Interleukin-2 variants and methods of use thereof

Abstract

The present application relates to interleukin-2 variants and methods of use thereof. The present invention relates to polypeptides that share a primary sequence with human IL-2 in addition to several mutated amino acids. A panel of IL-2 variants comprises mutations with profound impressive manufacturability that preferentially promote proliferation, survival, activation and / or function of immunosuppressive regulatory T cells (Treg: CD4 + CD25 + FoxP3 +) over effector T cells and NK cells. Also included is the therapeutic use of such IL-2 selective agents, alone or in combination with immunomodulators or disease tissue targeting antibodies, proteins or peptides, for the treatment of Treg cell deficiency, various autoimmune and inflammatory disorders, organ transplantation and graft versus host disease. In another aspect, the invention relates to pharmaceutical compositions comprising the disclosed polypeptides. Finally, the invention relates to the therapeutic use of the disclosed polypeptides and pharmaceutical compositions due to their selective modulation of the immune system on diseases such as autoimmune and inflammatory disorders.

Description

[0001] This application is a divisional application of the application with the filing date of June 20, 2019, application number 201980054901.X, and invention title "Interleukin-2 Variants and Methods of Use".

[0002] Related patent applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 755,016, filed on November 2, 2018, and U.S. Provisional Application No. 62 / 689,055, filed on June 22, 2018, each of which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

[0004] Interleukin 2 (IL-2) was the first described growth factor for T cells. Since its discovery, IL-2 has been shown to promote the proliferation and survival of T cells in vitro (Smith, K A. (1988) Science. 240, 1169-76) and to enhance the immune response in the case of T viral infection (Blattman, J N, et al. (2003) Nat Med 9, 540-7) or vaccination (Fishman, M., et al. (2008) JImmunother. 31, 72-80, Kudo-Saito, C., et al. (2007) Cancer Immunol Immunother. 56, 1897-910; Lin, C T., et al. (2007) Immunol Lett. 114, 86-93).

[0005] IL-2 has been used in cancer treatment. Recombinant human IL-2 is an effective immunotherapy for metastatic melanoma and renal cell carcinoma, and approximately 10% of patients have a durable response. However, the short half-life and severe toxicity limit the optimal administration of IL-2. In addition, IL-2 also binds to its heterotrimeric receptor IL-2Rαβγ with high affinity, preferentially expanding immunosuppressive regulatory T cells (Tregs) that express high constitutive levels of IL-2Rα. The expansion of Tregs represents an undesirable effect of IL-2 on cancer immunotherapy. However, the ability of IL-2 to stimulate Treg cells even at low doses can be used to treat autoimmune disorders and various inflammatory disorders.

[0006] Tregs are central to immune system homeostasis and play a major role in maintaining peripheral immune tolerance by suppressing (auto-reactive) effector T cells. A variety of autoimmune and inflammatory diseases have been shown to have defects in Treg cell numbers or Treg function. Thus, there is great interest in developing therapies that enhance Treg cell numbers and / or function. One approach being investigated for the treatment of autoimmune diseases is the use of low-dose IL-2 to target Treg cells, as Treg cells have higher constitutive levels of the IL-2Rα and thus respond to lower concentrations of IL-2 compared to many other immune cell types (Klatzmann D, 2015 Nat Rev Immunol. 15:283-94). Clinical trials of low-dose IL-2 for the treatment of various GvHD (Koreth, J., et al., 2011, N Engl J Med., 365:2055-66) and HCV-related autoimmune vasculitis patients (Saadoum, D., et al., 2011, N Engl J Med., 365:2067-77) have shown signs of increased Treg levels and clinical efficacy. However, even these lower doses can lead to serious safety and tolerability issues. Thus, there is a need for an effective autoimmune / inflammatory disease therapy that more specifically targets Treg cells than IL-2 and is capable of enhancing Treg cell numbers and function.

[0007] It has recently been discovered that IL-2 can be modified to selectively stimulate cytotoxic effector T cells or Treg cells. A variety of methods have led to the production of IL-2 variants with improved and selective immunostimulatory capabilities (see, for example, U.S. Patent No. 7,186,804, U.S. Patent No. 7,105,653, U.S. Patent No. 6,955,807, U.S. Patent No. 5,229,109, U.S. Patent Application 20050142106). In the present invention, IL-2 variants are designed to increase the ability of the molecule to signal primarily through the high-affinity receptor (α-chain, β-chain, and γ-chain) rather than through the intermediate-affinity receptor (β-chain and γ-chain). The basic idea is to promote signaling in Treg cells rather than in effector T cells and NK cells, which are thought to be the cause of the observed toxic effects. Importantly, the prior art disclosures do not relate to IL-2 variants that have greater in vivo therapeutic efficacy than native IL-2 based on the reduced ability of the IL-2 variant to stimulate natural regulatory T cells. However, since the initial studies of IL-2 variants, research in the field has more fully established that Treg cells constitutively express high levels of IL-2Rα (CD25) as well as IL-2Rβ and γ C, an IL-2 variant as an IL-2Rαβγ selective agonist should be selective for Treg cells.

[0008] In summary, IL-2 is a highly pleiotropic cytokine that is closely related to the biological activities of different cell populations. This property makes IL-2 an important node in regulating the immune response, which makes IL-2 an attractive target for therapy and complex immune regulation. In addition, receptor subunit-biased IL-2 variants can be prepared to achieve IL-2-mediated selective immune regulation to promote the expansion and activity of regulatory T cells (Tregs), while minimizing helper and cytotoxic T effector cells (Teffs) and resulting in lower levels of pro-inflammatory signaling molecules.

[0009] Disclosure of the invention

[0010] In one aspect, the present invention relates to the generation of mutant variants of IL-2. These variants are characterized in that they are more selective in stimulating Treg (T CD4+CD25+FoxP3+) cells than in stimulating conventional CD4+ T cells, cytotoxic effector CD8+ T lymphocytes, and NK cells. In particular, these variants will provide practical solutions for improving IL-2 therapy for autoimmune and inflammatory disorders. The present invention relates to polypeptides that share their primary sequence with human IL-2 except for one to several mutated amino acids. These variants have amino acid substitutions at positions corresponding to the interaction of IL-2 with IL-2Rβ and / or γ C and thus these variants have reduced affinity for the IL-2Rβγ receptor complex and have reduced or eliminated ability to activate IL-2Rβγ-expressing cells, but retain the ability to bind IL-2Rα and the ability to bind and activate the IL-2Rαβγ receptor complex. The present invention also includes the therapeutic use of these mutant variants for treating autoimmune disorders and various inflammatory disorders.

[0011] In one aspect, the present invention relates to the generation of mutant variants of IL-2. These variants have a combination of properties of Treg-selective activity, reduced aggregation, increased expression, improved manufacturability and developability. These variants also provide improved protein biophysical properties and reduced immunogenic risk associated with the IL-2 molecule. These variants provide a superior solution for improving IL-2 therapy and reducing toxicity in autoimmune and inflammatory disorders. The present invention relates to polypeptides that share their primary sequence with human IL-2 except for one to several mutated amino acids.

[0012] In one aspect, the present invention relates to the generation of IL-2 mutant variants, which are characterized by the removal of a component that has been proposed to be similar to a bacterial toxin19 The LDL’ motif (Baluna R, Rizo et al., Proc Natl Acad Sci 1999; 96:3957 - 62). This ‘toxic motif’ is partly responsible for the direct vascular toxicity of IL-2. Introducing mutations to remove the key residue D20 or two residues flanking the toxin-like domain is expected to abolish this toxic motif, prevent endothelial cell damage and significantly reduce VLS. Importantly, since this motif lies at the interface with IL-2Rβ, amino acid substitutions to this motif reduce their affinity for IL-2Rβ, and the resulting molecules are expected to have two beneficial properties, including selectivity for activating Treg cells and reduced endothelial damage. The present invention relates to polypeptides that share their primary sequence with human IL-2 except for one to several mutated amino acids. The present invention also includes the therapeutic use of these mutant variants for treating Treg cell-deficient autoimmune disorders and various inflammatory disorders.

[0013] The present invention allows for a substantial improvement of the current strategies of IL-2-based immunomodulation in the therapy of autoimmune disorders and various inflammatory disorders. In particular, the replacement of native IL-2 with the mutant variants described herein will result in a CD25-biased selective stimulation of Treg cells. In various embodiments, the IL-2 variant (or mutant) comprises a sequence of an IL-2 variant (or mutant) derived from the mature human IL-2 polypeptide sequence listed in SEQ ID NO:3. In various embodiments, the IL-2 variant acts as an IL-2 agonist. In various embodiments, the IL-2 variant acts as an IL-2 antagonist. In various embodiments, the IL-2 variant comprises the sequences listed in SEQ ID NOs: 4 - 43, 108 - 146, and 193 - 197.

