Interleukin-2 / Interleukin-2 receptor alpha fusion protein and method of use

Abstract

Disclosed herein is a fusion protein comprising: (a) a first polypeptide comprising interleukin-2 (IL2); and (b) a second polypeptide fused in-frame to the first polypeptide, wherein the second polypeptide comprises an extracellular domain of interleukin-2 receptor alpha (IL2Rα), wherein IL2 or IL2Rα comprises at least one less glycosylation site than native IL2 or native IL2Rα. Also disclosed are methods for producing the fusion protein and methods for therapeutic use.

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 649,379, filed March 28, 2018, which is incorporated herein by reference in its entirety.

[0003] 2. Citation of a sequence listing submitted electronically via EFS-WEB

[0004] The contents of the electronically submitted Sequence Listing in the ASCII text file (Name: 3338_100PC01_Seqlisting_ST25.txt; Size: 354,542 bytes; and Creation Date: March 27, 2019) submitted with this application are incorporated herein by reference in their entirety. Technical Field

[0005] The subject matter disclosed herein generally relates to methods and compositions for modulating immune responses using interleukin-2 / interleukin-2 receptor alpha fusion proteins. Background Art

[0006] Interleukin-2 (IL2 or IL-2) is a biological cytokine that regulates key aspects of the immune system. IL2 has been used in attempts to enhance the immune response in patients with cancer, inflammatory diseases, or autoimmune diseases. IL2 is a potent T cell growth factor that promotes immune responses (including clonal expansion of antigen-activated T cells), drives the development of CD4+ T helper (Th) 1 and Th2 cells, ultimately leading to the differentiation of CD8+ cytotoxic T lymphocytes (CTLs), and inhibits the development of CD4+ Th17 and T follicular helper cells (Tfh). IL2 also affects the recall response of T cell memory.

[0007] Clinical trials are taking advantage of the T cell-activating properties of IL2 in patients with cancer and HIV / AIDS by infusing high doses of IL2 (typically >500,000 units / kg, repeatedly) to enhance T cells and NK cells. Indeed, IL2 has been approved by the FDA for patients with melanoma and renal cell carcinoma because some of these patients (approximately 5%) have shown complete responses. However, response rates in these and other cancers are low, and this therapy is accompanied by severe toxicities. Similarly, IL2 is considered ineffective for enhancing immunity in patients with HIV / AIDS. In these cases, high-dose IL2 is less effective, in part because it is accompanied by the expansion of Tregs.

[0008] Recently, lower doses of IL2 have been used to selectively enhance tolerance to suppress unwanted immune responses associated with autoimmune-like attacks on one's own tissues. These low doses of IL2 have not yet shown any signs of enhancing or reactivating autoreactive T cells. Preclinical studies have shown that low IL2R signaling selectively promotes the key activity of Treg, but cannot promote the key activity of T effector (Teff) cells, and show that treatment of mice with low levels of IL2 prevents autoimmunity. Currently, many patients with excessive immune responses have been treated with low doses of IL2 (0.5-2 million units, regularly). The experience so far is that this therapy is safe, with no signs of reactivation of autoaggressive T cells, and in almost all patients, Treg is elevated, which is usually accompanied by clinical improvement. However, IL2 has important disadvantages as a therapeutic agent, including a very short half-life in the body (which limits its efficacy) and toxicity at high doses. For these reasons, there is a need for new IL2 biological preparations with improved pharmacokinetics and response persistence for use. Summary of the Invention

[0009] The present disclosure includes fusion proteins comprising: (a) a first polypeptide comprising an interleukin-2 (IL2) polypeptide; and (b) a second polypeptide comprising an extracellular domain of an interleukin-2 receptor alpha (IL2Rα) polypeptide; wherein (i) the extracellular domain of the IL2Rα polypeptide has at least one less glycosylation compared to the extracellular domain of native IL2Rα (SEQ ID NO: 7); and / or (ii) the IL2 polypeptide has at least one less glycosylation compared to native IL2 (SEQ ID NO: 2); and wherein the fusion protein has IL2 activity. In some embodiments, the extracellular domain of the IL2Rα polypeptide has at least one less glycosylation, at least two less glycosylations, at least three less glycosylations, at least four less glycosylations, at least five less glycosylations, at least six less glycosylations, at least seven less glycosylations, at least eight less glycosylations, or at least nine less glycosylations compared to the extracellular domain of native IL2Rα (SEQ ID NO: 7). In some embodiments, the IL2 polypeptide has at least one fewer glycosylation compared to native IL2 (SEQ ID NO: 2).

[0010] In some embodiments, the fusion protein comprises a first polypeptide, wherein the first polypeptide comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 2. In some embodiments, the fusion protein comprises a second polypeptide, wherein the second polypeptide comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 12.

[0011] In some embodiments, the extracellular domain of at least one glycosylated IL2Rα polypeptide comprises a mutation that removes glycosylation. In some embodiments, the mutation removes O-glycosylation and / or N-glycosylation. In some embodiments, the mutation removes O-glycosylation. In some embodiments, the mutation removes N-glycosylation.

[0012] In some embodiments, the mutation in the extracellular domain of the IL2Rα polypeptide is a mutation corresponding to SEQ ID or amino acids 184 to 219, amino acids 185 to 219, amino acids 186 to 219, amino acids 187 to 219, amino acids 188 to 219, amino acids 189 to 219, amino acids 190 to 219, amino acids 191 to 219, or amino acids 192 to 219 of NO:7. In some embodiments, the mutation is a deletion corresponding to amino acids 167, 169, 171 through 192 to 219 of SEQ ID NO: 7. In some embodiments, the mutation does not include a deletion corresponding to amino acids 170 to 219 of SEQ ID NO: 7. In some embodiments, the second polypeptide is SEQ ID NO: 11. In some embodiments, the second polypeptide is SEQ ID NO: 12.

[0013] In some embodiments, the mutation is one or more substitutions of glycosylated amino acids with non-glycosylated amino acids. In some embodiments, the mutation is one or more substitutions of amino acids that allow glycosylation at nearby amino acids with amino acids that do not allow glycosylation at nearby amino acids. In some embodiments, the one or more substitutions are at amino acid N49, amino acid N68, amino acid T74, amino acid T85, amino acid T197, amino acid T203, amino acid T208, and amino acid T216, or any combination thereof, wherein the amino acid position corresponds to SEQ ID NO: 7. In some embodiments, the one or more substitutions are from asparagine to an amino acid selected from the group consisting of alanine, threonine, serine, arginine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. In some embodiments, the one or more substitutions are from threonine to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine.

[0014] In some embodiments, one of the substitutions is amino acid N 49. In some embodiments, N49 is mutated to an amino acid selected from the group consisting of alanine, threonine, serine, arginine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine.

[0015] In some embodiments, one of the substitutions is amino acid N 68. In some embodiments, N 68 is mutated to an amino acid selected from the group consisting of alanine, threonine, serine, arginine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine.

[0016] In some embodiments, one of the substitutions is amino acid T74. In some embodiments, T74 is mutated to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine.

[0017] In some embodiments, one of the substitutions is amino acid T85. In some embodiments, T85 is mutated to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine.

[0018] In some embodiments, one of the substitutions is amino acid T 197. In some embodiments, T197 is mutated to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine.

[0019] In some embodiments, one of the substitutions is amino acid T203. In some embodiments, T203 is mutated to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine.

[0020] In some embodiments, one of the substitutions is amino acid T208. In some embodiments, T208 is mutated to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine.

[0021] In some embodiments, one of the substitutions is amino acid T216. In some embodiments, T216 is mutated to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine.

[0022] In some embodiments, the one or more substitutions are at amino acid S50, amino acid S51, amino acid T69, amino acid T70, amino acid C192, or any combination thereof, wherein the amino acid position corresponds to SEQ ID NO:7.

[0023] In some embodiments, one of the substitutions is at amino acid S50. In some embodiments, S50 is mutated to proline.

[0024] In some embodiments, one of the substitutions is amino acid S51. In some embodiments, S51 is mutated to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine.

[0025] In some embodiments, one of the substitutions is amino acid T69. In some embodiments, T69 is mutated to proline.

[0026] In some embodiments, one of the substitutions is amino acid T70. In some embodiments, T70 is mutated to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, and valine.

[0027] In some embodiments, one of the substitutions is amino acid C 192. In some embodiments, C192 is mutated to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0028] In some embodiments, at least one of the glycosylated IL2 polypeptides comprises a mutation that removes glycosylation. In some embodiments, the mutation is one or more substitutions of a glycosylated amino acid with a non-glycosylated amino acid. In some embodiments, the mutation is one or more substitutions of an amino acid that permits glycosylation with an amino acid that does not permit glycosylation at the nearby amino acid. In some embodiments, the one or more substitutions are from alanine to an amino acid selected from the group consisting of arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, the one or more substitutions are from threonine to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. In some embodiments, the one or more substitutions are from cysteine ​​to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, the one or more substitutions are from cysteine ​​to serine. In some embodiments, the one or more substitutions are from cysteine ​​to alanine. In some embodiments, the one or more substitutions are from cysteine ​​to valine.

[0029] In some embodiments, the one or more substitutions are at amino acid T3, as compared to the corresponding sequence of SEQ ID NO: 2. In some embodiments, one of the substitutions is at amino acid C125. In some embodiments, the substitution at amino acid C125 is selected from C125S, C125A, and C125V.

[0030] In some embodiments, the mutation is a deletion. In some embodiments, the deletion is at amino acid A1.

[0031] In some embodiments, the fusion protein is enzymatically or chemically deglycosylated. In some embodiments, the fusion protein is deglycosylated by alkali, hydrazinolysis, PNGase F, Endo H, O-glycosidase, or any combination thereof.

[0032] In some embodiments, the fusion protein further comprises a linker fused in frame between the first polypeptide and the second polypeptide. In some embodiments, the linker is a glycine / serine linker. In some embodiments, the glycine / serine linker comprises (GS)n 、(GGS) n 、(GGGS) n 、(GGGGS) n or (GGGGS) n wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the glycine / serine linker comprises an amino acid sequence of (GGGS)3.

[0033] In some embodiments, the fusion protein further comprises a heterologous moiety fused to the first polypeptide and / or the second polypeptide. In some embodiments, the heterologous moiety is a half-life extending moiety. In some embodiments, the heterologous moiety comprises a non-polypeptide moiety. In some embodiments, the heterologous moiety comprises a polypeptide. In some embodiments, the heterologous moiety comprises an albumin, an immunoglobulin constant region or a portion thereof, an immunoglobulin binding polypeptide, immunoglobulin G (IgG), an albumin binding polypeptide (ABP), a PASylated moiety, a HESylated moiety, an XTEN, a PEGylated moiety, an Fc region, and any combination thereof.

[0034] In some embodiments, the fusion protein is more stable than a polypeptide consisting of SEQ ID NO: 2 or SEQ ID NO: 13. In some embodiments, the fusion protein has one or more properties selected from the group consisting of: (i) increased thermodynamic stability compared to a reference protein; (ii) increased TM compared to a reference protein; (iii) increased resistance to degradation compared to a reference protein; (iv) increased resistance to modification compared to a reference protein; (v) increased in vivo stability compared to a reference protein; and (vi) any combination thereof, wherein the reference protein comprises (i) a first polypeptide comprising an interleukin-2 (IL2) polypeptide; and (b) a second polypeptide comprising an extracellular domain of an interleukin-2 receptor alpha (IL2Rα) polypeptide; and the reference protein has at least one fewer glycosylation than the fusion protein.

[0035] In some embodiments, the fusion protein is a monomer. In some embodiments, the fusion protein is a dimer. In some embodiments, the dimer comprises two monomers, and the monomers are associated with each other via a covalent bond. In some embodiments, the dimer comprises two monomers, and the monomers are associated via a non-covalent bond.

[0036] In some embodiments, the fusion protein has one or more pharmacokinetic properties selected from the group consisting of: increased half-life, increased C max , increased AUC, increased C min, reduced clearance, improved bioavailability, and any combination thereof. In some embodiments, the fusion protein has an extended half-life. In some embodiments, the extended half-life is at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 16 times, at least about 17 times, at least about 18 times, at least about 19 times, at least about 20 times, at least about 21 times, or at least about 22 times compared to the half-life of a polypeptide consisting of SEQ ID NO: 2 or SEQ ID NO: 13.

[0037] In some embodiments, disclosed herein are one or more fusion proteins. In some embodiments, provided herein are compositions comprising one or more fusion proteins disclosed herein.

[0038] In some embodiments, disclosed herein are nucleic acids encoding any of the fusion proteins disclosed herein. In some embodiments, disclosed herein are vectors comprising nucleic acids encoding any of the fusion proteins disclosed herein. In some embodiments, disclosed herein are host cells comprising nucleic acids encoding any of the fusion proteins disclosed herein. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from mammalian cells, insect cells, yeast cells, transgenic mammalian cells, and plant cells. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is a bacterial cell.

[0039] In some embodiments, provided herein is a pharmaceutical composition comprising (a) a fusion protein disclosed herein, a composition disclosed herein, a nucleic acid disclosed herein, a vector disclosed herein, or a host cell disclosed herein; and (b) a pharmaceutically acceptable excipient.

[0040] In some embodiments, provided herein is a kit comprising a fusion protein disclosed herein, a composition disclosed herein, a nucleic acid disclosed herein, a vector disclosed herein, or a host cell disclosed herein, and instructions for administering the fusion protein to a subject in need thereof.

[0041] In some embodiments, there is provided herein a method for producing fusion protein disclosed herein, comprising: cultivating a host cell disclosed herein under suitable conditions and reclaiming the fusion protein. In some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the host cell is a mammalian cell, an insect cell, a fungal cell, a plant cell, a transgenic mammalian cell or a bacterial cell. In some embodiments, the host cell is selected from CHO cells, HEK 293 cells, NSO cells, Per C6 cells, BHK cells and COS cells. In some embodiments, the bacterial cell is Escherichia coli.

[0042] In some embodiments, there is provided herein a method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of a fusion protein disclosed herein, a composition disclosed herein, a nucleic acid disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is bladder cancer, breast cancer, uterine cancer, endometrial cancer, ovarian cancer, colorectal cancer, colon cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, squamous cell carcinoma, skin cancer, central nervous system tumors, lymphoma, leukemia, sarcoma, virus-related cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin or non-Hodgkin lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, myeloma, salivary gland cancer, kidney cancer, basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer, or head and neck cancer, and any combination thereof.

[0043] In some embodiments, the disease or disorder is an inflammatory disease or an autoimmune disease. In some embodiments, the inflammatory disease or autoimmune disease is selected from the group consisting of type 1 diabetes, multiple sclerosis, rheumatoid arthritis, celiac disease, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis or systemic sclerosis, graft-versus-host disease, psoriasis, alopecia areata, HCV-induced vasculitis, Sjögren's syndrome, pemphigus, ankylosing spondylitis, Behçet's disease, Wegener's granulomatosis, Takayasu's disease, autoimmune hepatitis, sclerosing cholangitis, Guillain-Strauss syndrome and macrophage activation syndrome.

[0044] In some embodiments, the disease or disorder is an infectious disease. In some embodiments, the infectious disease is caused by a pathogenic virus. In some embodiments, the pathogenic virus is selected from human immunodeficiency virus (HIV), hepatitis A, hepatitis B, hepatitis C, herpes virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus (vaccinia virus), human T-lymphotropic virus (HTL), dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, John Cunningham (JC) virus and arboviral encephalitis virus. In some embodiments, the infectious disease is caused by pathogenic bacteria. In some embodiments, the pathogenic bacteria is selected from the group consisting of chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis and Lyme disease bacteria. In some embodiments, the infectious disease is caused by a pathogenic fungus.In some embodiments, the pathogenic bacteria are selected from the genus Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum. In some embodiments, the infectious disease is caused by a pathogenic parasite. In some embodiments, the pathogenic parasite is selected from Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus brasiliensis.

[0045] In some embodiments, the methods disclosed herein further comprise administering a second agent to the subject. In some embodiments, the second agent is a PD-1 antagonist, a CTLA-4 antagonist, a TIM3 antagonist, a GITR antagonist, a KIR antagonist, a LAG3 antagonist, or any combination thereof. In some embodiments, the second agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-KIR antibody, an anti-GITR antibody, an anti-LAG3 antibody, or any combination thereof. In some embodiments, the second agent is a cytokine inhibitor. In some embodiments, the cytokine inhibitor targets one or more of IL-6, IL-10, TGF-β, VEGF, IFN-γ, or any combination thereof.

[0046] In some embodiments, the fusion protein is administered via a topical, intramucosal, intranasal, oral, vaginal, rectal, sublingual, local, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, or intrasternal route. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The molecular weight of SEQ ID NO: 16 was shown to be 93 kDa as measured by SEC / MALS static light scattering. The theoretical mass of the individual polypeptide chains was reduced by 37,812 Da. The observed solution mass indicated that it existed as a homogeneous homodimer with approximately 19% of the mass due to glycosylation.

[0048] Figure 2 Shown is the prototype binding of IL2-CD25 fusion protein (SEQ ID NO: 16) to human sCD25 measured using surface plasmon resonance.

[0049] Figure 3 Shown are the serum concentration-time curves of the indicated fusion proteins in Balb / C mice after a single intravenous (IV) or subcutaneous (SC) dose of 0.5 mg / kg. Each time point represents the mean value of samples from 3 mice. Error bars represent standard deviations.

[0050] Figure 4 Shown are the serum concentration-time curves of the indicated fusion proteins in Balb / C mice after a single intravenous (IV) or subcutaneous (SC) dose of 0.5 mg / kg. Each time point represents the mean value of samples from 3 mice. Error bars represent standard deviations.

[0051] Figure 5Shown is the serum concentration-time curve of hIL2-CD25 (22-212) in cynomolgus monkeys after a single subcutaneous (SC) dose of 0.075 mg / kg. Each time point represents the mean of samples from 3 monkeys. Error bars represent standard deviations.

[0052] Figure 6 The activity of IL2-CD25 fusion protein in inducing STAT5 phosphorylation in human PBMC from a representative donor is shown. + 、foxp3 + 、CD25 + ) or Tconv(CD4 + 、foxp3 - ), CD8 + and NK cells (CD3 - 、CD56 + ) were gated and the percentage of cells that stained positive for pSTAT5 after incubation was quantified. ECs for pSTAT5 induction in Tregs determined from these experiments were 50 They are 10ng / ml, 4.4ng / ml and 4.6ng / ml respectively.

[0053] Figure 7 The fusion protein of truncations in whole blood is shown to stimulate IL2R and result in the ability to induce phosphorylated STAT5 in various cell types. IL2R signaling is detected by measuring by flow cytometry after intracellular staining for pSTAT5 and by determining the percentage of cells that are positive for pSTAT5 staining in the whole blood mixture. In a representative donor, the efficacy of hIL2-CD25 (22-240) was compared with that of hIL2-CD25 (22-212). The protein was titrated in human whole blood, and the intensity of intracellular pSTAT5 staining was measured by flow cytometry. For hIL2-CD25 (22-240), the EC50 for pSTAT5 induction in Treg was 22 ng / ml, and for hIL2-CD25 (22-212) was 36 ng / ml.

[0054] Figures 8A-8C The results show that hIL2-CD25(22-240), hIL2-CD25(22-212) and hIL2-CD25(22-184) have similar abilities to increase T cells in mice with humanized immune systems. NSG-huCD34 transplanted mice were given (subcutaneously) fusion proteins every three days for three doses. Tregs ( Figure 8A )、CD8( Figure 8B ) and NK cells ( Figure 8C ) analysis.

[0055] Figure 9A and Figure 9B Shown in the presence of PBMCs ( Figure 9A ) or whole blood concentration ( Figure 9B ) in a mixed cell population, phosphorylation of STAT5 after 15 minutes when hIL2-CD25(22-212) (SEQ ID NO: 16) was used.

[0056] Figure 10 LC-MS / MS-based analysis of the stability of the (G3S)3 linker in IL2(21-153)-(G3S)3-CD25(22-212) in vitro in human or mouse serum is shown. Peak area ratios of IL2 and CD25 obtained by LC-MS / MS are reported after capture with an anti-IL2 antibody.

[0057] Figure 11 LC-MS / MS-based analysis of the stability of the (G3S)3 linker in IL2(21-153)-(G3S)3-CD25(22-212) in monkey serum following a single subcutaneous dose of 0.075 mg / kg is shown. Peak area ratios of IL2 and CD25 obtained using LC-MS / MS are reported after capture with an anti-IL2 antibody. DETAILED DESCRIPTION

[0058] The present disclosure is described more fully hereinafter with reference to the accompanying drawings, in which some, but not all, embodiments of the disclosure are shown. Indeed, this disclosure may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these examples are provided so that this disclosure will satisfy applicable legal requirements. Throughout, like numbers refer to like elements.

[0059] Many modifications and other embodiments of the disclosures listed herein will be apparent to those skilled in the art having the benefit of the teachings presented in the foregoing description and the associated drawings. Therefore, it will be understood that this disclosure is not limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0060] 7.1 Overview

[0061] Various methods and compositions that can be used to modulate the immune system are provided. The compositions include a fusion protein comprising: (a) a first polypeptide comprising an interleukin-2 (IL2) polypeptide; and (b) a second polypeptide comprising the extracellular domain of an interleukin-2 receptor alpha (IL2Rα) polypeptide; wherein (i) the extracellular domain of the IL2Rα polypeptide has at least one less glycosylation than the extracellular domain of native IL2Rα (SEQ ID NO: 7); and / or (ii) the IL2 polypeptide has at least one less glycosylation than native IL2 (SEQ ID NO: 2); and wherein the fusion protein has IL2 activity.

[0062] The present disclosure also describes nucleotides encoding the fusion proteins disclosed herein, vectors comprising the nucleotides, host cells comprising the nucleotides, and compositions comprising the fusion proteins, the nucleotides, the vectors, or the host cells. The present disclosure also relates to methods of preparing the fusion proteins, the nucleotides, the vectors, the host cells, or the compositions, or methods of using the fusion proteins, the nucleotides, the vectors, the host cells, or the compositions.

[0063] 7.2 Definitions

[0064] It should be noted that the term "a" or "an" entity refers to one or more of said entities: for example, "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" or "an", "one or more", and "at least one" are used interchangeably herein.

[0065] Furthermore, as used herein, “and / or” is taken as a specific disclosure of each of the two specified features or components with or without the other features or components. Thus, the term “and / or” as used herein with phrases such as “A and / or B” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term “and / or” as used herein with phrases such as “A, B, and / or C” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0066] Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is also understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for description.

[0067] It should be understood that whenever any aspect is described herein using the language "comprising," other similar aspects are also provided that are described as "consisting of" and / or "consisting essentially of."

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press, provide those skilled in the art with general explanations of many of the terms used in this disclosure.

[0069] Units, prefixes and symbols are all expressed in a form acceptable to the International System of Units (SI). Numerical ranges include numbers defining the ranges. Unless otherwise indicated, amino acid sequences are written from left to right in the direction from amino to carboxyl. The headings provided herein are not limitations on the various aspects of this disclosure, which can be obtained as a whole by reference to the specification. Therefore, the terms defined below can be more fully defined by reference to the specification as a whole.

[0070] The term "about" is used herein to mean approximately, roughly, roughly, or around. When the term "about" is used with a numerical range, it modifies the range by extending the boundaries above and below the numerical values ​​recited. Thus, "about 10-20" means "about 10 to about 20." In general, the term "about" can modify a numerical value (higher or lower) above and below the recited value by a variance (e.g., 10%, up or down).

[0071] As used herein, "interleukin-2," "IL2," or "IL-2" refers to any natural or recombinant IL2 from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), as well as domesticated or agricultural mammals, unless otherwise indicated. The term encompasses unprocessed IL2 as well as any form of IL2 produced by processing in cells (i.e., the mature form of IL2). The term also encompasses naturally occurring variants and fragments (e.g., splice variants or allelic variants) of IL2, as well as non-naturally occurring variants having the IL2 activity of naturally occurring IL2.

[0072] Other nucleic acid and amino acid sequences of IL2 are known. See, for example, GenBank Accession Nos. Q7JFM2 (Aotus lemurinus (gray-bellied night monkey)); Q7JFM5 (Aotus nancymaae (horse night monkey)); P05016 (Bas taurus (cattle)); Q29416 (Dog (Dog) Chinese field dog); P36835 (Caprahircus (goat)); and P37997 (Equus caballus (horse)).

[0073] Biologically active fragments and variants of IL2 retain IL2 activity. The phrase "biological activity of IL2" or "IL2 activity" refers to one or more biological activities of IL2, including but not limited to the ability to stimulate lymphocytes with IL2 receptors. This activity can be measured in vitro and in vivo. IL2 is an overall regulator of immune activity, and the effect seen here is the sum of such activities. For example, it regulates survival activity (Bcl-2), induces T effector activity (IFN-γ, granzyme B and perforin) and / or promotes T regulatory activity (FoxP3).

[0074] Biologically active variants of IL2 are known. See, for example, U.S. Application Publications 20060269515 and 20060160187 and WO 99 / 60128.

[0075] As used herein, the terms "CD25," "IL2 receptor alpha," "IL2Rα," or "IL2Ra" refer to any natural or recombinant IL2α from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), as well as domesticated or agricultural mammals, unless otherwise indicated. The terms also encompass naturally occurring variants of IL2Rα (e.g., splice variants or allelic variants), or non-naturally occurring variants having IL2Rα activity. Human IL2 exerts its biological effects via signaling through its receptor system, IL2R. IL2 and its receptor (IL2R) are required for T cell proliferation and other essential functions important for immune responses. IL2R consists of three non-covalently linked type I transmembrane proteins: α (p55), β (p75), and γ (p65) chains. The human IL2Rα chain contains an extracellular domain of 219 amino acids, a transmembrane domain of 19 amino acids, and an intracellular domain of 13 amino acids. The secreted extracellular domain of IL2Rα (IL2R-α) can be used in the fusion proteins described herein.

[0076] The nucleic acid and amino acid sequences of IL2Rα are known. See, for example, GenBank Accession Nos. NP_001030597.1 (chimpanzee (Pan troglodytes)); NP_001028089.1 (rhesus monkey (Macaca mulatta)); NM_001003211.1 (wolf (Canis lupus)); NP_776783.1 (cattle (Bos taurus)); NP_032393.3 (mouse (Mus musculus)); and NP_037295.1 (rat (Rattus norvegicus)).

[0077] Also provided are biologically active fragments and variants of the extracellular domain of IL2Rα. Such IL2Rα extracellular domain active variants or fragments will retain the IL2Rα extracellular domain activity. The phrase "biological activity of the IL2Rα extracellular domain" refers to one or more biological activities of the IL2Rα extracellular domain, including but not limited to the ability to bind to IL2 and / or enhance intracellular signal transduction in IL2 receptor-responsive cells. Non-limiting examples of biologically active fragments and variants of IL2Rα are disclosed in, for example, Robb et al., Proc. Natl. Acad. Sci. USA, 85: 5654-5658, 1988. In some embodiments, the biologically active fragments and variants of IL2Rα disclosed herein comprise at least one less glycosylation compared to the extracellular domain of native IL2Rα.

[0078] As used herein, the term "naturally occurring" as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by man in a laboratory is naturally occurring.

[0079] A "polypeptide" is a chain of at least two consecutively linked amino acid residues, with no upper limit to the length of the chain. One or more amino acid residues in a protein may contain modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" or "fusion protein" may comprise one or more polypeptides.

[0080] The present disclosure also includes fragments or variants of the polypeptides and any combination thereof. When referring to the polypeptide binding domains or binding molecules of the present disclosure, the term "fragment" or "variant" includes any polypeptide that retains at least some properties of the reference polypeptide (e.g., the binding activity of IL2 for IL2Rα). Polypeptide fragments include proteolytic fragments and deletion fragments, but do not include naturally occurring full-length polypeptides (or mature polypeptides). Variants of the polypeptide binding domains or binding molecules of the present disclosure include fragments as described above, as well as polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants may be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be produced using mutagenesis techniques known in the art. Variant polypeptides may contain conservative or non-conservative amino acid substitutions, deletions, or additions.

[0081] As described above, polypeptide variants include, for example, modified polypeptides. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-link formation, cysteine ​​formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation (Mei et al., Blood 116:270-79 (2010)), proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins (such as arginylation and ubiquitination).

