TW202132331A - 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 the use of interleukin-2 (IL-2) variants to regulate the biological activity of interleukin-2 and in particular to regulate specific receptor (specific receptor) interactions In the method, the IL-2 variant is conjugated to the polymer at the position where the IL-2 protein interacts with the interleukin-2 receptor in the amino acid sequence of the IL-2 protein.

Cancer is one of the most important health conditions. In the United States, cancer has the second-largest death rate after heart disease, accounting for a quarter of deaths. It is widely expected that the incidence of cancer will increase with the aging of the American population, further exacerbating the impact of this disease. The current treatment regimens for cancer established in the 1970s and 1980s have not changed much. When used in most common advanced cancers, these treatments, including chemotherapy, radiation, and other modalities (including the newer targeted therapies), show limited overall survival benefits. Because these therapies mainly target tumor bulk.

More specifically, to date, conventional cancer diagnosis and therapy have attempted to selectively detect and eradicate neoplastic cells (that is, cells that form tumor masses), which are mainly fast-growing neoplastic cells. Standard oncology treatment regimens are usually designed to administer the highest dose of radiation or chemotherapeutics without excessive toxicity, which is often referred to as "maximum tolerated dose (MTD)" or "not observed The level of adverse effects (no observed adverse effect level, NOAEL)". Many conventional cancer chemotherapy (e.g. alkylating agents such as cyclophosphamide, antimetabolites such as 5-fluorouracil, and plant alkaloids such as vincristine) and conventional radiotherapy mainly interfere with cellular mechanisms involved in cell growth and DNA replication To exert their toxic effects on cancer cells. Chemotherapy regimens usually also include the administration of a combination of chemotherapeutic agents in an attempt to improve the efficacy of the treatment. Although a large number of different chemotherapeutic agents are available, these therapies have many disadvantages. For example, chemotherapeutics are known to be toxic due to non-specific side effects on fast-growing cells, whether normal or malignant; for example, chemotherapeutics cause significant and often dangerous side effects, including bone marrow suppression, immunosuppression, and gastrointestinal Road is unwell and waiting.

Cancer stem cells

Cancer stem cells consist of a unique subpopulation of tumors (usually about 0.1-10%), which are more tumorigenic than the remaining 90% tumors (and tumor masses), and grow relatively slower or slower. Quiescent and usually relatively more chemoresistant than tumor masses. Considering that most conventional therapies and programs are designed to attack fast-proliferating cells (that is, those cancer cells that make up tumor masses), therefore, compared with fast-growing tumor masses, generally slow-growing cancer stem cells may be relatively more effective for conventional therapies and programs. Resistant. Cancer stem cells can exhibit other characteristics that make them relatively more resistant to chemotherapy, such as multidrug resistance and anti-apoptotic pathways. The above-mentioned factors constitute the key reason why standard tumor treatment regimens fail to ensure long-term benefits in most patients with advanced cancer, that is, fail to adequately target and eradicate cancer stem cells. In some cases, cancer stem cells are tumor-producing cells (that is, they are the progenitor of the cancer cells that constitute the tumor mass).

IL-2 has been used to treat several cancers, such as renal cell carcinoma and metastatic melanoma. The commercially available IL-2 Aldesleukin® is a recombinant protein that is non-glycosylated with alanine-1 removed and the residue cysteine-125 replaced with serine-125 (Whittington et al., 1993) . Although IL-2 is the earliest FDA-approved cytokine in cancer treatment, IL-2 has been shown to exhibit serious side effects when used in high doses. This greatly limits its application to 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. Generally, IL-2 can not only form 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) Heterotrimeric complex, and can form a heterodimeric complex with IL-2Rβ and IL-2Rγ. In a clinical setting, when high doses of IL-2 are used, IL-2 begins to bind IL-2αβγ, which is the main receptor form in Treg cells. The suppressive effect of Treg cells causes the undesirable effects of IL-2 in cancer immunotherapy. In order to reduce the side effects of IL-2, many methods have been used before. Nektar manufactured a form of IL-2 that uses 6 PEGylated lysine acids to mask the IL2Rα binding region on the surface of IL-2 (Charych et al., 2016). This PEGylated IL-2 form has an extended half-life, contains a mixture of mono- and multi-PEGylated forms, contains very large amounts of PEG, and shows improved side effects. However, the results from the activity study showed that the effective PEGylated IL-2 form in this heterogeneous 6-PEGylated IL-2 mixture is only the mono-PEGylated form. Therefore, there is a need for more effective PEGylated IL-2 with a homogenous and clear product composition that modulates the side effects of IL-2.

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

In addition to other problems, the present invention is dedicated to solving problems related to the activity and production of IL-2 polypeptide conjugates, and is also dedicated to producing products with improved biological or pharmacological properties, such as increased activity and/or improvement on tumors. The conjugated and/or improved therapeutic half-life of IL-2 polypeptides. The IL-2 polypeptide of the present invention targets both Treg cells known to express trimeric IL-2 receptors (α, β, and γ) and CD8 cells mainly expressing β and γ dimers of IL-2 receptors . The IL-2 polypeptide of the present invention reduces the binding to the α receptor of Treg cells and promotes the biased binding with the β and γ dimers of CD8 cells, thereby being a disease or disorder in which IL-2 receptor α is highly expressed Provides improved therapeutic applications and improved therapeutic prognosis (prognosis).

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 with 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 in 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 modulating the receptor interaction of the IL-2 polypeptide of the present invention. The present invention provides a method for using the PEGylated IL-2 polypeptide of the present invention to inhibit or reduce the interaction of PEGylated IL-2 with the IL2Rα subunit of the trimeric IL-2 receptor.

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 signal peptide) human interleukin-2 linked to a PEG polymer. In this or any embodiment of the invention, the PEG-IL-2 may comprise a full-length, mature (lacking signal peptide) human interleukin-2 covalently linked to a PEG polymer or other biologically active molecule . In certain embodiments, the biologically active molecule is modified. As a non-limiting example, the biologically active 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 biologically active molecule directly or through a linker. Suitable linkers include, for example, cleavable and non-cleavable linkers.

The present invention provides a method for treating cancer in mammals by administering an effective amount of PEG-IL-2 polypeptide, such as mammals including but not limited to those having one or more of the following conditions: lumps Solid tumor, hematological tumor, 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 alpha. In certain embodiments, the present invention provides a method of treating cancer or a disorder or disease by administering to an article an effective amount of a composition comprising the IL-2 polypeptide of the present invention. In certain embodiments, the present invention provides a method of treating genetic diseases by administering to a patient an effective amount of the IL-2 composition of the present invention. In certain embodiments, the condition or disease is characterized by high expression of IL-2 receptor alpha. In certain embodiments, the condition or disease is characterized by 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 decrease 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 a sequence similar to IL-2 (including IL-2 mutant protein, mature IL-2 sequence (ie lacking The secretory leader sequence) and the known sequence of IL-2 disclosed in SEQ ID NO: 1, 2, 3, 5 or 7 of the present application are basically identical to the known sequence of the amino acid sequence, and (b) have the origin At least one biological activity shared by native or wild-type IL-2. For the purposes of the present invention, 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 The two 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 an IL-2 polypeptide conjugated to one or more water-soluble polymers, wherein the PEGylated IL-2 polypeptide is also linked to another drug or biologically active molecule, And wherein the IL-2 polypeptide comprises one or more non-naturally encoded amino acids. The invention also provides monomers and dimers of IL-2 polypeptides. The present invention also provides a trimer of IL-2 polypeptide. The present invention provides multimers of IL-2 polypeptides. The present invention also provides IL-2 dimers containing one or more non-naturally encoded amino acids. The present invention provides IL-2 multimers containing 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 multimers, wherein at least one of the IL-2 polypeptides comprises at least one non-naturally encoded amino acid, wherein any or each of the multimers IL-2 polypeptides can have different amino acid sequences.

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

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

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

In certain embodiments, a non-naturally encoded amino acid is incorporated into one or more of the following positions of IL-2 or its variants: first Before the bit (ie at the N-end), the first 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, or any combination thereof ( SEQ ID NO: 2, or the corresponding amino acid in SEQ ID NO: 3, 5, or 7). In certain embodiments, one or more 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 invention is connected to a linker. In certain embodiments, the IL-2 variant linked to the linker further comprises a biologically active molecule. In certain embodiments of the invention, the linker is connected to a non-naturally encoded amino acid.

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: 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 SEQ ID NO: 3, 5 or The corresponding amino acid position in 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: 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 amine in SEQ ID NO: 3, 5 or 7 Base acid). 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: 35th, 37th, 42nd, 45th, 49th , 61 or 65, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 45, 61, and 65, and Any combination (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in IL-2 or a variant thereof: positions 45 and 65, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7). 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 the third position. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at the 32nd position in the IL-2 or variants thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 35 in the IL-2 or variants thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 37 in the IL-2 or variants thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 38 in the IL-2 or variants thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 41 in the IL-2 or variants thereof of the present invention. 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 42. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 43 in the IL-2 or variants thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 44 in the IL-2 or variants thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 45 in the IL-2 or variants thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 48 in the IL-2 or variants thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 49 in the IL-2 or variants thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 61 in IL-2 or a variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 62 in the IL-2 or variants thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at the 64th position in the IL-2 or variants thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 65 in the IL-2 or variants thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 68 in IL-2 or a variant thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 72 in the IL-2 or variants thereof of the present invention. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at position 76 in IL-2 or a variant thereof of the present invention. 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, and the position includes but not Limited to the following positions: before the first position (ie at the N-end), the first 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 carboxy terminus of the protein, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid in SEQ ID NO: 3, 5, or 7).

In certain embodiments, the non-naturally occurring amino acid at one or more of these positions in IL-2 or a variant thereof is linked to a drug or other biologically active molecule, and the position includes but not Limited to the position of hydrophobic interactions, at or near the position of interaction with IL-2 receptor subunits (including IL2Rα), within the 3rd or 35th to 45th amino acid positions, in the first 107 Within one N-terminal amino acid, within the 61-72th amino acid position; each of said positions is the position of SEQ ID NO: 2 or the corresponding amino acid position in SEQ ID NO: 3, 5 or 7 . In certain embodiments, the non-naturally occurring amino 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, and the position includes but not Limited to the following positions: before the first position of SEQ ID NO: 2 (ie at the N-terminal), the first 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 acid in SEQ ID NO: 3, 5, or 7. In certain embodiments, the non-naturally occurring amino acid at one or more of these positions in IL-2 or a variant thereof is linked to a drug or other biologically active molecule, and the position includes but not Limited to the following positions of IL-2 or its variants: the 44th, 45th, 46th, 47th, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, SEQ ID NO: 2 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 bit , Or added to the carboxyl end of the protein, and any combination thereof; 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 in one or more of the following positions and linked to drugs or other biologically active molecules: 3. Positions 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 SEQ ID NO: 3 , 5 or 7 corresponding amino acid).

In certain embodiments, the non-naturally occurring amino acid at one or more of these positions in IL-2 or a variant thereof is attached to the linker, and the positions include, but are not limited to, the following positions: Before the first position (ie at the N-end), the first 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 carboxy terminus of the protein, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid in SEQ ID NO: 3, 5 or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof in one or more of the following positions and connected to the linker: 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 SEQ ID NO: 3, 5 or 7 The corresponding amino acid).

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 biologically active molecules, the positions include but are not limited to the following positions: before the first position (ie at the N-terminus), the first 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 bits, or add to The carboxyl end of the protein, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid in SEQ ID NO: 3, 5, or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof in one or more of the following positions and connected to a linker, which is further connected For water-soluble polymers or biologically active molecules, the positions include but are not limited to the following positions: 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, Before positions 72 and 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid in SEQ ID NO: 3, 5, or 7).

In certain embodiments, the non-naturally occurring amino acid at one or more of these positions in IL-2 or a variant thereof is attached to the water-soluble polymer, and the positions include but are not limited to the following The position: before the first position (ie at the N-end), the first 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 carboxy terminus of the protein, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid in SEQ ID NO: 3, 5 or 7). In certain embodiments, one or more non-naturally encoded amino acids are incorporated into IL-2 or a variant thereof in one or more of the following positions and connected to a linker, which is further connected For water-soluble polymers, the positions include but are not limited to the following positions: positions 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72, and 107 , And any combination thereof (SEQ ID NO: 2, or the corresponding amino acid in SEQ ID NO: 3, 5, or 7).

In certain embodiments, the IL-2 or a variant thereof comprises a substitution, an addition that modulates the affinity of the IL-2 to the IL-2 receptor subunit or its variant. ) Or deletion (deletion). In certain embodiments, the IL-2 or its variants include modulating the IL-2 or its variants to IL-2 receptor or binding partner (including but not limited to protein, polypeptide, Lipid, fatty acid, small molecule or nucleic acid) affinity substitution, addition or deletion. In certain embodiments, the IL-2 or a variant thereof comprises a substitution, addition or deletion that modulates the stability of the IL-2 compared to the stability of the corresponding IL-2 without the substitution, addition or deletion . Stability and/or solubility can be measured using a 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 replacement or addition 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 replacement, addition or deletion. Or missing.

In certain embodiments, the IL-2 or variants thereof comprise substitutions that increase the water solubility of the IL-2 compared to the water solubility of the corresponding IL-2 or variants without substitutions, additions or deletions , Added or missing. In certain embodiments, the IL-2 or its variants comprise a compound that is produced in a host cell (host cell) compared to the solubility of the corresponding IL-2 or its variant without substitutions, additions or deletions. The substitution, addition or deletion of the solubility of the IL-2 or its variants. In certain embodiments, the IL-2 or its variants comprise an increase in the expression or synthesis of the IL-2 or its variants in the host cell compared to the expression or synthesis of the corresponding IL-2 or its variants without substitutions, additions or deletions. The expression or increase the substitution, addition or deletion of in vitro synthesis. The IL-2 or its variant containing this replacement retains agonist activity or retains or increases the expression level in the host cell. In certain embodiments, the IL-2 or its variants comprise increased protease resistance of the IL-2 or its variants compared to the protease resistance of the corresponding IL-2 or its variants without substitutions, additions or deletions Substitution, addition or deletion of protease resistance. In certain embodiments, the IL-2 or a variant thereof comprises a modulating effect compared to the activity of the IL-2 receptor after interacting with the corresponding IL-2 or a variant thereof that does not contain substitutions, additions or deletions. The replacement, addition or deletion of the signal transduction activity of the IL-2 receptor. In certain embodiments, the IL-2 or its variants comprise substitutions, additions, or substitutions that modulate its binding to another molecule, such as a receptor, compared to the binding of the corresponding IL-2 without substitutions, additions, or deletions. Missing.

In certain embodiments, the present invention provides the use of PEG-IL-2 and at least one additional therapeutic or diagnostic agent to treat proliferative conditions, cancer, tumors, or precancerous conditions such as abnormal proliferation Methods. The additional therapeutic agent may be, for example, a cytokine or cytokine antagonist such as IL-12, interferon-α or anti-epidermal growth factor receptor antibody, doxorubicin, epirubicin, antifolate Agents such as methotrexate or fluorouracil, irinotecan, cyclophosphamide, radiotherapy, hormonal or antihormonal therapy such as androgen, estrogen, anti-estrogens, flutamide or diethyldiethylstilbestrol, surgery, tamoxifen Tamoxifen (tamoxifen), ifosfamide, dulc bromide, alkylating agents such as melphalan or cisplatin, etoposide, vinorelbine, vinblastine, vindesine, sugar Corticosteroids, 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, clone antibodies against another tumor antigen, clone antibodies and biologically active molecules Complexes, T cell adjuvants, bone marrow transplants, or antigen presenting cells such as dendritic cell therapy. The vaccine can be provided as, for example, a soluble protein or a nucleic acid encoding the protein (see, for example, Le et al., supra; Greco and Zellefsky editors (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) .

A method of extramedullary hematopoiesis (EMH) for the treatment of cancer is also provided. EMH has been described (see, for example, 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 a variant thereof comprises modulating its binding activity compared to the receptor or receptor subunit binding activity of the corresponding IL-2 or its variant without substitution, addition or deletion. Substitution, addition, or deletion of receptor or receptor subunit binding. In certain embodiments, the IL-2 or a variant thereof comprises a receptor or receptor subunit binding activity of the corresponding IL-2 or a variant thereof without substitution, addition or deletion. Substitution, addition, or deletion of receptor or receptor subunit binding-related activities.

In certain embodiments, the IL-2 or a variant thereof comprises an increased compatibility of the IL-2 or a variant thereof with a drug compared to the compatibility of the corresponding wild-type IL-2 without substitution, addition or deletion. Replacement, addition or deletion of compatibility of preservatives (for example, m-cresol, phenol, benzyl alcohol). This increased compatibility enables the preparation of well-preserved pharmaceutical formulations that maintain the physicochemical properties and biological activity of the protein during storage.

In certain embodiments, one or more non-natural amino acids are used to create one or more engineered bonds. The intramolecular bond can be produced in many ways, including but not limited to the reaction between two amino acids in the protein under suitable conditions (one or two amino acids may be non-natural amino acids), Reaction with two amino acids (each amino acid may be naturally coded or non-naturally coded), with linkers, polymers or other molecules under suitable conditions.

In certain embodiments, one or more amino acid substitutions in IL-2 or a variant thereof may use one or more naturally-occurring or non-naturally-occurring amino acids. In certain embodiments, the amino acid replacement in IL-2 or its variants may use a naturally-occurring or non-naturally-occurring amino acid, as long as at least one replacement is a non-naturally-encoded amino acid. . In certain embodiments, one or more amino acid substitutions in the IL-2 or its variants may use one or more naturally-occurring amino acids, and at least one substitution is non-naturally encoded Amino acid. In some embodiments, the amino acid substitution in IL-2 or its variants can use any naturally-occurring amino acid, and at least one substitution is the use of a non-naturally encoded amino acid. In certain embodiments, one or more natural amino acids can be substituted at one or more of the following positions of IL-2 or a variant thereof: before the first position (ie at the N-terminus), the first , 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 SEQ ID NO: the corresponding amino acid position in 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: 44th, 45th, 46th, 47th, 48th, 49th, 50th, 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 end of the protein, and any combination thereof (SEQ ID NO: 2, or SEQ ID NO: 3, 5 or 7 Corresponding amino acid position). In certain embodiments, the amino acid substitution in IL-2 or its variants may use at least one naturally-occurring amino acid, and at least one substitution is the use of a non-naturally encoded amino acid. In certain embodiments, the amino acid substitution in IL-2 or its variants may use at least two naturally-occurring amino acids, and at least one substitution is the use of a non-naturally encoded amino acid. In certain embodiments, the one or more naturally occurring or encoded amino acids may be any of 20 common amino acids, including but not limited to alanine, arginine, and 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 substitution may be at the following position of IL-2 or a variant thereof: position 38, 46, or 65. In certain embodiments, the naturally occurring amino acid substitution may be at position 38 of IL-2 or a variant thereof. In certain embodiments, the naturally-occurring amino acid substitution at position 38 of the IL-2 or a variant thereof can be selected from any one of 20 common natural amino acids, including but not limited to propylamine Acid, arginine, aspartic acid, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucamine Acid, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. In certain embodiments, the naturally-occurring amino acid substitution at position 38 of the IL-2 or a variant thereof may be an alanine substitution. In certain embodiments, the naturally occurring amino acid substitution may be at position 46 of IL-2 or a variant thereof. In certain embodiments, the naturally-occurring amino acid substitution at position 46 of the IL-2 or a variant thereof can be selected from any one of 20 common natural amino acids, including but not limited to propylamine Acid, arginine, aspartic acid, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucamine Acid, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. In certain embodiments, the naturally-occurring amino acid substitution at position 46 of the IL-2 or a variant thereof may be a leucine or isoleucine substitution. In certain embodiments, the naturally occurring amino acid substitution may be at position 65 of IL-2 or a variant thereof. In certain embodiments, the naturally-occurring amino acid substitution at position 65 of the IL-2 or a variant thereof can be selected from any one of 20 common natural amino acids, including but not limited to propylamine Acid, arginine, aspartic acid, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucamine Acid, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. In certain embodiments, the naturally-occurring amino acid substitution at position 65 of the IL-2 or a variant thereof may be an arginine substitution. In certain embodiments, the amino acid substitution in the IL-2 or a variant thereof may be a naturally-occurring amino acid substitution at position 38, 46, or 65, and at least one of the substitutions is incorporated into Non-naturally encoded amino acids in one or more of the following positions of IL-2 or its variants: 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, Positions 65, 68, 72, and 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitution in IL-2 or a variant thereof may be a naturally-occurring amino acid substitution at position 38, and at least one of the substitutions is incorporated into IL-2 or Non-naturally encoded amino acids in one or more of the following positions of its variants: 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, Position 72 and 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitution in IL-2 or a variant thereof may be a naturally-occurring amino acid substitution at position 46, and at least one of the substitutions is incorporated into IL-2 or Non-naturally encoded amino acids in one or more of the following positions of its variants: 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, Position 72 and 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitution in IL-2 or a variant thereof may be a naturally-occurring amino acid substitution at position 65, and at least one of the substitutions is incorporated into IL-2 or Non-naturally encoded amino acids in one or more of the following positions of its variants: 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, Position 72 and 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitution in the IL-2 or its variant may be a naturally occurring amino acid substitution at positions 38 and/or 46 and/or 65, and at least one of the substitutions is Use non-naturally encoded amino acids incorporated into IL-2 or its variants in one or more of the following positions: 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, Position 62, 64, 65, 68, 72, and 107, and any combination thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitution in IL-2 or a variant thereof may be a naturally-occurring amino acid substitution at position 38 and incorporated into IL-2 or it at position 42. The non-naturally encoded amino acid in the variant (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitutions in IL-2 or its variants may be naturally occurring amino acid substitutions at positions 38 and 46 and incorporated into IL-2 at position 42 Or a non-naturally encoded amino acid in a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitutions in IL-2 or its variants may be naturally occurring amino acid substitutions at positions 38 and 65 and incorporated into IL-2 at position 42 Or a non-naturally encoded amino acid in a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitutions in IL-2 or its variants may be naturally occurring amino acid substitutions at positions 38, 46, and 65 and incorporated into IL at position 42 -2 or a variant thereof in a non-naturally encoded amino acid (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitution in IL-2 or a variant thereof may be a naturally occurring amino acid substitution at position 38 and incorporation into IL-2 at position 45 or The non-naturally encoded amino acid in its variant (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitutions in IL-2 or its variants may be naturally occurring amino acid substitutions at positions 38 and 46 and incorporated into IL-2 at position 45. 2 or a non-naturally encoded amino acid in a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitutions in IL-2 or its variants may be naturally occurring amino acid substitutions at positions 38 and 65 and incorporated into IL-2 at position 45. 2 or a non-naturally encoded amino acid in a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitutions in the IL-2 or variants thereof may be naturally-occurring amino acid substitutions at positions 38, 46, and 65 and incorporated into position 45 A non-naturally encoded amino acid in IL-2 or a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitution in IL-2 or a variant thereof may be a naturally occurring amino acid substitution at position 38 and incorporation into IL-2 at position 65 or The non-naturally encoded amino acid in its variant (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7). In certain embodiments, the amino acid substitutions in IL-2 or its variants may be naturally occurring amino acid substitutions at positions 38 and 46 and incorporated into IL-2 at position 65. 2 or a non-naturally encoded amino acid in a variant thereof (SEQ ID NO: 2, or the corresponding amino acid position in SEQ ID NO: 3, 5, or 7).

In certain embodiments, the non-naturally encoded amino acid comprises a carbonyl group, an acetyl group, an aminooxy group, a hydrazine group (hydrazine), a hydrazide group (hydrazide group), a semicarbazide group (semicarbazide group), Azido or alkynyl.

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; R ₁ is alkyl, aryl, substituted alkyl or substituted aryl; R ₂ is H, alkyl, aryl, substituted alkyl and substituted aryl; R ₃ is H, an amino acid, polypeptide, or amino terminal modification group, and R ₄ is H, an amino acid, polypeptide, or a carboxy terminal modification group.

In certain embodiments, the non-naturally encoded amino acid comprises an aminooxy group. In certain embodiments, the non-naturally encoded amino acid comprises a hydrazine group. In certain embodiments, the non-naturally encoded amino acid comprises a hydrazine group. In certain embodiments, the non-naturally encoded amino acid residue comprises an aminourea group.

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

Wherein n is 0-10; R ₁ is alkyl, aryl, substituted alkyl, substituted aryl or absent; X is O, N, S or absent; m is 0-10; R ₂ is H , Amino acid, polypeptide, or amino terminal modification group, and R ₃ is H, amino acid, polypeptide, or carboxy terminal modification group.

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

Wherein n is 0-10; R ₁ is alkyl, aryl, substituted alkyl or substituted aryl; X is O, N, S or not present; m is 0-10, R ₂ is H, amino group An acid, polypeptide, or amino terminal modification group, and R ₃ is H, an amino acid, polypeptide, or a carboxy terminal modification group.

In certain embodiments, the polypeptide is an IL-2 agonist, partial agonist, antagonist, partial antagonist, or inverse agonist. In certain embodiments, the IL-2 agonist, partial agonist, antagonist, partial antagonist or inverse agonist comprises a non-naturally encoded amino acid linked to a water-soluble polymer. In certain embodiments, the water-soluble polymer comprises polyethylene glycol components. In certain embodiments, the IL-2 agonist, partial agonist, antagonist, partial antagonist or inverse agonist comprises a non-naturally encoded amino acid and one or more post-translational modifications, linkers, and Substances 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 encoding SEQ ID NO: 1 under stringent conditions. , 2, 3, 5, or 7 polypeptide hybridizes the polynucleotide (polynucleotide). 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 (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 those 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 the group consisting of amber codon, ochre codon, opal codon, unique codon, rare codon, five base codon Five-base codons and four-base codons.

The present invention also provides a method for manufacturing IL-2 or a variant thereof linked to a biologically active molecule. In certain embodiments, the method includes combining the isolated IL-2 containing a non-naturally encoded amino acid or a variant thereof with a biologically active molecule containing a component that reacts with the non-naturally encoded amino acid To contact. In certain embodiments, the non-naturally encoded amino acid incorporated into IL-2 or a variant thereof has a biologically active molecule that is originally non-reactive to any of the 20 commonly used amino acids. Reactive. In certain embodiments, the non-naturally encoded amino acid incorporated into IL-2 has a linker, Polymers or biologically active molecules are reactive.

In certain embodiments, the IL-2 or a variant thereof linked to a water-soluble polymer or a biologically active molecule is obtained by combining IL-2 or a variant thereof containing a carbonyl-containing amino acid with an aminooxy group, a hydrazine , Hydrazine or aminourea-based water-soluble polymers or biologically active molecules are produced by reaction. In certain embodiments, the aminooxy group, hydrazine, hydrazine or aminourea group is connected to the biologically active molecule through an amide bond. In certain embodiments, the amineoxy group, hydrazine, hydrazine or aminourea group is connected to the water-soluble polymer or biologically active molecule through a urethane bond.

The present invention also provides a method for manufacturing the IL-2 conjugate linked to the water-soluble polymer. In certain embodiments, the method includes combining an isolated IL-2-biologically active molecule conjugate containing a non-naturally encoded amino acid with a component that contains a component that reacts with the non-naturally encoded amino acid. The water-soluble polymers are in contact. In certain embodiments, the non-naturally encoded amino acid incorporated into the IL-2 conjugate has an effect on water-soluble polymers that are originally non-reactive to any of the 20 commonly used amino acids. Reactive. In certain embodiments, the non-naturally encoded amino acid incorporated into the IL-2 conjugate has a linker, polymer, or linker that is originally non-reactive to any of the 20 commonly used amino acids. Biologically active molecules are reactive.

The present invention also provides a method for manufacturing IL-2 or a variant thereof linked to a water-soluble polymer. In certain embodiments, the method includes a water-soluble polymerization of an isolated IL-2 containing a non-naturally encoded amino acid or a variant thereof with a component that reacts with the non-naturally encoded amino acid. Matter contact. In certain embodiments, the non-naturally encoded amino acid incorporated into IL-2 or a variant thereof is a water-soluble polymer that is originally non-reactive to any of the 20 commonly used amino acids Reactive. In certain embodiments, the non-naturally encoded amino acid incorporated into IL-2 has a linker, polymer, or linker that is originally unreactive to any of the 20 commonly used amino acids. Biologically active molecules are reactive.

In certain embodiments, the IL-2 or a variant thereof linked to a water-soluble polymer is obtained by combining IL-2 or a variant thereof containing a carbonyl-containing amino acid with an aminooxy group, hydrazine, hydrazine or Aminourea-based polyethylene glycol molecules are produced by reacting. In certain embodiments, the aminooxy group, hydrazine, hydrazine, or aminourea group is connected to the polyethylene glycol molecule through an amide bond. In certain embodiments, the amineoxy group, hydrazine, hydrazine, or aminourea group is connected to the polyethylene glycol molecule through a urethane bond.

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

In certain embodiments, the IL-2 or a variant thereof linked to a water-soluble polymer is obtained by combining IL-2 containing alkynyl amino acid with polyethylene containing an azide moiety. Alcohol molecules react to produce. In certain embodiments, the azide or alkynyl group is connected to the polyethylene glycol molecule through an amide bond.

In certain embodiments, the IL-2 or a variant thereof linked to a water-soluble polymer is obtained by combining IL-2 or a variant thereof containing an azido-containing amino acid with a polyethylene containing an alkynyl component. Diol molecules react to produce. In certain embodiments, the azide or alkynyl group is connected to the polyethylene glycol molecule through an amide bond.

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

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

In certain embodiments, the water-soluble polymer connected to IL-2 or a variant thereof comprises a polyalkylene glycol moiety. In certain embodiments, the non-naturally encoded amino acid residue incorporated into IL-2 comprises a carbonyl group, an aminooxy group, a hydrazine group, a hydrazine, an aminoureido group, an azide group, or an alkynyl group. . In certain embodiments, the non-naturally encoded amino acid residue incorporated into IL-2 or a variant thereof includes a carbonyl component, and the water-soluble polymer includes an aminooxy group, hydrazine, Component of hydrazine or aminourea. In certain embodiments, the non-naturally encoded amino acid residue incorporated into IL-2 or a variant thereof includes an alkynyl moiety, and the water-soluble polymer includes an azide moiety. In certain embodiments, the non-naturally encoded amino acid residue incorporated into IL-2 or a variant thereof includes an azide moiety, and the water-soluble polymer includes 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, which comprises a polynucleotide comprising a selector codon that encodes IL-2 or an IL-2 variant. In certain embodiments, the cell contains orthogonal RNA synthetase and/or orthogonal Trna (orthogonal tRNA) for replacing non-naturally encoded amino acids into the IL-2 .

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

The present invention also provides methods for manufacturing PEG-IL-2, IL-2 or any variants thereof containing non-naturally encoded amino acids. In certain embodiments, the method includes placing a cell containing one or more polynucleotides encoding IL-2, an orthogonal RNA synthetase, and/or an orthogonal tRNA in a cell that allows the IL-2 or a variant thereof Culturing under expression conditions; and purifying the IL-2 or variants thereof from the cells and/or the 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 modulating the immunogenicity of IL-2 or its variants. In certain embodiments, the method includes replacing any one or more amino acids in naturally-occurring IL-2 or its variants with non-naturally-encoded amino acids and/or replacing the IL-2 Or a variant thereof is attached to a linker, polymer, water-soluble polymer or biologically active molecule. In one embodiment of the invention, the linker is long enough to allow flexibility and allow dimer formation. In one embodiment of the present invention, the length of the linker is at least 3 amino acids or 18 atoms in order to allow the formation of dimers.

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 of the present invention or a variant thereof. In certain embodiments, the method includes administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising PEG-IL-2 or a variant thereof containing a non-naturally encoded amino acid and Pharmaceutically acceptable carrier. In certain embodiments, the method includes administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising a non-naturally encoded amino acid and a natural amino acid replacement PEG-IL- 2 or its variants and pharmaceutically acceptable carriers. In certain embodiments, the non-naturally encoded amino acid is linked to a water-soluble polymer. In certain embodiments, the PEG-IL-2 or variants thereof are glycosylated. In certain embodiments, the PEG-IL-2 or variants thereof are not glycosylated.

The present invention also provides a method for treating a patient in need of the treatment using an effective amount of the IL-2 or IL-2 variant molecule of the present invention. In certain embodiments, the method includes administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising IL-2 or an IL-2 variant molecule containing a non-naturally encoded amino acid And a pharmaceutically acceptable carrier. In certain embodiments, the method includes administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising one or more non-naturally encoded amino acids and one or more natural amines. Base acid-substituted IL-2 or its variants and a pharmaceutically acceptable carrier. In certain embodiments, the non-naturally encoded amino acid is linked to a water-soluble polymer. In certain embodiments, the natural amino acid is linked to a water-soluble polymer. In certain embodiments, the IL-2 is glycosylated. In certain embodiments, the IL-2 is not glycosylated. In certain embodiments, the patient in need of treatment suffers from a cancer, disorder or disease characterized by high expression of IL-2 receptor alpha, but is not limited thereto. In certain embodiments, the present invention provides a method of treating cancer or a disorder or disease by administering to an article a therapeutically effective amount of the IL-2 composition of the present invention. In certain embodiments, the present invention provides a method of treating genetic diseases by administering to a patient a therapeutically effective amount of the IL-2 composition of the present invention. The IL-2 polypeptide of the present invention is used to treat diseases or disorders in cells with high expression of IL-2 receptor alpha. In certain embodiments, the cancer, disorder or disease is treated by reducing, blocking or silencing IL-2 receptor alpha expression. The IL-2 polypeptides or variants of the present invention are used to manufacture drugs for the treatment of cancers, diseases or disorders related to the high expression of IL-2 receptor alpha. The IL-2 polypeptides or variants of the present invention are used to manufacture drugs for the treatment of cancer. The IL-2 polypeptides or variants of the present invention are used to manufacture drugs for the treatment of 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 certain embodiments, the non-naturally encoded amino acid is linked to a water-soluble polymer. In certain embodiments, the water-soluble polymer comprises polyethylene glycol components. In certain embodiments, the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazino group, a hydrazine group, an aminoureido group, an azide group, or an alkynyl group.