[0014] The present invention allows for a substantial improvement of the current strategies of IL-2-based immunomodulation in the therapy of Treg cell-deficient autoimmune disorders and various inflammatory disorders. In particular, the replacement of native IL-2 with the mutant variants described herein will result in a CD25-biased selective stimulation of Treg cells, and is expected to abolish the toxic motif, prevent endothelial cell damage and significantly reduce VLS. In various embodiments, the IL-2 variant (or mutant) comprises a sequence of an IL-2 variant (or mutant) derived from the mature human IL-2 polypeptide sequence listed in SEQ ID NO:3. In various embodiments, the IL-2 variant acts as an IL-2 agonist. In various embodiments, the IL-2 variant acts as an IL-2 antagonist. In various embodiments, the IL-2 variant comprises SEQ ID NOs: 5 - 14, 26 - 43, 108 - 111, 125 - 146, and 193 - 197.

[0015] In another aspect, the IL-2 variant of the invention is attached to at least one heterologous protein. In various embodiments, the IL-2 variant is fused to at least one polypeptide that confers an extended half-life to the fusion molecule. Such polypeptides include IgG Fc or other polypeptides that bind the neonatal Fcγ / receptor, human serum albumin, or polypeptides that bind proteins with an extended serum half-life, including IgG, non-IgG immunoglobulins, proteins, and non-protein agents that have an increased in vivo half-life due to the presence of an IgG constant domain or portion thereof that binds FcRn, and that have one or more amino acid modifications that increase the affinity of the constant domain or fragment for FcRn. Such proteins and molecules with an increased half-life have the advantage that less amounts and / or less frequent dosing are required in the therapeutic, prophylactic, or diagnostic use of such molecules (see, e.g., U.S. Patent No. 7,658,921).

[0016] In various embodiments, the IL-2 variant can be linked to the N-terminus or C-terminus of the heterologous protein.

[0017] In various embodiments, the IL-2 variant is fused to an IgG Fc molecule. In various embodiments, the Fc domain is a human IgG Fc domain. In various embodiments, the Fc domain is derived from the human IgG1 heavy chain constant domain sequence set forth in SEQ ID NO:44. In various embodiments, the Fc domain is an Fc domain having the amino acid sequence set forth in SEQ ID NO:45. In various embodiments, the Fc domain is an Fc domain having the amino acid sequence set forth in SEQ ID NO:46. In various embodiments, the Fc domain is an Fc domain having the amino acid sequence set forth in SEQ ID NO:47. In various embodiments, the Fc domain is derived from a human IgG2 heavy chain constant domain sequence. In various embodiments, the Fc domain is derived from a human IgG4 heavy chain constant domain sequence.

[0018] The term "Fc" refers to a molecule or sequence that comprises the sequence of a non-antigen-binding fragment of a full antibody, whether in monomeric or multimeric form. The original immunoglobulin source of native Fc is preferably of human origin and can be any immunoglobulin disclosed in the art. Native Fc consists of monomeric polypeptides that can be joined into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent associations. The number of intermolecular disulfide bonds between the monomeric subunits of a native Fc molecule ranges from 1 to 4 depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). An example of native Fc is the disulfide-bonded dimer produced by papain digestion of IgG (see Ellison et al. (1982), Nucleic Acids Res. 10:4071-9). As used herein, the term "native Fc" is a general term for monomeric, dimeric, and multimeric forms. The Fc domain contains binding sites for protein A, protein G, various Fc receptors, and complement proteins.

[0019] In various embodiments, the term "Fc variant" refers to a molecule or sequence that is modified from native Fc but still contains the binding site for the salvage receptor FcRn. International applications WO 97 / 34631 (published September 25, 1997) and WO 96 / 32458 describe exemplary Fc variants and their interactions with the salvage receptor and are hereby incorporated by reference. In addition, native Fc contains sites that can be removed because they provide structural features or biological activities that are not required for the fusion molecules of the present invention. Thus, in various embodiments, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues that affect or participate in (1) disulfide bond formation, (2) incompatibility with the selected host cell, (3) N-terminal heterogeneity upon expression in the selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor, or (7) antibody-dependent cell cytotoxicity (ADCC).

[0020] The term "Fc domain" includes native Fc and Fc variant molecules and sequences as defined above. Like Fc variants and native Fc, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from a full antibody or produced by recombinant gene expression or by other means. In various embodiments, the "Fc domain" refers to a dimer of two Fc domain monomers (SEQ ID NO:44), which typically includes all or part of the hinge region. In various embodiments, the Fc domain can be mutated to lack effector function. In various embodiments, each Fc domain monomer in the Fc domain contains an amino acid substitution in the CH2 antibody constant domain to reduce the interaction or binding between the Fc domain and Fcγ receptors. In various embodiments, each subunit of the Fc domain contains two amino acid substitutions that reduce binding to activating Fc receptors and / or effector function, wherein the amino acid substitutions are L234A, L235A. In various embodiments, each subunit of the Fc domain contains three amino acid substitutions that reduce binding to activating Fc receptors and / or effector function, wherein the amino acid substitutions are L234A, L235A, and G237A (SEQ ID NO:45).

[0021] In various embodiments, the Fc domain can be mutated to further extend the in vivo half-life. In various embodiments, each subunit of the Fc domain contains three amino acid substitutions that enhance binding to human FcRn, wherein the amino acid substitutions are M252Y, S254T, and T256E, as disclosed in U.S. Patent Publication No. 7,658,921. In various embodiments, each subunit of the Fc domain contains one amino acid substitution that enhances binding to human FcRn, wherein the amino acid substitution is N434A, as disclosed in U.S. Patent Publication No. 7,371,826. In various embodiments, each subunit of the Fc domain contains one amino acid substitution that enhances binding to human FcRn, wherein the amino acid substitutions are M428L and N434S, as disclosed in U.S. Patent Publication No. 8,546,543. In various embodiments, the half-life extension mutations can be combined with amino acid substitutions that reduce binding to activating Fc receptors and / or effector function.

[0022] In various embodiments, each of the two Fc domain monomers in the Fc domain contains an amino acid substitution that promotes heterodimerization of the two monomers. In various other embodiments, heterodimerization of the Fc domain monomers can be promoted by introducing different but compatible substitutions in the two Fc domain monomers, such as "knob-into-hole" residue pairs. The "knob-into-hole" technique is also disclosed in U.S. Patent Publication No. 8,216,805. In yet another embodiment, one Fc domain monomer contains the knob mutation T366W, and the other Fc domain monomer contains the hole mutations T366S, L358A, and Y407V. In various embodiments, two Cys residues that form a stabilizing disulfide bridge are introduced (S354C on the "knob" side and Y349C on the "hole" side). The use of heterodimeric Fc can produce monovalent IL-2 variant constructs.

[0023] In various embodiments, the IL-2 variant Fc-fusion protein will be monomeric, i.e., it contains only a single IL-2 mutant protein molecule. In such embodiments, the fusion protein is co-expressed with a heterodimeric Knob-Fc and a matching heterodimeric Hole Fc linked to the IL-2 variant. When the heterodimer of the two Fc-containing polypeptides forms, the resulting protein contains only a single IL-2 variant.

[0024] In various embodiments, the IL-2 variant is used to prepare the Fc-IL-2 fusion proteins listed in SEQ ID NOs: 69-107, 147-189, and 198-208.

[0025] In various embodiments, the IL-2 variant of the present invention can be attached to an antibody that confers an extended half-life to the fusion molecule, such as an anti-keyhole limpet hemocyanin (KLH) antibody. Such antibodies recognize foreign antigens, confer a longer half-life, but have no biological function or harm in the human body. The IgG class can be IgG, IgA, IgE, or a subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgA2).

[0026] In various embodiments, the heterologous protein is attached to the IL-2 variant via a linker and / or a hinge linker peptide. The linker or hinge linker can be an artificial sequence that is relatively free of secondary structure and is between 5, 10, 15, 20, 30, 40, or more amino acids.

[0027] In various embodiments, the heterologous protein is attached to the IL-2 variant via a rigid linker peptide between 10, 15, 20, 30, 40 or more amino acids, the rigid linker peptide exhibiting an α-helical conformation and serving as a rigid spacer between protein domains.

[0028] In another aspect, the IL-2 variant can be linked to various non-protein polymers, including but not limited to various polyols such as polyethylene glycol, polypropylene glycol or polyalkylene oxide, in the manner described in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 or 4,179,337. In various embodiments, amino acid substitutions can be made at different positions within the IL-2 variant to facilitate the addition of polymers such as PEG. In various embodiments, such PEGylated proteins can have an increased half-life and / or reduced immunogenicity compared to non-PEGylated proteins.

[0029] "Polyethylene glycol" or "PEG" means a polyalkylene glycol compound or a derivative thereof, with or without a coupling agent or derivatization, the derivatization having a coupling moiety or an activating moiety (e.g., having an aldehyde, hydroxysuccinimide, hydrazide, thiol, trifluoromethanesulfonate, tresylate, aziridine, ethylene oxide, orthopyridyldisulphide, vinyl sulfone, iodoacetamide or maleimide moiety). In various embodiments, PEG includes substantially linear, straight-chain PEG, branched PEG or dendritic PEG. PEG is a well-known water-soluble polymer, commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pp. 138-161).

[0030] In various embodiments, the Il-2 variant can be non-covalently or covalently linked at the N-terminus or C-terminus to IgGFc or other polypeptides (the other polypeptides binding to neonatal Fcγ / receptors, human serum albumin or polypeptides that bind to proteins with an extended serum half-life) or various non-protein polymers.