[0082] As used herein, "corresponding," "corresponding amino acids," "corresponding sites," or "equivalent amino acids" in a protein or nucleotide sequence are identified by alignment to maximize the identity or similarity between a first protein sequence (e.g., an IL2 sequence) and a second protein sequence (e.g., a second IL2 sequence). The number used to identify equivalent amino acids in the second protein sequence is based on the number used to identify corresponding amino acids in the first protein sequence. In some embodiments, the term "corresponding" refers to a relationship between mutations at one or more amino acids in a polypeptide or one or more nucleotides in a polynucleotide. As a non-limiting example, as disclosed herein, a particular amino acid (e.g., S50) of a polynucleotide (e.g., SEQ ID NO: 7) refers to the 50th amino acid in SEQ ID NO: 7, serine.

[0083] As used herein, the term "associated with " refers to a covalent or non-covalent bond formed between a first amino acid chain and a second amino acid chain. In one embodiment, the term "associated with " refers to a covalent non-peptide bond or a non-covalent bond. This association can be represented by a colon, i.e. (:). In another embodiment, it refers to a covalent bond other than a peptide bond. For example, the amino acid cysteine ​​contains a thiol group that can form a disulfide bond or a disulfide bridge with the thiol group on the second cysteine ​​residue. In most naturally occurring IgG molecules, the CH1 region and the CL region are associated by a disulfide bond, and the two heavy chains are associated by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system). Examples of covalent bonds include, but are not limited to, peptide bonds, metal bonds, hydrogen bonds, disulfide bonds, sigma bonds, pi bonds, delta bonds, glycosidic bonds, cleavage hydrogen bonds, bent bonds, dipole bonds, feedback pi bonds, double bonds, triple bonds, quadruple bonds, pentaplasm bonds, sextuple bonds, conjugated, hyperconjugated, aromatic, Haptor numbers, or antibonding. Non-limiting examples of non-covalent bonds include ionic bonds (e.g., cation-pi bonds or salt bonds), metal bonds, hydrogen bonds (e.g., dihydrogen bonds, molecular hydrogen complexes, low-barrier hydrogen bonds, or symmetric hydrogen bonds), van der Waals forces, London dispersion forces, mechanical bonds, halogen bonds, aurophilic interactions, intercalation, stacking, entropic forces, or chemical polarity.

[0084] As used herein, the term "comparable" means that the comparison rate or level, produced using, for example, a fusion protein, is equal to, substantially equal to, or similar to a reference rate or level. As used herein, the term "similar" means that the comparison rate or level differs from the reference rate or level by no more than 10% or no more than 15%. The term "substantially equal" means that the comparison rate or level differs from the reference rate or level by no more than 0.01%, 0.5%, or 1%.

[0085] As used herein, the term "expression" refers to the process by which a polynucleotide produces a gene product (eg, RNA or polypeptide).

[0086] A "fusion" or "fusion" protein comprises a first amino acid sequence linked in frame to a second amino acid sequence, with the first amino acid sequence not naturally linked to the second amino acid sequence in nature. Amino acid sequences that normally occur in separate proteins can be brought together in a fusion polypeptide, or amino acid sequences that normally occur in the same protein can be placed in a fusion polypeptide in a novel arrangement, for example, the fusion of IL2 protein with IL2-Rα protein. Fusion proteins can be produced, for example, by chemical synthesis or by generating and translating polynucleotides in which the peptide regions are encoded in a desired relationship. The fusion protein can further comprise a second amino acid sequence associated with the first amino acid sequence by a covalent, non-peptide, or non-covalent bond. Following transcription / translation, a single protein is produced. In this manner, multiple proteins or fragments thereof can be incorporated into a single polypeptide. "Operably linked" is intended to mean a functional connection between two or more elements. For example, an operably linked protein fuses the two polypeptides in frame to produce a single polypeptide fusion protein. In certain aspects, the fusion protein further comprises a third polypeptide, which, as discussed in further detail below, can include a linker sequence.

[0087] "Fc region" (fragment crystallizable region), "Fc domain" or "Fc" refers to the C-terminal region of an antibody's heavy chain, which mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells), or binding to the first component (C1q) of the classical complement system. Thus, the Fc region comprises the constant region of the antibody, excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region comprises two identical protein fragments, derived from the second (CH2) and third (CH3) constant domains of the antibody's two heavy chains, respectively; IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. IgG isotypes are divided into subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. For IgG, Fc district comprises immunoglobulin domain CH2 and CH3 and the hinge between CH1 and CH2 domain.As defined herein, although the definition of the boundary in the Fc district of immunoglobulin heavy chain may change, the Fc district of human IgG heavy chain is defined as extending to the carboxyl terminus of heavy chain from the amino acid residue of D221 of IgG1, V222 of IgG2, L221 of IgG3 and P224 of IgG4, wherein numbering is according to the EU index as in Kabat.The CH2 domain in human IgG Fc district extends from amino acid 237 to amino acid 340, and the CH3 domain is located at the C-terminal side of the CH2 domain in Fc district, that is, it extends from the amino acid 341 of IgG to amino acid 447 or 446 (if there is no C-terminal lysine residue) or 445 (if there is no C-terminal glycine and lysine residue).As used herein, Fc district can be native sequence Fc, including any allotype variant or variant Fc (for example, non-naturally occurring Fc).

[0088] "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. The FcR that binds to IgG antibodies comprises receptors of the FcγR family, including allelic variants of these receptors and alternatively including splice forms. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). Various properties of human FcγRs are known in the art. Most innate effector cell types co-express one or more activating FcγRs and inhibitory FcγRIIB, while natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans), but do not express inhibitory FcγRIIB in mice and humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to murine IgG2a with respect to the type of activating Fc receptor it binds.

[0089] As used herein, the terms "inserted," "is inserted," "inserted into," or grammatically related terms refer to the position of a heterologous moiety (e.g., a half-life extending moiety) in a fusion polypeptide relative to an analogous position in a particular protein. As used herein, the terms refer to characteristics of the recombinant polypeptides disclosed herein and do not indicate, suggest, or imply any method or process for making a fusion polypeptide.

[0090] " Heterologous " and " heterologous moiety " relating to polypeptides or polynucleotides are polypeptides or polynucleotides derived from different proteins or polynucleotides. The other components of the fusion protein can be derived from the same organism as the other polypeptide components of the fusion protein, or the other components can be from different organisms than the other polypeptide components of the fusion protein. For example, heterologous polypeptides can be synthetic or derived from different species of matter, different cell types, or the same or different cell types of different individuals. On the one hand, a heterologous moiety is a polypeptide fused to another polypeptide to produce a fusion polypeptide or protein. On the other hand, a heterologous moiety is a non-polypeptide conjugated to a polypeptide or protein, such as PEG. A non-limiting example of a heterologous moiety disclosed herein is a glycine / serine linker (e.g., GGGSGGGSGGGS (SEQ ID NO: 71) (also referred to as (Gly3Ser)3)).

[0091] A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include a native sequence human IgG1 Fc region; a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; and a native sequence human IgG4 Fc region and naturally occurring variants thereof. Native sequence Fc encompasses various allotypes of Fc (see, e.g., Jefferis et al. (2009) mAbs 1:1).

[0092] In the context of in vitro or in vivo assays using fusion proteins, the term "EC 50 "" refers to the concentration of the fusion protein that induces a response that is 50% of the maximal response (ie, halfway between the maximal response and the baseline).

[0093] "Conservative amino acid substitutions" refer to replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with the following terms: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, a predicted non-essential amino acid residue in the IL2 / IL2Rα fusion protein is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

[0094] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded and can be a cDNA.

[0095] As used herein, the term "regulatory region" refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region that affect transcription, RNA processing, stability, or translation of the associated coding region. A regulatory region may include a promoter, a translation leader sequence, introns, a polyadenylation recognition sequence, an RNA processing site, an effector binding site, and a stem-loop structure. If the coding region is intended to be expressed in eukaryotic cells, a polyadenylation signal and a transcription termination sequence will typically be located 3' of the coding region.

[0096] A polynucleotide encoding a gene product (e.g., a polypeptide) may include a promoter and / or other transcriptional or translational control elements operably associated with one or more coding regions. In addition to the promoter, other transcriptional control elements such as enhancers, operators, repressors, and transcription termination signals may also be operably associated with the coding region to direct gene product expression.

[0097] A variety of transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (immediate early promoter, together with intron A), simian virus 40 (early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes, such as actin, heat shock protein, bovine growth hormone, and rabbit beta-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers and lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

[0098] Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly internal ribosome entry sites, or IRES, also known as CITE sequences).

[0099] The terms "percentage of sequence identity," "percentage of identity," "sequence identity," or "identity" are used interchangeably and refer to the number of identical matched positions shared between two polynucleotide or polypeptide sequences over the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced to achieve optimal alignment of the two sequences. A matched position is any position where the same nucleotide or amino acid is present in the target and reference sequences. Since gaps are not nucleotides or amino acids, gaps present in the target sequence are not counted. Similarly, since target sequence nucleotides or amino acids are counted and nucleotides or amino acids in the reference sequence are not counted, gaps present in the reference sequence are not counted.

[0100] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0101] The percentage ratio of sequence identity is calculated in the following manner: determine the number of positions where identical amino acid residues or nucleic acid bases occur in the two sequences to obtain the number of positions of coupling, divide the number of positions of coupling by the total number of positions in the comparison window, and then multiply the result by 100 to obtain the sequence identity percentage ratio. The determination of the sequence identity percentage ratio between the comparison of the sequence and the two sequences can be completed using the software online that is easily available and downloading. Suitable software programs can be obtained from various sources, and can be used for comparing proteins and nucleotide sequences. A suitable program for determining sequence identity percentage ratio is bl2seq, which is a part of the BLAST suite of programs that can be obtained from the National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov) of the U.S. government. Bl2seq uses BLASTN or BLASTP algorithms to carry out the comparison between the two sequences. BLASTN is used for comparing nucleic acid sequences, and BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher (part of the EMBOSS suite of bioinformatics programs), also available from the European Bioinformatics Institute (EBI) website www.ebi.ac.uk / Tools / psa.

[0102] Different regions within a single polynucleotide or polypeptide target sequence aligned to a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. It should be noted that percent sequence identity values ​​are rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It should also be noted that length values ​​will always be integers.

[0103] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com) using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)), incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)), which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0104] The nucleic acid and protein sequences described herein can also be used as a "query sequence" to search public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25 (17): 3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0105] Nucleic acids can exist in intact cells, cell lysates, or partially purified or substantially pure forms. A nucleic acid is "isolated" or becomes "substantially pure" when it is purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of chromosomes), or proteins, by standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other methods well known in the art. See F. Ausubel, et al., eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0106] As used herein, the term "vector" is intended to refer to a nucleic acid molecule that can transport another nucleic acid to which it is connected. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which another DNA segment can be connected. Another type of vector is a viral vector, in which another DNA segment can be connected to a viral genome. Some vectors can replicate autonomously in the host cell in which they are introduced (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicate together with the host genome. In addition, some vectors can guide the expression of the gene operably connected thereto. Such vectors are referred to as "recombinant expression vectors" (or simply "expression vectors") in this article. Typically, expression vectors useful in recombinant DNA technology are typically plasmid forms. In this specification sheets, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector forms. However, other forms of expression vectors that play equivalent functions are also included, such as viral vectors (for example, replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0107] As used herein, the term "in vitro host cell" (or simply referred to as "host cell") is intended to refer to a cell comprising a non-natural nucleic acid present in a cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended not only to refer to a specific subject's cell, but also to the progeny of such a cell. Because some modifications may occur in the offspring due to mutation or environmental influences, such progeny may actually be different from the parental cell, but are still included in the scope of the term "host cell" as used herein. Exemplary host cells include, but are not limited to, prokaryotic cells (e.g., Escherichia coli (E. coli)), or alternatively, eukaryotic cells, such as fungal cells (e.g., yeast cells such as Saccharomyces cerevisiae, Pichia pastoris, or Schizosaccharomyces pombe), and various animal cells, such as insect cells (e.g., Sf-9) or mammalian cells (e.g., HEK293F, CHO, COS-7, NIH-3T3).

[0108] As used herein, the phrase "immediately downstream of an amino acid" refers to a position immediately adjacent to the terminal carboxyl group of an amino acid. Similarly, the phrase "immediately upstream of an amino acid" refers to a position immediately adjacent to the terminal amine group of an amino acid. Thus, as used herein, the phrase "between two amino acids of an insertion site" refers to a position at which a heterologous moiety (e.g., a half-life extending moiety) is inserted between two adjacent amino acids.

[0109] As used herein, the term "administer" refers to the physical introduction of a composition comprising a therapeutic agent into a subject using any of the various methods and delivery systems known to those skilled in the art. The different routes of administration of the IL2 / IL2Rα fusion proteins described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" means, in addition to enteral and topical administration, a mode of administration typically by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardial, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. Alternatively, the antibodies described herein can be administered via non-parenteral routes (such as topical, epidermal or mucosal administration routes), such as intranasal, oral, vaginal, rectal, sublingual or topical administration. Administration can also be performed, for example, once, multiple times, and / or over one or more extended time periods.

[0110] "Immune response" is as understood in the art, and generally refers to a biological response in vertebrates to an external factor (agent) or abnormality such as cancer cells, which protects the organism from these factors and the diseases caused therefrom. An immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, which results in selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells in the vertebrate body, or in the case of autoimmune or pathological inflammation, selective targeting, binding, damage, destruction, and / or elimination of normal human cells or tissues. An immune response includes, for example, T cells (e.g., effector T cells, Th cells, CD4 + cells, CD8 + T cells or Treg cells), or the activation or inhibition of any other cells of the immune system (e.g., NK cells).

[0111] An "immunomodulator" ("immunomodulator" or "immunoregulator") refers to an agent, such as an agent that targets a component of a signaling pathway, that can participate in modulating, regulating, or modifying an immune response. "Modulating," "regulating," or "modifying" an immune response refers to any change in the activity of a cell of the immune system or such a cell (e.g., an effector T cell, such as a Th1 cell). More particularly, as used herein, the term "modulating" includes inducing, inhibiting, enhancing, increasing, increasing, or decreasing a given activity or response. Such modulation includes stimulation or inhibition of the immune system, which can be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other changes that may occur within the immune system. Inhibitory and stimulatory immunomodulators have been identified, some of which may have enhanced function in the tumor microenvironment. In some embodiments, the immunomodulator targets molecules on the surface of T cells. An "immunomodulatory target" or "immunoregulatory target" is a molecule (e.g., a cell surface molecule) that is targeted for binding to a substance, agent, moiety, compound, or molecule, and the activity of which is altered by the binding of the substance, agent, moiety, compound, or molecule. Immunomodulatory targets include, for example, receptors on the cell surface ("immunomodulatory receptors") and receptor ligands ("immunomodulatory ligands").

[0112] "Immunotherapy" refers to the treatment of a subject having a disease or at risk of contracting a disease or suffering recurrence of a disease by methods that include inducing, enhancing, suppressing, or otherwise modifying the immune system or immune response.

[0113] "Immunostimulatory therapy" or "immunostimulatory therapy" refers to therapy that results in an increase (induction or enhancement) of an immune response in a subject, eg, to treat cancer.

[0114] "Enhancing an endogenous immune response" means increasing the effectiveness or efficacy of an existing immune response in a subject. This increase in effectiveness and efficacy can be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response or stimulating mechanisms that enhance the endogenous host immune response.

[0115] “T effect” eff ”) cells refer to T cells with cytolytic activity (such as CD4 + and CD8 + T cells) and T helper (Th) cells that secrete cytokines and activate and guide other immune cells, such as Th1 cells, but not regulatory T cells (Treg cells). Certain IL2 / IL2Rα fusion proteins described herein activate T eff cells, such as CD4 + and CD8 + T eff cells and Th1 cells.

[0116] The increased ability to stimulate an immune response or the immune system may be due to enhanced agonist activity of T cell co-stimulatory receptors and / or enhanced antagonist activity of inhibitory receptors. The increased ability to stimulate an immune response or the immune system can be measured by EC in assays that measure immune responses, such as assays that measure changes in cytokine or chemokine release, cytolytic activity (measured directly on target cells or indirectly via detection of CD107a or granzymes), and proliferation. 50 The ability to stimulate an immune response or immune system activity can be enhanced by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold or more.

[0117] As used herein, the terms "connection" and "fusion" refer to that a first amino acid sequence or nucleotide sequence is covalently or non-covalently connected to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid or nucleotide sequence can be directly connected or juxtaposed to the second amino acid or nucleotide sequence, or alternatively, an intervening sequence can covalently connect the first sequence to the second sequence. The term "connection" not only means that the first amino acid sequence is fused to the second amino acid sequence at the C-terminus or N-terminus, but also includes inserting the complete first amino acid sequence (or second amino acid sequence) between any two amino acids in the second amino acid sequence (or correspondingly, the first amino acid sequence). In one embodiment, the first amino acid sequence is connected to the second amino acid sequence through a peptide bond or a linker. The first nucleotide sequence can be connected to the second nucleotide sequence through a phosphodiester bond or a linker. The linker can be a peptide or polypeptide (for a polypeptide chain) or a nucleotide or nucleotide chain (for a nucleotide chain) or any chemical part (for both polypeptide and polynucleotide chains). The term "connection" is also represented by a hyphen (-).

[0118] As used herein, the term "T cell-mediated response" refers to a response mediated by T cells, including effector T cells (e.g., CD8 + cells) and helper T cells (such as CD4 + T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0119] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. CTL responses are primarily induced by CD8 + T cell mediated.

[0120] As used herein, the terms "inhibit" or "block" (e.g., referring to inhibition / blocking of the binding of IL2 to IL2Rα on a cell) are used interchangeably and encompass both partial and complete inhibition / blocking. In some embodiments, the IL2 / IL2Rα fusion protein inhibits the binding of IL2 to IL2Rα by at least about 50%, e.g., about 60%, 70%, 80%, 90%, 95%, 99% or 100%, e.g., as determined further described herein. In some embodiments, the IL2 / IL2Rα fusion protein inhibits the binding of IL2 to IL2Rα by no more than 50%, e.g., about 40%, 30%, 20%, 10%, 5% or 1%, e.g., as determined further described herein.

[0121] As used herein, the phrase "inhibiting the growth of a tumor" includes any measurable decrease in the growth of a tumor, for example, inhibiting the growth of a tumor by at least about 10%, such as at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or 100%.

[0122] As used herein, "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells that invade neighboring tissues and can metastasize to distant parts of the body through the lymphatic system or bloodstream.

[0123] As used herein, the terms "treat," "treating," and "treatment" refer to any type of intervention or process performed on a subject for the purpose of reversing, alleviating, relieving, inhibiting, slowing down, or preventing the progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical markers associated with the disease, or improving overall survival. Treatment can be for a subject with the disease or a subject without the disease (e.g., for prevention). When provided prophylactically, the fusion protein disclosed herein is provided before any symptoms. Prophylactic administration of the substance is used to prevent or alleviate any subsequent symptoms.

[0124] "Enhancing efficacy" or "enhancing immunogenicity" with respect to a fusion protein, pharmaceutical composition, or vaccine is intended to improve results, e.g., as measured by a change in a specific value (e.g., an increase or decrease in a specific parameter of the activity of a fusion protein, pharmaceutical composition, or vaccine associated with protective immunity). In one embodiment, enhancing refers to an increase in a specific parameter by at least 5%, 10%, 25%, 50%, 100%, or greater than 100%. In another embodiment, enhancing refers to a decrease in a specific parameter by at least 5%, 10%, 25%, 50%, 100%, or greater than 100%. In one example, the enhancement of vaccine efficacy / immunogenicity refers to an improvement in the ability of a vaccine to inhibit or treat disease progression, e.g., for this purpose, an increase in the effectiveness of a vaccine by at least 5%, 10%, 25%, 50%, 100%, or greater than 100%. In further examples, enhancement of vaccine efficacy / immunogenicity refers to an improvement in the ability of the vaccine to recruit a subject's natural defenses against an already developed cancer, such as an increase in the effectiveness of the fusion protein, pharmaceutical composition, or vaccine for this purpose by at least 5%, 10%, 25%, 50%, 100%, or greater than 100%.

[0125] Similarly, "overcoming a suppressed immune response" with respect to a fusion protein, pharmaceutical composition, or vaccine is intended to improve an outcome, e.g., as measured by a change in a specific value (e.g., a return to a previous positive value for a specific parameter of vaccine activity associated with protective immunity). In one embodiment, overcoming refers to an increase in a specific parameter by at least 5%, 10%, 25%, 50%, 100%, or greater than 100%. In one example, overcoming a suppressed immune response to a fusion protein, pharmaceutical composition, or vaccine refers to the ability of the fusion protein, pharmaceutical composition, or vaccine to inhibit or treat disease progression, e.g., for this purpose, a return to vaccine effectiveness by at least 5%, 10%, 25%, 50%, 100%, or greater than 100%.

[0126] As used herein (interchangeably), "therapeutically effective amount", "therapeutic dose", "dose", "effective dose", "effective dosage" or "administered amount" means a dose that achieves the therapeutic goals as described herein. In some embodiments, a "therapeutic dose" means a dose that induces immune tolerance in a subject. In certain embodiments, a "therapeutic dose" means a dose that induces immune tolerance in a subject within a specified tolerance time period, for example, within 12 weeks of administration of the first dose. A "therapeutically effective amount" of an IL2 / IL2Rα fusion protein refers to an amount of the IL2 / IL2Rα fusion protein sufficient to elicit a desired biological response. As will be understood by one of ordinary skill in the art, the absolute amount of a particular IL2 / IL2Rα fusion protein that is effective can vary depending on factors such as the desired biological endpoint, the IL2 / IL2Rα fusion protein to be delivered, the target cell or tissue, etc. It will further be understood by one of ordinary skill in the art that an effective amount can be administered in a single dose, or an effective amount can be achieved by administering multiple doses (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses). The ability of a therapeutic agent to promote disease regression or inhibit disease progression or recurrence can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the activity of the agent in in vitro assays.

[0127] As used herein, "treat," "treatment," or "treating" refers to, for example, a decrease in the severity of a disease or condition; a decrease in the duration of the disease; an improvement or elimination of one or more symptoms associated with the disease or condition; providing a beneficial effect to a subject having the disease or condition, but not necessarily a cure for the disease or condition.

[0128] For example, an anticancer agent is a drug that promotes cancer regression in a subject. In some embodiments, a therapeutically effective amount of a drug promotes cancer regression to the extent of eliminating cancer. "Promoting cancer regression" means administering an effective amount of a drug alone or in combination with an antitumor agent to cause a decrease in tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of the disease-free period, the prevention of damage or disability caused by the disease, or the improvement of disease symptoms in other ways in the patient. In addition, the terms "effective" and "effectiveness" for treatment include pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the toxicity level or other adverse physiological effects (adverse effects) at the cell, organ and / or biological level caused by the administration of the drug.

[0129] For the treatment of tumors, for example, a therapeutically effective amount or dose of a drug inhibits cell growth or tumor growth by at least about 20%, at least about 40%, at least about 60%, or at least about 80% relative to an untreated subject. In some embodiments, a therapeutically effective amount or dose of a drug completely inhibits cell growth or tumor growth, i.e., 100% inhibition of cell growth or tumor growth. The ability of a compound to inhibit tumor growth can be evaluated using the assays described below. Alternatively, this property of a composition can be evaluated by examining the ability of a compound to inhibit cell growth, and this inhibition can be measured in vitro by assays known to skilled practitioners. In some embodiments described herein, tumor regression can be observed and persists for a period of at least about 20 days, at least about 40 days, or at least about 60 days.

[0130] The term "patient" includes humans and other mammalian subjects receiving prophylactic or therapeutic treatment.

[0131] As used herein, the term "subject" includes any human or non-human animal. For example, the methods and compositions described herein can be used to treat a subject suffering from cancer. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0132] As used herein, the term "weight-based" dosage or administration means that the dosage administered to a patient is calculated based on the patient's weight. For example, if a 60 kg patient requires 3 mg / kg of anti-IL2 antibody, an appropriate amount of IL2 / IL2Rα fusion protein (i.e., 180 mg) can be calculated and administered.

[0133] With respect to the methods and doses described herein, the term "flat dose" is used to refer to a dose that is administered to a patient without regard to the patient's weight or body surface area (BSA). Thus, a flat dose is not provided as a mg / kg dose, but rather as an absolute amount of an agent (e.g., an IL2 / IL2Rα fusion protein). For example, a 60 kg human and a 100 kg human would receive the same dose of an antibody (e.g., 480 mg of an IL2 / IL2Rα fusion protein).

[0134] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μM," respectively.

[0135] Various aspects described herein are described in further detail in the following subsections.

[0136] References to amino acid numbering of immunoglobulins or immunoglobulin fragments or regions are based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, US Department of Public Health, Bethesda, MD. (FcRn receptors have been isolated from several mammalian species, including humans. The sequences of human FcRn, rat FcRn, and mouse FcRn are known (Story et al., J. Exp. Med. 180:2377 (1994)). Fc can comprise the CH2 and CH3 domains of an immunoglobulin, with or without the hinge region of an immunoglobulin. Exemplary Fc variants are provided in WO 2004 / 101740 and WO 2006 / 074199.

[0137] 7.3 Interleukin-2 / interleukin-2 receptor α fusion protein

[0138] Disclosed herein are fusion proteins comprising at least two components: (a) a first polypeptide comprising an interleukin-2 (IL2) polypeptide; and (b) a second polypeptide comprising the extracellular domain of an interleukin-2 receptor alpha (IL2Rα) polypeptide. In some embodiments, the extracellular domain of the IL2Rα polypeptide has at least one less glycosylation compared to the extracellular domain of native IL2Rα (SEQ ID NO: 7); and / or (ii) the IL2 polypeptide has at least one less glycosylation compared to native IL2 (SEQ ID NO: 2). In some embodiments, the fusion protein has IL2 activity.

[0139] 7.3.1 Interleukin-2

[0140] In some embodiments, a fusion protein is provided, comprising a first polypeptide fused in frame to a second polypeptide, the first polypeptide comprising interleukin-2 (IL2), the second polypeptide comprising or consisting of the extracellular domain of an interleukin-2 receptor alpha (IL2Rα) polypeptide. In some embodiments, the fusion protein comprises a first polypeptide comprising IL2 having SEQ ID NO: 2. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 2.

[0141] In one embodiment, the IL2 polypeptide is native or recombinant IL2 from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), as well as domesticated or agricultural mammals, unless otherwise indicated.

[0142] The IL2 polypeptides useful in the present disclosure are expressed in fusion proteins. The fusion proteins described herein specifically bind to human IL2R, more specifically, specific domains (e.g., functional domains) within the extracellular domain of human IL2Rα. In some embodiments, the fusion protein comprising IL2 is an antagonist. In some embodiments, the fusion protein comprising IL2 binds to human IL2Rα with high affinity.

[0143] The term IL2 encompasses unprocessed IL2 and any form of IL2 (i.e., mature form of IL2) produced by processing in cells. The term also encompasses naturally occurring variants and fragments (e.g., splice variants or allelic variants) of IL2, as well as non-naturally occurring variants. The amino acid sequence of an exemplary mature form of human IL2 (with a 20-amino acid signal sequence) is shown in SEQ ID NO: 2. Unprocessed human IL2 also includes a signal peptide (SEQ ID NO: 1) at the N-terminal 20 amino acids that is not present in the mature IL2 molecule. The amino acid sequence of an exemplary mature form of mouse IL2 (with a 20-amino acid signal sequence) is shown in SEQ ID NO: 4. Unprocessed mouse IL2 also includes a signal peptide (SEQ ID NO: 3) at the N-terminal 20 amino acids that is not present in the mature IL2 molecule. "Native IL2" is also referred to as "wild-type IL2," meaning naturally occurring or recombinant IL2.

[0144] Other nucleic acid and amino acid sequences of IL2 are known. See, for example, GenBank Accession Nos. Q7JFM2 (Aotus lemurinus (gray-bellied night monkey)); Q7JFM5 (Aotus nancymaae (horse night monkey)); P05016 (Bas taurus (cattle)); Q29416 (Dog (Dog) Chinese field dog); P36835 (Caprahircus (goat)); and P37997 (Equus caballus (horse)).

