TWI839476B - Interleukin-2 polypeptide conjugates and their uses - Google Patents

Abstract

The present invention provides methods for targeting interleukin-2 receptor-expressing cells, and, in particular, inhibiting the growth of such cells by using an interleukin-2 (IL-2) variant conjugated to a biologically active molecule that will affect cells expressing the interleukin-2 receptor.

Description

Interleukin-2 polypeptide conjugate and its use

The present invention provides a method for modulating the biological activity of interleukin-2 (IL-2) and in particular modulating specific receptor interactions by using an interleukin-2 variant, wherein the IL-2 variant is conjugated to a polymer at a position in the amino acid sequence of the IL-2 protein that interacts with the interleukin-2 receptor.

Cancer is one of the most significant health conditions. In the United States, cancer has the second-leading mortality rate after heart disease, accounting for one in four deaths. The incidence of cancer is widely expected to increase as the U.S. population ages, further exacerbating the impact of this condition. Current treatment options for cancer, established in the 1970s and 1980s, have not changed much. These treatments, which include chemotherapy, radiation, and other modalities (including newer targeted therapies), show limited overall survival benefit when used in most advanced common cancers because these therapies primarily target the tumor bulk.

More specifically, conventional cancer diagnosis and treatment to date have attempted to selectively detect and eradicate primarily rapidly growing neoplastic cells (i.e., cells that form tumor masses). Standard oncology treatment regimens are usually designed to administer the highest dose of radiation or chemotherapy that can be given without excessive toxicity, often referred to as the "maximum tolerated dose (MTD)" or "no observed adverse effect level (NOAEL)". Many conventional cancer chemotherapies (e.g., alkylating agents such as cyclophosphamide, anti-metabolites such as 5-fluorouracil, and plant alkaloids such as vincristine) and conventional radiation exert their toxic effects on cancer cells primarily by interfering with cellular machinery involved in cell growth and DNA replication. Chemotherapy regimens also often include the administration of combinations of chemotherapeutics in an attempt to improve the efficacy of treatment. Despite the availability of a large number of different chemotherapeutics, these therapies have a number of disadvantages. For example, chemotherapeutic agents are notoriously toxic due to nonspecific side effects on rapidly growing cells, both normal and malignant; for example, chemotherapeutic agents cause significant and often dangerous side effects, including bone marrow suppression, immunosuppression, and gastrointestinal upset.

Cancer stem cells

Cancer stem cells comprise a unique subpopulation of tumors (usually around 0.1-10%) that are more tumorigenic, relatively slower growing or quiescent, and generally relatively more chemoresistant than tumor masses relative to the remaining 90% or so of tumors (and tumor masses). Considering that conventional therapies and regimens are mostly designed to attack rapidly proliferating cells (i.e., those cancer cells that make up tumor masses), cancer stem cells that generally grow slowly may be relatively more resistant to conventional therapies and regimens than rapidly growing tumor masses. Cancer stem cells can exhibit other characteristics that make them relatively more chemoresistant, such as multidrug resistance and anti-apoptotic pathways. These factors constitute the key reason why standard cancer treatment regimens fail to ensure long-term benefit in most patients with advanced cancer, i.e., fail to adequately target and eradicate cancer stem cells. In some cases, cancer stem cells are tumor-generating cells (i.e., it is the progenitor of 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 Aldesleukin® is a recombinant protein that is non-glycosylated, has alanine-1 removed and replaces residual cysteine-125 with serine-125 (Whittington et al., 1993). Although IL-2 is the earliest FDA-approved cytokine in cancer treatment, it has been shown to exhibit severe side effects when used in high doses. This greatly limits its application in potential patients. The potential mechanism for the severe side effects has been attributed to the binding of IL-2 to one of its receptors, IL-2Rα. Normally, IL-2 can form heterotrimeric complexes not only with its receptors including IL-2Rα (or CD25), IL-2Rβ (or CD122), and IL-2Rγ (or CD132) (when all three receptors are present in the tissue), but also with IL-2Rβ and IL-2Rγ. In a clinical setting, when high doses of IL-2 are used, IL-2 begins to bind to IL-2αβγ, which is the main receptor form in Treg cells. The inhibitory effect of Treg cells causes an unwanted effect of the application of IL-2 in cancer immunotherapy. In order to reduce the side effects of IL-2, many approaches have been used previously. Nektar made a form of IL-2 that uses 6 PEGylated lysines to mask the IL2Rα binding region on the surface of IL-2 (Charych et al., 2016). This PEGylated IL-2 form has an extended half-life, comprises a mixture of mono- and poly-PEGylated forms, contains very high amounts of PEG, and exhibits improved side effects. However, results from activity studies show that the only effective PEGylated IL-2 form in this heterogeneous 6-PEGylated IL-2 mixture is the mono-PEGylated form. Therefore, there is a need for more effective 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 for the introduction of chemical functional groups that can provide valuable alternatives to naturally occurring functional groups such as the epsilon-NH2 of lysine, the hydroxyl-SH of cysteine, the imino group of histidine, etc. Certain chemical functional groups are known to be inert to the functional groups present in the 20 common genetically encoded amino acids, but react cleanly and efficiently to form stable bonds. For example, azide groups and acetylene groups are known in the art to undergo a Huisgen [3+2] cycloaddition reaction under aqueous conditions in the presence of catalytic amounts of copper. See, e.g., Tornoe et al., (2002) J. Org. Chem. 67:3057-3064; and Rostovtsev et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599. By, for example, introducing an azido moiety into a protein structure, one is able to incorporate functional groups that are chemically inert to amines, alkyls, carboxyls, hydroxyls present in proteins, but react smoothly and efficiently with acetylene moieties to form cycloaddition products. Importantly, in the absence of the acetylene moiety, the azido group remains chemically inert and unreactive in the presence of other protein side chains and under physiological conditions.

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

The present invention relates to interleukin-2 (IL-2) polypeptides having one or more non-naturally encoded amino acids. The present invention also relates to IL-2 polypeptide conjugates having one or more non-naturally encoded amino acids. The present invention also relates to an IL-2 polypeptide conjugate, wherein a water-soluble polymer such as PEG is conjugated to the IL-2 variant via one or more non-naturally encoded amino acids within the IL-2 variant. The present invention also relates to IL-2 polypeptide conjugates having one or more non-naturally encoded amino acids and one or more natural amino acid substitutions. The present invention also relates to 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 present invention provides a method for regulating the receptor interaction of the IL-2 polypeptide of the present invention. The present invention provides a method for inhibiting or reducing the interaction between PEGylated IL-2 and the IL2Rα subunit of the trimeric IL-2 receptor using the PEGylated IL-2 polypeptide of the present invention.

In one embodiment, the PEG-IL-2 is monopegylated. In one embodiment, the PEG-IL-2 is dipegylated. In one embodiment, the PEG-IL-2 has more than two (2) polyethylene glycol molecules attached to it. Another embodiment of the present invention provides a method of using the PEG-IL-2 polypeptide of the present 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 full-length, mature (lacking a signal peptide) human interleukin-2 linked to a PEG polymer. In this or any embodiment of the invention, the PEG-IL-2 may comprise full-length, mature (lacking a signal peptide) human interleukin-2 covalently linked to a PEG polymer or other bioactive molecule. In certain embodiments, the bioactive molecule is modified, and as a non-limiting example, the bioactive molecule may include one or more non-naturally encoded amino acids.

In the PEG-IL2 conjugate, the PEG or other water-soluble polymer can 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 by administering an effective amount of a PEG-IL-2 polypeptide, such as a mammal including but not limited to those having one or more of the following conditions: solid tumors, hematological tumors, colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, glioblastoma, and leukemia. In certain embodiments, the cancer is characterized by high levels of Treg cells. In certain embodiments, the cancer is characterized by high expression of IL-2 receptor α. In certain embodiments, the present invention provides a method of treating cancer or a condition or disease by administering to a subject an effective amount of a composition comprising an IL-2 polypeptide of the present invention. In certain embodiments, the present invention provides a method of treating a genetic disease by administering to a patient an effective amount of an IL-2 composition of the present invention. In certain embodiments, the 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 the expression of IL-2 receptor alpha. In certain embodiments, the cancer, disorder or disease is treated by reducing the 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.

When used in the present invention, interleukin 2 or IL-2 is defined as a protein having the following properties: (a) having an amino acid sequence that is substantially identical to the known sequence of IL-2 (including IL-2 mutant proteins, mature IL-2 sequences (i.e., lacking a secretory leader sequence) and IL-2 disclosed in SEQ ID NO: 1, 2, 3, 5 or 7 of the present application), and (b) having at least one biological activity shared by 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. coli) IL-2 are equivalent and can be used interchangeably. Other mutants and other analogs that retain the biological activity of IL-2 are also included, including viral IL-2.

The present invention provides IL-2 polypeptides conjugated to one or more water-soluble polymers, wherein the PEGylated IL-2 polypeptide is also linked to another drug or biologically active molecule, and wherein the IL-2 polypeptide comprises one or more non-naturally encoded amino acids. The present invention also provides monomers and dimers of IL-2 polypeptides. The present invention also provides trimers of IL-2 polypeptides. The present invention provides multimers of IL-2 polypeptides. The present invention also provides IL-2 dimers comprising one or more non-naturally encoded amino acids. The present invention provides IL-2 multimers comprising one or more non-naturally encoded amino acids. The present invention provides homogenous IL-2 multimers comprising one or more non-naturally encoded amino acids, wherein each IL-2 polypeptide has the same amino acid sequence. The present invention provides heterogenous IL-2 polymers, 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 polymer may 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, a polymer, or a biologically active molecule. In certain embodiments, the IL-2 monomers are homologous. In certain embodiments, the IL-2 dimers are homologous. In certain embodiments, the IL-2 multimers are conjugated to one water-soluble polymer. In certain embodiments, the IL-2 multimers are conjugated to two water-soluble polymers. In certain embodiments, the IL-2 multimers are conjugated to three water-soluble polymers. In certain embodiments, the IL-2 multimers are conjugated to more than three water-soluble polymers. In certain embodiments, the IL-2 polypeptide is linked to a linker long enough to allow dimer formation. In some embodiments, the IL-2 polypeptide is linked to a linker long enough to allow trimer formation. In some embodiments, the IL-2 polypeptide is linked to a linker long enough to allow multimer formation. In some embodiments, the IL-2 polypeptide is linked to a bifunctional polymer, a bifunctional linker, or at least one additional IL-2 polypeptide. In some embodiments, the IL-2 polypeptide comprises one or more post-translational modifications. In some embodiments, the IL-2 polypeptide is linked to a linker, a polymer, or a biologically active molecule.

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

In one embodiment, the IL-2 or PEG-IL-2 of the present invention is linked to a therapeutic agent such as an immunomodulator. The immunomodulator can be any agent that exerts a therapeutic effect on immune cells, which can be used as a therapeutic agent for conjugation to IL-2, PEG-IL-2 or IL-2 variants.

In some embodiments, a non-naturally 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, 5, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106 06, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or added to the carboxyl terminus of the protein, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5 or 7). In certain embodiments, one or more biologically active molecules are directly conjugated to the IL-2 variant. In certain embodiments, the one or more biologically active molecules are conjugated to the one or more non-naturally encoded amino acids in the IL-2 polypeptide. In certain embodiments, the IL-2 variant of the present invention is linked to a linker. In certain embodiments, the IL-2 variant linked to a linker also comprises a biologically active molecule. In certain embodiments of the present 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 at one or more of the following positions: 3, 32, 35, 37, 38, 42, 43, 44, 45, 48, 49, 61, 62, 64, 65, 68, 72, 76, and 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5, or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following positions: before position 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, and 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5, or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following positions: 35, 37, 42, 45, 49, 61, or 65, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5, or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following positions: 45, 61, and 65, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5, or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or its variants at one or more of the following positions: positions 45 and 65, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5, or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or its variants of the present invention at position 3. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or its variants of the present invention at position 32. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or its variants of the present invention at position 35. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or its variants of the present invention at position 37. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the 38th position of IL-2 or its variants of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the 41st position of IL-2 or its variants of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the 42nd position of IL-2 or its variants of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the 43rd position of IL-2 or its variants of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the 44th position of IL-2 or its variants of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the 45th position of IL-2 or its variants of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the 48th position of IL-2 or its variants of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the 49th position of IL-2 or its variants of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the 61st position of IL-2 or its variants of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the 62nd position of IL-2 or its variants of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 of the present invention or its variant at the 64th position. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 of the present invention or its variant at the 65th position. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 of the present invention or its variant at the 68th position. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 of the present invention or its variant at the 72nd position. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 of the present invention or its variant at the 76th position. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into the IL-2 or variants thereof of the present invention at position 107.

In certain embodiments, the non-naturally occurring amino acid at one or more of these positions in IL-2 or a variant thereof is linked to a drug or other biologically active molecule, including but not limited to the following positions: before position 1 (i.e., at the N-terminus), 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 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106 , 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or added to the carboxyl terminus of the protein, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5 or 7).

In certain embodiments, the non-naturally occurring amino acid at one or more of these positions in IL-2 or its variants is linked to a drug or other biologically active molecule, including but not limited to positions at hydrophobic interactions, at or near positions that interact with IL-2 receptor subunits (including IL2Rα), within the 3rd or 35 to 45th amino acid positions, within the first 107 N-terminal amino acids, and within the 61st-72nd amino acid positions; each of the positions is a position in SEQ ID NO: 2 or a corresponding amino acid position in SEQ ID NO: 3, 5 or 7. In certain embodiments, the non-naturally occurring amino acid at one or more of these positions in IL-2 or a variant thereof is linked to a drug or other biologically active molecule, including but not limited to the following positions: before position 1 (i.e., at the N-terminus), positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, and any combination thereof of SEQ ID NO: 2; or the corresponding amino acids in SEQ ID NO: 3, 5, or 7. In certain embodiments, the non-naturally occurring amino acid at one or more of these positions in IL-2 or a variant thereof is linked to a drug or other biologically active molecule, including but not limited to the following positions of IL-2 or a variant thereof: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or added to the carboxyl terminus of the protein, and any combination thereof; or the corresponding amino acids in SEQ ID NO: 3, 5 or 7. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or its variants at 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 acids in SEQ ID NO: 3, 5 or 7).

In certain embodiments, the non-naturally occurring amino acid at one or more of these positions in IL-2 or a variant thereof is linked to a linker, including but not limited to the following positions: before position 1 (i.e., at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 , 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 07, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or added to the carboxyl terminus of the protein, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5 or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof and linked to a linker at one or more of the following positions: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, and 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5, or 7).

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

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, 65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or added to the carboxyl terminus of the protein, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 3, 5 or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof and linked to a linker, which 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 acids in SEQ ID NO: 3, 5 or 7).

In certain embodiments, the IL-2 or its variant comprises a substitution, addition or deletion that modulates the affinity of the IL-2 to the IL-2 receptor subunit or its variant. In certain embodiments, the IL-2 or its variant comprises a substitution, addition or deletion that modulates the affinity of the IL-2 or its variant to the IL-2 receptor or binding partner (including but not limited to proteins, polypeptides, lipids, fatty acids, small molecules or nucleic acids). In certain embodiments, the IL-2 or its variant comprises a substitution, addition or deletion that modulates the stability of the IL-2 compared to the stability of the corresponding IL-2 without substitution, addition or deletion. Stability and/or solubility can be measured using a large number of different assays known to those of ordinary skill in the art. These assays include but are not limited to SE-HPLC and RP-HPLC. In certain embodiments, the IL-2 comprises a substitution, addition, or deletion that modulates the immunogenicity of the IL-2 compared to the immunogenicity of the corresponding IL-2 without the substitution, addition, or deletion. In certain embodiments, the IL-2 comprises a substitution, addition, or deletion that modulates the serum half-life or circulation time of the IL-2 compared to the serum half-life or circulation time of the corresponding IL-2 without the substitution, addition, or deletion.

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

In certain embodiments, the invention provides methods of treating a proliferative condition, cancer, tumor, or precancerous condition, such as dysplasia, using PEG-IL-2 and at least one additional therapeutic or diagnostic agent. The additional therapeutic agent can be, for example, a cytokine or cytokine antagonist such as IL-12, interferon-α or anti-epidermal growth factor receptor antibody, doxorubicin, epirubicin, antifolates such as methotrexate or fluorouracil, irinotecan, cyclophosphamide, radiation therapy, hormone or anti-hormonal therapy such as androgens, estrogens, anti-estrogen antibodies, flutamide or diethylstilbestrol, surgery, tamoxifen, isocyclophosphamide, dibromostilbestrol, Alkylating agents such as melphalan or cisplatin, etoposide, vinorelbine, vinblastine, vindesine, glucocorticoids, histamine receptor antagonists, angiogenesis inhibitors, radiation, radiosensitizers, anthracyclines, vinca alkaloids, taxanes such as paclitaxel and docetaxel, cell cycle inhibitors such as cell cycle protein-dependent kinase inhibitors, checkpoint inhibitors, immunomodulatory drugs, immunostimulatory drugs, monoclonal antibodies directed against another tumor antigen, complexes of monoclonal antibodies and biologically active molecules, T cell adjuvants, bone marrow transplants, or antigen presenting cells such as dendritic cell therapy. The vaccine can be provided, for example, as a soluble protein or as a nucleic acid encoding the protein (see, e.g., Le et al., supra; Greco and Zellefsky, eds. (2000) Radiotherapy of Prostate Cancer, Harwood Academic, Amsterdam; Shapiro and Recht (2001) New Engl. J. Med. 344: 1997-2008; Hortobagyi (1998) New Engl. J. Med. 339: 974-984; Catalona (1994) New Engl. J. Med. 331: 996-1004; Naylor and Hadden (2003) Int. Immunopharmacol. 3: 1205-1215; The Int. Adjuvant Lung Cancer Trial Collaborative Group (2004) New Engl. J. Med. 350: 351-360; Slamon et al., (2001) New Engl. J. Med. 344: 783-792; Kudelka et al., (1998) New Engl. J. Med. 338: 991-992; van Netten et al., (1996) New Engl. J. Med. 334: 920-921).

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

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

In certain embodiments, the IL-2 or variant thereof comprises substitutions, additions or deletions that improve the compatibility of the IL-2 or variant thereof with pharmaceutical preservatives (e.g., m-cresol, phenol, benzyl alcohol) compared to the compatibility of the corresponding wild-type IL-2 without the substitutions, additions or deletions. This improved compatibility enables the preparation of a well-preserved pharmaceutical formulation that maintains the physicochemical properties and biological activity of the protein during storage.

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

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

In certain embodiments, the non-naturally encoded amino acid comprises a carbonyl group, an acetyl group, an amine group, a hydrazine group, a hydrazide group, a semicarbazide group, an azido group, or an alkynyl 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 0-10; _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 carboxyl terminal modifying group.

In some embodiments, the non-naturally encoded amino acid comprises an amineoxy group. In some embodiments, the non-naturally encoded amino acid comprises a hydrazide group. In some embodiments, the non-naturally encoded amino acid comprises a hydrazine group. In some embodiments, the non-naturally encoded amino acid residue comprises a semicarbazide group.

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

wherein n is 0-10; _R1 is alkyl, aryl, substituted alkyl, substituted aryl or absent; X is O, N, S or absent; m is 0-10; _R2 is H, an amino acid, a polypeptide or an amino-terminal modifying group, and _R3 is H, an amino acid, a polypeptide or a carboxyl-terminal modifying 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 0-10; _R1 is alkyl, aryl, substituted alkyl or substituted aryl; X is O, N, S or absent; m is 0-10, _R2 is H, an amino acid, a polypeptide or an amino-terminal modifying group, and _R3 is H, an amino acid, a polypeptide or a carboxyl-terminal modifying group.

In some embodiments, the polypeptide is an IL-2 agonist, partial agonist, antagonist, partial antagonist, or inverse agonist. In some 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 some embodiments, the water-soluble polymer comprises a polyethylene glycol component. In some 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 present invention also provides an isolated nucleic acid comprising a polynucleotide encoding a polypeptide of SEQ ID NO: 1, 2, 3, 5 or 7, and the present invention provides an isolated nucleic acid comprising a polynucleotide that hybridizes with a polynucleotide encoding a polypeptide of SEQ ID NO: 1, 2, 3, 5 or 7 under strict conditions. The present invention also provides an isolated nucleic acid comprising a polynucleotide encoding a polypeptide shown as SEQ ID NO: 1, 2, 3, 5 or 7, wherein the polynucleotide comprises at least one selector codon. The present invention also provides an isolated nucleic acid comprising a polynucleotide encoding a polypeptide shown as SEQ ID NO: 1, 2, 3, 5 or 7 and having one or more non-naturally encoded amino acids. It is obvious to a person of ordinary skill in the art that many different polynucleotides can encode any polypeptide of the present invention.

In certain embodiments, the selector codon is selected from an amber codon, an ochre codon, an opal codon, a unique codon, a rare codon, a five-base codon, and a tetrabase codon.

The present invention also provides a method for making IL-2 or a variant thereof connected to a biologically active molecule. In certain embodiments, the method comprises contacting an isolated IL-2 or a variant thereof comprising a non-naturally encoded amino acid with a biologically active molecule comprising a component 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 to a biologically active molecule that is originally unreactive to any of the 20 commonly used amino acids. In certain embodiments, the non-naturally encoded amino acid incorporated into IL-2 is reactive to a linker, polymer, or biologically active molecule that is originally unreactive to any of the 20 commonly used amino acids connected to the biologically active molecule.

In some embodiments, the IL-2 or variant thereof linked to a water-soluble polymer or a bioactive molecule is produced by reacting IL-2 or a variant thereof containing a carbonyl amino acid with a water-soluble polymer or a bioactive molecule containing an amineoxy group, a hydrazine, a hydrazide or a amine urea group. In some embodiments, the amineoxy group, a hydrazine, a hydrazide or a amine urea group is linked to the bioactive molecule via an amide bond. In some embodiments, the amineoxy group, a hydrazine, a hydrazide or a amine urea group is linked to the water-soluble polymer or a bioactive molecule via a carbamate bond.

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

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

In some embodiments, the IL-2 or variant thereof linked to the water-soluble polymer is produced by reacting the IL-2 or variant thereof containing a carbonyl amino acid with a polyethylene glycol molecule containing an aminooxy group, a hydrazine, a hydrazide or a amine urea group. In some embodiments, the aminooxy group, a hydrazine, a hydrazide or a amine urea group is linked to the polyethylene glycol molecule via an amide bond. In some embodiments, the aminooxy group, a hydrazine, a hydrazide or a amine urea group is linked to the polyethylene glycol molecule via a carbamate bond.

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

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

In some embodiments, the IL-2 or variant thereof linked to a water-soluble polymer is produced by reacting an IL-2 or variant thereof containing an azido group-containing amino acid with a polyethylene glycol molecule containing an alkynyl component. In some embodiments, the azido group or alkynyl group is linked to the polyethylene glycol molecule via an amide bond.

In certain embodiments, the polyethylene glycol molecule has a molecular weight between about 0.1kDa and about 100kDa. In certain embodiments, the polyethylene glycol molecule has a molecular weight between 0.1kDa and 50kDa. In certain embodiments, the polyethylene glycol has a molecular weight between 1kDa and 25kDa or between 2 and 22kDa or between 5kDa and 20kDa. For example, the molecular weight of the polyethylene glycol polymer can be about 5kDa or about 10kDa or about 20kDa. For example, the molecular weight of the polyethylene glycol polymer can be 5kDa or 10kDa or 20kDa. In certain embodiments, the polyethylene glycol molecule is 20K2-branched PEG. In certain embodiments, the polyethylene glycol molecule is a straight chain (linear) 5KPEG. In certain embodiments, the polyethylene glycol molecule is a straight chain (linear) 10KPEG. In some embodiments, the polyethylene glycol molecule is a linear 20KPEG. In some embodiments, the molecular weight of the polyethylene glycol polymer is an average molecular weight. In some embodiments, the average molecular weight is a number average molecular weight (Mn). The average molecular weight can be determined or measured using GPC or SEC, SDS/PAGE analysis, RP-HPLC, mass spectrometry or capillary electrophoresis. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or its variants at one or more of the following positions: 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72 or 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5 or 7), and the IL-2 or its variants are linked to a linear 20K polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or its variants at one or more of the following positions: 35, 37, 42, 45, 49, 61 or 65, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5 or 7), and the IL-2 or its variants are linked to a linear 20K polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into IL-2 or its variants at position 65 (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5 or 7), and the IL-2 or its variants are linked to a linear 20K polyethylene glycol molecule. In certain embodiments, the non-naturally encoded amino acid is incorporated into IL-2 or a variant thereof at position 61 (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7), and the IL-2 or a variant thereof is linked to a linear 20K polyethylene glycol molecule. In certain embodiments, the non-naturally encoded amino acid is incorporated into IL-2 or a variant thereof at position 49 (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7), and the IL-2 or a variant thereof is linked to a linear 20K polyethylene glycol molecule. In certain embodiments, the 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 a variant thereof is linked to a linear 20K polyethylene glycol molecule. In certain embodiments, the 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 a variant thereof is linked to a linear 20K polyethylene glycol molecule. In certain embodiments, the non-naturally encoded amino acid is incorporated into position 37 of IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7), and the IL-2 or a variant thereof is linked to a linear 20K polyethylene glycol molecule. In certain embodiments, the non-naturally encoded amino acid is incorporated into position 35 of IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7), and the IL-2 or a variant thereof is linked to a linear 20K polyethylene glycol molecule.

In some embodiments, the polyethylene glycol molecule is a branched polymer. In some embodiments, each branch of the polyethylene glycol branched polymer has a molecular weight between 1 kDa and 100 kDa or between 1 kDa and 50 kDa. In some embodiments, each branch of the polyethylene glycol branched polymer has a molecular weight between 1 kDa and 25 kDa or between 2 and 22 kDa or between 5 kDa and 20 kDa. For example, the molecular weight of each branch of the polyethylene glycol branched polymer can be about 5 kDa or about 10 kDa or about 20 kDa. For example, the molecular weight of each branch of the polyethylene glycol branched polymer can be 5 kDa or 10 kDa or 20 kDa. In some embodiments, the polyethylene glycol molecule is 20K2-branched PEG. In certain embodiments, the polyethylene glycol molecule is 20K4-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 can be determined or measured using GPC or SEC, SDS/PAGE analysis, RP-HPLC, mass spectrometry or capillary electrophoresis. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or its variants: the 3rd, 35th, 37th, 38th, 41st, 42nd, 43rd, 44th, 45th, 61st, 62nd, 64th, 65th, 68th, 72nd or 107th position, and any combination thereof (SEQ ID NO: 2, or SEQ ID NO: 3, the corresponding amino acid position in 5 or 7), and the IL-2 or its variants are connected to the 20K2-branched polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or its variants at one or more of the following positions: 35, 37, 42, 45, 49, 61 or 65, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5 or 7), and the IL-2 or its variants are linked to a 20K2-branched polyethylene glycol molecule. In certain embodiments, non-naturally encoded amino acids are incorporated into IL-2 or its variants at position 65 (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5 or 7), and the IL-2 or its variants are linked to a 20K2-branched polyethylene glycol molecule. In certain embodiments, the non-naturally encoded amino acid is incorporated into IL-2 or its variant at position 61 (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5 or 7), and the IL-2 or its variant is linked to a 20K2-branched polyethylene glycol molecule. In certain embodiments, the non-naturally encoded amino acid is incorporated into IL-2 or its variant at position 49 (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5 or 7), and the IL-2 or its variant is linked to a 20K2-branched polyethylene glycol molecule. In certain embodiments, the non-naturally encoded amino acid is incorporated into position 45 of IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7), and the IL-2 or a variant thereof is linked to a 20K2-branched polyethylene glycol molecule. In certain embodiments, the non-naturally encoded amino acid is incorporated into position 42 of IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7), and the IL-2 or a variant thereof is linked to a 20K2-branched polyethylene glycol molecule. In certain embodiments, the non-naturally encoded amino acid is incorporated into IL-2 or its variant 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 its variant is linked to a 20K2-branched polyethylene glycol molecule. In certain embodiments, the non-naturally encoded amino acid is incorporated into IL-2 or its variant 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 its variant is linked to a 20K2-branched polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or its variants at one or more of the following positions: 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72 or 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5 or 7), and the IL-2 or its variants are linked to a 20K4-branched polyethylene glycol molecule. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or its variants at one or more of the following positions: 35, 37, 42, 45, 49, 61 or 65, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5 or 7), and the IL-2 or its variants are linked to a 20K4-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into IL-2 or its variants at position 65 (SEQ ID NO: 2, or the corresponding amino acid positions in SEQ ID NO: 3, 5 or 7), and the IL-2 or its variants are linked to a 20K4-branched polyethylene glycol molecule. In certain embodiments, the non-naturally encoded amino acid is incorporated into position 61 of IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5 or 7), and the IL-2 or a variant thereof is linked to a 20K4-branched polyethylene glycol molecule. In certain embodiments, the non-naturally encoded amino acid is incorporated into position 49 of IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5 or 7), and the IL-2 or a variant thereof is linked to a 20K4-branched polyethylene glycol molecule. In certain embodiments, the non-naturally encoded amino acid is incorporated into IL-2 or its variant 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 its variant is linked to a 20K4-branched polyethylene glycol molecule. In certain embodiments, the non-naturally encoded amino acid is incorporated into IL-2 or its variant 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 its variant is linked to a 20K4-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 37 of IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7), and the IL-2 or a variant thereof is linked to a 20K4-branched polyethylene glycol molecule. In certain embodiments, a non-naturally encoded amino acid is incorporated into position 35 of IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7), and the IL-2 or a variant thereof is linked to a 20K4-branched polyethylene glycol molecule.

In certain embodiments, the water-soluble polymer attached to IL-2 or its variant comprises a polyalkylene glycol moiety. In certain embodiments, the non-naturally encoded amino acid residue incorporated into IL-2 comprises a carbonyl group, an amineoxy group, a hydrazide group, a hydrazine group, an aminourea group, an azido group, or an alkynyl group. In certain embodiments, the non-naturally encoded amino acid residue incorporated into IL-2 or its variant comprises a carbonyl moiety, and the water-soluble polymer comprises an amineoxy group, a hydrazide group, a hydrazine group, or an aminourea moiety. In certain embodiments, the non-naturally encoded amino acid residue incorporated into IL-2 or its variant comprises an alkynyl moiety, and the water-soluble polymer comprises an azido group 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 present invention also provides compositions comprising IL-2 or a variant thereof containing a non-naturally encoded amino acid and a pharmaceutically acceptable carrier. In certain embodiments, the non-naturally encoded amino acid is linked to a water-soluble polymer.

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

The present 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 for replacing a non-naturally encoded amino acid into the IL-2 or a variant thereof.

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

The present invention also provides methods for increasing the therapeutic half-life, serum half-life or circulation time of IL-2 or its variants. The present invention also provides methods for regulating the immunogenicity of IL-2 or its variants. In certain embodiments, the method comprises replacing any one or more amino acids in naturally occurring IL-2 or its variants with non-naturally encoded amino acids and/or connecting the IL-2 or its variants to a linker, a polymer, a water-soluble polymer or a bioactive molecule. In one embodiment of the present invention, the linker is long enough to allow flexibility and allow dimer formation. In one embodiment of the present invention, the length of the linker is at least 3 amino acids or 18 atoms to allow dimer formation.

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

The present invention also provides a method for treating a patient who needs the treatment using an effective amount of IL-2 or IL-2 variant molecules of the present invention. In certain embodiments, the method includes administering a drug composition of an effective amount to the patient, the drug composition comprising an IL-2 or IL-2 variant molecule containing non-naturally encoded amino acids and a pharmaceutically acceptable carrier. In certain embodiments, the method includes administering a drug composition of an effective amount to the patient, the drug composition comprising IL-2 or its variant and a pharmaceutically acceptable carrier containing one or more non-naturally encoded amino acids and one or more natural amino acids. In certain embodiments, the non-naturally encoded amino acids are connected to a water-soluble polymer. In certain embodiments, the natural amino acids are connected to a water-soluble polymer. In certain embodiments, the IL-2 is glycosylated. In certain embodiments, the IL-2 is not glycosylated. In certain embodiments, the patient in need of treatment suffers from a cancer, disorder or disease characterized by high expression of IL-2 receptor α, but is not limited thereto. In certain embodiments, the present invention provides a method for treating cancer or a disorder or disease by administering a therapeutically effective amount of the IL-2 composition of the present invention to an object. In certain embodiments, the present invention provides a method for treating a genetic disease by administering a therapeutically effective amount of the IL-2 composition of the present invention to a patient. The IL-2 polypeptide of the present invention is used to treat a disease or disorder in cells with high expression of IL-2 receptor α. In certain embodiments, the cancer, disorder or disease is treated by reducing, blocking or silencing IL-2 receptor α expression. The IL-2 polypeptide or variant of the present invention is used to manufacture a drug for treating cancer, disease or condition associated with high expression of IL-2 receptor α. The IL-2 polypeptide or variant of the present invention is used to manufacture a drug for treating cancer. The IL-2 polypeptide or variant of the present invention is used to manufacture a drug for treating genetic diseases.

The present invention also provides IL-2 comprising the sequence shown in SEQ ID NO: 1, 2, 3, 5 or 7 or any other IL-2 sequence, except that at least one amino acid is replaced by a non-naturally encoded amino acid. In some embodiments, the non-naturally encoded amino acid is linked to a water-soluble polymer. In some embodiments, the water-soluble polymer comprises a polyethylene glycol component. In some embodiments, the non-naturally encoded amino acid comprises a carbonyl group, an amine group, a hydrazide group, a hydrazine group, a uridine group, an azido group or an alkynyl group.

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

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

The present invention provides an IL-2 or a variant thereof comprising at least one linker, polymer or bioactive molecule, wherein the linker, polymer or bioactive molecule is attached to the polypeptide via a functional group of a non-naturally encoded amino acid that is incorporated into the polypeptide via the ribosome. In certain embodiments, the IL-2 or a variant thereof is monoPEGylated. The present invention also provides an IL-2 or a variant thereof, comprising a linker, polymer or bioactive molecule attached to one or more non-naturally encoded amino acids, wherein the non-naturally encoded amino acids are incorporated into the polypeptide via the ribosome at a pre-selected position.