[0031] In another aspect, the present disclosure provides a pharmaceutical composition comprising an isolated IL-2 variant mixed with a pharmaceutically acceptable carrier.

[0032] In another aspect, the IL-2 variant can be formulated for sustained release, such as being covalently or non-covalently attached to a hydrogel, nanoparticles, etc.

[0033] In another aspect, the present disclosure provides a method for treating an autoimmune disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of the present invention. In one embodiment, the subject is a human subject. An autoimmune disease associated with the present invention is a disease or disorder that originates from and targets the subject's own tissues, or a condition co-segregating with, manifested by, or resulting from the subject's own tissues. In various embodiments, the autoimmune disease includes, but is not limited to, graft-versus-host disease (GvHD), immune-related adverse events (irAE), arthritis (including rheumatoid arthritis, reactive arthritis), systemic lupus erythematosus (SLE), atopic dermatitis, psoriasis, and inflammatory bowel disease (IBD), encephalomyelitis, uveitis, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes (type 1 diabetes), various dermatitides, Addison's disease, celiac disease, chronic fatigue syndrome, autoimmune hepatitis, alopecia areata, ankylosing spondylitis, ulcerative colitis, Crohn's disease, fibromyalgia, pemphigus vulgaris, Sjogren's syndrome, Kawasaki disease, hyperthyroidism / Graves' disease, hypothyroidism / Hashimoto's disease, endometriosis, scleroderma, pernicious anemia, Goodpasture's syndrome, Guillain-Barré syndrome, Wegener's disease, glomerulonephritis, aplastic anemia (including aplastic anemia patients with multiple blood transfusions), paroxysmal nocturnal hemoglobinuria, myelodysplastic syndrome, idiopathic thrombocytopenic purpura (IPT), autoimmune hemolytic anemia (AIHA), Evan's syndrome, Factor VIII inhibitor syndrome, systemic vasculitis, dermatomyositis, polymyositis, and rheumatic fever, autoimmune lymphoproliferative syndrome (ALPS), autoimmune bullous pemphigoid, Parkinson's disease, sarcoidosis, vitiligo, primary biliary cirrhosis, and autoimmune myocarditis.

[0034] In another aspect, the present disclosure provides a method for treating an autoimmune disease in a subject, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention in combination with a second therapeutic agent capable of treating the autoimmune disease.

[0035] In another aspect, the present disclosure provides a method for treating an inflammatory disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of the present invention. In one embodiment, the subject is a human subject. In various embodiments, the inflammatory diseases to be treated include, but are not limited to, Crohn's disease, colitis, dermatitis, psoriasis, diverticulitis, hepatitis, irritable bowel syndrome (IBS), lupus, nephritis, Parkinson's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's syndrome, and indeterminate colitis, multiple sclerosis (MS), Alzheimer's disease, arthritis, rheumatoid arthritis, asthma, and various cardiovascular diseases such as atherosclerosis and vasculitis. In various embodiments, the inflammatory disease is selected from the group consisting of rheumatoid arthritis, diabetes, gout, cryopyrin-associated periodic syndromes, and chronic obstructive pulmonary disease.

[0036] In another aspect, the present disclosure provides a method for treating an inflammatory disease in a subject, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention in combination with a second therapeutic agent capable of treating the inflammatory disease.

[0037] In another aspect, the present disclosure provides a method for organ transplantation or related graft-versus-host disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of the present invention. In one embodiment, the subject is a human subject. In various embodiments, the transplantation is an organ transplantation selected from the heart, kidney, liver, lung, pancreas, intestine, and thymus, or a tissue transplantation selected from bone, tendon, cornea, skin, heart valve, nerve, and blood vessel.

[0038] In another aspect, the present disclosure provides the use of an IL-2 variant for the preparation of a medicament for treating an autoimmune disease.

[0039] In another aspect, the present disclosure provides the use of an IL-2 variant for the preparation of a medicament for treating organ transplantation and GvHD.

[0040] In another aspect, the present disclosure provides the use of an IL-2 variant for the preparation of a medicament for treating an inflammatory disorder.

[0041] In another aspect, the present disclosure provides an isolated nucleic acid molecule comprising a polynucleotide encoding an IL-2 variant of the present disclosure. In another aspect, the present disclosure provides a vector comprising the nucleic acid described herein. In various embodiments, the vector is an expression vector. In another aspect, the present disclosure provides an isolated cell comprising the nucleic acid of the present disclosure. In various embodiments, the cell is a host cell comprising the expression vector of the present disclosure. In another aspect, a method of preparing an IL-2 variant is provided by culturing a host cell under conditions that promote the expression of a protein or polypeptide.

[0042] This application provides the following:

[0043] 1. An isolated interleukin-2 (IL-2) variant polypeptide, wherein the IL-2 variant polypeptide comprises an amino acid sequence of SEQ ID NO:3 in which one or more of the amino acid residue positions L19, D20, L21, Q22, R38, F42, N88, S125 or Q126 is replaced by another amino acid, and wherein the IL-2 variant polypeptide has a reduced ability to activate the IL-2Rβγ receptor complex compared to the polypeptide represented by SEQ ID NO:3, while retaining the ability to activate the IL-2Rαβγ receptor complex.

[0044] 2. The IL-2 variant polypeptide according to item 1, wherein the amino acid substitution is selected from the group consisting of: substitutions at position 19 of SEQ ID NO:3 of L19D, L19H, L19N, L19R, L19S, L19P and L19Y, substitutions at position 20 of D20E, D20I, D20N, D20Q, D20S, D20T and D20Y, substitutions at position 21 of L21S, L21R and L21N, substitutions at position 22 of Q22N, Q22H, Q22K, Q22Y, Q22I, substitutions at position 88 of N88E, N88G, N88T, N88M, N88Q, N88R and N88I, substitutions at position 125 of S125E, S125K, S125H, S125W and S125I, and substitutions at position 126 of Q126D, Q126E, Q126H, Q126K, Q126L, Q126M, Q126N and Q126Y, or any combination of these substitutions.

[0045] 3. The IL-2 variant polypeptide according to any one of items 1 and 2, wherein the IL-2 variant polypeptide comprises two amino acid substitutions at amino acid residue positions L19 and Q126 of SEQ ID NO:3.

[0046] 4. The IL-2 variant polypeptide according to any one of Items 1 and 2, wherein the IL-2 variant polypeptide comprises three amino acid substitutions at amino acid residue positions L19, S125, and Q126 of SEQ ID NO:3.

[0047] 5. The IL-2 variant polypeptide according to any one of Items 3 and 4, wherein the amino acid substitutions are selected from the group consisting of: substitutions L19D, L19H, L19N, L19R, L19S, L19P, and L19Y at position 19 of SEQ ID NO:3, substitutions S125E, S125K, S125H, S125W, and S125I at position 125, and substitutions Q126D, Q126E, Q126H, Q126K, Q126L, Q126M, Q126N, and Q126Y at position 126.

[0048] 6. The IL-2 variant polypeptide according to any one of Items 1 to 5, wherein the IL-2 variant polypeptide comprises an amino acid sequence selected from the group consisting of the amino acid sequences listed in SEQ ID NO:4-43, 108-146, and 193-197.

[0049] 7. The IL-2 variant polypeptide according to Item 6, wherein the IL-2 variant polypeptide comprises an amino acid sequence selected from the group consisting of the amino acid sequences listed in SEQ ID NO:5-14, 26-43, 108-111, 125-146, and 193-197.

[0050] 8. An isolated IL-2 variant polypeptide, the isolated IL-2 variant polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3, and wherein compared to the polypeptide represented by SEQ ID NO:3, the IL-2 variant polypeptide has a reduced ability to activate the IL-2Rβγ receptor complex while retaining the ability to activate the IL-2Rαβγ receptor complex.

[0051] 9. An isolated fusion protein, the isolated fusion protein comprising 1) the IL-2 variant polypeptide according to any one of Items 1-8 and 2) a heterologous protein.

[0052] 10. The isolated fusion protein according to Item 9, wherein the IL-2 variant polypeptide is fused to the C-terminal amino acid of the heterologous protein at its N-terminal amino acid, optionally via a peptide linker.

[0053] 11. The isolated fusion protein according to item 9, wherein the IL-2 variant polypeptide is fused to the N-terminal amino acid of the heterologous protein at its C-terminal amino acid, optionally via a peptide linker.

[0054] 12. The isolated fusion protein according to any one of items 9 to 11, wherein the heterologous protein increases the circulating half-life of the IL-2 variant polypeptide.

[0055] 13. The isolated fusion protein according to item 12, wherein the heterologous protein is selected from the group consisting of full-length inert antibodies or antibody fragments that provide extended half-life.

[0056] 14. The isolated fusion protein according to any one of items 9 to 11, wherein the heterologous protein enhances the expression level and overall purity of the IL-2 variant polypeptide.

[0057] 15. The isolated fusion protein according to any one of items 9 to 11, wherein the heterologous protein serves as a marker or tag or targeting moiety.