[0145] Also provided are biologically active fragments and variants of IL2. Such IL2 active variants or fragments will retain IL2 activity. The biological activity of IL2 can refer to the ability to stimulate lymphocytes with IL2 receptors. This activity can be measured in vitro and in vivo. IL2 is an overall regulator of immune activity, and the effect seen here is the sum of such activities. For example, it regulates survival activity (Bcl-2), induces T effector activity (IFN-γ, granzyme B and perforin) and promotes T regulatory activity (FoxP3). See, for example, Malek et al. (2010) Immunity 33(2): 153-65.

[0146] Biologically active variants of IL2 are known. See, for example, U.S. Application Publications 2006 / 0269515 and 2006 / 0160187 and WO 99 / 60128.

[0147] Biologically active fragments and variants of IL2 can be used in the fusion proteins disclosed herein. Such functional fragments can comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 75, 100, 125, 150 or more consecutive amino acids of SEQ ID NO: 2. Alternatively, the functional variant can comprise at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 2.

[0148] Further provided are active variants and fragments of polynucleotides encoding IL2 proteins. Such polynucleotides may comprise at least 100, 200, 300, 400, 500, 600, or 700 contiguous nucleotides encoding the polypeptide of SEQ ID NO: 2, and continue to encode a protein having IL2 activity. Alternatively, a functional polynucleotide may comprise at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 2, and continue to encode a functional IL2 polypeptide.

[0149] Exemplary polypeptide sequences of IL2 are listed in Table 1 below.

[0150] Table 1

[0151]

[0152] In some embodiments, the fusion proteins provided herein can comprise at least one mutation within the extracellular domain of IL2Rα. In some embodiments, the IL2 polypeptide has at least one less glycosylation site compared to native IL2 (SEQ ID NO: 2). In some embodiments, the at least one less glycosylation site is due to one or more mutations that remove glycosylation.

[0153] In other embodiments, the fusion protein comprises a mutation that replaces an amino acid having a glycosylation site with an amino acid that does not have a glycosylation site. In some embodiments, the mutation removes O-glycosylation and / or N-glycosylation. In one embodiment, the mutation removes O-glycosylation at amino acid 3 of SEQ ID NO: 2, for example, at threonine. In another embodiment, the mutation removes N-glycosylation.

[0154] In some embodiments, the mutation is one or more substitutions of glycosylated amino acids of IL2 with non-glycosylated amino acids. In some embodiments, the mutation is one or more substitutions of amino acids that allow glycosylation at nearby amino acids of IL2 with amino acids that do not allow glycosylation at nearby amino acids.

[0155] In some embodiments, the one or more substitutions of amino acids of IL2 are from alanine to an amino acid selected from the group consisting of arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0156] In some embodiments, the one or more substitutions of amino acids of IL2 are from threonine to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine.

[0157] In some embodiments, the one or more amino acid substitutions of IL2 are from a reactive amino acid such as cysteine ​​to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, the one or more substitutions are from cysteine ​​to serine. In some embodiments, the one or more substitutions are from cysteine ​​to alanine. In some embodiments, the one or more substitutions are from cysteine ​​to valine.

[0158] In some embodiments, the one or more substitutions are at amino acid T3 of IL2, compared to the corresponding sequence in SEQ ID NO:2.

[0159] In some embodiments, one of the substitutions is at amino acid C125 of SEQ ID NO: 2. In specific embodiments, the substitution at amino acid C125 is selected from C125S, C125A, and C125V.

[0160] In some embodiments, the mutation is a deletion. In specific embodiments, the deletion is at amino acid A1 of SEQ ID NO:2.

[0161] The present disclosure also includes any other mutations of the IL2 polypeptide. In other embodiments, the mutations also include one or more substitutions that improve the properties of IL2, for example, improving IL2 activity, improving IL2 half-life, improving stability, etc.

[0162] As disclosed below in this section, the mutations listed herein are mutations at amino acid positions relative to SEQ ID NO: 2. According to the present invention, any of the mutations described below, alone or in combination with other disclosed mutations or any mutations known in the art, can be used in one or more IL2 fusion proteins described herein.

[0163] In some embodiments, IL2 comprises one or more mutations disclosed in Carmenate et al., J Immunol, 200 (10) 3475-3484 (2018) and / or US 8,759,486: for example, at amino acid residues Q22, Q126, I129, S130, or any combination thereof, for example, Q22V, Q126A, I129D, S130G, or any combination thereof. In some embodiments, IL2 comprises one or more mutations as disclosed in US 8,759,486 B2, including L18N, Q126Y, and S130R. In some embodiments, IL2 comprises one or more mutations as disclosed in US 8,759,486 B2, including Q13Y, Q126Y, I129D, and S130R. In some embodiments, IL2 comprises one or more mutations of K35E, K35D, and K35Q as disclosed in WO 2018 / 091003A1.

[0164] In some embodiments, IL2 comprises one or more mutations disclosed in Epstein et al. Blood, 101(12):4853-61] (2003) and / or US 7,371,371: for example, at amino acid residue R38, e.g., R38W. In some embodiments, IL2 comprises an R38W mutation as disclosed in US 7,371,371 B2 and one or more mutations outside of amino acid positions 22 to 58 of IL2.

[0165] In some embodiments, IL2 comprises one or more mutations disclosed in Wittrup et al. J Immunother. 32(9):887-94(2009) and / or US 8,906,356: for example, amino acid residues 91, 126, or both, for example, V91R, Q126T, or both. In some embodiments, IL2 comprises an E15W mutation as disclosed in Wittrup et al. J Immunother. 32(9):887-94(2009) and also in US 8,906,356. In some embodiments, IL2 comprises one or both mutations as disclosed in Wittrup et al. J Immunother. 32(9):887-94(2009) and also in US 8,906,356, N88R and V91R. In some embodiments, IL2 comprises a Q126T or Q126I mutation as disclosed in Wittrup et al. J Immunother. 32(9):887-94 (2009) and / or US 8,906,356.

[0166] In some embodiments, IL2 comprises one or more mutations disclosed in US 8,906,356 B2: for example, at amino acids 69, 74, 91, 126, or any combination thereof. In some embodiments, the mutation is V91R, Q126T, Q126L, Q127T, or any combination thereof as disclosed in US 8,906,356 B2.

[0167] In some embodiments, IL2 comprises one or more mutations disclosed in Wittrup et al. J Immunother. 32(9):887-94 (2009) and / or US 7,569,215B2: for example, at amino acid residues E15, N30, E68, V69, N71, S75, N90, or any combination thereof, e.g., N30S, E68D, V69A, N71A, S75P, N90H, or any combination thereof. In some embodiments, IL2 comprises an E15W mutation as disclosed in Wittrup et al. Biochemistry, Vol. 44, No. 31 (2005). In some embodiments, the mutation is V69A as disclosed in US 7,569,215B2.

[0168] In some embodiments, IL2 comprises one or more mutations disclosed in Wittrup et al. J Immunother. 32(9):887-94 (2009) and / or US 7,951,360B2: for example, at amino acid residues N29, Y31, K35, T37, K48, V69, N71, N88, or any combination thereof, e.g., N29S, Y31H, K35R, T37A, K48E, V69A, N71R, N88D, or any combination thereof. In some embodiments, IL2 comprises an E15W mutation as disclosed in Wittrup et al. Biochemistry, Vol. 44, No. 31 (2005).

[0169] In some embodiments, IL2 comprises one or more mutations disclosed in Wittrup et al. J Immunother. 32(9):887-94 (2009) and / or US 8,349,311 B2: e.g., at amino acid residues 69, 74, 128, or any combination thereof, e.g., V69A, I128P, or any combination thereof.

[0170] In some embodiments, IL2 comprises one or more mutations disclosed in Wittrup et al. J Immunother. 32(9): 887-94 (2009): e.g., at amino acid residues S4, T10, Q11, V69, N88, T133, or any combination thereof, e.g., S4P, T10A, Q11R, V69A, N88D, T133A, or any combination thereof.

[0171] In some embodiments, IL2 comprises one or more mutations disclosed in Wittrup et al. J Immunother. 32(9):887-94 (2009): e.g., at amino acid residues N30, V69, I128, or any combination thereof, e.g., N30S, V69A, I128T, or any combination thereof.

[0172] In some embodiments, IL2 comprises one or more mutations disclosed in Wittrup et al. J Immunother. 32(9): 887-94 (2009): e.g., at amino acid residues K8, Q13, N26, N30, K35, T37, V69, or any combination thereof, e.g., K8R, Q13R, N26D, N30T, K35R, T37R, V69A, or any combination thereof.

[0173] In some embodiments, IL2 comprises one or more mutations disclosed in Shanafelt et al., Nat Biotechnol., 18(11): 1197-202 (2000), eg, at amino acid residue N88, eg, N88R.

[0174] In some embodiments, IL2 comprises one or more mutations disclosed in US 9,616,105 B2: for example, at amino acid residues 20, 88, 126, or any combination thereof, e.g., N88R, N88G, or N88I. In some embodiments, IL2 comprises an N88R, N88G, or N88I mutation as disclosed in US 9,616,105 B2. In some embodiments, IL2 comprises a D20H, D20I, or D20Y mutation as disclosed in US 9,616,105 B2. In some embodiments, IL2 comprises a Q126L mutation as disclosed in US 9,616,105 B2.

[0175] In some embodiments, IL2 comprises one or more mutations disclosed in US 2018 / 0125941 A1: for example, D20H, N88I, N88G, N88R, Q126L, Q126F, or any combination thereof. In some embodiments, IL2 comprises one or more mutations as disclosed in US 2018 / 0037624 A1: T3A, N88G, N88R, D20H, C125S, Q126L, and Q126F.

[0176] In some embodiments, IL2 comprises one or more mutations disclosed in US 2017 / 0327555 A1: e.g., at amino acid residues N88, D20, C125, Q126, or any combination thereof, e.g., N88G, N88R, D20H, C125S, Q126L, Q126F, or any combination thereof.

[0177] In some embodiments, IL2 comprises one or more mutations disclosed in WO 2016 / 025385 A1, for example, at amino acid residues D109, C125, or both, e.g., D109C, C125S, or both. In some embodiments, IL2 comprises one or more mutations disclosed in WO 2016 / 025385 A1, for example, at amino acid residues D20, N88, Q126, C125, Q126, or any combination thereof, e.g., D20H, N88I, N88G, N88R, Q126L, C125S, Q126F, or any combination thereof.

[0178] In some embodiments, IL2 comprises one or more mutations disclosed in WO 2016 / 164937A1: e.g., at amino acid residues L12, Q13, E15, H16, L19, D20, M23, D84, S87, N88, V91, E95, or any combination thereof, e.g., L12G, L12K, L12Q, L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, or any combination thereof. , L19E, L19G, L19N, L19S, L19T, L19V, D20A, D20E, D20F, D20G, D20T, D20W, M23R, D84A, D84E, D84G, D84I, D84M, D84Q, D84R, D84S, D84T, S87E, N88A, N88D, N88E, N88F, N88G, N88M, N88R, N88S, N88V, N88W, V91D, V91E, V91G, V91S, E95G, or any combination thereof.

[0179] In some embodiments, IL2 comprises one or more mutations disclosed in US Pat. No. 9,932,380 B2 or US Pat. No. 9,580,486: for example, at amino acid residue V91, e.g., V91K. In some embodiments, IL2 further comprises a C125A or C125S mutation. In some embodiments, IL2 further comprises a mutation at T3. In some embodiments, the mutation at T3 is one of T3A or T3N. In some embodiments, IL2 comprises a mutation at S5. In some embodiments, the mutation is S5T.

[0180] In some embodiments, IL2 comprises one or more mutations disclosed in US 9,732,134 B2: eg, E15, H16, Q22, D84, N88, E95, or any combination thereof.

[0181] In some embodiments, IL2 comprises one or more mutations disclosed in US 2015 / 0218260 A1, for example, N88D. In some embodiments, IL2 comprises a mutation disclosed in US 9,266,938 B2, for example, at amino acid residues 42, 45, 72, or any combination thereof, for example, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, or L72K. In some embodiments, IL2 comprises F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, and F42K mutations. In some embodiments, IL2 comprises Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, and Y45K mutations.

[0182] In some embodiments, IL2 comprises one to four mutations: a first mutation: L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, or L72K; a second mutation: F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, or F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, or Y45K; a third mutation: T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, or T3P; and / or a fourth mutation: C125A, C125S, C125T, or C125V. The mutations listed herein or disclosed in the patents / patent publications or any other references cited herein are incorporated by reference in their entirety.

[0183] 7.3.2 Interleukin-2 receptor alpha

[0184] The fusion protein comprises a second polypeptide comprising the extracellular domain of interleukin-2 receptor alpha (IL2Rα). In some embodiments, the extracellular domain of IL2Rα comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 7.

[0185] As used herein, the terms "CD25" or "IL2 receptor alpha", "IL2Rα", "IL2Ra", "IL2-Rα" and "IL2-Ra" refer to any natural or recombinant IL2α from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), as well as domesticated or agricultural mammals, unless otherwise indicated. The terms also encompass naturally occurring variants (e.g., splice variants or allelic variants), or non-naturally occurring variants of IL2Rα. Human IL2 exerts its biological effects via signaling through its receptor system, IL2R. IL2 and its receptor (IL2R) are required for T cell proliferation and other essential functions important for immune responses. IL2R consists of three non-covalently linked type I transmembrane proteins: α (p55), β (p75) and γ (p65) chains. The human IL2Rα chain contains an extracellular domain of 219 amino acids, a transmembrane domain of 19 amino acids and an intracellular domain of 13 amino acids. The secreted extracellular domain of IL2Rα (IL2Rα) can be used in the fusion proteins described herein.

[0186] The amino acid sequence of an exemplary mature form of human IL2Rα is shown in SEQ ID NO: 6. Unprocessed human IL2Rα is shown in SEQ ID NO: 5. The extracellular domain of SEQ ID NO: 5 and / or SEQ ID NO: 6 is shown in SEQ ID NO: 7. The amino acid sequence of an exemplary mature form of mouse IL2Rα is shown in SEQ ID NO: 9. Unprocessed mouse IL2Rα is shown in SEQ ID NO: 8. The extracellular domain of SEQ ID NO: 8 and / or SEQ ID NO: 9 is shown in SEQ ID NO: 10. "Native IL2Rα," also referred to as "wild-type IL2Rα," refers to naturally occurring or recombinant IL2Rα.

[0187] The nucleic acid and amino acid sequences of IL2Rα are known. See, for example, GenBank Accession Nos. NP_001030597.1 (chimpanzee (P. troglodytes)); NP_001028089.1 (rhesus monkey (M. mulatta)); NM_001003211.1 (wolf (C. lupus)); NP_776783.1 (cattle (B. taurus)); NP_032393.3 (mouse (M. musculus)); and NP_037295.1 (rat (R. norvegicus)).

[0188] Unless otherwise indicated, the extracellular domain of IL2Rα as used herein means a functional IL2Rα extracellular domain that performs its normal role in binding to IL2. The term IL2RαEC domain includes functional fragments, variants, analogs or derivatives that retain the function of full-length wild-type IL2RαEC in IL2 binding. The IL2RαEC domain can be a human, porcine, canine, rat or mouse IL2RαEC domain. The phrase "biological activity of the IL2RαEC domain" refers to one or more biological activities of the EC domain of IL2Rα, including but not limited to the ability to enhance intracellular signaling in IL2 receptor-responsive cells. Non-limiting examples of biologically active fragments and variants of the IL2RαEC domain are disclosed in, for example, Robb et al., Proc. Natl. Acad. Sci. USA, 85: 5654-5658, 1988. In some embodiments, the biologically active fragments and variants of the IL2RαEC domain disclosed herein comprise at least one less glycosylation compared to the extracellular domain of native IL2Rα.

[0189] Biologically active fragments and variants of the extracellular domain of IL2Rα can be used in the fusion proteins disclosed herein. Such functional fragments can comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 215 or more consecutive amino acids of the extracellular domain of any one of SEQ ID NO: 7. Alternatively, the functional variant can comprise at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 7.

[0190] Further provided are active variants and fragments of polynucleotides encoding the extracellular domain of IL2Rα. Such polynucleotides may comprise at least 100, 200, 300, 400, 500, 600, or more consecutive nucleotides encoding the polypeptide of SEQ ID NO: 7, and go on to encode a protein having the activity of the extracellular domain of IL2Rα. Alternatively, the functional polynucleotide may comprise at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 7, and go on to encode a protein having the activity of the extracellular domain of IL2Rα.

[0191] The polypeptide sequence of IL2Rα is listed in Table 2.

[0192] Table 2.

[0193]

[0194]

[0195] In some embodiments, the fusion proteins provided herein can comprise at least one mutation within the EC domain of IL2Rα.

[0196] In some embodiments, the EC domain of the IL2Rα polypeptide is at least one less glycosylated, at least two less glycosylated, at least three less glycosylated, at least four less glycosylated, at least five less glycosylated, at least six less glycosylated, at least seven less glycosylated, at least eight less glycosylated, or at least nine less glycosylated compared to the extracellular domain of native IL2Rα (SEQ ID NO: 7).

[0197] In some embodiments, the EC domain of at least one glycosylated IL2Rα polypeptide comprises a mutation that eliminates glycosylation. In other embodiments, the fusion protein comprises a mutation that replaces an amino acid that has a glycosylation site with an amino acid that does not have a glycosylation site. In some embodiments, the mutation eliminates O-glycosylation and / or N-glycosylation. In one embodiment, the mutation eliminates O-glycosylation. In another embodiment, the mutation eliminates N-glycosylation.

[0198] In some embodiments, the mutation in the fusion protein comprises a deletion of the C-terminus of IL2Rα. In some embodiments, the mutation is a deletion of amino acids 167 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 168 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 169 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 170 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 171 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 172 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 173 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 174 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 175 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 176 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 177 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 178 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 179 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 180 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 181 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 182 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 183 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 184 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 185 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 186 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 187 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 188 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 189 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 190 to 219 of SEQ ID NO: 7.In some embodiments, the mutation is a deletion of amino acids 191 to 219 of SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 192 to 219 of SEQ ID NO: 7.

[0199] In some embodiments, the mutation is a deletion corresponding to amino acids 167, 168, 169, or 171 through 192 to 219 of SEQ ID NO: 7. In some embodiments, the mutation does not include a deletion corresponding to 170 to 219 of SEQ ID NO: 7.

[0200] In some embodiments, the second polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 11. In other embodiments, the second polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 11 and +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, or +25 amino acids. In some embodiments, the second polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 11 and no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids. In some embodiments, the second polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 12.

[0201] In some embodiments, the fusion protein comprises one or more mutations. In some embodiments, the one or more mutations are one or more substitutions of a glycosylated amino acid of IL2Rα with a non-glycosylated amino acid.

[0202] In some embodiments, the one or more substitutions of amino acids of IL2Rα are at amino acid N49, amino acid N68, amino acid T74, amino acid T85, amino acid T197, amino acid T203, amino acid T208, and amino acid T216, or any combination thereof, wherein the amino acid positions correspond to SEQ ID NO: 7.

[0203] In some embodiments, the one or more substitutions are from asparagine to another amino acid. In some embodiments, the one or more substitutions are from asparagine to an amino acid selected from the group consisting of alanine, threonine, serine, arginine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine.

[0204] In some embodiments, the one or more substitutions are from threonine to another amino acid. In some embodiments, the one or more substitutions are from threonine to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine.

[0205] In some embodiments, the substitution is amino acid N49 of SEQ ID NO: 7. In some embodiments, amino acid N49 of SEQ ID NO: 7 is mutated to an amino acid selected from the group consisting of alanine, threonine, serine, arginine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine.

[0206] In some embodiments, the substitution is amino acid N68 of SEQ ID NO: 7. In some embodiments, amino acid N68 of SEQ ID NO: 7 is mutated to an amino acid selected from the group consisting of alanine, threonine, serine, arginine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine.

[0207] In some embodiments, the substitution is amino acid T74 of SEQ ID NO: 7. In some embodiments, amino acid T74 of SEQ ID NO: 7 is mutated to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine.

[0208] In some embodiments, the substitution is amino acid T85 of SEQ ID NO: 7. In some embodiments, amino acid T85 of SEQ ID NO: 7 is mutated to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine.

[0209] In some embodiments, the substitution is amino acid T197 of SEQ ID NO: 7. In some embodiments, amino acid T197 of SEQ ID NO: 7 is mutated to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine.

[0210] In some embodiments, the substitution is amino acid T203 of SEQ ID NO: 7. In some embodiments, amino acid T203 of SEQ ID NO: 7 is mutated to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine.

[0211] In some embodiments, the substitution is amino acid T208 of SEQ ID NO: 7. In some embodiments, amino acid T208 of SEQ ID NO: 7 is mutated to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine.

[0212] In some embodiments, the substitution is amino acid T216 of SEQ ID NO: 7. In some embodiments, amino acid T216 of SEQ ID NO: 7 is mutated to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine.

[0213] In some embodiments, the fusion protein comprises one or more mutations. In some embodiments, the one or more mutations are one or more substitutions of an amino acid of IL2Rα that allows glycosylation at a nearby amino acid with an amino acid that does not allow glycosylation at the nearby amino acid.

[0214] In some embodiments, the substitution is at amino acid S50, amino acid S51, amino acid T69, amino acid T70, amino acid C192, or any combination thereof, wherein the amino acid position corresponds to SEQ ID NO:7.

[0215] In some embodiments, the substitution is to amino acid S50 corresponding to SEQ ID NO: 7. In some embodiments, amino acid S50 corresponding to SEQ ID NO: 7 is mutated to proline.

[0216] In some embodiments, the substitution is with amino acid S51 corresponding to SEQ ID NO: 7. In some embodiments, amino acid S51 corresponding to SEQ ID NO: 7 is mutated to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine.

[0217] In some embodiments, the substitution is to amino acid T69 corresponding to SEQ ID NO: 7. In some embodiments, amino acid T69 corresponding to SEQ ID NO: 7 is mutated to proline.

[0218] In some embodiments, the substitution is with amino acid T70 corresponding to SEQ ID NO: 7. In some embodiments, amino acid T70 corresponding to SEQ ID NO: 7 is mutated to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, and valine.

[0219] In some embodiments, the substitution is with amino acid C192 corresponding to SEQ ID NO: 7. In some embodiments, amino acid C192 corresponding to SEQ ID NO: 7 is mutated to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0220] 7.3.3 Connectors

[0221] The fusion protein of the present disclosure may further comprise a linker. In some embodiments, the linker can connect the first polypeptide to the second polypeptide from the N-terminus to the C-terminus, for example, N-IL2-linker-IL2RαEC-C. In other embodiments, the linker can connect the second polypeptide to the first polypeptide from the N-terminus to the C-terminus, for example, N-IL2RαEC-linker-IL2-C.

[0222] In one embodiment, the IL2 / IL2Rα fusion protein comprises a linker sequence positioned between the IL2 polypeptide and the IL2Rα polypeptide. The linker can be of any length and can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, or 60 or more amino acids. In other embodiments, the joint for the present disclosure has at least one amino acid and is less than 100 amino acid, less than 90 amino acid, less than 80 amino acid, less than 70 amino acid, less than 60 amino acid, less than 50 amino acid, less than 40 amino acid, less than 30 amino acid, less than 20 amino acid, less than 19 amino acid, less than 18 amino acid, less than 17 amino acid, less than 16 amino acid, less than 15 amino acid, less than 14 amino acid, less than 13 amino acid or less than 12 amino acid. In one embodiment, the joint sequence comprises a glycine amino acid residue. In other cases, the joint sequence comprises a combination of glycine and serine amino acid residues.

[0223] In some embodiments, the fusion protein comprises a linker fused in frame between the first polypeptide and the second polypeptide. In some embodiments, the fusion protein comprises a linker that is a glycine / serine linker. Such glycine / serine linkers can comprise any combination of amino acid residues, including but not limited to the peptide GGGS (SEQ ID NO: 174) or GGGGS (SEQ ID NO: 72) or repeats thereof, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats of these given peptides. The glycine / serine linkers disclosed herein comprise (GS) n 、(GGS) n 、(GGGS) n 、(GGGGS) n or (GGGGS) nwherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In a specific embodiment, the linker sequence comprises GGGSGGGSGGGS (SEQ ID NO: 71) (also referred to as (Gly3Ser)3). In another embodiment, the linker sequence comprises GGGSGGGSGGGSGGGS (SEQ ID NO: 74) (also referred to as (Gly3Ser)4). In other embodiments, the linker sequence comprises one of (Gly3Ser)5(GGGSGGGSGGGSGGGSGGGS) (SEQ ID NO: 75), (Gly3Ser)6(GGGSGGGSGGGSGGGSGGGSGGGS) (SEQ ID NO: 76) or (Gly3Ser)7(GGGSGGGSGGGSGGGSGGGSGGGSGGGS) (SEQ ID NO: 77). In other embodiments, the linker sequence comprises (Gly4Ser)3(GGGGSGGGGSGGGGS), as shown in SEQ ID NO:78. In further embodiments, the linker sequence comprises GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:79) (also referred to as (Gly4Ser)4); GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:80) (also referred to as (Gly4Ser)5); (Gly4Ser)2(GGGGSGGGGS) (SEQ ID NO:81), (Gly4Ser)1(GGGGS) (SEQ ID NO:82), (Gly4Ser)6(GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS) (SEQ ID NO:83); (Gly4Ser)7(GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS) (SEQ ID NO:97); or (Gly4Ser)5(GGGGSGGGGSGGGGSGGGGSGGGGS) (SEQ ID NO:84).

[0224] 7.3.4 Heterogeneous parts

[0225] The fusion proteins of the present disclosure may further comprise additional elements, such as heterologous moieties. Such elements can aid in the expression of the fusion protein, aid in the secretion of the fusion protein, improve the stability of the fusion protein, allow for more effective protein purification, and / or modulate the activity of the fusion protein. In some embodiments, the heterologous moiety is a polypeptide moiety. In other embodiments, the heterologous moiety is a non-polypeptide moiety.

[0226] In some embodiments, the fusion protein comprises a heterologous portion fused to a first polypeptide. In some embodiments, the fusion protein comprises a heterologous portion fused to a second polypeptide. In some embodiments, the fusion protein comprises a heterologous portion fused to a first polypeptide and a second polypeptide.

[0227] In some embodiments, the fusion proteins disclosed herein comprise one or more additional heterologous moieties. In some embodiments, the heterologous moiety is a half-life extending moiety. In some embodiments, the heterologous moiety comprises albumin, an immunoglobulin constant region or portion thereof, an immunoglobulin binding polypeptide, immunoglobulin G (IgG), an albumin binding polypeptide (ABP), a PASylated moiety, a HESylated moiety, an XTEN, a PEGylated moiety, an Fc region, and any combination thereof.

[0228] 1) Immunoglobulin constant region or part thereof

[0229] The immunoglobulin constant region is composed of domains denoted as CH (constant heavy) domains (CH1, CH2, etc.). Depending on the isotype (i.e., IgG, IgM, IgA, IgD, or IgE), the constant region may be composed of three or four CH domains. Some isotypes (e.g., IgG) constant regions also contain a hinge region. See Janeway et al., 2001, Immunobiology, Garland Publishing, New York, NY.

[0230] The immunoglobulin constant region or its part for producing fusion protein of the present disclosure can be obtained from a variety of different sources. In a preferred embodiment, the immunoglobulin constant region or its part is derived from human immunoglobulin. However, it should be understood that the immunoglobulin constant region or its part can be derived from the immunoglobulin of another mammalian species, and the another mammalian species includes for example rodent (for example, mouse, rat, rabbit, guinea pig) or non-human primate (for example, chimpanzee, macaque) species. In addition, the immunoglobulin constant region or its part can be derived from any immunoglobulin classification (including IgM, IgG, IgD, IgA and IgE) and any immunoglobulin isotype (including IgG1, IgG2, IgG3 and IgG4). In one embodiment, human isotype IgG1 is used.

[0231] Multiple immunoglobulin constant region gene sequences (e.g., human constant region gene sequences) can be obtained in the form of publicly available deposits. Constant region domain sequences having specific effector functions (or lacking specific effector functions) or having specific modifications can be selected to reduce immunogenicity. The sequences of many antibodies and antibody encoding genes have been disclosed, and suitable Ig constant region sequences (e.g., hinge, CH2 and / or CH3 sequences or portions thereof) can be derived from these sequences using techniques recognized in the art. The genetic material obtained using any of the aforementioned methods can then be altered or synthesized to obtain the polypeptides of the present disclosure. It should be further understood that the scope of the present disclosure encompasses alleles, variants, and mutations of constant region DNA sequences.