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

The present invention also provides an IL-2 or a natural variant thereof, which comprises a water-soluble polymer linked to the IL-2 via a covalent bond at a single amino acid. In certain embodiments, the water-soluble polymer comprises polyethylene glycol components. 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 biologically active molecule, wherein the linker, polymer or biologically active molecule is incorporated into the polypeptide via a ribosome. The functional group of the encoded amino acid is attached to the polypeptide. In certain embodiments, the IL-2 or variants thereof are mono-PEGylated. The present invention also provides an IL-2 or a variant thereof, which comprises a linker, polymer or biologically active molecule attached to one or more non-naturally encoded amino acids, wherein the non-naturally encoded amino acid Incorporated into the polypeptide via the ribosome at a pre-selected location.

The scope of the present invention includes the leader or signal sequence (leader or signal sequence) of IL-2 or its variants linked to the IL-2 coding region and the heterologous signal sequence (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 secretion system of the host cell, and may be cleaved by the signal peptidase of the host cell. The method of using the IL-2 of the present invention to treat a disease or disorder implies treatment with IL-2 or a variant thereof with or without a signal or leader peptide.

In another embodiment, the IL-2 containing one or more non-naturally-occurring amino acids or a variant thereof is conjugated with another molecule (including but not limited to PEG) due to the The unique chemical reaction of non-natural amino acid conjugation provides substantially purified IL-2. Conjugation of IL-2 or its variants containing 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 A substantially pure IL-2 or a variant thereof is provided.

definition

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

When used in the present invention and in the scope of the accompanying patent application, the singular form without a specific number includes plural referents, unless the context clearly indicates otherwise. Therefore, for example, for "IL-2", "PEG-IL-2", "PEG-IL-2 conjugate" and various capitals, hyphenated and unlinked A reference in the form of a font size refers to one or more such proteins, and includes their equivalents known to those of ordinary skill in the art, and so on.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the art 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, preferred methods, devices and materials will now be described.

All publications and patents mentioned in the present invention are incorporated by reference into the present invention for the purpose of describing and disclosing, for example, the constructs and methods described in the publications that can be used in combination with the presently described invention. The publications discussed in this invention are provided only because their disclosure precedes the filing date of this application. Nothing in the present invention shall be construed as an admission that the inventor has no right to predate these disclosures by virtue of prior inventions or any other reasons.

The term "substantially purified" refers to IL-2 or a variant thereof, which may be substantially or essentially free from the naturally occurring environment, that is, in the native ) A component that usually accompanies or interacts with the protein found in the cell or in the host cell in the case of recombinantly produced IL-2. IL-2 that may be substantially free of cellular material includes 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 A protein preparation that contaminates protein at about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight). When the IL-2 or its variant is recombinantly produced by the host cell, the protein may be about 30%, about 25%, about 20%, about 15%, about 10%, about 5% of the net weight of the cell. , About 4%, about 3%, about 2%, or about 1% or less. When the IL-2 or its variant is recombinantly produced by the host cell, the protein may be about 5g/L, about 4g/L, about 3g/L, about 2g/L, about 1g/L of the net weight of the cell. 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 is present in the medium. Therefore, the "substantially purified" IL-2 produced by the method of the present invention may have 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% purity level, especially at least about 75%, 80%, 85% purity level, more particularly at least about 90% purity level, at least about 95% purity level The purity level, a purity level of at least about 99% or higher, as determined by suitable methods such as SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

"Recombinant host cell" or "host cell" refers to a cell that includes exogenous polynucleotides, regardless of the method used for insertion, such as direct uptake, transduction, f -Mating is also another method known in the art for the production of recombinant host cells. The exogenous polynucleotide can be maintained as a nonintegrated vector such as a plastid, or can be integrated into the host genome.

When used in the present invention, the term "medium" includes any medium, solution, solid, semi-solid or rigid support, which can support or contain any host cell, including bacterial host cells, yeast host cells, insect host cells, plants Host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, Escherichia coli or Pseudomonas host cells, and cell contents. Thus, the term can encompass the medium in which the host cell has grown, for example, the medium into which IL-2 has been secreted, including the medium before or after the proliferation step. The term can also encompass a buffer or reagent containing a host cell lysate, for example where IL-2 is produced in the cell and the host cell is lysed or disrupted to release IL-2.

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

When the use of protein refolding is involved in the present invention, "oxidant" is defined as any compound or material capable of removing electrons from the oxidized compound. Suitable oxidizing agents include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythritol, and oxygen. It is obvious to those of ordinary skill in the art that a wide range of oxidizing agents are suitable for use in the method of the present invention.

When used in the present invention, a "denaturing agent (or denaturant)" is defined as any compound or material that causes the reversible unfolding of a protein. The strength of the denaturant is determined by both the nature and concentration of the specific denaturant. Suitable denaturants can be chaotropic agents, detergents, organic solvents, water-miscible solvents, phospholipids, or a combination of two or more such agents. Suitable chaotropic agents include, but are not limited to, urea, guanidine, and sodium thiocyanate. Useful detergents can include, but are not limited to, strong detergents such as sodium lauryl sulfate or polyoxyethylene ethers (e.g. Tween or Triton detergents), Sarkosyl, mild non-ionic detergents (e.g., fur soil Xanthoside), mild cationic detergents such as N-›2,3-(dioleyloxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents (e.g. Sodium cholate or sodium deoxycholate) or zwitterionic detergents, including but not limited to sulfobetaine (Zwittergent), 3-(3-chloroaminopropyl) dimethyl ammonium-1-propane sulfuric acid Salt (CHAPS) and 3-(3-chloroaminopropyl)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 alkanols such as ethylene glycol) can be used as Denaturant. Phospholipids useful in the present invention may be naturally occurring phospholipids such as phospholipid ethanolamine, phospholipid choline, phospholipid serine and phospholipid inositol or synthetic phospholipid derivatives or variants such as dihexyl phospholipid choline Alkali or diheptanyl phospholipid choline.

When used in the present invention, "Refolding" describes the transition of a polypeptide containing disulfide bonds from an improperly folded or unfolded state to a native disulfide bond. ) Or any process, reaction, or method of a correctly folded conformation.

When used in the present invention, "cofolding" specifically refers to the refolding process of using at least two polypeptides that interact with each other and cause unfolding or incorrect folding of the polypeptide to convert the source, correctly folded polypeptide, Reaction or method.

When used in the present invention, "interleukin-2", "IL-2" and their hyphenated and unhyphenated forms include polypeptides and proteins with at least one biological activity of IL-2, and IL-2 analogs, IL-2 muteins, IL-2 variants, IL-2 isoforms, IL-2 mimetics, IL-2 fragments, hybrids Integrate IL-2 protein, fusion protein, oligomers and multimers, homologues, glycosylation pattern variants, variants, splice variants variants) and mutant proteins, regardless of their biological activity, and regardless of their synthesis and manufacturing methods, including but not limited to recombination (whether from cDNA, genomic DNA, synthetic DNA or other forms of nucleic acid production), in vitro , In vivo, through microinjection, synthesis, transgene and gene activation methods of nucleic acid molecules. The terms "IL-2", "IL-2 variant" and "IL-2 polypeptide" encompass IL-2 containing one or more amino acid substitutions, additions or deletions.

For the sequence of IL-2 lacking the leader sequence and without methionine at the N-terminus, see SEQ ID NO: 2 in the present invention. See SEQ ID NO: 3, 5, or 7 for the sequence of IL-2 without a leader sequence and having methionine at the N-terminus. In certain embodiments, the IL-2 of the present invention or a variant thereof 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 naturally occurring pharmaceutically acceptable salts and prodrugs of IL-2 and prodrugs, polymorphs, hydrates, solvates, biologically active fragments, and biologically active Variants and stereoisomers, as well as naturally-occurring IL-2 agonists, mimetics and antagonist variants and polypeptide fusions thereof.

Various references disclose the modification of polypeptides by polymer conjugated or glycosylation. The term "IL-2" includes polypeptides conjugated to polymers such as PEG, and may contain one or more additional derivatizations of cysteine, lysine, or other residues. In addition, the IL-2 may include a linker or a 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 use known in the art The technology is conjugated to naturally-encoded amino acids, such as lysine or cysteine.

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 as 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 polypeptides, proteins, carbohydrates, polymers, small molecules, linkers, ligands, or any type of other organisms that are linked by chemical means or expressed as fusion proteins. IL-2 heterodimers, homodimers, heteromultimers or homomultimers of active molecules, and polypeptide analogs that contain, for example, specific deletions or other modifications but still maintain biological activity.

When used in the present invention, whether it is conjugated to a biologically active molecule, conjugated to polyethylene glycol or in a non-conjugated form, "interleukin-2" or "IL-2" contains non-covalent linkages In order to form a homodimer of two subunits (subunit) of the protein. When used in the present invention, "interleukin-2" and "IL-2" can 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 with one or more polyethylene glycol molecules that Covalent attachment to one or more than one amino acid residues of the IL-2 protein through a linker makes the attachment stable. The terms "mono-PEGylated IL-2" and "mono-PEG-IL-2" mean that a polyethylene glycol molecule is covalently attached to a subunit of the IL-2 dimer through a linker A single amino acid residue on the top. The average molecular weight of the PEG component is preferably between about 5,000 to about 50,000 daltons (daltons). The method or site of attachment of PEG to IL-2 is not important, but preferably the pegylation does not change or only minimally changes the activity of the bioactive molecule. Preferably, the increase in half-life exceeds any decrease in biological activity.

All references to the amino acid positions in IL-2 described in the present invention are based on the positions in SEQ ID NO: 2, unless otherwise specified (that is, when it is stated that the comparison is based on SEQ ID NO: 3, 5, or 7 or other IL-2). Those skilled in the art will recognize that the amino acid position corresponding to the position in SEQ ID NO: 2 in any other IL-2, such as SEQ ID NO: 3, 5, or 7, can be easily identified. Those skilled in the art will recognize that in any other IL-2 molecules such as IL-2 fusions, variants, fragments, etc., the same as those in SEQ ID NO: 2, 3, 5 or 7 or any other IL-2 sequence The position of the amino acid corresponding to the position can be easily identified. For example, a sequence alignment program such as BLAST can be used to align and identify specific positions in the protein that correspond 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 IL described in the present invention or known in the art -2 Substitutions, deletions or additions in corresponding positions in fusions, variants, fragments, etc., and are clearly covered by the present invention.

IL-2 (IL3): Any form of IL-2 known in the art can be used in the composition described in the present invention. 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 changed without affecting its activity, and these modified forms of IL2 can also be linked to a carrier and used in the method described in the present invention.

The term "IL-2" encompasses IL-2 containing 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 combined 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 regulating drug stability, regulating one or more biological activities of IL-2 polypeptides such as but not limited to improving Agonist activity, increase the solubility of the polypeptide, reduce protease sensitivity, convert the polypeptide into an antagonist, etc., and are covered 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 variants thereof further comprise additions, substitutions or deletions that modulate the biological activity of the IL-2 or variant polypeptides. In certain embodiments, the IL-2 or variants further include additions, substitutions, or deletions of known and research-proven traits that modulate IL-2, such as treatment or alleviation of one or more symptoms of cancer. The addition, substitution or deletion can modulate one or more properties or activities of IL-2 or variants. For example, the addition, substitution or deletion can adjust the affinity to the IL-2 receptor or one or more subunits of the receptor, adjust the circulatory half-life, adjust the therapeutic half-life, and adjust the stability of the polypeptide. Sex, adjust the cleavage by the protease, adjust the dosage, adjust the release or bioavailability, promote purification, or improve or change the specific route of administration. Similarly, 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), which improve the detection (including but not limited to GFP), purification or other properties of the polypeptide.

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

When used in the present invention, the terms "conjugate of the present invention", "IL-2-biologically active molecule conjugate" or "PEG-IL-2" refer to the conjugate to a biologically active molecule, Interleukin-2 or a part, analogue or derivative thereof that binds to the interleukin-2 receptor or its subunit of a part or its analogue. Unless otherwise indicated, the terms "compound of the present invention" and "composition of the present invention" are used as alternatives to the term "conjugate of the present invention".

When used in the present invention, the term "cytotoxic agent" can mean that it has a therapeutic effect on cancer cells or activated immune cells, and can be used as a therapeutic agent with IL-2, PEG-IL-2, or IL-2 variants. Any medicament used in combination (see, for example, WO 2004/010957, "Drug Conjugates and Their Use for Treating Cancer, An Autoimmune Disease or an Autoimmune Disease or an Infectious Disease)). The categories of cytotoxic or immunosuppressive agents used in the present invention include, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes (complexes) such as Cisplatin, monoplatinum, diplatinum and trinuclear platinum complexes (complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, becarcinomycins, etoposide Etoposides, fluoropyrimidines, ionophores, lexitropsins, nitrosoureas, platinum alcohols, preformed compounds, purine antimetabolites, puromycins, radiation sensitizers, steroids , Taxanes, topoisomerase inhibitors, vinca alkaloids, etc.

Individual cytotoxic or immunosuppressive agents include, for example, androgen, antoxin (AMC), aspartame, 5-azacytidine, azathioprine, bleomycin, busulfan, butylthiamine Suthylene, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, Cytochalasin B, dacarbazine, dactinomycin (formerly known as actinomycin), daunorubicin, dacarbazine, docetaxel, doxorubicin, estrogen, 5-fluorodeoxyuridine , 5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), methylchloroethylamine, melphalan, 6-mercaptopurine, methyl Methotrexate, Mitoshin, Mitomycin C, Mitoxantrone, Nitroimidazole, Paclitaxel, Pracamycin, Procarbazine, Streptozotocin, Teniposide, 6-thioguan Purine, thioTEPA, 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 minor groove binders (e.g., enediynes and lexitropsins), beonomycins, violet Taxanes (e.g., paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin, risol New, cyanomorpholino-doxorubicin (cyanomorpholino-doxorubicin), echinomycin, compretin, fusiformin, epothilone A and B, estramustine, cryptophyrins, western Madotin, maytansinoids, discomerolide (discodermolide), arbutin, and mitoxantrone (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 can be used synonymously with the term "non-naturally encoded amino acid" are "non-natural amino acid", "non-naturally occurring amino acid" and their various hyphenated and unhyphenated forms . The term "non-naturally encoded amino acid" also includes but is not limited to the modification of naturally-encoded amino acids (including but not limited to 20 common amino acids or pyrrolysine and selenocysteine). , But not naturally incorporated into the growing polypeptide chain by the translation complex itself. Examples of such non-naturally occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyramine acid.

"Amino end modification group" refers to any molecule that can be attached to the amino end of a polypeptide. Likewise, "carboxyl terminal modification group" refers to any molecule that can be attached to the carboxyl terminal 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 peptides.

The terms "functional group", "active component", "activating group", "leaving group", "reactive site", "chemically reactive group" and "chemically reactive component" are in the art and In the present invention, it is used to refer to a unique and definable part or unit of a molecule. The terms are somewhat synonymous in the field of chemistry, and are used in the present invention to indicate parts of molecules that perform certain functions or activities and are reactive with other molecules.

The terms "linkage", "linkage" or "linker" are used in the present invention to refer to groups or bonds that are usually formed as a result of a chemical reaction, and are usually covalent bonds. A hydrolytically stable bond means that the bond is substantially stable in water and does not react with water for a long time, even possibly indefinitely, at a useful pH value (including but not limited to under physiological conditions). A hydrolytically unstable or degradable bond means that the bond is degradable in water or aqueous solutions (including, for example, blood). An enzyme-labile or degradable bond means that the bond can be degraded by one or more enzymes. As understood in the art, PEG and related polymers may include degradable bonds in the polymer backbone or in the linker group between the polymer backbone and one or more terminal functional groups of the polymer molecule. For example, an ester bond formed by the reaction of a PEG carboxylic acid or an activated PEG carboxylic acid with an alcohol group on a biologically active agent is usually hydrolyzed under physiological conditions to release the agent. Other hydrolytically degradable bonds include, but are not limited to, carbonate bonds, imine bonds derived from the reaction of amines and aldehydes, phosphate bonds formed by the reaction of alcohols and phosphoric acid groups, and hydrazone as a by-product of hydrazine and aldehydes. Bond, acetal bond as the reaction product of aldehyde and alcohol, orthoester bond as the reaction product of formic acid and alcohol, peptides formed from the amine group at the end of a polymer such as PEG and the carboxyl group of the peptide A bond, and an oligonucleotide bond formed by a phosphoramidite group at the end of the polymer, including but not limited to, and the 5'hydroxyl group of the oligonucleotide.

When used in the present invention, the terms "biologically active molecule", "biologically active component" or "biologically active agent" means any physical or biological activity that can affect biological systems, pathways, molecules, or interactions related to the organism. Any substance of chemical nature, the organisms include but are not limited to viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals and humans. Specifically, when used in the present invention, biologically active molecules include, but are not limited to, those intended to diagnose, cure, alleviate, treat or prevent diseases in humans or other animals, or otherwise enhance the physical or mental health of humans or animals. Any substance that is healthy. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, vaccines, immunogens, addictive drugs, non-addictive drugs, sugars, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclei Nucleic acids, oligonucleotides, toxoids, biologically active molecules, prokaryotic and eukaryotic cells, viruses, polysaccharides, nucleic acids derived from or derived from viruses, bacteria, insects, animals, or any other cell or cell type and their parts , Liposomes, microparticles and micelles. The categories 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, anti-anxiety drugs, hormones, growth factors, steroid drugs, biologically active molecules derived from microorganisms, etc. Bioactive agents also include amido compounds such as those described in Yamamori et al. Patent Application Publication No. 20080221112, which are administered before, after, and/or co-administered with the IL-2 polypeptide of the present invention.

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

When the substituents are described by their conventional chemical formulas written from left to right, they also cover the chemically consistent substituents obtained by writing the structure from right to left, for example, the structure -CH ₂ O-is equivalent to Structure -OCH ₂ ‑.

The term "substituents" includes but is not limited to "non-interfering substituents". "Non-interfering substituents" are groups that produce stable compounds. Suitable non-interfering substituents or residues include, but are not limited to, 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, tolyl, 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 alkylsulfinyl, --(CH ₂ ) _m --O--(C ₁ -C ₁₀ alkyl) (where m is 1 to 8) , Aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclyl, substituted heterocyclyl, 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) -CF3, --C(O)--CF3, --C(O)NR2, --(C ₁ -C ₁₀ aryl) -S--(C ₆ -C ₁₀ aryl), --C(O )--(C ₁ -C ₁₀ aryl), --(CH ₂ ) _m --O--(--(CH ₂ ) _m --O--(C ₁ -C ₁₀ alkyl) (where each m-1 to 8), - C (O) NR 2, - C (S) NR 2, - SO 2 NR 2, - NRC (O) NR 2, - NRC (S) NR 2 , Its salts, etc. When used in the present invention, each R is H, alkyl or substituted alkyl, aryl or substituted aryl (aryl), aralkyl or alkaryl (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 linear or branched or cyclic hydrocarbon group or a combination thereof, which may be fully saturated, mono- or polyunsaturated, and may Including divalent and multivalent residues, with the specified number of carbon atoms (ie, C ₁ -C ₁₀ means 1 to 10 carbon atoms). Examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl , Cyclopropylmethyl, homologues and isomers such as n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. An unsaturated alkyl group is a group having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1 ,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl and higher homologues and isomers. Unless otherwise indicated, the term "alkyl" is also meant to include derivatives of alkyl defined in more detail below, such as "heteroalkyl". Alkyl groups limited to hydrocarbyl groups are called "homoalkyl groups".

The term "alkylene" by itself or as part of another substituent means a divalent residue derived from an alkane, such as but not limited to the structures -CH ₂ CH ₂ -and -CH ₂ CH ₂ CH ₂ CH ₂ -, and also include groups described below as "heteroalkylene". Generally, alkyl groups (or alkylene groups) have 1 to 24 carbon atoms, and groups with 10 or fewer carbon atoms are specific examples of the methods and compositions described in this invention. "Lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, usually having 8 or fewer carbon atoms.

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

Unless otherwise stated, the term "heteroalkyl" by itself or in combination with another term means composed of the stated number of carbon atoms and at least one heteroatom selected from O, N, Si and S A stable linear or branched or cyclic hydrocarbon group or a combination thereof, and wherein the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. The heteroatoms O, N, S, and Si may be placed at any internal position of the heteroalkyl group or the position where the alkyl group is attached to the remainder of the molecule. Examples include but are not limited to -CH ₂ -CH ₂ -O-CH ₃ , -CH ₂ -CH ₂ -NH-CH ₃ , -CH ₂ -CH ₂ -N(CH ₃ )-CH ₃ , -CH ₂ -S -CH ₂ -CH ₃ , -CH ₂ -CH ₂ -S(O)-CH ₃ , -CH ₂ -CH ₂ -S(O) ₂ -CH ₃ , -CH=CH-O-CH ₃ , -Si (CH ₃ ) ₃ , -CH ₂ -CH=N-OCH ₃ and -CH=CH-N(CH ₃ )-CH ₃ . Up to two heteroatoms can be continuous, for example -CH ₂ -NH-OCH ₃ and -CH ₂ -O-Si(CH ₃ ) ₃ . Likewise, the term "heteroalkylene" by itself or as part of another substituent means a divalent residue derived from a heteroalkyl group, such as but not limited to -CH ₂ -CH ₂ -S-CH ₂ ‑CH ₂ -and -CH ₂ -S-CH ₂ -CH ₂ -NH-CH ₂ -. For heteroalkylene groups, the same or different heteroatoms can also occupy either or both chain ends (including but not limited to alkyleneoxy, alkylene dioxy, alkylene amino, alkylene Diamino, 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) ₂ R'‑ represents both –C(O) ₂ R'‑ and –R'C(O) ₂ ‑.

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

When used in the present invention, the term "water-soluble polymer" refers to any polymer that is soluble in an aqueous solvent. The connection of the 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, its mono C1-C10 alkoxy or aryloxy derivatives (described in U.S. Patent No. 5,252,714, which is incorporated by reference into the present invention ), monomethoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acid, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran Sugars, dextran derivatives (including dextran sulfate), polypropylene glycol, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols, heparin, heparin fragments, polysaccharides, oligosaccharides, polysaccharides, cellulose And cellulose derivatives (including but not limited to methyl cellulose and carboxymethyl cellulose), starch and starch derivatives, polypeptides, polyalkylene glycols and their derivatives, polyalkylene glycols and their derivatives Polyvinyl ethyl ether and α-β-poly(2-hydroxyethyl)-DL-aspartamide, 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 derivatives thereof. The term "polyalkylene glycol" covers both linear and branched chain polymers, and has an average molecular weight between 0.1 kDa and 100 kDa. Other exemplary embodiments are listed, for example, in catalogs of commercial suppliers, 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 fused together or covalently linked (including but not limited to 1 to 3 Rings). 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 Is quaternized. Heteroaryl groups can be attached to the rest of the molecule through heteroatoms. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazole Group, 2-imidazolyl (2-imidazolyl), 4-imidazolyl, pyrazinyl, 2-oxazolyl (2-oxazolyl,), 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furan Base, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purine Group, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl (2-quinoxalinyl), 5-quinoxalinyl, 3- 3-quinolyl and 6-quinolyl. For each of the above-mentioned aryl and heteroaryl ring systems, the substituents are selected from the acceptable substituents described below.

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

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

Used for alkyl and heteroalkyl residues (including often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl and heterocyclic The substituent of the alkenyl group) may be one or more selected from but not limited to the following various groups: -OR', =O, =NR', =N-OR', -NR'R",-SR', -halogen, -SiR'R"R"', -OC(O)R', -C(O)R', -CO ₂ R', -CONR'R", -OC( O)NR'R”,-NR”C(O)R', -NR'‑C(O)NR”R”', -NR”C(O) ₂ R', -NR-C(NR'R ”R'”)=NR””, ‑NR‑C(NR'R”)=NR'”, -S(O)R', -S(O) ₂ R', -S(O) ₂ NR'R", -NRSO ₂ R', -CN and -NO ₂ , the number of which ranges from 0 to (2m'+1), where m'is the total number of carbon atoms in this residue. R', R", R"' and R"" each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (including but not limited to aryl substituted with 1-3 halogens) Group), substituted or unsubstituted alkyl, alkoxy or thioalkoxy or aralkyl. When the compound of the present invention includes more than one R group, for example, each of the R groups is independently selected, and when there is more than one R', R”, R'” and R”” group, each The same is true for these groups. 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" means including but not limited to 1-pyrrolidine And 4-morpholinyl. From the discussion of the above 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 haloalkyl groups (including but not limited to- CF ₃ and -CH ₂ CF ₃ ) and acyl groups (including but not limited to -C(O)CH ₃ , -C(O)CF ₃ , -C(O)CH ₂ OCH _3, etc.).

Similar to the substituents described for alkyl residues, the substituents for aryl and heteroaryl are variable and are selected from but not limited to: halogen, -OR', =O, =NR', = N-OR', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(O)R', -C(O)R', -CO ₂ R',- CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O) ₂ R', ‑NR-C(NR'R”R’”)=NR””, ‑NR‑C(NR'R”)=NR'”, -S(O)R', -S(O) ₂ R', -S(O) ₂ NR'R", -NRSO ₂ R', -CN and -NO ₂ , -R', -N ₃ , -CH(Ph) ₂ , fluoro (C ₁ -C ₄ ) alkoxy And fluoro (C ₁ -C ₄ ) alkyl groups, the number of which is in the range of 0 to the total number of open valences on the aromatic ring system; and wherein R', R", R"' and R"" are independently It is selected from hydrogen, alkyl, heteroalkyl, aryl and heteroaryl. When the compound of the present invention includes more than one R group, for example, each of the R groups is independently selected, and when there is more than one R', R”, R'” and R”” group, each The same is true for these groups.

When used in the present invention, the term "regulated serum half-life" means a positive or negative change in the circulating half-life of modified IL-2 relative to its unmodified form. The serum half-life is measured by taking blood samples at various time points after IL-2 administration and determining the concentration of the molecule in each sample. The correlation between serum concentration and time allows calculation of serum half-life. The increased serum half-life is ideally at least about twice as long, but a smaller increase may also be useful, for example where it can achieve a satisfactory dosing regimen or avoid toxic effects. In certain embodiments, the increase is at least about three times, at least about five times, or at least about ten times.

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

The term "isolated" when applied to a nucleic acid or protein means that the nucleic acid or protein does not contain at least certain cellular components that accompany it in its natural state, or that the nucleic acid or protein has been concentrated to a level higher than The level of its concentration produced in vivo or in vitro. It can be in a homogeneous state. The separated substance can be in a dry or semi-dry state, or in a solution (including but not limited to an aqueous solution). It may be a component of a pharmaceutical composition that additionally contains pharmaceutically acceptable carriers and/or excipients. Purity and homogeneity are usually determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The protein, which is the main substance present in the preparation, is substantially purified. Specifically, the isolated gene is separated from the open reading frame flanking the gene and encoding a protein other than the gene of interest. The term "purified" means that the nucleic acid or protein essentially produces a band in the electrophoresis gel. Specifically, this may mean that the nucleic acid or protein is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or more pure.

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

The terms "polypeptide", "peptide" and "protein" are used interchangeably in the present invention to refer to polymers of amino acid residues. That is to say, the description of the polypeptide is equally applicable to the description of the peptide and the description of the protein, and vice versa. The term applies to naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. When used in the present invention, the term encompasses amino acid chains of any length, including full-length proteins, in which the amino acid residues are connected 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 similar manner to the naturally occurring amino acids. Naturally encoded amino acids are 20 common amino acids (alanine, arginine, aspartic acid, aspartic acid), cysteine, glutamic acid, glutamine, Glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine Amino acid) as well as pyrrole lysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, that is, they are bound to the alpha carbon of hydrogen, carboxyl, amino and R groups, such as homoserine, ortholeucine, Sulfide methionine, methyl thiomethionine. These analogs have modified R groups (for example, ortholeucine) 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, and naturally occurring non-protein amino acids such as β -Alanine, ornithine, etc., as well as chemically synthesized compounds known in the art to be unique to amino acids. Examples of non-naturally occurring amino acids include, but are not limited to, α-methylamino 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), amine groups with additional methylene groups in the side chain Acids ("homo" amino acids) and amino acids in which the carboxylic acid functional group in the side chain is replaced by a sulfonic acid group (e.g., cysteic acid). The incorporation of non-natural amino acids (including synthetic non-natural amino acids, substituted amino acids, or one or more D-amino acids) in the protein of the present invention may be advantageous in a number of different ways. D-amino acid-containing peptides and the like exhibit improved in vitro or in vivo stability compared to their counterparts containing L-amino acid. Therefore, when higher intracellular stability is desired or required, the configuration of peptides and the like incorporating D-amino acids may be particularly useful. More specifically, D-peptides and the like are resistant to endogenous peptidases and proteases, and therefore provide improved bioavailability and extended lifespan of the molecule when these properties are desirable . In addition, D-peptides, etc. cannot be efficiently processed for presentation to T helper cells restricted by type II major histocompatibility complexes, so it is unlikely to induce humoral immune responses in intact organisms. responses ).

Amino acids can be referred to in the present invention by their well-known three-letter symbols or the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Likewise, nucleotides can be referred to by their generally accepted one-letter codes.

"Conservatively modified variants" apply to both amino acid and nucleic acid sequences. For a specific nucleic acid sequence, "conservatively modified variant" refers to a nucleic acid that encodes an amino acid sequence that is identical or substantially identical, or when the nucleic acid does not encode an amino acid sequence, it refers to substantially identical sequence. 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 alanine. Therefore, at each position where alanine is specified by a codon, the codon can be changed to any corresponding codon described without changing the encoded polypeptide. These nucleic acid variations are "silent variations", which are one of conservatively modified variations. In the present invention, every nucleic acid sequence encoding a polypeptide also describes every possible silent variation of the nucleic acid. Those of ordinary skill in the art will recognize that every codon in a nucleic acid (except AUG, which is usually the only codon for methionine and TGG, which is usually the only codon for tryptophan) can be modified. To produce functionally consistent molecules. Therefore, every silent variation of the nucleic acid encoding the polypeptide is implied in every described sequence.

With regard to amino acid sequences, those of ordinary skill in the art will recognize that changes, additions, or deletions to nucleic acid, peptide, polypeptide, or protein sequences have a single amino acid or a small percentage of the encoded sequence. Each substitution, deletion or addition of an amino acid is a "conservatively modified variant" in which the change causes 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 that provide functionally similar amino acids are known to those of ordinary skill in the art. These conservatively modified variants are also and do not exclude polymorphic variants, interspecies homologs and alleles of the present invention.

Conservative substitution tables that provide functionally similar amino acids are known to those of ordinary skill in the art. The following 8 groups each contain amino acids that are conservative substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamine (E); 3) Aspartic acid (N), glutamic acid (Q); 4) Arginine (R), lysine (K); 5) Isoleucine (I), leucine Acid (L), Methionine (M), Valine (V); 6) Amphetamine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S) , Threonine (T); and 8) cysteine (C), methionine (M); (see, for example, Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co., second edition (December 1993)).

In the case 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. It is measured by using one of the following sequence comparison algorithms (or other algorithms available to those of ordinary skill in the art) or by manual alignment and visual inspection. When the sequences are compared and aligned in a comparison window or a designated area to obtain the maximum Correspondence, if they have a certain percentage of the same amino acid residues or nucleotides (that is, about 60% identity, about 65%, about 70%, about 75%, about 80%, About 85%, about 90%, or about 95% identity), then the sequence is "substantially identical." This definition also refers to the complementary sequence of the test sequence. The identity may exist in a region of at least about 50 amino acids or nucleotides in length or in a region of 75-100 amino acids or nucleotides in length, or span across The entire sequence of a polynucleotide or polypeptide. The polynucleotide encoding the polypeptide of the present invention (including homologs from species other than humans) can be obtained by a method including the following steps: using the polynucleotide sequence of the present invention under stringent hybridization conditions or The labeled probes of the fragments are used to screen the library and isolate the full-length cDNA and genome clones containing the polynucleotide sequence. This hybridization technique is well known to professionals.

The phrase "selectively (or specifically) hybridizes to" refers to when a specific nucleotide sequence is present in a complex mixture (including but not limited to total cells or library DNA or RNA), Under stringent hybridization conditions, the molecule only binds to the specific sequence, forms a doublet or hybridizes.

As known in the art, the phrase "stringent hybridization conditions" refers to the hybridization of sequences of DNA, RNA, PNA or other nucleic acid analogs or combinations thereof under low ionic strength and high temperature conditions. Generally, under stringent conditions, the probe will hybridize to its target sequence in a complex mixture of nucleic acids (including but not limited to total cell or library DNA or RNA), but will not hybridize to other sequences in the complex mixture. Stringent conditions are sequence-dependent and 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.), ciliates, plants ( Including but not limited to monocotyledonous plants, dicotyledonous plants, algae, etc.), fungi, yeasts, flagellates, microsporidians, protists and the like.

When used in the present invention, the term "non-eukaryote" refers to a non-eukaryote. For example, non-eukaryotic organisms may belong to eubacteria (including but not limited to Escherichia coli , Thermus thermophilus , Bacillus stearothermophilus ), Pseudomonas fluorescens ( Pseudomonas fluorescens ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Pseudomonas putida (Pseudomonas putida, etc.) system domains or archaea (including but not limited to Methanococcus jannaschii ), Methanobacterium thermoautotrophicum , Halobacterium ( Halobacterium , such as Haloferax volcanii and Halobacterium species NRC-1 , Archaeoglobus fulgidus , Pyrococcus furiosus , Pyrococcus horikoshii , Aeuropyrum pernix, etc.) system domain.

When used in the present invention, the term "subject" refers to an animal that is the target of treatment, observation, or experiment, in some embodiments a mammal, and in other embodiments a human. Animals can be companion animals (for example, dogs, cats, etc.), farm animals (for example, cows, sheep, pigs, horses, etc.), or experimental animals (for example, rats, mice, guinea pigs, etc.).

When used in the present invention, the term "effective amount" refers to one of the modified non-natural amino acid polypeptides administered to alleviate the disease, disorder, or disorder to be treated. Or the amount of multiple symptoms. The composition containing the modified non-natural amino acid polypeptide described in the present invention can be administered for prevention, enhancement and/or therapeutic treatment.

The term "enhancement" means to increase or prolong the desired effect in either aspect of potency or duration. Therefore, in terms of enhancing the effect of a therapeutic agent, the term "enhancement" refers to the ability to increase or prolong the effect of other therapeutic agents on the system in terms of efficacy or duration. When used in the present invention, the "enhancing effective amount" refers to an amount sufficient to enhance the effect of another therapeutic agent in the desired system. When used in patients, the amount effective for this use depends 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.

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

The term "post-translationally modified" refers to any modification of a natural or unnatural amino acid after it has been incorporated into the polypeptide chain. Merely as an example, the term covers co-translational in vivo modification, co-translational in vitro modification (for example, in a cell-free translation system), post-translational in vivo modification, and post-translational in vitro modification.