The scope of the present invention includes a leader or signal sequence of IL-2 or its variants linked to the IL-2 coding region and a heterologous signal sequence linked to the IL-2 coding region. The selected heterologous leader or signal sequence should be, for example, a sequence that is recognized and processed for secretion by the host cell secretion system and may be cleaved by the host cell signal peptidase. The method of using the IL-2 of the present invention to treat a disease or disorder is meant to imply treatment with IL-2 or its variants with or without a signal or leader peptide.

In another embodiment, the conjugation of IL-2 or its variants comprising one or more non-naturally occurring amino acids with another molecule, including but not limited to PEG, provides substantially purified IL-2 due to a unique chemical reaction used for conjugation with the non-natural amino acids. The conjugation of IL-2 or its variants comprising one or more non-naturally encoded amino acids with 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 its variants.

1:IL-2 receptor α

2:IL-2 receptor β

3:IL-2 receptor γ

4:IL-2

5: Locations to be screened

6: PEG (polyethylene glycol)

CD8+:CD8 receptor protein

Kd: dissociation constant

Rβγ: receptor βγ

Rαβγ: receptor αβγ

Treg: regulatory T cells

IL-2: Interleukin-2

IL-2Rα:IL-2 receptor

IL-2Rβ:IL-2 receptor

IL-2Rγ:IL-2 receptor

JAK1: tyrosine protein kinase 1

JAK3: tyrosine protein kinase 3

P: Phosphorylation

STAT5: signal transducer and activator of transcription 5

Figure 1 - Model showing a schematic view of the IL-2 polypeptide, where potential receptor interaction sites are marked with the structure of IL-2Rα and its interface with IL-2.

Figure 2 - shows the plasmid map of the expression vector used to express IL-2 in E. coli.

Figure 3A-shows Western blot analysis of IL-2 protein expression in E. coli.

Figure 3B - shows the titer of IL-2 variants in E. coli.

Figure 4A - shows the binding kinetic sensorgram of wild-type IL-2 to CD25 and the model fit and calculated measurements.

Figure 4B - shows a plasmid map of the expression vector used to express IL-2 in mammalian cells.

Figure 5-shows the design of the UPF1 genomic DNA sequence and CRISPR gRNA site.

Figure 6-shows the sequence verification of the UPF1 knockout cell line.

Figure 7A - shows the transient expression of various IL-2 variants in mammalian cells.

Figure 7B - shows Western blot analysis of wild-type IL-2 and IL-2 variants produced in mammalian cells.

Figure 8 - shows the CTLL-2 expansion assay of the F42 variant of IL-2.

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

Figure 10A - shows the binding kinetic sensorgrams of wild-type IL-2 and F42 variants.

Figure 10B - shows the binding kinetics sensorgrams of the K35 and Y45 variants.

Figure 10C - shows the binding kinetics sensorgrams of T37 and P65 variants.

Figure 11-Illustration of the IL-2 receptor dimerization assay.

Figure 12 - Schematic illustration of the in vitro pSTAT5 assay.

Figure 13 - shows the clonal outgrowth and long-term propagation of CTLL-2 cells in the presence of glycosylated or non-glycosylated IL-2.

Figure 14 - shows the comparison of titer concentrations before and after the generation of stable pools of the corresponding wild-type IL-2 or its selected variants.

Figure 15A - shows the titer in mammalian cells expressing the F42-R38A variant.

Figure 15B - shows the CTLL-2 binding assay of the F42-R38A variant.

Figure 15C - shows the binding kinetic sensorgram of the F42-R38A variant.

Definition

It should be understood that the present invention is not limited to the specific methods, laboratory instructions (protocols), cell lines (cell lines), constructs (constructs) and reagents described in the present invention, and that they themselves may vary. It should also be understood that the terminology used in the present invention is only for the purpose of describing specific embodiments and is not intended to limit the scope of the present invention, which is limited only by the scope of the attached patent application.

When used in this invention and the appended claims, the singular without a specific number includes plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "IL-2," "PEG-IL-2," "PEG-IL-2 conjugate," and various capitalized, hyphenated, and unhyphenated forms refer to one or more such proteins and include equivalents thereof known to those of ordinary skill in the art, and so forth.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by ordinary technicians in the field to which the present invention belongs. Although any methods, devices and materials similar or equivalent to those described in the present invention can be used in the practice or testing of the present invention, the preferred methods, devices and materials will now be described.

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

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

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

As used in the present invention, the term "medium" includes any medium, solution, solid, semisolid 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, E. coli or Pseudomonas host cells, and cell contents. Thus, the term can encompass a medium in which host cells have been grown, such as a medium into which IL-2 has been secreted, including a medium before or after a proliferation step. The term may also encompass buffers or reagents containing host cell lysates, for example where IL-2 is produced intracellularly and the host cells are lysed or disrupted to release the IL-2.

When used in the present invention in connection with protein refolding, a "reducing agent" is defined as any compound or material that maintains hydroxyls in a reduced state and reduces intramolecular or intermolecular disulfide bonds. Suitable reducing agents include, but are not limited to, dithiothreitol (DTT), 2-hydroxyethanol, dithioerythreitol, cysteine, cysteamine (2-aminoethanethiol), and reduced glutathione. It will be apparent to one 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 in the present invention in connection with protein refolding, an "oxidant" is defined as any compound or material capable of removing electrons from a compound being oxidized. Suitable oxidants include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythritol, and oxygen. It will be apparent to one of ordinary skill in the art that a wide variety of oxidants are suitable for use in the methods of the present invention.

As used in the present invention, "denaturing agent" or "denaturant" is defined as any compound or material that causes reversible unfolding of proteins. The strength of the denaturant is determined by both the nature and concentration of the particular denaturant. Suitable denaturants may be isolating agents, detergents, organic solvents, water-miscible solvents, phospholipids, or a combination of two or more such agents. Suitable isolating agents include, but are not limited to, urea, guanidine, and sodium thiocyanate. Useful detergents may include, but are not limited to, strong detergents such as sodium dodecyl sulfate or polyoxyethylene ethers (e.g., Tween or Triton detergents), Sarkosyl, mild nonionic detergents (e.g., digitonin), mild cationic detergents such as N-2,3-(dioleyloxy)-propyl-N,N,N-trimethylammonium, mild Ionic detergents (e.g. sodium cholate or sodium deoxycholate) or zwitterionic detergents, including but not limited to sulfobetaines (Zwittergent), 3-(3-chloroamidopropyl) dimethylammonium-1-propane sulfate (CHAPS) and 3-(3-chloroamidopropyl) dimethylammonium-2-hydroxy-1-propane sulfonate (CHAPSO). Water-miscible organic solvents such as acetonitrile, lower alkanols (especially C2-C4 alkanols such as ethanol or isopropanol) or lower alkanediols (especially C2-C4 alkanediols such as ethylene glycol) can be used as denaturants. The phospholipids that can be used in the present invention can be naturally occurring phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine and phosphatidylinositol or synthetic phospholipid derivatives or variants such as dihexylphosphatidylcholine or diheptylphosphatidylcholine.

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

When used in the present invention, "cofolding" specifically refers to a refolding process, reaction or method using at least two polypeptides that interact with each other and result in the conversion of unfolded or incorrectly folded polypeptides into original, correctly folded polypeptides.

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

The sequence of IL-2 lacking a leader sequence and without methionine at the N-terminus is referred to as SEQ ID NO: 2 in the present invention. The sequence of IL-2 without a leader sequence and with methionine at the N-terminus is referred to as SEQ ID NO: 3, 5 or 7. In certain embodiments, the IL-2 or variant thereof of the present invention is substantially identical to SEQ ID NO: 2, 3, 5 or 7 or any other sequence of IL-2. Nucleic acid molecules encoding IL-2 (including mutant IL-2 and other variants) and methods of expressing and purifying these polypeptides are well known in the art.

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

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

The term "IL-2 polypeptide" also includes glycosylated IL-2, such as but not limited to polypeptides glycosylated at any amino acid position, N-linked or O-linked glycosylated forms of the polypeptide. Variants containing single nucleotide changes are also considered biologically active variants of IL-2 polypeptides. 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 any type of other biologically active molecule that is chemically linked or expressed as a fusion protein, and IL-2 heterodimers, homodimers, heteromultimers or homomultimers, as well as polypeptide analogs that contain, for example, specific deletions or other modifications but still maintain biological activity.

As used herein, "interleukin-2" or "IL-2" is a protein comprising two subunits non-covalently linked to form a homodimer, whether conjugated to a biologically active molecule, conjugated to polyethylene glycol, or in a non-conjugated 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 terms "PEGylated IL-2", "PEGylated IL-2" or "PEG-IL-2" are IL-2 molecules having one or more polyethylene glycol molecules covalently attached to one or more amino acid residues of the IL-2 protein via a linker such that the attachment is stable. The terms "monoPEGylated IL-2" and "monoPEG-IL-2" mean that one polyethylene glycol molecule is covalently attached to a single amino acid residue on one subunit of the IL-2 dimer via a linker. The average molecular weight of the PEG component is preferably between about 5,000 and about 50,000 daltons. The method or site of attachment of PEG to IL-2 is not important, but preferably the PEGylation does not alter or only minimally alters the activity of the biologically active molecule. Preferably, the increase in half-life outweighs any decrease in biological activity.

All references to amino acid positions in IL-2 described herein are based on positions in SEQ ID NO: 2, unless otherwise indicated (i.e., when the comparison is stated to be based on SEQ ID NO: 3, 5, or 7 or other IL-2). One skilled in the art will recognize that amino acid positions corresponding to positions in SEQ ID NO: 2 in any other IL-2, such as SEQ ID NO: 3, 5, or 7, can be readily identified. One skilled in the art will recognize that amino acid positions corresponding to positions in SEQ ID NO: 2, 3, 5, or 7 or any other IL-2 sequence in any other IL-2 molecule, such as IL-2 fusions, variants, fragments, etc. can be readily identified. For example, a sequence alignment program such as BLAST can be used to align and identify specific positions in a protein corresponding to positions in SEQ ID NO: 2, 3, 5, or 7 or other IL-2 sequences. The substitution, deletion or addition of amino acids described in the present invention with reference to SEQ ID NO: 2, 3, 5 or 7 or other IL-2 sequences is intended to also refer to the substitution, deletion or addition in the corresponding position in the IL-2 fusions, variants, fragments, etc. described in the present invention or known in the art, and is explicitly covered by the present invention.

IL-2 (IL3): 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 can be altered without affecting its activity, and these modified forms of IL2 can also be linked to vectors and used in the methods described herein.

The term "IL-2" encompasses IL-2 comprising one or more amino acid substitutions, additions or deletions. The IL-2 of the present invention may comprise one or more natural amino acid modifications in combination with one or more non-natural amino acid modifications. Exemplary substitutions in a wide range of amino acid positions in naturally occurring IL-2 polypeptides have been described, including but not limited to substitutions that modulate drug stability, modulate one or more biological activities of IL-2 polypeptides such as but not limited to increasing agonist activity, increasing polypeptide solubility, reducing protease sensitivity, converting the polypeptide into an antagonist, etc., and are encompassed by the term "IL-2 polypeptide". In certain embodiments, the IL-2 antagonist 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 an addition, substitution or deletion that modulates the biological activity of the IL-2 or variant polypeptide. In certain embodiments, the IL-2 or variant further comprises an addition, substitution or deletion that modulates a known and research-proven property of IL-2, such as the treatment or relief of one or more symptoms of cancer. The addition, substitution or deletion may modulate one or more properties or activities of IL-2 or a variant. For example, the addition, substitution or deletion may modulate affinity for an IL-2 receptor or one or more subunits of the receptor, modulate circulation half-life, modulate therapeutic half-life, modulate stability of the polypeptide, modulate cleavage by proteases, modulate dosage, modulate release or bioavailability, promote purification, or improve or alter a specific route of administration. Likewise, IL-2 or variants may contain protease cleavage sequences, reactive groups, antibody binding domains (including but not limited to FLAG or poly-His) or other affinity-based sequences (including but not limited to FLAG, poly-His, GST, etc.) or linked molecules (including but not limited to biotin) that improve detection (including but not limited to GFP), purification or other properties 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 chains, to naturally encoded amino acid side chains, or indirectly linked via linkers. 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.

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

As used in the present invention, the term "cytotoxic agent" can be any agent that has a therapeutic effect on cancer cells or activated immune cells and can be used as a therapeutic agent in combination with IL-2, PEG-IL-2 or IL-2 variants (see, for example, WO 2004/010957, "Drug Conjugates and Their Use for Treating Cancer, An Autoimmune Disease or an Infectious Disease"). Classes of cytotoxic or immunosuppressive agents useful in the present invention include, for example, antitubulin agents, auristatins, DNA groove binding agents, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-, mono-, di- and trinuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, anti-metabolites, chemosensitizers, duocarcinoids, etoposides, fluoropyrimidines, ion carriers, lexitropsins, nitrosoureas, platinum alcohols, preformed compounds, purine anti-metabolites, puromycins, radiation sensitizers, sensitizers), steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, etc.

Individual cytotoxic or immunosuppressive agents include, for example, androgens, ampicillin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoxide, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytosine arabinoside, cytosine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin), daunorubicin, dacarbazine, docetaxel, doxorubicin, Estrogen, 5-fluorodeoxyuridine, 5-fluorouracil, bacitracin D, hydroxyurea, idarubicin, isocyclophosphamide, irinotecan, lomustine (CCNU), methylchlorethylamine, melphalan, 6-hydroxypurine, methotrexate, mithraquinone, mitromycin C, mitoxantrone, nitroimidazole, paclitaxel, prucamycin, procarbazine, streptozotocin, teniposide, 6-thioguanine, thio-TEPA, topotecan, vinblastine, vincristine, vinorelbine, VP-16, and VM-26.

In certain typical embodiments, the therapeutic agent is a cytotoxic agent. Suitable cytotoxic agents include, for example, dolastatins (e.g., auristatin E, AFP, MMAF, MMAE), DNA groove binders (e.g., enediynes and lexitropsins), duocarcinoids, taxanes (e.g., paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin (morpholi no-doxorubicin), lisocin, cyanomorpholino-doxorubicin, echinomycin, compretin, fumarin, ebomycin A and B, estramustine, cryptophycins, simadotin, maytansinoids, discodermolide, acanthopanax, and mitoxantrone.

"Non-naturally encoded amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrrole lysine or selenocysteine. Other terms that may be used synonymously with the term "non-naturally encoded amino acid" are "unnatural amino acid", "non-naturally occurring amino acid" and various hyphenated and unhyphenated 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 pyrrole lysine and selenocysteine), but that are not themselves naturally incorporated by the translation complex into a growing polypeptide chain. 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.

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

The terms "functional group", "active moiety", "activating group", "leaving group", "reactive site", "chemically reactive group", and "chemically reactive moiety" are used in the art and in the present invention to refer to a unique, definable portion or unit of a molecule. The terms are more or less synonymous in the chemical art and are used in the present invention to indicate a portion of a molecule that performs some function or activity and is reactive with other molecules.

The terms "linkage", "linkage" or "linker" are used herein to refer to a group or bond that is generally formed as a result of a chemical reaction, and is generally a covalent bond. A hydrolytically stable bond means that the bond is substantially stable in water and does not react with water for a long time, perhaps even indefinitely, at useful pH values, including but not limited to under physiological conditions. A hydrolytically unstable or degradable bond means that the bond is degradable in water or an aqueous solution, including, for example, blood. An enzymatically unstable or degradable bond means that the bond is degradable by one or more enzymes. As understood in the art, PEG and related polymers may include degradable bonds in the polymer backbone or in a linker group between the polymer backbone and one or more terminal functional groups of the polymer molecule. For example, ester bonds formed by the reaction of PEG carboxylic acid or activated PEG carboxylic acid with an alcohol group on a biologically active agent typically hydrolyze under physiological conditions to release the agent. Other hydrolytically degradable bonds include, but are not limited to, carbonate bonds, imine bonds resulting from the reaction of amines with aldehydes, phosphate ester bonds formed by the reaction of alcohols with phosphate groups, hydrazone bonds as byproducts of hydrazides with aldehydes, acetal bonds as products of the reaction of aldehydes with alcohols, orthoester bonds as products of the reaction of formic acid with alcohols, peptide bonds formed by amine groups at the termini of polymers including but not limited to PEG and carboxyl groups of peptides, and oligonucleotide bonds formed by phosphoramidite groups at the termini of polymers including but not limited to the 5' hydroxyl group of oligonucleotides.

As used herein, the term "biologically active molecule", "biologically active moiety" or "biologically active 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, phages, transposons, prions, insects, fungi, plants, animals and humans. Specifically, as used herein, biologically active molecules include but are not limited to any substance intended to diagnose, cure, mitigate, treat or prevent disease in humans or other animals or otherwise enhance the physical or psychological health of humans or animals. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, vaccines, immunogens, addictive drugs, non-addictive drugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxoids, biologically active molecules, prokaryotic and eukaryotic cells, viruses, polysaccharides, nucleic acids obtained or derived from viruses, bacteria, insects, animals or any other cell or cell type and portions thereof, liposomes, microparticles and micelles. Classes of bioactive agents suitable for use in the present invention include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, bile acid resins, niacin and/or statins, anti-inflammatory drugs, anti-tumor drugs, cardiovascular drugs, antianxiety drugs, hormones, growth factors, steroid drugs, bioactive molecules of microbial origin, etc. Bioactive agents also include amide compounds such as those described in patent application publication No. 20080221112 of Yamamori et al., which are administered before, after and/or co-administered with the IL-2 polypeptide of the present invention.

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

When substituents are described by their conventional chemical formula written from left to right, they equally cover chemically identical substituents that would result from writing the structure from right to left, for example, the structure _-CH2O- is equivalent to the structure _-OCH2- .

The term "substituent" includes but is not limited to "non-interfering substituents.""Non-interferingsubstituents" are groups that produce stable compounds. Suitable non-interfering substituents or residues include, but are not limited to, halogen, C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, C ₁ -C ₁₀ alkoxy, C ₁ -C ₁₂ aralkyl, C _{1 -C 12 alkaryl, C 3 -C 12 cycloalkyl} , C ₃ -C ₁₂ cycloalkenyl, phenyl, substituted phenyl, toluyl, xylyl, biphenyl, C ₂ -C ₁₂ alkoxyalkyl, C 2 -C _{12 alkoxyaryl, C 7 -C 12 aryloxyalkyl, C 7 -C 12 oxyaryl, C 1 -C 6 alkylsulfinyl , C 1 -C 10 alkylsulfonyl , --} (CH ₂ ) _m --O--(C ₁ -C -C ₁₀ alkyl) (wherein m is 1 to 8), aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic group, substituted heterocyclic group, nitroalkyl, --NO ₂ , --CN, --NRC(O)--(C ₁ -C ₁₀ alkyl), --C(O)--(C ₁ -C ₁₀ alkyl), C ₂ -C ₁₀ alkylthioalkyl, --C(O)O--(C ₁ -C ₁₀ alkyl), --OH, --SO ₂ , =S, --COOH, --NR ₂ , carbonyl, --C(O)--(C ₁ -C ₁₀ alkyl)-CF 3 , --C(O)-CF 3 , --C(O)NR 2 , --(C ₁ -C ₁₀ aryl)-S--(C ₆ -C ₁₀ aryl), --C(O)--(C ₁ -C 10 ₁₀ aryl), --(CH ₂ ) _m --O--(--(CH ₂ ) _m --O--(C ₁ -C ₁₀ alkyl) (wherein 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. When used in the present invention, 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 combinations thereof, which may be fully saturated, mono- or polyunsaturated, and may include divalent and polyvalent residues, having the number of carbon atoms specified (i.e., _C1 - _C10 means 1 to 10 carbon atoms). Examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated alkyl groups are groups having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers. Unless otherwise indicated, the term "alkyl" is also meant to include derivatives of alkyl groups such as "heteroalkyl" as defined in more detail below. Alkyl groups limited to alkyl groups are referred to as "homoalkyl".

The term "alkylene" by itself or as part of another substituent means a divalent residue derived from an alkane, such as, but not limited to, the structures -CH ₂ CH ₂ - and -CH ₂ CH ₂ CH ₂ CH ₂ -, and also includes groups described below as "heteroalkylene". Typically, an alkyl (or alkylene) group has from 1 to 24 carbon atoms, with groups having 10 or fewer carbon atoms being a particular embodiment of the methods and compositions described herein. A "lower alkyl" or "lower alkylene" group is a shorter chain alkyl or alkylene group, 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 via an oxygen atom, an amine 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 alkyl group 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 be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom O, N, S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, _-CH2 - _CH2 -O- _CH3 , -CH2- _CH2 - _NH - _CH3 , -CH2 _{- CH2} -N( _CH3 ) _-CH3 , _{-CH2- S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2 - CH3 , -CH = CH - O} - _CH3 , -Si( _CH3 ) ₃ , _-CH2- CH=N- _OCH3 , and -CH=CH-N( _CH3 ) _-CH3 . Up to two impurity atoms may be consecutive, for example, _-CH2 -NH- _OCH3 and _-CH2 -O-Si( _CH3 ) ₃ . Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent residue derived from a heteroalkyl group, such as, but not limited to, _-CH2 - _CH2 -S-CH2- _CH2- and _{-CH2 -S - CH2-CH2} -NH- _CH2- . For heteroalkylene groups, the same or different heteroatoms may also occupy either or both chain ends (including, but not limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, etc.). In addition, for alkylene and heteroalkylene linking groups, the direction in which the structural formula of the linking group is written does not imply the orientation of the linking group. For example, the formula -C(O) _2R'- represents both -C(O) _2R'- and -R'C(O) _2- .

Unless otherwise stated, the terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, refer to cyclic versions of "alkyl" and "heteroalkyl", respectively. Thus, cycloalkyl or heterocycloalkyl includes saturated, partially unsaturated, and fully unsaturated cyclic linkages. Furthermore, for heterocycloalkyl, heteroatoms may occupy the position where the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl groups 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. In addition, the term encompasses bicyclic and tricyclic ring structures. Similarly, the term "heterocycloalkylene" by itself or as part of another substituent means a divalent residue derived from a heterocycloalkyl group, and the term "cycloalkylene" by itself or as part of another substituent means a divalent residue derived from a cycloalkyl group.

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

When used in the present invention, the term "polyalkylene glycol" refers to polyethylene glycol, polypropylene glycol, polybutylene glycol and their derivatives. The term "polyalkylene glycol" covers both linear and branched polymers and has an average molecular weight between 0.1 kDa and 100 kDa. Other exemplary embodiments are listed, for example, in commercial supplier catalogs, such as Shearwater Corporation's catalog "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001).

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

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

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 radicals (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of different groups selected from, but not limited to, -OR', =O, =NR', =N-OR', -NR'R", -SR', -halogen, -SiR'R"R''', -OC(O)R', -C(O)R', _-CO2R ', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R''', -NR"C(O) _2R ', -NR-C(NR'R"R''')=NR'''', -NR-C(NR'R")=NR''', -S(O)R', -S(O) _2R ', -S(O) _2NR'R ", _-NRSO2R ', -CN and _-NO2 , the number of which ranges from 0 to (2m'+1), where m' is the total number of carbon atoms in such a residue. R', R", R''' and R'''' each independently refers to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (including but not limited to aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy or aralkyl. When a compound of the present invention includes more than one R group, for example, each of the R groups is independently selected, as is each of these groups when there is more than one R', R", R''' and R'''' group. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6- or 7-membered ring. For example, -NR'R" is meant to include but not be limited to 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will understand that the term "alkyl" is meant to include groups containing carbon atoms bonded to groups other than hydrogen groups, such as halogenated alkyl groups (including but not limited _{to -CF3} and _-CH2CF3 ) and acyl groups (including but not limited to -C(O) _CH3 , -C(O) _CF3 , -C(O) _{CH2OCH3 ,} etc.).

Similar to the substituents described for the alkyl residues, the substituents for the aryl and heteroaryl groups are variable and are selected from, but are not limited to: halogen, -OR', =O, =NR', =N-OR', -NR'R", -SR', -halogen, -SiR'R"R''', -OC(O)R', -C(O)R', _-CO2R ', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R''', -NR"C(O) _2R ', -NR-C(NR'R"R''')=NR'''', -NR-C(NR'R")=NR''', -S(O)R', -S(O) _2R ', -S(O)2NR'R", -NRSO2R _{' ,} -CN and _-NO2 , -R', -N ₃ , -CH(Ph) ₂ , fluoro(C ₁ -C ₄ )alkoxy and fluoro(C ₁ -C ₄ )alkyl, the number of which ranges 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 hydrogen, alkyl, heteroalkyl, aryl and heteroaryl. When the compounds of the present invention include more than one R group, for example, each of the R groups is independently selected, as are each of these groups when more than one R', R", R''' and R'''' groups are present.

As used herein, the term "modulated serum half-life" means a positive or negative change in the circulating half-life of the modified IL-2 relative to its unmodified form. Serum half-life is measured by obtaining blood samples at various time points after IL-2 administration and determining the concentration of the molecule in each sample. The correlation of serum concentration with time allows the calculation of serum half-life. The increased serum half-life ideally has at least about two-fold, but smaller increases 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-fold, at least about five-fold, or at least about ten-fold.

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

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

The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single or double chain form. Unless otherwise specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to 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 PNA (peptide nucleic acid), analogs of DNA used in antisense technology (phosphorothioate, aminophosphoric acid, etc.). Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to degenerate codon substitutions) and complementary sequences as well as the sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

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

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, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) as well as pyrrolyl lysine and selenocysteine. Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids, i.e., alpha carbon bound to hydrogen, carboxyl, amine, and R groups, such as homoserine, norleucine, methionine sulfonate, and methionine methyl sulfonate. These analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. References to amino acids include, for example, naturally occurring protein L-amino acids, D-amino acids, chemically modified amino acids such as amino acid variants and derivatives, naturally occurring non-protein amino acids such as β-alanine, ornithine, etc., and chemically synthesized compounds having properties known in the art to be unique to amino acids. Examples of non-naturally occurring amino acids include, but are not limited to, α-methyl amino acids (e.g., α-methylalanine), D-amino acids, histidine-like amino acids (e.g., 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine, and α-methyl-histidine), amino acids with additional methylene groups in the side chains ("homo" amino acids), and amino acids in which the carboxylic acid functional groups in the side chains are replaced by sulfonic acid groups (e.g., sulfalanine). The incorporation of non-natural amino acids (including synthetic non-natural amino acids, substituted amino acids, or one or more D-amino acids) in the proteins of the present invention may be advantageous in a number of different ways. Peptides containing D-amino acids, etc., exhibit improved in vitro or in vivo stability compared to their L-amino acid-containing counterparts. Thus, constructs of peptides and the like incorporating D-amino acids may be particularly useful when greater intracellular stability is desired or required. More specifically, D-peptides and the like are resistant to endogenous peptidases and proteases, thus providing enhanced 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 major histocompatibility complex class II restricted presentation to T helper cells and are therefore unlikely to induce humoral immune responses in intact organisms.

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

"Conservatively modified variants" apply to both amino acid and nucleic acid sequences. For a particular nucleic acid sequence, "conservatively modified variants" refers to nucleic acids that encode identical or substantially identical amino acid sequences, or, in the case where the nucleic acid does not encode an amino acid sequence, to substantially 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. Therefore, at each position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. These nucleic acid variations are "silent variations," which are a type of conservatively modified variation. Each nucleic acid sequence encoding a polypeptide in the present invention also describes each possible silent variation of the nucleic acid. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG, which is generally the only codon for methionine, and TGG, which is generally the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implied in each described sequence.

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

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

In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or percent "identity" means that two or more sequences or subsequences are identical. Sequences are "substantially identical" if they have a certain percentage of identical amino acid residues or nucleotides (i.e., about 60% identity, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% identity over a specified region) when the sequences are 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 applies to the complementary sequence of a test sequence. The identity may exist over a region of at least about 50 amino acids or nucleotides in length or over a region of 75-100 amino acids or nucleotides in length, or, where not specified, across the entire sequence of a polynucleotide or polypeptide. Polynucleotides encoding the polypeptides of the present invention (including homologs from species other than humans) may be obtained by a method comprising the steps of screening a library under stringent hybridization conditions using probes labeled with the polynucleotide sequence of the present invention or fragments thereof, and isolating full-length cDNA and genomic clones containing the polynucleotide sequence. Such hybridization techniques are well known to those skilled in the art.

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

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

When used in the present invention, the term "eukaryote" refers to organisms belonging to the eukaryotic system domain, such as animals (including but not limited to mammals, insects, reptiles, birds, etc.), caterpillars, plants (including but not limited to monocots, dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia, protists, etc.

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

As used in the present invention, the term "subject" refers to an animal, in some embodiments a mammal, and in other embodiments a human, that is the target of treatment, observation, or experimentation. The animal may be a companion animal (e.g., dog, cat, etc.), a farm animal (e.g., cow, sheep, pig, horse, etc.), or an experimental animal (e.g., rat, mouse, guinea pig, etc.).

When used in the present invention, the term "effective amount" refers to the amount of the modified non-natural amino acid polypeptide administered to alleviate one or more symptoms of the disease, condition or disorder to be treated to a certain extent. The composition containing the modified non-natural amino acid polypeptide described in the present invention can be administered for preventive, enhancing and/or therapeutic treatment.

The term "enhancing" means to increase or prolong, either in potency or duration, a desired effect. Thus, with respect to enhancing the effect of a therapeutic agent, the term "enhancing" refers to the ability to increase or prolong, either in potency or duration, the effect of another therapeutic agent on a system. As used herein, an "enhancing-effective amount" refers to an amount sufficient to enhance the effect of another therapeutic agent in a desired system. When used in a patient, the amount effective for this use depends on the severity and course of the disease, disorder or condition, previous treatments, the patient's health condition and response to the medication, and the judgment of the treating physician.

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

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

In prophylactic applications, the composition containing the IL-2 is administered to a patient susceptible to or otherwise at risk for a particular disease, disorder or condition. This amount is defined as a "prophylactically effective amount". In this use, the exact amount also depends on the patient's health condition, weight, etc. It is considered to be well within the skill of the art to determine such a prophylactically effective amount by routine experiments (e.g., dose escalation clinical trials).

In therapeutic applications, compositions containing the modified non-natural amino acid polypeptides are administered to a patient already suffering from a disease, condition or disorder in an amount sufficient to cure or at least partially prevent the symptoms of the disease, condition or disorder. This 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 patient's health and response to the drug, and the judgment of the treating physician. It is considered to be well within the skill of the art to determine such a therapeutically effective amount by routine experimentation (e.g., a dose escalation clinical trial).

The term "treatment" is used to refer to either preventive and/or curative treatment.

The non-naturally encoded amino acid polypeptides proposed in the present invention may include isotope-labeled compounds, which have one or more atoms replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ O, ¹⁷ O, ³⁵ S, ¹⁸ F, ³⁶ Cl, respectively. Certain isotope-labeled compounds described in the present invention, such as compounds into which radioactive isotopes such as ³ H and ¹⁴ C are incorporated, may be useful in the determination of the tissue distribution of drugs and/or substances. Further, substitution with isotopes such as deuterium, ie, ² H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example 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 further or additional embodiments, the non-naturally encoded amino acid polypeptide is metabolized after administration to an organism in need thereof to produce a metabolite, which is then used to produce a desired effect, including a desired therapeutic effect. In other or additional embodiments, it is an active metabolite of a non-naturally encoded amino acid polypeptide.

In some cases, non-naturally encoded amino acid polypeptides can exist as tautomers. In addition, the non-naturally encoded amino acid polypeptides described in the present invention can exist in non-solubilized forms as well as solubilized forms with pharmaceutically acceptable solvents such as water, ethanol, etc. The solubilized forms are also considered to be disclosed in the present invention. A person of ordinary skill in the art will recognize that certain compounds in the present invention can exist in several tautomeric forms. All of these tautomeric forms are considered to be part of the compositions described in the present invention.

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

I. Introduction

The present invention provides an IL-2 molecule comprising at least one non-natural amino acid. In certain embodiments of the present invention, the IL-2 having at least one non-natural amino acid comprises at least one post-translational modification. In one embodiment, the at least one post-translational modification comprises attaching a molecule comprising a second reactive group to at least one unnatural amino acid comprising a first reactive group using chemical methods known to those of ordinary skill in the art to be suitable for the particular reactive group, the molecule including but not limited to a label, a dye, a polymer, a water-soluble polymer, a derivative of polyethylene glycol, a photocrosslinker, a radionuclide, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound, a resin, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, DNA, RNA, an antisense polynucleotide, a sugar, a water-soluble dendrimer, a cyclodextrin, an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or non-covalently interact with other molecules, photoclavable moieties, actinic radiation excitable moieties, photoisomerizable moieties, biotin, biotin derivatives, biotin analogs, heavy atom-incorporated moieties, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox active agents, amino thioacids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chemiluminescent groups, electron dense groups, magnetic groups, intercalating groups, chromophores, energy transfer agents, biologically active agents, detectable labels, small molecules, quantum dots, nanoemitters, radionucleotides, radioemitters, neutron capture agents, or any combination of the foregoing 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 non-natural amino acid, wherein the propargyl group is sometimes referred to as an acetylene moiety) and the second reactive group is an azido moiety, and [3+2] cycloaddition chemistry is used. In another example, the first reactive group is an azido moiety (including but not limited to p-azido-L-phenylalanine or pAZ in the non-natural amino acid, as it is sometimes referred to in this specification) and the second reactive group is an alkynyl moiety. In certain embodiments of the modified IL-2 of the present invention, at least one non-natural amino acid (including but not limited to a non-natural amino acid containing a keto functional group) comprising at least one post-translational modification is used, 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 can attach the molecule to the polypeptide. In another embodiment, the linker attached to IL-2 is long enough to allow dimer formation. The molecule can also be directly linked to the polypeptide.

