EA048704B1 - INTERLEUKIN-2/INTERLEUKIN-2 ALPHA RECEPTOR FUSION PROTEINS AND METHODS OF ADMINISTRATION - Google Patents

Description

1. References to related applications

The invention claims priority from U.S. Provisional Patent Application No. 62/649,379, filed March 28, 2018, the contents of which are incorporated herein by reference in their entirety.

2. Link to the sequence listing provided electronically via Efs-Web

The contents of the sequence listing, provided electronically in an ASCII text file (name: 3338_100PC01_Seqlisting_ST25.txt; size: 354542 bytes; and creation date: March 27, 2019), filed with the application, are hereby incorporated by reference in their entirety.

3. Field of technology to which the present invention relates

The presently disclosed subject matter generally relates to methods and compositions for modulating an immune response using an interleukin-2/interleukin-2 receptor alpha fusion protein.

4. Prior art of the present invention

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 immune responses in patients with cancer, inflammatory disease, or autoimmune disease. IL2 is a highly potent T-cell growth factor that stimulates immune responses including clonal expansion of antigen-activated T cells, triggers the development of CD4+ T helper (Th)1 and Th2 cells, triggers terminal differentiation of CD8+ cytotoxic T lymphocytes (CTL), and antagonizes the development of CD4+ Th17 and T follicular helper (Tfh) cells. IL2 is also involved in the generation of memory-based T cell responses.

Clinical trials have harnessed the T-cell activating properties of IL2 to stimulate T and NK cells in patients with cancer and HIV/AIDS by infusing high doses of IL2 (typically >500,000 units/kg, multiple times). Indeed, IL2 has been approved by the FDA for use in patients with melanoma and renal cell carcinoma because some (approximately 5%) patients experienced complete remission. However, response rates have been low in these and other cancers, and the therapy has been associated with severe toxicity. At the same time, IL2 has been considered ineffective in stimulating immunity in patients with HIV/AIDS. The low efficacy of high doses of IL2 in this setting is partly explained by the concomitant increase in Treg cells.

More recently, lower doses of IL2 have been used to selectively induce tolerance to suppress unwanted immune responses associated with an autoimmune-like attack on self tissue. At these low doses of IL2, no evidence of upregulation or reactivation of autoreactive T cells was observed. Preclinical studies showed that low levels of IL2R signaling selectively stimulated key Treg but not effector T cell (Teff) activities, and that treatment of mice with low levels of IL2 prevented the development of an autoimmune response. A number of patients with hyperactive immune responses are now being treated with low doses of IL2 (0.5-2 million units, intermittently). To date, experience shows that this therapy is safe, has no evidence of reactivation of autoaggressive T cells, and at the same time, almost all patients experience increases in Treg cell numbers, often accompanied by clinical improvement. However, IL2 as a therapeutic agent has important limitations, including a very short half-life in vivo, which limits its efficacy, and toxicity at high doses. For these reasons, new IL2 biologics with improved pharmacokinetics and response duration are needed for use.

5. Brief summary of the present invention

The present invention relates to a fusion protein comprising (a) a first polypeptide comprising an interleukin-2 (IL2) polypeptide; and (b) a second polypeptide comprising an extracellular domain of an interleukin-2 alpha receptor polypeptide (IL2Ra);

wherein (i) the extracellular domain of the IL2Ra polypeptide has at least one less glycosylation site compared to the extracellular domain of native IL2Ra (SEQ ID NO: 7); and/or (ii) the IL2 polypeptide has at least one less glycosylation site compared to native IL2 (SEQ ID NO: 2); and wherein the fusion protein has IL2 activity. According to some embodiments, the extracellular domain of the IL2Ra polypeptide has at least one less glycosylation site, at least two less glycosylation sites, at least three less glycosylation sites, at least four less glycosylation sites, at least five less glycosylation sites, at least six less glycosylation sites, at least seven less glycosylation sites, at least eight less glycosylation sites, or at least nine less glycosylation sites compared to the extracellular domain of native IL2Ra (SEQ ID NO: 7). According to some embodiments, the IL2 polypeptide has at least one less glycosylation site compared to native IL2 (SEQ ID NO: 2).

- 1 048704

According to 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 the sequence of SEQ ID NO: 2. According to 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 the sequence of SEQ ID NO: 12.

In some embodiments, the extracellular domain of the IL2Ra polypeptide that has at least one fewer glycosylation site comprises a mutation that removes a glycosylation site. In some embodiments, the mutation removes an O-glycosylation site and/or an N-glycosylation site. In some embodiments, the mutation removes an O-glycosylation site. In some embodiments, the mutation removes an N-glycosylation site.

According to some embodiments, the mutation in the extracellular domain of the IL2Ra polypeptide is a deletion of amino acids 167-219, amino acids 168-219, amino acids 169-219, amino acids 170-219, amino acids 171-219, amino acids 172-219, amino acids 173-219, amino acids 174-219, amino acids 175-219, amino acids 176-219, amino acids 177-219, amino acids 178-219, amino acids 179-219, amino acids 180-219, amino acids 181-219, amino acids 182-219, amino acids 183-219, amino acids 184-219, amino acids 185-219, amino acids 186-219, amino acids 187-219, amino acids 188-219, amino acids 189-219, amino acids 190-219, amino acids 191-219, or amino acids 192-219 corresponding to the sequence of SEQ ID NO: 7. According to some embodiments, the mutation is a deletion of amino acids 167, 169, 171 to and including 192-219 corresponding to the sequence of SEQ ID NO: 7. According to some embodiments, the mutation does not include a deletion of 170-219 corresponding to the sequence of SEQ ID NO: 7. According to some embodiments, the second polypeptide is represented by the sequence of SEQ ID NO: 11. According to some embodiments, the second polypeptide is represented by the sequence of SEQ ID NO: 12.

According to some embodiments, the mutation is one or more substitutions of an amino acid that is glycosylated with an amino acid that is not glycosylated. According to some embodiments, the mutation is one or more substitutions of an amino acid that allows glycosylation on an adjacent amino acid with an amino acid that does not allow glycosylation on an adjacent amino acid. According to 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 locations correspond to the sequence of SEQ ID NO: 7. According to some embodiments, the one or more substitutions are a substitution 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. According to some embodiments, one or more substitutions are a substitution 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.

In some embodiments, one of the substitutions is at amino acid N49. 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.

In some embodiments, one of the substitutions is at amino acid N68. In some embodiments, N68 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.

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In some embodiments, one of the substitutions is at 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.

In some embodiments, one of the substitutions is at 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.

In some embodiments, one of the substitutions is at amino acid T197. 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.

In some embodiments, one of the substitutions is at 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.

In some embodiments, one of the substitutions is at 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.

In some embodiments, one of the substitutions is at 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.

According to some embodiments, 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 locations correspond to the sequence of SEQ ID NO: 7.

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

In some embodiments, one of the substitutions is at 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.

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

In some embodiments, one of the substitutions is at 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.

In some embodiments, one of the substitutions is at amino acid C192. 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.

In some embodiments, the IL2 polypeptide that has at least one fewer glycosylation site comprises a mutation that removes the glycosylation site. In some embodiments, the mutation is one or more substitutions of an amino acid that is glycosylated to an amino acid that is not glycosylated. In some embodiments, the mutation is one or more substitutions of an amino acid that allows glycosylation on an adjacent amino acid to an amino acid that does not allow glycosylation on an adjacent amino acid. In some embodiments, the one or more substitutions are a substitution from alanine to an amino acid selected from the group consisting of arginine, asparagine,

- 3 048704 on, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. According to some embodiments, one or more substitutions are a substitution 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. According to some embodiments, one or more substitutions are a substitution 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. According to some embodiments, one or more substitutions are a substitution from cysteine to serine. According to some embodiments, one or more substitutions are a substitution from cysteine to alanine. According to some embodiments, one or more substitutions are a substitution from cysteine to valine.

According to some embodiments, one or more substitutions are at the amino acid T3 relative to the corresponding sequence of SEQ ID NO: 2. According to some embodiments, one of the substitutions is at the amino acid C125. According to some embodiments, the substitution at the amino acid C125 is selected from the group consisting of C125S, C125A, and C125V.

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

According to some embodiments, the fusion protein is deglycosylated enzymatically or chemically. According to some embodiments, the fusion protein is deglycosylated by alkali, hydrazinolysis, PNGase F, endo-H, O-glycosidase, or any combination thereof.

According to some embodiments, the fusion protein further comprises a linker fused in-frame between the first polypeptide and the second polypeptide. According to some embodiments, the linker is a glycine/serine linker. According to some embodiments, the glycine/serine linker comprises the amino acid sequence (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. According to some embodiments, the glycine/serine linker comprises the amino acid sequence (GGGS) ₃ .

According to some embodiments, the fusion protein further comprises a heterologous moiety fused to the first polypeptide and/or the second polypeptide. According to some embodiments, the heterologous moiety is a half-life enhancing moiety. According to some embodiments, the heterologous moiety comprises a non-polypeptide moiety. According to some embodiments, the heterologous moiety comprises a polypeptide. According to some embodiments, the heterologous moiety comprises albumin, an immunoglobulin constant region or a portion thereof, an immunoglobulin binding polypeptide, immunoglobulin G (IgG), an albumin binding polypeptide (ABP), a PASylation moiety, a HEKylation moiety, XTEN, a PEGylation moiety, an Fc region, and any combination thereof.

According to some embodiments, the fusion protein is more stable than a polypeptide consisting of the sequence of SEQ ID NO: 2 or the sequence of SEQ ID NO: 13. According to 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 modifications compared to a reference protein; (v) increased stability in vivo 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 from an interleukin-2 receptor alpha (IL2Ra) polypeptide; and has at least one fewer glycosylation site compared to the fusion protein.

According to some embodiments, the fusion protein is a monomer. According to some embodiments, the fusion protein is a dimer. According to some embodiments, the dimer comprises two monomers, and the monomers are linked to each other by covalent bonds. According to some embodiments, the dimer comprises two monomers, and the monomers are linked by non-covalent bonds.

According to some embodiments, the fusion protein has one or more pharmacokinetic properties selected from the group consisting of increased half-life, increased _Cmax , increased AUC, increased _Cm ; _n , decreased clearance, improved bioavailability, and any combination thereof, compared to the pharmacokinetic property

- 4,048,704 polypeptide consisting of the sequence of SEQ ID NO: 2 or the sequence of SEQ ID NO: 13. According to some embodiments, the fusion protein has an extended half-life. According to some embodiments, the half-life is extended by 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 from the sequence of SEQ ID NO: 2 or the sequence of SEQ ID NO: 13.

In some embodiments, one or more fusion proteins are disclosed herein. In some embodiments, the present invention relates to a composition comprising one or more fusion proteins disclosed herein.

In some embodiments, disclosed herein is a nucleic acid that encodes any of the fusion proteins disclosed herein. In some embodiments, disclosed herein is a vector comprising a nucleic acid that encodes any of the fusion proteins disclosed herein. In some embodiments, disclosed herein is a host cell comprising a nucleic acid that encodes 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 the group consisting of a mammalian cell, an insect cell, a yeast cell, a transgenic mammalian cell, and a plant cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a prokaryotic cell. According to some embodiments, the prokaryotic cell is a bacterial cell.

According to some embodiments, the present invention relates to a pharmaceutical composition comprising (a) a fusion protein as disclosed herein, a composition as disclosed herein, a nucleic acid as disclosed herein, a vector as disclosed herein, or a host cell as disclosed herein; and (b) a pharmaceutically acceptable excipient.

According to some embodiments, the present invention relates to 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.

According to some embodiments, the present invention relates to a method for producing a fusion protein disclosed herein, comprising culturing a host cell disclosed herein under suitable conditions and isolating the fusion protein. According to some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell. According to 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. According to some embodiments, the host cell is selected from the group consisting of a CHO cell, a HEK293 cell, an NS0 cell, a Per C6 cell, a BHK cell, and a COS cell. According to some embodiments, the bacterial cell is Escherichia coli.

According to some embodiments, the present invention provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of a fusion protein as disclosed herein, a composition as disclosed herein, a nucleic acid as disclosed herein, a vector as disclosed herein, a host cell as disclosed herein, or a pharmaceutical composition as disclosed herein. According to some embodiments, the disease or disorder is cancer. According to some embodiments, the cancer is bladder cancer, breast cancer, uterine cancer, endometrial carcinoma, ovarian cancer, colorectal cancer, colon cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, squamous cell carcinoma,

- 5 048704 skin cancer, neoplasm of the central nervous system, lymphoma, leukemia, sarcoma, cancer of viral origin, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's or non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, myeloma, salivary gland carcinoma, renal cancer, basal cell carcinoma, melanoma, prostate cancer, cancer of the external genitalia, thyroid cancer, testicular cancer, esophageal cancer, or head or neck cancer, and any combination thereof.

According to some embodiments, the disease or disorder is an inflammatory disease or an autoimmune disease. According to some embodiments, the inflammatory disease or autoimmune disease is selected from the group consisting of diabetes mellitus type 1, multiple sclerosis, rheumatoid arthritis, celiac disease, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis or scleroderma, graft-versus-host disease, psoriasis, focal alopecia, HCV-induced vasculitis, Sjogren's syndrome, pemphigus, ankylosing spondylitis, Behcet's disease, Wegener's granulomatosis, Takayasu's syndrome, autoimmune hepatitis, sclerosing cholangitis, Gougerot-Sjogren syndrome and macrophage activation.

According to some embodiments, the disease or disorder is an infectious disease. According to some embodiments, the infectious disease is caused by a pathogenic virus. According to some embodiments, the pathogenic virus is selected from the group consisting of human immunodeficiency virus (HIV), hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes virus, adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, human T-lymphotropic virus (HTL), dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, John Cunningham virus (JC virus) and arbovirus encephalitis virus. According to some embodiments, the infectious disease is caused by a pathogenic bacterium. According to some embodiments, the pathogenic bacterium 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 the bacterium causing Lyme disease. According to some embodiments, the infectious disease is caused by pathogenic fungi. According to some embodiments, the pathogenic fungus is selected from the group consisting of a member of the genus Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, a member of the genus Aspergillus (fumigatus, niger, etc.), a member of the genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum. According to some embodiments, the infectious disease is caused by a pathogenic parasite. According to some embodiments, the pathogenic parasite is selected from the group consisting of Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus brasiliensis.

According to some embodiments, the methods disclosed herein further comprise administering to the subject a second agent. According to 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. According to 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-KTR antibody, an anti-GITR antibody, an anti-LAG3 antibody, or any combination thereof. According to some embodiments, the second agent is a cytokine inhibitor. According to some embodiments, the cytokine inhibitor targets one or more of IL-6, IL-10, TGF-β, VEGF, IFN-γ, or any combination thereof.

According to some embodiments, the fusion protein is administered locally, through the epidermal mucosa, intranasally, orally, vaginally, rectally, sublingually, topically, intravenously, intraperitoneally, intramuscularly, intraarterially, intrathecally, intralymphatically, intralesionally, intracapsularly, intraorbitally, intracardiacly, intradermally, transtracheally, subcutaneously, intradermally, intraarticularly, subcapsularly, subarachnoidally, intraspinally, epidurally, or epigastrically.

6. Brief description of drawings

Fig. 1 shows that the molecular weight of the sequence under SEQ ID NO: 16 is 93 kDa, as measured by static light scattering SEC/MALS. The theoretical mass of one poly

- 6 048704 peptide chain decreased by 37812 Da. From the observed mass in solution it is evident that it exists as a homogeneous homodimer with a mass of approximately 19% due to glycosylation.

Figure 2 shows the prototypical binding of the IL2-CD25 fusion protein (SEQ ID NO: 16) to human sCD25 using surface plasmon resonance measurements.

Figure 3 shows serum concentration-time plots for the indicated fusion proteins in Balb/C mice following a single 0.5 mg/kg intravenous (IV) or subcutaneous (SC) dose. Each time point represents the mean of samples from 3 mice. Error bars represent standard deviation.

Figure 4 shows serum concentration-time plots for the indicated fusion proteins in Balb/C mice following a single 0.5 mg/kg intravenous (IV) or subcutaneous (SC) dose. Each time point represents the mean of samples from 3 mice. Error bars represent standard deviation.

Figure 5 shows serum concentration-time plots of hIL2-CD25 (22212) in cynomolgus monkeys following 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 deviation.

Figure 6 shows the activity of IL2-CD25 fusion proteins to induce STAT5 phosphorylation in human PBMCs from a representative donor. Cells were gated on Tregs (CD4 ⁺ , foxp3 ⁺ , CD25+) or Tconv (CD4 ⁺ , ^foxp3- ), CD8 ⁺ , and NK cells ( ^CD3- , CD56 ⁺ ), and the percentage of cells that stained positive for pSTAT5 after incubation was calculated. The _EC50 for pSTAT5 induction in Tregs determined from these experiments were 10 ng/ml, 4.4 ng/ml, and 4.6 ng/ml, respectively, for hIL2-CD25(22-240), hIL2-CD25(22-212), and hIL2-CD25(22-187).

Figure 7 shows the ability of the truncated fusion proteins to stimulate IL2R in whole blood, resulting in the induction of phosphorylated STAT5 in various cell types. IL2R signaling was detected by flow cytometric measurement after intracellular pSTAT5 staining and by determining the percentage of pSTAT5-positive cells in the whole blood mixture. In a representative donor, the potency 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. The _EC50 for pSTAT5 induction in Tregs was 22 ng/ml for hIL2-CD25(22-240) and 36 ng/ml for hIL2-CD25(22-212).

Figures 8A-8C show similar abilities of hIL2-CD25(22-240), hIL2-CD25(22-212), and hIL2CD25(22-184) to expand T cells in humanized immune mice. NSG-huCD34-inoculated mice were treated with SC fusion protein every three days for three doses. Tregs (Figure 8A), CD8 (Figure 8B), and NK cells (Figure 8C) from the spleens of treated mice were analyzed by flow cytometry.

Figures 9A and 9B show STAT5 phosphorylation after 15 minutes in a mixed cell population including PBMCs (Figure 9A) or whole blood concentration (Figure 9B) with hIL2CD25(22-212) (SEQ ID NO: 16).

Figure 10 shows the results of LC-MS/MS analysis of the stability of the (G3S)3 linker in IL2(21-153)(G3S)3-CD25(22-212) in human or mouse serum in vitro. The peak area ratio of IL2 and CD25 obtained by LC-MS/MS after capture with anti-IL2 antibody is shown.

Figure 11 shows the results of LC-MS/MS analysis of the stability of the (G3S)3 linker in IL2(21-153)(G3S)3-CD25(22-212) in monkey serum after a single subcutaneous dose of 0.075 mg/kg. The peak area ratio of IL2 to CD25 obtained by LC-MS/MS after capture with anti-IL2 antibody is shown.

7. Detailed disclosure of the present invention

The present invention is further described in more detail hereinafter with reference to the accompanying drawings, which illustrate some, but not all, embodiments of the present invention. In fact, the present invention may be embodied in many different forms and should not be considered as limited to the embodiments set forth herein; rather, these embodiments are provided so that the present invention may satisfy the requirements of law. Like reference numerals refer to all like elements.

Those skilled in the art to which the present invention pertains will, with the benefit of the teachings presented in the foregoing description and the associated drawings, come to mind many modifications and other embodiments of the present invention as set forth herein. It is therefore to be understood that the present invention is not to be 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 used herein, they are used in a general and descriptive sense only and not for purposes of limitation.

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7.1 General overview

Disclosed herein are various methods and compositions that can be used to modulate the immune system. The compositions include a fusion protein 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 (IL2Ra) polypeptide; wherein (i) the extracellular domain of the IL2Ra polypeptide has at least one fewer glycosylation site compared to the extracellular domain of native IL2Ra (SEQ ID NO: 7); and/or (ii) the IL2 polypeptide has at least one fewer glycosylation site compared to native IL2 (SEQ ID NO: 2); and wherein the fusion protein has IL2 activity.

The present invention also relates to a nucleotide sequence encoding a fusion protein disclosed herein, to a vector comprising such a nucleotide sequence, to a host cell comprising such a nucleotide sequence, and to a composition comprising a fusion protein, a nucleotide sequence, a vector, or a host cell. The present invention also relates to methods for producing a fusion protein, a nucleotide sequence, a vector, a host cell, or a composition, or to methods for using a fusion protein, a nucleotide sequence, a vector, a host cell, or a composition.

7.2 Definitions

It should be noted that the singular terms denote one or more such entities; for example, the term nucleotide sequence is understood to denote one or more nucleotide sequences. Therefore, the singular terms or the terms one or more and at least one may be used interchangeably herein.

Furthermore, and/or as used herein is to be understood as specifically disclosing each of the two stated features or components with or without the other. Thus, the term and/or as used in the context of a phrase such as A and/or B is herein intended to include A and B, A or B, A (only) and B (only). Similarly, the term and/or as used in a phrase such as A, B and/or C is intended to include 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 (only); B (only); and C (only).

Likewise, the word or is intended to include and unless the context clearly indicates otherwise. It is also to be understood that all base sizes or amino acid sizes, as well as all molecular weights or molecular masses, given for nucleic acids or polypeptides are exemplary and are presented for descriptive purposes.

It is understood that wherever aspects in this document are described by the phrase comprising, similar aspects described by the phrase consisting of and/or actually consisting of are also contemplated.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide those skilled in the art with a general dictionary of many of the terms used in this invention.

Units, prefixes and symbols are indicated in their SI accepted form. Numeric ranges include the numbers defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in an amino-terminal to carboxyl-terminal orientation. The headings provided herein are not limitations of the various aspects of the invention, as can be understood by reference to the description as a whole. Accordingly, terms defined immediately below are more fully defined in light of the description as a whole.

The term approximately is used herein to mean approximately, about, around, or in the vicinity of. When the term approximately is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the stated numerical values. Thus, approximately 10-20 means from approximately 10 to approximately 20. In general, the term approximately may modify a numerical value above and below the stated value by a deviation of, for example, 10 percent up or down (more or less).

As used herein, interleukin-2, IL2 or IL-2 refers to any natural or recombinant IL2 derived from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), as well as domestic or farm mammals, unless otherwise specified. The term includes unprocessed IL2 as well as any form of IL2 that results from processing within the cell (i.e., mature

- 8 048704 form of IL2). The term also encompasses naturally occurring variants and fragments of IL2, such as splice variants or allelic variants, and non-naturally occurring variants that have the IL2 activity of naturally occurring IL2.

Additional nucleic acid and amino acid sequences are known for IL2. See, for example, the sequences under GenBank accession numbers: Q7JFM2 (Aotus lemurinus (grey-bellied night monkey)); Q7JFM5 (Aotus papsutae (western Amazonian lemur)); P05016 (Bas taurus (ox)); Q29416 (Canis familiaris (dog) (Canis lupus familiaris)); P36835 (Capra hircus (goat)); and P37997 (Equus caballus (horse)).

Biologically active fragments and variants of IL2 retain IL2 activity. The phrase IL2 biological activity or IL2 activity refers to one or more biological activities of IL2, including but not limited to the ability to stimulate IL2 receptor-bearing lymphocytes. Such activity can be measured both in vitro and in vivo. IL2 is a global regulator of immune activity, and the effects observed are the sum of such activities. For example, it regulates survival activity (Bcl-2), induces T-effector activity (IFN-gamma, granzyme B, and perforin), and/or stimulates T-regulatory activity (FoxP3).

Biologically active variants of IL2 are known. See, for example, U.S. Patent Application Publication Nos. 20060269515 and 20060160187, and WO 99/60128.

As used herein, the term CD25, IL2a receptor, IL2Ra or IL2Ra refers to any natural or recombinant IL2Ra from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), as well as domestic or farm mammals, unless otherwise specified. The term also encompasses naturally occurring IL2Ra variants, such as splice variants or allelic variants, or non-naturally occurring variants that have IL2Rα activity. Human IL2 exerts its biological effects by signaling through its receptor system, the IL2R. IL2 and its receptor (IL2R) are required for T cell proliferation and other fundamental functions that are critical to the immune response. IL2R consists of 3 non-covalently associated type I transmembrane proteins, which are the alpha (p55), beta (p75), and gamma (p65) chains. The alpha chain of human IL2R contains a 219 amino acid extracellular domain, a 19 amino acid transmembrane domain, and a 13 amino acid intracellular domain. The secreted extracellular domain of IL2R alpha (IL2R-a) can be used in the fusion proteins described herein.

Nucleic acid and amino acid sequences are known for IL2Ra. See, for example, the sequences under GenBank accession numbers: NP_001030597.1 (Pan troglodytes); NP_001028089.1 (Macaca mulatto); NM_001003211.1 (Canis lupus); NP_776783.1 (Bos taurus); NP_032393.3 (Mus musculus); and NP_037295.1 (Rattus norvegicus).

Also provided are biologically active fragments and variants of the extracellular domain of IL2Ra. Such active variants or fragments of the extracellular domain of IL2Ra will retain the activity of the extracellular domain of IL2Ra. The phrase biological activity of the extracellular domain of IL2Ra refers to one or more biological activities of the extracellular domain of IL2Ra, including, but not limited to, the ability to bind IL2 and/or enhance intracellular signaling in cells capable of responding to the IL2 receptor. Non-limiting examples of biologically active fragments and variants of IL2Ra are disclosed, for example, in Robb et al., Proc. Natl. Acad. Sci. USA, 85:5654-5658, 1988. According to some embodiments, the biologically active fragments and variants of IL2Ra disclosed herein comprise at least one less glycosylation site compared to the extracellular domain of native IL2Ra.

As used in the context of this document, the term naturally occurring in relation to an object means the fact that the object can be found in nature.

For example, a naturally occurring protein is a polypeptide or polynucleotide sequence that is present in an organism (including viruses), that can be isolated from a source in nature, and that has not been intentionally modified by man in a laboratory.

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

The present invention also relates to fragments or variants of the polypeptides and any combination thereof. The term fragment or variant, when applied to the polypeptide binding domains or polypeptide binding molecules of the present invention, includes any polypeptides that retain at least some properties (e.g., IL2 binding activity to IL2Ra) of the reference polypeptide. Fragments of polypeptides include proteolytic fragments as well as deleted fragments, but do not include naturally occurring polypeptides.

- 9 048 704 nodular polypeptide (or mature polypeptide). Variants of the polypeptide-binding domains or polypeptide-binding molecules of the present invention include the fragments described above, as well as polypeptides with amino acid sequences altered by amino acid substitutions, deletions or insertions. Variants may be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be obtained using mutagenesis techniques known in the art. Polypeptide variants may contain conservative or non-conservative amino acid substitutions, deletions or additions.

As noted above, variants of polypeptides include, for example, modified polypeptides. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent addition of flavin, covalent addition of heme, covalent addition of nucleotide or nucleotide derivative, covalent addition of lipid or lipid derivative, covalent addition of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-linking, 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, RNA transfer-mediated addition of amino acids to proteins, such as arginylation and ubiquitination.

In this context, a corresponding, amino acid, a corresponding, site, a corresponding or equivalent amino acid in a protein or nucleotide sequence is identified by alignment to find the maximum identity or similarity between a first protein sequence, for example, an IL2 sequence, and a second protein sequence, for example, a second IL2 sequence. The number used to identify the equivalent amino acid in the second protein sequence is based on the number used to identify the corresponding amino acid in the first protein sequence. According to some embodiments, the term corresponding means the relationship of a mutation in one or more amino acids in a polypeptide or in one or more nucleotides in a polynucleotide. As a non-limiting example, a particular amino acid (e.g., S50) of a polynucleotide (e.g., SEQ ID NO: 7) as disclosed herein refers to the 50th amino acid, i.e., serine, in the sequence under SEQ ID NO: 7.

As used herein, the term "linked to" refers to a covalent or non-covalent bond formed between a first amino acid chain and a second amino acid chain. According to one embodiment, the term "linked to" means a covalent, non-peptide bond or a non-covalent bond. This bond may be indicated by a colon, i.e. (:). According to another embodiment, it means 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 and CL regions are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 according to the Kabat numbering system (position 226 or 229 according to the EU numbering system). Examples of covalent bonds include, but are not limited to, a peptide bond, a metallic bond, a hydrogen bond, a disulfide bond, a sigma bond, a pi bond, a delta bond, a glycosidic bond, an agnostic bond, a bent bond, a dipolar bond, a backbone pi bond, a double bond, a triple bond, a quadruple bond, a quinary bond, a sextuple bond, conjugation, hyperconjugation, aromaticity, hapticity, or an antibond. Non-limiting examples of non-covalent bonding include ionic bonding (e.g., cationic pi bonding or salt bonding), metallic bonding, hydrogen bonding (e.g., dihydrogen bonding, dihydrogen complex, low barrier hydrogen bonding, or symmetric hydrogen bonding), van der Waals force, London dispersion force, mechanical bonding, halogen bonding, aurophilicity, intercalation, stacking, entropic force, or chemical polarity.

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

As used herein, the term expression refers to the process by which a polynucleotide produces a gene product, such as RNA or a polypeptide.

The fusion product or fusion protein contains a first amino acid sequence linked in frame to a second amino acid sequence to which it is not normally linked in nature. Amino acid sequences that normally exist in separate

- 10 048704 proteins, can be combined into a fusion polypeptide, or amino acid sequences that normally exist in the same protein can be arranged in a new order in a fusion polypeptide, such as in a fusion product of the IL2 protein with the IL2-Ra protein. The fusion protein is created, for example, by chemical synthesis or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired ratio. The fusion protein can further comprise a second amino acid sequence linked to the first amino acid sequence by a covalent, non-peptide bond, or non-covalent bond. After transcription/translation, a single protein is formed. In this way, several proteins or fragments thereof can be included in a single polypeptide. The term operably linked is intended to mean a functional linkage between two or more elements. For example, a functional linkage between two polypeptides fuses both polypeptides together in-frame to form a single fusion protein of polypeptides. According to a particular aspect, the fusion protein further comprises a third polypeptide, which, as discussed in more detail below, may comprise a linker sequence.

The Fc region (crystallizable fragment of immunoglobulin), Fc domain, or Fc refers to the C-terminal region of an antibody heavy chain that mediates binding of immunoglobulin to tissues or host factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. Thus, the Fc region comprises the constant region of an antibody excluding the immunoglobulin domain of the first constant region (e.g., CH1 or CL). In the 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 two antibody heavy chains; the Fc regions of IgM and IgE contain three heavy chain constant domains (CH domains 2–4) in each polypeptide chain. In some species, the IgG isotype is divided into subclasses: IgG1, IgG2, IgG3, and IgG4 in humans and IgG1, IgG2a, IgG2b, and IgG3 in mice. In the case of IgG, the Fc region contains the CH2 and CH3 immunoglobulin domains and a hinge between the CH1 and CH2 domains. Although the definition of the boundaries of the Fc region of the immunoglobulin heavy chain may vary, as defined herein, the Fc region of the human IgG heavy chain is defined as extending from amino acid residues D221 for IgG1, V222 for IgG2, L221 for IgG3, and P224 for IgG4 to the carboxy terminus of the heavy chain, with numbering according to the EU system as in Kabat. The CH2 domain of the Fc region of human IgG 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 the Fc region, i.e., extends from amino acid 341 to amino acid 447 or 446 (if the C-terminal lysine residue is missing) or 445 (if the C-terminal lysine residue is missing) in IgG. As used herein, the Fc region may be a naturally occurring Fc, including any allotypic variant, or Fc variant (e.g., a non-naturally occurring Fc).