In prophylactic applications, the composition containing the IL-2 is administered to patients who are susceptible or otherwise at risk of a specific disease, disorder or condition. This amount is defined as the "prophylactically effective amount". In this use, the precise amount also depends on the patient's health, weight, etc. The determination of this preventive effective dose through routine experiments (such as a dose escalation clinical trial) is considered to be completely within the technical scope of the art.

In therapeutic applications, the composition containing the modified non-natural amino acid polypeptide is administered in an amount sufficient to cure or at least partially prevent the symptoms of the disease, disorder, or disorder to those already suffering from the disease, disorder, or disorder Of patients. 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 doctor. The determination of this therapeutically effective amount through routine experiments (such as a dose-escalation clinical trial) is considered to be completely within the technical scope of the art.

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

The non-naturally encoded amino acid polypeptides proposed in the present invention may include isotopically-labeled compounds having one or more atoms replaced by atoms having an atomic weight or mass number different from those commonly 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, respectively , ³⁵ S, ¹⁸ F, ³⁶ Cl. Certain isotope-labeled compounds described in the present invention, such as compounds incorporating radioisotopes such as ³ H and ¹⁴ C, may be useful in the determination of the tissue distribution of drugs and/or substances. In addition, replacement with isotopes such as deuterium or ² H can obtain certain therapeutic advantages resulting from higher metabolic stability, such as increased in vivo half-life or reduced dosage requirements.

All isomers, including but not limited to diastereomers, enantiomers and mixtures thereof, are considered part of the composition described in the present invention. In further or other embodiments, the non-naturally encoded amino acid polypeptide is metabolized to produce metabolites after administration to the organism in need, which is then used to produce the desired effect, including the 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 may exist as tautomers. In addition, the non-naturally encoded amino acid polypeptides described in the present invention may exist in unsolvated forms as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated form is also believed to be disclosed in the present invention. Those of ordinary skill in the art will recognize that certain compounds of the present invention may exist in several tautomeric forms. All these tautomeric forms are considered part of the composition described in this invention.

Unless otherwise specified, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the technical scope of the art are used.

I. Introduction

In the present invention, an IL-2 molecule containing at least one non-natural amino acid is provided. In certain embodiments of the present invention, the IL-2 with at least one unnatural amino acid includes at least one post-translational modification. In one embodiment, the at least one post-translational modification includes attaching a molecule containing a second reactive group to a molecule containing a second reactive group using chemical methods known to those of ordinary skill in the art to be suitable for a particular reactive group. At least one non-natural amino acid of a sexual group, the molecule includes but is not limited to markers, dyes, polymers, water-soluble polymers, polyethylene glycol derivatives, photocrosslinkers, radionuclides, cells Toxic compounds, drugs, affinity labels, photoaffinity labels, reactive compounds, resins, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelating agents, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, antisense polynucleotides, sugars, water-soluble dendrimers, cyclodextrins, inhibitory ribonucleic acids, biological materials, nanoparticles, spin markers, fluorophores, metal-containing components, radiation Active components, new functional groups, groups that covalently or non-covalently interact with other molecules, photocage components, components that can be excited by actinic radiation, components that can be photoisomerized, biotin, biological Derivatives, biotin analogs, heavy atom-doped components, chemically cleavable groups, photo-cleavable groups, extended side chains, carbon-linked sugars, redox active agents, aminothio acids , Toxic components, isotope-labeled components, biophysical probes, phosphorescent groups, chemiluminescent groups, electron dense groups, magnetic groups, intercalating groups, chromophores, energy transfer reagents, biologically active agents, Detectable labels, small molecules, quantum dots, nano-emitters, radionucleotides, radioactive emitters, neutron capture agents, or any combination of the above-mentioned molecules or any other desired compounds or substances. For example, the first reactive group is an alkynyl component (including but not limited to p-propargyloxy amphetamine in the non-natural amino acid, where the propargyl group is sometimes also referred to as acetylene Component) and the second reactive group is an azido component and utilizes [3+2] cycloaddition chemistry. In another example, the first reactive group is an azido component (including but not limited to p-azido-L-phenylalanine or pAZ in the non-natural amino acid, as in this specification It is sometimes referred to as) and the second reactive group is an alkynyl component. In certain embodiments of the modified IL-2 of the present invention, at least one non-natural amino acid (including but not limited to non-natural amino acid containing a ketone functional group) containing at least one post-translational modification is used, wherein the At least one post-translational modification contains a sugar component. In certain embodiments, the post-translational modification is made in vivo in eukaryotic cells or in non-eukaryotic cells. Linkers, polymers, water-soluble polymers, or other molecules can attach the molecule to the polypeptide. In another embodiment, the linker attached to IL-2 is long enough to allow the formation of dimers. The molecule can also be directly linked to the polypeptide.

In certain embodiments, the IL-2 protein contains at least one post-translational modification produced in vivo by one host cell, wherein the post-translational modification is not normally produced by another host cell type. In certain embodiments, the protein includes 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, palmitization, palmitic acid addition, phosphorylation, glycolipid linkage modification, and the like.

In certain embodiments, the IL-2 comprises one or more non-naturally encoded amino acids for glycosylation, acetylation, acylation, lipid modification, palmitization, palmitization of the polypeptide. Acid addition, phosphorylation or glycolipid linking modification. In certain embodiments, the IL-2 contains 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, palmitization, palmitic acid of the polypeptide Addition, phosphorylation or glycolipid linking modification. In certain embodiments, the IL-2 contains one or more naturally-encoded amino acids for glycosylation of the polypeptide.

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

In one embodiment, the post-translational modification includes attaching the oligosaccharide (including but not limited to the case where the oligosaccharide includes (GlcNAc-Man)2-Man-GlcNAc-GlcNAc, etc.) through a GlcNAc-aspartamide linkage. To aspartame. In another embodiment, the post-translational modification includes passing oligosaccharides (including but not limited to Gal-GalNAc, Gal-GlcNAc, etc.) through GalNAc-serine, GalNAc-threonine, GlcNAc-serine or GlcNAc -Threonine link is attached to serine or threonine. In certain embodiments, the protein or polypeptide of the present invention may include secretion or localization sequences, epitope tags, FLAG tags, polyhistidine tags, GST fusions, and the like. Examples of secretion signal sequences include, but are not limited to, prokaryotic secretion signal sequence, eukaryotic secretion signal sequence, 5'-optimized eukaryotic secretion signal sequence for bacterial expression, new secretion signal sequence, pectate lyase secretion signal sequence , Omp A secretion signal sequence and phage secretion signal sequence. Examples of secretion signal sequences include, but are not limited to, STII (prokaryotes), Fd GIII and M13 (phage), Bgl2 (yeast), and the signal sequence bla derived from a transposon. Any such sequence can be modified to provide the desired result for the polypeptide, including but not limited to replacing one signal sequence with a different signal sequence, replacing the leader sequence with a different leader sequence, and the like.

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 A non-natural amino acid. The non-natural amino acid 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 , Which contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different unnatural amino acids. In certain embodiments, at least one but less than all of the specific amino acids present in the naturally-occurring form of the protein are replaced by non-natural amino acids.

The present invention provides methods and compositions based on IL-2 containing at least one non-naturally encoded amino acid. The introduction of at least one non-naturally encoded amino acid in IL-2 may allow the use of those involving, but not limited to, reaction with one or more non-naturally encoded amino acids while not reacting with the common 20 amino acids Conjugation chemistry for specific chemical reactions. In certain embodiments, the IL-2 containing the non-naturally encoded amino acid is linked to a water-soluble polymer such as polyethylene glycol (PEG) through the side chain of the non-naturally encoded amino acid. The present invention provides an efficient method for selectively modifying proteins with PEG derivatives. The method includes responding to selector codons and translating non-genetically encoded amino acids (including but not limited to those contained in 20 naturally-incorporated Functional groups or substituents that are not present in amino acids (including but not limited to amino acids of ketone, azide, or acetylene components) are selectively incorporated into the protein, and then modified with suitable reactive PEG derivatives Amino acid. Once incorporated, the amino acid side chain can then be modified using chemical methods known to those of ordinary skill in the art that are suitable for the specific functional groups or substituents present in the non-naturally encoded amino acid. A wide variety of known chemical methods are suitable for use in the present invention to incorporate water-soluble polymers into the protein. These methods include, but are not limited to, Huisgen [3+2] cycloaddition reactions including but not limited to acetylene or azide derivatives, respectively (see, for example, Padwa, A., "Comprehensive Organic Synthesis", Vol. . 4, (1991) Ed. Trost, BM, Pergamon, Oxford, p. 1069-1109; and Huisgen, R., "1,3-Dipolar Cycloaddition Chemistry" (1,3-Dipolar Cycloaddition Chemistry), (1984) Ed. Padwa, A., Wiley, New York, p. 1-176).

Since the Huisgen [3+2] cycloaddition method involves a cycloaddition rather than a nucleophilic substitution reaction, the protein 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. The molecules that can be added to the protein of the present invention through [3+2] cycloaddition include virtually any molecule with suitable functional groups or substituents, including but not limited to azido or acetylene derivatives. These molecules can be added to non-natural amino acids with acetylene groups, including but not limited to p-propargyloxy amphetamine, or non-natural amino acids with azido groups, including but not limited to p-azido- Phenylalanine.

The 5-membered ring obtained from the Huisgen [3+2] cycloaddition is generally irreversible in a reducing environment, and is stable against hydrolysis in an aqueous environment for a long time. Therefore, the physical and chemical properties of a wide range of substances can be modified with the active PEG derivatives of the present invention under highly demanding aqueous conditions. Even more importantly, since the azide 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), the protein can be in one or more specific The site is modified with extremely high selectivity.

The present invention also provides water-soluble and hydrolytically stable PEG derivatives and related hydrophilic polymers with one or more acetylene or azide components. The PEG polymer derivative containing an acetylene component is highly selective for conjugation with an azide component that has been selectively introduced into a protein in response to a selector codon. Similarly, PEG polymer derivatives containing azido moieties are highly selective for conjugation with acetylene moieties that have been selectively introduced into proteins in response to selector codons.

More specifically, the azide component includes, but is not limited to, alkyl azide, aryl azide, and derivatives of these azides. The derivatives of the alkyl and aryl azide 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 azide specific reactivity is maintained.

The present invention provides conjugates of substances with a wide range of functional groups, substituents or components and other substances, including but not limited to markers, dyes, polymers, water-soluble polymers, polyethylene glycols Derivatives, photocrosslinkers, radionuclides, cytotoxic compounds, drugs, affinity labels, photoaffinity labels, reactive compounds, resins, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metals Chelating agents, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, antisense polynucleotides, sugars, water-soluble dendrimers, cyclodextrins, inhibitory ribonucleic acids, biological materials, nanoparticles, self Spin markers, fluorescent groups, metal-containing components, radioactive components, new functional groups, groups that interact covalently or non-covalently with other molecules, photocaged components, components that can be excited by actinic radiation , Photo-isomerizable components, biotin, biotin derivatives, biotin analogs, components doped with heavy atoms, chemically cleavable groups, photo-cleavable groups, extended side chains, carbon Connected sugars, redox active agents, amino thioacids, toxic components, isotopically labeled components, biophysical probes, phosphorescent groups, chemiluminescent groups, electron dense groups, magnetic groups, intercalating groups Groups, chromophores, energy transfer reagents, biologically active agents, detectable markers, small molecules, quantum dots, nano-emitters, radionucleotides, radioactive emitters, neutron capture agents, or any combination of the foregoing Any other desired compounds or substances. The present invention also includes a conjugate of a substance having an azide or acetylene component and a PEG polymer derivative having a corresponding acetylene or azide component. For example, a PEG polymer containing an azide 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 link that conjugates the PEG to the biologically active molecule includes, but is not limited to, Huisgen [3+2] cycloaddition products.

It is clear in the art that PEG can be used to modify the surface of biological materials (see, for example, U.S. Patent 6,610,281; Mehvar, R., J. Pharm Sci., 3(1): 125-136 (2000), which are incorporated herein by reference. invention). The present invention also includes one or more reactive azido or acetylene sites on the surface and one or more of the azido or acetylene-containing polymers of the present invention are conjugated to the azide or acetylene through Huisgen [3+2] cycloaddition connection. The biological material on the surface. Biomaterials and other substances can also be derived from azide or acetylene linkages other than linkages, for example, through linkages containing carboxylic acid, amine, alcohol or thiol moieties conjugated to the azide or acetylene-activated polymer In order to leave the azide or acetylene component that can be used in subsequent reactions.

The present invention includes a method for synthesizing the azide and acetylene-containing polymer of the present invention. In the case of the azido-containing PEG derivative, the azido group may be directly bonded to the 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 conventional activated polymer, so that the resulting polymer has the Azido-based component. In the case of the acetylene-containing PEG derivative, the acetylene may be directly bonded to the carbon atom of the polymer. Alternatively, the acetylene-containing PEG derivative can be prepared by attaching a linking reagent having an acetylene component to one end of a conventional activated polymer, so that the resulting polymer has the acetylene component at its end.

More specifically, in the case of the azide-containing PEG derivative, a water-soluble polymer having at least one reactive hydroxyl component undergoes a reaction to produce a component having a higher reactivity thereon, such as methanesulfonic acid. Ester, tribenzoate, tosylate or halogen leaving group substituted polymers. The preparation and use of PEG derivatives containing sulfonate halides, halogen atoms and other leaving groups are known to those of ordinary skill in the art. The resulting substituted polymer then undergoes a reaction to replace the azido component at the end of the polymer with the more reactive component. Alternatively, a water-soluble polymer having at least one active nucleophilic or electrophilic component undergoes a reaction with a linking agent having an azide group at one end, so as to form a covalent relationship between the PEG polymer and the linking agent Bond, and the azido component is located at the end of the polymer. Nucleophilic and electrophilic components, including amines, thiols, hydrazine, hydrazine, alcohols, carboxylic acid esters, aldehydes, ketones, thioesters, etc., are known to those of ordinary skill in the art.

More specifically, in the case of the acetylene-containing PEG derivative, the water-soluble polymer having at least one active hydroxyl component undergoes a reaction to replace the halogen or other activated leaving group from the precursor containing the acetylene component group. Alternatively, a water-soluble polymer having at least one active nucleophilic or electrophilic component undergoes a reaction with a linking agent having acetylene at one end, so as to form a covalent bond between the PEG polymer and the linking agent, And the acetylene component is located at the end of the polymer. The use of halogen components, activated leaving groups, nucleophilic and electrophilic components in the context of organic synthesis and in the preparation and use of PEG derivatives is clear to those skilled in the art.

The present invention also provides a method for selectively modifying a protein to add other substances to the modified protein, the other substances including but not limited to water-soluble polymers such as PEG and PEG derivatives containing azide or acetylene components . The azido and acetylene-containing PEG derivatives can be used to modify surface and molecular properties where biocompatibility, stability, solubility, and lack of immunogenicity are important, and at the same time provide the PEG derivatives Attachment to proteins is a more selective means than previously known in the art.

II. Universal recombinant nucleic acid method used in the present invention

In a large number of embodiments of the present invention, the nucleic acid encoding IL-2 (IL-2 of interest) will be isolated, cloned and usually altered using recombinant methods. These examples are used to include, but are not limited to, protein expression or during the production of variants, derivatives, expression cassettes, or other sequences derived from IL-2. In certain embodiments, the sequence encoding the polypeptide of the present invention is operably linked to a heterologous promoter.

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

Table 1-IL-2 protein and DNA sequence

The nucleotide sequence encoding IL-2 containing a non-naturally encoded amino acid can include, but is not limited to, the parent polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 5, or 7. The amino acid sequence is synthesized on the basis of, and then the nucleotide sequence is changed in order to perform the introduction (i.e., incorporation or substitution) or removal (i.e., deletion or substitution) of related amino acid residues. The nucleotide sequence can be conveniently modified by site-directed mutagenesis according to conventional methods. Alternatively, the nucleotide sequence can be prepared by chemical synthesis, including but not limited to by using an oligonucleotide synthesizer, wherein the oligonucleotide is 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 will be produced are selected. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and assembled by PCR, ligation, or ligation chain reaction. See, for example, Barany et al., Proc. Natl. Acad. Sci. 88: 189-193 (1991); U.S. Patent 6,521,427, which is incorporated herein by reference.

The DNA sequence of the synthetic human IL-2 gene cloned into the pKG0269 expression plastid 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 the general methods used in the present invention include Sambrook et al., "Clone Experiment Guide" (Molecular Cloning, A Laboratory Manual) (3rd Edition, 2001); Kriegler, "Gene Transfer and Expression Experiments" "Guide" (Gene Transfer and Expression: A Laboratory Manual) (1990); and "Current Protocols in Molecular Biology" (Ausubel et al., editor-in-chief, 1994).

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

Several well-known methods for introducing target nucleic acids into cells are available, and any of them can be used in the present invention. These methods include: fusion of recipient cells with bacterial protoplasts containing the DNA, electroporation, projectile bombardment, and infection with viral vectors (discussed further below). Bacterial cells can be used to amplify the number of plastids containing the DNA constructs of the invention. The bacteria are grown to log phase, and the plastids within the bacteria can be separated by various methods known in the art (see, for example, Sambrook). In addition, kits for purifying plastids from bacteria are commercially available (see, for example, EasyPrep™, FlexiPrep™ all from Pharmacia Biotech; StrataClean™ from Stratagene; and QIAprep™ from Qiagen). The isolated and purified plastids are then further manipulated to produce other plastids that can be used to transfect cells or be incorporated into related vectors to infect organisms. A typical vector contains transcription and translation terminator, transcription and translation initiation sequence, and a promoter that can be used to regulate the expression of the specific target nucleic acid. The vector optionally includes a universal expression cassette, which contains at least one independent terminator sequence, a sequence that allows the expression cassette to replicate in eukaryotes or prokaryotes or both (including but not limited to shuttle vectors), and Selection marker for both prokaryotic and eukaryotic systems. The vector is suitable for replication and integration in prokaryotes, eukaryotes, or both. See Gillam & Smith, Gene 8:81 (1979); Roberts et al., Nature, 328:731 (1987); Schneider, E. et al., Protein Expr. Purif. 6(1):10-14 (1995); Ausubel, Sambrook, Berger (all ibid). A catalog of bacteria and phage that can be used for clone is provided by, for example, ATCC, such as "ATCC Catalogue of Bacteria and Bacteriophage" (1992), edited by Gherna et al., published by ATCC. Other basic programs for sequencing, cloning and other aspects of molecular biology and implicit theoretical considerations are also in Watson et al. (1992), Recombinant DNA, Second Edition, Scientific American Books , Found in NY. In addition, virtually any nucleic acid (and virtually any labeled nucleic acid, whether standard or non-standard) can be customized or ordered from any of a variety of different commercial sources, such as Midland Certified Reagent Company (Midland, TX, available at the World Wide Web site mcrc.com), The Great American Gene Company (Ramona, CA, available at the World Wide Web site genco.com), ExpressGen Inc. (Chicago, IL, available at the World Wide Web site expressgen.com Obtained), Operon Technologies Inc. (Alameda, CA), and many other sources.

Selector codon (Selector codons)

The selector codon of the present invention expands the genetic codon framework of the protein biosynthesis machine. For example, selector codons include, but are not limited to, unique three-base codons, nonsense codons such as stop codons (including but not limited to amber codons (UAG), ocher codons, or pebble codons (UGA)) , Unnatural codons, four or more base codons, rare codons, etc. It is obvious to those of ordinary skill in the art that the number of selector codons that can be introduced into the desired gene or polynucleotide has a wide range, including but not limited to a single encoding at least a part of IL-2. One or more, two or more, three or more, 4, 5, 6, 7, 8, 9, 10 or more of the polynucleotides.

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 stop codons (including but not limited to UAG) and is acylated by O-RS with the desired unnatural amino acid. This O-tRNA is not naturally occurring The host's aminyl-tRNA synthetase recognizes it. Conventional site-directed mutagenesis can be used to introduce the stop codon (including but not limited to TAG) at the position of interest in the polypeptide of interest. See, for example, Sayers, JR et al., (1988), 5'-3' Exonucleases in phosphorothioate-based mutagenesis (mutagenesis) based on phosphorothioate oligonucleotides. oligonucleotide-directed mutagenesis), Nucleic Acids Res, 16:791-802. When the O-RS, O-tRNA, and the nucleic acid encoding the polypeptide of interest are combined in vivo, the UAG codon responds to the unnatural amino acid is incorporated to give the A polypeptide containing the non-natural amino acid at the position.

The in vivo incorporation of unnatural amino acids can be performed without significantly disturbing the eukaryotic host cell. For example, since the suppression efficiency of the UAG codon depends on the O-tRNA (including but not limited to the amber suppressor tRNA) and the eukaryotic release factor (including but not limited to eRF) (the peptide that binds to the stop codon and initiates growth) Release from the ribosome), so the inhibition efficiency can be adjusted 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 demonstrated that when the concentration of arginine is decreased in an in vitro protein synthesis reaction, the rare arginine codon AGG is efficiently inserted into Ala by 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 that exists in E. coli as a minor species. 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 components of the present invention can be produced to use these rare codons in vivo.

Selector codons also include extended codons, including but not limited to four or more base codons such as four, five, six or more base codons. Examples of four base codons include but are not limited to AGGA, CUAG, UAGA, CCCU, etc. Examples of five-base codons include, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC, etc. The features of the present invention include the use of extended codons based on frameshift suppression. Four or more base codons can insert, including but not limited to, one or more unnatural amino acids into the same protein. For example, in the presence of a mutated O-tRNA (including but not limited to a special frameshift suppressor tRNA) with an anticodon loop such as an anticodon loop of at least 8-10 nt, the four or more base codons The child is read as a single amino acid. In other embodiments, the anticodon loop can be decoded including but not limited to at least four base codons, at least five base codons, or at least six base codons or more base codons. Since there are 256 possible four-base codons, four or more base codons can be used to encode 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, extended genetic code: four base codons 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, four-base codons have been used to incorporate unnatural amino acids into proteins in in vitro biosynthetic methods. See, for example, Ma et al., Biochemistry, 32:7939, (1993); and Hohsaka et al., J. Am. Chem. Soc., 121:34 (1999). CGGG and AGGU were used to use two chemically acylated frameshift inhibitor tRNAs in vitro, while incorporating NBD derivatives of 2-naphthylalanine and lysine into streptavidin. See, for example, Hohsaka et al., J. Am. Chem. Soc., 121:12194 (1999). In an in vivo study, Moore et al. examined the ability of tRNALeu derivatives with NCUA anticodons to inhibit UAGN codons (N can be U, A, G, or C), and found that the quadruplex UAGA can be UCUA anticodons. The tRNALeu of the codon is decoded with an efficiency of 13 to 26%, and is rarely decoded in the 0 or -1 box. 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 can also include one of the natural three-base codons, where the endogenous system does not use (or rarely uses) the natural base codon. For example, this includes systems lacking tRNAs that recognize the natural three-base codons and/or systems where the three-base codons are rare codons.

The selector codon optionally includes unnatural base pairs. These unnatural base pairs further expand the existing genetic alphabet. An extra 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, high-fidelity and high-fidelity enzymatic incorporation of the polymer into DNA, and efficient continued primer extension after the synthesis of nascent unnatural base pairs. Descriptions of unnatural base pairs that can be adapted to methods and compositions include, for example, Hirao et al., An unnatural base pair for incorporating amino acid analogs into proteins (An unnatural base pair for incorporating amino acid analogs). 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 nucleoside is membrane permeable and phosphorylated to form the corresponding triphosphate. In addition, the increased genetic information is stable and is not destroyed by cell enzymes. Previous attempts by Benner and others used a different hydrogen bonding mode from the classic Watson-Crick pair. The most notable example 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 usually mispair with natural bases to some extent and cannot be copied enzymatically. Kool and collaborators 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 unnatural base pairs that meet all the above requirements, Schultz, Romesberg, and collaborators systematically synthesized and studied a series of unnatural hydrophobic bases. It is found that the PICS:PICS self-pair is more stable than the natural base pair, and can be efficiently incorporated into DNA by the Klenow fragment (KF) of E. coli DNA polymerase I. See, for example, McMinn et al., J. Am. Chem. Soc., 121:11585-6 (1999); and Ogawa et al., J. Am. Chem. Soc., 122:3274 (2000). The 3MN:3MN self-pair can be synthesized by KF with sufficient efficiency and selectivity for biological functions. See, for example, Ogawa et al., J. Am. Chem. Soc., 122:8803 (2000). However, both bases act as chain terminator for further replication. Recently, mutant DNA polymerases have been evolved, which can be used to replicate PICS self-pairs. In addition, the 7AI pair itself can be copied. See, for example, Tae et al., J. Am. Chem. Soc., 123:7439 (2001). A new metal base pair Dipic:Py has also been developed, which forms a stable pair after binding to 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 method of the present invention can take advantage of this property to generate orthogonal tRNAs for them.

A translational bypassing system can also be used to incorporate non-natural amino acids into the desired polypeptide. In the translation bypass system, large sequences are incorporated into genes but not translated into proteins. The sequence contains a structure that serves as a clue to induce the ribosome to skip the sequence and resume translation downstream of the insertion.

In certain embodiments, in the methods and/or compositions of the present invention, the protein or polypeptide of interest (or a portion thereof) is encoded by a nucleic acid. Generally, the nucleic acid contains 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, and at least six selector codons. Codons, at least seven selector codons, at least eight selector codons, at least nine selector codons, ten or more selector codons.

Genes encoding proteins of interest or polypeptides can be mutagenized using methods known to those of ordinary skill in the art and described in the present invention to include, for example, one or more non-natural amines for incorporation The selector codon of the base acid. For example, a nucleic acid for a protein of interest is mutagenized to contain one or more selector codons for the incorporation of one or more unnatural 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 unnatural amino acid. Likewise, the present invention also includes corresponding nucleic acids, that is, any nucleic acid having one or more selector codons encoding one or more unnatural amino acids.

A nucleic acid molecule encoding a protein of interest, such as IL-2, can be easily 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 range of other molecules on the protein of interest. Methods suitable for incorporating cysteine into the desired position of the polypeptide are known to those of ordinary skill in the art, such as those described and standard in U.S. Patent No. 6,608,183, which is incorporated by reference into the present invention. Of mutagenesis technology.

III. Non-naturally encoded amino acid

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 introduced non-naturally encoded amino acid has an effect on 20 common genetically encoded amino acids (ie alanine, arginine, aspartic acid, aspartic acid), Cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine , Threonine, tryptophan, tyrosine and valine) are essentially chemically inert. In certain embodiments, the non-naturally encoded amino acid includes functional groups that are not present in the 20 common amino acids (including but not limited to azide, ketone, aldehyde, and aminooxy) with high efficiency and selectivity. To form the side chain functional group of the conjugate. For example, IL-2 including a non-naturally encoded amino acid containing an azide functional group can react with polymers (including but not limited to polyethylene glycol or a second polypeptide containing an alkynyl component) to form a stable Conjugates, this is due to the selective reaction of the azide and alkyne functional groups to form Huisgen [3+2] cycloaddition products.

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

I

The non-naturally encoded amino acid is generally any structure having the structural formula listed above, wherein the R group is any substituent other than the substituents used in the 20 natural amino acids, and can be suitably used In the present invention. Since the non-naturally encoded amino acid of the present invention is usually only different from the natural amino acid in the structure of the side chain, the non-naturally encoded amino acid is different from other amino acids (including but not limited to natural or non-natural amino acids). The encoded amino acids) form amide bonds in the same manner as they are formed in naturally-occurring polypeptides. However, the non-naturally encoded amino acid has a different side chain group from the natural amino acid. For example, R optionally includes alkyl-, aryl-, acyl-, ketone-, azido-, hydroxy-, hydrazine, cyano-, halo-, hydrazine, alkenyl, alkynyl, ether , Thiol, seleno-, sulfonyl-, borate, boronic acid, phosphinyl, phosphinyl, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amine基, 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 crosslinking agents, spin-labeled amino acids, fluorescent amino acids, and bound metals Amino acids, metal-containing amino acids, radioactive amino acids, amino acids with new functional groups, amino acids that covalently or non-covalently interact with other molecules, photo-caged and/or photo-isomerizable Amino acids, amino acids containing biotin or biotin analogs, glycosylated amino acids such as sugar-substituted serine, other sugar-modified amino acids, keto-containing amino acids, containing poly Ethylene glycol or polyether amino acids, heavy atom-substituted amino acids, chemically cleavable and/or photo-cleavable amino acids, side chains with extended side chains (including but not limited to poly Ether or long-chain hydrocarbons, including but not limited to long-chain hydrocarbons greater than about 5 or greater than about 10 carbons) amino acids, carbon-linked sugar-containing amino acids, redox active amino acids, containing amino groups Amino acids of thioacids and amino acids containing one or more toxic components.

Exemplary non-naturally encoded amino acids that may be suitable for use in the present invention and that can be used to react with water-soluble polymers include, but are not limited to, having carbonyl, aminooxy, hydrazine, hydrazine, aminourea, azide, and A non-naturally encoded amino acid with an alkyne-reactive group. In certain embodiments, the non-naturally encoded amino acid comprises a sugar component. Examples of such amino acids include N -acetyl-L-glucosamine-L-serine, N -acetyl-L-galactosamine-L-serine, N -acetyl -L-Glucosamine-L-threonine, N -acetyl-L-glucosamine-L-aspartamide and O -mannosamine-L-serine. Examples of such amino acids also include the naturally occurring N- or O- linkages between the amino acids and sugars, which are covalently linked not common in nature (including but not limited to alkenes, oximes, thioethers, Amide, etc.) instead of examples. Examples of such amino acids also include sugars that are not common in naturally-occurring proteins such as 2-deoxyglucose, 2-deoxygalactose, and the like.

Many non-naturally encoded amino acids provided in the present invention 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) is commercially available. Those non-naturally encoded amino acids that are not commercially available are optionally synthesized as provided in the present invention or using standard methods known to those of ordinary skill in the art. For organic synthesis techniques, see, for example, Fessendon and Fessendon's "Organic Chemistry" Second Edition, Willard Grant Press, Boston Mass (1982); March's "Advanced Organic Chemistry" Third Edition, Wiley and Sons, New York (1985); and Carey and Sundberg's "Advanced Organic Chemistry" (Advanced Organic Chemistry) third 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 the non-natural amino acids containing new side chains, the non-natural amino acids that may be suitable for use in the present invention also optionally contain modified backbone structures, including but not limited to those shown by the structures of formula II and III Out of:

Wherein Z usually contains OH, NH ₂ , SH, NH-R' or S-R'; X and Y may be the same or different, usually contain S or O, and optionally the same or different R and R'are usually selected from The same list of components and hydrogen as the R group described above for the non-natural amino acid of formula I. For example, the non-natural amino acids of the present invention optionally contain substitutions in the amine or carboxyl groups, as shown in Formula II and III. This type of non-natural amino acid includes but is not limited to α-hydroxy acid, α-thio acid, α-amino thiocarboxylic acid ester, including but not limited to having a side corresponding to the common 20 kinds of natural amino acids Chain or non-natural side chain of these compounds. In addition, the substitution at the α-carbon optionally includes, but is not limited to, L, D or α-α-disubstituted amino acids such as D-glutamic acid, D-alanine, D-methyl-O -Tyrosine, aminobutyric acid, etc. Other structural alternatives include cyclic amino acids such as proline analogs and 3, 4, 6, 7, 8 and 9-membered cyclic proline analogs, β and γ amino acids such as substituted β-propylamine Acid and gamma-aminobutyric acid.

Many non-natural amino acids are based on natural amino acids such as tyrosine, glutamic acid, 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 includes, but is not limited to, keto groups (including But not limited to acetyl), benzyl, amine, hydrazine, hydroxylamine, thiol, carboxy, isopropyl, methyl, C6-C20 linear or branched hydrocarbons, saturated or unsaturated hydrocarbons , O-methyl, polyether, nitro, alkynyl, etc. In addition, polysubstituted aryl rings are also considered. The glutamic acid analogs that can be applied to the present invention include, but are not limited to, α-hydroxy derivatives, γ-substituted derivatives, cyclic derivatives, and amide-substituted glutamic acid derivatives. Examples of amphetamine analogs that can be applied to the present invention include, but are not limited to, para-substituted amphetamines, ortho-substituted amphetamines, and meta-substituted amphetamines, wherein the substituents include, but are not limited to, hydroxyl, methyl Oxy, methyl, allyl, aldehyde, azido, iodo, bromo, keto (including but not limited to acetyl), benzyl, alkynyl, etc. Specific examples of non-natural amino acids that can be applied to the present invention include, but are not limited to, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)propylamine Acid, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L -Dopa, fluoro-phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-aniline-L-phenylalanine, p-anisyl-L-phenylalanine, L-phosphoserine, phosphoserine, phosphotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-amphetamine Acid and p-propargyloxy-phenylalanine, etc. Examples of the structures of various unnatural amino acids that can be applied to 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, Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation, PNAS 99:19-24 (2002), which is incorporated into the present invention by reference. The present invention is incorporated by reference and is entitled "Compositions Containing, Methods Involving, and Uses of Non-natural Amino Acids and Polypeptides" (Compositions Containing, Methods Involving, and Uses of Non-natural Amino Acids and Polypeptides). Application number PCT/US06/47822 describes the reductive alkylation and reductive amination of aromatic amine components (including but not limited to p-amino-phenylalanine).

In another embodiment of the present invention, the IL-2 polypeptide with 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 a relatively latecomer in the synthetic chemistry family, these bio-orthogonal reactions have inspired new strategies for compound library synthesis, protein engineering, functional proteomics, and cell surface chemical remodeling. Azide plays an important role as a unique chemical treatment for bioconjugation. Staudinger linkages have been used with phosphonates to label azido sugars that are metabolically introduced into cellular sugar conjugates. The Staudinger connection can be performed in living animals and is not harmful to the physiology; however, the Staudinger reaction is not without disadvantages. The necessary phosphines are susceptible to air oxidation, and optimizations to increase water solubility and increase reaction rate have proven to be synthetically challenging.

