HK40074343A - Interleukin-2 polypeptide conjugates and methods of use thereof - Google Patents

Description

Interleukin-2 polypeptide conjugates and methods of use thereof

Cross-referencing

This application claims the benefit of U.S. provisional application No. 62/987,872, filed on 11/3/2020, the contents of which are incorporated herein by reference in their entirety.

Sequence listing

This application contains a sequence listing filed in ASCII format via EFS-Web and incorporated herein by reference in its entirety. The ASCII copy was made at 3 months and 3 days 2021, named AMBX _0232_00PCT _ST25.Txt, and was 27,704 bytes in size.

Technical Field

Embodiments of the present disclosure relate at least to the fields of immunotherapy, immunooncology, and cancer therapy. More specifically, the disclosure relates to interleukin-2 (IL-2) conjugates and their uses.

Background

Cancer is one of the most important health disorders. Cancer has the second largest mortality rate in the united states, second to heart disease, accounting for one-fourth of deaths. It is generally expected that the incidence of cancer will increase with the aging population of the united states, further exacerbating the effects of this condition. The current therapeutic regimens for cancer established in the 1970 s and 1980 s have not changed much. When used in most advanced stage common cancers, these therapies, including chemotherapy, radiation, and others (including newer targeted therapies), show limited overall survival benefit because these therapies target primarily the tumor mass.

More specifically, conventional cancer diagnostics and therapies have heretofore attempted to selectively detect and eradicate neoplastic cells (i.e., cells that form tumor masses) that are predominantly fast-growing. Standard oncological treatment protocols are generally designed primarily to administer the highest dose of radiation or chemotherapeutic agent without undue toxicity, i.e., what is commonly referred to as the "maximum tolerated dose" (MTD) or "no adverse effect observed 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 cellular mechanisms involved in cell growth and DNA replication. Chemotherapy regimens also typically involve administration of a combination of chemotherapeutic agents in an attempt to improve the efficacy of the treatment. Despite the availability of a large number of different chemotherapeutic agents, these therapies suffer from a number of disadvantages. For example, chemotherapeutic agents are well known to be toxic due to non-specific side effects on rapidly growing cells, whether normal or malignant; for example, chemotherapeutic agents cause significant and often dangerous side effects, including myelosuppression, immunosuppression, and gastrointestinal discomfort, among others.

Cancer stem cells

Cancer stem cells comprise a unique sub-population of tumors (typically around 0.1-10%), which are more tumorigenic, grow relatively slower or silent relative to the remaining 90% or so of tumors (and tumor mass), and are typically relatively more chemoresistant than tumor masses. Given that conventional therapies and protocols are largely designed to attack rapidly proliferating cells (i.e., those cancer cells that make up the tumor mass), cancer stem cells that are generally slow growing compared to rapidly growing tumor masses may be relatively more resistant to conventional therapies and protocols. Cancer stem cells may exhibit other characteristics that make them relatively more chemotherapy-resistant, such as multidrug resistance and anti-apoptotic pathways. The above factors constitute a key reason why standard tumor treatment regimens fail to ensure long-term benefit, i.e., fail to adequately target and eradicate cancer stem cells, in most patients with advanced cancer. In some cases, the cancer stem cell is a generating cell of a tumor (i.e., it is an ancestor of the cancer cells that make up the tumor mass).

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, having alanine-1 removed and the residue cysteine-125 replaced with serine-125 (Whittington et al, 1993). Although it is a matter of courseIL-2 is the earliest FDA-approved cytokine in cancer therapy, but IL-2 has been shown to exhibit severe side effects when used at high doses. This greatly limits its application to potential patients. The potential mechanism for such severe side effects has been attributed to the binding of IL-2 to one of its receptors, IL-2R α. In general, IL-2 can form heterotrimeric complexes not only with its receptors, including IL-2R α (or CD 25), IL-2R β (or CD 122), and IL-2R γ (or CD 132) (when all three receptors are present in tissue), but also with IL-2R β and IL-2R γ. In the clinical setting, when high doses of IL-2 are used, IL-2 begins to bind IL-2. Alpha. Beta.gamma., which is Tp _reg The major receptor form in cells. T is a unit of _reg The inhibitory effect of the cells causes an unwanted effect of the application of IL-2 in cancer immunotherapy. To alleviate the side effects of IL-2, a number of approaches have been used in the art. For example, one form of IL-2 manufactured by Nektar uses 6 PEGylated lysines to mask the IL2R α binding region on the surface of IL-2 (Charych et al, 2016). This pegylated form of IL-2 has an extended half-life, comprises a mixture of mono-and multi-pegylated forms, and contains a very high amount of PEG, and exhibits improved side effects. However, results from activity studies show that the pegylated IL-2 forms that are effective in this heterogeneous 6-pegylated IL-2 mixture are only mono-pegylated forms. Thus, there is a need for more efficient pegylated IL-2 with a homogenous, well-defined product composition that modulates the side effects of IL-2.

The ability to incorporate non-genetically encoded amino acids into proteins allows the introduction of epsilon-NH which may be a naturally occurring functional group such as lysine ₂ thiol-SH of cysteine, imino of histidine, etc. provide valuable alternatives to chemical functionalities. 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 azido and ethynyl groups undergo Huisgen [3+2 in the presence of catalytic amounts of copper under aqueous conditions]And (3) performing cycloaddition reaction. See, e.g., tornoe et al, (2002) J.org.chem.67:3057-3064; and Rostovtsev et al, (2002) Angew. Chem. Int. Ed.41:2596-2599. By introducing, for example, an azido moiety into a protein structure, one can incorporate a functional group that is chemically inert to amines, sulfhydryls, carboxylic acids, hydroxyl groups present in the protein, but that reacts smoothly and efficiently with an acetylene moiety to form a cycloaddition product. Importantly, in the absence of an acetylene moiety, the azide remains chemically inert and non-reactive in the presence of other protein side chains and under physiological conditions.

The present invention addresses, among other problems, the problems associated with the activity and production of IL-2 polypeptide conjugates, and also the production of IL-2 polypeptides with improved biological or pharmacological properties, such as increased activity on tumors and/or improved conjugation and/or improved therapeutic half-life. The IL-2 polypeptides of the invention target both Treg cells known to express trimeric IL-2 receptors (α, β and γ) and CD8 cells that predominantly express β and γ dimers of the IL-2 receptor. The IL-2 polypeptides of the invention reduce binding to the alpha receptor of Treg cells and promote biased binding to the beta and gamma dimers of CD8 cells, thereby providing improved therapeutic applications and improved prognosis for diseases or conditions in which IL-2 receptor alpha is highly expressed.

Disclosure of Invention

In certain embodiments, the present disclosure provides a modified IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO:2, and comprises: a non-naturally encoded amino acid incorporated at position 42; in SEQ ID NO:2 at a selected position; and one or more PEG molecules; wherein the polypeptide is coupled to the one or more PEG molecules through the non-naturally encoded amino acid incorporated into the polypeptide. In certain embodiments, the present disclosure provides a modified IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO:2, and comprises: a non-naturally encoded amino acid incorporated at position 45; in SEQ ID NO:2 at a selected position; and one or more PEG molecules; wherein the polypeptide is coupled to the one or more PEG molecules through the non-naturally encoded amino acid incorporated into the polypeptide. In certain embodiments, the modified IL-2 polypeptide is comprised within a polypeptide corresponding to SEQ ID NO:2 at position 42 of the amino acid sequence of seq id No. 2. In certain embodiments, the modified IL-2 polypeptide is comprised in SEQ ID NO:2 at position 45 of the amino acid sequence. In certain embodiments, the present invention provides a modified IL-2 polypeptide comprising SEQ ID NO:2, and comprises: a non-naturally encoded amino acid incorporated at position 42; one or more PEG molecules; and optionally the amino acid sequence set forth in SEQ ID NO:2 at a selected position; wherein the polypeptide is coupled to the one or more PEG molecules through the non-naturally encoded amino acid incorporated into the polypeptide. In certain embodiments, the present invention provides a modified IL-2 polypeptide comprising SEQ ID NO:2, and comprises: a non-naturally encoded amino acid incorporated at position 45; one or more PEG molecules; and optionally the sequence set forth in SEQ ID NO:2 at a selected position; wherein the polypeptide is coupled to the one or more PEG molecules through the non-naturally encoded amino acid incorporated into the polypeptide. In certain embodiments, the modified IL-2 polypeptides of the invention are optionally substituted at the amino acid sequence of SEQ ID NO:2 comprises one or more amino acid substitutions at selected positions.

In certain embodiments, the modified IL-2 polypeptide comprises a non-naturally encoded amino acid, selected from the group consisting of para-acetylphenylalanine, para-nitrophenylalanine, para-sulfotyrosine, para-carboxyphenylalanine, ortho-nitrophenylalanine, meta-nitrophenylalanine, para-boronophenylalanine, ortho-boronophenylalanine, meta-boronophenylalanine, para-aminophenylalanine, ortho-aminophenylalanine, meta-aminophenylalanine, ortho-acylphenylalanine, meta-OMe-phenylalanine, p-OMe-phenylalanine, O-OMe-phenylalanine, m-OMe-phenylalanine, para-sulfophenylalanine, ortho-sulfophenylalanine, meta-sulfophenylalanine, 5-nitroHis, 3-nitroTyr, 2-nitroTyr, nitro-substituted Leu, and Leu nitro-substituted His, nitro-substituted De, nitro-substituted Trp, 2-nitroTrp, 4-nitroTrp, 5-nitroTrp, 6-nitroTrp, 7-nitroTrp, 3-aminotyrosine, 2-aminotyrosine, O-sulfotyrosine, 2-sulfooxyphenylalanine, 3-sulfooxyphenylalanine, 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-GlcNAc beta-serine, L-dopa beta-serine, fluorophenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propargyloxy-L-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine and p-azidomethyl-phenylalanine. In certain embodiments, the non-naturally encoded amino acid is para-acetylphenylalanine.

In certain embodiments, the modified IL-2 polypeptide is as set forth in SEQ ID NO:2 comprising one or more amino acid substitutions at positions R38 and P65. In certain embodiments, the modified IL-2 polypeptide is as set forth in SEQ ID NO:2 comprises one or more amino acid substitutions at positions 38 and 65. In certain embodiments, the modified IL-2 polypeptide is as set forth in SEQ ID NO:2 at position 38 or 65 comprising one or more amino acid substitutions. In certain embodiments, the modified IL-2 polypeptide is as set forth in SEQ ID NO:2 comprises one or more amino acid substitutions at position 38. In certain embodiments, the modified IL-2 polypeptide is as set forth in SEQ ID NO:2 comprises one or more amino acid substitutions at position 65. In certain embodiments, the polypeptide set forth in SEQ ID NO:2 is an alanine to amino acid substitution.

In certain embodiments, the modified IL-2 polypeptide comprises one or more PEG molecules, wherein the one or more PEG molecules are linear or branched or multi-armed. In certain embodiments, the one or more PEG molecules are linear. In certain embodiments, the one or more PEG molecules are branched. In certain embodiments, the one or more PEG molecules are multi-armed. In certain embodiments, the one or more PEG molecules have an average molecular weight of 5kDa, an average molecular weight of 10kDa, an average molecular weight of 15kDa, an average molecular weight of 20kDa, an average molecular weight of 25kDa, an average molecular weight of 30kDa, an average molecular weight of 35kDa, an average molecular weight of 40kDa, an average molecular weight of 45kDa, and an average molecular weight of 50kDa or greater. In certain embodiments, the one or more PEG molecules are 30kDa. In certain embodiments, the one or more PEG molecules is 40kDa. In certain embodiments, the one or more PEG molecules are linear 30kDa PEG molecules. In certain embodiments, the one or more PEG molecules are branched 30kDa PEG molecules. In certain embodiments, the one or more PEG molecules are linear 40kDa PEG molecules. In certain embodiments, the one or more PEG molecules are branched 40kDa PEG molecules. In certain embodiments, the modified IL-2 polypeptides of the invention comprise SEQ ID NO:2 comprising a site-specifically incorporated non-naturally encoded amino acid, a sequence set forth in SEQ ID NO:2 and one or more PEG molecules coupled through the site-specifically incorporated non-naturally encoded amino acid. In certain embodiments, the modified IL-2 polypeptides of the invention comprise SEQ ID NO:2 comprising a site-specifically incorporated non-naturally encoded amino acid and one or more PEG molecules coupled through 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-specifically incorporated non-naturally encoded amino acid is SEQ ID NO:10. in certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO:11. in certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO:12. in certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO:13. in certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO:14. in certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO:15. in certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO:16. in certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO:17. in certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO:18. in certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO:19. in certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO:20. in certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO:21. in certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO:22. in certain embodiments, the modified IL-2 polypeptide comprising a site-specifically incorporated non-naturally encoded amino acid is SEQ ID NO:23.

In certain embodiments, the invention relates to interleukin-2 (IL-2) polypeptides comprising one or more non-naturally encoded amino acids. In certain embodiments, the invention provides IL-2 polypeptide conjugates comprising one or more non-naturally encoded amino acids. In certain embodiments, the invention provides IL-2 polypeptide conjugates in which a water-soluble polymer, such as PEG, is coupled to an IL-2 variant via one or more non-naturally encoded amino acids within the IL-2 variant. In certain embodiments, the invention provides IL-2 polypeptide conjugates having one or more non-naturally encoded amino acid substitutions and one or more natural amino acid substitutions. In certain embodiments, the invention provides IL-2 polypeptide conjugates having one or more non-naturally encoded amino acids 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.

In one embodiment, the PEG-IL-2 is monopegylated. In one embodiment, the PEG-IL-2 is pegylated. In one embodiment, the PEG-IL-2 has more than two (2) polyethylene glycol molecules attached to it. Another embodiment of the invention provides the use of the PEG-IL-2 polypeptides of the invention to modulate the activity of cells of the immune system.

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

In PEG-IL2 conjugates, the PEG or other water-soluble polymer may be conjugated to the IL-2 protein or the bioactive molecule directly or through a linker. Suitable linkers include, for example, cleavable and non-cleavable linkers.

The present invention provides a method of treating cancer in a mammal, such as a mammal including but not limited to those having one or more of the following conditions, by administering an effective amount of a PEG-IL-2 polypeptide: solid tumors, hematologic 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, gastrointestinal pancreatic tumor, cervical cancer, esophageal cancer, colorectal cancer, cancer or tumor of epithelial origin, renal 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 invention provides a method of treating cancer or a disorder or disease by administering to a subject an effective amount of a composition comprising an IL-2 polypeptide of the invention. In certain embodiments, the invention provides a method of treating a genetic disorder by administering to a patient an effective amount of an IL-2 composition of the invention. In certain embodiments, the disorder or disease is characterized by high expression of IL-2 receptor alpha. In certain embodiments, the disorder or disease is characterized by high levels of Treg cells. In certain embodiments, the cancer, disorder or disease is treated by reducing, blocking or silencing expression of IL-2 receptor alpha. In certain embodiments, the cancer, disorder or disease is treated by reducing binding of IL-2 receptor alpha on the surface of Treg cells, resulting in a reduction in Treg cell proliferation in the cancer, disorder or disease to be treated.

As used herein, interleukin 2 or IL-2 is defined as a protein having the following properties: (a) Has an amino acid sequence substantially identical to known sequences of IL-2 (including IL-2 muteins, mature IL-2 sequences (i.e., lacking a secretory leader sequence) and IL-2 as disclosed in SEQ ID NOs: 1, 2, 3, 5, or 7 of the present application), and (b) has at least one biological activity common to native or wild-type IL-2. For the purposes of the present invention, both glycosylated (e.g., produced in eukaryotic cells such as yeast or CHO cells) and non-glycosylated (e.g., chemically synthesized or produced in e. Other mutants and other analogs that retain the biological activity of IL-2, including viral IL-2, are also included.

The present invention provides IL-2 polypeptides conjugated to one or more water-soluble polymers through one or more non-naturally encoded amino acids incorporated into the polypeptide. The present invention provides an IL-2 polypeptide 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 invention also provides monomers and dimers of the IL-2 polypeptides. The invention also provides trimers of IL-2 polypeptides. The present invention provides multimers of IL-2 polypeptides. The invention also provides IL-2 dimers comprising one or more non-naturally encoded amino acids. The invention provides IL-2 multimers comprising one or more non-naturally encoded amino acids. The 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 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 can have a different amino acid sequence.

In certain embodiments, the IL-2 polypeptide comprises one or more post-translational modifications. In certain embodiments, the IL-2 polypeptide is linked to a linker, polymer, or biologically active 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 coupled to a water-soluble polymer. In certain embodiments, the IL-2 multimer is coupled to two water-soluble polymers. In certain embodiments, the IL-2 multimer is coupled to three water-soluble polymers. In certain embodiments, the IL-2 multimer is coupled to more than three water-soluble polymers. In certain embodiments, the IL-2 polypeptide is linked to a linker that is long enough to allow dimer formation. In certain embodiments, the IL-2 polypeptide is linked to a linker that is long enough to allow trimer formation. In certain embodiments, the IL-2 polypeptide is linked to a linker that is long enough to allow multimer formation. In certain embodiments, the IL-2 polypeptide is linked to a bifunctional polymer, bifunctional linker, or at least one additional IL-2 polypeptide. In certain embodiments, the IL-2 polypeptide comprises one or more post-translational modifications. In certain embodiments, the IL-2 polypeptide is linked to a linker, polymer, or biologically active molecule.

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

In certain embodiments, the IL-2 or PEG-IL-2 of the invention is linked to a therapeutic agent, such as an immunomodulator. The immunomodulator may be any agent that exerts a therapeutic effect on immune cells, which may be used as a therapeutic agent for coupling to IL-2, PEG-IL-2 or IL-2 variants. In certain embodiments, the IL-2 or PEG-IL-2 of the invention is linked to a therapeutic agent, such as a cytokine, a chemotherapeutic agent, an immunotherapeutic agent, a hormonal agent, an antineoplastic agent, an immunostimulatory agent, or a combination thereof.

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

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

In certain embodiments, one or more non-naturally encoded amino acids are incorporated at any position in one or more of the following regions corresponding to secondary structure or specific amino acids as described below in IL-2 or variants thereof: at the site of hydrophobic interaction; at or near the site of interaction with the IL-2 receptor subunit (including IL2 ra); within amino acid positions 3 or 35 to 45; within the first 107N-terminal amino acids; within amino acid positions 61-72; each of said positions is SEQ ID NO:2 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: the amino acid sequence of SEQ ID NO:2 prior to bit 1 (i.e., at the N-terminus), bits 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, and any combination thereof; or 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: SEQ ID NO:2, position 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or to the carboxy terminus of the protein, and any combination thereof; or 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: <xnotran> 1 ( N- ), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133 , , (SEQ ID NO:2, SEQ ID NO:3, 5 7 ). </xnotran>

In certain embodiments, the non-naturally occurring amino acid at one or more of these positions in IL-2 or a variant thereof, including but not limited to at the site of hydrophobic interaction, at or near the site of interaction with an IL-2 receptor subunit (including IL2 ra), within amino acid positions 3 or 35 to 45, within the first 107N-terminal amino acids, within amino acid positions 61-72; each of said positions is SEQ ID NO:2 or 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: SEQ ID NO:2 (i.e., at the N-terminus), bits 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, and any combination thereof; or 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 of IL-2 or a variant thereof: SEQ ID NO:2, position 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or to the terminal end of the protein, and any combination thereof; 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 in one or more of the following positions and linked to a drug or other biologically active molecule: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72 and 107, and any combination thereof (SEQ ID NO:2, or the corresponding amino acid 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 linker at positions including, but not limited to, the following: <xnotran> 1 ( N- ), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133 , , (SEQ ID NO:2, SEQ ID NO:3, 5 7 ). </xnotran> In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof in one or more of the following positions and linked to a linker: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72 and 107, and any combination thereof (SEQ ID NO:2, or the corresponding amino acid 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 linker that is further linked to a water soluble polymer or a biologically active molecule, including but not limited to the following positions: <xnotran> 1 ( N- ), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133 , , (SEQ ID NO:2, SEQ ID NO:3, 5 7 ). </xnotran> In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof in one or more of the following positions and linked to a linker that is further linked to a water soluble polymer or biologically active molecule, including but not limited to the following positions: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72 and 107, and any combination thereof (SEQ ID NO:2, or the corresponding amino acid 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 water-soluble polymer, including, but not limited to, the following positions: <xnotran> 1 ( N- ), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133 , , (SEQ ID NO:2, SEQ ID NO:3, 5 7 ). </xnotran> In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof in one or more of the following positions and linked to a linker that is further linked to a water soluble polymer, including but not limited to the following positions: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72 and 107, and any combination thereof (SEQ ID NO:2, or the corresponding amino acid in SEQ ID NO:3, 5 or 7). In certain embodiments, the present disclosure provides a polypeptide corresponding to SEQ ID Nos: 9-23.

In certain embodiments, the IL-2 or variant thereof comprises a substitution, addition, or deletion that modulates the affinity of the IL-2 for an IL-2 receptor subunit or variant thereof. In certain embodiments, the IL-2 or variant thereof comprises a substitution, addition, or deletion that modulates the affinity of the IL-2 or variant thereof for an IL-2 receptor or binding partner (including but not limited to a protein, polypeptide, lipid, fatty acid, small molecule, or nucleic acid). In certain embodiments, the IL-2 or variant thereof comprises a substitution, addition, or deletion that modulates the stability of the IL-2 as compared to the stability of the corresponding IL-2 without the substitution, addition, or deletion. Stability and/or solubility can be measured using a 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 as compared to the immunogenicity of a corresponding IL-2 that does not comprise the substitution, addition, or deletion. In certain embodiments, the IL-2 comprises a substitution, addition, or deletion that modulates the serum half-life or circulation time of the IL-2 as compared to the serum half-life or circulation time of the corresponding IL-2 without the substitution, addition, or deletion.

In certain embodiments, the IL-2 or variant thereof comprises a substitution, addition, or deletion that increases the water solubility of the IL-2 as 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 the host cell as 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 synthesis in vitro as 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 the 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 as compared to the protease resistance of the corresponding IL-2 or variant thereof that does not comprise the substitution, addition, or deletion. In certain embodiments, the IL-2 or variant thereof comprises a substitution, addition, or deletion that modulates the signaling activity of the IL-2 receptor as compared to the activity of the IL-2 receptor following interaction with a corresponding IL-2 or variant thereof that does not comprise 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, as compared to the binding of the corresponding IL-2 without the substitution, addition, or deletion.

In certain embodiments, the invention provides methods of treating a proliferative disorder, cancer, tumor, or precancerous condition, such as abnormal hyperplasia, using PEG-IL-2 and at least one additional therapeutic or diagnostic agent. The additional therapeutic agent may be, for example, a cytokine or cytokine antagonist such as IL-12, interferon-alpha or anti-epidermal growth factor receptor antibody, doxorubicin, epirubicin, an antifolate such as methotrexate or fluorouracil, irinotecan, cyclophosphamide, radiation therapy, a hormone or anti-hormone therapy such as androgen, estrogen, anti-estrogen antibody, flutamide or diethylstilbestrol, surgery, tamoxifen, ifosfamide, dibromodulcitol, an alkylating agent such as melphalan or cisplatin, etoposide, vinorelbine, vinblastine, a glucocorticoid, a histamine receptor antagonist, an angiogenesis inhibitor, radiation, a radiosensitizer, anthracycline, a vinca alkaloid, a taxane such as paclitaxel and docetaxel, a cell cycle inhibitor such as a cyclin-dependent kinase inhibitor, a checkpoint inhibitor, an immunomodulatory drug, an immunostimulatory drug, a monoclonal antibody directed against another tumor antigen, a complex of a monoclonal antibody with a bioactive molecule, a T cell adjuvant, a bone marrow transplant, or an antigenic cell presenting cell such as dendritic cell therapy. Vaccines can be provided, for example, as soluble proteins or nucleic acids encoding such proteins (see, e.g., le et al, supra; greco and Zellefsky, eds. (2000) Radiotherapy for Prostate Cancer (radiothery of Prostate Cancer), harwood Academic, amsterdam; shapiro and Recht (2001) New Engl. J. Med.344: 1997-2008.

Methods of treating extramedullary hematopoiesis (EMH) in cancer are also provided. EMH has been described (see, e.g., rao et al, (2003) Leuk. Lymphoma 44.

In certain embodiments, the PEG-IL-2 or variant thereof comprises a substitution, addition, or deletion that modulates the binding of its receptor or receptor subunit 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 binding to a receptor or receptor subunit 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 increases the compatibility of the IL-2 or variant thereof with a pharmaceutical preservative (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 increased compatibility enables the preparation of well-preserved pharmaceutical formulations that maintain the physicochemical and biological activities of the protein during storage.

In certain embodiments, one or more engineered bonds are created using one or more unnatural amino acids. The intramolecular bond may be generated in a number of ways, including but not limited to a reaction between two amino acids in the protein (one or both amino acids may be non-natural amino acids) under suitable conditions, a reaction with two amino acids (each amino acid may be naturally encoded or non-naturally encoded), a linker, polymer, or other molecule, and the like.

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

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.

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

wherein n is 0 to 10; r ₁ Is alkyl, aryl, substituted alkyl or substituted aryl; r is ₂ Is H, alkyl, aryl, substituted alkyl and substituted aryl; r ₃ Is H, an amino acid, a polypeptide or an amino-terminal modifying group, and R ₄ Is H, an amino acid, a polypeptide, or a carboxy-terminal modifying group.

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.

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

Wherein n is 0 to 10; r ₁ Is alkyl, aryl, substituted alkyl, substituted aryl, or absent; x is O, N, S or is absent; m is 0 to 10; r ₂ Is H, an amino acid, a polypeptide or an amino-terminal modifying group, and R ₃ Is H, amino acid, polypeptide or carboxyl terminal modification group.

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

wherein n is 0 to 10; r ₁ Is alkyl, aryl, substituted alkyl or substituted aryl; x is O, N, S or absent; m is 0 to 10, R ₂ Is H, an amino acid, a polypeptide or an amino-terminal modifying group, and R ₃ Is H, an amino acid, a polypeptide, or a carboxy-terminal modifying group.

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 biologically active molecules.

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

In certain embodiments, the selector codon is selected from the group consisting of an amber codon, an ochre codon, an opal codon, a unique codon, a rare codon, a five base codon, and a four base codon.

The invention also provides methods of making IL-2 or variants thereof linked to a biologically active molecule. In certain embodiments, the method comprises contacting an isolated IL-2 or variant thereof comprising a non-naturally encoded amino acid with a biologically active molecule comprising a moiety that reacts with the non-naturally encoded amino acid. In certain embodiments, the non-naturally encoded amino acid incorporated into IL-2 or a variant thereof is reactive with a biologically active molecule that is otherwise non-reactive with any of the 20 commonly used amino acids. In certain embodiments, the non-naturally encoded amino acid incorporated into IL-2 is reactive with a linker, polymer, or biologically active molecule that is otherwise unreactive with any of the 20 common amino acids attached to the biologically active molecule.

In certain embodiments, the IL-2 or variant thereof linked to a water soluble polymer or biologically active molecule is made by reacting an IL-2 or variant thereof comprising a carbonyl-containing amino acid with a water soluble polymer or biologically active molecule comprising an aminooxy, hydrazine, hydrazide or semicarbazide group. In certain embodiments, the aminooxy, hydrazine, hydrazide or semicarbazide group is linked to the biologically active molecule through an amide linkage. In certain embodiments, the aminooxy, hydrazine, hydrazide or semicarbazide group is linked to the water soluble polymer or biologically active molecule through a urethane linkage.

The invention also provides methods of making IL-2 conjugates linked to water-soluble polymers. In certain embodiments, the method comprises contacting an isolated IL-2-bioactive molecule conjugate comprising a non-naturally encoded amino acid with a water soluble polymer comprising a moiety 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 with a water-soluble polymer that is otherwise unreactive with any of the 20 commonly used amino acids. In certain embodiments, the non-naturally encoded amino acid incorporated into the IL-2 conjugate is reactive with a linker, polymer, or biologically active molecule that is otherwise unreactive with any of the 20 commonly used amino acids.

The invention also provides methods of making IL-2 or variants thereof linked 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 moiety that reacts with the non-naturally encoded amino acid. In certain embodiments, the non-naturally encoded amino acid incorporated into IL-2 or a variant thereof is reactive with a water-soluble polymer that is otherwise non-reactive with any of the 20 commonly used amino acids. In certain embodiments, the non-naturally encoded amino acid incorporated into IL-2 is reactive with a linker, polymer, or biologically active molecule that is otherwise non-reactive with any of the 20 commonly used amino acids.

In certain embodiments, the IL-2 or variant thereof attached to a water-soluble polymer is made by reacting an IL-2 or variant thereof comprising a carbonyl-containing amino acid with a polyethylene glycol molecule comprising an aminooxy, hydrazine, hydrazide or semicarbazide group. In certain embodiments, the aminooxy, hydrazine, hydrazide or semicarbazide group is attached to the polyethylene glycol molecule through an amide linkage. In certain embodiments, the aminooxy, hydrazine, hydrazide or semicarbazide group is attached to the polyethylene glycol molecule through a urethane linkage.

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

In certain embodiments, the IL-2 or variant thereof attached to a water-soluble polymer is made by reacting an IL-2 comprising an alkynyl-containing amino acid with a polyethylene glycol molecule comprising an azido moiety. In certain embodiments, the azide or alkyne group is linked to the polyethylene glycol molecule through an amide linkage.

In certain embodiments, the IL-2 or variant thereof attached to a water soluble polymer is made by reacting an IL-2 or variant thereof comprising an azido-containing amino acid with a polyethylene glycol molecule comprising an alkynyl moiety. In certain embodiments, the azide or alkyne group is linked to the polyethylene glycol molecule through an amide linkage.

In certain embodiments, the polyethylene glycol molecule has a molecular weight between about 0.1kDa and about 100 kDa. In certain embodiments, the polyethylene glycol molecule has a molecular weight between 0.1kDa and 50 kDa. In certain embodiments, the polyethylene glycol has a molecular weight between 1kDa and 50kDa, between 1kDa and 25kDa, or between 2 and 22kDa or between 5kDa and 20kDa, or between 5kDa and 30kDa, or between 5kDa and 40kDa. For example, the polyethylene glycol polymer may have a molecular weight of about 5kDa or about 10kDa or about 20kDa or about 30kDa or about 40kDa. For example, the polyethylene glycol polymer may have a molecular weight of 5kDa or 10kDa or 20kDa or 30kDa or 40kDa. In certain embodiments, the polyethylene glycol molecule is a 20K 2-branched PEG. In certain embodiments, the polyethylene glycol molecule is 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 branched PEG of 40K or greater. 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 may 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 in one or more of the following positions: 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 position in SEQ ID NO:3, 5 or 7), and said IL-2 or variant thereof is attached 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 in 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 position in SEQ ID NO:3, 5 or 7), and said 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, the non-naturally encoded amino acid is incorporated into the polypeptide at position 65 in IL-2 or a variant thereof (SEQ ID NO:2, or the corresponding amino acid 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, the non-naturally encoded amino acid is incorporated into the polypeptide at position 61 in IL-2 or a variant thereof (SEQ ID NO:2, or the corresponding amino acid 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, the non-naturally encoded amino acid is incorporated into the polypeptide at position 49 in IL-2 or a variant thereof (SEQ ID NO:2, or the corresponding amino acid 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, the non-naturally encoded amino acid is incorporated into the polypeptide at position 45 in IL-2 or a variant thereof (SEQ ID NO:2, or the corresponding amino acid 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, the non-naturally encoded amino acid is incorporated into the polypeptide at position 42 in IL-2 or a variant thereof (SEQ ID NO:2, or the corresponding amino acid 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, the non-naturally encoded amino acid is incorporated into the polypeptide at position 37 in IL-2 or a variant thereof (SEQ ID NO:2, or the corresponding amino acid 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, the non-naturally encoded amino acid is incorporated into the polypeptide at position 35 in IL-2 or a variant thereof (SEQ ID NO:2, or the corresponding amino acid position in SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is linked to a 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 in one or more of the following positions: 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 position in SEQ ID NO:3, 5 or 7), and said IL-2 or variant thereof is attached 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 in 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 position in SEQ ID NO:3, 5 or 7), and said 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 in one or more of the following positions: 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 position in SEQ ID NO:3, 5 or 7), and said IL-2 or variant thereof is attached 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 in 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 position in SEQ ID NO:3, 5 or 7), and said 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 in one or more of the following positions: 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 position in SEQ ID NO:3, 5 or 7), and said IL-2 or variant thereof is attached 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 in 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 position in SEQ ID NO:3, 5 or 7), and said IL-2 or variant thereof is linked to a linear 40K polyethylene glycol molecule.

In certain embodiments, the polyethylene glycol molecule is a branched polymer. In certain embodiments, each branch of the polyethylene glycol branched polymer has a molecular weight between 1kDa and 100kDa or between 1kDa and 50 kDa. In certain embodiments, each branch of the polyethylene glycol branched polymer has a molecular weight between 1kDa and 25kDa or between 2 and 22kDa or between 5kDa and 20kDa or between 5kDa and 30kDa or between 5kDa and 40kDa or between 5kDa and 50kDa or between 5kDa and 60 kDa. For example, the molecular weight of each branch of the polyethylene glycol branched polymer may be about 5kDa or about 10kDa or about 20kDa or about 30kDa or about 40kDa or about 50kDa or about 60kDa or greater. For example, the molecular weight of each branch of the polyethylene glycol branched polymer may 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 greater. In certain embodiments, the polyethylene glycol molecule is a 20K 2-branched PEG. In certain embodiments, the polyethylene glycol molecule is a 20K 4-branched PEG. In certain embodiments, the polyethylene glycol molecule is 40K 2-branched PEG. In certain embodiments, the molecular weight of the polyethylene glycol polymer is an average molecular weight. In certain embodiments, the average molecular weight is a number average molecular weight (Mn). The average molecular weight may 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 in one or more of the following positions: 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 position in SEQ ID NO:3, 5 or 7), and said IL-2 or variant thereof is attached 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 in 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 position in SEQ ID NO:3, 5 or 7), and said 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 in one or more of the following positions: 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 position in SEQ ID NO:3, 5 or 7), and said IL-2 or variant thereof is attached 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 in 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 position in SEQ ID NO:3, 5 or 7), and said 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 in one or more of the following positions: 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 position in SEQ ID NO:3, 5 or 7), and said IL-2 or variant thereof is attached 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 in 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 position in SEQ ID NO:3, 5 or 7), and said 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 in one or more of the following positions: 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 position in SEQ ID NO:3, 5 or 7), and the IL-2 or variant thereof is attached 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 in 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 position in SEQ ID NO:3, 5 or 7), and the IL-2 or variant thereof is attached 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 position in SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is attached to a 20K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 61 (SEQ ID NO:2, or the corresponding amino acid position in SEQ ID NO:3, 5, or 7) in IL-2 or a variant thereof, and the IL-2 or variant thereof is attached to a 20K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 49 (SEQ ID NO:2, or the corresponding amino acid position in SEQ ID NO:3, 5, or 7) in IL-2 or a variant thereof, and the IL-2 or variant thereof is attached 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 position in SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is attached 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 position in SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is attached 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 position in SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is attached 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 position in SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is attached 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 in one or more of the following positions: 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 position in SEQ ID NO:3, 5 or 7), and said IL-2 or variant thereof is attached 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 in 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 position in SEQ ID NO:3, 5 or 7), and the IL-2 or variant thereof is attached 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 position in SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is attached to a 20K 4-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 61 (SEQ ID NO:2, or the corresponding amino acid position in SEQ ID NO:3, 5, or 7) in IL-2 or a variant thereof, and the IL-2 or variant thereof is attached to a 20K 4-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 49 (SEQ ID NO:2, or the corresponding amino acid position in SEQ ID NO:3, 5, or 7) in IL-2 or a variant thereof, and the IL-2 or variant thereof is attached to a 20K 4-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 45 (SEQ ID NO:2, or the corresponding amino acid position in SEQ ID NO:3, 5, or 7) in IL-2 or a variant thereof, and the IL-2 or variant thereof is attached 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 position in SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is attached 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 attached 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 attached 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 position in SEQ ID NO:3, 5, or 7), and the IL-2 or variant thereof is attached to a 40K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 61 (SEQ ID NO:2, or the corresponding amino acid position in SEQ ID NO:3, 5, or 7) in IL-2 or a variant thereof, and the IL-2 or variant thereof is attached to a 40K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 49 (SEQ ID NO:2, or the corresponding amino acid position in SEQ ID NO:3, 5, or 7) in IL-2 or a variant thereof, 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 position 45 (SEQ ID NO:2, or the corresponding amino acid position in SEQ ID NO:3, 5, or 7) in IL-2 or a variant thereof, and the IL-2 or variant thereof is attached to a 40K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 42 (SEQ ID NO:2, or the corresponding amino acid position in SEQ ID NO:3, 5, or 7) in IL-2 or a variant thereof, and the IL-2 or variant thereof is attached to a 40K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 37 (SEQ ID NO:2, or the corresponding amino acid position in SEQ ID NO:3, 5, or 7) in IL-2 or a variant thereof, and the IL-2 or variant thereof is attached to a 40K 2-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 35 (SEQ ID NO:2, or the corresponding amino acid position in SEQ ID NO:3, 5, or 7) in IL-2 or a variant thereof, and the IL-2 or variant thereof is attached to a 40K 2-branched polyethylene glycol molecule.

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

The invention also provides compositions comprising IL-2, or a variant thereof, comprising 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.

The 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.

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

In certain embodiments, the invention provides methods of modulating receptor interactions of the IL-2 polypeptides of the invention. In certain embodiments, the invention provides methods of inhibiting or reducing the interaction of pegylated IL-2 with the IL2R α subunit of a trimeric IL-2 receptor using the pegylated IL-2 polypeptides of the invention.

The invention also provides methods of making PEG-IL-2, or any variant thereof comprising a non-naturally encoded amino acid. In certain embodiments, the method comprises culturing a cell comprising one or more polynucleotides encoding IL-2, orthogonal RNA synthetases, and/or 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.

The invention also provides methods of increasing the therapeutic half-life, serum half-life, or circulation time of IL-2 or variants thereof. In certain embodiments, the half-life (t) of IL-2 or IL-2 variant or PEGylated IL-2 conjugate or glycosylated IL-2 conjugate _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 more. The invention also provides methods of modulating the immunogenicity of IL-2 or variants thereof. In certain embodiments, the method comprises non-naturally encoding any one or more amino acids of naturally occurring IL-2 or a variant thereofAmino acid substitutions of codes and/or linking said IL-2 or variant thereof to a linker, polymer, water soluble polymer or biologically active 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, so as to allow dimer formation.

The invention also provides methods of treating a patient in need of such treatment with an effective amount of a PEG-IL-2 conjugate of the 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 comprising 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 comprising a non-naturally encoded amino acid and a natural amino acid substitution, 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 variant thereof is glycosylated. In certain embodiments, the PEG-IL-2 or variant thereof is not glycosylated.

The invention also provides methods of treating a patient in need of such treatment with an effective amount of an IL-2 or IL-2 variant molecule of the 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 comprising 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 IL-2, or a variant thereof, comprising 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 has a cancer, disorder or disease characterized by high expression of IL-2 receptor alpha, but is not so limited. In certain embodiments, the invention provides a method of treating cancer or a disorder or disease by administering to a subject a therapeutically effective amount of an IL-2 composition of the invention. In certain embodiments, the invention provides a method of treating a genetic disorder by administering to a patient a therapeutically effective amount of an IL-2 composition of the invention. The IL-2 polypeptides of the invention are useful for treating diseases or disorders in cells having high expression of IL-2 receptor alpha. In certain embodiments, the cancer, disorder or disease is treated by reducing, blocking or silencing IL-2 receptor alpha expression. The IL-2 polypeptides or variants of the invention are used in the manufacture of a medicament for the treatment of a cancer, disease or disorder associated with high expression of IL-2 receptor alpha. The IL-2 polypeptides or variants of the invention are used in the manufacture of a medicament for the treatment of cancer. The IL-2 polypeptides or variants of the invention are used in the manufacture of a medicament for the treatment of genetic disorders.

The invention also provides a polypeptide comprising SEQ ID NO: 1. 2, 3, 5 or 7 or any other IL-2 sequence except IL-2 in which at least one amino acid is replaced by a non-naturally encoded amino acid. In certain embodiments, the present invention provides novel IL-2 polypeptides corresponding to SEQ ID NOs: 9. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23, and at least one amino acid is replaced with a non-naturally encoded amino acid. In certain embodiments, the present invention provides novel IL-2 polypeptides comprising SEQ ID NOs: 9. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23, and site-specifically incorporates a non-naturally encoded amino acid. 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.

The invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a peptide comprising SEQ ID NO: 1. 2, 3, 5 or 7 or any other IL-2 sequence, or a natural variant thereof, wherein at least one amino acid is replaced by a non-naturally encoded amino acid. The invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a peptide comprising SEQ ID NO: 1. 2, 3, 5 or 7 or a native variant thereof. 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 native variant thereof through a carbohydrate moiety. In certain embodiments, a linker, polymer, or biologically active molecule is linked to the IL-2 or native variant thereof through a carbohydrate moiety.

The invention also provides an IL-2, or a natural variant thereof, comprising a water soluble polymer covalently linked to said 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 attached to the water-soluble polymer is a non-naturally encoded amino acid present in the polypeptide.

The invention provides an IL-2 or variant thereof comprising at least one linker, polymer or biologically active molecule, wherein said linker, polymer or biologically active molecule is attached to said polypeptide through a functional group of a non-naturally encoded amino acid ribosomally incorporated into said polypeptide. In certain embodiments, the IL-2 or variant thereof is monopegylated. The invention also provides an IL-2 or variant thereof comprising a linker, polymer, or biologically active 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 a preselected site.