An Fc receptor or FcR is a receptor that binds to the Fc region of an immunoglobulin. The FcRs that bind to an IgG antibody include the FcyR family of receptors, including allelic variants and alternatively spliced forms of these receptors. The FcyR family consists of three activating (FcyRI, FcyRIII, and FcyRIV in mice, FcyRIA, FcyRIIA, and FcyRIIIA in humans) receptors and one inhibitory (FcyRIIB) receptor. Various properties of human FcyRs are known in the art. Most natural effector cell types coexpress one or more activating FcyRs and an inhibitory FcyRIIB, whereas natural killer (NK) cells selectively express one activating Fc receptor (FcyRIII in mice and FcyRIIIA in humans) but not the inhibitory FcyRIIB in mice and humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to mouse IgG2a with respect to the types of activating Fc receptors to which it binds.

As used herein, the terms inserted, 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 a similar position in the protein. As used herein, the terms refer to characteristics of the recombinant polypeptide disclosed herein and do not designate, imply, or suggest any method or process by which the fusion polypeptide was produced.

A heterologous and heterologous moiety, when applied to a polypeptide or polynucleotide, is a polypeptide or polynucleotide that is derived from another protein or polynucleotide. The additional components of the fusion protein may be derived from the same organism as the remaining polypeptide components of the fusion protein, or the additional components may be derived from a different organism than the remaining polypeptide components of the fusion protein. For example, a heterologous polypeptide may be synthetic or derived from a different species, a different cell type of an individual, or the same or different cell types of different individuals. In one aspect, a heterologous moiety is a polypeptide fused to another

- 11 048704 lipopeptide, to form a polypeptide or fusion protein. According to another aspect, the heterologous moiety is a non-polypeptide, such as PEG, conjugated to a polypeptide or protein. Non-limiting examples of heterologous moieties disclosed herein are glycine/serine linkers (e.g., GGGSGGGSGGGS (SEQ ID NO: 71) (also referred to as (Gly ₃ Ser) ₃ )).

A native sequence Fc region or native sequence Fc comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Human native sequence Fc regions include the human IgG1 native sequence Fc region; the human IgG2 native sequence Fc region; the human IgG3 native sequence Fc region; and the human IgG4 native sequence Fc region, as well as naturally occurring variants thereof. Native sequence Fc includes various Fc region allotypes (see, e.g., Jefferis et al. (2009) 1:1 mAbs).

The term EC ₅₀ in the context of an in vitro or in vivo assay using a fusion protein refers to the concentration of the fusion protein that induces a response that is 50% of the maximal response, i.e. midway between the maximal response and the baseline.

Conservative amino acid substitutions refer to substitutions of an amino acid residue with an amino acid residue having a similar side chain. The prior art provides a definition of families of amino acid residues having similar side chains. Such families include amino acids with 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), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). According to some embodiments, a putative accessory amino acid residue in the IL2/IL2R fusion protein is replaced with another amino acid residue from the same side chain family. Methods for identifying nucleotide and amino acid conservative substitutions that do not abolish 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)).

As used in the context of this document, the term nucleic acid molecule is understood to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded and may be cDNA.

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 influence transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions may include promoters, translational leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. If the coding region is intended for expression in a eukaryotic cell, the polyadenylation signal and transcription termination sequence will typically be located 3' of the coding sequence.

A polynucleotide encoding a gene product, such as a polypeptide, may include a promoter and/or other transcriptional or translational control elements operably linked to one or more coding regions. Other transcriptional control elements besides a promoter, such as enhancers, operators, repressors, and transcriptional termination signals, may also be operably linked to a coding region to direct expression of the gene product.

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

Similarly, various elements for translation control are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation start and stop codons, and elements derived from picornaviruses (in particular, the site

- 12 048704 internal ribosome entry site or IRES, also called the CITE sequence).

The terms percent sequence identity, percent identity, sequence identity, or identity are used interchangeably and refer to the number of identical matching positions between two polynucleotide or polypeptide sequences within a comparison interval, taking into account additions or deletions (i.e., gaps) that must be introduced to optimally align the two sequences. A matching position is any position at which an identical nucleotide or identical amino acid is present in both the target and reference sequences. Gaps present in the target sequence are not counted because gaps are not nucleotides or amino acids. Similarly, gaps present in the reference sequence are not counted because nucleotides or amino acids of the target sequence are counted, but not nucleotides or amino acids of the reference sequence.

Comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm as described in the non-limiting examples below.

Percent sequence identity is calculated by determining the number of positions at which an identical amino acid residue or identical nucleic acid base occurs in both sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison interval, and multiplying the result by 100 to yield the percent sequence identity. Sequence comparison and determination of percent sequence identity between two sequences can be accomplished using software that is readily available both online and for download. Suitable programs are available from a variety of sources and are designed to align both protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, part of the BLAST suite of programs available from the BLAST website of the U.S. government's National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov). B12seq compares two sequences using the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs, such as Needle, Stretcher, Water or Matcher, are part of the EMBOSS bioinformatics suite and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk/Tools/psa.

Each of the different regions within a single polynucleotide or polypeptide target sequence that matches the reference polynucleotide or polypeptide sequence when aligned may have its own percent sequence identity. It should be noted that the percent sequence identity value is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded to 80.2. It should also be noted that the length value will always be an integer.

The percentage identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available from global-web.gcg.com) using the NWSgapdna.CMP matrix and a gap opening penalty of 40, 50, 60, 70, or 80 and a gap extension penalty of 1, 2, 3, 4, 5, or 6. The percentage 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)), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 mutation probability matrix, a gap extension penalty of 12, and a gap opening penalty of 4. Additionally, the percentage identity between two amino acid sequences can be determined using the Needleman algorithm and Wunsch (J. Mol. Biol. (48):444–453 (1970)), which was incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either the Blossum 62 or PAM250 matrix and a gap-opening penalty of 16, 14, 12, 10, 8, 6, or 4, and a gap-extension penalty of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences described herein can also be used as query sequences to perform searches of public databases, for example, to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) Altschul, et al. (1990) J. Mol. Biol. 215:403-10. Nucleotide BLAST searches can be performed using the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. Protein BLAST searches can be performed using the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules described herein. Gapped BLAST can be used to obtain gapped alignment results for comparison purposes, as described

- 13 048704 in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

Nucleic acids may be present in whole cells, in a cell lysate, or in partially purified or substantially pure form. A nucleic acid is isolated or substantially purified if it is free from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of a chromosome) or proteins, by standard techniques including alkaline/SDS digestion, CsC1 coupling, column chromatography, agarose gel electrophoresis, and others well known in the art. See F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

As used herein, the term vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a plasmid, which refers to a circular double-stranded loop of DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the host cell genome upon introduction into the host cell and thereby replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as recombinant expression vectors (or simply expression vectors). In general, expression vectors used in recombinant DNA techniques are often in the form of plasmids. In the present specification, the terms plasmid and vector may be used interchangeably, since the plasmid is the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions, are also included.

The term in vitro host cell (or simply host cell), as used herein, is intended to refer to a cell that contains a nucleic acid that is not normally present in the cell, and possibly a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the specific cell of the subject, but also to the progeny of such a cell. Since certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but will still be within the scope of the term host cell as used herein. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., 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).

The phrase immediately below an amino acid in the context of the present document refers to the position immediately after the terminal carboxyl group of the amino acid. Similarly, the phrase immediately above an amino acid refers to the position immediately after the terminal amino group of the amino acid. Therefore, the phrase between two amino acids of an insertion site in the context of the present document refers to the position at which a heterologous moiety (e.g., a half-life extending moiety) is inserted between two adjacent amino acids.

As used herein, the term "administration" refers to the physical introduction of a composition comprising a therapeutic agent into a subject via any of the various delivery methods and systems known to those skilled in the art. Various routes of administration for the IL2/IL2Ra fusion protein 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 routes of administration other than enteral and local administration, typically by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intracranial, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspiral, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, an antibody described herein can be administered by a non-parenteral route, such as a topical, epidermal, or transmucosal route, such as intranasally, orally, vaginally, rectally, sublingually, or topically. Administration can also be, for example, single, multiple, and/or over one or more extended periods.

- 14 048704

The immune response has the same definition as in the present art and denotes a biological response in a vertebrate against foreign agents or pathological, such as cancer cells, wherein such a response protects the organism from these agents and the diseases they cause. The immune response is mediated by the action of one or more cells of the immune system (e.g., a T lymphocyte, a B lymphocyte, a natural killer (NK) cell, a macrophage, an eosinophil, a mast cell, a dendritic cell or a neutrophil) and soluble macromolecules produced by any of these cells or by the liver (including antibodies, cytokines and complement), which leads to selective targeting, binding to, damaging, destroying and/or eliminating from the vertebrate organism invading pathogens, cells or tissues infected with pathogens, cancer cells or other pathological cells or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. An immune response includes, for example, the activation or inhibition of a T cell, such as an effector T cell, a Th cell, a CD4+ cell, a CD8+ T cell, or a Treg cell, or the activation or inhibition of any other cell of the immune system, such as a NK cell.

An immunomodulator or immunoregulator refers to an agent, such as an agent targeting a component of a signaling pathway, that may be involved in modulating, regulating or modifying an immune response. Modulating, regulating or modifying an immune response refers to any change in a cell of the immune system or the activity of such a cell (e.g., an effector T cell, such as a Th1 cell). More specifically, as used herein, the term modulation includes inducing, inhibiting, potentiating, enhancing, increasing or decreasing a given activity or response. Such modulation includes stimulating or suppressing the immune system, which may be manifested by increasing or decreasing the number of different cell types, increasing or decreasing the activity of these cells, or any other changes that may occur in the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which may have enhanced function in the tumor microenvironment. According to some embodiments, an immunomodulator specifically targets a molecule on the surface of a T cell. An immunomodulatory target molecule or immunoregulatory target molecule is a molecule, such as a cell surface molecule, that acts as a target molecule for binding and whose activity is altered by binding a substance, agent, fragment, compound, or molecule. Immunomodulatory target molecules include, for example, cell surface receptors (immunomodulatory receptors) and receptor ligands (immunomodulatory ligands).

Immunotherapy refers to the treatment of a subject suffering from or at risk of contracting or recurring a disease by a method that involves inducing, enhancing, suppressing or otherwise modifying the immune system or immune response.

Immunostimulatory therapy or immunostimulatory therapy refers to therapy that results in an increase (induction or enhancement) of the immune response in a subject, such as for the treatment of cancer.

Potentiation of the endogenous immune response means increasing the effectiveness or efficiency of an existing immune response in a subject. Such an increase in effectiveness and efficiency can be achieved, for example, by overcoming mechanisms that suppress the endogenous immune response of the host or by stimulating mechanisms that enhance the endogenous immune response of the host.

T effector (T _eff ) cells refer to T cells (e.g., CD4+ and CD8+ T cells) with cytolytic activity, as well as T helper (Th) cells, such as Th1 cells, which secrete cytokines and activate and direct other immune cells, but do not include regulatory T cells (Treg cells). Certain IL2/IL2Ra fusion proteins described herein activate Teff cells, such as CD4+ and CD8+ Teff cells and Th1 cells.

The increased ability to stimulate an immune response or the immune system may be the result of enhanced agonist activity of costomotor T cell receptors and/or enhanced antagonist activity of inhibitory receptors. The increased ability to stimulate an immune response or the immune system may be reflected by a fold increase in _EC50 or peak activity in an assay that measures an immune response, such as an assay that measures changes in cytokine or chemokine release, cytolytic activity (as measured directly on target cells or indirectly through detection of CD107a or granzymes), and proliferation. The ability to stimulate an immune response or immune system activity may be increased by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold, or more.

The terms linked and fused as used herein refer to a first amino acid sequence or nucleotide sequence covalently or non-covalently linked to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid sequence or nucleotide sequence may be directly linked or fused to the second amino acid sequence or nucleotide sequence, or, alternatively,

- 15 048704 ternately, the intermediate sequence may covalently connect the first sequence to the second sequence. The term "linked" means not only the fusion of the first amino acid sequence to the second amino acid sequence at the C-terminus or N-terminus, but also includes the insertion of the entire first amino acid sequence (or the second amino acid sequence) at any two amino acids in the second amino acid sequence (or the first amino acid sequence, respectively). According to one embodiment, the first amino acid sequence is linked to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence may be linked to the second nucleotide sequence by a phosphodiester bond or a linker. The linker may be a peptide or a polypeptide (in the case of polypeptide chains), or a nucleotide or nucleotide chain (in the case of nucleotide chains), or any chemical moiety (in the case of both polypeptide and polynucleotide chains). The term "linked" is also denoted by a hyphen (-).

As used herein, the term T cell response refers to a response mediated by T cells, including effector T cells (e.g., CD8+ cells) and helper T cells (e.g., CD4+ cells). Examples of T cell responses include T cell cytotoxicity and proliferation.

As used in this document, the term cytotoxic T lymphocyte (CTL) response refers to an immune response induced by cytotoxic T cells. CTL responses are mediated primarily by CD8+ T cells.

As used herein, the terms inhibits or blocks (e.g., with respect to inhibiting/blocking the binding of IL2 to IL2Ra on cells) are used interchangeably and encompass both partial and complete inhibition/blocking. In some embodiments, the IL2/IL2Ra fusion protein inhibits the binding of IL2 to IL2Ra by at least about 50%, such as by about 60, 70, 80, 90, 95, 99%, or 100%, as determined, for example, as further described herein. In some embodiments, the IL2/IL2Ra fusion protein inhibits the binding of IL2 to IL2Ra by no more than 50%, such as by about 40, 30, 20, 10, 5, or 1%, as determined, for example, as further described herein.

As used herein, the phrase inhibits tumor growth includes any measurable reduction in tumor growth, such as inhibiting tumor growth 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%.

As used in this document, the term cancer refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may result in the formation of malignant tumors or cells that invade adjacent tissues and may metastasize to distant parts of the body through the lymphatic system or bloodstream.

As used herein, the terms treat, treat and cure refer to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, for the purpose of reversing, alleviating, ameliorating, inhibiting, or slowing, or preventing the progression, development, severity or recurrence of a symptom, complication, pathological condition or biochemical feature associated with a disease, or increasing overall survival. Treatment may be in a subject suffering from a disease or in a subject that does not have a disease (e.g., in the case of prophylaxis). In prophylactic administration, the fusion protein described herein is administered prior to the onset of any symptom. Prophylactic administration of the substance serves to prevent the development or ameliorate any subsequent symptom.

By enhancing the efficacy or enhancing the immunogenicity of a fusion protein, pharmaceutical composition or vaccine is meant an improvement in the result, for example, as measured by a change in a certain value, such as an increase or decrease in a specific parameter of the activity of the fusion protein, pharmaceutical composition or vaccine associated with protective immunity. According to one embodiment, the enhancement refers to at least a 5, 10, 25, 50, 100 or greater than 100% increase in a specific parameter. According to another embodiment, the enhancement refers to at least a 5, 10, 25, 50, 100 or greater than 100% decrease in a specific parameter. In one example, enhancing the efficacy/immunogenicity of a vaccine refers to an increase in the ability of the vaccine to inhibit or treat disease progression, such as at least a 5, 10, 25, 50, 100 or greater than 100% increase in the efficacy of the vaccine for

- 16 048704 of this purpose. In a further example, enhancing the efficacy/immunogenicity of a vaccine refers to increasing the ability of the vaccine to engage the subject's natural defenses against already developed forms of cancer, such as at least a 5%, 10%, 25%, 50%, 100% or greater than 100% increase in the efficacy of a fusion protein, pharmaceutical composition or vaccine for this purpose.

Similarly, the phrase "overcoming a suppressed immune response" with respect to a fusion protein, pharmaceutical composition or vaccine refers to an improvement in the result, for example, as measured by a change in a certain value, such as a return to a previously positive value of a specific parameter of vaccine activity associated with protective immunity. According to one embodiment, overcoming refers to at least a 5, 10, 25, 50%, 100 or more than 100% increase in a specific parameter. In one example, overcoming a suppressed immune response to a fusion protein, pharmaceutical composition or vaccine refers to a renewed ability of the radiance protein, pharmaceutical composition or vaccine to inhibit or treat disease progression, such as at least a 5, 10, 25, 50, 100 or more than 100% restoration of the effectiveness of the vaccine for this purpose.

A therapeutically effective amount, a therapeutic dose, a dose, an effective dose, an effective dosage, or an amount of a dose administered, as used herein (interchangeably), means a dose that achieves the therapeutic goal described herein. According to some embodiments, a therapeutic dose means a dose that induces immunological tolerance in a subject. According to certain embodiments, a therapeutic dose means a dose that induces immunological tolerance in a subject for a period of time ranging from a certain period of time to a period of tolerance, such as 12 weeks after the first dose. A therapeutically effective amount of an IL2/IL2Ra fusion protein refers to an amount of IL2/IL2Ra fusion protein sufficient to induce the desired biological response. As will be appreciated by one of ordinary skill in the art, the absolute amount of a particular IL2/IL2Ra fusion protein that is effective may vary depending on factors such as the desired biological endpoint, the IL2/IL2Ra fusion protein being delivered, the target cell or tissue, etc.

One of ordinary skill in the art will also appreciate that the effective amount may be administered as a single dose or may 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 the progression or recurrence of a disease may be assessed by a variety of methods known to the practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

As used in the context of this document, the terms treat, cure and treat refer to, for example, reducing the severity of a disease or pathological condition; shortening the duration of a pathological condition; alleviating or eliminating one or more symptoms associated with a disease or pathological condition; obtaining beneficial effects in a subject with a disease or pathological condition without necessarily curing the disease or pathological condition.

As an example, an anticancer agent is a drug that promotes regression of cancer in a subject. According to some embodiments, a therapeutically effective amount of the drug promotes regression of cancer to the point of eliminating the cancer. Promoting regression of cancer means that administration of an effective amount of the drug, alone or in combination with an anticancer agent, results in a decrease in tumor growth or size, tumor necrosis, a decrease in the severity of at least one symptom of the disease, an increase in the frequency and duration of disease-free periods, a prevention of functional impairment or disability due to the disease, or other relief of disease symptoms in a patient. In addition, the terms effective and efficacy, when applied to treatment, include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote regression of cancer in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects at the cellular, organ, and/or organismal level (adverse effects) resulting from administration of the drug.

As an example for treating tumors, a therapeutically effective amount or dosage 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 untreated subjects. According to some preferred embodiments, the therapeutically effective amount or dosage of a drug completely inhibits cell growth or tumor growth, i.e., inhibits

- 17 048704 cell growth or tumor growth by 100%. The ability of the compound to inhibit tumor growth can be assessed using the assays described below. Alternatively, this property of the composition can be assessed by studying the ability of the compound to inhibit cell growth, such inhibition can be measured in vitro using assays known to the skilled practitioner. According to some embodiments described herein, tumor regression can be observed and can continue for a period of at least about 20 days, at least about 40 days, or at least about 60 days.

The term patient includes humans and other mammals receiving prophylactic or therapeutic treatment.

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 having cancer. The term non-human animal includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

The term dose or weight-based dosing as referred to herein means that the dose administered to a patient is calculated based on the patient's weight. For example, if a patient weighing 60 kg requires 3 mg/kg of IL2 antibody, the corresponding amount of IL2/IL2Ra fusion protein (i.e., 180 mg) can be calculated and used for administration.

The use of the term constant dose with respect to the methods and dosages described herein means the dose that is administered to a patient without regard to the patient's weight or body surface area (BSA). Therefore, the constant dose is not reported as a mg/kg dose, but as an absolute amount of the agent (e.g., IL2/IL2Ra fusion protein). For example, a 60 kg human and a 100 kg human would receive the same dose of antibody (e.g., 480 mg of IL2/IL2Ra fusion protein).

As used in this document, the terms µg and µM are used interchangeably with microgram and micromole, respectively.

The various aspects described in this document are described in more detail in the following subsections.

All references to the numbering of amino acids in immunoglobulins or immunoglobulin fragments or regions are based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda; MD. (The FcRn receptor has been isolated from several mammalian species, including humans. Sequences for human FcRn, rat FcRn, and mouse FcRn are known (Story et al., J. Exp. Med. 180:2377 (1994)). Fc may comprise the CH2 and CH3 domains of an immunoglobulin, with or without the hinge region of an immunoglobulin. Exemplary Fc embodiments are provided in WO 2004/101740 and WO 2006/074199.

7.3 Interleukin-2/interleukin-2 receptor alpha fusion proteins

The present invention relates to a fusion protein comprising at least two components: (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 (IL2Ra) polypeptide. According to some embodiments, the extracellular domain of the IL2Ra polypeptide has at least one fewer glycosylation site compared to the extracellular domain of native IL2Ra (SEQ ID NO: 7); and/or (ii) the IL2 polypeptide has at least one fewer glycosylation site compared to native IL2 (SEQ ID NO: 2). According to some embodiments, the fusion protein has IL2 activity.

7.3.1 Interleukin-2

According to some embodiments, the present invention relates to a fusion protein that comprises a first polypeptide comprising interleukin-2 (IL2) fused in frame to a second polypeptide comprising or consisting of the extracellular domain of an interleukin-2 receptor alpha polypeptide (IL2Ra). According to some embodiments, the fusion protein comprises a first polypeptide comprising IL2 having the sequence of SEQ ID NO: 2. According to 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 the sequence of SEQ ID NO: 2.

According to one embodiment, the IL2 polypeptide is a natural or recombinant IL2 derived from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), as well as domestic or farm mammals, unless otherwise indicated.

The IL2 polypeptides useful in the present invention are expressed in a fusion protein.

- 18 048704 The fusion proteins described herein specifically bind human IL2R and, more particularly, a distinct domain (e.g., a functional domain) within the extracellular domain of human IL2Ra. According to some embodiments, the fusion protein comprising IL2 is an antagonist. According to some embodiments, the fusion protein comprising IL2 binds human IL2Ra with high affinity.

The term IL2 encompasses unprocessed IL2 as well as any form of IL2 that results from processing in the cell (i.e., mature IL2). The term also encompasses naturally occurring variants and fragments of IL2, such as splice variants or allelic variants, and non-naturally occurring variants. The amino acid sequence of an exemplary mature form of human IL2 (having a 20 amino acid signal sequence) is set forth in SEQ ID NO: 2. Unprocessed human IL2 further comprises an N-terminal 20 amino acid signal peptide (SEQ ID NO: 1), which is not present in the mature IL2 molecule. The amino acid sequence of an exemplary mature form of murine IL2 (having a 20 amino acid signal sequence) is shown as SEQ 4. Full-length murine IL2 additionally comprises a 20 amino acid N-terminal signal peptide (SEQ ID NO: 3) that is not present in the mature IL2 molecule. Native IL2, also referred to as wild-type IL2, refers to naturally occurring or recombinant IL2.

Additional nucleic acid and amino acid sequences are known for IL2. See, for example, the sequences under GenBank accession numbers: Q7JFM2 (Aotus lemurinus (grey-bellied night monkey)); Q7JFM5 (Aotus papsutae (western Amazonian lemur)); P05016 (Bas taurus (ox)); Q29416 (Canis familiaris (dog) (Canis lupus familiaris)); P36835 (Caprahircus (goat)); and P37997 (Equus caballus (horse)).

Biologically active fragments and variants of IL2 are also presented.

Such active IL2 variants and fragments will retain IL2 activity. Biological activity of IL2 may refer to the ability to stimulate the IL2 receptor on the surface of lymphocytes. Such activity can be measured both in vitro and in vivo. IL2 is a global regulator of immune activity and the effects observed are the sum of these activities. For example, it regulates survival activity (Bcl-2), induces T-effector activity (IFN-gamma, granzyme B and perforin) and stimulates T-regulatory activity (FoxP3). See, for example, Malek et al. (2010) Immunity 33(2): 153–65.

Biologically active variants of IL2 are known. See, for example, U.S. Publication Nos. 2006/0269515 and 2006/0160187 and WO 99/60128.

Biologically active fragments and variants of IL2 can be used in the fusion proteins disclosed herein. Such a functional fragment can comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 4θ, 50, 75, 100, 125, 150 or more consecutive amino acids with the sequence of SEQ ID NO: 2. Alternatively, the functional variant can have 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.

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

Exemplary IL2 polypeptide sequences are shown in Table 1 below.

- 19 048704

Table 1

SEQ II) NO Description Subsequence 1 IL2 (human, unprocessed) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLL DLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQC LEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK GSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 2 IL2 (human, mature form) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM LTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNF HLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEF LNRWITFCQSIISTLT 3 IL2 (mouse, unprocessed) MYSMQLAS CVTLTLVLLVNSAPT SS S T S S S TAEAQQQQQ QQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKF YLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDA ENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRW IAFCQSIISTSPQ 4 IL2 (mice, mature form) APTSSSTSSS TAEAQQQQQQQQQQQHLE QLLMDLQELL SRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELG PLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNT FECQFDDESATWDFLRRWIAFCQSIISTSPQ

In some embodiments, the fusion proteins provided herein may comprise at least one mutation within the extracellular domain of IL2Ra. In some embodiments, the IL2 polypeptide has at least one less glycosylation site compared to native IL2 (SEQ ID NO: 2). In some embodiments, having at least one less glycosylation site is due to one or more mutations that remove a glycosylation site.

According to other embodiments, the fusion protein comprises a mutation that consists of replacing an amino acid having a glycosylation site with an amino acid that does not have a glycosylation site. According to some embodiments, the mutation removes an O-glycosylation site and/or an N-glycosylation site. According to one embodiment, the mutation removes an O-glycosylation site, for example, threonine, which is the 3rd amino acid in the sequence under SEQ ID NO: 2. According to another embodiment, the mutation removes an N-glycosylation site.

In some embodiments, the mutation is one or more substitutions of an amino acid of IL2 that is glycosylated with an amino acid that is not glycosylated. In some embodiments, the mutation is one or more substitutions of an amino acid of IL2 that allows glycosylation on an adjacent amino acid with an amino acid that does not allow glycosylation on an adjacent amino acid.

According to some embodiments, one or more amino acid substitutions from IL2 are a substitution 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.

According to some embodiments, one or more amino acid substitutions from IL2 are a substitution 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.

According to some embodiments, the one or more amino acid substitutions of IL2 are a substitution from a reactive amino acid, such as cysteine, 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, threonine, tryptophan, tyrosine, and valine. According to some

- 20 048704 In some embodiments, one or more substitutions are a substitution from cysteine to serine. In some embodiments, one or more substitutions are a substitution from cysteine to alanine. In some embodiments, one or more substitutions are a substitution from cysteine to valine.

According to some embodiments, one or more substitutions are at the T3 amino acid of IL2 relative to the corresponding sequence of SEQ ID NO: 2.

According to some embodiments, one of the substitutions is at amino acid C125 of the sequence of SEQ ID NO: 2. According to a particular embodiment, the substitution at amino acid C125 is selected from the group consisting of C125S, C125A, and C125V.

According to some embodiments, the mutation is a deletion. According to particular embodiments, the deletion is at amino acid A1 of the sequence under SEQ ID NO: 2.

The present invention also relates to any other mutations in the IL2 polypeptide. According to other embodiments, the mutations also include one or more substitutions that improve the properties of IL2, such as increasing the activity of IL2, increasing the half-life of IL2, increasing stability, etc.

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

According to some embodiments, IL2 comprises one or more mutations described in Carmenate et al., J Immunol, 200 (10) 3475-3484 (2018) and/or U.S. Pat. No. 8,759,486: for example, at amino acid residue Q22, Q126, 1129, S130, or any combination thereof, such as Q22V, Q126A, I129D, S130G, or any combination thereof. According to some embodiments, IL2 comprises one or more mutations L18N, Q126Y, and S130R, which are disclosed in U.S. Pat. No. 8,759,486 B2. According to some embodiments, IL2 comprises one or more mutations Q13Y, Q126Y, I129D, and S130R, which are disclosed in U.S. Pat. According to some embodiments, IL2 comprises one or more of the K35E, K35D and K35Q mutations, which are disclosed in WO 2018/091003 A1.

In some embodiments, IL2 comprises one or more mutations disclosed in Epstein et al. Blood, 101(12):4853-61] (2003) and/or U.S. Pat. No. 7,371,371: for example, at amino acid residue R38, for example, R38W. In some embodiments, IL2 comprises the R38W mutation and one or more mutations outside amino acid positions 22-58 of IL2, as disclosed in U.S. Pat. No. 7,371,371 B2.

In some embodiments, IL2 comprises one or more mutations disclosed in Wittrup et al. J Immunother. 32(9):887-94 (2009) and/or US Pat. No. 8,906,356: e.g., amino acid residue 91, 126, or both, e.g., V91R, Q126T, or both. In some embodiments, IL2 comprises the El 5W mutation as disclosed in Wittrup et al. J Immunother. 32(9):887-94 (2009) and also US Pat. No. 8,906,356. In some embodiments, IL2 comprises one or both of the N88R and V91R mutations as disclosed in Wittrup et al. J Immunother. 32(9):887-94 (2009), and also in US Publication 8906356. According to some embodiments, IL2 comprises a Q126T or Q126I mutation as disclosed in Wittrup et al. J Immunother. 32(9):887-94 (2009) and/or in US Publication 8906356.

According to some embodiments, IL2 comprises one or more mutations disclosed in US 8906356 B2: for example, at amino acid 69, 74, 91, 126, or any combination thereof. According to some embodiments, the mutation is V91R, Q126T, Q126L, Q127T, or any combination thereof, as disclosed in US 8906356 B2.

In some embodiments, IL2 comprises one or more mutations disclosed in Wittrup et al. J Immunother. 32(9):887-94 (2009) and/or U.S. Pat. No. 7,569,215 B2: e.g., at amino acid residue 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 the E15W mutation as disclosed in Wittrup et al. Biochemistry, Vol. 44, No. 31 (2005). In some embodiments, the mutation is V69A as disclosed in U.S. Pat. No. 7,569,215 B2.

In some embodiments, IL2 comprises one or more mutations disclosed in Wittrup et al. J Immunother. 32(9):887-94 (2009) and/or US 7,951,360 B2: e.g., at amino acid residue 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 the E15W mutation as disclosed in Wittrup et al. Biochemistry, Vol. 44, No. 31 (2005).

