methotrexate or fluorouracil, irinotecan, cyclophosphamide, radiation therapy, hormone or anti-hormonal therapy, such as androgens, estrogens, anti-estrogen antibodies, flutamide, or diethylstilbestrol, surgery, tamoxifen, ifosfamide, dibromodole, an alkylating agent, such as melphalan or cisplatin, etoposide, vinorelbine, or dapoxetine. Vinblastine, vindesine, glucocorticoids, histamine receptor antagonists, angiogenesis inhibitors, radiation, radiosensitizers, anthracyclines, vinca alkaloids, taxanes such as paclitaxel and docetaxel, cell cycle inhibitors such as cyclin-dependent kinase inhibitors, checkpoint inhibitors, immunomodulatory drugs, immunostimulatory drugs, monoclonal antibodies directed against another tumor antigen, complexes of monoclonal antibodies with biologically active molecules, T cell adjuvants, bone marrow transplants, or antigen-presenting cells such as dendritic cell therapy. The vaccine can be provided, for example, as 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).

[0039] Also provided are methods of treating extramedullary hematopoiesis (EMH) in cancer. EMH has been described (see, eg, Rao et al., (2003) Leuk. Lymphoma 44:715-718; Lane et al., (2002) J. Cutan. Pathol. 29:608-612).

[0040] In certain embodiments, the PEG-IL-2 or variant thereof comprises a substitution, addition, or deletion that modulates its receptor or receptor subunit binding compared to the receptor or receptor subunit 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 associated with receptor or receptor subunit binding compared to the receptor or receptor subunit binding activity of the corresponding IL-2 or variant thereof without the substitution, addition, or deletion.

[0041] In certain embodiments, the IL-2 or variant thereof comprises a substitution, addition or deletion that improves the compatibility of the IL-2 or variant thereof with pharmaceutical preservatives (e.g., m-cresol, phenol, benzyl alcohol) compared to the compatibility of the corresponding wild-type IL-2 without the substitution, addition or deletion. This improved compatibility enables the preparation of well-preserved pharmaceutical formulations that maintain the physicochemical properties and biological activity of the protein during storage.

[0042] In certain embodiments, one or more engineered bonds are generated using one or more non-natural amino acids. The intramolecular bond can be generated in many ways, including but not limited to, by reaction between two amino acids in the protein under suitable conditions (one or both amino acids can be non-natural amino acids), by reaction with two amino acids (each of which can be naturally encoded or non-naturally encoded) under suitable conditions, with a linker, a polymer, or other molecule, etc.

[0043] In certain embodiments, one or more amino acid substitutions in the IL-2 or variants thereof may be with one or more naturally occurring or non-naturally occurring amino acids. In certain embodiments, the amino acid substitutions in the IL-2 or variants thereof may be with naturally occurring or non-naturally occurring amino acids, as long as at least one substitution is with a non-naturally encoded amino acid. In certain embodiments, one or more amino acid substitutions in the IL-2 or variants thereof may be with one or more naturally occurring amino acids, and at least one substitution is with a non-naturally encoded amino acid. In certain embodiments, the amino acid substitutions in the IL-2 or variants thereof may be with any naturally occurring amino acid, and at least one substitution is with a non-naturally encoded amino acid. In certain embodiments, one or more naturally occurring amino acids may be substituted at 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, 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 naturally occurring amino acid substitutions may be made at one or more of the following positions of IL-2 or a variant thereof: 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 1, 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, 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 variants thereof may be with at least one naturally occurring amino acid and at least one substitution is with a non-naturally encoded amino acid.In certain embodiments, the amino acid replacement in the IL-2 or its variant can use at least two naturally occurring amino acids, and at least one replacement is to use a non-naturally encoded amino acid. In certain embodiments, the one or more naturally occurring or encoded amino acids can be any one of 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 replacement can be at the following positions of IL-2 or its variant: the 38th and / or 46th and / or 65th or any combination thereof. In certain embodiments, the naturally occurring amino acid replacement can be at the 38th position of IL-2 or its variant. In certain embodiments, the naturally occurring amino acid replacement at position 38 of the IL-2 or its variants may be selected from any of the 20 common natural 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 replacement at position 38 of the IL-2 or its variants may be an alanine replacement. In certain embodiments, the naturally occurring amino acid replacement may be at position 46 of the IL-2 or its variants. In certain embodiments, the naturally occurring amino acid replacement at position 46 of the IL-2 or its variants may be selected from any of the 20 common natural 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 replacement at position 46 of the IL-2 or its variants may be a leucine or isoleucine replacement. In certain embodiments, the naturally occurring amino acid replacement may be at position 65 of IL-2 or its variants. In certain embodiments, the naturally occurring amino acid replacement at position 65 of the IL-2 or its variants may be selected from any one of 20 common natural 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 replacement at position 65 of the IL-2 or its variants may be an arginine replacement.In certain embodiments, the amino acid substitutions in the IL-2 or variants thereof may be with a naturally occurring amino acid at position 38, 46, or 65, and at least one substitution is with a non-naturally encoded amino acid incorporated into one or more of the following positions of IL-2 or a variant thereof: 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 acid positions in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitution in IL-2 or a variant thereof may be a naturally occurring amino acid substitution at position 38, and at least one substitution is with a non-naturally encoded amino acid incorporated into one or more of the following positions of IL-2 or a variant thereof: 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 acid positions in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitution in IL-2 or a variant thereof may be a naturally occurring amino acid substitution at position 46, and at least one substitution is with a non-naturally encoded amino acid incorporated into one or more of the following positions of IL-2 or a variant thereof: 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 acid positions in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitution in the IL-2 or variant thereof may be a naturally occurring amino acid substitution at position 65, and at least one substitution is with a non-naturally encoded amino acid incorporated into one or more of the following positions of IL-2 or a variant thereof: 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 acid positions in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitutions in the IL-2 or variants thereof may be substitutions with naturally occurring amino acids at positions 38 and / or 46 and / or 65, and at least one substitution is with a non-naturally encoded amino acid incorporated into one or more of the following positions of IL-2 or a variant thereof: 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 acid positions in SEQ ID NO: 3, 5, or 7).In certain embodiments, the amino acid substitutions in the IL-2 or variants thereof may be substitutions of a naturally occurring amino acid at position 38 and a non-naturally encoded amino acid incorporated into the IL-2 or variants thereof at position 42 (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 variants thereof may be substitutions of a naturally occurring amino acid at positions 38 and 46 and a non-naturally encoded amino acid incorporated into the IL-2 or variants thereof at position 42 (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 variants thereof may be substitutions of a naturally occurring amino acid at positions 38 and 65 and a non-naturally encoded amino acid incorporated into the IL-2 or variants thereof at position 42 (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 variants thereof may be naturally occurring amino acid substitutions at positions 38, 46, and 65 and a non-naturally encoded amino acid incorporated into the IL-2 or variants thereof at position 42 (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 variants thereof may be naturally occurring amino acid substitutions at position 38 and a non-naturally encoded amino acid incorporated into the IL-2 or variants thereof at position 45 (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 variants thereof may be naturally occurring amino acid substitutions at positions 38 and 46 and a non-naturally encoded amino acid incorporated into the IL-2 or variants thereof at position 45 (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 variants thereof may be naturally occurring amino acid substitutions at positions 38 and 65 and a non-naturally encoded amino acid incorporated into the IL-2 or variants thereof at position 45 (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 variants thereof may be naturally occurring amino acid substitutions at positions 38, 46, and 65 and a non-naturally encoded amino acid incorporated into the IL-2 or variants thereof at position 45 (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 variants thereof may be substitutions of a naturally occurring amino acid at position 38 and a non-naturally encoded amino acid incorporated into the IL-2 or variants thereof at position 65 (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 variants thereof may be substitutions of naturally occurring amino acids at positions 38 and 46 and a non-naturally encoded amino acid incorporated into the IL-2 or variants thereof at position 65 (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5, or 7).

[0044] In certain embodiments, the non-naturally encoded amino acid comprises a carbonyl group, an acetyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group.

[0045] In certain embodiments, the non-naturally encoded amino acid comprises a carbonyl group. In certain embodiments, the non-naturally encoded amino acid has the structure:

[0046]

[0047] wherein n is 0-10; R1 is alkyl, aryl, substituted alkyl or substituted aryl; R2 is H, alkyl, aryl, substituted alkyl and substituted aryl; R3 is H, an amino acid, a polypeptide or an amino terminus modification group, and R4 is H, an amino acid, a polypeptide or a carboxyl terminus modification group.

[0048] 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 hydrazine group. In certain embodiments, the non-naturally encoded amino acid residue comprises a semicarbazide group.

[0049] In certain embodiments, the non-naturally encoded amino acid residue comprises an azido group. In certain embodiments, the non-naturally encoded amino acid has the following structure:

[0050]

[0051] wherein n is 0-10; R1 is alkyl, aryl, substituted alkyl, substituted aryl, or absent; X is O, N, S, or absent; m is 0-10; R2 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R3 is H, an amino acid, a polypeptide, or a carboxyl terminus modification group.

[0052] In certain embodiments, the non-naturally encoded amino acid comprises an alkynyl group. In certain embodiments, the non-naturally encoded amino acid has the structure:

[0053]

[0054] wherein n is 0-10; R1 is alkyl, aryl, substituted alkyl, or substituted aryl; X is O, N, S, or absent; m is 0-10, R2 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R3 is H, an amino acid, a polypeptide, or a carboxyl terminus modification group.

[0055] 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.

[0056] The present invention also provides isolated nucleic acids comprising a polynucleotide encoding a polypeptide of SEQ ID NO: 1, 2, 3, 5, or 7, and the present invention provides isolated nucleic acids 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 isolated nucleic acids 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 isolated nucleic acids comprising a polynucleotide encoding a polypeptide shown as SEQ ID NO: 1, 2, 3, 5, or 7 having one or more non-naturally encoded amino acids. It will be apparent to one of ordinary skill in the art that many different polynucleotides can encode any polypeptide of the present invention.

[0057] In certain embodiments, the selector codon is selected from an amber codon, an ochre codon, an oval codon, a unique codon, a rare codon, a five-base codon, and a four-base codon.

[0058] The present invention also provides a method for producing IL-2 or a variant thereof linked to a bioactive molecule. In certain embodiments, the method comprises contacting an isolated IL-2 or variant thereof comprising a non-naturally encoded amino acid with a bioactive molecule comprising a component that reacts with the non-naturally encoded amino acid. In certain embodiments, the non-naturally encoded amino acid incorporated into the IL-2 or variant thereof is reactive toward a bioactive molecule that is otherwise unreactive toward any of the 20 commonly used amino acids. In certain embodiments, the non-naturally encoded amino acid incorporated into the IL-2 is reactive toward a linker, polymer, or bioactive molecule that is otherwise unreactive toward any of the 20 commonly used amino acids linked to the bioactive molecule.

[0059] In certain embodiments, the IL-2 or its variant connected to a water-soluble polymer or bioactive molecule is manufactured by reacting the IL-2 or its variant comprising a carbonyl amino acid with a water-soluble polymer or bioactive molecule comprising an aminooxy group, hydrazine, hydrazide or semicarbazide group. In certain embodiments, the aminooxy group, hydrazine, hydrazide or semicarbazide group is connected to the bioactive molecule via an amide bond. In certain embodiments, the aminooxy group, hydrazine, hydrazide or semicarbazide group is connected to the water-soluble polymer or bioactive molecule via a carbamate bond.

[0060] The present invention also provides a method for producing an IL-2 conjugate linked 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 component that reacts with the non-naturally encoded amino acid. In certain embodiments, the non-naturally encoded amino acid incorporated into the IL-2 conjugate is reactive toward a water-soluble polymer that is otherwise unreactive toward 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 toward a linker, polymer, or bioactive molecule that is otherwise unreactive toward any of the 20 commonly used amino acids.

[0061] The present invention also provides a method for producing IL-2 or a variant thereof connected to a water-soluble polymer. In certain embodiments, the method comprises contacting an isolated IL-2 or variant thereof comprising a non-naturally encoded amino acid with a water-soluble polymer comprising a component that reacts with the non-naturally encoded amino acid. In certain embodiments, the non-naturally encoded amino acid incorporated into the IL-2 or variant thereof is reactive toward a water-soluble polymer that is otherwise unreactive toward any of the 20 commonly used amino acids. In certain embodiments, the non-naturally encoded amino acid incorporated into the IL-2 is reactive toward a linker, polymer, or bioactive molecule that is otherwise unreactive toward any of the 20 commonly used amino acids.

[0062] In certain embodiments, the IL-2 or variant thereof connected to a water-soluble polymer is manufactured by reacting an IL-2 or variant thereof comprising a carbonyl amino acid with a polyethylene glycol molecule comprising an aminooxy group, a hydrazine, a hydrazide or a semicarbazide group. In certain embodiments, the aminooxy group, a hydrazine, a hydrazide or a semicarbazide group is connected to the polyethylene glycol molecule via an amide bond. In certain embodiments, the aminooxy group, a hydrazine, a hydrazide or a semicarbazide group is connected to the polyethylene glycol molecule via a carbamate bond.

[0063] In certain embodiments, the IL-2 or variant thereof linked to a water soluble polymer is produced by reacting a polyethylene glycol molecule comprising a carbonyl group with a polypeptide comprising a non-naturally encoded amino acid containing an aminooxy, hydrazine, hydrazide, or semicarbazide group.

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

[0065] In certain embodiments, the IL-2 or variant thereof linked to a water-soluble polymer is produced by reacting an IL-2 or variant thereof comprising an azide-containing amino acid with a polyethylene glycol molecule comprising an alkyne moiety. In certain embodiments, the azide or alkyne moiety is linked to the polyethylene glycol molecule via an amide bond.

[0066] In certain embodiments, the polyethylene glycol molecule has a molecular weight between about 0.1kDa and about 100kDa. In certain embodiments, the polyethylene glycol molecule has a molecular weight between 0.1kDa and 50kDa. In certain embodiments, the polyethylene glycol has a molecular weight between 1kDa and 50kDa, between 1kDa and 25kDa, between 2 and 22kDa, between 5kDa and 20kDa, between 5kDa and 30kDa, or between 5kDa and 40kDa. For example, the molecular weight of the polyethylene glycol polymer can be about 5kDa, about 10kDa, about 20kDa, about 30kDa, or about 40kDa. For example, the molecular weight of the polyethylene glycol polymer can be 5kDa, 10kDa, 20kDa, 30kDa, or 40kDa. In certain embodiments, the polyethylene glycol molecule is a 20K 2-branched PEG. In certain embodiments, the polyethylene glycol molecule is a 40K 2-branched PEG. In certain embodiments, the polyethylene glycol molecule is a 30K branched PEG. In certain embodiments, the polyethylene glycol molecule is a 40K or greater branched PEG. In certain embodiments, the polyethylene glycol molecule is a linear 5K PEG. In certain embodiments, the polyethylene glycol molecule is a linear 10K PEG. In certain embodiments, the polyethylene glycol molecule is a linear 15K PEG. In certain embodiments, the polyethylene glycol molecule is a linear 20K PEG. In certain embodiments, the polyethylene glycol molecule is a linear 25K PEG. In certain embodiments, the polyethylene glycol molecule is a linear 30K PEG. In certain embodiments, the polyethylene glycol molecule is a linear 35K PEG. In certain embodiments, the polyethylene glycol molecule is a linear 40K PEG. In certain embodiments, the polyethylene glycol molecule is a linear 45K PEG. In certain embodiments, the polyethylene glycol molecule is a linear 50K PEG. In certain embodiments, the polyethylene glycol molecule is a linear 60K PEG. In certain embodiments, the molecular weight of the polyethylene glycol polymer is an average molecular weight. In certain embodiments, the average molecular weight is a 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 IL-2 or its variants at one or more of the following positions: 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, or 107, 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 its variants are 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 IL-2 or a variant thereof at one or more of the following 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 (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 of 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 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 of 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 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 of 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 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 of 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 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 of 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 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 35 of 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, one or more non-naturally encoded amino acids are incorporated into IL-2, or a variant thereof, at one or more of the following positions: 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, or 107, 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 polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following 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 polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following positions: 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, or 107, 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 30K polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following 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 30K polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following positions: 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, or 107, 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 40K polyethylene glycol molecule.In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following 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 40K polyethylene glycol molecule.