[0232] The sequence of the immunoglobulin constant region or a portion thereof can be cloned, for example, using the polymerase chain reaction and selected primers for amplifying the domain of interest. To clone the sequence of the immunoglobulin constant region or a portion thereof from an antibody, mRNA can be isolated from a hybridoma, spleen, or lymphocytes, reverse transcribed into DNA, and the antibody gene amplified by PCR. The PCR amplification method is described in detail in U.S. Patent Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; and described in, for example, "PCR Protocols: A Guide to Methods and Applications" (Innis et al., eds., Academic Press, San Diego, CA (1990); Ho et al. 1989. Gene 77:51; Horton et al. 1993. Methods Enzymol. 217:270). PCR can be initiated with a consensus constant region primer or with a more specific primer based on the disclosed heavy and light chain DNA and amino acid sequences. As discussed above, PCR can also be used to separate DNA clones encoding antibody light and heavy chains. In this case, the library can be screened by common primers or larger homologous probes (such as mouse constant region probes). A plurality of primer sets suitable for amplifying antibody genes are known in the art (e.g., 5' primers based on the N-terminal sequence of purified antibodies (Benhar and Pastan.1994.Protein Engineering 7:1509); Rapid amplification of cDNA ends (Ruberti, F. et al. 1994.J.Immunol.Methods 173:33); Antibody leader sequences (Larrick et al. 1989Biochem.Biophys.Res.Commun.160:1250)). The cloning of antibody sequences is further described in U.S. Patent No. 5,658,570, filed by Newman et al. on January 25, 1995.

[0233] The immunoglobulin constant region used herein can include all domains and hinge region or part thereof.In one embodiment, the immunoglobulin constant region or part thereof comprises CH2 domain, CH3 domain and hinge region, ie, Fc region or FcRn binding partner.

[0234] As used herein, the term "Fc region" is defined as that portion of a polypeptide that corresponds to the Fc region of a native immunoglobulin, i.e., as formed by the dimeric association of the corresponding Fc domains of its two heavy chains. A native Fc region forms a homodimer with another Fc region.

[0235] In one embodiment, the "Fc region" refers to the portion of a single immunoglobulin heavy chain that begins at the hinge region immediately upstream of the papain cleavage site (i.e., residue 216 in IgG, with the first residue of the heavy chain constant region being 114) and ends at the C-terminus of the antibody. Thus, a complete Fc domain comprises at least the hinge domain, the CH2 domain, and the CH3 domain.

[0236] Depending on the immunoglobulin isotype, the Fc region of the immunoglobulin constant region can include CH2, CH3 and CH4 domains and a hinge region. Fusion proteins comprising the Fc region of an immunoglobulin confer several desired properties on the fusion protein, including increased stability, increased serum half-life (see Capon et al., 1989, Nature 337:525) and binding to Fc receptors such as neonatal Fc receptors (FcRn) (U.S. Patent numbers 6,086,875, 6,485,726, 6,030,613; WO 03 / 077834; US2003-0235536A1).

[0237] In some embodiments, the "Fc region" includes an Fc domain or an amino acid sequence derived from an Fc domain. In certain embodiments, the Fc region includes at least one of the following: a hinge (e.g., upper, middle, and / or lower hinge region) domain (about amino acids 216-230 in the antibody Fc region according to EU numbering), a CH2 domain (about amino acids 231-340 in the antibody Fc region according to EU numbering), a CH3 domain (about amino acids 341-438 in the antibody Fc region according to EU numbering), a CH4 domain, or a variant, portion, or fragment thereof. In other embodiments, the Fc region includes a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In some embodiments, the Fc region comprises, consists essentially of, or consists of a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), a hinge domain (or a portion thereof) fused to a CH2 domain (or a portion thereof), a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), or a CH2 domain (or a portion thereof) fused to a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In yet other embodiments, the Fc region lacks at least a portion of a CH2 domain (e.g., all or a portion of a CH2 domain). In specific embodiments, the Fc region comprises or consists of amino acids corresponding to EU numbering 221 to 447.

[0238] The Fc domains represented herein as F, F1 or F2 can be obtained from many different sources. In one embodiment, the Fc region of the polypeptide is derived from human immunoglobulin. However, it should be understood that the Fc region can be derived from the immunoglobulin of another mammalian species, including, for example, rodents (e.g., mice, rats, rabbits, guinea pigs) or non-human primates (e.g., chimpanzees, macaques) species. In addition, the polypeptide of the Fc domain or a portion thereof can be derived from any immunoglobulin class (including IgM, IgG, IgD, IgA and IgE) and any immunoglobulin isotype (including IgG1, IgG2, IgG3 and IgG4). In another embodiment, human isotype IgG1 is used.

[0239] In certain embodiments, the Fc variants confer a change in at least one effector function conferred by an Fc region comprising the wild-type Fc domain (e.g., an improvement or reduction in the ability of the Fc region to bind to an Fc receptor (e.g., FcγRI, FcγRII, or FcγRIII) or a complement protein (e.g., C1q), or to trigger antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC)). In other embodiments, the Fc variants provide engineered cysteine ​​residues.

[0240] The Fc regions of the present disclosure can utilize art-recognized Fc variants known to confer alterations (eg, enhancement or reduction) in effector function and / or FcR binding. In particular, the binding molecules of the present disclosure may include changes (e.g., substitutions) at one or more amino acid positions disclosed, for example, in International PCT Publications WO 88 / 07089 A1, WO 96 / 14339 A1, WO 98 / 05787 A1, WO 98 / 23289 A1, WO 99 / 51642 A1, WO 99 / 58572 A1, WO 00 / 09560 A2, WO 00 / 32767 A1, WO 00 / 42072 A2, WO 02 / 44215 A2, WO 02 / 060919 A2, WO 03 / 074569 A2, WO 04 / 016750 A2, WO 04 / 029207 A2, WO 04 / 035752 A2, WO 04 / 063351A2, WO 04 / 074455A2, WO 04 / 099249A2, WO 05 / 040217A2, WO 04 / 044859, WO 05 / 070963A1, WO 05 / 077981A2, WO 05 / 092925A2, WO 05 / 123780A2, WO 06 / 019447A1, WO 06 / 047350A2, and WO 06 / 085967A2; U.S. Patent Publication Nos. US 2007 / 0231329, US 2007 / 0231329, US 2007 / 0237765, US 2007 / 0237766, US 2007 / 0237767, US 2007 / 0243188, US 20070248603, US 20070286859, US 20080057056; or U.S. Patents 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; 7,083,784; 7,404,956 and 7,317,091. In one embodiment, a specific change can be made at one or more disclosed amino acid positions (e.g., a specific substitution of one or more amino acids disclosed in the art). In another embodiment, a different change can be made at one or more disclosed amino acid positions (e.g., a different substitution of one or more amino acid positions disclosed in the art).

[0241] The Fc region of IgG can be modified according to recognized procedures (such as site-directed mutagenesis, etc.) to produce modified IgG or Fc fragments or portions thereof that will be bound by Fc receptors. Such modifications include modifications away from the Fc receptor contact sites as well as modifications within the contact sites that retain or even enhance binding to the Fc receptor. For example, human IgG1 can be substituted without significantly losing the binding affinity of the Fc to the Fc receptor. The following single amino acid residues in Fc (Fcγ1): P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309 A. Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, P331A, E33 3A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K 360A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A and K447A, wherein, for example, P238A represents a substitution of wild-type proline by alanine at position number 238. As an example, a specific embodiment incorporates the N297A mutation to remove a highly conserved N-glycosylation site. At the positions specified above, in addition to alanine, wild-type amino acids can be substituted with other amino acids. Mutations can be introduced into Fc to generate more than one hundred Fc regions that are different from native Fc. Additionally, combinations of two, three, or more of these individual mutations can be introduced together, generating hundreds of additional Fc regions.Furthermore, one Fc region of a construct of the disclosure may be mutated without mutating the other Fc region of the construct, or both may be mutated but with different mutations.

[0242] Some of the above mutations can confer new functions to the Fc region. For example, one embodiment incorporates N297A, thereby removing a highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby enhancing the circulating half-life of the Fc region and rendering the Fc region unable to bind to FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA without compromising affinity (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632; Shields et al. 1995, J.Biol.Chem. 276:6591). As another example of new functions resulting from the above mutations, affinity for Fc receptors can be increased, in some cases exceeding wild-type affinity. This increased affinity can reflect an increased "association" rate, a decreased "dissociation" rate, or both an increased "association" rate and a decreased "dissociation" rate. Examples of mutations believed to confer increased affinity for Fc receptors include, but are not limited to, T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).

[0243] In addition, at least three human Fcγ receptors appear to recognize a binding site on IgG within the lower hinge region, typically amino acids 234-237. Thus, another example of novel function and potentially reduced immunogenicity can be generated from mutations in this region, such as by replacing amino acids 233-236 "ELLG" of human IgG1 with the corresponding sequence "PVA" from IgG2 (one amino acid deletion). It has been shown that when such mutations have been introduced, FcγRI, FcγRII, and FcγRIII, which mediate various effector functions, do not bind to IgG1. Ward and Ghetie 1995, Therapeutic Immunology 2:77; and Armour et al. 1999, Eur. J. Immunol. 29:2613.

[0244] In one embodiment, the immunoglobulin constant region or portion thereof, e.g., an Fc region, is a polypeptide comprising the sequence of PKNSSMISNTP (SEQ ID NO: 98), and optionally further comprises a sequence selected from the group consisting of HQSLGTQ (SEQ ID NO: 93), HQNLSDGK (SEQ ID NO: 94), HQNISDGK (SEQ ID NO: 95), or VISSHLGQ (SEQ ID NO: 96) (U.S. Pat. No. 5,739,277).

[0245] In certain embodiments, the immunoglobulin constant region or a portion thereof is hemiglycosylated. For example, a fusion protein comprising two Fc regions or FcRn binding partners may comprise a first glycosylated Fc region (e.g., a glycosylated CH2 region) and a second non-glycosylated Fc region (e.g., a non-glycosylated CH2 region). In one embodiment, a linker may be inserted between the glycosylated and non-glycosylated Fc regions. In another embodiment, the Fc region or FcRn binding partner is fully glycosylated, i.e., all Fc regions are glycosylated. In other embodiments, the Fc region may be non-glycosylated, i.e., no Fc portion is glycosylated.

[0246] In certain embodiments, the fusion proteins of the present disclosure comprise amino acid substitutions to an immunoglobulin constant region or portion thereof (e.g., Fc variants) that alter the antigen-independent effector functions of the Ig constant region, particularly the circulating half-life of the protein.

[0247] When compared with the protein lacking these substitutions, such protein shows the combination of the increase or decrease with Fc acceptors, therefore has the half-life of increase or decrease in serum respectively.The Fc variant with improved affinity to Fc acceptors is expected to have a longer serum half-life, and such molecules have useful applications in the method for treating mammals, in which the polypeptide given is required to have a long half-life such as to treat chronic diseases or obstacles (see, e.g., U.S. Patent numbers 7,348,004,7,404,956 and 7,862,820).On the contrary, the Fc variant with reduced Fc receptor binding affinity is expected to have a shorter half-life, and such molecules can also be used for example to give to mammals, and the circulation time shortened in the mammals may be advantageous, e.g., for in vivo diagnostic imaging or when the initial polypeptide is present in the circulation for a long time with toxic side effects. The Fc variant with reduced Fc receptor binding affinity is also unlikely to pass through the placenta, therefore can also be used for treating diseases or obstacles in pregnant women. In addition, other applications of Fc receptor binding affinity that may need to be reduced include those that need to be localized to the brain, kidney and / or liver. In an exemplary embodiment, the fusion protein of the present disclosure presents a reduction in transport from the vasculature through the glomerular epithelium. In another embodiment, the fusion protein of the present disclosure presents a reduction in transport from the brain through the blood-brain barrier (BBB) ​​into the vascular space. In one embodiment, the protein with altered Fc receptor binding is included in at least one Fc region (e.g., one or two Fc regions) with one or more amino acid substitutions within the "Fc receptor binding loop" of the Ig constant region. The Fc receptor binding loop is composed of amino acid residues 280-299 (according to EU numbering) of the wild-type full-length Fc region. In other embodiments, the Ig constant regions of the present disclosure, or portions thereof, with altered Fc receptor binding affinity comprise at least one Fc region having one or more amino acid substitutions at amino acid positions corresponding to any of the following EU positions: 256, 277-281, 283-288, 303-309, 313, 338, 342, 376, 381, 384, 385, 387, 434 (e.g., N434A or N434K), and 438. Exemplary amino acid substitutions that alter Fc receptor binding activity are disclosed in International PCT Publication No. WO 05 / 047327.

[0248] 2) Albumin or its fragments or variants

[0249] In certain embodiments, the heterologous moiety linked to the IL2 polypeptide and / or IL2Rα EC domain is albumin or a functional fragment thereof.

[0250] Human serum albumin (HSA or HA) is a 609 amino acid protein in its full-length form that is responsible for most of the osmotic pressure of serum and also serves as a carrier for endogenous and exogenous ligands. As used herein, the term "albumin" includes full-length albumin or a functional fragment, variant, derivative or analog thereof.

[0251] In one embodiment, the fusion protein comprises an IL2 polypeptide and an IL2RαEC domain as described herein, and albumin, a fragment or variant thereof, wherein the IL2 polypeptide is linked to the albumin, or a fragment or variant thereof. In another embodiment, the fusion protein comprises an IL2 polypeptide and an IL2RαEC domain as described herein, and albumin, a fragment or variant thereof, wherein the IL2RαEC is linked to the albumin, or a fragment or variant thereof.

[0252] In other embodiments, the heterologous moiety linked to the IL2 polypeptide and IL2Rα EC domain is albumin, or a fragment or variant thereof, which extends (or is capable of extending) the half-life of the IL2 polypeptide and IL2Rα EC domain. Additional examples of albumin, or fragments or variants thereof, are disclosed in U.S. Patent Publication Nos. 2008 / 0194481 A1, 2008 / 0004206 A1, 2008 / 0161243 A1, 2008 / 0261877 A1, or 2008 / 0153751 A1, or PCT Application Publication Nos. 2008 / 033413 A2, 2009 / 058322 A1, or 2007 / 021494 A2.

[0253] 3) Albumin binding fraction

[0254] In certain embodiments, the heterologous moiety linked to the IL2 polypeptide and IL2Rα EC domain is an albumin binding moiety comprising an albumin binding peptide, a bacterial albumin binding domain, an albumin binding antibody fragment, or any combination thereof. For example, the albumin binding protein can be a bacterial albumin binding protein, an antibody, or an antibody fragment, including a domain antibody (see U.S. Patent No. 6,696,245). For example, the albumin binding protein can be a bacterial albumin binding domain, such as a Streptococcal protein G (Konig, T. and Skerra, A. (1998) J. Immunol. Methods 218, 73-83). Other examples of albumin binding peptides that can be used as conjugation partners are, for example, those having the consensus sequence Cys-Xaa1-Xaa2-Xaa3-Xaa4-Cys, wherein Xaa1 is Asp, Asn, Ser, Thr or Trp; Xaa2 is Asn, Gln, His, Ile, Leu or Lys; Xaa3 is Ala, Asp, Phe, Trp or Tyr; and Xaa4 is Asp, Gly, Leu, Phe, Ser or Thr, as described in U.S. patent application 2003 / 0069395 or in Dennis et al. (Dennis et al. (2002) J. Biol. Chem. 277, 35035-35043).

[0255] 4) PAS sequence

[0256] In other embodiments, the heterologous moiety linked to the IL2 polypeptide and IL2RαEC domain is a PAS sequence.In one embodiment, the fusion protein comprises an IL2 polypeptide and an IL2RαEC domain as described herein and a PAS sequence, wherein the IL2 polypeptide and / or IL2RαEC domain is linked to the PAS sequence.

[0257] As used herein, PAS sequence means an amino acid sequence that mainly comprises Ala and Ser residues or mainly comprises Ala, Ser and Pro residues, and said amino acid sequence forms a random spiral conformation under physiological conditions. Therefore, the PAS sequence is a structural unit, amino acid polymer or a sequence box that comprises Ala, Ser and Pro, is essentially composed of said amino acid or is composed of said amino acid, which can be used as a part for heterologous part in the fusion protein. However, the technician knows that when adding the residue except Ala, Ser and Pro as the minor component in the PAS sequence, amino acid polymer can also form a random spiral conformation. As used herein, the term "minor component" means that amino acids other than alanine, serine and proline can be added to the PAS sequence to a certain extent, for example, up to about 12%, i.e., about 12 out of 100 amino acids of the PAS sequence; up to about 10%, i.e., about 10 out of 100 amino acids of the PAS sequence; up to about 9%, i.e., about 9 out of 100 amino acids; up to about 8%, i.e., about 8 out of 100 amino acids; about 6%, i.e., about 6 out of 100 amino acids; about 5%, i.e., about 5 out of 100 amino acids; about 4%, i.e., about 4 out of 100 amino acids; about 3%, i.e., about 3 out of 100 amino acids; about 2%, i.e., about 2 out of 100 amino acids; about 1%, i.e., about 1 out of 100 amino acids. Amino acids other than alanine, serine and proline may be selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr and Val.

[0258] Under physiological conditions, the PAS sequence segments form a random helical conformation, which can mediate increased in vivo and / or in vitro stability of the IL2 polypeptide. Because the random helical domain itself does not possess a stable structure or function, the biological activity mediated by the IL2 polypeptide and the fused IL2Rα EC domain is essentially retained. In other embodiments, the PAS sequence forming the random helical domain is biologically inert yet still biodegradable, particularly with respect to proteolysis in plasma, immunogenicity, isoelectric point / electrostatic behavior, binding to cell surface receptors, or internalization, offering significant advantages over synthetic polymers such as PEG.

[0259] Non-limiting examples of PAS sequences that form a random helical conformation include an amino acid sequence selected from the group consisting of ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 85), AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 86), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 87), APSSPSPSAPSSPSPASPS (SEQ ID NO: 88), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 89), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 90), and ASAAAPAAASAAASAPSAAA (SEQ ID NO: 91), or any combination thereof. Other examples of PAS sequences are known from, for example, U.S. Patent Publication No. 2010 / 0292130 A1 and PCT Application Publication No. WO 2008 / 155134 A1.

[0260] 5) HAP sequence

[0261] In certain embodiments, the heterologous moiety linked to the IL2 polypeptide and IL2RαEC domain is a glycine-rich homoamino acid polymer (HAP). The HAP sequence can comprise a repeating sequence of glycine that is at least 50 amino acids, at least 100 amino acids, 120 amino acids, 140 amino acids, 160 amino acids, 180 amino acids, 200 amino acids, 250 amino acids, 300 amino acids, 350 amino acids, 400 amino acids, 450 amino acids, or 500 amino acids in length. In one embodiment, the HAP sequence is capable of extending the half-life of the moiety fused to or linked to the HAP sequence. Non-limiting examples of HAP sequences include, but are not limited to, (Gly) n 、(Gly4Ser) n or S(Gly4Ser) n , wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In one embodiment, n is 20, 21, 22, 23, 24, 25, 26, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In another embodiment, n is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200.

[0262] 6) Transferrin or its fragments

[0263] In certain embodiments, the heterologous moiety connected to the IL2 polypeptide and the IL2RαEC domain is transferrin or a fragment thereof. Any transferrin can be used to prepare the fusion protein of the present disclosure. As an example, wild-type human Tf (Tf) is a 679 amino acid protein, approximately 75 kDa (excluding glycosylation), with two major domains N (about 330 amino acids) and C (about 340 amino acids) that appear to be derived from gene duplication. See GenBank accession numbers NM001063, XM002793, M12530, XM039845, XM 039847, and S95936 (www.ncbi.nlm.nih.gov / ). Transferrin comprises two domains, the N domain and the C domain. The N domain comprises two subdomains, the N1 domain and the N2 domain, and the C domain comprises two subdomains, the C1 domain and the C2 domain.

[0264] In one embodiment, the transferrins,iron complexes part of the fusion rotein comprises a transferrins,iron complexes splice variant. In one example, the transferrins,iron complexes splice variant can be a splice variant of human transferrins,iron complexes, such as Genbank accession number AAA61140. In another embodiment, the transferrins,iron complexes part of the fusion rotein comprises one or more domains of a transferrins,iron complexes sequence, such as N domain, C domain, N1 domain, N2 domain, C1 domain, C2 domain or its any combination.

[0265] 7) Polymers, such as polyethylene glycol (PEG)

[0266] In other embodiments, the heterologous moiety attached to the IL2 polypeptide and IL2Rα EC domain is a soluble polymer known in the art, including but not limited to polyethylene glycol, ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, or polyvinyl alcohol. The heterologous moiety, such as a soluble polymer, can be attached to any position within the IL2 polypeptide or IL2Rα EC domain or to the N- or C-terminus.

[0267] The disclosure also provides chemically modified derivatives of the fusion protein of the disclosure, which can provide other advantages, such as increased polypeptide solubility, stability and circulation time, or reduced immunogenicity (see U.S. Patent number 4,179,337). The chemical moiety for modification can be selected from water-soluble polymers, including but not limited to polyethylene glycol, ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran and polyvinyl alcohol. The fusion protein can be modified at a random position within the molecule or at the N or C-terminal end, or at a predetermined position within the molecule, and can include one, two, three or more attached chemical moieties.

[0268] The polymer can be of any molecular weight and can be branched or unbranched. For polyethylene glycol, in one embodiment, the molecular weight is between about 1 kDa and about 100 kDa for ease of handling and manufacturing. Other sizes can be used, depending on the desired properties (e.g., the duration of sustained release required, the effect on biological activity (if any), ease of handling, the degree of antigenicity or lack thereof, and other known effects of polyethylene glycol on proteins or the like). For example, the polyethylene glycol can have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

[0269] In some embodiments, the polyethylene glycol can have a branched structure. Branched polyethylene glycols are described in, for example, U.S. Patent No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999).

[0270] The number of polyethylene glycol moieties attached to each fusion protein, IL2 polypeptide, or IL2Rα EC domain of the present disclosure (i.e., the degree of substitution) can also vary. For example, the PEGylated proteins of the present disclosure can be linked to an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution ranges from, for example, 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

[0271] In other embodiments, the IL2 polypeptide and IL2RαEC domain used in the present disclosure are conjugated to one or more polymers. The polymer can be water-soluble and covalently or non-covalently attached to the IL2 polypeptide, IL2RαEC domain, or other moiety conjugated to the IL2 polypeptide or IL2RαEC domain. Non-limiting examples of polymers can be poly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, or poly(acryloylmorpholine).

[0272] 8) Hydroxyethyl starch (HES)

[0273] In certain embodiments, the heterologous moiety linked to the IL2 polypeptide and IL2RαEC domain is a polymer, e.g., hydroxyethyl starch (HES) or a derivative thereof. In one embodiment, the fusion protein comprises an IL2 polypeptide as described herein and HES, wherein the IL2 polypeptide and IL2RαEC domain are linked to HES.

[0274] Hydroxyethyl starch (HES) is a derivative of naturally occurring amylopectin and is degraded in vivo by α-amylase. HES is a substituted derivative of the carbohydrate polymer amylopectin, which is present in corn starch at concentrations up to 95% by weight. HES exhibits favorable biological properties and is used clinically as a blood volume substitute and for hemodilution therapy (Sommermeyer et al., Krankenhauspharmazie, 8(8), 271-278 (1987); and Weidler et al., Arzneim.-For schung / Drug Res., 41, 494-498 (1991)).

[0275] Amylopectin contains glucose moieties, with α-1,4-glycosidic bonds occurring in the main chain and α-1,6-glycosidic bonds found at branching sites. The physicochemical properties of this molecule are primarily determined by the type of glycosidic bonds. The nicked α-1,4-glycosidic bonds create a helical structure with approximately six glucose monomers per turn. The physicochemical and biochemical properties of the polymer can be modified through substitution. Hydroxyethyl groups can be introduced via alkaline hydroxyethylation. By adjusting the reaction conditions, the different reactivities of the individual hydroxyl groups in the unsubstituted glucose monomers can be exploited for hydroxyethylation. This fact allows the skilled artisan to influence the substitution pattern to a limited extent.

[0276] The main characteristics of HES are the molecular weight distribution and the degree of substitution. The degree of substitution, expressed as DS, is related to the molar substitution, as is known to the skilled person. See, for example, Sommermeyer et al., Krankenhauspharmazie, 8(8), 271-278 (1987), cited above, in particular page 273.

[0277] In one embodiment, the hydroxyethyl starch has an average molecular weight (weight average) of from 1 to 300 kD, 2 to 200 kD, 3 to 100 kD or 4 to 70 kD. With respect to the hydroxyethyl group, the hydroxyethyl starch may further exhibit a molar substitution of from 0.1 to 3, preferably from 0.1 to 2, more preferably from 0.1 to 0.9, preferably from 0.1 to 0.8, and a ratio between C2:C6 substitutions in the range of from 2 to 20. Non-limiting examples of HES with an average molecular weight of about 130 kD are HES with a degree of substitution of from 0.2 to 0.8, such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8, preferably from 0.4 to 0.7, such as 0.4, 0.5, 0.6 or 0.7. In a specific embodiment, the HES with an average molecular weight of about 130 kD is HES from Fresenius. It is an artificial colloid used for volume replacement in therapeutic indications such as the treatment and prevention of hypovolemia. is characterized by an average molecular weight of 130,000 + / - 20,000 D, a molar substitution of 0.4, and a C2:C6 ratio of about 9: 1. In other embodiments, the average molecular weight of hydroxyethyl starch ranges from, for example, 4 to 70 kD, or 10 to 70 kD, or 12 to 70 kD, or 18 to 70 kD, or 50 to 70 kD, or 4 to 50 kD, or 10 to 50 kD, or 12 to 50 kD, or 18 to 50 kD, or 4 to 18 kD, or 10 to 18 kD, or 12 to 18 kD, or 4 to 12 kD, or 10 to 12 kD, or 4 to 10 kD. In yet other embodiments, the average molecular weight of the hydroxyethyl starch employed is in the range of from greater than 4 kD to less than 70 kD, such as about 10 kD, or in the range of from 9 to 10 kD or from 10 to 11 kD or from 9 to 11 kD, or about 12 kD, or from 11 to 12 kD or from 12 to 13 kD or in the range of from 11 to 13 kD, or about 18 kD, or from 17 to 18 kD or from 18 to 19 kD or in the range of from 17 to 19 kD, or about 30 kD, or in the range of from 29 to 30, or from 30 to 31 kD, or about 50 kD, or in the range of from 49 to 50 kD or from 50 to 51 kD or from 49 to 51 kD.

[0278] In certain embodiments, the heterologous moiety can be a mixture of hydroxyethyl starches having different average molecular weights and / or different degrees of substitution and / or different C2:C6 substitution ratios. Thus, a mixture of hydroxyethyl starches having different average molecular weights and different degrees of substitution and different C2:C6 substitution ratios, or having different average molecular weights and different degrees of substitution and the same or approximately the same C2:C6 substitution ratios, or having different average molecular weights and the same or approximately the same degrees of substitution and different C2:C6 substitution ratios, or having the same or approximately the same average molecular weight and different degrees of substitution and different C2:C6 substitution ratios, or having different average molecular weights and the same or approximately the same degrees of substitution and the same or approximately the same C2:C6 substitution ratios, or having the same or approximately the same average molecular weight and different degrees of substitution and the same or approximately the same C2:C6 substitution ratios, or having the same or approximately the same average molecular weight and the same or approximately the same degrees of substitution and different C2:C6 substitution ratios, or having about the same average molecular weight and about the same degree of substitution and about the same C2:C6 substitution ratios can be used.