According to some embodiments, IL2 comprises one or more mut

- 21 048704 disclosed in Wittrup et al. J Immunother. 32(9): 887-94 (2009) and/or in US 8349311 B2: for example, at amino acid 69, 74, 128, or any combination thereof, for example, V69A, I128P, or any combination thereof.

According to 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 residue S4, T10, Q11, V69, N88, T133, or any combination thereof, e.g., S4P, T10A, Q11R, V69A, N88D, T133A, or any combination thereof.

According to 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 residue N30, V69, I128, or any combination thereof, e.g., N30S, V69A, I128T, or any combination thereof.

According to 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 residue K8, Q13, N26, N30, K35, T37, V69, or any combination thereof, e.g., K8R, Q13R, N26D, N30T, K35R, T37R, V69A, or any combination thereof.

According to some embodiments, IL2 comprises one or more mutations disclosed in Shanafelt et al., Nat Biotechnol., 18(11):1197-202 (2000), such as at amino acid residue N88, such as N88R

According to some embodiments, IL2 comprises one or more mutations disclosed in US 9,616,105 B2: for example, at amino acids 20, 88, 126, or any combination thereof, for example, N88R, N88G, or N88I. According to some embodiments, IL2 comprises an N88R, N88G, or N88I mutation as disclosed in US 9,616,105 B2. According to some embodiments, IL2 comprises a D20H, D20I, or D20Y mutation as disclosed in US 9,616,105 B2. According to some embodiments, IL2 comprises a Q126L mutation as disclosed in US 9,616,105 B2.

According to 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. According to some embodiments, IL2 comprises one or more mutations T3A, N88G, N88R, D20H, C125S, Q126L, and Q126F, as disclosed in US 2018/0037624 A1.

According to some embodiments, IL2 comprises one or more mutations disclosed in US 2017/0327555 A1: for example, at amino acid residue N88, D20, C125, Q126, or any combination thereof, for example, N88G, N88R, D20H, C125S, Q126L, Q126F, or any combination thereof.

According to some embodiments, IL2 comprises one or more mutations disclosed in WO 2016/025385 A1: for example, at amino acid residue D109, C125, or both, such as D109C, C125S, or both. According to some embodiments, IL2 comprises one or more mutations disclosed in WO 2016/025385 A1: for example; at amino acid residue D20, N88, Q126, C125, Q126, or any combination thereof, such as D20H, N88I, N88G, N88R, Q126L, C125S, Q126F, or any combination thereof.

According to some embodiments, IL2 comprises one or more mutations disclosed in WO 2016/164937 A1: for example, at amino acid residue L12, Q13, E15, H16, L19, D20, M23, D84, S87, N88, V91, E95, or any combination thereof, for example, L12G, L12K, L12Q, L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, 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.

According to some embodiments, IL2 comprises one or more mutations disclosed in US 9932380 B2 or US 9580486: for example, at amino acid residue V91, for example, V91K. According to some embodiments, IL2 further comprises a C125A or C125S mutation. According to some embodiments, IL2 further comprises a mutation in T3. According to some embodiments, the mutation in T3 is one of T3A or T3N. According to some embodiments, IL2 comprises a mutation in S5. According to some embodiments, the mutation is S5T.

According to some embodiments, IL2 comprises one or more mutations disclosed in US 9732134 B2: for example, in E15, H16, Q22, D84, N88, E95, or any combination thereof.

According to some embodiments, IL2 comprises one or more mutations disclosed in US 2015/0218260 A1: e.g., N88D. According to some embodiments, IL2 comprises a mutation disclosed in US 9266938 B2: e.g., at amino acid 42, 45, 72, or any combination thereof, e.g., L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D,

- 22 048704

L72R or L72K. According to some embodiments, IL2 comprises a mutation of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, and F42K. According to some embodiments, IL2 comprises a mutation of Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, and Y45K.

According to some embodiments, IL2 comprises one to four mutations: a first mutation of L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, or L72K, a second mutation of 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 of T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, or T3P, and/or a fourth mutation of C125A, C125S, C125T, or C125V. Mutations listed herein or disclosed in patents, patent publications, or any other source referenced herein are incorporated herein by reference in their entirety.

7.3.2 Interleukin-2 alpha receptor

The fusion protein comprises a second polypeptide comprising the extracellular domain of interleukin-2 receptor alpha (IL2Ra). According to some embodiments, the extracellular domain of IL2Ra comprises the amino acid sequence set forth in SEQ ID NO: 7. According to 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 the sequence set forth in SEQ ID NO: 7.

As used herein, the term CD25 or IL2 receptor a IL2Ra, IL2Ra, IL2-Ra and IL2-Ra refers to any natural or recombinant IL2Ra from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), as well as domestic or farm mammals, unless otherwise specified. The term also encompasses naturally occurring variants of IL2Ra, such as splice variants or allelic variants, or non-naturally occurring variants. Human IL2 exerts its biological effects by signaling through its receptor system, the IL2R IL2 and its receptor (IL2R) are required for proliferation and other fundamental functions of T cells that are critical for generating an immune response. IL2R consists of 3 non-covalently associated type I transmembrane proteins, which are the alpha (p55), beta (p75), and gamma (p65) chains. The alpha chain of human IL2R contains a 219 amino acid extracellular domain, a 19 amino acid transmembrane domain, and a 13 amino acid intracellular domain. The secreted extracellular domain of IL2R alpha (IL2Ra) can be used in the fusion proteins described herein.

The amino acid sequence of an exemplary mature form of human IL2Ra is shown as SEQ ID NO: 6. Unprocessed human IL2Ra is shown as SEQ ID NO: 5. The extracellular domain of SEQ ID NO: 5 and/or SEQ ID NO: 6 is shown as SEQ ID NO: 7. The amino acid sequence of an exemplary mature form of mouse IL2Ra is shown as SEQ ID NO: 9. Unprocessed mouse IL2Ra is shown as SEQ ID NO: 8. The extracellular domain of SEQ ID NO: 8 and/or SEQ ID NO: 9 is shown as SEQ ID NO: 10.

Natural IL2Ra, also called wild-type IL2Ra, refers to naturally occurring or recombinant IL2Ra.

Nucleic acid and amino acid sequences are known for IL2Ra. See, for example, sequences under GenBank accession numbers: NP_001030597.1 (P. troglodytes), NP_001028089.1 (M. mulatto), NM_001003211.1 (C. lupus), NP_776783.1 (B. taurus), NP_032393.3 (M. musculus), and NP_o37295.1 (R. norvegicus).

As used herein, the extracellular domain of IL2Ra means a functional extracellular domain of IL2Ra performing its normal role in binding to IL2, unless otherwise specified. The term IL2Ra EC domain includes a functional fragment, variant, analog or derivative thereof that retains the function of a full-length wild-type IL2Ra EC when binding to IL2. The IL2Ra EC domain may be a human, porcine, canine, rat or mouse IL2Ra EC domain. The phrase a biological activity of an IL2Ra EC domain refers to one or more biological activities of the IL2Ra EC domain, including but not limited to the ability to enhance intracellular signaling in cells capable of responding to the IL2 receptor. Non-limiting examples of biologically active fragments and variants of the IL2Ra EC domain are disclosed, for example, in Robb et al., Proc. Natl. Acad. Sci. USA, 85:5654-5658, 1988. According to some embodiments, the biologically active fragments and variants of the IL2Ra EC domain disclosed herein contain at least one fewer glycosylation site compared to the extracellular domain.

- 23 048704 nom natural IL2Ra.

Biologically active fragments and variants of the extracellular domain of IL2Ra can be used in the fusion proteins disclosed herein. Such a functional fragment 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 with any of the sequences of SEQ ID NO: 7. Alternatively, the functional variant can have 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.

Additionally provided are active variants and fragments of polynucleotides encoding the extracellular domain of IL2Ra. Such a polynucleotide may comprise at least 100, 200, 300, 400, 500, 600 or more consecutive nucleotides of the sequence under SEQ ID NO: 7 encoding the polypeptide and continue to encode a protein having the activity of the extracellular domain of IL2Ra. Alternatively, a functional polynucleotide may have 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 under SEQ ID NO: 7 and continue to encode a protein having the activity of the extracellular domain of IL2Ra.

The polypeptide sequences of IL2Ra are shown in Table 2.

Table 2

SEQ II) NO Description Subsequence 5 IL2Ra (human, unprocessed form) MDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHATFKAMAY KEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQC TSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPG HCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPA ESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEG RPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGC VFLLISVLLLSGLTWQRRQRKSRRTI 6 IL2Ra (human, mature form) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGS LYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKE RKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFW GQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICT GEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEM AATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRK SRRTI 7 IL2Ra (human, mature form of the extracellular domain of IL2Ra) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGS LYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKE RKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFW GQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICT

- 24 048704

GEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEM AATMETSIFTTEYQ 8 IL2Ra (mice, unprocessed form) MEPRLLMLGFLSLTIVPSCRAELCLYDPPEVPNATFKALSY KNGTILNCECKRGFRRLKELVYMRCLGNSWSSNCQCTSNSH DKSRKQVTAQLEHQKEQQTTTDMQKPTQSMHQENLTGHCRE PPPWKHEDSKRIYHFVEGQSVHYECIPGYKALQRGPAISIC KMKCGKTGWTQPQLTCVDEREHHRFLASEESQGSRNSSPES ETSCPITTTDFPQPTETTAMTETFVLTMEYKVAVASCLFLL ISILLLSGLTWQHRWRKSRRTI 9 IL2Ra (mice, mature form) ELCLYDPPEVPNATFKALSYKNGTILNCECKRGFRRLKELV YMRCLGNSWSSNCQCTSNSHDKSRKQVTAQLEHQKEQQTTT DMQKPTQSMHQENLTGHCREPPPWKHEDSKRIYHFVEGQSV HYECIPGYKALQRGPAISICKMKCGKTGWTQPQLTCVDERE HHRFLASEESQGSRNSSPESETSCPITTTDFPQPTETTAMT ETFVLTMEYKVAVASCLFLLISILLLSGLTWQHRWRKSRRT I 10 IL2Ra (mice, mature form of the extracellular domain of IL2Ra) ELCLYDPPEVPNATFKALSYKNGTILNCECKRGFRRLKELV YMRCLGNSWSSNCQCTSNILRASHDKSRKQVTAQLEHQKEQ QTTTDMQKPTQSMHQENLTGHCREPPPWKHEDSKRIYHFVE GQSVHYECIPGYKALQRGPAISICKMKCGKTGWTQPQLTCV DEREHHRFLASEESQGSRNSSPESETSCPITTTDFPQPTET TAMT E T FVL TME YK 11 IL2Ra (human, mature form of the extracellular domain of IL2Ra) - completely truncated ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGS LYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKE RKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFW GQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICT GE 12 IL2Ra (human, mature form of the extracellular domain of IL2Ra) - truncated by half ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGS LYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKE RKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFW GQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICT GEMETSQFPGEEKPQASPEGRPESETS

According to some embodiments, the fusion proteins provided herein may comprise at least one mutation within the EC domain of IL2Ra.

According to some embodiments, the EC domain of the IL2Ra polypeptide has at least one fewer glycosylation site, at least two fewer glycosylation sites, at least three fewer glycosylation sites, at least four fewer glycosylation sites, at least five fewer glycosylation sites, at least six fewer glycosylation sites, at least seven fewer glycosylation sites, at least eight fewer glycosylation sites, or at least nine fewer glycosylation sites compared to the extracellular domain of native IL2Ra (SEQ ID NO: 7).

According to some embodiments, the EC domain of the IL2Ra polypeptide having at least one less glycosylation site comprises a mutation that removes the glycosylation site. According to other embodiments, the fusion protein comprises a mutation that consists of replacing an amino acid having a glycosylation site with an amino acid that does not have a glycosylation site. According to some embodiments, the mutation removes an O-glycosylation site and/or an N-glycosylation site. According to one embodiment, the mutation removes an O-glycosylation site. According to another embodiment, the mutation removes an N-glycosylation site.

In some embodiments, the mutation in the fusion protein is a deletion of the C-terminus of IL2Ra. In some embodiments, the mutation is

- 25 048704 with a deletion of amino acids 167-219 of the sequence shown by SEQ ID NO: 7. According to some embodiments, the mutation is a deletion of amino acids 168-219 of the sequence shown by SEQ ID NO: 7. According to some embodiments, the mutation is a deletion of amino acids 169-219 of the sequence shown by SEQ ID NO: 7. According to some embodiments, the mutation is a deletion of amino acids 170-219 of the sequence shown by SEQ ID NO: 7. According to some embodiments, the mutation is a deletion of amino acids 171-219 of the sequence shown by SEQ ID NO: 7. According to some embodiments, the mutation is a deletion of amino acids 172-219 of the sequence shown by SEQ ID NO: 7. According to some embodiments, the mutation is a deletion of amino acids 173-219 of the sequence shown by SEQ ID NO: 7. According to some embodiments, the mutation is a deletion of amino acids 174-219 of the sequence shown by SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 175-219 of the sequence shown by SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 176-219 of the sequence shown by SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 177-219 of the sequence shown by SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 178-219 of the sequence shown by SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 179-219 of the sequence shown by SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 180-219 of the sequence shown by SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 181-219 of the sequence shown by SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 182-219 of the sequence shown by SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 183-219 of the sequence shown by SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 184-219 of the sequence shown by SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 185-219 of the sequence shown by SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 186-219 of the sequence shown by SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 187-219 of the sequence shown by SEQ ID NO: 7. In some embodiments, the mutation is a deletion of amino acids 188-219 of the sequence of SEQ ID NO: 7. According to some embodiments, the mutation is a deletion of amino acids 189-219 of the sequence of SEQ ID NO: 7. According to some embodiments, the mutation is a deletion of amino acids 190-219 of the sequence of SEQ ID NO: 7. According to some embodiments, the mutation is a deletion of amino acids 191-219 of the sequence of SEQ ID NO: 7. According to some embodiments, the mutation is a deletion of amino acids 192-219 of the sequence of SEQ ID NO: 7.

According to some embodiments, the mutation is a deletion of amino acids 167, 168, 169, or 171 to and including 192 or 219, corresponding to the sequence of SEQ ID NO: 7. According to some embodiments, the mutation does not include a deletion of 170219, corresponding to the sequence of SEQ ID NO: 7.

According to some embodiments, the second polypeptide comprises, consists essentially of, or has 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: 11. According to other embodiments, the second polypeptide comprises, consists essentially of, or has an amino acid sequence set forth in 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. According to some embodiments, the second polypeptide comprises the sequence of SEQ ID NO: 11 essentially consists of or consists of it, with 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 acid. According to 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.

In some embodiments, the fusion protein comprises one or more mutations. In some embodiments, the one or more mutations

- 26 048704 represents one or more substitutions of an amino acid from IL2Ra that is glycosylated with an amino acid that is not glycosylated.

According to some embodiments, the one or more amino acid substitutions of IL2Ra 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 locations correspond to the sequence of SEQ ID NO: 7.

In some embodiments, one or more substitutions are a substitution from asparagine to another amino acid. In some embodiments, one or more substitutions are a substitution 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, one or more substitutions are a substitution from threonine to another amino acid. In some embodiments, one or more substitutions are a substitution 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.

According to some embodiments, the substitution is at amino acid N49 of the sequence of SEQ ID NO: 7. According to some embodiments, amino acid N49 of the sequence 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.

According to some embodiments, the substitution is at amino acid N68 of the sequence of SEQ ID NO: 7. According to some embodiments, amino acid N68 of the sequence 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.

According to some embodiments, the substitution is at amino acid T74 of the sequence of SEQ ID NO: 7. According to some embodiments, amino acid T74 of the sequence 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.

According to some embodiments, the substitution is at amino acid T85 of the sequence of SEQ ID NO: 7. According to some embodiments, amino acid T85 of the sequence 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.

According to some embodiments, the substitution is at amino acid T197 of the sequence of SEQ ID NO: 7. According to some embodiments, amino acid T197 of the sequence 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.

According to some embodiments, the substitution is at amino acid T203 of the sequence of SEQ ID NO: 7. According to some embodiments, amino acid T203 of the sequence 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.

According to some embodiments, the substitution is at amino acid T208 of the sequence of SEQ ID NO: 7. According to some embodiments, amino acid T208 of the sequence 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.

In some embodiments, the substitution is for an amino acid.

- 27 048704

T216 of SEQ ID NO: 7. According to 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.

According to some embodiments, the fusion protein comprises one or more mutations. According to some embodiments, the one or more mutations are one or more substitutions of an amino acid from IL2Ra that allows glycosylation on an adjacent amino acid to an amino acid that does not allow glycosylation on an adjacent amino acid.

According to 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 locations correspond to the sequence of SEQ ID NO: 7.

According to some embodiments, the substitution is at amino acid S50 according to the sequence under SEQ ID NO: 7. According to some embodiments, amino acid S50 according to the sequence under SEQ ID NO: 7 is mutated to proline.

According to some embodiments, the substitution is at amino acid S51 according to the sequence of SEQ ID NO: 7. According to some embodiments, amino acid S51 according to the sequence 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.

According to some embodiments, the substitution is at amino acid T69 according to the sequence under SEQ ID NO: 7. According to some embodiments, amino acid T69 according to the sequence under SEQ ID NO: 7 is mutated to proline.

According to some embodiments, the substitution is at amino acid T70 according to the sequence of SEQ ID NO: 7. According to some embodiments, amino acid T70 according to the sequence 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, threonine, tryptophan, tyrosine and valine.

According to some embodiments, the substitution is at amino acid C192 according to the sequence of SEQ ID NO: 7. According to some embodiments, amino acid C192 according to the sequence of 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.

7.3.3 Linker

The fusion protein of the present invention may further comprise a linker. According to some embodiments, the linker may link the first polypeptide to the second polypeptide from the N-terminus to the C-terminus, for example, N-IL2-linker-IL2Ra EC-C. According to other embodiments, the linker may link the second polypeptide to the first polypeptide from the N-terminus to the C-terminus, for example, N-IL2Ra EC-linker-IL2-C.

According to one embodiment, the IL2/IL2Ra fusion protein comprises a linker sequence located between the IL2 polypeptide and the IL2Ra 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. According to other embodiments, a linker useful in the present invention has at least one amino acid and less than 100 amino acids, less than 90 amino acids, less than 80 amino acids, less than 70 amino acids, less than 60 amino acids, less than 50 amino acids, less than 40 amino acids, less than 30 amino acids, less than 20 amino acids, less than 19 amino acids, less than 18 amino acids, less than 17 amino acids, less than 16 amino acids, less than 15 amino acids, less than 14 amino acids, less than 13 amino acids, or less than 12 amino acids. According to one embodiment, the linker sequence comprises glycine amino acid residues. In other cases, the linker sequence comprises a combination of glycine and serine amino acid residues.

According to some embodiments, the fusion protein comprises a linker fused in-frame between the first polypeptide and the second polypeptide. According to 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 such said peptides. The presently disclosed

- 28 048704 document glycine/serine linkers comprise the amino acid sequence (GS) _n , (GGS) _n , (GGGS) _n , (GGGGS) _n or (GGGGS) _n , where n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. According to a particular embodiment, the linker sequence comprises GGGSGGGSGGGS (SEQ ID NO: 71) (also referred to as (Gly ₃ Ser) ₃ ). According to another embodiment, the linker sequence comprises GGGSGGGSGGGSGGGS (SEQ ID NO: 74) (also referred to as (Gly ₃ Ser) ₄ ). According to other embodiments, the linker sequence comprises one of (Gly ₃ Ser) ₅ (GGGSGGGSGGGSGGGSGGGSGGGS) (SEQ ID NO: 75), (Gly ₃ Ser) 6 (GGGSGGGSGGGSGGGSGGGSGGGS) (SEQ ID NO: 76), or (Gly 3 Ser) 7 (GGGSGGGSGGGSGGGSGGGSGGGSGGGS) (SEQ ID NO: 77). According to other embodiments, the linker sequence comprises (Gly 4 Ser) 3 (GGGGSGGGGSGGGGSGGGGS), which is set forth by SEQ ID NO: 78. According to further embodiments, the linker sequence comprises GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 79) (also referred to as (Gly ₄ Ser) ₄ ); GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 80) (also referred to as (Gly4Ser) ₅ ); (GlvSerl· (GGGGSGGGGS) (SEQ ID NO: 81), (Gly4Ser)1 (GGGGS) (SEQ ID NO: 82), (Gly4Ser)6 (GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 83); (G^Ser^ (GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 97); or (Gly4Ser)5 (GGGGSGGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 84).

7.3.4 Heterologous fragment

The fusion protein of the present invention may further comprise an additional element, such as a heterologous fragment. Such elements may promote expression of the fusion protein, promote secretion of the fusion protein, improve the stability of the fusion protein, allow for more efficient purification of the protein, and/or modulate the activity of the fusion protein. According to one embodiment, the heterologous fragment is a polypeptide fragment. According to other embodiments, the heterologous fragment is a non-polypeptide fragment.

According to some embodiments, the fusion protein comprises a heterologous fragment fused to a first polypeptide. According to some embodiments, the fusion protein comprises a polypeptide fragment fused to a second polypeptide. According to some embodiments, the fusion protein comprises a heterologous fragment fused to a first polypeptide and a second polypeptide.

According to some embodiments, the fusion proteins disclosed herein comprise one or more additional heterologous moieties. According to some embodiments, the heterologous moieties are half-life extending moieties. According to some embodiments, the heterologous moiety comprises albumin, an immunoglobulin constant region or a portion thereof, an immunoglobulin binding polypeptide, immunoglobulin G (IgG), an albumin binding polypeptide (ABP), a PASylation moiety, a HEKylation moiety, XTEN, a PEGylation moiety, an Fc region, and any combination thereof.

1. The constant region of an immunoglobulin or part of it.

The constant region of an immunoglobulin consists of domains designated CH (constant heavy) domains (CH1, CH2, etc.). Depending on the isotype (i.e., IgG, IgM, IgA, IgD, or IgE), the constant region may consist of three or four CH domains. In some isotypes (e.g., IgG), the constant regions also contain a hinge region. See Janeway et al. 2001, Immunobiology, Garland Publishing, N.Y., N.Y.

The immunoglobulin constant region or portion thereof for producing the fusion protein of the present invention can be obtained from a number of different sources. According to preferred embodiments, the immunoglobulin constant region or portion thereof is obtained from a human immunoglobulin. However, it is understood that the immunoglobulin constant region or portion thereof can be obtained from an immunoglobulin of another mammalian species, including, for example, a rodent species (e.g., mouse, rat, rabbit, guinea pig) or a non-human primate (e.g., chimpanzee, macaque). Moreover, the immunoglobulin constant region or portion thereof can be obtained from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4. According to one embodiment, the human IgG1 isotype is used.

Various immunoglobulin constant region gene sequences (e.g., human constant region gene sequences) are available as public depositories. It is possible to select a sequence of constant region domains with a particular effector function (or lacking a particular effector function) or with a particular modification to reduce immunogenicity. Many antibody and antibody-encoding gene sequences have been published, and suitable Ig constant region sequences (e.g., hinge, CH2, and/or CH3 sequences, or portions thereof) can be obtained from these sequences using art-recognized techniques. Genetic material obtained by any of the

- 29 048 704 of the above methods can then be modified or synthesized to produce the polypeptides of the present invention. It should also be understood that the scope of the present invention encompasses alleles, variants and mutations of the constant region DNA sequences.

Immunoglobulin constant region sequences or portions thereof can be cloned, for example, using the polymerase chain reaction and primers that are designed to amplify the domain of interest. To clone an immunoglobulin constant region sequence or portions thereof from an antibody, mRNA can be isolated from hybridoma, spleen, or lymph cells, reverse transcribed into DNA, and the antibody genes amplified using PCR. PCR amplification methods are described in detail in U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; and, for example, in 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 constant region consensus primers or more specific primers that are based on published DNA sequences and the amino acid sequences of the heavy and light chains. As discussed above, PCR can also be used to isolate DNA clones encoding the light and heavy chains of an antibody. In this case, the libraries can be screened with consensus primers or larger homologous probes such as mouse constant region probes. Numerous 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); for rapidly amplifying cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods 173:33); for antibody leader sequences (Larrick et al. 1989 Biochem. Biophys. Res. Commun. 160:1250). Cloning of antibody sequences is further described in Newman et al., U.S. Patent No. 5,658,570, filed January 25, 1995.

In the context of the present document, an immunoglobulin constant region may include all domains and a hinge region or parts thereof. According to one embodiment, the immunoglobulin constant region or part thereof comprises a CH2 domain, a CH3 domain and a hinge region, i.e., an Fc region or an FcRn binding partner.

In the context of this document, the term Fc region is defined as the portion of a polypeptide that corresponds to the Fc region of a natural immunoglobulin, i.e. formed by a dimeric association of the corresponding Fc domains of its two heavy chains. A natural Fc region forms a homodimer with another Fc region.

According to one embodiment, the Fc region refers to the portion of one immunoglobulin heavy chain beginning at the hinge region immediately before the papain cleavage site (i.e., residue 216 in IgG, taking the first residue of the heavy chain constant region as 114) and ending at the C-terminus of the antibody. Accordingly, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain.

The Fc region of an immunoglobulin constant region, depending on the immunoglobulin isotype, may include CH2, CH3, and CH4 domains, as well as a hinge region. Fusion proteins containing the Fc region of an immunoglobulin confer several desirable 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 the neonatal Fc receptor (FcRn) (U.S. Pat. Nos. 6,086,875, 6,485,726, 6,030,613; WO 03/077834; US 2003-0235536 A1).

According to some embodiments, the Fc region comprises an amino acid sequence of an Fc domain or a derivative of an Fc domain. According to some embodiments, the Fc region comprises at least one of a hinge (e.g., an upper, middle, and/or lower hinge region) domain (approximately amino acids 216-230 of the Fc region of the antibody according to EU numbering), a CH2 domain (approximately amino acids 231-340 of the Fc region of the antibody according to EU numbering), a CH3 domain (approximately amino acids 341-438 of the Fc region of the antibody according to EU numbering), a CH4 domain, or a variant, portion, or fragment thereof. According to other embodiments, the Fc region comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). According to 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), a CH2 domain (or a portion thereof) fused to both a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). According to still other embodiments, the Fc region lacks at least a portion of a CH2 domain (e.g., all or a portion of a CH2 domain). According to a particular embodiment, the Fc region comprises or consists of amino acids corresponding to EU numbers 221-447.

Fc domains, referred to herein as F, F1, or F2, may be obtained from a number of different sources. According to one embodiment, the Fc region of the polypeptide is derived from a human immunoglobulin. However, it is understood that the Fc region may be derived from an immunoglobulin from another mammalian species, including, for example, a rodent species (e.g., mouse, rat, rabbit,

- 30 048704 leopard, guinea pig) or non-human primates (e.g. chimpanzees, macaques). Moreover, the Fc domain polypeptide or portions thereof can be derived from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3 and IgG4. According to another embodiment, the human IgG1 isotype is used.

In some embodiments, the Fc variant introduces an alteration in at least one effector function conferred by an Fc region comprising said wild-type Fc domain (e.g., an improvement or decrease in the ability of the Fc region to bind to Fc receptors (e.g., FcγRI, FcγRII, or FcγRIII) or complement proteins (e.g., C1q), or to trigger antibody-dependent cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC)). In other embodiments, the Fc variant has an engineered altered cysteine residue.

The Fc regions of the present invention may utilize Fc variants known in the art that are known to alter (e.g., enhance or decrease) effector function and/or FcR binding. In particular, a binding molecule of the present invention may comprise, for example, an alteration (e.g., a substitution) at one or more amino acid positions disclosed 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 04/063351 A2, WO 04/074455 A2, WO 04/099249 A2, WO 05/040217 A2, WO 04/044859, WO 05/070963 A1, WO 05/077981 A2, WO 05/092925 A2, WO 05/123780 A2, WO 06/019447 A1, WO 06/047350 A2 and WO 06/085967 A2; 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. Patent Publication Nos. 5,648260; 5,739277; 5,834250; 5,869046; 6,096871; 6,121022; 6,194551; 6,242195; 6,277375; 6,528624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; 7,083,784; 7,404,956; and 7,317,091. According to one embodiment, a particular change (e.g., a particular substitution of one or more amino acids known in the art) can be made at one or more of the disclosed amino acid positions. According to another embodiment, a different change (e.g., a different substitution at one or more amino acid positions known in the art) can be made at one or more of the disclosed amino acid positions.

The Fc region of IgG can be modified by well-known procedures such as site-directed mutagenesis, etc., to yield modified IgG or Fc fragments or portions thereof that will bind to Fc receptors. Such modifications include modifications that are remote from the Fc receptor contact sites, as well as modifications within the contact sites that maintain or even enhance binding to Fc receptors. For example, the following individual amino acid residues in human IgG1 Fc (Fcy 1) can be substituted without significant loss of Fc binding affinity for Fc receptors: 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, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, P331A, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K36OA, 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, where, for example, P238A is a wild-type proline substituted with alanine at position 238. As an example, a specific embodiment includes a mutation N297A that removes a highly conserved N-glycosylation site. The wild-type amino acids at the above positions may be replaced by amino acids other than alanine. Mutations may be introduced into the Fc to produce more than one hundred Fc regions distinct from the native Fc. In addition, combinations of two, three, or more such individual mutations may be introduced together to produce hundreds of additional Fc regions. Furthermore, one Fc region of a construct of the present invention may be mutated while another Fc region of the construct is not mutated at all, or both may be mutated but with different mutations.

Some of the above mutations may confer new functionality to the Fc region. For example, one embodiment includes N297A, which removes a highly conserved N-glycosylation site. The result of this mutation is decreased immunogenicity, thereby increasing the circulating half-life of the Fc region, and rendering the Fc region unable to bind FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA without loss of affinity (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632; Shields et al. 1995, 1 Biol. Chem. 276:6591). As a further example of the new functionality that results from the above mutations,

- 31 048704 above mutations, in some cases the affinity for Fc receptors may be increased beyond that of the wild type. Such increased affinity may be reflected by an increased association constant, a decreased dissociation constant, or both an increased association constant and a decreased dissociation constant. Examples of mutations thought 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).

In addition, at least three human Fc gamma receptors appear to recognize a binding site on IgG in the lower hinge region, typically amino acids 234-237. Therefore, mutations in this region could provide another example of new functionality and potential reduction in immunogenicity, such as replacing amino acids 233-236 of human IgG1 ELLG with the corresponding sequence from IgG2 PVA (with a single amino acid deletion). When such mutations are introduced, FcyRI, FcyRII, and FcyRIII, which mediate different effector functions, have been shown to be unable to bind IgG1. Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613.

According to one embodiment, the immunoglobulin constant region or portion thereof, such as an Fc region, is a polypeptide comprising the sequence PKNSSMISNTP (SEQ ID NO: 98) and optionally further comprising a sequence selected from HQSLGTQ (SEQ ID NO: 93), HQNLSDGK (SEQ ID NO: 94), HQNISDGK (SEQ ID NO: 95), or VISSHLGQ (SEQ ID NO: 96) (U.S. Patent No. 5,739,277).