In certain embodiments, the IL-2 protein comprises at least one post-translational modification 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 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, palmitylation, palmitic acid addition, phosphorylation, glycolipid linkage modification, etc.

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

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

The protein or polypeptide of interest may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more non-natural amino acids. The non-natural amino acids may be the same or different, for example, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different sites in the protein containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different non-natural 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 by non-natural amino acids.

The present invention provides methods and compositions based on the IL-2 of the amino acid comprising at least one non-natural coding. The introduction of at least one non-natural coding amino acid in IL-2 can allow the use of conjugated chemistry involving a specific chemical reaction including but not limited to reacting with one or more non-natural coding amino acids and not reacting with 20 common amino acids simultaneously. In certain embodiments, the IL-2 comprising the amino acid of the non-natural coding is connected to a water-soluble polymer such as polyethylene glycol (PEG) by the side chain of the amino acid of the non-natural coding. The present invention provides a highly efficient method for selectively modifying proteins with PEG derivatives, the method comprising selectively incorporating non-genetically encoded amino acids (including but not limited to amino acids containing functional groups or substituents not present in the 20 naturally incorporated amino acids (including but not limited to ketone, azido or acetylene moieties)) 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 suitable for the specific functional groups or substituents present in the non-naturally encoded amino acids. A wide variety of known chemical methods are suitable for incorporating water-soluble polymers into the proteins in the present invention. These methods include, but are not limited to, Huisgen [3+2] cycloaddition reactions using, but are not limited to, acetylene or aziride derivatives, respectively (see, for example, Padwa, A., Comprehensive Organic Synthesis, Vol. 4, (1991) Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1109; and Huisgen, R., 1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, New York, p. 1-176).

Because the Huisgen [3+2] cycloaddition method involves a cycloaddition rather than a nucleophilic substitution reaction, proteins can be modified with extremely high selectivity. By adding a catalytic amount of Cu(I) salt to the reaction mixture, the reaction can be carried out at room temperature in aqueous conditions with excellent regioselectivity (1,4>1,5). See, for example, Tornoe et al., (2002) J. Org. Chem. 67:3057-3064; and Rostovtsev et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599; and WO 03/101972. Molecules that can be added to the proteins of the present invention by [3+2] cycloaddition include virtually any molecule with a suitable functional group or substituent, including but not limited to azido or acetylene derivatives. These molecules can be added to unnatural amino acids with an acetylene group, including but not limited to p-propargyloxyphenylalanine, or unnatural amino acids with an azido group, 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 is long-term stable against hydrolysis in an aqueous environment. Thus, the physical and chemical properties of a wide range of substances can be modified with the active PEG derivatives of the present invention under demanding aqueous conditions. Even more importantly, because the azido and acetylene components are specific to each other (and do not react with, for example, any of the 20 commonly used genetically encoded amino acids), proteins can be modified with extremely high selectivity in one or more specific sites.

The present 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 the acetylene moiety are highly selective for covalent bonding with the azido moiety that has been selectively introduced into the protein in response to the selector codon. Similarly, the PEG polymer derivatives containing the azido moiety are highly selective for covalent bonding with the acetylene moiety that has been selectively introduced into the protein in response to the selector codon.

More specifically, the azido component includes but is not limited to alkyl azido compounds, aryl azido compounds and derivatives of these azido compounds. The derivatives of the alkyl and aryl azido compounds may include other substituents as long as the acetylene specific reactivity is maintained. The acetylene component includes alkyl and aryl acetylene compounds and derivatives of each. The derivatives of the alkyl and aryl acetylene compounds may include other substituents as long as the azido specific reactivity is maintained.

The present invention provides conjugates of substances having a wide variety of functional groups, substituents or moieties with other substances, including but not limited to labels, dyes, polymers, water-soluble polymers, derivatives of polyethylene glycol, photocrosslinkers, radionuclides, cytotoxic compounds, drugs, affinity labels, photoaffinity labels, reactive compounds, resins, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelators, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, antisense polynucleotides, sugars, water-soluble dendrimers, cyclodextrins, inhibitory RNA, biomaterials, nanoparticles, spin labels, fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, and other molecules. covalently or non-covalently interacting groups, photoclavable moieties, actinic radiation excitable moieties, photoisomerizable moieties, biotin, biotin derivatives, biotin analogs, heavy atom-incorporated moieties, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox active agents, amino thioacids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chemiluminescent groups, electron dense groups, magnetic groups, intercalating groups, chromophores, energy transfer agents, biologically active agents, detectable labels, small molecules, quantum dots, nanoemitters, radionucleotides, radioemitters, neutron capture agents, or any combination of the foregoing or any other desired compound or substance. The present invention also includes conjugates of substances having an azido or acetylene moiety and PEG polymer derivatives having corresponding acetylene or azido moieties. For example, a PEG polymer containing an azido moiety can be conjugated to a biologically active molecule at a position in the protein containing a non-genetically encoded amino acid with an acetylene functional group. The conjugated connection of the PEG to the biologically active molecule includes, but is not limited to, a Huisgen [3+2] cycloaddition product.

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

The present invention includes methods for synthesizing polymers containing azido groups and acetylene of the present invention. In the case of the azido-containing PEG derivative, the azido group can be directly bonded to a carbon atom of the polymer. Alternatively, the azido-containing PEG derivative can be prepared by attaching a linking reagent having an azido component to one end of a conventionally activated polymer, so that the resulting polymer has the azido component at its end. In the case of the acetylene-containing PEG derivative, the acetylene can be directly bonded to a carbon atom of the polymer. Alternatively, the acetylene-containing PEG derivative can be prepared by attaching a linking reagent having an acetylene component to one end of a conventionally activated polymer, so that the resulting polymer has the acetylene component 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 are known to those of ordinary skill in the art. The resulting substituted polymer is then reacted to replace the azido moiety at the end of the polymer with the more reactive moiety. Alternatively, a water-soluble polymer having at least one reactive nucleophilic or electrophilic moiety is reacted with a linking agent having an azido at one end to form a covalent bond between the PEG polymer and the linking agent, and the azido moiety is located at the end of the polymer. Nucleophilic and electrophilic moieties, including amines, thiols, hydrazides, hydrazines, alcohols, carboxylates, aldehydes, ketones, thioesters, etc., 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 undergoes a reaction with a linking agent having an acetylene at one terminus to form a covalent bond between the PEG polymer and the linking agent, 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 in the preparation and use of PEG derivatives will be apparent to practitioners in the art.

The present invention also provides a method for selectively modifying proteins to add other substances to the modified proteins, including but not limited to water-soluble polymers such as PEG and PEG derivatives containing azido or acetylene components. 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, while providing a more selective means of attaching the PEG derivatives to proteins than previously known in the art.

II. General Recombinant Nucleic Acid Methods for Use in the Invention

In many embodiments of the present invention, nucleic acids encoding IL-2 of interest are isolated, cloned, and often altered using recombinant methods. These embodiments are used in, but are not limited to, protein expression or in the generation of variants, derivatives, expression cassettes, or other sequences derived from IL-2. In certain embodiments, sequences encoding polypeptides of the present invention are operably linked to heterologous promoters.

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

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

The DNA sequence of the synthetic human IL-2 gene cloned into the pKG0269 expression plasmid is shown as SEQ ID NO: 4 in Table 1 above. 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 general methods used in the present invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd edition, 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994).

The present invention also relates to eukaryotic host cells, non-eukaryotic host cells and organisms for incorporating non-natural amino acids in vivo via orthogonal tRNA/RS pairs. The host cell is genetically engineered (including but not limited to transformation, transduction or transfection) with a polynucleotide of the present invention or a construct comprising a polynucleotide of the present invention (including but not limited to a vector of the present invention), which may be, for example, a replication (clone) vector or an expression vector.

Several well-known methods are available for introducing target nucleic acids into cells, any of which can be used in the present invention. These methods include: fusion of recipient cells with bacterial protoplasts containing the DNA, electroporation, pellet bombardment, and infection with viral vectors (discussed further below), among others. Bacterial cells can be used to expand the number of plasmids containing the DNA constructs of the present invention. The bacteria are grown to logarithmic phase, and the plasmids within the bacteria can be isolated by a variety of different methods known in the art (see, e.g., Sambrook). In addition, kits for purifying plasmids from bacteria are commercially available (see, e.g., EasyPrep ^™ , FlexiPrep ^™ , both from Pharmacia Biotech; StrataClean ^™ from Stratagene; and QIAprep ^™ from Qiagen). The isolated and purified plasmids are then further manipulated to produce other plasmids for transfection of cells or incorporated into related vectors to infect organisms. Typical vectors contain transcriptional and translational terminators, transcriptional and translational initiation sequences, and promoters that can be used to regulate expression of the specific target nucleic acid. The vector optionally comprises a universal expression cassette containing at least one independent terminator sequence, sequences allowing replication of the expression cassette in eukaryotes or prokaryotes or both (including but not limited to shuttle vectors), and selection markers for both prokaryotic and eukaryotic systems. The vector is suitable for replication and integration in prokaryotes, eukaryotes, or both. See Gillam & Smith, Gene 8: 81 (1979); Roberts et al., Nature, 328: 731 (1987); Schneider, E. et al., Protein Expr. Purif. 6 (1): 10-14 (1995); Ausubel, Sambrook, Berger (all supra). Catalogs of bacteria and bacteriophages that can be used for cloning are provided, for example, by the ATCC, e.g., ATCC Catalogue of Bacteria and Bacteriophage (1992), edited by Gherna et al., published by the ATCC. Other basic programs for sequencing, cloning, and other aspects of molecular biology, as well as the underlying theoretical considerations, are also found in Watson et al., (1992), Recombinant DNA, 2nd edition, Scientific American Books, NY. In addition, virtually any nucleic acid (and virtually any labeled nucleic acid, whether standard or non-standard) can be custom or standard ordered from any of a variety of different commercial sources, such as Midland Certified Reagent Company (Midland, TX, available on the World Wide Web at mcrc.com), The Great American Gene Company (Ramona, CA, available on the World Wide Web at genco.com), ExpressGen Inc. (Chicago, IL, available on the World Wide Web at expressgen.com), Operon Technologies Inc. (Alameda, CA), and many others.

Selector codons

The selector codons of the present invention expand the genetic codon architecture of the protein biosynthetic machinery. For example, selector codons include but are not limited to unique tribasic codons, nonsense codons such as stop codons (including but not limited to amber codons (UAG), ochre codons or ovalite codons (UGA)), unnatural codons, tetrabasic or more codons, rare codons, etc. It is obvious to a person 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 includes using a selector codon as a stop codon to incorporate one or more unnatural amino acids in vivo. For example, an O-tRNA is produced that recognizes a stop codon (including but not limited to UAG) and is amine-acylated by an O-RS with a desired unnatural amino acid, and this O-tRNA is not recognized by the naturally occurring host's amidoacyl-tRNA synthetase. Conventional site-directed mutagenesis can be used to introduce the stop codon (including but not limited to TAG) into the position of interest in the polypeptide of interest. See, e.g., Sayers, J.R. et al., (1988), 5'-3' Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis, Nucleic Acids Res, 16: 791-802. When the O-RS, O-tRNA and nucleic acid encoding the polypeptide of interest are combined in vivo, the unnatural amino acid is incorporated in response to the UAG codon to give a polypeptide containing the unnatural amino acid at the specified position.

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

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

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

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

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

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

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

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

In certain embodiments, in the methods and/or compositions of the invention, the protein or polypeptide of interest (or a 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.

Genes encoding proteins or polypeptides of interest can be induced to include, for example, one or more selector codons for incorporating non-natural amino acids using methods known to those of ordinary skill in the art and described herein. For example, nucleic acids for proteins of interest are induced to include one or more selector codons for incorporation of one or more non-natural amino acids. The present invention includes any such variants of any protein, including but not limited to mutant forms, for example including at least one non-natural amino acid. Similarly, the present invention also includes corresponding nucleic acids, i.e., any nucleic acid having one or more selector codons encoding one or more non-natural amino acids.

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

III. Non-naturally encoded amino acids

A wide range of non-naturally encoded amino acids are suitable for use in the present invention. Any number of non-naturally encoded amino acids can be introduced into IL-2. Generally speaking, the non-naturally encoded amino acids introduced are substantially chemically inert to 20 common genetically encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). In certain embodiments, the non-naturally encoded amino acid includes 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 amine groups) to form a conjugate. For example, IL-2 comprising a non-naturally encoded amino acid containing an azido functional group can react with a polymer (including but not limited to polyethylene glycol or a second polypeptide containing an alkynyl component) to form a stable conjugate due to the selective reaction of the azido and alkyne functional groups to form a Huisgen [3+2] cycloaddition product.

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

Non-naturally encoded amino acids are generally any structure with the structural formula listed above, wherein the R group is any substituent other than the substituent used in the 20 kinds of natural amino acids, and can be suitably used in the present invention. Since the non-naturally encoded amino acids of the present invention are generally different from natural amino acids only in the structure of the side chain, the non-naturally encoded amino acids form amide bonds with other amino acids (including but not limited to natural or non-naturally encoded amino acids) in the same manner as they are formed in naturally occurring polypeptides. However, the non-naturally encoded amino acids have side chain groups different from the natural amino acids. For example, R optionally includes alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halogen-, hydrazide, alkenyl, alkynyl, ether, thiol, seleno-, sulfonyl-, borate, boronic acid, phosphino, phosphonyl, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amine, etc. or any combination thereof. Other non-naturally occurring amino acids of interest that may be suitable for use in the present invention include, but are not limited to, amino acids containing photoactivatable crosslinkers, spin-labeled amino acids, fluorescent amino acids, metal-binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or non-covalently interact with other molecules, photocaged and/or photoisomerizable amino acids, amino acids containing biotin or biotin analogs, glycosylated amino acids such as sugar-substituted serine, other sugars, amino acids, keto-containing amino acids, amino acids containing 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 greater than about 5 or greater than about 10 carbons), carbon-linked sugar-containing amino acids, redox-active amino acids, amino acids containing amino thioacids, and amino acids containing one or more toxic components.

The amino acid of exemplary non-natural coding that may be suitable for the present invention and can be used for reacting with water-soluble polymer includes but is not limited to the amino acid of non-natural coding with carbonyl, aminooxy group, hydrazine, hydrazide, semicarbazide, azido and alkyne reactive group.In certain embodiments, the amino acid of non-natural coding comprises sugar component.The example of such amino acid comprises 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 commonly found in nature (including but not limited to alkenes, oximes, thioethers, amides, etc.). Examples of such amino acids also include sugars not commonly found in naturally occurring proteins such as 2-deoxyglucose, 2-deoxygalactose, etc.

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

Wherein Z typically comprises OH, NH ₂ , SH, NH-R' or S-R'; X and Y can 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 components as the R group described above for the non-natural amino acid of formula I, as well as hydrogen. For example, the non-natural amino acid of the present invention optionally comprises substitution in the amine or carboxyl group, as shown in formula II and III. This type of non-natural amino acid includes, but is not limited to, α-hydroxy acids, α-thio acids, α-aminothiocarboxylates, including but not limited to these compounds having side chains or non-natural 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-glutamine, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, etc. Other structural alternatives include cyclic amino acids such as proline analogs and 3-, 4-, 6-, 7-, 8- and 9-membered ring proline analogs, β and γ amino acids such as substituted β-alanine and γ-aminobutyric acid.

Many non-natural amino acids are based on natural amino acids such as tyrosine, glutamine, phenylalanine, etc., and are suitable for use in the present invention. Tyrosine analogs include but are not limited to para-substituted tyrosine, ortho-substituted tyrosine, and meta-substituted tyrosine, wherein the substituted tyrosine contains but is not limited to keto groups (including but not limited to acetyl groups), benzyl groups, amine groups, hydrazines, hydroxylamines, thiol groups, carboxyl groups, isopropyl groups, methyl groups, C6-C20 straight or branched chain hydrocarbons, saturated or unsaturated hydrocarbons, O-methyl groups, polyether groups, nitro groups, alkynyl groups, etc. In addition, polysubstituted aryl rings are also contemplated. Glutamine analogs that can be used in the present invention include but are not limited to α-hydroxy derivatives, γ-substituted derivatives, cyclic derivatives and amide-substituted glutamine derivatives. Examples of phenylalanine analogs that can be used in the present invention include but are not limited to para-substituted phenylalanine, ortho-substituted phenylalanine and meta-substituted phenylalanine, wherein the substituents include but are not limited to hydroxyl, methoxy, methyl, allyl, aldehyde, azido, iodo, bromo, keto (including but not limited to acetyl), benzyl, alkynyl, etc. Specific examples of unnatural amino acids that can be used in the present invention include, but are not limited to, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-dopa, fluorophenyl The present invention also includes amino acid, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphoserine, phosphostyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine and p-propargyloxy-phenylalanine, etc. Examples of the structures of various unnatural amino acids that can be used in the present invention are provided in, for example, WO 2002/085923 entitled "In vivo incorporation of unnatural amino acids". For other methionine analogs, see also Kiick et al., Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation, PNAS 99:19-24 (2002), which is incorporated herein by reference. International Application No. PCT/US06/47822, entitled "Compositions Containing, Methods Involving, and Uses of Non-natural Amino Acids and Polypeptides," which is incorporated herein by reference, describes 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 the diverse functional groups of biological systems are considered to be important tools in chemical biology. As relative latecomers in the family of synthetic chemistry, these bioorthogonal reactions have inspired new strategies for compound library synthesis, protein engineering, functional proteomics, and chemical remodeling of cell surfaces. Azides have played an important role as unique chemical treatments for bioconjugation. Staudinger ligation has been used with phosphides to label azide sugars that are metabolically introduced into cellular sugar conjugates. The Staudinger ligation can be performed in living animals and is physiologically harmless; however, the Staudinger reaction is not without disadvantages. The necessary phosphines are susceptible to oxidation by air, and optimization for increased water solubility and improved reaction rates has proven synthetically challenging.

Azides have an alternative bioorthogonal mode of reactivity: a [3+2] cycloaddition to alkynes 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 collaborators overcame this obstacle by developing a form of copper(I) catalysis, termed "click chemistry," that is readily performed at physiological temperatures and in a richly functionalized biological environment. This discovery enabled the selective modification of viral particles, nucleic acids, and proteins from complex tissue lysates. Unfortunately, the obligatory copper catalysts are toxic to both bacterial and mammalian cells, thus preventing applications in which cells must remain viable. Uncatalyzed Huisgen cycloadditions of alkynes activated by electron-withdrawing substituents have been reported to occur at ambient temperature. However, these compounds undergo Michael reactions with biological nucleophiles.

In one embodiment, a composition of IL-2 comprising a non-natural amino acid (e.g., p-(propargyloxy)-phenylalanine) is provided. Various different compositions comprising p-(propargyloxy)-phenylalanine are also provided, including but not limited to proteins and/or cells. In one aspect, the composition comprising the p-(propargyloxy)-phenylalanine non-natural amino acid also comprises an orthogonal tRNA. The non-natural amino acid can be bonded (including but not limited to covalently) to the orthogonal tRNA, including but not limited to covalently bonding to the orthogonal tRNA via an amine-acyl bond, covalently bonding to the 3'OH or 2'OH of the terminal ribose of the orthogonal tRNA, etc.

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

The non-natural amino acid incorporated into the polypeptide at the amino terminus can be composed of an R group that is any substituent other than the substituents used in the 20 natural amino acids and a second reactive group different from the NH2 group normally present in α-amino acids (see Formula I). Similar non-natural amino acids can be incorporated at the carboxyl terminus, which has a second reactive group different from the COOH group normally present in α-amino acids (see Formula I).

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

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

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

其中：A是任選的，並且在存在時是低級亞烷基、取代的低級亞烷基、低級亞環烷基、取代的低級亞環烷基、低級亞烯基、取代的低級亞烯基、亞炔基、低級亞雜烷基、取代的亞雜烷基、低級亞雜環烷基、取代的低級亞雜環烷基、亞芳基、取代的亞芳基、亞雜芳基、取代的亞雜芳基、亞烷芳基、取代的亞烷芳基、亞芳烷基或取代的亞芳烷基；B是任選的，並且在存在時是選自低級亞烷基、取代的低級亞烷基、低級亞烯基、取代的低級亞烯基、低級亞雜烷基、取代的低級亞雜烷基、-O-、-O-(亞烷基或取代的亞烷基)-、-S-、-S-(亞烷基或取代的亞烷基)-、-S(O)_k-(其中k 是1、2或3)、-S(O)_k(亞烷基或取代的亞烷基)-、-C(O)-、-C(O)-(亞烷基或取代的亞烷基)-、-C(S)-、-C(S)-(亞烷基或取代的亞烷基)-、-N(R’)-、-NR’-(亞烷基或取代的亞烷基)-、-C(O)N(R’)-、-CON(R’)-(亞烷基或取代的亞烷基)-、-CSN(R’)-、-CSN(R’)-(亞烷基或取代的亞烷基)-、-N(R’)CO-(亞烷基或取代的亞烷基)-、-N(R’)C(O)O-、-S(O)_kN(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O)_kN(R’)-、-N(R’)-N=、-C(R’)=N-、-C(R’)=N-N(R’)-、-C(R’)=N-N=、-C(R’)₂-N=N-和-C(R’)₂-N(R’)-N(R’)-的接頭，其中每個R’獨立地是H、烷基或取代的烷基；J是

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、

或

；R是H、烷基、取代的烷基、環烷基或取代的環烷基；每個R”獨立地是H、烷基、取代的烷基或保護基團，或者當存在超過一個R”基團時，兩個R”任選地形成雜環烷基；R₁是任選的，並且在存在時是H、胺基保護基團、樹脂、胺基酸、多肽或多核苷酸；並且R₂是任選的，並且在存在時是OH、酯保護基團、樹脂、胺基酸、多肽或多核苷酸；每個R₃和R₄獨立地是H、鹵素、低級烷基或取代的低級烷基，或者R₃和R₄或兩個R₃基團任選地形成環烷基或雜環烷基； 或者-A-B-J-R基團一起形成包含至少一個羰基的雙環或三環環烷基或雜環烷基，所述羰基包括二羰基、保護的羰基(包括保護的二羰基)或掩蔽的羰基(包括掩蔽的二羰基)；或者-J-R基團一起形成包含至少一個羰基的單環或雙環環烷基或雜環烷基，所述羰基包括二羰基、保護的羰基(包括保護的二羰基)或掩蔽的羰基(包括掩蔽的二羰基)；前提是當A是亞苯基並且每個R₃是H時，B存在；並且當A是-(CH₂)₄-並且每個R₃是H時，B不是-NHC(O)(CH₂CH₂)-；並且當A和B不存在並且每個R₃是H時，R不是甲基。 Amino acids having electrophilic reactive groups allow the use of a variety of different reactions, especially to link molecules by nucleophilic addition reactions. Such electrophilic reactive groups include carbonyl groups (including keto groups and dicarbonyl groups), carbonyl-like groups (which have similar reactivity to carbonyl groups (including keto groups and dicarbonyl groups) and are structurally similar to carbonyl groups), masked carbonyl groups (which can be easily converted into carbonyl groups (including keto groups and dicarbonyl groups)) or protected carbonyl groups (which have similar reactivity to carbonyl groups (including keto groups and dicarbonyl groups) after deprotection). 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 lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) _- ; -(wherein k is 1, 2 or 3), -S(O) _k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) _kN (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')=NN(R')-, -C(R')=NN=, -C(R') ₂ -N=N-, and -C(R') ₂ -N(R')-N(R')-, wherein each R' is independently H, alkyl or substituted alkyl; J is

,

,

,

,

,

,

or

; 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, two R" optionally form a heterocycloalkyl; _R1 is optional and when present is H, an amine protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and _R2 is optional and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; each _R3 and _R4 is independently H, a halogen, a lower alkyl or a substituted lower alkyl, or _R3 and _R4 or two _R3 groups optionally form a cycloalkyl or a heterocycloalkyl; or -ABJR groups together form a bicyclic or tricyclic cycloalkyl or heterocyclic alkyl group containing at least one carbonyl group, including a dicarbonyl group, a protected carbonyl group (including a protected dicarbonyl group) or a masked carbonyl group (including a masked dicarbonyl group); or -JR groups together form a monocyclic or bicyclic cycloalkyl or heterocyclic alkyl group containing at least one carbonyl group, including a dicarbonyl group, a protected carbonyl group (including a protected dicarbonyl group) or a masked carbonyl group (including a masked dicarbonyl group); provided that when A is phenylene and each R ₃ is H, B is present; and when A is -(CH ₂ ) ₄ - and each R ₃ is H, B is not -NHC(O)(CH ₂ CH ₂ )-; and when A and B are not present and each R ₃ is H, R is not methyl.

其中：A是任選的，並且在存在時是低級亞烷基、取代的低級亞烷基、低級亞環烷基、取代的低級亞環烷基、低級亞烯基、取代的低級亞烯基、亞炔基、低級亞雜烷基、取代的亞雜烷基、低級亞雜環烷基、取代的低級亞雜環烷基、亞芳基、取代的亞芳基、亞雜芳基、取代的亞雜芳基、亞烷芳基、取代的亞烷芳基、亞芳烷基或取代的亞芳烷基；B是任選的，並且在存在時是選自低級亞烷基、取代的低級亞烷基、低級亞烯基、取代的低級亞烯基、低級亞雜烷基、取代的低級亞雜烷基、-O-、-O-(亞烷基或取代的亞烷基)-、-S-、-S-(亞烷基或取代 的亞烷基)-、-S(O)_k-(其中k是1、2或3)、-S(O)_k(亞烷基或取代的亞烷基)-、-C(O)-、-C(O)-(亞烷基或取代的亞烷基)-、-C(S)-、-C(S)-(亞烷基或取代的亞烷基)-、-N(R’)-、-NR’-(亞烷基或取代的亞烷基)-、-C(O)N(R’)-、-CON(R’)-(亞烷基或取代的亞烷基)-、-CSN(R’)-、-CSN(R’)-(亞烷基或取代的亞烷基)-、-N(R’)CO-(亞烷基或取代的亞烷基)-、-N(R’)C(O)O-、-S(O)_kN(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O)_kN(R’)-、-N(R’)-N=、-C(R’)=N-、-C(R’)=N-N(R’)-、-C(R’)=N-N=、-C(R’)₂-N=N-和-C(R’)₂-N(R’)-N(R’)-的接頭，其中每個R’獨立地是H、烷基或取代的烷基；R是H、烷基、取代的烷基、環烷基或取代的環烷基；R₁是任選的，並且在存在時是H、胺基保護基團、樹脂、胺基酸、多肽或多核苷酸；並且R₂是任選的，並且在存在時是OH、酯保護基團、樹脂、胺基酸、多肽或多核苷酸；前提是當A是亞苯基時，B存在；並且當A是-(CH₂)₄-時，B不是-NHC(O)(CH₂CH₂)-；並且當A和B不存在時，R不是甲基。 In addition, amino acids having the structure of formula (V) 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 lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) _- ; -(wherein k is 1, 2 or 3), -S(O) _k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) _kN (R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) _k wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, _{cycloalkyl ,} or substituted cycloalkyl; R1 is optional and, when present, is H, an amine protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and _R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; provided that when A is phenylene, B is present; and when A is -( _CH2 ) _4- , B is not _-NHC (O ₎ ( _CH2CH2 )-; and when A and B are not present, R is not methyl.

其中： B是選自低級亞烷基、取代的低級亞烷基、低級亞烯基、取代的低級亞烯基、低級亞雜烷基、取代的低級亞雜烷基、-O-、-O-(亞烷基或取代的亞烷基)-、-S-、-S-(亞烷基或取代的亞烷基)-、-S(O)_k-(其中k是1、2或3)、-S(O)_k(亞烷基或取代的亞烷基)-、-C(O)-、-C(O)-(亞烷基或取代的亞烷基)-、-C(S)-、-C(S)-(亞烷基或取代的亞烷基)-、-N(R’)-、-NR’-(亞烷基或取代的亞烷基)-、-C(O)N(R’)-、-CON(R’)-(亞烷基或取代的亞烷基)-、-CSN(R’)-、-CSN(R’)-(亞烷基或取代的亞烷基)-、-N(R’)CO-(亞烷基或取代的亞烷基)-、-N(R’)C(O)O-、-S(O)_kN(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O)_kN(R’)-、-N(R’)-N=、-C(R’)=N-、-C(R’)=N-N(R’)-、-C(R’)=N-N=、-C(R’)₂-N=N-和-C(R’)₂-N(R’)-N(R’)-的接頭，其中每個R’獨立地是H、烷基或取代的烷基；R是H、烷基、取代的烷基、環烷基或取代的環烷基；R₁是任選的，並且在存在時是H、胺基保護基團、樹脂、胺基酸、多肽或多核苷酸；並且R₂是任選的，並且在存在時是OH、酯保護基團、樹脂、胺基酸、多肽或多核苷酸；每個R_a獨立地選自H、鹵素、烷基、取代的烷基、-N(R’)₂、-C(O)_kR’(其中k是1、2或3)、-C(O)N(R’)₂、-OR’和-S(O)_kR’，其中每個R’獨立地是H、烷基或取代的烷基。 In addition, amino acids having the structure of formula (VI) are included:

wherein: B is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) _k- (wherein k is 1, 2 or 3), -S(O) _k -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) _kN (R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) _k wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, _{cycloalkyl ,} or substituted cycloalkyl; R1 is optional and, when present, is H, an amine protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and _R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; each Ra _is independently selected from H, halogen, alkyl, substituted alkyl, -N(R') _{2 ,} -C(O) _k R' (where k is 1, 2 or 3), -C(O)N(R') ₂ , -OR' and -S(O) _kR ', where each R' is independently H, alkyl or substituted alkyl.

In addition, the following amino acids are included:

These compounds are optionally amino protected, carboxyl protected or salts thereof. In addition, any of the following non-natural amino acids can be incorporated into non-natural amino acid polypeptides.

其中B是任選的，並且在存在時是選自低級亞烷基、取代的低級亞烷基、低級亞烯基、取代的低級亞烯基、低級亞雜烷基、取代的低級亞雜烷基、-O-、-O-(亞烷基或取代的亞烷基)-、-S-、-S-(亞烷基或取代的亞烷基)-、-S(O)_k-(其中k是1、2或3)、-S(O)_k(亞烷基或取代的亞烷基)-、-C(O)-、-C(O)-(亞烷基或取代的亞烷基)-、-C(S)-、-C(S)-(亞烷基或取代的亞烷基)-、-N(R’)-、-NR’-(亞烷基或取代的亞烷基)-、-C(O)N(R’)-、-CON(R’)-(亞烷基或取代的亞烷基)-、-CSN(R’)-、-CSN(R’)-(亞烷基或取代的亞烷基)-、-N(R’)CO-(亞烷基或取代的亞烷基)-、-N(R’)C(O)O-、 -S(O)_kN(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O)_kN(R’)-、-N(R’)-N=、-C(R’)=N-、-C(R’)=N-N(R’)-、-C(R’)=N-N=、-C(R’)₂-N=N-和-C(R’)₂-N(R’)-N(R’)-的接頭，其中每個R’獨立地是H、烷基或取代的烷基；R是H、烷基、取代的烷基、環烷基或取代的環烷基；R₁是任選的，並且在存在時是H、胺基保護基團、樹脂、胺基酸、多肽或多核苷酸；並且R₂是任選的，並且在存在時是OH、酯保護基團、樹脂、胺基酸、多肽或多核苷酸；每個R_a獨立地選自H、鹵素、烷基、取代的烷基、-N(R’)₂、-C(O)_kR’(其中k是1、2或3)、-C(O)N(R’)₂、-OR’和-S(O)_kR’，其中每個R’獨立地是H、烷基或取代的烷基；並且n是0至8；前提是當A是-(CH₂)₄-時，B不是-NHC(O)(CH₂CH₂)-。 In addition, the following amino acids having the structure of formula (VII) are included:

wherein B is optional and, when present, is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) _k- (wherein k is 1, 2 or 3), -S(O) _k -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) _kN (R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) _k wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, _{cycloalkyl ,} or substituted cycloalkyl; R1 is optional and, when present, is H, an amine protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and _R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; each Ra _is independently selected from H, halogen, alkyl, substituted alkyl, -N(R') _{2 ,} -C(O) _k R' (wherein k is 1, 2 or 3), -C(O)N(R') ₂ , -OR' and -S(O) _kR ', wherein each R' is independently H, alkyl or substituted alkyl; and n is 0 to 8; provided that when A is -( _CH2 ) _4- , B is not -NHC(O)( _{CH2CH2 )} -.

其中這些化合物任選地胺基被保護、任選地羧基被保護、任選地胺基被保護且羧基被保護，或者是其鹽。此外，這些非天然胺基酸和任何下述的非天然胺基酸可以被併入到非天然胺基酸多肽中。 In addition, the following amino acids are included:

Wherein these compounds are optionally protected by amine, optionally protected by carboxyl, optionally protected by amine and protected by carboxyl, or are salts thereof. In addition, these non-natural amino acids and any of the following non-natural amino acids can be incorporated into non-natural amino acid polypeptides.