[0058] 16. The isolated fusion protein according to any one of items 9 to 15, wherein the fusion protein is in the form of a dimer or monomer.

[0059] 17. The isolated fusion protein according to any one of items 9 to 11, wherein the heterologous protein is an Fc domain selected from the group consisting of: human IgG1 Fc domain, human IgG2 Fc domain, human IgG3 Fc domain, human IgG4 Fc domain, IgA Fc domain, IgD Fc domain, IgE Fc domain, IgG Fc domain, and IgM Fc domain.

[0060] 18. The isolated fusion protein according to item 17, wherein the Fc domain is an Fc domain having silent effector function and / or having an extended half-life function.

[0061] 19. The isolated fusion protein according to any one of items 17 to 18, wherein the Fc domain is an Fc domain having an amino acid sequence selected from the group consisting of the amino acid sequences listed in SEQ ID NO: 44 - 47 and 212 - 213.

[0062] 20. The isolated fusion protein according to any one of items 9 - 19, wherein the IL-2 variant polypeptide is fused to the heterologous protein via a peptide linker.

[0063] 21. The fusion protein according to item 20, wherein the peptide linker contains between 1 and 40 amino acids.

[0064] 22. The isolated fusion protein according to item 9, wherein the fusion protein comprises an amino acid sequence selected from the group consisting of the amino acid sequences listed in SEQ ID NO: 69-107, 147-189, and 198-211.

[0065] 23. A pharmaceutical composition comprising the IL-variant polypeptide or isolated fusion protein according to any one of items 1 to 22, mixed with a pharmaceutically acceptable carrier.

[0066] 24. A method of treating an autoimmune disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to item 23.

[0067] 25. The method according to item 24, wherein the method further comprises administering a second therapeutic agent or therapy capable of treating the autoimmune disease in the subject.

[0068] 26. A method of treating organ transplant rejection or related graft-versus-host disease (GvHD) in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to item 23.

[0069] 27. A method of treating an inflammatory disease, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, atopic dermatitis, diabetes, and immune-related adverse events (irAE) in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to item 23.

[0070] 28. The method according to item 27, wherein the method further comprises administering a second therapeutic agent or therapy capable of treating the inflammatory disease in the subject.

[0071] 29. An isolated nucleic acid molecule encoding the IL-2 variant polypeptide or fusion protein according to any one of items 1 to 22.

[0072] 30. An expression vector comprising the nucleic acid molecule according to item 29.

[0073] 31. A host cell comprising the nucleic acid molecule according to item 29 or the expression vector according to item 30.

[0074] 32. A method of producing the IL-2 variant polypeptide or fusion protein according to any one of items 1 to 22, the method comprising culturing the host cell according to item 31 under conditions that promote the expression of the IL-2 variant polypeptide or fusion protein, and recovering the IL-2 variant polypeptide or fusion protein.

[0075] 33. An isolated IL-2 variant polypeptide or fusion protein, said isolated IL-2 variant polypeptide or fusion protein being produced by the method according to item 32. Brief description of the drawings

[0077] Figures 1A - 1E Depicts the size exclusion chromatograms of exemplary IL-2Fc fusion proteins P-0250( Figure 1A ), P-0318( Figure 1B ), P-0317( Figure 1C ), P-0447( Figure 1D ) and P-0511( Figure 1E ) after protein A purification. Figure 1D And Figure 1E Also illustrates the SDS-PAGE of each sample in the absence (lane 2) and presence (lane 3) of a reducing agent.

[0078] Figures 2A - 2B Depicts the different effects of an Fc fusion protein of an IL-2 variant (D20X) with an aspartic acid amino acid substitution at position 20 on inducing STAT5 phosphorylation in CD4+ Treg cells( Figure 2A ) and Tconv cells( Figure 2B ) compared to the wild-type fusion protein (P-0250) in a human PBMC assay.

[0079] Figures 3A - 3B Depicts the different effects of an Fc fusion protein of an IL-2 variant P-0375 (N88Q) on inducing STAT5 phosphorylation in CD4+ Treg cells( Figure 3A ) and Tconv cells( Figure 3B ) compared to the wild-type (P-0250) and a reference protein in a human PBMC assay.

[0080] Figures 4A - 4D Depicts the different effects of an Fc fusion protein of an IL-2 variant with an amino acid substitution at position 19 on STAT5 phosphorylation compared to the wild-type (P-0250). In a human PBMC assay, the ability to induce STAT5 phosphorylation in CD4+ Treg cells( Figure 4A And Figure 4C ) and Tconv cells( Figure 4B And Figure 4D ) was determined by FACS analysis.

[0081] Figures 5A - 5FDepicted are the different effects of Fc fusion proteins of IL-2 variants with single amino acid substitutions or combined mutations (P-0447) at position 19 (P-0424) or position 126 (P-0303) on STAT5 phosphorylation compared to wild-type (P-0250) or benchmark-1 protein. Induction of CD4+ Treg cells ( Figure 5A , Figure 5C and Figure 5E ) and CD4+Tconv cells ( Figure 5B , Figure 5D and Figure 5F )'s ability to phosphorylate STAT5.

[0082] Figures 6A - 6D Depicted are the different effects of Fc fusion proteins of IL-2 variants containing different combinations of double amino acid substitutions (P-0419, P-0464, P-0471, P-0474, P-0417 and P-0322) on STAT5 phosphorylation compared to wild type (P-0250). The biological activities of P-0417 and P-0322 were also compared to their counterparts with single amino acid substitutions, P-0373 and P-0363, respectively. In human PBMC assays, the induction of CD4+ Treg cells ( Figure 6A and Figure 6C ) and Tconv cells ( Figure 6B and Figure 6D )'s ability to phosphorylate STAT5.

[0083] Figures 7A - 7D Depicted are the differential effects of IL-2 variants with single amino acid substitutions or combined mutations (P-0447) at position 19 (P-0424) or position 126 (P-0303) on STAT5 phosphorylation compared to wild type (P-0250), as well as the differential effects of IL-2 variants with different combined amino acid substitutions (P-0419, P-0447, P-0448, and P-0449) on STAT5 phosphorylation compared to wild type (P-0250) and benchmark Fc fusion proteins. In human PBMC assays, induction of CD4+ Treg cells ( Figure 7A and Figure 7C ) and CD4+Tconv cells ( Figure 7B and Figure 7D )'s ability to phosphorylate STAT5.

[0084] Figures 8A - 8FDepicts the comparison of the pSTAT5-stimulating activities of the IL-2 fusion proteins P-0250, P-0424, and P-447 with their respective counterparts P-0531, P-0491, and P-0511 containing the S125I substitution. In the human PBMC assay, the ability to induce STAT5 phosphorylation in CD4+ Treg cells ( Figure 8A , Figure 8C and Figure 8E ) and CD4+ Tconv cells ( Figure 8B , Figure 8D and Figure 8F ) was determined by FACS analysis.

[0085] Figures 9A - 9C Depicts the differential effects of the IL-2 variant Fc fusions (P-0511 and P-0512) on STAT5 phosphorylation in three CD4+ T cell subsets ( Figure 9A ) CD4+FoxP3+CD25+ Treg cells, Figure 9B ) CD4+FoxP3-CD25+ activated Tconv cells, and Figure 9C ) CD4+FoxP3-CD25- naive Tconv cells) compared to wild-type (P-0250) and three benchmark molecules. In the human PBMC assay, the ability to induce STAT5 phosphorylation was determined by FACS analysis.

[0086] Figures 10A - 10B Depicts the differential effects of P-0511 and P-0512 on the proliferation of stimulated CD8+ T cells ( Figure 10A ) and NK cells ( Figure 10B ) compared to wild-type (P-0250) and benchmark molecules. In the human PBMC assay, cell proliferation was determined by FACS analysis of CFSE dilution and expressed as the percentage of dividing cells.

[0087] Figures 11A - 11D Depicts the differential effects of the IL-2 variant Fc fusion P-0511 on STAT5 phosphorylation in different cell types compared to the wild-type equivalent P-0531. In the human PBMC assay, the ability to induce STAT5 phosphorylation in CD4+ Treg cells ( Figure 11A ), CD4+ Tconv cells ( Figure 11B ), CD8+ T cells ( Figure 11C ), and NK cells ( Figure 11D ) was determined by FACS analysis. Figure 11E Depicts the binding strength of P-511 to the IL-2Rβ and γc complex in an ELISA assay compared to P-0531 and Benchmark-1.

[0088] Figures 12A - 12C Depicts the proliferation and expansion of Treg cells in mice treated with an Fc fusion protein of an IL-2 variant and a benchmark after a single subcutaneous injection. Blood was collected at the indicated time points for proliferation measurement and lymphocyte phenotype analysis.( Figure 12A ) Percentage of Ki67-positive Treg cells;( Figure 12B ) Percentage of Treg cells in the total CD4+ T cell population;( Figure 12C ) Percentage of Treg cells in the total blood lymphocytes. Data are represented as mean ± SEM. Statistical analysis was performed by one-way ANOVA test followed by Tukey's post hoc test. ****p < 0.0001; ***p < 0.001, compared with the PBS group at the respective time points.