Included within the scope of the present invention are leader or signal sequences of IL-2 or variants thereof linked to the IL-2 coding region and heterologous signal sequences linked to the IL-2 coding region. The heterologous leader or signal sequence selected should be one that is recognized and processed for secretion by the host cell secretion system, for example, and that is likely to be cleaved by the host cell's signal peptidase. Methods of treating conditions or disorders using IL-2 of the invention are meant to imply treatment with IL-2 or variants thereof, with or without a signal or leader peptide.

In another embodiment, the conjugation of the IL-2 or variant thereof comprising one or more non-naturally occurring amino acids to another molecule (including but not limited to PEG) provides a substantially purified IL-2 due to the unique chemical reaction used to conjugate the non-natural amino acid. Conjugation of IL-2 or variants 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 variants thereof.

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.

Drawings

FIG. 1 depicts a model showing a view of an IL-2 polypeptide in which potential receptor interaction sites are labeled with the structure of IL-2R α and its interface with IL-2.

FIG. 2 depicts a plasmid map of an expression vector for expressing IL-2 in E.coli.

FIGS. 3A-3B depict Western blot analysis of IL-2 protein expression in E.coli (FIG. 3A) and IL-2 variant titers in E.coli (FIG. 3B).

FIGS. 4A-4B depict a sensorgram and model fit line and calculated measurements of wild-type IL-2 binding to CD25 (FIG. 4A), as well as a plasmid map of an expression vector for expressing IL-2 in mammalian cells (FIG. 4B).

Fig. 5 shows the design of the UPF1 genomic DNA sequence and CRISPR gRNA sites.

Figure 6 depicts sequence validation of a UPF1 knockout cell line.

FIGS. 7A-7B depict transient expression of various IL-2 variants in mammalian cells (FIG. 7A) and Western blot analysis of wild-type IL-2 and IL-2 variants produced in mammalian cells (FIG. 7B).

FIG. 8 depicts a CTLL-2 amplification assay for F42 variants of IL-2.

FIG. 9 shows screening of IL-2 variants by CTLL-2 proliferation assay.

FIGS. 10A-10C depict binding kinetics sensorgrams for IL-2 wild-type and F42 variants (FIG. 10A), K35 and Y45 variants (FIG. 10B), and T37 and P65 variants (FIG. 10C).

FIG. 11 shows a graphical illustration of an IL-2 receptor dimerization assay.

Figure 12 shows a graphical illustration of the ex vivo pSTAT5 assay.

FIG. 13 depicts clonal growth and long-term propagation of CTLL-2 cells in the presence of glycosylated or non-glycosylated IL-2.

FIG. 14 shows a comparison of titers before and after production of stable pools of the corresponding wild-type IL-2 or selected variants thereof.

FIGS. 15A-15C depict titers in mammalian cells expressing the F42-R38A variant (FIG. 15A), CTLL-2 binding assays for the F42-R38A variant (FIG. 15B), and a sensorgram of binding kinetics for the F42-R38A variant (FIG. 15C).

FIG. 16 depicts mean plasma concentrations of the Y45-PEG20K-BR2 and F42-R38A-PEG20K-BR2 variants as a function of time.

FIGS. 17A-17D depict sensorgrams of the binding kinetics of IL-2 wild-type (WT; FIG. 17A) with F42-R38A-P65R-PEG20K-BR2 (FIG. 17B), IL2-Y45-M46L-PEG20K-BR2 (FIG. 17C), and IL2-Y45-M46I-PEG20K-BR2 (FIG. 17D) variants.

FIG. 18 shows a CTLL-2 cell proliferation assay for PEGylated IL-2 variants.

Figure 19 depicts the mean plasma concentration of pegylated IL-2 variants over time.

FIGS. 20A-20B depict the activity of PEGylated IL-2 variants on tumor volume (FIG. 20A) and body weight (FIG. 20B).

FIGS. 21A-21B depict the effect of IL-2 variants F42-R38A-P65R-PEG30K-L, F-R38A-P65R-PEG 40K-BR2, Y45-PEG30K-L, and Y45-PEG40K-BR2 on B16F10 tumor growth inhibition in C57BL/6 mice at 2mg/kg (FIG. 21A) and 5-8mg/kg (FIG. 21B).

FIG. 22 depicts the final tumor volume in BALB/c mice bearing B16F10 tumors.

FIGS. 23A-23C depict the effect of PEGylated IL-2 variants F42-R38A-P65R-PEG30K-L (FIG. 23A) and Y45-PEG30K-L (FIG. 23B) on CT26 tumor growth inhibition and mouse body weight (FIG. 23C).

FIG. 24 depicts the final tumor volume in BALB/c mice bearing CT26 tumors.

FIGS. 25A-25C depict the effect of PEGylated IL-2 variants F42-R38A-P65R-PEG30K-L and Y45-PEG30K-L on CD8+ cells (FIG. 25A), CD4+ cells (FIG. 25B) and the CD8+/CD4+ ratio (FIG. 25C) in the blood of mice bearing CT26 tumors.

FIG. 26 depicts the melting temperature of wild-type IL-2 by DSF analysis.

Definition of

It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, constructs, and reagents described herein, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

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

Unless defined otherwise, 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 are now described.

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

The term "substantially purified" refers to IL-2 or variants thereof that may be substantially or essentially free of components normally associated with or interacting with the protein found in the naturally occurring environment, i.e., in the native cell or in the host cell in the case of recombinantly produced IL-2. IL-2, which may be substantially free of cellular material, includes preparations of protein 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 l% (by dry weight) of contaminating protein. When the IL-2 or variant thereof is recombinantly produced by a host cell, 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 cell. When the IL-2 or variant thereof is recombinantly produced by a host cell, the protein may be present in the culture medium in an amount of about 5g/L, about 4g/L, about 3g/L, about 2g/L, about 1g/L, about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L, about 10mg/L, or about 1mg/L of the dry weight of the cell, or less. Thus, a "substantially purified" IL-2 produced by a method of the 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 higher, as determined by suitable methods such as SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

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

As used herein, the term "culture medium" includes any medium, solution, solid, semi-solid, or rigid support that can support or contain any host cell, including bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, escherichia coli or Pseudomonas host cells, and cell contents. Thus, the term may encompass media in which the host cell has been grown, such as media into which IL-2 has been secreted, including media before or after the proliferation step. The term may also encompass buffers or reagents that contain host cell lysates, for example where IL-2 is produced in the cell and the host cell is lysed or disrupted to release IL-2.

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

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

As used herein, "denaturant" is defined as any compound or material that causes reversible unfolding of a protein. The strength of the denaturant is determined by both the nature and concentration of the particular denaturant. Suitable denaturants may be chaotropes, detergents, organic solvents, water-miscible solvents, phospholipids or combinations 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, sulfobetaine (Zwittergent), 3- (3-chloroaminopropyl) dimethylammonium-1-propane sulfate (CHAPS), and 3- (3-chloroaminopropyl) dimethylammonium-2-hydroxy-1-propane sulfonate (CHAPSO). Water-miscible organic solvents such as acetonitrile, lower alkanols (especially C) ₂ -C ₄ Alkanols, such as ethanol or isopropanol), or lower alkanediols, in particular C ₂ -C ₄ An alkylene glycol such as ethylene glycol) may be used as the denaturant. 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 dihexanylphosphatidylcholine or diheptanoylphosphatidylcholine.

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

As used herein, "co-folding" specifically refers to a refolding process, reaction or method that uses at least two polypeptides that interact with each other and results in the conversion of an unfolded or incorrectly folded polypeptide into an original, correctly folded polypeptide.

As used herein, "interleukin-2," "IL-2," and hyphenated and non-hyphenated forms thereof, include polypeptides and proteins having at least one biological activity of IL-2, and 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, splice variants, and muteins thereof, regardless of their biological activity, and regardless of their method of synthesis and manufacture, including, but not limited to, recombinant (whether produced from cDNA, genomic DNA, synthetic DNA, or other forms of nucleic acids), in vitro, in vivo, by microinjection, synthesis, transgenesis, and gene activation of nucleic acid molecules. The terms "IL-2", "IL-2 variant" and "IL-2 polypeptide" encompass IL-2 comprising one or more amino acid substitutions, additions or deletions.

The sequence of IL-2 lacking the leader sequence and lacking a methionine at the N-terminus is set forth in SEQ ID NO:2. the sequence of IL-2 without leader sequence and with a methionine at the N-terminus is shown in SEQ ID NO: 3. 5 or 7. In certain embodiments, the IL-2 or variant thereof of the invention is 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 for expressing and purifying such polypeptides are well known in the art.

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 such salts, as well as agonist, mimetic and antagonist variants of naturally occurring IL-2, and polypeptide fusions thereof.

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 comprise one or more additional derivatizations of cysteine, lysine, or other residues. Furthermore, the IL-2 may comprise 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 invention, or may be coupled to a naturally encoded amino acid using techniques known in the art, e.g. to lysine or cysteine.

The term "IL-2 polypeptide" also includes glycosylated IL-2, such as but not limited to glycosylation at any amino acid position of the polypeptide, the polypeptide N-linked or O-linked glycosylation form. Variants containing single nucleotide changes are also considered to be biologically active variants of the IL-2 polypeptide. In addition, splice variants are also included.

The term "IL-2" also includes 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, IL-2 heterodimers, homodimers, heteromultimers, or homomultimers that are chemically linked or expressed as a fusion protein, as well as polypeptide analogs that contain, for example, specific deletions or other modifications, but that still maintain biological activity.

As used herein, "interleukin-2" or "IL-2" is a protein comprising two subunits non-covalently joined to form a homodimer, whether coupled to a biologically active molecule, coupled 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".

The term "pegylated IL-2", "pegylated IL-2" or "PEG-IL-2" is an IL-2 molecule having one or more polyethylene glycol molecules covalently attached to one or more than one amino acid residue of the IL-2 protein by a linker such that the attachment is stable. The terms "monopegylated IL-2" and "monopegylated IL-2" mean that one polyethylene glycol molecule is covalently attached to a single amino acid residue on one subunit of the IL-2 dimer through a linker. The average molecular weight of the PEG moiety is preferably between about 5,000 and about 50,000 daltons. The method or site of attachment of PEG to IL-2 is not critical, but preferably the pegylation does not alter or only minimally alters the activity of the bioactive molecule. Preferably, the increase in half-life exceeds any decrease in biological activity.

All references to amino acid positions in IL-2 described herein are based on the amino acid sequences set forth in SEQ ID NO:2 unless otherwise indicated (i.e., when it is stated that the comparison is based on SEQ ID NOs 3, 5, or 7 or other IL-2). One skilled in the art will recognize that in any other IL-2, such as SEQ ID NO: 3. 5 or 7 and SEQ ID NO:2 can be readily identified. One skilled in the art will recognize that the sequence of the amino acid sequence of any other IL-2 molecule, e.g., IL-2 fusions, variants, fragments, etc., is identical to that of SEQ ID NO: 2. 3, 5 or 7 or any other amino acid position corresponding to a position in the IL-2 sequence can be readily identified. For example, a sequence alignment program such as BLAST can be used to align and identify proteins with SEQ ID NOs: 2. 3, 5 or 7 or other IL-2 sequence. Reference is made herein to SEQ ID NO: 2. 3, 5 or 7 or other IL-2 sequences, are intended to also refer to substitutions, deletions or additions in the corresponding positions in IL-2 fusions, variants, fragments, etc., described herein or known in the art, and are specifically encompassed by the invention.

IL-2 (IL 2): any form of IL-2 known in the art may 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 may be altered without affecting its activity, and that these modified forms of IL2 may also be linked to a vector and used in the methods described herein.

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 invention may comprise one or more modifications of a natural amino acid in combination with one or more modifications of a non-natural amino acid. Exemplary substitutions in a wide variety of amino acid positions in a naturally-occurring IL-2 polypeptide 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 solubility of the polypeptide, decreasing 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.

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 a known and study-confirmed trait of IL-2, such as the treatment or amelioration of one or more symptoms of cancer. The addition, substitution or deletion may modulate one or more properties or activities of IL-2 or the variant. For example, the addition, substitution, or deletion can modulate affinity for the IL-2 receptor or one or more subunits of the receptor, modulate circulatory half-life, modulate therapeutic half-life, modulate the stability of the polypeptide, modulate cleavage by proteases, modulate dosage, modulate release or bioavailability, facilitate purification, or improve or alter a particular route of administration. Likewise, IL-2 or variants 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 sequence (including but not limited to FLAG, poly-His, GST, etc.) or linked molecule (including but not limited to biotin) that improves detection (including but not limited to GFP), purification or other traits of the polypeptide.

The term "IL-2 polypeptide" also encompasses homodimers, heterodimers, homomultimers, or heteromultimers that are linked, including but not limited to those linked directly to the same or different non-naturally encoded amino acid side chain through a non-naturally encoded amino acid side chain, linked to a naturally encoded amino acid side chain, or linked indirectly through 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.

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

As used herein, the term "cytotoxic agent" may be any agent having a therapeutic effect on Cancer cells or activated immune cells, useful as a therapeutic agent in combination with IL-2, PEG-IL-2 or IL-2 variants (see, e.g., WO 2004/010957, "Drug Conjugates and Their Use for Treating Cancer, autoimmune Disease or Infectious Disease" (Drug Conjugates and the 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, antimicrotubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, monoplatin, biplatin, and trinuclear platinum complexes, and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemosensitizers, duocarmycins, etoposides, fluoropyrimidines, ionophores, lexitrophins, nitrosoureas, platinum alcohols, preformed compounds, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, and the like.

Individual cytotoxic or immunosuppressive agents include, for example, androgens, amtricin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, thiolutine 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, estrogen, 5-fluorodeoxyuridine, 5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel, plicamycin, procarbazine, teniposide, 6-mercaptopurine, vincristine, vinorelbine, 26-26, and vinorelbine.

In certain exemplary embodiments, the therapeutic agent is a cytotoxic agent. Suitable cytotoxic agents include, for example, urodolines (e.g., auristatin E, AFP, MMAF, MMAE), DNA minor groove binders (e.g., enediynes and lexitrophsins), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycin, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin, lisoproxil, cyanomorpholino-doxorubicin, echinomycin, combretastatin, fusioncin, epothilones a and B, estramustine, cryptophycins, cimadrol, maytansinoids, discodermolide, eleutherobin, and mitoxantrone.

By "non-naturally encoded amino acid" is meant 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 different 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.

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

The terms "functional group," "reactive 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 unique, definable portion or unit of a molecule. The terms are somewhat synonymous in the chemical arts and are used herein to indicate portions of a molecule that perform certain functions or activities and are reactive with other molecules.

The terms "bond", "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. Hydrolytically stable bonds means that the bonds are substantially stable in water and do not react with water for long periods of time, possibly even indefinitely, at useful pH values, including but not limited to physiological conditions. Hydrolytically unstable or degradable linkages means that the linkages are degradable in water or aqueous solutions, including, for example, blood. Enzymatically labile or degradable linkages mean that the linkages can be degraded by one or more enzymes. As understood in the art, PEG and related polymers may include degradable linkages in the polymer backbone or in linker groups between the polymer backbone and one or more terminal functional groups of the polymer molecule. For example, ester linkages formed by reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on biologically active agents are 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 amines with aldehydes, phosphate linkages formed by the reaction of alcohols with phosphate groups, hydrazone linkages as a by-product of hydrazides with aldehydes, acetal linkages as a reaction product of aldehydes with alcohols, orthoester linkages as a reaction product of formic acid with alcohols, peptide linkages formed from amine groups at termini including, but not limited to, polymers such as PEG and carboxyl groups of peptides, and oligonucleotide linkages formed from phosphoramidite groups at termini including, but not limited to, polymers and 5' hydroxyl groups of oligonucleotides.

As used herein, the term "bioactive molecule", "bioactive moiety" or "bioactive agent" means any substance that can affect any physical or biochemical property of a biological system, pathway, molecule or interaction associated with an organism, including but not limited to viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals and humans. In particular, as used herein, bioactive molecules include, but are not limited to, any substance intended for use in diagnosing, curing, alleviating, treating, or preventing a disease or otherwise enhancing the physical or mental well-being of a human or animal in that human or animal. 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 and portions thereof, liposomes, microparticles, and micelles derived or derived from viruses, bacteria, insects, animals, or any other cell or cell type. Classes of biologically active agents suitable for use in the present invention include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, bile acid resins, nicotinic acid and/or statins, anti-inflammatory drugs, antineoplastic agents, cardiovascular agents, anxiolytic drugs, hormones, growth factors, steroidal agents, biologically active molecules of microbial origin, and the like. Biologically active agents also include amide compounds such as those described in Yamamori et al, patent application publication No. 20080221112, which are administered before, after, and/or co-administered with the IL-2 polypeptides of the present invention.

"bifunctional polymer" refers to a polymer comprising two discrete functional groups capable of specifically reacting with other moieties (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 particular bioactive component and another group reactive with a group on a second bioactive component can be used to form conjugates that include the first bioactive component, bifunctional linker, and second bioactive component. Many procedures and linker molecules for attaching various compounds to peptides are known. See, e.g., european patent application No. 188,256, U.S. patent No. 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 capable of specifically reacting with other moieties (including but not limited to amino acid side groups) to form covalent or non-covalent bonds. The bifunctional or polyfunctional polymer may have any desired length or molecular weight, and may be selected to provide a particular desired spacing or conformation between one or more molecules attached to IL-2 and its receptor or IL-2.

When substituents are illustrated by their conventional formula, written from left to right, they likewise encompass chemically identical substituents resulting from writing the structure from right to left, e.g., the structure-CH ₂ O-is equivalent to the structure-OCH ₂ -。

The term "substituent" includes, but is not limited to, "non-interfering substituents". A "non-interfering substituent" is a group that results in a stable compound. Suitable non-interfering substituents or residues include, but are not limited to, halogen, C ₁ -C ₁₀ Alkyl radical, C ₂ -C ₁₀ Alkenyl radical, C ₂ -C ₁₀ Alkynyl, C ₁ -C ₁₀ Alkoxy radical, C ₁ -C ₁₂ Aralkyl radical, C ₁ -C ₁₂ Alkylaryl group, C ₃ -C ₁₂ Cycloalkyl, C ₃ -C ₁₂ Cycloalkenyl, phenyl, substituted phenyl, toluoyl, xylyl, biphenyl, C ₂ -C ₁₂ Alkoxyalkyl group, C ₂ -C ₁₂ Alkoxyaryl radical, C ₇ -C ₁₂ Aryloxyalkyl group, C ₇ -C ₁₂ Oxyaryl radical, C ₁ -C ₆ Alkylsulfinyl radical, C ₁ -C ₁₀ Alkylsulfonyl, - - (CH) ₂ ) _m --O--(C ₁ -C ₁₀ Alkyl) (where m is 1 to 8), aryl, substituted alkylOxy, fluoroalkyl, heterocyclic, substituted heterocyclic, nitroalkyl, - - -NO ₂ 、--CN、--NRC(O)--(C ₁ -C ₁₀ Alkyl), -C (O) - - (C) ₁ -C ₁₀ Alkyl group), C ₂ -C ₁₀ Alkylthio alkyl, - - -C (O) O- - (C) ₁ -C ₁₀ Alkyl), - -OH, - -SO ₂ 、＝S、--COOH、--NR ₂ Carbonyl, - - (C) C (O) - - - (C) ₁ -C ₁₀ Alkyl) -CF3, -C (O) NR2 ₁ -C ₁₀ Aryl) -S- - (C) ₆ -C ₁₀ Aryl), - -C (O) - - - (C) ₁ -C ₁₀ Aryl), - (CH) ₂ ) _m --O--(--(CH ₂ ) _m --O--(C ₁ -C ₁₀ Alkyl) (where each m is 1 to 8), -C (O) NR ₂ 、--C(S)NR ₂ 、--SO ₂ NR ₂ 、--NRC(O)NR ₂ 、--NRC(S)NR ₂ Salts thereof, and the like. As used herein, each R is H, alkyl or substituted alkyl, aryl or substituted aryl, aralkyl or alkaryl.

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

Unless otherwise stated, the term "alkyl" by itself or as part of another substituent means a straight or branched chain or cyclic hydrocarbon group or combination thereof, which may be fully saturated, mono or polyunsaturated, and may include divalent and polyvalent residues, having the indicated number of carbon atoms (i.e., C) ₁ -C ₁₀ Meaning 1 to 10 carbon atoms). Examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated alkyl is a group having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, ethenyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2,4-pentadienyl, 3- (1,4-pentadienyl), ethynyl, 1-and 3-propynyl, 3-butynyl, and higher homologs and isomers. Unless otherwise indicated, the term "alkyl" is also meant to be included under Derivatives of alkyl groups as defined in more detail herein are for example "heteroalkyl groups". Alkyl groups limited to hydrocarbyl groups are referred to as "homoalkyl groups".

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 structure-CH ₂ CH ₂ -and-CH ₂ CH ₂ CH ₂ CH ₂ And also includes groups described hereinafter as "heteroalkylene". Typically, alkyl (or alkylene) groups have 1 to 24 carbon atoms, with groups having 10 or fewer carbon atoms being particular embodiments of the methods and compositions described herein. "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene, typically having 8 or fewer carbon atoms.

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.

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 internal position of the heteroalkyl group or at a position where the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to-CH ₂ -CH ₂ -O-CH ₃ 、-CH ₂ -CH ₂ -NH-CH ₃ 、-CH ₂ -CH ₂ -N(CH ₃ )-CH ₃ 、-CH ₂ -S-CH ₂ -CH ₃ 、-CH ₂ -CH ₂ -S(O)-CH ₃ 、-CH ₂ -CH ₂ -S(O) ₂ -CH ₃ 、-CH＝CH-O-CH ₃ 、-Si(CH ₃ ) ₃ 、-CH ₂ -CH＝N-OCH ₃ and-CH = CH-N (CH) ₃ )-CH ₃ . Up to two heteroatoms may be consecutive, for exampleSuch as-CH ₂ -NH-OCH ₃ and-CH ₂ -O-Si(CH ₃ ) ₃ . Likewise, the term "heteroalkylene" by itself or as part of another substituent means a divalent residue derived from a heteroalkyl group, such as, but not limited to, -CH ₂ -CH ₂ -S-CH ₂ -CH ₂ -and-CH ₂ -S-CH ₂ -CH ₂ -NH-CH ₂ -. For heteroalkylene groups, the same or different heteroatoms may also occupy either or both of the 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 structural formula of the linking group is written does not imply orientation of the linking group. For example, of the formula-C (O) ₂ R' -represents-C (O) ₂ R '-and-R' C (O) ₂ -both.

Unless otherwise stated, the terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, mean the cyclic forms of "alkyl" and "heteroalkyl", respectively. Thus, cycloalkyl or heterocycloalkyl includes saturated, partially unsaturated, and fully unsaturated cyclic linkages. Further, for heterocycloalkyl, a heteroatom may occupy the position where the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl groups 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 term encompasses bicyclic and tricyclic ring structures. Likewise, the term "heterocycloalkylene" by itself or as part of another substituent means a divalent residue derived from heterocycloalkyl, and the term "cycloalkylene" by itself or as part of another substituent means a divalent residue derived from cycloalkyl.

As used herein, the term "water-soluble polymer" refers to any polymer that is soluble in an aqueous solvent. Attachment of the 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 may be used as a linker for attaching IL-2 to other substances, including but not limited to one or more IL-2 or one or more biologically active molecules. Suitable polymers include, but are not limited to, polyethylene glycol propionaldehyde, mono C1-C10 alkoxy or aryloxy derivatives thereof (described in U.S. patent No. 5,252,714, which is incorporated herein by reference), monomethoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N- (2-hydroxypropyl) -methacrylamide, dextran derivatives (including dextran sulfate), polypropylene glycol, polyoxypropylene/oxyethylene copolymers, polyoxyethylated polyols, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives (including, but not limited to, methyl cellulose and carboxymethyl cellulose), starch and starch derivatives, polypeptides, polyalkylene glycols and derivatives, copolymers of polyalkylene glycols and derivatives, polyvinyl ethyl ether and α - β -poly (2-hydroxyethyl) -DL-asparagine, and the like, or mixtures thereof. Examples of such water-soluble polymers include, but are not limited to, polyethylene glycol and serum albumin.

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

Unless otherwise stated, the term "aryl" means a polyunsaturated, aromatic hydrocarbon-based 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 (or ring) containing 1 to 4 heteroatoms selected from N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atoms are optionally quaternized. The heteroaryl group may be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2- Azolyl, 4-Azolyl, 2-phenyl-4-Azolyl, 5-Azolyl, 3-isoAzolyl, 4-isoAzolyl, 5-isoOxazolyl, 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-quinoxalyl, 5-quinoxalyl, 3-quinolyl, and 6-quinolyl. For each of the above-mentioned aryl and heteroFor aryl ring systems, the substituents are selected from the acceptable substituents described below.

Briefly, the term "aryl" when used in combination with other terms (including, but not limited to, aryloxy, arylsulfenoxy, aralkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term "aralkyl" is meant to include those residues in which the aryl group is attached to an alkyl group (including but not limited to benzyl, phenethyl, picolyl, and the like), 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, and the like).

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.

Substituents for alkyl and heteroalkyl residues (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) may be one or more of a variety of different 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 ', -CO ₂ R’，-CONR’R”，-OC(O)NR’R”，-NR”C(O)R’，-NR’-C(O)NR”R”’，-NR”C(O) ₂ R’，-NR-C(NR’R”R’”)＝NR””，-NR-C(NR’R”)＝NR’”，-S(O)R’，-S(O) ₂ R’，-S(O) ₂ NR’R”，-NRSO ₂ R', -CN and-NO ₂ The number of which is in the range of 0 to (2 m '+ 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 invention includes more than one R group, for example, each of the R groups is independently substituted Alternatively, when more than one of the R ', R ", R'" and R "" groups are present, the same is true for each of these groups. When R' and R "are attached to the same nitrogen atom, they may combine with the 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 above discussion of substituents, those skilled in the art will appreciate that the term "alkyl" is meant to include groups containing carbon atoms bonded to groups other than hydrogen radicals, such as haloalkyl (including, but not limited to-CF) ₃ and-CH ₂ CF ₃ ) And acyl (including but not limited to-C (O) CH ₃ 、-C(O)CF ₃ 、-C(O)CH ₂ OCH ₃ Etc.).

Like the substituents described for the alkyl residue, 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' ", -OC (O) R ', -C (O) R', -CO ₂ R’，-CONR’R”，-OC(O)NR’R”，-NR”C(O)R’，-NR’-C(O)NR”R”’，-NR”C(O) ₂ R’，-NR-C(NR’R”R’”)＝NR””，-NR-C(NR’R”)＝NR’”，-S(O)R’，-S(O) ₂ R’，-S(O) ₂ NR’R”，-NRSO ₂ R', -CN and-NO ₂ ，-R’，-N ₃ ，-CH(Ph) ₂ Fluoro (C) ₁ -C ₄ ) Alkoxy and fluoro (C) ₁ -C ₄ ) Alkyl groups in a number ranging from 0 to the total number of open valences on the aromatic ring system; and wherein R ', R ", R'" and R "" are independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, aryl and heteroaryl. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected, as is each of the R ', R ", R'", and R "" groups when more than one of these groups is present.

As used herein, the term "modulated serum half-life" means a positive or negative change in the circulatory half-life of the modified IL-2 relative to its unmodified form. Serum half-life is measured by taking blood samples at various time points after IL-2 administration and determining the concentration of the molecule in each sample. The correlation of serum concentration with time allows calculation of serum half-life. The increased serum half-life is desirably at least about two-fold, although a smaller increase may also be useful, for example where it enables a satisfactory dosing regimen or avoids toxic effects. In certain embodiments, the increase is at least about three times, at least about five times, or at least about ten times.

As used herein, the term "modulated therapeutic half-life" means a positive or negative change in the half-life of a therapeutically effective amount of IL-2 relative to its unmodified form. Therapeutic half-life is measured by measuring the pharmacokinetic and/or pharmacodynamic properties of the molecule at various time points after administration. The increased therapeutic half-life desirably enables a particularly beneficial dosing regimen, a particularly beneficial total dose, or avoids adverse effects. In certain embodiments, the increased therapeutic half-life results from an increase or decrease in potency, an increase or decrease in binding of the modified molecule to its target, an increase or decrease in breakdown of the molecule by an enzyme, e.g., 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.

The term "isolated" when used with respect 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 associated in nature, or that the nucleic acid or protein has been concentrated to a level above that at which it is produced in vivo or in vitro. It may be in a homogeneous state. The isolated material may be in a dry or semi-dry state, or in a solution (including but not limited to an aqueous solution). It may be a component of a pharmaceutical composition additionally comprising 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. The protein, which is the major material present in the preparation, is substantially purified. In particular, the isolated gene is separated from the open reading frames flanking the gene and encoding proteins other than the gene of interest. The term "purified" means that the nucleic acid or protein essentially produces a band in the electrophoresis gel. In particular, this may mean that the nucleic acid or protein is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or more pure.

The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the terms encompass nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also refers to oligonucleotide analogs, including PNAs (peptide nucleic acids), analogs of DNA used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more (or all) of the selected codons is replaced by 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 (1994)).

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, the description for polypeptides applies equally to the description for peptides and to the description for proteins, 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 are a non-naturally encoded amino acid. As used herein, the term encompasses amino acid chains of any length, including full length proteins, in which the amino acid residues are linked by covalent peptide bonds.

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. The 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 a naturally occurring amino acid, i.e., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. These analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. References to amino acids include, for example, naturally occurring protein L-amino acids, D-amino acids, chemically modified amino acids such as amino acid variants and derivatives, naturally occurring non-protein amino acids such as beta-alanine, ornithine, and the like, as well as 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, alpha-methyl amino acids (e.g., alpha-methyl alanine), D-amino acids, histidine-like amino acids (e.g., 2-amino-histidine, beta-hydroxy-histidine, homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine), amino acids having an additional methylene group in the side chain ("homo" amino acids), and amino acids in which the carboxylic acid functional group in the side chain is replaced with a sulfonic acid group (e.g., cysteic acid). Incorporation of unnatural amino acids, including synthetic unnatural amino acids, substituted amino acids, or one or more D-amino acids, into proteins of the invention can be advantageous in a number of different ways. Peptides containing D-amino acids and the like exhibit improved in vitro or in vivo stability as compared with the corresponding L-amino acid-containing ones. Therefore, the construction of a peptide or the like incorporating a D-amino acid may be particularly useful when higher intracellular stability is desired or required. More specifically, the D-peptide, etc., is resistant to endogenous peptidases and proteases, thus providing improved bioavailability and extended in vivo life of the molecule 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 unlikely to induce a humoral immune response in the whole organism.

Amino acids may be referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single letter codes.

"conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. These nucleic acid variations are "silent variations," which are one of the conservatively modified variations. Every possible silent variation of the nucleic acid is also described herein for each nucleic acid sequence encoding a polypeptide. 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 identical molecule. Thus, each silent variation of a nucleic acid encoding a polypeptide is implied in each described sequence.

With respect to amino acid sequences, one of ordinary skill in the art will recognize that each substitution, deletion, or addition of an alteration, addition, or deletion of a nucleic acid, peptide, polypeptide, or protein sequence by a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the deletion of an amino acid, addition of an amino acid, or substitution 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 invention.

Conservative substitution tables providing functionally similar amino acids are known to those of ordinary skill in the art. The following 8 groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (a), glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); 6) Phenylalanine (F), tyrosine (Y), tryptophan (W); 7) Serine (S), threonine (T); and 8) cysteine (C), methionine (M); (see, e.g., creighton, proteins: structural and Molecular Properties) (W H Freeman & Co., second edition (December 1993)).

The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same. Sequences are "substantially identical" if they have a percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity over a specified region) when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using one of the following sequence comparison algorithms (or other algorithms available to one of ordinary skill in the art), or by manual alignment and visual inspection. This definition also refers to the complement of the test sequence. The identity may exist over a region of at least about 50 amino acids or nucleotides in length or 75-100 amino acids or nucleotides in length, or, where not indicated, across the entire sequence of the polynucleotide or polypeptide. Polynucleotides encoding polypeptides of the invention (including homologues from species other than human) may be obtained by a method comprising the steps of: libraries are screened under stringent hybridization conditions using labeled probes having the polynucleotide sequences of the invention or fragments thereof, and full-length cDNA and genomic clones containing the polynucleotide sequences are isolated. Such hybridization techniques are well known to the skilled artisan.

The phrase "selectively (or specifically) hybridizes to" refers to the binding, duplex formation or hybridization of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that particular sequence is present in a complex mixture (including but not limited to total cellular or library DNA or RNA).

As known in the art, the phrase "stringent hybridization conditions" refers to 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 not to other sequences in the complex mixture. Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.

As used herein, the term "eukaryote" refers to organisms belonging to a domain of eukaryotic systems, 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, and the like.

As used herein, the term "non-eukaryotic organism" refers to an organism that is not eukaryotic. For example, the non-eukaryotic organism may belong to the eubacteria (including but not limited to Escherichia coli (Escherichia coli), thermus thermophilus (Thermus thermophilus), bacillus stearothermophilus (Bacillus stearothermophilus), pseudomonas fluorescens (Pseudomonas fluorescens), pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas putida (Pseudomonas putida), etc.) system domain or archaea (including but not limited to Methanococcus jannaschii, methanobacterium thermoautotrophicum, halobacterium (Halobacterium) such as Halobacterium halophilum (Halofax volcenii) and Halobacterium species NRC-1, areoglofurylus, pyrococcus furiosus (Pyrococcus furiosus), pyrococcus thermoroseus, aeuropus etc.) system domain.

As used herein, the term "subject" refers to an animal, in certain embodiments a mammal, and in other embodiments a human, who 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.).

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

The term "enhance" means to increase or prolong the desired effect in either efficacy or duration. Thus, with respect to enhancing the effect of a therapeutic agent, the term "enhance" refers to the ability to increase or prolong the effect of other therapeutic agents on the system, either in terms of efficacy or duration. As used herein, "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, an amount effective for such use will depend on the severity and course of the disease, disorder or condition, previous therapy, the health of the patient and response to the drug, and the judgment of the treating physician.

As used herein, the term "modified" refers to any change made to a given polypeptide, such as changes to the length, amino acid sequence, chemical structure, co-translational or post-translational modifications of the polypeptide. The term "(modified)" form means that the polypeptide in question is optionally modified, that is, the polypeptide in question may be modified or unmodified.

The term "post-translational modification" refers to any modification of a natural or unnatural amino acid that occurs at that amino acid after it has been incorporated into a polypeptide chain. Merely by way of example, 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.

In prophylactic applications, compositions containing the IL-2 are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder, or condition. Such an amount is defined as a "prophylactically effective amount". In this use, the exact amount will also depend on the health, weight, etc. of the patient. Determination of such a prophylactically effective amount by routine experimentation (e.g., dose escalation clinical trials) is considered to be well within the skill of the art.

In therapeutic applications, a composition containing the modified unnatural amino acid polypeptide is administered to a patient already suffering from a disease, condition, or disorder in an amount sufficient to cure or at least partially arrest the symptoms of the disease, condition, or disorder. Such an amount is defined as a "therapeutically effective amount" and will depend on the severity and course of the disease, disorder or condition, previous treatments, the health of the patient and response to the drug, and the judgment of the treating physician. Determination of such therapeutically effective amounts by routine experimentation (e.g., dose escalation clinical trials) is considered to be well within the skill of the art.

The term "treatment" is used to refer to any of prophylactic and/or therapeutic treatment.

Non-naturally encoded amino acid polypeptides set forth herein can 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 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、 ³⁶ And (4) Cl. Certain isotopically-labeled compounds described herein, for example, those into which a radioactive isotope such as ³ H and ¹⁴ compound of CAnd may be useful in determining the tissue distribution of drugs and/or substances. In addition, with isotopes such as deuterium ² H replacement may result in certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements.

All isomers, including but not limited to diastereomers, enantiomers, and mixtures thereof, are considered to be part of the compositions described herein. In additional or other embodiments, the non-naturally encoded amino acid polypeptide is metabolized upon administration to an organism in need thereof to produce a metabolite, which is subsequently used to produce a desired effect, including a desired therapeutic effect. In other or additional embodiments are active metabolites that are not naturally encoded amino acid polypeptides.

In certain instances, a non-naturally encoded amino acid polypeptide can exist as a tautomer. 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, and the like. The solvated forms are also considered disclosed herein. One of ordinary skill in the art will recognize that certain compounds herein may exist in several tautomeric forms. All of these tautomeric forms are considered part of the compositions described herein.

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

Detailed Description

I. Brief introduction to the drawings

In the present invention, IL-2 molecules are provided that comprise at least one unnatural amino acid. In certain embodiments of the invention, the IL-2 having at least one unnatural amino acid includes 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 chemistry known to one of ordinary skill in the art to be appropriate for the particular reactive group, such molecules include, but are 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, saccharides, polynucleotides, DNA, RNA, antisense polynucleotides, sugars, water-soluble dendrimers, cyclodextrins, inhibitory ribonucleic acids, biomaterials, nanoparticles, spin labels, fluorophores, metal-containing moieties, radioactive moieties, new functional groups, groups that interact covalently or non-covalently with other molecules, photocaging moieties, actinic radiation excitable moieties, photoisomerizable moieties, biotin, derivatives of biotin, biotin analogs, heavy atom doped moieties, chemical groups, compact photocleavable groups, extended side chains, carbon-linked sugars, redox active agents, aminothioacids, toxic moieties, physical probes, phosphorescent groups, chemical energy transfer groups, luminescent groups, magnetic energy transfer groups, chromophore groups, electron-cleavable nanoparticles, detectable agents, quantum dot emitting reagents, and other molecules, radioactive emitters, neutron capture agents, or any combination of the above molecules 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 the cycloaddition chemistry is utilized [3+2 ]. In another example, the first reactive group is an azido moiety (including but not limited to para-azido-L-phenylalanine (pAZ) in the unnatural amino acid) and the second reactive group is an alkynyl moiety. In certain embodiments of the modified IL-2 of the present invention, at least one unnatural amino acid (including but not limited to unnatural amino acids that contain 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 made in vivo in a eukaryotic cell or in a non-eukaryotic cell. A linker, polymer, water soluble polymer or other molecule may attach the molecule to the polypeptide. In another embodiment, the attachment to IL-2 link long enough to allow dimer formation. The molecule may also be linked directly to the polypeptide.

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

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

In one embodiment, the post-translational modification comprises modification of an oligosaccharide (including but not limited to wherein the oligosaccharide comprises (GlcNAc-Man) ₂ -Man-GlcNAc-GlcNAc, etc.) is attached to asparagine by a GlcNAc-asparagine linkage. In another embodiment, the post-translational modification comprises attachment of an oligosaccharide (including but not limited to Gal-GalNAc, gal-GlcNAc, etc.) to serine or threonine by a GalNAc-serine, galNAc-threonine, glcNAc-serine, or GlcNAc-threonine linkage. In certain embodiments, the proteins or polypeptides of the 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 for bacterial expression, novel secretion signal sequences, pectate lyase secretion signal sequences, omp a secretion signal sequences, and bacteriophage secretion signal sequences. Is divided intoExamples of secretory signal sequences include, but are not limited to, STII (prokaryotes), fd GIII and M13 (phages), bgl2 (yeast), and a signal sequence bla derived from a transposon. Any such sequence may be modified to provide the desired result for the polypeptide, including, but not limited to, replacement of one signal sequence with a different signal sequence, replacement of a leader sequence with a different leader sequence, and the like.

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, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different positions can be present in the protein, which comprises 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 unnatural amino acids.

The present invention provides methods and compositions based on IL-2 comprising at least one non-naturally encoded amino acid. The introduction of at least one non-naturally encoded amino acid into IL-2 may allow the use of coupling chemistry involving specific chemical reactions including, but not limited to, reaction with one or more non-naturally encoded amino acids while not reacting with the common 20 amino acids. In certain embodiments, IL-2 comprising the non-naturally encoded amino acid is linked to a water soluble polymer, such as polyethylene glycol (PEG), through a side chain of the non-naturally encoded amino acid. The present invention provides a highly efficient method for selective modification of proteins with PEG derivatives, said method comprising the selective incorporation of non-genetically encoded amino acids (including but not limited to amino acids containing functional groups or substituents (including but not limited to ketone, azido or acetylene moieties) not present among the 20 naturally incorporated amino acids) into proteins in response to selector codons, 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 appropriate for the particular functional group or substituent present in the non-naturally encoded amino acid. A wide variety of known chemical methods are suitable for use in the present invention for incorporating water-soluble polymers into the protein. These methods include, but are not limited to, the use of Huisgen [3+2] Cycloaddition reactions including, but not limited to, acetylene or azide derivatives, respectively (see, e.g., padwa, A., (Comprehensive Organic Synthesis), vol.4, (1991) Ed. Trost, B.M., pergamon, oxford, p.1069-1109; and Huisgen, R., (1,3-Dipolar Cycloaddition Chemistry), (1,3-Dipolar Cycloaddition Chemistry), (1984) Ed. Padwa., A., wiley, new York, p.1-176).

Since the Huisgen [3+2] cycloaddition method involves cycloaddition rather than nucleophilic substitution reactions, proteins can be modified with very high selectivity. The reaction can be carried out in aqueous conditions at room temperature with excellent regioselectivity (1, 4>, 1, 5) by adding catalytic amounts of a Cu (I) salt to the reaction mixture. See, e.g., tornoe et al, (2002) J.org.chem.67:3057-3064; and Rostovtsev et al, (2002) Angew. Chem. Int. Ed.41:2596-2599; and WO 03/101972. Molecules that can be added to the proteins of the invention by cycloaddition [3+2] include virtually any molecule with suitable functional groups or substituents, including but not limited to azide or acetylene derivatives. These molecules can be added to unnatural amino acids with acetylene groups, including but not limited to p-propargyloxyphenylalanine, or unnatural amino acids with azido groups, including but not limited to p-azido-phenylalanine.

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

The invention also provides water-soluble and hydrolytically stable PEG derivatives and related hydrophilic polymers having one or more acetylene or azido moieties. The PEG polymer derivatives containing acetylene moieties are highly selective for coupling to azido moieties that have been selectively introduced into proteins in response to selector codons. Likewise, PEG polymer derivatives containing an azido moiety are highly selective for coupling to acetylene moieties that have been selectively introduced into proteins in response to selector codons.