The azido group has an optional bio-orthogonal reactivity mode: [3+2] cycloaddition with alkynes described by Huisgen. In its classical form, this reaction has limited applicability in biological systems due to the increased temperature (or pressure) required for a reasonable reaction rate. Sharpless and collaborators overcome this obstacle by developing a copper (I) catalyzed form called "click chemistry", which is easy to perform at physiological temperatures and in a richly functionalized biological environment. This discovery enables selective modification of virus particles, nucleic acids and proteins from complex tissue lysates. Unfortunately, mandatory copper catalysts are toxic to both bacteria and mammalian cells, thus hindering applications where cells must remain viable. It has been reported that the catalyst-free Huisgen cycloaddition of alkynes activated by electron-withdrawing substituents occurs at ambient temperature. However, these compounds undergo a Michael reaction with biological nucleophiles.

In one embodiment, there is provided a composition of IL-2 comprising a non-natural amino acid (for example, p-(propargyloxy)-phenylalanine). Various different compositions containing 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 further comprises orthogonal tRNA. The non-natural amino acid may be bonded (including but not limited to covalently) to the orthogonal tRNA, including but not limited to covalently bonding to the orthogonal tRNA through an amino-acyl bond. The 3'OH or 2'OH or the like of the terminal ribose valently bonded to the orthogonal tRNA.

The chemical components that can be incorporated into proteins through non-natural amino acids provide the protein with various advantages and operations. For example, the unique reactivity of the ketone functional group allows 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 unnatural amino acids may be useful for phasing X-ray structure data. The site-specific introduction of heavy atoms using non-natural amino acids also provides selectivity and flexibility in choosing positions for 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, high-efficiency protein in vivo and in vitro Light cross-linking. Examples of photoreactive non-natural amino acids include, but are not limited to, p-azido-phenylalanine and p-anisyl-phenylalanine. Proteins with photoreactive non-natural amino acids can therefore be cross-linked at will by the excitation of the photoreactive groups, providing time control. In one example, the methyl group of the non-natural amino acid may be substituted with a methyl group including but not limited to isotope labeling, which can be used as a probe of local structure and dynamics when using nuclear magnetic resonance and vibrational spectroscopy including but not limited to. Alkynyl or azido functional groups allow for selective modification of proteins with molecules, for example by [3+2] cycloaddition reactions.

The non-natural amino acid incorporated into the polypeptide at the amino terminus can be made up of R groups that are any substituents other than those used in the 20 natural amino acids, and are different from those usually present in α-amines. The NH2 group in the base acid constitutes the second reactive group (see formula I). Similar non-natural amino acids can be incorporated at the carboxy terminus, which have a second reactive group different from the COOH group normally present in alpha-amino acids (see Formula I).

The non-natural amino acid of the present invention can be selected or designed to provide other characteristics 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 the protein, the following properties can be optionally modified: toxicity, biodistribution, solubility, stability such as heat, hydrolysis, oxidative stability, resistance to enzymatic degradation (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 covalent or non-covalent, etc. .

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

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

Amino acids with electrophilic reactive groups allow a variety of different reactions to be used, especially to link molecules through nucleophilic addition reactions. Such electrophilic reactive groups include carbonyl groups (including keto and dicarbonyl groups), carbonyl-like groups (which have similar reactivity to carbonyl groups (including keto and dicarbonyl groups) and are structurally similar to carbonyl groups), masking The carbonyl group (which can be easily converted into a carbonyl group (including keto and dicarbonyl)) or a protected carbonyl group (which has similar reactivity to the carbonyl group (including keto and dicarbonyl) after deprotection). These amino acids include those having the structure of formula (IV):

(IV)，

(IV),

in:

A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynyl, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted arylene Heteroaryl, alkylene, substituted alkylene, 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) _k -(where k is 1, 2 or 3), -S(O) _k (alkylene or substituted alkylene)-, ‑C(O)-, ‑C(O)-(alkylene or substituted alkylene) Group)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene) Alkyl)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R' )-(Alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-,- S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R ')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C (R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R')-, where each R'is independent Ground is H, alkyl or substituted alkyl;

、

、

、

、

、

、

或

；J is

,

,

,

,

,

,

or

；

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

Each R" is independently H, alkyl, substituted alkyl, or protecting group, or when more than one R" group is present, two R"s optionally form heterocycloalkyl;

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

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

Each R ₃ and R _{4 is} independently H, halogen, lower alkyl or substituted lower alkyl, or R ₃ and R ₄ or two R ₃ groups optionally form cycloalkyl or heterocycloalkyl;

Or -ABJR groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl 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);

Or -JR groups together form a monocyclic or bicyclic cycloalkyl or heterocycloalkyl 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);

The premise is 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 a methyl group.

In addition, amino acids having a structure of formula (V) are included:

(V)，

(V),

in:

A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynyl, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted arylene Heteroaryl, alkylene, substituted alkylene, 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) _k -(where k is 1, 2 or 3), -S(O) _k (alkylene or substituted alkylene)-, ‑C(O)-, ‑C(O)-(alkylene or substituted alkylene) Group)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene) Alkyl)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R' )-(Alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-,- S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R ')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C (R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R')-, where each R'is independent Ground is H, alkyl or substituted alkyl;

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

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

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

The premise is that when A is phenylene, B is present; and when A is -(CH ₂ ) ₄ -, B is not -NHC(O)(CH ₂ CH ₂ )-; and when A and B are not present, R is not a methyl group.

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

(VI)，

(VI),

in:

B is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-( Alkylene or substituted alkylene)-, -S-, -S- (alkylene or substituted alkylene)-, -S(O) _k- (where k is 1, 2 or 3), -S(O) _k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S) -, ‑C(S)-(alkylene or substituted alkylene)-, -N(R')-, ‑NR'-(alkylene or substituted alkylene)-, ‑C(O )N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted Alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) _k N(R' )-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) _k 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')-, where each R'is independently H, alkyl or substituted alkyl;

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

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

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

、

、

、

、

、

、

和

，Each R _a is independently selected from H, halogen, alkyl, substituted _{alkyl, -N (R ') 2,} -C (O) k R' ( where k is 1, 2 or 3), - C ( O) N(R') ₂ , -OR', and -S(O) _k R', where each R'is independently H, alkyl, or substituted alkyl. In addition, the following amino acids are included:

,

,

,

,

,

,

with

,

These compounds are optionally protected with amine group, protected carboxyl group or their salts. In addition, any of the following non-natural amino acids may be incorporated into the non-natural amino acid polypeptide.

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

(VII)

(VII)

in

B is optional, and when present, is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene , -O-, ‑O-(alkylene or substituted alkylene)-, -S-, ‑S-(alkylene or substituted alkylene)-, -S(O) _k -(where k is 1, 2 or 3), -S(O) _k (alkylene or substituted alkylene)-, ‑C(O)-, ‑C(O)-(alkylene or substituted alkylene) Group)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene) Alkyl)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R' )-(Alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-,- S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R ')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C (R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R')-, where each R'is independent Ground is H, alkyl or substituted alkyl;

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

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

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

Each R _a is independently selected from H, halogen, alkyl, substituted _{alkyl, -N (R ') 2,} -C (O) k R' ( where k is 1, 2 or 3), - C ( O) N(R') ₂ , -OR', and -S(O) _k R', wherein each R'is independently H, alkyl, or substituted alkyl; and n is 0 to 8;

The premise is that when A is -(CH ₂ ) ₄ -, B is not -NHC(O)(CH ₂ CH ₂ )-.

In addition, the following amino acids are included:

、

、

、

、

、

、

、

、

、

、

、

、

、

、

和

，

,

,

,

,

,

,

,

,

,

,

,

,

,

,

with

,

Wherein these compounds are optionally amine group protected, optionally carboxy group protected, optionally amine group protected and carboxy group protected, or are salts thereof. In addition, these non-natural amino acids and any of the following non-natural amino acids may be incorporated into the non-natural amino acid polypeptide.

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

(VIII)，

(VIII),

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, alkylaryl, substituted alkylaryl, 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) _k -(where k is 1, 2 or 3), -S(O) _k (alkylene or substituted alkylene)-, ‑C(O)-, ‑C(O)-(alkylene or substituted alkylene) Group)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene) Alkyl)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R' )-(Alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-,- S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R ')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C (R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R')-, where each R'is independent Ground is H, alkyl or substituted alkyl;

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

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

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

(IX)，

(IX),

B is optional, and when present, is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene , -O-, ‑O-(alkylene or substituted alkylene)-, -S-, ‑S-(alkylene or substituted alkylene)-, -S(O) _k -(where k is 1, 2 or 3), -S(O) _k (alkylene or substituted alkylene)-, ‑C(O)-, ‑C(O)-(alkylene or substituted alkylene) Group)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene) Alkyl)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R' )-(Alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-,- S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R ')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C (R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R')-, where each R'is independent Ground is H, alkyl or substituted alkyl;

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

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

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

Wherein each R _a is independently selected from H, halogen, alkyl, substituted _{alkyl, -N (R ') 2,} -C (O) k R' ( where k is 1, 2 or 3), - C (O)N(R') ₂ , -OR', and -S(O) _k R', where each R'is independently H, alkyl, or substituted alkyl.

In addition, the following amino acids are included:

、

、

、

、

、

、

和

，

,

,

,

,

,

,

with

,

Wherein these compounds are optionally amine group protected, optionally carboxy group protected, optionally amine group protected and carboxy group protected, or are salts thereof. In addition, these non-natural amino acids and any of the following non-natural amino acids may be incorporated into the non-natural amino acid polypeptide.

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

(X)，

(X),

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 Group, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) _k -( Where k is 1, 2 or 3), -S(O) _k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene) Alkyl)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted Alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R ')-(Alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N( R')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-,- C(R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R')-, where each R' Independently H, alkyl or substituted alkyl;

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

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

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

Each R _a is independently selected from H, halogen, alkyl, substituted _{alkyl, -N (R ') 2,} -C (O) k R' ( where k is 1, 2 or 3), - C ( O) N(R') ₂ , -OR', and -S(O) _k R', wherein each R'is independently H, alkyl, or substituted alkyl; and n is 0-8.

In addition, the following amino acids are included:

、

、

、

、

、

、

、和

，

,

,

,

,

,

,

,with

,

Wherein these compounds are optionally amine group protected, optionally carboxy group protected, optionally amine group protected and carboxy group protected, or are salts thereof. In addition, these non-natural amino acids and any of the following non-natural amino acids may be incorporated into the non-natural amino acid polypeptide.

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.

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

(XI)，

(XI),

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, alkylaryl, substituted alkylaryl, 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) _k -(where k is 1, 2 or 3), -S(O) _k (alkylene or substituted alkylene)-, ‑C(O)-, ‑C(O)-(alkylene or substituted alkylene) Group)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene) Alkyl)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R' )-(Alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-,- S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R ')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C (R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R')-, where each R'is independent Ground is H, alkyl or substituted alkyl;

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

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

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

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

(XII)，

(XII),

B is optional, and when present, is selected from lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene , -O-, ‑O-(alkylene or substituted alkylene)-, -S-, ‑S-(alkylene or substituted alkylene)-, -S(O) _k -(where k is 1, 2 or 3), -S(O) _k (alkylene or substituted alkylene)-, ‑C(O)-, ‑C(O)-(alkylene or substituted alkylene) Group)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene) Alkyl)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R' )-(Alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-,- S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R ')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C (R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R')-, where each R'is independent Ground is H, alkyl or substituted alkyl;

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

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

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

Wherein each R _a is independently selected from H, halogen, alkyl, substituted _{alkyl, -N (R ') 2,} -C (O) k R' ( where k is 1, 2 or 3), - C (O)N(R') ₂ , -OR', and -S(O) _k R', where each R'is independently H, alkyl, or substituted alkyl.

In addition, the following amino acids are included:

和

，

with

,

Wherein these compounds are optionally amine group protected, optionally carboxy group protected, optionally amine group protected and carboxy group protected, or are salts thereof. In addition, these non-natural amino acids and any of the following non-natural amino acids may be incorporated into the non-natural amino acid polypeptide.

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

(XIII)，

(XIII),

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 Group, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) _k -( Where k is 1, 2 or 3), -S(O) _k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene) Alkyl)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted Alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R ')-(Alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N( R')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-,- C(R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R')-, where each R' Independently H, alkyl or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R ₁ is optional, and when present, is H, an amine protecting group, resin, amino acid, polypeptide, or polynucleotide; and R ₂ is optional, and is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide when present; each R _a is independently selected from H, halo, alkyl, , Substituted alkyl, -N(R') ₂ , -C(O) _k R'(where k is 1, 2 or 3), -C(O)N(R') ₂ , -OR' and- S(O) _k R', where each R'is independently H, alkyl, or substituted alkyl; and n is 0-8.

In addition, the following amino acids are included:

、

、

、

、

、

、

、

、

、

、

、

、

、

、

和

，

,

,

,

,

,

,

,

,

,

,

,

,

,

,

with

,

Wherein these compounds are optionally amine group protected, optionally carboxy group protected, optionally amine group protected and carboxy group protected, or are salts thereof. In addition, these non-natural amino acids and any of the following non-natural amino acids may be incorporated into the non-natural amino acid polypeptide.

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

(XIV)；

(XIV);

in:

A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynyl, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted arylene Heteroaryl, alkylene, substituted alkylene, aralkylene or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R ₁ is optional, and when present, is H, an amine protecting group, resin, amino acid, polypeptide, or Polynucleotide; and R ₂ is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; X ₁ is C, S, or S(O); and L Is alkylene, substituted alkylene, N(R') (alkylene) or N(R') (substituted alkylene), where R'is H, alkyl, substituted alkyl, ring Alkyl or substituted cycloalkyl.

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

(XIV-A)

(XIV-A)

in:

A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynyl, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted arylene Heteroaryl, alkylene, substituted alkylene, aralkylene or substituted aralkylene;

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

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

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

(XIV-B)

(XIV-B)

in:

A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynyl, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted arylene Heteroaryl, alkylene, substituted alkylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R ₁ is optional, and when present, is H, an amine protecting group, resin, amino acid, polypeptide, or polynucleotide; and R ₂ is optional, and when present, is OH, an ester protecting group Group, resin, amino acid, polypeptide or polynucleotide; L is alkylene, substituted alkylene, N(R') (alkylene) or N(R') (substituted alkylene), Where R'is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.

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

(XV)；

(XV);

in:

A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynyl, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted arylene Heteroaryl, alkylene, substituted alkylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R ₁ is optional, and when present, is H, an amine protecting group, resin, amino acid, polypeptide, or polynucleotide; and R ₂ is optional, and when present, is OH, an ester protecting group Group, resin, amino acid, polypeptide or polynucleotide; X ₁ is C, S or S(O); and n is 0, 1, 2, 3, 4, or 5; and each CR ⁸ R ⁹ group Each of R ⁸ and R ^{9 on the above is} independently selected from H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R ⁸ and R ⁹ can together form =0 or cycloalkyl, or any Adjacent R ⁸ groups may together form a cycloalkyl group.

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

(XV-A)

(XV-A)

in:

A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynyl, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted arylene Heteroaryl, alkylene, substituted alkylene, aralkylene or substituted aralkylene;

R is H, 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 Polynucleotide; and R ₂ is optional and, when present, is OH, ester protecting group, resin, amino acid, polypeptide, or 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 together can form =O or cycloalkyl, or any adjacent R ⁸ groups can together form cycloalkyl.

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

(XV-B)

(XV-B)

in:

A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynyl, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted arylene Heteroaryl, alkylene, substituted alkylene, aralkylene or substituted aralkylene;

R is H, 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 Polynucleotide; and R ₂ is optional, and when present, is OH, ester protecting group, resin, amino acid, polypeptide, or 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 formed together =O or cycloalkyl, or any adjacent R ⁸ groups can together form a cycloalkyl.

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

(XVI)；

(XVI);

in:

A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynyl, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted arylene Heteroaryl, alkylaryl, substituted alkylaryl, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

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

R ₂ is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; X ₁ is C, S or S(O); and L is an alkylene, Substituted alkylene, N(R') (alkylene) or N(R') (substituted alkylene), where 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:

(XVI-A)

(XVI-A)

in:

A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynyl, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted arylene Heteroaryl, alkylaryl, substituted alkylaryl, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R ₁ is optional, and when present, is H, an amine protecting group, resin, amino acid, polypeptide, or polynucleotide; and R ₂ is optional, and when present, is OH, an ester protecting group Group, resin, amino acid, polypeptide or polynucleotide; L is alkylene, substituted alkylene, N(R') (alkylene) or N(R') (substituted alkylene), Where R'is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.

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

(XVI-B)

(XVI-B)

in:

A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynyl, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted arylene Heteroaryl, alkylene, substituted alkylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R ₁ is optional, and when present, is H, an amine protecting group, resin, amino acid, polypeptide, or polynucleotide; and R ₂ is optional, and when present, is OH, an ester protecting group Group, resin, amino acid, polypeptide or polynucleotide; L is alkylene, substituted alkylene, N(R') (alkylene) or N(R') (substituted alkylene), Where R'is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.

In addition, amino acids having a structure of formula (XVII) are included:

(XVII)，

(XVII),

in:

A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynyl, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted arylene Heteroaryl, alkylene, substituted alkylene, aralkylene or substituted aralkylene;

、

、

、

、

、

、

或

，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 The cycloalkyl group, 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 alkane Group, 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, resin, amino acid, polypeptide, or polynucleotide; and R ₂ is optional, and when present is OH , Ester protecting group, resin, amino acid, polypeptide or polynucleotide.

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

(XVIII)，

(XVIII),

in:

、

、

、

、

、

、

或

，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 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, resin, amino acid, polypeptide, or polynucleotide; and R ₂ is optional, and when present is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each R _a is independently selected from H, halogen, alkyl, substituted _{alkyl, -N (R ') 2,} -C (O) _k R'(where k is 1, 2 or 3), -C(O)N(R') ₂ , -OR' and -S(O) _k R', where each R'is independently H, alkyl or substituted alkyl.

In addition, amino acids having a structure of formula (XIX) are included:

(XIX)，

(XIX),

in:

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

T ₃ is O or S.

In addition, amino acids having a structure of formula (XX) are included:

(XX)，

(XX),

in:

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:

和

。

with

.

In certain embodiments, polypeptides containing non-natural amino acids are chemically modified to produce reactive carbonyl or dicarbonyl functional groups. For example, an aldehyde functional group that can be used for a conjugation reaction can be generated from a functional group having adjacent amine groups and hydroxyl groups. For example, in the case where the biologically active molecule is a polypeptide, the N-terminal serine or threonine (which may exist normally or may be exposed through chemical or enzymatic digestion) can be used to use high-density acid under mild oxidative cleavage conditions. Iodate produces aldehyde functional groups. 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, the methods known in the art are limited to amino acids at the N-terminus of peptides or proteins.

In the present invention, non-natural amino acids with adjacent hydroxyl and amino groups can be incorporated into the polypeptide as "masked" aldehyde functional groups. For example, 5-hydroxylysine has a hydroxyl group adjacent to the epsilon amine group. The reaction conditions used to produce aldehydes generally include the addition of a molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other locations within the polypeptide. The pH of the oxidation reaction is usually about 7.0. A typical reaction involves adding about 1.5-fold molar excess of sodium metaperiodate to a buffered solution of the polypeptide, followed by incubation in the dark for about 10 minutes. See, for example, U.S. Patent No. 6,423,685.

The carbonyl or dicarbonyl functional group can selectively react with the reagent containing hydroxylamine in an aqueous solution under mild conditions to form a corresponding oxime bond that is 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 allows selective modification in the presence of other amino acid side chains. See, for example, Cornish, VW, etc., J. Am. Chem. Soc. 118: 8150-8151 (1996); Geoghegan, KF & Stroh, JG, Bioconjug. Chem. 3:138-146 (1992); Mahal, LK, etc., Science 276: 1125-1128 (1997).

A. Carbonyl reactive group (Carbonyl reactive groups)

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

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

Wherein n is 0-10; R ₁ is alkyl, aryl, substituted alkyl or substituted aryl; R ₂ is H, alkyl, aryl, substituted alkyl, and substituted aryl; and R ₃ Is H, an amino acid, a polypeptide, or an amino terminal modification group, and R ₄ is H, an amino acid, a polypeptide, or a carboxy terminal modification group. In certain embodiments, n is 1, R ₁ is phenyl, and R ₂ is a simple alkyl group (ie, methyl, ethyl, or propyl), and the ketone component is positioned opposite to the side chain of the alkyl group. In place. In certain embodiments, n is 1, R ₁ is phenyl, and R ₂ is a simple alkyl group (ie, methyl, ethyl, or propyl), and the ketone component is located between the side chains of the alkyl group. In place.

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

In certain embodiments, polypeptides containing non-naturally encoded amino acids are chemically modified to produce reactive carbonyl functional groups. For example, the aldehyde functional group used for the conjugation reaction may be generated from a functional group having adjacent amine groups and hydroxyl groups. For example, in the case that the biologically active molecule is a polypeptide, N -terminal serine or threonine (which may exist normally or may be exposed through chemical or enzymatic digestion) can be used under mild oxidative cleavage conditions Periodate to generate aldehyde functional groups. 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, the methods known in the art are limited to the amino acid at the N-terminus of the peptide or protein.

In the present invention, non-naturally encoded amino acids with adjacent hydroxyl and amino groups can be incorporated into the polypeptide as "masked" aldehyde functional groups. For example, 5-hydroxylysine has a hydroxyl group adjacent to the epsilon amine group. The reaction conditions used to produce aldehydes generally include the addition of a molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other locations within the polypeptide. The pH of the oxidation reaction is usually about 7.0. A typical reaction involves adding about a 1.5-fold molar excess of sodium metaperiodate to a buffered solution of the polypeptide, and then 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 selectively react with reagents containing hydrazine, hydrazine, hydroxylamine or semicarbazide in an aqueous solution under mild conditions to form corresponding hydrazone, oxime or hemicarbazone bonds that are stable under physiological conditions, respectively. 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 selective modification in the presence of other amino acid side chains. See, for example, Cornish, VW, etc., J. Am. Chem. Soc. 118: 8150-8151 (1996); Geoghegan, KF & Stroh, JG, Bioconjug. Chem. 3:138-146 (1992); Mahal, LK, etc., Science 276: 1125-1128 (1997).

B. Hydrazine (hydrazine) Hydrazine (hydrazide) Aminourea (semicarbazide) Reactive group

Non-naturally encoded amino acids containing nucleophilic groups such as hydrazine, hydrazine, or aminourea are allowed to react with a variety of different electrophilic groups to form conjugates (including but not limited to PEG or other water-soluble polymer Conjugates of things).

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

Where n is 0-10; R ₁ is alkyl, aryl, substituted alkyl or substituted aryl or not present; X is O, N or S or not present; R ₂ is H, amino acid, polypeptide Or an amino terminal modification group, and R ₃ is H, an amino acid, a polypeptide, or a carboxy terminal modification group.

In certain embodiments, n is 4, R _{1 is} not present, and X is N. In certain embodiments, n is 2, R _{1 is} not present, and X is not present. In certain embodiments, n is 1, R ₁ is phenyl, X is O, and the oxygen atom is in the para position of the aliphatic group on the aryl ring.

Amino acids containing hydrazine, hydrazine and semicarbazide can be obtained from commercial sources. For example, L-glutamic acid-γ-hydrazine can be obtained from Sigma Chemical (St. Louis, MO). Other amino acids that are not commercially available can be prepared by those of ordinary skill in the art. See, for example, U.S. Patent No. 6,281,211, which is incorporated herein by reference.

Polypeptides containing non-naturally encoded amino acids with hydrazine, hydrazine, or aminourea functional groups can react efficiently and selectively with molecules containing aldehydes or other functional groups with similar chemical reactivity. See, for example, Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995). The unique reactivity of hydrazine, hydrazine and aminourea functional groups allows them to interact with the nucleophilic groups present on 20 common amino acids (including but not limited to the hydroxyl or lysine of serine or threonine and N- The amine group at the end) has a significantly higher reactivity to aldehydes, ketones and other electrophilic groups.

C. Containing aminooxy group (Aminooxy-containing) Amino acid

Non-naturally encoded amino acids containing aminooxy (also known as hydroxylamine) groups are allowed to react with a variety of different electrophilic groups to form conjugates (including but not limited to PEG or other water-soluble Conjugates of sexual polymers). Similar to hydrazine, hydrazine and aminourea, the high nucleophilicity of aminooxy allows it to react efficiently and selectively with various molecules containing aldehydes or other functional groups with similar chemical reactivity. See, for example, Shao, J. and Tam, J., J. Am. Chem. Soc. 117: 3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34: 727-736 (2001 ). Although the result of the reaction with a hydrazine group is the corresponding hydrazone, an oxime is usually obtained from the reaction of an aminooxy group with a carbonyl-containing group such as a ketone.

Exemplary amino acids containing amine groups can be represented as follows:

Wherein n is 0-10; R ₁ is alkyl, aryl, substituted alkyl or substituted aryl or not present; X is O, N, S or not present; m is 0-10; Y = C( O) or not present; R ₂ is H, an amino acid, a polypeptide, or an amino terminal modification group, and R ₃ is H, an amino acid, a polypeptide, or a carboxy terminal modification group. In certain embodiments, n is 1, R ₁ is phenyl, X is O, m is 1, and Y is present. In certain embodiments, n is 2, R ₁ and X are not present, m is 0, and Y is not present.

Amino acids containing aminooxy 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). Certain amino acids containing aminooxy 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 aminooxy groups can be prepared by those of ordinary skill in the art.

D. Azido and alkyne reactive groups

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

Because the Huisgen cycloaddition reaction involves a selective cycloaddition reaction (see, for example, Padwa, A., "Comprehensive Organic Synthesis" (Comprehensive Organic Synthesis), Vol. 4, Trost, BM editor (1991), p. 1069-1109; Huisgen, R., "1,3-DIPOLAR CYCLOADDITION CHEMISTRY" (1,3-DIPOLAR CYCLOADDITION CHEMISTRY), Padwa, A. Editor-in-Chief, (1984), p. 1-176) instead of nucleophilic substitution, therefore The incorporation of a non-naturally encoded amino acid with side chains containing azide and alkynyl groups allows the resulting polypeptide to be selectively modified at the position of the non-naturally encoded amino acid. Cycloaddition reactions involving IL-2 containing azide or alkynyl groups can be carried out at room temperature and under aqueous conditions by adding a catalytic amount of Cu in the presence of a reducing agent for reducing Cu(II) to Cu(I). (II) (including but not limited to in the form of a catalytic amount of CuSO ₄ ) is performed in situ. See, for example, Wang, Q., etc., J. Am. Chem. Soc. 125, 3192-3193 (2003); Tornoe, CW, etc., J. Org. Chem. 67:3057-3064 (2002); Rostovtsev, etc., 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 some cases, when the Huisgen [3+2] cycloaddition reaction between azide and alkyne is required, the IL-2 contains a non-naturally encoded amino acid containing an alkynyl component and a A water-soluble polymer containing an azide component attached to the amino acid. Alternatively, the reverse reaction (that is, using the azide component on the amino acid and the alkynyl component present on the water-soluble polymer) can also be carried out.

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

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

Where X can be O, N, S or not present, Ph is a phenyl group, W is a water-soluble polymer, and R can be H, an alkyl group, an aryl group, a substituted alkyl group, and a substituted aryl group. Exemplary R groups include but are not limited to -CH ₂ , -C(CH ₃ ) ₃ , -OR', -NR'R", -SR', -halogen, -C(O)R', -CONR'R", -S(O) ₂ R', -S(O) ₂ NR'R", -CN and -NO _2. R', R", R"' and R"" each independently refer to hydrogen, Substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (including but not limited to aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy Or aralkyl. When the compound of the present invention includes more than one R group, for example, each of the R groups is independently selected when there is more than one R', R”, R'” and R”” group When, the same is true for each of these groups. 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" means including but not limited to 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will understand that the term "alkyl" means including Groups to the carbon atoms of groups other than hydrogen groups such as haloalkyl groups (including but not limited to -CF ₃ and -CH ₂ CF ₃ ) and acyl groups (including but not limited to -C(O)CH ₃ ,- C(O)CF ₃ , -C(O)CH ₂ OCH _3, etc.).

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

Where n is 1-10; X can be O, N, S or not present, 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; R ₁ is alkyl, aryl, substituted alkyl or substituted aryl or not present; X is O, N, S or not present; m is 0-10, R ₂ is H , Amino acid, polypeptide, or amino terminal modification group, and R ₃ is H, amino acid, polypeptide, or carboxy terminal modification group. In certain embodiments, n is 1, R ₁ 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, R ₁ is phenyl, X is O, m is 1 and the propargyloxy group is located in the para position relative to the alkyl side chain (ie O-propargyl-tyramine acid). In certain embodiments, n is 1, R ₁ and X are not present, and m is 0 (ie, propargylglycine).

Alkynyl amino acids are commercially available. For example, propargylglycine is commercially available from Peptech (Burlington, MA). Alternatively, the alkynyl amino acid 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 It can be synthesized as described in Kayser, B. et al., Tetrahedron 53(7): 2475-2484 (1997). Other alkynyl-containing amino acids can be prepared by those of ordinary skill in the art.

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

Wherein n is 0-10; R ₁ is alkyl, aryl, substituted alkyl, substituted aryl or absent; X is O, N, S or absent; m is 0-10; R ₂ is H , Amino acid, polypeptide, or amino terminal modification group, and R ₃ is H, amino acid, polypeptide, or carboxy terminal modification group. In certain embodiments, n is 1, R ₁ is phenyl, X is absent, m is 0 and the azido moiety is located in the para position of the alkyl side chain. In some embodiments, n is 0-4 and R ₁ and X are not present, and m=0. In certain embodiments, n is 1, R ₁ is phenyl, X is O, m is 2 and the β-azidoethoxy moiety is in the para position relative to the alkyl side chain.

The azido-containing amino acid can be obtained from commercial sources. For example, 4-azidophenylalanine can be obtained from Chem-Impex International, Inc. (Wood Dale, IL). For those azido-containing amino acids that are not commercially available, the azido group can be prepared relatively easily using standard methods known to those of ordinary skill in the art, including but not limited to passing a suitable leaving group ( This includes, but is not limited to, replacement of halides, mesylate, tosylate) or opening by appropriately protected lactones. See, for example, March's "Advanced Organic Chemistry" (third edition, 1985, Wiley and Sons, New York).

E. Amino mercaptan (Aminothiol) Reactive group

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

F. Additional reactive groups

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

Polypeptides with non-natural amino acids

The incorporation of unnatural amino acids can be carried out for a variety of different purposes, including but not limited to modulating the interaction of a protein with its receptor or one or more subunits of its receptor, customizing protein structure and/ Or functional changes, changing size, acidity, nucleophilicity, hydrogen bond formation, hydrophobicity, accessibility of protease target sites, targeting components (including but not limited to protein arrays), adding biologically active molecules, Attach polymer, attach radionuclide, adjust serum half-life, adjust tissue penetration (such as tumor), adjust active transport, adjust tissue, cell or organ specificity or distribution, adjust immunogenicity, adjust protease resistance, etc. . Proteins that include unnatural amino acids can have enhanced or even completely new catalytic or biophysical properties. For example, by including non-natural amino acids in the protein, 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), the ability to react with other molecules (including but not limited to covalent or non-covalent reactions). A composition comprising a protein including at least one unnatural amino acid can be used to include but not limited to new therapeutic agents, diagnostic agents, catalytic enzymes, industrial enzymes, binding proteins (including but not limited to antibodies), and include but not limited to proteins Structure and function research. 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 present invention, the composition includes at least one protein, which has 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 acid 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 in the protein Sites, which contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different unnatural amino acids. On the other hand, the composition includes a protein in which at least one but less than all specific amino acids present in the protein are replaced by non-natural amino acids. For a given protein with more than one unnatural amino acid, the unnatural amino acid may be the same or different (including but not limited to, the protein may include two or more different types of unnatural amino acids). Amino acid, or may include two identical non-natural amino acids). For a given protein with more than two non-natural amino acids, the non-natural amino acids may be the same, different or multiple of the same type and at least one different non-natural amino acid The combination.

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

By producing a protein or polypeptide of interest with at least one non-natural amino acid in a eukaryotic cell, the protein or polypeptide will generally include eukaryotic post-translational modifications. In certain embodiments, the protein 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 through the non-natural amino acid. For example, the post-translational modification can be achieved through a nucleophilic-electrophilic reaction. Most of the reactions currently used for the selective modification of proteins involve the formation of covalent bonds between nucleophilic and electrophilic reaction partners, including but not limited to α-haloketones with histidine or cysteine side chains reaction. In these cases, the selectivity is determined by the number and accessibility of nucleophilic residues in the protein. In the protein of the present invention, other more selective reactions can be used in vitro and in vivo, such as the reaction of unnatural keto-amino acids with hydrazine or aminooxy 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 into the present invention by reference. This allows the selective labeling of virtually any protein with a large number of reagents including fluorophores, cross-linkers, sugar derivatives and cytotoxic molecules. See U.S. Patent No. 6,927,042 entitled "Glycoprotein synthesis", which is incorporated herein by reference. Post-translational modifications (including but not limited to post-translational modifications through azidoamino acids) can be made through Staudinger connection (including but not limited to the use of triarylphosphine reagents). See, for example, Kiick et al., Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation, PNAS 99:19-24 (2002).

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

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

The production methods of tRNA and tRNA synthetase 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 by reference into the present invention. These methods involve the production of translation machines that do not rely on syntheses and tRNAs endogenous to the translation system to function (and therefore are sometimes referred to as "orthogonal") translation machines. Generally, the translation system includes orthogonal tRNA (O-tRNA) and orthogonal amino tRNA synthetase (O-RS). Generally, the O-RS preferentially uses at least one non-naturally-occurring amino acid in the translation system to aminate the O-tRNA, and the O-tRNA recognizes at least those in the system that are not recognized by other tRNAs. A selector codon. Therefore, the translation system responds to the encoded selector codon by inserting the non-naturally encoded amino acid into the protein produced in the system, thereby “replacement” of the amino acid into the The position in the encoded polypeptide.

A wide variety of orthogonal tRNA and aminyl tRNA synthetases for inserting specific synthetic amino acids into polypeptides have been described in the art, and they are generally suitable for use in the present invention. For example, keto-specific O-tRNA/amido-tRNA synthetase is 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 by reference into the present invention. The corresponding O-tRNA molecules used with the O-RS are also described in U.S. Patent Nos. 7,045,337 and 7,083,970, which are incorporated herein by reference. Additional examples of O-tRNA/amido-tRNA synthetase pairs are described in WO 2005/007870, WO 2005/007624 and WO 2005/019415.