[0067] In some embodiments, the polyethylene glycol molecule is a branched polymer. In some embodiments, each side of the polyethylene glycol branched polymer has a molecular weight between 1kDa to 100kDa or between 1kDa to 50kDa. In some embodiments, each side of the polyethylene glycol branched polymer has a molecular weight between 1kDa to 25kDa or between 2 to 22kDa or between 5kDa to 20kDa or between 5kDa to 30kDa or between 5kDa to 40kDa or between 5kDa to 50kDa or between 5kDa to 60kDa. For example, the molecular weight of each side of the polyethylene glycol branched polymer can be about 5kDa or about 10kDa or about 20kDa or about 30kDa or about 40kDa or about 50kDa or about 60kDa or larger. In some embodiments, the molecular weight of each branch of the polyethylene glycol branched polymer can be 5kDa or 10kDa or 15kDa or 20kDa or 25kDa or 30kDa or 35kDa or 40kDa or 45kDa or 50kDa or 55kDa or 60kDa or larger. In some embodiments, the polyethylene glycol molecule is a 20K 2-branched PEG. In some embodiments, the polyethylene glycol molecule is a 20K 4-branched PEG. In some embodiments, the polyethylene glycol molecule is a 40K 2-branched PEG. In some embodiments, the molecular weight of the polyethylene glycol polymer is an average molecular weight. In some embodiments, the average molecular weight is a 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 IL-2 or a variant thereof at one or more of the following positions: 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, or 107, 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 branched 20K polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following 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 branched 20K polyethylene glycol molecule.In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following positions: 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, or 107, 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 branched 30K polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following 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 branched 30K polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following positions: 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, or 107, 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 branched 40K polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following 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 branched 40K polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following positions: 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, or 107, 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 2-branched or 40K 2-branched polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following 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 2-branched or 40K 2-branched polyethylene glycol molecule.In certain embodiments, a non-naturally encoded amino acid is incorporated into IL-2 or a variant thereof at position 65 (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 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into IL-2 or a variant thereof at position 61 (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 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into IL-2 or a variant thereof at position 49 (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 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into IL-2 or a variant thereof at position 45 (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 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into IL-2 or a variant thereof at position 42 (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 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into IL-2 or a variant thereof at position 37 (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 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into IL-2, or a variant thereof, at position 35 (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 2-branched polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2, or a variant thereof, at one or more of the following positions: 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, or 107, 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, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following 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 IL-2 or a variant thereof at position 65 (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 IL-2 or a variant thereof at position 61 (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 IL-2 or a variant thereof at position 49 (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 IL-2 or a variant thereof at position 45 (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 IL-2 or a variant thereof at position 42 (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 IL-2 or a variant thereof at position 37 (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 IL-2 or a variant thereof at position 35 (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 IL-2 or a variant thereof at position 65 (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 40K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into IL-2 or a variant thereof at position 61 (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 40K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into IL-2 or a variant thereof at position 49 (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 40K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into IL-2 or a variant thereof at position 45 (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 40K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into IL-2 or a variant thereof at position 42 (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 40K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into IL-2 or a variant thereof at position 37 (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 40K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into IL-2 or a variant thereof at position 35 (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 40K 2-branched polyethylene glycol molecule.

[0068] In certain embodiments, the water-soluble polymer attached to IL-2 or its variant comprises a polyalkylene glycol moiety. In certain embodiments, the non-naturally encoded amino acid residue incorporated into IL-2 comprises a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkynyl group. In certain embodiments, the non-naturally encoded amino acid residue incorporated into IL-2 or its variant comprises a carbonyl moiety, and the water-soluble polymer comprises an aminooxy group, a hydrazide group, a hydrazine group, or a semicarbazide group. In certain embodiments, the non-naturally encoded amino acid residue incorporated into IL-2 or its variant comprises an alkynyl 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 its variant comprises an azide moiety, and the water-soluble polymer comprises an alkynyl moiety.

[0069] 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.

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

[0071] 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 substituting a non-naturally encoded amino acid into the IL-2 or variant thereof.

[0072] In certain embodiments, the present invention provides methods for modulating receptor interactions of IL-2 polypeptides of the present invention. In certain embodiments, the present invention provides methods for inhibiting or reducing the interaction of PEGylated IL-2 with the IL2Rα subunit of the trimeric IL-2 receptor using PEGylated IL-2 polypeptides of the present invention.

[0073] The present invention also provides methods for producing PEG-IL-2, IL-2, or any variant thereof comprising a non-naturally encoded amino acid. In certain embodiments, the method comprises culturing cells comprising one or more polynucleotides encoding IL-2, an orthogonal RNA synthetase, and / or an orthogonal tRNA under conditions that allow expression of the IL-2 or variant thereof; and purifying the IL-2 or variant thereof from the cells and / or culture medium.

[0074] 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 a cycle time of 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 a method for regulating the immunogenicity of IL-2 or its variants. In certain embodiments, the method comprises replacing any one or more amino acids in naturally occurring IL-2 or its variants with non-naturally encoded amino acids and / or connecting the IL-2 or its variants to a linker, polymer, water-soluble polymer or bioactive molecule. In one embodiment of the invention, the linker is long enough to allow flexibility and allow dimer formation. In one embodiment of the invention, the linker is at least 3 amino acids or 18 atoms in length to allow dimer formation.

[0075] The present invention also provides a method for treating a patient in need of such treatment using an effective amount of a PEG-IL-2 conjugate of the present invention or a variant thereof. In certain embodiments, the method comprises administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising PEG-IL-2 or a variant thereof 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 PEG-IL-2 or a variant thereof substituted with a non-naturally encoded amino acid and a natural amino acid 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 PEG-IL-2 or a variant thereof is glycosylated. In certain embodiments, the PEG-IL-2 or a variant thereof is not glycosylated.

[0076] The present invention also provides methods for treating a patient in need of such treatment using an effective amount of an 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 IL-2 variant molecule containing one or more non-naturally encoded amino acids and one or more natural 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 natural 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 suffers from a cancer, condition, or disease characterized by, but not limited to, elevated expression of IL-2 receptor alpha. In certain embodiments, the present invention provides a method for treating cancer, a condition, or a disease by administering to a subject a therapeutically effective amount of an IL-2 composition of the present invention. In certain embodiments, the present invention provides a method for treating a genetic disease by administering to a patient a therapeutically effective amount of an IL-2 composition of the present invention. The IL-2 polypeptides of the present invention are used to treat a disease or condition in cells that have elevated expression of the IL-2 receptor alpha. In certain embodiments, the cancer, condition, or disease is treated by reducing, blocking, or silencing expression of the IL-2 receptor alpha. The IL-2 polypeptides or variants of the present invention are used in the manufacture of a medicament for treating a cancer, disease, or condition associated with elevated expression of the IL-2 receptor alpha. The IL-2 polypeptides or variants of the present invention are used in the manufacture of a medicament for treating cancer. The IL-2 polypeptides or variants of the present invention are used in the manufacture of a medicament for treating a genetic disease.

[0077] The present invention also provides IL-2 comprising the sequence set forth in SEQ ID NO: 1, 2, 3, 5, or 7, or any other IL-2 sequence, except 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, wherein 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, wherein a non-naturally encoded amino acid is site-specifically incorporated. 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.

[0078] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and PEG-IL-2 or a natural variant thereof comprising the sequence set forth in SEQ ID NO: 1, 2, 3, 5, or 7, or any other IL-2 sequence, wherein at least one amino acid is replaced with a non-naturally encoded amino acid. The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and IL-2 or a natural variant thereof comprising the sequence set forth in SEQ ID NO: 1, 2, 3, 5, or 7. In certain embodiments, the non-naturally encoded amino acid comprises a carbohydrate moiety. In certain embodiments, the water-soluble polymer is linked to the IL-2 or a natural variant thereof via a carbohydrate moiety. In certain embodiments, a linker, polymer, or biologically active molecule is linked to the IL-2 or a natural variant thereof via a carbohydrate moiety.

[0079] The present invention also provides an IL-2 or a natural 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.

[0080] The present invention provides an IL-2 or variant thereof comprising at least one linker, polymer, or bioactive molecule, wherein the linker, polymer, or bioactive molecule is attached to the polypeptide via a functional group of a non-naturally encoded amino acid that is ribosomally incorporated into the polypeptide. In certain embodiments, the IL-2 or variant thereof is monoPEGylated. The present invention also provides an IL-2 or 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 ribosomally incorporated into the polypeptide at preselected sites.

[0081] The present invention includes within its scope the leader or signal sequence of IL-2 or its variants linked to the IL-2 coding region, as well as heterologous signal sequences linked to the IL-2 coding region. The heterologous leader or signal sequence selected should be, for example, one that is recognized and processed for secretion by the host cell secretion system and that is potentially cleaved by the host cell's signal peptidase. The methods of treating a condition or disorder using IL-2 of the present invention are meant to imply treatment with IL-2 or its variants, with or without a signal or leader peptide.

[0082] In another embodiment, the conjugation of IL-2 or a variant thereof comprising one or more non-naturally occurring amino acids to another molecule, including but not limited to PEG, provides substantially purified IL-2 due to the unique chemical reaction used for conjugation to the non-natural amino acids. The conjugation of IL-2 or a variant thereof comprising one or more non-naturally encoded amino acids to another molecule, such as PEG, can be performed using other purification techniques performed before or after the conjugation step to provide substantially pure IL-2 or a variant thereof.

[0083] In certain embodiments, the present invention provides a modified IL-2 polypeptide for use in the preparation of 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

[0084] Figure 1 A model showing a view of the IL-2 polypeptide is depicted, where potential receptor interaction sites are labeled with the structure of IL-2Rα and its interface with IL-2.

[0085] Figure 2 Depicted is a plasmid map of the expression vector used to express IL-2 in E. coli.

[0086] Figures 3A-3B Western blot analysis of IL-2 protein expression in E. coli is depicted ( Figure 3A ) and the titers of IL-2 variants in E. coli ( Figure 3B ).

[0087] Figures 4A-4B The binding kinetics of wild-type IL-2 to CD25 are shown in the sensorgram and the model fitting line and the calculated measurement value ( Figure 4A ), and a plasmid map of an expression vector for expressing IL-2 in mammalian cells ( Figure 4B ).

[0088] Figure 5 The design of the UPF1 genomic DNA sequence and CRISPR gRNA sites is shown.

[0089] Figure 6 Depicted is the sequence validation of the UPF1 knockout cell line.

[0090] Figures 7A-7B Transient expression of various IL-2 variants in mammalian cells is described ( Figure 7A ) and Western blot analysis of wild-type IL-2 and IL-2 variants produced in mammalian cells ( Figure 7B ).

[0091] Figure 8 Depicted is a CTLL-2 amplification assay of the F42 variant of IL-2.

[0092] Figure 9 Screening of IL-2 variants by CTLL-2 proliferation assay is shown.

[0093] Figures 10A-10C The binding kinetics of IL-2 wild type and F42 variant are depicted ( Figure 10A ), binding kinetics sensorgrams of K35 and Y45 variants ( Figure 10B ) and binding kinetics of T37 and P65 variants ( Figure 10C ).

[0094] Figure 11 A schematic illustration of the IL-2 receptor dimerization assay is shown.

[0095] Figure 12 A schematic illustration of the ex vivo pSTAT5 assay is shown.

[0096] Figure 13 Depicted are the clonal growth and long-term proliferation of CTLL-2 cells in the presence of glycosylated or non-glycosylated IL-2.

[0097] Figure 14 Shown are comparisons of titers before and after production of stable pools of the corresponding wild-type IL-2 or its selected variants.

[0098] Figures 15A-15CTiters in mammalian cells expressing the F42-R38A variant are depicted ( Figure 15A ), CTLL-2 binding assay of F42-R38A variant ( Figure 15B ) and F42-R38A variants ( Figure 15C ).

[0099] Figure 16 Depicted are mean plasma concentrations of the Y45-PEG20K-BR2 and F42-R38A-PEG20K-BR2 variants over time.

[0100] Figures 17A-17D Depicted are IL-2 wild type (WT; Figure 17A ) and F42-R38A-P65R-PEG20K-BR2( Figure 17B )、IL2-Y45-M46L-PEG20K-BR2( Figure 17C ) and IL2-Y45-M46I-PEG20K-BR2 ( Figure 17D ) Binding kinetic sensorgrams of the variants.

[0101] Figure 18 Shown is a CTLL-2 cell proliferation assay of PEGylated IL-2 variants.

[0102] Figure 19 The mean plasma concentrations of the PEGylated IL-2 variants are plotted over time.

[0103] Figures 20A-20B The effect of PEGylated IL-2 variants on tumor volume ( Figure 20A ) and weight ( Figure 20B ) activity.

[0104] Figures 21A-21B Depicted are the effects of 2 mg / kg ( Figure 21A ) and 5-8 mg / kg ( Figure 21B Effects of the IL-2 variants F42-R38A-P65R-PEG30K-L, F42-R38A-P65R-PEG40K-BR2, Y45-PEG30K-L and Y45-PEG40K-BR2 on B16F10 tumor growth inhibition.

[0105] Figure 22 Depicted are final tumor volumes in BALB / c mice bearing B16F10 tumors.

[0106] Figures 23A-23C Depicted is the PEGylated IL-2 variant F42-R38A-P65R-PEG30K-L ( Figure 23A) and Y45-PEG30K-L( Figure 23B ) inhibited CT26 tumor growth and mouse body weight ( Figure 23C )’s impact.

[0107] Figure 24 Depicted are final tumor volumes in BALB / c mice bearing CT26 tumors.

[0108] Figures 25A-25C Figure 3 shows the effects of PEGylated IL-2 variants F42-R38A-P65R-PEG30K-L and Y45-PEG30K-L on CD8+ cells in the blood of mice bearing CT26 tumors. Figure 25A ), CD4+ cells ( Figure 25B ) and the effect of CD8+ / CD4+ ratio ( Figure 25C ).

[0109] Figure 26 Depicted are the melting temperatures of wild-type IL-2 analyzed by DSF.

[0110] definition

[0111] 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 these may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0112] As used herein and in the appended claims, the singular form "a", "an", and "an" includes plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "IL-2," "PEG-IL-2," "PEG-IL-2 conjugate," and variously capitalized, hyphenated, and unhyphenated forms refers to one or more such proteins and includes equivalents thereof known to those skilled in the art, and so forth.

[0113] 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.

[0114] 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 their disclosures precede the filing date of the present application. Nothing herein should be construed as an admission that the inventors are not entitled to antedate such disclosures by virtue of prior invention or for any other reason.

[0115] The term "substantially purified" refers to IL-2 or a variant thereof that is substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment, i.e., in native cells or, in the case of recombinantly produced IL-2, in host cells. IL-2 that may be 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 proteins. When the IL-2 or variant thereof is recombinantly produced by host cells, the protein may 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 cells. When the IL-2 or variant thereof is recombinantly produced by host cells, 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, based on the dry weight of the cells. Thus, "substantially purified" IL-2 produced by the methods of the present invention may 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 a purity level of at least about 75%, 80%, 85%, more particularly a purity level of at least about 90%, at least about 95%, at least about 99% or more, as determined by suitable methods such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0116] "Recombinant host cell" or "host cell" refers to a cell that includes an exogenous polynucleotide, regardless of the method used for insertion, such as direct uptake, transduction, f-mating, or other methods known in the art for producing recombinant host cells. The exogenous polynucleotide can be maintained as a non-integrating vector, such as a plasmid, or can be integrated into the host genome.