More specifically, the azide moieties include, but are not limited to, alkyl azides, aryl azides, and derivatives of these azides. The derivatives of alkyl and aryl azides may include other substituents as long as acetylene-specific reactivity is maintained. The acetylene moiety comprises 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.

The present invention provides conjugates of a substance 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 tags, photoaffinity tags, reactive compounds, resins, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelators, cofactors, fatty acids, saccharides, polynucleotides, DNA, RNA, antisense polynucleotides, sugars, water-soluble dendrimers, cyclodextrins, inhibitory ribonucleic acids, biological materials, nanoparticles, spin labels, fluorophores, metal-containing moieties, radioactive moieties, new functional groups, groups that interact covalently or non-covalently with other molecules, photocaging moieties, radionuclide excitable moieties, photoisomerizable dense moieties, biotin, derivatives of biotin, biotin analogs, moieties incorporating heavy atoms, chemical groups, photocleavable groups, extended side chains, carbon-linked saccharides, redox moieties, amino acid moieties, radiolabel moieties, detectable moieties, electron capture moieties, radioactive quantum capture agents, radioactive moieties, or any combination of the above-cleavable electron emission reagents, cleavable moieties, radioactive emission reagents, or neutron capture agents. The invention also includes conjugates of a substance having an azido or acetylene moiety and a PEG polymer derivative having a corresponding acetylene or azido moiety. For example, a PEG polymer containing an azido moiety may be coupled to a biologically active molecule at a position in the protein that contains a non-genetically encoded amino acid with an acetylene functional group. Linkages coupling the PEG to the bioactive molecule include, but are not limited to, huisgen [3+2] cycloaddition products.

It is well established in the art that PEG can be used to modify the surface of biomaterials (see, e.g., U.S. Pat. nos. 6,610,281, mehvar, r., j.pharm sci.,3 (1): 125-136 (2000), which are incorporated herein by reference). The invention also includes biomaterials having a surface with one or more reactive azido or acetylene sites and one or more azido or acetylene containing polymers of the invention coupled to the surface by Huisgen [3+2] cycloaddition. Biomaterials and other substances may also be coupled to the azide or acetylene activated polymer derivative through linkages other than azide or acetylene linkages, for example by linkages comprising carboxylic acid, amine, alcohol or thiol moieties, to leave the azide or acetylene moieties available for subsequent reactions.

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

More specifically, in the case of the azido-containing PEG derivatives, a water-soluble polymer having at least one reactive hydroxyl moiety undergoes a reaction to produce a substituted polymer having a more reactive moiety thereon, 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 is known to those of ordinary skill in the art. The resulting substituted polymer then undergoes a reaction to replace the azido moieties at the ends of the polymer with the more reactive moieties. Alternatively, a water soluble polymer having at least one active nucleophilic or electrophilic moiety and a linking reagent having an azido group at one terminus undergo a reaction to form a covalent bond between the PEG polymer and the linking reagent, and the azido moiety is located at the terminus of the polymer. Nucleophilic and electrophilic components, including amines, thiols, hydrazides, hydrazines, alcohols, carboxylates, aldehydes, ketones, thioesters, and the like, are known to those of ordinary skill in the art.

More specifically, in the case of the acetylene-containing PEG derivatives, a water-soluble polymer having at least one active hydroxyl moiety undergoes a reaction to displace a halogen or other activated leaving group from a precursor containing the acetylene moiety. Alternatively, a water soluble polymer having at least one active nucleophilic or electrophilic moiety and a linking reagent having an acetylene at one terminus undergo a reaction to form a covalent bond between the PEG polymer and the linking reagent, and the acetylene moiety is located 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 the preparation and use of PEG derivatives is well-defined to practitioners in the art.

The present invention also provides a method of selectively modifying a protein to add other substances to the modified protein, including but not limited to water soluble polymers such as PEG and PEG derivatives containing an azido or acetylene moiety. The azido 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, and at the same time provide a more selective means of attaching the PEG derivatives to proteins than previously known in the art.

General recombinant nucleic acid methods for use in the invention

In a number of embodiments of the invention, the nucleic acid encoding the IL-2 of interest will be isolated using recombinant methods, cloned, and typically altered. These embodiments are useful during production including, but not limited to, 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.

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

TABLE 1 IL-2 protein and DNA sequences

The nucleotide sequence encoding IL-2 comprising a non-naturally encoded amino acid can be found in a nucleic acid sequence including, but not limited to, a nucleic acid sequence having SEQ ID NO: 1. 2, 3, 5 or 7, and then altering the nucleotide sequence so as to perform the introduction (i.e. incorporation or substitution) or removal (i.e. deletion or substitution) of the relevant amino acid residue. The nucleotide sequence may be conveniently modified by site-directed mutagenesis in accordance with conventional procedures. Alternatively, the nucleotide sequence may be prepared by chemical synthesis, including but not limited to by using an oligonucleotide synthesizer, wherein oligonucleotides are designed on the basis of the amino acid sequence of the desired polypeptide and preferably selecting those codons which are advantageous in the host cell in which the recombinant polypeptide is to be produced. For example, several small oligonucleotides encoding portions of a desired polypeptide can be synthesized and assembled by PCR, ligation, or ligation chain reactions. See, e.g., barany et al, proc. Natl. Acad. Sci.88:189-193 (1991); U.S. Pat. No. 6,521,427, which is incorporated herein by reference.

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

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, A Laboratory Manual for Molecular Cloning (3 rd edition, 2001); kriegler, guide to Gene Transfer and Expression experiments (Gene Transfer and Expression: A Laboratory Manual) (1990); and Current Protocols in Molecular Biology (Current Protocols in Molecular Biology) (eds. Ausubel et al, 1994).

The invention also relates to eukaryotic host cells, non-eukaryotic host cells, and organisms for the in vivo incorporation of an unnatural amino acid by orthogonal tRNA/RS pairs. Host cells are genetically engineered (including but not limited to transformation, transduction, or transfection) with a polynucleotide of the invention or a construct comprising a polynucleotide of the invention (including but not limited to a vector of the invention), which may be, for example, a cloning vector or an expression vector.

Several well-known methods of introducing a target nucleic acid into a cell 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, projectile bombardment, and infection with viral vectors (discussed further below), among others. Bacterial cells can be used to amplify the number of plasmids containing the DNA constructs of the invention. The bacteria are grown to log phase and plasmids within the bacteria can be isolated by various methods known in the art (see, e.g., sambrook). In addition, kits for purifying plasmids from bacteria are commercially available (see, e.g., easyPrep, both from Pharmacia Biotech ^TM 、FlexiPrep ^TM (ii) a StrataClean from Stratagene ^TM (ii) a And QIAprep from Qiagen ^TM ). The isolated and purified plasmids are then further manipulated to generate additional plasmids for transfection into cells or incorporation into relevant vectors to infect an organism. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulating expression of the particular target nucleic acid. The vector optionally comprises a universal expression cassette containing at least one independent terminator sequence, sequences that allow replication of the expression cassette in eukaryotes or prokaryotes or both (including but not limited to shuttle vectors), and selectable markers for both prokaryotic and eukaryotic systems. The vector is suitable for replication and integration in prokaryotes, eukaryotes, or both. See Gillam&Smith, gene 8 (1979); roberts et al, nature,328, 731 (1987); schneider, E.et al, protein Expr. Purif.6 (1): 10-14 (1995); ausubel, sambrook, berger (all supra). A list of bacteria and phages available for cloning is provided, for example, by the ATCC (ATCC catalogues of bacteria and phages)Catalogue of Bacteria and Bacteriophage (1992), eds Gherna et al, published by ATCC. Other basic procedures for sequencing, cloning and other aspects of molecular biology and implicit theoretical considerations are also found in Watson et al, (1992), recombinant DNA, second edition, scientific American Books, NY. In addition, virtually any nucleic acid (and indeed any labeled nucleic acid, whether standard or nonstandard) can be custom-made or standard-ordered from any of a variety of different commercial sources, such as, for example, midland Certified Reagent Company (Midland, TX, available at The world wide web site mcrc.com), the Great American Gene Company (Ramona, CA, available at The world wide web site genco.com), expressGen inc. (Chicago, IL, available at The world wide web site expressgen.com), operon Technologies inc. (Alameda, CA), and many others.

Selector codon

The selector codons of the invention extend the genetic codon framework 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 codons (UAG), ochre codons, or ovalbumin codons (UGA), unnatural codons, four or more base codons, rare codons, and the like. 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.

In one embodiment, the method comprises using a selector codon that is a stop codon for the incorporation of one or more unnatural amino acid in vivo. For example, an O-tRNA is produced that recognizes a stop codon (including but not limited to UAG) and is aminoacylated by an O-RS that has the desired unnatural amino acid, which 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 polypeptide of interest at a site of interest. See, e.g., sayers, j.r. et al, (1988), 5'-3' exonucleases (5 '-3' exotases in phosphorothionate-based oligonucleotide-directed mutagenesis), nucleic Acids Res,16 791-802 in phosphorothioate-guided mutagenesis. 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 that contains the unnatural amino acid at the specified position.

In vivo incorporation of the unnatural amino acid can be performed without significant disruption of the eukaryotic host cell. For example, because the suppression efficiency of the UAG codon depends on the competition between the O-tRNA, including but not limited to the amber suppressor tRNA, and eukaryotic release factors, including but not limited to eRF, that bind to a stop codon and initiate release of the growing peptide from the ribosome, the suppression efficiency can be modulated by, including but not limited to, increasing the expression level of the O-tRNA and/or the suppressor tRNA.

Unnatural amino acids can also be encoded with rare codons. For example, it has been demonstrated that the rare arginine codon AGG is efficiently inserted into Ala by synthetic tRNA acylated with alanine when the concentration of arginine is reduced in an in vitro protein synthesis reaction. See, e.g., ma et al, biochemistry,32, 7939 (1993). In this case, the synthetic tRNA competes with naturally occurring tRNAArg, which is present in E.coli as a minor species. Some organisms do not use all triplet codons. The unassigned codon AGA in Micrococcus luteus (Micrococcus luteus) has been used for insertion of amino acids in vitro transcription/translation extracts. See, e.g., kowal and Oliver, nucl. The components of the invention can be produced to use these rare codons in vivo.

Selector codons also include extended codons including, but not limited to, four or more base codons, e.g., four, five, six or more base codons, examples of which include, but are not limited to, AGGA, CUAG, UAGA, CCCU, and the like. Examples of five base codons include, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC, and the like. Features of the 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 unnatural amino acid inserted into the same protein. For example, the four or more base codon is read as a single amino acid in the presence of a mutated O-tRNA (including but not limited to a special frameshift suppressor tRNA) that has an anticodon loop, e.g., an anticodon loop of at least 8-10 nt. In other embodiments, the anticodon loop can decode codons that include, but are not limited to, at least four base codons, at least five base codons, or at least six base codons or more. Because there are 256 possible four base codons, four or more base codons can be used to encode multiple unnatural amino acids in the same cell. See Anderson et al, exploring the restriction of codon and anti-codon sizes (expanding the Limits of Coden and Anticodon Size), chemistry and Biology, 9-237-244 (2002); magliery, expanded genetic code: selection of high efficiency Suppressors of Four base codons and Identification of "shifted" Four base codons in E.coli using a Library Approach (Expanding the Genetic Code: selection of Efficient supressors of Four-base codons and Identification of "Shifty" Four-base codons with a Library Approach in Escherichia coli), J.mol.biol.307:755-769 (2001).

For example, four base codons have been used to incorporate unnatural amino acids into proteins in vitro biosynthetic methods. See, e.g., ma et al, biochemistry,32, 7939, (1993); and Hohsaka et al, j.am.chem.soc., 121. CGGG and AGGU were used to simultaneously incorporate 2-naphthylalanine and NBD derivatives of lysine into streptavidin in vitro using two chemically acylated frameshift suppressor tRNAs. See, e.g., hohsaka et al, j.am.chem.soc., 121. In vivo studies, moore et al examined the ability of tRNALeu derivatives with NCUA anticodons to suppress UAGN codons (N could be U, A, G or C), and found that the quadruplet UAGA could be decoded by tRNALeu with UCUA anticodons with an efficiency of 13 to 26% and little decoding in the 0 or-1 box. See Moore et al, j.mol.biol.,298 (2000). In one embodiment, extended codons based on rare codons or nonsense codons can be used in the invention, which can reduce missense readthrough and frameshift suppression at other unwanted sites.

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

Selector codons optionally include unnatural base pairs. These unnatural base pairs further extend the existing genetic alphabet. One additional base pair increases the number of triplet codons from 64 to 125. The properties of the third base pair include stable and selective base pairing, efficient enzymatic incorporation into DNA with high fidelity by the polymer, and efficient continued primer extension after synthesis of the nascent unnatural base pair. Descriptions of unnatural base pairs that can be engineered to be useful in methods and compositions include, for example, hirao et al, unnatural base pairs for the incorporation of amino acid analogs into proteins (An unnatural base pair for the incorporation of amino acid analogs into proteins), nature Biotechnology, 20. See also Wu, Y, et al, J.am.chem.Soc.124:14626-14630 (2002). Other related publications are listed below.

For in vivo use, the non-natural nucleoside is membrane permeable and is phosphorylated to form the corresponding triphosphate. Furthermore, the increased genetic information is stable and not destroyed by cellular enzymes. Previous attempts by Benner and others have utilized different hydrogen bonding modes than the classical Watson-Crick pair, the most notable example of which is the iso-C: iso-G pair. See, e.g., switzer et al, j.am.chem.soc., 111; and Piccirilli et al, nature, 343; kool, curr, opin, chem, biol., 602 (2000). These bases are usually mismatched to some extent with the natural base and cannot be enzymatically replicated. Kool and co-workers demonstrated that hydrophobic stacking interactions between bases can drive base pair formation instead of hydrogen bonding. See Kool, curr, opin, chem, biol, 4; and Guckian and Kool, angelw.chem.int.ed.engl., 36,2825 (1998). In an attempt to develop an unnatural base pair that meets all of the above requirements, schultz, romesberg and collaborators systematically synthesized and studied a series of unnatural hydrophobic bases. PICS is found to be more stable to itself than to the natural base pairs and can be incorporated into DNA with high efficiency by the Klenow Fragment (KF) of E.coli DNA polymerase I. See, e.g., mcMinn et al, j.am.chem.soc., 121; and Ogawa et al, j.am.chem.soc., 122. 3mn self-pairing can be synthesized by KF with sufficient efficiency and selectivity for biological function. See, e.g., ogawa et al, j.am.chem.soc., 122. However, both bases act as chain terminators for further replication. Mutant DNA polymerases have recently evolved that can be used to replicate PICS self-pairing. In addition, the 7AI self-pairing can be replicated. See, e.g., tae et al, j.am.chem.soc., 123. A novel metal base pair Dipic: py has also been developed, which forms a stable pair upon binding Cu (II). See Meggers et al, j.am.chem.soc., 122. Since the extended codons and unnatural codons are inherently orthogonal to the natural codons, the methods of the invention can take advantage of this property to generate orthogonal trnas for them.

Translation bypass systems can also be used to incorporate unnatural amino acids into desired polypeptides. In the translational bypass system, large sequences are incorporated into genes but are not translated into proteins. The sequence contains a structure that serves as a cue to induce ribosomes to skip the sequence and restore translation downstream of the insertion.

In certain embodiments, in the methods and/or compositions of the present 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.

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 incorporation of the unnatural amino acid. For example, a nucleic acid for a protein of interest is mutagenized to include one or more selector codons for the incorporation of one or more unnatural amino acids. The invention includes any such variant, including but not limited to mutant forms, of any protein, e.g., including at least one unnatural amino acid. Likewise, the invention also includes the corresponding nucleic acids, i.e., any nucleic acid having one or more selector codons encoding one or more unnatural amino acid.

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

Non-naturally encoded amino acid

A wide variety 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, the introduced non-naturally encoded amino acid is 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 acid comprises a side chain functional group that reacts efficiently and selectively with functional groups not present in the 20 common amino acids (including, but not limited to, azido, ketone, aldehyde, and aminoxy groups) to form a conjugate. For example, IL-2 comprising a non-naturally encoded amino acid comprising an azido functional group can be reacted with a polymer (including, but not limited to, polyethylene glycol or a second polypeptide comprising an alkynyl moiety) to form a stable conjugate because of the selective reaction of the azido and alkyne functional groups to form a Huisgen [3+2] cycloaddition product.

The general structure of the α -amino acids is shown below (formula I):

I

the non-naturally encoded amino acid is generally any structure having the structural formula listed above, wherein the R group is any substituent other than those used in the 20 natural amino acids, and can be suitably used in the present invention. Since the non-naturally encoded amino acids of the invention typically differ from the 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, naturally or non-naturally encoded amino acids) in the same manner as they are formed in naturally occurring polypeptides. However, the non-naturally encoded amino acid has a different side chain group than the natural amino acid. For example, R optionally includes alkyl-, aryl-, acyl-, ketone-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynyl, ether, thiol, seleno-, sulfonyl-, borate, boronic acid, phosphoryl, phosphono, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino, and the like, 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 new functional groups, amino acids that interact covalently or non-covalently with other molecules, photocaged and/or photoisomerizable amino acids, amino acids comprising biotin or biotin analogs, glycosylated amino acids such as sugar-substituted serines, 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 extended side chains compared to natural amino acids (including but not limited to polyethers or long chain hydrocarbons, including but not limited to long chain hydrocarbons 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.

Exemplary non-naturally encoded amino acids that may be suitable for use in the present invention and that may be used to react with the water soluble polymer include, but are not limited to, non-naturally encoded amino acids having carbonyl, aminoxy, hydrazine, hydrazide, semicarbazide, azido, and alkyne reactive groups. In certain embodiments, the non-naturally encoded amino acid comprises a sugar moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine. Examples of such amino acids also include those in which the naturally occurring N-or O-linkage between the amino acid and the sugar is replaced by a covalent linkage not normally found in nature, including but not limited to alkenes, oximes, thioethers, amides, and the like. Examples of such amino acids also include saccharides such as 2-deoxyglucose, 2-deoxygalactose, and the like, which are not commonly found in naturally occurring proteins.

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-commercially available non-naturally encoded amino acids are optionally synthesized as provided herein or using standard methods known to those of ordinary skill in the art. For Organic synthesis techniques, see, e.g., fessendon and Fessendon, second edition Organic Chemistry, willad Grant Press, boston Mass (1982); march's third edition of Advanced Organic Chemistry, wiley and Sons, new York (1985); and third edition of Advanced Organic Chemistry, parts A and B, plenum Press, new York (1990), by Carey and Sundberg. See also U.S. Pat. nos. 7,045,337 and 7,083,970, which are incorporated herein by reference. In addition to unnatural amino acids containing novel side chains, unnatural amino acids that may be suitable for use in the invention also optionally comprise modified backbone structures, including but not limited to those illustrated by the structures of formulas II and III:

II

III

Wherein Z typically comprises OH, NH ₂ SH, NH-R 'or S-R'; x and Y, which may be the same or different, typically comprise S or O, and optionally the same or different R and R' are typically selected from the same list of constituents as the R groups described above for the unnatural amino acid of formula I and hydrogen. For example, the unnatural amino acids of the invention optionally comprise substitutions in the amino or carboxyl group, as shown for formulas II and III. This type of unnatural amino acid includes, but is not limited to, alpha-hydroxy acids, alpha-thio acids, alpha-aminothiocarboxylates, including, but not limited to, these compounds having side chains or unnatural side chains corresponding to the common 20 natural amino acids. In addition, substitutions at the α -carbon optionally include, but are not limited to, L, D or α - α -disubstituted amino acids such as D-glutamic acid, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Other structural alternatives include cyclic amino acids such as proline analogs and 3, 4, 6, 7, 8 and 9 membered ring prolidesAmino acid analogs, beta and gamma amino acids such as substituted beta-alanine and gamma-aminobutyric acid.

Many unnatural amino acids are based on natural amino acids such as tyrosine, glutamine, phenylalanine, and the like, 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 comprise, but are not limited to, keto groups (including, but not limited to, acetyl groups), benzoyl groups, amino groups, hydrazine, hydroxylamine, thiol groups, carboxyl groups, isopropyl groups, methyl groups, C ₆ -C ₂₀ Straight or branched chain hydrocarbons, saturated or unsaturated hydrocarbons, O-methyl, polyether groups, nitro groups, alkynyl groups, and the like. Also, polysubstituted aryl rings are contemplated. Glutamine analogs that may be suitable for use in the present invention include, but are not limited to, alpha-hydroxy derivatives, gamma-substituted derivatives, cyclic derivatives, and amide-substituted glutamine derivatives. Examples of phenylalanine analogs that may be suitable for use in the present invention include, but are not limited to, para-substituted phenylalanines, ortho-substituted phenylalanines, and meta-substituted phenylalanines, wherein the substituents include, but are not limited to, hydroxy, methoxy, methyl, allyl, aldehyde, azido, iodo, bromo, keto (including, but not limited to, acetyl), benzoyl, alkynyl, and the like. Specific examples of unnatural amino acids that can be suitable for use in the 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-GlcNAc β -serine, L-dopa, fluorophenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphinylserine, phosphotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, and p-propargyloxy-phenylalanine, and the like. Examples of the structure of various unnatural amino acids that can be used In the 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, azide Incorporation into recombinant proteins by Staudinger ligation for chemoselective modification (Incorporation of azides into recombinant proteins by chemoselective modification), PNAS 99 (2002), incorporated herein by reference. International application No. PCT/US06/47822 entitled "Compositions Containing unnatural Amino Acids and Polypeptides, methods relating to them, and Uses thereof" (Compositions containment, methods Involuting, and Uses of Non-natural Amino Acids and Polypeptides) which is incorporated herein by reference describes the reductive alkylation and reductive amination of aromatic amine moieties, including but not limited to p-Amino-phenylalanine.

In another embodiment of the invention, the IL-2 polypeptide having one or more non-naturally encoded amino acids is covalently modified. Selective chemical reactions orthogonal to diverse functional groups of biological systems are considered to be important tools in chemical biology. As a relative successor in the synthetic chemistry large family, these bioorthogonal reactions have motivated new strategies for compound library synthesis, protein engineering, functional proteomics, and cellular surface chemical remodeling. Azides play an important role as unique chemical treatments for bioconjugation. Staudinger ligation has been used with phosphonylates to label azido sugars metabolically introduced into cellular glycoconjugates. The Staudinger connection can be made in living animals and is not physiologically harmful; however, the Staudinger reaction is not without disadvantages. The requisite phosphonites are susceptible to air oxidation and optimization to increase water solubility and increase reaction rates has proven to be synthetically challenging.

The azido group has an alternative bioorthogonal reactivity pattern: cycloaddition with alkynes [3+2] as described by Huisgen. In its classical form, this reaction has limited applicability in biological systems due to the elevated temperatures (or pressures) required for reasonable reaction rates. Sharpless and co-workers overcome this obstacle by developing a copper (I) -catalyzed form known as "click chemistry," which is readily performed at physiological temperatures and in a rich functionalized biological environment. This finding enables selective modification of virions, nucleic acids and proteins from complex tissue lysates. Unfortunately, the mandatory copper catalysts are toxic to both bacteria and mammalian cells, thus precluding applications in which cells must remain viable. Catalyst-free Huisgen cycloadditions of alkynes activated by electron-withdrawing substituents have been reported to occur at ambient temperatures. However, these compounds undergo a michael reaction with the biological nucleophile.

In one embodiment, a composition is provided for IL-2 comprising an unnatural amino acid (e.g., p- (propargyloxy) -phenylalanine). Also provided are various compositions comprising p- (propargyloxy) -phenylalanine, including but not limited to proteins and/or cells. In one aspect, a 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 bonded to the orthogonal tRNA through an amino-acyl bond, covalently bonded to 3'OH or 2' OH of a terminal ribose sugar of the orthogonal tRNA, and the like.

Chemical moieties that can be incorporated into proteins by unnatural amino acids provide the proteins with a variety of advantages and manipulations. For example, the unique reactivity of the ketone functional group allows for selective modification of proteins in vitro or in vivo with any of a number of hydrazine-or hydroxylamine-containing reagents. For example, heavy atom unnatural amino acids may be useful for phasing of X-ray structural data. Site-specific introduction of heavy atoms using unnatural amino acids also provides selectivity and flexibility in selecting positions for heavy atoms. Photoreactive unnatural amino acids, including but not limited to amino acids with benzophenone and arylazides (including but not limited to phenylazide) side chains, allow for efficient in vivo and in vitro photocrosslinking of, for example, proteins. Examples of photoreactive unnatural amino acids include, but are not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine. Proteins with photoreactive unnatural amino acids can thus be cross-linked at will by excitation of the photoreactive groups, providing temporal control. In one example, the methyl group of the unnatural amino acid can be substituted with a methyl group including, but not limited to, isotopically labeled, as a probe of local structure and kinetics, including, but not limited to, the use of nuclear magnetic resonance and vibrational spectroscopy. The alkynyl or azido functional groups allow for selective modification of proteins with molecules, for example by the [3+2] cycloaddition reaction.

The unnatural amino acid incorporated into a polypeptide at the amino terminus can be made up of an R group as any substituent other than those used in the 20 natural amino acids and an NH group different from that normally present in an alpha-amino acid ₂ The second reactive group of the group (see formula I). Similar unnatural amino acids can be incorporated at the carboxy terminus, which have a second reactive group different from the COOH group typically present in α -amino acids (see formula I).

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

In certain embodiments, the invention provides IL-2 linked to a water-soluble polymer, such as PEG, by an oxime linkage. Many types of non-naturally encoded amino acids are suitable for forming oxime linkages. They include, but are not limited to, non-naturally encoded amino acids containing a carbonyl, dicarbonyl, or hydroxylamine group. 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 unnatural Amino Acids and Polypeptides, methods relating to them, and Uses of Non-natural Amino Acids and Polypeptides," which are incorporated by reference herein in their entirety. Non-naturally encoded amino acids are also described in U.S. Pat. No. 7,083,970 and U.S. Pat. No. 7,045,337, which are incorporated herein by reference in their entirety.

Certain embodiments of the invention utilize IL-2 polypeptides that are substituted at one or more positions with a p-acetylphenylalanine amino acid. The synthesis of acetyl- (+/-) -phenylalanine and meta-acetyl- (+/-) -phenylalanine is described in Zhang, Z, et al, biochemistry 42 (2003), incorporated by reference. Other carbonyl-or dicarbonyl-containing amino acids can be similarly prepared by one of ordinary skill in the art. Further, a non-limiting exemplary synthesis of unnatural amino acids included herein is presented in FIGS. 4, 24-34, and 36-39 of U.S. Pat. No. 7,083,970, which is incorporated by reference herein in its entirety.

Amino acids with electrophilic reactive groups allow the use of a variety of different reactions, in particular the attachment of molecules by nucleophilic addition reactions. Such electrophilic reactive groups include carbonyl groups (including keto and dicarbonyl), carbonyl-like groups (which have similar reactivity and are structurally similar to carbonyl groups (including keto and dicarbonyl)), masked carbonyl groups (which can be readily converted to carbonyl groups (including keto and dicarbonyl)), or protected carbonyl groups (which after deprotection have similar reactivity to carbonyl groups (including keto and dicarbonyl)). These amino acids include amino acids having the structure of formula (IV):

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;

b is optional and when present is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) _k - (where k is 1, 2 or 3), -S (O) _k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -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 ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) _k N(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O) _k N(R’)-、-N(R’)-N＝、-C(R’)＝N-、-C(R’)＝N-N(R’)-、-C(R’)＝N-N＝、-C(R’) ₂ -N = N-and-C (R') ₂ -N (R ') -, wherein each R' is independently H, alkyl or substituted alkyl;

j is

R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

each R "is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R" group is present, both R "optionally form a heterocycloalkyl group;

R ₁ is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and is

R ₂ Is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

each R ₃ And R ₄ Independently is H, halogen, lower alkyl or substituted lower alkyl, or R ₃ And R ₄ Or two R ₃ The groups optionally form a cycloalkyl or heterocycloalkyl group;

or-A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl containing at least one carbonyl group, including dicarbonyl, protected carbonyl (including protected dicarbonyl), or masked carbonyl (including masked dicarbonyl);

or-J-R groups together form a mono-or bicyclic cycloalkyl or heterocycloalkyl containing at least one carbonyl group, including dicarbonyl, protected carbonyl (including protected dicarbonyl), or masked carbonyl (including masked dicarbonyl);

provided that when A is phenylene and each R is ₃ When is H, B is present; and when A is- (CH) ₂ ) ₄ And each R ₃ When H, B is not-NHC (O) (CH) ₂ CH ₂ ) -; and when A and B are absent and each R is ₃ When H, R is not methyl.

Further, amino acids having the structure of formula (V):

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;

B is optional and when present is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, and mixtures thereofLower heteroalkylene radicals of the formulae, -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) - (alkylene or substituted alkylene) -, -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 ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) _k N(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O) _k N(R’)-、-N(R’)-N＝、-C(R’)＝N-、-C(R’)＝N-N(R’)-、-C(R’)＝N-N＝、-C(R’) ₂ -N = N-and-C (R') ₂ -N (R ') -wherein each R' is independently H, alkyl or substituted alkyl;

r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

R ₁ is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and is provided with

R ₂ Is optional and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;

Provided that when A is phenylene, B is present; and when A is- (CH) ₂ ) ₄ When B is not-NHC (O) (CH) ₂ CH ₂ ) -; and when A and B are absent, R is not methyl.

Further, amino acids having the structure of formula (VI):

wherein:

b is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene)Alkyl or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) _k - (where k is 1, 2 or 3), -S (O) _k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -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 ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) _k N(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O) _k N(R’)-、-N(R’)-N＝、-C(R’)＝N-、-C(R’)＝N-N(R’)-、-C(R’)＝N-N＝、-C(R’) ₂ -N = N-and-C (R') ₂ -N (R ') -, wherein each R' is independently H, alkyl or substituted alkyl;

r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

R ₁ Is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and is

R ₂ 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.

In addition, the following amino acids are included:

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

Further, the following amino acids having the structure of formula (VII) are included:

wherein

B is optional and when present is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) _k - (where k is 1, 2 or 3), -S (O) _k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -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 ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) _k N(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O) _k N(R’)-、-N(R’)-N＝、-C(R’)＝N-、-C(R’)＝N-N(R’)-、-C(R’)＝N-N＝、-C(R’) ₂ -N = N-and-C (R') ₂ -N (R ') -wherein each R' is independently H, alkyl or substituted alkyl;

r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

R ₁ is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and is

R ₂ 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'Independently is H, alkyl or substituted alkyl; and n is 0 to 8;

provided that when A is- (CH) ₂ ) ₄ When B is not-NHC (O) (CH) ₂ CH ₂ )-。

In addition, the following amino acids are included:

wherein the compounds are optionally amino-protected, optionally carboxy-protected, optionally amino-protected and carboxy-protected, or salts thereof. In addition, these unnatural amino acids, and any of the unnatural amino acids described below, can be incorporated into an unnatural amino acid polypeptide.

Further, the following amino acids having the structure of formula (VIII) are included:

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;

B is optional and when present is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) _k - (where k is 1, 2 or 3), -S (O) _k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -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 ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) _k N(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O) _k N(R’)-、-N(R’)-N＝、-C(R’)＝N-、-C(R’)＝N-N(R’)-、-C(R’)＝N-N＝、-C(R’) ₂ -N = N-and-C (R') ₂ -N (R ') -, wherein each R' is independently H, alkyl or substituted alkyl;

R ₁ is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and is

R ₂ Is optional, and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

Further, the following amino acids having the structure of formula (IX) are included:

b is an optional one, and B is, and when present is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) _k - (where k is 1, 2 or 3), -S (O) _k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -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 ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) _k N(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O) _k N(R’)-、-N(R’)-N＝、-C(R’)＝N-、-C(R’)＝N-N(R’)-、-C(R’)＝N-N＝、-C(R’) ₂ -N = N-and-C (R') ₂ -N (R ') -wherein each R' is independently H, alkyl or substituted alkyl;

r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

R ₁ is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and is

R ₂ Is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

wherein 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.

In addition, the following amino acids are included:

wherein these compounds are optionally amino-protected, optionally carboxy-protected, optionally amino-protected and carboxy-protected, or salts thereof. In addition, these unnatural amino acids, and any of the unnatural amino acids described below, can be incorporated into unnatural amino acid polypeptides.

Further, amino acids having the structure of formula (X) below are included:

wherein B is an optional group selected from the group consisting of, and when present is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (R) ((R))O) _k - (where k is 1, 2 or 3), -S (O) _k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -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 ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) _k N(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O) _k N(R’)-、-N(R’)-N＝、-C(R’)＝N-、-C(R’)＝N-N(R’)-、-C(R’)＝N-N＝、-C(R’) ₂ -N = N-and-C (R') ₂ -N (R ') -wherein each R' is independently H, alkyl or substituted alkyl;

r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

R ₁ is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and is

R ₂ 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.

In addition, the following amino acids are included:

wherein these compounds are optionally amino-protected, optionally carboxy-protected, optionally amino-protected and carboxy-protected, or salts thereof. In addition, these unnatural amino acids, and any of the unnatural amino acids described below, can be incorporated into unnatural amino acid polypeptides.

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

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

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;

b is optional and when present is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) _k - (where k is 1, 2 or 3), -S (O) _k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -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 ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) _k N(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O) _k N(R’)-、-N(R’)-N＝、-C(R’)＝N-、-C(R’)＝N-N(R’)-、-C(R’)＝N-N＝、-C(R’) ₂ -N = N-and-C (R') ₂ -N (R ') -, wherein each R' is independently H, alkyl or substituted alkyl;

r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

R ₁ is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and is

R ₂ Is optional, and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

Further, the following amino acids having the structure of formula (XII) are included:

b is an optional one, and B is, and when present is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) _k - (where k is 1, 2 or 3), -S (O) _k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -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 ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) _k N(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O) _k N(R’)-、-N(R’)-N＝、-C(R’)＝N-、-C(R’)＝N-N(R’)-、-C(R’)＝N-N＝、-C(R’) ₂ -N = N-and-C (R') ₂ -N (R ') -wherein each R' is independently H, alkyl or substituted alkyl;

r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

R ₁ is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and is

R ₂ Is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

wherein 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.

In addition, the following amino acids are included:

wherein the compounds are optionally amino-protected, optionally carboxy-protected, optionally amino-protected and carboxy-protected, or salts thereof. In addition, these unnatural amino acids, and any of the unnatural amino acids described below, can be incorporated into an unnatural amino acid polypeptide.

Further, amino acids having the following structure of formula (XIII) are included:

wherein B is optional and when present is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) _k - (where k is 1, 2 or 3), -S (O) _k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -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 ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) _k N(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O) _k N(R’)-、-N(R’)-N＝、-C(R’)＝N-、-C(R’)＝N-N(R’)-、-C(R’)＝N-N＝、-C(R’) ₂ -N = N-and-C (R') ₂ -N (R ') -, wherein each R' is independently H, alkyl or substituted alkyl;

r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; r is ₁ Is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R is ₂ 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.

In addition, the following amino acids are included:

wherein the compounds are optionally amino-protected, optionally carboxy-protected, optionally amino-protected and carboxy-protected, or salts thereof. In addition, these unnatural amino acids, and any of the unnatural amino acids described below, can be incorporated into an unnatural amino acid polypeptide.

Further, the following amino acids having the structure of formula (XIV) are included:

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;

r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; r is ₁ Is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R is ₂ Is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; x ₁ 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.

Further, the following amino acids having the structure of formulSup>A (XIV-A) are included:

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;

r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; r ₁ Is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R is ₂ Is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

l is alkylene, substituted alkylene, N (R ') (alkylene), or N (R ') (substituted alkylene), wherein R ' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Further, the following amino acids having the structure of formula (XIV-B) are included:

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; r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

R ₁ is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R is ₂ Is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; l is alkylene, substituted alkylene, N (R ') (alkylene), or N (R ') (substituted alkylene), wherein R ' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Further, the following amino acids having the structure of formula (XV) are included:

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; r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

R ₁ is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R is ₂ Is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; x ₁ Is C, S or S (O); and n is 0, 1, 2, 3, 4 or 5; and each CR ⁸ R ⁹ Each R in the group ⁸ And R ⁹ Independently selected from H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R ⁸ And R ⁹ May together form = O or cycloalkyl, or any adjacent R ⁸ The groups may together form a cycloalkyl group.

Also included are the following amino acids having the structure of formulSup>A (XV-A):

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;

r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; r ₁ Is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R is ₂ Is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotideAn acid;

n is 0, 1, 2, 3, 4 or 5; and each CR ⁸ R ⁹ Each R in the group ⁸ And R ⁹ Independently selected from H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R ⁸ And R ⁹ May together form = O or cycloalkyl, or any adjacent R ⁸ The groups may together form a cycloalkyl group.

Also included are the following amino acids having the structure of formula (XV-B):

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;

r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; r ₁ Is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R is ₂ 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 in the group ⁸ And R ⁹ Independently selected from H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R ⁸ And R ⁹ May together form = O or cycloalkyl, or any adjacent R ⁸ The groups may together form a cycloalkyl group.

Also included are the following amino acids having the structure of formula (XVI):

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; r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

R ₁ is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and is

R ₂ Is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; x ₁ 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.

Further, the following amino acids having the structure of formula (XVI-A) are included:

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; r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

R ₁ Is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R is ₂ Is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; l is alkylene, substituted alkylene, N (R ') (alkylene), or N (R ') (substituted alkylene), wherein R ' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Further, the following amino acids having the structure of formula (XVI-B) are included:

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; r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

R ₁ is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R is ₂ Is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; l is alkylene, substituted alkylene, N (R ') (alkylene), or N (R ') (substituted alkylene), wherein R ' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Also included are amino acids having the structure of formula (XVII):

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;

m is-C (R) ₃ )-、

Wherein (a) indicates bonding to the A group, and (b) indicates bonding to the corresponding carbonyl group, R ₃ And R ₄ Independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl, or R ₃ And R ₄ Or two R ₃ Radicals or two R ₄ The groups optionally form a cycloalkyl or heterocycloalkyl group; r is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; t is ₃ Is a bond, C (R) (R), O or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; r ₁ Is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R is ₂ Is optional, and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

Also included are amino acids having the structure of formula (XVIII):

wherein:

m is-C(R ₃ )-、

(a) (a) indicates bonding to the A group, and (b) indicates bonding to the corresponding carbonyl group, R ₃ And R ₄ Independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl, or R ₃ And R ₄ Or two R ₃ Radicals or two R ₄ The groups optionally form a cycloalkyl or heterocycloalkyl group; r is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; t is ₃ Is a bond, C (R) (R), O or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; r ₁ Is optional, and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R is ₂ 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.

Further, amino acids having the structure of formula (XIX):

wherein:

r is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; and is provided with

T ₃ Is O or S.

Further, amino acids having the structure of formula (XX) are included:

wherein:

r is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.

Further, the following amino acids having the structure of formula (XXI) are included:

in certain embodiments, a polypeptide comprising an unnatural amino acid is chemically modified to produce a reactive carbonyl or dicarbonyl function. For example, aldehyde functionality useful for the coupling reaction can be generated from functional groups having adjacent amino and hydroxyl groups. For example, where the biologically active molecule is a polypeptide, the N-terminal serine or threonine (which may be normally present or may be exposed by chemical or enzymatic digestion) can be used to generate an aldehyde function using periodate under mild oxidative cleavage conditions. See, e.g., gaertner et al, bioconjugate. Chem.3:262-268 (1992); geoghegan, K. & Stroh, J., bioconjugate. Chem.3:138-146 (1992); gaertner et al, J.biol.chem.269:7224-7230 (1994). However, the methods known in the art are limited to the amino acid at the N-terminus of the peptide or protein.

In the present invention, unnatural amino acids with adjacent hydroxyl and amino groups can be incorporated into polypeptides as "masked" aldehyde functionalities. For example, 5-hydroxylysine has a hydroxyl group adjacent to the epsilon amine group. Reaction conditions for aldehyde production typically include the addition of 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 an 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.

The carbonyl or dicarbonyl functional groups can be selectively reacted with hydroxylamine containing reagents in aqueous solution under mild conditions to form the corresponding oxime linkage which is stable under physiological conditions. See, e.g., jencks, W.P., J.Am.chem.Soc.81,475-481 (1959); shao, J and Tam, J.P., J.Am.chem.Soc.117:3893-3899 (1995). In addition, the unique reactivity of the carbonyl or dicarbonyl group allows for selective modification in the presence of other amino acid side chains. See, e.g., cornish, V.W., et al, J.Am.chem.Soc.118:8150-8151 (1996); geoghegan, K.F, & Stroh, J.G., bioconjug.Chem.3:138-146 (1992); mahal, L.K., et al, science 276.

A. Carbonyl reactive groups

Amino acids with carbonyl-reactive groups allow a variety of different reactions to be performed to attach molecules (including but not limited to PEG or other water-soluble molecules), especially by nucleophilic addition or aldol condensation reactions.

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

wherein n is 0 to 10; r ₁ Is alkyl, aryl, substituted alkyl or substituted aryl; r ₂ Is H, alkyl, aryl, substituted alkyl and substituted aryl; and R is ₃ Is H, an amino acid, a polypeptide or an amino-terminal modifying group, and R ₄ Is H, amino acid, polypeptide or carboxyl terminal modification group. In certain embodiments, n is 1,R ₁ Is phenyl and R ₂ Is a simple alkyl group (i.e., methyl, ethyl, or propyl), and the ketone moiety is located in the para position relative to the alkyl side chain. In certain embodiments, n is 1,R ₁ Is phenyl and R ₂ Is a simple alkyl group (i.e., methyl, ethyl, or propyl), and the ketone moiety is located in the meta position relative to the alkyl side chain.

The synthesis of acetyl- (+/-) -phenylalanine and meta-acetyl- (+/-) -phenylalanine is described in Zhang, z. Et al, biochemistry 42 (2003), which is incorporated herein by reference. Other carbonyl-containing amino acids can be similarly prepared by one of ordinary skill in the art.