其中A是任選的，並且在存在時是低級亞烷基、取代的低級亞烷基、低級亞環烷基、取代的低級亞環烷基、低級亞烯基、取代的低級亞烯基、亞炔基、低級亞雜烷基、取代的亞雜烷基、低級亞雜環烷基、取代的低級亞雜環烷基、亞芳基、取代的亞芳基、亞雜芳基、取代的亞雜芳基、亞烷芳基、取代的亞烷芳基、亞芳烷基或取代的亞芳烷基；B是任選的，並且在存在時是選自低級亞烷基、取代的低級亞烷基、低級亞烯基、取代的低級亞烯基、低級亞雜烷基、取代的低級亞雜烷基、-O-、-O-(亞烷基或取代的亞烷基)-、-S-、-S-(亞烷基或取代的亞烷基)-、-S(O)_k-(其中k是1、2或3)、-S(O)_k(亞烷基或取代的亞烷基)-、-C(O)-、-C(O)-(亞烷基或取代的亞烷基)-、-C(S)-、-C(S)-(亞烷基或取代的亞烷基)-、-N(R’)-、-NR’-(亞烷基或取代的亞烷基)-、-C(O)N(R’)-、-CON(R’)-(亞烷基或取代的亞烷基)-、-CSN(R’)-、-CSN(R’)-(亞烷基或取代的亞烷基)-、-N(R’)CO-(亞烷基或取代的亞烷基)-、-N(R’)C(O)O-、-S(O)_kN(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O)_kN(R’)-、-N(R’)-N=、-C(R’)=N-、-C(R’)=N-N(R’)-、-C(R’)=N-N=、-C(R’)₂-N=N- 和-C(R’)₂-N(R’)-N(R’)-的接頭，其中每個R’獨立地是H、烷基或取代的烷基；R₁是任選的，並且在存在時是H、胺基保護基團、樹脂、胺基酸、多肽或多核苷酸；並且R₂是任選的，並且在存在時是OH、酯保護基團、樹脂、胺基酸、多肽或多核苷酸。 In addition, 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 lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) _-; -(wherein k is 1, 2 or 3), -S(O) _k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) _kN (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')=NN(R')-, -C(R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R')-, wherein each R' is independently H, alkyl or substituted alkyl; _R1 is optional and when present is H, an amine protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and _R2 is optional and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide.

B是任選的，並且在存在時是選自低級亞烷基、取代的低級亞烷基、低級亞烯基、取代的低級亞烯基、低級亞雜烷基、取代的低級亞雜烷基、-O-、-O-(亞烷基或取代的亞烷基)-、-S-、-S-(亞烷基或取代的亞烷基)-、-S(O)_k-(其中k是1、2或3)、-S(O)_k(亞烷基或取代的亞烷基)-、-C(O)-、-C(O)-(亞烷基或取代的亞烷基)-、-C(S)-、-C(S)-(亞烷基或取代的亞烷基)-、-N(R’)-、-NR’-(亞烷基或取代的亞烷基)-、-C(O)N(R’)-、-CON(R’)-(亞烷基或取代的亞烷基)-、-CSN(R’)-、-CSN(R’)-(亞烷基或取代的亞烷基)-、-N(R’)CO-(亞烷基或取代的亞烷基)-、-N(R’)C(O)O-、-S(O)_kN(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O)_kN(R’)-、-N(R’)-N=、-C(R’)=N-、-C(R’)=N-N(R’)-、-C(R’)=N-N=、-C(R’)₂-N=N-和-C(R’)₂-N(R’)-N(R’)-的接頭，其中每個R’獨立地是H、烷基或取代的 烷基；R是H、烷基、取代的烷基、環烷基或取代的環烷基；R₁是任選的，並且在存在時是H、胺基保護基團、樹脂、胺基酸、多肽或多核苷酸；並且R₂是任選的，並且在存在時是OH、酯保護基團、樹脂、胺基酸、多肽或多核苷酸；其中每個R_a獨立地選自H、鹵素、烷基、取代的烷基、-N(R’)₂、-C(O)_kR’(其中k是1、2或3)、-C(O)N(R’)₂、-OR’和-S(O)_kR’，其中每個R’獨立地是H、烷基或取代的烷基。 In addition, the following amino acids having the structure of formula (IX) are included:

B is optional and, when present, is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) _k- (wherein k is 1, 2 or 3), -S(O) _k -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) _kN (R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) _k wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, _{cycloalkyl ,} or substituted cycloalkyl; R1 is optional and, when present, is H, an amine protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and _R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; wherein each Ra _is independently selected from H, halogen, alkyl, substituted alkyl, -N(R') _{2 ,} -C(O) _k R' (where k is 1, 2 or 3), -C(O)N(R') ₂ , -OR' and -S(O) _kR ', where each R' is independently H, alkyl or substituted alkyl.

其中這些化合物任選地胺基被保護、任選地羧基被保護、任選地胺基被保護且羧基被保護，或者是其鹽。此外，這些非天然胺基酸和任何下述的非天然胺基酸可以被併入到非天然胺基酸多肽中。 In addition, the following amino acids are included:

Wherein these compounds are optionally protected by amine, optionally protected by carboxyl, optionally protected by amine and protected by carboxyl, or are salts thereof. In addition, these non-natural amino acids and any of the following non-natural amino acids can be incorporated into non-natural amino acid polypeptides.

其中B是任選的，並且在存在時是選自低級亞烷基、取代的低級亞烷基、低級亞烯基、取代的低級亞烯基、低級亞雜烷基、取代的低級亞雜烷基、-O-、-O-(亞烷基或取代的亞烷基)-、-S-、-S-(亞烷基或取代的亞烷基)-、-S(O)_k-(其中k是1、2或3)、-S(O)_k(亞烷基或取代的亞烷基)-、-C(O)-、-C(O)-(亞烷基或取代的亞烷基)-、-C(S)-、-C(S)-(亞烷基或取代的亞烷基)-、-N(R’)-、-NR’-(亞烷基或取代的亞烷基)-、-C(O)N(R’)-、-CON(R’)-(亞烷基或取代的亞烷基)-、-CSN(R’)-、-CSN(R’)-(亞烷基或取代的亞烷基)-、-N(R’)CO-(亞烷基或取代的亞烷基)-、-N(R’)C(O)O-、-S(O)_kN(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O)_kN(R’)-、-N(R’)-N=、-C(R’)=N-、-C(R’)=N-N(R’)-、-C(R’)=N-N=、-C(R’)₂-N=N-和-C(R’)₂-N(R’)-N(R’)-的接頭，其中每個R’獨立地是H、烷基或取代的烷基；R是H、烷基、取代的烷基、環烷基或取代的環烷基；R₁是任選的，並且在存在時是H、胺基保護基團、樹脂、胺基酸、多肽或多核苷酸；並且R₂是任選的，並且在存在時是OH、酯保護基團、樹脂、胺基酸、多肽或多核苷酸；每個R_a獨立地選自H、鹵素、烷基、取代的烷基、-N(R’)₂、-C(O)_kR’(其中k是1、2或3)、-C(O)N(R’)₂、-OR’和-S(O)_kR’，其中每個 R’獨立地是H、烷基或取代的烷基；並且n是0至8。 In addition, the following amino acids having the structure of formula (X) are included:

wherein B is optional and, when present, is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) _k- (wherein k is 1, 2 or 3), -S(O) _k -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) _kN (R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) _k wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, _{cycloalkyl ,} or substituted cycloalkyl; R1 is optional and, when present, is H, an amine protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and _R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; each Ra _is independently selected from H, halogen, alkyl, substituted alkyl, -N(R') _{2 ,} -C(O) _k R' (wherein 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.

其中這些化合物任選地胺基被保護、任選地羧基被保護、任選地胺基被保護且羧基被保護，或者是其鹽。此外，這些非天然胺基酸和任何下述的非天然胺基酸可以被併入到非天然胺基酸多肽中。 In addition, the following amino acids are included:

Wherein these compounds are optionally protected by amine, optionally protected by carboxyl, optionally protected by amine and protected by carboxyl, or are salts thereof. In addition, these non-natural amino acids and any of the following non-natural amino acids can be incorporated into non-natural amino acid polypeptides.

In addition to the monocarbonyl structure, the non-natural amino acids described in the present invention may include groups such as dicarbonyl, dicarbonyl-like, masked dicarbonyl, and protected dicarbonyl.

其中A是任選的，並且在存在時是低級亞烷基、取代的低級亞烷基、低級亞環烷基、取代的低級亞環烷基、低級亞烯基、取代的低級亞烯基、亞炔基、低級亞雜烷基、取代的亞雜烷基、低級亞雜環烷基、取代的低級亞雜環烷基、亞芳基、取代的亞芳基、亞雜芳基、取代的亞雜芳基、亞烷芳基、取代的亞烷芳基、亞芳烷基或取代的亞芳烷基；B是任選的，並且在存在時是選自低級亞烷基、取代的低 級亞烷基、低級亞烯基、取代的低級亞烯基、低級亞雜烷基、取代的低級亞雜烷基、-O-、-O-(亞烷基或取代的亞烷基)-、-S-、-S-(亞烷基或取代的亞烷基)-、-S(O)_k-(其中k是1、2或3)、-S(O)_k(亞烷基或取代的亞烷基)-、-C(O)-、-C(O)-(亞烷基或取代的亞烷基)-、-C(S)-、-C(S)-(亞烷基或取代的亞烷基)-、-N(R’)-、-NR’-(亞烷基或取代的亞烷基)-、-C(O)N(R’)-、-CON(R’)-(亞烷基或取代的亞烷基)-、-CSN(R’)-、-CSN(R’)-(亞烷基或取代的亞烷基)-、-N(R’)CO-(亞烷基或取代的亞烷基)-、-N(R’)C(O)O-、-S(O)_kN(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O)_kN(R’)-、-N(R’)-N=、-C(R’)=N-、-C(R’)=N-N(R’)-、-C(R’)=N-N=、-C(R’)₂-N=N-和-C(R’)₂-N(R’)-N(R’)-的接頭，其中每個R’獨立地是H、烷基或取代的烷基；R是H、烷基、取代的烷基、環烷基或取代的環烷基；R₁是任選的，並且在存在時是H、胺基保護基團、樹脂、胺基酸、多肽或多核苷酸；並且R₂是任選的，並且在存在時是OH、酯保護基團、樹脂、胺基酸、多肽或多核苷酸。 For example, the following amino acids having the structure of formula (XI) 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 lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) _-; -(wherein k is 1, 2 or 3), -S(O) _k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) _kN (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')=NN(R')-, -C(R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R')-, wherein each R' is independently H, alkyl or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; _R1 is optional and when present is H, an amine protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and _R2 is optional and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide.

B是任選的，並且在存在時是選自低級亞烷基、取代的低 級亞烷基、低級亞烯基、取代的低級亞烯基、低級亞雜烷基、取代的低級亞雜烷基、-O-、-O-(亞烷基或取代的亞烷基)-、-S-、-S-(亞烷基或取代的亞烷基)-、-S(O)_k-(其中k是1、2或3)、-S(O)_k(亞烷基或取代的亞烷基)-、-C(O)-、-C(O)-(亞烷基或取代的亞烷基)-、-C(S)-、-C(S)-(亞烷基或取代的亞烷基)-、-N(R’)-、-NR’-(亞烷基或取代的亞烷基)-、-C(O)N(R’)-、-CON(R’)-(亞烷基或取代的亞烷基)-、-CSN(R’)-、-CSN(R’)-(亞烷基或取代的亞烷基)-、-N(R’)CO-(亞烷基或取代的亞烷基)-、-N(R’)C(O)O-、-S(O)_kN(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O)_kN(R’)-、-N(R’)-N=、-C(R’)=N-、-C(R’)=N-N(R’)-、-C(R’)=N-N=、-C(R’)₂-N=N-和-C(R’)₂-N(R’)-N(R’)-的接頭，其中每個R’獨立地是H、烷基或取代的烷基；R是H、烷基、取代的烷基、環烷基或取代的環烷基；R₁是任選的，並且在存在時是H、胺基保護基團、樹脂、胺基酸、多肽或多核苷酸；並且R₂是任選的，並且在存在時是OH、酯保護基團、樹脂、胺基酸、多肽或多核苷酸；其中每個R_a獨立地選自H、鹵素、烷基、取代的烷基、-N(R’)₂、-C(O)_kR’(其中k是1、2或3)、-C(O)N(R’)₂、-OR’和-S(O)_kR’，其中每個R’獨立地是H、烷基或取代的烷基。 In addition, the following amino acids having the structure of formula (XII) are included:

B is optional and, when present, is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) _k- (wherein k is 1, 2 or 3), -S(O) _k -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) _kN (R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) _k wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, _{cycloalkyl ,} or substituted cycloalkyl; R1 is optional and, when present, is H, an amine protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and _R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; wherein each Ra _is independently selected from H, halogen, alkyl, substituted alkyl, -N(R') _{2 ,} -C(O) _k R' (where k is 1, 2 or 3), -C(O)N(R') ₂ , -OR' and -S(O) _kR ', where each R' is independently H, alkyl or substituted alkyl.

其中這些化合物任選地胺基被保護、任選地羧基被保護、任選地胺基被保護且羧基被保護，或者是其鹽。此外，這些非天然胺基酸和任何下述的非天然胺基酸可以被併入到非天然胺基酸多肽中。 In addition, the following amino acids are included:

Wherein these compounds are optionally protected by amine, optionally protected by carboxyl, optionally protected by amine and protected by carboxyl, or are salts thereof. In addition, these non-natural amino acids and any of the following non-natural amino acids can be incorporated into non-natural amino acid polypeptides.

其中B是任選的，並且在存在時是選自低級亞烷基、取代的低級亞烷基、低級亞烯基、取代的低級亞烯基、低級亞雜烷基、取代的低級亞雜烷基、-O-、-O-(亞烷基或取代的亞烷基)-、-S-、-S-(亞烷基或取代的亞烷基)-、-S(O)_k-(其中k是1、2或3)、-S(O)_k(亞烷基或取代的亞烷基)-、-C(O)-、-C(O)-(亞烷基或取代的亞烷基)-、-C(S)-、-C(S)-(亞烷基或取代的亞烷基)-、-N(R’)-、-NR’-(亞烷基或取代的亞烷基)-、-C(O)N(R’)-、-CON(R’)-(亞烷基或取代的亞烷基)-、-CSN(R’)-、-CSN(R’)-(亞烷基或取代的亞烷基)-、-N(R’)CO-(亞烷基或取代的亞烷基)-、-N(R’)C(O)O-、-S(O)_kN(R’)-、-N(R’)C(O)N(R’)-、-N(R’)C(S)N(R’)-、-N(R’)S(O)_kN(R’)-、-N(R’)-N=、-C(R’)=N-、-C(R’)=N-N(R’)-、-C(R’)=N-N=、-C(R’)₂-N=N-和-C(R’)₂-N(R’)-N(R’)-的接頭，其中每個R’獨立地是H、烷基或取代的烷基； R是H、烷基、取代的烷基、環烷基或取代的環烷基；R₁是任選的，並且在存在時是H、胺基保護基團、樹脂、胺基酸、多肽或多核苷酸；並且R₂是任選的，並且在存在時是OH、酯保護基團、樹脂、胺基酸、多肽或多核苷酸；每個R_a獨立地選自H、鹵素、烷基、取代的烷基、-N(R’)₂、-C(O)_kR’(其中k是1、2或3)、-C(O)N(R’)₂、-OR’和-S(O)_kR’，其中每個R’獨立地是H、烷基或取代的烷基；並且n是0至8。 In addition, the following amino acids having the structure of formula (XIII) are included:

wherein B is optional and, when present, is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) _k- (wherein k is 1, 2 or 3), -S(O) _k -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) _kN (R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) _k wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, _{cycloalkyl ,} or substituted cycloalkyl; R1 is optional and, when present, is H, an amine protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and _R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; each Ra _is independently selected from H, halogen, alkyl, substituted alkyl, -N(R') _{2 ,} -C(O) _k R' (wherein 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.

其中這些化合物任選地胺基被保護、任選地羧基被保護、任選地胺基被保護且羧基被保護，或者是其鹽。此外，這些非天然胺基酸和任何下述的非天然胺基酸可以被併入到非天然胺基酸多肽中。 In addition, the following amino acids are included:

Wherein these compounds are optionally protected by amine, optionally protected by carboxyl, optionally protected by amine and protected by carboxyl, or are salts thereof. In addition, these non-natural amino acids and any of the following non-natural amino acids can be incorporated into non-natural amino acid polypeptides.

其中：A是任選的，並且在存在時是低級亞烷基、取代的低級亞烷基、低級亞環烷基、取代的低級亞環烷基、低級亞烯基、取代的低級亞烯基、亞炔基、低級亞雜烷基、取代的亞雜烷基、低級亞雜環烷基、取代的低級亞雜環烷基、亞芳基、取代的亞芳基、亞雜芳基、取代的亞雜芳基、亞烷芳基、取代的亞烷芳基、亞芳烷基或取代的亞芳烷基；R是H、烷基、取代的烷基、環烷基或取代的環烷基；R₁是任選的，並且在存在時是H、胺基保護基團、樹脂、胺基酸、多肽或多核苷酸；並且R₂是任選的，並且在存在時是OH、酯保護基團、樹脂、胺基酸、多肽或多核苷酸；X₁是C、S或S(O)；並且L是亞烷基、取代的亞烷基、N(R’)(亞烷基)或N(R’)(取代的亞烷基)，其中R’是H、烷基、取代的烷基、環烷基或取代的環烷基。 In addition, 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, cycloalkylene, or substituted cycloalkylene; R is optional and, when present, is H, an amine protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and _R is optional and, when present, is _OH , an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; X _R 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.

In addition, the following amino acids having the structure of formula (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 is optional and, when present, is H, an amine protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and _{R R} 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.

In addition, 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 is optional and, when present, is H, an amine protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and _{R R} 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.

其中：A是任選的，並且在存在時是低級亞烷基、取代的低級亞烷基、低級亞環烷基、取代的低級亞環烷基、低級亞烯基、取代的低級亞烯基、亞炔基、低級亞雜烷基、取代的亞雜烷基、低級亞雜環烷基、取代的低級亞雜環烷基、亞芳基、取代的亞芳基、亞雜芳基、取代的亞雜芳基、亞烷芳基、取代的亞烷芳基、亞芳烷基或取代的亞芳烷基；R是H、烷基、取代的烷基、環烷基或取代的環烷基；R₁是任選的，並且在存在時是H、胺基保護基團、樹脂、胺基酸、多肽或多核苷酸；並且R₂是任選的，並且在存在時是OH、酯保護基團、樹脂、胺基酸、多肽或多核苷酸；X₁是C、S或S(O)；並且n是0、1、2、3、4或5；並且每個CR⁸R⁹基團上的每個R⁸和R⁹獨立地選自H、烷氧基、烷基胺、鹵素、烷基、芳基，或者任何R⁸和R⁹可以一起形成=O或環烷基，或者任何相鄰的R⁸基團可以一起形成環烷基。 In addition, 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, cycloalkylene, or substituted cycloalkylene; R is optional and, when present, is H, an amine protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and _R 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 R ⁸ and R ⁹ on each CR ⁸ R ⁹ group is independently selected from H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R ⁸ and R ⁹ can be together to form =0 or cycloalkyl, or any adjacent R ⁸ groups can be together to form a cycloalkyl.

In addition, the following amino acids having the structure of formula (XV-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 is optional and, when present, is H, an amine protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and _{R 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 R ⁸ and R ⁹ on each CR ⁸ R ⁹ group is independently selected from H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R ⁸ and R ⁹ can be taken together to form =0 or a cycloalkyl, or any adjacent R ⁸ groups can be taken together to form a cycloalkyl.

In addition, the following amino acids having the structure of formula (XV-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 is optional and, when present, is H, an amine protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and _{R 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 R ⁸ and R ⁹ on each CR ⁸ R ⁹ group is independently selected from H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R ⁸ and R ⁹ can be taken together to form =0 or a cycloalkyl, or any adjacent R ⁸ groups can be taken together to form a cycloalkyl.

其中：A是任選的，並且在存在時是低級亞烷基、取代的低級亞烷基、低級亞環烷基、取代的低級亞環烷基、低級亞烯基、取代的低級亞烯基、亞炔基、低級亞雜烷基、取代的亞雜烷基、低級亞雜環烷基、取代的低級亞雜環烷基、亞芳基、取代的亞芳基、亞雜芳基、取代的亞雜芳基、亞烷芳基、取代的亞烷芳基、亞芳烷基或取代的亞芳烷基；R是H、烷基、取代的烷基、環烷基或取代的環烷基；R₁是任選的，並且在存在時是H、胺基保護基團、樹脂、胺基酸、多肽或多核苷酸；並且R₂是任選的，並且在存在時是OH、酯保護基團、樹脂、 胺基酸、多肽或多核苷酸；X₁是C、S或S(O)；並且L是亞烷基、取代的亞烷基、N(R’)(亞烷基)或N(R’)(取代的亞烷基)，其中R’是H、烷基、取代的烷基、環烷基或取代的環烷基。 In addition, the following amino acids having the structure of formula (XVI) 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, cycloalkylene, or substituted cycloalkylene; R is optional and, when present, is H, an amine protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and _R is optional and, when present, is _OH , an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; X _R 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.

In addition, 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 is optional and, when present, is H, an amine protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and _{R R} 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.

In addition, 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 is optional and, when present, is H, an amine protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and _{R R} 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.

其中：A是任選的，並且在存在時是低級亞烷基、取代的低級亞 烷基、低級亞環烷基、取代的低級亞環烷基、低級亞烯基、取代的低級亞烯基、亞炔基、低級亞雜烷基、取代的亞雜烷基、低級亞雜環烷基、取代的低級亞雜環烷基、亞芳基、取代的亞芳基、亞雜芳基、取代的亞雜芳基、亞烷芳基、取代的亞烷芳基、亞芳烷基或取代的亞芳烷基；M是-C(R₃)-、

、

、

、

、

、

、

或

，其中(a)指示鍵合到A基團，並且(b)指示鍵合到相應的羰基，R₃和R₄獨立地選自H、鹵素、烷基、取代的烷基、環烷基或取代的環烷基，或者R₃和R₄或兩個R₃基團或兩個R₄基團任選地形成環烷基或雜環烷基；R是H、鹵素、烷基、取代的烷基、環烷基或取代的環烷基；T₃是鍵、C(R)(R)、O或S，並且R是H、鹵素、烷基、取代的烷基、環烷基或取代的環烷基；R₁是任選的，並且在存在時是H、胺基保護基團、樹脂、胺基酸、多肽或多核苷酸；並且R₂是任選的，並且在存在時是OH、酯保護基團、樹脂、胺基酸、多肽或多核苷酸。 In addition, amino acids having the structure of formula (XVII) 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; M is -C(R ₃ )-,

,

,

,

,

,

,

or

, wherein (a) indicates bonding to the A group, and (b) indicates bonding to the corresponding carbonyl group, R ₃ and R ₄ are independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl, or R ₃ and R ₄ or two R ₃ groups or two R ₄ groups optionally form a cycloalkyl or heterocycloalkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; T 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 amine protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and R ₂ is optional, and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide.

其中：M是-C(R₃)-、

、

、

、

、

、

、

或

，(a)(a)指示鍵合到A基團，並且(b)指示鍵合到相應的羰基，R₃和R₄獨立地選自H、鹵素、烷基、取代的烷基、環烷基或取代的環烷基，或者R₃和R₄或兩個R₃基團或兩個R₄基團任選地形成環烷基或雜環烷基；R是H、鹵素、烷基、取代的烷基、環烷基或取代的環烷基；T₃是鍵、C(R)(R)、O或S，並且R是H、鹵素、烷基、取代的烷基、環烷基或取代的環烷基；R₁是任選的，並且在存在時是H、胺基保護基團、樹脂、胺基酸、多肽或多核苷酸；並且R₂是任選的，並且在存在時是OH、酯保護基團、樹脂、胺基酸、多肽或多核苷酸；每個R_a獨立地選自H、鹵素、烷基、取代的烷基、-N(R’)₂、-C(O)_kR’(其中k是1、2或3)、-C(O)N(R’)₂、-OR’和-S(O)_kR’，其中每個R’獨立地是H、烷基或取代的烷基。 In addition, amino acids having the structure of formula (XVIII) are included:

Wherein: M is -C(R ₃ )-,

,

,

,

,

,

,

or

, (a)(a) indicates bonding to the A group, and (b) indicates bonding to the corresponding carbonyl group, R ₃ and R ₄ are independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl, or R ₃ and R ₄ or two R ₃ groups or two R ₄ groups optionally form a cycloalkyl or heterocycloalkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; T 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 amine protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and R _R' is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; each R _{' is} independently selected from H, halogen, alkyl, substituted alkyl, -N(R') ₂ , -C(O) _kR ' (wherein k is 1, 2 or 3), -C(O)N(R') ₂ , -OR' and -S(O) _kR ', wherein each R' is independently H, alkyl or substituted alkyl.

其中： R是H、鹵素、烷基、取代的烷基、環烷基或取代的環烷基；並且T₃是O或S。 In addition, amino acids having the structure of formula (XIX) are included:

wherein: R is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; and T ₃ is O or S.

其中：R是H、鹵素、烷基、取代的烷基、環烷基或取代的環烷基。 In addition, amino acids having the structure of formula (XX) are included:

Wherein: R is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.

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

In certain embodiments, a polypeptide comprising a non-natural amino acid is chemically modified to generate a reactive carbonyl or dicarbonyl functional group. For example, an aldehyde functional group that can be used for a conjugated reaction can be generated from a functional group having adjacent amine and hydroxyl groups. For example, where the biologically active molecule is a polypeptide, an 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 functional group using a periodate salt under mild oxidative cleavage conditions. See, for example, Gaertner et al., Bioconjug. Chem. 3: 262-268 (1992); Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3: 138-146 (1992); Gaertner et al., J. Biol. Chem. 269: 7224-7230 (1994). However, methods known in the art are limited to the amino acids at the N-terminus of peptides or proteins.

In the present invention, non-natural amino acids with adjacent hydroxyl and amine groups can be incorporated into polypeptides as "masked" aldehyde functional groups. For example, 5-hydroxylysine has a hydroxyl group adjacent to the epsilon amine group. Reaction conditions for generating aldehydes typically include adding a molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other positions within the polypeptide. The pH of the oxidation reaction is typically about 7.0. A typical reaction includes adding about 1.5 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.

Carbonyl or dicarbonyl functional groups can react selectively with hydroxylamine-containing reagents under mild conditions in aqueous solution to form corresponding oxime bonds that are stable under physiological conditions. See, for example, 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 carbonyl or dicarbonyl groups allows for selective modification in the presence of other amino acid side chains. See, for example, Cornish, V.W. et al., J.Am.Chem.Soc.118:8150-8151 (1996); Geoghegan, K.F. & Stroh, J.G., Bioconjug.Chem.3:138-146 (1992); Mahal, L.K. et al., Science 276:1125-1128 (1997).

A. Carbonyl reactive groups

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

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

wherein n is 0-10; _R1 is alkyl, aryl, substituted alkyl, or substituted aryl; _R2 is H, alkyl, aryl, substituted alkyl, and substituted aryl; and _R3 is H, an amino acid, a polypeptide, or an amino-terminal modification group, and _R4 is H, an amino acid, a polypeptide, or a carboxyl-terminal modification group. In certain embodiments, n is 1, _R1 is phenyl and _R2 is a simple alkyl (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, _R1 is phenyl and _R2 is a simple alkyl (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 p-acetyl-(+/-)-phenylalanine and m-acetyl-(+/-)-phenylalanine is described in Zhang, Z. et al., Biochemistry 42: 6735-6746 (2003), which is incorporated herein by reference. Other carbonyl-containing amino acids can be similarly prepared by one of ordinary skill in the art.

In certain embodiments, a polypeptide comprising a non-naturally encoded amino acid is chemically modified to generate a reactive carbonyl functional group. For example, an aldehyde functional group for a conjugated reaction can be generated from a functional group having adjacent amine and hydroxyl groups. For example, where the biologically active molecule is a polypeptide, an 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 functional group using a periodate salt under mild oxidative cleavage conditions. See, e.g., Gaertner et al., Bioconjug. Chem. 3: 262-268 (1992); Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3: 138-146 (1992); Gaertner et al., J. Biol. Chem. 269: 7224-7230 (1994). However, methods known in the art are limited to 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 amine groups can be incorporated into polypeptides as "masked" aldehyde functional groups. For example, 5-hydroxylysine has a hydroxyl group adjacent to the epsilon amine group. Reaction conditions for generating aldehydes typically include adding a molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other positions within the polypeptide. The pH of the oxidation reaction is typically about 7.0. A typical reaction includes adding about 1.5 times molar excess of sodium metaperiodate to a buffered solution of the polypeptide, followed by incubation in the dark for about 10 minutes. See, for example, U.S. Patent No. 6,423,685, which is incorporated herein by reference.

The carbonyl functional group can react selectively with reagents containing hydrazine, hydrazide, hydroxylamine or semicarbazide under mild conditions in aqueous solution to form the corresponding hydrazone, oxime or semicarbazone bonds, respectively, which are stable under physiological conditions. See, for example, Jencks, 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, for example, Cornish, V.W. et al., J.Am.Chem.Soc.118:8150-8151 (1996); Geoghegan, K.F. & Stroh, J.G., Bioconjug.Chem.3:138-146 (1992); Mahal, L.K. et al., Science 276:1125-1128 (1997).

B. Hydrazine, hydrazide or semicarbazide reactive groups

Non-naturally encoded amino acids containing nucleophilic groups such as hydrazine, hydrazide or aminourea 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 0-10; _R1 is alkyl, aryl, substituted alkyl or substituted aryl or is absent; X is O, N or S or is absent; _R2 is H, an amino acid, a polypeptide or an amino terminal modifying group, and _R3 is H, an amino acid, a polypeptide or a carboxyl terminal modifying group.

In certain embodiments, n is 4, _R1 is absent, and X is N. In certain embodiments, n is 2, _R1 is absent, and X is absent. In certain embodiments, n is 1, _R1 is phenyl, X is O, and the oxygen atom is located in the para position to the aliphatic group on the aryl ring.

Amino acids containing hydrazides, hydrazides, and aminoureas can be obtained from commercial sources. For example, L-glutamine-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 aminourea 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 hydrazide, hydrazine, and aminourea functional groups makes them significantly more reactive toward aldehydes, ketones, and other electrophilic groups compared to nucleophilic groups present on the 20 common amino acids (including but not limited to the hydroxyl or lysine of serine or threonine and the amine group at the N-terminus).

C. Aminooxy-containing amino acids

Non-naturally encoded amino acids containing an amineoxy (also known as a hydroxylamine) group allow reaction with a variety of different electrophilic groups to form conjugates (including but not limited to conjugates with PEG or other water-soluble polymers). Similar to hydrazines, hydrazides, and aminoureas, the high nucleophilicity of the amineoxy 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). Although the result of the reaction with the hydrazine group is the corresponding hydrazone, the reaction of the amineoxy group with a carbonyl-containing group such as a ketone generally results in an oxime.

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

wherein n is 0-10; _R1 is alkyl, aryl, substituted alkyl or substituted aryl or absent; X is O, N, S or absent; m is 0-10; Y=C(O) or absent; _R2 is H, an amino acid, a polypeptide or an amino-terminal modifying group, and _R3 is H, an amino acid, a polypeptide or a carboxyl-terminal modifying group. In certain embodiments, n is 1, _R1 is phenyl, X is O, m is 1, and Y is present. In certain embodiments, n is 2, _R1 and X are absent, m is 0, and Y is absent.

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

D. Azide and Alkyne Reactive Groups

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

Since the Huisgen cycloaddition reaction involves a selective cycloaddition reaction (see, e.g., Padwa, A., Comprehensive Organic Synthesis, Vol. 4, Trost, BM, ed. (1991), p. 1069-1109; Huisgen, R., 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, Padwa, A., ed. (1984), p. 1-176) rather than a nucleophilic substitution, the incorporation of non-naturally encoded amino acids with azide- and alkynyl side chains allows the resulting polypeptide to be selectively modified at the position of the non-naturally encoded amino acid. Cycloaddition reactions involving halogen- or alkynyl-containing IL-2s can be carried out in situ at room temperature and under aqueous conditions by adding a catalytic amount of Cu(II) (including but not limited to in the form of a catalytic amount of CuSO ₄ ) in the presence of a reducing agent for reducing Cu(II) to Cu(I). 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 applied electric potential.

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

The azido functional group can also react selectively with a water-soluble polymer containing an aryl ester and suitably functionalized with an aryl phosphine moiety to produce an amide bond. The aryl phosphine group reduces the azido in situ, and the resulting amine then reacts efficiently with the adjacent ester bond 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 azido (including but not limited to 2-amino-6-azido-1-hexanoic acid) or an aryl azido (p-azido-phenylalanine).

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

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 refers to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (including but not limited to aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy, or aralkyl. When a compound of the present invention includes more than one R group, for example, each of the R groups is independently selected, as is each of these groups when there is more than one R', R", R''' and R'''' groups. When R' and R" are attached to the same nitrogen atom, they can combine with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the discussion of substituents above, one skilled in the art will understand that the term "alkyl" is meant to include groups containing carbon atoms bonded to groups other than hydrogen groups, such as halogenated alkyl groups (including, but not limited to, _-CF3 and _-CH2CF3 ) and acyl groups (including, but not limited to, _-C (O) _CH3 , -C(O) _CF3 , -C(O) _{CH2OCH3 ,} etc.).

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

wherein n is 1-10; X can 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 0-10; _R1 is alkyl, aryl, substituted alkyl or substituted aryl or is absent; X is O, N, S or is absent; m is 0-10, _R2 is H, an amino acid, a polypeptide or an amino terminal modifying group, and _R3 is H, an amino acid, a polypeptide or a carboxyl terminal modifying group. In certain embodiments, n is 1, _R1 is phenyl, X is absent, m is 0 and the acetylene moiety is located in the para position relative to the alkyl side chain. In certain embodiments, n is 1, _R1 is phenyl, X is O, m is 1 and the propargyloxy group is located in the para position relative to the alkyl side chain (i.e., O-propargyl-tyrosine). In certain embodiments, n is 1, _R1 and X are absent and m is 0 (i.e., propargylglycine).

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

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

wherein n is 0-10; _R1 is alkyl, aryl, substituted alkyl, substituted aryl or absent; X is O, N, S or absent; m is 0-10; _R2 is H, an amino acid, a polypeptide or an amino terminal modifying group, and _R3 is H, an amino acid, a polypeptide or a carboxy terminal modifying group. In certain embodiments, n is 1, _R1 is phenyl, X is absent, m is 0 and the azido moiety is located in the para position relative to the alkyl side chain. In certain embodiments, n is 0-4 and _R1 and X are absent, and m=0. In certain embodiments, n is 1, _R1 is phenyl, X is O, m is 2 and the β-azidoethoxy moiety is located in the para position relative to the alkyl side chain.