[0117] When used in this article, the term "culture medium" includes any culture medium, solution, solid, semisolid or rigid support, which 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, Chinese hamster ovary celI, prokaryotic host cells, Escherichia coli or Pseudomonas (Pseudomonas) host cells, and cell inclusions. Therefore, the term can encompass culture medium in which host cells have grown, such as culture medium in which IL-2 has been secreted, including culture medium before or after the proliferation step. The term can also encompass buffer or reagent containing host cell lysate, such as when IL-2 is produced in the cell and the host cell is cracked or broken to release IL-2.

[0118] When used in relation to protein refolding in this article, "reducing agent" is defined as any compound or material that maintains sulfhydryl groups in a reduced state and reduces intramolecular 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 range of reducing agents are suitable for use in the methods and compositions of the present invention.

[0119] When used in relation to protein refolding herein, an "oxidant" is defined as any compound or material capable of removing electrons from an oxidized compound. Suitable oxidants include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythritol, and oxygen. It will be apparent to one of ordinary skill in the art that a wide variety of oxidants are suitable for use in the methods of the present invention.

[0120] As used herein, a "denaturant" is defined as any compound or material that causes the 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 chaotropic agents, detergents, organic solvents, water-miscible solvents, phospholipids, or a combination of two or more such agents. Suitable chaotropic agents include, but are not limited to, urea, guanidine, and sodium thiocyanate. Useful detergents may 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-(dioleyloxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents (e.g., sodium cholate or sodium deoxycholate), or zwitterionic detergents including, but not limited to, sulfobetaines (Zwittergent), 3-(3-chloroaminopropyl)dimethylammonium-1-propane sulfate (CHAPS), and 3-(3-chloroaminopropyl)dimethylammonium-2-hydroxy-1-propanesulfonate (CHAPSO). Water-miscible organic solvents such as acetonitrile, lower alkanols (particularly C2-C4 alkanols such as ethanol or isopropanol), or lower alkane diols (particularly C2-C4 alkane diols such as ethylene glycol) may be used as denaturants. The phospholipids useful in the present invention may be naturally occurring phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine and phosphatidylinositol or synthetic phospholipid derivatives or variants such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

[0121] As used herein, "refolding" describes any process, reaction, or method that converts a disulfide bond-containing polypeptide from an improperly folded or unfolded state to a conformation that is native or correctly folded with respect to the disulfide bonds.

[0122] As used herein, "cofolding" specifically refers to a refolding process, reaction, or method that uses at least two polypeptides that interact with each other and results in the conversion of unfolded or incorrectly folded polypeptides to the original, correctly folded polypeptides.

[0123] As used herein, "interleukin-2," "IL-2," and hyphenated and unhyphenated forms thereof include polypeptides and proteins having at least one biological activity of IL-2, as well as IL-2 analogs, IL-2 muteins, IL-2 variants, IL-2 isoforms, IL-2 mimetics, IL-2 fragments, hybrid IL-2 proteins, fusion proteins, oligomers and multimers, homologs, glycosylation pattern variants, variants, splice variants, and muteins thereof, regardless of their biological activity, and regardless of the method of synthesis and manufacture, including but not limited to recombinant (whether produced from cDNA, genomic DNA, synthetic DNA, or other forms of nucleic acid), in vitro, in vivo, by microinjection of nucleic acid molecules, synthetic, transgenic, and gene activation methods. The terms "IL-2," "IL-2 variant," and "IL-2 polypeptide" encompass IL-2 comprising one or more amino acid substitutions, additions, or deletions.

[0124] The sequence of IL-2 lacking a leader sequence and without a methionine at the N-terminus is set forth herein as SEQ ID NO: 2. The sequence of IL-2 without a leader sequence and with a methionine at the N-terminus is set forth herein as SEQ ID NO: 3, 5, or 7. In certain embodiments, the IL-2 or variant thereof 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) and methods of expressing and purifying these polypeptides are well known in the art.

[0125] The term "IL-2" also includes pharmaceutically acceptable salts and prodrugs of naturally occurring IL-2 and prodrugs, polymorphs, hydrates, solvates, biologically active fragments, biologically active variants and stereoisomers of said salts, as well as agonist, mimetic and antagonist variants of naturally occurring IL-2 and polypeptide fusions thereof.

[0126] Various references disclose modifications of polypeptides by polymer coupling or glycosylation. The term "IL-2" includes polypeptides coupled to polymers such as PEG, and may include one or more additional derivatizations of cysteine, lysine, or other residues. In addition, the IL-2 may include a linker or polymer, wherein the amino acid to which the linker or polymer is coupled may be a non-natural amino acid according to the present invention, or may be coupled to a naturally encoded amino acid, such as lysine or cysteine, using techniques known in the art.

[0127] 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 the polypeptide. Variants containing single nucleotide changes are also considered biologically active variants of the IL-2 polypeptide. In addition, splice variants are also included.

[0128] The term "IL-2" also includes IL-2 heterodimers, homodimers, heteromultimers, or homomultimers comprising any one or more IL-2 or any other polypeptide, protein, carbohydrate, polymer, small molecule, linker, ligand, or other biologically active molecule of any type 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.

[0129] As used herein, "interleukin-2" or "IL-2" is a protein comprising two subunits non-covalently associated to form a homodimer, whether conjugated to a biologically active molecule, conjugated to polyethylene glycol, or in unconjugated form. As used herein, "interleukin-2" and "IL-2" may refer to human or mouse IL-2, which is also referred to as "hIL-2" or "mIL-2."

[0130] The terms "PEGylated IL-2," "PEGylated IL-2," or "PEG-IL-2" refer to an IL-2 molecule having one or more polyethylene glycol molecules covalently attached to one or more amino acid residues of the IL-2 protein via a linker such that the attachment is stable. The terms "monoPEGylated IL-2" and "monoPEG-IL-2" mean that one polyethylene glycol molecule is covalently attached via a linker to a single amino acid residue on 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 alter, or only minimally alters, the activity of the biologically active molecule. Preferably, the increase in half-life outweighs any decrease in biological activity.

[0131] All references to amino acid positions in IL-2 described herein are based on 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 another IL-2). Those skilled in the art will recognize that amino acid positions corresponding to 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 amino acid positions corresponding to positions in SEQ ID NO: 2, 3, 5, or 7, or any other IL-2 sequence, can be readily identified in any other IL-2 molecule, such as an IL-2 fusion, variant, fragment, etc. For example, sequence alignment programs such as BLAST can be used to align and identify specific positions in a protein that correspond to positions in SEQ ID NO: 2, 3, 5, or 7, or another IL-2 sequence. Amino acid substitutions, deletions, or additions described herein with reference to SEQ ID NO: 2, 3, 5, or 7, or another IL-2 sequence, are intended to also refer to substitutions, deletions, or additions at the corresponding positions in IL-2 fusions, variants, fragments, etc., described herein or known in the art, and are expressly encompassed by the present invention.

[0132] IL-2 (IL2): Any form of IL-2 known in the art can be used in the compositions described herein. For experimental work, the mouse 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 linked to vectors and used in the methods described herein.

[0133] The term "interleukin-2" or "IL-2" encompasses IL-2 comprising one or more amino acid substitutions, additions, or deletions. The IL-2 of the present invention may comprise one or more natural amino acid modifications in combination with one or more non-natural amino acid modifications. Exemplary substitutions at 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, increasing agonist activity, increasing polypeptide solubility, reducing protease sensitivity, converting the polypeptide into an antagonist, and the like, and are encompassed by the term "IL-2 polypeptide." In certain embodiments, the IL-2 antagonist comprises 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.

[0134] In certain embodiments, the IL-2 or variant thereof further comprises additions, substitutions, or deletions that modulate the biological activity of the IL-2 or variant polypeptide. In certain embodiments, the IL-2 or variant further comprises additions, substitutions, or deletions that modulate known and research-proven properties of IL-2, such as the treatment or alleviation of one or more symptoms of cancer. The additions, substitutions, or deletions may modulate one or more properties or activities of the IL-2 or variant. For example, the additions, substitutions, or deletions may modulate affinity for the IL-2 receptor or one or more subunits of the receptor, modulate circulating half-life, modulate therapeutic half-life, modulate stability of the polypeptide, modulate cleavage by proteases, modulate dosage, modulate release or bioavailability, facilitate purification, or improve or alter a specific route of administration. Similarly, the IL-2 or variant may 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 linked molecule (including but not limited to biotin) that improves detection (including but not limited to GFP), purification, or other properties of the polypeptide.

[0135] The term "IL-2 polypeptide" also encompasses linked homodimers, heterodimers, homomultimers, or heteromultimers, including but not limited to those linked directly via non-naturally encoded amino acid side chains to the same or different non-naturally encoded amino acid side chains, to naturally encoded amino acid side chains, or indirectly via a linker. Exemplary linkers include, but are not limited to, small organic compounds, water-soluble polymers of various lengths such as polyethylene glycol or dextran, or polypeptides of various lengths.

[0136] As used herein, the term "conjugate of the present invention," "IL-2-bioactive molecule conjugate," or "PEG-IL-2" refers to interleukin-2, or a portion, analog, or derivative thereof, that binds to an interleukin-2 receptor or a subunit thereof, conjugated to a bioactive molecule, portion, or analog thereof. Unless otherwise indicated, the terms "compound of the present invention" and "composition of the present invention" are used as alternatives to the term "conjugate of the present invention."

[0137] 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 IL-2 variants (see, for example, WO 2004 / 010957, "Drug Conjugates and Their Use for Treating Cancer, An Autoimmune Disease or an Infectious Disease"). Classes of cytotoxic or immunosuppressive agents useful in the present invention include, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, monoplatin, bisplatin, and trinuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemosensitizers, duocarmycins, etoposide, fluoropyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, preformed compounds, purine antimetabolites, puromycins, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, and the like.

[0138] Individual cytotoxic or immunosuppressive agents include, for example, androgens, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoxide, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly known as actinomycin), daunorubicin, dacarbazine, docetaxel, doxorubicin, Estrogens, 5-fluorodeoxyuridine, 5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazoles, paclitaxel, plicamycin, procarbazine, streptozotocin, teniposide, 6-thioguanine, thio-TEPA, topotecan, vinblastine, vincristine, vinorelbine, VP-16, and VM-26.

[0139] In some typical embodiments, the therapeutic agent is a cytotoxic agent. Suitable cytotoxic agents include, for example, Aplysias (such as auristatin E, AFP, MMAF, MMAE), DNA minor groove binders (such as enediynes and lexitropsins), duocarmycins, taxanes (such as paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin, lisocin, cyanomorpholino-doxorubicin, echinomycin, Compretin, spindle fungus, epothilone A and B, estramustine, cryptophycins, Cemadotin, maytansinoids, spondylolipids, acanthopanax and mitoxantrone.

[0140] "Non-naturally 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 "non-naturally encoded amino acid" are "non-natural amino acid," "non-naturally occurring amino acid," and various hyphenated and non-hyphenated forms thereof. The term "non-naturally encoded amino acid" also includes, but is not limited to, amino acids that are produced by modification of naturally encoded amino acids (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine), but are not themselves naturally incorporated into a 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.

[0141] An "amino-terminal modification group" refers to any molecule that can be attached to the amino terminus of a polypeptide. Similarly, a "carboxyl-terminal modification group" refers to any molecule that can be attached to the carboxyl terminus of a polypeptide. Terminal modification groups include, but are not limited to, various water-soluble polymers, peptides, or proteins such as serum albumin, or other moieties that increase the serum half-life of a peptide.

[0142] 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 a distinct, definable portion or unit of a molecule. The terms are somewhat synonymous in the chemistry arts and are used herein to indicate a portion of a molecule that performs some function or activity and is reactive with other molecules.

[0143] The terms "linkage," "linkage," or "linker" 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 hydrolytically stable bond means that the bond is substantially stable in water and does not react with water at useful pH values ​​(including, but not limited to, under physiological conditions) for an extended period, possibly even indefinitely. A hydrolytically unstable or degradable bond means that the bond is degradable in water or an aqueous solution (including, for example, blood). An enzymatically unstable or degradable bond means that the bond can be degraded by one or more enzymes. As understood in the art, PEG and related polymers can include degradable bonds in the polymer backbone or in a linker group 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 biologically active agent is typically hydrolyzed under physiological conditions to release the agent. Other hydrolytically degradable linkages include, but are not limited to, carbonate linkages, imine linkages resulting from the reaction of an amine with an aldehyde, phosphate linkages formed by the reaction of an alcohol with a phosphate group, hydrazone linkages that are byproducts of hydrazides with aldehydes, acetal linkages that are the reaction product of aldehydes with alcohols, orthoester linkages that are the reaction product of formic acid with alcohols, peptide linkages formed by an amine group at the terminus of a polymer, including but not limited to, PEG, and a carboxyl group of a peptide, and oligonucleotide linkages formed by a phosphoramidite group at the terminus of a polymer, including but not limited to, PEG, and the 5' hydroxyl group of an oligonucleotide.

[0144] As used herein, the terms "biologically active molecule," "biologically active moiety," or "biologically active 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, phages, transposons, prions, insects, fungi, plants, animals, and humans. Specifically, as used herein, biologically active molecules include, but are not limited to, any substance intended for use in diagnosing, curing, mitigating, treating, or preventing disease in humans or other animals, or otherwise enhancing the physical or psychological health of humans or animals. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, vaccines, immunogens, addictive drugs, non-addictive drugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxoids, biologically active molecules, prokaryotic and eukaryotic cells, viruses, polysaccharides, nucleic acids obtained or derived from viruses, bacteria, insects, animals, or any other cells or cell types, and portions thereof, 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, antifungals, bile acid resins, niacin and / or statins, anti-inflammatory drugs, anti-tumor drugs, cardiovascular agents, anxiolytics, hormones, growth factors, steroidal agents, bioactive molecules of microbial origin, etc. Bioactive agents also include amide compounds such as those described in Patent Application Publication No. 20080221112 to Yamamori et al., which can be administered before, after, and / or co-administered with the IL-2 polypeptides of the present invention.

[0145] "Bifunctional polymer" refers to a polymer comprising two discrete functional groups that can react specifically with other components (including but not limited to amino acid side groups) to form covalent or non-covalent bonds. Bifunctional linkers having one functional group reactive with a group on a specific biologically active component and another group reactive with a group on a second biological component can be used to form a conjugate comprising the first biologically active component, a bifunctional linker, and a second biologically active component. Many procedures and linker molecules are known for attaching a variety of different compounds to peptides. 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 comprising two or more discrete functional groups that can react specifically with other components (including but not limited to amino acid side groups) to form covalent or non-covalent bonds. The bifunctional or multifunctional polymer can be of any desired length or molecular weight and can be selected to provide a specific desired spacing or conformation between the molecule or molecules attached to IL-2 and its receptor or IL-2.

[0146] When substituents are described by their conventional chemical formulae written left to right, they likewise encompass chemically identical substituents resulting from writing the structure right to left, for example, the structure -CH2O- is equivalent to the structure -OCH2-.