In certain embodiments, a polypeptide comprising a non-naturally encoded amino acid is chemically modified to produce a reactive carbonyl functionality. For example, the aldehyde functionality for the coupling reaction can be generated from functional groups having adjacent amino and hydroxyl groups. For example, where the biologically active molecule is a polypeptide, the N-terminal serine or threonine (which may normally be present or may be exposed by chemical or enzymatic digestion) can be used to generate an aldehyde function using periodate under mild oxidative cleavage conditions. See, e.g., gaertner et al, bioconjugate. Chem.3:262-268 (1992); geoghegan, K. & Stroh, J., bioconjugate. Chem.3:138-146 (1992); gaertner et al, J.biol.chem.269:7224-7230 (1994). However, the methods known in the art are limited to the amino acid at the N-terminus of the peptide or protein.

In the present invention, non-naturally encoded amino acids with adjacent hydroxyl and amino groups can be incorporated into polypeptides as "masked" aldehyde functionalities. For example, 5-hydroxylysine has a hydroxyl group adjacent to the epsilon amine group. The reaction conditions for aldehyde production typically include the addition of 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 an 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, e.g., U.S. patent No. 6,423,685, which is incorporated herein by reference.

The carbonyl functional group can selectively react with a hydrazine, hydrazide, hydroxylamine or semicarbazide containing reagent in aqueous solution under mild conditions to form the corresponding hydrazone, oxime or semicarbazone bond, respectively, that is stable under physiological conditions. See, e.g., jencks, w.p., j.am.chem.soc.81,475-481 (1959); shao, J.and Tam, J.P., J.Am.chem.Soc.117:3893-3899 (1995). In addition, the unique reactivity of the carbonyl group allows for selective modification in the presence of other amino acid side chains. See, e.g., cornish, V.W., et al, J.Am.chem.Soc.118:8150-8151 (1996); geoghegan, K.F, & Stroh, J.G., bioconjugate. Chem.3:138-146 (1992); mahal, L.K. et al, science 276.

B. Hydrazine, hydrazide or semicarbazide reactive groups

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

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

wherein n is 0 to 10; r ₁ Is alkyl, aryl, substituted alkyl or substituted aryl or is absent; x is O, N or S or absent; r ₂ Is H, an amino acid, a polypeptide or an amino-terminal modifying group, and R ₃ Is H, an amino acid, a polypeptide, or a carboxy-terminal modifying group.

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

Hydrazide, hydrazine, and semicarbazide-containing amino acids are available 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.

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, e.g., shao, J. and Tam, J., J.am.chem.Soc.117:3893-3899 (1995). The unique reactivity of the hydrazide, hydrazine, and semicarbazide functional groups makes them significantly more reactive towards aldehydes, ketones, and other electrophilic groups than the nucleophilic groups present on the 20 common amino acids (including but not limited to the hydroxyl groups of serine or threonine or lysine and the amino group at the N-terminus).

C. Amino acids containing amino groups

Non-naturally encoded amino acids containing aminooxy (also referred to as hydroxylamine) groups allow for reaction with a variety of different electrophilic groups to form conjugates, including but not limited to conjugates with PEG or other water-soluble polymers. Like hydrazine, hydrazides, and semicarbazides, the high nucleophilicity of the aminoxy 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, e.g., 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). While the result of the reaction with a hydrazine group is the corresponding hydrazone, an oxime is generally obtained from the reaction of an aminooxy group with a carbonyl-containing group such as a ketone.

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

wherein n is 0 to 10; r is ₁ Is alkyl, aryl, substituted alkyl or substituted aryl or is absent; x is O, N, S or absent; m is 0 to 10; y = C (O) or absent; r ₂ Is H, an amino acid, a polypeptide or an amino-terminal modifying group, and R ₃ Is H, amino acid, polypeptide or carboxyl terminal modification group. In certain embodiments, n is 1,R ₁ Is phenyl, X is O, m is 1, and Y is present. In certain embodiments, n is 2,R ₁ And X is absent, m is 0, and Y is absent.

Amino acids containing an amino group can be prepared from readily available amino acid precursors (homoserine, serine and threonine). See, e.g., M.Carrasco and R.Brown, J.org.chem.68:8853-8858 (2003). Certain amino acid containing an aminooxy group such as L-2-amino-4- (aminooxy) butanoic acid have been isolated from natural sources (Rosenthal, G., life Sci.60:1635-1641 (1997)). Other amino acids containing an amino group can be prepared by one of ordinary skill in the art.

D. Azido and alkyne reactive groups

The unique reactivity of the azide and alkyne functional groups makes them extremely useful for the selective modification of polypeptides and other biomolecules. Organic azides, particularly aliphatic azides and alkynes, are generally stable to commonly used reactive chemical conditions. Specifically, both the azide and alkyne functional groups are inert to the side chains (i.e., R groups) of the 20 common amino acids found in naturally occurring polypeptides. However, when brought into proximity, the "spring-loaded" nature of the azido and alkynyl groups is revealed and they react selectively and efficiently by the Huisgen 3+2 cycloaddition reaction to produce the corresponding triazoles. See, e.g., chin J. Et al, science 301; wang, Q, et al, J.Am.chem.Soc.125,3192-3193 (2003); chin, J.W., et al, J.am.chem.Soc.124:9026-9027 (2002).

Since the Huisgen cycloaddition reaction involves selective cycloaddition reactions (see, e.g., padwa, a., "Comprehensive Organic Synthesis", vol.4, trost, b.m. eds. (1991), p.1069-1109, huisgen, r., "1,3-dipolar cycloaddition chemistry" (1,3-D) _IPOLAR CYCLOADDITION C _HEMISTRY ) Padwa, a. Eds. (1984), p.1-176), rather than nucleophilic substitution, thus incorporation of a non-naturally encoded amino acid bearing azido and alkynyl-containing side chains allows the resulting polypeptide to be selectively modified at the position of the non-naturally encoded amino acid. Cycloaddition reactions involving IL-2 containing an azido or alkynyl group can be carried out at room temperature and under aqueous conditions by adding catalytic amounts of Cu (II) (including but not limited to, in catalytic amounts of CuSO) in the presence of a reducing agent for reducing Cu (II) to Cu (I) ₄ In situ). See, e.g., wang, q, et al, j.am.chem.soc.125,3192-3193 (2003); tornoe, C.W., et al, J.org.chem.67:3057-3064 (2002); rostovtsev et al, angew.chem.Int.Ed.41:2596-2599 (2002). Exemplary reducing agents include, but are not limited to, ascorbate, metallic copper, quinine, hydroquinone, vitamin K, glutathione, cysteine, fe ²⁺ 、Co ²⁺ And the applied potential.

In certain instances, where 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 alkynyl moiety and a water soluble polymer containing an azido moiety to be attached to the amino acid. Alternatively, the reverse reaction may be carried out (i.e., using the azido moiety on the amino acid and the alkynyl moiety present on the water-soluble polymer).

The azido functional group can also be selectively reacted with water-soluble polymers containing aryl esters and suitably functionalized with aryl phosphine moieties to produce amide linkages. The arylphosphine group reduces the azide group in situ and the resulting amine then reacts efficiently with the adjacent ester linkage to produce the corresponding amide. See, e.g., e.saxon and c.bertozzi, science 287,2007-2010 (2000). The azido-containing amino acid can be an alkyl azide (including but not limited to 2-amino-6-azido-1-hexanoic acid) or an aryl azide (p-azido-phenylalanine).

Exemplary water-soluble polymers containing aryl ester and phosphine moieties can be represented as follows:

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-CH ₂ 、-C(CH ₃ ) ₃ -OR ', -NR ' R ", -SR ', -halogen, -C (O) R ', -CONR ' R", -S (O) ₂ R’、-S(O) ₂ NR' R ", -CN and-NO ₂ . 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 the R groups is independently selected, as is each of the R ', R ", R'" and R "" groups when more than one of these groups is present. When R 'and R' are attached to the same nitrogen atom, they may combine with the nitrogen atom to form 5, 6 orA 7-membered ring. For example, -NR' R "is meant to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will appreciate that the term "alkyl" is meant to include groups containing carbon atoms bonded to groups other than hydrogen radicals such as haloalkyl (including, but not limited to-CF) ₃ and-CH ₂ CF ₃ ) And acyl (including but not limited to-C (O) CH ₃ 、-C(O)CF ₃ 、-C(O)CH ₂ OCH ₃ Etc.).

The azido functional group can also be selectively reacted with a water-soluble polymer containing thioesters and suitably functionalized with arylphosphine moieties to produce amide linkages. The arylphosphine group reduces the azide group in situ and the resulting amine then reacts efficiently with a thioester bond to produce the corresponding amide. Exemplary water-soluble polymers containing thioester and phosphine moieties can be represented as follows:

Wherein n is 1 to 10; x may be O, N, S or absent, ph is phenyl, and W is a water soluble polymer.

Exemplary alkynyl-containing amino acids can be represented as follows:

wherein n is 0 to 10; r ₁ Is alkyl, aryl, substituted alkyl or substituted aryl or is absent; x is O, N, S or is absent; m is 0 to 10, R ₂ Is H, an amino acid, a polypeptide or an amino-terminal modifying group, and R ₃ Is H, an amino acid, a polypeptide, or a carboxy-terminal modifying group. In certain embodiments, n is 1,R ₁ Is phenyl, X is absent, m is 0 and the acetylene moiety is in the para position relative to the alkyl side chain. In certain embodiments, n is 1,R ₁ Is phenyl, X is O, m is 1 and the propargyloxy is in the para position relative to the alkyl side chain (i.e., O-propargyl-tyrosine). In certain embodiments, n is 1,R ₁ And X is absent and m is 0 (i.e. propargylglycine).

Alkynyl-containing amino acids are commercially available. For example, propargylglycine is commercially available from Peptech (Burlington, MA). Alternatively, the alkynyl-containing amino acids can be prepared according to standard methods. For example, p-propargyloxyphenylalanine may be synthesized, for example, as described in Deiters, A. Et al, J.Am.chem.Soc.125:11782-11783 (2003), and 4-alkynyl-L-phenylalanine may 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.

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

wherein n is 0 to 10; r ₁ Is alkyl, aryl, substituted alkyl, substituted aryl, or absent; x is O, N, S or absent; m is 0 to 10; r ₂ Is H, an amino acid, a polypeptide or an amino-terminal modifying group, and R ₃ Is H, amino acid, polypeptide or carboxyl terminal modification group. In certain embodiments, n is 1,R ₁ Is phenyl, X is absent, m is 0 and the azido moiety is para to the alkyl side chain. In certain embodiments, n is 0-4 and R ₁ And X is absent, and m =0. In certain embodiments, n is 1,R ₁ Is phenyl, X is O, m is 2 and the β -azidoethoxy moiety is located in the para position relative to the alkyl side chain.

The azido-containing amino acids are available from commercial sources. For example, 4-azidophenylalanine is available from Chem-Impex International, inc. (Wood Dale, IL). For those azido-containing amino acids that are not commercially available, the azido group can be prepared relatively easily using standard methods known to those of ordinary skill in the art, including but not limited to, displacement by a suitable leaving group (including but not limited to halides, mesylates, tosylates) or by opening of an appropriately protected lactone. See, for example, march's Advanced Organic Chemistry (third edition, 1985, wiley and sons, new York).

E. Aminothiol reactive groups

The unique reactivity of the β -substituted aminothiol functional groups makes them extremely useful for selective modification of aldehyde group-containing polypeptides and other biomolecules through the formation of thiazolidines. See, e.g., j.shao and j.tam, j.am.chem.soc.,117 (14) 3893-3899 (1995). In certain embodiments, a β -substituted aminothiol amino acid can be incorporated into an IL-2 polypeptide and then reacted with a water-soluble polymer comprising an aldehyde functional group. In certain embodiments, a water-soluble polymer, drug conjugate, or other payload can be conjugated to IL-2 comprising a β -substituted aminothiol amino acid through the formation of thiazolidine.

F. Additional reactive groups

Additional reactive groups and non-naturally encoded amino acids (including but not limited to p-amino-phenylalanine) that may be incorporated into the IL-2 polypeptides of the present invention are described in the following patent applications, all of which are incorporated herein by reference in their entirety: U.S. patent publication No. 2006/0194256, U.S. patent publication No. 2006/0217532, U.S. patent publication No. 2006/0217289, U.S. provisional patent No. 60/755,338; U.S. provisional patent No. 60/755,711; U.S. provisional patent nos. 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 nos. 60/743,040; international patent application No. PCT/US06/47822; U.S. provisional patent nos. 60/882,819; U.S. provisional patent nos. 60/882,500; and U.S. provisional patent No. 60/870,594. These applications also discuss reactive groups that may be present on the PEG or other polymer for coupling, including but not limited to hydroxylamine (aminooxy) groups.

Polypeptides with unnatural amino acids

Incorporation of unnatural amino acids can be performed for a variety of different purposes, including, but not limited to, modulating protein interaction with its receptor or one or more subunits of its receptor, tailoring changes in protein structure and/or function, altering size, acidity, nucleophilicity, hydrogen bond formation, hydrophobicity, accessibility of protease target sites, targeting moieties (including, but not limited to, for protein arrays), adding bioactive molecules, attaching polymers, attaching radionuclides, modulating serum half-life, modulating tissue penetration (e.g., tumors), modulating active transport, modulating tissue, cell, or organ specificity or distribution, modulating immunogenicity, modulating protease resistance, and the like. Proteins comprising unnatural amino acids can have enhanced or even entirely new catalytic or biophysical properties. For example, by including unnatural amino acids within a protein, the following properties are 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), ability to react with other molecules (including but not limited to covalent or non-covalent reactions), and the like. Compositions comprising proteins comprising at least one unnatural amino acid can be used for research including, but not limited to, novel therapeutics, diagnostics, catalytic enzymes, industrial enzymes, binding proteins (including, but not limited to, antibodies), and including, but not limited to, protein structure and function. See, e.g., dougherty, unnatural Amino Acids as Probes for Protein Structure and Function (Unnominal Amino Acids as Probes of Protein Structure and Function), current Opinion in Chemical Biology, 4.

In one aspect of the invention, a composition includes at least one protein having at least one unnatural amino acid including, but not limited to, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more. The unnatural 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 positions that can be present in the protein, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different unnatural amino acids. In another aspect, a composition includes a protein in which at least one but less than all of a particular amino acid present in the protein is replaced with an unnatural amino acid. For a given protein having more than one unnatural amino acid, the unnatural amino acids can be identical or different (including, but not limited to, the protein can include two or more different types of unnatural amino acids, or can include two identical unnatural amino acids). For a given protein having more than two unnatural amino acids, the unnatural amino acids can be the same, different, or a combination of multiple unnatural amino acids of the same type with at least one different unnatural amino acid.

Proteins or polypeptides of interest having at least one unnatural amino acid are a feature of the invention. The invention also includes polypeptides or proteins having at least one unnatural amino acid produced using the compositions and methods of the invention. Excipients (including but not limited to pharmaceutically acceptable excipients) may also be present with the protein.

By producing a protein or polypeptide of interest having at least one unnatural amino acid in a eukaryotic cell, the protein or polypeptide will typically include eukaryotic post-translational modifications. In certain embodiments, the protein comprises at least one unnatural amino acid and at least one post-translational modification that is made in vivo by a eukaryotic cell, where the post-translational modification is not made by a prokaryotic cell. For example, the post-translational modifications include, but are not limited to, acetylation, acylation, lipid modification, palmitoylation, palmitic acid addition, phosphorylation, glycolipid attachment modification, glycosylation, and the like.

One advantage of an unnatural amino acid is that it provides an additional chemical moiety that can be used to add other molecules. These modifications can be made in vivo in eukaryotic or non-eukaryotic cells or in vitro. Thus, in certain embodiments, the post-translational modification is effected by the unnatural amino acid. For example, the post-translational modification may be achieved by a nucleophilic-electrophilic reaction. Most reactions currently used for 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 nucleophilic residues in the protein. In the proteins of the invention, other more selective reactions, such as the reaction of an unnatural keto-amino acid with a hydrazide or aminooxy compound, can be used in vitro and in vivo. See, e.g., cornish et al, j.am.chem.soc.,118, 8150-8151 (1996); mahal et al, science, 276; wang et al, science 292; chin et al, J.Am.chem.Soc.124:9026-9027 (2002); chin et al, proc.Natl.Acad.Sci., 99; wang et al, proc.natl.acad.sci., 100; zhang et al, biochemistry, 42; 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 variety of reagents, including fluorophores, cross-linking agents, sugar derivatives, and cytotoxic molecules. See U.S. Pat. No. 6,927,042 entitled "Glycoprotein Synthesis", which is incorporated herein by reference. Post-translational modifications (including but not limited to those achieved through azido amino acids) can be made through Staudinger ligation (including but not limited to the use of triarylphosphine reagents). See, for example, kiick et al, for Incorporation of azides into recombinant proteins by Staudinger ligation for chemoselective modification (Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation), PNAS 99 (2002).

In vivo production of IL-2 comprising non-naturally encoded amino acids

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

Methods of producing tRNA and tRNA synthetases using amino acids that are not encoded in naturally occurring systems are described, for example, in U.S. Pat. nos. 7,045,337 and 7,083,970, which are incorporated herein by reference. These methods involve the production of a translation machine that functions independent of synthetases and tRNAs endogenous to the translation system (and thus are sometimes referred to as "orthogonal"). Typically, the translation system comprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS). Typically, the O-RS preferentially aminoacylates an O-tRNA with at least one non-naturally occurring amino acid in the translation system, and the O-tRNA recognizes at least one selector codon in the system that is not recognized by other tRNA's. Thus, the translation system, in response to the encoded selector codon, inserts the non-naturally encoded amino acid into the protein produced in the system, thereby "substituting" an amino acid into position in the encoded polypeptide.

A wide variety of orthogonal tRNA 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 invention. For example, keto-specific O-tRNA/aminoacyl-tRNA synthetases are described in Wang, L.et al, proc.Natl.Acad.Sci.USA 100, 56-61 (2003) and Zhang, Z.et al, biochem.42 (22): 6735-6746 (2003). Exemplary O-RSs, or portions thereof, are encoded by polynucleotide sequences and include the amino acid sequences disclosed in U.S. Pat. Nos. 7,045,337 and 7,083,970, each of which is incorporated herein by reference. Corresponding O-tRNA molecules for use with the O-RS are also described in U.S. Pat. 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.

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

Several other orthogonal pairs have been reported. Glutaminyl derived from Saccharomyces cerevisiae tRNA and synthetases (see, e.g., liu, D.R., and Schultz, P.G. (1999) Proc.Natl.Acad.Sci.U.S.A.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). Systems derived from E.coli glutaminyl (see, e.g., kowal, A.K., et al, (2001) Proc. Natl. Acad. Sci. U.S.A.98: 2268-2273) and tyrosyl (see, e.g., edwards, H. And Schimmel, P. (1990) mol. Cell. Biol.10: 1633-1641) synthetases have been described for use in s.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.

The use of O-tRNA/aminoacyl-tRNA synthetases involves the selection of a specific codon (selector codon) that encodes an amino acid that is not naturally encoded. Although any codon can be used, it is generally desirable to select codons that are rare or never used in cells expressing the O-tRNA/aminoacyl-tRNA synthetase. For example, exemplary codons include nonsense codons such as stop codons (amber, ochre, and ovalbumin), four or more base codons, and other rare or unused natural three base codons.

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, and the like).

Position of non-naturally occurring amino acid in IL-2

The present invention contemplates the incorporation of one or more non-naturally occurring amino acids into IL-2. One or more non-naturally occurring amino acids can be incorporated at specific locations that do not destroy 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.

Various biochemical and structural approaches can be used to select desired sites for substitution of non-naturally encoded amino acids within the IL-2. It will be apparent to one of ordinary skill in the art that any position of the polypeptide chain is suitable for selection to incorporate a non-naturally encoded amino acid, and that selection can be based on rational design or by random selection for any or no particular desired purpose. Selection of the desired site can be used to produce an IL-2 molecule having any desired property or activity, 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, no change in activity or property relative to the native molecule, or manipulation of any physical or chemical property of the polypeptide such as solubility, aggregation or stability. For example, the polypeptide of IL-2 biological activity required position, can use the known in the art of point mutation analysis, alanine scanning, saturation mutagenesis and biological activity screening or homologous scanning method to identify. Other methods can be used to identify residues for modification of IL-2, including but not limited to sequence profiling (Bowie and Eisenberg, science 253 (5016): 164-70 (1991)), rotamer library selection (Dahiyat and Mayo, protein Sci 5 (5): 895-903 (1996); dahiyat and Mayo, science 278 (5335): 82-7 (1997); desjarlais and Handel, protein Science 4 Rational design of the technology (see U.S. patent)National patent numbers 6,188,965, 6,269,312, 6,403,312, WO98/47089, incorporated herein by reference). Residues other than those identified as critical for biological activity by alanine or homology scanning mutagenesis may be good candidates for substitution with non-naturally encoded amino acids, depending on the desired activity sought for the polypeptide. Alternatively, sites identified as critical for biological activity may also be good candidates for substitution with non-naturally encoded amino acids, again depending on the desired activity sought for the polypeptide. Another alternative is to simply make a series of substitutions with non-naturally encoded amino acids in each position on the polypeptide chain and observe the effect on the activity of the polypeptide. It will be apparent to one of ordinary skill in the art that any means, technique, or method of selecting a position for substitution with an unnatural amino acid in any polypeptide is suitable for use in the invention.

Mutants of IL-2 polypeptides containing deletions may also be examined for structure and activity to determine regions of the protein that are likely to be tolerant to substitution with non-naturally encoded amino acids. In a similar manner, protease digestion and monoclonal antibodies can be used to identify the region of IL-2 responsible for binding to the IL-2 receptor. Once residues that may not be tolerated for 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.

One of ordinary skill in the art recognizes that such analysis of IL-2 enables the determination of which amino acid residues are surface exposed as compared to amino acid residues buried within the tertiary structure of the protein. Thus, an embodiment of the invention is the replacement of an amino acid that is a surface exposed residue with a non-naturally encoded amino acid.

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

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

In certain embodiments, one or more non-naturally encoded amino acids are incorporated at any position in one or more of the following regions corresponding to secondary structure or specific amino acids as described below in IL-2 or variants thereof: at the site of hydrophobic interaction; at or near the site of interaction with the IL-2 receptor subunit (including IL2 ra); within amino acid positions 3 or 35 to 45; within the first 107N-terminal amino acids; within amino acid positions 61-72; each of said positions is SEQ ID NO:2 or 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: SEQ ID NO:2 (i.e., at the N-terminus), bits 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, and any combination thereof; or 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: SEQ ID NO:2, position 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or to the terminal end of the protein, and any combination thereof; or SEQ ID NO: 3. 5 or 7.

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: bits 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: around positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72 and 107.

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 on the basis of: examination of the three-dimensional crystal structure of IL-2 polypeptides or other IL-2 family members and their receptors, the preference for conservative substitutions (i.e., replacement of Phe, tyr, or Trp with an aryl-based non-naturally encoded amino acid such as para-acetylphenylalanine or O-propargyl tyrosine), and the particular coupling chemistry one wishes to incorporate into the IL-2 (e.g., introduction of 4-azidophenylalanine if one wishes to effect Huisgen [3+2] cycloaddition with a water-soluble polymer bearing an alkynyl moiety or amide bond formation with a water-soluble polymer bearing an aryl ester and thus incorporating a phosphine moiety).

In one embodiment, the method further comprises: incorporating the unnatural amino acid in the protein, where the unnatural amino acid comprises a first reactive group; and contacting the protein with a molecule comprising a second reactive group (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 tag, a photoaffinity tag, a reactive compound, a resin, a second protein or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a saccharide, a polynucleotide, DNA, RNA, an antisense polynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin, an inhibitory ribonucleic acid, a biological material, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a new functional group, a group that interacts covalently or non-covalently with other molecules, a photocaged moiety, an actinic radiation excitable moiety, a photoisomerizable moiety, a radionuclide, a derivative of biotin, a compact biotin analog, a heavy atom-doped moiety, a chemical quantum group, a photoquantum group, an extended side chain, a carbon-linked saccharide, a redox active agent, an amino thioate moiety, a toxic moiety, a neutron-energy-transfer moiety, a chemical energy-emitting probe, a detectable agent, a small energy-emitting probe, a detectable agent, a light-emitting agent, or any combination thereof). The first reactive group reacts with the second reactive group via a [3+2] cycloaddition to attach the molecule to the unnatural amino acid. In one embodiment, the first reactive group is an alkyne group or an azide moiety and the second reactive group is an azide group or an alkyne moiety. For example, the first reactive group is an alkynyl moiety (including but not limited to that in 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 that in unnatural amino acid p-azido-L-phenylalanine) and the second reactive group is an alkynyl moiety.

In certain instances, the non-naturally encoded amino acid substitutions are combined with other additions, substitutions, or deletions within the IL-2 to affect other biological properties of the IL-2 polypeptide. In certain instances, the other additions, substitutions, or deletions may increase the stability (including but not limited to resistance to proteolytic degradation) of the IL-2 or increase the affinity of the IL-2 for its receptor. In certain instances, the additional additions, substitutions, or deletions may increase the pharmaceutical stability of the IL-2. In certain instances, the other additions, substitutions or deletions may enhance the tumor-inhibiting and/or tumor-reducing activity of the IL-2. In certain instances, the other additions, substitutions or deletions may increase the solubility of the IL-2 or variant (including but not limited to when expressed in e. In certain embodiments, the additions, substitutions, or deletions can increase the solubility of the IL-2 following expression in E.coli or other recombinant host cells. In certain embodiments, sites for substitution of a naturally encoded or unnatural amino acid are selected in addition to another site for incorporation of an unnatural amino acid that results in increased solubility of the polypeptide after expression in E.coli or other recombinant host cells. In certain embodiments, the IL-2 polypeptide comprises another addition, substitution, or deletion that modulates affinity for an IL-2 receptor, binding protein, or related ligand, modulates signal transduction upon binding to an IL-2 receptor, modulates circulating half-life, modulates release or bioavailability, facilitates purification, or modifies or alters a particular route of administration. In certain embodiments, the IL-2 polypeptide comprises an addition, substitution, or deletion that increases the affinity of the IL-2 variant for its receptor. In certain embodiments, the IL-2 comprises an addition, substitution, or deletion that increases the affinity of the IL-2 variant for IL-2-R1 and/or IL-2-R2. Likewise, an IL-2 polypeptide may comprise a chemical or enzymatic cleavage sequence, protease cleavage sequence, reactive group, antibody binding domain (including but not limited to FLAG or poly-His) or other affinity-based sequence (including but not limited to FLAG, poly-His, GST, etc.) or linked molecule (including but not limited to biotin) that improves detection (including but not limited to GFP), purification, transport through tissue or cell membranes, prodrug release or activation, IL-2 size reduction, or other traits of the polypeptide.

In certain embodiments, the substitution of the non-naturally encoded amino acid results in an IL-2 antagonist. In certain embodiments, the non-naturally encoded amino acid is substituted or added in a region associated with receptor binding. In certain embodiments, the IL-2 antagonist comprises at least one substitution that results in IL-2 acting as an antagonist. In certain embodiments, the IL-2 antagonist comprises a non-naturally encoded amino acid linked to a water-soluble polymer present in the receptor binding region of the IL-2 molecule.

In certain instances, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids are replaced with one or more non-naturally encoded amino acids. In certain instances, the IL-2 further comprises the substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more naturally occurring amino acids with one or more non-naturally encoded amino acids. For example, in certain embodiments, one or more residues in IL-2 are replaced with one or more non-naturally encoded amino acids. In certain instances, the one or more non-naturally encoded residues are linked to one or more lower molecular weight linear or branched PEGs, thereby increasing binding affinity and comparable serum half-life relative to substances attached to a single higher molecular weight PEG.

Expression in non-eukaryotes and eukaryotes

To obtain high levels of expression of the cloned IL-2 polynucleotide, one typically subclones the polynucleotide encoding the IL-2 polypeptide of the invention into an expression vector containing a strong promoter to direct transcription, a transcription/translation terminator, and a ribosome binding site for translation initiation if used for a nucleic acid encoding a protein. Suitable bacterial promoters are known to those of ordinary skill in the art and are described, for example, in Sambrook et al and Ausubel et al.

Bacterial expression systems for expression of IL-2 of the invention are available in a range including, but not limited to, E.coli (E.coli), bacillus (Bacillus sp.), pseudomonas fluorescens (Pseudomonas fluorescens), pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas putida (Pseudomonas putida), and Salmonella (Salmonella) (Palva et al, gene 22-229-235 (1983); mosbach et al, nature 302. Kits for use with these expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast and insect cells are known to those of ordinary skill in the art and are also commercially available. In the case where orthogonal tRNA's and aminoacyl-tRNA synthetases (described above) are used to express an IL-2 polypeptide of the invention, the host cells for expression are selected based on their ability to use the orthogonal components. Exemplary host cells include gram positive bacteria (including but not limited to bacillus brevis (b. Brevis), bacillus subtilis (b. Subtilis), or Streptomyces (Streptomyces)) and gram negative bacteria (e. Coli), pseudomonas fluorescens (Pseudomonas fluorescens), pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas putida (Pseudomonas putida)), as well as yeast and other eukaryotic cells. Cells comprising an O-tRNA/O-RS pair can be used as described herein.

The eukaryotic or non-eukaryotic host cells of the invention provide the ability to synthesize proteins comprising unnatural amino acids in useful quantities. In one instance, the composition optionally comprises a protein comprising an unnatural amino acid, including but not limited to at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10 milligrams, at least 100 milligrams, at least 1 gram or more, or an amount that can be obtained using in vivo protein production methods (details regarding recombinant protein production and purification are provided herein). In another instance, the protein is optionally present in the composition at a concentration including, but not limited to, at least 10 micrograms of protein per liter, at least 50 micrograms of protein per liter, at least 75 micrograms of protein per liter, at least 100 micrograms of protein per liter, at least 200 micrograms of protein per liter, at least 250 micrograms of protein per liter, at least 500 micrograms of protein per liter, at least 1 milligram of protein per liter, or at least 10 milligrams of protein per liter or more, including but not limited to, in a cell lysate, buffer, pharmaceutical buffer, or other liquid suspension (including but not limited to, in any volume including but not limited to between about 1nl to about 100L or more). The production of proteins comprising at least one unnatural amino acid in large quantities, including but not limited to larger quantities than are generally possible using other methods, including but not limited to in vitro translation, in eukaryotic cells is a feature of the invention.

The eukaryotic host cells or non-eukaryotic host cells of the invention provide the ability to biosynthesize proteins comprising unnatural amino acids in useful quantities. For example, proteins comprising unnatural amino acids can be produced in cell extracts, cell lysates, media, buffers, etc., at protein concentrations that include, but are not limited to, at least 10 μ g/liter, at least 50 μ g/liter, at least 75 μ g/liter, at least 100 μ g/liter, at least 200 μ g/liter, at least 250 μ g/liter, or at least 500 μ g/liter, at least 1 mg/liter, at least 2 mg/liter, at least 3 mg/liter, at least 4 mg/liter, at least 5 mg/liter, at least 6 mg/liter, at least 7 mg/liter, at least 8 mg/liter, at least 9 mg/liter, at least 10 mg/liter, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 mg/liter, 1 g/liter, 5 g/liter, 10 g/liter, or more.

A number of vectors suitable for the expression of IL-2 are commercially available. Useful expression vectors for eukaryotic hosts include, but are not limited to, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Such vectors include pCDNA3.1 (+) \ Hyg (Invitrogen, carlsbad, calif., USA) and pCI-neo (Stratagene, la Jolla, calif., USA). Bacterial plasmids such as those from E.coli, including pBR322, pET3a and pET12a, broad host range plasmids such as RP4, phage DNA such as lambda phage such as NM989 and numerous derivatives of other DNA phages such as M13 and filamentous single stranded DNA phages may be used. The 2 μ plasmid and its derivatives, POT1 vector (U.S. Pat. No. 4,931,373 incorporated herein by reference), pJSO37 vector described in (Okkels, ann.new York cited. Sci.782,202 207,1996), and pPICZ a, B or C (Invitrogen), can be used with yeast host cells. For insect Cells, vectors include, but are not limited to, pVL941, pBG (Cate et al, "Isolation of Bovine And Human Genes for Mullerian inhibition substances And Expression of said Human Genes In Animal Cells" (Isolation of the Bovine And Human Genes for Mullerian inhibition And Expression of the Human Gene In Animal Cells), pBluebac 4.5, and pMelbac (Invitrogen, carlsbad, calif.).

The nucleotide sequence encoding IL-2 or a variant thereof may or may not also include a sequence encoding a signal peptide. The signal peptide is present when the polypeptide is to be secreted from the cell in which it is expressed. These signal peptides may be of any sequence. The signal peptide may be prokaryotic or eukaryotic. Coloma, M (1992) j.imm. Methods 152. Other signal peptides include, but are not limited to, the alpha-factor signal peptide from Saccharomyces cerevisiae (U.S. Pat. No. 4,870,008, incorporated herein by reference), the signal peptide of mouse salivary amylase (O.Hagenbuchle et al, nature 289,1981, pp.643-646), the modified carboxypeptidase signal peptide (L.A.Valls et al, cell 48,1987, pp.887-897), the Yeast BAR1 signal peptide (WO 87/02670, incorporated herein by reference), and the Yeast aspartic protease 3 (YAP 3) signal peptide (see M.Egel-Mitani et al, yeast 6,1990, pp.127-137).

Examples of suitable mammalian host cells are known to those of ordinary skill in the art. These host cells can be Chinese Hamster Ovary (CHO) cells (e.g., CHO-K1; ATCC CCL-61), green monkey Cells (COS) (e.g., COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)), mouse cells (e.g., NS/O), baby Hamster Kidney (BHK) cell lines (e.g., ATCC CRL-1632 or ATCC CCL-10), and human cells (e.g., HEK 293 (ATCC CRL-1573)), and plant cells in tissue culture. These and other cell lines are available from public collections such as the American Type Culture Collection (Rockville, md.). To provide for increased glycosylation of IL-2 polypeptides, mammalian host cells can be modified to express sialyltransferases, e.g., 1,6-sialyltransferase, e.g., as described in U.S. patent No. 5,047,335, which is incorporated herein by reference.

Methods for introducing exogenous DNA into mammalian host cells include, but are not limited to, calcium phosphate-mediated transfection, electroporation, DEAE-dextran-mediated transfection, liposome-mediated transfection, viral vectors, and the transfection method described by Life Technologies Ltd, paisley, UK using lipofectamine 2000 and Roche Diagnostics Corporation, indianapolis, USA using FuGENE 6. These methods are well known in the art and are described by Ausbel et al, eds 1996, modern methods of Molecular Biology (Current Protocols in Molecular Biology), john Wiley & Sons, new York, USA. Mammalian Cell Culture can be performed according to established Methods, such as those disclosed in (Animal Cell Biotechnology, methods and Protocols), edited by Nigel Jenkins, 1999, human Press Inc. Totowa, N.J., USA, and Harrison Mass., and Rae IF, general Techniques for Cell Culture (General technologies of Cell Culture), cambridge University Press 1997).

I.Coli (e.coli), pseudomonas species (Pseudomonas species) and other prokaryotesBacterial expression techniques are known to those of ordinary skill in the art. A wide variety of different vectors are available for use in bacterial hosts. The vector may be a single copy or a low or high multiple copy vector. The vectors may be used for cloning and/or expression. In view of the large body of literature on vectors, the commercial availability of many vectors and even manuals describing vectors and their limiting patterns and characteristics, a thorough discussion is not necessary here. As is well known, the vector will generally comprise a marker which allows selection to be performed, said marker being Can provide cytotoxic drug resistance, prototrophy or immunity. There are often multiple markers that provide different characteristics.

A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating transcription of a downstream (3') coding sequence (e.g., a structural gene) into mRNA. Promoters have a transcriptional initiation region, which is usually located near the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. Bacterial promoters may also have a second domain, known as an operon, which may overlap with the adjacent RNA polymerase binding site at the beginning of RNA synthesis. The operon allows for negatively regulated (inducible) transcription, since the gene repressor protein can bind to the operon and thus inhibit transcription of a specific gene. Constitutive expression may occur in the absence of negative regulatory elements such as operons. In addition, positive regulation can be achieved by gene activator protein binding sequences, if present, usually in the vicinity of the RNA polymerase binding sequence (5'). An example of a gene activator is the Catabolite Activator Protein (CAP), which helps initiate transcription of the lac operon in E.coli (Raibaud et al, A) _NNU .R _EV .G _ENET . (1984) 18:173). Thus, the expression may be regulated positively or negatively, thereby enhancing or attenuating transcription.

Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include those derived from sugar-metabolizing enzymes such as galactose, lactose (lac) (Chang et al, N _ATURE (1977) 198. Other examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) (Goeddel et al, N _UC .A _CIDS R _ES (1980) 8; yelvton et al, N _UCL .A _CIDS R _ES (1981) 9; U.S. Pat. No. 4,738,921; european patent publication nos. 036 776 and 121 775, which are incorporated herein by reference). Beta-galactosidase (bla) promoter system (Weissmann (1981), cloning and other errors of Interferon (The cloning of Interferon and other mismatches), interferon 3 (I.Gresser's major edition)), phage lambda PL (Shimatake et al, N. _ATURE (1981)292:128 And T5 (U.S. Pat. No. 4,689,406, which is incorporated herein by reference) promoter systems also provide useful promoter sequences. Preferred methods of the invention utilize strong promoters, such as the T7 promoter, to induce IL-2 polypeptides at high levels. Examples of such vectors are known to those of ordinary skill in the art and include the pET29 series from Novagen and the popp vector described in WO1999/05297, which is incorporated herein by reference. These expression systems produce high levels of IL-2 polypeptide in a host without compromising host cell viability or growth parameters. pET19 (Novagen) is another vector known in the art.

In addition, synthetic promoters not found in nature also function as bacterial promoters. For example, a transcriptional activation sequence of one bacterial or bacteriophage promoter may be joined to an operator sequence of another bacterial or bacteriophage promoter to produce a synthetic hybrid promoter (U.S. Pat. No. 4,551,433, incorporated herein by reference). For example, the tac promoter is a hybrid trp-lac promoter, comprising both the trp promoter and a lac operator sequence which is regulated by a lac repressor protein (Amann et al, G) _ENE (1983) 25, 167; de Boer et al, P _ROC .N _ATL .A _CAD .S _CI . (1983) 80:21). In addition, bacterial promoters may include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. Naturally occurring promoters of non-bacterial origin may also be coupled with compatible RNA polymerases to produce high levels of expression of certain genes in prokaryotes. Phage T7 RNA polymerase/promoter systems are examples of coupled promoter systems (Studier et al, J.M) _OL .B _IOL (1986) 189; tabor et al, proc Natl.Acad.Sci. (1985) 82. In addition, hybrid promoters may also comprise a phage promoter and an E.coli operator region (European patent publication No. 267 851).

In addition to a functional promoter sequence, a highly efficient ribosome binding site is also useful for expression of foreign genes in prokaryotes. In E.coli, the ribosome binding site is called Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a nucleotide sequence located atA sequence 3-9 nucleotides in length 3-11 nucleotides upstream of the start codon (Shine et al, N _ATURE (1975) 254:34). The SD sequence is thought to promote binding of mRNA to ribosomes by base pairing between the SD sequence and the 3' end of E.coli 16S rRNA (Steitz et al, genetic signals and nucleotide sequences in messenger RNA (Genetic signals and nucleotide sequences in messenger RNA), ` Biological Regulation and Development ` (Gene Expression) (R.F.Goldberger eds., 1979)). For Expression of eukaryotic and prokaryotic genes with weak ribosome binding sites (Sambrook et al, expression of cloned genes in Escherichia coli (Molecular Cloning: A Laboratory Manual), 1989).

The term "bacterial host" or "bacterial host cell" refers to a bacterium that can be used or has been used as a recipient for a recombinant vector or other transfer of DNA. The term includes progeny of the original bacterial host cell that has been transfected. It will be appreciated that, due to accidental or deliberate mutation, progeny of a single parent cell may not necessarily be identical in morphology or in the entire genome or total DNA to the original parent. Progeny of the parent cell that are sufficiently similar to the parent cell, as characterized by the presence of the relevant property, e.g., nucleotide sequence encoding an IL-2 polypeptide, are included within the progeny intended for this definition.

The choice of a suitable host bacterium for expressing the IL-2 polypeptide is known to those of ordinary skill in the art. Among the bacterial hosts selected for expression, suitable hosts may include those that exhibit inter alia good inclusion body formation, low proteolytic activity and overall robustness. Bacterial hosts are commonly available from a variety of sources, including, but not limited to, the Bacterial Genetic Stock Collection (Bacterial Genetic Stock Center, department of Biophysics and Medical Physics, university of California) and the American Type Culture Collection ("ATCC") (Manassas, va.). Industrial/pharmaceutical fermentations typically use bacteria derived from strain K (e.g., W3110) or from strain B (e.g., BL 21). These strains are particularly useful because their growth parameters are well understood and robust. Furthermore, these strains are nonpathogenic, which is commercially important for safety and environmental reasons. Other examples of suitable E.coli hosts include, but are not limited to, the BL21, DH10B strains or derivatives thereof. In another embodiment of the method of the invention, the E.coli strain is a protease-attenuated strain, including but not limited to OMP-and LON-. The host cell strain may be a Pseudomonas species, including but not limited to Pseudomonas fluorescens, pseudomonas aeruginosa and Pseudomonas putida. Pseudomonas fluorescens biovar 1, designated MB101 strain, is known to be useful for recombinant production and can be used in therapeutic protein production processes. Examples of pseudomonas expression systems include those available from The Dow Chemical Company as host strains (Midland, MI, available at The world wide web site dow.com).