[0279] 9) Polysialic acid (PSA)

[0280] In certain embodiments, the non-polypeptide heterologous moiety linked to the IL2 polypeptide and / or IL2Rα EC domain is a polymer, for example, polysialic acid (PSA) or a derivative thereof. Polysialic acid (PSA) is a naturally occurring unbranched polymer of sialic acid produced by certain bacterial strains and certain mammalian cells, Roth J., et al. (1993), Polysialic Acid: From Microbes to Man, ed. Roth J., Rutishauser U., Troy FA ( Verlag, Basel, Switzerland), pp. 335–348. They can be produced by limited acid hydrolysis or by digestion with neuraminidase, or by fractionating bacterially derived forms of natural polymers, with varying degrees of polymerization, ranging from n = about 80 or more sialic acid residues down to n = 2. The composition of the different polysialic acids also varies, so that there are homopolymeric forms, i.e., α-2,8-linked polysialic acids, which comprise the capsular polysaccharides of Escherichia coli strain K1 and group B meningococci, and which are also found in the embryonic form of the neural cell adhesion molecule (N-CAM). There are also heteropolymeric forms, such as the alternating α-2,8α-2,9 polysialic acids of the polysaccharides of Escherichia coli strain K92 and group C Neisseria meningitidis. Sialic acid can also be found in alternating copolymers with monomers other than sialic acid, such as the W135 group or Y group of Neisseria meningitidis. Although there are no known polysialic acid receptors in mammals, polysialic acid has important biological functions, including evasion of the immune system and complement system by pathogenic bacteria, and regulation of glial adhesion of immature neurons during fetal development (where the polymer has an anti-adhesive function) Cho and Troy, PNAS, USA, 91 (1994) 11427-11431. The α-2,8-linked polysialic acid of Escherichia coli strain K1 is also known as "polyacetylneuraminic acid" and is used (in various lengths) to illustrate the present disclosure. Various methods for attaching or conjugating polysialic acid to polypeptides have been described, for example, see U.S. Patent No. 5,846,951; WO-A-0187922 and US 2007 / 0191597 A1.

[0281] 10) XTEN sequences

[0282] As used herein, "XTEN sequence" refers to an extended length polypeptide having a non-naturally occurring, substantially non-repetitive sequence composed primarily of small hydrophilic amino acids and having little or no secondary or tertiary structure under physiological conditions. As a fusion protein partner, XTEN can serve as a carrier that, when linked to the IL2 polypeptide and / or IL2Rα EC domain of the present disclosure to produce a fusion protein, imparts certain desirable pharmacokinetic, physicochemical, and pharmaceutical properties. These desirable properties include, but are not limited to, enhanced pharmacokinetic parameters and solubility characteristics.

[0283] In some embodiments, the XTEN sequences of the present disclosure are peptides or polypeptides having greater than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues. In certain embodiments, the XTEN is a peptide or polypeptide having greater than about 20 to about 3000 amino acid residues, greater than 30 to about 2500 residues, greater than 40 to about 2000 residues, greater than 50 to about 1500 residues, greater than 60 to about 1000 residues, greater than 70 to about 900 residues, greater than 80 to about 800 residues, greater than 90 to about 700 residues, greater than 100 to about 600 residues, greater than 110 to about 500 residues, or greater than 120 to about 400 residues.

[0284] The XTEN sequences of the present disclosure can comprise one or more sequence motifs having 9 to 14 amino acid residues, or an amino acid sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence motifs, wherein the motifs comprise, consist essentially of, or consist of 4-6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P). See US 2010-0239554 A1.

[0285] In some embodiments, the XTEN sequence comprises multiple units of non-overlapping sequence motifs of the AD motif family, or the AE motif family, or the AF motif family, or the AG motif family, or the AM motif family, or the AQ motif family, or the BC family, or the BD family, and the resulting XTEN exhibits a range of homology. In other embodiments, the XTEN comprises multiple units of motif sequences from two or more motif families. These sequences can be selected to achieve desired physical / chemical characteristics, including properties such as net charge, hydrophilicity, lack of secondary structure, or lack of repetitiveness, which are conferred by the amino acid composition of the motif, as will be described more fully below. In other embodiments, motifs incorporated into the XTEN can be selected and assembled using methods described herein to obtain XTEN of about 36 to about 3000 amino acid residues.

[0286] In further embodiments, the XTEN sequences used in the present disclosure affect the physical or chemical properties of the fusion proteins of the present disclosure, such as pharmacokinetics. The XTEN sequences used in the present disclosure can exhibit one or more of the following advantageous properties: conformational flexibility, enhanced water solubility, high protease resistance, low immunogenicity, low binding to mammalian receptors, or increased hydrodynamic (or Stokes) radius. In specific embodiments, the XTEN sequences linked to the IL2 polypeptide and / or IL2Rα EC domain of the present disclosure enhance pharmacokinetic properties, such as a longer terminal half-life or increased area under the curve (AUC), such that the fusion proteins described herein remain in vivo for an increased period of time compared to wild-type IL2 polypeptide. In further embodiments, the XTEN sequences used in the present disclosure enhance pharmacokinetic properties, such as a longer terminal half-life or increased area under the curve (AUC), such that the IL2 polypeptide remains in vivo for an increased period of time compared to wild-type IL2.

[0287] Various methods and assays can be used to determine the physical / chemical properties of proteins comprising XTEN sequences. These methods include, but are not limited to, analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion, HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Raman spectroscopy, refractometry, and UV / visible spectroscopy. Additional methods are disclosed in Amau et al., Prot Expr and Purif 48, 1-13 (2006).

[0288] Additional examples of XTEN sequences can be used in accordance with the present disclosure and are disclosed in U.S. Patent Application Nos. 2010 / 0239554 Al, 2010 / 0323956 Al, 2011 / 0046060 Al, 2011 / 0046061 Al, 2011 / 0077199 Al, or 2011 / 0172146 Al or International Patent Application Nos. WO 2010091122 Al, WO 2010144502 A2, WO2010144508 Al, WO 2011028228 Al, WO 2011028229 Al, or WO 2011028344 A2.

[0289] 11) Immunoglobulin binding peptide (or polypeptide)

[0290] In certain embodiments, the non-heterologous moiety linked to the IL2 polypeptide and / or IL2Rα EC domain is an immunoglobulin binding peptide. The immunoglobulin binding peptide can bind to the Fc region and can improve the half-life of the fusion proteins described herein.

[0291] In some embodiments, immunoglobulin binding peptides useful in the present disclosure are peptides or polypeptides having greater than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acid residues.

[0292] In some embodiments, the immunoglobulin binding peptides useful in the present disclosure include 13-mer IgG-Fc domain binding peptides (IgGBPs). DeLano WL, et al. (2000) Science 287: 1279–1283. In other embodiments, the immunoglobulin binding peptides useful in the present disclosure include peptides disclosed in U.S. Patent Publication No. 20170334954, U.S. Patent Publication No. 20170210777, or PCT Publication No. WO / 2017 / 069158.

[0293] 7.3.5 Fusion Proteins

[0294] In some embodiments, the fusion protein comprises any one of SEQ ID NO: 13 to SEQ ID NO: 70 and SEQ ID NO: 202 to SEQ ID NO: 204 described in Table 3.

[0295] Table 3:

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308] Unless otherwise stated, IL2 is mature (IL2(21-153)). Unless otherwise stated, the linker between IL2 and CD25 is (G3S)3.

[0309] In some embodiments, the fusion protein of the present disclosure comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any one of SEQ ID NO: 13 to SEQ ID NO: 70 and SEQ ID NO: 202 to SEQ ID NO: 204.

[0310] The IL2-IL2Rα fusion proteins of the present disclosure may have one or more of the following properties / activities: (1) increase regulatory T cell (Treg) activity and / or increase immune tolerance in low-dose IL2-based therapies; (2) increase immune response and memory in higher-dose therapies; (3) increase IL2 utilization when compared to recombinant IL2; and / or (4) increase sustained IL2 stimulation of IL2R-bearing lymphocytes in vivo.

[0311] In some embodiments, the fusion proteins disclosed herein comprise one or more pharmacokinetic properties selected from the group consisting of: increased half-life, increased C max , increased AUC, increased C min , decreased clearance, increased bioavailability, and any combination thereof.

[0312] In one embodiment, the fusion proteins disclosed herein have an extended half-life compared to IL2 (SEQ ID NO: 2) or SEQ ID NO: 204 (wt IL2-CD25 sequence without truncation with a 12-mer linker). In some embodiments, the extended half-life is at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, or at least about 22-fold compared to the half-life of a polypeptide consisting of IL2 (SEQ ID NO: 2) or SEQ ID NO: 204 (wt IL2-CD25 sequence without any truncation with a 12-mer linker).

[0313] In some embodiments, the increase in Treg activity caused by the IL2 / IL2Rα fusion protein can be measured in a variety of ways, including, for example, (1) increased Treg presence and number in the CD4+ T cell compartment; (2) IL2-dependent upregulation of CD25; (3) increased proliferation as assessed by expression of the proliferation marker Ki67; (4) increased proportion of the IL2-dependent terminally differentiated Klrg1+ Treg subpopulation. This effect on Tregs can be seen, for example, in the spleen and / or inflamed pancreas.

[0314] In some embodiments, the IL2 / IL2Rα fusion protein of the present disclosure increases tolerogenic and immunosuppressive Tregs and immunity by increasing T effector / memory responses, and in further embodiments, it exhibits improved pharmacokinetics by: (1) delivering such responses at lower effective levels of IL2 activity compared to native or recombinant IL2; and / or (2) showing a more sustained biological response than native or recombinant IL2.

[0315] In specific embodiments, the fusion protein has improved activity relative to natural or recombinant IL2. For example, the effect of the IL2 / IL-2Rα fusion protein can increase the tolerogenic Treg activity by about 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold or less compared to natural or recombinant IL2. In other embodiments, the IL2 / IL2Rα fusion protein is more effective than natural or recombinant IL2 in inducing a sustained increase in Tregs and related properties.

[0316] Various IL2 and IL2Rα fragments and variants from various organisms can be used to generate the IL2 / IL2Rα extracellular domain fusion proteins provided herein. Such components are discussed in further detail elsewhere herein. Non-limiting examples of unprocessed or mature IL2 / IL2Rα extracellular domain fusion proteins are set forth in the following SEQ ID NOs: SEQ ID NO: 13 to SEQ ID NO: 70 and SEQ ID NO: 202 to SEQ ID NO: 204.

[0317] The term "secretory signal sequence" refers to a polynucleotide sequence encoding a polypeptide ("secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through the secretory pathway of the cell in which it is synthesized. The larger polypeptide is typically cleaved during transport through the secretory pathway to remove the secretory peptide. As used herein, a "mature" form of a fusion protein or polypeptide includes a processed form of the polypeptide from which the secretory peptide has been removed. As used herein, an "unprocessed" form of a fusion protein retains the secretory peptide sequence.

[0318] Also provided are biologically active fragments and variants of mature and unprocessed forms of IL2 / IL-Ra EC domain fusion proteins, and polynucleotides encoding the same. Such functional polypeptide fragments can comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more contiguous amino acids of any one of SEQ ID NOs: 13 to 70 and 202 to 204. Alternatively, the functional polypeptide variant may comprise at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NO: SEQ ID NO: 13 to SEQ ID NO: 70 and SEQ ID NO: 202 to SEQ ID NO: 204.

[0319] Further provided are active variants and fragments of polynucleotides encoding IL2 / IL-Ra extracellular domain fusion proteins. Such polynucleotides may comprise at least 100, 200, 300, 400, 500, 600, 700, 800, 1000, 1100, 1200, 1300, 1500, 1800, 2000 contiguous nucleotides encoding a polypeptide as set forth in SEQ ID NOs: 13 to 70 and 202 to 204 and continue to encode a functional IL2 / IL-Ra extracellular domain fusion protein.

[0320] It is further recognized that the components of the IL2 / IL2Rα fusion protein may be seen in any order.In one embodiment, the IL2 polypeptide is at the N-terminus of the fusion protein and the extracellular domain of IL2Rα is at the C-terminus of the fusion protein.

[0321] In some embodiments, the fusion protein forms a dimer. In other embodiments, the fusion protein is a monomer. Still, in some embodiments, the dimer comprises two monomers, and the monomers are associated with each other via a covalent bond. In some embodiments, the dimer comprises two monomers, and the monomers are associated via a non-covalent bond.

[0322] In some embodiments of the present disclosure, the fusion protein is more stable than a polypeptide consisting of IL2 (SEQ ID NO: 2) or SEQ ID NO: 204 (a wt IL2-CD25 sequence without truncation and having a 12-mer linker). In some embodiments, the fusion protein has one or more properties selected from the group consisting of: (i) increased thermodynamic stability compared to a reference protein; (ii) increased TM compared to a reference protein; (iii) increased resistance to degradation compared to a reference protein; (iv) increased resistance to modification compared to a reference protein; (v) increased in vivo stability compared to a reference protein; and (vi) any combination thereof, wherein the reference protein comprises (i) a first polypeptide comprising an interleukin-2 (IL2) polypeptide; and (b) a second polypeptide comprising the extracellular domain of an interleukin-2 receptor alpha (IL2Rα) polypeptide; and the reference protein has at least one more glycosylation than the fusion protein.

[0323] Any of the glycosylation sites of the fusion proteins disclosed herein can be removed by other mechanisms. In some embodiments, the fusion protein is enzymatically deglycosylated or chemically deglycosylated. In some embodiments, the fusion protein is deglycosylated by alkali, hydrazinolysis, peptide-N-glycosidase F (PNGase F), endo-β-N-acetylglucosaminidase H (Endo H), O-glycosidase, or any combination thereof.

[0324] In some embodiments, removal of one or more glycosylation sites of the fusion protein is achieved by treating the fusion protein with an alkali. In some embodiments, glycans are removed from glycosylated polypeptides by alkali borohydride treatment. In other embodiments, glycosylation sites of the fusion proteins disclosed herein can be removed using alkali metal carbonates such as sodium carbonate and potassium carbonate. In some embodiments, the alkali is used for β-elimination treatment.

[0325] In some embodiments, the removal of one or more glycosylation sites of the fusion protein is achieved by chemical treatment of the fusion protein by hydrazinolysis. In one embodiment, glycosylation is released from the fusion protein disclosed herein by subjecting the fusion protein to hydrazinolysis, and the released sugar chains are subjected to fluorescent labeling with 2-aminopyridine. See Hase et al. J. Biochem., 95, 197 (1984). In some embodiments, hydrazinolysis is performed using an instrument (GlycoPrep 1000) provided by Oxford GlycoSystems.

[0326] In another embodiment, removal of one or more glycosylation sites of the fusion protein is achieved by subjecting the fusion protein to trifluoromethanesulfonic acid (TFMS).

[0327] In some embodiments, the removal of one or more glycosylation sites of the fusion protein is achieved by treating the fusion protein with an enzyme. In some embodiments, the enzyme is a glycosidase. In some embodiments, the removal of one or more glycosylation sites of the fusion protein is achieved using peptide-N-glycosidase F (PNGase F). The concentration of PNGase F can vary and needs to be determined empirically. In some embodiments, the glycosidase is PNGase F. PNGase F is a commercially available enzyme (e.g., New England Biolabs, Ipswich, Massachusetts, catalog number P0704 or P0710). In some embodiments, the PNGase F is a fusion protein. For example, PNGase F can be a PNGase F or a PNGase F-SNAP fusion protein labeled with a chitin binding domain (CBD). In some embodiments, the glycosidase is lyophilized. In some embodiments, the glycosidase is lyophilized PNGase F. In some embodiments, the glycosidase is substantially free of animal-derived reagents.

[0328] In some embodiments, removal of one or more glycosylation sites of the fusion protein is achieved by treating the fusion protein with endo-β-N-acetylglucosaminidase H (Endo H). Endo-H is a glycosidase secreted by Streptomyces plicatus and some other Streptomyces species (Tarentino et al., 1976). It cleaves the β-1,4-glycosidic bond of the N-acetylglucosamine core of the oligosaccharide and leaves an N-acetylchitobiose attached to an asparagine residue of the glycoprotein (Trimble et al., 1978; Muramatsu 1971). The Endo H gene of S. plicatus is 939 bp (GenBank accession number AAA26738.1) and encodes a 28.9-kDa protein. Recently, Endo H from Streptomyces plicatilis was expressed in Pichia pastoris, and the deglycosylation activity of Pichia pastoris-produced Endo H was demonstrated in vitro by co-fermentation and post-fermentation treatment (Wang et al., 2015).

[0329] In some embodiments, removal of one or more glycosylation sites of a fusion protein is achieved by treating the fusion protein with O-glycosidase (New England Biolabs, Ipswich, MA). O-glycosidase, also known as endo-α-N-acetylgalactosaminidase, catalyzes the removal of Core 1 and Core 3 O-linked disaccharides from glycoproteins. In some embodiments, it releases unsubstituted Ser- and Thr-links from glycoproteins.

[0330] Removal of one or more glycosylation sites of the fusion protein can be achieved after the IL2 / IL2Rα fusion protein is produced in cell culture (e.g., a bioreactor), when the IL2 / IL2Rα fusion protein is produced in cell culture, after the fusion protein is harvested, and / or when the fusion protein is purified. In some embodiments, the removal of one or more glycosylation sites can be achieved by adding one or more removal agents when the fusion protein is expressed during cell culture. In other embodiments, the removal of one or more glycosylation sites can be achieved by selecting a specific cell type as a host cell that eliminates glycosylation or has reduced glycosylation (e.g., Escherichia coli or Streptomyces species). In certain embodiments, the removal of one or more glycosylation sites is achieved by co-expressing a gene encoding the fusion protein with a gene encoding an enzyme that removes one or more glycosylation.

[0331] The following Table 4 lists various IL-2 amino acid sequences. In some embodiments, the fusion protein described herein comprises any one of SEQ ID NO: 101 to SEQ ID NO: 115 as described in Table 4.

[0332] Table 4:

[0333]

[0334]

[0335] Various linker amino acid sequences are listed in Table 5 below. In some embodiments, the fusion protein disclosed herein comprises a plurality of tandem sequences selected from any one of SEQ ID NO: 116 to SEQ ID NO: 127 as shown in Table 5.

[0336] Table 5:

[0337]

[0338] Various CD25 amino acid sequences are listed in Table 6 below. In some embodiments, the fusion protein described herein comprises any one of SEQ ID NO: 128 to SEQ ID NO: 169 as described in Table 6.

[0339] Table 6:

[0340]

[0341]

[0342]

[0343]

[0344]

[0345] Table 7 below lists various linker amino acid sequences. In some embodiments, the fusion protein described herein comprises any one of SEQ ID NO: 170 to SEQ ID NO: 186 as described in Table 7. In some embodiments, there is no linker sequence (i.e., "TL0" as described in Table 7).

[0346] Table 7:

[0347]

[0348] The following Table 8 lists various enhancer amino acid sequences. In some embodiments, the fusion protein described herein comprises any one of SEQ ID NO: 187 to SEQ ID NO: 201 as described in Table 8.

[0349] Table 8:

[0350]

[0351]

[0352]

[0353] In some embodiments, the fusion protein disclosed herein comprises an IL-2 sequence selected from Table 4 (i.e., one of SEQ ID NO: 101 to SEQ ID NO: 115) and a CD25 sequence from Table 6 (i.e., one of SEQ ID NO: 128 to SEQ ID NO: 169). In some embodiments, the fusion protein further comprises a sequence or multiple tandem sequences selected from Table 5 (i.e., one or more of SEQ ID NO: 116 to SEQ ID NO: 127).

[0354] In some embodiments, the fusion protein disclosed herein comprises an IL-2 sequence selected from Table 4 above (i.e., one of SEQ ID NO: 101 to SEQ ID NO: 115) and a CD25 sequence from Table 6 (i.e., one of SEQ ID NO: 128 to SEQ ID NO: 169), a sequence or multiple tandem sequences selected from Table 5 (i.e., one or more of SEQ ID NO: 116 to SEQ ID NO: 127), and optionally a linker comprising a sequence or multiple tandem sequences from Table 7 (i.e., one or more of SEQ ID NO: 170 to SEQ ID NO: 186).

[0355] In some embodiments, the fusion protein disclosed herein comprises an IL-2 sequence selected from Table 4 above (i.e., one of SEQ ID NO: 101 to SEQ ID NO: 115) and a CD25 sequence from Table 6 (i.e., one of SEQ ID NO: 128 to SEQ ID NO: 169), one sequence or multiple tandem sequences selected from Table 5, and optionally a linker comprising one sequence or multiple tandem sequences from Table 8 (i.e., one of SEQ ID NO: 187 to SEQ ID NO: 201).

[0356] In some embodiments, the fusion proteins disclosed herein comprise, in order, an enhancer sequence from Table 8 (i.e., one of SEQ ID NO: 187 to SEQ ID NO: 201), a sequence or multiple tandem sequences selected from Table 7 (i.e., one or more of SEQ ID NO: 170 to SEQ ID NO: 186), an IL-2 sequence selected from Table 4 (i.e., one of SEQ ID NO: 101 to SEQ ID NO: 115), and a CD25 sequence from Table 6 (i.e., one of SEQ ID NO: 128 to SEQ ID NO: 169). In some embodiments, the fusion protein comprises a sequence or multiple tandem sequences selected from Table 7 (i.e., one or more of SEQ ID NO: 170 to SEQ ID NO: 186). In some embodiments, the fusion protein comprises a sequence or multiple tandem sequences selected from Table 5 (i.e., one or more of SEQ ID NO: 116 to SEQ ID NO: 127).

[0357] 7.4 Polynucleotides

[0358] In certain aspects, provided herein are polynucleotides, such as DNA or RNA, comprising nucleotide sequences encoding fusion proteins having IL-2 activity as described herein, and vectors comprising such polynucleotide sequences, such as expression vectors for efficient expression in host cells, such as mammalian cells. In some embodiments, provided herein are polynucleotide sequences encoding the polypeptide sequences of SEQ ID NO: 1 to SEQ ID NO: 70 and SEQ ID NO: 202 to SEQ ID NO: 204.

[0359] As used herein, "isolated" polynucleotides or nucleic acid molecules are polynucleotides or nucleic acid molecules that are separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule (e.g., in mice or humans). For example, "isolated" nucleic acid molecules, such as cDNA molecules, can be substantially free of other cellular materials or culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the language "substantially free" includes polynucleotides or nucleic acid molecule preparations having less than about 15%, 10%, 5%, 2%, 1%, 0.5% or 0.1% (particularly less than about 10%) of other materials (e.g., cellular materials, culture medium, other nucleic acid molecules, chemical precursors and / or other chemicals). In specific embodiments, one or more nucleic acid molecules encoding the fusion protein described herein are isolated or purified.

[0360] Polynucleotide can be obtained by any method known in the art, and the nucleotide sequence of polynucleotide is determined.Method well known in the art can be used to determine the nucleotide sequence of the modified form of coding fusion protein as herein described (such as fusion protein described in Table 3) and these fusion proteins, that is, the nucleotide codons of known coding specific amino acids are assembled in a manner to produce the nucleic acid encoding the fusion protein.The polynucleotide of this coding fusion protein can be assembled by chemically synthesized oligonucleotides (for example, as in Kutmeier G et al., (1994), BioTechniques 17: described in 242-6), which briefly relate to the overlapping oligonucleotides, annealing and connection of the part of the sequence containing the fusion protein encoding the fusion protein and then the oligonucleotides connected by pcr amplification.

[0361] Alternatively, polynucleotides encoding the fusion proteins described herein can be generated from nucleic acids from suitable sources (e.g., hybridomas) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, genomic DNA obtained from hybridoma cells producing the fusion protein of interest can be used to perform PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of known sequences. Such PCR amplification methods can be used to obtain nucleic acids comprising sequences encoding, for example, IL2, linker sequences, or IL2-Rα. The amplified nucleic acids can be cloned into vectors for expression in host cells and further cloned, for example, to produce fusion proteins.

[0362] If a clone containing a nucleic acid encoding a particular fusion protein is not available, but the sequence of the fusion protein molecule is known, a nucleic acid encoding the fusion protein can be obtained by chemical synthesis from a suitable source (e.g., a cDNA library, or a cDNA library generated from any tissue or cell expressing the protein of interest (e.g., a hybridoma cell selected to express a fusion protein described herein), or nucleic acid (preferably poly A + RNA) isolated from any tissue or cell), or by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence or by cloning oligonucleotide probes specific for a particular gene sequence to identify, for example, a cDNA clone encoding the fusion protein from a cDNA library. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

[0363] DNA encoding the fusion proteins described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the fusion proteins described herein). Hybridoma cells can be used as a source of such DNA. Once isolated, the DNA can be placed in expression vectors, which are then transfected into host cells (e.g., E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., from CHO GS System TM (Lonza) or myeloma cells that do not originally produce immunoglobulins) to obtain synthesis of the fusion protein in the recombinant host cells.

[0364] It is further recognized that the polynucleotide encoding the IL2 / IL2Rα fusion protein may contain other elements that facilitate translation of the fusion protein. Such sequences include, for example, a Kozak sequence attached to the 5' end of the polynucleotide encoding the fusion protein. The Kozak consensus sequence is a sequence that occurs on eukaryotic mRNA that plays a role in the initiation of the translation process and has the consensus sequence (gee)gccRccAUGG (SEQ ID NO: 92); wherein: (1) lowercase letters represent the most common base at a position where the base can still vary; (2) uppercase letters represent highly conserved bases, i.e., the 'AUGG' sequence is constant or rarely (if ever) varies, except for the IUPAC ambiguity code 'R', which indicates that a purine (adenine or guanine) is usually observed at this position; (3) the sequence in brackets ((gee)) is of uncertain meaning.

[0365] In one non-limiting embodiment, the IL2 / IL2Rα fusion protein comprises an IL2 leader sequence-optimized Kozak sequence as set forth in SEQ ID NO: 92 (gccaccATGGACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGT), or a functional variant or fragment thereof. A functional variant or fragment of the Kozak sequence will retain the ability to increase protein translation when compared to the translation level of a sequence lacking the leader sequence. Such a functional fragment may comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, or 40 consecutive nucleotides of the Kozak sequence or the sequence set forth in SEQ ID NO: 92 or SEQ ID NO: 99 (gccaccATGG). Alternatively, the functional variant may comprise at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the Kozak sequence or the sequence shown in SEQ ID NO:92 or SEQ ID NO:99.

[0366] 7.5 Cells and Vectors

[0367] In certain aspects, provided herein are cells (e.g., host cells) expressing (e.g., recombinantly) fusion proteins described herein and expression vectors comprising nucleotides encoding the fusion proteins described herein. Provided herein are vectors (e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding the fusion proteins for recombinant expression in host cells.

[0368] In some embodiments, the host cell comprises a nucleic acid described herein.

[0369] In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from mammalian cells, insect cells, yeast cells, transgenic mammalian cells and plant cells. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is a bacterial cell.

[0370] In some embodiments, the host cell is a mammalian cell. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSCl, BSC40, YB / 20, BMT10, and HsS78Bst cells.

[0371] As used herein, an expression vector refers to any nucleic acid construct that contains the necessary elements for transcription and translation of an inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation when introduced into a suitable host cell. Expression vectors may include plasmids, phagemids, viruses, and derivatives thereof.

[0372] As used herein, a gene expression control sequence is any regulatory nucleotide sequence, such as a promoter sequence or a promoter-enhancer combination, that promotes efficient transcription and translation of a coding nucleic acid to which it is operably linked. A gene expression control sequence can be, for example, a mammalian or viral promoter, such as a constitutive promoter or an inducible promoter. Constitutive mammalian promoters include, but are not limited to, promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, β-actin promoter, and other constitutive promoters. Exemplary viral promoters that constitutively function in eukaryotic cells include, for example, promoters from the long terminal repeats (LTRs) of cytomegalovirus (CMV), simian viruses (e.g., SV40), papillomaviruses, adenoviruses, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, Moloney leukemia virus, and other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art. Promoters that can be used as gene expression sequences for the present disclosure also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced in the presence of certain metal ions to promote transcription and translation. Other inducible promoters are known to those of ordinary skill in the art.

[0373] For the purposes of this disclosure, many expression vector systems can be used. These expression vectors can typically be replicated in host organisms as episomes or as components of host chromosomal DNA. Expression vectors can include expression control sequences, including but not limited to promoters (such as natural associations or heterologous promoters), enhancers, signal sequences, splicing signals, enhancer elements, and transcription termination sequences. Preferably, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting eukaryotic host cells. Expression vectors can also utilize DNA elements derived from animal viruses, such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV or MOMLV), cytomegalovirus (CMV) or SV40 virus. Others relate to the use of polycistronic systems with internal ribosome binding sites.