An example of an azido-specific O-tRNA/amido-tRNA synthetase system is 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 disclosed in U.S. Patent No. 7,083,970, which is incorporated herein by reference. -32 and amino acid sequences of SEQ ID NO: 46-48 and 61-64. Exemplary O-tRNA sequences suitable for use in the present invention include, but are not limited to, the nucleotide sequence SEQ ID NO: 1-3 disclosed in U.S. Patent No. 7,083,970, which is incorporated by reference into the present invention. Other examples of O-tRNA/amino-tRNA synthetase pairs specific to specific 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 incorporating both ketone and azide amino acids in S. cerevisiae are described in Chin, JW et al., Science 301:964-967 (2003) .

Several other orthogonal pairs have been reported. Derived from the glutamic acid base of Saccharomyces cerevisiae tRNA and synthetase (see, for example, Liu, DR and Schultz, PG (1999) Proc. Natl. Acad. Sci. USA 96: 4780-4785), aspartame base (See, for example, Pastrnak, M., et al., (2000) Helv. Chim. Acta 83: 2277-2286) and tyramine groups (see, for example, Ohno, S., et al., (1998) J. Biochem. (Tokyo, Jpn.) 124:1065-1068; and Kowal, AK et al. (2001) Proc. Natl. Acad. Sci. USA 98:2268-2273) system has been described for the potential incorporation of unnatural amino acids in E. coli. Derived from Escherichia coli glutamate group (see, for example, Kowal, AK, etc., (2001) Proc. Natl. Acad. Sci. USA 98: 2268-2273) and tyrosinyl group (see, for example, Edwards, H. and Schimmel, P. (1990) Mol. Cell. Biol. 10:1633-1641) The system of synthetase has been described for use in Saccharomyces cerevisiae. The E. coli tyrosine-based 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/amino-tRNA synthetase involves the selection of specific codons (selector codons) encoding non-naturally encoded amino acids. Although any codons can be used, it is generally desirable to select codons that are rare or never used in cells expressing the O-tRNA/amino-tRNA synthetase. For example, exemplary codons include nonsense codons such as stop codons (amber, ocher, and pebble), four or more base codons, and other rare or unused natural three-base codons.

Specific selector codons can be introduced into the IL-2 coding sequence using mutagenesis methods known in the art (including but not limited to site-specific mutagenesis, cassette mutagenesis, restricted selection mutagenesis, etc.) Suitable for location.

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

The present invention contemplates the incorporation of one or more non-naturally occurring amino acids into IL-2. One or more non-naturally occurring amino acids may be incorporated at specific locations that do not destroy the activity of the polypeptide. This can be achieved by making "conservative" substitutions, including but not limited to replacing hydrophobic amino acids with hydrophobic amino acids, replacing bulk amino acids with bulk amino acids, replacing hydrophilic amino acids with hydrophilic amino acids, and /Or the non-naturally occurring amino acid is inserted into a position not required for activity.

Various 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 non-naturally encoded amino acids, and the selection can be based on rational design or by random selection for any or no specific The desired purpose. The selection of the desired site can be used to produce IL-2 molecules with any desired properties or activities, including but not limited to modulating receptor binding or one or more subunits, agonists, and super agonists. The combination of an agent, inverse agonist, antagonist, receptor binding modifier, receptor activity modifier, dimer or multimer formation, does not change the activity or properties of the original molecule, or manipulates any of the polypeptide Physical or chemical properties such as solubility, aggregation or stability. For example, the position required for the biological activity of IL-2 in the polypeptide can be identified using point mutation analysis, alanine scanning, saturation mutagenesis and biological activity screening or homologous 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 the potential pairing residues (Jones, Protein Science 3: 567-574 (1994)) and the use of Protein design Automation ^® rational design techniques (see U.S. Patent No. 6,188,965,6,269,312,6,403,312, WO98 / 47089, which is incorporated by reference invention). Residues other than those identified as critical for biological activity by alanine or homology scanning mutagenesis may be good candidates for substitution with non-naturally encoded amino acids, depending on the polypeptide The desired activity sought. Alternatively, sites identified as critical for biological activity may also be good candidates for substitution with non-naturally encoded amino acids, again depending on the desired activity sought for the polypeptide. Another alternative is to simply make a series of substitutions with non-naturally encoded amino acids in each position on the polypeptide chain and observe the effect on the activity of the polypeptide. It is obvious to a person of ordinary skill in the art that any means, technique, or method for selecting positions for substitution of non-natural amino acids in any polypeptide is 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 resistant to substitution with non-naturally encoded amino acids. In a similar manner, protease digestion and clone antibodies can be used to identify the region of IL-2 responsible for binding to the IL-2 receptor. Once residues that may be intolerant to substitutions with non-naturally encoded amino acids have been eliminated, the impact of the proposed substitutions at each remaining position can be examined. Therefore, those of ordinary skill in the art can easily identify amino acid positions that can be replaced with non-naturally encoded amino acids.

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

In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the following positions in LI-2: before position 1 (ie 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, 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 end of the protein, and any combination thereof (SEQ ID NO: 2, or SEQ ID NO: 3, 5 or 7 Corresponding amino acid).

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: 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 amine in SEQ ID NO: 3, 5 or 7 Base acid).

In certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more of the secondary structures or specific amino acids described below in IL-2 or its variants. Any position in the region: at the position of hydrophobic interaction; at or near the position of interaction with the IL-2 receptor subunit (including IL2Rα); within the 3 or 35 to 45 amino acid positions; Within the first 107 N-terminal amino acids; within the 61-72th amino acid positions; each of the positions is the position of SEQ ID NO: 2 or the corresponding amine in SEQ ID NO: 3, 5 or 7 Base acid position. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at one or more of the following positions of IL-2 or a variant thereof: before position 1 of SEQ ID NO: 2 ( That is at the N-end), 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 acid in SEQ ID NO: 3, 5 or 7. In certain embodiments, one or more non-naturally encoded amino acids are incorporated at one or more of the following positions of IL-2 or a variant thereof: the 44th, 45th, and 45th positions of SEQ ID NO: 2 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 carboxy terminus of the protein, and any combination thereof; or SEQ ID NO: 3, 5 Or the corresponding amino acid in 7.

In certain embodiments, the IL-2 polypeptide is an agonist, and the non-naturally occurring amino acid in one or more of these regions is linked to a water-soluble polymer, including but not limited to: No. 3, 35 , 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 72 and 107 bits. In certain embodiments, the IL-2 polypeptide is an agonist, and the non-naturally occurring amino acid in one or more of these regions is linked to a water-soluble polymer, including but not limited to: Around 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. Generally, the specific non-naturally encoded amino acid used for incorporation is selected on the basis of the following: the examination of the three-dimensional crystal structure of IL-2 polypeptides or other IL-2 family members and their receptors, the preference of conservative substitutions (ie Non-naturally encoded amino acids based on aryl groups, such as p-acetylphenylalanine or O-propargyl tyrosine instead of Phe, Tyr or Trp), and specific conjugates that people wish to introduce into the IL-2 Chemistry (for example, if one wants to achieve Huisgen [3+2] cycloaddition with water-soluble polymers with alkynyl components or achieve amides with water-soluble polymers with aryl esters and thus incorporated into phosphine components The bond is formed, then 4-azidophenylalanine is introduced).

In one embodiment, the method further comprises: incorporating the non-natural amino acid in the protein, wherein the non-natural amino acid comprises a first reactive group; and combining the protein with The molecules of the second reactive group are in contact (the molecules include but are not limited to markers, 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 chelating agents, cofactors, fatty acids, carbohydrates, polynucleosides Acid, DNA, RNA, antisense polynucleotide, sugar, water-soluble dendrimer, cyclodextrin, inhibitory ribonucleic acid, biological material, nanoparticle, spin label, fluorophore, containing Metal components, radioactive components, new functional groups, groups that covalently or non-covalently interact with other molecules, photocaged components, components that can be excited by actinic radiation, components that can be photoisomerized , Biotin, biotin derivatives, biotin analogs, heavy atom-incorporated components, chemically cleavable groups, photo-cleavable groups, extended side chains, carbon-linked sugars, redox active agents, Aminothio acids, toxic components, isotope-labeled components, biophysical probes, phosphorescent groups, chemiluminescent groups, electron dense groups, magnetic groups, intercalating groups, chromophores, energy transfer reagents , Biologically active agents, detectable labels, small molecules, quantum dots, nano emitters, 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 through a [3+2] cycloaddition to attach the molecule to the non-natural amino acid. In one embodiment, the first reactive group is an alkynyl or azido component, and the second reactive group is an azido or an alkynyl component. For example, the first reactive group is an alkynyl component (including but not limited to the non-natural amino acid p-propargyloxyphenylalanine), and the second reactive group is an azide component part. In another example, the first reactive group is an azido component (including but not limited to the non-natural amino acid p-azido-L-phenylalanine), and the second reactive group The group is an alkynyl component.

In some cases, the non-naturally encoded amino acid substitution is combined with other additions, substitutions or deletions in the IL-2 to affect other biological properties of the IL-2 polypeptide. In some cases, the other additions, substitutions or deletions can increase 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 suppressive and/or tumor reducing activity of the IL-2. In some cases, the other additions, substitutions or deletions can increase the solubility of the IL-2 or variant (including but not limited to when expressed in E. coli or other host cells). In some embodiments, addition, substitution or deletion can increase the solubility of the IL-2 after expression in E. coli or other recombinant host cells. In certain embodiments, in addition to another site used to incorporate non-natural amino acids that lead to increased solubility of the polypeptide after expression in E. coli or other recombinant host cells, it is also selected for use in natural The site of substitution of encoded or non-natural amino acids. In certain embodiments, the IL-2 polypeptide includes another addition, substitution or deletion, which modulates the affinity for IL-2 receptor, binding protein or related ligands, and modulates the binding to IL-2 receptor. Signal transduction, adjust the circulatory half-life, adjust the release or bioavailability, promote purification, or improve or change the 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 includes additions, substitutions, or deletions that increase the affinity of the IL-2 variant for IL-2-R1 and/or IL-2-R2. Similarly, IL-2 polypeptides may contain 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), which improve detection (including but not limited to GFP), purification, transport through tissues or cell membranes, prodrug release or activation, IL- 2 Reduced size or other traits of the polypeptide.

In certain embodiments, the replacement of the non-naturally encoded amino acid produces an IL-2 antagonist. In certain embodiments, non-naturally encoded amino acids are replaced or added in regions 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 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 with 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 that are modified by one or more non-natural amino acids. Replacement of encoded amino acids. For example, in certain embodiments, one or more residues in IL-2 are replaced with one or more non-naturally encoded amino acids. In some cases, the one or more non-naturally encoded residues are linked to one or more lower molecular weight linear or branched PEGs, thereby relative to the one attached to a single higher molecular weight PEG. The substance improves binding affinity and comparable serum half-life.

VI. Expression in non-eukaryotes and eukaryotes

In order to obtain high-level expression of cloned IL-2 polynucleotides, people usually clone the polynucleotide encoding the IL-2 polypeptide of the present invention in an expression vector containing a transcription guide. A strong promoter, transcription/translation terminator, and ribosome binding site for translation initiation if used for protein-encoding nucleic acids. Suitable bacterial promoters are known to those of ordinary skill in the art and are described in, for example, Sambrook et al. and Ausubel et al.

Include but not limited to Escherichia coli ( E. coli ), Bacillus sp. , Pseudomonas fluorescens , Pseudomonas aeruginosa , Pseudomonas putida ) and Salmonella (Salmonella), a bacterial expression system of the invention for expression of IL-2 is available (like Palva, Gene 22: 229-235 (1983) ; Mosbach et, 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 tRNA and aminyl tRNA synthetase (described above) to express the IL-2 polypeptide of the present invention, host cells for expression are selected on the basis of their ability to use the orthogonal components. Exemplary host cells include Gram-positive bacteria (including but not limited to Bacillus brevis ( B. brevis ), Bacillus subtilis ( B. subtilis ) or Streptomyces ( Streptomyces )) and Gram-negative bacteria (E. coli ( E. coli , Pseudomonas fluorescens , Pseudomonas aeruginosa , Pseudomonas putida ), yeast and other eukaryotic cells. Cells containing O-tRNA/O-RS pairs can be used as described in the present invention.

The eukaryotic host cell or non-eukaryotic host cell of the present invention provides the ability to synthesize proteins containing unnatural amino acids in a useful large amount. In one case, the composition optionally includes, but is 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 mg, at least 10 mg, at least 100 mg, at least 1 gram or more of protein containing unnatural amino acids, or an amount that can be obtained using in vivo protein production methods (details on recombinant protein production and purification are provided in the present invention). In another case, the protein optionally includes but is not limited to at least 10 micrograms protein/liter, at least 50 micrograms protein/liter, at least 75 micrograms protein/liter, at least 100 micrograms protein/liter, at least 200 micrograms protein/liter. Liter, at least 250 microgram protein/liter, at least 500 microgram protein/liter, at least 1 mg protein/liter, or at least 10 mg protein/liter or higher is present in the composition, including but not limited to cell lysates , Buffer, drug buffer or other liquid suspension (including but not limited to any volume including but not limited to about 1 nl to about 100 L or more). The production of proteins including at least one unnatural amino acid in eukaryotic cells in large amounts (including but not limited to larger amounts than usually possible using other methods (including but not limited to in vitro translation)) is a feature of the present invention.

The eukaryotic host cell or non-eukaryotic host cell of the present invention provides the ability to biosynthesize proteins containing unnatural amino acids in a useful large amount. For example, a protein containing an unnatural amino acid may include, but is 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. 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, at least 8 mg/L, at least 9 mg/L, at least 10 mg/L, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 Protein concentrations of mg/liter, 1 g/liter, 5 g/liter, 10 g/liter or higher 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 papillomavirus, adenovirus, and cytomegalovirus. Such vectors include pCDNA3.1(+)\Hyg (Invitrogen, Carlsbad, Calif., USA) and pCI-neo (Stratagene, La Jolla, Calif., USA). Bacterial plastids such as those derived from Escherichia coli, including pBR322, pET3a and pET12a, wide host range plastids such as RP4, phage DNA such as lambda phage such as NM989 and other DNA phage such as M13 and filamentous single-stranded DNA phage can be used. Lots of derivatives. 2μ plastids and derivatives thereof, POT1 vector (U.S. Patent No. 4,931,373 incorporated by reference into the present invention), pJSO37 vector and pPICZ described in (Okkels, Ann. New York Aced. Sci. 782, 202 207, 1996) 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 bovine and human genes for Mullerian inhibitory substances and expression of the human genes in animal cells" (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 a variant thereof may or may not 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 can be any sequence. The signal peptide may be prokaryotic or eukaryotic. Coloma, M (1992) J. Imm. Methods 152:89 104 describes a signal peptide (murine Ig κ light chain signal peptide) used in mammalian cells. Other signal peptides include, but are not limited to, the α-factor signal peptide from Saccharomyces cerevisiae (US Patent No. 4,870,008, incorporated by reference in the present invention), the signal peptide of mouse salivary amylase (O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646), modified carboxypeptidase signal peptide (LA Valls et al., Cell 48, 1987, pp. 887-897), yeast BAR1 signal peptide (WO 87/02670, which is incorporated by reference into the present invention) and Yeast aspartic acid protease 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 can be Chinese Hamster Ovary (CHO) cells (e.g. CHO-K1; ATCC CCL-61), green monkey cells (COS) (e.g. COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)) , Mouse cells (such as NS/O), baby hamster kidney (BHK) cell lines (such as ATCC CRL-1632 or ATCC CCL-10) and human cells (such as HEK 293 (ATCC CRL-1573)), and tissue culture Plant cell. These cell lines and other cell lines can be obtained from public collections such as the American Type Culture Collection (Rockville, Md). In order to provide for increased glycosylation of IL-2 polypeptides, mammalian host cells can be modified to express sialyltransferases such as 1,6-sialyltransferases, for example in the U.S. Patent, which is incorporated by reference into the present invention. As described in No. 5,047,335.

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 The vector and the transfection method described by Life Technologies Ltd, Paisley, UK using Lipofectamin 2000 and the FuGENE 6 described by Roche Diagnostics Corporation, Indianapolis, USA. These methods are well known in the art and are described by Ausbel et al., 1996, Current Protocols in Molecular Biology, John Wiley & Sons, New York, USA. Cultivation of mammalian cells can be carried out in accordance with established methods, for example in ("Animal Cell Biotechnology, Methods and Protocols" (Animal Cell Biotechnology, Methods and Protocols), edited by Nigel Jenkins, 1999, Human Press Inc. Totowa, NJ , USA and Harrison Mass. and Rae IF, "General Techniques of Cell Culture" (General Techniques of Cell Culture, Cambridge University Press 1997).

I. Escherichia coli (E. Coli) , Pseudomonas species (Pseudomonas species) and other prokaryotic bacteria 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 vector can be a single copy or a low or high multiple copy vector. The vector can be used for cloning 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, no further discussion is needed here. As is well known, vectors usually contain markers that allow selection, which may provide cytotoxic agent resistance, prototrophy, or immunity. There are often multiple marks that provide different characteristics.

A bacterial promoter is any DNA sequence that can bind to bacterial RNA polymerase and initiate the transcription of downstream (3') coding sequences (such as structural genes) into mRNA. A promoter has a transcription initiation region, which is usually located near the 5'end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. Bacterial promoters can also have a second domain called an operon, which can overlap with the adjacent RNA polymerase binding site at the beginning of RNA synthesis. The operon allows negatively regulated (inducible) transcription because the gene repressor protein can bind to the operon and thus inhibit the transcription of specific genes. Constitutive expression may occur in the absence of negative regulatory elements such as operons. In addition, positive regulation can be achieved by a gene promoter protein binding sequence, which, if present, is usually near (5') the RNA polymerase binding sequence. An example of a gene promoter protein is the catabolic metabolite promoter protein (CAP), which helps initiate transcription of the lac operon in E. coli (Raibaud et al., ANNU. REV. GENET. (1984) 18:173). Therefore, regulatory expression can be positive or negative, thereby enhancing or attenuating transcription.

Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes 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). ; US Patent No. 4,738,921; European Patent Publication Nos. 036 776 and 121 775, which are incorporated into the present invention by reference). β-galactosidase (bla) promoter system (Weissmann (1981), clone of interferon and other mistakes, Interferon 3 (edited by I. Gresser)), phage λ The PL (Shimatake et al., NATURE (1981) 292:128) and T5 (U.S. Patent No. 4,689,406, which is incorporated herein by reference) promoter systems also provide useful promoter sequences. The preferred method of the present invention utilizes strong promoters such as the T7 promoter to induce IL-2 polypeptides at high levels. Examples of these 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-level IL-2 polypeptides in the host without compromising the viability or growth parameters of the host cell. pET19 (Novagen) is another vector known in the art.

In addition, synthetic promoters (synthetic promoters) that do not exist in nature also function as bacterial promoters. For example, the transcription initiation 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 into the present invention by reference). For example, the tac promoter is a hybrid trp-lac promoter that contains both the trp promoter and the lac operator sequence 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 may include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. Naturally occurring promoters of non-bacterial origin can also be coupled with compatible RNA polymerases to produce high-level expression of certain genes in prokaryotes. The phage 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 include phage promoters and E. coli operon regions (European Patent Publication No. 267 851).

In addition to functional promoter sequences, efficient ribosome binding sites are also useful for the expression of foreign genes in prokaryotes. In E. coli, the ribosome binding site is called Shine-Dalgarno (SD) sequence, and includes the start codon (ATG) and a length of 3-11 nucleotides upstream of the start codon It is a sequence of 3-9 nucleotides (Shine et al., NATURE (1975) 254:34). The SD sequence is believed to promote the binding of mRNA and ribosomes through the base pairing between the SD sequence and the 3'end of E. coli 16S rRNA (Steitz et al., Genetic signals and nucleotide sequences in messenger RNA (Genetic signals) and nucleotide sequences in messenger RNA), "Biological Regulation and Development: Gene Expression" (RF Goldberger editor in chief, 1979)). In order to express eukaryotic and prokaryotic genes with weak ribosome binding sites (Sambrook et al., Expression of cloned genes in Escherichia coli), "Clone experiment" Guide" (Molecular Cloning: A Laboratory Manual), 1989).

The term "bacterial host" or "bacterial host cell" refers to bacteria that can be or have been used as recipients for recombinant vectors or other transfer DNA. The term includes the progeny of the original bacterial host cell that has been transfected. It should be understood that due to accidental or deliberate mutations, the progeny of a single parent cell may not necessarily be exactly the same as the original parent in morphology or the entire genome or total DNA. The progeny of the parent cell that is sufficiently similar to the parent, characterized by related properties such as 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 the expression of IL-2 polypeptides is known to those of ordinary skill in the art. Among the bacterial hosts selected for expression, suitable hosts may include those that show particularly good inclusion body forming ability, low proteolytic activity, and overall robustness. Bacterial hosts can usually be obtained 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 American Type Culture Collection ("ATCC") (Manassas, VA)). Industrial/pharmaceutical fermentation usually uses bacteria derived from K strain (for example, W3110) or bacteria derived from B strain (for example, BL21). These strains are particularly useful because their growth parameters are well understood and robust. In addition, these strains are non-pathogenic, which is commercially important for safety and environmental reasons. Other examples of suitable E. coli hosts include, but are not limited to, BL21, DH10B strains or derivatives thereof. 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 may be a species of Pseudomonas (Pseudomonas ), including but not limited to Pseudomonas fluorescens , Pseudomonas aeruginosa and Pseudomonas putida ). Pseudomonas fluorescens (Pseudomonas fluorescens) biological variant 1, named MB101 strain, is known to be useful for recombinant production and can be used in therapeutic protein production processes. Examples of Pseudomonas expression systems include those available from The Dow Chemical Company as a host strain (Midland, MI, available at dow.com on the World Wide Web).

Once the recombinant host cell strain has been established (that is, the expression construct has been introduced into the host cell and the host cell with the correct expression construct has been isolated), the recombinant host cell strain is suitable for the production of IL-2 Culture under the condition of peptide. As is obvious to those 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 usually cultivated using methods known to those 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 vitamins, amino acids, growth factors, and other protein culture supplements known to those of ordinary skill in the art. The liquid medium used for host cell culture may optionally contain antibiotics or antifungal agents for preventing the growth of unwanted microorganisms and/or compounds for selecting host cells containing expression vectors, including but not limited to antibiotics.

Recombinant host cells can be cultured in a batch or continuous manner, and cell harvest (in the case where the IL-2 polypeptide accumulates in the cell) or culture supernatant is harvested in a batch or continuous manner. For production in prokaryotic host cells, batch culture and cell harvesting are preferred.

The IL-2 polypeptide of the present invention is usually purified after being expressed in a recombinant system. The IL-2 polypeptide can be purified from the host cell or culture medium by various methods known in the art. The IL-2 polypeptide produced in bacterial host cells may be poorly soluble or insoluble (in the form of inclusion bodies). In an embodiment of the present invention, the method disclosed in the present invention and known in the art can be used to easily perform amino acid substitution in the IL-2, and the substitution is selected to improve recombinant production. The purpose of the solubility of the protein. In the case of insoluble protein, the protein can be collected from the host cell lysate by centrifugation, and then further homogenization of the cells can be performed. 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. Recombinant host cells can be disrupted or homogenized by various methods known to those of ordinary skill in the art to release the inclusion bodies from the cells. The host cell disruption or homogenization can be performed using well-known techniques, including but not limited to enzymatic cell disruption, sonication, dunes 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 body of the IL-2 polypeptide. When processing inclusion bodies of IL-2 polypeptides, 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.

Any of a variety of suitable solubilizers known in the art can then be used to solubilize the insoluble or precipitated IL-2 polypeptide. The IL-2 polypeptide can be dissolved with urea or guanidine hydrochloride. The volume of the dissolved IL-2 polypeptide should be minimized so that a manageable batch size can be used to produce a large number of batches. This factor may be important in the context of large-scale commercialization where recombinant hosts may be grown in batches of thousands of liters in volume. In addition, when IL-2 polypeptides are manufactured in the context of large-scale commercialization, especially for human pharmaceutical use, if possible, the use of harsh chemicals that can damage machinery and containers or the protein product itself should be avoided. In the method of the present invention, it has been shown that the milder denaturant urea can replace the harsher denaturant guanidine hydrochloride for the solubilization of IL-2 polypeptide inclusion bodies. The use of urea significantly reduces the risk of damaging the stainless steel equipment used in the manufacturing and purification of IL-2 polypeptides, while effectively dissolving IL-2 polypeptide 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 the host cell. It may be desirable to concentrate the soluble IL-2 before proceeding to the purification step. Standard techniques known to those of ordinary skill in the art can be used to concentrate soluble IL-2 from, for example, cell lysates or culture media. In addition, standard techniques known to those of ordinary skill in the art can be used to disrupt the host cell and release soluble IL-2 from the cytoplasm or periplasmic space of the host cell.

Generally speaking, it is sometimes desirable to denature and reduce the expressed polypeptide, and then refold the polypeptide into a preferred conformation. For example, guanidine, urea, DTT, DTE, and/or chaperone protein can be added to the translation product of interest. Methods of reducing, denaturing and renaturing proteins are known to those of ordinary skill in the art (see above references, and 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. described the denaturation and reduction of inclusion body proteins in guanidine-DTE. The protein can be refolded in a redox buffer containing, but not limited to, oxidized glutathione and L-arginine. The refolding reagent can be made to flow or otherwise move to contact one or more polypeptides or other expression products, and 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". In general, misfolded IL-2 polypeptides are prepared by using, for example, one or more chaotropic agents (such as urea and/or guanidine) and reducing agents capable of reducing disulfide bonds (such as dithiothreitol DTT or 2-mercaptoethanol). 2-ME) dissolving (where the IL-2 polypeptide is also insoluble), unfolding and reducing the polypeptide chain to refold. An oxidizing agent (such as oxygen, cystine or cystamine) that allows the re-formation of disulfide bonds is then added under a moderate concentration of chaotropic agent. IL-2 polypeptides can be refolded using standard methods known in the art, such as the methods described in U.S. Patent Nos. 4,511,502, 4,511,503, and 4,512,922, which are incorporated herein by reference. The IL-2 polypeptide can also be co-folded 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 various techniques known to those of ordinary skill in the art, including hydrophobic interaction chromatography, pore 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 step of drying or precipitation of the purified protein.

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

The purified IL-2 may be at least 90% pure (as measured by reversed-phase high performance liquid chromatography RP-HPLC or sodium lauryl 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 higher pure. Regardless of the exact value of the purity of the IL-2, the IL-2 is sufficiently pure for use as a medicine or for further processing such as conjugation with water-soluble polymers such as PEG.

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

It has previously been shown that by adding a chemically aminated inhibitor tRNA to a protein synthesis reaction programmed with a gene containing the desired amber nonsense mutation, unnatural amino acids can be site-specifically incorporated into proteins in vitro middle. Using these methods, people can use strains that are auxotrophic to specific amino acids to replace many common 20 amino acids with similar structural homologues, such as replacing amphetamine with fluoroamphetamine. See, for example, 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, non 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, JA, Mendel, D., Anthony-Cahill, S., Noren, CJ, Schultz, PG, used to site-specifically unnatural amino acids Biosynthetic method for introducing unnatural amino acids site-specifically into proteins , Methods in Enz., vol. 202, 301-336 (1992); and Mendel, D., Cornish, VW & Schultz , PG, 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 aminated 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 oligonucleotide-guided mutagenesis (5'-3' Exonucleases in phosphorothioate- based olignoucleotide-directed mutagensis ), Nucleic Acids Res, 16(3):791-802 (1988). When the acylated suppressor tRNA and mutant genes are combined in an in vitro transcription/translation system, the unnatural amino acid is incorporated into the unnatural amino acid in response to the UAG codon, giving it at the specific position The protein containing the amino acid. The experiment using [ ³ H]-Phe and the experiment using α-hydroxy acid confirmed that only the required amino acid was incorporated at the position specified by the UAG codon, and this amino acid was not incorporated in At any other position in the protein. See, for example, Noren et al., supra; Kobayashi et al., (2003) Nature Structural Biology 10(6):425-432; and Ellman, JA, Mendel, D., Schultz, PG, Site specificity of new framework structures in proteins Incorporated ( 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 amination.

Amidation can be accomplished by aminyl tRNA synthetase or other enzyme molecules (including but not limited to ribozymes). The term "ribozyme" can be interchanged with "catalytic RNA". Cech and collaborators (Cech, 1987, Science, 236:1532-1539; McCorkle et al., 1987, Concepts Biochem. 64:221-226) confirmed the existence of naturally occurring RNA (ribozyme) that can act as a catalyst. However, although these natural RNA catalysts have only been shown to act on ribonucleic acid reaction substrates for cutting and splicing, the latest developments in the artificial evolution of ribozymes have expanded the catalytic spectrum to a variety of different chemical reactions. Research has identified RNA molecules (Illangakekare et al. 1995 Science 267:643-647) that can catalyze amine-RNA bonds on their own (2')3'-ends and can remove amino acids from an RNA molecule An RNA molecule that is transferred to another RNA molecule (Lohse et al., 1996, Nature 381:442-444).

US Patent Application Publication 2003/0228593, which is incorporated by reference into the present invention, describes methods for constructing ribozymes and their use in the amination of tRNAs with naturally-encoded and non-naturally-encoded amino acids. The matrix-immobilized form of enzyme molecules (including but not limited to ribozymes) that can aminate tRNA amidation may be able to perform high-efficiency affinity purification of the amidation product. Examples of suitable matrices include agarose, sepharose, and magnetic beads. The production of matrix-immobilized forms of ribozymes and their use for amination are described in Chemistry and Biology 2003, 10:1077-1084 and U.S. Patent Application Publication 2003/0228593, which are incorporated into the present invention by reference.

Chemical amination methods include but are not limited to those introduced by the following researchers: Hecht and collaborators (Hecht, SM Acc. Chem. Res. 1992, 25, 545; Heckler, TG; Roesser, JR; Xu, C. ; Chang, P.; Hecht, SM Biochemistry 1988, 27, 7254; Hecht, SM; Alford, BL; Kuroda, Y.; Kitano, SJ Biol. Chem. 1978, 253, 4517); Schultz, Chamberlin, Dougherty, etc. (Cornish, VW; Mendel, D.; Schultz, PG Angew. Chem. Int. Ed. Engl. 1995, 34, 621; Robertson, SA; Ellman, JA; Schultz, PGJ Am. Chem. Soc. 1991, 113, 2722; Noren, CJ; Anthony-Cahill, SJ; Griffith, MC; Schultz, PG Science 1989, 244, 182; Bain, JD; Glabe, CG; Dix, TA; Chamberlin, ARJ Am. Chem. Soc. 1989, 111 , 8013; Bain, JD et al., Nature 1992, 356, 537; Gallivan, JP; Lester, HA; Dougherty, DA Chem. Biol. 1997, 4, 740; Turcatti et al., J. Biol. Chem. 1996, 271, 19991 ; Nowak, MW, etc., Science, 1995, 268, 439; Saks, ME, etc., J. Biol. Chem. 1996, 271, 23169; Hohsaka, T., etc., J. Am. Chem. Soc. 1999, 121, 34 ), said document is incorporated into the present invention by reference to avoid the use of synthase in amination. These methods or other chemical amination methods can be used to aminate tRNA molecules.

The method for generating catalytic RNA may include generating separate pools of randomized ribozyme sequences, subjecting the pools to directed evolution, screening the pools for the desired amination activity, and selecting those that exhibit The sequence of the ribozyme for the desired amination activity.

A refactored translation system can also be used. A mixture of purified translation factors has also been successfully used to translate mRNA into protein, and a combination of lysates or supplemented with purified translation factors such as initiation factor -1 (IF-1), IF-2, IF-3 ( The same is true for lysates of α or β), extension factor T (EF-Tu) or termination factor. A cell-free system can also be coupled with a transcription/translation system, in which DNA is introduced into the system, transcribed into mRN and translated into the mRNA, as in the "Modern Methods of Molecular Biology" specifically incorporated by reference of the present invention ( Current Protocols in Molecular Biology ) (FM Ausubel et al., editor-in-chief, Wiley Interscience, 1993). The RNA transcribed in the eukaryotic transcription system can take the form of heteronuclear RNA (hnRNA) or mature mRNA with 5′-end capped (7-methylguanosine) and 3′-end poly A tailed. It may be advantageous in a translation system. For example, mRNA capped in the reticulocyte lysate system is translated with high efficiency.

IX. Conjugate to IL-2 Polypeptide macromolecular polymer (Macromolecular Polymers)

Various modifications to the non-natural amino acid polypeptides described in the present invention can be implemented using the compositions, methods, techniques, and strategies described in the present invention. These modifications include the incorporation of other functional groups into the non-natural amino acid component of the polypeptide. The other functional groups include, but are not limited to, markers, dyes, polymers, water-soluble polymers, and derivatives of polyethylene glycol. , Photocrosslinking agent, radionuclide, cytotoxic compound, drug, affinity label, photoaffinity label, reactive compound, resin, second protein or polypeptide or polypeptide analogue, antibody or antibody fragment, metal chelator, Cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, antisense polynucleotides, sugars, water-soluble dendrimers, cyclodextrins, inhibitory ribonucleic acids, biological materials, nanoparticles, spin markers , Fluorescent groups, metal-containing components, radioactive components, new functional groups, groups that covalently or non-covalently interact with other molecules, photocage components, components that can be excited by actinic radiation, Isomerized components, biotin, biotin derivatives, biotin analogs, components doped with heavy atoms, chemically cleavable groups, photo-cleavable groups, extended side chains, carbon-linked sugars , Redox active agents, amino thioacids, toxic components, isotope-labeled components, biophysical probes, phosphorescent groups, chemiluminescent groups, electron dense groups, magnetic groups, intercalating groups, hair Chromophores, energy transfer reagents, biologically active agents, detectable markers, small molecules, quantum dots, nano emitters, radionucleotides, radioactive emitters, neutron capture agents or any combination of the above substances or any other Need compound or substance. As an illustrative and non-limiting example of the compositions, methods, techniques, and strategies described in the present invention, the following description will focus on the addition of macromolecular polymers to non-natural amino acid polypeptides, and it should be understood that the composition , Methods, techniques, and strategies are also applicable (appropriate modifications if necessary, and those skilled in the art can use this disclosure to make these modifications) to add other functional groups, including but not limited to those listed above.