Once the recombinant host cell strain has been established (i.e., the expression construct has been introduced into the host cell and isolated into the host cell with the correct expression construct), the recombinant host cell strain is cultured under conditions suitable for the production of the IL-2 polypeptide. As will be apparent to those of ordinary skill in the art, the method of culturing the recombinant host cell strain will depend on the nature of the expression construct used and the identity of the host cell. Recombinant host strains are typically cultured using methods known to those of ordinary skill in the art. Recombinant host cells are typically cultured in liquid media containing assimilable sources of carbon, nitrogen and inorganic salts, and optionally vitamins, amino acids, growth factors and other protein culture supplements known to those of ordinary skill in the art. The liquid medium used for the cultivation of the host cells may optionally contain antibiotics or antifungal agents for preventing the growth of unwanted microorganisms and/or compounds for selecting host cells containing the expression vector, including but not limited to antibiotics.

Recombinant host cells may be cultured in a batch or continuous manner, and cell harvesting (in the case of intracellular accumulation of the IL-2 polypeptide) or culture supernatant harvesting is performed in a batch or continuous manner. For production in prokaryotic host cells, batch culture and cell harvest are preferred.

The IL-2 polypeptides of the invention are typically purified after expression in recombinant systems. The IL-2 polypeptide can be purified from the host cell or culture medium by a variety of different methods known in the art. IL-2 polypeptides produced in bacterial host cells may be poorly soluble or insoluble (in the form of inclusion bodies). In one embodiment of the invention, amino acid substitutions in the IL-2 can be readily made using the methods disclosed herein and known in the art, the substitutions being selected for the purpose of increasing the solubility of the recombinantly produced protein. In the case of insoluble proteins, the protein may be collected from the host cell lysate by centrifugation, and then further homogenization of the cells may be performed. In the case of poorly soluble proteins, compounds, including but not limited to Polyethyleneimine (PEI), may be added to cause precipitation of the partially soluble protein. The precipitated protein may then be conveniently collected by centrifugation. Recombinant host cells can be disrupted or homogenized by a variety of different methods known to those of ordinary skill in the art to release the inclusion bodies from the cells. Host cell disruption or homogenization can be performed using well known techniques including, but not limited to, enzymatic cell disruption, sonication, dounce homogenization, or high pressure release disruption. In one embodiment of the methods of the invention, high pressure release techniques are used to disrupt E.coli host cells to release the inclusion bodies of the IL-2 polypeptide. In processing inclusion bodies of IL-2 polypeptides, it may be advantageous to minimize homogenization repetition time in order to maximize the yield of inclusion bodies without losses due to factors such as solubilization, mechanical shearing, or proteolysis.

Any of a variety of suitable solubilizing agents known in the art can then be used to solubilize the insoluble or precipitated IL-2 polypeptide. The IL-2 polypeptide may be solubilized with urea or guanidine hydrochloride. The volume of the solubilized IL-2 polypeptide should be minimized so that large batches can be produced using easily manageable batch sizes. This factor may be important in a large-scale commercial context where recombinant hosts may be grown in batches of thousands of liters in volume. Furthermore, when manufacturing IL-2 polypeptides in a large-scale commercial setting, especially for human pharmaceutical use, harsh chemicals that can damage machinery and containers or the protein product itself should be avoided if possible. In the method of the invention has shown, milder denaturant urea can replace harsher denaturant guanidine hydrochloride for dissolving IL-2 polypeptide inclusion body. The use of urea significantly reduces the risk of damage to stainless steel equipment used in the manufacture and purification of IL-2 polypeptides, while effectively solubilizing IL-2 polypeptide inclusion bodies.

In the case of soluble IL-2 proteins, the IL-2 may be secreted into the periplasmic space or into the culture medium. In addition, soluble IL-2 may be present in the cytoplasm of the host cell. It may be desirable to concentrate the soluble IL-2 prior to performing the purification step. Soluble IL-2 can be concentrated from, for example, cell lysates or culture media using standard techniques known to those of ordinary skill in the art. In addition, the use of ordinary skill in the art known standard techniques to break up host cells, and from the host cell cytoplasm or periplasmic space release soluble IL-2.

In general, it is sometimes desirable to denature and reduce the expressed polypeptide and then refold the polypeptide into a preferred conformation. For example, guanidine, urea, DTT, DTE, and/or chaperone proteins may be added to the translation product of interest. Methods for reducing, denaturing and renaturing proteins are known to those of ordinary skill in the art (see the above references, and Debinski et al, (1993) J.biol.chem.,268, 14065-14070, kreitman and Pastan, (1993) bioconjug.chem., 4. For example, debinski et al describe the denaturation and reduction of inclusion body proteins in guanidine-DTE. The protein may be refolded in a redox buffer containing, but not limited to, oxidized glutathione and L-arginine. The refolding agent can be flowed or otherwise moved into contact with one or more polypeptides or other expression products, or vice versa.

In the case of prokaryotic production of IL-2 polypeptides, the IL-2 polypeptides produced thereby may be misfolded and thus lack or have reduced biological activity. The biological activity of the protein can be restored by "refolding". In general, misfolded IL-2 polypeptides are refolded by solubilizing (wherein the IL-2 polypeptide is also insoluble), unfolding, and reducing the polypeptide chain using, for example, one or more chaotropic agents (e.g., urea and/or guanidine) and a reducing agent capable of reducing disulfide bonds (e.g., dithiothreitol DTT or 2-mercaptoethanol 2-ME). An oxidizing agent (e.g., oxygen, cystine, or cystamine) that allows disulfide bond reformation is then added at moderate concentrations of chaotropic agent. The IL-2 polypeptide may be refolded using standard methods known in the art, such as the methods described in U.S. patent nos. 4,511,502, 4,511,503, and 4,512,922, which are incorporated by reference herein. The IL-2 polypeptides may also be co-folded with other proteins to form heterodimers or heteromultimers.

After refolding, the IL-2 can be further purified. Purification of IL-2 can be accomplished using a variety of different techniques known to those of ordinary skill in the art, including hydrophobic interaction chromatography, pore size exclusion chromatography, ion exchange chromatography, reverse phase high performance liquid chromatography, affinity chromatography, and the like, or any combination thereof. Additional purification may also include a step of drying or precipitation of the purified protein.

After purification, the IL-2 may be exchanged and/or concentrated in different buffers by any of a variety of different methods known in the art, including but not limited to diafiltration and dialysis. IL-2 provided as a single purified protein may undergo aggregation and precipitation.

The purified IL-2 may be at least 90% pure (as measured by reverse phase high performance liquid chromatography RP-HPLC or sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE) or at least 95% pure or at least 96% pure or at least 97% pure or at least 98% pure or at least 99% or more pure. Regardless of the exact numerical value of the purity of the IL-2, the IL-2 is sufficiently pure for use as a pharmaceutical product or for further processing, e.g., coupling to a water-soluble polymer such as PEG.

Certain IL-2 molecules may be used as therapeutic agents in the absence of other active ingredients or proteins (in addition to excipients, carriers and stabilizers, serum albumin, etc.), or they may be complexed with another protein or polymer.

It has been previously shown that unnatural amino acids can be site-specifically incorporated into proteins in vitro by adding a chemically aminoacylated suppressor tRNA to a protein synthesis reaction programmed with a gene containing a desired amber nonsense mutation. Using these methods, one can replace many of the common 20 amino acids with structurally similar homologues, such as replacement of phenylalanine with fluorophenylalanine, using strains that are auxotrophic for a particular amino acid. See, e.g., noren, C.J., anthony-Cahill, griffith, M.C., schultz, P.G., general methods for site-specific incorporation of unnatural amino acids in proteins (A general method for site-specific incorporation of unnatural amino acids in proteins), science,244, 182-188 (1989); M.W.Nowak et al, science 268, 439-42 (1995); bain, j.d., glabe, c.g., dix, t.a., chamberlin, a.r., diala, e.s., biosynthetic site-specific Incorporation of unnatural amino acids in polypeptides (Biosynthetic site-specific Incorporation of an unnatural amino acid into a polypeptide), j.am Chem Soc,111, 8013-8014 (1989); N.Budis et al, FASEB J.13:41-51 (1999); ellman, J.A., mendel, D., anthony-Cahill, S., noren, C.J., schultz, P.G., biosynthetic Methods for the site-specific introduction of unnatural amino acids into proteins (Biosynthetic method for the introduction of unnatural amino acids site-specific in proteins), methods in Enz., vol.202,301-336 (1992); and Mendel, D., cornish, V.W, & Schultz, P.G., site-Directed Mutagenesis using the Expanded Genetic Code (Site-Directed Mutagenesis with an Expanded Genetic Code), annu Rev Biophys.Biomol Structure.24, 435-62 (1995).

For example, a compound which recognizes the stop codon UAG and is chemically aminoacylated with an unnatural amino acid is preparedSuppressor tRNA. The stop codon TAG is introduced at a site of interest in the protein gene using conventional site-directed mutagenesis. See, e.g., sayers, J.R., schmidt, W.Eckstein, F., 5'-3' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis, nucleic Acids Res,16 (3): 791-802 (1988). Incorporation of the unnatural amino acid in response to the UAG codon when the acylated suppressor tRNA and mutant gene are incorporated in an in vitro transcription/translation system gives a protein containing the amino acid at the specified position. Use of ³ H]Experiments with-Phe and experiments with alpha-hydroxy acids confirmed that only the desired amino acid was incorporated at the position specified by the UAG codon and that this amino acid was not incorporated at any other site in the protein. See, e.g., noren et al, supra; kobayashi et al, (2003) Nature Structural Biology 10 (6): 425-432; and Ellman, J.A., mendel, D., schultz, P.G., site-specific incorporation of novel framework structures in proteins (Site-specific incorporation of novel backbone structures in proteins), science,255 (5041): 197-200 (1992).

the tRNA can be aminoacylated with the desired amino acid by any method or technique, including, but not limited to, chemical or enzymatic aminoacylation.

Aminoacylation can be accomplished by aminoacyl-tRNA synthetases or other enzyme molecules, including but not limited to ribozymes. The term "ribozyme" is interchangeable with "catalytic RNA". The presence of naturally occurring RNA (ribozymes) that can act as catalysts was confirmed by Cech and co-workers (Cech, 1987, science,236, 1532-1539, mcCorkle et al, 1987, concepts biochem.64. However, although these natural RNA catalysts have been shown to act only on ribonucleic acid substrates for cleavage and splicing, recent developments in ribozyme evolution have expanded the catalytic spectrum to a variety of different chemical reactions. Studies have identified RNA molecules that can catalyze aminoacyl-RNA bonds at their own (2 ') 3' -end (Illangakekare et al, 1995 Science 267.

U.S. patent application publication No. 2003/0228593, which is incorporated herein by reference, describes methods for constructing ribozymes and their use in aminoacylating tRNA's with naturally encoded and non-naturally encoded amino acids. The substrate-immobilized forms of enzyme molecules, including but not limited to ribozymes, that aminoacylate tRNA's may enable high-efficiency affinity purification of the aminoacylated product. Examples of suitable matrices include agarose, agarose gels, and magnetic beads. The production of matrix-immobilized forms of ribozymes and uses for aminoacylation are described in Chemistry and Biology 2003,10 and U.S. patent application publication 2003/0228593, which are incorporated herein by reference.

Chemical aminoacylation methods include, but are not limited to, those described by the following investigators: hecht and co-workers (Hecht, s.m. acc. Chem. Res.1992,25,545, heckler, t.g.; roesser, j.r.; xu, c.; chang, p.; hecht, s.m. biochemistry 1988,27,7254, hecht, s.m.; alford, b.l.; kuroda, y.; kitano, s.j.biol. Chem.1978,253, 4517); <xnotran> Schultz, chamberlin, dougherty (Cornish, V.W.; mendel, D.; schultz, P.G.Angew.Chem.Int.Ed.Engl.1995,34,621;Robertson,S.A.; ellman, J.A.; schultz, P.G.J.Am.Chem.Soc.1991,113,2722; noren, C.J.; anthony-Cahill, S.J.; griffith, M.C.; schultz, P.G.Science 1989,244,182;Bain,J.D.; glabe, C.G.; dix, T.A.; chamberlin, A.R.J.Am.Chem.Soc.1989,111,8013; bain, J.D. , nature 1992,356,537;Gallivan,J.P.; lester, H.A.; dougherty, D.A.Chem.Biol.1997,4,740;Turcatti , J.Biol.Chem.1996,271,19991; nowak, M.W. , science,1995,268,439;Saks,M.E. , J.Biol.Chem.1996,271,23169; hohsaka, T. , J.Am.Chem.Soc.1999,121, 34), , . </xnotran> These methods or other chemical aminoacylation methods can be used to aminoacylate the tRNA molecule.

Methods for generating catalytic RNA may include generating separate pools of randomized ribozyme sequences, directed evolution of the pools, screening the pools for a desired aminoacylation activity, and selecting those sequences of ribozymes that exhibit the desired aminoacylation activity.

A reconstructed translation system may also be used. Mixtures of purified translation factors have also been successfully used to translate mRNA into protein, as have lysates combined or supplemented with purified translation factors such as initiation factor-1 (IF-1), IF-2, IF-3 (α or β), elongation factor T (EF-Tu), or termination factor. Cell-free systems may also be coupled with transcription/translation systems in which DNA is introduced into the system, transcribed into mrns and the mrnas translated, as described in "modern methods of Molecular Biology" (f.m. ausubel et al eds., wiley Interscience, 1993), which is specifically incorporated herein by reference. RNA transcribed in eukaryotic transcription systems may take the form of heteronuclear RNA (hnRNA) or mature mRNA capped at the 5 '-end (7-methylguanosine) and tailed at the 3' -end by poly A, which may be advantageous in certain translation systems. For example, mRNA capped in reticulocyte lysate systems is translated with high efficiency. IX. macromolecular polymers conjugated to IL-2 Polypeptides

Various different modifications to the unnatural amino acid polypeptides described herein can be affected using the compositions, methods, techniques, and strategies described herein. These modifications include the incorporation of other functional groups onto the unnatural amino acid component of the polypeptide, including, but not limited to, labels, dyes, polymers, water-soluble polymers, derivatives of polyethylene glycol, photocrosslinkers, radionuclides, cytotoxic compounds, drugs, affinity tags, photoaffinity tags, reactive compounds, resins, second proteins or polypeptide analogs, antibodies or antibody fragments, metal chelators, cofactors, fatty acids, saccharides, polynucleotides, DNA, RNA, antisense polynucleotides, sugars, water-soluble dendrimers, cyclodextrins, inhibitory ribonucleic acids, biomaterials, nanoparticles, spin tags, fluorophores, metal-containing components, radioactive components, new functional groups, groups that interact covalently or non-covalently with other molecules, photocaged components, actinic radiation excitable components, photoisomerizable components, dense biotin, derivatives of biotin, biotin analogs, heavy atom doped components, chemical groups, photocleavable side chains, carbon-linked sugars, redox active moieties, amino acid moieties, thioluminescent moieties, radioactive moieties, neutron emitting moieties, quantum emitters, radioactive emission reagents, or any combination thereof. As illustrative, but non-limiting examples of the compositions, methods, techniques and strategies described herein, the following description will focus on the addition of macromolecular polymers to unnatural amino acid polypeptides, and it is to be understood that the compositions, methods, techniques and strategies are also applicable (with appropriate modifications if necessary, and that those modifications can be made by one skilled in the art using the present disclosure) to the addition of other functional groups, including but not limited to those listed above.

A wide variety of macromolecular polymers and other molecules may be attached to the IL-2 polypeptides of the invention to modulate the biological properties of the IL-2 polypeptides and/or to provide new biological properties to the IL-2 molecules. These macromolecular polymers may be linked to the IL-2 polypeptide by naturally encoded amino acids, by non-naturally encoded amino acids, or any functional substituent of a natural or non-natural amino acid, or any substituent or functional group added to a natural or non-natural amino acid. The molecular weight of the polymer can have a wide range including, but not limited to, between about 100Da to about 100,000Da or greater. The molecular weight of the polymer may be between about 100Da to about 100,000da, including but not limited to 100,000da, 95,000da, 90,000da, 85,000da, 80,000da, 75,000da, 70,000da, 65,000da, 60,000da, 55,000da, 50,000da, 45,000da, 40,000da, 35,000da, 30,000da, 25,000da, 20,000da, 15,000da, 10,000da, 9,000da, 8,000da, 7,000da, 6,000da, 5,000da, 4,000da, 3,000da, 2,000da, 1,000da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da. In certain embodiments, the molecular weight of the polymer is between about 100Da to about 50,000da. In certain embodiments, the molecular weight of the polymer is between about 100Da to about 40,000da. In certain embodiments, the molecular weight of the polymer is between about 1,000da to about 40,000da. In certain embodiments, the molecular weight of the polymer is between about 5,000da to about 40,000da. In certain embodiments, the molecular weight of the polymer is between about 10,000da to about 40,000da.

The present invention provides a polymer: a substantially homogeneous preparation of a protein conjugate. As used herein, "substantially homogeneous" means that the polymer is observed: the protein conjugate molecule is more than half of the total protein. The polymer: protein conjugates are biologically active, and the "substantially homogeneous" pegylated IL-2 polypeptide preparations provided herein are homogeneous preparations sufficient to exhibit the advantages of homogeneous preparations, such as ease of obtaining predictability of pharmacokinetics between batches in clinical applications.

One can also choose to make a polymer: a mixture of protein conjugate molecules, and the advantages provided herein are that a human can select a single polymer comprised in the mixture: ratio of protein conjugates. Thus, if desired, one can prepare a mixture of various proteins having various different numbers (i.e., two, three, four, etc.) of polymer moieties attached thereto, and combine the conjugate with a monopolymer prepared using the method of the invention: the protein conjugates are combined to obtain a single polymer having a predetermined ratio: a mixture of protein conjugates.

The polymer selected may be water soluble such that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. The polymer may be branched or unbranched. For therapeutic use of the final product preparation, the polymer will be pharmaceutically acceptable.

Examples of polymers include, but are not limited to, polyalkyl ethers and alkoxy-terminated analogs thereof (e.g., polyoxyethylene glycols, polyoxyethylene/propylene glycols and their especially polyoxyethylene glycolMethoxy or ethoxy-terminated analogs of alcohols, polyoxyethylene glycols also known as polyethylene glycols or PEG), polyvinylpyrrolidone, polyvinyl alkyl ethers, polyvinylsAzolines, polyalkyl radicalsOxazoline and polyhydroxyalkylOxazoline, polyacrylamide, polyalkylacrylamide and polyhydroxyalkylacrylamide (e.g., polyhydroxypropylmethacrylamide and derivatives thereof), polyhydroxyalkylacrylate, polysialic acid and analogs thereof, hydrophilic peptide sequences, polysaccharides and derivatives thereof, including dextran and dextran derivatives such as carboxymethyl dextran, dextran sulfate, aminodextran, cellulose and derivatives thereof such as carboxymethyl cellulose, hydroxyalkyl cellulose, chitin and derivatives thereof such as chitosan, succinylchitin, carboxymethyl chitin, carboxymethyl chitosan, hyaluronic acid and derivatives thereof, starch, alginate, chondroitin sulfate, albumin, pullulan and carboxymethyl pullulan, polyamino acids and derivatives thereof such as polyglutamic acid, polylysine, polyaspartic acid, polyaspartamide, maleic anhydride copolymers such as styrene maleic anhydride copolymers, divinyl ether maleic anhydride copolymers, polyvinyl alcohol, copolymers thereof, terpolymers thereof, mixtures thereof, and derivatives thereof.

The ratio of polyethylene glycol molecules to protein molecules may vary, as may their concentration in the reaction mixture. In general, the optimum ratio (in terms of reaction efficiency, since there is little excess unreacted protein or polymer) can be determined by the molecular weight of the polyethylene glycol selected and the number of reactive groups available. With respect to molecular weight, generally the higher the molecular weight of the polymer, the fewer the number of polymer molecules that can be attached to the protein. Likewise, when optimizing these parameters, branching of the polymer should be taken into account. Generally, the higher the molecular weight (or more branching), the polymer: the higher the protein ratio.

As used herein, and when considering PEG: IL-2 polypeptide conjugates, the term "therapeutically effective amount" refers to an amount that provides the desired benefit to the patient. The amount varies from individual to individual and depends on many factors, including the overall physical condition of the patient and the underlying cause of the condition to be treated. The amount of IL-2 polypeptide used in the therapy provides an acceptable rate of change and maintains the desired response at a beneficial level. A therapeutically effective amount of a composition of the invention can be readily determined by one of ordinary skill in the art using publicly available materials and procedures.

The water-soluble polymer may have any structural form including, but not limited to, linear, branched, or branched. Typically, the water soluble polymer is a polyalkylene glycol such as polyethylene glycol (PEG), but other water soluble polymers may also be used. As an example, PEG is used to describe certain embodiments of the invention.

PEG is a well-known water-soluble Polymer which is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods known to those of ordinary skill in the art (Sandler and Karo, polymer Synthesis, academic Press, new York, vol.3, pp.138-161). The term "PEG" is used broadly to encompass any polyethylene glycol molecule, regardless of size or whether there is a modification at the end of the PEG, and when attached to an IL-2 polypeptide can be represented by the formula:

XO-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -Y

wherein n is 2 to 10,000 and X is H or a terminal modification, including but not limited to C _1-4 Alkyl, protecting group or terminal functional group.

In some cases, the PEG used in the present invention is terminated at one end with a hydroxyl or methoxy group, i.e. X is H or CH ₃ ("methoxy PEG"). Alternatively, the PEG may be capped with a reactive group, thereby forming a bifunctional polymer. Typical reactive groups may be included Including reactive groups commonly used to react with functional groups present in 20 common amino acids (including but not limited to maleimide groups, activated carbonates (including but not limited to p-nitrophenyl esters), activated esters (including but not limited to N-hydroxysuccinimide, p-nitrophenyl esters), and aldehydes), and functional groups inert to the 20 common amino acids but reactive specifically with complementary functional groups present in non-naturally encoded amino acids (including but not limited to azide groups, alkyne groups). Notably, the other end of the PEG shown with Y in the above formula will be attached directly or indirectly to the IL-2 polypeptide through a naturally occurring or non-naturally encoded amino acid. For example, Y may be an amide, carbamate, or urea linkage to an amine group of the polypeptide, including but not limited to the epsilon amine group or the N-terminal amine group of lysine. Alternatively, Y may be a maleimide bond linked to a thiol group (including but not limited to that of cysteine). Alternatively, Y may be a bond to a residue that is not normally accessible to the 20 common amino acids. For example, the azido group on the PEG can react with an alkynyl group on the IL-2 polypeptide to form Huisgen [3+2 ]Cycloaddition products. Alternatively, the alkynyl group on the PEG can react with the azido group present in the non-naturally encoded amino acid to form a similar product. In certain embodiments, where applicable, strong nucleophiles (including but not limited to hydrazine, hydrazide, hydroxylamine, semicarbazide) may react with aldehyde or ketone groups present in the non-naturally encoded amino acid to form a hydrazone, oxime or semicarbazone, which in some cases may be further reduced by treatment with a suitable reducing agent. Alternatively, the strong nucleophilic agent can be incorporated into the IL-2 polypeptide by a non-naturally encoded amino acid and used to preferentially react with ketone or aldehyde groups present in the water-soluble polymer.

For PEG, any molecular mass can be used as is actually desired, including but not limited to about 100 daltons (Da) to 100,000Da or more (including but not limited to sometimes 0.1-50kDa or 10-40 kDa) as desired. The molecular weight of the PEG can have a wide range including, but not limited to, between about 100Da to about 100,000da or greater. The PEG may be between about 100Da to about 100,000Da including, but not limited to, 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da, and 100Da. In certain embodiments, the PEG is between about 100Da to about 50,000da. In certain embodiments, the PEG is between about 100Da to about 40,000da. In certain embodiments, the PEG is between about 1,000da to about 40,000da. In certain embodiments, the PEG is between about 5,000da to about 40,000da. In certain embodiments, the PEG is between about 10,000da to about 40,000da. Branched PEGs can also be used, including but not limited to PEG molecules having a MW in the range of 1-100kDa (including but not limited to 1-50kDa or 5-20kDa or 5-30kDa or 5-40 kDa) per chain. The molecular weight of each chain of the branched PEG can include, but is not limited to, between about 1,000da to about 100,000da or greater. The molecular weight of each chain of the branched PEG may be between about 1,000da to about 100,000da, including but not limited to 100,000da, 95,000da, 90,000da, 85,000da, 80,000da, 75,000da, 70,000da, 65,000da, 60,000da, 55,000da, 50,000da, 45,000da, 40,000da, 35,000da, 30,000da, 25,000da, 20,000da, 15,000da, 10,000da, 9,000da, 8,000da, 7,000da, 6,000da, 5,000da, 4,000da, 3,000da, 2,000da, and 1,000da. In certain embodiments, each chain of the branched PEG has a molecular weight between about 1,000da to about 50,000da. In certain embodiments, each chain of the branched PEG has a molecular weight between about 1,000da to about 40,000da. In certain embodiments, each chain of the branched PEG has a molecular weight between about 5,000da to about 40,000da. In certain embodiments, each chain of the branched PEG has a molecular weight between about 5,000da to about 20,000da. A wide range of PEG molecules are described including, but not limited to, shearwater Polymers, inc.

Typically, at least one end of the PEG molecule is available for reaction with the non-naturally encoded amino acid. For example, PEG derivatives bearing alkyne and azide moieties and used to react with amino acid side chains can be used to attach PEG to non-naturally encoded amino acids described herein. If the non-naturally encoded amino acid comprises an azide group, the PEG will typically contain an alkynyl moiety to perform the formation of a [3+2] cycloaddition product, or an activated PEG species (i.e., ester, carbonate) containing a phosphine group to perform the formation of an amide bond. Alternatively, if the non-naturally encoded amino acid comprises an alkyne, the PEG will typically contain an azido moiety to perform the formation of the [3+2] Huisgen cycloaddition product. If the non-naturally encoded amino acid comprises a carbonyl group, the PEG will typically comprise a strong nucleophilic agent (including, but not limited to, a hydrazide, hydrazine, hydroxylamine, or semicarbazide functional group) in order to effect formation of the corresponding hydrazone, oxime, and semicarbazone bonds, respectively. In other alternatives, the reversal of the orientation of the reactive groups described above may be used, i.e., the azido moiety in the non-naturally encoded amino acid may be reacted with an alkyne-containing PEG derivative.

In certain embodiments, the PEG derivative bearing variant IL-2 polypeptide contains a chemical functional group that is reactive with a chemical functional group present on a side chain of the non-naturally encoded amino acid.

In certain embodiments, the present invention provides azide and acetylene containing polymer derivatives comprising a water soluble polymer backbone having an average molecular weight of from about 800Da to about 100,000da. The polymer backbone of the water-soluble polymer may be polyethylene glycol. However, it should be understood that a wide variety of water-soluble polymers, including but not limited to polyethylene glycol and other related polymers, including polydextrose and polypropylene glycol, are also suitable for use in the practice of the present invention, and that the use of the term PEG or polyethylene glycol is intended to encompass and include all such molecules. The term PEG includes, but is not limited to, polyethylene glycol in any form, including bifunctional PEG, multi-armed PEG, derivatized PEG, branched PEG, pendant PEG (i.e., PEG or related polymer having one or more functional groups pendant from the polymer backbone), or PEG having degradable linkages therein.

PEG is usually permeableClear, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, not hydrolyzed or denatured, and generally non-toxic. Polyethylene glycol is considered to be biocompatible, that is to say PEG is able to coexist with living tissue or organisms without causing damage. More specifically, PEG is substantially non-immunogenic, that is PEG is not prone to generate an immune response in vivo. When attached to a molecule having a certain desired function in the body, such as a bioactive agent, PEG tends to mask the agent and may reduce or eliminate any immune response so that the organism may tolerate the presence of the agent. PEG conjugates are not prone to produce significant immune responses or cause clotting or other adverse effects. Having the formula-CH ₂ CH ₂ O--(CH ₂ CH ₂ O) _n --CH ₂ CH ₂ PEGs in which n is from about 3 to about 4000, typically from about 20 to about 2000, are suitable for use in the present invention. In certain embodiments of the invention, PEG having a molecular weight of about 800Da to about 100,000Da is particularly useful as the polymer backbone. The molecular weight of the PEG can have a wide range, including but not limited to between about 100Da to about 100,000Da or higher. The molecular weight of the PEG may be between about 100Da to about 100,000Da including, but not limited to, 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 000300 Da, 200Da, and 100Da. In certain embodiments, the PEG has a molecular weight between about 100Da and about 50,000da. In certain embodiments, the PEG has a molecular weight between about 100Da and about 40,000da. In certain embodiments, the PEG has a molecular weight between about 1,000da and about 40,000da. In certain embodiments, the PEG has a molecular weight between about 5,000da and about 40,000da. In certain embodiments, the PEG has a molecular weight between about 10,000da and about 40,000da.

The polymer backbone may be linear or branched. Branched polymer scaffolds have generally been known in the artAs is known. Typically, the branched polymer has a central branched core component and a plurality of linear polymer chains attached to the central branched core. PEG is often used in branched form, which can be prepared by the addition of ethylene oxide to a variety of different polyols such as glycerol, glycerol oligomers, pentaerythritol and sorbitol. The central branch moieties may also be derived from several amino acids such as lysine. The branched polyethylene glycol may be represented in general form as R (-PEG-OH) _m Wherein R is derived from a core moiety such as glycerol, glycerol oligomer or pentaerythritol and m represents the number of arms. Multi-arm PEG molecules, such as those described in U.S. Pat. Nos. 5,932,462, 5,643,575, 5,229,490, 4,289,872, U.S. patent application 2003/0143596, WO 96/21469, and WO 93/21259, each of which is incorporated herein by reference in its entirety, may also be used as the polymer backbone.

Branched PEG may also be PEG (- -YCHZ) ₂ ) _n The form of the branched PEG is represented, where Y is a linking group and Z is an activated terminal group, which is linked to CH through a chain of atoms of defined length.

Another branched form of pendant PEG has reactive groups, such as carboxyl groups, along the PEG backbone rather than at the ends of the PEG chain.

In addition to these forms of PEG, the polymers can also be prepared with weak or degradable bonds in the backbone. For example, PEG can be prepared with readily hydrolyzable ester linkages in the polymer backbone. This hydrolysis results in the polymer being cleaved into lower molecular weight fragments as shown below:

-PEG-CO ₂ -PEG-+H ₂ O→PEG-CO ₂ H+HO-PEG-

it will be understood by those of ordinary skill in the art that the term polyethylene glycol or PEG represents or encompasses all forms known in the art, including but not limited to those disclosed herein.

Many other polymers are also suitable for use in the present invention. In certain embodiments, water-soluble, polymeric scaffolds having from 2 to about 300 termini are particularly useful in the present invention. Examples of suitable polymers include, but are not limited to, other polyalkylene glycols such as polypropylene glycol ("PPG"), copolymers thereof (including, but not limited to, copolymers of ethylene glycol and propylene glycol), terpolymers thereof, mixtures thereof, and the like. Although the molecular weight of each chain of the polymer backbone can vary, it typically ranges from about 800Da to about 100,000da, typically from about 6,000da to about 80,000da. The molecular weight of each chain of the polymer backbone may be between about 100Da to about 100,000da, including but not limited to 100,000da, 95,000da, 90,000da, 85,000da, 80,000da, 75,000da, 70,000da, 65,000da, 60,000da, 55,000da, 50,000da, 45,000da, 40,000da, 35,000da, 30,000da, 25,000da, 20,000da, 15,000da, 10,000da, 9,000da, 8,000da, 7,000da, 6,000da, 5,000da, 4,000da, 3,000da, 2,da, 1,000da, 900Da, 800Da, 000700 Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da. In certain embodiments, each chain of the polymer backbone has a molecular weight between about 100Da and about 50,000da. In certain embodiments, each chain of the polymer backbone has a molecular weight between about 100Da and about 40,000da. In certain embodiments, each chain of the polymer backbone has a molecular weight between about 1,000da to about 40,000da. In certain embodiments, each chain of the polymer backbone has a molecular weight between about 5,000da and about 40,000da. In certain embodiments, each chain of the polymer backbone has a molecular weight between about 10,000da and about 40,000da.

Those skilled in the art will recognize that the aforementioned list of substantially water-soluble backbones is by no means exhaustive, but merely illustrative, and that all polymeric materials having the above-described properties are deemed suitable for use in the present invention.

In certain embodiments of the invention, the polymer derivative is "multifunctional," meaning that the polymer backbone has at least two ends and may have up to about 300 ends functionalized or activated with functional groups. Polyfunctional polymer derivatives include, but are not limited to, linear polymers having two termini, each of which is bonded to a functional group that may be the same or different.

In one embodiment, the polymer derivative has the following structure:

X—A—POLY—B—N＝N＝N

wherein:

n = N is an azido moiety;

b is a linking moiety, which may or may not be present;

POLY is a water-soluble non-antigenic polymer;

a is a linking moiety, which may or may not be present and may be the same or different from B; and is provided with

And X is a second functional group.

Examples of linking moieties A and B include, but are not limited to, polyfunctional alkyl groups containing up to 18 carbon atoms, and may contain between 1 and 10 carbon atoms. Heteroatoms such as nitrogen, oxygen or sulfur may be included within the alkyl chain. The alkyl chain may also be branched at a heteroatom. Other examples of linking moieties A and B include, but are not limited to, polyfunctional aryl groups containing up to 10 carbon atoms, and may contain 5 to 6 carbon atoms. The aryl group may be substituted with one or more carbon, nitrogen, oxygen or sulfur atoms. Other examples of suitable linking groups include those described in U.S. Pat. Nos. 5,932,462, 5,643,575 and U.S. patent application publication 2003/0143596, each incorporated herein by reference. Those skilled in the art will recognize that the foregoing list of connected components is by no means exhaustive, but merely illustrative, and that all connected components having the above properties are deemed suitable for use in the present invention.

Examples of functional groups suitable for use as X include, but are not limited to, hydroxyl, protected hydroxyl, alkoxy, active esters such as N-hydroxysuccinimidyl esters and 1-benzotriazolyl esters, active carbonates such as N-hydroxysuccinimidyl carbonate and 1-benzotriazolyl carbonate, acetals, aldehydes, aldehyde hydrates, alkenyl, acrylates, methacrylates, acrylamides, active sulfones, amines, aminoxy, protected amines, hydrazides, protected thiols, carboxylic acids, protected carboxylic acids, isocyanates, isothiocyanates, maleimides, vinyl sulfones, dithiopyridines, vinyl pyridines, iodoacetamides, epoxides, glyoxals, diketones, methanesulfonates, tosylates, tribenzoates, olefins, ketones, and azides. As will be understood by those of ordinary skill in the art, the X moieties selected should be compatible with the azide group such that no reaction with the azide group occurs. The azide-containing polymer derivative may be homobifunctional, meaning that the second functional group (i.e., X) is also an azide moiety, or heterobifunctional, meaning that the second functional group is a different functional group.

The term "protected" refers to the presence of a protecting group or moiety that prevents a chemically reactive functional group from reacting under certain reaction conditions. The protecting group varies depending on the type of chemically reactive group to be protected. For example, if the chemically reactive group is an amine or hydrazide, the protecting group may be selected from tert-butoxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). If the chemically reactive group is a thiol, the protecting group may be an ortho-pyridyl disulfide. If the chemically reactive group is a carboxylic acid such as butyric or propionic acid or a hydroxyl group, the protecting group may be a benzyl group or an alkyl group such as methyl, ethyl or tert-butyl. Other protecting groups known in the art may also be used in the present invention.

Specific examples of terminal functional groups in the literature include, but are not limited to, N-succinimidyl carbonate (see, e.g., U.S. Pat. Nos. 5,281,698, 5,468,478), amines (see, e.g., buckmann et al, makromol. Chem.182:1379 (1981), zalipsky et al, eur. Polym.J.19:1177 (1983)), hydrazides (see, e.g., andresz et al, makromol. Chem.179:301 (1978)), succinimidyl propionate and succinimidyl butyrate (see, e.g., olson et al, polyethylene glycol Chemistry and biology Applications (Poly (ethylene glycol) Chemistry & Biological Applications), pp 170-181, harris and Zalipsky master eds, ACS, washington, D.C.,1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (see, e.g., abuchowski et al, cancer biochem. Biophys.7:175 (1984) and Joppich et al, makromol. Chem.180:1381 (1979)), succinimidyl ester (see, e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see, e.g., U.S. Pat. No. 5,650,234), glycidyl ether (see, e.g., pitha et al, eur. J biochem.94:11 (1979), elling et al, biotech. Appl. Biochem.13:354 (1991)), oxycarbonylimidazoles (see, e.g., beauchamp et al, anal. Biochem.131:25 (1983), tondelli et al, J.Controled Release 1 251 (1985)), p-nitrophenyl carbonate (see, e.biochem. 19811, biochem. 141, tondel. J.J.J.J.J.12: and Sartore et al, appl.biochem.Biotech, 27 (1991)), aldehydes (see, e.g., harris et al, J.Polym.Sci.chem.Ed.22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No. 5,252,714), maleimides (see, e.g., goodson et al, biotechnology (NY) 8, romani et al, chemistry of Peptides and Proteins 2 (1984) and Kogan, synthetic Comm.22:2417 (1992)), orthopyridyl disulfide (see, e.g., woghiren et al, bioconj. Chem.4:314 (1993)), allyl alcohol (see, e.g., sawhney et al, macromolecules,26 (1993)), vinyl sulfone (see, e.g., U.S. Pat. No. 5,900,461). All of the above references and patents are incorporated herein by reference.

In certain embodiments of the present invention, the polymer derivatives of the present invention comprise a polymer backbone having the structure:

X—CH ₂ CH ₂ O--(CH ₂ CH ₂ O) _n --CH ₂ CH ₂ –N＝N＝N

wherein:

x is a functional group as described above; and is

n is from about 20 to about 4000.

In another embodiment, the polymer derivative of the present invention comprises a polymer backbone having the structure:

X—CH ₂ CH ₂ O--(CH ₂ CH ₂ O) _n --CH ₂ CH ₂ –O-(CH ₂ ) _m -W-N＝N＝N

wherein:

w is an aliphatic or aromatic linker moiety comprising between 1-10 carbon atoms;

n is from about 20 to about 4000; and is

X is a functional group as described above. m is between 1 and 10.

The azido-containing PEG derivatives of the invention can be prepared by a variety of different methods known in the art and/or disclosed herein. In one method, a water-soluble polymer backbone having an average molecular weight of about 800Da to about 100,000da, the polymer backbone having a first end bonded to a first functional group and a second end bonded to a suitable leaving group, is reacted with an azide anion, which may be paired with any one of a number of suitable counterions, including sodium, potassium, tert-butylammonium, and the like, as shown below. The leaving group undergoes nucleophilic displacement and is replaced by the azido moiety, resulting in the desired azido-containing PEG polymer.

X-PEG-L+N ₃ ^- →X-PEG-N ₃

As shown, the polymer backbone suitable for use in the present invention has the formula X-PEG-L, wherein PEG is polyethylene glycol, X is a functional group that does not react with azide, and L is a suitable leaving group. Examples of suitable functional groups include, but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl, amine, aminoxy, protected amine, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine, and vinylpyridine and copper. Examples of suitable leaving groups include, but are not limited to, chloride, bromide, iodide, mesylate, tribenzoate, and tosylate.

In another method for preparing the azido-containing polymer derivatives of the present invention, a linking reagent bearing an azido functional group is contacted with a water-soluble polymer backbone having an average molecular weight of about 800Da to about 100,000da, wherein the linking reagent bears a chemical functional group that selectively reacts with a chemical functional group on the PEG polymer to form an azido-containing polymer derivative product wherein the azido group is separated from the polymer backbone by a linking group.

An exemplary reaction scheme is as follows:

X-PEG-M + N-linker-N = N → PG-X-PEG-linker-N =

Wherein:

PEG is polyethylene glycol, X is a capping group such as alkoxy or a functional group as described above; and is

M is a functional group that is not reactive with the azido functional group but will react efficiently and selectively with the N functional group.

Examples of suitable functional groups include, but are not limited to, if N is an amine, M is a carboxylic acid, a carbonate, or an activated ester; m is a ketone if N is a hydrazide or aminooxy moiety; if N is a nucleophile, M is a leaving group.

Purification of the crude product may be accomplished by known methods, including but not limited to precipitation of the product followed by chromatography if desired.

More specific examples are illustrated below for PEG diamines, where one amine group is protected by a protecting group moiety such as tert-butyl-Boc, and the resulting mono-protected PEG diamine is reacted with a linking moiety bearing an azido functional group:

BocHN-PEG-NH ₂ +HO ₂ C-(CH ₂ ) ₃ -N＝N＝N

in this case, the amine group may be coupled to the carboxylic acid group using a variety of different activating agents such as thionyl chloride or carbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazole to create an amide bond between the monoamine PEG derivative and the azide bearing linker moiety. After successful formation of the amide bond, the resulting N-tert-butyl-Boc protected azido-containing derivative can be used directly to modify a biologically active molecule, or it can be further elaborated to install other useful functional groups. For example, the N-t-Boc group can be hydrolyzed by treatment with a strong acid to produce an ω -amino-PEG-azide. The resulting amines can be used as synthetic handles to install other useful functional groups such as maleimide groups, activated disulfides, activated esters, etc., for generating valuable heterobifunctional reagents.

Heterobifunctional derivatives are particularly useful when it is desired to attach different molecules to each end of the polymer. For example, ω -N-amino-N-azido PEG allows for attaching molecules with activated electrophilic groups, such as aldehydes, ketones, activated esters, activated carbonates, and the like, to one end of the PEG and attaching molecules with acetylene groups to the other end of the PEG.

In another embodiment of the present invention, the polymer derivative has the following structure:

X—A—POLY—B—C≡C-R

wherein:

r may be H or alkyl, alkenyl, alkoxy or aryl or substituted aryl;

b is a linking moiety, which may or may not be present;

POLY is a water-soluble non-antigenic polymer;

a is a linking moiety, which may or may not be present, and may be the same or different from B; and is

And X is a second functional group.