The azido amino acids can be obtained from commercial sources. For example, 4-azidophenylalanine can be obtained from Chem-Impex International, Inc. (Wood Dale, IL). For those azido 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 a suitably protected lactone. See, for example, March's Advanced Organic Chemistry (3rd edition, 1985, Wiley and Sons, New York).

E. Aminothiol Reactive Group

The unique reactivity of β-substituted aminothiol functional groups makes them extremely useful for the selective modification of polypeptides and other biomolecules containing aldehyde groups 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, β-substituted aminothiol amino acids can be incorporated into IL-2 polypeptides and then reacted with water-soluble polymers containing aldehyde functional groups. In certain embodiments, water-soluble polymers, drug conjugates or other effective loads can be conjugated to IL-2 containing β-substituted aminothiol amino acids through the formation of thiazolidines.

F. Additional Reactive Groups

Additional reactive groups and non-naturally encoded amino acids (including but not limited to p-amino-phenylalanine) that can be incorporated into the IL-2 polypeptides of the 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 No. 60/755,018; International Patent Application No. PCT/US06/49397; WO 2006/069246; U.S. Provisional Patent No. 60/743,041; U.S. Provisional Patent No. 60/743,040; International Patent Application No. PCT/US06/47822; U.S. Provisional Patent No. 60/882,819; U.S. Provisional Patent No. 60/882,500; and U.S. Provisional Patent No. 60/870,594. These applications also discuss reactive groups that may be present on PEG or other polymers for conjugation, including but not limited to hydroxylamine (amino) groups.

Peptides with unnatural amino acids

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

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

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

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

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

IV. In vivo production of IL-2 containing non-naturally encoded amino acids

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

Methods for producing tRNA and tRNA synthetases using amino acids that are not encoded in naturally occurring systems are described in, for example, U.S. Patent Nos. 7,045,337 and 7,083,970, which are incorporated herein by reference. These methods relate to the production of a translation machine that does not rely on synthetases and tRNAs endogenous to the translation system to function (and is therefore sometimes referred to as "orthogonal") . Typically, the translation system comprises an orthogonal tRNA (O-tRNA) and an orthogonal amidoyl tRNA synthetase (O-RS). Typically, the O-RS preferentially amines the O-tRNA with at least one non-naturally occurring amino acid in the translation system, and the O-tRNA recognizes at least one selector codon in the system that is not recognized by other tRNAs. Thus, the translation system responds to the encoded selector codon by inserting the non-naturally encoded amino acid into a protein produced in the system, thereby "substituting" the amino acid into a position in the encoded polypeptide.

A wide variety of orthogonal tRNAs and amidoyl tRNA synthetases for inserting specific synthetic amino acids into polypeptides have been described in the art and are generally suitable for use in the present invention. For example, keto-specific O-tRNA/amidoyl-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-RS or portions thereof are encoded by polynucleotide sequences and include amino acid sequences disclosed in U.S. Patent Nos. 7,045,337 and 7,083,970, each of which is incorporated herein by reference. Corresponding O-tRNA molecules for use with the O-RS are also described in U.S. Patent Nos. 7,045,337 and 7,083,970, which are incorporated herein by reference. Additional examples of O-tRNA/amidyl-tRNA synthetase pairs are described in WO 2005/007870, WO 2005/007624, and WO 2005/019415.

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

Several other orthogonal pairs have been reported. Glutamine acyl groups from tRNAs and synthetases of Saccharomyces cerevisiae (see, e.g., Liu, D. R. and Schultz, P. G. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:4780-4785), aspartate acyl groups (see, e.g., Pastrnak, M. et al., (2000) Helv. Chim. Acta 83:2277-2286) and tyrosyl (see, e.g., Ohno, S. et al., (1998) J. Biochem. (Tokyo, Jpn.) 124:1065-1068; and Kowal, A.K. et al., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2268-2273) systems have been described for the potential incorporation of unnatural amino acids in E. coli. Systems derived from E. coli glutamic acid acyl (see, e.g., Kowal, A.K., et al., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2268-2273) and tyrosinyl (see, e.g., Edwards, H. and Schimmel, P. (1990) Mol. Cell. Biol. 10:1633-1641) synthetases have been described for use in brewing yeast. The E. coli tyrosinyl 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/amidyl-tRNA synthetases involves the selection of specific codons (selector codons) encoding non-naturally encoded amino acids. Although any codon can be used, it is generally desirable to select codons that are rarely or never used in cells expressing the O-tRNA/amidyl-tRNA synthetase. For example, exemplary codons include nonsense codons such as stop codons (amber, ochre, and ovalite), four or more basic codons, and other rare or unused natural tribasic 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, etc.).

V. Location of non-naturally occurring amino acids in IL-2

The present invention contemplates incorporating one or more non-naturally occurring amino acids into IL-2. One or more non-naturally occurring amino acids can be incorporated at specific positions that do not disrupt the activity of the polypeptide. This can be achieved by making "conservative" substitutions, including but not limited to replacing hydrophobic amino acids with hydrophobic amino acids, replacing bulky amino acids with bulky amino acids, replacing hydrophilic amino acids with hydrophilic amino acids, and/or inserting the non-naturally occurring amino acids into positions that are not required for activity.

A variety of different biochemical and structural methods can be used to select the desired site for the replacement of the non-naturally encoded amino acid in the IL-2. It is obvious to those of ordinary skill in the art that any position of the polypeptide chain is suitable for selection to incorporate the non-naturally encoded amino acid, and the selection can be based on rational design or by random selection, for any or no specific desired purpose. Selection of a desired site can be used to generate an IL-2 molecule with any desired property or activity, including but not limited to modulating receptor binding or binding to one or more subunits of its receptor, agonists, superagonists, inverse agonists, antagonists, receptor binding modulators, receptor activity modulators, dimer or multimer formation, without changing the activity or properties relative to the native molecule, or manipulating any physical or chemical property of the polypeptide such as solubility, aggregation or stability. For example, the position of the polypeptide required for the biological activity of IL-2 can be identified using point mutation analysis, alanine scanning, saturation induction and biological activity screening or homology scanning methods known in the art. Other methods can be used to identify modified residues for IL-2, including but not limited to sequence analysis (Bowie and Eisenberg, Science 253(5016): 164-70, (1991)), rotamer library selection (Dahiyat and Mayo, Protein Sci 5(5): 895-903 (1996); Dahiyat and Mayo, Science 278(5335): 82-7 (1997); Desjarlais and Handel, Protein Science 4: 2006-2018 (1995); Harbury et al., PNAS USA 92(18): 8408-8412 (1995); Kono et al., Proteins: Structure, Function and Genetics 19:244-255 (1994); Hellinga and Richards, PNAS USA 91:5803-5807 (1994)) and residue pairing potential (Jones, Protein Science 3:567-574 (1994)) and rational design using Protein Design ^Automation® technology (see U.S. Patent Nos. 6,188,965, 6,269,312, 6,403,312, WO98/47089, which are incorporated herein by reference). Residues other than those identified as critical for biological activity by alanine or homology scanning induced mutagenesis may be good candidates for substitution with non-naturally encoded amino acids, depending on the desired activity sought for the polypeptide. Alternatively, positions identified as being critical for biological activity may also be good candidates for substitutions using 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 effects on the activity of the polypeptide. It will be apparent to one of ordinary skill in the art that any means, techniques or methods for selecting positions for substitutions using non-natural amino acids in any polypeptide are suitable for use in the present invention.

The structure and activity of IL-2 polypeptide mutants containing deletions can also be examined to determine regions of the protein that may be tolerant to substitution with non-naturally encoded amino acids. In a similar manner, protease digestion and single-copy (clone) antibodies can be used to identify regions of IL-2 responsible for binding to the IL-2 receptor. Once residues that may be intolerant to substitution with non-naturally encoded amino acids have been eliminated, the effect of the proposed substitution at each remaining position can be examined. Thus, one of ordinary skill in the art can readily identify amino acid positions that can be substituted with non-naturally encoded amino acids.

One of ordinary skill in the art recognizes that analysis of such IL-2 is able to determine which amino acid residues are surface exposed compared to those buried within the tertiary structure of the protein. Thus, embodiments of the present invention are to replace amino acids that are surface exposed residues with non-naturally encoded amino acids.

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

In certain embodiments, one or more non-naturally encoded amino acids are incorporated into any position in one or more of the following regions corresponding to the secondary structure or specific amino acids in IL-2 or its variants as described below: at a position of hydrophobic interaction; at or near a position of interaction with an IL-2 receptor subunit (including IL2Rα); within the 3rd or 35 to 45th amino acid position; within the first 107 N-terminal amino acids; within the 61st-72nd amino acid position; each of the positions is a position in SEQ ID NO: 2 or a 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 at one or more of the following positions: before position 1 (i.e., at the N-terminus), positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, and any combination thereof; or the corresponding amino acids in SEQ ID NO: 3, 5, or 7. In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof at one or more of the following positions: before position 1 (i.e., at the N-terminus), positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, and any combination thereof; or the corresponding amino acids in SEQ ID NO: 3, 5, or 7. 44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or added to the carboxyl terminus of the protein, and any combination thereof; or the corresponding amino acids in SEQ ID NO: 3, 5 or 7.

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

A wide variety of non-naturally encoded amino acids can be substituted or incorporated into a given position in IL-2. Typically, the specific non-naturally encoded amino acid for incorporation is selected based on inspection of the three-dimensional crystal structure of the IL-2 polypeptide or other IL-2 family member with its receptor, preference for conservative substitutions (i.e., substitution of Phe, Tyr, or Trp for an aryl-based non-naturally encoded amino acid such as p-acetylphenylalanine or O-propargyltyrosine), and the specific conjugation chemistry one wishes to introduce into the IL-2 (e.g., introduction of 4-azidophenylalanine if one wishes to effect a Huisgen [3+2] cycloaddition with a water-soluble polymer bearing an alkynyl moiety or 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 non-natural amino acid into the protein, wherein the non-natural amino acid comprises a first reactive group; and contacting the protein with a molecule comprising a second reactive group (the molecule includes but is not limited to a label, a dye, a polymer, a water-soluble polymer, a derivative of polyethylene glycol, a photocrosslinker, a radionuclide, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound, a resin, a second protein or a polypeptide or polypeptide analog, an antibody or an antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, an antisense polynucleotide, a sugar, a water-soluble dendrimer, a cyclodextrin, an inhibitory RNA, a biomaterial, a nanoparticle, a spin Labels, fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or non-covalently interact with other molecules, photoclavable moieties, actinic radiation excitable moieties, photoisomerizable moieties, biotin, biotin derivatives, biotin analogs, heavy atom-incorporated moieties, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox active agents , amino thioacids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chemiluminescent groups, electron dense groups, magnetic groups, intercalating groups, chromophores, energy transfer reagents, biologically active agents, detectable markers, small molecules, quantum dots, nanoemitters, radionucleotides, radioactive emitters, neutron capture agents, or any combination of the above molecules or any other desired compounds or substances). 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 alkynyl or azido moiety, and the second reactive group is an azido or alkynyl moiety. For example, the first reactive group is an alkynyl moiety (including but not limited to the unnatural amino acid p-propargyloxyphenylalanine), and the second reactive group is an azido moiety. In another example, the first reactive group is an azido moiety (including but not limited to the unnatural amino acid p-azido-L-phenylalanine), and the second reactive group is an alkynyl moiety.

In some cases, 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 some cases, the other additions, substitutions or deletions can improve the stability of the IL-2 (including but not limited to resistance to proteolytic degradation) or increase the affinity of the IL-2 for its receptor. In some cases, the other additions, substitutions or deletions can improve the pharmaceutical stability of the IL-2. In some cases, the other additions, substitutions or deletions can enhance the tumor inhibition and/or tumor reduction activity of the IL-2. In some cases, the other additions, substitutions or deletions can improve the solubility of the IL-2 or variant (including but not limited to when expressed in E. coli or other host cells). In certain embodiments, the addition, substitution or deletion can improve the solubility of the IL-2 after expression in E. coli or other recombinant host cells. In certain embodiments, the site for substitution of a naturally encoded or non-natural amino acid is selected in addition to another site for incorporation of a non-natural amino acid that results in improved 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 after binding to an IL-2 receptor, modulates circulation half-life, modulates release or bioavailability, facilitates purification, or improves or alters a specific route of administration. In certain embodiments, the IL-2 polypeptide comprises additions, substitutions or deletions that increase the affinity of the IL-2 variant for its receptor. In certain embodiments, the IL-2 comprises additions, substitutions or deletions that increase the affinity of the IL-2 variant for IL-2-R1 and/or IL-2-R2. Similarly, the IL-2 polypeptide may comprise chemical or enzymatic cleavage sequences, protease cleavage sequences, reactive groups, antibody binding domains (including but not limited to FLAG or poly-His) or other affinity-based sequences (including but not limited to FLAG, poly-His, GST, etc.) or linked molecules (including but not limited to biotin) that improve detection (including but not limited to GFP), purification, transport through tissue or cell membranes, prodrug release or activation, IL-2 size reduction, or other properties of the polypeptide.

In certain embodiments, the substitution of the non-naturally encoded amino acid produces 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 causes IL-2 to act as an antagonist. In certain embodiments, the IL-2 antagonist comprises a non-naturally encoded amino acid linked to a water-soluble polymer that is present in the receptor binding region of the IL-2 molecule.

In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids are replaced by one or more non-naturally encoded amino acids. In some cases, the IL-2 also includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more naturally occurring amino acids replaced by one or more non-naturally encoded amino acids. For example, in some embodiments, one or more residues in IL-2 are replaced by one or more non-naturally encoded amino acids. In some cases, the one or more non-naturally encoded residues are attached to one or more lower molecular weight linear or branched PEGs, thereby improving binding affinity and comparable serum half-life relative to substances attached to a single higher molecular weight PEG.

VI. Expression in non-eukaryotic and eukaryotic organisms

In order to obtain high levels of expression of cloned IL-2 polynucleotides, one typically clones the polynucleotide encoding the IL-2 polypeptide of the present invention into an expression vector containing a strong promoter to direct transcription, a transcription/translation terminator, and, if used for protein-encoding nucleic acids, a ribosome binding site for initiation of translation. 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 expressing the IL-2 of the present invention are available in, including but not limited to, E. coli, Bacillus sp. , Pseudomonas fluorescens , Pseudomonas aeruginosa , Pseudomonas putida , and Salmonella (Palva et al., Gene 22: 229-235 (1983); Mosbach et al., Nature 302: 543-545 (1983)). Kits for 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 of using orthogonal tRNAs and amidyl tRNA synthetases (described above) to express the IL-2 polypeptides of the present invention, the host cells used 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 B. brevis, B. subtilis or Streptomyces ) and Gram-negative bacteria ( E. coli, Pseudomonas fluorescens , Pseudomonas aeruginosa , Pseudomonas putida ), as well as yeast and other eukaryotic cells. Cells containing O-tRNA/O-RS pairs can be used as described in the present invention.

The eukaryotic host cells or non-eukaryotic host cells of the present invention provide the ability to synthesize proteins comprising non-natural amino acids in useful quantities. In one instance, the composition optionally comprises, 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 of a protein comprising a non-natural amino acid, or an amount obtainable using an in vivo protein production method (details on recombinant protein production and purification are provided herein). In another aspect, the protein is optionally present in the composition at a concentration of, including but not limited to, at least 10 μg protein/L, at least 50 μg protein/L, at least 75 μg protein/L, at least 100 μg protein/L, at least 200 μg protein/L, at least 250 μg protein/L, at least 500 μg protein/L, at least 1 mg protein/L, or at least 10 mg protein/L or more, including but not limited to, in a cell lysate, a buffer, a drug buffer, or other liquid suspension (including but not limited to, in any volume between, including but not limited to, about 1 nl to about 100 L or more). The production of proteins comprising at least one unnatural amino acid in eukaryotic cells in large quantities (including but not limited to, greater quantities than would normally be possible using other methods (including but not limited to in vitro translation)) is a feature of the present invention.

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

A large 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 containing 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 plasmids from Escherichia coli, including pBR322, pET3a and pET12a, broad host range plasmids such as RP4, phage DNA such as lambda phage such as NM989 and other DNA phages such as M13 and a large number of derivatives of filamentous single-chain DNA phages can be used. The 2μ plasmid and its derivatives, the POT1 vector (U.S. Patent No. 4,931,373, incorporated herein by reference), the pJS037 vector described in (Okkels, Ann. New York Aced. 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, pBG311 (Cate et al., "Isolation of the Bovine and Human Genes for Mullerian Inhibiting Substance And Expression of the Human Gene In Animal Cells", Cell, 45, pp. 685 98 (1986)), pBluebac 4.5 and pMelbac (Invitrogen, Carlsbad, CA).

The nucleotide sequence encoding IL-2 or its variant 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: 89 104 describes a signal peptide used in mammalian cells (murine Ig kappa light chain signal peptide). Other signal peptides include, but are not limited to, α-factor signal peptide from brewing yeast (U.S. Patent No. 4,870,008, incorporated herein by reference), signal peptide of mouse salivary amylase (O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646), improved carboxypeptidase signal peptide (L. A. Valls et al., Cell 48, 1987, pp. 887-897), yeast BAR1 signal peptide (WO 87/02670, incorporated herein by reference), and yeast aspartic acid proteinase 3 (YAP3) 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 may be Chinese hamster ovary (CHO) cells (e.g., CHO-K1; ATCC CCL-61), green monkey cells (COS) (e.g., COS1 (ATCC CRL-1650), COS7 (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)), as well as plant cells in tissue culture. These cell lines and other cell lines can be obtained from public depositories such as the American Type Culture Collection, Rockville, Md. To provide enhanced glycosylation of the IL-2 polypeptide, the mammalian host cells can be modified to express a sialyltransferase such as 1,6-sialyltransferase, such as described in U.S. Patent No. 5,047,335, which is incorporated herein by reference.

Methods for introducing foreign 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 transfection methods using Lipofectamin 2000 described by Life Technologies Ltd, Paisley, UK, and FuGENE 6 described by Roche Diagnostics Corporation, Indianapolis, USA. These methods are well known in the art and are described in Ausbel et al., eds., 1996, Current Protocols in Molecular Biology, John Wiley & Sons, New York, USA. Cultivation of mammalian cells can be carried out 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 of Cell Culture, Cambridge University Press 1997).

I. E. coli, Pseudomonas species and other prokaryotes Bacterial expression techniques are known to those of ordinary skill in the art. A large variety of different vectors are available for use in bacterial hosts. The vectors can be single copy or low or high multi-copy vectors. Vectors can be used for replication (clone) and/or expression. In view of the large amount of literature on vectors, the commercial availability of many vectors and even manuals describing vectors and their restriction maps and characteristics, there is no need for an in-depth discussion here. As is well known, vectors usually contain markers that allow selection, which may provide resistance to cytotoxic agents, prototrophy or immunity. There are usually multiple markers that provide different characteristics.

A bacterial promoter is any DNA sequence that is capable of binding to a bacterial RNA polymerase and initiating transcription of a downstream (3') coding sequence (e.g., a structural gene) into mRNA. A promoter has a transcription initiation region, which is typically 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. A bacterial promoter may also have a second domain, called an operator, that may overlap with a neighboring RNA polymerase binding site at the start of RNA synthesis. The operator allows negatively regulated (inducible) transcription because a gene repressor protein can bind to the operator and thereby inhibit transcription of a specific gene. Constitutive expression may occur in the absence of a negative regulatory element, such as an operator. In addition, positive regulation can be achieved by gene promoter binding sequences, which, if present, are usually near (5') the RNA polymerase binding sequence. An example of a gene promoter protein is the catabolite promoter protein (CAP), which helps initiate transcription of the lac operon in E. coli (Raibaud et al., ANNU. REV. GENET. (1984) 18: 173). Thus, regulatory expression can be positive or negative, thereby enhancing or weakening transcription.

Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolites such as galactose, lactose (lac) (Chang et al., NATURE (1977) 198: 1056) and maltose. Other examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) (Goeddel et al., NUC. ACIDS RES. (1980) 8: 4057; Yelverton et al., NUCL. ACIDS RES. (1981) 9: 731; U.S. Patent No. 4,738,921; European Patent Publication Nos. 036 776 and 121 775, which are incorporated herein by reference). The β-galactosidase (bla) promoter system (Weissmann (1981), The cloning of interferon and other mistakes, Interferon 3 (I. Gresser, ed.), bacteriophage λPL (Shimatake et al., NATURE (1981) 292: 128) and T5 (U.S. Patent No. 4,689,406, which is incorporated herein by reference) promoter system also provide useful promoter sequences. Preferred methods of the present 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 pPOP vectors described in WO99/05297, which is incorporated herein by reference. These expression systems produce high levels of IL-2 polypeptides in the host without compromising host cell viability or growth parameters. pET19 (Novagen) is another vector known in the art.

In addition, synthetic promoters that do not occur in nature also function as bacterial promoters. For example, the transcriptional promoter sequence of one bacterial or phage promoter can be linked to the operator sequence of another bacterial or phage promoter to produce a synthetic hybrid promoter (U.S. Patent No. 4,551,433, which is incorporated herein by reference). For example, the tac promoter is a hybrid trp-lac promoter that contains both the trp promoter and the lac operator sequence that is regulated by the lac repressor protein (Amann et al., GENE (1983) 25: 167; de Boer et al., PROC. NATL. ACAD. SCI. (1983) 80: 21). In addition, bacterial promoters can 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 can also be coupled with compatible RNA polymerases to produce high levels of expression of certain genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system (Studier et al., J.MOL.BIOL. (1986) 189: 113; Tabor et al., Proc Natl.Acad.Sci. (1985) 82: 1074). In addition, hybrid promoters can also contain a bacteriophage promoter and an Escherichia coli operator region (European Patent Publication No. 267 851).

In addition to a functional promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes. In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes a start codon (ATG) and a sequence of 3-9 nucleotides in length located 3-11 nucleotides upstream of the start codon (Shine et al., NATURE (1975) 254: 34). The SD sequence is believed to promote the binding of mRNA to the ribosome by base pairing between the SD sequence and the 3' end of the E. coli 16S rRNA (Steitz et al., Genetic signals and nucleotide sequences in messenger RNA, Biological Regulation and Development: Gene Expression (R.F. Goldberger, ed., 1979)). To express 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 recombinant vectors or other transfer of DNA. The term includes the progeny of the original bacterial host cell that has been transfected. It should be understood that the progeny of a single parental cell may not necessarily be completely identical to the original parent in morphology or in the entire genome or total DNA due to accidental or deliberate mutations. Progeny of the parental cell that are sufficiently similar to the parent, as characterized by the presence of a nucleotide sequence encoding an IL-2 polypeptide, are included in the progeny intended by this definition.

The selection of suitable host bacteria for expression of IL-2 polypeptides is known to those of ordinary skill in the art. In selecting bacterial hosts for expression, suitable hosts may include those shown to have, inter alia, good inclusion body formation ability, low proteolytic activity, and overall stability. Bacterial hosts are generally available from a variety of different sources, including, but not limited to, the Bacterial Genetic Stock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA) and the American Type Culture Collection ("ATCC") (Manassas, VA). Industrial/pharmaceutical fermentations typically use bacteria derived from K strains (e.g., W3110) or bacteria derived from B strains (e.g., BL21). These strains are particularly useful because their growth parameters are extremely well understood and robust. In addition, these strains are non-pathogenic, which is important commercially for safety and environmental reasons. Other examples of suitable E. coli hosts include, but are not limited to, BL21, DH10B strains or their derivatives. In another embodiment of the method of the present invention, the E. coli strain is a protease-attenuated strain, including, but not limited to, OMP- and LON-. The host cell strain can be a species of the genus Pseudomonas , including, but not limited to, Pseudomonas fluorescens , Pseudomonas aeruginosa , and Pseudomonas putida . Pseudomonas fluorescens biovar 1, designated strain MB101, 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 as host strains from The Dow Chemical Company (Midland, MI, available on the World Wide Web at 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 to the host cell with the correct expression construct), the recombinant host cell strain is cultured under conditions suitable for production of the IL-2 polypeptide. As will be apparent to one of ordinary skill in the art, the method of culturing the recombinant host cell strain depends 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 one of ordinary skill in the art. Recombinant host cells are typically cultured in a liquid medium containing assimilable sources of carbon, nitrogen, and inorganic salts and optionally containing vitamins, amino acids, growth factors, and other protein culture supplements known to one of ordinary skill in the art. Liquid media used for host cell culture may optionally contain antibiotics or antifungals to prevent 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 can be cultured in batch or continuous mode, and cell harvesting (in the case where the IL-2 polypeptide accumulates intracellularly) or culture supernatant harvesting can be performed in batch or continuous mode. For production in prokaryotic host cells, batch culture and cell harvesting are preferred.

The IL-2 polypeptides of the present invention are generally purified after expression in a recombinant system. The IL-2 polypeptides can be purified from host cells or culture media by various 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 present invention, amino acid substitutions can be easily made in the IL-2 using methods disclosed in the present invention and known in the art, and the substitutions are selected for the purpose of improving the solubility of the recombinantly produced protein. In the case of insoluble protein, the protein can be collected from the host cell lysate by centrifugation, and the cells can then be further homogenized. In the case of poorly soluble proteins, compounds (including but not limited to polyethyleneimine (PEI)) can be added to cause precipitation of partially soluble proteins. The precipitated protein can then be conveniently collected by centrifugation. The recombinant host cells can be disrupted or homogenized by various 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, ultrasonic treatment, dounce homogenization, or high-pressure release disruption. In one embodiment of the method of the present invention, high-pressure release technology is used to disrupt E. coli host cells to release the inclusion bodies of the IL-2 polypeptide. When processing inclusion bodies of IL-2 polypeptide, it may be advantageous to minimize the homogenization repetition time in order to maximize the yield of inclusion bodies without losses due to factors such as solubilization, mechanical shearing or proteolysis.

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

In the case of soluble IL-2 protein, 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 host cells. It may be desirable to concentrate the soluble IL-2 prior to performing the purification step. Standard techniques known to those of ordinary skill in the art may be used to concentrate soluble IL-2 from, for example, cell lysates or culture medium. In addition, standard techniques known to those of ordinary skill in the art may be used to disrupt host cells and release soluble IL-2 from the cytoplasm or periplasmic space of the host cells.

In general, it is sometimes desirable to denature and reduce an expressed polypeptide and then allow the polypeptide to refold into a preferred conformation. For example, guanidine, urea, DTT, DTE, and/or a chaperone protein may be added to the translation product of interest. Methods for reducing, denaturing, and refolding proteins are known to those of ordinary skill in the art (see the references above, as well as Debinski et al., (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan, (1993) Bioconjug. Chem., 4: 581-585; and Buchner et al., (1992) Anal. Biochem., 205: 263-270). For example, Debinski et al. describe the denaturation and reduction of inclusion body proteins in guanidine-DTE. The protein can be refolded in a redox buffer containing, including but not limited to, oxidized glutathione and L-arginine. The refolding reagent can be flowed or otherwise moved to contact 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 thus produced may be misfolded and therefore lack or have reduced biological activity. The biological activity of the protein can be restored by "refolding". Generally speaking, the misfolded IL-2 polypeptide is refolded by solubilizing (where the IL-2 polypeptide is also insoluble), unfolding and reducing the polypeptide chain using, for example, one or more isolating agents (e.g., urea and/or guanidine) and a reducing agent capable of reducing disulfide bonds (e.g., dithiothreitol DTT or 2-hydroxyethanol 2-ME). An oxidizing agent (e.g., oxygen, cystine or cystamine) is then added at a moderate concentration of the isolating agent, which allows the reformation of disulfide bonds. The IL-2 polypeptide can be refolded using standard methods known in the art, such as those described in U.S. Patent Nos. 4,511,502, 4,511,503, and 4,512,922, which are incorporated herein by reference. The IL-2 polypeptide can also be cofolded with other proteins to form heterodimers or heteromultimers.

After refolding, the IL-2 can be further purified. The 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, size exclusion chromatography, ion exchange chromatography, reversed-phase high performance liquid chromatography, affinity chromatography, etc. or any combination thereof. Additional purification may also include a drying or precipitation step of the purified protein.

After purification, IL-2 can be exchanged in different buffers and/or concentrated by any of a variety of different methods known in the art, including but not limited to filtration 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 value of the purity of the IL-2, the IL-2 is sufficiently pure for use as a pharmaceutical or for further processing such as conjugation with a water-soluble polymer such as PEG.

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

It has been shown previously that unnatural amino acids can be site-specifically incorporated into proteins in vitro by adding a chemically amine-acylated suppressor tRNA to a protein synthesis reaction programmed with a gene containing the desired amber nonsense mutation. Using these approaches, one can replace many of the 20 common amino acids with structurally close homologues, such as phenylalanine with fluorophenylalanine, using strains deficient for specific amino acids. See, e.g., Noren, CJ, Anthony-Cahill, Griffith, MC, Schultz, PG, A general method for site-specific incorporation of unnatural amino acids into proteins , Science, 244: 182-188 (1989); MW Nowak et al., Science 268: 439-42 (1995); Bain, JD, Glabe, CG, Dix, TA, Chamberlin, AR, Diala, ES, Biosynthetic site-specific incorporation of a unnatural amino acid into a polypeptide , J. Am Chem Soc, 111: 8013-8014 (1989); N. Budisa et al., FASEB J. 13: 41-51 (1999); Ellman, J. A., Mendel, D., Anthony-Cahill, S., Noren, C. J., Schultz, P. G., Biosynthetic method for introducing unnatural amino acids site-specifically into proteins, Methods in Enz., vol. 202, 301-336 (1992); and Mendel, D., Cornish, V. W. & Schultz, P. G., Site-Directed Mutagenesis with an Expanded Genetic Code , Annu Rev Biophys. Biomol Struct. 24, 435-62 (1995).

For example, a suppressor tRNA that recognizes the stop codon UAG and is chemically amine-acylated with an unnatural amino acid is prepared. The stop codon TAG is introduced at the position of interest in the protein gene using conventional site-directed mutagenesis. See, for example, Sayers, JR, Schmidt, W. Eckstein, F., 5'-3' Exonucleases in phosphorothioate-based olignoucleotide-directed mutagensis , Nucleic Acids Res, 16(3): 791-802 (1988). When the acylated suppressor tRNA and mutant gene are combined in an in vitro transcription/translation system, the unnatural amino acid is incorporated in response to the UAG codon, giving a protein containing the amino acid at the specific position. Experiments using [ ^3H ]-Phe and experiments using α-hydroxy acids confirmed that only the desired amino acid is incorporated at the position specified by the UAG codon, and this amino acid is not incorporated at any other position in the protein. See, e.g., Noren et al., supra; Kobayashi et al., (2003) Nature Structural Biology 10(6):425-432; and Ellman, JA, Mendel, D., Schultz, PG, Site-specific incorporation of novel backbone structures into proteins , Science, 255(5041):197-200 (1992).

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

Amine acylation can be accomplished by amidyl tRNA synthetases or other enzyme molecules, including but not limited to ribozymes. The term "ribozyme" can be interchangeably used with "catalytic RNA". Cech and coworkers (Cech, 1987, Science, 236: 1532-1539; McCorkle et al., 1987, Concepts Biochem. 64: 221-226) demonstrated the existence of naturally occurring RNA (ribozymes) that can act as catalysts. However, although these natural RNA catalysts have only been shown to act on RNA reaction substrates for cutting and splicing, recent developments in the artificial evolution of ribozymes have expanded the catalytic spectrum to a variety of different chemical reactions. Studies have identified RNA molecules that can catalyze amido-RNA bonds at their own (2')3'-ends (Illangakekare et al., 1995 Science 267:643-647) and RNA molecules that can transfer amino acids from one RNA molecule to another (Lohse et al., 1996, Nature 381:442-444).

U.S. Patent Application Publication 2003/0228593, which is incorporated herein by reference, describes methods for constructing ribozymes and their use in amine acylation of tRNAs with naturally encoded and non-naturally encoded amino acids. Matrix-immobilized forms of enzyme molecules (including but not limited to ribozymes) that amine acylate tRNAs may enable efficient affinity purification of the amine acylated products. Examples of suitable matrices include agarose, agarose gels, and magnetic beads. The production of matrix-immobilized forms of ribozymes and their use for amine acylation are described in Chemistry and Biology 2003, 10: 1077-1084 and U.S. Patent Application Publication 2003/0228593, which are incorporated herein by reference.

Chemical amine acylation methods include but are not limited to the methods described by the following researchers: 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); Schultz, Chamberlin, Dougherty, et al. (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.,et al.,Nature 1992,356,537; Gallivan, J.P.; Lester, H.A.; Dougherty, D.A. Chem. Biol. 1997,4,740; Turcatti et al., J.Biol. Chem. 1996,271,19991; Nowak, M.W. et al., Science, 1995,268,439; Saks, M.E. et al., J.Biol. Chem. 1996,271,23169; Hohsaka, T. et al., J.Am. Chem. Soc. 1999,121,34), which are incorporated by reference into the present invention to avoid the use of synthetases in the acylation. These methods or other chemical acylation methods can be used to acylate tRNA molecules.

Methods for generating catalytic RNA may include generating separate consortia of randomized ribozyme sequences, performing directed evolution on the consortia, screening the consortia for desired amine acylation activity, and selecting those ribozyme sequences that exhibit the desired amine acylation activity.

Reconstructed translation systems can also be used. Purified mixtures of translation factors have also been successfully used to translate mRNA into protein, as have combinations of lysates or lysates 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 factors. Cell-free systems can also be coupled to transcription/translation systems, in which DNA is introduced into the system, transcribed into mRNA and the mRNA is translated, as described in Current Protocols in Molecular Biology (FM Ausubel et al., ed., Wiley Interscience, 1993), which is specifically incorporated by reference into the present invention. RNA transcribed in eukaryotic transcription systems can take the form of heterokaryotic RNA (hnRNA) or mature mRNA with a 5' -end cap (7-methylguanosine) and a 3 ' end poly A tail, which can be advantageous in certain transcription systems. For example, in the reticulocyte lysate system, capped mRNA is transcribed with high efficiency.