A wide variety of macromolecular polymers and other molecules can be linked to the IL-2 polypeptide of the present invention to adjust the biological properties of the IL-2 polypeptide and/or provide new biological properties for the IL-2 molecule . These macromolecular polymers can be through naturally encoded amino acids, through non-naturally encoded amino acids, or any functional substituents of natural or non-natural amino acids, or added to natural or non-natural amino acids. Any substituent or functional group is connected to the IL-2 polypeptide. The molecular weight of the polymer can have a wide range, including but not limited to between about 100 Da to about 100,000 Da or greater. 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, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In certain embodiments, the molecular weight of the polymer is between about 100 Da and about 50,000 Da. In certain embodiments, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In certain embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In certain embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In certain 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 molecule is observed to exceed half of the total protein. The polymer: protein conjugate has biological activity, and the "substantially homogenous" PEGylated IL-2 polypeptide preparation provided in the present invention is sufficient to show the advantages of a homogeneous preparation, for example, In clinical applications, it is easy to obtain homogeneous preparations with predictable pharmacokinetics between batches.

People can also choose to prepare a mixture of polymer:protein conjugate molecules, and the advantage provided in the present invention is that humans can choose the ratio of single polymer:protein conjugate contained in the mixture. Therefore, if necessary, one can prepare a mixture of various proteins with various numbers of polymer components attached (ie, two, three, four, etc.), and combine the conjugates with those prepared using the method of the present invention. The single polymer: protein conjugates are combined to obtain a mixture of single polymer: protein conjugates having a predetermined ratio.

The selected polymer 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 their alkoxy-terminated analogs (e.g., polyoxyethylene glycol, polyoxyethylene glycol/propylene glycol and their especially methoxy or polyoxyethylene glycol Ethoxy-terminated analogs, polyoxyethylene glycol (also known as polyethylene glycol or PEG), polyvinylpyrrolidone, polyvinyl alkyl ether, polyoxazoline, polyalkyloxazoline and polyhydroxy Alkyloxazoline, polyacrylamide, polyalkylacrylamide and polyhydroxyalkylacrylamide (for example, polyhydroxypropylmethacrylamide and its derivatives), polyhydroxyalkyl acrylate, poly Sialic acid and its analogs, hydrophilic peptide sequences, polysaccharides and their derivatives, including dextran and dextran derivatives, such as carboxymethyl dextran, dextran sulfate, aminodextran, fiber And its derivatives such as carboxymethyl cellulose, hydroxyalkyl cellulose, chitin and its derivatives such as chitosan, succinochitosan, carboxymethyl chitin, carboxymethyl chitosan, Hyaluronic acid and its derivatives, starch, alginate, chondroitin sulfate, albumin, pullulan and carboxymethyl pullulan, polyamino acids and their derivatives such as polyglutamic acid, polylysine , Polyaspartic acid (aspartic acid), polyaspartic acid amide, maleic anhydride copolymers such as styrene maleic anhydride copolymer, divinyl ether maleic anhydride copolymer, polyvinyl alcohol, their copolymers Substances, their terpolymers, their mixtures, and their derivatives.

The ratio of polyethylene glycol molecules to protein molecules can be varied, as is their concentration in the reaction mixture. Generally, the optimal ratio (in terms of reaction efficiency, because there is a very small excess of unreacted protein or polymer) can be determined by the molecular weight of the selected polyethylene glycol and the number of available reactive groups. In terms of molecular weight, generally, the higher the molecular weight of the polymer, the smaller the number of polymer molecules that can be attached to the protein. Likewise, when optimizing these parameters, the branching of the polymer should be taken into account. Generally speaking, the higher the molecular weight (or the more branches), the higher the polymer:protein ratio.

When 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 individual to individual 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. The therapeutically effective amount of the composition of the present invention can be easily determined by a person of ordinary skill in the art using publicly available materials and procedures.

The water-soluble polymer may have any structural form, including but not limited to linear, branched or branched chain. Generally, 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 invention.

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

XO-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -Y

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

In some cases, PEG used in the present invention at one end with hydroxy or methoxy-terminated, i.e., X is H or CH ₃ ( "methoxy PEG"). Alternatively, the PEG can be end-capped 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 20 common amino acids (including but not limited to maleimide groups, activated carbonates (including but not limited to Nitrophenyl esters), activated esters (including but not limited to N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes), as well as inert to 20 common amino acids but with non-natural codes The complementary functional groups (including but not limited to azido and alkynyl) that exist in the amino acid that specifically react. It is worth noting that the other end of the PEG shown by Y in the above formula will be directly or indirectly attached to the IL-2 polypeptide through a naturally occurring or non-naturally encoded amino acid. For example, Y may be an amide, urethane, or urea bond connected to the amine group of the polypeptide (including but not limited to the epsilon amine group or the N-terminal amine group of lysine). Alternatively, Y may be a maleimine bond connected to a thiol group (including but not limited to the thiol group of cysteine). Alternatively, Y can be a bond to residues that are not normally accessible to the 20 common amino acids. For example, the azide group on the PEG can react with the alkynyl group on the IL-2 polypeptide to form a Huisgen [3+2] cycloaddition product. Alternatively, the alkynyl group on the PEG can react with the azide group present in the non-naturally encoded amino acid to form a similar product. In certain embodiments, when applicable, strong nucleophiles (including but not limited to hydrazine, hydrazine, hydroxylamine, aminourea) can react with aldehyde or ketone groups present in non-naturally encoded amino acids to form Hydrazone, oxime or semicarbazone, 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 through a non-naturally encoded amino acid and used to preferentially react with the ketone or aldehyde groups present in the water-soluble polymer.

For PEG, any molecular quality can be used according to actual needs, including but not limited to about 100 Daltons (Da) to 100,000 Da or greater (including but not limited to sometimes 0.1-50 kDa or 10-40 kDa). The molecular weight of the PEG can have a wide range, including but not limited to between about 100 Da to about 100,000 Da or greater. 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, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da , 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da and 100 Da. In certain embodiments, PEG is between about 100 Da and about 50,000 Da. In certain embodiments, PEG is between about 100 Da and about 40,000 Da. In certain embodiments, PEG is between about 1,000 Da and about 40,000 Da. In certain embodiments, PEG is between about 5,000 Da and about 40,000 Da. In certain embodiments, PEG is between about 10,000 Da and about 40,000 Da. Branched chain PEGs can also be used, including but not limited to PEG molecules having a MW in the range of 1-100 kDa (including but not limited to 1-50 kDa or 5-20 kDa) per chain. The molecular weight of each chain of the branched PEG may include, but is not limited to, between about 1,000 Da to about 100,000 Da or greater. The molecular weight of each chain of the branched PEG may 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, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In certain embodiments, the molecular weight of each chain of the branched chain PEG is between about 1,000 Da and about 50,000 Da. In certain embodiments, the molecular weight of each chain of the branched PEG is between about 1,000 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 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 Shearwater Polymers, Inc. catalog, Nektar Therapeutics catalog, which catalogs are incorporated into the present invention by reference.

Generally, at least one end of the PEG molecule can be used to react with the non-naturally encoded amino acid. For example, PEG derivatives that have alkyne and azide components and are used to react with amino acid side chains can be used to attach PEG to the non-naturally encoded amino acids described in the present invention. If the non-naturally encoded amino acid contains an azide group, the PEG will usually contain an alkynyl moiety to perform the formation of a [3+2] cycloaddition product, or an activated PEG containing a phosphine group Substances (ie esters, carbonates) to perform the formation of amide bonds. Optionally, if the non-naturally encoded amino acid contains an alkyne, the PEG will usually contain an azide moiety to perform the formation of the [3+2] Huisgen cycloaddition product. If the non-naturally encoded amino acid contains a carbonyl group, the PEG will usually contain a strong nucleophile (including but not limited to hydrazine, hydrazine, hydroxylamine or semicarbazide functional groups) in order to perform the corresponding hydrazone, oxime, respectively And the formation of a semi-carba hydrazone bond. In another alternative manner, the above-mentioned reversal of the direction of the reactive group can be used, that is, the azide component of the non-naturally encoded amino acid can react with the PEG derivative containing an alkyne.

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 azide and acetylene, which comprise a water-soluble polymer backbone with an average molecular weight of about 800 Da to about 100,000 Da. The polymer backbone of the water-soluble polymer may be polyethylene glycol. However, it should be understood that a wide variety of water-soluble polymers, including but not limited to polyethylene glycol and other related polymers, including polydextrose and polypropylene glycol, are also suitable for the practice of the present invention, and the term PEG or polyethylene glycol The use is intended to encompass and include all such molecules. The term PEG includes, but is not limited to, polyethylene glycol in any form, including bifunctional PEG, multi-arm PEG, derivatized PEG, bifurcated PEG, branched PEG, pendant PEG (that is, having one or more pendants from the polymer backbone). A functional group of PEG or related polymers) or a PEG with a degradable bond therein.

PEG is usually 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 to be biocompatible, which means that PEG can coexist with living tissues or organisms without causing damage. More specifically, PEG is basically 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. PEG having the formula --CH2CH2O--(CH2CH2O)n--CH2CH2-- (where n is about 3 to about 4000, usually about 20 to about 2000) is suitable for use in the present invention. In certain embodiments of the present invention, PEG having a molecular weight of about 800 Da to about 100,000 Da is particularly useful as a polymer backbone. 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 higher. 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, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In certain embodiments, the molecular weight of PEG is between about 100 Da and about 50,000 Da. In certain embodiments, the molecular weight of PEG is between about 100 Da and about 40,000 Da. In certain embodiments, the molecular weight of PEG is between about 1,000 Da and about 40,000 Da. In certain embodiments, the molecular weight of PEG is between about 5,000 Da and about 40,000 Da. In certain embodiments, the molecular weight of PEG is between about 10,000 Da and about 40,000 Da.

The polymer backbone can be linear or branched. Branched polymer backbones are generally known in the art. Generally, a branched polymer has a central branched core component and a plurality of linear polymer chains connected to the central branched core. PEG is often used in a branched form, which can be prepared by adding ethylene oxide to various polyols such as glycerol, glycerol oligomers, pentaerythritol, and sorbitol. The central branch component can also be derived from several amino acids such as lysine. The branched polyethylene glycol can be represented in a general form as R(-PEG-OH)m, where R is derived from a core component such as glycerol, glycerol oligomer or pentaerythritol, and m represents the number of arms. Multi-arm PEG molecules such as those described in U.S. Patent Nos. 5,932,462, 5,643,575, 5,229,490, 4,289,872, U.S. Patent Applications 2003/0143596, WO 96/21469, and WO 93/21259, each of which is incorporated by reference in its entirety, may also be used. Used as the polymer backbone.

The branched PEG can also take the form of a forked PEG represented by PEG(--YCHZ2)n, where Y is a linking group and Z is an activated terminal group, which is connected to CH through a chain of atoms of a certain length.

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

In addition to these forms of PEG, the polymers can also be prepared with weak or degradable bonds in the backbone. For example, PEG can be prepared to have ester bonds in the polymer backbone that are easily hydrolyzed. As shown below, this hydrolysis causes the polymer to be cleaved into lower molecular weight fragments:

-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 with 2 to about 300 ends 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 and so on. Although the molecular weight of each chain of the polymer backbone is variable, it is usually in the range of about 800 Da to about 100,000 Da, and usually about 6,000 Da to about 80,000 Da. The molecular weight of each chain of the polymer backbone 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, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In certain embodiments, the molecular weight of each chain of the polymer backbone is between about 100 Da and about 50,000 Da. In certain embodiments, the molecular weight of each chain of the polymer backbone is between about 100 Da and about 40,000 Da. In certain embodiments, the molecular weight of each chain of the polymer backbone is between about 1,000 Da and about 40,000 Da. In certain embodiments, the molecular weight of each chain of the polymer backbone is between about 5,000 Da and about 40,000 Da. In certain 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 frameworks is by no means exhaustive, but merely illustrative, and all polymeric materials having the aforementioned 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 possibly up to about 300 ends are 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

in:

N=N=N is the azide group component;

B is a connected component, which can exist or not exist;

POLY is a water-soluble non-antigenic polymer;

A is a connected component, which may or may not be present and may be the same or different from B; and

X is the second functional group.

Examples of linking components A and B include, but are not limited to, polyfunctionalized alkyl groups, which contain 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 branch at heteroatoms. Other examples of linking components A and B include, but are not limited to, polyfunctionalized aryl groups, which contain 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 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 all connection components having the above properties are considered suitable for use in the present invention.

Examples of functional groups suitable for use as X include, but are not limited to, hydroxyl groups, protected hydroxyl groups, alkoxy groups, active esters such as N-hydroxysuccinimidyl ester and 1-benzotriazolyl ester, active carbonates such as N- Hydroxysuccinimidyl carbonate and 1-benzotriazole carbonate, acetals, aldehydes, aldehyde hydrates, alkenyl groups, acrylates, methacrylates, acrylamides, activated moss, amines, amine oxides Group, protected amine, hydrazine, protected hydrazine, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimine, vinyl sulfide, dithiopyridine, Vinylpyridine, iodoacetamide, epoxides, glyoxal, diketones, mesylate, tosylate, tribenzoate, alkenes, ketones, and azides. As understood by those of ordinary skill in the art, the selected X component should be compatible with the azido group so that no reaction with the azido group occurs. The azido-containing polymer derivative may be homobifunctional, meaning that the second functional group (ie X) is also a component of the azido group, or heterobifunctional, meaning that the second functional group Are different functional groups.

The term "protected" refers to the presence of protective groups or components that prevent chemically reactive functional groups from reacting under certain reaction conditions. The protecting group varies according to the type of chemically reactive group to be protected. For example, if the chemically reactive group is an amine or hydrazine, the protecting group may be selected from tert-butoxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). If the chemically reactive group is a thiol, the protecting group may be an 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 methyl, ethyl or tert-butyl. Other protecting groups known in the art can 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, for example, U.S. Patent Nos. 5,281,698, 5,468,478), amines (see, for example, Buckmann et al., Makromol. Chem. 182:1379 (1981), Zalipsky et al., Eur. Polym. J. 19:1177 (1983)), hydrazine (see, for example, Andresz et al., Makromol. Chem. 179:301 (1978)), succinimidyl propionate and succinimidyl Butyrate (see, for example, Olson et al., Poly(ethylene glycol) Chemistry & Biological Applications), pp 170-181, edited by Harris & Zalipsky, ACS, Washington, DC, 1997 ; See also U.S. Patent No. 5,672,662), succinimidyl succinate (see, for example, Abuchowski et al., Cancer Biochem. Biophys. 7:175 (1984) and Joppich et al., Makromol. Chem. 180:1381 (1979)), Succinimidyl ester (see, e.g., U.S. Patent No. 4,670,417), benzotriazole carbonate (see, e.g., U.S. Patent No. 5,650,234), glycidyl ether (see, e.g., Pitha et al., Eur. J Biochem. 94: 11 (1979) ), Elling et al., Biotech. Appl. Biochem. 13:354 (1991)), oxycarbonylimidazole (see, for example, Beauchamp et al., Anal. Biochem. 131:25 (1983), Tondelli et al., J. Controlled Release 1:251 (1985)), p-nitrophenyl carbonate (see, for example, Veronese et al., Appl. Biochem. Biotech., 11: 141 (1985); and Sartore et al., Appl. Biochem. Biotech., 27:45 (1991)) , Aldehydes (see, for example, Harris et al., J. Polym. Sci. Chem. Ed. 22:341 (1984), U.S. Patent No. 5,824,784, U.S. Patent No. 5,252,714), maleimines (see, e.g., Goodson et al., Biotechnology (NY ) 8:343 (1990), Romani et al., Chemistry of Peptides a nd Proteins 2:29 (1984) and Kogan, Synthetic Comm. 22:2417 (1992)), o-pyridyl disulfides (see, for example, Woghiren et al., Bioconj. Chem. 4:314 (1993)), allyl alcohol (see For example, Sawhney et al., Macromolecules, 26:581 (1993)), vinyl chloride (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 includes a polymer backbone having the following structure:

X--CH2CH2O--(CH2CH2O)n--CH2CH2 --N=N=N

in:

X is a functional group as described above; and

n is about 20 to about 4000.

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

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

in:

W is an aliphatic or aromatic linker component containing between 1-10 carbon atoms;

n is 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, 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 leaving group The water-soluble polymer backbone reacts with the azido anion (which can be paired with any of a large number of suitable counterions (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, a polymer backbone suitable for use in the present invention has the formula X-PEG-L, where PEG is polyethylene glycol, X is a functional group that does not react with azide 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 hydrazine, protected thiol, carboxylic acid, protected carboxylic acid, horse Leximine, dithiopyridine and vinylpyridine and copper. Examples of suitable leaving groups include, but are not limited to, chlorine, bromine, iodine, mesylate, tribenzoate, and tosylate.

In another method for preparing the azido-containing polymer derivative of the present invention, the linking reagent with 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 reagent has a chemical functional group that selectively reacts with the chemical functional group on the PEG polymer to form an azide-containing group in which the azide group is separated from the polymer backbone by a linking group Polymer derivative products.

An exemplary reaction scheme is shown below:

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

in:

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 has no reactivity with the azide 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 hydrazine or an aminooxy component, M is a ketone; if N is a nucleophile , M is a leaving group.

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

A more specific example is shown below by taking PEG diamine as an example. One of the amine groups is protected by a protective group component such as tert-butyl-Boc, and the resulting mono-protected PEG diamine is combined with the one with azide functional group. Connection component reaction:

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

In this case, various activators such as sulfite chloride or carbodiimide reagents and N-hydroxysuccinimidyl or N-hydroxybenzotriazole can be used to conjugate the amine group to the carboxylic acid. An acid group to generate an amide bond between the monoamine PEG derivative and the linker component with an azide group. After the successful formation of the amide bond, the resulting N-tert-butyl-Boc-protected azido-containing derivative can be directly used to modify biologically active molecules, or it can be further elaborately designed to install other useful functional groups. For example, the N-t-Boc group can be hydrolyzed by treatment with a strong acid to produce ω-amino-PEG-azide. The resulting amine can be used as a synthetic handle to install other useful functional groups such as maleimide groups, activated disulfides, activated esters, etc., for the production of 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 molecules with activated electrophilic groups such as aldehydes, ketones, activated esters, activated carbonates, etc., to be attached to one end of the PEG, And attach a molecule with an acetylene group 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

in:

R can be H or alkyl, alkenyl, alkoxy or aryl or substituted aryl;

B is a connected component, which can exist or not exist;

POLY is a water-soluble non-antigenic polymer;

A is a connected component, which can be present or absent, and can be the same or different from B; and

X is the second functional group.

Examples of linking components A and B include, but are not limited to, polyfunctionalized alkyl groups, which contain 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 branch at heteroatoms. Other examples of linking components A and B include, but are not limited to, polyfunctionalized aryl groups, which contain 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 2003/0143596, each of which is incorporated herein by reference. Those of ordinary skill in the art will recognize that the foregoing list of connecting components is by no means exhaustive, but merely illustrative, and a wide variety of connecting components having the above-mentioned properties are considered useful in the present invention.

Examples of functional groups suitable for use as X include hydroxyl groups, protected hydroxyl groups, alkoxy groups, activated esters such as N-hydroxysuccinimidyl ester and 1-benzotriazolyl ester, activated carbonates such as N-hydroxysuccinyl ester Imino carbonate and 1-benzotriazole carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, activated ash, amine, aminooxy, protection Amine, hydrazine, protected hydrazine, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimide, vinyl pyridine, dithiopyridine, vinyl pyridine , Iodoacetamide, epoxide, glyoxal, diketone, methanesulfonate, tosylate, tribenzoate, alkene, ketone and acetylene. As will be understood, the selected X component should be compatible with the ethynyl group so that no reaction with the ethynyl group occurs. The acetylene-containing polymer derivative may be homobifunctional, meaning that the second functional group (ie X) is also an acetylene component, 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—CH ₂ CH ₂ O--(CH ₂ CH ₂ O) _n --CH ₂ CH ₂ – O-(CH ₂ ) _m -C≡CH

in:

X is a functional group as described above;

n is 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 in the present invention. In one method, a water-soluble polymer 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 The backbone reacts with a compound bearing both an acetylene functional group and a leaving group suitable for reacting with the nucleophilic group on the PEG. When the PEG polymer with a nucleophilic component is combined with the molecule with a leaving group, the leaving group undergoes nucleophilic substitution and is replaced by the nucleophilic component to obtain the The required acetylene-containing polymer.

X-PEG-Nu + L-A-C → X-PEG-Nu-A-C≡CR’

As shown, the preferred polymer backbone for the reaction has the formula X-PEG-Nu, where PEG is polyethylene glycol, Nu is a nucleophilic component, and X is not reactive with Nu, L or acetylene functional groups Functional group.

Examples of Nu include, but are not limited to, amines, alkoxy groups, aryloxy groups, sulfhydryl groups, imino groups, carboxylic acids, hydrazine, and amineoxy groups that react mainly through SN2-type mechanisms. Other examples of Nu groups include functional groups that react mainly through nucleophilic addition reactions. Examples of L groups include chlorine, bromine, iodine, mesylate, tribenzoate, and tosylate and other groups expected to undergo nucleophilic displacement, as well as ketones, aldehydes, thioesters, olefins, α- Beta-unsaturated carbonyls, carbonates, and other electrophilic groups that are expected to undergo addition through 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 an azide group, and L is a suitable leaving group.

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

An exemplary reaction scheme is shown below:

X-PEG-L + -C≡CR’ → X-PEG-C≡CR’

in:

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 have sufficient reactivity to undergo SN2 type displacement when in contact with a sufficient concentration of acetylene anion. The reaction conditions required to achieve the SN2 displacement of the leaving group by the acetylene anion are known to those of ordinary skill in the art.

The purification of the crude product can usually be completed by methods known in the art, including but not limited to precipitation of the product, followed by chromatography if necessary.

A water-soluble polymer can be linked to the IL-2 polypeptide of the present invention. The water-soluble polymer can be incorporated into the IL-2 polypeptide by the non-naturally encoded amino acid, or any functional group or substituent of the non-naturally encoded or naturally encoded amino acid, or added to the non-naturally encoded amino acid. Any functional group or substituent of the encoded or naturally encoded amino acid is attached. Optionally, the water-soluble polymer is connected to an amine incorporating a non-naturally encoded amino acid through a naturally-occurring amino acid (including but not limited to cysteine, lysine, or the amine group of the N-terminal residue). IL-2 polypeptide based on acid. In some cases, the IL-2 polypeptide of the present invention contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-natural amino acids, of which one or more non-naturally encoded amines The base acid is attached to a water-soluble polymer (including but not limited to PEG and/or oligosaccharide). 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 amines linked to a water-soluble polymer. Base acid. 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 certain embodiments, the water-soluble polymer used in the present invention increases the serum half-life of the IL-2 polypeptide relative to the unconjugated form.

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

Contains strong nucleophilic groups ( Namely hydrazine, hydrazine, hydroxylamine or aminourea ) of PEG derivative

In one embodiment of the present invention, IL-2 polypeptides containing carbonyl-containing non-naturally encoded amino acids are derivatized with PEG containing terminal hydrazine, hydroxylamine, hydrazine or semicarbazide components directly linked to the PEG backbone物modification.

In certain embodiments, the hydroxylamine-terminal 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 (that is, the average molecular weight is between 5-40 kDa).

In some embodiments, the PEG derivative containing hydrazine or hydrazine 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) that may or may not be present.

In certain embodiments, the aminourea-containing PEG derivative 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 present invention, the IL-2 polypeptide containing a carbonyl amino acid is used as a PEG derivative containing a terminal hydroxylamine, hydrazine, hydrazine or semicarbazide moiety linked to the PEG backbone by an amide bond Retouch.

In certain embodiments, the hydroxylamine-terminal 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 (that is, the average molecular weight is between 5-40 kDa).

In some embodiments, the PEG derivative containing hydrazine or hydrazine 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) that may or may not be present.

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

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

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, the IL-2 polypeptide comprising a carbonyl-containing amino acid is modified with a branched PEG derivative containing a terminal hydrazine, hydroxylamine, hydrazine or aminourea component, wherein the branched chain Each chain of PEG has 10-40 kDa and can have a MW in the range of 5-20 kDa.

In another embodiment of the present invention, the IL-2 polypeptide containing a non-naturally encoded amino acid is modified with a PEG derivative having a branched chain structure. For example, in certain embodiments, the PEG derivative at the end of the hydrazine or hydrazine 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 or may not be present.

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

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

Where R is a simple alkyl group (methyl, ethyl, propyl, etc.), X is optionally NH, O, S, C(O) or not present, 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 not present, m is 2-10, and n is 100-1,000.

The extent and position of the water-soluble polymer being attached to the IL-2 polypeptide can regulate the binding of the IL-2 polypeptide to the IL-2 receptor. In certain embodiments, the linkage is arranged such that the IL-2 polypeptide has a K _{d of} about 400 nM or less, a K d of 150 nM or less, _{and in some cases a K d} of 100 nM or less The lower K _d binds to the IL-2 receptor, which K _d is measured by a balanced binding assay, such as described in Spencer et al., J. Biol. Chem. , 263:7862-7867 (1988).

Methods and chemistry for activation of polymers and conjugation of peptides have been described in the literature and are known in the art. Commonly used methods for polymer activation include, but are not limited to, the use of cyanogen bromide, periodate, glutaraldehyde, diepoxide, epichlorohydrin, divinyl chloride, carbodiimide, sulfonate halogen, trichloro Triazine and other functional group activation (see RF Taylor, (1991), "Protein Immobilization: Fundamental and Applications" (PROTEIN IMMOBILISATION. FUNDAMENTAL AND APPLICATIONS), Marcel Dekker, NY; SS Wong, (1992), "Protein Conjugation and "CHEMISTRY OF PROTEIN CONJUGATION AND CROSSLINKING", CRC Press, Boca Raton; GT Hermanson et al., (1993), "IMMOBILIZED AFFINITY LIGAND TECHNIQUES", Academic Press, NY; Dunn, Editor-in-chief such as RL, "POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS", ACS Symposium Series Vol. 469, American Chemical Society, Washington, DC 1991).

Regarding the functionalization and conjugation of PEG, several reviews and leaflets are available. See, for example, Harris, M acromol. 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 Methods found in WO 93/15189 and used for conjugation between activated polymers and enzymes include but are not limited to coagulation factor VIII (WO 94/15625), hemoglobin (WO 94/09027), oxygen-carrying molecules (US Patent No. 4,412,989), ribonuclease and superoxide dismutase (Veronese et al., App. Biochem. Biotech . 11: 141-52 (1985)). All references and patents cited are incorporated into the present invention by reference.

The PEGylation (ie, the 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-terminal mPEG derivative. _{Briefly, at room temperature, an excess amount of solid mPEG(5000)-O-CH 2} -C≡CH is added to the aqueous solution of the IL-2 polypeptide containing p-azido-L-Phe under stirring. Typically, the pH of the aqueous solution during the reaction to be carried out with a pK _a close (generally about pH 4-10) buffered with a buffer. Examples of suitable buffers for PEGylation at, for example, pH 7.5 include, but are not limited to, HEPES, phosphate, borate, TRIS-HCl, EPPS, and TES. The pH is continuously monitored and adjusted if necessary. The reaction is generally allowed to continue between about 1-48 hours.

The reaction product was then subjected to hydrophobic interaction chromatography to combine the PEGylated IL-2 polypeptide variant with free mPEG(5000)-O-CH ₂ -C≡CH and the PEGylated IL-2 polypeptide Any high-molecular-weight complex of PEG is separated, and the complex may be formed when the unblocked PEG is activated at both ends of the molecule, thereby cross-linking the IL-2 polypeptide variant molecule. The conditions in the hydrophobic interaction chromatography process are such that free mPEG(5000)-O-CH ₂ -C≡CH flows through the column, and any cross-linked PEGylated IL-2 polypeptide variant complexes are in the desired form. Elution, the desired form contains an IL-2 polypeptide variant molecule conjugated to one or more PEG groups. Suitable conditions vary with the relative size of the cross-linked complex relative to the desired conjugate, and are easily determined by those of ordinary skill in the art. The eluent containing the desired conjugate is concentrated by ultrafiltration and desalted by diafiltration.

Using the elution method outlined above can produce substantially purified PEG-IL-2, wherein the produced PEG-IL-2 has 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% purity level, especially at least about 75%, 80%, 85% purity level, more particularly at least about 90% A purity level of at least about 95%, a purity level of at least about 99% or higher, as determined by suitable methods such as SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis. If necessary, 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 (including but not limited to DEAE SEPHAROSE), silica gel chromatography, reverse phase HPLC, gel filtration (including but not limited to SEPHADEX G-75), hydrophobic interaction chromatography, pore exclusion chromatography, metal Chelating agent chromatography, ultrafiltration/diafiltration, ethanol precipitation, ammonium sulfate precipitation, chromatographic focusing, displacement chromatography, electrophoresis programs (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 (Editor-in-Chief Harris & Angal), 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 polymer linked to the amino acid of the IL-2 polypeptide of the present invention can be further derivatized or substituted without limitation.

Containing azide group PEG derivative

In another embodiment of the present invention, the IL-2 polypeptide is modified with a PEG derivative containing an azide component that will react with the alkynyl component present on the side chain of the non-naturally encoded amino acid. Generally, 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-terminal 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 (that is, the average molecular weight is between 5-40 kDa).

In another embodiment, the azido-terminal 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 (that is, the average molecular weight is between 5-40 kDa).

In another embodiment of the present invention, the IL-2 polypeptide containing an alkynyl amino acid is modified with a branched PEG derivative containing a terminal azide component, and each chain of the branched PEG has 10 MW in the range of -40 kDa and can be in the range of 5-20 kDa. For example, in certain embodiments, the azido-terminal 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 N ₃

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 may or may not be present in each case.

Alkynyl PEG derivative

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

In certain embodiments, the alkynyl-terminal 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 (that is, the average molecular weight is between 5-40 kDa).

In another embodiment of the present invention, the IL-2 polypeptide containing alkynyl non-naturally encoded amino acid is used with a PEG containing a terminal azide or terminal alkynyl component linked to the PEG backbone by an alkamine bond. Derivative modification.

In certain embodiments, the alkynyl-terminal 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 present invention, the IL-2 polypeptide comprising an azido-containing amino acid is modified with a branched PEG derivative containing a terminal alkynyl moiety, and each chain of the branched PEG has 10 MW in the range of -40 kDa and can be in the range of 5-20 kDa. For example, in certain embodiments, the alkynyl-terminal 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 does not exist.

Phosphine PEG derivative

In another embodiment of the present invention, the IL-2 polypeptide contains activated functional groups (including but not limited to esters and carbonates) and further includes an azide group on the side chain of a non-naturally encoded amino acid. Modification of PEG derivatives of aryl phosphine groups that are part of the reaction. Generally, 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:

Where n is 1-10; X can be O, N, S or not present, Ph is phenyl, and W is a water-soluble polymer.

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

Where X can be O, N, S or not present, Ph is a phenyl group, W is a water-soluble polymer, and R can be H, an alkyl group, an aryl group, a substituted alkyl group, and a substituted aryl group. Exemplary R groups include but are not limited to -CH ₂ , -C(CH ₃ ) ₃ , -OR', -NR'R", -SR', -halogen, -C(O)R', -CONR'R", -S(O) ₂ R', -S(O) ₂ NR'R", -CN and -NO _2. R', R", R"' and R"" each independently refer to hydrogen, Substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (including but not limited to aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy , Or aralkyl. When, for example, the compound of the present invention includes more than one R group, each of the R groups is independently selected, when there is more than one R', R", R'" and R"" group When, the same is true for each of these groups. 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" means including but not limited to 1-pyrrolidinyl and 4-morpholinyl. From the discussion of the above substituents, those skilled in the art will understand that the term "alkyl" means including a bond Groups bonded to carbon atoms of groups other than hydrogen groups, such as haloalkyl groups (including but not limited to -CF ₃ and -CH ₂ CF ₃ ) and acyl groups (including but not limited to -C(O)CH _3. -C(O)CF ₃ , -C(O)CH ₂ OCH _3, etc.).

other PEG Derivatives and universal PEG Chemical technology

Other exemplary PEG molecules and PEGylation methods that can be linked to IL-2 polypeptides include, but are not limited to, those described in, for example, the following documents: 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, 2003/0105224, 2003/0023023, 2002/0156047, 2002/0099133, 2002/0086939, 2002/0082345, 2002 /0072573, 2002/0052430, 2002/0040076, 2002/0037949, 2002/0002250, 2001/0056171, 2001/0044526, 2001/0021763, US Patent No. 6,646,110, 5,824,778, 5,476,653, 5,219,564, 5,629,384, 5,736,625, 4,902,502, 5,281,698 5,122,614,5,473,034,5,516,673,5,382,657,6,552,167,6,610,281,6,515,100,6,461,603,6,436,386,6,214,966,5,990,237,5,900,461,5,739,208,5,672,662,5,446,090,5,808,096,5,612,460,5,324,844,5,252,714,6,420,339,6,201,072,6,451,346,6,306,821,5,559,213,5,747,646, 5,834,594, 5,849,860, 5,980,948, 6,004,573, 6,129,912, WO 97/32607, EP 229,108, EP 402,378, WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024 , WO 95/00162, WO 95/11924, WO95/13090, WO 95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO 99/32140, WO 96/4079 1. 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 into the present invention by reference. Any PEG molecule described in the present invention can be used in any form, including but not limited to single chain, branched chain, multiarm chain, monofunctional, bifunctional, multifunctional or any combination thereof.

Other polymers and PEG derivatives include, but are not limited to, hydroxylamine (aminooxy) PEG derivatives, which are described in the following patent applications, all of which are incorporated by reference into the present invention in their entirety: U.S. Patent Publication No. 2006/0194256, U.S. Patent Publication No. 2006/0217532, US Patent Publication No. 2006/0217289, US Provisional Patent No. 60/755,338, US Provisional Patent No. 60/755,711, US 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. IL-2 Polypeptide glycosylation

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

The sugar (including but not limited to glycosyl) components can be added to the IL-2 polypeptide in vivo or in vitro. In certain embodiments of the present invention, IL-2 polypeptides containing carbonyl-containing non-naturally encoded amino acids are modified with sugars derivatized with aminooxy groups to produce corresponding glycosylated polypeptides linked by oxime bonds . Once attached to the non-naturally encoded amino acid, the sugar can be further elaborated by treatment with glycosyltransferase and other enzymes to produce oligosaccharides that are bound to the IL-2 polypeptide. See, for example, H. Liu et al., J. Am. Chem. Soc. 125: 1702-1703 (2003).