Examples of linking moieties A and B include, but are not limited to, polyfunctional alkyl groups containing up to 18 carbon atoms, and may contain between 1 and 10 carbon atoms. Heteroatoms such as nitrogen, oxygen or sulfur may be included within the alkyl chain. The alkyl chain may also be branched at a heteroatom. Other examples of linking moieties A and B include, but are not limited to, polyfunctional aryl groups containing up to 10 carbon atoms, and may contain 5 to 6 carbon atoms. The aryl group may be substituted with one or more carbon, nitrogen, oxygen or sulfur atoms. Other examples of suitable linking groups include those described in U.S. Pat. Nos. 5,932,462, 5,643,575 and U.S. patent application publication 2003/0143596, each incorporated herein by reference. Those skilled in the art will recognize that the foregoing list of connected components is by no means exhaustive, but merely illustrative, and that a wide variety of connected components having the above-described properties are contemplated as being useful in the present invention.

Examples of functional groups suitable for use as X include hydroxy, protected hydroxy, alkoxy, active esters such as N-hydroxysuccinimidyl ester and 1-benzotriazolyl ester, active carbonates such as N-hydroxysuccinimidyl carbonate and 1-benzotriazolyl carbonate, acetals, aldehydes, aldehyde hydrates, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, amine, aminoxy, protected amine, hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimide, vinyl sulfone, dithiopyridine, vinyl pyridine, iodoacetamide, epoxide, glyoxal, diketone, mesylate, tosylate, tribenzoate, olefin, ketone, and acetylene. As will be appreciated, the selected moiety X should be compatible with the ethynyl group such that no reaction with the ethynyl group occurs. The acetylene-containing polymer derivative may be homobifunctional, meaning that the second functional group (i.e., X) is also an acetylene moiety, or heterobifunctional, meaning that the second functional group is a different functional group.

In another embodiment of the present invention, the polymer derivative comprises a polymer backbone having the structure:

X—CH ₂ CH ₂ O--(CH ₂ CH ₂ O) _n --CH ₂ CH ₂ –O-(CH ₂ ) _m -C≡CH

Wherein:

x is a functional group as described above;

n is from about 20 to about 4000; and is

m is between 1 and 10.

Specific examples of each heterobifunctional PEG polymer are shown below.

The acetylene-containing PEG derivatives of the invention can be prepared using methods known to those of ordinary skill in the art and/or disclosed herein. In one method, a water soluble polymer backbone having an average molecular weight of about 800Da to about 100,000da, the polymer backbone having a first end bonded to a first functional group and a second end bonded to a suitable nucleophilic group, is reacted with a compound bearing both an acetylene functional group and a leaving group suitable for reaction with the nucleophilic group on the PEG. When the PEG polymer with the nucleophilic moiety is combined with the molecule with the leaving group, the leaving group undergoes nucleophilic substitution and is replaced by the nucleophilic moiety, resulting in the desired acetylene-containing polymer.

X-PEG-Nu+L-A-C→X-PEG-Nu-A-C≡CR’

As shown, a preferred polymer backbone for the reaction has the formula X-PEG-Nu, where PEG is polyethylene glycol, nu is a nucleophilic moiety, and X is a functional group that does not react with Nu, L, or acetylene functional groups.

Examples of Nu include, but are not limited to, amines, alkoxy, aryloxy, sulfhydryl, imino, carboxylic acid, hydrazide, aminoxy, which react primarily by SN 2-type mechanisms. Other examples of Nu groups include functional groups that react primarily by nucleophilic addition reactions. Examples of L groups include chlorine, bromine, iodine, mesylate, tribenzoate, and tosylate and other groups intended to undergo nucleophilic displacement, as well as ketones, aldehydes, thioesters, olefins, α - β unsaturated carbonyl, carbonates, and other electrophilic groups intended to undergo addition by nucleophiles.

In another embodiment of the invention, A is an aliphatic linker between 1 and 10 carbon atoms or a substituted aryl ring between 6 and 14 carbon atoms. X is a functional group that does not react with the azide group and L is a suitable leaving group.

In another method for preparing the acetylene-containing polymer derivatives of the present invention, a PEG polymer having an average molecular weight of about 800Da to about 100,000da with a protected functional group or capping agent at one end and a suitable leaving group at the other end is contacted with an acetylene anion.

An exemplary reaction scheme is shown below:

X-PEG-L+-C≡CR’→X-PEG-C≡CR’

wherein:

PEG is polyethylene glycol, X is a capping group such as alkoxy or a functional group as described above; and is

R' is H, alkyl, alkoxy, aryl or aryloxy or substituted alkyl, alkoxy, aryl or aryloxy.

In the above examples, the leaving group L should be sufficiently reactive to undergo a SN2 type displacement when contacted with a sufficient concentration of acetylene anion. The reaction conditions required to achieve displacement of the leaving group by SN2 of the acetylene anion are known to those of ordinary skill in the art.

Purification of the crude product can generally be accomplished by methods known in the art, including but not limited to precipitation of the product followed by chromatography if desired.

Water soluble polymers may be attached to the IL-2 polypeptides of the invention. The water-soluble polymer may be attached by, or added to, a non-naturally encoded or naturally encoded amino acid, or any functional group or substituent of a non-naturally encoded or naturally encoded amino acid that is incorporated into the IL-2 polypeptide. Alternatively, the water-soluble polymer is linked to the IL-2 polypeptide incorporating the non-naturally encoded amino acid through a naturally occurring amino acid (including but not limited to an amine group of cysteine, lysine, or N-terminal residues). In certain instances, the IL-2 polypeptides of the invention comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 unnatural amino acids, where one or more of the unnatural encoded amino acids are linked to a water soluble polymer (including but not limited to PEG and/or oligosaccharides). In certain instances, the IL-2 polypeptides of the invention further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more naturally encoded amino acids linked to a water soluble polymer. In certain instances, the IL-2 polypeptides of the invention comprise one or more non-naturally encoded amino acids linked to a water soluble polymer and one or more naturally occurring amino acids linked to a water soluble polymer. In certain embodiments, the water-soluble polymer used in the present invention increases the serum half-life of the IL-2 polypeptide relative to the unconjugated form.

The number of water-soluble polymers attached to the IL-2 polypeptides of the invention (i.e., the degree of pegylation or glycosylation) can be adjusted to provide altered (including but not limited to increased or decreased) pharmacological, pharmacokinetic or pharmacodynamic characteristics such as in vivo half-life. In certain embodiments, the half-life of IL-2 is increased at least about 10, 20, 30, 40, 50, 60, 70, 80, 90%, 2-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, or at least about 100-fold relative to the unmodified polypeptide.

PEG derivatives containing strong nucleophilic groups (i.e. hydrazides, hydrazines, hydroxylamines or semicarbazides)

In one embodiment of the invention, an IL-2 polypeptide comprising a carbonyl-containing non-naturally encoded amino acid is modified with a PEG derivative comprising a terminal hydrazine, hydroxylamine, hydrazide or semicarbazide moiety attached directly to the PEG backbone.

In certain embodiments, the hydroxylamine-terminated PEG derivative has the following structure:

RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) _m -O-NH ₂

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10, and n is 100-1,000 (i.e., average molecular weight between 5-40 kDa).

In certain embodiments, the hydrazine or hydrazide-containing PEG derivative has the following structure:

RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) _m -X-NH-NH ₂

wherein R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, and n is 100-1,000,x is optionally a carbonyl that may or may not be present (C = O).

In certain embodiments, the semicarbazide-containing PEG derivative has the following structure:

RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) _m -NH-C(O)-NH-NH ₂

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10, and n is 100-1,000.

In another embodiment of the invention, an IL-2 polypeptide comprising a carbonyl-containing amino acid is modified with a PEG derivative comprising a terminal hydroxylamine, hydrazide, hydrazine, or semicarbazide moiety attached to the PEG backbone with an amide bond.

In certain embodiments, the hydroxylamine-terminated PEG derivative has the following structure:

RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) ₂ -NH-C(O)(CH ₂ ) _m -O-NH ₂

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10, and n is 100-1,000 (i.e., average molecular weight between 5-40 kDa).

In certain embodiments, the hydrazine or hydrazide-containing PEG derivative has the following structure:

RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) ₂ -NH-C(O)(CH ₂ ) _m -X-NH-NH ₂

wherein R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, n is 100-1,000, and X is optionally a carbonyl group (C = O) which may or may not be present.

In certain embodiments, the semicarbazide-containing PEG derivative has the following structure:

RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) ₂ -NH-C(O)(CH ₂ ) _m -NH-C(O)-NH-NH ₂

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10, and n is 100-1,000.

In another embodiment of the invention, an IL-2 polypeptide comprising a carbonyl-containing amino acid is modified with a branched PEG derivative containing a terminal hydrazine, hydroxylamine, hydrazide or semicarbazide moiety, wherein each chain of the branched PEG has a MW of 10-40kDa and may be in the range of 5-20 kDa.

In another embodiment of the invention, an IL-2 polypeptide comprising a non-naturally encoded amino acid is modified with a PEG derivative having a branched chain structure. For example, in certain embodiments, the hydrazine or hydrazide terminated PEG derivative has the following structure:

[RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) ₂ -NH-C(O)] ₂ CH(CH ₂ ) _m -X-NH-NH ₂

wherein R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, and n is 100-1,000, and X is optionally a carbonyl group (C = O) that may or may not be present.

In certain embodiments, the semicarbazide-group containing PEG derivative has the following structure:

[RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) ₂ -C(O)-NH-CH ₂ -CH ₂ ] ₂ CH-X-(CH ₂ ) _m -NH-C(O)-NH-NH ₂

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), X is optionally NH, O, S, C (O), or absent, m is 2 to 10, and n is 100 to 1,000.

In certain embodiments, the PEG derivative containing a hydroxylamine group has the following structure:

[RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) ₂ -C(O)-NH-CH ₂ -CH ₂ ] ₂ CH-X-(CH ₂ ) _m -O-NH ₂

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), X is optionally NH, O, S, C (O), or absent, m is 2 to 10, and n is 100 to 1,000.

The extent and site of attachment of the water-soluble polymer to the IL-2 polypeptide can modulate the binding of the IL-2 polypeptide to an IL-2 receptor. In certain embodiments, the linkage is arranged such that the IL-2 polypeptide has a K of about 400nM or less _d At a K of 150nM or less _d And in some cases with a K of 100nM or less _d Binds to the IL-2 receptor, said K _d Measured by an equilibrium binding assay, such as described in Spencer et al, j.biol.chem., 263.

Methods and chemistries for activation of polymers and for coupling of peptides have been described in the literature and are known in the art. Common methods for polymer activation include, but are not limited to, the use of cyanogen bromide, highActivation of functional groups such as iodates, glutaraldehyde, diepoxides, epichlorohydrin, divinyl sulfone, carbodiimides, sulfonyl halides, trichlorotriazines (see r.f. taylor, (1991); protein immobilization: base & applications (P) _ROTEIN I _{MMOBILISATION} .F _{UNDAMENTAL AND} A _PPLICATIONS ) Marcel Dekker, n.y.; wong (1992), chemistry of protein coupling and Cross-linking (C) _{HEMISTRY OF} P _ROTEIN C _{ONJUGATION AND} C _ROSSLINKING ) CRC Press, boca Raton; hermanson et al, (1993) immobilized affinity ligand technology (I) _MMOBILIZED A _FFINITY L _IGAND T _ECHNIQUES ) Academic Press, n.y.; dunn, R.L. et al, polymer DRUG AND DRUG delivery System (POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS), ACS Symposium Series Vol.469, american Chemical Society, washington, D.C. 1991).

There are several reviews and single books available regarding functionalization and conjugation of PEG. See, e.g., harris, macromol. Chem. Phys. C25:325-373 (1985); scouten, methods in Enzymology 135 (1987); wong et al, enzyme Microb.Technol.14:866-874 (1992); delgado et al, clinical Reviews in Therapeutic Drug Carrier Systems 9 (1992); zalipsky, bioconjugate chem.6:150-165 (1995).

Methods for polymer activation can also be found in WO 94/17039, U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No. 5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No. 5,281,698 and WO 93/15189, and methods for coupling between activated polymers and enzymes include, but are not limited to, coagulation factor VIII (WO 94/15625), hemoglobin (WO 94/09027), oxygen-carrying molecules (U.S. Pat. No. 4,412,989), ribonucleases and superoxide dismutase (App. Biochem. Biotech.11:141-52 (1985)). All references and patents cited are incorporated herein by reference.

PEGylation of IL-2 polypeptides containing non-naturally encoded amino acids, such as p-azido-L-phenylalanine (i.e., addition of any water soluble polymer) is performed by any convenient method.For example, an IL-2 polypeptide is PEGylated with an alkynyl-terminated mPEG derivative. Briefly, an excess of solid mPEG (5000) -O-CH is added to an aqueous solution of an IL-2 polypeptide containing p-azido-L-Phe under stirring at room temperature ₂ -C ≡ CH. Typically, the aqueous solution is treated with pK _a A buffer near the pH at which the reaction is to proceed (typically about pH 4-10). Examples of suitable buffers for pegylation, e.g., at pH 7.5, include, but are not limited to, HEPES, phosphate, borate, TRIS-HCl, EPPS, and TES. The pH was continuously monitored and adjusted as necessary. The reaction is generally allowed to continue for between about 1 and 48 hours.

The reaction product is then subjected to hydrophobic interaction chromatography to bind the pegylated IL-2 polypeptide variant to free mPEG (5000) -O-CH ₂ -C.ident.CH and said PEGylated IL-2 polypeptide, said complexes possibly being formed when unblocked PEG is activated at both ends of the molecule, thereby cross-linking the IL-2 polypeptide variant molecule. The conditions during hydrophobic interaction chromatography are such that free mPEG (5000) -O-CH ₂ -C ≡ CH flows through the column, while any cross-linked pegylated IL-2 polypeptide variant complexes elute after the desired form containing one IL-2 polypeptide variant molecule coupled to one or more PEG groups. Suitable conditions vary with the relative size of the crosslinked complex to the desired conjugate, and are readily determined by one of ordinary skill in the art. The eluate containing the desired conjugate is concentrated by ultrafiltration and desalted by diafiltration.

Substantially purified PEG-IL-2 can be produced using the elution methods outlined above, wherein the PEG-IL-2 produced has 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%, a purity level of at least about 95%, a purity level of at least about 99% or higher, as determined by suitable methods such as SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis. If necessary, from the hydrophobic chromatographyThe pegylated IL-2 polypeptides of (a) can be further purified by one or more procedures known to those of ordinary skill in the art including, but not limited to, affinity chromatography, anion or cation exchange chromatography (using procedures including, but not limited to, DEAE SEPHAROSE), silica gel chromatography, reverse phase HPLC, gel filtration (using procedures including, but not limited to, SEPHADEX G-75), hydrophobic interaction chromatography, pore size exclusion chromatography, metal chelator chromatography, ultrafiltration/diafiltration, ethanol precipitation, ammonium sulfate precipitation, chromatofocusing, displacement chromatography, electrophoresis procedures (including, but not limited to, preparative isoelectric focusing), differential solubility (including, but not limited to, ammonium sulfate precipitation), or extraction. The apparent molecular weight can be estimated by GPC by comparison with globular protein standards (Preneta, AZ, protein purification method: practical method (P) _{ROTEIN PURIFICATION METHODS,APRACTICAL APPROACH} )(Harris&Angal major), IRL Press 1989, 293-306). The purity of the IL-2-PEG conjugate can be assessed by proteolytic degradation (including but not limited to trypsin cleavage) followed by mass spectrometry. Pepinsky RB., j.&Exp.Ther.297(3):1059-66(2001)。

The water-soluble polymer of an amino acid attached to an IL-2 polypeptide of the present invention may be further derivatized or substituted without limitation.

PEG derivative containing azido group

In another embodiment of the invention, an IL-2 polypeptide is modified with a PEG derivative that contains an azido moiety that will react with an alkynyl moiety present on the side chain of a non-naturally encoded amino acid. Typically, the PEG derivative will have an average molecular weight in the range of 1-100kDa and in certain embodiments in the range of 10-40 kDa.

In certain embodiments, the azido-terminated PEG derivative has the following structure:

RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) _m -N ₃

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10, and n is 100-1,000 (i.e., average molecular weight between 5-40 kDa).

In another embodiment, the azido-terminated PEG derivative has the following structure:

RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) _m -NH-C(O)-(CH ₂ ) _p -N ₃

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10, p is 2-10, and n is 100-1,000 (i.e., average molecular weight between 5-40 kDa).

In another embodiment of the invention, an IL-2 polypeptide comprising an alkynyl-containing amino acid is modified with a branched PEG derivative containing a terminal azido moiety, each strand of which has a MW in the range of 10-40kDa, and can be in the range of 5-20 kDa. For example, in certain embodiments, the PEG derivative at the azido terminus has the following structure:

[RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) ₂ -NH-C(O)] ₂ CH(CH ₂ ) _m -X-(CH ₂ ) _p N ₃

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10, p is 2-10, n is 100-1,000, and X is optionally O, N, S or a carbonyl (C = O), which may or may not be present in each case.

Alkynyl-containing PEG derivative

In another embodiment of the invention, the IL-2 polypeptide is modified with a PEG derivative containing an alkynyl moiety that will react with an azido moiety present on the side chain of a non-naturally encoded amino acid.

In certain embodiments, the alkynyl-terminated PEG derivative has the following structure:

RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) _m -C≡CH

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10, and n is 100-1,000 (i.e., average molecular weight between 5-40 kDa).

In another embodiment of the invention, an IL-2 polypeptide comprising an alkynyl-containing non-naturally encoded amino acid is modified with a PEG derivative comprising a terminal azido or terminal alkynyl moiety linked to a PEG backbone by an amide bond.

In certain embodiments, the alkynyl-terminated PEG derivative has the following structure:

RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) _m -NH-C(O)-(CH ₂ ) _p -C≡CH

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10, p is 2-10, and n is 100-1,000.

In another embodiment of the invention, an IL-2 polypeptide comprising an azido-containing amino acid is modified with a branched PEG derivative containing a terminal alkynyl moiety, each chain of the branched PEG having a MW in the range of 10-40kDa and may be in the range of 5-20 kDa. For example, in certain embodiments, the alkynyl-terminated PEG derivative has the following structure:

[RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) ₂ -NH-C(O)] ₂ CH(CH ₂ ) _m -X-(CH ₂ ) _p C≡CH

wherein R is simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is 2-10, n is 100-1,000, and X is optionally O, N, S or carbonyl (C = O) or absent.

Phosphine-containing PEG derivatives

In another embodiment of the invention, the IL-2 polypeptide is modified with PEG derivatives containing activated functional groups (including but not limited to esters, carbonates) and further comprising an arylphosphine group that will react with an azido moiety on the side chain of a non-naturally encoded amino acid. Typically, the PEG derivative has an average molecular weight in the range of 1-100kDa and in certain embodiments in the range of 10-40 kDa.

In certain embodiments, the PEG derivative has the following structure:

wherein n is 1 to 10; x may be O, N, S or absent, ph is phenyl, and W is a water soluble polymer.

In certain embodiments, the PEG derivative has the following structure:

wherein X may be O, N, S or absent, ph is phenyl, W is a water soluble polymer, and R may be H, alkyl, aryl, substituted alkyl, and substituted aryl. Exemplary R groups include, but are not limited to-CH ₂ 、-C(CH ₃ ) ₃ -OR ', -NR ' R ", -SR ', -halogen, -C (O) R ', -CONR ' R", -S (O) ₂ R’、-S(O) ₂ NR' R ", -CN and-NO ₂ . 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, for example, a compound of the invention comprises more than one R group, each said R group is independently selected, as is each of these groups when more than one R ', R ", R'" and R "" groups are present. When R' and R "are attached to the same nitrogen atom, they may combine with the 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 above discussion of substituents, those skilled in the art will appreciate that the term "alkyl" is meant to include groups containing carbon atoms bonded to groups other than hydrogen radicals, such as haloalkyl (including, but not limited to-CF) ₃ and-CH ₂ CF ₃ ) And acyl (including but not limited to-C (O) CH ₃ 、-C(O)CF ₃ 、-C(O)CH ₂ OCH ₃ Etc.).

Other PEG derivatives and general PEGylation techniques

Other exemplary PEG molecules that can be attached to IL-2 polypeptides and methods of PEGylation include, but are not limited to, those described in, for example, the following documents: <xnotran> 2004/0001838, 2002/0052009, 2003/0162949, 2004/0013637, 2003/0228274, 2003/0220447, 2003/0158333, 2003/0143596, 2003/0114647, 2003/0105275, 2003/0105224, 2003/0023023, 2002/0156047, 2002/0099133, 2002/0086939, 2002/0082345, 2002/0072573, 2002/0052430, 2002/0040076, 2002/0037949, 2002/0002250, 2001/0056171, 2001/0044526, 2001/0021763, 6,646,110,5,824,778,5,476,653,5,219,564,5,629,384,5,736,625,4,902,502,5,281,698,5,122,614,5,473,034,5,516,673,5,382,657,6,552,167,6,610,281,6,515,100,6,461,603,6,436,386,6,214,966,5,990,237,5,900,461,5,739,208,5,672,662,5,446,090,5,808,096,5,612,460,5,324,844,5,252,714,6,420,339,6,201,072,6,451,346,6,306,821,5,559,213,5,747,646,5,834,594,5,849,860,5,980,948,6,004,573,6,129,912,WO 97/32607,EP 229,108,EP 402,378,WO 92/16555,WO 94/04193,WO 94/14758,WO 94/17039,WO 94/18247,WO 94/28024,WO 95/00162,WO 95/11924,WO95/13090,WO 95/33490,WO 96/00080,WO 97/18832,WO 98/41562,WO 98/48837,WO 99/32134,WO 99/32139,WO 99/32140,WO 96/40791,WO 98/32466,WO 95/06058,EP 439 508,WO 97/03106,WO 96/21469,WO 95/13312,EP 921 131,WO 98/05363,EP 809 996,WO 96/41813,WO 96/07670,EP 605 963,EP 510 356,EP 400 472,EP 183 503 EP 154 316, . </xnotran> Any PEG molecule described herein can be used in any form, including but not limited to single chain, branched, multi-armed, mono-functional, di-functional, multi-functional, or any combination thereof.

Other polymers and PEG derivatives, including but not limited to hydroxylamine (aminooxy) PEG derivatives, are described in the following patent applications, all incorporated herein by reference in their entirety: U.S. patent publication No. 2006/0194256, U.S. patent publication No. 2006/0217532, U.S. patent publication No. 2006/0217289, U.S. provisional patent No. 60/755,338, U.S. provisional patent No. 60/755,711, U.S. provisional patent No. 60/755,018, international patent application No. PCT/US06/49397,WO 2006/069246, U.S. provisional patent No. 60/743,041, U.S. provisional patent No. 60/743,040, international patent application No. PCT/US06/47822, U.S. provisional patent No. 60/882,819, U.S. provisional patent No. 60/882,500, and U.S. provisional patent No. 60/870,594.

Glycosylation of IL-2 polypeptides

The invention includes IL-2 polypeptides incorporating one or more non-naturally encoded amino acids with sugar residues. The sugar residues may be natural (including but not limited to N-acetylglucosamine) or non-natural (including but not limited to 3-fluorogalactose). The sugar may be linked to the non-naturally encoded amino acid by an N-or O-linked glycosidic linkage (including but not limited to N-acetylgalactosamine-L-serine) or a non-natural linkage (including but not limited to an oxime or a corresponding C-or S-linked glycoside).

The sugar (including but not limited to glycosyl) component can be in vivo or in vitro added to IL-2 polypeptide. In certain embodiments of the invention, an IL-2 polypeptide comprising a carbonyl-containing non-naturally encoded amino acid is modified with a sugar derivatized with an aminooxy group to produce a corresponding glycosylated polypeptide linked by an oxime linkage. Once attached to the non-naturally encoded amino acid, the sugar can be further elaborated by treatment with glycosyltransferases and other enzymes to produce an oligosaccharide that is bonded to the IL-2 polypeptide. See, e.g., H.Liu et al, J.am.chem.Soc.125:1702-1703 (2003).

In certain embodiments of the invention, an IL-2 polypeptide comprising a carbonyl-containing non-naturally encoded amino acid is directly modified with a glycan of defined structure prepared as an aminooxy derivative. One of ordinary skill in the art will recognize that other functional groups (including azide, alkyne, hydrazide, hydrazine, and semicarbazide) may be used to attach the sugar to the non-naturally encoded amino acid. In certain embodiments of the invention, an IL-2 polypeptide comprising an azide or alkyne-containing non-naturally encoded amino acid can then be modified with, but not limited to, alkyne or azide derivatives, respectively, by cycloaddition reactions including, but not limited to, huisgen [3+2 ]. This method allows modification of proteins with very high selectivity.

IL-2 dimers and multimers

The invention also provides IL-2 and IL-2 analog combinations such as homodimers, heterodimers, homomultimers, or heteromultimers (i.e., trimers, tetramers, etc.), wherein IL-2 containing one or more non-naturally encoded amino acids is bound to another IL-2 variant or any other polypeptide that is not an IL-2 variant, either directly to the polypeptide backbone or through a linker. Due to the increased molecular weight compared to the monomer, the IL-2 dimer or multimer conjugates may exhibit novel or desirable properties, including but not limited to, different pharmacological, pharmacokinetic, pharmacodynamic properties, modulated therapeutic half-life, or modulated plasma half-life relative to monomeric IL-2. In certain embodiments, the IL-2 dimers of the invention modulate signal transduction of the IL-2 receptor. In other embodiments, the IL-2 dimers or multimers of the invention will act as IL-2 receptor antagonists, agonists, or modulators.

In certain embodiments, one or more IL-2 molecules present in the IL-2-containing dimer or multimer comprise a non-naturally encoded amino acid linked to a water-soluble polymer. In certain embodiments, the IL-2 polypeptides are directly linked by including, but not limited to, an Asn-Lys amide bond or a Cys-Cys disulfide bond. In certain embodiments, the IL-2 polypeptide and/or linked non-IL-2 molecule comprises different non-naturally encoded amino acids to promote dimerization, including, but not limited to, an alkynyl group in one non-naturally encoded amino acid of a first IL-2 polypeptide and an azido group in a second non-naturally encoded amino acid of a second molecule would be coupled by Huisgen [3+2] cycloaddition. Alternatively, an IL-2 and/or linked non-IL-2 molecule comprising a ketone-containing non-naturally encoded amino acid can be coupled to a second polypeptide comprising a hydroxylamine-containing non-naturally encoded amino acid, and the polypeptides reacted by formation of the corresponding oxime.

Optionally, the two IL-2 polypeptides and/or linked non-IL-2 molecules are linked by a linker. Any iso-or homobifunctional linker may be used to link the two molecules, which may have the same or different primary sequences, and/or the linked non-IL-2 molecule. In certain instances, the linker used to tether the IL-2 and/or linked non-IL-2 molecules together can be a bifunctional PEG reagent. The linker may have a wide range of molecular weights or molecular lengths. Linkers of greater or lesser molecular weight can be used to provide a desired spatial relationship or conformation between IL-2 and the linked entity or between IL-2 and its receptor or, if present, the linked entity and its binding partner. Linkers with longer or shorter molecular lengths can also be used to provide the desired space or flexibility between IL-2 and the linked entity or between the linked entity and its binding partner, if any.

In certain embodiments, the present invention provides a water-soluble bifunctional linker having a dumbbell structure, comprising: a) An azide, alkyne, hydrazine, hydrazide, hydroxylamine or carbonyl containing moiety on at least a first end of the polymer backbone; and b) at least one second functional group on a second end of the polymer backbone. The second functional group may be the same as or different from the first functional group. In certain embodiments, the second functional group is non-reactive with the first functional group. In certain embodiments, the present invention provides water-soluble compounds comprising at least one arm of a branched molecular structure. For example, the branched molecular structure may be dendritic.

In certain embodiments, the invention provides multimers comprising one or more IL-2 polypeptides formed by reaction with a water-soluble activated polymer having the structure:

R-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) _m -X

wherein n is about 5 to 3,000,m is 2-10,x can be a moiety containing azide, alkyne, hydrazine, hydrazide, aminoxy, hydroxylamine, acetyl or carbonyl groups and R is a capping group, functional group or leaving group which can be the same or different from X. R may be, for example, a functional group selected from the group consisting of hydroxyl, protected hydroxyl, alkoxy, N-hydroxysuccinimidyl ester, 1-benzotriazolyl ester, N-hydroxysuccinimidyl carbonate, 1-benzotriazolyl carbonate, acetal, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, amine, aminoxy, protected amine, hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimide, vinyl sulfone, dithiopyridine, vinyl pyridine, iodoacetamide, epoxide, glyoxal, diketone, mesylate, tosylate, tribenzoate, olefin, and ketone.

Measurement of IL-2 polypeptide Activity and affinity of IL-2 Polypeptides for the IL-2 receptor

IL-2 polypeptide activity can be used standard or known in vitro or in vivo determination to determine. PEG-IL-2 can be through the field known in the appropriate method to analyze biological activity. These assays include, but are not limited to, activation of IL-2 responsive genes, receptor binding assays, assays for antiviral activity, inhibition of cytopathic effects assays, anti-proliferation assays, immunomodulatory assays, and assays for monitoring induction of MHC molecules.

The ability of PEG-IL-2 polypeptides to activate IL-2 sensitive signal transduction pathways can be assayed. One example is an Interferon Stimulated Response Element (ISRE) assay. Cells constitutively expressing the IL-2 receptor were transiently transfected with ISRE-luciferase vector (pISRE-luc, clontech). After transfection, the cells are treated with an IL-2 polypeptide. A number of protein concentrations, for example 0.0001-10ng/mL, were tested to generate dose response curves. If the IL-2 polypeptide binds to and activates the IL-2 receptor, the resulting signal transduction cascade induces luciferase expression. Luminescence can be measured in a number of ways, for example by using TopCount ^TM Or Fusion ^TM Microplate reader and Steady-Glo ^R Luciferase assay system (Promega).

The ability of an IL-2 polypeptide to bind to an IL-2 receptor can be assayed. For unpegylated or pegylated IL-2 polypeptides comprising unnatural amino acids, the affinity of IL-2 for its receptor can be determined using BIAcore ^TM Biosensor (Pharmacia). Suitable binding assays include, but are not limited to, the BIAcore assay (Pearce et al, biochemistry 38-89 (1999)) and acscreen ^TM Assay (Perkinelmer).

Regardless of the method used to produce the IL-2 polypeptide, the IL-2 polypeptide is subjected to biological activity assays. Generally, assays for biological activity should provide assays for desired results, such as increased or decreased biological activity (as compared to modified IL-2), different biological activity (as compared to modified IL-2), receptor or binding partner affinity assays, conformational or structural changes in IL-2 itself or its receptor (as compared to modified IL-2), or serum half-life assays. Measurement of potency, functional in vivo half-life and pharmacokinetic parameters XIII

An important aspect of the invention is the extended biological half-life achieved by constructing an IL-2 polypeptide with or without a water-soluble polymer moiety coupled thereto. The rapid decline in IL-2 polypeptide serum concentration following administration makes it important to assess the biological response to treatment with conjugated and unconjugated IL-2 polypeptides and variants thereof. Conjugated and unconjugated IL-2 polypeptides and variants thereof of the invention may also have an extended serum half-life after administration, e.g. by subcutaneous or i.v. administration, making measurement possible by e.g. ELISA methods or by prescreening assays. ELISA or RIA kits from commercial sources such as Invitrogen (Carlsbad, CA) may be used. Measurement of in vivo biological half-life is performed as described herein.

The potency and functional in vivo half-life of an IL-2 polypeptide comprising a non-naturally encoded amino acid can be determined according to protocols known to those of ordinary skill in the art.

Pharmacokinetic parameters of IL-2 polypeptides comprising non-naturally encoded amino acids can be assessed in normal Sprague-Dawley male rats (N =5 animals per treatment group). The animals received a single dose of 25 ug/rat iv or 50 ug/rat sc and approximately 5-7 blood samples were taken according to a predetermined time course that generally covered about 6 hours for an IL-2 polypeptide comprising a non-naturally encoded amino acid that was not conjugated to a water-soluble polymer to about 4 days for an IL-2 polypeptide comprising a non-naturally encoded amino acid and conjugated to a water-soluble polymer. Pharmacokinetic data for IL-2 without the non-naturally encoded amino acid can be directly compared to data obtained for IL-2 polypeptides comprising the non-naturally encoded amino acid.

Administration and pharmaceutical compositions

The polypeptides or proteins of the invention (including but not limited to IL-2, synthetases, proteins comprising one or more unnatural amino acid, etc.) are optionally for therapeutic use, including but not limited to in combination with a suitable pharmaceutical carrier. These compositions comprise, for example, a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier or excipient. Such carriers or excipients include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and/or combinations thereof. The formulation is made to be suitable for the mode of administration. In general, methods of administering proteins are known to those of ordinary skill in the art and may be applied to the administration of polypeptides of the invention. The compositions may take a water-soluble form, for example, present as a pharmaceutically acceptable salt, which is meant to include both acid and base addition salts.

Therapeutic compositions comprising one or more polypeptides of the invention are optionally tested in one or more suitable in vitro and/or in vivo animal models of disease according to methods known to those of ordinary skill in the art to confirm efficacy, tissue metabolism, and estimate dosage. In particular, dosages can be initially determined by comparison of the activity, stability, or other suitable measure of non-natural polypeptide to natural amino acid homolog herein (including, but not limited to, comparison of an IL-2 polypeptide modified to include one or more non-natural amino acids to a natural amino acid IL-2 polypeptide and comparison of an IL-2 polypeptide modified to include one or more non-natural amino acids to currently available IL-2 treatments), i.e., in a related assay.

Administration is by any route commonly used to introduce molecules for eventual contact with blood or tissue cells. The unnatural amino acid polypeptides of the invention are administered in any suitable manner, optionally with one or more pharmaceutically acceptable carriers. Suitable methods of administering these polypeptides to a patient are available in the context of the present invention, and while more than one route may be used to administer a particular composition, a particular route may generally provide a more immediate and more effective action or response than another route.

The pharmaceutically acceptable carrier will be determined in part by the particular composition to be administered and by the particular method of administering the composition. Thus, there is a wide variety of suitable formulations for the pharmaceutical compositions of the present invention.

The IL-2 polypeptides of the invention may be administered by any conventional route suitable for proteins or peptides, including but not limited to parenteral, e.g., injection, including but not limited to subcutaneous or intravenous or any other form of injection or infusion. The polypeptide compositions can be administered by a variety of routes including, but not limited to, oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal means. Compositions comprising modified or unmodified unnatural amino acid polypeptides can also be administered via liposomes. These routes of administration and suitable formulations are well known to those skilled in the art. The IL-2 polypeptide may be used alone or in combination with other suitable components, such as pharmaceutical carriers. The IL-2 polypeptide can be used in combination with other agents or therapeutic agents.

IL-2 polypeptides comprising unnatural amino acids, alone or in combination with other suitable components, can also be manufactured as aerosol formulations (i.e., they can be "nebulized") for administration by inhalation. The aerosol formulation may be placed in an acceptable pressurized propellant such as dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, e.g., by the intra-articular, intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which can include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The preparation of IL-2 may be presented in single-dose or multi-dose sealed containers such as ampoules and vials.

Parenteral and intravenous administration are preferred methods of administration. In particular, routes of administration that have been used for natural amino acid homolog therapeutics (including, but not limited to, the routes of administration of proteins commonly used for EPO, GH, G-CSF, GM-CSF, IFNs such as IL-2, interleukins, antibodies, FGF, and/or any other drug delivery) as well as the formulations currently in use, provide preferred routes of administration and formulations for the polypeptides of the invention.

In the context of the present invention, the dose administered to a patient is sufficient to obtain a beneficial therapeutic response or other suitable activity in said patient over time, depending on the application. The dosage will be determined by the efficacy of the particular carrier or formulation, the activity, stability or serum half-life of the unnatural amino acid polypeptide employed, the condition of the patient, and the weight or surface area of the patient to be treated. The size of the dose is also determined by the presence, nature and extent of any adverse side effects associated with the administration of a particular carrier, formulation or the like in a particular patient.

In determining the effective amount of a carrier or formulation to be administered in the treatment or prevention of a disease (including, but not limited to, neutropenia, aplastic anemia, periodic neutropenia, idiopathic neutropenia, chdiak-Higashi syndrome, systemic Lupus Erythematosus (SLE), leukemia, myelodysplastic syndrome, myelofibrosis, and the like), physicians assess circulating plasma levels, formulation toxicity, disease progression, and/or, where relevant, the production of anti-unnatural amino acid polypeptide antibodies.

The dose administered to a patient of, for example, 70 kg is generally in the range equivalent to the dose of a commonly used therapeutic protein and is adjusted according to the altered activity or serum half-life of the relevant composition. The vector or pharmaceutical preparation of the present invention may supplement the treatment conditions by any known conventional therapy including administration of antibodies, administration of vaccines, administration of cytotoxic agents, natural amino acid polypeptides, nucleic acids, nucleotide analogs, biological response modifiers, and the like.

For administration, the formulations of the invention are administered at a rate determined by observation of any side effects of the LD-50 or ED-50 and/or unnatural amino acid polypeptide of the relevant formulation at various concentrations, including but not limited to, body weight and overall health, as appropriate for the patient. Administration can be accomplished in a single dose or in divided doses.

If a patient undergoing infusion of the formulation develops fever, chills or muscle aches, he/she receives the appropriate dose of aspirin, ibuprofen, acetaminophen or other pain/fever controlling medication. Patients who experience reactions to infusions such as fever, muscle aches and chills are pre-dosed 30 minutes prior to future infusions with aspirin, acetaminophen or include, but are not limited to, phenheimine. Pethidine is used for more severe chills and muscle pain that do not respond rapidly to antipyretics and antihistamines. Slowing or interrupting cell infusion depending on the severity of the response.

The human IL-2 polypeptides of the invention can be administered directly to a mammalian subject. Administration is by any route commonly used to introduce IL-2 polypeptides into a subject. IL-2 polypeptide compositions according to embodiments of the present invention include compositions suitable for oral, rectal, topical, inhalation (including but not limited to by aerosol), buccal (including but not limited to sublingual), vaginal, parenteral (including but not limited to subcutaneous, intramuscular, intradermal, intra-articular, intrapleural, intraperitoneal, intracerebral, intraarterial or intravenous), topical (and both skin and mucosal surfaces, including airway surfaces), pulmonary, intraocular, intranasal and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated. Administration may be local or systemic. The formulations of the compounds may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials. The IL-2 polypeptides of the invention may be prepared in a unit dose injectable form as a mixture (including but not limited to a solution, suspension or emulsion) with a pharmaceutically acceptable carrier. The IL-2 polypeptides of the invention can also be through continuous infusion (using including but not limited to small pump such as osmotic pump), single bolus injection or slow release reservoir preparation to drug delivery.

Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Freeze-drying is a common technique used to present proteins for removal of water from a protein preparation of interest. Freeze-drying or lyophilization is the process by which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment. Excipients may be included in the formulation prior to lyophilization to improve stability during lyophilization and/or to improve the stability of the lyophilized product upon storage. Pikal, M.Biopharm.3 (9) 26-30 (1990) and Arakawa et al, pharm.Res.8 (3): 285-291 (1991).

Spray drying of drugs is also known to those of ordinary skill in the art. See, e.g., broadhead, j, et al, spray Drying of drugs (The Spray Drying of Pharmaceuticals), drug dev. Ind. Pharm,18 (11 and 12), 1169-1206 (1992). In addition to small molecule drugs, a variety of different biomaterials have been spray dried, including: enzymes, serum, plasma, microorganisms and yeasts. Spray drying is a useful technique because it can convert a liquid pharmaceutical formulation into a fine, non-dusting or agglomerated powder in a one-step process. The basic technology comprises the following four steps: a) Atomizing the feed solution into a spray; b) Spray-air contact; c) Drying the spray; and d) separation of the dried product from the drying air. U.S. patent nos. 6,235,710 and 6,001,800, which are incorporated herein by reference, describe the preparation of recombinant erythropoietin by spray drying.

The pharmaceutical compositions and formulations of the present invention may comprise pharmaceutically acceptable carriers, excipients or stabilizers. The pharmaceutically acceptable carrier will be determined in part by the particular composition to be administered and by the particular method used to administer the composition. Thus, there exists a wide variety of suitable formulations of the Pharmaceutical compositions of the present invention (including optional pharmaceutically acceptable carriers, excipients or stabilizers) (see, e.g., remington's Pharmaceutical Sciences, 17 th edition, 1985).

Suitable carriers include, but are not limited to, buffers including succinate, phosphateBorate, HEPES, citrate, histidine, imidazole, acetate, bicarbonate, and other organic acids; antioxidants, including but not limited to ascorbic acid; low molecular weight polypeptides, including but not limited to polypeptides of less than about 10 residues; proteins, including but not limited to serum albumin, gelatin, or immunoglobulins; hydrophilic polymers including, but not limited to, polyvinylpyrrolidone; amino acids, including but not limited to glycine, glutamine, asparagine, arginine, histidine or histidine derivatives, methionine, glutamic acid or lysine; monosaccharides, disaccharides, and other sugars including, but not limited to trehalose, sucrose, glucose, mannose, or dextrins; chelating agents, including but not limited to EDTA and disodium EDTA; divalent metal ions including, but not limited to, zinc, cobalt, or copper; sugar alcohols, including but not limited to mannitol or sorbitol; salt-forming counterions including, but not limited to, sodium and sodium chloride; fillers such as microcrystalline cellulose, lactose, corn and other starches; a binder; sweeteners and other flavoring agents; a colorant; and/or nonionic surfactants, including but not limited to Tween ^TM (including but not limited to Tween 80 (polysorbate 80) and Tween 20 (polysorbate 20), pluronics ^TM And other pluronic acids, including but not limited to pluronic acid F68 (poloxamer 188) or PEG. Suitable surfactants include, for example, but are not limited to, polyethers based on polyethylene oxide-polypropylene oxide-polyethylene oxide, i.e. (PEO-PPO-PEO) or polypropylene oxide-polyethylene oxide-polypropylene oxide, i.e. (PPO-PEO-PPO), or combinations thereof. PEO-PPO-PEO and PPO-PEO-PPO are available under the trade name Pluronics ^TM 、R-Pluronics ^TM 、Tetronics ^TM And R-Tetronics ^TM Commercially available (BASF Wyandotte corp., wyandotte, mich.), and is further described in U.S. patent No. 4,820,352, which is incorporated herein by reference in its entirety. Other ethylene/polypropylene block polymers may be suitable surfactants. A surfactant or combination of surfactants can be used to stabilize pegylated IL-2 against one or more stresses, including but not limited to stresses caused by agitation. Some of the above may be referred to as "extenders". Certain substances may also be referred to as "tonicity adjustmentAgents ". Antimicrobial preservatives can also be used to achieve product stability and antimicrobial effectiveness; suitable preservatives include, but are not limited to, benzyl alcohol, benzalkonium chloride, m-cresol, methyl/propyl paraben, cresol and phenol or combinations thereof. U.S. patent No. 7,144,574, which is incorporated herein by reference, describes other materials that may be suitable for the pharmaceutical compositions and formulations of the present invention and other delivery formulations.