[0147] The term "substituent" includes, but is not limited to, "non-interfering substituents". A "non-interfering substituent" is a group that produces a stable compound. Suitable non-interfering substituents or residues include, but are not limited to, halogen, 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, xylyl, 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 10alkyl) (wherein m is 1 to 8), aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic, substituted heterocyclic, 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) (wherein each m is 1 to 8), --C(O)NR2, --C(S)NR2, --SO2NR2, --NRC(O)NR2, --NRC(S)NR2, salts thereof, and the like. When used herein, each R is H, alkyl or substituted alkyl, aryl or substituted aryl, aralkyl or alkaryl.

[0148] The term "halogen" includes fluorine, chlorine, iodine and bromine.

[0149] Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent means a straight or branched chain or cyclic hydrocarbon radical or combinations thereof, which may be fully saturated, mono- or polyunsaturated, and may include divalent and polyvalent radicals, having the specified number of carbon atoms (i.e., C1-C 10 Meaning 1 to 10 carbon atoms). The example of saturated hydrocarbon radical includes but is not limited to groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, such as homologues and isomers of n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl is a group with one or more double bonds or triple bonds. The example of unsaturated alkyl includes but is 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 indicated, the term "alkyl" also means derivatives of the alkyl defined in more detail below such as "heteroalkyl". Alkyl groups limited to hydrocarbon radicals are referred to as "homoalkyl".

[0150] 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 below as "heteroalkylene". Typically, an alkyl (or alkylene) group has from 1 to 24 carbon atoms, with groups having 10 or fewer carbon atoms being a particular embodiment of the methods and compositions described herein. "Lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, typically having 8 or fewer carbon atoms.

[0151] The terms "alkoxy," "alkylamino," and "alkylthio" (or thioalkoxy) are used in their conventional sense 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.

[0152] Unless otherwise stated, the term "heteroalkyl" by itself or in combination with another term means a stable straight or branched chain or cyclic hydrocarbon radical, or combinations 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 optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N, S and Si may be placed at any interior position of the heteroalkyl radical or at the position at which the alkyl radical 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 radical derived from a heteroalkyl radical, such as, but not limited to, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, the same or different heteroatoms may also occupy either or both chain termini (including, but not limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, and the like). Furthermore, for alkylene and heteroalkylene linking groups, the direction in which the formula of the linking group is written does not imply an orientation of the linking group. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-.

[0153] Unless otherwise indicated, the terms "cycloalkyl" and "heterocycloalkyl," by themselves or in combination with other terms, refer to cyclic versions of "alkyl" and "heteroalkyl," respectively. Thus, cycloalkyl or heterocycloalkyl include saturated, partially unsaturated, and fully unsaturated cyclic linkages. Furthermore, for heterocycloalkyl, heteroatoms may 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-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. Furthermore, the terms encompass bicyclic and tricyclic ring structures. Likewise, the term "heterocycloalkylene" by itself or as part of another substituent means a divalent radical derived from heterocycloalkyl, and the term "cycloalkylene" by itself or as part of another substituent means a divalent radical derived from cycloalkyl.

[0154] As used herein, the term "water-soluble polymer" refers to any polymer that is soluble in an aqueous solvent. Linking 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 biologically active 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. Pat. No. 5,252,714, which is incorporated herein by reference), monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran derivatives (including dextran sulfate), polypropylene glycol, polyoxypropylene / 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 derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers and α-β-poly(2-hydroxyethyl)-DL-asparagine, etc., or mixtures thereof. Examples of these water-soluble polymers include, but are not limited to, polyethylene glycol and serum albumin.

[0155] As used herein, the term "polyalkylene glycol" refers to polyethylene glycol, polypropylene glycol, polybutylene glycol, and their derivatives. 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 commercial supplier catalogs, such as Shearwater Corporation's catalog "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001).

[0156] Unless otherwise stated, the term "aryl" means a polyunsaturated aromatic hydrocarbon substituent which can be a single ring or multiple rings (including but not limited to 1 to 3 rings) fused together or covalently linked. The term "heteroaryl" refers to an aryl group (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 atom is optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule via a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2- Azolyl, 4- Azolyl, 2-phenyl-4- Azolyl, 5- Azolyl, 3-iso oxazolyl, 4-iso Azolyl, 5-iso oxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 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, substituents are selected from the group consisting of the acceptable substituents described below.

[0157] 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.), including 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.).

[0158] 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 residue. Exemplary substituents for each type of residue are provided below.

[0159] 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 groups selected from, but not limited to, -OR', =O, =NR', =N-OR', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(O)R', -C(O)R', -CO2R', -CONR'R", -O C(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 ranges from 0 to (2m'+1), where m' is the total number of carbon atoms in such 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 a compound of the present invention includes more than one R group, for example, each of said R groups is independently selected, as is each of said R', R", R'", and R"" groups when there is more than one of these groups. When R' and R" are attached to the same nitrogen atom, they can be combined with said nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the discussion of substituents above, one skilled in the art will understand that the term "alkyl" is meant to include groups containing carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl groups (including but not limited to -CF3 and -CH2CF3) and acyl groups (including but not limited to -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0160] Similar to the substituents described for the alkyl residues, the substituents for the aryl and heteroaryl groups 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', -C02R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -NR-C(N wherein R', R", R"' and R"" are independently selected from hydrogen, alkyl, heteroalkyl, aryl and heteroaryl. When a compound of the invention includes more than one R group, for example, each of said R groups is independently selected, as are each of said R', R", R'" and R"" groups when more than one R', R", R'" and R"" group is present.

[0161] When used in this article, the term "adjusted serum half-life" means the positive or negative change in the circulating half-life of the 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 between serum concentration and time allows the calculation of serum half-life. The improved serum half-life ideally has at least about twice, but smaller improvements may also be useful, for example, when it can achieve a satisfactory dosage regimen or avoid toxic effects. In certain embodiments, the improvement is at least about three times, at least about five times, or at least about ten times.

[0162] 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. The 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 ideally 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 efficacy, 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 of action or mechanism of action of the unmodified molecule, or an increase or decrease in receptor-mediated clearance of the molecule.

[0163] The term "isolated," when applied to a nucleic acid or protein, means that the nucleic acid or protein is at least free of certain cellular components with which it is naturally associated, or that the nucleic acid or protein has been concentrated to a level above its concentration upon in vivo or in vitro production. It can be in a homogeneous state. The isolated material can be in a dry or semi-dry state, or in a solution (including but not limited to an aqueous solution). It can be a component of a pharmaceutical composition that also includes 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 predominant species present in a preparation is substantially purified. Specifically, an isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest. The term "purified" means that a nucleic acid or protein produces essentially a single band in an electrophoretic gel. Specifically, this can mean that the nucleic acid or protein is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% pure, or more.

[0164] The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and the polymers thereof in single-strand or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have a binding property similar to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, the term also refers to oligonucleotide analogs, including PNA (peptide nucleic acid), the analogs (phosphorothioate, phosphoramidate etc.) of the DNA used in antisense technology. Unless otherwise specified, specific nucleic acid sequences also implicitly encompass variants (including but not limited to degenerate codon replacements) and complementary sequences of their conservative modifications and the sequences of the clear instructions. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed-base 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)).

[0165] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. That is, a description directed to a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally encoded amino acid. When used herein, the terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0166] 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 function 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 refer to compounds having the same basic chemical structure as naturally occurring amino acids, i.e., an alpha carbon bound to a hydrogen, carboxyl, amino, and R groups, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. References to amino acids include, for example, naturally occurring proteinogenic L-amino acids, D-amino acids, chemically modified amino acids such as amino acid variants and derivatives, naturally occurring non-proteinogenic amino acids such as β-alanine, ornithine, and chemically synthesized compounds having properties known in the art to be unique to amino acids. Examples of non-naturally occurring amino acids include, but are not limited to, α-methyl amino acids (e.g., α-methylalanine), 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 with additional methylene groups 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., cysteic acid). 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 and the like containing D-amino acids exhibit improved in vitro or in vivo stability compared to their L-amino acid-containing counterparts. Therefore, when greater intracellular stability is desired or required, constructs of peptides and the like incorporating D-amino acids may be particularly useful. More specifically, D-peptides and the like are resistant to endogenous peptidases and proteases, thereby providing enhanced bioavailability and extended in vivo lifespan of the molecules when these properties are desirable. Furthermore, D-peptides and the like cannot be efficiently processed for class II major histocompatibility complex-restricted presentation to T helper cells and are therefore less likely to induce a humoral immune response in intact organisms.

[0167] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their generally accepted single-letter codes.

[0168] "Conservatively modified variants" are applicable to both amino acid and nucleic acid sequences. For a particular nucleic acid sequence, a "conservatively modified variant" refers to a nucleic acid encoding a consistent or substantially consistent amino acid sequence, or, in the case where the nucleic acid does not encode an amino acid sequence, refers to a substantially consistent sequence. Due to the degeneracy of the genetic code, a large number of functionally consistent nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. These nucleic acid variations are "silent variations," which are one type of conservatively modified variation. Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variation of the nucleic acid. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce a functionally consistent molecule. Therefore, each silent variation of a nucleic acid encoding a polypeptide is implied in each described sequence.

[0169] With respect to amino acid sequences, one of ordinary skill in the art will recognize that alterations, additions, or deletions of a single amino acid or a small percentage of amino acids in a nucleic acid, peptide, polypeptide, or protein sequence are "conservatively modified variants," wherein the alterations result in the deletion of an amino acid, the addition of an amino acid, or the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are known to those of ordinary skill in the art. These conservatively modified variants are also, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the present invention.

[0170] Conservative substitution tables providing functionally similar amino acids are known to those of ordinary skill in the art. The following eight groups each contain amino acids that are conservative substitutions for each other: 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 (WH Freeman & Co., 2nd ed. (December 1993)).

[0171] In the case of two or more nucleic acid or peptide sequences, the term "consistent" or percentage "identity" refers to that two or more sequences or subsequences are identical. 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 to measure, when sequences are compared and aligned to obtain maximum correspondence in a comparison window or a specified region, 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 a specified region), the sequence is "substantially identical". This definition is also for the complementary sequence of a test sequence. The identity can be present in a region of at least about 50 amino acids or nucleotides or in a region of 75-100 amino acids or nucleotides in length, or across the entire sequence of a polynucleotide or polypeptide when not specified. Polynucleotides encoding the polypeptides of the present invention (including homologs from species other than humans) can be obtained by a method comprising the following steps: screening a library under stringent hybridization conditions using a labeled probe having the polynucleotide sequence of the present invention or a fragment thereof, and isolating full-length cDNA and genomic clones containing the polynucleotide sequence. Such hybridization techniques are well known to those skilled in the art.

[0172] The phrase "selectively (or specifically) hybridizes to" means that when a specific nucleotide sequence is present in a complex mixture (including but not limited to total cellular or library DNA or RNA), a molecule binds, forms a duplex or hybridizes only to that specific sequence under stringent hybridization conditions.

[0173] As known in the art, the phrase "stringent hybridization conditions" refers to the hybridization of sequences of DNA, RNA, PNA or other nucleic acid mimics or combinations thereof under conditions of low ionic strength and high temperature. Typically, under stringent conditions, a probe will hybridize to its target subsequence in a complex mixture of nucleic acids (including but not limited to total cell or library DNA or RNA), but will not hybridize to other sequences in the complex mixture. Stringent conditions are sequence-dependent and vary in different situations. Longer sequences will hybridize specifically at higher temperatures.

[0174] As used herein, the term "eukaryote" refers to organisms belonging to the Eukaryota system domain, 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, yeasts, flagellates, microsporidia, protists, etc.

[0175] As used herein, the term "non-eukaryotic organism" refers to an organism that is not a eukaryotic organism. 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.) system 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.) system domain.

[0176] As used herein, the term "subject" refers to an animal, in certain embodiments a mammal, and in other embodiments a human, that is the target of treatment, observation, or experiment. The animal can be a companion animal (e.g., dog, cat, etc.), a farm animal (e.g., cow, sheep, pig, horse, etc.), or a laboratory animal (e.g., rat, mouse, guinea pig, etc.).

[0177] As used herein, the term "effective amount" refers to an amount of the modified non-natural amino acid polypeptide administered that alleviates one or more symptoms of the disease, condition, or disorder to be treated to a certain extent. Compositions containing the modified non-natural amino acid polypeptides described herein can be administered for prophylactic, enhancing, and / or therapeutic treatment.

[0178] The term "enhancing" means increasing or prolonging a desired effect, either in potency or duration. Thus, with respect to enhancing the effect of a therapeutic agent, the term "enhancing" refers to the ability to increase or prolong, either in potency or duration, the effect of another therapeutic agent on a system. As used herein, an "enhancing-effective amount" refers to an amount sufficient to enhance the effect of another therapeutic agent in a 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 treatments, the patient's health status and response to the drug, and the judgment of the treating physician.

[0179] 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 "(modified)" form of the term means that the polypeptide in question is optionally modified, that is, the polypeptide in question can be modified or unmodified.

[0180] The term "post-translational modification" refers to any modification of a natural or non-natural amino acid after it has been incorporated into a polypeptide chain. By way of example only, the term encompasses co-translational in vivo modifications, co-translational in vitro modifications (e.g., in a cell-free translation system), post-translational in vivo modifications, and post-translational in vitro modifications.

[0181] In prophylactic applications, a composition containing the IL-2 is administered to a patient who is susceptible to or otherwise at risk for a particular disease, disorder, or condition. This amount is defined as a "prophylactically effective amount." In this application, the precise amount also depends on the patient's health, weight, and the like. Determining such a prophylactically effective amount by routine experimentation (e.g., a dose escalation clinical trial) is considered to be well within the skill of the art.

[0182] In therapeutic applications, a composition containing the modified non-natural amino acid polypeptide is administered to a patient already suffering from the disease, disorder, or disorder in an amount sufficient to cure or at least partially prevent the symptoms of the disease, disorder, or disorder. This amount is defined as a "therapeutically effective amount" and will depend on the severity and course of the disease, disorder, or disorder, previous treatment, the patient's health status, and the response to the drug, as well as the judgment of the treating physician. It is considered to be well within the skill of the art to determine this therapeutically effective amount by routine experimentation (e.g., a dose escalation clinical trial).

[0183] The term "treatment" is used to refer to either prophylactic and / or therapeutic treatment.

[0184] The non-naturally encoded amino acid polypeptides presented herein may include isotopically labeled compounds having one or more atoms replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the 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. 36Certain isotopically labeled compounds described herein, for example, into which radioactive isotopes such as 3 H and 14 C compounds may be useful in drug and / or substance tissue distribution assays. In addition, isotopes such as deuterium, i.e. 2 H substitutions may provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements.

[0185] All isomers, including but not limited to diastereomers, enantiomers, and mixtures thereof are considered to be part of the compositions described herein. In further or additional embodiments, the non-naturally encoded amino acid polypeptides are metabolized after administration to an organism in need thereof to produce metabolites, which are then used to produce a desired effect, including a desired therapeutic effect. In other or additional embodiments, they are active metabolites of the non-naturally encoded amino acid polypeptides.

[0186] In some cases, non-naturally encoded amino acid polypeptides can exist as tautomers. In addition, the non-naturally encoded amino acid polypeptides described herein can exist in unsolvated forms as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, etc. Such solvated forms are also considered to be disclosed herein. One of ordinary skill in the art will recognize that certain compounds herein can exist in several tautomeric forms. All of these tautomeric forms are considered to be part of the compositions described herein.