[0374] In general, expression vectors contain selective markers (such as ampicillin resistance, hygromycin resistance, tetracycline resistance or neomycin resistance) to allow detection of those cells transformed with the desired DNA sequence (see, for example, Itakura et al., U.S. Patent number 4,704,362). The cell into which DNA has been integrated into its chromosome can be selected by introducing one or more markers that allow selection of transfected host cells. The marker can provide prototrophy, biocide resistance (such as antibiotic) or resistance to heavy metals such as copper to auxotrophic hosts. Selectable marker gene can be directly connected to the DNA sequence to be expressed, or introduced into the same cell by co-transformation.

[0375] An example of a vector that can be used to optimize expression of the fusion proteins used in the methods of the present disclosure is NEOSPLA (U.S. Patent No. 6,159,730). This vector contains a cytomegalovirus promoter / enhancer, a mouse beta-globin major promoter, an SV40 origin of replication, a bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, a dihydrofolate reductase gene, and a leader sequence. Vector systems are also taught in U.S. Patent Nos. 5,736,137 and 5,658,570. This system provides high expression levels, e.g., >30 pg / cell / day. Other exemplary vector systems are disclosed, e.g., in U.S. Patent No. 6,413,777.

[0376] In other embodiments, polycistronic constructs are used to express the polypeptides of the present disclosure. In these expression systems, multiple target gene products, such as multiple polypeptides of multimeric binding proteins, can be produced from a single polycistronic construct. These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of polypeptides in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. Patent No. 6,193,980.

[0377] In some embodiments, the present invention relates to a method for the expression of a polypeptide of interest. The method comprises the steps of: a) expressing a polypeptide of interest in a host cell and b) expressing a polypeptide of interest in a cell. The method comprises the steps of: a) expressing a polypeptide of interest in a host cell and b) expressing a polypeptide of interest in a cell. More generally, once a vector or DNA sequence encoding a polypeptide is prepared, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. As discussed above, various techniques well known to those skilled in the art can be used to achieve the introduction of a plasmid into a host cell. The transformed cells are grown under conditions suitable for producing the fusion protein, and the fusion protein synthesis assay is performed. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence activated cell sorter analysis (FACS), immunohistochemistry, etc.

[0378] 7.6 Pharmaceutical Compositions

[0379] The various IL2 / IL2Rα fusion proteins disclosed herein (also referred to herein as “active compounds”) can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the fusion protein and a pharmaceutically acceptable carrier. As used herein, the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, their use in the composition is contemplated. Supplementary active compounds may also be incorporated into the composition.

[0380] In some embodiments, a pharmaceutical composition is disclosed, comprising (a) an IL2 / IL2Rα fusion protein as described herein and (b) a pharmaceutically acceptable excipient.

[0381] In some embodiments, a pharmaceutical composition is disclosed comprising (a) a composition comprising an IL2 / IL2Rα fusion protein as described herein and (b) a pharmaceutically acceptable excipient.

[0382] In some embodiments, a pharmaceutical composition is disclosed comprising (a) a nucleic acid as described herein and (b) a pharmaceutically acceptable excipient.

[0383] In some embodiments, a pharmaceutical composition is disclosed comprising (a) a carrier as described herein and (b) a pharmaceutically acceptable excipient.

[0384] In some embodiments, a pharmaceutical composition is disclosed comprising (a) a host cell as described herein and (b) a pharmaceutically acceptable excipient.

[0385] The pharmaceutical composition of the present disclosure is prepared to be compatible with its expected route of administration. The example of route of administration includes parenteral, such as intravenous, intradermal, subcutaneous, oral (such as inhalation), transdermal (topical) and transmucosal. In addition, it may be necessary to locally administer the pharmaceutical composition of a therapeutically effective amount to the region in need of treatment. This can be achieved by, for example, local or regional infusion or perfusion, topical application, injection, catheter, suppository or implant (for example, by porous, non-porous or gelatinous material (including film, such as salivary elastic membrane, or fiber) formed implant) etc. during surgery. In another embodiment, in vesicle, such as liposome, deliver the pharmaceutical composition of a therapeutically effective amount (see, for example, Langer, Science 249:1527-33,1990 and Treat et al., Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (editor), Liss, New York, 353-65 pages, 1989).

[0386] In yet another embodiment, a therapeutically effective amount of a pharmaceutical composition can be delivered in a controlled release system. In one example, a pump can be used (see, e.g., Langer, Science 249: 1527-33, 1990; Sefton, Crit. Rev. Biomed. Eng. 14: 201-40, 1987; Buchwald et al., Surgery 88: 507-16, 1980; Saudek et al., N Engl. J Med. 321: 574-79, 1989). In another example, a polymeric material can be used (see, e.g., Levy et al., Science 228: 190-92, 1985; During et al., Ann. Neural. 25: 351-56, 1989; Howard et al., J Neurosurg. 71: 105-12, 1989). Other controlled-release systems, such as those discussed by Langer (Science 249: 1527-33, 1990), may also be used.

[0387] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as TWEEN®. TM 、PLURONICS TM or polyethylene glycol (PEG).

[0388] The pharmaceutically acceptable carrier used in the parenteral preparation includes aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending agents and dispersants, emulsifiers, chelating agents (sequestering agent or chelating agent) and other pharmaceutically acceptable substances. The example of an aqueous vehicle includes sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils of plant origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents with antibacterial or antifungal concentrations can be added to the parenteral preparation packaged in multiple dose containers, and the antimicrobial agents include phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinyl pyrrolidone. Emulsifiers include polysorbate 80 ( 80). Metal ion chelators include EDTA. Pharmaceutical carriers also include ethanol, polyethylene glycol, and propylene glycol for water-miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0389] Solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methyl paraben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer (such as acetate, citrate or phosphate), and an agent for adjusting tonicity (such as sodium chloride or dextrose). The pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. The parenteral formulation can be packaged in an ampoule, disposable syringe or multiple-dose vial made of glass or plastic.

[0390] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (water-soluble) or dispersions, as well as sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELS (BASF, Parsippany, New Jersey), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that it is easy to inject. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating effects of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. For example, appropriate fluidity can be maintained by using a coating (e.g., lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. Protection against the effects of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols (such as mannitol, sorbitol), sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0391] Sterile injectable solutions can be prepared by incorporating the active compound in the desired amount into an appropriate solvent optionally with one or a combination of the ingredients listed above, followed by sterilization by filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing an alkaline dispersion medium and the desired other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying, which produce a powder of the active ingredient and any other desired ingredients from a previously sterile-filtered solution.

[0392] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.Systemic administration can also be by transmucosal or transdermal means.

[0393] For transmucosal or transdermal administration, penetrants suitable for the barrier to be penetrated are used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives with respect to transmucosal administration. Transmucosal administration can be accomplished by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into an ointment, salves, gel, or cream as is commonly known in the art. Compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas (for rectal delivery).

[0394] In one embodiment, the active compound is prepared with a carrier that protects the compound from rapid removal from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The preparation methods of such formulations are clear to those skilled in the art. The materials can also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as those described in U.S. Patent No. 4,522,811.

[0395] It is particularly advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the present disclosure is determined by or directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of synthesizing such functional compounds for use in treating individuals. The pharmaceutical composition can be contained in a container, package, or dispenser together with instructions for administration.

[0396] The effective amount of IL2 / IL2Rα fusion protein that can be used to modulate such functions will depend on the subject being treated, the severity of the affliction, and the mode of administration of the IL2 / IL2Rα fusion protein. Exemplary dosages include about 104 to about 107 IU of IL2 activity per adult, about 104 to 105 IU of IL2 activity per adult, about 105 to about 106 IU of IL2 activity per adult, and about 106 to about 107 IU of IL2 activity per adult. In other cases, the therapeutically effective dose of the IL2 / IL2Rα fusion protein is about 105 IU of IL2 activity ± 100 times, about 105 IU of IL2 activity ± 10 times, about 105 IU of IL2 activity ± 2 times, about 105 IU of IL2 activity ± 20 times, about 105 IU of IL2 activity ± 30 times, about 105 IU of IL2 activity ± 40 times, about 105 IU of IL2 activity ± 50 times, about 105 IU of IL2 activity ± 60 times, about 105 IU of IL2 activity ± 70 times, about 105 IU of IL2 activity ± 80 times, or about 105 IU of IL2 activity ± 90 times. In specific, non-limiting embodiments, the human IL2 fusion protein is administered at this dose.

[0397] 7.7 Purpose and Method

[0398] 7.7.1 Methods of Making the Compositions Disclosed Herein

[0399] As discussed above, the IL2 / IL2Rα fusion proteins disclosed herein can be used to generate new IL2 / IL2Rα fusion proteins by modifying IL2, IL2Rα, or any heterologous partial sequence described herein. Thus, in another aspect described herein, the structural features of the IL2 / IL2Rα fusion proteins described herein are used to generate structurally related IL2 / IL2Rα fusion proteins that retain at least one functional property of the IL2 / IL2Rα fusion proteins described herein, such as binding to human IL2Rα and cynomolgus monkey IL2Rα. For example, as discussed above, one or more of the IL2, IL2Rα, or any heterologous partial sequences described herein can be recombinantly combined with known framework regions and / or other proteins to generate additional recombinantly engineered fusion proteins discussed herein.

[0400] In some embodiments, disclosed herein is a method for producing an IL2 / IL2Rα fusion protein, the method comprising culturing a host cell comprising the fusion protein under suitable conditions and recovering the fusion protein. In some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the host cell is a mammalian cell, an insect cell, a fungal cell, a plant cell, a transgenic mammalian cell, or a bacterial cell. In some embodiments, the host cell is selected from CHO cells, HEK 293 cells, NSO cells, Per C6 cells, BHK cells, and COS cells. In one embodiment, the host cell is a bacterial cell. In a specific embodiment, the bacterial cell is Escherichia coli.

[0401] Other types of modifications include those described in the previous section. The starting material for the engineering method is one or more of the IL2 or IL2Rα sequences provided herein. In order to produce an engineered fusion protein, it is not necessary to actually prepare (i.e., express as a protein) a fusion protein having one or more IL2 or IL2Rα sequences provided herein. Instead, the information contained in one or more sequences is used as a starting material to produce one or more "second generation" sequences derived from one or more original sequences, and then one or more "second generation" sequences are prepared and expressed as proteins.

[0402] Thus, provided herein is a method for preparing a fusion protein, comprising: (a) providing IL2 and IL2Rα; (b) altering at least one amino acid residue within IL2 and IL2Rα to produce at least one altered fusion protein sequence; and (c) expressing the altered fusion protein sequence as a protein.

[0403] Also provided herein is a method for preparing a fusion protein, the method comprising: (a) providing IL2 and IL2Rα; (b) altering at least one glycosylation within IL2 and IL2Rα to produce at least one altered glycosylation site; and (c) expressing the altered fusion protein sequence as a protein.

[0404] The altered antibody may exhibit one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all of the functional properties described in (1) to (10) above. The functional properties of the altered antibody can be assessed using standard assays available in the art.

[0405] In some embodiments of the methods for engineering IL2 / IL2Rα fusion proteins described herein, mutations can be introduced randomly or selectively along all or part of the IL2 / IL2Rα fusion protein coding sequence, as described herein, and the resulting modified IL2 / IL2Rα fusion protein can be screened for binding activity and / or other functional properties. Mutation methods have been described in the art. For example, PCT Publication WO 02 / 092780 by Short describes methods for generating and screening mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Alternatively, PCT Publication WO 03 / 074679 by Lazar et al. describes methods for optimizing the physicochemical properties of proteins using computer screening methods.

[0406] The compositions further include isolated polynucleotides encoding the various fusion proteins and variants and fragments thereof described above. Further disclosed are vectors and expression cassettes comprising the polynucleotides described herein. The expression cassette will typically include a promoter linked to the polynucleotide and transcription and translation termination regions.

[0407] The use of the term "polynucleotide" is not intended to limit the present disclosure to polynucleotides comprising DNA. One of ordinary skill in the art will recognize that a polynucleotide can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs.

[0408] In constructs containing more than one processing or cleavage site, it will be understood that these sites may be the same or different.

[0409] An "isolated" or "purified" polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free of components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide is free of sequences that naturally flank the polynucleotide (optimally, protein-coding sequences) in the genomic DNA of the organism from which the polynucleotide is derived (i.e., sequences located at the 5' and 3' ends of the polynucleotide). For example, in various embodiments, an isolated polynucleotide may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived.

[0410] A protein that is substantially free of cellular material includes preparations of the protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When a protein of the disclosure, or a biologically active portion thereof, is recombinantly produced, optimal culture media represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or chemicals other than the protein of interest.

[0411] Conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art may be employed herein. Such techniques are fully described in the literature. See, e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual” (1989); “Current Protocols in Molecular Biology” Volumes I-III [Ausubel, RM, ed. (1994)]; “Cell Biology: A Laboratory Handbook” Volumes I-III [JECelis, ed. (1994)]; “Current Protocols in Immunology” Volumes I-III [Coligan, JE, ed. (1994)]; “Oligonucleotide Synthesis” (MJ Gaited. 1984); “Nucleic Acid Hybridization” [BD Hames & S.J. Higgins, eds. (1985)]; “Transcription And Translation” [BD Hames & S.J. Higgins, eds. (1984)]; “Animal Cell Culture” [RI Freshney, ed. (1986)]; “Immobilized Cells and Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

[0412] Also provided herein is a vector comprising the above-mentioned polynucleotide operably connected to a promoter. When an expression control sequence controls and regulates the transcription and translation of the sequence, a nucleotide sequence is "operably connected" to an expression control sequence (e.g., a promoter). When it comes to a nucleotide sequence, the term "operably connected" is included in a sequence to be expressed with a suitable start signal (e.g., ATG) in front of the nucleotide sequence, and maintains a correct reading frame to allow the sequence to be expressed under the control of the expression control sequence and to produce the desired product encoded by the sequence. If the gene that people wish to be inserted into the recombinant nucleic acid molecule does not contain a suitable start signal, this start signal can be inserted in front of the gene." vector " is a replicon, such as a plasmid, phage, or clay, to which another nucleic acid segment can be attached, to achieve the replication of the attached fragment. The promoter can be a promoter from bacteria, yeast, insects, or mammals, or the same as thereto. In addition, the vector can be a plasmid, clay, yeast artificial chromosome (YAC), phage, or eukaryotic viral DNA. Many other vector skeletons known in the art for expressing protein can be used. Such vectors include, but are not limited to, adenovirus, simian virus 40 (SV40), cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), Moloney murine leukemia virus, DNA delivery systems (i.e., liposomes), and expression plasmid delivery systems. In addition, one class of vectors comprises DNA elements derived from viruses such as bovine papilloma virus, polyoma virus, baculovirus, retrovirus, or Semliki Forest virus. Such vectors can be obtained commercially or assembled from sequences by methods well known in the art.

[0413] Provided herein is a host-vector system for producing polypeptides, the host-vector system comprising a vector of a suitable host cell. Suitable host cells include, but are not limited to, prokaryotic or eukaryotic cells, for example, bacterial cells (including gram-positive cells), yeast cells, fungal cells, insect cells and zooblasts. Many mammalian cells can be used as hosts, including but not limited to mouse fibroblasts NIH 3T3, Chinese hamster ovary celI, HeLa cells, Ltk cells etc. Other zooblasts can also be used, such as R1.1, BW and LM cells, African green monkey kidney cells (such as COS 1, COS 7, BSC1, BSC40 and BMT10), insect cells (such as Sf9) and human cells and plant cells in tissue culture.

[0414] A variety of host / expression vector combinations can be used to express the polynucleotide sequences presented herein. Useful expression vectors, for example, can be composed of segments of chromosomal, non-chromosomal, and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, for example, E. coli plasmids col E1, pCR1, pBR322, pMB9, and derivatives thereof, plasmids such as RP4; phage DNA, for example, various derivatives of phage A (e.g., NM989), and other phage DNA (e.g., M13 and filamentous single-stranded phage DNA); yeast plasmids, such as 2! 1 plasmids or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from a combination of plasmids and phage DNA, such as plasmids modified to use phage DNA or other expression control sequences; and the like.

[0415] Any of a variety of expression control sequences (sequences that control the expression of a nucleotide sequence to which it is operably linked) can be used in these vectors to express the polynucleotide sequences provided herein. Such useful expression control sequences include, for example, early and late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter region of bacteriophage A, the control region of the fd coat protein, the promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter of acid phosphatase (e.g., Pho5), the promoter of yeast α-mating factor, and other sequences known to control gene expression in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

[0416] Should be understood that not all carriers, expression control sequences and hosts will express the polynucleotide sequence provided herein just as well.On the identical expression system, all hosts all can not play a role just as well.Yet, those skilled in the art will be able to select suitable carrier, expression control sequence and host, and need not excessive experiment to complete required expression, and do not deviate from the scope of this disclosure.For example, when selecting carrier, must consider host, because described carrier must work therein.Also will consider the copy number of carrier, the ability of controlling this copy number and by the expression of any other protein such as antibiotic marker of described carrier encoding.

[0417] In selecting the expression control sequence, many factors are generally considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the specific nucleotide sequence or gene to be expressed, particularly with respect to potential secondary structure. Suitable unicellular hosts are selected by considering, for example, the compatibility of the host with the selected vector, the secretion characteristics of the host, the ability of the host to correctly fold proteins and its fermentation requirements, the toxicity of the product encoded by the nucleotide sequence to be expressed to the host, and the ease with which the expression product can be purified.

[0418] In the preparation of expression cassettes, various polynucleotides can be manipulated to provide polynucleotide sequences in the correct orientation and, as the case may be, in the appropriate reading frame. For this reason, adapters or joints can be used to connect polynucleotides, or other operations can be involved to provide convenient restriction sites, remove unnecessary DNA, remove restriction sites, etc. For example, a joint such as two glycines can be added between the polypeptides. A methionine residue encoded by the ATG nucleotide sequence can be added to allow the start of gene transcription. For this purpose, in vitro mutagenesis, primer repair, restriction enzyme digestion, annealing, replacement, such as conversion and transversion, can be involved.

[0419] Also provided is a method of producing a polypeptide, the method comprising expressing a polynucleotide encoding a fusion protein disclosed herein in a host cell under suitable conditions to allow production of the polypeptide, and recovering the polypeptide so produced.

[0420] 7.7.2 Therapeutic uses and methods

[0421] The fusion proteins and methods described herein have many in vitro and in vivo utilities related to the following: for example, such as enhancing immune responses or detecting IL2 by inhibiting (or antagonizing) IL2Rα (e.g., signaling). For example, the IL2 / IL2Rα fusion proteins described herein can be administered to cells in culture in vitro or ex vivo, or, for example, to human subjects in vivo to enhance immunity in a variety of diseases. Thus, provided herein is a method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject an IL2 / IL2Rα fusion protein described herein, thereby altering the immune response in the subject. In some embodiments, the response is enhanced, stimulated, or upregulated.

[0422] Subjects suitable for the methods of the present invention include human patients who desire to enhance their immune response. The methods are particularly suitable for treating human patients with disorders that can be treated by increasing an immune response (e.g., a T cell-mediated immune response, such as an antigen-specific T cell response). In some embodiments, the methods are particularly suitable for treating cancer in vivo. In order to achieve antigen-specific enhancement of immunity, the IL2 / IL2Rα fusion protein described herein can be administered together with the target antigen, or the antigen can already be present in the subject to be treated (e.g., a subject with a tumor or a subject with a virus). When the IL2 / IL2Rα fusion protein described herein is administered together with another agent, the two can be administered separately or simultaneously.

[0423] Also encompassed are methods for detecting the presence of human IL2 or human IL2Rα in a sample or measuring the amount of human IL2 antigen, the method comprising contacting the sample and a control sample with a fusion protein or monoclonal antibody, such as a human monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to human IL2 or IL2Rα, under conditions that allow formation of a complex between the IL2 / IL2Rα fusion protein described herein and human IL2Rα. The formation of the complex is then detected, wherein differential complex formation between the sample and the control sample indicates the presence of human IL2 or IL2Rα in the sample. Furthermore, the IL2 / IL2Rα fusion proteins described herein can be used to purify human IL2 or IL2Rα via immunoaffinity purification.

[0424] In view of the fact that the IL2 / IL2Rα fusion protein described herein has the ability to stimulate or co-stimulate T cell responses (e.g., antigen-specific T cell responses) by inhibiting the negative effects of IL2 or IL2Rα, provided herein are in vitro and in vivo methods for stimulating, enhancing, or upregulating antigen-specific T cell responses (e.g., anti-tumor T cell responses) using the IL2 / IL2Rα fusion protein described herein. In some embodiments, CD3 stimulation (e.g., by co-incubation with cells expressing cell membrane CD3) is also provided, which can be provided simultaneously with, before, or after stimulation with the IL2 / IL2Rα fusion protein described herein. For example, provided herein is a method for stimulating antigen-specific T cell responses, the method comprising contacting the T cells with the IL2 / IL2Rα fusion protein described herein and, optionally, with an anti-CD3 antibody, thereby stimulating antigen-specific T cell responses.

[0425] Any suitable indication of an antigen-specific T cell response can be used to measure an antigen-specific T cell response. Non-limiting examples of such suitable indications include T cell proliferation increased in the presence of an antibody and / or cytokine production increased in the presence of an antibody. In some embodiments, the antigen-specific T cells stimulate the production of interleukin-2 and / or interferon-γ.

[0426] T cells including CD4 + T cells and CD8 + T cells. The T cells may be Teff cells, such as CD4 + Teff cells, CD8 + Teff cells, T helper (Th) cells (eg, Th1 cells), or T cytotoxic (Tc) cells.

[0427] Also contemplated are methods for stimulating an immune response (e.g., an antigen-specific T cell response) in a subject, comprising administering to the subject an IL2 / IL2Rα fusion protein as described herein to stimulate an immune response (e.g., an antigen-specific T cell response) in the subject. In some embodiments, the subject is a tumor-bearing subject, and an immune response against the tumor is stimulated. The tumor may be a solid tumor or a liquid tumor, such as a hematological malignancy. In some embodiments, the tumor is an immunogenic tumor. In some embodiments, the tumor is non-immunogenic. In some embodiments, the tumor is PD-L1 positive. In some embodiments, the tumor is PD-L1 negative. The subject may also be a subject carrying a virus, and an immune response against the virus is stimulated.

[0428] Further provided are methods for inhibiting tumor cell growth in a subject, comprising administering to the subject an IL2 / IL2Rα fusion protein as described herein, thereby inhibiting tumor growth in the subject. Further provided are methods for treating a viral infection in a subject, comprising administering to the subject an IL2 / IL2Rα fusion protein as described herein, thereby treating the viral infection in the subject.

[0429] In some embodiments, the IL2 / IL2Rα fusion protein described herein is administered to a subject as an adjuvant therapy. Treatment of a subject with cancer with the IL2 / IL2Rα fusion protein described herein can result in prolonged survival, such as a long-term durable response relative to the current standard of care; long-term survival of at least 3 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years or more, or recurrence-free survival of at least 3 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years or 10 years or more. In some embodiments, treatment of a subject with cancer with the IL2 / IL2Rα fusion protein described herein prevents the recurrence of cancer or delays the recurrence of cancer for, for example, 3 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years or 10 years or more. IL2 / IL2Rα fusion protein treatment can be used as a first-line, second-line or third-line treatment.

[0430] Treatment of a subject having cancer with an IL2 / IL2Rα fusion protein described herein can result in, for example, stable disease, partial response, increased overall survival, increased disease-free survival, or increased progression-free survival.

[0431] In some embodiments, the IL2 / IL2Rα fusion proteins described herein are not significantly toxic. For example, as determined, for example, in clinical trials, the IL2 / IL2Rα fusion proteins described herein are not significantly toxic to one or more of the human organs, such as the liver, kidney, brain, lung, and heart. In some embodiments, the IL2 / IL2Rα fusion proteins described herein do not significantly trigger an undesirable immune response, such as autoimmunity or inflammation.

[0432] In some embodiments, treatment of a subject with an IL2 / IL2Rα fusion protein described herein does not result in overstimulation of the immune system to the extent that the subject's immune system subsequently attacks the subject itself (e.g., an autoimmune response) or results in, for example, an allergic reaction. Thus, in some embodiments, the IL2 / IL2Rα fusion protein described herein does not cause an allergic reaction.

[0433] In some embodiments, treatment of a subject with an IL2 / IL2Rα fusion protein as described herein does not result in significant inflammatory reactions, such as immune-mediated pneumonitis, immune-mediated colitis, immune-mediated hepatitis, immune-mediated nephritis or renal insufficiency, immune-mediated hypophysitis, immune-mediated hypothyroidism and hyperthyroidism, or other immune-mediated adverse reactions. In some embodiments, treatment of a subject with an IL2 / IL2Rα fusion protein as described herein does not result in significant cardiac disorders, such as ventricular arrhythmias; eye disorders, such as iridocyclitis; infusion-related reactions; increased amylase, increased lipase; nervous system disorders, such as vertigo, peripheral neuropathy, and sensory neuropathy; skin and subcutaneous tissue disorders, such as rash, pruritus, exfoliative dermatitis, erythema multiforme, vitiligo, or psoriasis; respiratory, thoracic, and mediastinal disorders, such as cough; fatigue; nausea; decreased appetite; constipation; joint pain; or diarrhea.

[0434] In some embodiments, the IL2 / IL2Rα fusion proteins described herein provide a synergistic anti-tumor effect in combination with another cancer therapy, such as a compound that stimulates the immune system (e.g., an immuno-oncology agent), such as a compound described herein, or a compound that modulates a target described herein.

[0435] In some embodiments, the IL2 / IL2Rα fusion protein described herein is administered via a topical, mucosal, intranasal, oral, vaginal, rectal, sublingual, local, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, or intrasternal route.

[0436] Various methods for increasing an immune response in a subject are provided. Such methods comprise administering a therapeutically effective amount of an IL2 / IL2Rα fusion protein to a subject in need of an increased immune response. Thus, in specific embodiments, transient administration of a higher dose of IL2 is employed to enhance immune and memory responses.

[0437] Various methods for reducing an immune response in a subject are provided. Such methods include administering a therapeutically effective amount of an IL2 / IL2Rα fusion protein to a subject in need of a reduced immune response. Exploiting the suppressive ability of Tregs to suppress unwanted immune responses has aroused great interest. Data in mice and humans show that enhancing IL2R signaling with low doses of IL2 selectively enhances Tregs and strengthens immune tolerance mechanisms. The IL2 / IL2Rα fusion protein provided herein represents a new and improved form of IL2 that is more likely to enhance Tregs. Therefore, the IL2 / IL2Rα fusion protein can be administered to patients with autoimmune diseases, chronic graft-versus-host disease, transplant rejection, and other conditions where the purpose is to suppress autoreactivity.

[0438] These and other methods described herein are discussed in further detail below.

[0439] 7.7.2.1 Cancer

[0440] In some embodiments, disclosed herein are methods for treating cancer. The inhibition of IL2Rα by IL2 / IL2Rα fusion protein can enhance the immune response of patients with cancer to cancer cells. Provided herein are methods for treating subjects with cancer, comprising administering to the subject an IL2 / IL2Rα fusion protein as described herein, such that the subject is treated, for example, such that the growth of a cancerous tumor is inhibited or reduced and / or the tumor regresses and / or an extended survival period is achieved. The IL2 / IL2Rα fusion protein can be used alone to inhibit the growth of a cancerous tumor. Alternatively, the IL2 / IL2Rα fusion protein can be used in combination with another agent (e.g., other immunogenic agents), a standard cancer treatment, or another antibody.

[0441] Thus, provided herein are methods for treating cancer, for example, by inhibiting tumor cell growth in a subject, the methods comprising administering to the subject a therapeutically effective amount of an IL2 / IL2Rα fusion protein disclosed herein. Cancers whose growth can be inhibited using the antibodies of the present disclosure include cancers that are typically responsive to immunotherapy and cancers that are typically unresponsive to immunotherapy. The cancer can be a solid tumor or a hematological malignancy (liquid tumor). In some embodiments, the cancer is bladder cancer, breast cancer, uterine cancer, endometrial cancer, ovarian cancer, colorectal cancer, colon cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, squamous cell carcinoma, skin cancer, a central nervous system tumor, lymphoma, leukemia, sarcoma, a virus-related cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin or non-Hodgkin lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, myeloma, salivary gland cancer, kidney cancer, basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer, or head and neck cancer, and any combination thereof.