The IL-2 polypeptides of the invention, including IL-2 polypeptides linked to a water soluble polymer such as PEG, can also be administered by or as part of a sustained release system. Sustained release compositions include, but are not limited to, semipermeable polymer matrices in the form of shaped articles, including, but not limited to, films or microcapsules. Sustained release matrices include polymers derived from biocompatible materials such as poly (2-hydroxyethyl methacrylate) (Langer et al, J.biomed.Mater.Res.,15, J.Biomed.15, 15, 1981), langer, chem.Tech.,12, 98-105 (1982)), ethylene vinyl acetate (Langer et al, supra) or poly D- (-) -3-hydroxybutyric acid (EP 133,988), polylactide (polylactic acid) (U.S. Pat. Nos. 3,773,919), polyglycolide (a polymer of glycolic acid), polylactide-co-glycolide (a copolymer of lactic acid and glycolic acid), polyanhydrides, copolymers of L-glutamic acid and γ -ethyl-L-glutamic acid (Sidman et al, biopolymers,22,547-556 (1983)), polyorthoesters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acid, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as isoleucine, phenylalanine, poly (MPS), poly (OPP (PE), and poly (vinylpyrrolidone). Sustained release compositions also include liposome-encapsulated compounds. Liposomes containing the compounds are prepared by methods known per se: DE 3,218,121; eppstein et al, proc.natl.acad.sci.u.s.a., 82; hwang et al, proc.natl.acad.sci.u.s.a., 77; EP 52,322; EP 36,676; U.S. Pat. No. 4,619,794; EP 143,949; U.S. Pat. No. 5,021,234; japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. All references and patents cited are incorporated herein by reference.

Liposome-encapsulated ILThe-2 polypeptide can be prepared, for example, by the methods described in the following documents: DE 3,218,121; eppstein et al, proc.natl.acad.sci.u.s.a., 82; hwang et al, proc.natl.acad.sci.u.s.a., 77; EP 52,322; EP 36,676; U.S. Pat. No. 4,619,794; EP 143,949; U.S. Pat. No. 5,021,234; japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. The composition and size of liposomes are well known or can be readily determined empirically by one of ordinary skill in the art. Some examples of liposomes are described, for example, in the following documents: park JW et al, proc.Natl.Acad.Sci.USA 92; lasic D and Papapapaahad jopoulos D, eds. & medical applications of liposomes (M) _EDICAL A _{PPLICATIONS OF} L _IPOSOMES ) (1998); drummond DC et al, liposomal drug delivery systems for cancer therapy (Liposomal drug delivery systems for cancer therapy), ed. Teicher B, ed. Eds., (C) cancer drug discovery and development _ANCER D _RUG D _{ISCOVERY AND} D _EVELOPMENT ) (2002); park JW et al, clin. Cancer Res.8:1172-1181 (2002); nielsen UB et al, biochim.Biophys.acta 1591 (1-3): 109-118 (2002); mamot C et al, cancer res.63:3154-3161 (2003). All references and patents cited are incorporated herein by reference.

In the context of the present invention, the dose administered to the patient should be sufficient to elicit a beneficial response in said subject over time. Typically, the total therapeutically effective amount of the IL-2 polypeptide of the invention administered parenterally per dose will be in the range of about 0.01 μ g/kg to about 100 μ g/kg or about 0.05mg/kg to about 1mg/kg of patient body weight per day, although this will depend on the therapeutic judgment. In particular instances of this embodiment, the conjugate can be administered at a dosage in the range of greater than 4 μ/kg per day to about 20 μ g/kg per day. In other cases, the conjugate may be administered at a dosage in the range of greater than 4 μ g/kg per day to about 9 μ g/kg per day. In other cases, the conjugate can be administered at a dosage in the range of about 4 μ g/kg per day to about 12.5 μ g/kg per day. In particular instances, the conjugate may be administered at a dose equal to or lower than the highest dose tolerated without undue toxicity. Further, the conjugate may be administered at least twice a week, or the conjugate may be administered at least three times a week, at least four times a week, at least five times a week, at least six times a week, or seven times a week. In certain instances, where the conjugate is administered more than once, the conjugate may be administered at a dose of greater than 4 μ g/kg per day. In particular, the conjugate may be administered over a period of two weeks or more. In certain instances, the growth of cells expressing interleukin-2 receptor may be inhibited by at least 50%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to a reference sample, i.e., a sample of cells not contacted with a conjugate of the invention. In particular instances of this embodiment, the conjugate can be administered at a dose of about 5.3 μ g/kg per day or at a dose of about 7.1 μ g/kg per day or at a dose of about 9.4 μ g/kg per day or at a dose of about 12.5 μ g/kg per day. The frequency of administration also depends on the therapeutic judgment and can be more or less frequent than commercially available IL-2 polypeptide products approved for use in humans. In general, the IL-2 polypeptide, PEGylated IL-2 polypeptide, conjugated IL-2 polypeptide, or PEGylated conjugated IL-2 polypeptide of the invention may be administered by any of the routes of administration described above.

XV. therapeutic uses of IL-2 polypeptides of the invention

The IL-2 polypeptides of the invention are useful in the treatment of a wide range of disorders. The invention also includes methods of treating a mammal at risk for, suffering from, and/or having cancer responsive to IL-2, CD8+ T-cell stimulation, and/or IL-2 agents. The administration of an IL-2 polypeptide may produce a short-term effect, i.e. an immediate beneficial effect, which may be 12 or 24 hours from the administration, and on the other hand, may also produce a long-term effect, beneficially slowing the progression of tumor growth, reducing tumor size and/or increasing circulating CD8+ T cell levels, and the IL-2 polypeptides of the present invention may be administered by any means known to those skilled in the art, and may advantageously be administered by infusion, e.g. by arterial, intraperitoneal or intravenous injection and/or infusion, at a dose sufficient to obtain the desired pharmacological effect.

The dose of the IL-2 polypeptide may be in the range of 10-200mg or 40-80mg IL-2 polypeptide per kg body weight per treatment. For example, the dose of IL-2 polypeptide administered may be about 20-100mg IL-2 polypeptide per kg body weight, provided as a bolus injection and/or as an infusion over a clinically necessary period of time, e.g., from several minutes to several hours, e.g., up to a 24 hour period. If necessary, the IL-2 polypeptide administration can be repeated once or several times. Administration of the IL-2 polypeptide may be combined with administration of other agents, such as chemotherapeutic agents. Furthermore, the present invention relates to a method for preventing and/or treating cancer, comprising administering to a subject in need thereof an effective amount of an IL-2 polypeptide.

The average amount of IL-2 may vary and in particular should be based on recommendations and prescriptions of qualified physicians. The exact amount of IL-2 is a matter of preference, depending on factors such as the exact type of disorder to be treated, the condition of the patient to be treated, and other ingredients in the composition. The invention also provides for the administration of a therapeutically effective amount of another active agent. The amount to be administered can be readily determined by one of ordinary skill in the art based on the therapy with IL-2.

Examples

The following examples are provided to illustrate, but not to limit, the claimed invention.

Example 1 determination of residue positions to be mutated to an amber stop codon for incorporation of unnatural amino acids in IL-2

IL-2 has been used to treat several cancers such as renal cell carcinoma and metastatic melanoma. Commercially available IL-2Is a recombinant protein that has not been glycosylated, has had alanine-1 removed and has replaced the residue cysteine-125 with serine-125 (Whittington et al, drugs,46 (3): pp:446-514 (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 at high doses. This greatly limits its application to potential patients. The underlying mechanism of the severe side effects has been Is due to the binding of IL-2 to one of its receptors, IL-2R α. In general, when all three receptors, including IL-2R α (or CD 25), IL-2R β (or CD 122), and IL-2R γ (or CD 132), are present in a tissue, IL-2 can form heterotrimeric complexes not only with these receptors, but also with IL-2R β and IL-2R γ. In the clinical setting, when high doses of IL-2 are used, IL-2 begins to bind IL-2. Alpha. Beta.gamma., which is Tp _reg The major receptor form in cells. T is _reg The inhibitory effect of the cells causes an unwanted effect of the use of IL-2 in cancer immunotherapy. To alleviate the side effects of IL-2, a number of approaches have been previously used. One of the major breakthroughs is the invention from Nektar, which uses 6 PEGylated lysines to mask the IL2R α binding region on the surface of IL-2 (Charych et al, clin Cancer Res,22 (3): pp:680-90 (2016)). PEGylated IL-2 not only has an extended half-life, but also shows dramatically reduced side effects. However, results from activity studies show that the effective form of pegylated IL-2 in this heterogeneous 6-pegylated IL-2 mixture is only a mono-pegylated form. Thus, there is a need for more efficient pegylated IL-2 with homogeneous products.

In the present application, the incorporated unnatural amino acid provides a unique coupling site for use in IL-2 PEGylation. The resulting pegylated IL-2 muteins had a homogeneous product rather than the heterogeneous 6-pegylated IL-2 previously derived from Nektar.

The sites used in the production of IL-2 muteins can be selected by analyzing the existing crystal structure of IL-2 and its heterotrimeric receptor. In particular, the structure of IL-2R α and its interface with IL-2 has been investigated in detail (FIG. 1). The interface is divided into two regions comprising a hydrophobic center and a polarizing region. The hydrophobic center is at IL-2R alpha residue Leu-2 ^α 、Met-25 ^α 、Leu-42 ^α And Tyr-43 ^α With IL-2 residues Phe-42 ^IL-2 、Phe-44 ^IL-2 、Tyr-45 ^IL-2 、Pro-65 ^IL-2 And Leu-72 ^IL-2 Is formed between the two. The polarization region is formed by 5 ion pairs (including Lys-38) of IL-2R alpha and IL-2 ^α /Glu-61 ^IL-2 、Arg-36 ^α /Glu-62 ^IL-2 、Glu-1 ^α /Lys-35 ^IL-2 、Asp-6 ^α /Arg-38 ^IL-2 And Glu-29 ^α /Lys-43 ^IL-2 ) Is formed in between. In addition, electrostatic mapping suggests that certain other residues may play a role in mediating the interaction between IL-2R α and IL-2. These residues are Thr-37 ^IL-2 、Thre-41 ^IL-2 、Lys-64 ^IL-2 、Glu-68 ^IL-2 And Tyr-107 ^IL-2 . Thus, a site which may be used is Phe-42 ^IL-2 、Phe-44 ^IL-2 、Tyr-45 ^IL-2 、Pro-65 ^IL-2 、Leu-72 ^IL-2 、Glu-61 ^IL-2 、Glu-62 ^IL-2 、Lys-35 ^IL-2 、Arg-38 ^IL-2 、Lys-43 ^IL-2 、Thr-37 ^IL-2 、Thr-3 ^IL-2 、Lys-64 ^IL-2 、Glu-68 ^IL-2 And Tyr-107 ^IL-2 . A list of protein sequences used to produce muteins with unnatural amino acids is listed in Table 2 below:

TABLE 2 IL-2 protein sequences with potential sites for use in PEGylation

* U: unnatural amino acids

Example 2: human IL-2 expression system

This section describes expression methods for IL-2 polypeptides comprising unnatural amino acids. The host cell is transfected with an orthogonal tRNA, an orthogonal aminoacyl-tRNA synthetase, and a nucleic acid sequence as set forth in SEQ ID NO: 4. 6 or 8 or a polynucleotide encoding an IL-2 polypeptide as set forth in SEQ ID NO: 1. 2, 3, 5, 7 and 9 to 23.

Construction of E.coli expression vector and sequence verification: this example details the cloning and expression of human IL-2 (hIL-2) comprising non-naturally encoded amino acids in E.coli. All human IL-2 expression plasmids were constructed in the escherichia coli NEB5 α clonal strain (New England Biolabs, MA) by recombinant-based cloning methods using either the Gibson assembly kit (New England Biolabs (NEB), ipswich, MA) or the QuikChange mutagenesis kit (Agilent Technologies, santa Clara, CA) as described below. The E.coli expression plasmid is shown in FIG. 2.

Gibson assembly: primers used to amplify the various genes of interest (GOIs) containing donor fragments have sequences overlapping with the acceptor vector sequence of about 18-24 base pairs (bp) at their 5' -end for homologous recombination and were synthesized in Integrated DNA Technologies (IDT) (San Diego, CA). The PCR fragment was amplified using the high fidelity DNA polymerase mix Pfu Ultra II Hotstart PCR master mix (Cat. No. 600852, agilent Technologies). The PCR product was digested with Dpn1 restriction enzyme (NEB # R0176L) for 2 hours at 37 ℃ to remove plasmid background, then column purified using Qiagen PCR column purification kit (Qiagen, valencia, CA; # 28104), and quantified by Nanodrop (ThermoFisher, carlsbad, CA). The recipient vector was linearized by digestion with restriction enzymes (NEB, MA) unique to the vector at the temperature recommended by the supplier for 3 to 5 hours, purified on a PCR column and quantified. The donor insert and an appropriately prepared recipient vector were mixed at a molar ratio of 3:1, incubated for 15min at 50 ℃ using the Gibson assembly kit (NEB # E2611S), and then used for transformation into the e.coli NEB5 α strain (NEB # 2987).

Recombinants were recovered by plating the Gibson assembly mix on LB agar plates containing the appropriate antibiotic. The next day, 4 to 6 well separated single colonies were inoculated into 5mL LB + 50. Mu.g/mL kanamycin sulfate (Sigma, st Louis, MO; cat # K0254) medium and grown overnight at 37 ℃. Recombinant plasmids were isolated using the Qiagen plasmid DNA miniprep kit (Qiagen # 27104) and verified by DNA sequencing (Eton Biosciences, san Diego, calif.). The complete GOI region plus 100bp upstream and 100bp downstream sequences were verified using gene-specific sequencing primers.

QuickChange mutagenesis (QCM):all amber variants containing a TAG stop codon were generated using the QuickChange Lightning site-directed mutagenesis kit (Agilent Technologies # 201519). All QCM oligonucleotides were designed using QuickChange Web Portal (Agilent Technologies) and ordered from IDT (San Diego, CA). QCM PCR mix contained 5. Mu.l 10 Xbuffer, 2.5. Mu.l dNTP mix, 1. Mu.l (100 ng) plasmid template, 1. Mu.l oligo mix (10. Mu.M each), 1. Mu.l QuickChange Lightning enzyme, 2.5. Mu.l Quick solution and 37. Mu.l Distilled Water (DW). The DNA was amplified for only 18 cycles using the PCR procedure recommended by the kit.

After the PCR reaction was completed, the mixture was digested with DpnI enzyme with a kit (Agilent Technologies) at 37 ℃ for 2-3 hours and run on a gel to check for the presence of amplified PCR products. Subsequently, 2.5 to 5. Mu.l of the PCR product was transformed into the E.coli NEB 5. Alpha. Strain. Recombinant plasmids from 4 to 6 colonies were then isolated and sequence verified as described in the Gibson assembly section above.

Construction and validation of expression strains (AXID):to prepare the AXID production strain, chemocompetent E.coli W3110B60 host cells were transformed with sequence-verified plasmid DNA (50 ng), recombinant cells were selected on 2xYT +1% glucose agar plates containing 50. Mu.g/mL kanamycin sulfate (Sigma, cat # K0254), and incubated overnight at 37 ℃. A single colony from a freshly transformed plate was then propagated three times by serial streaking and incubation at 37 ℃ overnight on 2xYT +1% glucose agar plates containing 50. Mu.g/mL kanamycin sulfate. Finally, single colonies from the third streaking plate were inoculated into 20mL of Super Broth (Fisher-Optigrow) containing 50. Mu.g/mL kanamycin sulfate (Sigma, cat # K0254) ^TM # BP1432-10B 1) and incubated overnight at 37 ℃ and 250 rpm. The overnight grown cultures were then diluted with glycerol to a final glycerol concentration of 20% (w/v) (KIC, ref #67790-GL99 UK). The cell suspension was then dispensed into 1mL aliquots, placed in several cryovials and frozen at-80 ℃ for AXID production And (4) strain tubes.

After glycerol tubes of the AXID producing strains were generated as described above, they were further validated by DNA sequencing and phenotypic characterization of antibiotic resistance markers. To confirm that the AXID producer tubes have the correct plasmid in the production host, the plasmid was sequence verified. 20mL of LB containing 50. Mu.g/mL kanamycin sulfate was inoculated with a puncture from a glycerol tube of an AXID clone and grown overnight at 37 ℃ and 250 rpm. Plasmid DNA was isolated using the Qiagen miniprep kit (# 27104) and the presence of the complete GOI ORF in the isolated plasmid was confirmed by DNA sequencing (Eton Biosciences, CA).

To further verify the strain genotype of the AXID producing strain, cells from the same tube were streaked on 4 independent LB plates: LB containing 50ug/mL kanamycin sulfate, LB containing 15ug/mL tetracycline, LB containing 34ug/mL chloramphenicol, and LB containing 75ug/mL trimethoprim. They were then examined for positive growth on all these plates, as expected for the strain genotype of the W3110B60 production host strain.

Expression system:the amino acid sequence of hIL-2 and the E.coli codon optimized DNA sequences encoding hIL-2 are shown in tables 1 and 2. A translation system comprising the introduction of an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS) was used to express hIL-2 containing the non-naturally encoded amino acid (see plasmid map pKG0269; FIG. 2). The O-RS preferentially aminoacylates the O-tRNA with a non-naturally encoded amino acid. In turn, the translation system inserts the non-naturally encoded amino acid into IL-2 or an IL-2 variant in response to the encoded selector codon. Suitable O-RS and O-tRNA sequences are described in WO2006/068802 entitled "Aminoacyl-tRNA Synthetase Compositions and Uses Thereof" (Compositions of Aminoacyl-tRNA synthetases and Uses Thereof Thereof) and WO2007/021297 entitled "Compositions of tRNA and Uses Thereof" (Compositions of tRNA and Uses Thereof), which patent applications are incorporated by reference herein in their entirety.

Coli transformed with a plasmid containing the modified IL-2 variant polynucleotide sequence and an orthogonal aminoacyl-tRNA synthetase/tRNA pair (specific for a desired non-naturally encoded amino acid) allows site-specific incorporation of the non-naturally encoded amino acid into the IL-2 polypeptide. Expression of the IL-2 variant polypeptide was under the control of the T7 promoter and induced by addition of arabinose in the culture medium (see plasmid map pKG0269; FIG. 2).

Inhibition with acetyl-phenylalanine (pAF):the plasmid for expression of the IL-2 polypeptide was transformed into W3110B60 E.coli cells. Para-acetyl-phenylalanine (pAF) was added to the cells and protein expression was induced by the addition of arabinose. SDS-PAGE analysis of the expression of the IL-2 polypeptide was performed and the IL-2 polypeptide was observed. Multiple lanes are run for comparison between the original wild-type IL-2 polypeptide and the pAF-substituted IL-2 polypeptide, which is IL-2 with a substitution made at a particular amino acid residue, e.g., para-acetylphenylalanine. Expression of the T7 polymerase is under the control of an arabinose inducible T7 bacteriophage promoter. Para-acetyl-phenylalanine (pAF) was added to the cells and protein expression was induced by the addition of arabinose (final 0.2%). The cultures were incubated at 37 ℃ for several hours (3-5 hours).

Additional constructs for increasing hIL-2 expression in E.coli: in order to improve hIL-2 production in Escherichia coli, in addition to the herein reported optimization of the DNA sequence based on E.coli codon usage, the following expression parameters can be further optimized: testing different promoters other than the T7 phage promoter, such as arabinose B (araB), pTrc and phage T5 promoter; stabilization of hIL-2 mRNA; screening of different E.coli host strains other than the standard W3110B60 strain; production process parameter optimization such as temperature, medium, inducer concentration, etc.; transcription and translation control element optimization, such as start and stop codons, ribosome Binding Sites (RBSs), transcription terminators, and the like; optimizing the copy number and stability of the plasmid; translation Initiation Region (TIR) optimization.

Example 3-This example details E.coli shake flask expression assay and high cell density fermentation

Shake flask expression test: hIL-2 expression was tested in shake flask experiments using the AXID producing strain as described above. Briefly, inoculum from AXID glycerol tubes was placed in 5mL Super Broth (Fisher-Optigrow) containing 50. Mu.g/mL kanamycin sulfate (Sigma, MO) ^TM # BP1432-10B 1) medium and grown overnight at 37 ℃ with shaking. The overnight cultures were incubated in Super Broth (Fisher-Optigrow) containing 50. Mu.g/mL kanamycin sulfate (Sigma, MO) ^TM # BP1432-10B 1) medium 1. When the culture density reached an OD600 of 0.6-0.8, it was induced with 0.2% arabinose and pAF added, followed by harvesting after several hours of production (typically 3 to 5 hours). Aliquots of the harvested cells were removed and analyzed by SDS-PAGE. Optimal expression of hIL-2 was normalized by varying temperature, duration of induction, and inducer concentration. The expression of hIL-2 was confirmed by immunoblotting of the crude extract with standard monoclonal antibodies against hIL-2, following the following Western assay for hIL2 expression (FIG. 3A): harvested cell deposits were normalized to an OD600 of 5 and dissolved in a calculated amount of B-PER solution (ThermoFisher) containing lysozyme (100. Mu.g/ml) and DNase 1 (1U/ml). The sediment was mixed by vortexing at high speed for 2-5 minutes and by incubating the mixture at 37 ℃ and 250 rpm. The sample was mixed with the manufacturer's sample buffer (4X) and sample reducing agent (10X) to adjust the final concentration to 1X. A total of 20. Mu.l of sample was mixed with hIL2 standard (R) &D Systems, minneapolis, MN) were loaded together onto a pre-formed polyacrylamide gel (ThermoFisher) and electrophoretically separated in 1x MES buffer (ThermoFisher). Protein samples were transferred to nitrocellulose membranes using an iBlot apparatus and a gel transfer stack. Goat anti-human IL-2 antigen antibody (R) for hIL2&D Systems) and passed through an HRP conjugated anti-goat IgG secondary antibody (R)&D Systems) were detected using opti 4CN colorimetric substrates (Bio-Rad, hercules, calif.).

High cell density fermentation: the fermentation process for the production of hIL-2 consists of two stages: (i) inoculum preparation, and (ii) fermentor production. The inoculum was started from a single glycerin tube, thawed,in a 250mL baffled Erlenmeyer flask in 50mL defined seed medium 1 (v/v) dilution, and at 37 ℃ and 250rpm temperature. Prior to use, the fermenters were cleaned and autoclaved. A specific amount of basal medium was added to the fermentor and steam sterilized. To the basal medium were added specific amounts of kanamycin sulfate solution, feed medium and P2000 antifoam prior to inoculation. All solutions added to the fermentor after autoclaving were either 0.2 μm filtered or autoclaved before sterile addition.

The fermentor was charged in batch 4L of chemically defined medium using glycerol as carbon source. Seed culture was added to the fermentor to an initial OD600nm of 0.05. Dissolved oxygen was maintained at 30% air saturation using 480 to 1200rpm agitation and an oxygen enrichment at 6psig head pressure and 5slpm air flow. The temperature and pH were controlled at 37 ℃ and 7.0, respectively. When the culture reached an OD600nm of 35. + -.5, feeding was started at a rate of 0.25 mL/L/min. Therefore, L-Ala-pAcF (also referred to as L-Ala-pAF) dipeptide was added in an amount of 0.4 g/L. 15 minutes after dipeptide addition, cultures were induced with L-arabinose at a final concentration of 2 g/L. Cultures were harvested 6h after induction.

Reverse phase HPLC titer analysis:1.0mL of E.coli fermentation sample (cell paste) was first dried and weighed to determine the mass used for sample preparation. Lysonase Bioprocessing reagent (EMD Millipore # 71230) and Benzonase nuclease reagent (EMD Millipore # 70664) were each diluted 1. To 1.0mL of the dried cell paste was added 1.0mL of the Bugbuster-Lysonase-Benzonase mixture and the resulting mixture was vortexed vigorously. The mixture was then placed on an Eppendorf Thermomixer R shaker and shaken at 1000rpm for 20 minutes at 22 ℃. After incubation, the cell lysate was centrifuged at 16,050rcf for 5 minutes to deposit cell debris. A200 μ L aliquot of the cell lysate supernatant was then filtered by centrifugation through a 0.22 μm PVDF spin-on-tube filter (EMD Millipore # UFC30 GVNB) for 1 minute at 16,050rcf. The filtered product was then analyzed by reverse phase chromatography to determine the amount of hIL2 present in the fermentation sample. Is filled with A4.6X 150mm Zorbax 300SB-C3 (Agilent # 863973-909) reverse phase column of 3.5 μm particles separated hIL2 from host cell protein contaminants. Mobile phase a containing 0.1% trifluoroacetic acid in water was used to bind hIL2. Mobile phase B containing 0.1% trifluoroacetic acid in acetonitrile was used to elute the hIL2 from the column. The amount of hIL2 in the sample was determined by comparing the observed area counts from the fixed injection volumes against a linear equation obtained from a standard curve generated using purified hIL2. Several of the tested IL-2 amber variants, as exemplified in figure 3B, showed high titer expression in the range of about 65 to 150mg/L in high cell density e.

Example 4-This example details inclusion body preparation, refolding, purification and PEGylation

Inclusion body preparation and dissolution:cell paste harvested from high cell density fermentations was resuspended to final 10% solids by mixing in Inclusion Body (IB) buffer I (50mM Tris pH 8.0 100mm edta 1 ÷ triton X-100). The cells were lysed by passing the resuspended material through a microfluidizer a total of two times. The sample was then centrifuged (14,000g 15 min; 4 ℃) and the supernatant decanted. Inclusion body deposits were washed by resuspending in an additional volume of IB buffer I (50mM Tris pH 8.0 100mm nacl 1% > -triton X-100 ℃) and the resuspended material was passed through a microfluidizer a total of two times. The sample was then centrifuged (14,000g 15 min; 4 ℃) and the supernatant decanted. The inclusion body sediments were each resuspended in 1 volume of buffer II (50mM Tris pH 8.0 100mm nacl 1mm edta 4 ℃). The sample was centrifuged (14,000g 15 min; 4 ℃) and the supernatant decanted. The inclusion body sediment was resuspended in 1/2 volume of buffer II (50mM Tris pH 8.0, 100mM NaCl, 1mM EDTA 4 ℃). The inclusion bodies are then divided into aliquots in suitable containers. The sample was centrifuged (14,000g, 15 min; 4 ℃) and the supernatant decanted. The inclusion bodies are dissolved or stored at-80 ℃ until further use.

The inclusion bodies were dissolved in solubilization buffer (20mM Tris, pH 8.0, 8M guanidine; 10 mM. Beta. -ME) to a final concentration of between 10-15 mg/mL. The solubilized inclusion bodies were then incubated at room temperature with constant mixing for 1 hour or until complete solubilization. The samples were then centrifuged (10,000g for 20 min; 4 ℃) to remove any undissolved material. The protein concentration of each sample was then adjusted by dilution with additional solubilization buffer if the protein concentration was high.

Refolding and purification:by subjecting the sample to conditions of 20mM Tris, pH 8.0;60% sucrose; refolding was performed by dilution at 4 ℃ to a final concentration of 0.5 mg/mL. Refolding was allowed to proceed at 4 ℃ for 5 days. Refolding the material with Milli-Q H ₂ O1:1. The material was filtered through 0.22 μmPES filter and loaded into 20mm tris, ph 8.0; blue Sepharose FF column (GE Healthcare) equilibrated in 0.15M NaCl (buffer A). The column was washed with 5 column volumes of 30% buffer B (20mM tris, ph 8.0, 50% ethylene glycol). The IL-2 polypeptide was eluted by washing the column with 10 column volumes of 100% buffer B.

PEGylation and post-PEGylation purification: IL-2 pools were taken and diluted 10X with Milli-Q water. The pH of each sample was adjusted to 4.0 with 50% glacial acetic acid. The sample was concentrated to-1.0 mg/mL. To each sample was added 1. The samples were then incubated at 27 ℃ for 48-72 hours. Taking out the sample and subjecting the sample to water: ( <8 m/S) and loaded in buffer a (50mM naac, ph 6.0;50mM NaCl; 0.05%) on an equilibrated SP HP column (GE Healthcare). The IL-2 polypeptide was eluted with 5 column volumes of buffer B (50mM NaAc, pH 6.0, 500mM NaCl 0.05% zwittergent 3-14). Fractions of IL-2 were combined and run on a Superdex 200 pore size exclusion column equilibrated in IL-2 storage buffer (20mM NaAc, pH 5.0;150mM NaCl 3-14). The PEGylated material was collected and stored at 4 ℃.

Example 5-This example details the purification of IL-2 from E.coli and mammalian expression systems. This example also discloses PEGylation, site-specific conjugation, and PEG-IL-2 purification procedures.

Preparation from E.coli inclusion bodies:IL-2 inclusion bodies were isolated by a series of washing steps. The frozen cell paste was resuspended in wash buffer I (50mM Tris, pH 8.0 1% triton X-100, 1M Urea, 5mM EDTA,1mM PMSF) to a concentration of 10% (W/V), homogenized at 4 ℃ and then centrifuged (15, 000g,30 min, 4 ℃). The supernatant was discarded and the inclusion body sediment was resuspended in washing buffer II (50mM Tris, pH 8.0 1% triton X-100, 5mM EDTA). The resuspended inclusion bodies were centrifuged at 15,000g for 30 minutes at 4 ℃. The supernatant was discarded and the inclusion body pellet was resuspended in wash buffer III (50mM Tris, pH 8.0; sodium deoxycholate, 5mM EDTA). The resuspended inclusion bodies were centrifuged at 15,000g for 30 minutes at 4 ℃. The supernatant was discarded and the inclusion body pellet was resuspended in water and then centrifuged at 15,000g for 30 min at 4 ℃. The washed inclusion bodies were stored at-80 ℃ until further use.

Refolding:IL-2 inclusion bodies were solubilized by resuspending in water and adjusting the pH of the mixture to pH 12.2. Insoluble material was removed by centrifugation (12,000g, 15 min). Solubilized IL-2 was refolded by adjusting the pH down to pH 8.8. + -. 0.2. Appropriate disulfide bond formation was promoted by adding 50 μ M cystine to the refolding reaction. The refolding reaction was allowed to stand at room temperature for 16-22 hours. Host cell contaminants were precipitated by adjusting the refolding reaction to pH 6.8 with hydrochloric acid. The precipitate was removed by centrifugation (12,000g, 15 min), and the clear supernatant was adjusted to pH7.8 with sodium hydroxide and filtered at 0.22 μm.

Column purification: refolded IL-2 was loaded onto a Capto Adhere (GE Healthcare) column equilibrated in buffer A (20 mM sodium phosphate, pH 7.8). After loading, the column was washed with buffer a (20 mM sodium phosphate, pH 7.8) and IL-2 was eluted from the column using a linear pH gradient up to 100% buffer B (20 mM sodium phosphate, 20mM citric acid, pH 4.0) in 20 column volumes. Fractions containing IL-2 were collected, adjusted to pH 4.0 with 10% acetic acid, and buffer exchanged in 20mM sodium acetate, 2.5% trehalose, pH 4.0. IL-2 was concentrated to 1-10mg/mL, filtered at 0.22. Mu.M, and stored at-80 ℃.

Purification of IL-2 from eukaryotic expression systems:the cell culture medium containing the His-tagged IL-2 was adjusted to pH 7.8 with sodium hydroxide and loaded onto a Ni Excel column (GE Healthcare) equilibrated in 20mM sodium phosphate, pH 7.8. After loading, the column was washed with buffer a (20 mM sodium phosphate, pH 7.8) and then with wash buffer B (20 mM sodium phosphate, 1.0M sodium chloride, 30mM imidazole, pH 7.8) to remove host cell contaminants. IL-2 was eluted from the column with elution buffer (10 mM sodium phosphate, 300mM imidazole, pH 7.8) and the fractions containing IL-2 were combined. The IL-2 pool was loaded onto a Capto Adhere Impres (GE Healthcare) column equilibrated in buffer A (20 mM sodium phosphate, pH 7.8). After loading, the column was washed with buffer a (20 mM sodium phosphate, pH 7.8) and IL-2 was eluted from the column using a linear pH gradient up to 100% buffer B (20 mM sodium phosphate, 20mM citric acid, pH 4.0) in 20 column volumes. Fractions containing IL-2 were collected, adjusted to pH 4.0 with 10% acetic acid, and buffer exchanged in 20mM sodium acetate, 2.5% trehalose, pH 4.0. IL-2 was concentrated to 1-10mg/mL,0.22 μ M filtered, and stored at-80 ℃ until further use.

Site-specific conjugation and PEG-IL-2 purification:the IL-2 variant containing the unnatural amino acid (nnaA) p-acetylphenylalanine was buffer exchanged in coupling buffer (20 mM sodium acetate, pH 4.0) and concentrated to 1-10mg/mL. To the reaction was added a final 100mM of acethydrazide followed by a 10-fold molar excess of aminoxy functionalized PEG. The coupling reaction was incubated at 25-30 ℃ for 18-20 hours. After coupling, the pegylated IL-2 was diluted with 20mM sodium acetate 1, pH 5.0, and loaded onto Capto SP Impres column. After loading, the column was washed with buffer a (20 mM sodium acetate, pH 5.0) and pegylated IL-2 was eluted from the column using a linear gradient up to 100% buffer B (20 mM sodium acetate, 1.0M sodium chloride, pH 5.0) over 20 column volumes. Fractions containing PEGylated IL-2 were collected and buffer exchanged in 10mM sodium phosphate, 100mM sodium chloride, 2.5% trehalose, pH 7.0. IL-2 was concentrated to 1-2mg/mL, filtered at 0.22. Mu.M, and stored at-80 ℃ untilTo further use.

IL-2/CD-25 binding assay by biolayer interferometer:IL-2/CD25 multiple concentration binding kinetics experiments were performed on an Octet RED96 (PALL/ForteBio) instrument at 30 ℃. An anti-human Fc antibody capture biosensor (PALL/ForteBio, cat. No. 18-5063) was loaded with purified CD25-Fc fusion protein in 1 XHBS-P + buffer (GE Healthcare, cat. No. BR-1008-27). A level of immobilization between 0.8nm and 1.0nm is achieved. The loaded biosensor was washed with 1X HBS-P + buffer to remove any unbound protein prior to measuring the binding and dissociation kinetics. For monitoring of the binding phase, IL-2 analyte samples were diluted with 1 XHBS-P + buffer and transferred to solid black 96-well plates (Greiner Bio-One, cat. No. 655209). IL-2 samples were allowed to bind to the CD25-Fc loaded biosensor for 60 seconds. The dissociation phase was recorded for 90 seconds in wells of a solid black 96-well plate containing 1X HBS-P + buffer. Data were referenced using parallel buffer blank subtraction, baseline was aligned with y-axis, and smoothed by Savitzky-Golay filter in Octet data analysis software version 10.0 (PALL/ForteBio). Use description 1:1 global fit to the treated kinetic sensorgrams in combination with the chemometric Langmuir model (fig. 4A).

Example 6-This example details the cloning and expression of IL-2 in mammalian systems, including non-naturally encoded amino acids. This example also describes methods for assessing the biological activity of modified IL-2.

IL-2 variants are produced in mammalian cells.Native human IL-2 is a glycosylated protein with O-linked glycosylation at Thr-3 (Conradt et al, eur J Biochem,153 (2): pp:255-61 (1985)). Although non-glycosylated IL-2 has been shown to have similar activity as glycosylated IL-2, glycosylated human IL-2 has been shown to have better activity in clonal growth and long-term propagation of activated human T cells. There are also reports indicating that native IL-2 has a more specific activity. It has also been shown that IL-2 expression in mammalian cells is superior to its expression in E.coli (Kim et al, J Microbiol B)iotechnol,14 (4), 810-815 (2004)). In the present invention, wild-type IL-2 and its various different mutant proteins, respectively, designed in tables 1 and 2 above, can be produced in CHO cells (as described in the examples herein).

To produce IL-2 muteins containing an unnatural amino acid at a desired position, each mutein is produced in a stable pool or stable cell line derived from a transfected platform cell line containing an engineered orthogonal tRNA/tRNA synthetase pair (Tian et al, proc Natl Acad Sci U S A,111 (5): pp:1766-71 (2014) and PCT/2018US/035764: each of which is incorporated by reference in its entirety). Briefly, CHOK1 cells were engineered to stably express a platform cell line that is a proprietary orthogonal tRNA synthetase (O-RS) and its cognate amber suppressor tRNA (O-tRNA), for efficient incorporation of an unnatural amino acid, e.g., pAF, into a therapeutic protein, e.g., IL-2, e.g., in CHO cells. The platform cell line was then pre-adapted to suspension growth for rapid development in a bioreactor. The platform cell lines have been well characterized and evolved with improved unnatural amino acid incorporation efficiency and clonal selection efficiency. The platform cell line is used as a maternal cell to produce therapeutic proteins incorporating unnatural amino acids for use in early studies at titers above 100mg/L by rapid and efficient transient expression. Transient transfection and stable pool production were performed to assess the expression of candidate molecules and provide material for functional assays to identify lead molecules. A production cell line is generated by transfecting a gene of interest containing an amber nonsense codon in a GS expression system into a platform cell line to produce an IL-2 protein incorporating an unnatural amino acid. A stable cell line development strategy was performed using the platform cell line as a maternal cell to obtain a producer cell line with 5-10PCD for 3-4 months and 20-30PCD for 6 months.

In the present invention, a human IL-2cDNA with its native signal peptide sequence (NM — 000586.3) was synthesized and cloned into a mammalian expression vector containing the GS selection marker (fig. 4B). As shown in Table 1, the cloned wild-type human IL-2cDNA retained its original DNA sequence of each amino acid without any mutations. In contrast, during the production of IL-2 variants (table 2), each of the 15 mutant proteins had a unique position mutated to an amber stop codon (TAG), which can be suppressed and expressed in engineered cells to produce nnAA-containing proteins.

Establishment of engineered CHO cells for expression of IL-2 variants.Engineered CHO cells were derived from the gene knockout of previously established proprietary platform cells (PCT/2018 US/035764, which is incorporated herein by reference in its entirety). Briefly, the network-based target discovery tool, CRISPy, was used to quickly identify gRNA target sequences in the early exons with zero off-target in CHO-K1 cells. The grnas were cloned into a mammalian expression vector pGNCV co-expressing a CHO codon-optimized form of Cas 9. Production cell lines were transfected with protein expression vectors to generate cell pools, which were then cloned to identify single cell isolates with gene knockouts. The frequency of indels (indels) from the combined results of the multiple projects was 30-90% and 50-80% for cell pools and single cell isolates, respectively. CRISPR was used to knock out target genes in CHO cells. Specifically, in order to increase the mRNA stability of IL-2, the UPF1 gene was knocked out using CRISPR technology. Grnas for knockdown are shown in fig. 5. Two UPF1-KO cell lines were obtained after 192 clones were screened and confirmed to have a UPF1 knock-out by sequencing (fig. 6). The resulting UPF1-KO cell line was then used to transiently express IL-2 variants.

Transient expression of IL-2 in engineered CHO cells.IL-2 variants were transiently expressed in UPF1-KO cell lines obtained as disclosed in the above examples. Transfection was performed using electroporation using Amaxa kit (Lonza) for suspension cells. 6ug of plasmid prepared as disclosed in the above example was transfected into 2X 10 ⁶ In engineered CHO cells. After transfection, cells were incubated at 37 ℃ for 4 days, and then titers were analyzed by ELISA using a commercial kit from Invitrogen (Carlsbad, CA). As shown in fig. 7A and 7B, variant F42 exhibited the highest expression level among the 15 variants during transient expression.

T cell expansion assay of IL-2 variants in CTLL-2 cells.CTLL-2 cell expansion assays were performed using transiently expressed F42 variant supernatants from transfected engineered CHO cells. During the cell proliferation assay, wild-type IL-2 was used as a control for 100% proliferation (shown in figure 8). Variant F42 was prepared as serial dilutions, 10ng/mL, 3.33ng/mL, 1.11ng/mL, 0.37ng/mL, 0.12ng/mL and 0.04ng/mL in the assay. Cell Titer Glo (Promega, WI) was used for Cell proliferation. The luminescence signal is read on TECAN genes pro. As shown in FIG. 8, F42 showed an EC of around 0.24ng/mL ₅₀ While retaining 95% of its function compared to its wild-type control. The general procedure used to study the IL-2 variants of the invention is shown below:

example 7Screening of IL-2 variants by CTLL-2 cell amplification

Using the CTLL-2 cell expansion assay as disclosed in the examples, 20 different IL-2 variants were screened (Charych, d. Et al, PLoS One,12 (7): p.e0179431, 2017) including 16 initially selected sites known in the art (including wild-type) and 4 other sites (K32, K48, K49, K76). As shown in figure 9 and table 3, most variants retained their activity after mutagenesis. Due to the property of CTLL-2 cells to have residual IL-2R α expression, variants with the lowest binding to CTLL2 cells at mutagenesis still showed some intrinsic binding to IL-2R α, although this binding was very low. For example, the P65 IL-2 variant that exhibits the lowest binding to IL-2 Ra was observed to exhibit some inherent biased binding to IL-2 Ra. The identified variants were further analyzed for their binding capacity after PEGylation.