[0187] Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art are employed. Detailed description

[0188] I. Introduction

[0189] Provided herein are IL-2 molecules comprising at least one unnatural amino acid. In certain embodiments of the invention, the IL-2 comprising 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 to be suitable for specific reactive groups, the molecule including but not limited to a label, a dye, a polymer, a water-soluble polymer, a derivative of polyethylene glycol, a photocrosslinker, 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 analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, 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 moieties, radioactive moieties, novel functional groups, groups that covalently or non-covalently interact with other molecules, photocaged moieties, actinic radiation excitable moieties, photoisomerizable moieties, biotin, derivatives of biotin, biotin analogs, moieties incorporating heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox-active agents, amino thioacids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chemiluminescent groups, electron dense groups, magnetic groups, intercalating groups, chromophores, energy transfer agents, biologically active agents, detectable labels, small molecules, quantum dots, nanoemitters, radionucleotides, radioemitters, neutron capture agents, or any combination thereof, or any other desired compound or substance. 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 acetylene moiety) and the second reactive group is an azido moiety, and [3+2] cycloaddition chemistry is utilized. In another example, the first reactive group is an azido moiety (including but not limited to para-azido-L-phenylalanine (pAZ) in the non-natural 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 non-natural amino acid (including but not limited to non-natural amino acids containing a ketone functional group) is used that comprises at least one post-translational modification, wherein the at least one post-translational modification comprises a sugar moiety. In certain embodiments, the post-translational modification is produced in vivo in a eukaryotic cell or in a non-eukaryotic cell. A linker, polymer, water-soluble polymer, or other molecule can attach the molecule to the polypeptide. In another embodiment, the linker attached to the IL-2 is long enough to allow dimer formation.The molecule may also be linked directly to the polypeptide.

[0190] In certain embodiments, the IL-2 protein comprises at least one post-translational modification produced in vivo by a host cell type, wherein the post-translational modification is not normally produced by another host cell type. In certain embodiments, the protein comprises at least one post-translational modification produced in vivo by a eukaryotic cell type, wherein the post-translational modification is not normally produced by a non-eukaryotic cell type. 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 a different amino acid in the polypeptide. In certain embodiments, the IL-2 comprises one or more deletions that enhance glycosylation at a different amino acid 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 a non-naturally encoded amino acid 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 a naturally encoded amino acid 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 a different amino acid 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 a naturally encoded amino acid 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 the non-naturally encoded amino acid in the polypeptide.

[0193] In one embodiment, the post-translational modification comprises attaching an oligosaccharide (including but not limited to the case where the oligosaccharide comprises (GlcNAc-Man)2-Man-GlcNAc-GlcNAc, etc.) to an asparagine via a GlcNAc-asparagine linkage. In another embodiment, the post-translational modification comprises attaching an oligosaccharide (including but not limited to Gal-GalNAc, Gal-GlcNAc, etc.) to a 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, or the like. Examples of secretion signal sequences include but are not limited to prokaryotic secretion signal sequences, eukaryotic secretion signal sequences, 5'-optimized eukaryotic secretion signal sequences for bacterial expression, novel secretion signal sequences, pectate lyase secretion signal sequences, Omp A secretion signal sequences, and bacteriophage secretion signal sequences. Examples of secretory signal sequences include, but are not limited to, STII (prokaryotes), Fd GIII and M13 (phages), Bgl2 (yeast), and the signal sequence bla derived from a transposon. Any of these sequences may 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 can 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 unnatural amino acids. The unnatural amino acids can be the same or different, for example, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different sites in the protein containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different unnatural amino acids. In certain embodiments, at least one, but less than all, of the specific amino acids present in the naturally occurring form of the protein are replaced with an unnatural amino acid.

[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 in IL-2 can allow the use of coupling chemistries involving, but not limited to, specific chemical reactions that react 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 proteins with PEG derivatives, the method comprising selectively incorporating non-genetically encoded amino acids (including but not limited to amino acids containing functional groups or substituents that are not present in the 20 naturally incorporated amino acids (including but not limited to ketone, azide or acetylene moieties)) into proteins in response to a selector codon, followed by modification of those amino acids with suitable reactive PEG derivatives. 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 acids. A wide range of known chemical methods are suitable for use in the present invention in incorporating water-soluble polymers into the protein. These methods include, but are not limited to, the Huisgen [3+2] cycloaddition reaction (see, e.g., Padwa, A., Comprehensive Organic Synthesis, Vol. 4, (1991) Ed. Trost, BM, Pergamon, Oxford, p. 1069-1109; and Huisgen, R., 1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, New York, p. 1-176) using, but not limited to, acetylene or azide derivatives, respectively.

[0196] Because the Huisgen [3+2] cycloaddition method involves a cycloaddition rather than a nucleophilic substitution reaction, proteins can be modified with extremely high selectivity. By adding a catalytic amount of Cu(I) salt to the reaction mixture, the reaction can be carried out at room temperature in aqueous conditions with excellent regioselectivity (1,4>1,5). 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 virtually any molecule with a suitable functional group or substituent, including but not limited to an azido or acetylene derivative. These molecules can be added to non-natural amino acids with an acetylene group, including but not limited to p-propargyloxyphenylalanine, or non-natural amino acids with an azido group, including but not limited to p-azido-phenylalanine.

[0197] The 5-membered ring obtained from the Huisgen [3+2] cycloaddition is generally irreversible in a reducing environment and is stable to hydrolysis in an aqueous environment for a long time. Therefore, the physical and chemical properties of a wide range of substances can be modified with the active PEG derivatives of the present invention under demanding aqueous conditions. Even more importantly, because the azide and acetylene components 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 in one or more specific sites.

[0198] The present invention also provides water-soluble and hydrolytically stable PEG derivatives and related hydrophilic polymers having one or more acetylene or azide moieties. The PEG polymer derivatives containing acetylene moieties are highly selective for coupling with azide moieties that have been selectively introduced into proteins in response to a selector codon. Similarly, the PEG polymer derivatives containing azide moieties are highly selective for coupling with acetylene moieties that have been selectively introduced into proteins 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 may 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 may include other substituents as long as the azide-specific reactivity is maintained.

[0200] The present invention provides conjugates of substances having a wide variety of functional groups, substituents, or moieties with other substances, including, but not limited to, labels, dyes, polymers, water-soluble polymers, derivatives of polyethylene glycol, photocrosslinkers, 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, carbohydrates, polynucleotides, DNA, RNA, antisense polynucleotides, sugars, water-soluble dendrimers, cyclodextrins, inhibitory ribonucleic acids, biomaterials, nanoparticles, spin labels, fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, and other molecules. Covalently or non-covalently interacting groups, photocaged moieties, actinic radiation excitable moieties, photoisomerizable moieties, biotin, derivatives of biotin, biotin analogs, moieties incorporating heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox-active agents, amino thioacids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chemiluminescent groups, electron dense groups, magnetic groups, intercalating groups, chromophores, energy transfer agents, biologically active agents, detectable labels, small molecules, quantum dots, nanoemitters, radionucleotides, radioemitters, neutron capture agents, or any combination of the foregoing or any other desired compound or substance. The present invention also includes conjugates of substances having an azide or acetylene moiety and PEG polymer derivatives having the corresponding acetylene or azide moiety. For example, a PEG polymer containing an azide moiety can be coupled to a biologically active molecule at a position in the protein containing a non-genetically encoded amino acid bearing an acetylene functional group. Linkages for coupling the PEG to the biologically active molecule include, but are not limited to, Huisgen [3+2] cycloaddition products.

[0201] It is well known in the art that PEG can be used to modify the surface of biomaterials (see, for example, U.S. Patent No. 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 acetylene sites on their surfaces and one or more azide- or acetylene-containing polymers of the present invention coupled to the surface via a Huisgen [3+2] cycloaddition linkage. Biomaterials and other substances can also be coupled to the azide- or acetylene-activated polymer derivatives via linkages other than azide or acetylene linkages, such as linkages comprising carboxylic acid, amine, alcohol, or thiol moieties, to leave the azide or acetylene moiety available for subsequent reactions.

[0202] The present invention includes methods for synthesizing the azide- and acetylene-containing polymers of the present invention. In the case of the azide-containing PEG derivatives, the azide group can be directly bonded to a carbon atom of the polymer. Alternatively, the azide-containing PEG derivatives can be prepared by attaching a linking reagent having an azide moiety to one end of a conventional activated polymer, such that the resulting polymer has the azide moiety at its end. In the case of the acetylene-containing PEG derivatives, the acetylene can be directly bonded to a carbon atom of the polymer. Alternatively, the acetylene-containing PEG derivatives can be prepared by attaching a linking reagent having an acetylene moiety to one end of a conventional 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. 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 is then reacted 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 is reacted with a linking reagent having an azide group at one end to form a covalent bond between the PEG polymer and the linking reagent, with the azide moiety located at the end of the polymer. Nucleophilic and electrophilic moieties include amines, thiols, hydrazides, hydrazines, alcohols, carboxylates, aldehydes, ketones, thioesters, and the like, and are known to those of ordinary skill in the art.

[0204] More specifically, in the case of the acetylene-containing PEG derivatives, a water-soluble polymer having at least one reactive hydroxyl moiety undergoes a reaction to displace a halogen or other activated leaving group from a precursor containing the acetylene moiety. Alternatively, a water-soluble polymer having at least one reactive nucleophilic or electrophilic moiety undergoes a reaction with a linking reagent having an acetylene at one terminus to form a covalent bond between the PEG polymer and the linking reagent, with the acetylene moiety positioned at the terminus 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 well known to practitioners in the art.

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

[0206] II. General Recombinant Nucleic Acid Methods Used in the Present Invention

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

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

[0209] Table 1 - IL-2 protein and DNA sequences

[0210]

[0211]

[0212] Nucleotide sequences encoding IL-2 comprising non-naturally encoded amino acids can be synthesized based on the amino acid sequence of a parent polypeptide, including but not limited to a parent polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1, 2, 3, 5, or 7, and then altered to introduce (i.e., incorporate or replace) or remove (i.e., delete or replace) the relevant amino acid residues. The nucleotide sequence can be conveniently modified by site-directed mutagenesis according to conventional methods. Alternatively, the nucleotide sequence can be prepared by chemical synthesis, including but not limited to by using an oligonucleotide synthesizer, wherein oligonucleotides are designed based on the amino acid sequence of the desired polypeptide and preferably selected for codons that are favorable in the host cell in which the recombinant polypeptide is to be produced. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and assembled by PCR, ligation, or ligation chain reaction. See, for example, Barany et al., Proc. Natl. Acad. Sci. 88: 189-193 (1991); U.S. Patent 6,521,427, which are incorporated herein by reference.

[0213] The DNA sequence of the synthetic human IL-2 gene cloned into the pKG0269 expression plasmid is shown above in Table 1 as SEQ ID NO: 4. This DNA sequence has been codon-optimized for E. coli.

[0214] The present invention utilizes conventional techniques in the field of recombinant genetics. Basic texts disclosing the general methods used in the present invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd edition, 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994).

[0215] The present invention also relates to eukaryotic host cells, non-eukaryotic host cells, and organisms for incorporating unnatural amino acids in vivo by orthogonal tRNA / RS. The host cell is genetically engineered (including but not limited to, transformed, transduced, or transfected) using a polynucleotide of the present invention or a construct comprising a polynucleotide of the present invention (including but not limited to a vector of the present invention), such as a cloning vector or an expression vector.

[0216] 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, pellet bombardment, and infection with viral vectors (discussed further below). 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 methods known in the art (see, for example, Sambrook). In addition, kits for purifying plasmids from bacteria are commercially available (see, for example, EasyPrep, both from Pharmacia Biotech). TM 、FlexiPrep TM StrataClean from Stratagene TM ; and QIAprep from Qiagen TM). The isolated and purified plasmid is then further manipulated to produce other plasmids for transfecting cells or incorporating into related vectors to infect 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 comprises a universal expression cassette containing at least one independent terminator sequence, a sequence that allows the expression cassette to replicate in eukaryotes or prokaryotes or both (including but not limited to a shuttle vector), and a selection marker 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 the same). Catalogs of bacteria and bacteriophages that can be used for cloning are provided, for example, by the ATCC, such as in "ATCC Catalog of Bacteria and Bacteriophage" (1992), edited by Gherna et al., published by the ATCC. Other basic procedures for sequencing, cloning, and other aspects of molecular biology, as well as underlying theoretical considerations, are also found in Watson et al. (1992), "Recombinant DNA," 2nd edition, Scientific American Books, NY. In addition, virtually any nucleic acid (and virtually any labeled nucleic acid, whether standard or non-standard) can be custom or standard ordered from any of a variety of commercial sources, such as Midland Certified Reagent Company (Midland, TX, available on the World Wide Web at mcrc.com), The Great American Gene Company (Ramona, CA, available on the World Wide Web at genco.com), ExpressGen Inc. (Chicago, IL, available on the World Wide Web at expressgen.com), Operon Technologies Inc. (Alameda, CA), and many others.

[0217] Selector codon

[0218] The selector codons of the present invention expand the genetic codon architecture of the protein biosynthetic 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 oval codon (UGA)), unnatural codons, four or more base codons, rare codons, etc. It will be apparent to one 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.

[0219] In one embodiment, the method includes using a selector codon as a stop codon for incorporating one or more non-natural amino acids in vivo. For example, a kind of O-tRNA is produced, which recognizes a stop codon (including but not limited to UAG) and is aminoacylated by the O-RS with the desired non-natural amino acid, and this O-tRNA is not recognized by the aminoacyl-tRNA synthetase of the naturally occurring host. 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, JR 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 specified position.

[0220] In vivo incorporation of unnatural amino acids can be performed without significantly disrupting 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, but not limited to, increasing the expression level of the O-tRNA and / or suppressor tRNA.

[0221] Unnatural amino acids can also be encoded with rare codons. For example, it has been shown that when the concentration of arginine is reduced in an in vitro protein synthesis reaction, the rare arginine codon AGG is efficiently inserted into Ala by a synthetic tRNA acylated with alanine. See, for example, Ma et al., Biochemistry, 32:7939 (1993). In this case, the synthetic tRNA competes with the naturally occurring tRNAArg present in Escherichia coli as a minor species. Some organisms do not use all triplet codons. The unassigned codon AGA in Micrococcus luteus has been used for inserting amino acids in in vitro transcription / translation extracts. See, for example, Kowal and Oliver, Nucl.Acid.Res., 25:4685 (1997). The components of the present invention can be produced to use these rare codons in vivo.

[0222] 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. Four or more base codons can include but are not limited to one or more non-natural amino acids inserted into the same protein. For example, in the presence of an anticodon loop, such as an O-tRNA (including but not limited to a special frameshift suppressor tRNA) with a mutation of an anticodon loop of at least 8-10nt, the four or more base codons are read as a single amino acid. In other embodiments, the anticodon loop can decode including but not limited to at least four base codons, at least five base codons or at least six base codons or more base codons. Since there are 256 possible four base codons, four or more base codons can be used to encode a variety of 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).

[0223] For example, four-base codons have been used to incorporate unnatural amino acids into proteins 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 were used in vitro to incorporate 2-naphthylalanine and NBD derivatives of lysine into streptavidin using two chemically acylated frameshift suppressor tRNAs. See, for example, Hohsaka et al., J. Am. Chem. Soc., 121:12194 (1999). In an in vivo study, Moore et al. examined the ability of tRNALeu derivatives with NCUA anticodons to suppress UAGN codons (N can be U, A, G, or C) and found that the quadruple UAGA could be decoded by tRNALeu with UCUA anticodons with an efficiency of 13 to 26%, and rarely decoded in 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.

[0224] 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 three-base codon. For example, this includes systems that lack a tRNA that recognizes the natural three-base codon and / or systems where the three-base codon is a rare codon.

[0225] The selector codon optionally includes a non-natural base pair. These non-natural 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, being efficiently enzymatically incorporated into DNA with high fidelity by polymers, and efficiently continuing primer extension after synthesis of the nascent non-natural base pair. The description of the non-natural base pair that can be modified to be suitable for methods and compositions includes, 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 related publications are listed below.