[0442] Other non-limiting examples of cancers for treatment include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon cancer, and head and neck cancer (or carcinomas), stomach cancer, and ovarian cancer. carcinoma, germ cell tumor, pediatric sarcoma, sinonasal natural killer cells, melanoma (e.g., metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethra cancer, penile cancer, solid tumors in children, ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord tumors, brain cancer, brainstem glioma, pituitary gland adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), virus-related cancers or cancers of viral origin (e.g., human papillomavirus (HPV-associated or HPV-derived tumors)), and hematological malignancies (e.g., all types of leukemias, lymphomas, and myeloma, such as acute, chronic, lymphocytic, and / or myeloid leukemias) derived from either of the two major blood cell lineages, i.e., the myeloid lineage (which produces granulocytes, erythrocytes, platelets, macrophages, and mast cells) or the lymphoid lineage (which produces B, T, NK, and plasma cells). , such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML), undifferentiated AML (MO), myeloid leukemia (M1), myeloid leukemia (M2; with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma and chloroma);Lymphomas, such as Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), B-cell hematological malignancies (e.g., B-cell lymphoma), T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma Systemic lymphoma, primary effusion lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic neoplasms of the lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sézary syndrome), and leukemia with Waldenström's giant cell lymphoma Lymphoplasmacytoid lymphoma (LPL) with hyperglobulinemia; myelosuppressive myeloma, such as IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also known as indolent myeloma), solitary plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of the myeloid lineage, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; seminoma, teratoma, central nervous system tumors and peripheral nervous system tumors, including astrocytomas, neuroblastomas, and leukemia. Transthecal tumors; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma and teratocarcinoma, hematopoietic tumors of the lymphoid lineage, such as T-cell and B-cell tumors, including but not limited to T-cell disorders such as T-cell prolymphocytic leukemia (T-PLL), including small cell and cerebroid cell types; T-cell large granular lymphocytic leukemia (LGL); a / d T-NHL hepatosplenic lymphoma; peripheral / postthymic T-cell lymphoma (polymorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head and neck cancer, renal cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma, and any combination of the above cancers. The methods described herein can also be used to treat metastatic cancer, unresectable cancer, refractory cancer (e.g., cancer that is refractory to previous immunotherapy (e.g., using blocking CTLA-4 or PD-1 antibodies)), and / or recurrent cancer.

[0443] In some embodiments, the IL2 / IL2Rα fusion proteins disclosed herein are administered to patients with cancer that has exhibited an inadequate response or progression to a previous treatment (e.g., a previous treatment with an immuno-oncology or immunotherapy drug), or with a cancer that is refractory or resistant (intrinsically refractory or resistant (e.g., refractory to a PD-1 pathway antagonist), or in whom a resistant or refractory state has been acquired. For example, subjects who did not respond or did not respond adequately to a first therapy, or who saw disease progression after treatment, such as an anti-PD-1 therapy, can be treated by administering an IL2 / IL2Rα fusion protein disclosed herein, alone or in combination with other therapies (e.g., with an anti-PD-1 therapy).

[0444] In some embodiments, an IL2 / IL2Rα fusion protein described herein is administered to a patient who has not previously received (ie, has not been treated with) an immuno-oncology agent (eg, a PD-1 pathway antagonist).

[0445] Methods for treating a subject with cancer using an IL2 / IL2Rα fusion protein disclosed herein may comprise administering a therapeutically effective amount of an IL2 / IL2Rα fusion protein disclosed herein to a subject having cancer cells expressing IL2 or IL2Rα. Also provided herein are methods for predicting whether a subject will respond to treatment with an IL2 / IL2Rα fusion protein disclosed herein, wherein the method comprises determining the level of IL2 or IL2Rα in a patient, and if the subject is IL2 or IL2Rα positive, the subject is likely to respond to treatment with an IL2 / IL2Rα fusion protein disclosed herein.

[0446] In some embodiments, a method of treating cancer in a subject comprises first determining whether the subject is PD-L1 or PD-1 positive, e.g., having tumor cells or TILs expressing PD-L1 or PD-1, and if the subject has PD-L1 or PD-1 positive cancer cells or TIL cells, administering to the subject an IL2 / IL2Rα fusion protein disclosed herein (and optionally a PD-1 or PD-L1 antagonist). A method of treating a subject having cancer with an IL2 / IL2Rα fusion protein disclosed herein (and optionally a PD-1 or PD-L1 antagonist) may comprise administering a therapeutically effective amount of an IL2 / IL2Rα fusion protein disclosed herein (and optionally a PD-1 or PD-L1 antagonist) to a subject having cancer cells or TIL cells expressing PD-L1 or PD-1. Also provided herein are methods of predicting whether a subject will respond to treatment with an IL2 / IL2Rα fusion protein (and optionally a PD-1 or PD-L1 antagonist) disclosed herein, wherein the method comprises determining the level of PD-L1 or PD-1 in the patient's cancer cells or TIL cells, and if the subject's cancer cells or TIL cells are PD-L1 or PD-1 positive, the subject is likely to respond to treatment with an IL2 / IL2Rα fusion protein (and optionally a PD-1 or PD-L1 antagonist) disclosed herein.

[0447] In some embodiments, an IL2 / IL2Rα fusion protein disclosed herein is administered with standard of care treatment.In some embodiments, an IL2 / IL2Rα fusion protein disclosed herein is administered as maintenance therapy, e.g., as a therapy intended to prevent tumor development or recurrence.

[0448] In some embodiments, the IL2 / IL2Rα fusion proteins disclosed herein are administered together with another treatment, such as radiation, surgery, or chemotherapy. For example, in some embodiments, adjuvant therapy using the IL2 / IL2Rα fusion proteins disclosed herein is administered when there may be a risk of micrometastasis and / or to reduce the risk of recurrence.

[0449] In some embodiments, the IL2 / IL2Rα fusion protein disclosed herein is administered as a monotherapy or as the sole immunostimulatory therapy. Antibodies to IL2Rα can also be combined with immunogenic agents such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immunostimulatory cytokines (He et al., (2004) J. Immunol. 173: 4919-28). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens (such as gp100, MAGE antigens, Trp-2, MART1, and / or tyrosinase peptides) or tumor cells transfected to express the cytokine GM-CSF (discussed further below).

[0450] In some embodiments, the IL2 / IL2Rα fusion protein disclosed herein is combined with a vaccination regimen. Many experimental strategies for vaccination against tumors have been designed (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C, 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; also see Restifo, N. and Sznol, M., Cancer Vaccines, Chapter 61, pages 3023-3043, in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition). In one of these strategies, autologous or allogeneic tumor cells are used to prepare a vaccine. It has been shown that these cellular vaccines are most effective when tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci USA 90:3539-43).

[0451] Studies of gene expression and large-scale gene expression patterns in various tumors have led to the definition of so-called tumor-specific antigens (Rosenberg, SA (1999) Immunity 10: 281-7). In many cases, these tumor-specific antigens are differentiation antigens expressed in tumors and tumor-producing cells, such as melanocyte antigen gp100, MAGE antigens, and Trp-2. More importantly, many of these antigens can be shown to be targets of tumor-specific T cells found in the host. In some embodiments, the IL2 / IL2Rα fusion protein disclosed herein is used in combination with a collection of recombinant proteins and / or peptides expressed in tumors to generate an immune response against these proteins. These proteins are generally regarded as self-antigens by the immune system and can therefore be tolerated by the immune system. Tumor antigens can include the protein telomerase, which is required for the synthesis of chromosome telomeres and is expressed in more than 85% of human cancers and only a limited number of somatic tissues (Kim et al. (1994) Science 266: 2011-2013). Tumor antigens can also be “neoantigens” expressed in cancer cells due to somatic mutations that alter the protein sequence or generate a fusion protein between two unrelated sequences (i.e., bcr-abl in the Philadelphia chromosome), or from the idiotype of a B-cell tumor.

[0452] Other tumor vaccines can include proteins from viruses implicated in human cancers, such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's herpes sarcoma virus (KHSV). Another form of tumor-specific antigen that can be used in conjunction with the fusion proteins disclosed herein is purified heat shock proteins (HSPs) isolated from the tumor tissue itself. These heat shock proteins contain protein fragments from tumor cells, and these HSPs are highly effective in eliciting tumor immunity when delivered to antigen-presenting cells (Suot & Srivastava (1995) Science 269: 1585-1588; Tamura et al. (1997) Science 278: 117-120).

[0453] Dendritic cells (DCs) are potent antigen-presenting cells that can be used to elicit antigen-specific responses. DCs can be generated in vitro and loaded with various protein and peptide antigens and tumor cell extracts (Nestle et al. (1998) Nature Medicine 4:328-332). DCs can also be transduced by genetic means to express these tumor antigens. For immunization purposes, DCs have also been directly fused with tumor cells (Kugler et al. (2000) Nature Medicine 6:332-336). As a vaccination method, DC immunity can be effectively combined with the IL2 / IL2Rα fusion protein disclosed herein to activate a more potent anti-tumor response.

[0454] In some embodiments, the method of treating cancer using the IL2 / IL2Rα fusion protein disclosed herein is combined with standard cancer treatment (e.g., surgery, radiation, and chemotherapy). An example of such a combination is a combination of an anti-TIM3 antibody and an IL2 / IL2Rα fusion protein disclosed herein for the treatment of melanoma. The scientific principle behind the combined use of the IL2 / IL2Rα fusion protein disclosed herein and chemotherapy is that cell death as a result of the cytotoxic effect of most chemotherapeutic compounds should lead to an increase in the level of tumor antigens in the antigen presentation pathway. Other combination therapies that may result in synergy with the IL2 / IL2Rα fusion protein disclosed herein through cell death are radiation, surgery, and hormone deprivation. Each of these regimens produces a source of tumor antigens in the host. Angiogenesis inhibitors can also be combined with the IL2 / IL2Rα fusion protein disclosed herein. Inhibiting angiogenesis leads to tumor cell death, which can supply tumor antigens to the host antigen presentation pathway.

[0455] The IL2 / IL2Rα fusion proteins disclosed herein as described herein can also be used in combination with bispecific antibodies that target effector cells expressing Fcα or Fcγ receptors to tumor cells (see, e.g., U.S. Patent Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. For example, anti-Fc receptor / anti-tumor antigen (e.g., Her-2 / neu) bispecific antibodies have been used to target macrophages to tumor sites. This targeting can more effectively activate tumor-specific responses. Alternatively, antigens can be delivered directly to DCs using bispecific antibodies that bind to tumor antigens and dendritic cell-specific cell surface markers.

[0456] Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivation of proteins expressed by tumors and immunosuppressive. These also include TGF-β (Kehrl et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13: 198-200) and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365). Antibodies against each of these entities can be used in combination with the IL2 / IL2Rα fusion protein disclosed herein to counteract the effects of immunosuppressive agents and facilitate the host's tumor immune response.

[0457] Other antibodies that activate the host immune response can be used in combination with the IL2 / IL2Rα fusion proteins disclosed herein. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Anti-CD40 antibodies can effectively replace T cell helper activity (Ridge et al. (1998) Nature 393:474-478) and can be used in combination with the IL2 / IL2Rα fusion proteins disclosed herein. Activating antibodies against T cell co-stimulatory molecules such as CTLA-4 (e.g., U.S. Pat. No. 5,811,097), OX-40 (Weinberg et al. (2000) Immunol 164:2160-2169), 4-1BB (Melero et al. (1997) Nature Medicine 3:682-685 (1997), and ICOS (Hutloff et al. (1999) Nature 397:262-266) can also provide increased levels of T cell activation. Inhibitors of PD1 or PD-L1 can also be used in combination with the IL2 / IL2Rα fusion proteins disclosed herein. Other combinations are provided elsewhere herein.

[0458] Bone marrow transplantation is currently used to treat a variety of hematopoietic tumors. Although graft-versus-host disease is a consequence of this treatment, therapeutic benefit can be obtained from a graft-versus-tumor response. In some embodiments, the IL2 / IL2Rα fusion proteins disclosed herein are used to increase the effectiveness of transplanted tumor-specific T cells from the donor.

[0459] There are also several experimental treatment options that involve ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells into recipients to stimulate antigen-specific T cells to fight tumors (Greenberg & Riddell (1999) Science 285: 546-51). These methods can also be used to activate T cells in response to infectious agents (such as CMV). In some embodiments, ex vivo activation in the presence of the IL2 / IL2Rα fusion protein disclosed herein can increase the frequency and activity of adoptively transferred T cells.

[0460] 7.7.2.2 Inflammatory or autoimmune diseases

[0461] In some embodiments, disclosed herein are methods of treating a disease or disorder in a subject in need thereof, wherein the disease or disorder is an inflammatory disease or an autoimmune disease. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an IL2 / IL2Rα fusion protein disclosed herein.

[0462] In some embodiments, an IL2 / IL2Rα fusion protein disclosed herein is administered to a patient with an inflammatory disease or autoimmune disease that has exhibited an inadequate response or progression to a previous treatment. In some embodiments, an IL2 / IL2Rα fusion protein described herein is administered to a patient who has not previously received (i.e., not been treated with) a treatment for an inflammatory disease or autoimmune disease.

[0463] In some embodiments, the IL2 / IL2Rα fusion proteins disclosed herein are administered with standard of care treatment for an inflammatory disease or autoimmune disease. In some embodiments, the IL2 / IL2Rα fusion proteins disclosed herein are administered as maintenance therapy for an inflammatory disease or autoimmune disease, e.g., as a therapy intended to prevent the onset or recurrence of inflammation.

[0464] In some embodiments, the IL2 / IL2Rα fusion proteins disclosed herein are administered as a monotherapy for treating an inflammatory disease or an autoimmune disease, or as the sole immunostimulatory therapy for treating an inflammatory disease or an autoimmune disease. In some embodiments, the IL2 / IL2Rα fusion proteins disclosed herein are combined with a vaccination regimen for treating an inflammatory disease or an autoimmune disease. In some embodiments, the IL2 / IL2Rα fusion proteins disclosed herein are combined with an antibody for treating an inflammatory disease or an autoimmune disease.

[0465] In some embodiments, the inflammatory disease or autoimmune disease is selected from the group consisting of type 1 diabetes, multiple sclerosis, rheumatoid arthritis, celiac disease, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis or systemic sclerosis, graft-versus-host disease, psoriasis, alopecia areata, HCV-induced vasculitis, Sjögren's syndrome, pemphigus, ankylosing spondylitis, Behçet's disease, Wegener's granulomatosis, Takayasu's disease, autoimmune hepatitis, sclerosing cholangitis, Guillain-Strauss syndrome and macrophage activation syndrome.

[0466] 7.7.2.3 Infectious diseases

[0467] The methods described herein can also be used to treat patients who have been exposed to specific toxins or pathogens. Thus, in some embodiments, disclosed herein are methods for treating a disease or disorder in a subject in need thereof, wherein the disease or disorder is an infectious disease. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an IL2 / IL2Rα fusion protein disclosed herein to treat the infectious disease.

[0468] Similar to its application to tumors as discussed above, methods of treatment include using the IL2 / IL2Rα fusion proteins disclosed herein, alone or in combination as adjuvants with vaccines, to stimulate immune responses against pathogens, toxins, and self-antigens. Examples of pathogens for which such treatments may be particularly useful include pathogens for which there are currently no effective vaccines or for which conventional vaccines are not fully effective. These include, but are not limited to, HIV, hepatitis viruses (type A, B, and C), influenza viruses, herpes viruses, Giardia, Malaria, Leishmania, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0469] Some examples of pathogenic viruses that cause infections treatable by the methods described herein include HIV, hepatitis (A, B, or C), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0470] Some examples of pathogenic bacteria that cause infections that can be treated by the methods described herein include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme disease bacteria.

[0471] Some examples of pathogenic fungi that cause infections treatable by the methods described herein include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0472] Some examples of pathogenic parasites that cause infections treatable by the methods described herein include Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lamblia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus brasiliensis.

[0473] In all of the above methods, treatment with the IL2 / IL2Rα fusion proteins described herein can be combined with other forms of immunotherapy, such as those described herein, such as cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL2), or bispecific antibody therapy, which provides enhanced tumor antigen presentation (see, e.g., Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2:1121-1123).

[0474] 7.7.2.4 Vaccines

[0475] By co-administering the IL2 / IL2Rα fusion protein disclosed herein with an antigen of interest (e.g., a vaccine), the IL2 / IL2Rα fusion protein disclosed herein can be used to stimulate an antigen-specific immune response. Thus, provided herein is a method for enhancing a subject's immune response to an antigen, comprising administering to the subject: (i) an antigen; and (ii) an IL2 / IL2Rα fusion protein disclosed herein, such that the subject's immune response to the antigen is enhanced. The antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen from a pathogen. Non-limiting examples of such antigens include those discussed in the above sections, such as the tumor antigens (or tumor vaccines) discussed above, or antigens from the above-mentioned viruses, bacteria, or other pathogens.

[0476] In some embodiments, a peptide or fusion protein comprising an epitope bound by an IL2 / IL2Rα fusion protein disclosed herein is used as a vaccine instead of, or in addition to, the IL2 / IL2Rα fusion protein disclosed herein.

[0477] Suitable routes for in vivo and in vitro administration of the antibody compositions described herein (e.g., human monoclonal antibodies, multispecific and bispecific molecules, and immunoconjugates) are well known in the art and can be selected by one of ordinary skill. For example, the antibody compositions can be administered by injection (e.g., intravenously or subcutaneously). The appropriate dosage of the molecule used will depend on the age and weight of the subject and the concentration and / or formulation of the antibody composition.

[0478] 7.7.2.5 Co-administration with a second agent

[0479] As previously described, the IL2 / IL2Rα fusion protein disclosed herein can be co-administered with one or more other therapeutic agents (e.g., cytotoxic agents, radiotoxic agents, or immunosuppressants). The IL2 / IL2Rα fusion protein disclosed herein can be linked to the agent (as an immune complex), or can be administered separately from the agent. In the latter case (separate administration), the IL2 / IL2Rα fusion protein disclosed herein can be administered before, after, or simultaneously with the other agent, or can be co-administered with other known therapies (e.g., anticancer therapies, such as radiation). Such therapeutic agents include, in particular, anti-tumor agents such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil, dacarbazine, and cyclophosphamide hydroxyurea, which themselves are only effective at levels that are toxic or subtoxic to the patient. Cisplatin is administered intravenously at a dose of 100 mg / ml once every four weeks, and adriamycin is administered intravenously at a dose of 60-75 mg / ml once every 21 days. Co-administration of the IL2 / IL2Rα fusion proteins disclosed herein with chemotherapeutic agents provides two anticancer agents that act through different mechanisms, which produce cytotoxic effects on human tumor cells. Such co-administration can address the problem of tumor cells becoming unreactive with antibodies due to the development of drug resistance or changes in their antigenicity.

[0480] Provided herein are methods of combination therapy, wherein the IL2 / IL2Rα fusion proteins disclosed herein are co-administered with one or more additional agents (second therapeutic agents) effective to stimulate an immune response (e.g., small molecule drugs, antibodies, or antigen-binding portions thereof), thereby further enhancing, stimulating, or upregulating the subject's immune response.

[0481] Typically, the IL2 / IL2Rα fusion proteins disclosed herein can be combined with (i) stimulatory (e.g., co-stimulatory) molecules (e.g., receptors or ligands) agonists and / or (ii) antagonists of inhibitory signals or molecules (e.g., receptors or ligands) on immune cells (e.g., T cells), both of which result in an amplified immune response (e.g., antigen-specific T cell response). In some aspects, the immuno-oncology agent is an agonist of (i) stimulatory (including co-stimulatory) molecules (e.g., receptors or ligands) or (ii) an antagonist of inhibitory (including co-inhibitory) molecules (e.g., receptors or ligands) on cells (e.g., cells that inhibit T cell activation or cells that participate in innate immunity, e.g., NK cells), and wherein the immuno-oncology agent enhances innate immunity. Such immuno-oncology agents are generally referred to as immune checkpoint regulators, such as immune checkpoint inhibitors or immune checkpoint stimulators.

[0482] In some embodiments, the IL2 / IL2Rα fusion protein disclosed herein is administered together with an agent that targets a stimulatory or inhibitory molecule that is a member of the immunoglobulin superfamily (IgSF). For example, the IL2 / IL2Rα fusion protein disclosed herein can be administered to a subject together with an agent that targets an IgSF family member to increase the immune response. For example, the IL2 / IL2Rα fusion protein disclosed herein can be administered together with an agent that targets (or specifically binds to) a B7 family member of a membrane-bound ligand or an agent that targets a co-stimulatory or co-inhibitory receptor or ligand that specifically binds to a B7 family member, wherein the B7 family member of the membrane-bound ligand includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA) and B7-H6.

[0483] The IL2 / IL2Rα fusion proteins disclosed herein can also be administered with agents that target members of the TNF and TNFR family of molecules (ligands or receptors), such as CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137, TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR / Fn 14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTpR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3, EDAl, EDA2, TNFR1, lymphotoxin alpha / TNFp, TNFR2, TNFa, LTpR, lymphotoxin alpha 1β2, FAS, FASL, RELT, DR6, TROY, and NGFR (see, e.g., Tansey (2009) Drug Discovery Today 00:1).

[0484] In some embodiments, the IL2 / IL2Rα fusion protein disclosed herein is administered together with an agent comprising an anti-PD-1 antibody. The anti-PD-1 antibody can be any antibody that binds to PD-1 and inhibits the interaction between PD-1 and PD-L1. In some embodiments, the anti-PD-1 antibody is any anti-PD-1 antibody known in the art. In some embodiments, the second therapeutic agent comprises nivolumab. In some embodiments, the second therapeutic agent comprises pembrolizumab.

[0485] In some embodiments, the second therapeutic agent comprises an anti-PD-L1 antibody. The anti-PD-L1 antibody can be any antibody that binds to PD-L1 and inhibits the interaction between PD-1 and PD-L1. In some embodiments, the anti-PD-L1 antibody is any anti-PD-L1 antibody known in the art. In some embodiments, the second therapeutic agent comprises atezolizumab. In some embodiments, the second therapeutic agent comprises durvalumab. In some embodiments, the second therapeutic agent comprises avelumab.

[0486] In some embodiments, the second therapeutic agent comprises an anti-CTLA-4 antibody. The anti-CTLA-4 antibody can be any antibody that binds to CTLA-4 and inhibits its activity. In some embodiments, the anti-CTLA-4 antibody is any anti-CTLA-4 antibody known in the art. In some embodiments, the second therapeutic agent comprises tremelimumab. In some embodiments, the second therapeutic agent comprises ipilimumab.

[0487] In some embodiments, the second therapeutic agent comprises an anti-LAG3 antibody. The anti-LAG3 antibody can be any antibody that binds to LAG-3 and inhibits its activity. In some embodiments, the anti-LAG3 antibody is any anti-LAG3 antibody known in the art. In some embodiments, the second therapeutic agent comprises 25F7.

[0488] In some embodiments, wherein the second therapeutic agent comprises an anti-CD137 antibody. The anti-CD137 antibody can be any antibody that binds CD137 and inhibits its activity. In some embodiments, the anti-CD137 antibody is any anti-CD137 antibody known in the art. In some embodiments, the second therapeutic agent comprises Urelumab.

[0489] In some embodiments, the second therapeutic agent comprises an anti-KIR antibody. The anti-KIR antibody can be any antibody that binds to KIR and inhibits its activity. In some embodiments, the anti-KIR antibody is any anti-KIR antibody known in the art. In some embodiments, the second therapeutic agent comprises lirilumab.

[0490] In some embodiments, wherein the second therapeutic agent comprises an anti-GITR antibody. The anti-GITR antibody can be any antibody that binds to GITR and inhibits its activity. In some embodiments, the anti-GITR antibody is any anti-GITR antibody known in the art. In some embodiments, the second therapeutic agent comprises MK4166. In some embodiments, the second therapeutic agent comprises TRX518.

[0491] In other embodiments, the second therapy comprises administering an anti-TIM3 antibody. The anti-TIM3 antibody can be any antibody that binds to TIM3 and inhibits its activity. In some embodiments, the anti-TIM3 antibody is any anti-TIM3 antibody known in the art.

[0492] In certain embodiments, the second therapy comprises administering a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is selected from a proteasome inhibitor, an immunomodulatory drug (IMiD), a Bet inhibitor, and any combination thereof. In some embodiments, the proteasome inhibitor is selected from bortezomib, ixazomib, carfilzomib, oprozomib, and marizomib. In certain embodiments, the proteasome inhibitor comprises bortezomib.

[0493] In some embodiments, the second therapy comprises radiation therapy.Any radiation therapy known in the art can be used as the second therapy.

[0494] In some embodiments, the second therapy includes administering an agent that activates innate immune cells. In some embodiments, the agent that activates innate immune cells includes an NLRP3 agonist. In some embodiments, the NLRP3 agonist comprises sodium monourate monohydrate (MSU) and / or the vaccine adjuvant alum. In some embodiments, the agent that activates innate immune cells is a toll-like receptor 7 (TLR7) agonist. In some embodiments, the TLR7 agonist includes imiquimod (R837), GS-9620 (see Tsai et al., J.Virology doi:10.1128 / JVI.02166-16 (February 8, 2017)), ORN R-2336 (Miltenyl Biotec) or any combination thereof.

[0495] In some embodiments, the second therapy comprises administering a drug that enhances natural killer (NK) cells, CD8 + T cells, or both.

[0496] In certain embodiments, the second therapy comprises administering an agent selected from the group consisting of: doxorubicin Cisplatin, carboplatin, bleomycin sulfate, carmustine, chlorambucil Cyclophosphamide Lenalidomide Bortezomib Dexamethasone, mitoxantrone, etoposide, cytarabine, bendamustine Rituximab Ifosfamide, vincristine Fludarabine Thalidomide Alemtuzumab Ofatumumab Everolimus Carfilzomib (KYPROLIST™) and any combination thereof.

[0497] Exemplary agents that modulate one of the above proteins and that can be used in combination with the fusion proteins described herein to treat cancer include: (ipilimumab) or tremelimumab (anti-CTLA-4), galiximab (anti-B7.1), BMS-936558 (anti-PD-1), MK-3475 (anti-PD-1), atezolizumab AMP224 (anti-B7DC), BMS-936559 (anti-B7-H1), MPDL3280A (anti-B7-H1), MEDI-570 (anti-ICOS), AMG557 (anti-B7H2), MGA271 (anti-B7H3), IMP321 (anti-LAG-3), BMS-663513 (anti-CD137), PF-05082566 (anti-CD137), CDX-1127 (anti-CD27), anti-OX40 (Providence Health Services), huMAbOX40L (anti-OX40L), atacicept (anti-TACI), CP-870893 (anti-CD40), rucatumumab (anti-CD40), dacetuzumab (anti-CD40), muromonab-CD3 (anti-CD3); anti-GITR antibodies MK4166, TRX518, Medi1873, INBRX-110, LK2-145, GWN-323, GITRL-Fc, or any combination thereof.

[0498] Other molecules that can be combined with the fusion protein for treating a disease or disorder include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, the fusion protein described herein can be combined with a KIR antagonist (e.g., lirilumab).

[0499] T cell activation is also regulated by soluble cytokines, and the fusion proteins described herein can be administered to a subject, for example, with cancer, along with a cytokine antagonist that inhibits T cell activation or a cytokine agonist that stimulates T cell activation.

[0500] In some embodiments, the fusion proteins described herein can be used in combination with: (i) antagonists (or inhibitors or blockers) of IgSF family, B7 family, or TNF family proteins that inhibit T cell activation or antagonists of cytokines that inhibit T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF; "immunosuppressive cytokines") and / or (ii) agonists of IgSF family, B7 family, or TNF family stimulating receptors or cytokine agonists that stimulate T cell activation to stimulate an immune response, e.g., to treat a proliferative disease, such as cancer.

[0501] Still other agents for use in combination therapy include agents that inhibit or deplete macrophages or monocytes, including but not limited to CSF-1R antagonists, such as CSF-1R antagonist antibodies, including RG7155 (WO 11 / 70024, WO 11 / 107553, WO 11 / 131407, WO 13 / 87699, WO 13 / 119716, WO 13 / 132044) or FPA-008 (WO 11 / 140249; WO13169264; WO 14 / 036357).