[0089] Figures 13A - 13C Depicts the proliferation of effector T cells and NK cells in mice treated with an IL-2 mutant Fc fusion protein and a benchmark after a single subcutaneous injection. Blood was collected at the indicated time points to measure lymphocyte proliferation.( Figure 13A ) Percentage of Ki67-positive CD4+ T conventional (Tconv) cells;( Figure 13B ) Percentage of Ki67-positive CD8+ T cells;( Figure 13C ) Percentage of Ki67-positive NK cells. Data are represented as mean ± SEM. Statistical analysis was performed by one-way ANOVA test followed by Tukey's post hoc test. ****p < 0.0001; ***p < 0.001, compared with the PBS group at the respective time points.

[0090] Figures 14A - 14D Depicts the expansion of effector T cells and NK cells in mice treated with an IL-2 mutant Fc fusion protein and a benchmark after a single subcutaneous injection.( Figures 14A - 14B ) Total CD4+ T cells( Figure 14A ) and total blood lymphocytes( Figure 14B ) Percentage of CD4+ T conventional (Tconv) cells.( Figure 14C ) Percentage of CD8+ T cells in the total blood lymphocytes;( Figure 14D ) Percentage of NK cells in the total blood lymphocytes. Data are represented as mean ± SEM.

[0091] Figures 15A - 15B Depicts the Treg to Tconv cell ratio in mice treated with an IL-2 mutant Fc fusion protein and a benchmark, based on: Figure 15A ) Percentage of Ki67-positive expression and Figure 15B) Cell number. Data were obtained by FACS and presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test. ****p < 0.0001; *p < 0.05, compared with the PBS group at the respective time points.

[0092] Figures 16A - 16B Depicts the expression of CD25 and Foxp3 on Treg cells in mice treated with IL-2 mutant Fc fusion protein and vehicle after a single subcutaneous injection. The expression levels of Foxp3 ( Figure 16A ) and CD25 ( Figure 16B ) were analyzed by FACS and presented as mean fluorescence intensity (MFI). Data are presented as mean ± SEM. ****p < 0.0001, compared with the PBS group at the respective time points.

[0093] Figures 17A - 17D Depicts the dose-dependent increase in the proliferation and expansion of Treg cells in mice after a single injection of IL-2 variant Fc fusion protein P-0511. Blood was collected at the indicated time points for lymphocyte phenotype analysis and measurement of the Ki67 proliferation marker. ( Figure 17A ) Percentage of Ki67-positive Treg cells, the proliferation marker; ( Figure 17B ) Percentage of Treg cells among total CD4+ T cells; ( Figure 17C ) Number of Treg cells per microliter of whole blood; ( Figure 17D ) Fold change in the number of Treg cells from baseline for each group. Data are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, compared with the PBS group at the respective time points.

[0094] Figures 18A - 18D Depicts the dose-dependent effect of IL-2 variant Fc fusion protein P-0511 on the percentage of Treg cells ( Figure 18A ), CD4+ Tconv cells ( Figure 18B ), CD8 T cells ( Figure 18C ), and NK cells ( Figure 18D ) in the total lymphocytes of mice after a single injection. Blood was collected at the indicated time points for lymphocyte phenotype analysis. Data were determined by FACS analysis and presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test. ****p < 0.0001, **p < 0.01, *p < 0.05, compared with the PBS group at the respective time points.

[0095] Figures 19A - 19C Depicts the dose-dependent increase in the ratio of Treg to T conv cell numbers ([ Figure 19A ), the expression of CD25 on Treg cells ([ Figure 19B ), and the expression of Foxp3 on Treg cells ([ Figure 19C ) in mice after a single injection of P-0511. Data were determined by FACS analysis and are expressed as mean ± SEM. Statistical analysis was performed by one-way ANOVA test, followed by Tukey's post hoc test. ****p<0.0001, ***p<0.001, **p<0.01, compared to the PBS group at the respective time points.

[0096] Figures 20A - 20C Depicts the sustained proliferation and expansion of Treg cells in mice receiving repeated doses of the Fc fusion proteins of IL-2 variants (P-0511 and P-0512), but not wild-type (P-0531) and benchmark. The compounds were administered s.c. once every three days (Q3D), and blood was collected 3 days after the 1st and 3rd injections for lymphocyte phenotyping analysis and measurement of the proliferation marker Ki67. ([ Figure 20A ) Percentage of Ki67-positive Treg cells; ([ Figure 20B ) Percentage of Treg cells among total CD4+ T cells; ([ Figure 20C ) Percentage of Treg cells among total blood lymphocytes. Data were determined by FACS analysis and are expressed as mean ± SEM. Statistical analysis was performed by one-way ANOVA test, followed by Tukey's post hoc test. ****p<0.0001, *p<0.05, compared to the respective PBS group.

[0097] Figures 21A - 21B Depicts the sustained increase in Treg cell counts in mice receiving repeated doses of the Fc fusion proteins of IL-2 variants (P-0511 and P-0512), but not wild-type (P-0531) and benchmark. The compounds were administered s.c. once every three days (Q3D), and blood was collected 3 days after the 1st and 3rd injections for lymphocyte phenotyping analysis and measurement of the proliferation marker Ki67. ([ Figure 21A ) Number of Treg cells per microliter of whole blood; ([ Figure 21B ) Fold change in Treg cell number compared to the PBS control group. Data were determined by FACS analysis and are expressed as mean ± SEM. Statistical analysis was performed by one-way ANOVA test, followed by Tukey's post hoc test. ****p<0.0001, ***p<0.001, **p<0.01, compared to the respective PBS group.

[0098] Figure 22Depicts the maintenance of an elevated Treg-to-Tconv ratio in mice receiving repeated doses of the Fc fusion proteins of IL-2 variants (P-0511 and P-0512), but not wild-type (P-0531) and a benchmark. The compounds were administered s.c. once every three days (Q3D), and blood was collected 3 days after the 1st and 3rd injections for Treg and Tconv cell phenotype analysis. The ratio was calculated based on Treg% and Tconv% in total CD4 cells. Data were determined by FACS analysis and represented as mean ± SEM. Statistical analysis was performed by one-way ANOVA test followed by Tukey's post hoc test. ****p < 0.0001, compared to the respective PBS group.

[0099] Figures 23A - 23B Depicts in mice In a delayed-type hypersensitivity (DTH) mouse model induced by keyhole Figure 23A ) and Q5D ( Figure 23B ) dosing regimens, the kinetics of the DTH response were illustrated using the change in ear thickness relative to the baseline value (Δ ear thickness) at different times after KLH challenge. Data were represented as mean ± SEM. Statistical analysis was performed by one-way ANOVA test followed by Tukey's post hoc test. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, compared to the respective PBS group at the respective time points.

[0100] Figure 24 Depicts the inhibition of antigen-driven inflammation by P-0511 compared to a benchmark molecule (benchmark-1) in a mouse model of DTH induced by KLH antigen. Mice were immunized with KLH on day 0 and rechallenged in the right ear on day 5. From day -2, mice were treated with the compound Q5D. The kinetics of the DTH response were illustrated using the change in ear thickness relative to the baseline value (Δ ear thickness) at different times after KLH challenge. Data were represented as mean ± SEM. Statistical analysis was performed by one-way ANOVA test followed by Tukey's post hoc test. ****p < 0.0001, **p < 0.01, *p < 0.05, compared to the respective PBS group at the respective time points.

[0101] Ways to carry out the present disclosure

[0102] The present invention relates to polypeptides that share the primary sequence with human IL-2 except for one to several mutated amino acids. A group of IL-2 variants contain mutations that preferentially promote the proliferation, survival, activation, and / or function of immunosuppressive regulatory T cells (TCD4+CD25+FoxP3+) relative to effector T cells and NK cells. The present invention also includes the therapeutic use of such IL-2 selective agonists alone or in combination with disease tissue-targeting proteins or peptides to treat autoimmune disorders and various inflammatory disorders. Another group of IL-2 variants contain mutations that substantially reduce the ability of these polypeptides to stimulate Treg cells or Tconv cells, while may have receptor-binding activity and render them effective as IL-2 antagonists. In another aspect, the present invention relates to pharmaceutical compositions comprising the disclosed polypeptides. Finally, the present invention relates to the therapeutic use of the disclosed polypeptides and pharmaceutical compositions due to their selective regulatory effects on the immune system against diseases such as autoimmune disorders and inflammatory disorders.

[0103] Definition

[0104] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to polymers of amino acid residues. In various embodiments, a "peptide", "polypeptide", and "protein" is a chain of amino acids whose α-carbons are linked by peptide bonds. Thus, the terminal amino acid at one end (amino terminus) of the chain has a free amino group, while the terminal amino acid at the other end (carboxyl terminus) of the chain has a free carboxyl group. As used herein, the term "amino terminus" (abbreviated as N-terminus) refers to the free α-amino group on the amino acid at the amino terminus of a peptide, or to the α-amino group of an amino acid at any other position in the peptide (the amino group when participating in a peptide bond). Similarly, the term "carboxyl terminus" refers to the free carboxyl group on the carboxyl terminus of a peptide, or to the carboxyl group of an amino acid at any other position in the peptide. Peptides also include substantially any polyamino acids, including but not limited to peptide mimetics, such as amino acids linked by ether bonds rather than amide bonds.