[0226] For in vivo use, the non-natural nucleosides are membrane permeable and phosphorylated to form the corresponding triphosphates. In addition, the increased genetic information is stable and not destroyed by cellular enzymes. Previous attempts by Benner and others have utilized hydrogen bonding patterns different from those in the classical Watson-Crick pair, the most notable example of which is 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 are usually mispaired with natural bases to a certain extent and cannot be enzymatically replicated. Kool and collaborators have demonstrated that hydrophobic stacking interactions between bases can drive the formation of base pairs instead of hydrogen bonding. See Kool, Curr. Opin. Chem. Biol., 4: 602 (2000); and Guckian and Kool, Angew. Chem. Int. Ed. Engl., 36, 2825 (1998). In an attempt to develop non-natural base pairs that meet all of the above requirements, Schultz, Romesberg and collaborators systematically synthesized and studied a series of non-natural hydrophobic bases. It was found that PICS: PICS self-pairing is more stable than natural base pairs and can 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). 3MN: 3MN self-pairing can 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, the two bases act as chain terminators for further replication. Recently, a mutant DNA polymerase has evolved that can be used to replicate PICS self-pairing. In addition, 7AI self-pairing can be replicated. See, for example, Tae et al., J.Am.Chem.Soc., 123:7439 (2001). A new metallobase pair, Dipic:Py, has also been developed that forms a stable pair after binding Cu(II). See, for example, Meggers et al., J.Am.Chem.Soc., 122:10714 (2000). Since the extended codons and non-natural codons are inherently orthogonal to the natural codons, the method of the present invention can utilize this property to produce orthogonal tRNAs for them.

[0227] Translational bypass systems can also be used to incorporate non-natural amino acids into desired polypeptides. In a translational bypass system, a large sequence is incorporated into a gene but is not translated into protein. The sequence contains a structure that acts as a cue to induce the ribosome to skip over the sequence and resume translation downstream of the insertion.

[0228] In certain embodiments, in the methods and / or compositions of the invention, the protein or polypeptide of interest (or portion thereof) is encoded by a nucleic acid. Typically, 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.

[0229] 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 include, for example, one or more selector codons for incorporating non-natural amino acids. For example, a nucleic acid for a protein of interest is mutagenized to include one or more selector codons for incorporation of one or more non-natural amino acids. The present invention includes any such variants of any protein, including but not limited to mutant forms, for example, including at least one non-natural amino acid. Similarly, the present invention also includes corresponding nucleic acids, i.e., any nucleic acid having one or more selector codons encoding one or more non-natural amino acids.

[0230] Nucleic acid molecules encoding proteins of interest, such as IL-2, can be easily mutated to introduce cysteine ​​at any desired position of the polypeptide. Cysteine ​​is widely used to introduce reactive molecules, water-soluble polymers, proteins, or other molecules of a wide variety of types on proteins of interest. Methods suitable for incorporating cysteine ​​into the desired position of a polypeptide are known to those of ordinary skill in the art, such as those described in U.S. Patent No. 6,608,183, incorporated herein by reference, and standard mutagenesis techniques.

[0231] III. Non-naturally encoded amino acids

[0232] A wide range of non-naturally encoded amino acids are suitable for use in the present invention. Any number of non-naturally encoded amino acids can be introduced into IL-2. Generally speaking, the non-naturally encoded amino acids introduced 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 functional groups that efficiently and selectively react with functional groups that are not present in the 20 common amino acids (including but not limited to azido, ketone, aldehyde, and aminooxy groups) to form side chain functional groups of conjugates. For example, IL-2 comprising a non-naturally encoded amino acid containing an azido functional group can react with a polymer (including but not limited to polyethylene glycol or a second polypeptide containing an alkynyl component) to form a stable conjugate, because the selective reaction of the azide and alkyne functional groups forms a Huisgen [3+2] cycloaddition product.

[0233] The general structure of an α-amino acid is shown below (Formula I):

[0234] I

[0235]

[0236] Non-naturally encoded amino acids are generally any structure with the structural formula listed above, wherein the R group is any substituent other than the substituent used in the 20 kinds of natural amino acids, and can be suitably used in the present invention. Since the non-naturally encoded amino acids of the present invention are generally different from natural amino acids only in the structure of the side chain, the non-naturally encoded amino acids form amide bonds with other amino acids (including but not limited to natural or non-naturally encoded amino acids) in the same manner as they are formed in naturally occurring polypeptides. However, the non-naturally encoded amino acids have side chain groups different from the natural amino acids. For example, R optionally includes alkyl-, aryl-, acyl-, ketone-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynyl, ether, thiol, seleno-, sulfonyl-, borate, boric acid group, phosphoryl, phosphonyl, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino group etc. or any combination thereof. Other non-naturally occurring amino acids of interest that may be suitable for use in the present invention include, but are not limited to, amino acids comprising photoactivatable cross-linkers, spin-labeled amino acids, fluorescent amino acids, metal-binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or non-covalently interact with other molecules, photocaged and / or photoisomerizable amino acids, amino acids comprising biotin or a biotin analog, glycosylated amino acids such as sugar-substituted serine, other carbohydrate-modified amino acids, keto-containing amino acids, amino acids comprising polyethylene glycol or polyethers, heavy atom-substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids with elongated side chains compared to naturally occurring amino acids (including but not limited to polyethers or long chain hydrocarbons, including but not limited to long chain hydrocarbons of greater than about 5 or greater than about 10 carbons), carbon-linked sugar-containing amino acids, redox-active amino acids, amino thioacid-containing amino acids, and amino acids comprising one or more toxic moieties.

[0237] Exemplary non-naturally encoded amino acids that may be suitable for use in the present invention and can be used to react with water-soluble polymers include, but are not limited to, non-naturally encoded amino acids with carbonyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide, and alkyne reactive groups. In certain embodiments, non-naturally encoded amino acids include sugar components. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosamine-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosamine-L-serine. Examples of such amino acids also include examples in which the naturally occurring N- or O-connections between the amino acid and the sugar are replaced by covalent connections (including but not limited to olefins, oximes, thioethers, amides, etc.) that are uncommon in nature. Examples of such amino acids also include sugars such as 2-deoxyglucose, 2-deoxygalactose, etc. that are uncommon in naturally occurring proteins.

[0238] 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, Fessendon and Fessendon's Organic Chemistry, 2nd edition, Willard Grant Press, Boston Mass (1982); March's Advanced Organic Chemistry, 3rd edition, Wiley and Sons, New York (1985); and Carey and Sundberg's Advanced Organic Chemistry, 3rd edition, Parts A and B, Plenum Press, New York (1990). See also U.S. Patent Nos. 7,045,337 and 7,083,970, which are incorporated herein by reference. In addition to non-natural amino acids containing novel side chains, non-natural amino acids that may be suitable for use in the present invention also optionally contain modified backbone structures, including but not limited to those shown by the structures of Formula II and III:

[0239] II

[0240]

[0241] III

[0242]

[0243] wherein Z typically comprises OH, NH2, SH, NH-R', or SR'; X and Y may be the same or different and typically comprise S or O, and R and R', which are optionally the same or different, are typically selected from the same list of moieties as the R groups described above for the non-natural amino acids of formula I, as well as hydrogen. For example, the non-natural amino acids of the present invention optionally comprise substitutions in the amino or carboxyl groups, as shown for formulas II and III. This type of non-natural amino acid includes, but is not limited to, α-hydroxy acids, α-thioacids, and α-aminothiocarboxylates, including but not limited to those compounds having side chains corresponding to the common 20 natural amino acids or non-natural side chains. Furthermore, 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, and the like. Other structural alternatives include cyclic amino acids such as proline analogs and 3-, 4-, 6-, 7-, 8-, and 9-membered ring proline analogs, and β- and γ-aminobutyric acid, such as substituted β-alanine and γ-aminobutyric acid.

[0244] Many non-natural amino acids are based on natural amino acids such as tyrosine, glutamine, phenylalanine, etc. and are suitable for use in the present invention. Tyrosine analogs include, but are not limited to, para-substituted tyrosines, ortho-substituted tyrosines, and meta-substituted tyrosines, wherein the substituted tyrosines include, but are not limited to, keto groups (including, but not limited to, acetyl groups), benzoyl groups, amino groups, hydrazine groups, hydroxylamine groups, thiol groups, carboxyl groups, isopropyl groups, methyl groups, C6-C 20Straight or branched hydrocarbons, saturated or unsaturated hydrocarbons, O-methyl, polyether, nitro, alkynyl, etc. In addition, multi-substituted aromatic rings are also considered. Glutamine analogs that can be applied 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 that can be applied 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, azido, iodo, bromo, keto (including but not limited to acetyl), benzoyl, alkynyl, etc. Specific examples of unnatural amino acids that may be suitable for use in 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, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, and p-propargyloxy-phenylalanine, among others. Examples of the structures of various unnatural amino acids that may be suitable for use in the present invention are provided, for example, in WO 2002 / 085923, entitled "In vivo incorporation of unnatural amino acids." For other methionine analogs, see also 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 AminoAcids 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.

[0245] 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 relative newcomers to the family of synthetic chemistry, 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 treatments for bioconjugation. Staudinger ligation has been used with phosphine compounds to label azido sugars that are metabolically introduced into cellular glycoconjugates. The Staudinger ligation can be performed in living animals and is physiologically harmless; however, the Staudinger reaction is not without disadvantages. The required phosphine compounds are susceptible to air oxidation, and optimization to increase water solubility and improve reaction rate has proven to be synthetically challenging.

[0246] The azide group has an optional bioorthogonal reactivity mode: 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 collaborators overcame this obstacle by developing a copper (I)-catalyzed form known as "click chemistry," which is readily performed at physiological temperatures and in a biological environment rich in functionalization. This discovery enables the selective modification of viral particles, nucleic acids, and proteins from complex tissue lysates. Unfortunately, the mandatory copper catalyst is toxic to both bacteria and mammalian cells, thus hindering applications where 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.

[0247] In one embodiment, a composition of IL-2 comprising an unnatural amino acid (e.g., p-(propargyloxy)-phenylalanine) is provided. Various compositions comprising p-(propargyloxy)-phenylalanine are also provided, including but not limited to proteins and / or cells. In one aspect, the composition comprising the p-(propargyloxy)-phenylalanine unnatural amino acid further comprises an orthogonal tRNA. The unnatural 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 via an amino-acyl bond, covalently bonding to the 3'OH or 2'OH of the terminal ribose of the orthogonal tRNA, and the like.

[0248] Chemical components that can be incorporated into proteins via non-natural amino acids provide a variety of advantages and manipulations for the proteins. For example, the unique reactivity of the ketone functional group allows for selective modification of proteins using a variety of reagents containing hydrazine or hydroxylamine in vitro or in vivo. For example, heavy atom non-natural amino acids may be useful for phasing X-ray structural data. Site-specific introduction of heavy atoms using non-natural amino acids also provides selectivity and flexibility in selecting positions for heavy atoms. Photoreactive non-natural amino acids (including but not limited to amino acids with benzophenone and aryl azide (including but not limited to phenyl azide) side chains) allow, for example, efficient in vivo and in vitro photocrosslinking of proteins. Examples of photoreactive non-natural amino acids include but are not limited to p-azido-phenylalanine and p-benzoyl-phenylalanine. Proteins with photoreactive non-natural amino acids can therefore be crosslinked at will by excitation of the photoreactive groups, providing temporal control. In one example, the methyl groups of non-natural amino acids can be substituted with, but are not limited to, isotopically labeled methyl groups, serving as probes of local structure and dynamics when using, including but not limited to, nuclear magnetic resonance and vibrational spectroscopy. Alkynyl or azido functional groups allow for the selective modification of proteins with molecules, for example via [3+2] cycloaddition reactions.

[0249] Unnatural amino acids incorporated into polypeptides at the amino terminus can be composed of an R group that is any substituent other than those used in the 20 natural amino acids and a second reactive group that is different from the NH2 group typically present in α-amino acids (see Formula 1). Similar unnatural amino acids can be incorporated at the carboxyl terminus with a second reactive group that is different from the COOH group typically present in α-amino acids (see Formula 1).

[0250] The non-natural amino acids of the present invention can be selected or designed to provide additional properties not available in 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 protein into which they are incorporated. For example, by including non-natural amino acids in proteins, 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.

[0251] In certain embodiments, the invention provides IL-2 that is connected to a water-soluble polymer such as PEG by an oxime bond. Many types of non-naturally encoded amino acids are suitable for forming oxime bonds. They include, but are not limited to, non-naturally encoded amino acids containing carbonyl, dicarbonyl or hydroxylamine groups. These amino acids are described in U.S. Patent Publication Nos. 2006 / 0194256, 2006 / 0217532 and 2006 / 0217289 and WO 2006 / 069246 entitled "Compositions Containing, Methods Involving, and Uses of Non-natural Amino Acids and Polypeptides," which are incorporated herein 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 incorporated herein by reference in their entirety.

[0252] Certain embodiments of the present invention utilize IL-2 polypeptides that have been replaced with p-acetylphenylalanine amino acids 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 similarly prepared by one of ordinary skill in the art. In addition, non-limiting exemplary syntheses of the unnatural amino acids included herein are presented in Figures 4, 24-34, and 36-39 of U.S. Patent No. 7,083,970, which is incorporated herein by reference in its entirety.

[0253] Amino acids with electrophilic reactive groups allow the use of a variety of reactions, particularly to link molecules via nucleophilic addition reactions. Such electrophilic reactive groups include carbonyls (including keto and dicarbonyls), carbonyl-like groups (which have similar reactivity to carbonyls (including keto and dicarbonyls) and are structurally similar to carbonyls), masked carbonyls (which can be easily converted to carbonyls (including keto and dicarbonyls)), or protected carbonyls (which have similar reactivity to carbonyls (including keto and dicarbonyls) after deprotection). These amino acids include amino acids having the structure of formula (IV):

[0254]

[0255] in:

[0256] 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;

[0257] 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')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl;

[0258] J is

[0259] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0260] 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;

[0261] R1 is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0262] R2 is optional, and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

[0263] Each R3 and R4 are independently H, halogen, lower alkyl or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or heterocycloalkyl group;

[0264] or the -ABJR groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl group containing at least one carbonyl group, including a dicarbonyl, a protected carbonyl (including a protected dicarbonyl), or a masked carbonyl (including a masked dicarbonyl);

[0265] or the -JR groups taken together form a monocyclic or bicyclic cycloalkyl or heterocycloalkyl group containing at least one carbonyl group, including a dicarbonyl, a protected carbonyl (including a protected dicarbonyl), or a masked carbonyl (including a masked dicarbonyl);

[0266] 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 not present and each R3 is H, R is not methyl.

[0267] Additionally, amino acids having the structure of Formula (V) are included:

[0268]

[0269] in:

[0270] 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;

[0271] 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')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl;

[0272] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0273] R1 is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0274] R2 is optional, and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

[0275] 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 not present, R is not methyl.

[0276] Additionally, amino acids having the structure of Formula (VI) are included:

[0277]

[0278] in:

[0279] 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')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl;

[0280] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0281] R1 is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0282] R2 is optional, and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

[0283] Each R a 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', wherein each R' is independently H, alkyl, or substituted alkyl.

[0284] In addition, the following amino acids are included:

[0285]

[0286] Wherein these compounds are optionally amino protected, carboxyl protected or are salts thereof.In addition, any of the following non-natural amino acids can be incorporated into the non-natural amino acid polypeptide.

[0287] In addition, the following amino acids having the structure of formula (VII) are included:

[0288]

[0289] in

[0290] 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')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl;

[0291] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0292] R1 is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0293] R2 is optional, and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

[0294] Each R a 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', wherein each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8;

[0295] The premise is that when A is –(CH2)4-, B is not –NHC(O)(CH2CH2)-.