IX. Macromolecular Polymers Conjugated to IL-2 Polypeptide

A variety of modifications to the non-natural amino acid polypeptides described herein can be performed using the compositions, methods, techniques and strategies described herein. These modifications include incorporating other functional groups into the non-natural amino acid component of the polypeptide, and the other functional groups 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, carbohydrates, polynucleotides, DNA, RNA, antisense polynucleotides, sugars, water-soluble dendrimers, cyclodextrins, inhibitory RNA, biomaterials, nanoparticles, spin labels, fluorophores, metal-containing moieties, radioactive moieties, new functional groups, covalent or non-covalently interacting groups, photoclavable moieties, actinic radiation excitable moieties, photoisomerizable moieties, biotin, biotin derivatives, biotin analogs, heavy atom-incorporated moieties, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox active agents, amino thioacids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chemiluminescent groups, electron dense groups, magnetic groups, intercalating groups, chromophores, energy transfer agents, biologically active agents, detectable labels, small molecules, quantum dots, nanoemitters, radionucleotides, radioemitters, neutron capture agents, or any combination of the foregoing or any other desired compound or substance. As an illustrative but not limiting example of the compositions, methods, techniques and strategies described in the present invention, the following description will focus on adding macromolecular polymers to non-natural amino acid polypeptides, and it should be understood that the compositions, methods, techniques and strategies are also applicable (if necessary, with appropriate modifications, and those skilled in the art can make these modifications using this disclosure) to add other functional groups, including but not limited to those listed above.

A wide variety of macromolecular polymers and other molecules can be attached to the IL-2 polypeptides of the present 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 can be attached to the IL-2 polypeptides through naturally encoded amino acids, through non-naturally encoded amino acids, or any functional substituents of natural or non-natural amino acids, or any substituents or functional groups added to natural or non-natural amino acids. The molecular weight of the polymers can have a wide range, including but not limited to between about 100 Da to about 100,000 Da or more. The molecular weight of the polymer may be between about 100 Da and about 100,000 Da, including but not limited to 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30 In some embodiments, the molecular weight of the polymer is between about 100Da and about 50,000Da. In some embodiments, the molecular weight of the polymer is between about 100Da and about 40,000Da. In some embodiments, the molecular weight of the polymer is between about 1,000Da and about 40,000Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da.

The present invention provides substantially homogenous preparations of polymer: protein conjugates. When used in the present invention, "substantially homogenous" means that the polymer: protein conjugate molecules are observed to be more than half of the total protein. The polymer: protein conjugate has biological activity, and the "substantially homogenous" PEGylated IL-2 polypeptide preparations provided in the present invention are homogenous preparations that are sufficient to show the advantages of homogenous preparations, such as the ease of obtaining predictability of drug metabolism kinetics between batches in clinical applications.

One may also choose to prepare a mixture of polymer:protein conjugate molecules, and the advantage provided in the present invention is that one can choose the ratio of the single polymer:protein conjugates to be included in the mixture. Thus, if desired, one can prepare a mixture of various different proteins with various numbers of polymer components attached (i.e., two, three, four, etc.) and combine the conjugates with the single polymer:protein conjugates prepared using the method of the present invention to obtain a mixture of single polymer:protein conjugates having a predetermined ratio.

The polymer selected may be water soluble so 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 glycol, polyoxyethylene glycol/propylene glycol and their methoxy- or ethoxy-terminated analogs, particularly polyoxyethylene glycol, also known as polyethylene glycol or PEG), polyvinyl pyrrolidone, polyvinyl alkyl ethers, polyoxazolines, polyalkyloxazolines and polyhydroxyalkyloxazolines, polyacrylamide, polyalkylacrylamide and polyhydroxyalkylacrylamide (e.g., polyhydroxypropylmethacrylamide and its derivatives), polyhydroxyalkyl acrylates, polysialic acid and its derivatives. Analogs, hydrophilic peptide sequences, polysaccharides and their derivatives, including dextran and dextran derivatives, such as carboxymethyl dextran, dextran sulfate, amino dextran, cellulose and its derivatives such as carboxymethyl cellulose, hydroxyalkyl cellulose, chitin and its derivatives such as chitosan, succinylchotan, carboxymethyl chitosan, carboxymethyl chitosan, hyaluronic acid and its derivatives, starch, alginate, chondroitin sulfate, albumin, pullulan and carboxymethyl pullulan, polyamino acids and their derivatives such as polyglutamine, polylysine, polyaspartic acid, polyaspartamide, maleic anhydride copolymers such as styrene maleic anhydride copolymer, divinyl ether maleic anhydride copolymer, polyvinyl alcohol, their copolymers, their terpolymers, their mixtures, and derivatives of the above substances.

The ratio of polyethylene glycol molecules to protein molecules can vary, as can their concentrations in the reaction mixture. In general, the optimal 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 chosen and the number of reactive groups available. With respect to molecular weight, generally the higher the molecular weight of the polymer, the fewer polymer molecules can be attached to the protein. Similarly, branching of the polymer should be taken into account when optimizing these parameters. In general, the higher the molecular weight (or the more branches), the higher the polymer:protein ratio.

As used in the present invention, 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 one individual to another and depends on many factors, including the patient's general physical condition and the underlying cause of the condition to be treated. The amount of IL-2 polypeptide used in therapy provides an acceptable rate of change and maintains the desired response at a beneficial level. Therapeutically effective amounts of the compositions of the present invention can be readily determined by one of ordinary skill in the art using publicly available materials and procedures.

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

PEG is a well-known water-soluble polymer that 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 cover any polyethylene glycol molecule, regardless of size or with modifications at the termini of the PEG, and when attached to an IL-2 polypeptide can be represented by the formula: XO-( _CH2CH2O ) _{n - CH2CH2 -} Y

wherein n is 2 to 10,000, and X is H or a terminal modification, including but not limited to a C _1-4 alkyl group, a protecting group or a 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 can be terminated with a reactive group, thereby forming a bifunctional polymer. Typical reactive groups can include reactive groups commonly used to react with functional groups present in the 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 that are inert to the 20 common amino acids but specifically react with complementary functional groups present in non-naturally encoded amino acids (including but not limited to azido groups, alkynyl groups). It is noteworthy that the other end of the PEG, shown as 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 can be an amide, carbamate, or urea bond linked 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 can be a maleimide bond linked to a thiol group (including but not limited to the thiol group of cysteine). Alternatively, Y can be a bond to a residue that is usually inaccessible to the 20 common amino acids. For example, an azido group on the PEG can react with an alkynyl group on the IL-2 polypeptide to form a Huisgen [3+2] cycloaddition product. Alternatively, an alkynyl group on the PEG can react with an azido group present in a non-naturally encoded amino acid to form a similar product. In certain embodiments, strong nucleophiles (including but not limited to hydrazines, hydrazides, hydroxylamines, semicarbazides) can react with aldehyde or ketone groups present in non-naturally encoded amino acids to form hydrazones, oximes or semicarbazones, which in some cases can be further reduced by treatment with a suitable reducing agent. Alternatively, the strong nucleophile can be incorporated into the IL-2 polypeptide via the 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 desired, including but not limited to about 100 daltons (Da) to 100,000 Da or more (including but not limited to 0.1-50 kDa or 10-40 kDa sometimes) as desired. The molecular weight of the PEG can have a wide range, including but not limited to about 100 Da to about 100,000 Da or more. PEG can be between about 100 Da and about 100,000 Da, including but not limited to 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, In some embodiments, PEG is between about 100Da and about 50,000Da. In some embodiments, PEG is between about 100Da and about 40,000Da. In some embodiments, PEG is between about 1,000Da and about 40,000Da. In certain embodiments, PEG is between about 5,000Da and about 40,000Da. In certain embodiments, PEG is between about 10,000Da and about 40,000Da. Branched PEGs can also be used, including but not limited to PEG molecules with a MW in the range of 1-100kDa (including but not limited to 1-50kDa or 5-20kDa) for each chain. The molecular weight of each chain of the branched PEG can include but not limited to between about 1,000Da and about 100,000Da or more. The molecular weight of each chain of the branched PEG can be between about 1,000 Da and about 100,000 Da, including but not limited to 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, In some embodiments, the molecular weight of each chain of the branched-chain PEG is between about 1,000Da and about 50,000Da. In some embodiments, the molecular weight of each chain of the branched-chain PEG is between about 1,000Da and about 40,000Da. In certain embodiments, the molecular weight of each chain of the branched PEG is between about 5,000 Da and about 40,000 Da. In certain embodiments, the molecular weight of each chain of the branched PEG is between about 5,000 Da and about 20,000 Da. A wide range of PEG molecules are described in, including but not limited to, the Shearwater Polymers, Inc. catalog, the Nektar Therapeutics catalog, which are incorporated herein by reference.

Typically, at least one end of the PEG molecule can be used to react with the non-naturally encoded amino acid. For example, a PEG derivative with an alkyne and an azide component and used to react with the amino acid side chain can be used to attach PEG to the non-naturally encoded amino acid described in the present invention. If the non-naturally encoded amino acid contains an azide, the PEG will typically contain an alkyne component to perform the formation of a [3+2] cycloaddition product, or contain an activated PEG substance (i.e., ester, carbonate) containing a phosphine group to perform the formation of an amide bond. Alternatively, if the non-naturally encoded amino acid contains an alkyne, the PEG will typically contain an azide component to perform the formation of a [3+2] Huisgen cycloaddition product. If the non-naturally encoded amino acid contains a carbonyl group, the PEG will typically contain a strong nucleophile (including but not limited to hydrazide, hydrazine, hydroxylamine or semicarbazide functional groups) to carry out the formation of the corresponding hydrazone, oxime and semicarbazone bonds, respectively. In other alternatives, the inversion of the orientation of the reactive groups described above can be used, that is, the azido moiety of the non-naturally encoded amino acid can react with the alkyne-containing PEG derivative.

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

In certain embodiments, the present invention provides polymer derivatives containing azides and acetylene, which contain a water-soluble polymer backbone having an average molecular weight of about 800Da to about 100,000Da. The polymer backbone of the water-soluble polymer can 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 the practice of the present invention, and the use of the term PEG or polyethylene glycol is intended to cover and include all such molecules. The term PEG includes but is not limited to polyethylene glycol in any form, including bifunctional PEG, multi-arm PEG, derivatized PEG, bifurcated PEG, branched PEG, pendant PEG (i.e., PEG or related polymers having one or more functional groups pendant from the polymer backbone) or PEG having degradable bonds therein.

PEG is generally transparent, colorless, odorless, soluble in water, stable to heat, inert to many chemical reagents, does not hydrolyze or deteriorate, and is generally non-toxic. Polyethylene glycol is considered biocompatible, which means that PEG can coexist with living tissues or organisms without causing damage. More specifically, PEG is essentially non-immunogenic, which means that PEG does not tend to produce an immune response in the body. When attached to a molecule that has a certain desired function in the body, such as a biologically active agent, PEG tends to mask the agent and can reduce or eliminate any immune response so that the organism can tolerate the presence of the agent. PEG conjugates do not tend to produce a significant immune response or cause coagulation or other adverse effects. PEGs having the formula --CH2CH2O--(CH2CH2O)n--CH2CH2-- (wherein n is about 3 to about 4000, typically about 20 to about 2000) are suitable for use in the present invention. In certain embodiments of the present invention, PEGs having a molecular weight of about 800 Da to about 100,000 Da are particularly useful as polymer backbones. The molecular weight of PEG can have a wide range, including but not limited to between about 100 Da to about 100,000 Da or more. The molecular weight of PEG can be between about 100 Da and about 100,000 Da, including but not limited to 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 5 In some embodiments, the molecular weight of PEG is between about 100Da and about 50,000Da. In some embodiments, the molecular weight of PEG is between about 100Da and about 40,000Da. In some embodiments, the molecular weight of PEG is between about 1,000Da and about 40,000Da. In some embodiments, the molecular weight of PEG is between about 1,000Da and about 40,000Da. In some embodiments, the molecular weight of PEG is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of PEG is between about 10,000 Da and about 40,000 Da.

The polymer backbone can be straight chain or branched. Branched polymer backbones are generally known in the art. Typically, branched polymers have a central branched core component and multiple linear polymer chains connected to the central branched core. PEG is often used in branched form, which can be prepared by adding ethylene oxide to various polyols such as glycerol, glycerol oligomers, pentaerythritol and sorbitol. The central branched component can also be derived from several amino acids such as lysine. The branched polyethylene glycol can be expressed in a general form as R(-PEG-OH)m, where R is derived from a core component such as glycerol, glycerol oligomers or pentaerythritol, and m represents the number of arms. Multi-arm PEG molecules such as those described in U.S. Patent Nos. 5,932,462, 5,643,575, 5,229,490, 4,289,872, U.S. Patent Application Nos. 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 can also take the form of a forked PEG represented by PEG(--YCHZ2)n, where Y is the linking group and Z is the activated terminal group connected to CH through an atomic chain of a defined length.

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

In addition to these forms of PEG, the polymer can also be prepared with weak or degradable bonds in the backbone. For example, PEG can be prepared with ester bonds in the polymer backbone that are susceptible to hydrolysis. 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-

Those of ordinary skill in the art should understand that the term polyethylene glycol or PEG represents or includes all forms known in the art, including but not limited to those disclosed in the present invention.

Many other polymers are also suitable for use in the present invention. In certain embodiments, water-soluble polymer backbones having 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 is typically in the range of about 800 Da to about 100,000 Da, typically about 6,000 Da to about 80,000 Da. The molecular weight of each chain of the polymer backbone can be between about 100 Da and about 100,000 Da, including but not limited to 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35 ... In some embodiments, the molecular weight of each chain of the polymer backbone is between about 100 Da and about 50,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 100 Da and about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 10,000 Da and about 40,000 Da.

Those skilled in the art will recognize that the foregoing 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 considered suitable for use in the present invention.

In certain embodiments of the present 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. Multifunctional polymer derivatives include, but are not limited to, linear polymers having two ends, each end being 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

Where: N=N=N is an azido component; B is a linking component, which may be present or absent; POLY is a water-soluble non-antigenic polymer; A is a linking component, which may be present or absent and may be the same as or different from B; and X is a second functional group.

Examples of linking components A and B include, but are not limited to, polyfunctionalized alkyl groups containing up to 18 carbon atoms and may contain between 1-10 carbon atoms. Heteroatoms such as nitrogen, oxygen or sulfur may be included in the alkyl chain. The alkyl chain may also be branched at heteroatoms. Other examples of linking components A and B include, but are not limited to, polyfunctionalized aryl groups containing up to 10 carbon atoms and may contain 5-6 carbon atoms. The aryl group may be substituted with one or more carbon atoms, nitrogen, oxygen or sulfur atoms. Other examples of suitable linking groups include those described in U.S. Patent Nos. 5,932,462, 5,643,575 and U.S. Patent Application Publication No. 2003/0143596, each of which is incorporated herein by reference. Those of ordinary skill in the art will recognize that the foregoing list of connection components is by no means exhaustive but merely illustrative, and that all connection components having the properties described above are considered suitable for use with the present invention.

Examples of suitable functional groups 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, alkenyls, acrylates, methacrylates, acrylamides, active sulfones, amines, aminooxy groups, protected amines, hydrazides, protected hydrazides, protected thiols, carboxylic acids, protected carboxylic acids, isocyanates, isothiocyanates, maleimides, vinyl sulfones, dithiopyridines, vinyl pyridines, iodoacetamides, epoxides, glyoxals, diones, mesylates, tosylates, tribenzoates, olefins, ketones, and azides. As will be understood by those of ordinary skill in the art, the selected X component should be compatible with the azido group so as not to react with the azido group. The azido-containing polymer derivative may be homobifunctional, meaning that the second functional group (i.e., X) is also an azido component, 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 the 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-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). If the chemically reactive group is a thiol, the protecting group may be an o-pyridyl disulfide. If the chemically reactive group is a carboxylic acid such as butyric acid or propionic acid or a hydroxyl group, the protecting group may be a benzyl group or an alkyl group such as a methyl, ethyl or tert-butyl group. 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. Patent 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., Poly(ethylene glycol) Chemistry & Biological Applications, pp 170-181, Harris & Zalipsky, ed., ACS, Washington, D.C., 1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinates (see, e.g., Abuchowski et al., Cancer Biochem. Biophys. 7:175 (1984) and Joppich et al., Makromol. Chem. 180:1381 (1979)), succinimidyl esters (see, e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonates (see, e.g., U.S. Pat. No. 5,650,234), glycidyl ethers (see, e.g., Pitha et al., Eur. J. Chem. 1996:1341-1357). Biochem. 94: 11 (1979), Elling et al., Biotech. Appl. Biochem. 13: 354 (1991)), oxycarbonyl imidazoles (see, for example, Beauchamp et al., Anal. Biochem. 131: 25 (1983), Tondelli et al., J. Controlled Release 1:251 (1985)), p-nitrophenyl carbonate (see, e.g., Veronese et al., Appl. Biochem. Biotech., 11:141 (1985); and Sartore et al., Appl. Biochem. Biotech., 27:45 (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), maleimide (see, e.g., Goodson et al., Biotechnology (NY) 8:343 (1990), Romani et al., Chemistry of Peptides and Proteins 2:29 (1984) and Kogan, Synthetic Comm.22:2417(1992)), o-pyridyl disulfide (see, for example, Woghiren et al., Bioconj.Chem.4:314(1993)), allyl alcohol (see, for example, Sawhney et al., Macromolecules, 26:581(1993)), vinyl sulfide (see, for example, U.S. Patent No. 5,900,461). All of the above references and patents are incorporated into the present invention by reference.

In certain embodiments of the present invention, the polymer derivative of the present invention comprises a polymer backbone having the following structure: X-CH2CH2O--(CH2CH2O)n--CH2CH2-N=N=N

wherein: X is a functional group as described above; and n is from about 20 to about 4000.

In another embodiment, the polymer derivative of the present invention comprises a polymer backbone having _the following structure: X- _CH2CH2O --( _CH2CH2O ) _{n -- CH2CH2 -} O-( _CH2 ) _m -WN=N=N

Wherein: W is an aliphatic or aromatic linker moiety containing between 1 and 10 carbon atoms; n is from about 20 to about 4000; and X is a functional group as described above. m is between 1 and 10.

The azido-containing PEG derivatives of the present invention can be prepared by various methods known in the art and/or disclosed in the present invention. In one method, as shown below, a water-soluble polymer backbone having an average molecular weight of about 800Da to about 100,000Da, a 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 azido anion (which can be paired with any of a large number of suitable balance ions (including sodium, potassium, tert-butylammonium, etc.)). The leaving group undergoes nucleophilic displacement and is replaced by the azido component to obtain the desired azido-containing PEG polymer.

X-PEG-L+N ₃ ^- →X-PEG-N ₃

As shown, polymer backbones suitable for use in the present invention have the formula X-PEG-L, where PEG is polyethylene glycol, X is a functional group that does not react with azido groups, and L is a suitable leaving group. Examples of suitable functional groups include, but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl, amine, aminooxy, 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, chloro, bromo, iodo, mesylate, tribenzoate and tosylate.

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

An exemplary reaction scheme is shown below: X-PEG-M+N-Linker-N=N=N→PG-X-PEG-Linker-N=N=N

Wherein: PEG is polyethylene glycol, X is a capping group such as an alkoxy group or a functional group as described above; and M is a functional group that is unreactive 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, carbonate, or active ester; if N is a hydrazide or an amineoxy moiety, M is a ketone; if N is a nucleophile, M is a leaving group.

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

A more specific example is shown below using PEG diamine as an example, wherein one amine group is protected by a protecting group component such as tert-butyl-Boc, and the resulting mono-protected PEG diamine is reacted with a linking component having an azido functional group: BocHN-PEG- _NH2 + _HO2C- ( _CH2 ) ₃ -N=N=N

In this case, the amine group can be conjugated to the carboxylic acid group using a variety of different activating agents such as sulfinyl chloride or carbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazole to generate an amide bond between the monoamine PEG derivative and the linker component with an azido group. After the amide bond is successfully formed, the resulting N-tert-butyl-Boc protected azido derivative can be used directly to modify biologically active molecules, 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 generate an ω-amine-PEG-azido compound. The resulting amines can be used as synthetic handles to install other useful functional groups such as maleimide groups, activated disulfides, activated esters, etc., to generate 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 the attachment of molecules with activated electrophilic groups such as aldehydes, ketones, activated esters, activated carbonates, etc. to one end of the PEG and 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 can be H or alkyl, alkenyl, alkoxy or aryl or substituted aryl; B is a linking component, which may be present or absent; POLY is a water-soluble non-antigenic polymer; A is a linking component, which may be present or absent and may be the same as or different from B; and X is a second functional group.

Examples of linking components A and B include, but are not limited to, polyfunctionalized 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 in the alkyl chain. The alkyl chain may also be branched at heteroatoms. Other examples of linking components A and B include, but are not limited to, polyfunctionalized aryl groups containing up to 10 carbon atoms and may contain 5-6 carbon atoms. The aryl group may be substituted with one or more carbon atoms, nitrogen, oxygen or sulfur atoms. Other examples of suitable linking groups include those described in U.S. Patent Nos. 5,932,462, 5,643,575 and U.S. Patent Application Publication No. 2003/0143596, each of which is incorporated herein by reference. Those of ordinary skill in the art will recognize that the foregoing list of connection components is by no means exhaustive but is merely illustrative, and that a wide variety of connection components having the properties described above are contemplated as being useful in the present invention.

Examples of functional groups suitable for use as X include 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, alkenyls, acrylates, methacrylates, acrylamides, active sulfones, amines, aminooxy groups, protected amines, hydrazides, protected hydrazides, protected thiols, carboxylic acids, protected carboxylic acids, isocyanates, isothiocyanates, maleimides, vinyl sulfones, dithiopyridines, vinyl pyridines, iodoacetamides, epoxides, glyoxals, diones, mesylates, tosylates, tribenzoates, olefins, ketones, and acetylene. As will be appreciated, the selected X moiety should be compatible with the acetylene group such that no reaction with the acetylene 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 following structure: X- _CH2CH2O --( _CH2CH2O ) _n -- _{CH2CH2 -} O-( _CH2 ) _m -C≡CH

wherein: X is a functional group as described above; n is from about 20 to about 4000; and m is between 1 and 10.

Specific examples of each heterobifunctional PEG polymer are shown below.

The acetylene-containing PEG derivatives of the present 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 800 Da to about 100,000 Da, a 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 having both an acetylene functional group and a leaving group suitable for reacting with the nucleophilic group on the PEG. When the PEG polymer having the nucleophilic component is combined with the molecule having the leaving group, the leaving group undergoes nucleophilic substitution and is replaced by the nucleophilic component to obtain 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, aminooxy groups that react primarily via SN2-type mechanisms. Other examples of Nu groups include functional groups that react primarily via nucleophilic addition reactions. Examples of L groups include chloro, bromo, iodo, mesylate, tribenzoate, and tosylate and other groups that are expected to undergo nucleophilic displacement, as well as ketones, aldehydes, thioesters, alkenes, α-β unsaturated carbonyls, carbonates, and other electrophilic groups that are expected to undergo addition by nucleophiles.

In another embodiment of the present invention, A is an aliphatic linker between 1-10 carbon atoms or a substituted aryl ring between 6-14 carbon atoms. X is a functional group that does not react with the azido 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 800 Da to about 100,000 Da, having a protective functional group or end-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 an alkoxy group or a functional group as described above; and R' is H, alkyl, alkoxy, aryl or aryloxy or substituted alkyl, alkoxy, aryl or aryloxy.

In the above example, the leaving group L should be sufficiently reactive to undergo an SN2 type of displacement when in contact with a sufficient concentration of acetylene anions. The reaction conditions required to achieve SN2 displacement of the leaving group by acetylene anions 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.

The water-soluble polymer can be linked to the IL-2 polypeptide of the present invention. The water-soluble polymer can be linked by a non-naturally encoded amino acid, or any functional group or substituent of a non-naturally encoded or naturally encoded amino acid, or any functional group or substituent added to a non-naturally encoded or naturally encoded amino acid incorporated into the IL-2 polypeptide. Alternatively, the water-soluble polymer is linked to an IL-2 polypeptide incorporating a non-naturally encoded amino acid via a naturally occurring amino acid (including but not limited to the amine group of cysteine, lysine, or an N-terminal residue). In some cases, the IL-2 polypeptide of the present invention comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-natural amino acids, wherein one or more non-naturally encoded amino acids are linked to a water-soluble polymer (including but not limited to PEG and/or oligosaccharides). In some cases, the IL-2 polypeptide of the present invention further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more naturally encoded amino acids linked to a water-soluble polymer. In some cases, the IL-2 polypeptide of the present invention comprises 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 some 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 present 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 by 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., hydrazide, hydrazine, hydroxylamine, or semicarbazide)

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 amine urea moiety directly linked 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

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

In certain embodiments, the PEG derivative containing hydrazine or hydrazide has the following structure: RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) _m -X-NH-NH ₂

Where R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10, and n is 100-1,000, and X is optionally a carbonyl group (C=O) which may be present or absent.

In certain embodiments, the PEG derivative containing a semicarbazide has the following structure: RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) _m -NH-C(O)-NH-NH ₂

Where 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 aminourea moiety linked to the PEG backbone via 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

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

In certain embodiments, the PEG derivative containing hydrazine or hydrazide 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

Where R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10, n is 100-1,000, and X is optionally a carbonyl group (C=O) which may be present or absent.

In certain embodiments, the PEG derivative containing a semicarbazide 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

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

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

Where R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10, and n is 100-1,000, and X is optionally a carbonyl group (C=O) which may be present or absent.

In certain embodiments, the PEG derivative containing a semicarbazide 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 -NH-C(O)-NH-NH ₂

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

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

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

The extent and location of the water-soluble polymer attached to the IL-2 polypeptide can modulate the binding of the IL-2 polypeptide to the IL-2 receptor. In certain embodiments, the attachment is arranged such that the IL-2 polypeptide binds to the IL-2 receptor with a K _d of about 400 nM or less, with a K _d of 150 nM or less, and in some cases with a K _d of 100 nM or less, as measured by an equilibrium binding assay, such as described in Spencer et al _. , J. Biol. Chem. , 263: 7862-7867 (1988).

Methods and chemistries for activation of polymers and for conjugation 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, functional group activation using cyanogen bromide, periodate, glutaraldehyde, bis(epoxy), epichlorohydrin, divinyl sulfide, carbodiimide, sulfonyl halides, trichlorotriazines, etc. (see R.F. Taylor, (1991), PROTEIN IMMOBILISATION. FUNDAMENTAL AND APPLICATIONS, Marcel Dekker, N.Y.; S.S. Wong, (1992), CHEMISTRY OF PROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; G.T. Hermanson et al., (1993), IMMOBILIZED AFFINITY LIGAND TECHNIQUES, Academic Press, NY). Press, N.Y.; Dunn, R.L. et al., eds., POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series Vol. 469, American Chemical Society, Washington, D.C. 1991).

Several reviews and separate books are available on the functionalization and conjugation of PEG. See, for example, Harris, Macromol. Chem. Phys. C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987); Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992); Zalipsky, Bioconjugate Chem. 6: 150-165 (1995).

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

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

The reaction products are then subjected to hydrophobic interaction chromatography to separate the PEGylated IL-2 polypeptide variant from free mPEG(5000)-O- _CH2 -C≡CH and any high molecular weight complexes of the PEGylated IL-2 polypeptide that may form when unblocked PEG is activated at both ends of the molecule to crosslink IL-2 polypeptide variant molecules. The conditions during the hydrophobic interaction chromatography are such that free mPEG(5000)-O- _CH2 -C≡CH flows through the column, while any crosslinked PEGylated IL-2 polypeptide variant complexes elute after the desired form, which contains one IL-2 polypeptide variant molecule conjugated to one or more PEG groups. Suitable conditions vary with the relative size of the crosslinked complex relative to the desired conjugate, and are readily determined by one of ordinary skill in the art. The eluent 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 produced PEG-IL-2 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%, at least about 95%, at least about 99% or more, as determined by appropriate methods such as SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis. If necessary, the PEGylated IL-2 polypeptide obtained from the hydrophobic chromatography 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, including but not limited to, DEAE SEPHAROSE), silica gel chromatography, reverse phase HPLC, gel filtration (using, including but not limited to, SEPHADEX G-75), hydrophobic interaction chromatography, size exclusion chromatography, metal chelate chromatography, ultrafiltration/filtration, ethanol precipitation, ammonium sulfate precipitation, chromatography focusing, 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 METHODS, A PRACTICAL APPROACH (Harris & Angal, eds.), 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 analysis. Pepinsky RB. et al., J. Pharmcol. & Exp. Ther. 297(3): 1059-66 (2001).

The water-soluble polymers linked to the amino acids of the IL-2 polypeptides of the present invention can be further derivatized or substituted without limitation.

PEG derivatives containing aziridine groups

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

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

Where 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., the average molecular weight is 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 chain of the branched PEG having a MW in the range of 10-40 kDa and may be in the range of 5-20 kDa. For example, in certain embodiments, the azido-terminal PEG derivative has the _following structure: [RO-( _CH2CH2O ) _n -O-( _CH2 ) ₂ - _NH -C(O)] _2CH ( _CH2 ) _m -X-( _CH2 ) _pN3

Where 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 carbonyl (C=O), which in each case may be present or absent.

Alkyne-containing PEG derivatives

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 the 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

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

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

Where 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-chain PEG derivative containing a terminal alkynyl moiety, each chain of the branched PEG having a MW in the range of 10-40 kDa 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

Where 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 carbonyl (C=O) or absent.

Phosphine-containing PEG derivatives

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

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

wherein n is 1-10; X can 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 refers 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 present invention includes more than one R group, each of the R groups is independently selected, as are each of the R', R", R''' and R'''' groups when there is more than one. 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 discussion of substituents above, one skilled in the art will understand that the term "alkyl" is meant to include groups containing carbon atoms bonded to groups other than hydrogen groups, such as halogenated alkyl groups (including, but not limited to, _-CF3 and _-CH2CF3 ) and acyl groups (including, but not limited to, -C(O) _CH3 , -C(O) _CF3 , -C(O) _{CH2OCH3 , etc.} ).

Other PEG Derivatives and General PEGylation Techniques

Other exemplary PEG molecules and PEGylation methods that can be attached to the IL-2 polypeptide include, but are not limited to, those described in, for example, U.S. Patent Publication Nos. 2004/0001838, 2002/0052009, 2003/0162949, 2004/0013637, 2003/0228274, 2003/0220447, 2003/0158333, 2003/0143596, 2003/0114647, 2003/0105275, 200 3/0105224, 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, U.S. Patent No. 6,64 6,110,5,824,778,5,476,653,5,219,564,5,629,384,5,736,625,4,902,502,5,281,698,5,122,614,5,473,034,5,516,673,5,382,657,6,552,167,6,610,281,6,515,100,6,461,603,6,436,386,6,214,966,5,990,237,5,9 00,461, 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, WO 95/13090, 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 and EP 154 316, which are incorporated herein by reference. Any PEG molecule described in the present invention may be used in any form, including but not limited to single chain, branched chain, multi-arm chain, monofunctional, bifunctional, multifunctional or any combination thereof.

Other polymers and PEG derivatives include, but are not limited to, hydroxylamine (amino) PEG derivatives, 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 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.

X. Glycosylation of IL-2 polypeptide

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

The sugar (including but not limited to glycosyl) moiety can be added to the IL-2 polypeptide in vivo or in vitro. 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 amineoxy group to produce a corresponding glycosylated polypeptide linked via an oxime bond. 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 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 non-naturally encoded amino acid containing a carbonyl group is directly modified with a polysaccharide of a defined structure that is prepared as an aminooxy derivative. One of ordinary skill in the art will recognize that other functional groups, including azides, alkynes, hydrazides, hydrazines, and aminoureas, can be used to link the sugar to the non-naturally encoded amino acid. In certain embodiments of the invention, an IL-2 polypeptide comprising a non-naturally encoded amino acid containing an azido or alkynyl group can then be modified with, including but not limited to, alkynyl or azido derivatives, respectively, by, including but not limited to, the Huisgen [3+2] cycloaddition reaction. This approach allows for modification of proteins with extremely high selectivity.

XI.IL-2 Dimers and Multimers

The present invention also provides IL-2 and IL-2 analog combinations such as homodimers, heterodimers, homopolymers or heteropolymers (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, and the binding is directly bound to the polypeptide backbone or through a linker. Due to the increased molecular weight compared to the monomer, the IL-2 dimer or multimer conjugate may exhibit new or desired properties, including but not limited to different pharmacology, pharmacokinetic, pharmacodynamic properties, regulated therapeutic half-life or regulated plasma half-life relative to monomeric IL-2. In certain embodiments, the IL-2 dimer of the present invention regulates signal transduction of the IL-2 receptor. In other embodiments, the IL-2 dimer or multimer of the present invention will act as an IL-2 receptor antagonist, agonist or modulator.

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 polypeptides and/or linked non-IL-2 molecules comprise 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 to be conjugated via a Huisgen [3+2] cycloaddition. Alternatively, an IL-2 and/or a linked non-IL-2 molecule comprising a ketone-containing non-naturally encoded amino acid can be conjugated to a second polypeptide comprising a hydroxylamine-containing non-naturally encoded amino acid and the polypeptides reacted via formation of a corresponding oxime.

Optionally, the two IL-2 polypeptides and/or the linked non-IL-2 molecules are linked via a linker. Any hetero- 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 molecules. In some cases, the linker used to tether the IL-2 and/or the linked non-IL-2 molecules together may be a bifunctional PEG agent. The linker may have a wide range of molecular weights or molecular lengths. Linkers of greater or lesser molecular weight may 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 any, between the linked entity and its binding partner. Linkers of greater or lesser molecular length may also be used to provide a desired spatial relationship or flexibility between IL-2 and the linked entity or, if any, between the linked entity and its binding partner.