In certain embodiments of the present invention, IL-2 polypeptides containing carbonyl-containing non-naturally encoded amino acids are directly modified with glycans with defined structures prepared as aminooxy derivatives. Those of ordinary skill in the art will recognize that other functional groups (including azides, alkynes, hydrazine, hydrazine, and aminoureas) can be used to link the sugar to the non-naturally encoded amino acid. In certain embodiments of the present invention, IL-2 polypeptides containing non-naturally encoded amino acids containing azide or alkynyl groups can then be used including but not limited to alkynyl or azido derivatives, respectively, by Including but not limited to Huisgen [3+2] cycloaddition reaction for modification. This method allows the modification of proteins with extremely high selectivity.

XI. IL-2 Dimer ( Dimers) And multimer (Multimers)

The present invention also provides a combination of IL-2 and IL-2 analogs such as homodimers, heterodimers, homomultimers or heteromultimers (ie trimers, tetramers, etc.), which contains one The IL-2 of or multiple 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 relative to the monomer IL-2 , Pharmacokinetics, pharmacodynamic properties, adjusted therapeutic half-life or adjusted plasma half-life. In certain embodiments, the IL-2 dimers of the invention modulate signal transduction of IL-2 receptors. 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 a dimer or multimer containing IL-2 comprise a non-naturally encoded amino acid linked to a water-soluble polymer. In certain embodiments, the IL-2 polypeptides are directly connected by including but not limited to Asn-Lys amide bond or Cys-Cys disulfide bond. In certain embodiments, the IL-2 polypeptide and/or the linked non-IL-2 molecule contains different non-naturally encoded amino acids to promote dimerization, including but not limited to the first IL-2 polypeptide The alkynyl group in one non-naturally encoded amino acid and the azide group in the second non-naturally encoded amino acid of the second molecule will be conjugated by Huisgen [3+2] cycloaddition. Alternatively, IL-2 containing a ketone-containing non-naturally encoded amino acid and/or a linked non-IL-2 molecule can be conjugated to a second polypeptide containing a hydroxylamine-containing non-naturally encoded amino acid, and The polypeptide reacts through the formation of the corresponding oxime.

Optionally, the two IL-2 polypeptides and/or the connected non-IL-2 molecules are connected by a linker. Any hetero- or homo-bifunctional linker may be used to connect the two molecules that may have the same or different primary sequence and/or the connected non-IL-2 molecules. In some cases, the linker used to tether the IL-2 and/or linked non-IL-2 molecules together may be a bifunctional PEG reagent. The linker can have a wide range of molecular weight or molecular length. A linker of larger or smaller molecular weight can be used to provide the requirements 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. Spatial relationship or conformation. Linkers with longer or shorter molecular lengths can also be used to provide the required space or flexibility between IL-2 and the attached entity or, if any, between the attached entity and its binding partner.

In certain embodiments, the present invention provides a water-soluble bifunctional linker with a dumbbell structure, which includes: a) an azide, alkyne, hydrazine, and hydrazide on at least the first end of the polymer backbone A component of hydrazine, hydroxylamine or carbonyl; and b) at least one second functional group on the 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 not reactive with the first functional group. In certain embodiments, the present invention provides water-soluble compounds comprising at least one arm of a branched molecular structure. For example, the branched molecular structure may be dendritic.

In certain embodiments, the present invention provides a multimer comprising one or more IL-2 polypeptides, which is 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 component containing azide, alkyne, hydrazine, hydrazine, aminooxy, hydroxylamine, acetyl or carbonyl, and R can be combined with X The same or different end-capping groups, functional groups or leaving groups. R can be, for example, selected from hydroxyl, protected hydroxyl, alkoxy, N-hydroxysuccinimidyl ester, 1-benzotriazolyl ester, N-hydroxysuccinimidyl carbonate, 1-benzo Triazolyl carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, activated sulfonium, amine, aminooxy, protected amine, hydrazine, protected hydrazine , Protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimide, vinyl sulfide, dithiopyridine, vinyl pyridine, iodoacetamide, epoxide, Functional groups for glyoxal, diketone, mesylate, tosylate, tribenzoate, alkene and ketone.

XII. IL-2 Peptide activity and IL-2 Peptide pair IL-2 Receptor affinity measurement

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, anti-proliferation assays, immunomodulatory assays, and monitoring the induction of MHC molecules The determination method.

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

The ability of IL-2 polypeptides to bind to IL-2 receptors can be analyzed. For unPEGylated or PEGylated IL-2 polypeptides containing 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 biological activity of the IL-2 polypeptide is determined. Generally, the test for biological activity should provide analysis of the desired results, such as increase or decrease in biological activity (compared to modified IL-2), different biological activity (compared to modified IL-2), Body or binding partner affinity analysis, conformational or structural changes of IL-2 itself or its receptor (compared to modified IL-2), or serum half-life analysis.

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

An important aspect of the present invention is to obtain an extended biological half-life by constructing an IL-2 polypeptide conjugated with or without a water-soluble polymer component. The rapid decline of IL-2 polypeptide serum concentration after administration makes it important to evaluate the biological response to treatments using conjugated and non-conjugated IL-2 polypeptides and their variants. 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, for example, subcutaneously or iv administration, so that measurement can be performed, for example, by ELISA method or by preliminary screening assay. become possible. ELISA or RIA kits from commercial sources such as Invitrogen (Carlsbad, CA) can be used. The measurement of the biological half-life in vivo is carried out as described in the present invention.

The potency and functional in vivo half-life of IL-2 polypeptides containing non-naturally encoded amino acids can be determined according to protocols known to those of ordinary skill in the art.

The pharmacokinetic parameters of IL-2 polypeptides containing non-naturally encoded amino acids can be evaluated in normal Sprague-Dawley male rats (N=5 animals per treatment group). The animal receives a single dose of 25 ug/rat iv or 50 ug/rat sc, and obtains about 5-7 blood samples according to a predetermined time course, which usually covers the unconjugated water-soluble polymer It takes about 6 hours for the IL-2 polypeptide containing non-naturally encoded amino acid to about 4 days for the IL-2 polypeptide containing non-naturally encoded amino acid and conjugated to a water-soluble polymer. The pharmacokinetic data of IL-2 without non-naturally encoded amino acids can be directly compared with the data obtained for IL-2 polypeptides containing non-naturally encoded amino acids.

XIV. Administration and pharmaceutical composition

The polypeptide or protein of the present invention (including but not limited to IL-2, synthetase, protein containing one or more unnatural amino acids, etc.) is optionally used for therapeutic purposes, including but not limited to a suitable pharmaceutical carrier Phase combination. These compositions include, 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 preparation is made to suit 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 may take a water-soluble form, for example as a pharmaceutically acceptable salt, which is meant to include 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 the dose. Specifically, the dosage can be initially compared with the activity, stability or other suitable measures of the non-natural polypeptides of the present invention and natural amino acid homologues (including but not limited to being modified to include one or more non-natural polypeptides). Comparison of amino acid IL-2 polypeptide with natural amino acid IL-2 polypeptide and comparison of IL-2 polypeptide modified to include one or more non-natural amino acid with currently available IL-2 therapy), That is, it is determined in the relevant measurement method.

Administration is carried out by any route commonly used to introduce molecules for eventual contact with blood or tissue cells. The non-natural amino acid polypeptides of the present invention are administered in any suitable manner, optionally together with one or more pharmaceutically acceptable carriers. In the context of the present invention, suitable methods of administering these polypeptides to patients are available, and although more than one route can be used to administer a particular composition, a particular route can generally provide a more immediate and more effective way than another route. Action or reaction.

The pharmaceutically acceptable carrier is determined in part by the specific composition to be administered and by the specific method of administration of the composition. Therefore, there are a wide variety of suitable formulations for the pharmaceutical composition of the present invention.

The IL-2 polypeptide of the present invention can be administered by any conventional route suitable for protein or peptide, including but not limited to parenteral, 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) administration. 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 pharmaceutical carriers. The IL-2 polypeptide can be used in combination with other pharmaceuticals or therapeutic agents.

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

Preparations suitable for parenteral administration, for example, by intra-articular, intravenous, intramuscular, intradermal, intraperitoneal and subcutaneous routes include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, inhibitors Bacteria and solutes that impart isotonicity with the blood of the target recipient to the formulation, and also include aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Formulations of IL-2 can be presented in single-dose or multi-dose sealed containers such as ampoules and vials.

Parenteral administration and intravenous administration are the preferred methods of administration. Specifically, it has been used for the administration route of natural amino acid homologue therapeutics (including but not limited to commonly used EPO, GH, G-CSF, GM-CSF, IFN such as IL-2, interleukin, antibody , FGF and/or any other drug-delivered protein administration route) and the formulations currently in use provide a preferred route of administration and formulation for the polypeptide of the present invention.

In the context of the present invention, the dose administered to the patient is sufficient to obtain a beneficial therapeutic response or other suitable activity in the patient over time, depending on the application. The dosage is determined by the efficacy of the specific carrier or preparation, 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 existence, nature and extent of any adverse side effects associated with the administration of a specific carrier, preparation, etc. in a specific patient.

In the future diseases (including but not limited to neutropenia, aplastic anemia, cyclic neutropenia, idiopathic neutropenia, Chdiak-Higashi syndrome, systemic lupus erythematosus (SLE), leukemia, myelodysplastic syndrome, myelofibrosis, etc.) In determining the effective amount of the carrier or preparation administered in the treatment or prevention, doctors evaluate the circulating plasma level, preparation toxicity, disease progression, and/ Or, when relevant, the production of antibodies against non-natural amino acid polypeptides.

The dose administered to a patient of, for example, 70 kg is usually within a range equivalent to the dose of a commonly used therapeutic protein, and is adjusted according to the altered activity or serum half-life of the relevant composition. The carrier or pharmaceutical preparation of the present invention can be administered by any known conventional therapies (including antibody administration, vaccine administration, cytotoxic agents, natural amino acid polypeptides, nucleic acids, nucleotide analogs, biological response modifiers, etc.). Medicine) to supplement the treatment condition.

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

If the patient undergoing the preparation infusion develops fever, chills or muscle pain, he/she receives a suitable dose of aspirin, ibuprofen, paraacetaminophen or other pain/fever control drugs. Patients who experience reactions to infusions such as fever, muscle pain, and chills are pre-administered with aspirin, acetaminophen, or including but not limited to diphenhydramine, 30 minutes before future infusions. Gutidine is used for more severe chills and muscle pain that do not respond quickly to antipyretics and antihistamines. Depending on the severity of the reaction, the cell infusion is slowed down or interrupted.

The human IL-2 polypeptide of the present invention can be directly administered to a mammalian object. Administration is by any route commonly used to introduce IL-2 polypeptides into an article. The IL-2 polypeptide composition according to the embodiments of the present invention includes suitable for oral, rectal, topical, inhalation (including but not limited to aerosol), buccal (including but not limited to sublingual), vagina, parenteral ( Including but not limited to subcutaneous, intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, intracerebral, intraarterial or intravenous), local (and both skin and mucosal surfaces, including airway surfaces), lungs, intraocular The composition for intranasal and transdermal administration, although the most suitable route in any given situation depends on the nature and severity of the condition to be treated. Administration can be local or systemic. The formulations of the compounds may be presented in single-dose or multi-dose sealed containers such as ampoules and vials. The IL-2 polypeptide of the present invention can be prepared as a mixture (including but not limited to solution, suspension or emulsion) with a pharmaceutically acceptable carrier in the form of unit dose injection. The IL-2 polypeptide of the present invention can also be administered by continuous infusion (using including but not limited to a small pump such as an osmotic pump), a single bolus injection or a slow release depot preparation.

Preparations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that impart isotonicity to the formulation, and may contain suspending agents, solubilizers, and thickeners. , Aqueous and non-aqueous sterile suspensions of stabilizers and preservatives. Solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.

Freeze drying is a commonly used technique for protein presentation, which is used to remove water from protein preparations 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. Excipients may be included in the preparation before lyophilization to improve the stability during the lyophilization process and/or to improve the stability of the lyophilized 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 drugs 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 transform a liquid pharmaceutical formulation into a fine, dust-free or agglomerated powder in a one-step process. The basic technology includes the following four steps: a) atomization of the feed solution into a spray; b) spray-air contact; c) drying of the spray; and d) separation of the dried product from the dry air. US 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 pharmaceutically acceptable carriers, excipients or stabilizers. 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. Accordingly, the pharmaceutical compositions of the invention is present in a wide variety of suitable formulations (including optional pharmaceutically acceptable carriers, excipients, or stabilizers) (see e.g. "Remington Pharmaceutical Science"(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 of less than about 10 residues; proteins, including but not limited to serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, including but not limited to polyvinylpyrrolidone; amines Base acids, including but not limited to glycine, glutamic acid, 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 edetate; divalent metal ions, including but not limited to Zinc, cobalt, or copper; sugar alcohols, including but not limited to mannitol or sorbitol; salt-forming counterions, including but not limited to sodium and sodium chloride; fillers such as microcrystalline cellulose, lactose, corn and others Starches; binders; sweeteners and other flavoring agents; 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™ and other pluronic acid (including but not limited to pluronic acid F68 (Poloxamer 188)) or PEG. Suitable surfactants include, for example, but not limited to, polyoxygenated Ethylene-polyoxypropylene-polyoxyethylene (PEO-PPO-PEO) or polyoxypropylene-polyoxyethylene-polyoxypropylene (PPO-PEO-PPO) polyether or a combination thereof. PEO-PPO-PEO and PPO-PEO-PPO is 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 its entirety and incorporated by reference. Invented in U.S. Patent No. 4,820,352. Other ethylene/polypropylene block polymers may be suitable surfactants. Surfactants or combinations of surfactants can be used to make PEGylated IL-2 against one or more stresses (including But not limited to stress caused by agitation) stability. Some of the above substances can be referred to as "extenders." Certain substances can also be referred to as "tonicity modifiers." Antimicrobial preservatives can also be used to obtain product stability. And antimicrobial effectiveness; suitable preservatives include but are not limited to benzyl alcohol, benzalkonium chloride, m-cresol, methyl/propyl paraben, cresol and phenol or combinations thereof. The present invention is incorporated by reference. US Patent No. 7,144,574 of US Patent No. 7,144,574 describes other materials that may be suitable for the pharmaceutical compositions and formulations of the present invention and other delivery formulations.

The IL-2 polypeptides of the present invention, including IL-2 polypeptides linked to a water-soluble polymer such as PEG, can also be administered through 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), polyanhydride, L-glutamic acid and γ- Ethyl-L-glutamic acid copolymer (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, polyvinylpyrrolidone, and silicones. Sustained release compositions also include liposome-encapsulated compounds. Liposomes containing compounds 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 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and EP 102,324. All references and patents cited are incorporated into the present invention by reference.

The liposome-encapsulated IL-2 polypeptide can be prepared by, for example, the method described in the following documents: 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 83-118008; U.S. Patent No. 4,485,045 and 4,544,545; and EP 102,324. The composition and size of liposomes are well known or can be easily determined by those of ordinary skill in the art empirically. Some examples of liposomes are described in, for example, the following documents: Park JW et al., Proc. Natl. Acad. Sci. USA 92:1327-1331 (1995); Lasic D and Papahadjopoulos D editors, "Medical Applications of Liposomes" ( MEDICAL APPLICATIONS OF LIPOSOMES (1998); Drummond DC, etc., Liposomal drug delivery systems for cancer therapy, edited by Teicher B, "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 into the present invention by reference.

In the context of the present invention, the dose administered to the patient should be sufficient to elicit a beneficial response in the article over time. Generally, the total therapeutically effective amount of the IL-2 polypeptide of the present invention administered parenterally per 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 the patient’s body weight per day Inside, although it depends on treatment judgment. In the specific case of this embodiment, the conjugate may be administered at a dose in the range of greater than 4 μ/kg per day to about 20 μg/kg per day. In other cases, the conjugate may be administered at a dose ranging from greater than 4 μg/kg per day to about 9 μg/kg per day. In other cases, the conjugate may be administered at a dose ranging from about 4 μg/kg per day to about 12.5 μg/kg per day. In certain cases, the conjugate may be administered at a dose equal to or lower than the highest dose tolerated without excessive toxicity. In addition, the conjugate may be administered at least twice a week, or the conjugate may be administered at least three times a week, at least four times a week, at least five times a week, at least six times a week, or seven times a week . In certain cases, where the conjugate is administered more than once, the conjugate may 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 some cases, the growth of cells expressing the interleukin-2 receptor can be inhibited by at least 50%, at least 65% compared to a reference sample, that is, a sample of cells that are not in contact with the conjugate of the present invention. , At least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. In the specific case of this example, the conjugate may be 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 per day. /kg dose administration. The frequency of dosing also depends on therapeutic judgments, and can be more frequent 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 by any of the above-mentioned administration routes.

XV. Of the invention IL-2 Therapeutic use of peptides

The IL-2 polypeptides of the present invention can be used to treat a wide range of disorders. The present invention also includes treatment methods for mammals who are at risk, are currently suffering from, and/or have suffered from cancers that are responsive to IL-2, CD8+ T-cell stimulation and/or IL-2 preparations. The administration of IL-2 polypeptide can produce short-term effects on several clinical parameters observed, that is, immediate beneficial effects, which can be 12 or 24 hours from the administration, and on the other hand, may also produce long-term effects, which are beneficial. To slow down the progression of tumor growth, reduce tumor size and/or increase the level of circulatory CD8+ T cells, and the IL-2 polypeptide 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, is administered by arterial, intraperitoneal or intravenous injection and/or infusion at a dose sufficient to obtain the desired pharmacological effect.

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

The average amount of IL-2 can be changed, and specifically should be based on the recommendations and prescriptions of qualified physicians. The exact amount of IL-2 is a matter of preference and depends on factors such as the exact type of condition to be treated, the condition of the patient to be treated, and other ingredients in the composition. The present invention also provides the administration of another active agent in a therapeutically effective amount. The amount to be administered can be easily determined by a person of ordinary skill in the art according to the therapy using IL-2.

Example

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

Example 1-Determination of residue positions in IL-2 to be mutated into an amber stop codon to incorporate non-natural amino acids

IL-2 has been used to treat several cancers such as renal cell carcinoma and metastatic melanoma. The commercially available IL-2 Aldesleukin® is a recombinant protein that is not glycosylated, has removed alanine-1 and replaced the residue cysteine-125 with serine-125 (Whittington et al., Drugs, 46(3): pp: 446-514 (1993)). Although IL-2 is the first cytokine approved by the FDA in cancer treatment, IL-2 has been shown to exhibit serious side effects when used in high doses. This greatly limits its application to 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. Generally, 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 not only differ from these receptors. Trimeric complexes, and can form heterodimeric complexes with IL-2Rβ and IL-2Rγ. In a clinical setting, when high doses of IL-2 are used, IL-2 begins to bind IL-2αβγ, which is the main receptor form _{in T reg cells.} The inhibitory effect of T _reg cells causes the unwanted effects of the application 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 main breakthroughs comes from the invention of Nektar, which uses 6 PEGylated lysine acids 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 a prolonged half-life, but also shows drastically reduced side effects. However, the results from the activity study show that the effective form of PEGylated IL-2 in this heterogeneous 6-PEGylated IL-2 mixture is only the mono-PEGylated form. Therefore, there is a need for 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 instead of the heterogenous 6-PEGylated IL-2 previously derived from Nektar.

The site used in the production of IL-2 mutant protein can be selected by analyzing the existing crystal structure 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 including a hydrophobic center and a polarization zone. The IL-2Rα center hydrophobic residues ^{Leu-2 α, Met-25} α, Leu-42 α and ^Tyr-43 α and IL-2 residues ^{Phe-42 IL-2, Phe} -44 IL-2, Tyr -45 ^IL-2 , Pro-65 ^IL-2 and Leu-72 ^IL-2 are formed. The polarization zone is in the 5 ion pairs of IL-2Rα and IL-2 (including Lys- ^38α /Glu-61 ^IL-2 , Arg- ^36α /Glu-62 ^IL-2 , Glu- ^1α /Lys -35 ^IL-2 , Asp-6 ^α /Arg-38 ^IL-2 and Glu-29 ^α /Lys-43 ^IL-2 ). In addition, electrostatic mapping indicates 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 , Thr-41 ^IL-2 , Lys-64 ^IL-2 , Glu-68 ^IL-2 and Tyr-107 ^IL-2 . Therefore, 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 . The list of protein sequences used to produce mutant proteins with unnatural amino acids is listed in Table 2 below:

Table 2. IL-2 protein sequence with potential sites used in PEGylation

*U: Unnatural amino acid

Example 2: Human IL-2 expression system

This section describes the expression methods for IL-2 polypeptides containing non-natural amino acids. Orthogonal tRNA, orthogonal aminyl tRNA synthetase and a polynucleotide encoding an IL-2 polypeptide as shown in SEQ ID NO: 4, 6 or 8 containing a selector codon or a polynucleotide encoding SEQ ID are used for the host cell NO: 1,2,3,5,7, and 9 to 23 amino acid sequences shown in the construct transformation of polynucleotides.

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

Gibson assembly : Primers used to amplify various genes of interest (GOI) containing donor fragments have about 18-24 base pairs (bp) at their 5'-ends and acceptor vectors Sequences with overlapping sequences were used for homologous recombination and synthesized at Integrated DNA Technologies (IDT) (San Diego, CA). The PCR fragment was amplified using the high-fidelity DNA polymerase mix Pfu Ultra II Hotstart PCR master mix (catalog number 600852, Agilent Technologies). The PCR product was digested with Dpn1 restriction enzyme (NEB# R0176L) at 37°C for 2 hours to remove the plastid background, then the Qiagen PCR Column Purification Kit (Qiagen, Valencia, CA; # 28104) was used for column purification and passed through Nanodrop (ThermoFisher, Carlsbad, CA) quantification. The acceptor vector is linearized by digesting with unique restriction enzymes (NEB, MA) in the vector at the temperature recommended by the supplier for 3 to 5 hours, and then purified and quantified by a PCR column. The donor insert and the acceptor vector suitable for preparation were mixed at a molar ratio of 3:1, and incubated at 50°C for 15 min using Gibson assembly kit (NEB # E2611S), and then used to transform into E. coli NEB5α strain (NEB # 2987).

Recombinants were recovered by plating the Gibson assembly mixture on LB agar plates containing appropriate antibiotics. On the second day, 4 to 6 well-separated 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 . The recombinant plastids were isolated using the Qiagen plastid DNA mini preparation kit (Qiagen #27104) and verified by DNA sequencing (Eton Biosciences, San Diego, CA). The complete GOI region plus 100 bp upstream and 100 bp downstream sequences were verified using gene-specific sequencing primers.

QuickChange Mutagenesis (QuickChange Mutagenesis , QCM) : All amber variants containing a TAG stop codon were produced using the QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies # 201519). All QCM oligonucleotides were designed using QuickChange Web Portal (Agilent Technologies) and ordered from IDT (San Diego, CA). The QCM PCR mix contains 5 µl 10x buffer, 2.5 µl dNTP mix, 1 µl (100 ng) plastid template, 1 µl oligomer mix (10 uM each), 1 µl QuickChange Lightning enzyme, 2.5 µl Quick solution and 37 µl of distilled water (DW). The DNA was amplified 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 supplied with the kit (Agilent Technologies) at 37°C for 2-3 hours, and run on a gel to check the presence of the amplified PCR product. Subsequently, 2.5 to 5 µl of PCR product was transformed into E. coli NEB5α strain. Recombinant plastids from 4 to 6 colonies were then isolated, and sequence verification was performed as described in the Gibson assembly section above.

Expression strain (AXID) Construction and validation (construction and verification): To prepare AXID production strain, the E. coli host cells W3110B60 sequence verified using chemically competent (chemically competent) plasmid DNA (50 ng) transformation, cells containing the recombinant Select 50 µg/mL kanamycin sulfate (Sigma, cat# K0254) on 2xYT+1% glucose agar plates and incubate overnight at 37°C. Then, a single colony from the freshly transformed plate was multiplied three times on a 2xYT+1% glucose agar plate containing 50 µg/mL kanamycin sulfate and streaked three times and incubated at 37°C overnight. Finally, a single colony from the third streaking plate was inoculated into 20 mL Super Broth (Fisher-Optigrow ^TM , #BP1432-10B1) containing 50 µg/mL kanamycin sulfate (Sigma, cat# K0254) And incubate 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 divided into 1 mL aliquots, placed in several cryovials and frozen at -80°C, as AXID production strain tubes.

After the glycerol tubes of the AXID production strain were produced as described above, they were further verified by DNA sequencing and phenotypic characterization of antibiotic resistance markers. In order to confirm that the AXID production strain tube has the correct plastid in the production host, sequence verification of the plastid is performed. 20 mL LB containing 50 µg/mL kanamycin sulfate was inoculated with a puncture from a glycerol tube cloned from AXID and grown overnight at 37°C and 250 rpm. The plastid DNA was isolated using Qiagen Mini Preparation Kit (#27104), and DNA sequencing (Eton Biosciences, CA) was used to confirm the presence of intact GOI ORF in the isolated plastids.

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

Expression system: The amino acid sequence of hIL-2 and the codon-optimized DNA sequence of E. coli encoding hIL-2 are shown in Tables 1 and 2. The translation system containing the introduction of orthogonal tRNA (O-tRNA) and orthogonal amino acid tRNA synthetase (O-RS) was used to express hIL-2 containing non-naturally encoded amino acids (see pKG0269 ;figure 2). The O-RS preferentially uses non-naturally encoded amino acids to aminate the O-tRNA. Furthermore, the translation system responds to the encoded selector codon by inserting the non-naturally encoded amino acid into the IL-2 or IL-2 variant. Suitable O-RS and O-tRNA sequences are described in WO2006/068802 entitled "Compositions of Aminoacyl-tRNA Synthetase and Uses Thereof" and "Combination of tRNA""Compositions of tRNA and Uses Thereof" (Compositions of tRNA and Uses Thereof) in WO2007/021297, the patent application is incorporated into the present invention by reference in its entirety.

Transform E. coli with a plastid containing the modified IL-2 variant polynucleotide sequence and an orthogonal amino acid tRNA synthetase/tRNA pair (specific to the desired non-naturally encoded amino acid), allowing the Non-naturally encoded amino acids are site-specifically incorporated into the IL-2 polypeptide. The expression of IL-2 variant polypeptides is under the control of the T7 promoter and is induced by adding arabinose to the culture medium (see the plastid map pKG0269; Figure 2).

Using the inhibition of acetyl- phenylalanine (pAF) : transform the plastids used to express IL-2 polypeptide into W3110B60 E. coli cells. Add p-acetyl-phenylalanine (pAF) to the cells, and induce protein expression by adding arabinose. Perform SDS-PAGE analysis of the expression of IL-2 polypeptide, and observe the IL-2 polypeptide. Multiple lanes were run for comparison between the original wild-type IL-2 polypeptide and the IL-2 polypeptide replaced by pAF, the latter being made at specific amino acid residues such as p-acetanilide replacement IL-2. The expression of T7 polymerase is under the control of the arabinose-inducible T7 phage promoter. Add p-acetyl-phenylalanine (pAF) to the cells and induce protein expression by adding arabinose (0.2% final). The culture was incubated at 37°C for several hours (3-5 hours).

HIL-2 for enhancing the expression of other constructs in E. coli: In order to improve the production of hIL-2 in E. coli, in addition to the present invention in E. coli are reported in addition to the password based on the DNA sequence of the sub-optimized usage, The following expression parameters can be further optimized: test different promoters other than the T7 phage promoter, such as arabinose B (araB), pTrc and phage T5 promoter; stabilization of hIL-2 mRNA; in addition to the standard W3110B60 strain Screening of different E. coli host strains outside; optimization of production process parameters such as temperature, medium, inducer concentration, etc.; optimization of transcription and translation control elements, such as start and stop codons, ribosome binding site (RBS), transcription termination Sub, etc.; optimization of plastid copy number and plastid stability; optimization of translation initiation region (TIR).

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

Shake flask expression assays (Shake-flask expression testing): AXID producing strain using the above test hIL-2 expression in shake flask experiments. Simply put, the inoculum from the AXID glycerol tube is placed in 5 mL Super Broth (Fisher-Optigrow ^TM , #BP1432-10B1) medium containing 50 μg/mL kanamycin sulfate (Sigma, MO), and in Grow overnight at 37°C with shaking. ^{The overnight culture was diluted 1:100 in Super Broth (Fisher-Optigrow TM} , #BP1432-10B1) medium containing 50 μg/mL kanamycin sulfate (Sigma, MO), and grown with shaking at 37°C . When the density of the culture 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 taken and analyzed by SDS-PAGE. The optimal expression of hIL-2 was standardized by changing temperature, induction duration and inducer concentration. According to the following Western assay for analyzing the expression of hIL2, immunoblotting of the crude extract using a standard clone antibody against hIL-2 confirmed the expression of hIL-2 (Figure 3A): The harvested cells The sediment was normalized to an OD600 of 5 and dissolved in a calculated amount of B-PER solution (ThermoFisher) containing lysozyme (100µg/ml) and DNase 1 (1U/ml). The sediment was mixed by vortexing at high speed for 2-5 minutes and by incubating the mixture at 37°C and 250 rpm. Mix the sample with the sample buffer (4X) and sample reducing agent (10x) provided by the manufacturer, and adjust the final concentration to 1x. A total of 20 µl samples were loaded on a precast polyacrylamide gel (ThermoFisher) together with hIL2 standards (R&D Systems, Minneapolis, MN), and separated by electrophoresis in 1x MES buffer (ThermoFisher). Use the iBlot device and gel transfer stack to transfer the protein sample to the nitrocellulose membrane. HIL2 was captured with goat anti-human IL-2 antigen antibody (R&D Systems), and HRP conjugated with an anti-goat IgG secondary antibody (R&D Systems) using opti 4CN colorimetric reaction substrate (Bio-Rad, Hercules) , CA) to detect.

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

The fermenter was batch-loaded with 4L of a chemically defined medium using glycerol as a carbon source. The seed culture was added to the fermentor to an initial OD600nm of 0.05. Using 480 to 1200 rpm stirring, 6 psig head pressure and 5 slpm air flow oxygen enrichment, the dissolved oxygen is maintained at 30% air saturation. The temperature and pH were controlled at 37°C and 7.0, respectively. When the culture reaches an OD600nm of 35 ± 5, start feeding at a rate of 0.25 mL/L/min. Therefore, L-Ala-pAcF (also known as L-Ala-pAF) dipeptide was added in an amount of 0.4 g/L. 15 minutes after the dipeptide addition, the culture was induced with L-arabinose at a final concentration of 2 g/L. The culture was harvested 6 h after induction.

Reverse phase HPLC titer (Titer) Analysis: First, 1.0 mL E. coli fermentation samples (cell paste) was dried and weighed to determine the quality of 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 at 22°C for 20 minutes. After incubation, the cell lysate was centrifuged at 16,050 rcf for 5 minutes to deposit cell debris. Then a 200 μL aliquot of the cell lysate supernatant was filtered through a 0.22 μm PVDF centrifuge tube screening program (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. A 4.6 x 150 mm Zorbax 300SB-C3 (Agilent #863973-909) reversed-phase column packed with 3.5 µm particles was used to separate hIL2 from host cell protein contaminants. Mobile phase A containing 0.1% trifluoroacetic acid in water was used to bind hIL2. Mobile phase B containing 0.1% trifluoroacetic acid in acetonitrile was used to elute hIL2 from the column. The amount of hIL2 in the sample was determined by comparing the observed area counts from a fixed injection volume against 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 range of about 65 to 150 mg/L in high cell density E. coli fermentation.

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

Comprising a body (Inclusion body) was prepared and dissolved: from a high cell density fermentation cell paste harvested at 4 ℃ of inclusion bodies (IB) Buffer I (50 mM Tris pH 8.0; 100 mM NaCl; 1 mM EDTA; 1% Triton X-100; 4℃) was mixed and resuspended to the final 10% solids. The cells were lysed by passing the resuspended material through the microfluidizer twice in total. The sample was then centrifuged (14,000 g; 15 minutes; 4°C) and the supernatant was decanted. The inclusion body sediment was washed by resuspension in another volume of IB buffer I (50 mM Tris pH 8.0; 100 mM NaCl; 1 mM EDTA; 1% Triton X-100; 4°C), and resuspended The suspended material passes through the microfluidizer twice in total. The sample was then centrifuged (14,000 g; 15 minutes; 4°C) and the supernatant was decanted. The inclusion body deposits were each resuspended in 1 volume of buffer II (50 mM Tris pH 8.0; 100 mM NaCl; 1 mM EDTA; 4°C). The sample was centrifuged (14,000 g; 15 minutes; 4°C) and the supernatant was decanted. The inclusion body sediment was resuspended in ½ volume of buffer II (50 mM Tris pH 8.0; 100 mM NaCl; 1 mM EDTA; 4°C). The inclusion bodies are then divided into aliquots in suitable containers. The sample was centrifuged (14,000 g; 15 minutes; 4°C) and the supernatant was decanted. The inclusion bodies were dissolved or stored at -80°C until further use.

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

Refolding and purification: Refolding was performed by diluting the sample in 20 mM Tris, pH 8.0; 60% sucrose; 4°C to a final concentration of 0.5 mg/mL. Allow refolding to proceed for 5 days at 4°C. The refolded material was diluted 1:1 with Milli-Q H _{2 O.} 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 flushing 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). The IL-2 polypeptide was eluted by washing the column with 10 column volumes of 100% buffer B.

Purification after PEGylation and PEGylation : Take the IL-2 pool and dilute it with Milli-Q water 10X. Adjust the pH of each sample to 4.0 with 50% glacial acetic acid. Concentrate the sample to ~1.0 mg/mL. A 1:12 molar excess of activated PEG (hydroxylamine PEG) was added to each sample. The samples were then incubated at 27°C for 48-72 hours. Take out the sample and dilute the sample 8-10 times with water (>8 m/S), and load it into SP HP equilibrated in buffer A (50 mM NaAc, pH 6.0; 50 mM NaCl; 0.05% Zwittergent 3-14) Column (GE Healthcare). The 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). The fractions of IL-2 were combined and run on a Superdex 200 pore 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 the process of PEGylation, Site Specific conjugation and PEG-IL-2 purification.