[0296] Additionally, the following amino acids are included:

[0297]

[0298]

[0299] Wherein these compounds optionally amino is protected, optionally carboxyl is protected, optionally amino is protected and carboxyl is protected, or are salts thereof.In addition, these alpha-non-natural amino acids and any following alpha-non-natural amino acids can be incorporated into the alpha-non-natural amino acid polypeptide.

[0300] In addition, the following amino acids having the structure of formula (VIII) are included:

[0301]

[0302] 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, aralkylene, or substituted aralkylene;

[0303] 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')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl;

[0304] R1 is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0305] R2 is optional, and when present is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide.

[0306] In addition, the following amino acids having the structure of formula (IX) are included:

[0307]

[0308] 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')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl;

[0309] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0310] R1 is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0311] R2 is optional, and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

[0312] Each R a 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', wherein each R' is independently H, alkyl, or substituted alkyl.

[0313] Additionally, the following amino acids are included:

[0314]

[0315] Wherein these compounds optionally amino is protected, optionally carboxyl is protected, optionally amino is protected and carboxyl is protected, or are salts thereof.In addition, these alpha-non-natural amino acids and any following alpha-non-natural amino acids can be incorporated into the alpha-non-natural amino acid polypeptide.

[0316] In addition, the following amino acids having the structure of formula (X) are included:

[0317]

[0318] 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')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl;

[0319] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0320] R1 is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0321] R2 is optional, and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

[0322] Each R a 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', wherein each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8.

[0323] Additionally, the following amino acids are included:

[0324]

[0325] Wherein these compounds optionally amino is protected, optionally carboxyl is protected, optionally amino is protected and carboxyl is protected, or are salts thereof.In addition, these alpha-non-natural amino acids and any following alpha-non-natural amino acids can be incorporated into the alpha-non-natural amino acid polypeptide.

[0326] In addition to single carbonyl structures, the unnatural amino acids described herein can include groups such as dicarbonyl, dicarbonyl-like, masked dicarbonyl, and protected dicarbonyl groups.

[0327] For example, the following amino acids having the structure of formula (XI) are included:

[0328]

[0329] 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, aralkylene, or substituted aralkylene;

[0330] 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')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl;

[0331] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0332] R1 is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0333] R2 is optional, and when present is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide.

[0334] In addition, the following amino acids having the structure of formula (XII) are included:

[0335]

[0336] 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')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl;

[0337] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0338] R1 is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0339] R2 is optional, and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

[0340] Each R a 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', wherein each R' is independently H, alkyl, or substituted alkyl.

[0341] Additionally, the following amino acids are included:

[0342]

[0343] Wherein these compounds optionally amino is protected, optionally carboxyl is protected, optionally amino is protected and carboxyl is protected, or are salts thereof.In addition, these alpha-non-natural amino acids and any following alpha-non-natural amino acids can be incorporated into the alpha-non-natural amino acid polypeptide.

[0344] In addition, the following amino acids having the structure of formula (XIII) are included:

[0345]

[0346] 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 linkers of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl;

[0347] 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 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', wherein each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8.

[0348] Additionally, the following amino acids are included:

[0349]

[0350] Wherein these compounds optionally amino is protected, optionally carboxyl is protected, optionally amino is protected and carboxyl is protected, or are salts thereof.In addition, these alpha-non-natural amino acids and any following alpha-non-natural amino acids can be incorporated into the alpha-non-natural amino acid polypeptide.

[0351] In addition, the following amino acids having the structure of formula (XIV) are included:

[0352]

[0353] in:

[0354] 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;

[0355] 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.

[0356] In addition, the following amino acids having the structure of formula (XIV-A) are included:

[0357]

[0358] in:

[0359] 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;

[0360] 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;

[0361] L is alkylene, substituted alkylene, N(R')(alkylene), or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0362] In addition, the following amino acids having the structure of formula (XIV-B) are included:

[0363]

[0364] in:

[0365] 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;

[0366] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; L is alkylene, substituted alkylene, N(R')(alkylene), or N(R')(substituted alkylene), wherein R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0367] Additionally, the following amino acids having the structure of Formula (XV) are included:

[0368]

[0369] in:

[0370] 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;

[0371] R1 is optional and, when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; X1 is C, S, or S(O); and n is 0, 1, 2, 3, 4, or 5; and each CR 8 R 9 Each R on the group 8 and R 9 are independently selected from H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 and R 9 can together form =O or cycloalkyl, or any adjacent R 8 The groups may together form a cycloalkyl group.

[0372] Additionally, the following amino acids having the structure of Formula (XV-A) are included:

[0373]

[0374] in:

[0375] 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;

[0376] 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;

[0377] n is 0, 1, 2, 3, 4, or 5; and each CR 8 R 9 Each R on the group 8 and R 9 are independently selected from H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 and R 9 can together form =O or cycloalkyl, or any adjacent R 8 The groups may together form a cycloalkyl group.

[0378] Additionally, the following amino acids having the structure of Formula (XV-B) are included:

[0379]

[0380] in:

[0381] 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;

[0382] 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 Each R on the group 8 and R 9 are independently selected from H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 and R 9 can together form =O or cycloalkyl, or any adjacent R 8 The groups may together form a cycloalkyl group.

[0383] Additionally, the following amino acids having the structure of Formula (XVI) are included:

[0384]

[0385] in:

[0386] 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;

[0387] R1 is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0388] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or 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.

[0389] In addition, the following amino acids having the structure of formula (XVI-A) are included:

[0390]

[0391] in:

[0392] 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;

[0393] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; L is alkylene, substituted alkylene, N(R')(alkylene), or N(R')(substituted alkylene), wherein R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0394] In addition, the following amino acids having the structure of formula (XVI-B) are included:

[0395]

[0396] in:

[0397] 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;

[0398] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; L is alkylene, substituted alkylene, N(R')(alkylene), or N(R')(substituted alkylene), wherein R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0399] Additionally, amino acids having the structure of Formula (XVII) are included:

[0400]

[0401] in:

[0402] 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;

[0403] M is -C(R3)-,

[0404] wherein (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 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.

[0405] Additionally, amino acids having the structure of Formula (XVIII) are included:

[0406]

[0407] in:

[0408] M is -C(R3)-,

[0409] (a) (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 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 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', wherein each R' is independently H, alkyl, or substituted alkyl.

[0410] Additionally, amino acids having the structure of Formula (XIX) are included:

[0411]

[0412] in:

[0413] R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; and

[0414] T3 is O or S.

[0415] Additionally, amino acids having the structure of formula (XX) are included:

[0416]

[0417] in:

[0418] R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0419] Additionally, the following amino acids having the structure of formula (XXI) are included:

[0420]

[0421] In some embodiments, the polypeptide comprising non-natural amino acids is chemically modified to produce a reactive carbonyl or dicarbonyl functional group. For example, an aldehyde functional group that can be used for a coupling reaction can be produced 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 normally exist or may be exposed by chemical or enzymatic digestion) can be used to produce an aldehyde functional group using periodate under mild oxidative cleavage conditions. See, for example, 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.

[0422] In the present invention, non-natural amino acids with adjacent hydroxyl and amino groups can be incorporated into polypeptides as "masked" aldehyde functional groups. For example, 5-hydroxylysine has a hydroxyl group adjacent to the epsilon amine group. The reaction conditions for generating the aldehyde typically 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 a 1.5-fold molar excess of sodium metaperiodate to a buffered solution of the polypeptide, followed by incubation in the dark for about 10 minutes. See, for example, U.S. Patent No. 6,423,685.

[0423] Carbonyl or dicarbonyl functional groups can react selectively with hydroxylamine-containing reagents under mild conditions in aqueous solution to form the corresponding oxime bonds that are stable under physiological conditions. See, for example, Jencks, WP, J. Am. Chem. Soc. 81, 475-481 (1959); Shao, J. and Tam, JP, J. Am. Chem. Soc. 117: 3893-3899 (1995). In addition, the unique reactivity of carbonyl or dicarbonyl groups allows for selective modification in the presence of other amino acid side chains. See, eg, Cornish, VW et al., J. Am. Chem. Soc. 118:8150-8151 (1996); Geoghegan, KF & Stroh, JG, Bioconjug. Chem. 3:138-146 (1992); Mahal, LK et al., Science 276:1125-1128 (1997).

[0424] A. Carbonyl Reactive Group

[0425] Amino acids with carbonyl reactive groups allow for a variety of reactions to attach molecules (including but not limited to PEG or other water-soluble molecules), particularly via nucleophilic addition or aldol condensation reactions.

[0426] Exemplary carbonyl-containing amino acids can be represented as follows:

[0427]

[0428] wherein 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 terminus modification group, and R4 is H, an amino acid, a polypeptide, or a carboxyl terminus modification group. In certain embodiments, n is 1, R1 is phenyl, and R2 is a simple alkyl group (i.e., methyl, ethyl, or propyl), and the ketone moiety is positioned 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 group (i.e., methyl, ethyl, or propyl), and the ketone moiety is positioned in the meta position relative to the alkyl side chain.

[0429] 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 similarly prepared by one of ordinary skill in the art.

[0430] In certain embodiments, the polypeptide comprising a non-naturally encoded amino acid is chemically modified to produce a reactive carbonyl functional group. For example, the aldehyde functional group for the coupling reaction can be produced from a functional group having adjacent amino and hydroxy groups. For example, in the case where the bioactive molecule is a polypeptide, an N-terminal serine or threonine (which may normally exist or may be exposed by chemical or enzymatic digestion) can be used to produce an aldehyde functional group using periodate under mild oxidative cleavage conditions. See, for example, 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 the amino acid at the N-terminus of the peptide or protein.

[0431] In the present invention, non-naturally encoded amino acids with adjacent hydroxyl and amino groups can be incorporated into polypeptides as "masked" aldehyde functional groups. For example, 5-hydroxylysine has a hydroxyl group adjacent to the epsilon amine group. The reaction conditions for producing aldehydes typically 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 includes adding about 1.5-fold molar excess of sodium metaperiodate to a buffered solution of the polypeptide, followed by incubation in the dark for about 10 minutes. See, for example, U.S. Patent No. 6,423,685, which is incorporated herein by reference.

[0432] 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, respectively, which are stable under physiological conditions. See, for example, Jencks, WP, J. Am. Chem. Soc. 81, 475-481 (1959); Shao, J. and Tam, JP, J. Am. Chem. Soc. 117: 3893-3899 (1995). In addition, the unique reactivity of the carbonyl group allows for selective modification in the presence of other amino acid side chains. See, for example, Cornish, VW et al., J. Am. Chem. Soc. 118:8150-8151 (1996); Geoghegan, KF & Stroh, JG, Bioconjug. Chem. 3:138-146 (1992); Mahal, LK et al., Science 276:1125-1128 (1997). B. Hydrazine, hydrazide or semicarbazide reactive groups

[0433] Non-naturally encoded amino acids containing nucleophilic groups such as hydrazine, hydrazide, or semicarbazide allow for reaction with a variety of electrophilic groups to form conjugates (including but not limited to conjugates with PEG or other water soluble polymers).

[0434] Exemplary hydrazine-, hydrazide-, or semicarbazide-containing amino acids can be represented as follows:

[0435]

[0436] wherein n is 0-10; R1 is alkyl, aryl, substituted alkyl or substituted aryl or is absent; X is O, N or S or is absent; R2 is H, an amino acid, a polypeptide or an amino terminus modification group, and R3 is H, an amino acid, a polypeptide or a carboxyl terminus modification group.

[0437] 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 located in the para position relative to the aliphatic group on the aryl ring.

[0438] 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 amino acids that are not commercially available can be prepared by one of ordinary skill in the art. See, for example, U.S. Patent No. 6,281,211, which is incorporated herein by reference.

[0439] Polypeptides containing non-naturally encoded amino acids with hydrazide, hydrazine, or semicarbazide functional groups can react efficiently and selectively with molecules containing aldehydes 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 toward aldehydes, ketones, and other electrophilic groups than the nucleophilic groups present on the 20 common amino acids (including but not limited to the hydroxyl group of serine or threonine or the amino group at the N-terminus of lysine).

[0440] C. Aminooxy-containing amino acids

[0441] Non-naturally encoded amino acids containing an aminooxy (also known as a hydroxylamine) group allow reaction with a variety of electrophilic groups to form conjugates (including but not limited to conjugates with PEG or other water-soluble polymers). Similar to hydrazines, hydrazides, and semicarbazides, the high nucleophilicity of the aminooxy group allows it to react efficiently and selectively with a variety of different molecules containing aldehydes 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 result of the reaction with a hydrazine group is the corresponding hydrazone, oximes are generally obtained from the reaction of the aminooxy group with a carbonyl-containing group such as a ketone.

[0442] Exemplary amino acids containing an aminooxy group can be represented as follows:

[0443]

[0444] wherein n is 0-10; R1 is alkyl, aryl, substituted alkyl, or substituted aryl, or is absent; X is O, N, S, or is absent; m is 0-10; Y═C(O) or is absent; R2 is H, an amino acid, a polypeptide, or an amino-terminal modification group, and R3 is H, an amino acid, a polypeptide, or a carboxyl-terminal modification 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.

[0445] 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 one of ordinary skill in the art.

[0446] D. Azide and Alkyne Reactive Groups

[0447] 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 commonly used 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 brought into close 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, JW et al., J. Am. Chem. Soc. 124:9026-9027 (2002).

[0448] Since the Huisgen cycloaddition reaction involves a selective cycloaddition reaction (see, for example, Padwa, A., Comprehensive Organic Synthesis, Vol. 4, edited by Trost, BM (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, thus the incorporation of non-naturally encoded amino acids with azide and alkynyl side chains allows the resulting polypeptide to be selectively modified at the position of the non-naturally encoded amino acid. Cycloaddition reactions involving azide- or alkynyl-containing IL-2s can be carried out in situ at room temperature and under aqueous conditions by adding a catalytic amount of Cu(II) (including but not limited to in the form of a catalytic amount 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, CW 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.

[0449] In some cases, when a Huisgen [3+2] cycloaddition reaction between an azide and an alkyne is desired, the IL-2 comprises a non-naturally encoded amino acid containing an alkyne moiety and a water-soluble polymer containing an azide moiety to be attached to the amino acid. Alternatively, the reverse reaction (i.e., using an azide moiety on the amino acid and an alkyne moiety present on the water-soluble polymer) can also be performed.

[0450] The azido functional group can also react selectively with a water-soluble polymer containing an aryl ester and suitably functionalized with an aryl phosphine moiety to produce an amide bond. The aryl phosphine group reduces the azido group in situ, and the resulting amine then reacts efficiently with the adjacent ester bond to produce the corresponding amide. See, for example, E. Saxon and C. Bertozzi, Science 287, 2007-2010 (2000). The azide-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).

[0451] An exemplary water-soluble polymer containing an aryl ester and a phosphine moiety can be represented as follows:

[0452]

[0453] 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 a compound of the invention includes more than one R group, for example, each of said R groups is independently selected, as are each of said R', R", R'", and R"" groups when more than one is present. When R' and R" are attached to the same nitrogen atom, they may be combined with said nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include, but not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the discussion of substituents above, one skilled in the art will understand that the term "alkyl" is meant to include 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.).

[0454] The azide functional group can also react selectively with water-soluble polymers containing a thioester and suitably functionalized with an arylphosphine moiety to produce an amide bond. The arylphosphine group reduces the azide group in situ, and the resulting amine then reacts efficiently with the thioester bond to produce the corresponding amide. An exemplary water-soluble polymer containing a thioester and a phosphine moiety can be represented as follows:

[0455]

[0456] wherein n is 1-10; X can be O, N, S or absent, Ph is phenyl, and W is a water-soluble polymer.