[0105] The polypeptides of the present disclosure include polypeptides that have been modified in any way and for any reason, for example, to: (1) reduce sensitivity to proteolysis, (2) reduce sensitivity to oxidation, (3) alter the binding affinity to form protein complexes, (4) alter the binding affinity, and (5) confer or alter other physicochemical or functional properties.

[0106] As used herein, an amino acid "substitution" refers to the replacement of one amino acid at a specific position in a parental polypeptide sequence in a polypeptide with a different amino acid. Amino acid substitutions can be generated using genetic methods or chemical methods well known in the art. For example, single amino acid substitutions or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in a naturally occurring sequence (e.g., in a polypeptide portion other than a domain that forms intermolecular contacts). A "conservative amino acid substitution" refers to the replacement of an amino acid in a polypeptide with an amino acid that is functionally similar. The following six groups each contain amino acids that are conservative substitutions for one another:

[0107] 1) Alanine (A), Serine (S), and Threonine (T)

[0108] 2) Aspartic acid (D) and Glutamic acid (E)

[0109] 3) Asparagine (N) and Glutamine (Q)

[0110] 4) Arginine (R) and Lysine (K)

[0111] 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V)

[0112] 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W)

[0113] A "non-conservative amino acid substitution" refers to the replacement of a member of one of these classes with a member from another class. When making such a change, the hydropathic index of the amino acid can be considered according to various embodiments. Based on the hydrophobicity and charge characteristics of the amino acids, each amino acid has been assigned a hydropathic index. They are: Isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / Cystine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamic acid (-3.5); Glutamine (-3.5); Aspartic acid (-3.5); Asparagine (-3.5); Lysine (-3.9); and Arginine (-4.5).

[0114] Those skilled in the art understand the importance of the hydrophilic amino acid index in conferring biological functions of protein interactions (see, for example, Kyte et al., 1982, J. Mol. Biol. 157: 105-131). It is known that certain amino acids can be replaced by other amino acids having similar hydrophilic indices or scores and still retain similar biological activities. When making changes based on the hydrophilic index, in various embodiments, substitutions of amino acids with hydrophilic indices within ±2 are included. In various embodiments, those within ±1 are included, and in various embodiments, those within ±0.5 are included.

[0115] Those skilled in the art also understand that substitutions of similar amino acids can be effectively made based on hydrophilicity, especially when the resulting biofunctional protein or peptide is intended for use in immunological embodiments as disclosed herein. In various embodiments, the maximum local average hydrophilicity of the protein (determined by the hydrophilicity of its adjacent amino acids) is related to its immunogenicity and antigenicity, i.e., to the biological properties of the protein.

[0116] The following hydrophilicity values are assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). When making changes based on similar hydrophilicity values, in various embodiments, substitutions of amino acids with hydrophilicity values within ±2 are included, in various embodiments, those within ±1 are included, and in various embodiments, those within ±0.5 are included.

[0117] Exemplary amino acid substitutions are listed in Table 1.

[0118] Table 1

[0119]

[0120] Skilled artisans will be able to use known techniques to identify suitable polypeptide variants as listed herein. In various embodiments, those skilled in the art can identify regions of a molecule that can be altered without disrupting activity by targeting regions that are not considered important for activity. In other embodiments, those skilled in the art can identify residues and portions of a molecule that are conserved among similar polypeptides. In additional embodiments, even regions that may be important for biological activity or for structure can undergo conservative amino acid substitutions without disrupting biological activity or unduly affecting polypeptide structure.

[0121] In addition, those skilled in the art can review structure - function studies that identify residues important for activity or structure in similar polypeptides. Given such comparisons, those skilled in the art can predict the importance of amino acid residues in a polypeptide corresponding to amino acid residues important for activity or structure in similar polypeptides. Those skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0122] Those skilled in the art can also analyze the three - dimensional structure and amino acid sequence of similar polypeptides in relation to that structure. Given such information, those skilled in the art can predict the arrangement of the amino acid residues of a polypeptide in terms of its three - dimensional structure. In various embodiments, those skilled in the art can choose not to make radical changes to amino acid residues predicted to be on the surface of a polypeptide, as such residues may be involved in important interactions with other molecules. In addition, those skilled in the art can generate test variants that contain a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. These variants can be used to gather information about suitable variants. For example, if it is found that a change in a particular amino acid residue results in disrupted, undesirably reduced, or inappropriate activity, then variants having such a change can be avoided. In other words, based on the information collected from such routine experiments, those skilled in the art can readily determine the amino acids at which further substitutions, either alone or in combination with other mutations, should be avoided.

[0123] As used herein, the terms "polypeptide fragment" and "truncated polypeptide" refer to polypeptides having an amino-terminal deletion and / or a carboxyl-terminal deletion compared to the corresponding full-length protein. In various embodiments, the length of the fragment can be, for example, at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 amino acids. In various embodiments, the length of the fragment can also be, for example, at most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 450, at most 400, at most 350, at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 25, at most 10, or at most 5 amino acids. The fragment can also contain one or more additional amino acids at either or both of its termini, for example, an amino acid sequence from a different naturally occurring protein (e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence).

[0124] As used herein, the terms "polypeptide variant", "hybrid polypeptide", and "polypeptide mutant" refer to polypeptides that contain an amino acid sequence in which one or more amino acid residues have been inserted into, deleted from, and / or substituted into the amino acid sequence relative to another polypeptide sequence. In various embodiments, the number of amino acid residues to be inserted, deleted, or substituted can be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids in length. The hybrids of the present disclosure include fusion proteins.

[0125] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, such as conjugated to another chemical moiety (such as, for example, polyethylene glycol, albumin (e.g., human serum albumin)), phosphorylation, and glycosylation.

[0126] The terms “% sequence identity” and “% identity” are used interchangeably herein and refer to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% identity and 80% sequence identity as determined by a defined algorithm mean the same thing and mean that a given sequence is at least 80% identical to another sequence of another length. In various embodiments, the % identity is selected from, for example, sequence identities of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or greater with a given sequence. In various embodiments, the % identity is in the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[0127] The terms “% sequence homology” and “% homology” are used interchangeably herein and refer to the level of amino acid sequence homology between two or more peptide sequences or the level of nucleotide sequence homology between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% homology and 80% sequence homology as determined by a defined algorithm mean the same thing and thus a homolog of a given sequence has a sequence homology greater than 80% relative to the length of the given sequence. In various embodiments, the % homology is selected from, for example, sequence homologies of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or greater with a given sequence. In various embodiments, the % homology is in the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[0128] Exemplary computer programs that can be used to determine identity between two sequences include, but are not limited to, a suite of BLAST programs publicly available on the Internet at the NCBI website, such as BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN. See also Altschul et al., J. Mol. Biol. 215:403-10, 1990 (specifically refer to the published default settings, i.e., parameters w = 4, t = 17) and Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997. When evaluating a given amino acid sequence relative to GenBank protein sequences and amino acid sequences in other public databases, the BLASTP program is typically used for sequence retrieval. The BLASTX program is preferably used to retrieve nucleic acid sequences that have been translated in all reading frames against amino acid sequences in GenBank protein sequences and other public databases. Both BLASTP and BLASTX are run using default parameters of an open gap penalty of 11.0 and an extended gap penalty of 1.0 and using the BLOSUM-62 matrix.

[0129] In addition to calculating the percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)), which gives an indication of the probability that a match between two nucleotide sequences or amino acid sequences occurs by chance. For example, if in a comparison of a test nucleic acid with a reference nucleic acid, the minimum total probability is, for example, less than about 0.1, less than about 0.01, or less than about 0.001, then the nucleic acid is considered similar to the reference sequence.

[0130] As used herein, the term "modification" refers to any manipulation of the peptide backbone (e.g., amino acid sequence) or post-translational modification of a polypeptide (e.g., glycosylation).

[0131] As used herein, the term "knob-into-hole modification" refers to a modification within the interface between the CH3 domains of two immunoglobulin heavy chains. In one embodiment, a "knob-into-hole modification" comprises the amino acid substitution T366W and optionally the amino acid substitution S354C in one antibody heavy chain, and the amino acid substitutions T366S, L368A, Y407V and optionally Y349C in the other antibody heavy chain. The knob-into-hole technology is described, for example, in U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).

[0132] As used herein, the term "fusion protein" refers to a fusion polypeptide molecule comprising two or more genes that originally encoded different proteins, wherein the components of the fusion protein are directly linked to each other by peptide bonds or are linked to each other by a peptide linker. As used herein, the term "fusion" refers to components that are directly linked by peptide bonds or components that are linked via one or more peptide linkers.

[0133] A "linker" is a molecule that covalently or by ionic bonds, van der Waals forces or hydrogen bonds links two other molecules, such as a nucleic acid molecule that hybridizes to a complementary sequence at the 5' end and hybridizes to another complementary sequence at the 3' end to link two non-complementary sequences. A "cleavable linker" is a linker that can be degraded or otherwise cleaved to separate two components linked by the cleavable linker. Cleavable linkers are typically cleaved by enzymes, usually peptidases, proteases, nucleases, lipases, etc. Cleavable linkers can also be cleaved by changes in environmental factors, such as, for example, temperature, pH, salt concentration, etc.