According to certain embodiments, the immunoglobulin constant region or a portion thereof is semiglycosylated. 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, aglycosylated Fc region (e.g., an aglycosylated CH2 region). According to one embodiment, a linker may be inserted between the glycosylated and aglycosylated Fc regions. According to another embodiment, the Fc region or FcRn binding partner is fully glycosylated, i.e., all of the Fc regions are glycosylated. According to other embodiments, the Fc region may be aglycosylated, i.e., none of the Fc moieties are glycosylated.

According to some embodiments, the fusion protein of the present invention comprises an amino acid substitution in an immunoglobulin constant region or portion thereof (e.g., Fc variants) that alters the antigen-dependent effector functions of the Ig constant region, in particular the half-life of the protein in the bloodstream.

Such proteins have increased or decreased binding to the Fc receptor compared to proteins lacking such substitutions and, therefore, have increased or decreased serum half-lives, respectively. Fc variants with improved affinity for the Fc receptor are expected to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where a long half-life of the administered polypeptide is desired, such as for the treatment of a chronic disease or disorder (see, e.g., U.S. Patent Nos. 7,348,004, 7,404,956, and 7,862,820). In contrast, Fc variants with reduced binding affinity for the Fc receptor are expected to have shorter half-lives, and such molecules are also useful, for example, for administration to mammals where a shortened residence time in the bloodstream may be advantageous, such as for in vivo diagnostic imaging or in situations where the parent polypeptide has toxic side effects if it is present in the bloodstream for extended periods of time. Fc variants with reduced binding affinity for the Fc receptor are also less likely to cross the placenta and are therefore also useful in the treatment of diseases or disorders in pregnant women. Furthermore, other applications that may require reduced binding affinity for the Fc receptor include those that require localization to the brain, kidney, and/or liver. According to one exemplary embodiment, a fusion protein of the present invention is characterized by reduced transport from the vasculature across the glomerular epithelium of the kidney. According to another embodiment, the fusion protein of the present invention is characterized by reduced transport across the blood-brain barrier (BBB) from the brain to the vascular space. According to one embodiment, the protein with altered Fc receptor binding comprises at least one Fc region (e.g., one or two Fc regions) having one or more amino acid substitutions in the Fc receptor binding loop of the Ig constant region. The Fc receptor binding loop consists of amino acid residues 280-299 (according to EU numbering) of the full-length wild-type Fc region. According to other embodiments, an Ig constant region or portion thereof of the present invention having altered binding affinity for an Fc receptor comprises at least one Fc region having one or more amino acid substitutions at an amino acid position 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

- 32 048704 which alter Fc receptor binding activity are disclosed in International PCT Publication No. WO 05/047327.

2. Albumin or its fragment or variant.

According to certain embodiments, the heterologous fragment linked to the IL2 polypeptide and/or the IL2Ra EC domain is albumin or a functional fragment thereof.

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

According to one embodiment, the fusion protein comprises an IL2 polypeptide and an IL2Ra EC domain as described herein, and albumin, a fragment or variant thereof, wherein the IL2 polypeptide is linked to albumin or a fragment or variant thereof. According to another embodiment, the fusion protein comprises an IL2 polypeptide and an IL2Ra EC domain as described herein, and albumin, a fragment or variant thereof, wherein the IL2 EC is linked to albumin or a fragment or variant thereof.

According to other embodiments, the heterologous fragment linked by the IL2 polypeptide and the IL2Ra EC domain is albumin or a fragment or variant thereof that extends (or can extend) the half-life of the IL2 polypeptide and the IL2Ra 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 in PCT Application Publication Nos. 2008/033413 A2, 2009/058322 A1, or 2007/021494 A2.

3. Albumin-binding fragment.

According to certain embodiments, the heterologous moiety associated with the IL2 polypeptide and the IL2Ra EC domain is an albumin binding moiety that comprises an albumin binding peptide, a bacterial albumin binding domain, an albumin binding fragment of an antibody, or any combination thereof. For example, the albumin binding protein can be a bacterial albumin binding protein, an antibody, or an antibody fragment, including domain antibodies (see U.S. Patent No. 6,696,245). The albumin binding protein, for example, can be a bacterial albumin binding domain, such as that from 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 a conjugation partner include, for example, peptides with the consensus sequence Cys-Xaa ₁ -Xaa2 -Xaa ₃ -Xaa4 -Cys, wherein Xaa1 is Asp, Asn, Ser, Thr, or Trp; Xaa ₂ is Asn, Gln, His, Ile, Leu, or Lys; Xaa ₃ is Ala, Asp, Phe, Trp, or Tyr; and Xaa ₄ is Asp, Gly, Leu, Phe, Ser, or Thr, as described in U.S. Patent Application Publication No. 2003/0069395 or in Dennis et al. (Dennis et al. (2002) J. Biol. Chem. 277, 35035-35043).

4. PAS sequence.

According to other embodiments, the heterologous fragment linked to the IL2 polypeptide and the IL2Ra EC domain is a PAS sequence. According to one embodiment, the fusion protein comprises the IL2 polypeptide and IL2Ra EC domain described herein, as well as a PAS sequence, wherein the IL2 polypeptide and/or IL2Ra EC domain are linked to a PAS sequence.

In the context of the present document, a PAS sequence means an amino acid sequence comprising predominantly alanine and serine residues or comprising predominantly alanine, serine and proline residues, wherein such an amino acid sequence under physiological conditions forms a random helical conformation. Accordingly, a PAS sequence is a structural element, an amino acid polymer or a cassette of sequences comprising, essentially consisting of or consisting of alanine, serine and proline, which can be used as part of a heterologous moiety in a fusion protein. In this regard, it is known to a person skilled in the art that an amino acid polymer can also form a random helical conformation by introducing residues other than alanine, serine and proline into the PAS sequence as a minor component. The term minor component in the context of the present document means that amino acids other than alanine, serine and proline can be introduced into the PAS sequence to a certain extent, for example up to about 12%, i.e. approximately 12 of the 100 amino acids in the PAS sequence, up to approximately 10%, i.e. approximately 10 of the 100 amino acids in the PAS sequence, up to approximately 9%, i.e. approximately 9 of the 100 amino acids, up to approximately 8%, i.e. approximately 8 of the 100 amino acids, approximately 6%, i.e. approximately 6 of the 100 amino acids, approximately 5%, i.e. approximately 5 of the 100 amino acids, approximately 4%, i.e. approximately 4 of the 100 amino acids, approximately 3%, i.e. at

- 33 048704 approximately 3 of 100 amino acids, approximately 2%, i.e. approximately 2 of 100 amino acids, approximately 1%, i.e. approximately 1 of 100 amino acids. The amino acids other than alanine, serine and proline can be selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr and Val.

Under physiological conditions, the PAS sequence region forms a random helical conformation and, due to this, can mediate increased in vivo and/or in vitro stability of the IL2 polypeptide. Since the random helical conformation domain does not adopt a stable structure or function by itself, the biological activity mediated by the IL2 polypeptide and the IL2Ra EC domain to which it is fused is essentially preserved. According to other embodiments, PAS sequences that form a random helical conformation domain are biologically inert, especially with respect to proteolysis in blood plasma, immunogenicity, isoelectric point/electrostatic behavior, binding to cell surface receptors, or internalization, but are still biodegradable, which provides distinct advantages over synthetic polymers such as PEG.

Non-limiting examples of PAS sequences that form a random coil 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 combinations thereof. Additional examples of PAS sequences are known, for example, from US Patent Publication No. 2010/0292130 A1 and PCT Application No. WO 2008/155134 A1.

5. Sequence of NAR.

According to some embodiments, the heterologous fragment linked to the IL2 polypeptide and the IL2Ra EC domain is a glycine-rich homo-amino acid polymer (HAP). The HAP sequence may include a repeating glycine sequence that is at least 50 amino acids long, at least 100 amino acids long, 120 amino acids long, 140 amino acids long, 160 amino acids long, 180 amino acids long, 200 amino acids long, 250 amino acids long, 300 amino acids long, 350 amino acids long, 400 amino acids long, 450 amino acids long, or 500 amino acids long. According to one embodiment, the HAP sequence is capable of extending the half-life of a fragment fused or linked to the HAP sequence. Non-limiting examples of a HAP sequence include, but are not limited to, (Gly) _n , (Gly ₄ Ser) _n , or S(Gly ₄ Ser) _n , where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. According to 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. According to another embodiment, n is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 19θ or 200.

6. Transferrin or its fragment.

According to some embodiments, the heterologous moiety linked to the IL2 polypeptide and the IL2Ra EC domain is transferrin or a fragment thereof. Any transferrin can be used to produce the fusion proteins of the present invention. As an example, wild-type human Tf (Tf) is a 679-amino acid protein of approximately 75 kDa (excluding glycosylation) with two major domains, N (approximately 330 amino acids) and C (approximately 340 amino acids), which are believed to result from gene duplication. See GenBank Accession Numbers NM001063, XM002793, M12530, XM039845, XM 039847, and S95936 (www.ncbi.nlm.nih.gov/). Transferrin is composed of two domains, i.e., N domain and C domain. N domain consists of two subdomains such as N1 domain and N2 domain, and C domain consists of two subdomains such as C1 domain and C2 domain.

According to one embodiment, the transferrin portion of the fusion protein comprises a splice variant of transferrin. In one example, the splice variant of transferrin may be a splice variant of human transferrin, such as under Genbank accession number AAA61140. According to another embodiment, the transferrin portion of the hybrid protein comprises one or more domains of a transferrin sequence, such as an N domain, a C domain, an N1 domain, an N2 domain, a C1 domain, a C2 domain, or any combination thereof.

7) Polymer, such as polyethylene glycol (PEG)

According to other embodiments, the heterologous moiety attached to the IL2 polypeptide and the IL2Ra EC domain is a soluble polymer known in the art, including but not limited to polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, or polyvinyl alcohol. The heterologous moiety, such as a soluble polymer, can be attached to any positions in the IL2 polypeptide, or in the IL2Ra EC domain, either at the N- or C-terminus.

The present invention also provides chemically modified derivatives of the sli

- 34 048 704 of the protein of the present invention, which may provide additional advantages such as increased solubility, stability and residence time in the bloodstream of the polypeptide or reduced immunogenicity (see U.S. Patent No. 4,179,337). Chemical moieties for modification may be selected from the group consisting of water-soluble polymers including, but not limited to, polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran and polyvinyl alcohol. The fusion protein may be modified at random positions within the molecule, either at the N- or C-terminus, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

The polymer may have any molecular weight and may be branched or unbranched. In the case of polyethylene glycol, according to one embodiment, the molecular weight is from about 1 kDa to about 100 kDa for ease of handling and production. Other sizes may be used, depending on the desired profile (e.g., the duration of sustained release desired, the effects, if any, on biological activity, ease of handling, the degree or lack of antigenicity, and other known effects of polyethylene glycol on a protein or analog). For example, polyethylene glycol may 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, 10000, 10500, 11000, 11500, 12000, 12500, 13000, 13500, 14000, 14500, 15000, 15500, 16000, 16500, 17000, 17500, 18000, 18500, 19000, 19500, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 55000, 60000, 65000, 70000, 75000, 80000, 85000, 90000, 95000 or 100000 kDa.

According to some embodiments, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in 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).

The number of polyethyleneglycol moieties attached to each fusion protein, IL2 polypeptide, or IL2Ra EC domain of the present invention (i.e., the degree of substitution) may also vary. For example, the PEGylated proteins of the present invention may be linked to an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethyleneglycol molecules. Similarly, the average degree of substitution is in the ranges of 1-3, 2-4, 3-5, 4-6, 5-7, 68, 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 polyethyleneglycol units per protein molecule. Methods for determining the degree of substitution are reviewed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

According to other embodiments, the IL2 polypeptide and the IL2Ra EC domain, in the context of the present invention, are conjugated to one or more polymers. The polymer may be water-soluble and covalently or non-covalently attached to the IL2 polypeptide, the IL2Ra EC domain, or other moieties conjugated to the IL2 polypeptide or the IL2Ra EC domain. Non-limiting examples of the polymer may be poly(alkylene oxide), poly(vinylpyrrolidone), poly(vinyl alcohol), polyoxazoline, or poly(acryloylmorpholine).

8. Hydroxyethyl starch (HES).

According to some embodiments, the heterologous moiety linked to the IL2 polypeptide and the IL2Ra EC domain is a polymer, such as hydroxyethyl starch (HES) or a derivative thereof. According to one embodiment, the fusion protein comprises the IL2 polypeptide described herein and HES, wherein the IL2 polypeptide and the IL2Ra EC domain are linked to HES.

Hydroxyethyl starch (HES) is a derivative of the naturally occurring amylopectin and is broken down in the body by alpha-amylase. HES is a substituted derivative of the carbohydrate polymer amylopectin, which is present in corn starch at a concentration of up to 95% by weight. HES has beneficial biological properties and is used as a blood volume replacement agent and in hemodilution therapy in clinical settings (Sommermeyer et al., Krankenhauspharmazie, 8(8), 271–278 (1987); and Weidler et al., Arzneim.-Forschung/Drug Res., 41, 494–498 (1991)).

Amylopectin contains glucose units in which alpha1,4-glycosidic bonds are present in the main chain and alpha-1,6-glycosidic bonds are found at the branching sites. The physicochemical properties of this molecule are mainly determined by the type of glycosidic bonds. Due to the broken alpha-1,4-glycosidic bond, helical structures with approximately six glucose monomers per turn are formed. The physicochemical as well as the biochemical properties of the polymer can be modified by substitution. The introduction of a hydroxyethyl group can be carried out by alkaline hydroxyethylation. By adapting the reaction conditions, it is possible to exploit the different reactivity of the corresponding hydroxy group in the unsubstituted glucose monomer with respect to hydroxyethylation. Thanks to this, a person skilled in the art can influence the nature of the substitution to a limited extent.

HES is mainly characterized by its molecular weight distribution and degree of substitution.

- 35 048704

The degree of substitution, denoted as DS, refers to molar substitution and is known to those skilled in the art. See Sommermeyer et al., Krankenhauspharmazie, 8(8), 271-278 (1987), cited above, in particular on page 273.

According to one embodiment, the hydroxyethyl starch has an average molecular weight (weight average) of 1 to 300 kDa, 2 to 200 kDa, 3 to 100 kDa, or 4 to 70 kDa. The hydroxyethyl starch may further have a molar degree of substitution of 0.1 to 3, preferably 0.1 to 2, more preferably 0.1 to 0.9, preferably 0.1 to 0.8, and a C2:C6 substitution ratio in the range of 2 to 20 with respect to the hydroxyethyl groups. A non-limiting example of HES with an average molecular weight of about 130 kDa is HES with a degree of substitution of 0.2 to 0.8, such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8, preferably 0.4 to 0.7, such as 0.4, 0.5, 0.6 or 0.7. According to a specific embodiment, an example of HES with an average molecular weight of about 130 kDa is VOLUVEN® from Fresenius. VOLUVEN® is an artificial colloid used, for example, to replace usable volume in therapeutic indications for the treatment and prevention of hypovolemia. The characteristics of VOLUVEN® are as follows: an average molecular weight of 130,000+/-20,000 Da, a molar substitution of 0.4 and a C2:C6 ratio of about 9:1. According to other embodiments, the average molecular weight ranges of the hydroxyethyl starch are, for example, 4-70 kDa, or 10-70 kDa, or 12-70 kDa, or 18-70 kDa, or 50-70 kDa, or 4-50 kDa, or 10-50 kDa, or 12-50 kDa, or 18-50 kDa, or 4-18 kDa, or 10-18 kDa, or 12-18 kDa, or 4-12 kDa, or 10-12 kDa, or 4-10 kDa. According to still other embodiments, the average molecular weight of the hydroxyethyl starch used is in the range of greater than 4 kDa and less than 70 kDa, such as about 10 kDa, or in the range of 9 to 10 kDa, or 10 to 11 kDa, or 9 to 11 kDa, or about 12 kDa, or in the range of 11 to 12 kDa, or 12 to 13 kDa, or 11 to 13 kDa, or about 18 kDa, or in the range of 17 to 18 kDa, or 18 to 19 kDa, or 17 to 19 kDa, or about 30 kDa, or in the range of 29 to 30, or 30 to 31 kDa, or about 50 kDa, or in the range of 49 to 50 kDa, or 50 to 51 kDa, or from 49 to 51 kDa.

According to some embodiments, the heterologous moiety may be mixtures of hydroxyethyl starch species having different average molecular weights and/or different degrees of substitution and/or different C2:C6 substitution ratios. Therefore, it is possible to use mixtures of hydroxyethyl starch varieties having different average molecular weight values and different degrees of substitution, as well as different C2:C6 substitution ratios, or having different average molecular weight values and different degrees of substitution, as well as the same or approximately the same C2:C6 substitution ratio, or having different average molecular weight values and the same or approximately the same degrees of substitution, as well as different C2:C6 substitution ratios, or having the same or approximately the same average molecular weight values and different degrees of substitution, as well as different C2:C6 substitution ratios, or having different average molecular weight values and the same or approximately the same degrees of substitution, as well as the same or approximately the same C2:C6 substitution ratio, or having the same or approximately the same average molecular weight values and different degrees of substitution, as well as the same or approximately the same C2:C6 substitution ratio, or having the same or approximately the same average molecular weight values and the same or approximately the same degree of substitution values, as well as different C2:C6 substitution ratios, or having approximately the same average molecular weight values and approximately the same degree of substitution values, as well as approximately the same C2:C6 substitution ratio.

9. Polysialic acids (PSA).

According to some embodiments, the non-polypeptide heterologous moiety linked to the IL2 polypeptide and/or the IL2Ra EC domain is a polymer, such as polysialic acids (PSA) or a derivative thereof. Polysialic acids (PSA) are naturally occurring unbranched polymers of sialic acid produced by certain bacterial strains and mammals in certain cells Roth J., et al. (1993) in Polysialic Acid: From Microbes to Man, eds Roth J., Rutishauser U., Troy F. A. (Birkhauser Verlag, Basel, Switzerland), pp 335-348. They can be prepared with varying degrees of polymerization from n = about 80 or more sialic acid residues to n = 2 by limited acid hydrolysis or neuraminidase cleavage or by fractionation of natural bacterial forms of the polymer. The composition of the various polysialic acids also varies, so that homopolymeric forms exist, i.e., alpha-2,8-linked polysialic acid, which is part of the capsular polysaccharide of E. coli strain K1 and group B meningococci, and which is also found on the embryonic form of the neuronal cell adhesion molecule (N-CAM). Heteropolymeric forms also exist, such as the alternating alpha-2,8-alpha-2,9 polysialic acid of E. coli strain K92 and the group C polysaccharides of N. meningitidis. Sialic acid

- 36 048704 may also occur in alternating copolymers with monomers other than sialic acid, such as the W135 group or the Y group of N. meningitidis. Polysialic acids have important biological functions, including evasion of the immune and complement systems by pathogenic bacteria and regulation of glial adhesion of immature neurons during fetal development (with the polymer having an anti-adhesive function) Cho and Troy, P.N.A.S., USA, 91 (1994) 11427-11431, although there are no known receptors for polysialic acids in mammals. The alpha-2,8-linked polysialic acid of E. coli strain K1 is also known as colominic acid, and is used (at various lengths) to illustrate the present invention. Various methods for attaching or conjugating polysialic acids to a polypeptide have been described, see, for example, U.S. Patent No. 5,846,951; WO-A-0187922; and US 2007/0191597 A1.

10. XTEN sequences.

As used herein, the term XTEN sequence refers to extended length polypeptides with non-naturally occurring, substantially non-repetitive sequences that consist primarily of small hydrophilic amino acids, wherein such a sequence under physiological conditions has a secondary or tertiary structure with a low degree of completion or does not have any secondary or tertiary structure at all. As a partner of a fusion protein, XTEN can serve as a carrier, imparting certain desired pharmacokinetic, physicochemical and pharmaceutical properties when linked to an IL2 polypeptide and/or an IL2Ra EC domain of the present invention to create a fusion protein. Such desired properties include, but are not limited to, improved pharmacokinetic parameters and solubility characteristics.

According to some embodiments, the XTEN sequence of the present invention is a peptide or polypeptide 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. According to some embodiments, XTEN is a peptide or polypeptide having from greater than about 20 to about 3000 amino acid residues, from greater than 30 to about 2500 residues, from greater than 40 to about 2000 residues, from greater than 50 to about 1500 residues, from greater than 60 to about 1000 residues, from greater than 70 to about 900 residues, from greater than 80 to about 800 residues, from greater than 90 to about 700 residues, from greater than 100 to about 600 residues, from greater than 110 to about 500 residues, or from greater than 120 to about 400 residues.

The XTEN sequence of the present invention may comprise one or more sequence motifs of 9-14 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 a sequence motif, wherein the motif comprises, consists essentially of or consists of 4-6 types of amino acids selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P). See US 2010-0239554 A1.

According to some embodiments, the XTEN sequence comprises multiple units of non-overlapping sequence motifs from 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, with the resulting XTEN having a defined range of homology. According to other embodiments, the XTEN comprises multiple units of sequence motifs from two or more motif families. Such sequences can be selected to achieve desired physical/chemical characteristics, including properties such as net charge, hydrophilicity, lack of secondary structure, or lack of repetition, which are imparted by the amino acid composition of the motifs, as described in more detail below. According to other embodiments, the motifs included in the XTEN can be selected and assembled using the methods described herein to produce XTENs of from about 36 to about 3000 amino acid residues in length.

According to further embodiments, the XTEN used in the present invention affects the physical or chemical properties, such as the pharmacokinetics, of the fusion protein of the present invention. The XTEN sequence in the context of the present invention may be characterized by one or more of the following advantageous properties: conformational flexibility, increased aqueous solubility, high resistance to proteases, low immunogenicity, low binding to mammalian receptors, or increased hydrodynamic (or Stokes) radii. According to a particular embodiment, the XTEN sequence linked to the IL2 polypeptide and/or the IL2Ra EC domain in the present invention enhances pharmacokinetic properties such as a longer terminal half-life or an increased area under the curve (AUC), such that the fusion protein described herein is retained in vivo for

- 37 048 704 an extended period of time compared to a wild-type IL2 polypeptide. According to further embodiments, the XTEN sequence in the context of the present invention increases pharmacokinetic properties such as a longer terminal half-life or an increased area under the curve (AUC), such that the IL2 polypeptide persists in vivo for an extended period of time compared to wild-type IL2.

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

Additional examples of XTEN sequences that can be used in accordance with the present invention are disclosed in U.S. Patent Publication Nos. 2010/0239554 A1, 2010/0323956 A1, 2011/0046060 A1, 2011/0046061 A1, 2011/0077199 A1, or 2011/0172146 A1 or International Patent Publication Nos. WO 2010091122 A1, WO 2010144502 A2, WO 2010144508 A1, WO 2011028228 A1, WO 2011028229 A1, or WO 2011028344 A2.

11. Immunoglobulin-binding peptide (or polypeptide).

According to some embodiments, the non-heterologous moiety associated with the IL2 polypeptide and/or the IL2Ra EC domain is an immunoglobulin binding peptide. Immunoglobulin binding peptides can bind to the Fc region and can increase the half-life of the fusion protein described herein.

According to some embodiments, an immunoglobulin-binding peptide useful in the present invention is a peptide or polypeptide having more 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.

According to some embodiments, an immunoglobulin-binding peptide useful in the present invention comprises a 13-monomeric IgG-Fc domain-binding peptide (IgGBP). DeLano WL, et al. (2000) Science 287:1279-1283. According to other embodiments, an immunoglobulin-binding peptide useful in the present invention comprises the peptides disclosed in U.S. Patent Publication No. 20170334954, U.S. Patent Publication No. 20170210777, or PCT Publication No. WO/2017/069158.

7.3.5 Fused protein

According to some embodiments, the fusion proteins comprise any of the sequences of SEQ ID NO: 13 to SEQ ID NO: 70 and SEQ ID NO: 202 to SEQ ID NO: 204, which are set forth in Table 3.

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Table 3

SEQ II) NO: Design Subsequence 204 IL2(21-153)(G3S)3-CD25(22240) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAA TMETSIFTTEYQ 13 IL2(C145S)- CD25(22-240) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAA TMETSIFTTEYQ 14 IL2(C145A)- CD25(22-240) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN

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INVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAA TMETSIFTTEYQ 15 IL2(C145V)- CD25(22-240) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFVQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAA TMETSIFTTEYQ 16 IL2-CD25(22- 212) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETS 17 IL2-CD25(22187) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK TGE

- 40 048704

18 IL2(C145A)CD25(C213S, 22240) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETSSLVTTTDFQIQTEMAA TMETSIFTTEYQ 19 HSA-( _G4S ) _3IL2 -CD25(22187) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVN EVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMA DCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEE TFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAA CLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLS QRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYI CENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAAD FVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSWLLLRLAKT YETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKH PEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNR RPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTA LVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEG KKLVAASQAALGLGGGGSGGGGSGGGGS APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTL TGGGSGGGSGGGS ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYM LCTGNSHSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEM QS PMQPVDQAS L P GHCRE P P PWENEATE RI YHF WGQMVYYQC V QGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE

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20 21 IL2(C145A)CD25(22-212)- GG-HSA IL2(C145A)CD25(22-192)GGC APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTL TGGGSGGGSGGGS ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYM LCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEM QSPMQPVDQASLPGHCREPPPWENEATERIYHFWGQMVYYQCV QGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEE KPQASPEGRPESETSGGDAHKSEVAHRFKDLGEENFKALVLIAF AQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFG DKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRL VRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAK RYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASL QKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCH GDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIA EVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYA RRHPDYSWLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKP LVE EPQNLIKQNCEL EE QL GE YK FQNAL LVRYT KKVP QVS T P T L VEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKT PVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHA DICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFV EKCCKADDKETCFAEEGKKLVAASQAALGL APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQGGC 22 IL2(C145A)CD25(22-213) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN

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INVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETSC 23 IL2(C145A)CD25(22-187, N70C) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGCSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGE 24 IL2(C145A)CD25(22-187, N89C) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK TGE 25 IL2(C145A)- CD25(22-187)-C APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEC 26 Human Fc 1.1(f)-AZI- DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVW DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV

- 43 048704

IL2(C145S)CD25(22-187)-G first side Fc for getting into heterodimeric form LHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYVYP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGAAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLR PRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFS QSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGT MLNCECKRGFRRIKSGSLYMLCTGNSSHSWDNQCQCTSSATRN TTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWEN EATERIYHFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRW TQPQLICTGEG 27 IL2(C145S)CD25(22-187)HuFc 1.1(f)-AZI first side Fc for heterodimer pair production APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR WSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVYVYPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG 29 IL2-C145S- CD25(22-187) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK TGE

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30 IL2-T23AC145S-CD25(22187) APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK TGE 31 IL2-CD25(22240)-C213S APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETSSLVTTTDFQIQTEMAA TMETSIFTTEYQ 32 IL2-T23AC145S-CD25(22240)-C213S APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETSSLVTTTDFQIQTEMAA TMETSIFTTEYQ 33 NativeSigPepIL2-CD25(22187) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMI LNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEE VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTEMCEYADETA TIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIP HATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSW DNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQAS

- 45 048704

LPGHCREPPPWENEATERIYHFWGQMVYYQCVQGYRALHRGPA ESVCKMTHGKTRWTQPQLICTGE 34 NativeSigPepHuIL2-CD25(22212)-PP MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMI LNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEE VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETA TIVEFLNRWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIP HATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSW DNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQAS LPGHCREPPPWENEATERI YHFWGQMVYYQCVQGYRALHRGPA ESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPE SETSPP 35 M-IL2(C145S)- CD25(C213S) MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFK FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLIS NINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIIST LTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCEC KRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVT PQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLI CTGEMETSQFPGEEKPQASPEGRPESETSSLVTTTDFQIQTEMA ATMETSIFTTEYQ 36 M-IL2(C145S)- CD25(C213S) MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFK FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLIS NINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIIST LTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCEC KRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVT PQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLI CTGEMETSQFPGEEKPQASPEGRPESETSSLVTTTDFQIQTEMA ATMETSIFTTEYQ 37 M-IL2-C145ACD25(22-212) MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFK FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLIS NINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIIST

- 46 048704

LTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCEC KRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVT PQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLI CTGEMETSQFPGEEKPQASPEGRPESETS 38 M-IL2-C145ACD25(22-212) MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFK FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLIS NINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIIST LTGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCEC KRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVT PQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLI CTGEMETSQFPGEEKPQASPEGRPESETS 39 Del-A21-IL2- CD(22-212) PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY MPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNI NVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT GGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKR GFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQ PEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYH FWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICT GEMETSQFPGEEKPQASPEGRPESETS 40 IL2-T23A-L2- CD25(22-212) APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGSSGGAGGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETS 41 IL2-T3A-L3CD(22-212) APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL

- 47 048704

TGGGSGGGSGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEG TMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATR NTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWE NEATERIYHFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTR WTQPQLICTGEMETSQFPGEEKPQASPEGRPESETS 43 IL2-T23AC145S-CD25(22187) APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK TGE 44 IL2-T23A- C145S-CD25(22240)-C213S APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETSSLVTTTDFQIQTEMAA TMETSIFTTEYQ 45 IL2-T23A- CD25(22-187) APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK TGE

- 48 048704

46 IL2-T23A- CD25(22-212)- T106A APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKATEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETS 47 IL2-T23ACD25(22-212)- T95A APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVAP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETS 49 IL2-T23A- CD25(22-212) APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETS 50 IL2-T23AC145S-CD25(22212) APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETS

- 49 048704

51 IL2-C145S- CD25(22-212) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETS 52 IL2-T23ACD25(22-212, T95A-T1O5A) APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVAP QPEEQKERKATEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETS 53 IL2-T23ACD25(22-212)PG APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETSPG 54 IL2-CD25(22187) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK TGE

- 50 048704

55 IL2-T23AC145S-CD25(22187) APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK TGE 56 IL2-CD25(22240, C213S) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETSSLVTTTDFQIQTEMAA TMETSIFTTEYQ 57 Del-A21-IL2- CD(22-212) PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY MPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNI NVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT GGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKR GFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQ PEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYH FWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICT GEMETSQFPGEEKPQASPEGRPESETS 58 IL2-T23A-L2- CD25(22-212) APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGSSGGAGGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC

- 51 048704

TGEMETSQFPGEEKPQASPEGRPESETS 59 IL2-T23A-L3- CD25(22-212) APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGGSGGGSGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEG TMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATR NTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWE NEATERIYHFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTR WTQPQLICTGEMETSQFPGEEKPQASPEGRPESETS 60 IL2-T23A- C145S-CD25(22- 187)-N70Q-N89Q APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK TGE 61 IL2-C145S- CD25(22-187)- N70Q-N89Q APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGQSSHSSWDNQCQCTSSATRQTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGE 62 IL2-T23A C145S-CD25(22187)-N89Q APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRQTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC

- 52 048704

TGE 63 IL2-T23A C145S-CD25(22187)-N70Q APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGQSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGE 64 IL2-C145S- CD25(22-187)- N89Q APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK TGE 65 IL2-C145S- CD25(22-187)- N70Q APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGQSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGE 66 IL2-T23A C145S-CD25(22187) APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC

- 53 048704

TGE 67 Fcl.l-71inker- IL2-CD25(22212) Gives Fc homodimer DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVW DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV LHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGGSGGSGGAPTSSSTKKTQLQLEHLLLDLQMILNGINNY KNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQS KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN RWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAM AYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCT SSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCRE PPPWENEATERIYHFWGQMVYYQCVQGYRALHRGPAESVCKMT HGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETS 68 IL2-CD25(22- 212)-Fcl.l Produces Fc homodimer APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETSDKTHTCPPCPAPEAEG APSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVS NKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 69 IL2(V111K)- CD25(22-212) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INKIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSWDNQCQCTSSATRNTTKQVTP

- 54 048704

QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETS 70 IL2(D40T)- CD25(22-212) APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTP QPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIY HFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLIC TGEMETSQFPGEEKPQASPEGRPESETS 202 IL2-T3A- CD25(22-187)- N89Q-T95A- T106A APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TGGGSGGGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK TGE 203 Fc(IL2(V91K)CD25)2, divalent DKTHTCPPCPAPELLGGKSVFLFPPKPKDTLMISRTPEVTCVW DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGGSGGSGGAPTSSSTKKTQLQLEHLLLDLQMILNGINNY KNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQS KNFHLRPRDLISNINKIVLELKGSETTFMCEYADETATIVEFLN RWITFCQSIISTLTGGGSGGGSGGGSELCDDDPPEIPHATFKAM AYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCT SSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCRE PPPWENEATERIYHFWGQMVYYQCVQGYRALHRGPAESVCKMT HGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETS

IL2 represents mature (IL2(21-153) unless otherwise noted.