In certain embodiments, the present invention provides a water-soluble bifunctional linker having a dumbbell structure, comprising: a) a component containing an azide, an alkyne, a hydrazine, a hydrazide, a hydroxylamine, or a carbonyl group at at least a first end of a polymer backbone; and b) at least one second functional group at 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 unreactive with the first functional group. In certain embodiments, the present invention provides a water-soluble compound comprising at least one arm of a branched molecular structure. For example, the branched molecular structure may be tree-like.

In certain embodiments, the present invention provides a multimer comprising one or more IL-2 polypeptides formed by reaction with a water-soluble activated polymer having the following 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 an azide, an alkyne, a hydrazine, a hydrazide, an amineoxy group, a hydroxylamine group, an acetyl group, or a carbonyl group, and R is a capping group, a functional group, or a leaving group which can be the same as or different from X. R can be, for example, a functional group selected from hydroxyl, protected hydroxyl, alkoxy, N-hydroxysuccinimidyl ester, 1-benzotriazolyl ester, N-hydroxysuccinimidyl carbonate, 1-benzotriazolyl carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, activated sulfonate, amine, aminooxy, protected amine, hydrazide, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimide, vinyl sulfonate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, tribenzoate, olefin and ketone.

XII. Measurement of IL-2 polypeptide activity and affinity of IL-2 polypeptides for IL-2 receptors

IL-2 polypeptide activity can be determined using standard or known in vitro or in vivo assays. PEG-IL-2 can be analyzed for biological activity by suitable methods known in the art. These assays include, but are not limited to, activation of IL-2 responsive genes, receptor binding assays, antiviral activity assays, cytopathic effect inhibition assays, antiproliferative assays, immunomodulatory assays, and assays monitoring the induction of MHC molecules.

The ability of PEG-IL-2 polypeptides to activate IL-2 sensitive signaling pathways can be analyzed. One example is the interferon stimulated response element (ISRE) assay. Cells constitutively expressing the IL-2 receptor are transiently transfected with an ISRE-luciferase vector (pISRE-luc, Clontech). Following transfection, the cells are treated with the IL-2 polypeptide. A number of protein concentrations, such as 0.0001-10 ng/mL, are tested to generate a dose response curve. If the IL-2 polypeptide binds to and activates the IL-2 receptor, the resulting signaling cascade induces luciferase expression. Luminescence can be measured in a number of ways, such as by using a TopCount ^™ or Fusion ^™ microplate reader and the Steady-Glo ^R luciferase assay system (Promega).

The ability of the IL-2 polypeptide to bind to the IL-2 receptor can be analyzed. For unpegylated or pegylated IL-2 polypeptides comprising non-natural amino acids, the affinity of IL-2 for its receptor can be measured using a BIAcore ^™ biosensor (Pharmacia). Suitable binding assays include, but are not limited to, the BIAcore assay (Pearce et al., Biochemistry 38:81-89 (1999)) and the AScreen ^™ assay (PerkinElmer).

Regardless of the method used to produce the IL-2 polypeptide, the IL-2 polypeptide is assayed for biological activity. Generally, the assay for biological activity should provide an analysis of the desired result, such as an increase or decrease in biological activity (compared to a modified IL-2), a different biological activity (compared to a modified IL-2), a receptor or binding partner affinity analysis, a conformational or structural change in IL-2 itself or its receptor (compared to a modified IL-2), or a serum half-life analysis.

XIII. Measurement of potency, functional in vivo half-life and pharmacokinetic parameters

An important aspect of the present invention is to obtain extended biological half-life by constructing IL-2 polypeptides with or without conjugated water-soluble polymer components. The rapid decline in IL-2 polypeptide serum concentration after administration makes it important to evaluate the biological response to treatment with conjugated and non-conjugated IL-2 polypeptides and variants thereof. The conjugated and non-conjugated IL-2 polypeptides and variants thereof of the present invention may also have an extended serum half-life after administration, such as by subcutaneous or i.v. administration, making it possible to measure by, for example, ELISA methods or by primary screening assays. ELISA or RIA kits from commercial sources such as Invitrogen (Carlsbad, CA) can be used. Measurement of in vivo biological half-life is performed as described in the present invention.

The potency and functional in vivo half-life of IL-2 polypeptides comprising non-naturally encoded amino acids 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 evaluated in normal Sprague-Dawley male rats (N=5 animals per treatment group). Animals receive a single dose of 25ug/rat iv or 50ug/rat sc, and approximately 5-7 blood samples are obtained over a predetermined time course that typically covers about 6 hours for IL-2 polypeptides comprising non-naturally encoded amino acids that are not conjugated to a water-soluble polymer to about 4 days for IL-2 polypeptides comprising non-naturally encoded amino acids and conjugated to a water-soluble polymer. Pharmacokinetic data for IL-2 without non-naturally encoded amino acids can be directly compared to data obtained for IL-2 polypeptides comprising non-naturally encoded amino acids.

XIV. Administration and Drug Compositions

The polypeptides or proteins of the present invention (including but not limited to IL-2, synthetases, proteins containing one or more non-natural amino acids, etc.) are optionally used for therapeutic purposes, including but not limited to combination with suitable pharmaceutical carriers. These compositions contain, 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 manufactured to be suitable for the mode of administration. In general, methods for administering proteins are known to those of ordinary skill in the art and can be applied to the administration of the polypeptides of the present invention. The composition can be in a water-soluble form, for example, as a pharmaceutically acceptable salt, which means both acid and base addition salts.

The therapeutic composition comprising one or more polypeptides of the present invention is optionally tested in one or more suitable in vitro and/or in vivo disease animal models according to methods known to those of ordinary skill in the art to confirm efficacy, tissue metabolism and estimate dosage. Specifically, the dosage can be initially determined by comparing the activity, stability or other suitable metrics of the non-natural polypeptides of the present invention with natural amino acid homologs (including but not limited to IL-2 polypeptides modified to include one or more non-natural amino acids with natural amino acid IL-2 polypeptides and IL-2 polypeptides modified to include one or more non-natural amino acids with currently available IL-2 treatments), i.e., in relevant assays.

Administration is performed by any route commonly used to introduce molecules for ultimate contact with blood or tissue cells. The non-natural amino acid polypeptides of the present invention are administered in any suitable manner, optionally with one or more pharmaceutically acceptable carriers. Suitable methods for administering these polypeptides to patients in the context of the present invention are available, and although 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 is determined in part by the specific composition to be administered and by the specific 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 polypeptide of the present invention can be administered by any conventional route suitable for proteins or peptides, including but not limited to parenteral administration, such as injection, including but not limited to subcutaneous or intravenous or any other form of injection or infusion. The polypeptide composition can be administered by a variety of routes (including but not limited to oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual or rectal). Compositions containing modified or unmodified non-natural 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 can be used alone or in combination with other suitable components such as drug carriers. The IL-2 polypeptide can be used in combination with other agents or therapeutic agents.

IL-2 polypeptides containing unnatural amino acids, alone or in combination with other suitable components, can also be formulated into aerosol formulations (i.e., they can be "nebulized") for administration by inhalation. Aerosol formulations can be placed in an acceptable pressurized propellant such as dichlorodifluoromethane, propane, nitrogen, etc.

Formulations suitable for parenteral administration, such as by intraarticular, intravenous, intramuscular, intradermal, intraperitoneal and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats and solutes that impart isotonicity to the preparation with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickening agents, stabilizers and preservatives. IL-2 formulations may be present in single-dose or multi-dose sealed containers such as ampoules and vials.

Parenteral administration and intravenous administration are preferred methods of administration. Specifically, the administration routes that have been used for natural amino acid homolog therapeutics (including but not limited to the administration routes commonly used for EPO, GH, G-CSF, GM-CSF, IFN such as IL-2, interleukin, antibodies, FGF and/or any other drug delivery proteins) and the formulations currently in use provide preferred administration routes and formulations for the polypeptides of the present 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 the patient over time, depending on the application. The dose is determined by the efficacy of the particular carrier or formulation, the activity, stability or serum half-life of the non-natural amino acid polypeptide used, 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, etc. in a particular patient.

In determining the effective amount of a vector or formulation to be administered in the treatment or prevention of a disease including but not limited to neutropenia, aplastic anemia, cyclical neutropenia, idiopathic neutropenia, Chdiak-Higashi syndrome, systemic lupus erythematosus (SLE), leukemia, myelodysplastic syndrome, and myelofibrosis, physicians assess circulating plasma levels, formulation toxicity, disease progression, and/or the development of anti-unnatural amino acid polypeptide antibodies when relevant.

The dosage administered to a patient weighing, for example, 70 kg is generally within the range equivalent to the dosage of commonly used therapeutic proteins and is adjusted according to the altered activity or serum half-life of the relevant composition. The vector or drug formulation of the present invention can be supplemented with any known conventional therapy (including administration of antibodies, vaccines, cytotoxic agents, natural amino acid peptides, nucleic acids, nucleotide analogs, biological response improvers, etc.) to supplement the therapeutic conditions.

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

If a patient undergoing infusion of the formulation develops fever, chills, or muscle aches, he/she receives appropriate doses of aspirin, ibuprofen, acetaminophen, or other pain/fever control medications. Patients who experience reactions to infusions such as fever, muscle aches, and chills are premedicated 30 minutes prior to future infusions with aspirin, acetaminophen, or medications including but not limited to diphenhydramine. Perthidine is used for more severe chills and muscle aches that do not respond quickly to antipyretics and antihistamines. Cell infusions are slowed or interrupted depending on the severity of the reaction.

The human IL-2 polypeptides of the present invention can be administered directly to a mammalian subject. Administration is by any route commonly used to introduce IL-2 polypeptides to a subject. The IL-2 polypeptide compositions described in accordance with the 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, intraarticular, 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 depends on the nature and severity of the condition to be treated. Administration can be local or systemic. The formulation of the compound may be present in single-dose or multi-dose sealed containers such as ampoules and vials. The IL-2 polypeptide of the present invention may be prepared as a mixture (including but not limited to a solution, suspension or emulsion) with a pharmaceutically acceptable carrier in a unit dose injection form. The IL-2 polypeptide of the present invention may also be administered by continuous infusion (using, including but not limited to, a small pump such as an osmotic pump), a single rapid concentration injection or a slow release depot formulation.

Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions that may contain antioxidants, buffers, bacteriostats and solutes that impart isotonicity to the formulation, 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 types previously described.

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

Spray drying of pharmaceuticals is also known to those of ordinary skill in the art. See, for example, Broadhead, J. et al., The Spray Drying of Pharmaceuticals, Drug Dev. Ind. Pharm, 18 (11 & 12), 1169-1206 (1992). In addition to small molecule drugs, a variety of different biological materials have been spray dried, including enzymes, serum, plasma, microorganisms and yeast. Spray drying is a useful technique because it can convert liquid pharmaceutical formulations into fine, dust-free or agglomerated powders in a one-step process. The basic technology includes the following four steps: a) atomization of feed solution into spray; b) spray-air contact; c) drying of 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 contain a pharmaceutically acceptable carrier, excipient or stabilizer. The pharmaceutically acceptable carrier is determined in part by the specific composition to be administered and by the specific method used to administer the composition. Thus, there are a wide variety of suitable formulations (including optional pharmaceutically acceptable carriers, excipients or stabilizers) for the pharmaceutical compositions of the present invention (see, e.g., Remington 's Pharmaceutical Sciences , 17th edition, 1985).

Suitable carriers include, but are not limited to, buffers, including succinate, phosphate, borate, 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 with 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, glutamine or lysine; monosaccharides, disaccharides and other sugars, including but not limited to trehalose, sucrose, glucose, mannose or dextrin; chelating agents, including but not limited to EDTA and disodium ethylenediaminetetraacetate; divalent metal ions, including but not limited to zinc, cobalt or copper; sugar alcohols, including but not limited to mannitol or sorbitol; salt-forming counter ions, including but not limited to sodium and sodium chloride; fillers such as microcrystalline cellulose, lactose, corn and other starches; binders; sweeteners and other flavorings; coloring agents; and/or non-ionic surfactants, including but not limited to Tween ^™ (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 not limited to, polyethers based on polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO) or polypropylene oxide-polyethylene oxide-polypropylene oxide (PPO-PEO-PPO) or combinations thereof. PEO-PPO-PEO and PPO-PEO-PPO are commercially available under the trade names Pluronics ^TM , R-Pluronics ^TM , Tetronics ^TM and R-Tetronics ^TM (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. Surfactants or combinations of surfactants may be used to stabilize PEGylated IL-2 against one or more stresses, including but not limited to stress caused by agitation. Some of the above substances may be referred to as "enhanced surfactants." Dosing agent". Certain substances may also be referred to as "tonicity adjusters". Antimicrobial preservatives may also be used to achieve product stability and antimicrobial effectiveness; suitable preservatives include, but are not limited to, benzyl alcohol, ammonium benzoate, m-cresol, methyl/propyl paraben, cresol and phenol or combinations thereof. U.S. Patent No. 7,144,574, incorporated herein by reference, describes other materials that may be suitable for the pharmaceutical compositions and formulations and other delivery formulations of the present invention.

The IL-2 polypeptides of the present invention, including IL-2 polypeptides linked to water-soluble polymers such as PEG, can also be administered via 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 those derived from biocompatible materials such as poly(2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. , 15:267-277 (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. Patent No. 3,773,919; EP 58,481), polyglycolide (polymer of glycolic acid), polylactide-co-glycolide (copolymer of lactic acid and glycolic acid), polyanhydrides, copolymers of L-glutamine and γ-ethyl-L-glutamine (Sidman et al., BioPolymers , 22, 547-556 (1983)), polyorthoesters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinyl pyrrolidone and silicone. Sustained release compositions also include liposome-encapsulated compounds. Liposomes containing the compound are prepared by methods known per se: DE 3,218,121; Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad . Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; U.S. Patent No. 4,619,794; EP 143,949; U.S. Patent No. 5,021,234; Japanese Patent Application No. 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and EP 102,324. All references and patents cited are incorporated herein by reference.

Liposome-encapsulated IL-2 polypeptide can be prepared by the methods described in, for example, DE 3,218,121; Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc . Natl. Acad. Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; U.S. Patent No. 4,619,794; EP 143,949; U.S. Patent No. 5,021,234; Japanese Patent Application No. 83-118008; U.S. Patent 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 in, for example, the following references: Park JW et al., Proc. Natl. Acad. Sci. USA 92: 1327-1331 (1995); Lasic D and Papahadjopoulos D, eds., MEDICAL APPLICATIONS OF LIPOSOMES (1998); Drummond DC et al., Liposomal drug delivery systems for cancer therapy, Teicher B, ed., CANCER DRUG DISCOVERY AND DEVELOPMENT (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 induce a beneficial response in the subject over time. Typically, the total therapeutically effective amount of the IL-2 polypeptide of the present invention per parenteral dose is in the range of about 0.01 μg/kg to about 100 μg/kg or about 0.05 mg/kg to about 1 mg/kg of patient body weight per day, although this depends on therapeutic judgment. In certain cases of this embodiment, the conjugate may be administered in a dose ranging from greater than 4 μg/kg per day to about 20 μg/kg per day. In other cases, the conjugate may be administered in a dose ranging from greater than 4 μg/kg per day to about 9 μg/kg per day. In other cases, the conjugate can be administered in a dose ranging from about 4 μg/kg per day to about 12.5 μg/kg per day. In certain cases, the conjugate can be administered at a dose equal to or less than the maximum dose tolerated without excessive toxicity. In addition, the conjugate can be administered at least twice per week, or the conjugate can be administered at least three times per week, at least four times per week, at least five times per week, at least six times per week, or seven times per week. In certain cases, where the conjugate is administered more than once, the conjugate can be administered at a dose greater than 4 μg/kg per day. Specifically, the conjugate can be administered over a period of two weeks or longer. In certain instances, the growth of cells expressing the 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 the conjugate of the present invention. In certain instances of this embodiment, the conjugate may be administered at a dose of about 5.3 μg/kg per day, or at a dose of about 7.1 μg/kg per day, or at a dose of about 9.4 μg/kg per day, or at a dose of about 12.5 μg/kg per day. The frequency of administration also depends on therapeutic judgment and may be more or less frequent than commercially available IL-2 polypeptide products approved for use in humans. Generally, the IL-2 polypeptide, PEGylated IL-2 polypeptide, conjugated IL-2 polypeptide or PEGylated conjugated IL-2 polypeptide of the present invention can be administered via any of the above-mentioned administration routes.

XV. Therapeutic Uses of the IL-2 Polypeptides of the Present Invention

The IL-2 polypeptides of the present invention can be used to treat a wide range of disorders. The present invention also includes methods of treating mammals at risk for, suffering from, and/or suffering from cancer that is responsive to IL-2, CD8+ T-cell stimulation, and/or IL-2 preparations. Administration of IL-2 polypeptides can produce short-term effects on several observed clinical parameters, i.e., immediate beneficial effects, which can be 12 or 24 hours from administration, and on the other hand, long-term effects, 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 can be administered by any means known to those skilled in the art, and can advantageously be administered by infusion, for example by arterial, intraperitoneal or intravenous injection and/or infusion in an amount sufficient to obtain the desired pharmacological effect.

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

The average amount of IL-2 can vary and should be based on the 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 condition to be treated, the condition of the patient to be treated, and the other ingredients in the composition. The present 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 a person of ordinary skill in the art based on the therapy using IL-2.

Embodiment

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

Example 1 - Determination of the residue position in IL-2 to be mutated into an amber stop codon to incorporate a non-natural amino acid

IL-2 has been used to treat several cancers such as renal cell carcinoma and metastatic melanoma. Commercially available IL-2 Aldesleukin® is a recombinant protein that is not glycosylated, has alanine-1 removed and has cysteine-125 replaced with serine-125 (Whittington et al., Drugs, 46(3): pp: 446-514 (1993)). Although IL-2 is the earliest cytokine approved by the FDA for cancer treatment, it has been shown to exhibit severe side effects when used in high doses. This greatly limits its use in potential patients. The underlying mechanism of the severe side effects has been attributed to the binding of IL-2 to one of its receptors, IL-2Rα. Normally, when all three receptors including IL-2Rα (or CD25), IL-2Rβ (or CD122), and IL-2Rγ (or CD132) are present in tissues, IL-2 can form heterotrimeric complexes not only with these receptors, but also with IL-2Rβ and IL-2Rγ. In a clinical setting, when high doses of IL-2 are used, IL-2 begins to bind to IL-2αβγ, which is the main receptor form in T _reg cells. The inhibitory effect of T _reg cells causes an unwanted effect of the use of IL-2 in cancer immunotherapy. In order to reduce the side effects of IL-2, many methods have been used before. One of the major breakthroughs came from the invention of Nektar, which uses 6 PEGylated lysines to mask the IL2Rα 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 exhibits dramatically reduced side effects. However, results from activity studies showed that the effective form of PEGylated IL-2 in this heterogeneous 6-PEGylated IL-2 mixture is only the monoPEGylated form. Therefore, there is a need for a more effective PEGylated IL-2 with a homogeneous product.

In this application, the incorporated non-natural amino acids provide unique conjugation sites for use in IL-2 PEGylation. The resulting PEGylated IL-2 mutant protein has a homogeneous product rather than the previous heterogenous 6-PEGylated IL-2 from Nektar.

*U：非天然胺基酸 The sites used in generating IL-2 mutant proteins can be selected by analyzing the existing crystal structures of IL-2 and its heterotrimeric receptor. Specifically, the structure of IL-2Rα and its interface with IL-2 has been investigated in detail (Figure 1). The interface is divided into two regions containing a hydrophobic center and a polarized region. The hydrophobic center is formed between the IL-2Rα residues Leu-2 ^α , Met-25 ^α , Leu-42 ^α and Tyr-43 ^α and the IL-2 residues Phe-42 ^IL-2 , Phe-44 ^IL-2 , Tyr-45 ^IL-2 , Pro-65 ^IL-2 and Leu-72 ^IL-2 . The polarization region is formed between IL-2Rα and five ion pairs of IL-2, including Lys-38 ^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 . In addition, electrostatic mapping suggests that certain other residues may play a role in mediating the interaction between IL-2Rα 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, the sites that can be used are 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 generate mutant proteins with non-natural amino acids is listed in Table 2 below: Table 2. IL-2 protein sequences with potential sites for use in PEGylation

*U: Unnatural amino acid

Example 2: Human IL-2 expression system

This section describes methods for expressing IL-2 polypeptides comprising non-natural amino acids. Host cells are transformed with an orthogonal tRNA, an orthogonal amidyl tRNA synthetase, and a construct comprising a selector codon encoding a polynucleotide encoding an IL-2 polypeptide as shown in SEQ ID NO: 4, 6, or 8, or a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO: 1, 2, 3, 5, 7, and 9 to 23.

E. coli expression vector construction and sequence verification : This example details the replication and expression of human IL-2 (hIL-2) including non-naturally encoded amino acids in E. coli. All human IL-2 expression plasmids were constructed in E. coli NEB5α clone strain (New England Biolabs, MA) by a recombination-based cloning method using a Gibson assembly kit (New England Biolabs (NEB), Ipswich, MA) or using a QuikChange mutation kit (Agilent Technologies, Santa Clara, CA) as described below. The E. coli expression plasmid is shown in Figure 2.

Gibson assembly : Primers for amplification of various genes of interest (GOI) containing donor fragments had approximately 18-24 base pairs (bp) of sequence overlapping with the acceptor vector sequence at their 5'-end for homologous recombination and were synthesized at Integrated DNA Technologies (IDT) (San Diego, CA). The PCR fragments were amplified using a high-fidelity DNA polymerase mixture Pfu Ultra II Hotstart PCR Master Mix (Catalog No. 600852, Agilent Technologies). The PCR products were digested with Dpn1 restriction enzyme (NEB # R0176L) at 37°C for 2 hours to remove plastid 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 a restriction enzyme unique to the vector (NEB, MA) at the temperature recommended by the supplier for 3 to 5 hours, PCR column purified and quantified. The donor insert fragment and the appropriately prepared recipient vector were mixed at a molar ratio of 3:1, incubated at 50°C for 15 minutes using a Gibson assembly kit (NEB # E2611S), and then used for transformation into E. coli NEB5α strain (NEB # 2987).

Recombinants were recovered by plating the Gibson assembly mixture on LB agar plates containing the appropriate antibiotics. The next day, 4 to 6 well-isolated single colonies were inoculated into 5 mL LB + 50 μg/mL kanamycin sulfate (Sigma, St Louis, MO; cat# K0254) medium and grown overnight at 37°C. Recombinant plasmids were isolated using the Qiagen plasmid DNA miniprep kit (Qiagen #27104) and verified by DNA sequencing (Eton Biosciences, San Diego, CA). The entire GOI region plus 100 bp upstream and 100 bp 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 the QuickChange Web Portal (Agilent Technologies) and ordered from IDT (San Diego, CA). The QCM PCR mix contained 5 μl 10x buffer, 2.5 μl dNTP mix, 1 μl (100 ng) plasmid template, 1 μl oligo mix (10 uM concentration each), 1 μl QuickChange Lightning enzyme, 2.5 μl Quick solution, and 37 μl distilled water (DW). The DNA was amplified for only 18 cycles using the PCR program recommended by the kit.

After the PCR reaction was completed, the mixture was digested with the DpnI enzyme that came with the kit (Agilent Technologies) at 37°C for 2-3 hours and run on a gel to check for the presence of the amplified PCR product. Subsequently, 2.5 to 5 μl of the PCR product was transformed into the E. coli NEB5α strain. Recombinant plasmids from 4 to 6 colonies were then isolated and sequence verified as described in the Gibson assembly section above.

Construction and verification of expression strain (AXID): To prepare the AXID production strain, chemically competent E. coli W3110B60 host cells were transformed with sequence-verified plasmid DNA (50 ng), and recombinant cells were selected on 2xYT+1% glucose agar plates containing 50 μg/mL kanamycin sulfate (Sigma, cat# K0254) and incubated overnight at 37°C. Single colonies from fresh transformation plates were then propagated three times by streaking three times on 2xYT+1% glucose agar plates containing 50 μg/mL kanamycin sulfate and incubated overnight at 37°C. Finally, a single colony from the third streak plate was inoculated into 20 mL Super Broth (Fisher-Optigrow ^™ , #BP1432-10B1) containing 50 μg/mL kanamycin sulfate (Sigma, cat# K0254) and cultured overnight at 37°C and 250 rpm. The overnight culture was then diluted with glycerol to a final glycerol concentration of 20% (w/v) (KIC, Ref# 67790-GL99UK). The cell suspension was then distributed into 1 mL aliquots, placed in several cryovials and frozen at -80°C as AXID production strain tubes.

After glycerol tubes of the AXID production strain were generated as described above, they were further verified by DNA sequencing and phenotypic characterization of antibiotic resistance markers. To confirm that the AXID production strain tubes had the correct plasmid in the production host, the plasmids were sequence verified. 20 mL LB containing 50 μg/mL kanamycin sulfate was inoculated with a stab from a glycerol tube of the AXID clone and grown overnight at 37°C and 250 rpm. Plasmid DNA was isolated using a Qiagen miniprep kit (#27104) and the presence of the intact GOI ORF in the isolated plasmid was confirmed by DNA sequencing (Eton Biosciences, CA).

To further verify the strain genotype of the AXID production strain, cells from the same tube were streaked on 4 separate 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 checked for positive growth on all of 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 sequence encoding hIL-2 are shown in Tables 1 and 2. A translation system comprising an orthogonal tRNA (O-tRNA) and an orthogonal amido-tRNA synthetase (O-RS) is used to express hIL-2 containing non-naturally encoded amino acids (see plasmid map pKG0269; Figure 2). The O-RS preferentially amines the O-tRNA with non-naturally encoded amino acids. Furthermore, the translation system responds to the encoded selector codons to insert the non-naturally encoded amino acids into IL-2 or IL-2 variants. Suitable O-RS and O-tRNA sequences are described in WO2006/068802 entitled "Compositions of Aminoacyl-tRNA Synthetase and Uses Thereof" and WO2007/021297 entitled "Compositions of tRNA and Uses Thereof", which are incorporated herein by reference in their entirety.

E. coli was transformed with a plasmid containing the modified IL-2 variant polynucleotide sequence and an orthogonal amidyl tRNA synthetase/tRNA pair specific for the desired non-naturally encoded amino acid, allowing 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 the addition of arabinose to the culture medium (see plasmid map pKG0269; Figure 2).

Inhibition using p-acetyl-phenylalanine (pAF): Plasmids for expression of IL-2 polypeptides are transformed into W3110B60 E. coli cells. p-acetyl-phenylalanine (pAF) is added to the cells, and protein expression is induced by the addition of arabinose. SDS-PAGE analysis of expression of IL-2 polypeptide is performed and observed. Multiple lanes are run for comparison between the original wild-type IL-2 polypeptide and the pAF-substituted IL-2 polypeptide, which is an IL-2 with a substitution made at a specific amino acid residue, such as p-acetylphenylalanine. Expression of T7 polymerase is under the control of the arabinose-inducible T7 phage promoter. p-Acetyl-phenylalanine (pAF) was added to the cells and protein expression was induced by the addition of arabinose (final 0.2%). The culture was incubated at 37°C for several hours (3-5 hours).

Other constructs for improving hIL-2 expression in E. coli : To improve the production of hIL-2 in E. coli, in addition to the DNA sequence optimization based on the codon usage of E. coli reported in the present invention, the following expression parameters can be further optimized: testing different promoters besides 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 in addition to the standard W3110B60 strain; optimization of production process parameters such as temperature, culture medium, inducer concentration, etc.; optimization of transcription and translation control elements, such as start and stop codons, ribosome binding site (RBS), transcription terminator, etc.; optimization of plastid copy number and plastid stability; optimization of translation initiation region (TIR).

Example 3 - This example describes in detail the shake flask expression assay and high cell density fermentation of E. coli

Shake-flask expression testing : hIL-2 expression was tested in a shake-flask assay using the AXID producer strain as described above. Briefly, inocula from AXID glycerol tubes were placed in 5 mL Super Broth (Fisher-Optigrow™, #BP1432-10B1) containing 50 μg/mL kanamycin sulfate (Sigma, MO) and grown overnight at 37°C with shaking. The overnight culture was diluted 1:100 in Super Broth ( ^Fisher -Optigrow ^™ , #BP1432-10B1) containing 50 μg/mL kanamycin sulfate (Sigma, MO) and grown at 37°C with shaking. When the culture density reached an OD600 of 0.6-0.8, it was induced with added 0.2% arabinose and pAF and then harvested after a few hours of production (usually 3 to 5 hours). An aliquot of the harvested cells was removed and analyzed by SDS-PAGE. The optimal expression of hIL-2 was standardized by varying the temperature, duration of induction, and inducer concentration. The expression of hIL-2 was confirmed by immunoblotting of crude extracts using a standard single clone antibody against hIL-2 according to the following Western assay for analysis of hIL-2 expression (Figure 3A): The harvested cell pellets were normalized to an OD600 of 5 and dissolved in a calculated amount of B-PER solution (ThermoFisher) containing lysozyme (100 μg/ml) and DNase 1 (1 U/ml). The pellets were mixed by vortexing at high speed for 2-5 minutes and by incubation of the mixture at 37°C and 250 rpm. The samples were mixed with the sample buffer (4X) and sample reducing agent (10x) provided by the manufacturer, and the final concentration was adjusted to 1x. A total of 20 μl of sample was loaded onto a pre-made polyacrylamide gel (ThermoFisher) along with hIL2 standards (R&D Systems, Minneapolis, MN) and separated by electrophoresis in 1× MES buffer (ThermoFisher). Protein samples were transferred to a nitrocellulose membrane using an iBlot device and gel transfer stacking. hIL2 was captured with a goat anti-human IL-2 antigen antibody (R&D Systems) and detected by an HRP-conjugated anti-goat IgG secondary antibody (R&D Systems) using opti 4CN colorimetric reaction substrate (Bio-Rad, Hercules, CA).

High cell density fermentation : The fermentation process for the production of hIL-2 consists of two stages: (i) inoculum preparation, and (ii) fermenter production. The inoculum was started from a single glycerol tube, thawed, diluted 1:1000 (v/v) in 50 mL defined seed medium in a 250 mL baffled Erlenmeyer flask, and incubated at 37°C and 250 rpm. Prior to use, the fermenter was cleaned and autoclaved. A specific amount of basal medium was added to the fermenter and autoclaved. A specific amount of kanamycin sulfate solution, feed medium, and P2000 defoamer were added to the basal medium prior to inoculation. All solutions added to the fermenter after autoclaving were either 0.2 μm filtered or autoclaved before aseptic addition.

The fermenter was batch-charged with 4 L of chemically defined medium using glycerol as a carbon source. Seed culture was added to the fermenter to an initial OD600nm of 0.05. Dissolved oxygen was maintained at 30% air saturation using agitation of 480 to 1200 rpm and oxygen enrichment with a head pressure of 6 psig and an air flow of 5 slpm. Temperature and pH were controlled at 37°C and 7.0, respectively. When the culture reached an OD600nm of 35±5, feeding began 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 the dipeptide was added, the culture was induced with L-arabinose at a final concentration of 2 g/L. Cultures were harvested 6 h after induction.

Reverse phase HPLC titer analysis: 1.0 mL of E. coli fermentation sample (cell paste) was first dried and weighed to determine the quality used for sample preparation. Lysonase Bioprocessing Reagent (EMD Millipore #71230) and Benzonase Nuclease Reagent (EMD Millipore #70664) were each diluted 1:500 in BugBuster Protein Extraction Reagent (EMD Millipore #70584) and used for chemical lysis of cell paste. 1.0 mL of the Bugbuster-Lysonase-Benzonase mixture was added to 1.0 mL of dried cell paste, and the resulting mixture was vortexed vigorously. The mixture was then placed on an Eppendorf Thermomixer R shaker and shaken at 1000 rpm for 20 minutes at 22°C. After incubation, the cell lysate was centrifuged at 16,050 rcf for 5 minutes to sediment cell debris. A 200 μL aliquot of the cell lysate supernatant was then filtered through a 0.22 μm PVDF centrifugal tube screen (EMD Millipore #UFC30GVNB) at 16,050 rcf for 1 minute. The filtered product was then analyzed by reverse phase chromatography to determine the amount of hIL2 present in the fermentation sample. hIL2 was separated from host cell protein contaminants using a 4.6 x 150 mm Zorbax 300SB-C3 (Agilent #863973-909) reverse phase column packed with 3.5 μm particles. 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 hIL2 from the column. The amount of hIL2 in the sample was determined by comparing the observed area counts from a fixed injection volume to a linear equation obtained from a standard curve generated using purified hIL2. Several tested IL-2 amber variants, as exemplified in Figure 3B, showed high titer expression in the high cell density E. coli fermentation ranging from approximately 65 to 150 mg/L.

Example 4 - This example describes in detail the preparation, refolding, purification and PEGylation of inclusion bodies.

Inclusion body preparation and lysis: Cell paste harvested from high cell density fermentation was resuspended in inclusion body (IB) buffer I (50 mM Tris pH 8.0; 100 mM NaCl; 1 mM EDTA; 1% Triton X-100; 4°C) at 4°C with mixing to a final 10% solids. Cells were lysed by passing the resuspended material through a microfluidizer a total of two times. Samples were then centrifuged (14,000 g; 15 min; 4°C) and the supernatant was decanted. The inclusion body pellet was washed by resuspending in an additional volume of IB Buffer I (50 mM Tris pH 8.0; 100 mM NaCl; 1 mM EDTA; 1% Triton X-100; 4°C) and passing the resuspended material through the microfluidizer a total of two times. The samples were then centrifuged (14,000 g; 15 min; 4°C) and the supernatant was decanted. The inclusion body pellets were each resuspended in 1 volume of Buffer II (50 mM Tris pH 8.0; 100 mM NaCl; 1 mM EDTA; 4°C). The samples were centrifuged (14,000 g; 15 min; 4°C) and the supernatant was decanted. Resuspend the inclusion body pellet in ½ volume of Buffer II (50 mM Tris pH 8.0; 100 mM NaCl; 1 mM EDTA; 4°C). Then divide the inclusion bodies into aliquots in suitable containers. Centrifuge the samples (14,000 g; 15 min; 4°C) and decant the supernatant. Dissolve the inclusion bodies or store at -80°C until further use.