[0134] As used herein, the term "peptide linker" refers to a peptide comprising one or more amino acids, usually about 2 to 20 amino acids. Peptide linkers are known in the art or are described herein. Suitable non-immunogenic linker peptides include, for example, (G4S) n 、(SG4) n or G4(SG4)n peptide linkers. "n" is typically a number between 1 and 10, usually between 2 and 4.

[0135] "Pharmaceutical composition" means a composition suitable for pharmaceutical use in animals. A pharmaceutical composition contains a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. "Pharmacologically effective amount" means the amount of an agent effective to produce the desired pharmacological result. "Pharmaceutically acceptable carrier" means any standard pharmaceutical carrier, vehicle, buffer, and excipient, such as phosphate buffered saline solution, aqueous solution of 5% dextrose, and emulsions, such as oil / water emulsion or water / oil emulsion, and various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21st Edition 2005, Mack Publishing Co, Easton. "Pharmaceutically acceptable salt" is a salt of a compound that can be formulated for pharmaceutical use, including, for example, salts of metals (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or salts of organic amines.

[0136] As used herein, "treatment" (and its grammatical variations such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of a disease in an individual being treated, and the clinical intervention can be used prophylactically or during a clinical pathologic process. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, alleviating any direct or indirect pathologic consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or ameliorating the disease state, and relieving or improving the prognosis. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder, or condition. Additionally, reference to "treatment" herein includes reference to curative, palliative, and prophylactic treatment.

[0137] As used herein, the term "effective amount" or "therapeutically effective amount" means the amount of a compound or composition sufficient to treat a particular disorder, condition, or disease, such as to improve, ameliorate, alleviate, and / or delay one or more of its symptoms. With reference to cancer or other unwanted cell proliferation, an effective amount includes an amount sufficient to achieve: (i) a reduction in the number of cancer cells; (ii) a reduction in tumor size; (iii) to some extent inhibit, retard, slow down, and preferably stop the infiltration of cancer cells into peripheral organs; (iv) inhibit (i.e., to some extent slow down and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of a tumor; and / or (vii) to some extent alleviate one or more cancer-related symptoms. An effective amount can be administered in one or more administrations.

[0138] The term "administer" or "cause to be administered" refers to the action of a medical professional (e.g., a physician) or a person controlling the medical care of a patient to control and / or permit the administration of the agent / compound under discussion to the patient. Causing administration can include diagnosing and / or determining an appropriate treatment regimen, and / or prescribing a particular agent / compound for the patient. Such prescribing can include, for example, drafting a prescription form, annotating medical records, etc. "Causing administration" is also contemplated when administration is described herein.

[0139] The terms "patient", "individual", and "subject" are used interchangeably and refer to a mammal, preferably a human or non-human primate, but also to domestic mammals (e.g., canines or felines), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., equines, bovines, pigs, ovine). In various embodiments, the patient can be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other healthcare worker in a hospital, a psychiatric care facility such as an outpatient clinic, or other clinical setting. In various embodiments, the patient can be an immunocompromised patient or a patient with a weakened immune system, including but not limited to patients with primary immunodeficiencies, AIDS; cancer patients and transplant patients taking certain immunosuppressive drugs; and patients with genetic diseases affecting the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency). In various embodiments, the patient has an immunogenic cancer, including but not limited to bladder cancer, lung cancer, melanoma, and other cancers reported to have a high mutation rate (Lawrence et al., Nature, 499(7457):214-218, 2013).

[0140] As used herein, the term "Fc domain" or "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region includes the IgG CH2 and IgG CH3 domains. The CH3 region herein can be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced "protuberance" ("knob") in one of its chains and a corresponding introduced "cavity" ("hole") in its other chain; see U.S. Patent No. 5,821,333, which is hereby expressly incorporated by reference). Such variant CH3 domains can be used to facilitate heterodimerization of two different immunoglobulin heavy chains as described herein. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system.

[0141] As used herein, the term "effector function" refers to those biological activities that can be attributed to the Fc region of an immunoglobulin, which varies with immunoglobulin isotype. Examples of immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, antigen uptake by immune complex-mediated antigen-presenting cells, downregulation of cell surface receptors (such as the B cell receptor), and B cell activation.

[0142] As used herein, the term "regulatory T cell" or "Treg cell" refers to a specialized type of CD4+ T cell that can inhibit the responses of other T cells (effector T cells). Treg cells are characterized by the expression of CD4, the α subunit of the interleukin-2 receptor (CD25), and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)), and play a key role in inducing and maintaining peripheral self-tolerance to antigens, including antigens expressed by tumors.

[0143] As used herein, the term "conventional CD4+ T cell" refers to a CD4+ T cell other than a regulatory T cell.

[0144] As used herein, the term "selective activation of Treg cells" refers to the activation of Treg cells that is substantially not accompanied by the activation of other T cell subsets, such as CD4+ helper T cells, CD8+ cytotoxic T cells, or natural killer (NK) cells. Methods for identifying and differentiating these cell types are described in the Examples. Activation can include induction of interleukin-2 receptor signaling (measured, for example, by detecting phosphorylated STAT5), induction of proliferation (measured, for example, by detecting Ki-67), and / or upregulation of the expression of activation markers, such as CD25, as well as expansion of cell numbers.

[0145] As used herein, "specifically binds" means that the binding to an antigen is selective and can be distinguished from unwanted or non-specific interactions. The ability of an immunoglobulin to bind a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology.

[0146] As used herein, the term "affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (such as an antibody) and its binding partner (such as an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (koff and kon, respectively). A particular method for measuring affinity is surface plasmon resonance (SPR).

[0147] As used herein, the term "reduced binding" refers to a decrease in the affinity of the respective interaction, as measured, for example, by SPR. In contrast, "increased binding" refers to an increase in the binding affinity of the respective interaction.

[0148] As used herein, the term "polymer" generally includes, but is not limited to, homopolymers; copolymers, such as, for example, block, graft, random, and alternating copolymers; and terpolymers; as well as mixtures and modifications thereof. Additionally, unless otherwise explicitly defined, the term "polymer" shall include all possible geometric configurations of the material. These configurations include, but are not limited to, isotactic, syndiotactic, and atactic symmetries.

[0149] "Polynucleotide" refers to a polymer containing nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs. Nucleic acid analogs include those containing: non-naturally occurring bases, nucleotides that engage with other nucleotides via linkages other than the naturally occurring phosphodiester bond, or nucleotides containing bases attached via linkages other than the phosphodiester bond. Thus, nucleotide analogs include, for example and without limitation, phosphorothioates, dithiophosphates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using automated DNA synthesizers. The term "nucleic acid" generally refers to large polynucleotides. The term "oligonucleotide" generally refers to short polynucleotides, usually no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes the RNA sequence (i.e., A, U, G, C) in which "U" replaces "T".

[0150] This text uses conventional symbols to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is called the 5'-direction. The direction in which nucleotides are added to a nascent RNA transcript from 5' to 3' is called the transcription direction. The DNA strand having the same sequence as the mRNA is called the "coding strand"; the sequence at the 5' that is on the DNA strand having the same sequence as the mRNA transcribed from that DNA and is located 5' of the 5'-end of the RNA transcript is called the "upstream sequence"; the sequence at the 3' that is on the DNA strand having the same sequence as the RNA and is at the 3'-end of the coding RNA transcript is called the "downstream sequence".

[0151] "Complementary" refers to the topological compatibility or fitting together of the interacting surfaces of two polynucleotides. Thus, the two molecules can be described as complementary, and further, the contact surface features are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially the same as the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize with the second polynucleotide under stringent hybridization conditions.

[0152] "Specific hybridization with", "specific hybridization", or "selective hybridization with" means that when a specific nucleotide sequence is present in a complex mixture (e.g., total cellular) DNA or RNA, the nucleic acid molecule preferentially binds, duplexes, or hybridizes with that sequence under stringent conditions. The term "stringent conditions" refers to conditions under which a probe will preferentially hybridize with its target sequence and to a lesser extent with other sequences or not at all with other sequences. In the context of nucleic acid hybridization experiments such as DNA hybridization and RNA hybridization, "stringent hybridization" and "stringent hybridization wash conditions" are sequence-dependent and vary under different environmental parameters. Extensive guidance on nucleic acid hybridization can be found in: Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, N.Y.; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3rd ed. Suppl., NY; and Ausubel et al., eds., current ed., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

[0153] Typically, highly stringent hybridization and wash conditions are selected to be about 5 °C lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength and pH. The Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe (under defined ionic strength and pH conditions). Very stringent conditions are selected to be equal to the Tm of a particular probe. An example of stringent hybridization conditions for hybridizing complementary nucleic acids having more than about 100 complementary residues on a filter in a Southern or Northern blot is hybridization overnight at 42 °C in 50% formamide with 1 mg heparin. An example of highly stringent wash conditions is washing at 72 °C in 0.15 M NaCl for about 15 minutes. An example of stringent wash conditions is washing at 65 °C in 0.2x SSC for 15 minutes. See Sambrook et al. for a description of SSC buffer. Low stringency washes can be performed to remove background probe signal, followed by high stringency washes. An exemplary moderately stringent wash for a duplex of more than about 100 nucleotides, for example, is washing at 45 °C in 1x SSC for 15 minutes. An exemplary low stringency wash for a duplex of more than about 100 nucleotides, for example, is washing at 40 °C in 4 - 6x SSC for 15 minutes. Typically, in a particular hybridization assay, a signal - to - noise ratio of 2x (or higher) compared to that observed for an unrelated probe indicates specific hybridization detected.