The linker between IL2 and CD25 is (G3S)3 unless otherwise noted.

According to some embodiments, a fusion protein of the present invention 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 of SEQ ID NO: 13 through SEQ ID NO: 70 and SEQ ID NO: 202 through SEQ ID NO: 204.

The IL2-IL2Ra fusion protein of the present invention may have one or more of the following properties/activities: (1) increasing regulatory T cell (Treg) activity and/or increasing immune tolerance with low dose IL2 therapy; (2) increasing immune response and memory with higher dose therapy; (3) increasing IL2 availability compared to recombinant IL2; and/or (4) increasing sustained stimulation of IL2R-bearing lymphocytes by IL2 in vivo.

According to one embodiment, the fusion proteins disclosed herein have one or more pharmacokinetic properties selected from the group consisting of increased half-life, increased C _max , increased AUC, increased C _min , decreased clearance, improved bioavailability, and any combination thereof, compared to a pharmacokinetic property of a polypeptide consisting of the IL2 sequence (SEQ ID NO: 2) or

- 55 048704 sequences under SEQ ID NO: 204 (wild-type IL2-CD25 sequences with a 12-monomer linker without truncation).

According to one embodiment, the fusion proteins disclosed herein have an extended half-life compared to IL2 (SEQ ID NO: 2) or the sequence of SEQ ID NO: 204 (the wild-type IL2-CD25 sequence with a 12-monomer linker without truncation). According to some embodiments, the half-life is extended by 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 from the IL2 sequence (SEQ ID NO: 2) or the sequence under SEQ ID NO: 204 (wild-type IL2CD25 sequence with a 12-monomer linker without truncation).

According to some embodiments, the increased Treg activity that results from exposure to the IL2/IL2Ra fusion protein can be analyzed in a variety of ways, including, for example, (1) increased abundance and number of Tregs in the CD4+ T cell compartment; (2) upregulation of IL2-dependent CD25; (3) increased proliferation as assessed by expression of the proliferative marker Ki67; and (4) an increased proportion of IL2-dependent fully differentiated Klrg1+ Tregs. Such effects on Tregs can be observed, for example, in the spleen and/or inflamed pancreas.

In some embodiments, the IL2/IL2Ra fusion protein of the present invention increases the number of tolerogenic and immunosuppressive Treg cells and immunity by increasing T effector/memory cell responses, and in further embodiments, has improved pharmacokinetics by generating such responses at (1) lower effective levels of IL2 activity compared to natural or recombinant IL2; and/or (2) elicits more robust biological responses than natural or recombinant IL2.

According to certain embodiments, the fusion protein has improved activity compared to natural or recombinant IL2. For example, the effect of the IL2/IL-2Ra fusion protein can be to increase the number of tolerogenic Treg cells 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 a lower effective level of IL2 activity compared to natural or recombinant IL2. According to other embodiments, the L2/IL2Ra fusion protein is more effective than natural or recombinant IL2 in inducing a sustained increase in the number of Treg cells and the properties associated therewith.

Various fragments and variants of IL2 and IL2Ra from various organisms can be used to create the IL2/IL2Ra extracellular domain fusion proteins provided herein. Such components are discussed in more detail elsewhere herein. Non-limiting examples of full-length or mature IL2Ra/IL2Ra extracellular domain fusion proteins are provided by the following SEQ ID NOs: SEQ ID NO: 13 through SEQ ID NO: 70 and SEQ ID NO: 202 through SEQ ID NO: 204.

The term secretory signal sequence refers to a polynucleotide sequence that encodes a polypeptide (a 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 to remove the secretory peptide during passage through the secretory pathway. As used herein, the mature form of a fusion protein or polypeptide includes the processed form of the polypeptide from which the secretory peptide has been removed. As used herein, the unprocessed form of the fusion protein retains the secretory peptide sequence.

Also presented are biologically active fragments and variants of the mature and unprocessed form of the IL-Ra IL2/EC domain fusion protein and the polynucleotide that encodes it. Such a functional polypeptide fragment may 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 consecutive amino acids of any sequence of the following SEQ ID NOs: SEQ ID NO: 13 to SEQ ID NO: 70 and SEQ ID NO: 202 to SEQ ID NO: 204. Alternatively, the functional polypeptide variant may have at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth under the following SEQ ID NOs: SEQ ID NO: 13 - SEQ ID NO: 70

- 56 048704 and SEQ ID NO: 202 - SEQ ID NO: 204.

Additionally, active variants and fragments of polynucleotides encoding IL-Ra IL2/extracellular domain fusion proteins are provided. Such a polynucleotide can contain at least 100, 200, 300, 400, 500, 600, 700, 800, 1000, 1100, 1200, 1300, 1500, 1800, 2000 consecutive nucleotides encoding the polypeptides presented under the following SEQ ID NOs: SEQ ID NO: 13 - SEQ ID NO: 70 and SEQ ID NO: 202 - SEQ ID NO: 204, and continue to encode a functional IL-Ra IL2/extracellular domain fusion protein.

In addition, it is understood that the components of the IL2/IL2Ra fusion protein can be in any order. According to one embodiment, the IL2 polypeptide is at the N-terminus and the IL2Ra extracellular domain is at the C-terminus of the fusion protein.

According to some embodiments, the fusion protein forms a dimer. According to other embodiments, the fusion protein is a monomer. In this case, according to some embodiments, the dimer comprises two monomers, and the monomers are linked to each other by covalent bonds. According to some embodiments, the dimer comprises two monomers, and the monomers are linked by non-covalent bonds.

According to some embodiments of the present invention, the fusion protein is more stable than a polypeptide consisting of IL2 (SEQ ID NO: 2) or the sequence of SEQ ID NO: 204 (the wild-type IL2-CD25 sequence with a 12-monomer linker without truncation). According to 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 modifications compared to a reference protein; (v) increased stability in vivo 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 from an interleukin-2 receptor alpha (IL2Ra) polypeptide; and has at least one more glycosylation site compared to the fusion protein.

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

According to some embodiments, the removal of one or more glycosylation sites of the fusion protein is accomplished by treating the fusion protein with alkali. According to some embodiments, glycans are removed from glycosylated polypeptides by treatment with an alkali metal borohydride. According to other embodiments, the glycosylation sites of the fusion proteins disclosed herein can be removed using alkali metal carbonates, such as sodium carbonate and potassium carbonate. According to some embodiments, alkali is used for the P-cleavage treatment.

In some embodiments, the removal of one or more glycosylation sites of the fusion protein is accomplished by chemically treating the fusion protein with hydrazinolysis. In one embodiment, the glycosylation sites of the fusion protein disclosed herein are deglycosylated by subjecting the fusion protein to hydrazinolysis and the released sugar chain is fluorescently labeled with 2-aminopyridine. See Hase et al. J. Biochem., 95, 197 (1984). In some embodiments, hydrazinolysis is performed using an instrument available from Oxford GlycoSystems (GlycoPrep 1000).

According to another embodiment, removal of one or more glycosylation sites from the fusion protein is accomplished by exposing the fusion protein to trifluoromethanesulfonic acid (TFMS).

In some embodiments, the removal of one or more glycosylation sites from the fusion protein is accomplished 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 from the fusion protein is accomplished using peptide N-glycosidase F (PNGase F). The concentration of PNGase F can vary and is determined empirically. In some embodiments, the glycosidase is PNGase F. PNGase F is a commercially available enzyme (e.g., New England Biolabs, Ipswich, MA, Cat. No. P0704 or No. P0710). In some embodiments, PNGase F is a fusion protein. For example, PNGase F can be PNGase F labeled with a chitin-binding

- 57 048 704 domain (CBD), or a fusion protein of PNGase F and SNAP. According to some embodiments, the glycosidase is lyophilized. According to some embodiments, the glycosidase is lyophilized PNGase F. According to some embodiments, the glycosidase is substantially free of animal-derived reagents.

According to some embodiments, the removal of one or more glycosylation sites from the fusion protein is accomplished by treating the fusion protein with endo-β-N-acetylglucosaminidase H (endo-H). Endo-H is a glycohydrolase secreted by Streptomyces plicatus and some other Streptomyces species (Tarentino et al., 1976). It cleaves the β-1,4-glycosidic bond in the N-acetylglucosamine core of the oligosaccharides and leaves a single N-acetylchitobiose attached to the asparagine residue of the glycoprotein (Trimble et al., 1978; Muramatsu 1971). The endo-H gene from S. plicatus is 939 bp in length (GenBank accession number AAA26738.1) and encodes a 28.9 kDa protein. Endo-H from S. plicatus was recently expressed in Pichiapastoris, and the deglycosylation activity of endo-H produced by P. pastoris was demonstrated in vitro under both co-fermentation and post-fermentation treatments (Wang et al., 2015).

In some embodiments, removing one or more glycosylation sites from the fusion protein is accomplished by treating the fusion protein with O-glycosidase (New England Biolabs, Ipswich, MA). O-glycosidase, also called endo-alpha-N-acetylgalactosaminidase, catalyzes the removal of O-linked Core 1 and Core 3 disaccharides from glycoproteins. In some embodiments, it releases unsubstituted Ser- and Thr-linked glycoproteins.

The removal of one or more glycosylation sites from the fusion protein can be accomplished after the IL2/IL2Ra fusion protein has been produced in cell culture (e.g., in a bioreactor), while the IL2/IL2Ra fusion protein is being produced in cell culture, after the fusion protein has been harvested, and/or during purification of the fusion protein. In some embodiments, the removal of one or more glycosylation sites can be accomplished by adding one or more removal agents during cell culturing during expression of the fusion protein. In other embodiments, the removal of one or more glycosylation sites can be accomplished by selecting a particular cell type as a host cell that does not perform glycosylation or has reduced glycosylation (e.g., E. coli or Streptomyces species). According to some embodiments, the removal of one or more glycosylation sites is accomplished by co-expressing a gene encoding a fusion protein with a gene encoding an enzyme that removes one or more glycosylation sites.

Table 4 below lists various amino acid sequences of IL2. According to some embodiments, the fusion proteins described herein comprise any of the sequences of SEQ ID NO: 101 - SEQ ID NO: 115, which are listed in Table 4.

Table 4

SEQ II) NO: Design Subsequence 101 IL2(21-153) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRD LISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQ SIISTLT 102 IL2(21- 153)(C145S) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRD LISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQ SIISTLT 103 IL2(21- 153)(C145A) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRD LISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQ SIISTLT

- 58 048704

104 IL2(21153)(C145V) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRD LISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFVQ SIISTLT 105 IL2(21- 153)(T23A) APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRD LISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQ SIISTLT 106 IL2(21153)(T23A+C 145S) APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRD LISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQ SIISTLT 107 IL2(21153)(T23A+C 145 A) APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRD LISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQ SIISTLT 108 NativeSigPep- IL2(1-153) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQ MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK PLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFCQSIISTLT 109 M-IL2(C145S) MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPR DLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFS QSIISTLT BY M- IL2(C145A) MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPR DLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFA QSIISTLT 111 Del-A21-IL2 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFK FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDL ISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQS IISTLT

- 59 048704

112 Del-A21- IL2(C145S) PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFK FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDL ISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQS IISTLT FROM Del-A21- IL2(C145A) PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFK FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDL ISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQS IISTLT 114 IL2(V111K) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRD LISNINKIVLELKGSETTFMCEYADETATIVEFLNRWITFCQ SIISTLT 115 IL2(D40T) APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTF KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRD LISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQ SIISTLT

Table 5 below lists various amino acid sequences of a linker. According to some embodiments, the fusion protein disclosed herein includes multiple sequentially linked sequences selected from any of SEQ ID NO: 116 - SEQ ID NO: 127, which are listed in Table 5.

___________________________________________________________Table 5

SEQ ID NO: Construction Subsequence 116 L1-(G3S)3 GGGSGGGSGGGS 117 L2-G2S2 GGSSGGAGGGGS 118 L3-(G3S)5 GGGSGGGSGGGSGGGSGGGS 119 L4-(G2S)3 GGSGGSGGS 120 L5-(G2S)5 GGSGGSGGSGGSGGS 121 L6-GGEEE GGEEEGGEEEGS 122 L712mer_Stiff ESPEPETPEDES 123 L8- 22mer_Helix GRGGEEKKKEKEKEEQEERETK 124 L9-G G 125 L10-GG GG 126 Lll-S S 127 L12-GS GS

Table 6 below lists various amino acid sequences of CD25. According to some embodiments, the fusion proteins described herein comprise any of the sequences of SEQ ID NO: 128 - SEQ ID NO: 169, which are listed in Table 6.

- 60 048704

Table 6

SEQ II) NO: Design Subsequence 128 CD25(22-240) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERIYHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA SPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQ 129 CD25(22-212) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA SPEGRPESETS 130 CD25(22-187) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGE

- 61 048704

131 CD25(22240)(C213S) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERIYHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA SPEGRPESETSSLVTTTDFQIQTEMAATMETSIFTTEYQ 132 CD25(22-192)GGC ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQGGC 133 CD25(22-213) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA SPEGRPESETSC 134 CD25(22187)(N70C) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGCSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGE 135 CD25(22187)(N89C) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRCTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGE 136 CD25(22-187)C ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEC 137 CD25(22-187)G ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEG 138 CD25(22-212)- PP ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSHSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS

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PMQPVDQASLPGHCREPPPWENEATERIYHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA SPEGRPESETSPP 139 CD25(22212)(T106A) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKATEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA SPEGRPESETS 140 CD25(22212)(T95A) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVAPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA SPEGRPESETS 141 CD25(22212)(T95A+T1 06A) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVAPQPEEQKERKATEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA SPEGRPESETS 142 CD25(22-212)PG ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA SPEGRPESETSPG 143 CD25(22187)(N70Q+N89Q) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGQSSHSSWDNQCQCTSSATRQTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGE 144 CD25(22187)(N89Q) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHHSSWDNQCQCTSSATRQTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGE

- 63 048704

145 CD25(22187)(N70Q) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGQSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERIYHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGE 146 CD25(22-211) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA SPEGRPESET 147 CD25(22-210) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA SPEGRPESE 148 CD25(22-209) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA SPEGRPES 149 CD25(22-208) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA SPEGRPE 150 CD25(22-207) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA SPEGRP 151 CD25(22-206) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSHSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY

- 64 048704

RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA SPEGR 152 CD25(22-205) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERIYHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA SPEG 153 CD25(22-204) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA SPE 154 CD25(22-203) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA SP 155 CD25(22-202) S 156 CD25(22-201) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQA 157 CD25(22-200) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQ 158 CD25(22-199) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSHSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS

- 65 048704

PMQPVDQASLPGHCREPPPWENEATERIYHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKP 159 CD25(22-198) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEK 160 CD25(22-197) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEE 161 CD25(22-196) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGE 162 CD25(22-195) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPG 163 CD25(22-194) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFP 164 CD25(22-193) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQF 165 CD25(22-192) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQ 166 CD25(22-191) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSHSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMETS 167 CD25(22-190) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEMET 168 CD25(22-189) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEME 169 CD25(22-188) ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYML CTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERI YHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTGEM

Table 7 below lists various linker amino acid sequences. In some embodiments, the fusion proteins described herein comprise any of SEQ ID NO: 170 through SEQ ID NO: 186, which are listed in Table 7. In some embodiments, there are no linker sequences (i.e., TL0, as listed in Table 7).

____________________________________________________________Table 7

SEQ ID NO: Design Subsequence TL0 - 170 TL1 G 171 TL2 GG 172 TL3 GS 173 TL4 GGS 174 TL5 GGGS 175 TL6 GGSGG 176 TL7 GGGSGG 177 TL8 GGSGGSG 178 TL9 GGSGGGSG 179 TL10 GGGSGGGSG 180 TL11 GGGGSGGGGS 181 TL12 GGGGSGGGGSG 182 TLI3 EPKS 183 TL14 PKSS 184 TL15 KSS 185 TL16 SS 186 TL17 S

Table 8 below lists various enhancer amino acid sequences. According to some embodiments, the fusion proteins described herein comprise any of the sequences of SEQ ID NO: 187 through SEQ ID NO: 201, which are listed in Table 8.

Table 8

SEQ ID NO: Design Subsequence 187 IgGl-Fc DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 188 IgGl.1-Fc DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWL NGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

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189 IgGl.3-Fc DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 190 IgGl- Fc(P238K) DKTHTCPPCPAPELLGGKSVFLFPPKPKDTLMISRTPEVTCVWDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 191 IgG4-Fc ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWD VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 192 IgG4. 1-F c ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCVWD VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 193 IgGl.3-Fcknob DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 194 IgGl. 3-Fc-hole DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

- 68 048704

195 IgGl- Fc(P238K)knob DKTHTCPPCPAPELLGGKSVFLFPPKPKDTLMISRTPEVTCVWDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 196 IgGl- Fc(P238K)-hole DKTHTCPPCPAPELLGGKSVFLFPPKPKDTLMISRTPEVTCVWDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 197 IgGl. 1- Fc(AZl) DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWL NGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYVYPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 198 IgGl. 1- Fc(AZ2) DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWL NGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYVLPPSREEMTKN QVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 199 HSA DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVT EFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQ EPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYE IARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDE GKAS SAKQRLKCAS L QKFGE RAFKAWAVARL S QRF PKAE FAEVS KLV TDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKP LLEKS NSIAEVENDEMPADL P S LAADFVE S KDVCKNYAEAKDVFL GM FLYEYARRHPDYSWLLLRLAKTYETTLEKCCAAADPHECYAKVFDE FKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTL VEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTPVS DRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLS

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EKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDK ETCFAEEGKKLVAASQAALGL 200 HSA(C34S) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVT EFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQ EPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYE IARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDE GKAS SAKQRLKCAS L QKFGE RAFKAWAVARL S QRF PKAE FAEVS KLV TDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKP LLEKS NSIAEVENDEMPADL P S LAADFVE S KDVCKNYAEAKDVFL GM FLYEYARRHPDYSWLLLRLAKTYETTLEKCCAAADPHECYAKVFDE FKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTL VEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTPVS DRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLS EKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDK ETCFAEEGKKLVAASQAALGL 201 HSA(C35A) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVT EFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQ EPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYE IARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDE GKAS SAKQRLKCAS L QKFGE RAFKAWAVARL S QRF PKAE FAEVS KLV TDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKP LLEKS NSIAEVENDEMPADL P S LAADFVE S KDVCKNYAEAKDVFL GM FLYEYARRHPDYSWLLLRLAKTYETTLEKCCAAADPHECYAKVFDE FKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTL VEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTPVS DRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLS EKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDK ETCFAEEGKKLVAASQAALGL

According to 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). According to some embodiments, the fusion protein also comprises a sequence or more consecutively linked sequences selected from Table 5 (i.e., one or more of SEQ ID NO: 116 to SEQ ID NO: 127).

According to 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 more sequentially linked sequences selected from Table 5 (i.e., one or more of SEQ ID NO: 116 to SEQ ID NO: 127), and an optional linker comprising a sequence or more sequentially linked sequences from Table 7 (i.e., one or more of SEQ ID NO: 170 to SEQ ID NO: 186).

According to 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 more sequentially linked sequences selected from Table 5, and an optional linker comprising a sequence or more sequentially linked sequences from Table 8 (i.e., one of SEQ ID NO: 187 to SEQ ID NO: 201).

According to some embodiments, the fusion protein disclosed herein comprises, in the following order: the enhancer sequence of Table 8 (i.e., one of the sequences of SEQ ID NO: 187 - SEQ ID NO: 201), the sequence or more following

- 70 048 704 sequentially linked sequences selected from Table 7 (i.e., one or more of the sequences under SEQ ID NO: 170 to SEQ ID NO: 186), an IL-2 sequence selected from Table 4 (i.e., one of the sequences under SEQ ID NO: 101 to SEQ ID NO: 115), and a CD25 sequence from Table 6 (i.e., one of the sequences under SEQ ID NO: 128 to SEQ ID NO: 169). According to some embodiments, the fusion protein comprises a sequence or more sequentially linked sequences selected from Table 7 (i.e., one or more of the sequences under SEQ ID NO: 170 to SEQ ID NO: 186). According to some embodiments, the fusion protein comprises a sequence or more sequentially linked sequences selected from Table 5 (i.e. one or more of the sequences under SEQ ID NO: 116 - SEQ ID NO: 127).

7.4 Polynucleotides

In certain aspects, provided herein are polynucleotides, such as DNA or RNA, comprising a nucleotide sequence encoding a fusion protein having IL2 activity as described herein, as well as vectors comprising such polynucleotide sequences, such as expression vectors for efficiently expressing them in host cells, such as mammalian cells. In accordance with some embodiments, provided herein are polynucleotide sequences that encode the polypeptide sequences of SEQ ID NO: 1 to SEQ ID NO: 70 and of SEQ ID NO: 202 to SEQ ID NO: 204.

As used herein, an isolated polynucleotide or nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source (e.g., a mouse or a human) of the nucleic acid molecule. Moreover, an isolated nucleic acid molecule, such as a cDNA molecule, may be 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. For example, the term substantially free includes preparations of a polynucleotide or nucleic acid molecule that contain less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (particularly less than about 10%) of other material, such as cellular material, culture medium, other nucleic acid molecules, chemical precursors, and/or other chemicals. According to a specific embodiment, one or more nucleic acid molecules encoding a fusion protein described herein are isolated or purified.

Polynucleotides can be obtained and the nucleotide sequence of the polynucleotides can be determined by any method known in the art. Nucleotide sequences encoding the fusion proteins described herein, for example, the fusion proteins described in Table 3, and modified versions of these fusion proteins can be determined by methods well known in the art, i.e., nucleotide codons known to encode specific amino acids are assembled to produce a nucleic acid encoding a fusion protein. Such a polynucleotide encoding a fusion protein can be assembled from chemically synthesized oligonucleotides (e.g., those described in Kutmeier G et al., (1994), BioTechniques 17: 242-6), which, briefly, involves synthesizing overlapping oligonucleotides containing portions of the fusion protein encoding sequence, hybridizing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding a fusion protein described herein can be prepared from a nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning techniques). For example, PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of a known sequence can be performed using genomic DNA prepared from hybridoma cells producing a fusion protein of interest. Such PCR amplification methods can be used to obtain nucleic acids containing a sequence encoding, for example, IL2, a linker sequence, or IL2-Ra. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to produce fusion proteins.

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 such a fusion protein can be synthesized chemically or obtained from a suitable source (e.g., a cDNA library or a cDNA library prepared from nucleic acid, preferably poly A+ RNA, isolated from any tissue or cells expressing the proteins of interest, such as hybridoma cells selected for expression of the fusion protein described herein) by PCR amplification using synthetic primers hybridizing to the 3' and 5' ends of the sequence, or by cloning using an oligonucleotide

- 71 048704 a probe specific for a particular gene sequence to detect, for example, a cDNA clone from a cDNA library that encodes fusion proteins. The amplified nucleic acids obtained by PCR can then be cloned into replicable cloning vectors by any method well known in the art.

DNA encoding the fusion proteins described herein can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the fusion proteins described herein). Such DNA can be obtained from hybridoma cells. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)) or myeloma cells that do not otherwise produce immunoglobulin protein, to produce the fusion proteins in the recombinant host cells.

In addition, it is known that the polynucleotide encoding the IL2/IL2Ra fusion protein may contain additional elements that facilitate translation of the fusion protein. Such sequences include, for example, Kozak sequences attached to the 5' end of the polynucleotide encoding the fusion protein. The Kozak consensus sequence is a sequence that occurs on eukaryotic mRNA and plays a role in the initiation of the translation process, and has the consensus sequence (gee)gccRccAUGG (SEQ ID NO: 92); where (1) the lowercase letter denotes the most common base at the position at which the base may nevertheless vary; (2) the capital letters indicate highly conserved bases, i.e., the sequence AUGG is unchanged or rarely, if ever, varies, with the exception of the IUPAC ambiguity code R, which indicates that a purine (adenine or guanine) is commonly found at this position; and (3) the sequence in parentheses ((gee)) has undefined meaning.

In one non-limiting embodiment, the IL2/IL2Ra fusion protein comprises a Kozak sequence optimized for the IL2 leader sequence as set forth in SEQ ID NO: 92 (gccaccATGGACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAA ACAGT), or a functional variant or fragment thereof. A functional variant or fragment of a Kozak sequence will retain the ability to increase translation of the protein compared to the level of translation from a sequence without a 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, 40 consecutive nucleotides of a Kozak sequence or a sequence presented under SEQ ID NO: 92 or under SEQ ID NO: 99 (gccaccATGG). Alternatively, the functional variant may have at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to a Kozak sequence or a sequence presented under SEQ ID NO: 92 or SEQ ID NO: 99.

7.5 Cells and Vectors

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

According to some embodiments, the host cell comprises the nucleic acids described herein.

According to some embodiments, the host cell is a eukaryotic cell. According to some embodiments, the host cell is selected from the group consisting of a mammalian cell, an insect cell, a yeast cell, a transgenic mammalian cell, and a plant cell. According to some embodiments, the host cell is a prokaryotic cell. According to some embodiments, the prokaryotic cell is a bacterial cell.

According to some embodiments, the host cell is a mammalian cell. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20, BMT10, and HsS78Bst cells.

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

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

Numerous expression vector systems can be used for the present invention. Such expression vectors are typically replicated in host organisms either as episomes or as an integral part of the host chromosomal DNA. The expression vectors can include expression control sequences, including, but not limited to, promoters (e.g., naturally associated or heterologous promoters), enhancers, signal sequences, splicing signals, enhancer elements, and transcription termination sequences. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. DNA elements derived from animal viruses such as bovine papillomavirus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV, or MOMLV), cytomegalovirus (CMV), or SV40 virus can also be used in expression vectors. Others may use polycistronic systems with internal ribosome entry sites.

Expression vectors typically contain selectable markers (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, or neomycin resistance) that allow detection of those cells that have been transformed with the desired DNA sequences (see, e.g., Itakura et al., U.S. Patent No. 4,704,362). Cells that have integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. The marker may provide for auxotrophy of the host, resistance to biocides (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene may either be directly linked to the DNA sequences to be expressed or introduced into the same cell by co-transformation.

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

According to other embodiments, the polypeptides of the present invention are expressed using polycistronic constructs. In these expression systems, multiple gene products of interest, such as multiple polypeptides of a multimeric binding protein, can be produced from a single polycistronic construct. These systems advantageously utilize 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.

More generally, once a vector or DNA sequence encoding a polypeptide has been obtained, the expression vector can be introduced into a suitable host cell. That is, the host cells can be transformed. Introduction of the plasmid into the host cell can be accomplished by a variety of techniques well known to those skilled in the art, as discussed above. The transformed cells are grown under conditions suitable for the production of the fusion protein and assayed for fusion protein synthesis. Exemplary assay techniques include solid phase

- 73 048704 enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or fluorescence-activated cell sorting (FACS) analysis, immunohistochemistry, etc.

7.6 Pharmaceutical compositions

The various IL2/IL2Ra fusion proteins disclosed herein (also referred to herein as active compounds) can be included in pharmaceutical compositions suitable for administration. Such compositions typically comprise the fusion protein and a pharmaceutically acceptable carrier. As used herein, the phrase pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions contemplate the use of any conventional medium or agent, except where it is incompatible with the active compound. Additional active compounds can also be included in the compositions.

According to some embodiments, the present invention relates to a pharmaceutical composition comprising (a) the IL2/IL2Ra fusion protein described herein and (b) a pharmaceutically acceptable excipient.

According to some embodiments, the present invention relates to a pharmaceutical composition comprising (a) a composition comprising the IL2/IL2Ra fusion protein described herein and (b) a pharmaceutically acceptable excipient.

According to some embodiments, the present invention relates to a pharmaceutical composition comprising (a) a nucleic acid described herein and (b) a pharmaceutically acceptable excipient.

According to some embodiments, the present invention relates to a pharmaceutical composition comprising (a) a vector described herein and (b) a pharmaceutically acceptable excipient.

According to some embodiments, the present invention relates to a pharmaceutical composition comprising (a) a host cell described herein and (b) a pharmaceutically acceptable excipient.

The pharmaceutical composition of the present invention is formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g. inhalation), transdermal (topical) and transmucosal. In addition, it may be necessary to administer a therapeutically effective amount of the pharmaceutical composition locally to the area in need of treatment. This can be accomplished, for example, by local or regional infusion or perfusion during surgery, topical application, injection, catheter, suppository or implant (e.g. implants formed from porous, non-porous or gelatinous materials, including membranes such as sialast membranes or fibers) and the like. According to another embodiment, a therapeutically effective amount of the pharmaceutical composition is delivered in a vesicle, such as liposomes (see, e.g., Langer, Science 249:1527-33, 1990 and Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, N. Y., pp. 35-365, 1989).