[0457] Exemplary alkyne-containing amino acids can be represented as follows:

[0458]

[0459] wherein n is 0-10; R1 is alkyl, aryl, substituted alkyl, or substituted aryl, or is absent; X is O, N, S, or is absent; m is 0-10, R2 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R3 is H, an amino acid, a polypeptide, or a carboxyl terminus modification group. In certain embodiments, n is 1, R1 is phenyl, X is absent, m is 0, and the acetylene moiety is positioned 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 moiety is positioned 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).

[0460] Alkynyl-containing amino acids are commercially available. For example, propargylglycine can be purchased commercially from Peptech (Burlington, MA). Alternatively, alkynyl-containing amino acids can be prepared according to standard methods. For example, p-propargyloxyphenylalanine can be synthesized, for example, as described 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 one of ordinary skill in the art.

[0461] Exemplary azide-containing amino acids can be represented as follows:

[0462]

[0463] wherein n is 0-10; R1 is alkyl, aryl, substituted alkyl, substituted aryl, or absent; X is O, N, S, or absent; m is 0-10; R2 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group. In certain embodiments, n is 1, R1 is phenyl, X is absent, m is 0, and the azido moiety is positioned in the para position relative to the alkyl side chain. In certain embodiments, n is 0-4, 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 positioned in the para position relative to the alkyl side chain.

[0464] 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 opening by a suitably protected lactone. See, for example, March's Advanced Organic Chemistry (3rd edition, 1985, Wiley and Sons, New York).

[0465] E. Aminothiol Reactive Groups

[0466] The unique reactivity of β-substituted aminothiol functional groups makes them extremely useful for the selective modification of polypeptides and other biomolecules containing aldehyde groups via thiazolidine formation. 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 IL-2 polypeptides and then reacted with water-soluble polymers containing aldehyde functional groups. In certain embodiments, water-soluble polymers, drug conjugates, or other payloads can be coupled to IL-2 containing β-substituted aminothiol amino acids via thiazolidine formation.

[0467] F. Additional Reactive Groups

[0468] Additional reactive groups and non-naturally encoded amino acids that can be incorporated into the IL-2 polypeptides of the invention, including but not limited to para-amino-phenylalanine, are described in the following patent applications, all of which are incorporated herein by reference in their entireties: 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 (aminooxy) groups.

[0469] Peptides with unnatural amino acids

[0470] The incorporation of unnatural amino acids can be performed for a variety of 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 to protease target sites, targeting components (including but not limited to use in 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 that include unnatural amino acids can have enhanced or even completely new catalytic or biophysical properties. For example, by including unnatural amino acids in proteins, the following properties can be optionally modified: receptor binding, toxicity, biodistribution, structural properties, spectroscopic 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 comprising proteins comprising at least one unnatural amino acid can be used for, but are not limited to, new therapeutic agents, diagnostic agents, catalytic enzymes, industrial enzymes, binding proteins (including but not limited to antibodies), and include but are not limited to research on protein structure and function. See, for example, Dougherty, Unnatural Amino Acids as Probes of Protein Structure and Function, Current Opinion in Chemical Biology, 4: 645-652 (2000).

[0471] In one aspect of the invention, the composition includes at least one protein having at least one, 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 can be the same or different, including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different sites in the protein containing 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different non-natural amino acids. On the other hand, the composition includes proteins in which at least one but less than all of the specific amino acids present in the protein are replaced by non-natural amino acids. For a given protein with more than one non-natural amino acid, the non-natural amino acids can be consistent or different (including but not limited to the protein can include two or more different types of non-natural amino acids, or can include two identical non-natural amino acids). For a given protein with more than two non-natural amino acids, the non-natural amino acids can be the same, different or a combination of multiple non-natural amino acids of the same type and at least one different non-natural amino acid.

[0472] Proteins or polypeptides of interest having at least one unnatural amino acid are a feature of the present invention. The present invention also includes polypeptides or proteins having at least one unnatural 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.

[0473] By producing a protein or polypeptide of interest with at least one unnatural amino acid in a eukaryotic cell, the protein or polypeptide will typically include a eukaryotic post-translational modification. In certain embodiments, the protein includes at least one unnatural amino acid and at least one post-translational modification produced in vivo by a eukaryotic cell, wherein the post-translational modification is not produced 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, and the like.

[0474] An advantage of non-natural amino acids is that they provide additional chemical building blocks that can be used to add other molecules. These modifications can be made in eukaryotic or non-eukaryotic cells or in vitro. Therefore, in certain embodiments, the post-translational modification is achieved by the non-natural amino acids. For example, the post-translational modification can be achieved by nucleophilic-electrophile reactions. Most reactions currently used for the selective modification of proteins involve covalent bond formation between nucleophilic and electrophilic reaction partners, including but not limited to the reaction of α-haloketones with histidine or cysteine ​​side chains. In these cases, 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 non-natural keto-amino acids with hydrazides or aminooxy compounds. 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., Pr . 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 wide range 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 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).

[0475] IV. In Vivo Production of IL-2 Comprising Non-Naturally Encoded Amino Acids

[0476] The IL-2 polypeptides of the present invention can be produced in vivo using modified tRNAs and tRNA synthetases to add or substitute amino acids that are not encoded in naturally occurring systems.

[0477] The production method of tRNA and tRNA synthetase using amino acids that are not encoded in naturally occurring systems is described in, for example, U.S. Patent Nos. 7,045,337 and 7,083,970, which are incorporated herein by reference. These methods relate to the production of (and therefore sometimes referred to as "orthogonal") translation machinery that does not rely on the endogenous synthetase and tRNA of the translation system. Typically, the translation system comprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl-tRNA synthetase (O-RS). Typically, the O-RS preferentially aminoacylates the O-tRNA with at least one non-natural 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. Therefore, the translation system responds to the selector codon of the encoding, inserts the non-naturally encoded amino acid into the protein produced in the system, thereby "replacing" the amino acid in the position in the encoded polypeptide.

[0478] 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 amino acid sequences disclosed in U.S. Patent Nos. 7,045,337 and 7,083,970, each of which is incorporated herein by reference. Corresponding O-tRNA molecules used with the O-RSs 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.

[0479] Examples of azide-specific O-tRNA / aminoacyl-tRNA synthetase systems are described in Chin, JW et al., J. Am. Chem. Soc. 124:9026-9027 (2002). Exemplary O-RS sequences for p-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. Pat. No. 7,083,970, 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. Pat. No. 7,083,970, 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. Pat. No. 7,045,337, incorporated herein by reference. O-RS and O-tRNA that incorporate both keto- and azido-containing amino acids in S. cerevisiae are described in Chin, JW et al., Science 301:964-967 (2003).

[0480] Several other orthogonal pairs have been reported. Glutaminyl (see, e.g., Liu, DR, and Schultz, PG (1999) Proc. Natl. Acad. Sci. USA 96:4780-4785), aspartyl (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, AK, et al. (2001) Proc. Natl. Acad. Sci. USA 98:2268-2273) systems derived from Saccharomyces cerevisiae tRNA and synthetase have been described for potential incorporation of unnatural amino acids in E. coli. Systems derived from E. coli glutaminyl (see, e.g., Kowal, A.K., et al., (2001) Proc. Natl. Acad. Sci. USA 98:2268-2273) and tyrosyl (see, e.g., Edwards, H. and Schimmel, P. (1990) Mol. Cell. Biol. 10:1633-1641) synthetases have been described for use in Saccharomyces cerevisiae. The E. coli tyrosyl system has been used to incorporate 3-iodo-L-tyrosine in vivo in mammalian cells. See Sakamoto, K., et al., (2002) Nucleic Acids Res. 30:4692-4699.

[0481] The use of O-tRNA / aminoacyl-tRNA synthetase involves the selection of a specific codon (selector codon) encoding a non-naturally 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 expressing the O-tRNA / aminoacyl-tRNA synthetase. For example, exemplary codons include nonsense codons such as stop codons (amber, ochre, and oval), four or more base codons, and other rare or unused natural three-base codons.

[0482] Specific selector codons can be introduced into appropriate positions in the IL-2 coding sequence using mutagenesis methods known in the art (including but not limited to site-specific mutagenesis, cassette mutagenesis, restriction-selection mutagenesis, etc.).

[0483] V. Position of non-naturally occurring amino acids in IL-2

[0484] 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 that do not disrupt the activity of the polypeptide. This can be achieved by making "conservative" substitutions, including but not limited to replacing a hydrophobic amino acid with a hydrophobic amino acid, replacing a bulky amino acid with a bulky amino acid, replacing a hydrophilic amino acid with a hydrophilic amino acid, and / or inserting the non-naturally occurring amino acid into a position that is not required for activity.

[0485] A variety of biochemical and structural methods can be used to select 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 for incorporation of non-naturally encoded amino acids, and selection can be based on rational design or by random selection, for any or no specific desired purpose. The selection of desired sites can be used to generate IL-2 molecules with any desired properties or activities, including but not limited to modulation of 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, without altering activity or properties relative to the native molecule, or manipulating any physical or chemical properties of the polypeptide, such as solubility, aggregation, or stability. For example, positions in the polypeptide required for the biological activity of IL-2 can be identified using point mutation analysis, alanine scanning, saturation mutagenesis, and bioactivity screening or homology scanning methods known in the art. Other methods can be used to identify residues for modification of IL-2, including but not limited to sequence dissection (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, Protein Science 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 In some embodiments, the present invention provides a method for the rational design of the present invention (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 residues identified as key to biological activity by alanine or homology 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 key to biological activity may also be good candidates for replacement with non-naturally encoded amino acids, 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 in each position on the polypeptide chain and observe the effect on the activity of the polypeptide. It is obvious to those of ordinary skill in the art that any means, techniques or methods for selecting positions for the replacement of non-natural amino acids in any polypeptide are suitable for use in the present invention.

[0486] The structure and activity of IL-2 polypeptide mutants containing deletions can also be examined to determine regions of the protein that may be resistant to substitution 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 may be resistant to substitution with non-naturally encoded amino acids have been eliminated, the effect of the proposed substitution at each remaining position can be examined. Thus, one of ordinary skill in the art can readily identify amino acid positions that can be substituted with non-naturally encoded amino acids.

[0487] One of ordinary skill in the art will recognize that such analysis of IL-2 can determine which amino acid residues are surface exposed compared to those buried within the tertiary structure of the protein. Thus, an embodiment of the present invention is to replace amino acids that are surface exposed residues with non-naturally encoded amino acids.

[0488] 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, 100 124, 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: 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 ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5, or 7).

[0489] In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following positions: before position 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).

[0490] In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at any position in one or more of the following regions corresponding to secondary structure or specific amino acids as described below: at a site of hydrophobic interaction; at or near a site of interaction with an IL-2 receptor subunit (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 of which is a position in SEQ ID NO: 2 or the corresponding amino acid positions 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 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, and any combination thereof of 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 at 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, 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 3, 5 or 7; or the corresponding amino acids in SEQ ID NO: 3, 5 or 7.

[0491] In certain embodiments, the IL-2 polypeptide is an agonist and the non-naturally occurring amino acids in one or more of these regions are linked 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 the non-naturally occurring amino acids in one or more of these regions are linked 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.

[0492] A wide variety of non-naturally encoded amino acids can be substituted or incorporated into a given position in IL-2. Typically, the specific non-naturally encoded amino acid for incorporation is selected based on an examination of the three-dimensional crystal structure of the IL-2 polypeptide or other IL-2 family member and its receptor, a preference for conservative substitutions (i.e., aryl-based non-naturally encoded amino acids such as p-acetylphenylalanine or O-propargyltyrosine for Phe, Tyr, or Trp), and the specific 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 alkynyl moiety or an amide bond formation with a water-soluble polymer bearing an aryl ester and thus incorporating a phosphine moiety).

[0493] In one embodiment, the method further comprises: 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 photocrosslinker, 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 analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, 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 , radioactive moieties, novel functional groups, groups that covalently or non-covalently interact with other molecules, photocaged moieties, actinic radiation excitable moieties, photoisomerizable moieties, biotin, derivatives of biotin, biotin analogs, moieties incorporated with heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox-active agents, amino thioacids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chemiluminescent groups, electron dense groups, magnetic groups, intercalating groups, chromophores, energy transfer agents, biologically active agents, detectable labels, small molecules, quantum dots, nanoemitters, radionucleotides, radioemitters, neutron capture agents, or any combination of the foregoing or any other desired compound or substance). The first reactive group reacts with the second reactive group via a [3+2] cycloaddition to attach the molecule to the non-natural amino acid. In one embodiment, the first reactive group is an alkynyl or azido moiety and the second reactive group is an azido or alkynyl moiety. For example, the first reactive group is an alkynyl moiety (including but not limited to, in the unnatural amino acid p-propargyloxyphenylalanine), and the second reactive group is an azido moiety. In another example, the first reactive group is an azido moiety (including but not limited to, in the unnatural amino acid p-azido-L-pheny...

Claims

1. 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 coupled to the one or more PEG molecules via the non-naturally encoded amino acid incorporated into the polypeptide.

2. The modified IL-2 polypeptide of claim 1, wherein the non-naturally encoded amino acid is incorporated at position 45.

3. The modified IL-2 polypeptide according to claim 2, optionally comprising one or more amino acid substitutions at selected positions within SEQ ID NO:

2.

4. The modified IL-2 polypeptide of claim 1 or 2, wherein the non-naturally encoded amino acid is p-acetylphenylalanine, p-nitrophenylalanine, p-sulfotyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-boronophenylalanine, o-boronophenylalanine, m-boronophenylalanine, 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-aminotyr amino acid, 2-aminotyrosine, o-sulfotyrosine, 2-sulfoxyphenylalanine, 3-sulfophenylalanine, 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, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propargyloxy-L-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine, and p-azidomethyl-phenylalanine.

5. The modified IL-2 polypeptide of claim 1 or 2, wherein the non-naturally encoded amino acid is p-acetylphenylalanine.

6. The modified IL-2 polypeptide according to claim 1 or 3, wherein the one or more amino acid substitutions are at positions 38 and / or 65 of SEQ ID NO:

2.

7. The modified IL-2 polypeptide according to claim 1 or 3, wherein the amino acid substitution at position 38 is alanine.

8. The modified IL-2 polypeptide according to claim 1 or 3, wherein the amino acid substitution at position 65 is arginine.

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

10. The modified IL-2 polypeptide of 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, and 50 kDa.

11. The modified IL-2 polypeptide of claim 1, wherein the one or more PEG molecules are 30 kDa.

12. The modified IL-2 polypeptide of claim 1, wherein the one or more PEG molecules are 40 kDa.

13. A method of treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of the modified IL-2 polypeptide according to any one of claims 1 to 12.

14. The method of claim 13, wherein the cancer is breast cancer, small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, gastroenteropancreatic tumor, cervical cancer, esophageal cancer, colon cancer, colorectal cancer, a cancer or tumor of epithelial origin, kidney cancer, brain cancer, glioblastoma, pancreatic cancer, thyroid cancer, endometrial cancer, pancreatic cancer, head and neck cancer, or skin cancer.

15. The method of claim 13, further comprising administering a therapeutic agent.

16. The method of claim 15, wherein the therapeutic agent is a chemotherapeutic agent, a hormonal agent, an anti-tumor agent, an immunostimulatory agent, an immunotherapeutic agent, or a combination thereof.

17. Use of a modified IL-2 polypeptide according to any one of the preceding claims in the preparation of a medicament.

18. A pharmaceutical composition comprising a therapeutically effective amount of IL-2 according to any one of the preceding claims and a pharmaceutically acceptable carrier or excipient.

19. An IL-2 polypeptide of SEQ ID NOs: 9 or 11.

20. A glycosylated IL-2 polypeptide according to any one of the preceding claims.

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