[0502] The fusion proteins of the present disclosure can also be administered with agents that inhibit TGF-β signaling.

[0503] Additional agents that can be combined with the fusion proteins described herein include agents that enhance tumor antigen presentation (e.g., dendritic cell vaccines, GM-CSF-secreting cellular vaccines, CpG oligonucleotides, and imiquimod) or therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines).

[0504] Another therapy that can be combined with the fusion proteins described herein is one that inhibits metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase.

[0505] Another class of agents that can be used with the fusion proteins described herein include agents that inhibit adenosine formation (e.g., CD73 inhibitors) or agents that inhibit adenosine A2A receptors.

[0506] Other therapies that can be used in combination with the fusion proteins described herein to treat a disease or disorder (e.g., cancer) include therapies that reverse / prevent T cell anergy or exhaustion and therapies that trigger innate immune activation and / or inflammation at tumor sites.

[0507] Other therapies that can be used in combination with the fusion proteins described herein to treat a disease or disorder (eg, cancer) include therapies that block IL-8 (eg, using HuMax-IL8).

[0508] The fusion proteins described herein can be combined with more than one immuno-oncology agent and can be combined, for example, with combinatorial approaches that target multiple elements of the immune pathway, such as one or more of the following: therapies that enhance tumor antigen presentation (e.g., dendritic cell vaccines, cellular vaccines that secrete GM-CSF, CpG oligonucleotides, imiquimod); therapies that inhibit negative immune regulation, for example, by inhibiting the CTLA-4 and / or PD1 / PD-L1 / PD-L2 pathways and / or depleting or blocking Treg or other immunosuppressive cells; therapies that stimulate positive immune regulation, for example, using agonists that stimulate the CD-137, OX-40 and / or CD40 or GITR pathways and / or stimulate T cell effector function; therapies that systemically increase the frequency of anti-tumor T cells ; therapies that deplete or suppress Tregs (such as Tregs in tumors), for example, using antagonists of CD25 (such as daclizumab) or by ex vivo anti-CD25 bead depletion; therapies that affect suppressive myeloid cell function in tumors; therapies that enhance the immunogenicity of tumor cells (such as anthracyclines); adoptive T cell or NK cell transfer, including genetically modified cells, such as cells modified by chimeric antigen receptors (CAR-T therapy); therapies that inhibit metabolic enzymes (such as indoleamine dioxygenase (IDO), dioxygenase, arginase or nitric oxide synthase); therapies that reverse / prevent T cell anergy or exhaustion; therapies that trigger innate immune activation and / or inflammation at the tumor site; administration of immunostimulatory cytokines; or blocking immunosuppressive cytokines.

[0509] The fusion proteins described herein can be used with one or more agonists that bind to positive co-stimulatory receptors, blockers that attenuate signaling through inhibitory receptors, antagonists, and one or more agents that systemically increase the frequency of anti-tumor T cells, overcome different immunosuppressive pathways in the tumor microenvironment (e.g., blocking inhibitory receptor engagement (e.g., PD-L1 / PD-1 interaction), deplete or inhibit Tregs (e.g., using anti-CD25 monoclonal antibodies (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion), inhibit metabolic enzymes (such as IDO), or reverse / prevent T cell anergy or exhaustion), and agents that trigger innate immune activation and / or inflammation at the tumor site.

[0510] In some embodiments, if the subject is BRAF V600 mutation positive, the fusion protein of the present disclosure is administered to the subject together with a BRAF inhibitor.

[0511] For example, the fusion proteins of the present disclosure and the combination therapies described herein can be used in combination (e.g., simultaneously or separately) with other therapies, such as irradiation and / or chemotherapy, for example, using camptothecin (CPT-11), 5-fluorouracil (5-FU), cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin, paclitaxel, carboplatin-paclitaxel (Taxol), doxorubicin, or camptothecin + apo21 / TRAIL (6X combination)), one or more proteasome inhibitors (e.g., bortezomib or MG132), one or more Bcl-2 inhibitors (e.g., BH3I- 2' (Bcl-xl inhibitors), indoleamine dioxygenase-1 inhibitors (e.g., INCB24360, indomod, NLG-919, or F001287), AT-101 (R-(-)-gossypol derivative), ABT-263 (small molecule), GX-15-070 (obatoclax), or MCL-1 (myeloid leukemia cell differentiation protein-1) antagonists), iAP (inhibitor of apoptosis protein) antagonists (e.g., smac7, smac4, small molecule smac mimetics, synthetic smac peptides (see Fulda et al., Nat Med 2002;8:808-15), ISIS23722 (LY2181308) or AEG-35156 (GEM-640)), HDAC (histone deacetylase) inhibitors, anti-CD20 antibodies (e.g., rituximab), angiogenesis inhibitors (e.g., bevacizumab), anti-angiogenic agents targeting VEGF and VEGFR (e.g., Avastin), synthetic triterpenes (see Hyer et al., Cancer Research 2005;65:4799-808), c-FLIP (cellular FLICE inhibitory protein) modulators (e.g., natural and synthetic ligands of PPARy (peroxisome proliferator-activated receptor gamma), 5809354 or 5569100), kinase inhibitors (e.g., sorafenib), trastuzumab, cetuximab, temsirolimus, mTOR inhibitors (e.g., rapamycin and temsirolimus), bortezomib, JAK2 inhibitors, HSP90 inhibitors, PI3K-AKT inhibitors, lenalidomide, GSK3P inhibitors, IAP inhibitors, and / or genotoxic drugs.

[0512] The fusion proteins of the present disclosure and the combination therapies described herein can be further used in combination with one or more anti-proliferative cytotoxic agents. The types of compounds that can be used as anti-proliferative cytotoxic agents include, but are not limited to, the following:

[0513] Alkylating agents (including but not limited to nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes): uracil mustard, chlorambucil, cyclophosphamide Phosphamide, melphalan, chlorambucil, guanidine, triethylene melamine, triethylene thiophosphamide, busulfan, carmustine, lomustine, streptozotocin, dacarbazine, and temozolomide.

[0514] Antimetabolites (including but not limited to folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors): methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, and gemcitabine.

[0515] Suitable antiproliferative agents for use in combination with the fusion proteins of the present disclosure include, but are not limited to, taxanes, paclitaxel (paclitaxel is available as TAXOL TM commercially available), docetaxel, dimorphostatin (DDM), dictyostatin (DCT), Peloruside A, Epothilone, Epothilone A, Epothilone B, Epothilone C, Epothilone D, Epothilone E, Epothilone F, Furoxepothilone D, Deoxyepothilone B1,

[17] -Dehydrodeoxyepothilone B,

[18] -Dehydrodeoxyepothilone B, C12,13-cyclopropyl-epothilone A, C6-C8 bridged epothilone A, trans-9,10-dehydroepothilone D, cis-9,10-dehydroepothilone D, 16-Demethylepothilone B, Epothilone BIO, discoderomolide, Padupirone (EPO-906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA296A (soblidotin), TZT-1027 (soblidotin), ILX-651 (tasidotin hydrochloride), Halichondrin B, Eribulin mesylate (E-7389), Hamitrin (HTI-286), E-7974, Cyrptophycins, LY-355703, Maytansinoid immunoconjugate (DM-1), MKC-1, ABT-751, Tl-38067, T-900607, SB-715992 (Epinaxin), SB-743921, MK-0731, STA-5312, and Softcoralin (eleutherobin), 17β-acetoxy-2-ethoxy-6-oxo-B-homo-estra-l,3,5(10)-trien-3-ol, cyclostreptin, isolaulimalide, laulimalide, 4-epi-7-dehydroxy-14,16-didemethyl-(+)-discinolide and cryptothilone 1, as well as other microtubule stabilizers known in the art.

[0516] Where it is desired to render abnormally proliferating cells quiescent, in conjunction with or prior to treatment with the fusion proteins of the present disclosure as described herein, hormones and steroids (including synthetic analogs) such as 17a-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, methylandrostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlortrisone, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, When using the methods or compositions described herein, other agents used in clinical settings to modulate tumor growth or metastasis, such as antimimetic drugs, can also be administered as needed.

[0517] In some embodiments, the combination of the fusion rotein of the present disclosure discussed herein and the second medicament can be given simultaneously as a single composition in a pharmaceutically acceptable carrier, or as a separated composition, wherein the fusion rotein of the present disclosure and the second medicament are in a pharmaceutically acceptable carrier. In some embodiments, the combination sequence of the fusion rotein of the present disclosure and the second medicament can be given. The giving of two kinds of medicament can start at a distance of for example 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 5 days, 7 days or a week or many weeks, or the giving of the second medicament can start after having given the first medicament for example 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 5 days, 7 days or a week or many weeks.

[0518] In some embodiments, anti-tumor antibodies that can be combined with the fusion proteins of the present disclosure and / or the second agent include (rituximab), (trastuzumab), (tositumomab), (Eretomazumab), (alemtuzumab), (eprtuzumab), (bevacizumab) and (erlotinib) or any combination thereof. In some embodiments, the second antibody that can be used in combination therapy with the fusion proteins of the present disclosure can be an antibody drug conjugate.

[0519] In some embodiments, the fusion proteins of the present disclosure, alone or in combination with another agent, are used simultaneously or sequentially with bone marrow transplantation to treat a variety of tumors of hematopoietic origin.

[0520] Provided herein are methods for altering adverse events associated with the treatment of hyperproliferative diseases (e.g., cancer) with immunostimulants, comprising administering a fusion protein of the present disclosure to a subject with or without a second agent. For example, the methods described herein provide methods for reducing the incidence of colitis or diarrhea induced by immunostimulatory therapeutic antibodies by administering a non-absorbable steroid to a patient. As used herein, a "non-absorbable steroid" is a glucocorticoid that exhibits extensive first-pass metabolism such that after metabolism in the liver, the bioavailability of the steroid is low, i.e., less than about 20%. In some embodiments described herein, the non-absorbable steroid is budesonide. Budesonide is a locally acting glucocorticoid that is extensively metabolized primarily by the liver after oral administration. ENTOCORT (Astra-Zeneca) is a pH- and time-dependent oral formulation of budesonide developed to optimize drug delivery to the ileum and throughout the colon. Approved in the United States for the treatment of mild to moderate Crohn's disease involving the ileum and / or ascending colon. In some embodiments, the fusion protein of the present disclosure in combination with a non-absorbable steroid can be further combined with a salicylate. Salicylates include 5-ASA agents such as, for example, sulfasalazine ( Pharmacia & Up John); Olsalazine ( Pharmacia & Up John); balsalazide ( Salix Pharmaceuticals, Inc.); and mesalazine ( Procter & Gamble Pharmaceuticals; Shire US; Axcan Scandipharm, Inc.; Solvay).

[0521] 7.8 Reagent Kit

[0522] As used herein, the kit comprises an IL2 / IL2Rα fusion protein for modulating an immune response, as described elsewhere herein. As used herein, the terms "kit" and "system" are intended to refer to at least one or more IL2 / IL2Rα fusion proteins, in particular embodiments, in combination with one or more other types of elements or components (e.g., other types of biochemical reagents, containers, packaging (e.g., packaging intended for commercial sale), instructions for use, etc.).

[0523] In some embodiments, a kit is disclosed comprising (a) one or more of the following: an IL2 / IL2Rα fusion protein as described herein, a composition comprising an IL2 / IL2Rα fusion protein as described herein, a nucleic acid encoding an IL2 / IL2Rα fusion protein as described herein, a vector, and / or a host cell; and (b) instructions for administering the fusion protein to a subject in need thereof. In some embodiments, a kit is disclosed comprising (a) an IL2 / IL2Rα fusion protein as described herein and (b) instructions for administering the fusion protein to a subject in need thereof. In some embodiments, a kit is disclosed comprising (a) a composition comprising an IL2 / IL2Rα fusion protein as described herein and (b) instructions for administering the composition to a subject in need thereof. In some embodiments, a kit is disclosed comprising (a) a nucleic acid encoding an IL2 / IL2Rα fusion protein as described herein and (b) instructions for administering the nucleic acid to a subject in need thereof. In some embodiments, a kit is disclosed comprising (a) a vector as described herein and (b) instructions for administering the vector to a subject in need thereof. In some embodiments, a kit is disclosed comprising (a) a host cell as described herein and (b) instructions for administering the host cell to a subject in need thereof.

[0524] In a specific embodiment, a pharmaceutical package or test kit is provided herein, which includes one or more containers, and the container is filled with one or more ingredients of a pharmaceutical composition as described herein, such as one or more fusion proteins provided herein. In some embodiments, the test kit contains a pharmaceutical composition as described herein and any prophylactic or therapeutic agent, such as those described herein. In certain embodiments, the test kit may contain a T cell mitogen, such as, for example, phytohemagglutinin (PHA) and / or phorbol myristate acetate (PMA), or a TCR complex stimulating antibody (such as an anti-CD3 antibody and an anti-CD28 antibody). Optionally associated with one or more containers may be an announcement in the form of a government agency regulating the manufacture, use or sale of a drug or biological product, reflecting approval of manufacture, use or sale by the agency for administration to the human race.

[0525] Also provided herein are kits that can be used for the above methods. In one embodiment, the kit comprises a fusion protein as described herein in one or more containers, preferably a purified fusion protein. In a specific embodiment, the kit as described herein contains a substantially isolated fusion protein as a control. In another specific embodiment, the kit as described herein further comprises a control antibody or fusion protein that does not react with the IL2 and / or IL2-Rα antigen. In another specific embodiment, the kit as described herein contains one or more elements for detecting the binding of the fusion protein to the IL2 and / or IL2-Rα antigen (e.g., the fusion protein can be conjugated to a detectable substrate (e.g., a fluorescent compound, an enzyme substrate, a radioactive compound, or a luminescent compound), or a second antibody that recognizes the first antibody can be conjugated to a detectable substrate). In a specific embodiment, the kit provided herein may include recombinantly produced or chemically synthesized fusion proteins. Antigens for the fusion proteins disclosed herein as provided in the kit may also be attached to a solid support. In a more specific embodiment, the detection device of the above kit includes a solid support to which the antigen of the fusion protein is attached. Such a kit may also include unattached anti-human antibodies or anti-mouse / rat antibodies labeled with a reporter. In this embodiment, binding of the fusion protein to the antigen can be detected by binding of the reporter-labeled antibody.

[0526] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are fully described in the literature. See, e.g., Sambrook et al., eds. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., eds. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, New York); D. N. Glover, ed., (1985) DNA Cloning, Vols. I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al., U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning. Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., New York); Miller and Calos, eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds. (1986) Handbook Of Experimental Immunology, vols. I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, (1986); Crooks, Antisense drug Technology: Principles, strategies and applications, 2nd ed. CRC Press (2007); and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0527] All references cited above, as well as all references and amino acid or nucleotide sequences (eg, GenBank numbers and / or Uniprot numbers) cited herein, are incorporated herein by reference in their entirety.

[0528] The following examples are offered by way of illustration and not by way of limitation.

[0529] 8. Examples

[0530] Example 1. IL2-CD25 fusion protein forms a stable homogeneous homodimer

[0531] The size of fusion protein and oligomeric state are studied by size exclusion chromatography (SEC-MALS) coupled with in-situ multi-angle light scattering detector.Prepare sample by injecting 30 μ g of reserve protein sample.In the buffer (adding 0.02% sodium azide and filtering through 0.1um, running with the flow velocity of 0.75mL / min) that contains 40mM Tris, 200mMNaCl (pH 7.5), on the GE Healthcare Superdex 200Increase 10 / 300GL post (10mm * 300mm) that is connected with Prominence Shimadzu UFLC system (described system is made up of degasser, isocratic pump, cooling sample rack, UV / vis detector and column oven with syringe), carry out isocratic separation. The sample was injected into the column using a Shimadzu autosampler, and data were acquired from three online detectors in series: a Shimadzu SPD-20 dual wavelength UV / vis spectrophotometer set to collect at 280 nm, followed by a Wyatt Technologies mini-Dawn TREOS triangular laser light scattering detector, and then a Wyatt Optilab T-rEX interferometer refractometer. Data were collected and analyzed using Astra 6 (Wyatt) and LabSolutions Lite (Shimadzu) software.

[0532] Figure 1 The data in show a typical absolute mass versus elution time relationship for analytical size exclusion chromatography. Figure 1 The sample analyzed was for IL2-CD25 (22-212) (SEQ ID NO: 16) fused to a His tag (GGHHHHHH (SEQ ID NO: 100)), with a theoretical molecular weight of 37,812 (reduced). The data indicated that IL2-CD25 (22-212) formed a homogeneous species throughout the elution profile with an absolute mass of 93 kDa. The elution peak showed no evidence of monomeric species or oligomeric species that were orders of magnitude higher than the main peak. The mass value of 93 kDa indicated that the molecule formed a homodimer and was also glycosylated (approximately 18% of the mass from the known N- and O-linked glycosylation sites on IL2 and CD25).

[0533] Example 2: IL2-CD25 fusion protein binds equivalently to sCD25 and sIL2Rβ / IL2Rγ heteroreceptors, with reduced affinity observed.

[0534] Surface plasmon resonance (SPR) studies were performed on Biacore T100 and / or T200 instruments (GE Healthcare) at 25° C. Binding of fusion protein analytes was tested in phosphate-buffered saline (PBS-T) (pH 7.1) on surfaces consisting of low-density (approximately 300 RU) biot-hCD25-BioP-TVMV-His, which had been cleaved of His (hCD25) and captured on a streptavidin SA sensor chip, or hCD122(27-241)-hFc-D / hCD132(23-263)-hFc-κ heterodimeric Fc fusion (hIL2-Rb / g), which had been captured on a protein A-immobilized CM5 sensor chip surface using standard ethyl(dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (NHS) chemistry and blocked with ethanolamine. Protein analytes were injected in a titration series and regenerated back to baseline by 2 x 8 s injections of low pH buffer. Data were analyzed using Biacore T-200 evaluation software.

[0535] Figure 2 Prototype binding of IL2-CD25 fusion protein (SEQ ID NO: 16) to human sCD25 is shown. The observed apparent equilibrium dissociation constant of 4.2 micromolar is approximately 150-fold weaker than the affinity of isolated IL2 for sCD25 measured by surface plasmon resonance (Liparoto, SF, Myszka, DG, Wu, Z., Goldstein, B., Laue, TM, and Ciardelli, TL (2002) Biochemistry 41, 2543-51.).

[0536] Table 9: Pharmacokinetics of IL2-CD25 fusion protein (SEQ ID NO: 16) binding to human sCD25.

[0537] ka(1 / Ms) kd(1 / s) KD(M) 1.29E+4 0.054 4.2E-6

[0538] Table 10 below shows the observed equilibrium K for binding of the short and long forms of IL2-CD25 to the prototype of human sCD25 and sIL2Rβ / IL2Rγ heterodimers. D The observed apparent equilibrium dissociation constants for various IL2-CD25 fusion proteins were significantly reduced compared to IL2. The IL2-CD25 fusions bound to sCD25 approximately 100-fold weaker than IL2. Similarly, the IL2-CD25 fusions bound to the β-γ heterodimer approximately 150-fold weaker than IL2.

[0539] Table 10: Observed equilibrium K for binding of IL2 and IL2-CD25 constructs to sCD25 or sIL2Rβ / IL2Rγ heterodimers as measured by surface plasmon resonance D value.

[0540]

[0541]

[0542] Example 3: Truncated forms of IL2-CD25 have improved stability against aggregation: accelerated stability testing

[0543] The stability of fusion protein is tested by hatching in the buffer of pH range 4-8 at 40 ℃.Fusion protein is concentrated into about 15mg / ml, and at 4 ℃, is dialyzed into following buffer: 1) 20mM acetate, 250mM sucrose, pH 4,2) 20mM citrate, 250mM sucrose, pH 5,3) 20mM histidine, 250mM sucrose, pH 6,4) 20mM phosphate, 250mM sucrose, pH 7, and 5) 20mM Tris, 250mM sucrose, pH 8.4 (room temperature). After recovering from dialysis, by diluting with dialysis buffer, the concentration of fusion protein is normalized to 10mg / ml, and is placed in the incubator monitored at 40 ℃ all around, wherein just before hatching (t0), 40 ℃ are hatched one week (1w) and after four weeks (4w), aliquots are taken out. Each time point was analyzed by size exclusion chromatography on a Shodex KW403-4F column connected to an Agilent 1260 HPLC system, running at a flow rate of 0.30 mL / min in a buffer containing 100 mM sodium phosphate, 150 mM sodium chloride, pH 7.3 (0.2 μm filtered).

[0544] As shown in Tables 11 and 12, after accelerated stability studies, the truncated form of IL2-CD25 exhibited a better high and low molecular weight profile than the longer form. This was particularly true for pH values ​​of 4, 5, 6, and 7. The samples were maintained at 40 degrees Celsius for four weeks under different pH conditions. The results, shown as the percentage of the high and low molecular weight fractions for each construct, indicate that the stability of the short construct was significantly improved compared to the long construct. The results, shown as the percentage of the main peak fraction for each construct, indicate that the stability of the short construct was significantly improved compared to the long construct.

[0545] Table 11: Accelerated stability profiles of IL2-CD25 fusion proteins.

[0546]

[0547] For the purpose of easy purification, the IL2-CD25 fusion protein herein contains a His tag (GGHHHHHH, SEQ ID NO: 100).

[0548] Table 12: Accelerated stability profiles of IL2-CD25 fusion proteins.

[0549]

[0550] Example 4: Pharmacokinetic studies in non-human animals

[0551] Pharmacokinetic Studies All animal experimental protocols have been approved by the New Jersey Central Institutional Animal Care and Use Committee, and animals were housed according to guidelines. Female Balb / C mice weighing 19-20 grams were purchased from Charles-River (Wilmington, Massachusetts). Non-fasted Balb / C mice were given a single 0.5 mg / kg dose of the indicated molecules via either the intravenous (IV) route or the subcutaneous (SC) route via the tail vein. The IL2(21-153)-(G3S)3-CD25(22-240) used in the pharmacokinetic studies had a His6 tag. Blood was collected from the tail vein at the following time points after administration: 5 minutes (IV only), 1 hour, 7 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, and 168 hours. For monkey PK studies, male cynomolgus monkeys were obtained from Buckshire Corporation (Perkasie, Pennsylvania). Monkeys (N=3, mean body weight 7.8 kg) were administered a single 0.075 mg / kg subcutaneous dose of hIL2-CD25 (22-212). Serial blood samples were collected from the femoral artery of conscious and seated monkeys at 5, 24, 48, 72, 96, 168, 192, and 240 hours post-dose. Blood samples were allowed to clot and centrifuged at 4°C (1500–2000...

Claims

1. A fusion protein, comprising: (a) a first polypeptide comprising an interleukin-2 (IL2) polypeptide, wherein the first polypeptide consists of the amino acid sequence shown in SEQ ID NO: 2; and (b) a second polypeptide comprising the extracellular domain of an interleukin-2 receptor α (IL2Rα) polypeptide, wherein the second polypeptide consists of the amino acid sequence shown in SEQ ID NO: 12; wherein the extracellular domain of the IL2Rα polypeptide does not include amino acids 192-219 of the C-terminal portion of the extracellular domain of native IL2Rα as shown in SEQ ID NO: 7; wherein the fusion protein comprises a linker between the first polypeptide and the second polypeptide, wherein the first polypeptide is at the N-terminal side of the second polypeptide; and The fusion protein has IL2 activity.

2. The fusion protein according to claim 1, wherein the fusion protein is enzymatically or chemically deglycosylated.

3. The fusion protein according to claim 2, wherein the fusion protein is deglycosylated by alkali, hydrazinolysis, PNGase F, EndoH, O-glycosidase or any combination thereof. The fusion protein according to claim 1 , wherein the linker is a glycine / serine linker.

5. The fusion protein of claim 4, wherein the glycine / serine linker comprises (GS) n 、(GGS) n 、(GGGS) n 、(GGGGS) n or (GGGGS) n An amino acid sequence of wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

6. The fusion protein of claim 4, wherein the glycine / serine linker comprises (GGGS) 3 The amino acid sequence of 7. The fusion protein according to claim 1, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:

16.

8. The fusion protein according to claim 7, wherein the fusion protein consists of the amino acid sequence shown in SEQ ID NO:

16.

9. A fusion protein comprising the fusion protein of claim 1 and further comprising a heterologous portion fused to the first polypeptide and / or the second polypeptide, wherein the heterologous portion comprises a heterologous polypeptide or a non-polypeptide portion.

10. The fusion protein of claim 9, wherein the heterologous moiety is a half-life extending moiety.

11. The fusion protein of claim 9, wherein the heterologous portion comprises a non-polypeptide portion.

12. The fusion protein of claim 9, wherein the heterologous moiety comprises a polypeptide.

13. The fusion protein of claim 9, wherein the heterologous moiety comprises albumin, an immunoglobulin constant region or a portion thereof, an immunoglobulin binding polypeptide, immunoglobulin G (IgG), an albumin binding polypeptide, a PASylation moiety, a HESylation moiety, XTEN, a PEGylated moiety, an Fc region, or any combination thereof.

14. The fusion protein according to claim 1, wherein the fusion protein is more stable than a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO:

13.

15. The fusion protein of claim 14, wherein the fusion protein has one or more properties selected from the group consisting of: (i) increased thermodynamic stability compared to a reference protein; (ii) increased TM compared to a reference protein; (iii) enhanced resistance to degradation compared to a reference protein; (iv) enhanced resistance to modification compared to a reference protein; (v) increased in vivo stability compared to a reference protein; and (vi) any combination thereof, wherein the reference protein comprises (a) a first polypeptide comprising an interleukin-2 (IL2) polypeptide; and (b) a second polypeptide comprising an extracellular domain of an interleukin-2 receptor α (IL2Rα) polypeptide; and the reference protein has at least one more glycosylation than the fusion protein. The fusion protein according to claim 1 , which is a monomer. The fusion protein according to claim 1 , which is a dimer. The fusion protein according to claim 17 , wherein the dimer comprises two monomers, and the monomers are associated with each other via a covalent bond. The fusion protein of claim 17 , wherein the dimer comprises two monomers, and the monomers are associated via a non-covalent bond.

20. The fusion protein according to claim 1, which has one or more pharmacokinetic properties selected from the group consisting of an increased half-life, an increased C max , increased AUC, increased C min , decreased clearance, increased bioavailability, and any combination thereof. The fusion protein according to claim 20 , which has a prolonged half-life.

22. The fusion protein of claim 21, wherein the extended half-life is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 21 times, or at least 22 times compared to the half-life of a polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO:

13.

23. A composition comprising the fusion protein according to claim 16 and the fusion protein according to claim 17.

24. A nucleic acid encoding the fusion protein according to any one of claims 1 to 22.

25. A vector comprising the nucleic acid according to claim 24.

26. A host cell comprising the nucleic acid according to claim 24.

27. The host cell according to claim 26, which is a eukaryotic cell.

28. The host cell of claim 26, wherein the host cell is selected from the group consisting of a mammalian cell, an insect cell, and a yeast cell.

29. The host cell of claim 28, wherein the host cell is a mammalian cell.

30. The host cell of claim 26, which is a prokaryotic cell.

31. The host cell of claim 30, wherein the prokaryotic cell is a bacterial cell.

32. A pharmaceutical composition comprising (a) the fusion protein according to any one of claims 1 to 22; and (b) a pharmaceutically acceptable excipient.

33. A kit comprising the fusion protein of any one of claims 1 to 22, and instructions for administering the fusion protein to a subject in need thereof.

34. A method for producing a fusion protein, wherein include: The host cell according to claim 26 is cultured under suitable conditions, and the fusion protein is recovered.

35. The method of claim 34, wherein the host cell is a eukaryotic cell or a prokaryotic cell.

36. The method of claim 34, wherein the host cell is a mammalian cell, an insect cell, or a fungal cell.

37. The method of claim 34, wherein the host cell is selected from the group consisting of CHO cells, HEK 293 cells, NSO cells, Per C6 cells, BHK cells and COS cells.

38. The method of claim 34, wherein the host cell is a bacterial cell.

39. The host cell according to claim 38, wherein the bacterial cell is Escherichia coli ( Escherichia coli ).

40. The method of claim 36, wherein the host cell is a transgenic mammalian cell.

41. The cell of claim 29, wherein the host cell is a transgenic mammalian cell.

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