According to another embodiment, a therapeutically effective amount of the pharmaceutical composition can be delivered in a controlled release system. According to 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). According to another example, polymeric materials 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 reviewed by Langer (Science 249:1527-33, 1990), can also be used.

Acceptable carriers, excipients, or stabilizers are nontoxic to patients receiving them at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol, and mcresol); low molecular weight polypeptides (less than about 10 residues); 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;

- 74 048704 non-ionic counterions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethyleneglycol (PEG).

Pharmaceutically acceptable carriers useful in parenteral preparations include aqueous media, non-aqueous media, antimicrobial agents, isotonicity agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, complexing or chelating agents, and other pharmaceutically acceptable substances. Examples of aqueous media include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water for injection, dextrose injection, and lactated Ringer's injection. Non-aqueous parenteral media include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Parenteral preparations packaged in multidose containers may be treated with antimicrobial agents in bacteriostatic or fungistatic concentrations and include phenols or cresols, mercury, benzyl alcohol, chlorobutanol, methyl and propyl parahydroxybenzoate esters, 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 polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN® 80). Complexing or chelating agents include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible media; and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

Solutions or suspensions for parenteral, intradermal, or subcutaneous use may contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethyleneglycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The parenteral preparation may be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor ELS (BASF; Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents in the composition, for example, sugars, polyhydric alcohols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of injection compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with, if necessary, one or a combination of the ingredients enumerated above, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile drug medium which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional required ingredient from a previously filter sterilized solution thereof.

- 75 048704

When administered by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as carbon dioxide, or a nebulizer. Systemic administration can also be accomplished by the transmucosal or transdermal route.

In the case of transmucosal or transdermal administration, wetting agents suitable for the barrier to be penetrated are used in the formulation. Such wetting agents are generally known in the art and include, for example, transmucosal agents, detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be carried out using nasal sprays or suppositories. In the case of transdermal administration, the active compounds are formulated into ointments, balms, gels or creams, as is generally known in the art. The compounds can also be prepared in the form of suppositories (for example with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

According to one embodiment, the active compounds are prepared with carriers that will protect the compound from rapid elimination from the body, such as in the form of 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. Methods for preparing such formulations will be apparent to those skilled in the art. The materials can also be purchased commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. They can be prepared according to methods known to those skilled in the art, such as described in U.S. Patent No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit refers to physically discrete units suitable as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. The specification of the dosage unit forms of the present invention is dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect sought to be achieved, as well as the limitations inherent in the art of preparing mixtures of such functional compound for the treatment of individuals. The pharmaceutical compositions may be enclosed in a container, package or dispenser together with instructions for use.

An effective amount of the IL2/IL2Ra fusion protein suitable for modulating such functions will depend on the subject being treated, the severity of the disease, and the route of administration of the IL2/IL2Ra fusion protein. Exemplary doses 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, about 106 to about 107 IU of IL2 activity per adult. In other cases, a therapeutically effective dose of the IL2/IL2Ra fusion protein is approximately 105 IU of IL2 activity ± 100-fold, approximately 105 IU of IL2 activity ± 10-fold, approximately 105 IU of IL2 activity ± 2-fold, approximately 105 IU of IL2 activity ± 20-fold, approximately 105 IU of IL2 activity ± 30-fold, approximately 105 IU of IL2 activity ± 40-fold, approximately 105 IU of IL2 activity ± 50-fold, approximately 105 IU of IL2 activity ± 60-fold, approximately 105 IU of IL2 activity ± 70-fold, approximately 105 IU of IL2 activity ± 80-fold, or approximately 105 IU of IL2 activity ± 90-fold. According to a specific non-limiting embodiment, the human IL2 fusion protein is administered at such a dosage.

7.7 Applications and methods

7.7.1 Methods for preparing the compositions disclosed herein

As discussed above, the IL2/IL2Ra fusion proteins disclosed herein can be used to create new IL2/IL2Ra fusion proteins by modifying IL2, IL2Ra, or any heterologous fragment sequence described herein. Thus, in another aspect, the structural features of the IL2/IL2Ra fusion protein described herein are used to create structurally related IL2/IL2Ra fusion proteins that retain at least one functional property of the IL2/IL2Ra fusion protein described herein, such as binding to human IL2Ra and cynomolgus monkey IL2Ra. For example, one or more of IL2, IL2Ra, or any heterologous fragment sequence described herein can be recombinantly combined with known framework regions and/or other proteins to create an additional recombinantly engineered IL2 described herein.

- 76 048704 ment of the fused protein, as discussed above.

According to some embodiments, disclosed herein are methods for producing an IL2/IL2Ra fusion protein comprising culturing a host cell comprising the fusion protein under suitable conditions and isolating the fusion protein. According to some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell. According to 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. According to some embodiments, the host cell is selected from the group consisting of a CHO cell, a HEK293 cell, an NS0 cell, a Per C6 cell, a BHK cell, and a COS cell. According to one embodiment, the host cell is a bacterial cell. According to a specific embodiment, the bacterial cell is Escherichia coli.

Other types of modifications include those described in the previous section. The starting material for the design methods is one or more of the IL2 or IL2Ra sequences provided herein. To create the designed fusion protein, it is not necessary to actually produce (i.e., express as a protein) a fusion protein having one or more of the IL2 or IL2Ra sequences provided herein. Instead, the information contained in the sequence(s) is used as a starting material to create second-generation sequence(s) derived from the original sequence(s), and then the second-generation sequence(s) is produced and expressed as a protein.

Accordingly, the present invention relates to methods for producing a fusion protein comprising: (a) providing IL2 and IL2Ra; (b) altering at least one amino acid residue in IL2 and IL2Ra to produce at least one altered fusion protein sequence; and (c) expressing the altered fusion protein sequence as a protein.

The present invention also relates to methods for producing a fusion protein comprising: (a) providing IL2 and IL2Ra; (b) altering at least one glycosylation site in IL2 and IL2Ra to obtain at least one altered glycosylation site; and (c) expressing the altered fusion protein sequence as a protein.

The altered antibody may be characterized by 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 set forth above under paragraphs (1) (10). The functional properties of the altered antibodies can be assessed using standard assays available in the art.

According to some embodiments of the methods for constructing the IL2/IL2Ra fusion proteins described herein, mutations can be introduced randomly or selectively throughout all or part of the IL2/IL2Ra fusion protein coding sequence, and the resulting modified IL2/IL2Ra fusion proteins can be screened for binding activity and/or other functional properties as described herein. Methods for introducing mutations have been described in the prior art. For example, PCT Publication No. WO 02/092780 to Short describes methods for generating and screening mutations using saturation mutagenesis, synthetic assembly by ligation, or a combination thereof. Alternatively, PCT Publication No. WO 03/074679 to Lazar et al. describes methods for using computational screening methods to optimize the physicochemical properties of proteins.

The compositions further include isolated polynucleotides that encode the various fusion proteins and variants and fragments thereof described herein above. Vectors and expression cassettes comprising the polynucleotides described herein are further disclosed. Expression cassettes will typically include a promoter operably linked to the polynucleotide and a transcription and translation termination site.

The use of the term polynucleotide is not intended to limit the present invention to polynucleotides containing DNA. Those skilled in the art will appreciate that polynucleotides may contain ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs.

It will be appreciated that in constructs that include more than one processing or cleavage site, such sites may be the same or different.

An isolated or purified polynucleotide or protein, or a biologically active portion thereof, is substantially or substantially free of components that normally accompany or interact with the polynucleotide or protein and that are found in its natural 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

- 77 048704 contains chemical precursors or other chemicals in chemical synthesis. Optimally, the isolated polynucleotide does not contain sequences (optimally protein-coding sequences) that normally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which such polynucleotide is obtained. For example, according to various embodiments, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that normally flank the polynucleotide in the genomic DNA of the cell from which such polynucleotide is obtained.

A protein that is substantially free of cellular material includes protein preparations that contain less than about 30, 20, 10, 5, or 1% (dry weight) of contaminating protein. If the protein of the present invention or a biologically active portion thereof is produced recombinantly, the culture medium optimally comprises less than about 30, 20, 10, 5, or 1% (dry weight) of chemical precursors or chemicals unrelated to the protein of interest.

In this case, conventional techniques of molecular biology, microbiology and recombinant DNA known to those skilled in the art can be used. Such techniques are fully explained in the literature. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (1989); Current Protocols in Molecular Biology Volumes I-III [Ausubel, R. M., ed. (1994)]; Cell Biology: A Laboratory Handbook Volumes I-III [J. E. Celis, ed. (1994)]; Current Protocols in Immunology Volumes I-III [Coligan, J. E., ed. (1994)]; Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984).

The present invention also relates to a vector that comprises the above-described polynucleotides operably linked to a promoter. A nucleotide sequence is operably linked to an expression control sequence (e.g., a promoter) if the expression control sequence controls and regulates transcription and translation of the sequence. The term operably linked, when applied to a nucleotide sequence, includes the presence of an appropriate start signal (e.g., ATG) upstream of the nucleotide sequence to be expressed, and the maintenance of the correct reading frame to ensure expression of the sequence under the control of the expression control sequence, and the production of the desired product encoded by such a sequence. If the gene to be inserted into the recombinant nucleic acid molecule does not contain an appropriate start signal, such a start signal can be inserted upstream of the gene. The vector is a replicon, such as a plasmid, phage, or cosmid, to which another nucleic acid segment can be attached so as to cause replication of the attached segment. The promoter may be or is identical to a promoter from bacteria, yeast, insects, or mammals. In addition, the vector may be a plasmid, cosmid, yeast artificial chromosome (YAC), bacteriophage, or eukaryotic viral DNA. Numerous other vector backbones known in the art to be useful for protein expression may 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, in one class of vectors, the vectors contain DNA elements derived from viruses such as bovine papillomavirus, polyoma virus, baculovirus, retroviruses, or Semliki Forest virus. Such vectors may be purchased commercially or assembled from the disclosed sequences using methods well known in the art.

The present document provides a host vector system for producing a polypeptide, which includes a suitable host cell vector. Suitable host cells include, but are not limited to, prokaryotic or eukaryotic cells, such as bacterial cells (including gram-positive cells), yeast cells, fungal cells, insect cells, and animal cells. Numerous mammalian cells can be used as hosts, including, but not limited to, NIH 3T3 mouse fibroblast, CHO cells, HeLa cells, Ltk cells, etc. Additional animal cells can also be used, such as R1.1, B-W and L-M cells, African green monkey kidney cells (e.g., COS1, COS7, BSC1, BSC40, and BMT10), insect cells (e.g., Sf9), as well as human cells and plant cells in tissue culture.

A wide range of host/expression vector combinations can be used to express the polynucleotide sequences provided herein. For example, suitable expression vectors can be comprised of segments of chromosomal, non-chromosomal, and synthetic DNA sequences. Suitable vectors include SV40 derivatives and known bacterial plasmids, such as E. coli col E1, pCR1, pBR322, pMB9, and their derivatives, such as RP4; phage DNAs, such as numerous derivatives of phage A, such as NM989, and others.

- 78 048704 phage DNAs, such as M13 and filamentous single-stranded phage DNA; yeast plasmids, such as plasmid 2!l or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to use phage DNA or other sequences to control expression; etc.

For expression of the polynucleotide sequences provided herein, any of a wide variety of expression control sequences (sequences that control expression of an operably linked nucleotide sequence) can be used in these vectors. Suitable expression control sequences include, for example, the early or 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 regions of phage A, the regulatory regions of the fd coat protein gene, the promoter of the 3-phosphoglycerate kinase gene or other glycolytic enzymes, the promoters of the acid phosphatase gene (e.g., Pho5), the promoters of the yeast mating factors a gene and other sequences that are known to control expression of genes of prokaryotic or eukaryotic cells or their viruses, as well as various combinations thereof.

It will be appreciated that not all vectors, expression control sequences, and hosts will function equally well for the expression of the polynucleotide sequences disclosed herein. Nor will all hosts function equally well with the same expression system. However, one skilled in the art, without undue experimentation, will be able to select suitable vectors, expression control sequences, and hosts to achieve the desired expression without departing from the scope of the present invention. For example, when selecting a vector, the host must be considered because the vector must function in it. Also considered will be the copy number of the vector, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers.

In selecting an expression control sequence, a number of factors will typically be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular nucleotide sequence or gene to be expressed, especially with respect to potential secondary structures. Suitable unicellular hosts will be selected based on, for example, their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the nucleotide sequences to be expressed, and the ease of purification of the expression products.

When producing an expression cassette, various polynucleotides can be manipulated to ensure the correct orientation of the polynucleotide sequences and, if necessary, to obtain the proper reading frame. For this purpose, adapters or linkers can be used to attach polynucleotides, or other manipulations can be performed to obtain convenient restriction sites, remove excess DNA, remove restriction sites, etc. For example, linkers such as two glycines can be added between polypeptides. Methionine residues encoded by ATG nucleotide sequences can be added to initiate gene transcription. For this purpose, in vitro mutagenesis, primer repair, restriction, hybridization, repeated substitutions such as transitions and transversions can be used.

Furthermore, the present invention relates to a method for producing a polypeptide, which comprises expressing a polynucleotide encoding the fusion protein described herein in a host cell under suitable conditions allowing the production of the polypeptide, and isolating the polypeptide thus obtained.

7.7.2 Therapeutic uses and methods

The fusion proteins and methods described herein have a variety of uses in vitro and in vivo, including, for example, enhancing an immune response, such as by inhibiting (or antagonizing) IL2Ra (e.g., signaling thereof) or detecting IL2. For example, the IL2/IL2Ra fusion proteins described herein can be administered to cells in culture, in vitro or ex vivo, or to human subjects, such as in vivo, to enhance immunity in various diseases. Accordingly, the present invention provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an IL2/IL2Ra fusion protein described herein such that the immune response in the subject is modified. According to some embodiments, the response is enhanced, stimulated, or upregulated.

Suitable subjects for the present methods include human patients in whom enhancing the immune response would be desirable. The methods are particularly suitable for treating human patients suffering from a disorder that can be treated by enhancing the immune response (e.g., T

- 79 048704 cellular immune response, such as an antigen-specific T cell response). According to some embodiments, the methods are particularly suitable for treating cancer in vivo. To achieve antigen-specific immune enhancement, the IL2/IL2Ra fusion proteins described herein can be administered together with an antigen of interest, or the antigen can already be present in the subject to be treated (e.g., a subject harboring a tumor or a virus). If the IL2/IL2Ra fusion proteins described herein are co-administered with another agent, the two can be administered separately or simultaneously.

Also encompassed by the present invention are methods for detecting the presence of human IL2 or human IL2Ra in a sample or measuring the amount of human IL2 antigen, comprising contacting the sample and a control sample with a fusion protein or a monoclonal antibody, such as a human monoclonal antibody or an antigen-binding portion thereof, that specifically binds to human IL2 or IL2Ra, under conditions that allow the formation of a complex between the IL2/IL2Ra fusion proteins and human IL2Ra described herein. The formation of the complex is then detected, wherein a differential formation of the complex between the sample relative to the control sample indicates the presence of human IL2 or IL2Ra antigen in the sample. Moreover, the IL2/IL2Ra fusion proteins described herein can be used to purify human IL2 or IL2Ra by immunoaffinity purification.

Given the ability of the IL2/IL2Ra fusion proteins described herein to stimulate or co-stimulate T cell responses, such as antigen-specific T cell responses, such as by inhibiting the negative effects of IL2 or IL2Ra, provided herein are methods in vitro and in vivo of using the IL2/IL2Ra fusion proteins described herein to stimulate, enhance, or increase antigen-specific T cell responses, such as anti-tumor T cell responses. In some embodiments, stimulation of CD3 is also provided (e.g., by co-incubation with a cell expressing membrane CD3), which stimulation can be performed concurrently with, prior to, or subsequent to stimulation with the IL2/IL2Ra fusion proteins described herein. For example, the present invention relates to methods for stimulating an antigen-specific T cell response comprising contacting said T cell with the IL2/IL2Ra fusion proteins described herein and, optionally, with an anti-CD3 antibody so as to stimulate the antigen-specific T cell response.

Any suitable indicator of an antigen-specific T cell response can be used to measure an antigen-specific T cell response. Non-limiting examples of such suitable indicators include increased proliferation of T cells in the presence of an antibody and/or increased cytokine production in the presence of an antibody. According to some embodiments, the production of interleukin 2 and/or interferon-y by antigen-specific T cells is stimulated.

T cells that can be expanded or co-stimulated using the IL2/IL2Ra fusion proteins described herein include CD4+ T cells and CD8+ T cells. The T cells can be Teff cells, such as CD4+ Teff cells, CD8+ Teff cells, Th helper (Th) cells (such as Th1 cells), or cytotoxic T (Tc) cells.

Additionally, the present invention relates to methods for stimulating an immune response (e.g., an antigen-specific T cell response) in a subject, comprising administering an IL2/IL2Ra fusion protein described herein to the subject to stimulate an immune response (e.g., an antigen-specific T cell response) in the subject. In some embodiments, the subject is a tumor carrier and an immune response to the tumor is stimulated in the subject. 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 virus carrier and an immune response to the virus is stimulated in the subject.

In addition, the present invention relates to methods of inhibiting tumor cell growth in a subject, comprising administering to the subject the IL2/IL2Ra fusion protein described herein so that tumor growth in the subject is inhibited. The present invention also relates to methods of treating a viral infection in a subject, comprising administering to the subject the IL2/IL2Ra fusion protein described herein so that the viral infection in the subject is treated.

According to some embodiments, the IL2/IL2Ra fusion protein described herein is administered to a subject as an adjunctive therapy. Treatment of subjects having

- 80 048704 cancer, the IL2/IL2Ra fusion protein described herein can result in prolongation of survival, such as a long-term durable response compared to the current standard of care; prolongation of survival by at least 3 months, 6 months, 9 months, 1, 2, 3, 4, 5, 10 or more years, or relapse-free survival of at least 3 months, 6 months, 9 months, 1, 2, 3, 4, 5, or 10 or more years. According to some embodiments, treating a subject having cancer with the IL2/IL2Ra fusion proteins described herein prevents recurrence of cancer or delays recurrence of cancer, such as by 3 months, 6 months, 9 months, 1, 2, 3, 4, 5, or 10 or more years. Treatment with the IL2/IL2Ra fusion protein can be used as first-, second-, or third-line therapy.

Treatment of a subject having cancer with the IL2/IL2Ra fusion protein described herein may result in, for example, disease stabilization, a partial response, an increase in overall survival, an increase in disease-free survival, or an increase in disease-free survival.

In some embodiments, the IL2/IL2Ra fusion protein described herein does not exhibit significant toxicity. For example, the IL2/IL2Ra fusion protein described herein does not exhibit significant toxicity to a human organ, such as one or more of the liver, kidney, brain, lung, and heart, as determined, for example, in clinical trials. In some embodiments, the IL2/IL2Ra fusion protein described herein does not significantly affect the initiation of an undesirable immune response, such as autoimmunity or inflammation.

In some embodiments, treating a subject with an IL2/IL2Ra fusion protein described herein does not overstimulate the immune system to the extent that the subject's immune system then attacks the subject itself (e.g., an autoimmune response) or results in, for example, anaphylaxis. Thus, in some embodiments, the IL2/IL2Ra fusion proteins described herein do not cause anaphylaxis.

In some embodiments, treatment of a subject with an IL2/IL2Ra fusion protein described herein does not cause significant inflammatory reactions, such as immune-mediated pneumonitis, immune-mediated colitis, immune-mediated hepatitis, immune-mediated nephritis or renal dysfunction, immune-mediated hypophysitis, immune-mediated hypothyroidism and hyperthyroidism, or other immune-mediated adverse reactions. In some embodiments, treatment of a subject with the IL2/IL2Ra fusion proteins described herein does not cause significant cardiac disorders, such as ventricular arrhythmia; ocular disorders, such as iridocyclitis; infusion reactions; increased amylase levels, increased lipase levels; nervous system disorders, such as dizziness, peripheral 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; arthralgia; or diarrhea.

In some embodiments, the IL2/IL2Ra fusion proteins described herein provide synergistic anti-tumor effects when combined with another anti-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.

According to some embodiments, the IL2/IL2Ra fusion proteins described herein are administered locally, mucosally, intranasally, orally, vaginally, rectally, sublingually, topically, intravenously, intraperitoneally, intramuscularly, intraarterially, intrathecally, intralymphaticly, intralesionally, intracapsularly, intraorbitally, intracardiacly, intradermally, transtracheally, subcutaneously, intradermally, intraarticularly, subcapsularly, subarachnoidally, intraspinally, epidurally, or intrasternally.

Various methods for enhancing an immune response in a subject are provided. Such methods include administering to a subject in need of an enhanced immune response a therapeutically effective amount of an IL2/IL2Ra fusion protein. Also, in accordance with particular embodiments, transient administration of higher doses of IL2 is used to enhance responses involving immune effector cells and memory cells.

Various methods have been proposed for reducing the immune response in a subject. Such methods include administering to a subject in need of a reduced immune response a therapeutically effective amount of an IL2/IL2Ra fusion protein. Of particular interest is the use of the suppressive power of Tregs to inhibit unwanted immune responses. Data in mice and humans suggest that increased IL2R signaling following exposure to low

- 81 048704 dose of IL2 selectively stimulates Tregs and enhances immune tolerogenic mechanisms. The IL2/IL2Ra fusion proteins presented herein represent a new and improved form of IL2 that has the potential to enhance Tregs. Thus, IL2/IL2Ra fusion proteins can be administered to patients with autoimmune diseases, chronic graft-versus-host disease, transplant rejection reactions, and other pathological conditions in which the goal is to suppress self-reactivity.

These and other methods described in this document are discussed in more detail below.

7.7.2.1 Cancer

According to some embodiments, the present invention relates to methods of treating cancer. Inhibition of IL2Ra by an IL2/IL2Ra fusion protein can enhance an immune response to cancer cells in a patient with cancer.

The present invention relates to methods of treating a subject having a cancer comprising administering to the subject an IL2/IL2Ra fusion protein as described herein in a manner that treats the subject, for example, in a manner that inhibits or reduces the growth of cancer tumors and/or in a manner that achieves tumor regression and/or prolongs survival. The IL2/IL2Ra fusion protein can be used alone to inhibit the growth of cancer tumors. Alternatively, the IL2/IL2Ra fusion protein can be used in combination with another agent, for example, another immunogenic agent, a standard anticancer agent, or another antibody, as described below.

Accordingly, the present invention relates to methods of treating cancer, for example by inhibiting the growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of the IL2/IL2Ra fusion protein described herein. Cancers whose growth can be inhibited by the antibodies of the present invention include cancers that are typically amenable to immunotherapy and those that are not typically amenable to immunotherapy. Cancers can be cancers with solid tumors or with malignant neoplasms of the blood (hemoblastosis). According to some embodiments, the cancer is bladder cancer, breast cancer, uterine cancer, endometrial carcinoma, 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 neoplasm, lymphoma, leukemia, sarcoma, viral cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's or non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, myeloma, salivary gland carcinoma, renal cancer, basal cell carcinoma, melanoma, prostate cancer, cancer of the female external genitalia, thyroid cancer, testicular cancer, esophageal cancer, or head or neck cancer, and any combinations thereof.

Other non-limiting examples of cancers to be treated include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, non-small cell squamous cell lung cancer (NSCLC), non-squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colon cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory adenocarcinoma of the prostate), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer (or carcinoma), gastric cancer, germinoma, childhood sarcoma, sinonasal tumor natural killer cells, melanoma (eg, metastatic malignant melanoma such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, small bowel cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, ureteral cancer, renal pelvis carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal cord tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, extrinsic cancers, including asbestos-induced cancers, virus-associated cancers or cancers of viral origin (e.g., from human papillomavirus (HPV-associated tumors or HPV-induced tumors)) and hematological malignancies derived from either of the two lineages of hemocyte development, i.e., the myeloid cell line (which produces granulocytes, red blood cells, platelets, macrophages, and mast cells) or the lymphoid cell line (which produces B, T, NK cells, and plasma cells), such as all types of leukemia, lymphoma, and myelomas, e.g., acute, chronic, lymphocytic, and/or myelogenous forms of leukemia such as acute leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia

- 82 048704 (CML), undifferentiated AML (M0), myeloblastic leukemia (M1), myeloblastic 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's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematological malignancies such as B-cell lymphomas, T-cell lymphomas, 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, progenitor cell T-lymphoblastic lymphoma, T-lymphoblastic lymphoma; and lymphomas/leukemias (T-Lbly/T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic tumors of the lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, B-lymphoblastic progenitor cell lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sezary syndrome), and lymphoplasmacytoid lymphoma (LPL) with macroglobulinemia Waldenstrom's; myelomas such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma), solitary plasmacytoma and multiple myelomas, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of the myeloid lineage, tumors of mesenchymal origin including fibrosarcoma and rhabdomyosarcoma; spermatocytoma, teratocarcinoma, tumors of the central and peripheral nervous system including astrocytoma, schwannomas; tumors of mesenchymal origin including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors including melanoma, xeroderma pigmentosum, keratoacanthoma, spermatocytoma, follicular thyroid cancer 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 medullary cell types; large granular lymphocyte leukemia (LGL) of the T-cell type; a/d T-cell non-Hodgkin's lymphoma of the liver and spleen; peripheral/postthymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); (nasal) T-cell lymphangioma; head and neck cancer, renal cancer, colorectal cancer, thyroid cancer; acute myeloid lymphoma, as well as any combination of these cancers. The methods described herein can also be used to treat metastatic cancers, inoperable, refractory cancers (e.g., cancers resistant to previous immunotherapy, such as with blocking antibodies to CTLA-4 or PD-1) and/or recurrent cancers.

In some embodiments, the IL2/IL2Ra fusion protein disclosed herein is administered to patients having a cancer that has responded inadequately to or has progressed following prior treatment, such as prior treatment with an immuno-oncology or immunotherapeutic drug, or to patients having a cancer that is refractory or resistant, or is inherently refractory or resistant (e.g., refractory to a PD-1 pathway antagonist), or that has become refractory or resistant. For example, subjects who fail to respond or respond adequately to a first therapy or who have progressed following treatment, such as treatment with an anti-PD-1 antibody, can be treated by administering the IL2/IL2Ra fusion protein described herein alone or in combination with another therapy (e.g., with anti-PD-1 antibody therapy).

In some embodiments, the IL2/IL2Ra fusion protein disclosed herein is administered to patients who have not previously received (i.e., have not been treated with) an immuno-oncology agent, such as a PD-1 pathway antagonist.

A method of treating a subject having a cancer with the IL2/IL2Ra fusion protein described herein may comprise administering to a subject having cancer cells expressing IL2 or IL2Ra a therapeutically effective amount of the IL2/IL2Ra fusion protein disclosed herein. The present invention also relates to methods of predicting whether a subject will respond to treatment with the IL2/IL2Ra fusion protein disclosed herein, wherein the methods comprise determining the level of IL2 or IL2Ra in the patient, and if the subject is positive for IL2 or IL2Ra, then the subject is likely to respond to treatment with the IL2/IL2Ra fusion protein disclosed herein.

According to some embodiments, the method of treating cancer in a subject comprises

- 83 048704 involves first determining whether a subject is PD-L1 or PD-1 positive, such as whether the subject has tumor cells or TILs that express PD-L1 or PD-1, and if the subject has PD-L1 or PD-1 positive cancer cells or TILs, then administering to the subject the IL2/IL2Ra fusion protein disclosed herein (and optionally a PD-1 or PDL1 antagonist). A method of treating a subject having a cancer with the IL2/IL2Ra fusion protein disclosed herein (and optionally a PD-1 or PD-L1 antagonist) may comprise administering to a subject having cancer cells or TIL cells expressing PD-L1 or PD-1 a therapeutically effective amount of the IL2/IL2Ra fusion protein disclosed herein (and optionally a PD-1 or PD-L1 antagonist). The present invention also relates to methods for predicting whether a subject will respond to treatment with the IL2/IL2Ra fusion protein disclosed herein (and, optionally, a PD-1 or PD-L1 antagonist), wherein the methods comprise determining the level of PD-L1 or PD-1 in cancer cells or TIL cells of the subject, and if the cancer cells or TIL cells of the subject are PD-L1 or PD-1 positive, then the subject is likely to respond to treatment with the IL2/IL2Ra fusion protein disclosed herein (and, optionally, a PD-1 or PD-L1 antagonist).

In some embodiments, the IL2/IL2Ra fusion protein disclosed herein is administered with a standard of care. In some embodiments, the IL2/IL2Ra fusion protein disclosed herein is administered as a maintenance therapy, such as a therapy that is intended to prevent the occurrence or recurrence of tumors.

In some embodiments, the IL2/IL2Ra fusion protein disclosed herein is administered with another treatment, such as radiation therapy, surgery, or chemotherapy. For example, in some embodiments, adjuvant therapy using the IL2/IL2Ra fusion protein described herein is administered if there is a risk that micrometastases may be present and/or to reduce the risk of recurrence.

In some embodiments, the IL2/IL2Ra fusion protein disclosed herein is administered as a monotherapy or as a single immunostimulatory therapy. Anti-IL2Ra antibodies can be combined with an immunogenic agent such as cancer 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 melanoma antigen peptides such as gp100 peptides, MAGE antigens, Trp-2, MART1, and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF (discussed further below).

According to some embodiments, the IL2/IL2Ra fusion protein disclosed herein is combined with a vaccination protocol. Many experimental tumor vaccination strategies have been developed (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; see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition). According to one of these strategies, the vaccine is prepared using autologous or allogeneic tumor cells. Such cell-based vaccines have been shown to be most effective when tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation in tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci U.S.A. 90: 3539-43).

The study of gene expression and large-scale patterns of gene expression in various tumors has led to the identification of so-called tumor-specific antigens (Rosenberg, S A (1999) Immunity 10: 281-7). In many cases, these tumor-specific antigens are differentiation antigens that are expressed in tumors and in the cell from which the tumor arose, such as the melanocyte antigens gp100, MAGE and Trp-2 antigens. More importantly, many of these antigens can be demonstrated to be targets for tumor-specific T cells in the host. According to some embodiments, the IL2/IL2Ra fusion protein disclosed herein is used in combination with a panel of recombinant proteins and/or peptides expressed in the tumor to generate an immune response to such proteins. These proteins are typically recognized by the immune system as its antigens and are therefore tolerated by the immune system. Tumor antigens 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 in a limited number of somatic tissues (Kim et al. (1994) Science 266: 2011–2013). Tumor antigens can also be neoantigens, which are expressed in cancer cells due to somatic mutations that alter the protein sequence or create fusion proteins between two unrelated sequences (i.e.,

- 84 048704 bcr-abl in the Philadelphia chromosome), or an idiotype from B-cell tumors.