Preparation from E. coli inclusion body preparations: The IL-2 inclusion bodies are isolated through a series of washing steps. Resuspend the frozen cell paste in washing buffer I (50mM Tris, pH 8.0; 1% triton X-100; 1M urea, 5mM EDTA, 1mM PMSF) to a concentration of 10% (W/V) at 4°C Homogenize, then centrifuge (15,000g, 30 minutes, 4°C). The supernatant was discarded and the inclusion body sediment was resuspended in washing buffer II (50mM Tris, pH 8.0; 1% triton X-100; 1M urea, 5mM EDTA). The resuspended inclusion bodies were centrifuged at 15,000 g for 30 minutes at 4°C. The supernatant was discarded, and the inclusion body sediment was resuspended in washing 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 minutes at 4°C. The supernatant was discarded, and the inclusion body sediment was resuspended in water, and then centrifuged at 15,000 g for 30 minutes at 4°C. Store the washed inclusion bodies at -80°C until further use.

Refolding: Dissolve IL-2 inclusion bodies by resuspending in water and adjusting the pH of the mixture to pH 12.2. The insoluble material was removed by centrifugation (12,000 g, 15 minutes). The dissolved IL-2 is refolded by lowering the pH to pH 8.8±0.2. Promotes proper disulfide bond formation by adding 50 µM cystine to the refolding reaction. The refolding reaction is allowed to stand at room temperature for 16-22 hours. The 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 minutes), and the clear supernatant was adjusted to pH 7.8 with sodium hydroxide and filtered at 0.22 μm.

Column purification (Column Purification): The refolded IL-2 was loaded into the balance in Buffer A (20mM sodium phosphate, pH 7.8) Capto Adhere Impres ( GE Healthcare) column. After loading, the column was washed with buffer A (20mM sodium phosphate, pH 7.8) and used within 20 column volumes up to 100% buffer B (20mM sodium phosphate, 20mM citric acid, pH 4.0) linearity The pH gradient eluted IL-2 from the column. The fractions containing IL-2 were collected, adjusted to pH 4.0 with 10% acetic acid, and then buffer exchanged in 20 mM sodium acetate, 2.5% trehalose, pH 4.0. Concentrate IL-2 to 1-10 mg/mL, filter at 0.22 µM, and store at -80°C.

Purification of IL-2 from eukaryotic expression system : The 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). After loading, the column was washed with buffer A (20mM sodium phosphate, pH 7.8), and then washed with washing buffer B (20mM sodium phosphate, 1.0M sodium chloride, 30mM imidazole, pH 7.8) to remove the host Cell contaminants. The IL-2 was eluted from the column with an elution buffer (10 mM sodium phosphate, 300 mM imidazole, pH 7.8), and the fractions containing IL-2 were combined. The IL-2 pooled material was loaded on 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 (20mM sodium phosphate, pH 7.8) and used within 20 column volumes up to 100% buffer B (20mM sodium phosphate, 20mM citric acid, pH 4.0) linearity The pH gradient eluted IL-2 from the column. The fractions containing IL-2 were collected, adjusted to pH 4.0 with 10% acetic acid, and then buffer exchanged in 20 mM sodium acetate, 2.5% trehalose, pH 4.0. Concentrate IL-2 to 1-10 mg/mL, filter at 0.22 µM, and store at -80°C until further use.

Site-specific conjugate (Site Specific Conjugation) PEG-IL 2- purification: The non-natural amino acids containing (nnAA) of IL-2 variant of the acetyl group in the amphetamine conjugate acid buffer (20 mM acetic acid Sodium, pH 4.0) was buffer exchanged and concentrated to 1-10 mg/mL. A final 100 mM acethydrazine 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 20 mM sodium acetate, pH 5.0, and loaded on a Capto SP Impres column. After loading, the column was washed with buffer A (20mM sodium acetate, pH 5.0) and used in 20 column volumes up to 100% buffer B (20mM sodium acetate, 1.0M sodium chloride, pH 5.0) The linear gradient of PEGylated IL-2 eluted from the column. The 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. Concentrate IL-2 to 1-2 mg/mL, filter at 0.22 µM, and store at -80°C until further use.

The IL-2/CD-25 binding assay was performed by Bio-Layer Interferometery : The IL-2/CD25 multi-concentration binding kinetics experiment was performed on an Octet RED96 (PALL/ForteBio) instrument at 30°C. The anti-human Fc antibody capture biosensor (PALL/ForteBio, catalog number 18-5063) was loaded with purified CD25-Fc fusion protein in 1X HBS-P+ buffer (GE Healthcare, catalog number BR-1008-27) . Achieve an immobilization level between 0.8 nm and 1.0 nm. The loaded biosensor was washed with 1X HBS-P+ buffer to remove any unbound protein before measuring the binding and dissociation kinetics. For monitoring of the binding phase, the IL-2 analyte sample was diluted with 1X HBS-P+ buffer and transferred to a solid black 96-well plate (Greiner Bio-One, catalog number 655209). Allow the IL-2 sample to bind to the CD25-Fc loaded biosensor for 60 seconds. The dissociation phase was recorded in the wells of a solid black 96-well plate containing 1X HBS-P+ buffer for 90 seconds. Use the parallel buffer blank subtraction method to quote the data, align the baseline with the y-axis, and smooth it with the Savitzky-Golay screening program in the Octet data analysis software version 10.0 (PALL/ForteBio). The Langmuir model with description 1:1 combined with chemometrics was used to perform global fitting on the processed kinetic sensorgrams (Figure 4A).

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

Production of IL-2 variants in mammalian cells. Natural 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 it has been shown that non-glycosylated IL-2 has similar activity to glycosylated IL-2, glycosylated human IL-2 has been shown to have better clone growth and long-term reproduction of activated human T cells. Good activity. There are also some reports that natural IL-2 has higher specific activity. It has also been shown that the expression of IL-2 in mammalian cells has advantages over their expression in E. coli (Kim et al., J Microbiol Biotechnol, 14(4), 810-815 (2004)). In the present invention, wild-type IL-2 and various mutant proteins designed in Tables 1 and 2 above can be produced in CHO cells (as described in the Examples of the present invention).

In order to produce IL-2 mutant proteins containing unnatural amino acids at the desired location, each mutant protein is produced in a stable pool or a stable cell line derived from transfected platform cells Strains containing engineered orthogonal tRNA/tRNA synthetase pairs (Tian et al., Proc Natl Acad Sci USA, 111(5): pp: 1766-71 (2014) and PCT/2018US/035764: each of which is incorporated by reference in its entirety Incorporated into the present invention). In simple terms, CHOK1 cells are engineered into a platform cell line that stably expresses proprietary orthogonal tRNA synthetase (O-RS) and its homologous amber inhibitory tRNA (O-tRNA), for example in CHO cells Efficient incorporation of non-natural amino acids such as pAF into therapeutic proteins such as IL-2. The platform cell strain is then adapted to suspension growth in advance for rapid development in a bioreactor. The platform cell line has been fully characterized and evolved, with improved non-natural amino acid incorporation efficiency and clone selection efficiency. The platform cell line is used as a parent cell to produce treatments incorporating unnatural amino acids for early research at a titer higher than 100 mg/L through rapid and efficient transient expression Sex protein. Perform transient transfection and stable pool production to evaluate the expression of candidate molecules and provide materials for functional assays to identify lead molecules. The production cell line is 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 incorporating unnatural amino acids. The platform cell line is used as a parent cell to implement a stable cell line development strategy to obtain a production cell line with 5-10 PCD within 3-4 months and 20-30 PCD within 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 mutations. In contrast, during the production of IL-2 variants (Table 2), each of the 15 mutant proteins has a unique position to be mutated into an amber stop codon (TAG), which can be suppressed in engineered cells And expression to produce nnAA-containing proteins.

Establishment of engineered CHO cells for IL-2 variant expression. The engineered CHO cells are derived from the knockout of a previously established proprietary platform cell (PCT/2018US/035764, the entirety of which is incorporated into the present invention by reference). Simply put, CRISPy, a web-based target discovery tool, is used to quickly identify gRNA target sequences that have zero off-targets in CHO-K1 cells, preferably in early exons. The gRNA is cloned into the mammalian expression vector pGNCV that co-expresses the CHO codon-optimized form of Cas9. The production cell line was transfected with the protein expression vector to produce a cell pool, and then cloned to identify single cell isolates with gene knockout. The frequency of indels (insertions/deletions) from the combined results of multiple projects is 30-90% and 50-80% for cell pools and single-cell isolates, respectively. Use CRISPR to knock out target genes in CHO cells. Specifically, in order to improve the mRNA stability of IL-2, CRISPR technology was used to knock out the UPF1 gene. The gRNA used for knockout is shown in FIG. 5. After screening 192 clones, two UPF1-KO cell lines were obtained and confirmed by sequencing that they had UPF1 knockout (Figure 6). Then the obtained UPF1-KO cell line is used for transient expression of IL-2 variants.

Transient expression of IL-2 in engineered CHO cells . The IL-2 variant was transiently expressed in the UPF1-KO cell line obtained as disclosed in the above example. Using the Amaxa kit (Lonza) for suspension cells, electroporation was used for transfection. 6 ug of the plastids prepared as disclosed in the above example were transfected into 2 ^{×10 6} engineered CHO cells. After transfection, the cells were incubated (incubation) at 37°C for 4 days, and then the titer was analyzed by ELISA using a commercial kit from Invitrogen (Carlsbad, CA). As shown in Figures 7A and 7B, during the transient expression period, variant F42 showed the highest expression level among the 15 variants.

IL-2 變體在 CTLL-2 細胞中的 T 細胞擴增試驗 (expansion test) 。 使用來自於被轉染的工程化CHO細胞的暫態表達的F42變體上清液進行CTLL-2細胞擴增測定法。在所述細胞增殖測定法期間，將野生型IL-2用作100%增殖的對照(示出在圖8中)。在所述測定法中將變體F42製備成連續稀釋液，10 ng/mL、3.33 ng/mL、1.11 ng/mL、0.37 ng/mL、0.12 ng/mL和0.04 ng/mL。使用Cell Titer Glo(Promega, WI)來進行細胞增殖。在TECAN genios pro上讀取發光信號。正如在圖8中所示，F42顯示出0.24 ng/mL左右的EC₅₀ ，同時與其野生型對照相比保留95%的功能。用於研究本發明的IL-2變體的通用程式顯示如下：

T cell expansion test of IL-2 variants in CTLL-2 cells . The CTLL-2 cell expansion assay was performed using the transiently expressed F42 variant supernatant from the 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). In the assay, the variant F42 was prepared as serial dilutions of 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 Titer Glo (Promega, WI) was used for cell proliferation. Read the luminous signal on TECAN genios pro. As shown in Figure 8, F42 shows about 0.24 ng / mL EC _50, while compared to its wild type controls retain 95% of the function. The general formula 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, the screening included 16 initially selected sites (including wild-type) and 4 other sites known in the art ( K32, K48, K49, K76) 20 different IL-2 variants (Charych, D., etc., PLoS One, 12(7): p. e0179431, 2017). As shown in Figure 9 and Table 3, most of the variants retain their activity after mutagenesis. Because CTLL-2 cells have the property of residual IL-2Rα expression, the variants with the lowest binding to CTLL2 cells still show some inherent binding to IL-2Rα, although this binding is extremely low. For example, it was observed that the P65 IL-2 variant that exhibited the lowest binding to IL-2Rα exhibited a certain inherent biased binding with IL-2Rα. The identified variants were further analyzed for their binding ability after PEGylation.

Table 3. Activity of IL-2 variants using CTLL2 proliferation assay

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

The analysis of selected variants P65, Y45, E61, F42, K35, K49, and T37 was performed using the in vitro binding assay described in the above examples and the biolayer interferometer assay. Each variant is conjugated with 20K PEG at their specific position. The PEGylated variants were then analyzed by the BLI (Biological Layer 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 of the 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 blockade of more than 99%, Table 4.

Table 4. In vitro binding activity of IL-2 variants

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, which have been engineered to have complementary binding domains for enzymes, once the previously separated receptors are dimerized by the addition of IL-2 molecules. After being chemically activated, a chemiluminescence signal is generated (Figure 11). Two cell lines were produced 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 value of each variant (EC 50-} βγ/EC _50- αβγ) was used to estimate their relative retained binding capacity. As shown in Table 5, the best possible variants have a value of 1, meaning that 100% of their βγ binding capacity is retained, while α binding is 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 These two PEGylated variants will be the best candidates for further evaluation.

Table 5. Binding activity of IL-2 variants using dimerization assay-Experiment 1

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

Table 6. Improved binding activity of IL-2 variants using dimerization assay-Experiment 2

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

In order to further evaluate the in vitro function of the PEGylated variant, an ex vivo assay using PBMC was used. As shown in Figure 12, the binding of IL-2 to its receptor triggered an increase in the phosphorylation of STAT5 (pSTAT5). Therefore, the detection of pSTAT5 levels will be an indicator of the binding of IL-2 variants to the endogenous IL-2 receptor. Use selected PEGylated variants of human whole PBMC 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 (a variant P65 conjugated with linear 20K PEG), and then separated into two populations of CD8+ T cells and CD4+ Treg cells. As shown in Table 7, all three variants showed greatly improved activity for their retained βγ binding activity and blocked α binding activity. These results are further supported by the variants tested in another pSTAT5 assay as shown in Table 8. The results from this pSTAT5 assay (Table 8) show that many variants have drastically improved activities for their reduced ability to bind to Treg cells and relatively maintained binding to CD8+ cells. In Table 8, the calculated CD8+/Treg ratio is used to indicate the ranking of the variants so that the PathHunter assay results can be directly compared with the pSTAT5 assay results through a similar ranking system.

Table 7-Binding activity of IL-2 variants using in vitro assay-Experiment 1

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

Example 11- Comparison of clone growth and long-term reproduction of CTLL-2 cells in the presence of glycosylated IL-2 produced in CHO mammalian cells and non-glycosylated IL-2 produced in E. coli

It has been reported that native human IL-2 is a glycosylated protein with O-linked glycosylation on Thr-3 (Conradt et al., Eur J Biochem 153(2): 255-261 (1985)). Compared with non-glycosylated IL-2, this glycosylation function is associated with higher solubility at physiological pH, slower clearance in the body, and lower immunogenicity in cancer therapy (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, it has been shown that glycosylated IL-2 is superior to non-glycosylated IL-2 in promoting clone growth and long-term reproduction of human T cells activated by the same species (Pawelec et al., Immunobiology 174(1) : 67-75 (1987)), indicating that glycosylated IL-2 is a better choice in therapeutic applications.

In order to further analyze the biological functions of glycosylated IL-2 and non-glycosylated IL-2, experiments were performed to analyze the clone growth rate and long-term reproduction frequency of CTLL-2 cells (Figure 13). Place a single CTLL-2 cell on a 96-well plate (Thermo Fisher, Waltham, MA, CAT#A34180) with a pre-coated feeder cell layer of γ-irradiated CF1-MEF (mouse embryo fibroblast) cells middle. 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 and the number of colonies grown The percentage of colonies that survived at the end of the 19-day incubation was counted and analyzed. As shown in Figure 13 (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 0.5 nM IL-2 concentration, which is the optimal cell culture condition for CTLL-2 cell growth, glycosylated IL-2 promotes clone growth as much as non-glycosylated IL-2. Times higher. After long-term incubation (~19 days), the survival rate of colonies from glycosylated IL-2 treatment was 4 times higher than that of non-glycosylated IL-2 treatment. The data clearly proves that glycosylated IL-2 has superior activity in promoting the clone growth and long-term reproduction of IL-2 responsive cells, and further supports 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 art to increase 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, increasing the expression of proteins containing unnatural amino acids in industry is challenged by relatively low yields in mammalian cells. In order to solve this problem in the present invention, the proprietary technology disclosed in PCT/2018US/035764 (the entirety of which is incorporated into the present invention by reference) for improving the production of protein titer in eukaryotic cell lines is utilized. Technology to generate stable pooled cells of IL-2 and its variants, and use them in the resulting stable IL-2 cell lines.

To put it simply, it was found that different fifth-generation platform cell lines expressing Bax/Bak knockout dramatically increased the protein expression of IL-2 and increased the IL-2 protein production to about 40% higher than that of the parent cell line. In addition to inhibiting apoptosis in these cells by Bax/Bak knockout, UPF1 knockout was also found to further increase IL-2 expression.

Both wild-type IL-2 and its variants (F42, Y45 and P65) have been tested by generating stable pools of them. As shown in Figure 13, the three IL-2 variants F42, Y45 and P65, including the stable combination of wild-type IL-2, have a greatly improved expression level compared to the existing level in the art (see, for example, 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 for the F42 variant after generating the respective stable pools /L (shown in Figure 14). The data show that by generating a new CHO cell line with highly-incorporated non-natural amino acids, the production or yield of IL-2 protein can be improved or increased. It has also been shown that expression level and function are site-specifically related.

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

As disclosed in the present invention, the substitution of the non-naturally encoded amino acid is 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 increasing the affinity of the IL-2 for its receptor, and improving the drug stability of the IL-2 To enhance the tumor suppressor and/or tumor reduction activity of the IL-2, and improve the solubility of the IL-2 or variants (including but not limited to when expressed in E. coli or other host cells), Improve the solubility of the IL-2 after expression in E. coli or other recombinant host cells, increase the solubility of the polypeptide after expression in E. coli or other recombinant host cells, and regulate the IL-2 receptor and binding protein Or the affinity of related ligands, regulate signal transduction after binding to IL-2 receptor, regulate circulatory half-life, regulate release or bioavailability, promote purification, or improve or change specific routes of administration to increase IL-2 transformation The affinity of the body to its receptor increases the affinity of IL-2 variants to IL-2-Rβ and/or IL-2-Rγ.

Therefore, in order to improve the function of variant F42, a new variant with additional mutation R38A was prepared in CHO cells. As shown in Figure 15A, during the production of a stable cell line, the combination of the non-natural amino acid and the natural amino acid replacement in IL-2 variant F42 was used in the 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. 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 exhibited an EC50 of 15.9 nM, compared with F42 exhibited an EC50 of 3.6 nM, so the binding resistance The breaking efficiency is increased by more than 4 times (Figure 15B). Based on the 0.025 nM EC50 of wild-type IL-2, the binding and blocking efficiency exceeds 99.9%. In terms of high protein expression level and efficiency of blocking IL-2Rα binding, this variant shows great potential for therapeutic applications.

Table 9. CTLL-2 binding assay of PEGylated F42-R38A variants

The binding kinetics of F42pAF variants, R38A-F42pAF variants (including unnatural amino acids and point mutations), and F42-R38A-PEG20K-BR2 were evaluated by BLI to determine the effect of 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 seen in Table 10, IL-2-F42pAF has an IL-2Rα binding KD of 20 nM. After adding the R38A mutation, IL-2-F42-R38ApAF has an IL-2Rα binding KD of 233 nM, which corresponds to a 12-fold decrease in IL-2Rα binding. After IL-2-R38A-F42pAF was conjugated with 20K 2-branched PEG molecules, IL-2Rα binding was prevented. The results clearly demonstrated that the added mutation effectively blocked the binding of F42-R38A to its receptor IL-2Rα.

Table 10. Binding of IL-2 PEGylated variants with natural and non-natural amino acid substitutions

It should be understood that the embodiments and embodiments described in the present invention are for illustrative purposes only, and various modifications or changes made according to them will be suggested by those of ordinary skill in the art, and will be included in the spirit and scope of the present application and subsequent Within the scope of the attached patent application. All publications, patents, patent applications and/or other documents cited in this application are incorporated into the present invention by reference in their entirety for all purposes, to the same extent as each individual publication, patent, patent application and/or other documents It is individually indicated to be incorporated into the present invention by reference for all purposes.

The invention is further described by the following numbered examples.

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

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

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

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

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

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

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

10. The IL-2 of embodiment 1, wherein the non-naturally encoded amino acid is replaced at a position selected from the following residues: before position 1 (ie at the N-terminus), first and second , 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 carboxy terminus of the protein, and any combination thereof.

11. The IL-2 of embodiment 10, wherein compared with wild-type IL-2, the IL-2 comprises one or more amino acids that modulate the affinity of the IL-2 polypeptide for its IL2Rα receptor subunit Replacement, addition or deletion.

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 increase the expression of the IL-2 polypeptide in a recombinant host cell or synthesis in vitro.

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

15. The IL-2 of embodiment 10, wherein the non-naturally encoded amino acid has a linker, polymer, or biological activity that is originally non-reactive to any of the 20 common amino acids in the polypeptide The molecules are reactive.

16. The IL-2 of embodiment 10, wherein the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazine group, a hydrazino group, an aminoureido group, an azide 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 biologically active molecule, cytotoxic agent, or immunostimulant is connected to the IL-2 through a linker.

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

22. The IL-2 of embodiment 18, wherein the bioactive molecule, cytotoxic agent, or immunostimulant 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, amine A base acid, a polypeptide, or an amino terminal modification group; and R4 is H, an amino acid, a polypeptide, or a carboxy terminal modification group.

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

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

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

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

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

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

Where n is 0-10; R1 is alkyl, aryl, substituted alkyl, substituted aryl or not present; X is O, N, S or not present; m is 0-10; R2 is H, amine A base acid, a polypeptide, or an amino terminal modification group, and R3 is H, an amino acid, a polypeptide, or a carboxy terminal modification group.

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

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

Wherein n is 0-10; R1 is alkyl, aryl, substituted alkyl or substituted aryl; X is O, N, S or not present; m is 0-10; R2 is H, amino acid, A polypeptide or amino terminal modification group, and R3 is H, an amino acid, a polypeptide, or a carboxy terminal modification group.

32. The IL-2 of embodiment 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 embodiment 16, wherein the aminooxy group, hydrazine, hydrazine or aminourea group is connected to the water-soluble polymer through an amide bond.

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

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

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

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

39. A method of manufacturing the IL-2 of Example 1, the method comprising combining an isolated IL-2 polypeptide comprising a non-naturally encoded amino acid with a component that reacts with the non-naturally encoded amino acid Contact with the linker, polymer or biologically active molecule.

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

41. The method of embodiment 39, wherein the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazino group, a hydrazine group, an aminoureido group, an azide 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 aminooxy, hydrazine, hydrazine, or semicarbazide component.

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

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

45. The method of embodiment 39, wherein the non-naturally encoded amino acid comprises an azide moiety, and the linker, polymer, or bioactive molecule comprises an alkyne moiety.

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

47. The IL-2 polypeptide of embodiment 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 with a disorder modulated by IL-2, the method comprising administering to the patient a therapeutically effective amount of the composition of Example 42.

51. A composition comprising the 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, the IL-2 further comprising a linker and a conjugate.

53. A method for producing IL-2 containing non-naturally encoded amino acids, the method comprising combining one or more polynucleotides containing selector codons that encode IL-2 polypeptides, an orthogonal RNA synthetase, and Orthogonal tRNA cells are cultured under conditions that allow the expression of the IL-2 polypeptide containing non-naturally encoded amino acids; and the polypeptide is purified.

54. A method for regulating the serum half-life or cycle time of an IL-2 polypeptide, the method comprising replacing any one or more naturally-occurring amino groups in the polypeptide with one or more non-naturally encoded amino acids acid.

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 incorporated into the polypeptide via a ribosome through a non-naturally encoded amine group The functional group of the acid is attached to 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 Ribosomes are incorporated into the polypeptide.

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

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.

1: IL-2 receptor alpha 2: IL-2 receptor β 3: IL-2 receptor γ 4: IL-2 5: Site to be screened 6: PEG (polyethylene glycol) CD8+: CD8 receptor protein Kd: Dissociation constant (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-shows a model of the IL-2 polypeptide view, 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-Western blot analysis showing the expression of IL-2 protein 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 and CD25, model fitting line and calculated measurement values.

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

Figure 5-shows the UPF1 genomic DNA sequence and the design of the 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 the 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 the CTLL-2 proliferation assay.

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

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

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

Figure 11-Schematic 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 a comparison of the titer before and after the production 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 for the F42-R38A variant.

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

1: IL-2 receptor alpha

2: IL-2 receptor β

3: IL-2 receptor γ

4: IL-2

5: Site 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 (67)

An interleukin-2 (IL-2) polypeptide comprising one or more non-naturally encoded (non-naturally encoded) amino acids, wherein the IL-2 polypeptide is compared with wild-type IL-2 to its receptor Receptor subunit interaction is reduced. The IL-2 polypeptide according to claim 1, wherein the IL-2 polypeptide is 90% homologous to SEQ ID NO: 2 or SEQ ID NO: 3. The IL-2 polypeptide of claim 1, wherein the IL-2 polypeptide is at least 95% homologous to SEQ ID NO:2. The IL-2 polypeptide of claim 1, wherein the IL-2 polypeptide is at least 98% homologous to SEQ ID NO:2. The IL-2 polypeptide of claim 1, wherein the IL-2 polypeptide is at least 99% homologous to SEQ ID NO:2. The IL-2 polypeptide according to claim 1, wherein the IL-2 polypeptide is conjugated to one or more water-soluble polymers. The IL-2 polypeptide according to claim 6, wherein at least one of the water-soluble polymer is linked to at least one of the non-naturally encoded amino acid. The IL-2 polypeptide according to claim 7, wherein the water-soluble polymer is PEG. The IL-2 polypeptide according to claim 8, wherein the PEG has a molecular weight between 10 and 50. The IL-2 polypeptide according to claim 1, wherein the non-naturally encoded amino acid is replaced at a position selected from the following residues: before the first position (ie at the N-terminus), the first , 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 carboxy terminus of the protein and any combination thereof. The IL-2 polypeptide according to claim 10, wherein compared with wild-type IL-2, the IL-2 polypeptide comprises one or more amine groups that modulate the affinity of the IL-2 polypeptide for its IL2Rα receptor subunit Acid substitution, addition or deletion. The IL-2 polypeptide according to claim 10, 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 according to claim 10, wherein the IL-2 polypeptide comprises one or more amino acid substitutions, additions or deletions that increase the expression of the IL-2 polypeptide polypeptide in recombinant host cells or synthesis in vitro. The IL-2 polypeptide according to claim 10, wherein the non-naturally encoded amino acid is substituted in selected from the group 3, 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, The positions of residues 65, 68, 72 and 107 and any combination thereof. The IL-2 polypeptide according to claim 10, wherein the non-naturally encoded amino acid has an unreactive linker to any of the 20 common amino acids in the polypeptide, Polymers or biologically active molecules are reactive. The IL-2 polypeptide according to claim 10, wherein the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazine group, a hydrazine group, a semicarbazide group, an azide group or an alkyne group. The IL-2 polypeptide of claim 16, wherein the non-naturally encoded amino acid comprises a carbonyl group. The IL-2 polypeptide according to claim 10, wherein the IL-2 polypeptide is linked to a biologically active molecule, a cytotoxic agent, a water-soluble polymer or an immunostimulant. The IL-2 polypeptide according to claim 18, wherein the conjugated IL-2 polypeptide is attached to one or more water-soluble polymers. The IL-2 polypeptide according to claim 18, wherein the biologically active molecule, cytotoxic agent or immunostimulant is connected to the IL-2 polypeptide through a linker. The IL-2 polypeptide according to claim 18, wherein the biologically active molecule, cytotoxic agent or immunostimulant is connected to the IL-2 polypeptide through a cleavable or non-cleavable linker. The IL-2 polypeptide of claim 18, wherein the biologically active molecule, cytotoxic agent or immunostimulant is directly conjugated to one or more of the non-naturally encoded amino acids in the IL-2 polypeptide. The IL-2 polypeptide according to claim 10, wherein the non-naturally encoded amino acid has the following structure:

Wherein n is 0-10; R ₁ is alkyl, aryl, substituted alkyl or substituted aryl; R ₂ is H, alkyl, aryl, substituted alkyl and substituted aryl; R ₃ is H, an amino acid, a polypeptide, or an amino terminal modification group; and R ₄ is H, an amino acid, a polypeptide, or a carboxy terminal modification group. The IL-2 polypeptide of claim 23, wherein the non-naturally encoded amino acid comprises an aminooxy group. The IL-2 polypeptide of claim 23, wherein the non-naturally encoded amino acid comprises a hydrazine group. The IL-2 polypeptide of claim 23, wherein the non-naturally encoded amino acid comprises a hydrazine group. The IL-2 polypeptide of claim 23, wherein the non-naturally encoded amino acid residue comprises a semicarbazide. The IL-2 polypeptide of claim 23, wherein the non-naturally encoded amino acid residue comprises an azide group. The IL-2 polypeptide according to claim 1, wherein the non-naturally encoded amino acid has the following structure:

Wherein n is 0-10; R ₁ is alkyl, aryl, substituted alkyl, substituted aryl or absent; X is O, N, S or absent; m is 0-10; R ₂ is H , Amino acid, polypeptide, or amino terminal modification group, and R ₃ is H, amino acid, polypeptide, or carboxy terminal modification group. The IL-2 polypeptide of claim 29, wherein the non-naturally encoded amino acid comprises an alkynyl group. The IL-2 polypeptide according to claim 1, wherein the non-naturally encoded amino acid has the following structure:

Wherein n is 0-10; R ₁ is alkyl, aryl, substituted alkyl or substituted aryl; X is O, N, S or not present; m is 0-10; R ₂ is H, amino group An acid, polypeptide, or amino terminal modification group, and R ₃ is H, an amino acid, polypeptide, or a carboxy terminal modification group. The IL-2 polypeptide according to claim 7, wherein the water-soluble polymer has a molecular weight between about 0.1 kDa and about 100 kDa. The IL-2 polypeptide polypeptide of claim 32, wherein the water-soluble polymer has a molecular weight between about 0.1 kDa and about 50 kDa. The IL-2 polypeptide according to claim 16, wherein the aminooxy group, hydrazine, hydrazine or aminourea group is connected to the water-soluble polymer through an amide bond. The IL-2 polypeptide according to claim 19, which is performed by reacting a water-soluble polymer containing a carbonyl group with a polypeptide containing a non-naturally encoded amino acid containing an aminooxy group, a hydrazine group, a hydrazine group, or an aminourea group To make. The IL-2 polypeptide of claim 1, wherein the IL-2 polypeptide is glycosylated. The IL-2 polypeptide of claim 1, wherein the IL-2 polypeptide further comprises a linker, polymer or biologically active molecule linked to the polypeptide through the non-naturally encoded amino acid. The IL-2 polypeptide according to claim 37, wherein the linker, polymer or biologically active molecule is linked to the IL-2 polypeptide through a saccharide moiety. A method for producing the IL-2 polypeptide according to claim 1, the method comprising combining the isolated IL-2 polypeptide comprising a non-naturally encoded amino acid with a component that reacts with the non-naturally encoded amino acid Contact with the linker, polymer or biologically active molecule. The method according to claim 39, wherein the polymer comprises a moiety selected from the group consisting of water-soluble polymers and polyethylene glycol. The method according to claim 39, wherein the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazine group, a hydrazine group, an aminoureido group, an azide group, or an alkynyl group. The method according to claim 39, wherein the non-naturally encoded amino acid contains a carbonyl component, and the linker, polymer or biologically active molecule contains an aminooxy, hydrazine, hydrazine or aminourea component (moiety ). The method according to claim 39, wherein the aminooxy group, hydrazine, hydrazine or aminourea component is connected to the linker, polymer or biologically active molecule through an amide bond. The method of claim 39, wherein the non-naturally encoded amino acid comprises an alkynyl component, and the linker, polymer or biologically active molecule comprises an azide component. The method of claim 39, wherein the non-naturally encoded amino acid comprises an azide group component, and the linker, polymer or biologically active molecule comprises an alkynyl group component. The IL-2 polypeptide according to claim 7, wherein the water-soluble polymer is a component of polyethylene glycol. The IL-2 polypeptide of claim 46, wherein the polyethylene glycol component is a branched or multi-arm polymer. A composition comprising the IL-2 polypeptide described in claim 10 and a pharmaceutically acceptable carrier. The composition of claim 48, wherein the non-naturally encoded amino acid is linked to a water-soluble polymer. A method of treating a patient with a disorder modulated by IL-2, the method comprising administering to the patient a therapeutically-effective amount of the composition of claim 42. A composition comprising the IL-2 polypeptide described in claim 10 conjugated to a biologically active molecule and a pharmaceutically acceptable carrier. A composition comprising the IL-2 polypeptide according to claim 10 and a pharmaceutically acceptable carrier, the IL-2 polypeptide further comprising a linker and a conjugate. A method for producing the IL-2 polypeptide comprising a non-naturally encoded amino acid, the method comprising combining one or more polynucleotides comprising a selector codon (selector codon) encoding an IL-2 polypeptide, orthogonal RNA synthetase (orthogonal RNA synthetase) and orthogonal tRNA (orthogonal tRNA) cells are cultured under conditions that allow the expression of the IL-2 polypeptide polypeptide containing non-naturally encoded amino acids; and purification of the IL- 2 Polypeptides. A method for regulating the serum half-life or serum circulation time of IL-2 polypeptides, the method comprising replacing any one or more of the polypeptides with one or more non-naturally encoded amino acids A naturally occurring amino acid. 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. An IL-2 polypeptide comprising at least one linker, polymer or biologically active molecule, wherein the linker, polymer or biologically active molecule is incorporated into the IL-2 polypeptide via a non-naturally encoded amino acid via a ribosome The functional group is attached to the polypeptide. 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 acid is incorporated into a ribosome at a preselected position To the IL-2 polypeptide. A method of reducing the number of tumor cells in a person diagnosed with cancer, the method comprising administering a pharmaceutical composition comprising the PEG-IL-2A of claim 56 to a person in need of such reduction. The method of claim 58, wherein the conjugate is administered at a dose of about 0.1 μ/kg to about 50 μ/kg. The IL-2 polypeptide according to any one of claims 1-38, 46-47 and 55-57, wherein the IL-2 polypeptide further comprises at least one natural amine group at one or more positions selected from Natural amino acid substitution: 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, Residues 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133. The method according to any one of claims 39-45, 53-54 and 58-59 or the composition according to any one of claims 48-49, wherein the method or the composition is still in one or more At least one natural amino acid substitution is included at positions selected from the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 , 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 , 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 , 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117 , 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133 residues. The IL-2 polypeptide according to claim 60, and the method or composition according to claim 61, wherein the natural amino acid is substituted at positions 38, 46 and/or 65. The IL-2 polypeptide according to claim 60, and the method or composition according to claim 61, wherein the natural amino acid is substituted at positions 38 and 46. The IL-2 polypeptide according to claim 60, and the method or composition according to claim 61, wherein the natural amino acid is substituted at positions 38 and 65. The IL-2 polypeptide or method or composition according to any one of claims 62-64, wherein the natural amino acid substitution at position 38 is alanine. The IL-2 polypeptide or method or composition according to any one of claims 62 to 63, wherein the natural amino acid substitution at position 46 is leucine or isoleucine. The IL-2 polypeptide or method or composition according to any one of claim 62 or 64, wherein the natural amino acid substitution at position 65 is arginine.

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