TABLE 3 Activity of IL-2 variants Using CTLL2 proliferation assay

Example 8-Analysis of selected variants using in vitro binding assays

Analysis of selected variants P65, Y45, E61, F42, K35, K49 and T37 was performed using the in vitro binding assay biolayer interferometer assay described in the examples above. Each variant was coupled with 20K PEG at their specific site, respectively. The pegylated variants were then analyzed by BLI (bio-layer interferometer) assay described elsewhere in the examples. As shown in FIGS. 10A-10C, the PEGylated variants were tested for binding to IL-2R α on Octet. Wild-type IL-2 was used as a positive control in the assay. After pegylation, most variants showed a sharp decrease in binding to IL-2 ra between 92.9% and 99.9%. In the pegylated variants tested, P65 and Y45 showed activity blockages of more than 99%, table 4.

TABLE 4 in vitro binding Activity of IL-2 variants

IL-2 variants	Steady state Kd (nM)	Blockade of binding to IL-2R alpha
			IL-2WT	11	0％
P65-PEG20K	32000	99.9％
			Y45-PEG20K	1900	99.4％
E61-EPG20K	1400	99.0％
			F42-PEG20K	1100	99.0％
K35-PEG20K	840	98.7％
			K49-PEG20K	180	93.8％
T37-PEG20K	155	92.9％

Example 9-Analysis of selected variants using PathHunter dimerization assay

To find the optimal site for PEG coupling, a PathHunter dimerization assay developed by discover x (Fremont, CA) was used. In general, the assay system uses an exogenously expressed IL-2 receptor that has been engineered to have complementary binding domains for the enzyme, which generates a chemiluminescent signal once the previously separated receptor is activated by dimerization with the addition of an IL-2 molecule (FIG. 11). Two cell lines were generated in U2OS cells. One cell line expresses three receptors, IL-2R α, IL-2R β and IL-2R γ. Another cell lineExpressing IL-2R beta and IL-2R gamma. Binding EC for each variant ₅₀ Ratio of values (EC) ₅₀ -βγ/EC ₅₀ α β γ) were used to estimate their relative retained binding capacity. As shown in table 5, the best possible variants have a value of 1, meaning that 100% of their β γ binding capacity is retained, while α binding is 100% blocked. As noted, the variants Y45-BR4 (variant Y45 coupled with 20K 4-branched PEG) and P65-PEG20K (variant P65 coupled with 20K-linear PEG) showed the lowest values, indicating that these two pegylated variants would be the best candidates for further evaluation.

TABLE 5 binding Activity of IL-2 variants Using dimerization assay-experiment 1

As shown in Table 6, in another experiment performed, in addition to the variants Y45-BR4 and P65-PEG20K, variants P65-BR4 (variant P65 coupled with 20K 4-branched PEG) and P65-BR2 (variant P65 coupled with 20K 2-branched PEG) were also selected as candidates for further evaluation.

TABLE 6 improved binding Activity of IL-2 variants Using dimerization assay-experiment 2

Compound (I)	βγEC50(nM)	αβγEC50(nM)	Beta gamma/alpha beta gamma ratio
				The best possible	0.41	0.41	1
P65-BR4	8.50	4.86	1.75
				P65-BR2	13.06	4.80	2.96
Y45-BR4	3.67	0.84	4.31
				P65-PEG20K	5.21	1.10	5.06
Y45-BR2	3.39	0.40	8.34
				IL-2WT	0.41	0.03	16.67
Y45-PEG20K	2.34	0.04	30.87

Example 10-Ex vivo pSTAT5 assay for IL-2 variants

To further evaluate the in vitro function of the pegylated variants, ex vivo assays using PBMCs were used. As shown in figure 12, binding of IL-2 to its receptor triggers an increase in phosphorylation of STAT5 (pSTAT 5). Thus, detection of pSTAT5 levels would be an indicator of IL-2 variant binding to endogenous IL-2 receptors. Human whole PBMCs were treated with selected pegylated variants such as Y45-BR2 (variant Y45 coupled with 20K 2-branched PEG), Y45-BR4 (variant Y45 coupled with 20K 4-branched PEG), and P65-PEG20K (variant P65 coupled with linear 20 KPEG), and then separated into two populations of CD8+ T cells and CD4+ Treg cells. As shown in table 7, all three variants showed greatly improved activity with respect to their retained β γ binding activity and blocked α binding activity. These results are further supported by variants tested in another pSTAT5 assay as shown in table 8. The results from this pSTAT5 assay (table 8) show that many variants have dramatically improved activity with respect to their reduced ability to bind Treg cells and relatively maintained binding to CD8+ cells. In table 8, the calculated CD8+/Treg ratio is used to indicate the ranking of the variants so that the PathHunter assay results can be directly compared to the pSTAT5 assay results by a similar ranking system.

Table 7-binding activity of IL-2 variants using ex vivo assay-experiment 1

Table 8-improved binding activity of IL-2 variants using ex vivo assay-experiment 2

Compound (I)	EC50-CD8(nM)	EC50-Treg(nM)	Ratio (CD 8/Treg)
				IL-2WT	0.03377	0.0002857	118.2
Y45-BR2	4.604	36.003	0.13
				Y45-PEG20K	3.367	5.377	0.61
P65-BR2	41.467	111.644	0.37
				Y45-BR4	7.462	3.398	2.20
P65-PEG20K	10.643	4.514	2.36
				P65-BR4	23.961	4.351	5.51

Example 11-Comparison of clonal growth and Long-term reproduction of CTLL-2 cells in the Presence of glycosylated IL-2 produced in CHO mammalian cells and non-glycosylated IL-2 produced in E.coli

The native human IL-2 has been reported to be a glycosylated protein with O-linked glycosylation at Thr-3 (Conradt et al, eur J Biochem 153 (2): 255-261 (1985)). This glycosylation function is associated with higher solubility at physiological pH, slower clearance in vivo and lower immunogenicity in cancer therapy compared to non-glycosylated IL-2 (Robb et al, proc Natl Acad Sci U S A81 (20): 6486-6490 (1984); goodson et al, biotechnology (NY) 8 (4): 343-346 (1990)). More importantly, it has been shown that glycosylated IL-2 is superior to non-glycosylated IL-2 in promoting clonal growth and long-term propagation of homologously activated human T cells (Pawelec et al, immunology 174 (1): 67-75 (1987)), suggesting that glycosylated IL-2 is a better choice for therapeutic applications.

To further analyze the biological function of both glycosylated IL-2 and non-glycosylated IL-2, experiments were performed to analyze the clonal growth rate and long-term proliferation frequency of CTLL-2 cells (FIG. 13). Individual CTLL-2 cells were placed in pre-feeder cell-coated 96-well plates (Thermo Fisher, waltham, MA, CAT # A34180) with gamma-irradiated CF1-MEF (mouse embryonic fibroblasts) cells. The percentage of colony numbers that grew out and the percentage of colonies that survived at the end of the 19 day incubation were counted and analyzed during the 19 day growth period with a single treatment with various concentrations (0.005 nM, 0.05nM, 0.5nM and 5 nM) of wild type IL-2 produced from CHO cells or E.coli. As shown in FIG. 13 (with 0.5nM treatment as an example), glycosylated IL-2 showed superior activity in promoting clone growth compared to non-glycosylated IL-2. On average, glycosylated IL-2 promoted clonal growth twice as much as non-glycosylated IL-2 in the presence of 0.5nM IL-2 concentration as the optimal cell culture conditions for CTLL-2 cell growth. After long-term incubation (-19 days), colony survival from glycosylated IL-2 treatment was 4-fold higher than that of non-glycosylated IL-2 treatment. The data clearly demonstrate that glycosylated IL-2 has superior activity in promoting clonal growth and long-term proliferation of IL-2 responsive cells, and further supports its promising therapeutic applications.

Example 12Increased titer of IL-2 expression in a novel stable host CHO cell line.

A number of methods have been tried in the art to increase the expression of wild type IL-2 and variants thereof in CHO cells (see, e.g., kim et al, J Microbiol Biotechnol,14 (4), 810-815 (2004)). However, increasing the expression of proteins containing unnatural amino acids in the industry is challenged by relatively low yields in mammalian cells. To address this challenge in the present invention, a proprietary technique for improving protein titer production in eukaryotic cell lines disclosed in PCT/2018US/035764 (which is incorporated herein by reference in its entirety) is utilized to generate stable pooled cells of IL-2 and variants thereof and to use them in the generated stable IL-2 cell lines.

Briefly, it was found that a different five generation platform cell line expressing Bax/Bak knockdown dramatically increased protein expression of IL-2 and increased IL-2 protein production to about 40% higher than the parental cell line. In addition to inhibiting apoptosis in these cells by Bax/Bak knock-out, UPF1 knock-out was also found to further increase the expression of IL-2.

Both wild-type IL-2 and its variants (F42, Y45 and P65) have been tested by generating stable pools thereof. As shown in FIG. 13, stable pools of the three IL-2 variants F42, Y45 and P65, including wild-type IL-2, have greatly improved expression levels compared to levels already in the art (see, e.g., kim et al, J Microbiol Biotechnol.,14 (4), 810-815, (2004)), up to about 740mg/L for wild-type and up to 120mg/L for the F42 variant (shown in FIG. 14) after generation of the respective stable pool. The data show that by generating novel CHO cell lines with highly efficient incorporation of unnatural amino acids, production or yields of IL-2 proteins can be improved or enhanced. It has also been shown that expression levels and function are site-specifically related.

Example 13IL-2 variant F42-R38A shows complete blocking of IL-2R alpha binding

As disclosed herein, the non-naturally encoded amino acid substitutions are combined with other additions, substitutions, or deletions within IL-2 to affect other biological traits of the IL-2 polypeptide, including, but not limited to, increasing the stability of IL-2 (including, but not limited to, resistance to proteolytic degradation) or increasing the affinity of IL-2 for its receptor, increasing the drug stability of IL-2, enhancing the tumor suppression and/or tumor reduction activity of IL-2, increasing the solubility of IL-2 or variants (including, but not limited to, when expressed in escherichia coli or other host cells), increasing the solubility of IL-2 after expression in escherichia coli or other recombinant host cells, increasing the solubility of the polypeptide after expression in escherichia coli or other recombinant host cells, modulating the affinity for an IL-2 receptor, a binding protein, or a related ligand, modulating signal transduction after binding to an IL-2 receptor, modulating the circulating half-life, modulating release or bioavailability, facilitating purification, or improving or altering a particular route of administration, increasing the affinity of an IL-2 variant for its receptor, increasing the affinity for IL-2 β -R and/or γ -R variants.

Thus, to improve the function of variant F42, a new variant with an additional mutation R38A was prepared in CHO cells. As shown in figure 15A, titers increased to 118mg/L during stable cell line production using a combination of unnatural and natural amino acid substitutions in IL-2 variant F42 in stable pools. In the presence of the R38A mutation, the protein expression level of variant F42 was not only maintained, but also showed a 20% increase. To test the function of the PEGylated F42-R38A variant, a CTLL-2 cell binding assay was performed. As shown in the table 9 below, the following examples,the F42-R38A-20K 2-branched PEG (variant F42-R38-BR 2) conjugate showed an EC of 15.9nM ₅₀ In contrast, F42 showed an EC of 3.6nM ₅₀ Therefore, the binding blocking efficiency was improved more than 4-fold (fig. 15B). EC of 0.025nM based on wild type IL-2 ₅₀ The binding blocking efficiency was over 99.9%. Such variants show great potential for therapeutic applications in terms of high protein expression levels and efficiency of blocking binding to IL-2 ra.

TABLE 9 CTLL-2 binding assay for PEGylated F42-R38A variants

	WT-IL2	F42-PEG20K-BR2	F42-R38A-PEG20K-BR2
				EC50	0.025nM	3.6nM	15.9nM

The binding kinetics of the F42pAF variant, the R38A-F42pAF variant (containing unnatural amino acids and point mutations), and the F42-R38A-PEG20K-BR2 were evaluated by BLI assays to determine the effect of the R38A mutation on the binding of IL-2R α. FIG. 15C shows the binding sensorgram for the three constructs, and the associated binding constants (KD) are shown in Table 10. As seen in Table 10, IL-2-F42pAF had an IL-2R α binding KD of 20 nM. After addition of the R38A mutation, the IL-2-F42-R38ApAF has an IL-2 Ra binding KD of 233nM, which corresponds to a 12-fold decrease in IL-2 Ra binding. After coupling IL-2-R38A-F42pAF to a 20K 2-branched PEG molecule, IL-2R α binding was prevented. The results clearly demonstrate that the added mutation effectively blocks the binding of F42-R38A to its receptor IL-2R α.

TABLE 10 conjugation of IL-2 PEGylated variants with natural and unnatural amino acid substitutions

	F42pAF	F42-PEG20K-BR2	F42-R38A-PEG20K-BR2
				K D	20nM	233nM	Without bonding

Example 14Pharmacokinetic (PK) studies in naive CD-1 mice

Group 3 female CD-1 mice IV were given a single dose of wild type IL-2 (IL-2-WT) or the PEGylated IL-2 variant Y45-PEG20K-BR2 or F42-R38A-PEG20K-BR2 by bolus injection and plasma concentrations were assessed over time. The study design is summarized in table 11. The study included 14 time points (0, 0.08, 0.25, 0.5, 1, 2.5, 5, 7, 24, 72, 168, 240, 336, 408 hours) and 5 mice were sacrificed at each time point. Bioanalysis of plasma samples was performed using ELISA assays. PK data analysis was performed using WinNonlin software. The results are summarized in fig. 16 and table 12, which depict the mean plasma concentration over time. PEGylated IL-The 2 variant Y45-PEG20K-BR2 shows a t of 8.5 _1/2 In contrast thereto the PEGylated IL-2 variant F42-R38A-PEG20K-BR2 showed a t of 7.6 _1/2 。

TABLE 11 PK Studies of IL-2 variants in naive CD-1 mice

TABLE 12 PK parameters of IL-2 variants in CD-1 female mice

Parameter(s)	Unit of	Y45-PEG20K-BR2	F42-R38A-PEG20K-BR2	IL-WT
					Cmax	ng/mL	1457	1190	63
AUC 0-t	h*ng/mL	11170	8572	11
					R 2		0.999	0.985	0.991
AUC INF	h*ng/mL	12955	8939	19.1
					t 1/2	Hour(s)	8.5	7.6	50.13

Example 15In vitro binding assays for IL-2 conjugates

The kinetics of binding of IL-2 wild type (IL-2 WT; FIG. 17A), IL2-F42-R38A-P65R-PEG20K-BR2 (FIG. 17B), IL2-Y45-M46L-PEG20K-BR2 (FIG. 17C), and IL2-Y45-M46I-PEG20K-BR2 (FIG. 17D) were evaluated using the BLI assay described in the examples above to determine the binding of pegylated variants to IL-2R α. FIGS. 17A-17D depict binding sensorgrams for wild-type IL-2 and three variants. As seen in FIGS. 17A-17C, none of the three PEGylated variants showed binding to IL-2R α.

Example 16 CTLL-2 proliferation assay for IL-2 variants F42-R38A-P65R

To further improve the function of the variant F42-R38A, a new variant with an additional mutation P65R was prepared in CHO cells. To test the function of the F42-R38A-P65R variants, CTLL-2 cell binding assays were performed as described in the examples above. As shown in FIG. 18 and Table 13, the PEGylated F42-R38A-P65R showed an EC of 140.2nM ₅₀ In contrast, PEGylated F42-R38A has an EC of 7.6nM ₅₀ . This shows an increase in binding blocking efficiency of more than 18-fold. EC of 0.025nM based on wild type IL-2 ₅₀ The binding blocking efficiency was over 99.9%. Thus, the pegylated F42-R38A-P65R variant shows great potential for in vivo applications, based on its high protein expression level and excellent blocking of binding to IL-2 ra.

TABLE 13 EC of PEGylated IL-2 variants ₅₀ (nM)

Variants	EC 50 (nM)
		Y45-PEG20K-BR2	4.1
Y45-M46I-PEG20K-BR2	9.8
		Y45-M46LPEG20K-BR2	11.1
P65-PEG20K-BR2	74.3
		F42-R38A-PEG20K-BR2	7.6
F42-R38A-P65R-PEG20K-BR2	140.2
		F42-PEG20K-BR2	2.8
IL-2WT	0.020
		IL-2 commercial control	0.025

Example 17Pharmacokinetic (PK) study of IL-2 variants in naive CD-1 mice

To improve PK parameters, a new PK study was performed using a larger size 40K pegylated IL-2 variant. Groups of 4 groups of 5 female CD-1 mice were IV bolus-administered with a single dose of wild-type IL-2 (IL-2-WT) or pegylated IL-2 variant Y45-PEG40K-BR2 or F42-R38A-P65R-PEG30K-L (L = linear) or F42-R38A-P65R-PEG40K-BR2 (BR = branched). Plasma concentrations were assessed at 14 time points (0, 0.08, 0.25, 0.5, 1, 2.5, 5, 7, 24, 72, 168, 240, 336, 408 hours). Bioanalysis of plasma samples was performed using ELISA assays. PK data analysis was performed using WinNonlin software. The results are summarized in fig. 18 and table 14, which depict the mean plasma concentration over time. PEGylated IL-2 variants Y45-PEG40K-BR2, F42-R38A-P65R-PEG30K-L and F42-R38A-P65R-PEG40K-BR2 show t values of 24.2, 12.9 and 26.5, respectively _1/2 。

TABLE 14 PK parameters of PEGylated IL-2 variants in CD-1 female mice

Example 18Study of efficacy in C57BL/6 mice

PEGylated IL-2 variants Y45-PEG40K-BR2, F42-R38A-P65R-PEG30K-L and F42-R38A-P65R-PEG40K-BR2 were tested for anti-tumor efficacy in B16-F10 tumor bearing C57BL/6 mice. When the tumor is about 80-100mm ³ All mice were dosed intravenously at 10 mg/kg. Tumor growth of the animals was monitored by caliper measurement (fig. 20A) and body weight was monitored (fig. 20B). The results shown in FIGS. 20A and 20B respectively indicate the pairsSignificant tumor size reduction and weight loss for all pegylated IL-2 variants tested.

Additional studies were conducted to further investigate the anti-tumor efficacy and cytotoxicity of pegylated IL-2 variants Y45-PEG40K-BR2, F42-R38A-P65R-PEG30K-L, and F42-R38A-P65R-PEG40K-BR2 in B16-F10 tumor-bearing mice, including intravenous doses ranging from about 0.01mg/kg to about 5mg/kg, including 0.01mg/kg, 0.03mg/kg, 0.1mg/kg, 0.3mg/kg, and 5mg/kg.

Example 19Efficacy Studies of B16F10 tumor model in BALB/c mice

PEGylated IL-2 variants F42-R38A-P65R-PEG30K-L, F-R38A-P65R-PEG 40K-BR2, Y45-PEG30K-L and Y45-PEG40K-BR2 were tested for anti-tumor efficacy in BALB/c mice bearing B16F10 tumors (Table 15). When the tumor is about 100mm ³ All mice were dosed intravenously and tumor growth was monitored. As shown in fig. 21A and 21B, the data indicate that tumor size is significantly reduced using all of the pegylated IL-2 variants tested. The individual final tumor volumes are shown in fig. 22, and the final Tumor Growth Inhibition (TGI) at day 14 is summarized in table 15.

TABLE 15 efficacy Studies of the B16F10 tumor model in BALB/c mice

Example 20Efficacy Studies of the CT26 tumor model in BALB/c mice

PEGylated IL-2 variants F42-R38A-P65R-PEG30K-L and Y45-PEG30K-L were tested for anti-tumor efficacy in BALB/c mice bearing CT26 tumors (Table 16). When the tumor is about 100mm ³ All mice were dosed intravenously at 0.3mg/kg, 1mg/kg and 3 mg/kg. Tumor growth was monitored by caliper measurement and body weight was monitored. The data shown in figures 23A and 23B indicate that tumor size was significantly reduced without weight loss using all of the pegylated IL-2 variants tested (figure 23C). The individual final tumor volumes are shown in figure 24, and the final Tumor Growth Inhibition (TGI) at day 17) Summarized in table 16.

TABLE 16 efficacy study of CT26 tumor model in BALB/c mice

Example 21Effect of PEGylated IL2 on CD8+ and CD4+ cells in PBMCs

Blood was drawn from 5 mice in each treatment group on day 7 post-dose and analyzed by FACS analysis for CD45, CD3, CD8 and CD4. The resulting image representations are shown in fig. 25A-25C. The percentage of CD8+ cells in the CD3+ population is shown in fig. 25A, indicating a significant increase in CD8+ cells treated by pegylated IL 2. The percentage of CD4+ cells in the CD45+ population shown in figure 25B indicates that there was no significant increase in CD4+ cells by pegylated IL2 treatment. The ratio of CD8+/CD4+ cells shown in fig. 25C indicates that the ratio of CD8+/CD4+ increases significantly in a dose-responsive manner.

Example 22Effect of PEGylated IL2 on CD8+ TIL in CT26 tumors

Immunohistochemistry (IHC) was performed to assess the effect of Y45-PEG30K-L on Tumor Infiltrating Lymphocytes (TILs) in CT26 tumors in BALB/c mice. CT26 tumor tissue was collected from BALB/c mice 7 days after treatment with 3mg/kg Y45-PEG 30K-L. CD8+ T cells were stained and analyzed by IHC. The results showed a dramatic increase in CD8+ TIL aggregation of approximately 5-fold in the area of CT26 tumors treated with 3mg/kg Y45-PEG30K-L compared to vehicle controls (data not shown).

Example 23Melting temperature analysis by DSF

Scanning fluorescence measurements (DSF) were performed to analyze the melting temperature of wild-type IL-2 from different sources, e.g., E.coli and CHO cells. As shown in FIG. 26, the results indicate that the expression of CHO cells in the wild type IL-2 has a higher melting temperature, compared to the expression of IL-2 in Escherichia coli by up to 6.2 ℃. This increased thermostability clearly demonstrates the advantage of glycosylated IL-2 of the invention expressed in CHO cells.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication, patent application, and/or other document were individually indicated to be incorporated by reference for all purposes.

The invention is further described by the following numbered embodiments:

1. an IL-2 polypeptide comprising one or more non-naturally encoded amino acids, wherein the IL-2 polypeptide has reduced interaction with its receptor subunit compared to wild-type IL-2.

2. The IL-2 of embodiment 1, wherein the IL-2 polypeptide is identical to SEQ ID NO:2 or SEQ ID NO: 3% homology.

3. The IL-2 of embodiment 1, wherein the IL-2 polypeptide is identical to SEQ ID NO:2 at least 95% homologous.

4. The IL-2 of embodiment 1, wherein the IL-2 polypeptide is identical to SEQ ID NO:2 is at least 98% homologous.

5. The IL-2 of embodiment 1, wherein the IL-2 polypeptide is identical to SEQ ID NO:2 at least 99% homologous.

6. The IL-2 of embodiment 1, wherein the IL-2 is coupled to one or more water soluble polymers.

7. The IL-2 of embodiment 6, wherein at least one of the water-soluble polymers is linked to at least one of the non-naturally encoded amino acids.

8. The IL-2 of embodiment 7, wherein the water soluble polymer is PEG.

9. The IL-2 of embodiment 8, wherein the PEG has a molecular weight between 10 and 50.

10. The IL-2 of embodiment 1, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of: <xnotran> 1 ( N- ), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133 , , . </xnotran>

11. The IL-2 of embodiment 10, wherein the IL-2 comprises one or more amino acid substitutions, additions or deletions as compared to wild-type IL-2 that modulate the affinity of the IL-2 polypeptide for its IL-2 ra receptor subunit.

12. The IL-2 of embodiment 10, wherein the IL-2 comprises one or more amino acid substitutions, additions or deletions that increase the stability or solubility of the IL-2.

13. The IL-2 of embodiment 10, wherein the IL-2 comprises one or more amino acid substitutions, additions or deletions that increase the expression or in vitro synthesis of the IL-2 polypeptide in a recombinant host cell.

14. The IL-2 of embodiment 10, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, and 107, and any combination thereof.

15. The IL-2 of embodiment 10, wherein the non-naturally encoded amino acid is reactive to a linker, polymer, or biologically active molecule that is otherwise unreactive to any of the 20 common amino acids in the polypeptide.

16. The IL-2 of embodiment 10, wherein the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group.

17. The IL-2 of embodiment 16, wherein the non-naturally encoded amino acid comprises a carbonyl group.

18. The IL-2 of embodiment 10, wherein the IL-2 is linked to a bioactive molecule, a cytotoxic agent, a water soluble polymer, or an immunostimulatory agent.

19. The IL-2 of embodiment 18, wherein the conjugated IL-2 is attached to one or more water soluble polymers.

20. The IL-2 of embodiment 18, wherein the bioactive molecule, cytotoxic agent, or immunostimulatory agent is linked to the IL-2 by a linker.

21. The IL-2 of embodiment 18, wherein the bioactive molecule, cytotoxic agent, or immunostimulatory agent is linked to the IL-2 by a cleavable or non-cleavable linker.

22. The IL-2 of embodiment 18, wherein the bioactive molecule, cytotoxic agent, or immunostimulatory agent is directly coupled to one or more of the non-naturally encoded amino acids in the IL-2.

23. The IL-2 of embodiment 10, wherein the non-naturally encoded amino acid has the structure:

wherein n is 0 to 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-terminal modifying group; and R4 is H, an amino acid, a polypeptide, or a carboxy-terminal modifying group.

24. The IL-2 of embodiment 23, wherein the non-naturally encoded amino acid comprises an aminooxy group.

25. The IL-2 of embodiment 23, wherein the non-naturally encoded amino acid comprises a hydrazide group.

26. The IL-2 of embodiment 23, wherein the non-naturally encoded amino acid comprises a hydrazine group.

27. The IL-2 of embodiment 23, wherein the non-naturally encoded amino acid residue comprises a semicarbazide group.

28. The IL-2 polypeptide of embodiment 23, wherein the non-naturally encoded amino acid residue comprises an azide group.

29. The IL-2 of embodiment 1, wherein the non-naturally encoded amino acid has the structure:

wherein n is 0 to 10; r1 is alkyl, aryl, substituted alkyl, substituted aryl, or absent; x is O, N, S or absent; m is 0 to 10; r2 is H, an amino acid, a polypeptide, or an amino-terminal modifying group, and R3 is H, an amino acid, a polypeptide, or a carboxy-terminal modifying group.

30. The IL-2 of embodiment 29, wherein the non-naturally encoded amino acid comprises an alkynyl group.

31. The IL-2 of embodiment 1, wherein the non-naturally encoded amino acid has the structure:

Wherein n is 0 to 10; r1 is alkyl, aryl, substituted alkyl or substituted aryl; x is O, N, S or absent; m is 0 to 10; r2 is H, an amino acid, a polypeptide, or an amino-terminal modifying group, and R3 is H, an amino acid, a polypeptide, or a carboxy-terminal modifying group.

32. The IL-2 of embodiment 7, wherein the water soluble polymer has a molecular weight between about 0.1kDa and about 100 kDa.

33. The IL-2 polypeptide of embodiment 32, wherein the water soluble polymer has a molecular weight between about 0.1kDa and about 50 kDa.

34. The IL-2 of embodiment 16, wherein the aminooxy, hydrazine, hydrazide or semicarbazide group is linked to the water-soluble polymer through an amide linkage.

35. The IL-2 of embodiment 19, which is made by reacting a water-soluble polymer comprising a carbonyl group with a polypeptide comprising a non-naturally encoded amino acid comprising an aminooxy, hydrazine, hydrazide or semicarbazide group.

36. The IL-2 of embodiment 1, wherein the IL-2 is glycosylated.

37. The IL-2 of embodiment 1, wherein the IL-2 polypeptide further comprises a linker, polymer, or biologically active molecule linked to the polypeptide through the non-naturally encoded amino acid.

38. The IL-2 of embodiment 37, wherein the linker, polymer, or biologically active molecule is linked to the polypeptide through a sugar moiety.

39. A method of making an IL-2 polypeptide according to embodiment 1, the method comprising contacting an isolated IL-2 polypeptide comprising a non-naturally encoded amino acid with a linker, polymer, or biologically active molecule comprising a moiety that reacts with the non-naturally encoded amino acid.

40. The method of embodiment 39, wherein the polymer comprises a moiety selected from the group consisting of a water soluble polymer and polyethylene glycol.

41. The method of embodiment 39, wherein 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.

42. The method of embodiment 39, wherein the non-naturally encoded amino acid comprises a carbonyl moiety and the linker, polymer, or biologically active molecule comprises an aminooxy, hydrazine, hydrazide or semicarbazide moiety.

43. The method of embodiment 39, wherein the aminooxy, hydrazine, hydrazide or semicarbazide moiety is linked to the linker, polymer, or biologically active molecule through an amide linkage.

44. The method of embodiment 39, wherein the non-naturally encoded amino acid comprises an alkynyl moiety and the linker, polymer, or biologically active molecule comprises an azido moiety.

45. The method of embodiment 39, wherein the non-naturally encoded amino acid comprises an azido moiety and the linker, polymer, or biologically active molecule comprises an alkynyl moiety.

46. The IL-2 polypeptide of embodiment 7, wherein the water soluble polymer is a polyethylene glycol moiety.

47. The IL-2 polypeptide of embodiment 46, wherein the polyethylene glycol moiety is a branched or multiarmed polymer.

48. A composition comprising IL-2 according to embodiment 10 and a pharmaceutically acceptable carrier.

49. The composition of embodiment 48, wherein the non-naturally encoded amino acid is linked to a water soluble polymer.

50. A method of treating a patient having a disorder modulated by IL-2, comprising administering to the patient a therapeutically effective amount of the composition of embodiment 42 or 36.

51. A composition comprising IL-2 according to embodiment 10 coupled to a biologically active molecule and a pharmaceutically acceptable carrier.

52. A composition comprising IL-2 according to embodiment 10 and a pharmaceutically acceptable carrier, said IL-2 further comprising a linker and a conjugate.

53. A method of making an IL-2 comprising a non-naturally encoded amino acid, the method comprising culturing a cell comprising one or more polynucleotides encoding an IL-2 polypeptide comprising a selector codon, an orthogonal RNA synthetase and an orthogonal tRNA under conditions that allow expression of the IL-2 polypeptide comprising the non-naturally encoded amino acid; and purifying the polypeptide.

54. A method of modulating serum half-life or circulation time of an IL-2 polypeptide, comprising replacing any one or more naturally occurring amino acids in said polypeptide with one or more non-naturally encoded amino acids.

55. An IL-2 polypeptide comprising one or more amino acid substitutions, additions or deletions that increase the expression of said IL-2 polypeptide in a recombinant host cell.

56. An IL-2 polypeptide comprising at least one linker, polymer, or biologically active molecule, wherein the linker, polymer, or biologically active molecule is attached to the polypeptide through a functional group of a non-naturally encoded amino acid ribosomally incorporated into the polypeptide.

57. An IL-2 polypeptide comprising a linker, polymer, or biologically active molecule attached to one or more non-naturally encoded amino acids, wherein said non-naturally encoded amino acids are ribosomally incorporated into said polypeptide at preselected sites.

58. A method of reducing the number of tumor cells in a human diagnosed with cancer, the method comprising administering to a human in need of such reduction a pharmaceutical composition comprising PEG-IL-2A according to embodiment 56.

59. The method of embodiment 58, wherein the conjugate is administered at a dose of about 0.1 μ/kg to about 50 μ/kg.

60. IL-2 according to any one of embodiments 1-38, 46-47 and 55-57, wherein the IL-2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, etc 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, or 133, and further comprises at least one natural amino acid substitution.

61. The method of any one of embodiments 39-45, 53-54, and 58-59 or the composition of any one of embodiments 48-49, wherein the method or composition is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133, and further comprises at least one natural amino acid substitution.

62. The IL-2 of embodiment 60, the method or composition of embodiment 61, wherein the natural amino acid substitution is at position 38, 46 and/or 65.

63. The IL-2 of embodiment 60, the method or composition of embodiment 61, wherein the natural amino acid substitution is at position 38 and/or 46.

64. The IL-2 of embodiment 60, the method or composition of embodiment 61, wherein the natural amino acid substitution is at position 38 and/or 65.

65. The IL-2 or the method or composition of any one of embodiments 62-64, wherein the natural amino acid substitution at position 38 is an alanine.

66. The IL-2 or the method or composition of any one of embodiments 62-63, wherein the natural amino acid substitution at position 46 is leucine or isoleucine.

67. The IL-2 or the method or composition of any one of embodiments 62 or 64, wherein the natural amino acid substitution at position 65 is arginine.

68. A glycosylated IL-2 polypeptide comprising one or more non-naturally encoded amino acids.

69. The glycosylated IL-2 polypeptide of embodiment 68, wherein the non-naturally encoded amino acid is para-acetylphenylalanine, para-nitrophenylalanine, para-sulfotyrosine, para-carboxyphenylalanine, ortho-nitrophenylalanine, meta-nitrophenylalanine, para-boronophenylalanine, ortho-boronophenylalanine, meta-boronophenylalanine, para-aminophenylalanine, ortho-aminophenylalanine, meta-acylphenylalanine, p-OMe phenylalanine, O-OMe phenylalanine, m-OMe phenylalanine, para-sulfophenylalanine, ortho-sulfophenylalanine, meta-sulfophenylalanine, 5-nitroHis, 3-nitroTyr, 2-nitroTyr, nitro-substituted Leu, p-nitrophenyl, O-OMe phenylalanine, m-OMe phenylalanine, p-sulfophenylalanine, O-sulfophenylalanine, m-sulfophenylalanine, 5-nitroHis, 3-nitroTyr, 2-nitroTyr, or nitro-substituted His, nitro-substituted De, nitro-substituted Trp, 2-nitroTrp, 4-nitroTrp, 5-nitroTrp, 6-nitroTrp, 7-nitroTrp, 3-aminotyrosine, 2-aminotyrosine, O-sulfotyrosine, 2-sulfooxyphenylalanine, 3-sulfooxyphenylalanine, 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-GlcNAc beta-serine, L-dopa beta-serine, fluorophenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propargyloxy-L-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine and p-azidomethyl-phenylalanine.

70. The glycosylated IL-2 polypeptide of embodiment 68, further comprising one or more natural amino acids.

71. The glycosylated IL-2 polypeptide of embodiment 68, further comprising one or more linkers, polymers, or biologically active molecules, wherein the linkers, polymers, or biologically active molecules are attached to the polypeptide through a functional group of a non-naturally encoded amino acid ribosomally incorporated into the polypeptide.

72. The glycosylated IL-2 polypeptide of embodiment 71, wherein the polymer is a water soluble polymer.

73. The glycosylated IL-2 polypeptide of embodiment 72, wherein the water soluble polymer is a polyethylene glycol moiety.

74. The glycosylated IL-2 polypeptide of embodiment 73, wherein the polyethylene glycol moiety is a branched or multiarmed polymer.

75. Use of an IL-2 polypeptide according to any one of the preceding embodiments in the manufacture of a medicament.

76. A modified IL-2 polypeptide comprising a) at least one non-naturally encoded amino acid with a linker, polymer, or biologically active molecule comprising a moiety that reacts with the non-naturally encoded amino acid; and b) at least one naturally occurring amino acid.

Sequence listing

<110> Ambrx, Inc.

<120> Interleukin-2 polypeptide conjugates and methods of use thereof

<130> AMBX-0232.00PCT

<150> 62/987,872

<151> 2020-03-11

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Met Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 8

<211> 396

<212> DNA

<213> human

<400> 8

atgaccagca gtagcaccaa gaaaactcag ctgcagctgg agcatctgct gctggattta 60

cagatgattc tgaatggcat taataattac aaaaatccga aactgacccg catgctgacc 120

ttcaagttct acatgccgaa gaaggccacc gaactgaagc atctgcagtg tttagaagag 180

gaactgaagc cgctggaaga ggtgctgaat ttagcccaga gcaaaaactt ccatctgcgc 240

ccgcgcgatt taattagcaa tattaacgtg attgtgctgg aactgaaagg cagcgagacc 300

acctttatgt gcgagtacgc agatgagacc gccaccatcg tggaattttt aaaccgctgg 360

atcaccttca gccagagtat cattagcact ttaacc 396

<210> 9

<211> 133

<212> PRT

<213> human

<220>

<221> MISC_FEATURE

<222> (42)..(42)

<223> Xaa = U = unnatural amino acid

<400> 9

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Xaa Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 10

<211> 133

<212> PRT

<213> human

<220>

<221> misc_feature

<222> (44)..(44)

<223> Xaa = U = unnatural amino acid

<400> 10

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Xaa Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 11

<211> 133

<212> PRT

<213> human

<220>

<221> misc_feature

<222> (45)..(45)

<223> Xaa = U = unnatural amino acid

<400> 11

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Xaa Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 12

<211> 133

<212> PRT

<213> human

<220>

<221> misc_feature

<222> (65)..(65)

<223> Xaa = U = unnatural amino acid

<400> 12

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Xaa Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 13

<211> 133

<212> PRT

<213> human

<220>

<221> misc_feature

<222> (72)..(72)

<223> Xaa = U = unnatural amino acid

<400> 13

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Xaa Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 14

<211> 133

<212> PRT

<213> human

<220>

<221> misc_feature

<222> (61)..(61)

<223> Xaa = U = unnatural amino acid

<400> 14

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Xaa Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 15

<211> 133

<212> PRT

<213> human

<220>

<221> misc_feature

<222> (62)..(62)

<223> Xaa = U = unnatural amino acid

<220>

<221> misc_feature

<222> (68)..(68)

<223> Xaa = U = unnatural amino acid

<400> 15

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Xaa Leu Lys

50 55 60

Pro Leu Glu Xaa Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 16

<211> 133

<212> PRT

<213> human

<220>

<221> misc_feature

<222> (35)..(35)

<223> Xaa = U = unnatural amino acid

<400> 16

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 17

<211> 133

<212> PRT

<213> human

<220>

<221> misc_feature

<222> (38)..(38)

<223> Xaa = U = unnatural amino acid

<400> 17

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Xaa Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 18

<211> 133

<212> PRT

<213> human

<220>

<221> misc_feature

<222> (43)..(43)

<223> Xaa = U = unnatural amino acid

<400> 18

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Xaa Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 19

<211> 133

<212> PRT

<213> human

<220>

<221> misc_feature

<222> (37)..(37)

<223> Xaa = U = unnatural amino acid

<400> 19

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Xaa Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 20

<211> 133

<212> PRT

<213> human

<220>

<221> misc_feature

<222> (3)..(3)

<223> Xaa = U = unnatural amino acid

<400> 20

Ala Pro Xaa Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 21

<211> 133

<212> PRT

<213> human

<220>

<221> misc_feature

<222> (64)..(64)

<223> Xaa = U = unnatural amino acid

<400> 21

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Xaa

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 22

<211> 133

<212> PRT

<213> human

<220>

<221> misc_feature

<222> (68)..(68)

<223> Xaa = U = unnatural amino acid

<400> 22

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Xaa Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 23

<211> 133

<212> PRT

<213> human

<220>

<221> misc_feature

<222> (107)..(107)

<223> Xaa = U = unnatural amino acid

<400> 23

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Xaa Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

Claims (20)

1. A modified IL-2 polypeptide comprising SEQ ID NO:2, and comprises: a non-naturally encoded amino acid incorporated at position 42; in SEQ ID NO:2 at a selected position; and one or more PEG molecules; wherein the polypeptide is coupled to the one or more PEG molecules through a non-naturally encoded amino acid incorporated into the polypeptide.

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

3. The modified IL-2 polypeptide of claim 2, optionally comprising the amino acid sequence set forth in SEQ ID NO:2 at a selected position within the group.

4. The modified IL-2 polypeptide according to claim 1 or 2, wherein the non-naturally encoded amino acid is para-acetylphenylalanine, para-nitrophenylalanine, para-sulfotyrosine, para-carboxyphenylalanine, ortho-nitrophenylalanine, meta-nitrophenylalanine, para-boronophenylalanine, ortho-boronophenylalanine, meta-boronophenylalanine, para-aminophenylalanine, ortho-aminophenylalanine, meta-acylphenylalanine, p-OMe phenylalanine, O-OMe phenylalanine, m-OMe phenylalanine, para-sulfophenylalanine, ortho-sulfophenylalanine, meta-sulfophenylalanine, 5-nitroHis, 3-nitroTyr, 2-nitroTyr, nitro-substituted Leu, p-nitrophenyl, O-OMe phenylalanine, m-OMe phenylalanine, p-sulfophenylalanine, O-sulfophenylalanine, m-sulfophenylalanine, 5-nitroHis, 3-nitroTyr, 2-nitroTyr, or nitro-substituted His, nitro-substituted De, nitro-substituted Trp, 2-nitroTrp, 4-nitroTrp, 5-nitroTrp, 6-nitroTrp, 7-nitroTrp, 3-aminotyrosine, 2-aminotyrosine, O-sulfotyrosine, 2-sulfooxyphenylalanine, 3-sulfooxyphenylalanine, 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-GlcNAc beta-serine, L-dopa beta-serine, fluorophenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphotyrosine, 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 para-acetylphenylalanine.

6. The modified IL-2 polypeptide of claim 1 or 3, wherein the one or more amino acid substitutions is as set forth in SEQ ID NO:2 at position 38 and/or 65.

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

8. The modified IL-2 polypeptide of 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 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, and 50 kDa.

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

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

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

14. The method of claim 13, wherein the cancer is breast cancer, small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, gastrointestinal pancreatic tumor, cervical cancer, esophageal cancer, colon cancer, colorectal cancer, cancer or tumor of epithelial origin, renal 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 antineoplastic agent, an immunostimulant, an immunomodulator, 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 manufacture 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. A polypeptide of SEQ ID NOs:9 or 11.

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