Other tumor vaccines may include proteins from viruses associated with the development of human cancers, such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's sarcoma herpes virus (KHSV). Another form of tumor-specific antigen that may be used in combination with the fusion protein of the present invention is purified heat shock proteins (HSPs) isolated from the tumor tissue itself. These heat shock proteins contain fragments of proteins from tumor cells, and such HSPs are highly efficient in delivery to antigen-presenting cells to induce tumor immunity (Suot & Srivastava (1995) Science 269: 1585-1588; Tamura et al. (1997) Science 278: 117-120).

Dendritic cells (DCs) are highly efficient antigen-presenting cells that can be used to initiate antigen-specific responses. DCs can be generated ex vivo and loaded with a variety of protein and peptide antigens, as well as 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. DCs have also been fused directly to tumor cells for the purpose of immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As a vaccination method, DC immunization can be effectively combined with the IL2/IL2Ra fusion protein disclosed herein to activate more highly potent antitumor responses.

In some embodiments, the methods of treating cancer using the IL2/IL2Ra fusion protein disclosed herein are combined with standard cancer treatments (e.g., surgery, radiation, and chemotherapy). An example of such a combination is an anti-TIM3 antibody in combination with the IL2/IL2Ra fusion protein disclosed herein for the treatment of melanoma. The scientific rationale for the combined use of the IL2/IL2Ra fusion protein disclosed herein and chemotherapy is that cell death, which is a consequence of the cytotoxic effect of most chemotherapeutic compounds, should result in increased levels of tumor antigen in the antigen-presenting pathway. Other combination therapies that can synergize with the IL2/IL2Ra fusion protein disclosed herein through cell death include radiation, surgery, and endocrine shutdown. Each of these protocols creates a source of tumor antigen in the host. Angiogenesis inhibitors can also be combined in combination with the IL2/IL2Ra fusion protein disclosed herein. Inhibition of angiogenesis results in tumor cell death, which can serve as a source of tumor antigen in the host antigen-presenting pathways.

The IL2/IL2Ra fusion protein disclosed and described herein can also be used in combination with bispecific antibodies that target effector cells expressing the Fca or Fcy receptor 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, Fc receptor/tumor antigen (e.g., Her-2/neu) bispecific antibodies have been used to target macrophages to tumor sites. Such targeting can more effectively activate tumor-specific responses. Alternatively, antigen can be delivered directly to DCs using bispecific antibodies that bind to a tumor antigen and a cell surface marker that is specific for dendritic cells.

Tumors evade host immunosurveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating proteins expressed by tumors that are immunosuppressive. These include, among others, 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 to each of these molecules can be used in combination with the IL2/IL2Ra fusion protein disclosed herein to counteract the actions of immunosuppressants and stimulate antitumor immune responses generated by the host.

Other antibodies that activate host immunological reactivity may be used in combination with the IL2/IL2Ra fusion protein disclosed herein. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Antibodies to CD40 are able to effectively displace helper T cell activity (Ridge et al. (1998) Nature 393: 474-478) and may be used in combination with the IL2/IL2Ra fusion protein disclosed herein. Activation of antibodies to T cell costimulatory molecules such as CTLA-4 (e.g., U.S. Patent No. 5,811,097), OX-40 (Weinberg et al. (2000) Immunol 164: 21602169), 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. PD1 or PD-L1 inhibitors can also be used in combination with the IL2/IL2Ra fusion protein disclosed herein. Other combinations are described elsewhere herein.

- 85 048704

Bone marrow transplantation is currently used to treat various tumors of hematopoietic origin. Although this treatment results in graft-versus-host disease, graft-versus-tumor responses can provide a therapeutic effect. According to some embodiments, the IL2/IL2Ra fusion protein disclosed herein is used to enhance the efficacy of donor-grafted tumor-specific T cells.

There are also several experimental treatment protocols 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 against a tumor (Greenberg & Riddell (1999) Science 285: 546-51). These methods can also be used to activate T cell responses to infectious agents such as CMV. In some embodiments, ex vivo activation in the presence of the IL2/IL2Ra fusion protein disclosed herein can increase the frequency and activity of adoptively transferred T cells.

7.7.2.2 Inflammatory disease or autoimmune disease

According to some embodiments, the present invention relates to 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. According to some embodiments, the methods comprise administering to the subject a therapeutically effective amount of the IL2/IL2Ra fusion protein disclosed herein.

In some embodiments, the IL2/IL2Ra fusion protein disclosed herein is administered to patients having an inflammatory disease or an autoimmune disease that has had an inadequate response to or continues to progress after prior treatment. In some embodiments, the IL2/IL2Ra fusion protein disclosed herein is administered to patients who have not previously received (i.e., have not undergone) treatment for an inflammatory disease or an autoimmune disease.

In some embodiments, the IL2/IL2Ra fusion protein disclosed herein is administered with a standard of care for an inflammatory disease or an autoimmune disease. In some embodiments, the IL2/IL2Ra fusion protein disclosed herein is administered as a maintenance therapy for an inflammatory disease or an autoimmune disease, such as a therapy that is intended to prevent the onset or recurrence of inflammation.

In some embodiments, the IL2/IL2Ra fusion protein disclosed herein is administered as a monotherapy for treating an inflammatory disease or an autoimmune disease or as an immunostimulatory therapy alone for treating an inflammatory disease or an autoimmune disease. In some embodiments, the IL2/IL2Ra fusion protein disclosed herein is combined with a vaccination protocol for treating an inflammatory disease or an autoimmune disease. In some embodiments, the IL2/IL2Ra fusion protein disclosed herein is combined with an antibody used to treat an inflammatory disease or an autoimmune disease.

According to some embodiments, the inflammatory disease or autoimmune disease is selected from the group consisting of type 1 diabetes mellitus, multiple sclerosis, rheumatoid arthritis, celiac disease, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis or scleroderma, graft versus host disease, psoriasis, focal alopecia, HCV-induced vasculitis, Sjogren's syndrome, pemphigus, ankylosing spondylitis, Behcet's disease, Wegener's granulomatosis, Takayasu's syndrome, autoimmune hepatitis, sclerosing cholangitis, Gougerot-Sjogren syndrome and macrophage activation.

7.7.2.3 Infectious disease

The methods described herein may also be used to treat patients exposed to certain toxins or pathogens. Accordingly, in some embodiments, the present invention provides 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 methods comprise administering to the subject a therapeutically effective amount of the IL2/IL2Ra fusion protein disclosed herein to treat an infectious disease.

Similar to its use in tumors, as discussed above, methods involving treatment with the IL2/IL2Ra fusion protein disclosed herein are used alone or as adjuncts in combination with vaccines to stimulate immune responses to pathogens, toxins, and autoantigens. Examples of pathogens for which this therapeutic approach may be particularly useful include pathogens for which there is currently no effective vaccine.

- 86 048704 cins, or pathogens, against which conventional vaccines are not fully effective. These include, but are not limited to, HIV, hepatitis (A, B and C), influenza, herpes, giardia, malaria, leishmania, Staphylococcus aureus, Pseudomonas aeruginosa.

Some examples of pathogenic viruses that cause infections that can be treated with the methods described herein include HIV, hepatitis virus (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flaviviruses, 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 arbovirus encephalitis virus.

Some examples of pathogenic bacteria that cause infections that can be treated with the methods described herein include chlamydia, rickettsialpox, mycobacterium, staphylococcus, streptococcus, pneumococcus, meningococcus and gonococcus, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulism, anthrax, plague, leptospirosis and the bacterium that causes Lyme disease.

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

Some examples of pathogenic parasites that cause infections that can be treated with the methods described herein include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinri, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus brasiliensis.

In all of the above methods, treatment with the IL2/IL2Ra fusion protein disclosed herein can be combined with other forms of immunotherapy, such as those described herein, such as cytokine therapy (e.g., interferons, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy that provides improved presentation of tumor antigens (see, e.g., Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2: 1121-1123).

7.7.2.4 Vaccines

The IL2/IL2Ra fusion proteins disclosed herein can be used to stimulate antigen-specific immune responses by co-administering the IL2/IL2Ra fusion protein disclosed herein with an antigen of interest (e.g., a vaccine). Accordingly, the present invention provides methods for enhancing an immune response to an antigen in a subject, comprising administering to the subject: (i) the antigen; and (ii) the IL2/IL2Ra fusion protein disclosed herein so as to enhance an immune response to the antigen in the subject. The antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or a pathogen antigen. Non-limiting examples of such antigens include those discussed in the sections above, such as the tumor antigens (or tumor vaccines) discussed above, or antigens of viruses, bacteria, or other pathogens described above.

According to some embodiments, a peptide or fusion protein comprising an epitope to which the IL2/IL2Ra fusion protein disclosed herein binds is used as a vaccine in place of or in addition to the IL2/IL2Ra fusion protein disclosed herein.

Suitable routes of administration of the antibody compositions (e.g., human monoclonal antibodies, multispecific and bispecific molecules, and immunoconjugates) disclosed herein in vivo and in vitro are well known in the art and can be selected by those skilled in the art. For example, the antibody compositions can be administered by injection (e.g., intravenously or subcutaneously). Suitable dosages of the molecules used will depend on the age and weight of the subject, as well as the concentration and/or composition of the antibody composition.

7.7.2.5 Co-administration with a second agent

As described previously, the IL2/IL2Ra fusion protein disclosed herein can be co-administered with one or more other therapeutic agents, such as a cytotoxic agent, a radiotoxic agent, or an immunosuppressant. The IL2/IL2Ra fusion protein disclosed herein can be associated with the agent (as an immunocomplex), or it can be administered separately from the agent. In the latter case (separate administration), the IL2/IL2Ra fusion protein disclosed herein can be administered before, after, or simultaneously with the agent, or can be co-administered with other known therapeutic agents, such as an anti-cancer agent, such as radiation. Such therapeutic agents include, but are not limited to, anti-tumor

- 87 048704 left-handed agents such as doxorubicin (Adriamycin), cisplatin, bleomycin sulfate, carmustine, chlorambucil, dacarbazine, and cyclophosphamide-hydroxyurea, which are themselves effective only at levels that are toxic or subtoxic to the patient. Cisplatin is administered intravenously at a dose of 100 mg/ml every four weeks, and Adriamycin is administered intravenously at a dose of 60-75 mg/ml every 21 days.

Co-administration of the IL2/IL2Ra fusion protein disclosed herein with chemotherapeutic agents provides the presence of two anti-cancer agents that act through different mechanisms to produce a cytotoxic effect on human tumor cells. Such co-administration may overcome problems associated with the development of drug resistance or changes in the antigenicity of tumor cells that would render them inert to the antibody.

The present invention relates to methods of combination therapy in which the IL2/IL2Ra fusion protein disclosed herein is administered together with one or more additional agents (a second therapeutic agent), such as small molecule drugs, antibodies or antigen-binding portions thereof, that are effective in stimulating immune responses to further enhance, stimulate or positively regulate immune responses in a subject.

In general, the IL2/IL2Ra fusion protein disclosed herein may be combined with (i) an agonist of a stimulatory (e.g., co-stimulatory) molecule (e.g., a receptor or ligand) and/or (ii) an antagonist of an inhibitory signal or molecule (e.g., a receptor or ligand) on immune cells, such as T cells, which in both cases results in enhanced immune responses, such as antigen-specific T cell responses. In some aspects, the immuno-oncology agent is (i) an agonist of a stimulatory (including costimulatory) molecule (e.g., a receptor or ligand) or (ii) an antagonist of an inhibitory (including coinhibitory) molecule (e.g., a receptor or ligand) on cells, such as on cells that inhibit T cell activation or on cells that participate in innate immunity, such as NK cells, and wherein the immuno-oncology agent enhances innate immunity. Such immuno-oncology agents are often referred to as immune checkpoint regulators, such as immune checkpoint inhibitors or immune checkpoint stimulators.

According to some embodiments, the IL2/IL2Ra fusion protein disclosed herein is administered with an agent that targets a stimulatory or inhibitory molecule that is a member of the immunoglobulin superfamily (IgSF). For example, the IL2/IL2Ra fusion protein disclosed herein can be administered to a subject with an agent that targets a member of the IgSF family to enhance an immune response. For example, the IL2/IL2Ra fusion protein disclosed herein can be administered with an agent that targets (or specifically binds to) a member of the B7 family of membrane-associated ligands, which includes B71, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6, or a costimulatory or coinhibitory receptor or ligand that specifically binds to a member of the B7 family.

The IL2/IL2Ra fusion protein disclosed herein may also be administered with an agent that targets a member 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, EDA1, EDA2, TNFR1, lymphotoxin-a/TNFp, TNFR2, TNFa, LTpR, lymphotoxin 1β2, FAS, FASL, RELT, DR6, TROY and NGFR see, for example, Tansey (2009) Drug Discovery Today 00:1).

In some embodiments, the IL2/IL2Ra fusion protein disclosed herein is administered with an agent comprising an anti-PD-1 antibody. The anti-PD-1 antibody may be any antibody that binds PD-1 and inhibits the interaction of 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 is nivolumab. In some embodiments, the second therapeutic agent is pembrolizumab.

In some embodiments, the second therapeutic agent is an anti-PD-L1 antibody. The anti-PD-L1 antibody may be any antibody that binds PD-L1 and inhibits the interaction of 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 is atezolizumab. In some embodiments, the second therapeutic agent is durvalumab. In some embodiments, the second therapeutic agent is avelumab.

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In some embodiments, the second therapeutic agent is an anti-CTLA-4 antibody. The anti-CTLA-4 antibody may be any antibody that binds 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 is tremelimumab. In some embodiments, the second therapeutic agent is ipilimumab.

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

In some embodiments, the second therapeutic agent is an anti-CD137 antibody. The anti-CD137 antibody may 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 is urelumab.

In some embodiments, the second therapeutic agent is an antibody to KIR. The antibody to KIR may be any antibody that binds KIR and inhibits its activity. In some embodiments, the antibody to KIR is any antibody to KIR known in the art. In some embodiments, the second therapeutic agent is lirilumab.

In some embodiments, the second therapeutic agent is an anti-GITR antibody. The anti-GITR antibody may be any antibody that binds 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 is MK4166. In some embodiments, the second therapeutic agent is TRX518.

According to other embodiments, the second therapy comprises administering an antibody to TIM3. The antibody to TIM3 may be any antibody that binds TIM3 and inhibits its activity. According to some embodiments, the antibody to TIM3 is any antibody to TIM3 known in the art.

In some 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 some embodiments, the proteasome inhibitor is bortezomib.

According to some embodiments, the second therapy comprises radiation therapy. Any radiation therapy known in the art can be used as the second therapy.

In some embodiments, the second therapy comprises administering an agent that activates innate immune cells. In some embodiments, the agent that activates innate immune cells is an NLRP3 agonist. In some embodiments, the NLRP3 agonist is monosodium urate monohydrate (MSU) and/or alum, which act as a vaccine adjuvant. In some embodiments, the agent that activates innate immune cells is a Toll-like receptor 7 (TLR7) agonist. According to some embodiments, the TLR7 agonist is imiquimod (R837), GS9620 (see Tsai et al., J. Virology doi: 10.1128/JVI.02166-16 (Feb. 8, 2017)), ORN R-2336 (Miltenyl Biotec), or any combination thereof.

In some embodiments, the second therapy comprises administering an agent that enhances the survival of natural killer (NK) cells, CD8+ T cells, or both.

According to some embodiments, the second therapy comprises administering an agent selected from the group consisting of doxorubicin (ADRIAMYCIN®), cisplatin, carboplatin, bleomycin sulfate, carmustine, chlorambucil (LEUKERAN®), cyclophosphamide (CY

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TOXAN®; NEOSAR®), lenalidomide (NEOSAR®), bortezomib (VELCADE®), dexamethasone, mitoxantrone, etoposide, cytarabine, bendamustine (TREANDA®), rituximab (RITUXAN®), ifosfamide, vincristine (ONCOVIN®), fludarabine (FLUDARA®), thalidomide (THALOMID®), alemtuzumab (CAMPRATH®), ofatumumab (ARZERRA®), everolimus (AFINITOR®, ZORTRESS®), carfilzomib (KYPROLISTM), and any combination thereof.

Illustrative agents that modulate one of the proteins described above and that can be combined with the fusion protein described herein for the treatment of cancer include: YERVOY® (ipilimumab) or tremelimumab (anti-CTLA-4), galiximab (anti-B7.1), BMS-936558 (anti-PD-1), MK-3475 (anti-PD-1), atezolizumab (TECENTRIQ®), 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 antibody (Providence Health Services), huMAbOX40L (anti-OX40L), atacept (anti-TACI), CP-870893 (anti-CD40), lucatumumab (anti-CD40), dacetuzumab (anti-CD40), muromonab-CD3 (anti-CD3); MK4166 antibodies to GiTR, TRX518, Medi1873, INBRX-110, LK2-145, GWN-323, GITRL-Fc, or any combination thereof.

Other molecules that may be combined with the fusion proteins to treat a disease or disorder include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, the fusion proteins described herein may be combined with KIR antagonists (e.g., lirilumab).

T cell activation is also regulated by soluble cytokines, and the fusion proteins described herein can be administered to a subject, such as one with cancer, with cytokine antagonists that inhibit T cell activation or cytokine agonists that stimulate T cell activation.

According to some embodiments, the fusion proteins described herein can be used in combination with (i) antagonists (or inhibitors or blocking agents) of IgSF family or B7 family proteins 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 stimulatory receptors or cytokines that stimulate T cell activation, to stimulate an immune response, such as to treat proliferative diseases such as cancer.

Further combination therapy agents include agents that inhibit or reduce the number of macrophages or monocytes, including but not limited to CSF1R 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; WO 13169264; WO 14/036357).

The fusion protein of the present invention can also be administered with agents that inhibit TGF-β signaling.

Additional agents that may be combined with the fusion protein described herein include agents that enhance the presentation of tumor antigens, such as dendritic cell-based vaccines, GMCSF-secreting cell-based vaccines, CpG oligonucleotides, and imiquimod, or therapeutic agents that enhance the immunogenicity of tumor cells (e.g., anthracyclines).

Another therapeutic agent that can be combined in combination with the fusion protein described herein is a therapeutic agent that inhibits a metabolic enzyme such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthetase.

Another class of agents that can be used with the fusion protein described herein includes agents that inhibit adenosine formation, such as CD73 inhibitors, or inhibit the adenosine A2A receptor.

Other therapeutic agents that may be combined with the fusion protein described herein to treat a disease or disorder, such as cancer, include therapeutic agents that reverse/prevent T cell anergy or exhaustion, and therapeutic agents that trigger innate immune activation and/or inflammation at the tumor site.

Other therapeutic agents that may be combined with the fusion protein described herein to treat a disease or disorder, such as cancer, include therapeutic agents that block IL-8, such as with HuMax-IL8.

The fusion protein described herein may be combined with more than one immuno-oncology agent and may, for example, be combined with a combinatorial approach that specifically targets multiple elements of the immune pathway, such as one or more of the following: a therapeutic agent that enhances tumor antigen presentation (e.g., a dendritic cell-based vaccine, GM-secreting cell-based vaccines,

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CSF, CpG oligonucleotides, imiquimod); a therapeutic agent that inhibits negative immune regulation, such as by inhibiting the CTLA-4 and/or PD1/PD-L1/PD-L2 pathway and/or by reducing the number of or blocking Treg cells or other immunosuppressive cells; a therapeutic agent that stimulates positive immune regulation, such as with agonists that stimulate the CD-137, OX-40 and/or CD40 or GITR pathway and/or stimulate T cell effector function; a therapeutic agent that systemically increases the frequency of anti-tumor T cells; a therapeutic agent that reduces the number of or inhibits Treg cells, such as Treg cells in a tumor, such as using a CD25 antagonist (e.g. daclizumab) or by ex vivo depletion using CD25 antibody microcarriers; a therapeutic agent that interferes with the function of suppressor myeloid cells in a tumor; a therapeutic agent that enhances the immunogenicity of tumor cells (e.g., anthracyclines); adoptive transfer of T cells or NK cells, including genetically modified cells such as cells modified with chimeric antigen receptors (CAR-T therapy); a therapeutic agent that inhibits a metabolic enzyme such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthetase; a therapeutic agent that reverses/prevents T cell anergy or exhaustion; a therapeutic agent that triggers activation of innate immunity and/or inflammation at the site of a tumor; administration of immunostimulatory cytokines; or blockade of immunorepressor cytokines.

The fusion protein described herein can be administered in combination with one or more agonist agents that ligate positive costimulatory receptors, blocking agents that attenuate signaling through inhibitory receptors, antagonists and one or more agents that systemically increase the frequency of anti-tumor T cells, agents that offset various immunosuppressive pathways in the tumor microenvironment (e.g., block inhibitory receptor engagement (e.g., PD-L1/PD-1 interactions), reduce the number or inhibit Treg cells (e.g., with an anti-CD25 monoclonal antibody (e.g., daclizumab) or by ex vivo depletion with anti-CD25 antibody microcarriers), 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 tumor sites.

According to some embodiments, the fusion protein of the present invention is administered to a subject together with a BRAF inhibitor if the subject is positive for the BRAF V600 mutation.

For example, the fusion protein and combination therapies described herein can be used in combination (e.g., simultaneously or separately) with an additional treatment, such as radiation and/or chemotherapy (e.g., using camptothecin (CPT-11), 5-fluorouracil (5-FU), cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin, paclitaxel, carboplatin-paclitaxel (Taxol), doxorubicin, or camptothecin + apo21/TRAIL (6X combo)), one or more proteasome inhibitors (e.g., bortezomib or MG132), one or more Bcl-2 inhibitors (e.g., BH3I-2' (bcl-x1 inhibitor), indoleamine dioxygenase-1 inhibitor (e.g., INCB24360, indoximod, NLG-919 or F001287), AT-101 (R(-)-derivative of gossypol), 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, CD20 antibodies (e.g., rituximab), angiogenesis inhibitors (e.g., bevacizumab), antiangiogenic agents targeting VEGF and VEGFR (e.g., avastin), synthetic triterpenoids (see Hyer et al., Cancer Research 2005;65:4799-808), c-FLIP (cellular FLICE-inhibitory protein) modulators (e.g., natural and synthetic PPARy (peroxisome proliferator-activated receptor-γ) ligands, 5809354 or 5569100), kinase inhibitors (eg, sorafenib), trastuzumab, cetuximab, temsirolimus, mTOR inhibitors such as rapamycin and temsirolimus, bortezomib, JAK2 inhibitors, HSP90 inhibitors, PI3K-AKT inhibitors, lenalildomide, GSK3P inhibitors, IAP inhibitors, and/or genotoxic drugs.

The fusion protein of the present invention and combination therapies described herein may further be used in combination with one or more antiproliferative cytotoxic agents. Classes of compounds that may be used as antiproliferative cytotoxic agents include, but are not limited to, the following classes.

Alkylating agents (including but not limited to nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): uramustine, chlormethine, cyclophosphamide (CYTOXAN®), melphalan, chlorambucil, pipropromane, triethylenemelamine, triethylenethiophosphoramine,

- 91 048704 busulfan, carmustine, lomustine, streptozocin, dacarbazine and temozolomide.

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

Suitable antiproliferative agents for combination with the fusion protein of the present invention include, but are not limited to, taxanes, paclitaxel (paclitaxel is commercially available under the name TAXOL™), docetaxel, discodermolide (DDM), dictyostatin (DCT), peloruside A, epothilones, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, furnoepothilone D, deoxyepothilone B1, [17]-dehydrodeoxyepothilone B, [18]-dehydrodeoxyepothilones B, C12,13-cyclopropylepothilone A, C6-C8-bridged epothilone A, trans-9,10-dehydroepothilone D, cis9,10-dehydroepothilone D, 16-desmethylepothilone B, epothilone BIO, discoderomolide, patupilone (EPO-906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA296A (discoderomolide), TZT-1027 (soblidotin), ILX-651 (tasidotin hydrochloride), halichondrin B, eribulin mesylate (E-7389), hemiasterlin (HTI-286), E-7974, cirptophycins, LY-355703, maytansinoid immunoconjugates (DM-1), MKC-1, ABT-751, TI-38067, T900607, SB-715992 (ispinesib), SB-743921, MK-0731, STA-5312, eleutherobin, 17-beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol, cyclostreptin, isolaulimalide, laulimalide, 4-epi-7-dehydroxy-14,16-didemethyl-(+)-discodermolide and cryptotilone 1, in addition to other microtubulin stabilizing agents that are known in the art.

In cases where it is desirable to render aberrantly proliferating cells inactive in combination with or prior to treatment with the fusion protein of the present invention described herein, hormones and steroids (including synthetic analogs) such as 17a-ethinyl estradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, ZOLADEX® can also be administered to the patient. When using the methods or compositions described herein, other agents used in modulating tumor growth or metastasis in clinical settings, such as antimimetics, may be administered if necessary.

According to some embodiments, the combination of the fusion protein of the present invention and the second agent contemplated herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier or simultaneously as separate compositions with the fusion protein of the present invention and the second agent in a pharmaceutically acceptable carrier. According to some embodiments, the combination of the fusion protein of the present invention and the second agent can be administered sequentially. The administration of the two agents may be started at different times, such as 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 one or more weeks apart, or the administration of the second agent may be started, such as 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 one or more weeks after the first agent was administered.

In some embodiments, an anti-tumor antibody that can be combined with a fusion protein of the present invention and/or a second agent includes RITUXAN® (rituximab), HERCEPTIN® (transtuzumab), BEXXAR® (tositumomab), ZEVALIN® (ibritumomab), CAMPARTH® (alemtuzumab), LYMPHOCIDE® (eprtuzumab), AVASTIN® (bevacizumab), and TARCEVA® (erlotinib), or any combination thereof. In some embodiments, a second antibody useful in combination therapy with a fusion protein of the present invention can be an antibody-drug conjugate.

According to some embodiments, the fusion protein of the present invention, alone or in combination with another agent, is administered simultaneously or sequentially with bone marrow transplantation to treat various tumors of hematopoietic origin.

The present invention provides methods for altering an adverse event associated with the treatment of a hyperproliferative disease (e.g., cancer) with an immunostimulatory agent comprising administering to a subject a fusion protein of the present invention, with or without a second agent. For example, the methods described herein provide a method for reducing the incidence of immunostimulatory therapeutic antibody-induced colitis or diarrhea by administering to the patient a non-absorbable steroid. As used herein, the term non-absorbable steroid refers to a glucocorticoid that is characterized by extensive first-pass metabolism such that, following metabolism in the liver, the bioavailability of the steroid is low, i.e., less than about 20%. According to some embodiments described herein, the non-absorbable steroid is budesonide. Budesonide is a locally acting glucocorticosteroid that is extensively metabolized, primarily by the

- 92 048704 following oral administration. ENTOCORT EC® (Astra-Zeneca) is a pH and time dependent oral formulation of budesonide designed to optimize drug delivery to the ileum and along the entire colon. ENTOCORT EC® is approved in the United States for the treatment of mild to moderate Crohn's disease involving the ileum and/or ascending colon. According to some embodiments, the fusion protein of the present invention in combination with a non-absorbable steroid can be further combined with a salicylate. Salicylates include 5-ASA agents such as, for example: sulfasalazine (AZULFIDINE®, Pharmacia & Up John); olsazaline (DJPENTUM®, Pharmacia & Up John); balsalazide (COLAZAL®, Salix Pharmaceuticals, Inc.); and mesalamine (ASACOL®, Procter & Gamble Pharmaceuticals; PENTASA®, Shire, USA; CANASA®, Axcan Scandipharm, Inc.; ROWASA®, Solvay).

7.8 Sets

In the context of the present document, a kit comprises an IL2/IL2Ra fusion protein for use in modulating an immune response, as described elsewhere herein. The terms kit and system as used herein are intended to refer to at least one or more IL2/IL2Ra fusion proteins, which, according to particular embodiments, are presented in combination with one or more elements or components of another type (e.g., biochemical reagents, containers, other types of packaging, such as packaging material intended for commercial sale, instructions for use, and the like).

According to some embodiments, the present invention relates to a kit comprising (a) one or more IL2/IL2Ra fusion proteins as described herein, a composition comprising an IL2/IL2Ra fusion protein as described herein, a nucleic acid encoding an IL2/IL2Ra fusion protein as described herein, a vector, and/or a host cell; and (b) and instructions for administering the fusion protein to a subject in need thereof. According to some embodiments, the present invention relates to a kit comprising (a) an IL2/IL2Ra fusion protein as described herein, and (b) instructions for administering the fusion protein to a subject in need thereof. In some embodiments, the present invention provides a kit comprising (a) a composition comprising an IL2/IL2Ra fusion protein as described herein, and (b) instructions for administering the composition to a subject in need thereof. In some embodiments, the present invention provides a kit comprising (a) a nucleic acid encoding an IL2/IL2Ra fusion protein as described herein, and (b) instructions for administering the nucleic acid to a subject in need thereof. In some embodiments, the present invention provides a kit comprising (a) a vector as described herein, and (b) instructions for administering the vector to a subject in need thereof. In some embodiments, the present invention provides a kit comprising (a) a host cell as described herein, and (b) instructions for administering the host cell to a subject in need thereof.

According to a particular embodiment, the present invention relates to a pharmaceutical package or kit that comprises one or more containers filled with one or more ingredients of the pharmaceutical compositions described herein, such as one or more fusion proteins provided herein. According to some embodiments, the kits comprise a pharmaceutical composition described herein and any prophylactic or therapeutic agent, such as those described herein. According to certain embodiments, the kits can comprise a T-cell mitogen, such as, for example, phytohemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or an antibody that stimulates formation of a complex with a TCR, such as an anti-CD3 antibody and an anti-CD28 antibody. Such one or more containers may optionally be associated with a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products, such notice indicating approval by the agency of manufacture, use, or sale for administration to humans.

Also provided herein are kits that can be used in accordance with the methods described above. According to one embodiment, a kit comprises a fusion protein, preferably a purified fusion protein, as described herein in one or more containers. According to a specific embodiment, the kits described herein comprise a substantially isolated fusion protein as a control. According to another specific embodiment, the kits described herein further comprise a control antibody or a control fusion protein that does not react with an IL2 and/or IL2-Ra antigen. According to another specific embodiment, the kits described herein comprise one or more

- 93 048704 for detecting the binding of a fusion protein to an IL2 and/or IL2-Ra antigen (e.g., the fusion protein can be conjugated to a detectable substrate, such as 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 the detectable substrate). According to specific embodiments, the kit provided herein can include a recombinantly produced or chemically synthesized fusion protein. An antigen for the fusion protein disclosed herein, which is provided in the kit, can also be attached to the solid support. According to a more specific embodiment, the detecting means of the kit described above includes a solid support to which the fusion protein antigen is attached. Such a kit may also include an unattached, reporter-labeled antibody to human antigens or antibodies to mouse/rat antigens. According to this embodiment, binding of the fusion protein to the antigen can be detected by binding of said reporter-labeled antibody.