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

Refolding and Purification: Refolding was performed by diluting the sample to a final concentration of 0.5 mg/mL in 20 mM Tris, pH 8.0; 60% sucrose; 4°C. Refolding was allowed to proceed for 5 days at 4°C. The refolded material was diluted 1:1 with Milli-Q _H2O . The material was filtered through a 0.22 μm PES filter and loaded onto a Blue Sepharose FF column (GE Healthcare) equilibrated in 20 mM Tris, pH 8.0; 0.15 M NaCl (Buffer A). In the wash flow, the column was washed with 5 column volumes of 30% Buffer B (20 mM Tris, pH 8.0; 2 M NaCl; 50% ethylene glycol). IL-2 polypeptide was eluted by flushing the column with 10 column volumes of 100% buffer B.

PEGylation and post-PEGylation purification : Take the IL-2 pool and dilute 10X with Milli-Q water. Adjust the pH of each sample to 4.0 with 50% glacial acetic acid. Concentrate the samples to ~1.0 mg/mL. Add a 1:12 molar excess of activated PEG (hydroxylamine PEG) to each sample. Then incubate the samples at 27°C for 48-72 hours. Remove the samples and dilute the samples 8-10 times with water (<8m/S) and load onto a SP HP column (GE Healthcare) equilibrated in buffer A (50mM NaAc, pH 6.0; 50mM NaCl; 0.05% Zwittergent 3-14). IL-2 polypeptide was eluted with 5 column volumes of buffer B (50 mM NaAc, pH 6.0; 500 mM NaCl; 0.05% Zwittergent 3-14). IL-2 fractions were combined and run on a Superdex 200 size exclusion column equilibrated in IL-2 storage buffer (20 mM NaAc, pH 5.0; 150 mM NaCl; 0.05% Zwittergent 3-14). The PEGylated material was collected and stored at 4°C.

Example 5 - This example describes in detail 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 processes.

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 (50 mM Tris, pH 8.0; 1% triton X-100; 1 M urea, 5 mM EDTA, 1 mM PMSF) to a concentration of 10% (W/V), homogenized at 4°C, and then centrifuged (15,000 g, 30 min, 4°C). The supernatant was discarded and the inclusion body pellet was resuspended in wash buffer II (50 mM Tris, pH 8.0; 1% triton X-100; 1 M urea, 5 mM EDTA). The resuspended inclusion bodies were centrifuged at 15,000 g for 30 min at 4°C. The supernatant was discarded and the inclusion body pellet was resuspended in wash buffer III (50 mM Tris, pH 8.0; sodium deoxycholate, 5 mM EDTA). The resuspended inclusion bodies were centrifuged at 15,000 g for 30 min at 4°C. The supernatant was discarded and the inclusion body pellet was resuspended in water and then centrifuged at 15,000 g for 30 min at 4°C. The washed inclusion bodies were stored at -80°C 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 (12,000 g, 15 min). The solubilized IL-2 was refolded by lowering the pH to pH 8.8 ± 0.2. Appropriate disulfide bond formation was promoted by adding 50 μM 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 (12,000 g, 15 min), and the clear supernatant was adjusted to pH 7.8 with sodium hydroxide and filtered through 0.22 μm.

Column Purification: The refolded IL-2 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 to 100% buffer B (20 mM sodium phosphate, 20 mM citric acid, pH 4.0) over 20 column volumes. Fractions containing IL-2 were collected, the pH was adjusted to 4.0 with 10% acetic acid, and then buffer exchanged in 20 mM sodium acetate, 2.5% trehalose, pH 4.0. IL-2 was concentrated to 1-10 mg/mL, filtered at 0.22 μM, and stored at -80°C.

Purification of IL-2 from eukaryotic expression systems: Cell culture medium containing His-tagged IL-2 was adjusted to pH 7.8 with sodium hydroxide and loaded onto a Ni Excel column (GE Healthcare) equilibrated in 20 mM 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, 1.0 M sodium chloride, 30 mM imidazole, pH 7.8) to remove host cell contaminants. IL-2 was eluted from the column with elution buffer (10 mM sodium phosphate, 300 mM imidazole, pH 7.8) and fractions containing IL-2 were pooled. The IL-2 pooled material 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 to 100% buffer B (20 mM sodium phosphate, 20 mM citric acid, pH 4.0) over 20 column volumes. Fractions containing IL-2 were collected, the pH was adjusted to 4.0 with 10% acetic acid, and then buffer exchanged in 20 mM sodium acetate, 2.5% trehalose, pH 4.0. IL-2 was concentrated to 1-10 mg/mL, filtered at 0.22 μM, and stored at -80 °C until further use.

Site Specific Conjugation and PEG-IL-2 Purification: IL-2 variants containing the non-natural amino acid (nnAA) p-acetylphenylalanine were buffer exchanged in conjugation buffer (20mM sodium acetate, pH 4.0) and concentrated to 1-10mg/mL. A final 100mM acetic acid hydrazide was added to the reaction, followed by a 10-fold molar excess of amineoxy-functionalized PEG. The conjugation reaction was incubated at 25-30°C for 18-20 hours. After conjugation, the PEGylated IL-2 was diluted 1:10 with 20mM sodium acetate, pH 5.0, and loaded onto a 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 to 100% buffer B (20 mM sodium acetate, 1.0 M sodium chloride, pH 5.0) in 20 column volumes. Fractions containing PEGylated IL-2 were collected and buffer exchanged in 10 mM sodium phosphate, 100 mM sodium chloride, 2.5% trehalose, pH 7.0. IL-2 was concentrated to 1-2 mg/mL, filtered at 0.22 μM, and stored at -80°C until further use.

IL-2/CD-25 binding assay by Bio-Layer Interferometery: IL-2/CD25 multiplex binding kinetics experiments were performed on an Octet RED96 (PALL/ForteBio) instrument at 30°C. Anti-human Fc antibody capture biosensors (PALL/ForteBio, catalog number 18-5063) were loaded with purified CD25-Fc fusion protein in 1X HBS-P+ buffer (GE Healthcare, catalog number BR-1008-27). Immobilization levels between 0.8nm and 1.0nm were achieved. The loaded biosensors were washed with 1X HBS-P+ buffer to remove any unbound protein before measuring association and dissociation kinetics. For monitoring of the binding phase, IL-2 analyte samples were diluted with 1X HBS-P+ buffer and transferred to a solid black 96-well plate (Greiner Bio-One, catalog number 655209). The 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 the wells of the solid black 96-well plate containing 1X HBS-P+ buffer. Data were referenced using parallel buffer blank subtraction, baseline aligned to the y-axis, and smoothed by the Savitzky-Golay filter in Octet data analysis software version 10.0 (PALL/ForteBio). The processed kinetic sensorgrams were globally fitted using a Langmuir model describing 1:1 binding chemometrics (Figure 4A).

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

IL-2 variants are prepared in mammalian cells. Native human IL-2 is a glycosylated protein with O-linked glycosylation on 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 to glycosylated IL-2, glycosylated human IL-2 has been shown to have better activity in clone growth and long-term proliferation of activated human T cells. There are also some reports indicating that native IL-2 has higher specific activity. It has also been shown that IL-2 expression in mammalian cells has advantages over their expression in Escherichia coli (Kim et al., J Microbiol Biotechnol, 14(4), 810-815 (2004)). In the present invention, wild-type IL-2 and various mutant proteins thereof designed in Tables 1 and 2 above, respectively, can be produced in CHO cells (as described in the examples of the present invention).

To produce IL-2 mutant proteins containing non-natural amino acids at desired positions, each mutant protein is produced in a stable pool or a 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 USA, 111(5): pp: 1766-71 (2014) and PCT/2018US/035764: each of which is incorporated herein by reference in its entirety). Briefly, CHOK1 cells are engineered into a platform cell line that stably expresses a proprietary orthogonal tRNA synthetase (O-RS) and its cognate amber inhibitory tRNA (O-tRNA) for efficient incorporation of unnatural amino acids, such as pAF, into therapeutic proteins, such as IL-2, for example, in CHO cells. The platform cell line is then pre-adapted to suspension growth for rapid development in a bioreactor. The platform cell line has been fully characterized and evolved with improved unnatural amino acid incorporation efficiency and clone selection efficiency. The platform cell line is used as a parent cell to produce therapeutic proteins with unnatural amino acids incorporated for early research use at titers above 100 mg/L by rapid and efficient transient expression. Transient transfection and stable incorporation production are performed to evaluate the expression of candidate molecules and provide material for functional assays to identify lead molecules. Production cell lines are generated by transfecting the gene of interest containing amber nonsense codons in the GS expression system into the platform cell line to produce IL-2 protein with unnatural amino acids incorporated. Use the platform cell lines as parent cells to implement stable cell line development strategies to obtain production cell lines with 5-10 PCD in 3-4 months and 20-30 PCD in 6 months.

In the present invention, human IL-2 cDNA (NM_000586.3) with its natural signal peptide sequence was synthesized and cloned into a mammalian expression vector containing a GS selection marker (Figure 4B). As shown in Table 1, the cloned wild-type human IL-2 cDNA retains its original DNA sequence of each amino acid without any mutation. In contrast, during the generation of IL-2 variants (Table 2), each of the 15 mutant proteins has a unique position mutated into an amber stop codon (TAG), which can be inhibited and expressed in engineered cells to produce proteins containing nnAA.

Establishment of engineered CHO cells for expression of IL-2 variants. Engineered CHO cells were derived from a previously established proprietary platform cell (PCT/2018US/035764, which is incorporated herein by reference in its entirety) for gene knockout. Briefly, the web-based target discovery tool CRISPy was used to rapidly identify gRNA target sequences that had zero off-targets in CHO-K1 cells, preferably in early exons. The gRNAs were cloned into the mammalian expression vector pGNCV that co-expressed a CHO codon-optimized form of Cas9. Production cell lines were transfected with protein expression vectors to generate cell amalgams, which were then cloned to identify single cell isolates with gene knockouts. The indel (indel) frequencies of the combined results from multiple projects were 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 improve the mRNA stability of IL-2, the UPF1 gene was knocked out using CRISPR technology. The gRNA used for knockout is shown in Figure 5. Two UPF1-KO cell lines were obtained after screening 192 clones and confirmed to have UPF1 knockout by sequencing (Figure 6). The UPF1-KO cell lines obtained were then used to transiently express IL-2 variants.

Transient expression of IL-2 in engineered CHO cells. IL-2 variants were transiently expressed in the UPF1-KO cell line obtained as disclosed in the above examples. Transfection was performed using electroporation using the Amaxa kit (Lonza) for suspended cells. 6ug of the plasmid prepared as disclosed in the above examples was transfected into 2 x 10 ⁶ engineered CHO cells. After transfection, the cells were incubated at 37°C for 4 days and then titrated by ELISA analysis using a commercial kit from Invitrogen (Carlsbad, CA). As shown in Figures 7A and 7B, variant F42 showed the highest expression level among the 15 variants during transient expression.

T cell expansion test of IL-2 variants in CTLL-2 cells. CTLL-2 cell expansion assay was 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 into serial dilutions in the assay, 10 ng/mL, 3.33 ng/mL, 1.11 ng/mL, 0.37 ng/mL, 0.12 ng/mL and 0.04 ng/mL. Cell proliferation was performed using Cell Titer Glo (Promega, WI). Luminescent signals were read on TECAN genios pro. As shown in Figure 8, F42 showed an _EC50 of around 0.24 ng/mL while retaining 95% of its function compared to its wild-type control. The general protocol used to study the IL-2 variants of the present invention is shown below:

Example 7 - Screening of IL-2 variants by CTLL-2 cell expansion

Using the CTLL-2 cell expansion assay as disclosed in the Examples, 20 different IL-2 variants including 16 initially selected sites known in the art (including wild type) and 4 other sites (K32, K48, K49, K76) were screened (Charych, D. et al., PLoS One, 12 (7): p. e0179431, 2017). 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-2Rα expression, variants with the lowest induced binding to CTLL2 cells still showed some intrinsic binding to IL-2Rα, although this binding was extremely low. For example, the P65 IL-2 variant that was observed to show the lowest binding to IL-2Rα showed some inherent biased binding to IL-2Rα. The identified variants were further analyzed for their binding ability after PEGylation.

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

The selected variants P65, Y45, E61, F42, K35, K49 and T37 were analyzed using the in vitro binding assay biolayer interferometer assay described in the above examples. Each variant was conjugated with 20K PEG at their specific positions. The PEGylated variants were then analyzed by the BLI (biolayer interferometer) assay described elsewhere in the examples. As shown in Figures 10A-10C, the binding of the PEGylated variants to IL-2Rα was tested on Octet. Wild-type IL-2 was used as a positive control in the assay. After PEGylation, most variants showed a drastically reduced binding to IL-2Rα between 92.9% and 99.9%. Among the PEGylated variants tested, P65 and Y45 showed activity blocking of more than 99%, Table 4.

Example 9 - Analysis of selected variants using the PathHunter Dimerization assay

To find the best site for PEG conjugation, the PathHunter dimerization assay developed by DiscoverX (Fremont, CA) was used. In general, the assay system uses exogenously expressed IL-2 receptors that have been engineered to have complementary binding domains for enzymes, and once the previously separated receptors are activated by dimerization with the addition of IL-2 molecules, a chemiluminescent signal is generated (Figure 11). Two cell lines were generated in U2OS cells. One cell line expresses three receptors IL-2Rα, IL-2Rβ, and IL-2Rγ. Another cell line expresses IL-2Rβ and IL-2Rγ. The ratio of the binding EC ₅₀ values of each variant (EC ₅₀ -βγ/EC ₅₀ -αβγ) is used to estimate their relative retention binding ability. As shown in Table 5, the best possible variants had a value of 1, meaning that 100% of their βγ binding ability was retained, while α binding was 100% blocked. As noted, variants Y45-BR4 (variant Y45 conjugated with 20K 4-branched PEG) and P65-PEG20K (variant P65 conjugated with 20K-linear PEG) showed the lowest values, indicating that these two PEGylated variants would be the best candidates for further evaluation.

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

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

To further evaluate the in vitro function of the PEGylated variants, an ex vivo assay using PBMCs was used. As shown in Figure 12, the binding of IL-2 to its receptor triggers an increase in the phosphorylation of STAT5 (pSTAT5). Therefore, detecting pSTAT5 levels will be an indicator of the binding of IL-2 variants to endogenous IL-2 receptors. Human whole PBMCs were treated with selected PEGylated variants such as Y45-BR2 (variant Y45 conjugated with 20K 2-branched PEG), Y45-BR4 (variant Y45 conjugated with 20K 4-branched PEG) and P65-PEG20K (variant P65 conjugated with linear 20K PEG) and then separated into two populations, CD8+ T cells and CD4+ Treg cells. As shown in Table 7, all three variants showed greatly improved activity with respect to their retained βγ binding activity and blocked α binding activity. These results were further supported by the variants tested in another pSTAT5 assay as shown in Table 8. The results from this pSTAT5 assay (Table 8) showed that many variants had drastically improved activity with respect to their reduced ability to bind to Treg cells and relatively maintained binding to CD8+ cells. In Table 8, the calculated CD8+/Treg ratio was used to indicate the ranking of the variants so that the PathHunter assay results could be directly compared to the pSTAT5 assay results through a similar ranking system.

Table 8 - Improved binding activity of IL-2 variants using in vitro assay - Experiment 2

Example 11 - Comparison of clone growth and long-term proliferation 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

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)). The function of this glycosylation 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 USA 81(20):6486-6490 (1984); Goodson et al., Biotechnology (NY) 8(4):343-346 (1990)). More importantly, glycosylated IL-2 has been shown to be superior to non-glycosylated IL-2 in promoting clone growth and long-term proliferation of allogeneic activated human T cells (Pawelec et al., Immunobiology 174(1):67-75(1987)), suggesting that glycosylated IL-2 is a better choice for therapeutic applications.

To further analyze the biological functions of glycosylated IL-2 and non-glycosylated IL-2, experiments were performed to analyze the replication (clone) growth rate and long-term proliferation frequency of CTLL-2 cells (Figure 13). Single CTLL-2 cells were placed in 96-well plates (Thermo Fisher, Waltham, MA, CAT#A34180) with pre-coated culture cell layers of γ-irradiated CF1-MEF (mouse embryonic fibroblast) cells. During the 19-day growth period of a single treatment with various concentrations (0.005 nM, 0.05 nM, 0.5 nM and 5 nM) of wild-type IL-2 produced from CHO cells or E. coli, the percentage of the number of colonies grown and the percentage of colonies that survived at the end of the 19-day incubation were counted and analyzed. As shown in FIG13 (taking 0.5 nM treatment as an example), glycosylated IL-2 showed superior activity in promoting clone growth compared to non-glycosylated IL-2. On average, in the presence of a 0.5 nM IL-2 concentration, which is the optimal cell culture condition for CTLL-2 cell growth, the ability of glycosylated IL-2 to promote clone growth was twice as high as that of non-glycosylated IL-2. After long-term incubation (~19 days), the survival rate of colonies from glycosylated IL-2 treatment was 4 times higher than that from non-glycosylated IL-2 treatment. These data clearly demonstrate that glycosylated IL-2 has superior activity in promoting clone growth and long-term proliferation of IL-2-responsive cells and further support its promising therapeutic application.

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

Many methods have been tried in the field to improve the expression of wild-type IL-2 and its variants in CHO cells (see, for example, Kim et al., J Microbiol Biotechnol, 14(4), 810-815 (2004)). However, the improvement of the expression of proteins containing non-natural amino acids in industry is challenged by the relatively low yield in mammalian cells. In order to solve this problem in the present invention, the proprietary technology disclosed in PCT/2018US/035764 (which is incorporated by reference in its entirety) for improving protein titer production in eukaryotic cell lines is used to produce stable fusion cells of IL-2 and its variants, and used in the generated stable IL-2 cell lines.

Briefly, different five-passage platform cell lines expressing Bax/Bak knockout were found to dramatically increase IL-2 protein expression and boost IL-2 protein production to approximately 40% higher than the parental cell lines. In addition to inhibiting apoptosis in these cells by Bax/Bak knockout, UPF1 knockout was found to further increase IL-2 expression.

Both wild-type IL-2 and its variants (F42, Y45 and P65) have been tested by generating their stable incorporations. As shown in FIG13 , three IL-2 variants, F42, Y45 and P65, including stable incorporations of wild-type IL-2, have greatly improved expression levels compared to existing levels in the art (see, e.g., Kim et al., J Microbiol Biotechnol., 14(4), 810-815, (2004)), up to about 740 mg/L for the wild-type and up to 120 mg/L for the F42 variant after generating their respective stable incorporations (shown in FIG14 ). The data show that the production or yield of IL-2 protein can be improved or increased by generating new CHO cell lines with highly efficiently incorporated non-natural amino acids. It has also been shown that expression levels and function are site-specifically related.

Example 13 - IL-2 variant F42-R38A shows complete blockade of IL-2Rα binding

As disclosed in the present invention, the non-naturally encoded amino acid substitutions are combined with other additions, substitutions or deletions in the IL-2 to affect other biological properties of the IL-2 polypeptide, including but not limited to improving the stability of the IL-2 (including but not limited to resistance to proteolytic degradation) or improving the affinity of the IL-2 for its receptor, improving the drug stability of the IL-2, enhancing the tumor inhibition and/or tumor reduction activity of the IL-2, improving the solubility of the IL-2 or variants (including but not limited to when in E. coli or other host bacteria) cells), improve the solubility of the IL-2 after being expressed in E. coli or other recombinant host cells, improve the solubility of the polypeptide after being expressed in E. coli or other recombinant host cells, regulate the affinity for IL-2 receptors, binding proteins or related ligands, regulate signal transduction after binding to IL-2 receptors, regulate the circulation half-life, regulate release or bioavailability, promote purification, or improve or change a specific route of administration, increase the affinity of IL-2 variants for their receptors, and increase the affinity of IL-2 variants for IL-2-Rβ and/or IL-2-Rγ.

Therefore, in order 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, during the stable cell line generation, the combination of non-natural amino acids and natural amino acid substitutions in IL-2 variant F42 was used in a stable pool, and the titer was increased to 118 mg/L. In the presence of the R38A mutation, the protein expression level of variant F42 was not only maintained, but also showed a 20% increase. In order to test the function of the PEGylated F42-R38A variant, a CTLL-2 cell binding assay was performed. As shown in Table 9, the F42-R38A 20K 2-branched PEG (variant F42-R38-BR2) conjugate showed an EC50 of 15.9 nM, compared to 3.6 nM for F42, thus increasing the binding blocking efficiency by more than 4 times ( FIG. 15B ). Based on the EC50 of 0.025 nM for wild-type IL-2, the binding blocking efficiency exceeded 99.9%. In terms of high protein expression levels and efficiency in blocking binding to IL-2Rα, this variant shows great potential for therapeutic applications.

The binding kinetics of the F42pAF variant, the R38A-F42pAF variant (containing non-natural amino acids and point mutations), and F42-R38A-PEG20K-BR2 were evaluated by BLI to determine the effect of the R38A mutation on the binding of IL-2Rα. Figure 15C shows the binding sensorgrams of the three constructs, and the associated binding constants (KD) are shown in Table 10. As can be seen in Table 10, IL-2-F42pAF has a 20nM IL-2Rα binding KD. After adding the R38A mutation, IL-2-F42-R38ApAF has a 233nM IL-2Rα binding KD, which corresponds to a 12-fold decrease in IL-2Rα binding. After conjugating IL-2-R38A-F42pAF with a 20K 2-branched PEG molecule, IL-2Rα binding was blocked. The results clearly demonstrate that the added mutations effectively block the binding of F42-R38A to its receptor IL-2Rα.

It should be understood that the embodiments and examples described in the present invention are for illustrative purposes only, and that various modifications or variations therefrom will be suggested by persons of ordinary skill in the art and will be included within the spirit and scope of the present application and the scope of the accompanying patent applications. All publications, patents, patent applications and/or other documents cited in this application are incorporated herein by reference for all purposes to the same extent as if each individual publication, patent, patent application and/or other document was individually indicated as being incorporated herein by reference for all purposes.

The present 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 Example 1, wherein the IL-2 polypeptide is 90% homologous to SEQ ID NO: 2 or SEQ ID NO: 3.

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

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

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

6. The IL-2 of Example 1, wherein the IL-2 is conjugated 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 IL-2 of Example 7, wherein the water-soluble polymer is PEG.

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

11. The IL-2 of embodiment 10, wherein the IL-2 comprises one or more amino acid substitutions, additions or deletions that regulate the affinity of the IL-2 polypeptide to its IL2Rα receptor subunit compared to wild-type IL-2.

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

14. The IL-2 of embodiment 10, wherein the non-naturally encoded amino acid is replaced at a position selected from the residues at positions 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 originally 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 amineoxy group, a hydrazine group, a hydrazide group, a amine urea group, an azido group or an alkynyl 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 biologically active molecule, a cytotoxic agent, a water-soluble polymer or an immunostimulant.

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 immunostimulant is linked to the IL-2 via a linker.

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

22. The IL-2 of embodiment 18, wherein the biologically active molecule, cytotoxic agent or immunostimulatory agent is directly conjugated 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 following structure:

Where n is 0-10; R1 is alkyl, aryl, substituted alkyl or substituted aryl; R2 is H, alkyl, aryl, substituted alkyl and substituted aryl; R3 is H, amino acid, polypeptide or amino terminal modification group; and R4 is H, amino acid, polypeptide or carboxyl terminal modification group.

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

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

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

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

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

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

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

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

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

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

34. The IL-2 of Example 16, wherein the aminooxy group, hydrazine, hydrazide or aminourea group is connected to the water-soluble polymer via an amide bond.

35. The IL-2 of Example 19, which is produced by reacting a water-soluble polymer comprising a carbonyl group with a polypeptide comprising a non-naturally encoded amino acid containing an amineoxy group, a hydrazine, a hydrazide or an aminourea group.

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

37. The IL-2 of embodiment 1, wherein the IL-2 polypeptide further comprises a linker, a polymer or a biologically active molecule connected to the polypeptide via 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 via a sugar component.

39. A method of producing the IL-2 of 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 component that reacts with the non-naturally encoded amino acid.

40. The method of embodiment 39, wherein the polymer comprises a component selected from 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 amine group, a hydrazide group, a hydrazine group, a amine urea group, an azido group, or an alkynyl group.

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

43. The method of embodiment 39, wherein the aminooxy, hydrazine, hydrazide or aminourea component is connected to the linker, polymer or bioactive molecule via an amide bond.

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

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

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

47. The IL-2 polypeptide of Example 46, wherein the polyethylene glycol component is a branched or multi-arm polymer.

48. A composition comprising the IL-2 of Example 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 regulated by IL-2, the method comprising administering to the patient a therapeutically effective amount of the composition of Example 42.

51. A composition comprising IL-2 of Example 10 conjugated to a biologically active molecule and a pharmaceutically acceptable carrier.

52. A composition comprising the IL-2 of Example 10 and a pharmaceutically acceptable carrier, wherein the IL-2 further comprises a linker and a conjugate.

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

54. A method for regulating the serum half-life or circulation time of an IL-2 polypeptide, the method comprising replacing any one or more naturally occurring amino acids in the 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 the 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 via a functional group of a non-naturally encoded amino acid that is 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 the non-naturally encoded amino acids are ribosomally incorporated into the polypeptide at a preselected position.

58. A method for 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 of Example 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.

<110> Beijing Tide Pharmaceuticals Co Ltd, a Chinese company

<120> Interleukin-2 polypeptide conjugate and its use

<140> TW 109106336

<141> 2020-02-26

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<213> Human

<400> 2

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<211> 133

<212> PRT

<213> E.Coli

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<210> 4

<211> 399

<212> DNA

<213> E.Coli

<400> 4

<210> 5

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1:IL-2 receptor α

2:IL-2 receptor β

3:IL-2 receptor γ

4:IL-2

5: Locations to be screened

6: PEG (polyethylene glycol)

CD8+:CD8 receptor protein

Kd: dissociation constant

Rβγ: receptor βγ

Rαβγ: receptor αβγ

Treg: Regulatory T Cells

Claims (41)

An interleukin-2 (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, the IL-2 polypeptide is at least 99% homologous to SEQ ID NO: 2, and wherein the non-naturally encoded amino acid is substituted at the position of residue 45, wherein the IL-2 polypeptide is conjugated to one or more PEGs, and the IL-2 polypeptide is glycosylated. The IL-2 polypeptide as described in claim 1, wherein at least one PEG is linked to at least one of the non-naturally encoded amino acids. The IL-2 polypeptide as described in claim 1, wherein the PEG has a molecular weight between 10 and 50 kDa. The IL-2 polypeptide as described in claim 1, wherein the IL-2 polypeptide comprises one or more amino acid substitutions, additions or deletions that regulate the affinity of the IL-2 polypeptide to its IL2Rα receptor subunit compared to wild-type IL-2. The IL-2 polypeptide as described in claim 1, wherein the IL-2 polypeptide comprises one or more amino acid substitutions, additions or deletions that improve the stability or solubility of the IL-2 polypeptide. The IL-2 polypeptide as described in claim 1, wherein the IL-2 polypeptide comprises one or more amino acid substitutions, additions or deletions that improve the expression of the IL-2 polypeptide in recombinant host cells or in vitro synthesis. The IL-2 polypeptide as described in claim 1, wherein the non-naturally encoded amino acid is reactive to a linker, polymer or biologically active molecule that is originally unreactive to any of the 20 common amino acids in the polypeptide. The IL-2 polypeptide as described in claim 1, wherein the non-naturally encoded amino acid comprises a carbonyl group, an amine group, a hydrazine group, a hydrazide group, a semicarbazide group, an azido group or an alkynyl group. The IL-2 polypeptide as described in claim 8, wherein the non-naturally encoded amino acid comprises a carbonyl group. The IL-2 polypeptide as described in claim 1, wherein the IL-2 polypeptide is linked to a biologically active molecule, a cytotoxic agent or an immunostimulant. The IL-2 polypeptide as described in claim 10, wherein the biologically active molecule, cytotoxic agent or immunostimulant is linked to the IL-2 polypeptide via a cleavable or non-cleavable linker. The IL-2 polypeptide as described in claim 10, wherein the biologically active molecule, cytotoxic agent or immunostimulatory agent is directly conjugated to one or more of the non-naturally encoded amino acids in the IL-2 polypeptide. The IL-2 polypeptide of claim 1, wherein the non-naturally encoded amino acid has the following structure:

wherein n is 0-10; _R1 is alkyl, aryl, substituted alkyl or substituted aryl; _R2 is H, alkyl, aryl, substituted alkyl and substituted aryl; _R3 is H, an amino acid, a polypeptide or an amino terminal modifying group; and _R4 is H, an amino acid, a polypeptide or a carboxyl terminal modifying group. The IL-2 polypeptide as described in claim 13, wherein the non-naturally encoded amino acid comprises an amine group. The IL-2 polypeptide as described in claim 13, wherein the non-naturally encoded amino acid comprises a hydrazide group. The IL-2 polypeptide as described in claim 13, wherein the non-naturally encoded amino acid comprises a hydrazide group. The IL-2 polypeptide as described in claim 13, wherein the non-naturally encoded amino acid residue comprises a semicarbazide group. The IL-2 polypeptide as described in claim 13, wherein the non-naturally encoded amino acid residue comprises an azido group. The IL-2 polypeptide of claim 1, wherein the non-naturally encoded amino acid has the following structure:

wherein n is 0-10; _R1 is alkyl, aryl, substituted alkyl, substituted aryl or absent; X is O, N, S or absent; m is 0-10; _R2 is H, an amino acid, a polypeptide or an amino-terminal modifying group, and _R3 is H, an amino acid, a polypeptide or a carboxyl-terminal modifying group. The IL-2 polypeptide as described in claim 19, wherein the non-naturally encoded amino acid comprises an alkynyl group. The IL-2 polypeptide of claim 1, wherein the non-naturally encoded amino acid has the following structure:

wherein n is 0-10; _R1 is alkyl, aryl, substituted alkyl or substituted aryl; X is O, N, S or absent; m is 0-10; _R2 is H, an amino acid, a polypeptide or an amino-terminal modifying group, and _R3 is H, an amino acid, a polypeptide or a carboxyl-terminal modifying group. The IL-2 polypeptide as described in claim 8, wherein the aminooxy group, hydrazine group, hydrazide group or aminourea group is linked to the water-soluble polymer via an amide bond. The IL-2 polypeptide as described in claim 1 is produced by reacting a water-soluble polymer comprising a carbonyl group with a polypeptide comprising a non-naturally encoded amino acid containing an amineoxy group, a hydrazine group, a hydrazide group or an aminourea group. The IL-2 polypeptide as described in claim 1, wherein the IL-2 polypeptide further comprises a linker, a polymer or a biologically active molecule connected to the polypeptide via the non-naturally encoded amino acid. The IL-2 polypeptide as described in claim 24, wherein the linker, polymer or biologically active molecule is linked to the IL-2 polypeptide via a saccharide moiety. A method for producing an IL-2 polypeptide as described in claim 1, the method comprising contacting the isolated IL-2 polypeptide comprising a non-naturally encoded amino acid with a linker, polymer or biologically active molecule comprising a component that reacts with the non-naturally encoded amino acid. The method as described in claim 26, wherein the non-naturally encoded amino acid comprises a carbonyl group, an amine group, a hydrazide group, a hydrazine group, a semicarbazide group, an azido group or an alkynyl group. The method of claim 26, wherein the non-naturally encoded amino acid comprises a carbonyl moiety, and the linker, polymer or bioactive molecule comprises an aminooxy, hydrazine, hydrazide or aminourea moiety. The method as described in claim 26, wherein the aminooxy, hydrazine, hydrazide or aminourea moiety is linked to the linker, polymer or bioactive molecule via an amide bond. The method of claim 26, wherein the non-naturally encoded amino acid comprises an alkynyl moiety and the linker, polymer or biologically active molecule comprises an azido moiety. The method of claim 26, wherein the non-naturally encoded amino acid comprises an azido moiety and the linker, polymer or biologically active molecule comprises an alkynyl moiety. A composition comprising the IL-2 polypeptide as described in claim 1 and a pharmaceutically acceptable carrier. A composition as described in claim 32, wherein the non-naturally encoded amino acid is linked to a water-soluble polymer. A therapeutically-effective amount of a composition as claimed in claim 32 for use in the preparation of a medicament for treating a patient with a disorder regulated by IL-2. A composition comprising an IL-2 polypeptide as described in claim 1 conjugated to a biologically active molecule and a pharmaceutically acceptable carrier. A composition comprising the IL-2 polypeptide as described in claim 1 and a pharmaceutically acceptable carrier, wherein the IL-2 polypeptide further comprises a linker and a conjugate. A method for producing an IL-2 polypeptide as described in claim 1, the method comprising culturing cells comprising one or more polynucleotides comprising a selector codon encoding an IL-2 polypeptide, an orthogonal RNA synthetase, and an orthogonal tRNA under conditions that allow expression of the IL-2 polypeptide comprising a non-naturally encoded amino acid; and purifying the IL-2 polypeptide. A method for regulating the serum half-life or circulation time of an IL-2 polypeptide, the method comprising replacing any one or more naturally occurring amino acids in the polypeptide with one or more non-naturally encoded amino acids as defined in claim 1. A use of the composition as described in claim 36 in the preparation of a medicament, wherein the medicament is used to reduce the number of tumor cells in a person diagnosed with cancer. The use as described in claim 39, wherein the drug composition is administered at a dose of 0.1μ/kg to 50μ/kg. A composition comprising the IL-2 polypeptide as described in claim 1 and a pharmaceutically acceptable carrier, wherein the composition is used in the preparation of a medicament, wherein the medicament is used for treating cancer.

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