The practice of the present invention will employ, unless otherwise indicated, 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 explained in the literature. See, for example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. Patent 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; the treatise, Methods In Enzymology (Academic Press, Inc., NY); 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, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986); Crooks, Antisense drug technology: Principles, strategies and applications, 2 ^nd Ed. CRC Press (2007), and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

All references mentioned above, as well as all references and amino acid or nucleotide sequences (e.g., GenBank accession numbers and/or Uniprot accession numbers) mentioned herein, are hereby incorporated by reference in their entirety.

The examples below are provided for illustrative purposes and not as limitations.

8. Examples

Example 1

The IL2-CD25 fusion protein forms a stable homogeneous homodimer.

The size and oligomeric state of the fusion proteins were studied by size exclusion chromatography coupled to an integrated multi-angle light scattering detector (SEC-MALS). Samples were prepared by injecting 30 μg of the starting protein sample. Isocratic separation was performed on a GE Healthcare Superdex 200 Increase 10/300 GL column (10 mm x 300 mm) coupled to a Prominence Shimadzu UFLC system consisting of a degasser, isocratic pump, chilled sample holder with injector, UV/VIS detector, and column oven in a buffer containing 40 mM Tris, 200 mM NaCl (pH 7.5) with 0.02% sodium azide added and filtered through a 0.1 μm filter at a flow rate of 0.75 ml/min. Samples were injected onto the column using a Shimadzu autosampler, and data were acquired from three online detectors connected in series: a Shimadzu SPD-20 dual-wavelength UV/Vis spectrophotometer set to collect data at 280 nm, followed by a Wyatt Technologies mini-Dawn TREOS three-angle laser light scattering detector, and a Wyatt T-rEX Optilab interferometric refractometer. Data were collected and analyzed using Astra 6 (Wyatt) and Lab Solutions Lite (Shimadzu) software.

The data presented in Fig. 1 show a typical absolute mass versus elution time profile obtained from analytical size exclusion chromatography. The sample analyzed in Fig. 1 is IL2-CD25(22-212) (SEQ ID NO: 16) fused to a His-tag (GGHHHHHH) (SEQ ID NO: 100), which has a theoretical molecular weight of 37,812 (reduced) for the polypeptide chain. The data show that IL2-CD25(22-212) forms a homogeneous form throughout the elution profile with a measured absolute mass of 93 kDa. No higher order monomeric or oligomeric forms than the main peak were observed in the elution peak. The 93 kDa mass value indicated that the molecule forms a homodimer and is also glycosylated (at approximately 18 wt% from known N- and O-linked glycosylation sites on IL2 and CD25).

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Example 2.

IL2-CD25 fusion proteins bind equivalently to sCD25 and sIL2Rp/IL2RY heteroreceptors with reduced apparent affinity

Surface plasmon resonance (SPR) studies were performed on a Biacore T100 and/or T200 instrument (GE Healthcare) at 25°C. Binding of the fusion protein analytes was tested in phosphate-buffered saline (PBS-T) (pH 7.1) onto surfaces consisting of (~300RU) low-density biot-hCD25-BioPTVMV-His, from which His (hCD25) had been cleaved and captured on a streptavidin-based, i.e., SA, a sensor chip, or from a heterodimeric Fc-site of the hCD122(27241)-hFc-D/hCD132(23-263)-hFc-K fusion (hIL2-Rb/g) that was captured by protein A immobilized on the surface of the CM5 sensor chip using standard ethyl(dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (NHS) chemistry with ethanolamine blocking. Protein analytes were injected in serial titers and regenerated to baseline by 2x8 injection of low pH buffer. Data were analyzed using Biacore T-200 Evaluation software.

Figure 2 shows the prototypical binding of the IL2-CD25 fusion protein (SEQ ID NO: 16) to human sCD25. The observed apparent equilibrium dissociation constant of 4.2 micromolar was approximately 150-fold weaker than the affinity of isolated IL2 for sCD25 measured by surface plasmon resonance (Liparoto, S. F., Myszka, D. G, Wu, Z., Goldstein, B., Laue, T. M., and Ciardelli, T. L. (2002) Biochemistry 41, 2543-51.).

Table 9

Pharmacokinetics of binding of IL2-CD25 fusion protein (SEQ ID NO: 16) to human sCD25

ka (1/Ms) kd (1/s) KD (M) 1.29E+4 0,054 4.2E-6

Table 10 below shows the observed equilibrium Kd values for the prototype short and long versions of IL2-CD25 to human sCD25 and the sIL2Rp/IL2RY heterodimer. The apparent equilibrium dissociation constants observed for the various IL2-CD25 fusion proteins were markedly weaker compared to IL2. Binding to sCD25 was approximately 100-fold weaker for the IL2-CD25 fusion molecules compared to IL2. Similarly, binding of the IL2-CD25 fusion proteins was approximately 150-fold weaker to the beta-gamma heterodimer than to IL2.

Table 10

Observed equilibrium KD values for binding of IL2 and IL2-CD25 constructs to sCD25 or the sIL2Rp/IL2Ry heterodimer as measured by _______________surface plasmon resonance_______________

Receptor K _D c sCD25, mkM K _D with sIL2R3/IL2Ry heterodimer, mkM IL2 0,025 0,0004 IL2-CD25(22-187) 1,6 0,054 IL2-CD25(22-212) 2,3 0,065 IL2-CV-CD25(22-240) 2,1 0,063 IL2-CD25(22-240) 2,4 0,044 Fcl. l-71inker-IL2-CD25(22-212), divalent, SEQ ID NO:67 5,6 ND IL2-CD25(22-212)-Fcl.l SEQ ID NO:68 2,5 0,1 IL2-T3A-CD25(22-187)-N89Q-T95A-T106A (SEQ ID NO:202) 1,9 ND.

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Fc- (IL2(V91 K) CD2 5)2, divalent SEQ ID NO:203 3.0 HS A-(G4S)3 -IL2-CD25(22-187) SEQ GO NO: 19 4.0 0,03 IL2(C 145 A)-CD25(22-212)-GG-HS A SEQ ID NO:20 2,1 0,03 Human Fc 1.1 (f)-AZ 1 -IL2(C 145 S)CD25(22-187)-G SEQ ID NO:26 3.0 0,15 IL2(C 145S)-CD25(22-187)- HuFcl. 1(f)-AZI SEQ ID NO:27 2.0 0,15 IL2(V111K)-CD25 SEQ ID NO:69 1,9 IL2(D40T)-CD25 SEQ ID NO:70 2.8

Example 3.

Shortened versions of IL2-CD25 have improved stability against aggregation; accelerated stability testing

The stability of the fusion proteins was tested by incubation at 40°C in buffers with a pH range of 4-8. The fusion proteins were concentrated to approximately 15 mg/ml and dialyzed at 4°C into the following buffers: 1) 20 mM acetate, 250 mM sucrose, pH 4, 2) 20 mM citrate, 250 mM sucrose, pH 5, 3) 20 mM histidine, 250 mM sucrose, pH 6, 4) 20 mM phosphate, 250 mM sucrose, pH 7 and 5) 20 mM Tris, 250 mM sucrose, pH 8.4 (room temperature). Following isolation, the resulting fusion protein concentration was normalized to 10 mg/mL by dilution with dialysis buffer and incubated at 40°C for four weeks, with aliquots taken immediately before incubation (t0), one week (1w), and four weeks (4w) after the start of incubation at 40°C. An aliquot of each time point was analyzed by size exclusion chromatography on a Shodex KW403-4F column connected to an Agilent 1260 HPLC system in a buffer containing 100 mM sodium phosphate, 150 mM sodium chloride, pH 7.3 (filtered through a 0.2 μm filter), carried out at a flow rate of 0.30 mL/min.

As shown in Tables 11 and 12, the truncated versions of IL2-CD25 had significantly better high-molecular weight and low-molecular weight profiles than the longer versions after accelerated stability studies. This was particularly true for pH values of 4, 5, 6, and 7. The samples were incubated at different pH conditions for four weeks and forty degrees Celsius. The results, presented as percentages of the high-molecular weight and low-molecular weight fractions of each construct, indicated significantly improved stability for the short constructs compared to the long ones. The results, presented as percentages of the major peak fractions of each construct, indicated significantly improved stability for the short constructs compared to the long ones.

Table 11

Stability profiles from accelerated testing of IL2-CD25 fusion proteins

IL2-CD25(22-187) IL2-CD25(22-212) IL2-C145V- CD25(22-240) IL2-CD25(22- 240) pH HMW (%) LMW (%) HMW (%) LMW (%) HMW (%) LMW (%) HMW (%) LMW (%) 4 2,3 6.3 5.3 3,4 19.4 5.7 13,0 13.4 5 1.9 3.9 1,8 1,9 12.5 0,3 14.3 2.6 6 1,3 2,2 1,9 1,2 13.3 0,0 15.9 o,o 7 5.1 3.8 2.6 2,5 9.4 2,3 10,5 2,5 8 5.5 4.2 2.6 2.7 6.2 2,3 7,8 2.7

The IL2-CD25 fusion proteins of the present invention contain a His tag (GGHHHHHH, SEQ ID NO: 100) to facilitate purification.

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Table 12

Stability profiles from accelerated testing of IL2-CD25 fusion proteins

pH IL2-CD25(22-187) IL2-CD25(22- 212) IL2-C145V- CD25(22-240) IL2-CD25(22- 240) 4 91.4 91.3 74.9 73.6 5 94.2 96.4 87.2 83.1 6 96.6 97,0 86.7 84.1 7 91.1 94.9 88.3 87,0 8 90.3 94.7 91.6 89.5

Example 4.

Pharmacokinetic studies in non-human animals.

Pharmacokinetic studies: All animal protocols were approved by the New Jersey Central 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, MA). Fed Balb/C mice were administered a single 0.5 mg/kg dose of the indicated molecule either by the intravenous (IV) route via the tail vein or by the subcutaneous (SC) route. IL2(21-153)-(G3S)3-CD25(22-240) used in the pharmacokinetic studies carried a His6 tag. Post-dose, tail vein blood was collected at the following time points: 5 min (IV only), 1 h, 7 h, 24 h, 48 h, 72 h, 96 h, and 168 h. For the monkey PK study, male cynomolgus macaques were purchased from Buckshire Corporation (Perkasie, PA). Monkeys (N=3, mean body weight 7.8 kg) were injected with a single 0.075 mg/kg subcutaneous dose of the hIL2-CD25(22-212) construct. Conscious monkeys restrained in a chair had serial femoral artery blood samples collected at 5 h, 24 h, 48 h, 72 h, 96 h, 168 h, 192 h, and 240 h post-dose. Blood samples were allowed to clot and centrifuged at 4°C (1500–2000 x g) to obtain serum. Serum samples were stored at -80°C until further analysis.

Detection of fusion protein in serum: A ligand binding assay was used to detect fusion protein levels in mouse or monkey serum samples. Briefly, Nunc MaxdiSorp 96-well flat-bottomed plates (Thermo Fisher Scientific, Denmark) were coated with capture reagent (rat anti-human IL2, monoclonal clone: MQ1-17H12; Thermo Fisher Scientific, San Diego, CA) at 2 μg/mL in PBS overnight at 4°C. Plates were blocked with blocking buffer (5% BSA in PBS) and incubated at 25°C for one hour. Standards, quality controls (QCs), and samples were diluted 20-fold in assay buffer (PBS with 1% BSA and 0.05% Tween 20), added to washed plates in duplicate, and incubated at 4°C overnight. Washed plates were incubated with detection reagent (human biotinylated anti-CD25 antibody, R&D Systems, Minneapolis, MN, at 250 ng/mL in assay buffer) and incubated for 2 h at 25°C. Washed plates were sequentially loaded with 100 ng/mL neutravidin-HRP complex (Thermo Scientific, Rockford, IL) in assay buffer at 25°C for 45 min, followed by chemiluminescent substrate mix (Thermo Scientific, Rockford, IL) for 1 min before reading the plates on a SpectraMax plate reader in luminescence mode. The analyte concentration of the serum samples was calculated using a standard curve generated using the Log-Log linear curve fitting algorithm of the Softmax analysis software (Molecular Devices).

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Table 13

Pharmacokinetic parameters of the indicated molecules in Balb/C mice

Dose Path CL Vss AUC (θafter DAY time point) Old X Tta X T1/2 Bioavailability mg/kg ml/h/kg ml/k g ηΜΝ nM h h % IL2(21-153)(G3S)3-CD25(22240) 0,5 IV 1,3 46.3 8046,3 NA NA 19.4 NA 0,5 sc NA NA 3639,6 79 24 31 45% IL2(21-153)(G3S)3-CD25(22212) 0,5 IV 4 94.2 3053,1 NA NA 19.4 NA 0,5 sc NA NA 2033,1 54.6 7 23 67% IL2(21-153)(G3S)3-CD25(22187) 0,5 IV 23.1 200.4 610.4 NA NA 11.6 NA 0,5 sc NA NA 261.3 13.2 7 10.9 43% Fcl. 1GSGGSGGIL2(21-153)(G3S)3-CD25(22212) 0,5 IV 1.96 167.6 1442 NA NA 66.6 NA 0,5 sc NA NA 886,5 12.02 24 68.8 61% IL2(21-153)(G3S)3-CD25(22212)-Fcl. 1 0,5 IV 1,2 194.4 1771,3 NA NA 120 NA 0,5 sc NA NA 1478,6 16.2 24 61.3 83%

NA = not applicable, ND = not determined.

Table 14

Pharmacokinetic parameters of hIL2-CD25(22-212) in cynomolgus monkeys

Dose Path AUC (0last time) Stakh Ttah T1/2 mg/kg nM*h nM h day IL2(21-153)(G3S)3-CD25(22212) 0,075 SC 1200 7.6 24 2,1

Example 5.

Activity of IL2-CD25 fusion protein on Kit225 cells.

The biological activity of the modified fusion proteins was determined by measuring the ability to stimulate IL2R, which is endogenously expressed on the Kit225 T-cell line (an IL2-dependent human T-cell line from a patient with T-cell chronic lymphocytic leukemia with an OKT3+, -T4+, -T8 phenotype). The activity was measured using a reporter system sensitive to the IL2R signaling cascade. Human Kit225 T cells with a stably integrated firefly luciferase reporter gene under the control of an IFNY-activating sequence (IRF1GAS-Luc) were grown in a medium containing RPMI + glutamax, 10% heat-inactivated FBS, 20 ng / ml recombinant IL2 (Invitrogen PHC0023), 1% penicillin / streptavidin mixture (Pen / Strep) and 0.7

- 98 048704 mg/ml geneticin. The day before the assay, reporter cells were washed and resuspended in assay medium (RPMI without phenol red + L-glutamine, 10% heat-inactivated FBS, 1% Pen/Strep) to remove IL2 present in the growth medium, then incubated overnight at 37°C. On the day of the assay, IL2 molecules were serially diluted in assay buffer to obtain a 3-fold 11-point concentration-dependence curve, IL2 molecules were added at various concentrations to the CulturPlate-384 assay plate (Perkin Elmer, cat. No. 6007688) followed by the addition of 70,000 cells/well. Assay plates were incubated for 5 hours in a 37°C _CO2 incubator, then equilibrated to room temperature for 20 minutes before addition of ONE-GLO™ substrate (Promega E6120). Plates were sealed and fluorescent signals were measured on a Perkin Elmer Envision. Eleven-point IL2 dose-response fluorescent signals were plotted using GraphPad Priam. The _EC50 was defined as the concentration of IL2 fusion proteins tested that corresponded to 50% activation and was obtained from the 11-point fitted curve, which was generated by a four-parameter logistic regression model. The potency of the fusion proteins on IL2R signaling in primary cells was determined by stimulating either human peripheral blood mononuclear cells (PBMCs) or human whole blood with the fusion proteins. Tyrosine phosphorylation of pSTAT5 is a direct consequence of IL2R signaling and was detected in various cell populations of whole blood or PBMCs by flow cytometry. Blood or PBMC samples were treated with serial dilutions of ML2-CD25 fusion proteins for 15 min at 37°C. Following incubation, cells were fixed and blood was lysed with Lyse/Fix buffer for 10 min (BD Phosflow). Cells were washed twice and then permeabilized with ice-cold methanol on ice for 30 min and washed twice more. Samples were then treated with Human Fc Block (Ebioscience) followed by staining with labeled antibodies to CD3, CD4, CD8, CD25, pSTAT5, Foxp3, and CD56. All samples were then analyzed by flow cytometry.

In in vitro cell-based assays, the fusion protein exhibited a potency for IL2 receptor activation that was significantly shifted to the right (towards lower potency) relative to wild-type (wt) IL2. This was consistent with the reduced apparent affinity of the fusion protein in the non-covalent dimeric form. Addition of an Fc tail to the fusion protein did result in an additional shift toward 20- to 30-fold lower potency. Removal of the His tag or truncation of the C terminus had minimal effects on the potency of the fusion proteins. Similar results for relative potency were obtained with whole blood.

Table 15A

Activity of IL2-CD25 fusion protein on Kit225 cells

Fused protein ESeo (ng/ml) IL2 WT 0.41 N=9 hIL2-CD25 (22-240)-His 138 n=2 hIL2-C145V-CD25 (22-240)-His 166 n=2 hIL2-CD25(22-212)-His 210 n=2 hIL2-CD25(22-212) 139 n=12 hIL2-CD25(22-187)-His 145 n=2 hIL2-CD25(22-187) 49 n=2 cl. 1 -71inker-IL2-CD25(22-212 (bivalent) 3414 n=2 )25(22-212)-Fcl. 1 (bivalent) 3413 n=2

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Table 15B

Activity of IL2-CD25 fusion protein on Kit225 cells

Protein SEQ Efficiency Ymax (%) EC50, ng/ml STDEV η % response IL2 wt wt-hIL2 0.42 0.23 9 N/A hIL2-CD25(22-212) 16 139 112 12 103% HSA-hIL2-CD25(22187) 19 972 856 4 104% hIL2-CD25(22-187)- HSA 20 205 78 4 95% Fc-hIL2-CD25(22187) 26 327 90 3 91% hIL2-CD25(22-187)- Fc 27 257 95 3 93% Fc-(hIL2-CD25(22187))2, divalent 67 737 264 4 97% hIL2-CD25 (aglycosylated at four residues) 202 39.4 3.1 3 85%

The selectivity of hIL2-CD25 proteins, including hIL2-CD25(22-212) (SEQ ID NO: 16) signaling for Tregs compared to other cell types, can be measured by monitoring STAT5 phosphorylation after 15 min in a mixed cell population including PBMCs or whole blood (Figs. 9A and 9B, respectively). Freshly collected blood samples were treated with serial dilutions of hIL2-CD25(22-212) (SEQ ID NO: 16) for 15 min at 37°C. Following incubation, cells were processed (fixed and permeabilized), stained with labeled antibodies to CD3, CD4, CD8, CD25, pSTAT5, Foxp3, and CD56, and analyzed by flow cytometry. Tregs are highly responsive to IL2R stimulation by hIL2-CD25(22-212) (SEQ ID NO: 16), with a maximal efficiency of 90% of Tregs responding to stimulation with pSTAT5 activation and a potency of 7 +/- 11 ng/ml. In contrast to the robust activation observed in Tregs, CD4 non-Treg cells (foxp3-), CD8, and NK cells were less efficiently stimulated by hIL2-CD25(22-212) (SEQ ID NO: 16); less than 50% of these cells activated pSTAT5 production even at concentrations up to 2700 ng/ml on average for all other cell types (Table 16). Overall, IL2R selectivity for Tregs over other cell types was maintained or increased in hIL2-CD25(22212) (SEQ ID NO: 16) compared to rhIL2. IL2-CD25 Fc or HSA fusion proteins retained high selectivity for Tregs in whole blood, albeit with lower potency (Table 16).

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Table 16

Efficacy and effectiveness of stimulation of different cell types in human whole blood

Treg CD4 foxp3Other than Treg CD 8 T cells NK cells % pSTAT5 positive cells EC50 ng/ml Maximum at the highest tested Maximum at the highest concentration tested Maximum at the highest concentration tested Maximum at the highest concentration tested hIL2-CD25(22- 212) (SEQ 16) 7.2 (+ 10.4, n=10) 90% (+/-8, n=10) 48% (+/-14, n=10) at 2.7 μg/ml 27% (+/-11, n=10) at 2.7 μg/ml 22% (+/-19, n=8) at 2.7 µg/ml Fc-(IL2-CD25) ₂ (SEQ 67), divalent 88 (±74, n=4) 95% (+/-2, n=4) 50% (p-3) in quantity 24 µg/ml 33% (p-3) in the amount of 24 μg/ml 8% (n-2) in a quantity of 24 μg/ml Fc-(IL2-CD25) (SEQ 26), monovalent 25 (± 12, n=3) 94% (+/-1,n=3) 60% (n=3) in quantity 8.1 µg/ml 36% (n=3) in quantity 8.1 µg/ml 6% (n=3) in a quantity of 8.1 μg/ml HSA-(G4S)3- IL2-CD25-FT (SEQ 19) 83 (±53, n=3) 95% (+/-3, n=3) 59% (n=2) in quantity 24 µg/ml 38% (n=2) in quantity 24 µg/ml 6% (n=2) at 24 µg/ml IL2-CD25 FT- GG-HSA (SEQ 20) 43 (±35, n=2) 92% (+/-5, n=2) 60% (n=2) in quantity 8.1 µg/ml 39% (n=2) in quantity 8.1 µg/ml 10% (n=2) in quantity 8.1 µg/ml (IL2-CD25)-Fc (SEQ 27), monovalent 11.4 (± 1.6, n=2) 94% (+/-5, n=2) 67% n=2 in quantity 8.1 µg/ml 49% n=2 in quantity 8.1 µg/ml 25% n=2 in quantity 8.1 µg/ml

Data are presented as means of 2-10 donors on freshly collected blood from normal donors. The maximum % of pSTAT5 positive cells was determined at concentrations ~200-400 times higher than the potency for Tregs.

Example 6.

In vivo activity in increasing leukocyte counts when treated with truncated IL2-CD25 fusion proteins.

The ability of the fusion proteins to expand white blood cell counts in vivo was tested in mice with humanized immune systems. Sixteen- to 20-week-old female NSG-huCD34 (NOD.Cg-Prkdc ^sCid IL2rg ^tmlWj1 /SzJ, in which human CD34+ hematopoietic stem cells have been reconstituted) engrafted mice were purchased from Jackson Laboratory. Fourteen weeks after engraftment, each humanized mouse was analyzed for the presence of hCD45+ cells and murine CD45+ cells (mCD45+) in peripheral blood by flow cytometry. Mice with at least 25% human CD45 engraftment were selected. The engrafted human hematopoietic stem cells were tested negative for HIV, HBV, HCV, and LCMV (lymphocytic choriomeningitis virus).

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Claims (25)

Animals were fed standard rodent chow and purified water ad libitum and acclimated for at least 1 week prior to use. All animal procedures were reviewed and approved by the BMS Animal Care and Use Committee. NSG-huCD34-transplanted mice aged 16–20 weeks were injected every third day with either PBS or the huIL2-CD25(22-187), huIL2-CD25(22-212), and HuIL2-CD25(22-240) fusion protein at 10 μg/mouse in 200 μl subcutaneously while the animals were anesthetized. A total of three subcutaneous doses were administered: on days 0, 3, and 6. Twenty-four hours after the last dose, all treated groups were anesthetized and spleens were collected in HBSS for FACS analysis of Tregs, CD8 T cells, or NK cells. All three fusion proteins showed similar ability to expand spleen cells. Example 7. Stability of the peptide linker (G3S) 3 in IL2(21-153)-(G3S)3-CD25(22-212) in human or mouse serum. A liquid chromatography tandem mass spectrometry (LC-MS/MS)-based bioanalytical method was developed to confirm the stability results of the (G3S)3 linker obtained from the in vitro serum stability studies and the in vivo PK study in monkeys. Following capture of IL2(21-153)-(G3S)3-CD25(22-212) (i.e., SEQ ID NO: 16) with rat anti-IL-2 antibody, the peak area ratio of the two representative peptides, i.e., DLISNINVIVLELK (from the IL-domain of IL2(21-153)-(G3S)3-CD25(22-212)) and EPPPWENEATER (from the CD25 domain of IL2(21-153)-(G3S)3-CD25(22212)) were used to assess the stability of the linker between IL-2 and CD25. Cleavage of the linker and the resulting circulating serum cleaved product would result in a systematic increase in the IL2/CD25 area ratio >1. To test the cleavability of the linker of IL2(21-153)-(G3S)3-CD25(22-212) in vitro, 0.5 μM IL2(21-153)-(G3S)3-CD25(22-212) was incubated in human serum (Bioreclamation, Cat.# HMSRM-M; Lot# BRH1332647) or mouse serum (Bioreclamation, Cat.# MSESRM-BALB-M; Lot# MSE264349) in a total volume of 1.5 mL for up to 72 hours at 3°C. Samples (200 μl serum) were collected at 0, 4, 24, 48 and 72 h and stored at -80°C until peak area ratio analysis by LC-MS/MS. To detect the presence of circulating cleavage product resulting from in vivo linker cleavage following a single dose of IL2(21-153)-(G3S)3-CD25(22-212) in monkeys, serum samples collected at 5, 24, 48, 72, 96 and 168 h post-dose were analyzed for peak area ratio by LC-MS/MS. As shown in Fig. 10, the peak area ratio of the characteristic peptides in the IL2 and CD25 domains of IL2(21-153)-(G3S)3-CD25(22-212) remained close to unity during 72 hours of in vitro incubation in human or mouse serum at 37°C, indicating minor cleavage of the linker in IL2(21-153)-(G3S)3-CD25(22-212) in both human and mouse serum. In addition, as shown in Fig. 11, in serum samples collected from monkeys administered a single subcutaneous dose of 0.075 mg/kg IL2(21-153)-(G3S)3-CD25(22-212), the ratio of IL2 to CD25 surrogate peptides remained close to unity after immunocapture with an anti-IL2 antibody. These data indicated that there was no linker cleavage in the serum of monkeys dosed with IL2(21-153)-(G3S)3-CD25(22-212) (i.e., SEQ ID NO: 16). CLAUSE OF THE INVENTION 1. Fusion protein containing: (a) a first polypeptide comprising an interleukin-2 (IL2) polypeptide, wherein the first polypeptide comprises an amino acid sequence having at least 85% identity to the sequence of SEQ ID NO: 2; and (b) a second polypeptide comprising an extracellular domain of an interleukin-2 receptor alpha (IL2Ra) polypeptide, wherein the second polypeptide comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 12; wherein the extracellular domain of the IL2Ra polypeptide does not include amino acids 192-219 of the extracellular domain of the natural IL2Ra under SEQ ID NO: 7; and wherein the fusion protein has IL2 activity. 2. The fusion protein of claim 1, wherein the first polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO: 2. 3. The fusion protein according to claim 1 or 2, wherein the second polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO: 12. - 102 048704 4. The fusion protein of any one of claims 1 to 3, wherein the fusion protein comprises a mutation in the second polypeptide, wherein a) the mutation is one or more substitutions, wherein 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 locations correspond to the sequence of SEQ ID NO: 7; and/or b) the mutation is one or more substitutions, wherein 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 locations correspond to the sequence of SEQ ID NO: 7. 5. The fusion protein according to any one of claims 1 to 4, wherein the fusion protein comprises a mutation in the first polypeptide, wherein the mutation in the first polypeptide is one or more substitutions at the amino acid T3 and/or C125 relative to the corresponding sequence under SEQ ID NO: 2. 6. The fusion protein of any one of claims 1 to 4, wherein the fusion protein comprises a mutation in the first polypeptide, wherein the mutation in the first polypeptide is a deletion at amino acid A1 relative to the corresponding sequence under SEQ ID NO: 2. 7. The fusion protein of any one of claims 1 to 6, further comprising a linker fused in frame between the first polypeptide and the second polypeptide. 8. The fusion protein of claim 7, wherein the linker is a glycine/serine linker. 9. The fusion protein of claim 8, wherein the glycine/serine linker comprises the amino acid sequence (GGGS) ₃ . 10. The fusion protein according to any one of claims 1 to 9, wherein the fusion protein further comprises a heterologous fragment fused to the first polypeptide and/or the second polypeptide, wherein the heterologous fragment comprises a polypeptide. 11. The fusion protein of claim 10, wherein the heterologous fragment is a fragment that increases half-life. 12. The fusion protein of claim 11, wherein the heterologous fragment comprises albumin or a functional variant thereof, an immunoglobulin constant region or a portion thereof, an immunoglobulin binding polypeptide, immunoglobulin G (IgG), an albumin binding polypeptide (ABP), a PAS sequence, a HAP sequence, transferrin or a fragment thereof, an XTEN polypeptide, an Fc region, or any combination thereof. 13. The fusion protein of claim 1, wherein the fusion protein comprises an amino acid sequence as set forth in SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 34, SEQ ID NOs: 39-41, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49-53, SEQ ID NO: 57-59, or SEQ ID NO: 67-70. 14. The fusion protein of any one of claims 1 to 13, wherein the fusion protein further comprises a heterologous fragment fused to the first polypeptide and/or the second polypeptide, wherein the heterologous fragment comprises a non-polypeptide. 15. The fusion protein of claim 14, wherein the heterologous fragment is a fragment that increases half-life. 16. The fusion protein of claim 15, wherein the heterologous fragment comprises a polymer or derivative thereof, and wherein the polymer comprises polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, hydroxyethyl starch (HES), polysialic acid (PSA), or any combinations thereof. 17. A fusion protein according to any one of claims 1 to 16, which is a monomer or a dimer. 18. A nucleic acid that encodes a fusion protein according to any one of claims 1 to 13. 19. An expression vector containing a nucleic acid according to claim 18. 20. A host cell containing the nucleic acid of claim 18. 21. A pharmaceutical composition comprising (a) a fusion protein according to any one of claims 1 to 17, a nucleic acid according to claim 18, a vector according to claim 19; and (b) a pharmaceutically acceptable excipient. 22. A kit for treating a disease or disorder modulated by IL-2, comprising a fusion protein according to any one of claims 1-17, a nucleic acid according to claim 18, an expression vector according to claim 19, a host cell according to claim 20, or a pharmaceutical composition according to claim 21, and instructions for use. 23. A method for producing a fusion protein, comprising: culturing the host cell according to claim 20 under suitable conditions and isolating the fusion protein. 24. A method for treating a disease or disorder modulated by IL-2 in a subject in need thereof, comprising administering to the subject an effective amount of a fusion protein according to any one of claims 1-17, a nucleic acid according to claim 18, an expression vector according to claim 19, a host cell according to claim 20, or a pharmaceutical composition according to claim 21. 25. The method according to claim 24, wherein the disease or disorder modulated by IL-2 includes an infectious disease caused by a pathogenic virus, pathogenic bacteria, pathogenic fungi, or pathogenic parasite. - 103 -