[0154] A "primer" refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a starting point for the synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions that induce synthesis (i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as DNA polymerase). Primers are typically single - stranded, but can be double - stranded. Primers are generally deoxyribonucleic acids, but many synthetic and naturally occurring primers can be used for many applications. A primer is complementary to the template, and the primer is designed to hybridize to the template to serve as a site for the start of synthesis, but does not need to reflect the exact sequence of the template. In such cases, the specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, for example, chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.

[0155] When used in reference to polynucleotide usage, a "probe" refers to a polynucleotide that is capable of specifically hybridizing to a designated sequence of another polynucleotide. A probe specifically hybridizes to a target complementary polynucleotide, but does not need to reflect the exact complementary sequence of the template. In such cases, the specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. Probes can be labeled with, for example, chromogenic, radioactive, or fluorescent moieties and used as detectable moieties. In cases where the probe provides a starting point for the synthesis of a complementary polynucleotide, the probe can also be a primer.

[0156] "Vector" refers to a polynucleotide that can be used to introduce another nucleic acid linked thereto into a cell. One type of vector is a "plasmid", which refers to a linear or circular double-stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), into which additional DNA segments can be introduced into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell and are thereby replicated along with the host genome. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide.

[0157] "Regulatory sequence" refers to a nucleic acid that affects the expression (e.g., level, timing, or location of expression) of a nucleic acid operably linked thereto. A regulatory sequence can, for example, act directly on the nucleic acid being regulated or act through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and / or the nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Additional examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. and Baron et al., 1995, Nucleic Acids Res. 23: 3605-06. A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression of the nucleotide sequence (e.g., level, timing, or location of expression).

[0158] "Host cell" is a cell that can be used to express a polynucleotide of the present disclosure. The host cell can be a prokaryote, such as Escherichia coli (E. coli), or the host cell can be a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., human cell, monkey cell, hamster cell, rat cell, mouse cell, or insect cell), or a hybridoma. Generally, the host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, which can then be expressed in the host cell. The phrase "recombinant host cell" can be used to denote a host cell that has been transformed or transfected with the nucleic acid to be expressed. The host cell can also be a cell that contains the nucleic acid but does not express the nucleic acid at a desired level, unless regulatory sequences are introduced into the host cell such that the regulatory sequences become operably linked to the nucleic acid. It should be understood that the term host cell refers not only to a particular subject cell but also to the progeny or potential progeny of such a cell. Since certain modifications may occur in subsequent generations due to, for example, mutations or environmental influences, such progeny may not actually be identical to the parental cell but are still included within the scope of the term as used herein.

[0159] The term "isolated molecule" (where the molecule is, for example, a polypeptide or a polynucleotide) is a molecule that, by virtue of its origin or source of derivation, (1) is not associated with the components that are naturally associated with it in its natural state, (2) is substantially free of other molecules from the same species, (3) is expressed by cells from a different species, or (4) does not exist in nature. Thus, a molecule that is chemically synthesized or expressed in a cell system different from the cell of its natural origin will be "isolated" from its naturally associated components. Molecules can also be isolated by using purification techniques well known in the art to render the molecule substantially free of naturally associated components. The purity or homogeneity of a molecule can be determined by many means well known in the art. For example, the purity of a polypeptide sample can be determined using techniques well known in the art using polyacrylamide gel electrophoresis and staining the gel to visualize the polypeptide. For certain purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification.

[0160] A protein or polypeptide is "substantially pure", "substantially homogeneous" or "substantially purified" when at least about 60% to 75% of the sample exhibits a single species of polypeptide. The polypeptide or protein can be monomeric or polymeric. A substantially pure polypeptide or protein will generally comprise about 50%, 60%, 70%, 80% or 90% w / w of the protein sample, more typically about 95% and preferably will exceed 99% pure. Protein purity or homogeneity can be indicated by many means well known in the art, such as polyacrylamide gel electrophoresis of the protein sample followed by visualization of individual polypeptide bands after staining the gel with a stain well known in the art. For certain purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification.

[0161] As used herein, the term "label" or "labeled" refers to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable marker, such as a polypeptide incorporating a radiolabeled amino acid or attached to a biotin moiety that can be detected by labeled avidin (e.g., streptavidin incorporating a fluorescent marker or an enzyme activity that can be detected by optical methods or calorimetry). In another embodiment, the label or marker can be therapeutic, such as a drug conjugate or toxin. A variety of methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131I); fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores); enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a second reporter (e.g., leucine zipper pairing sequences, binding sites of second antibodies, metal binding domains, epitope tags); magnetic agents, such as gadolinium chelates; toxins such as pertussis toxin, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogs or homologs. In various embodiments, the label is attached via spacer arms of various lengths to reduce potential steric hindrance.

[0162] As used herein, the term "heterologous" refers to a combination or state that is not natural or does not exist in nature, which combination or state can be achieved, for example, by replacing existing natural components or states with components or states from another source. Similarly, protein expression in an organism other than the organism in which the protein is naturally expressed constitutes a heterologous expression system and a heterologous protein.

[0163] It should be understood that aspects and embodiments of the present disclosure described herein include "consisting of these aspects and embodiments" and / or "consisting essentially of these aspects and embodiments".

[0164] As used herein, the recitation of "about" a value or parameter includes (and describes) variations that are directed to that value or parameter itself. For example, the recitation of "about X" includes the description of "X".

[0165] Unless the context clearly dictates otherwise, the singular forms "a", "or", and "the" as used herein and in the appended claims include plural referents. It should be understood that aspects and variations of the present disclosure described herein include "consisting of these aspects and variations" and / or "consisting essentially of these aspects and variations".

[0166] IL - 2

[0167] Interleukin-2 (IL-2) is a classical Th1 cytokine produced by T cells upon activation through the T cell antigen receptor and the co-stimulatory molecule CD28. Regulation of IL-2 occurs through signal transduction pathways and activation of transcription factors acting on the IL-2 promoter to generate new gene transcription, but also involves regulation of the stability of IL-2 mRNA. IL-2 binds to a multi-chain receptor, including a highly regulated α chain that mediates signal transduction through the Jak-STAT pathway, as well as β and γ chains. IL-2 delivers activation, growth, and differentiation signals to T cells, B cells, and NK cells. IL-2 is also important in mediating the cell death of activated-induced T cells, a function that provides a key mechanism for terminating the immune response. An unglycosylated recombinant human IL-2 product, aldesleukin (available from Prometheus Laboratories Inc., San Diego, Calif. as des-alanyl-1, serine-125 human interleukin-2 under the trademark ), has been approved for administration to patients with metastatic renal cell carcinoma and metastatic melanoma. IL-2 has also been proposed for use in patients with or infected with hepatitis C virus (HCV), human immunodeficiency virus (HIV), acute myeloid leukemia, non-Hodgkin lymphoma, cutaneous T cell lymphoma, juvenile rheumatoid arthritis, atopic dermatitis, breast cancer, and bladder cancer. Unfortunately, the short half-life and severe toxicity limit the optimal administration of IL-2.

[0168] As used herein, the terms "native IL-2" and "native interleukin-2" in the context of a protein or polypeptide refer to any naturally occurring mammalian interleukin-2 amino acid sequence, including immature or precursor forms and mature forms. Non-limiting examples of GenBank accession numbers for the amino acid sequences of native mammalian interleukin-2 for various species include NP_032392.1 (Mus musculus, immature form), NP_001040595.1 (Macaca mulatta, immature form), NP_000577.2 (human, precursor form), CAA01199.1 (human, immature form), AAD48509.1 (human, immature form), and AAB20900.1 (human). In various embodiments of the present invention, native IL-2 is an immature or precursor form of naturally occurring mammalian IL-2. In other embodiments, native IL-2 is a mature form of naturally occurring mammalian IL-2. In various embodiments, native IL-2 is a precursor form of naturally occurring human IL-2. In various embodiments, native IL-2 is a mature form of naturally occurring human IL-2. In various embodiments, IL-2 is derived from the amino acid sequence of the human IL-2 precursor sequence set forth in SEQ ID NO:1:

[0169]

[0170] In various embodiments, IL-2 comprises the amino acid sequence of the wild-type sequence of the mature form of human IL-2 containing a cysteine to serine substitution at position 125 set forth in SEQ ID NO:3, but does not alter IL-2 receptor binding as compared to naturally occurring IL-2:

[0171]

[0172] IL - 2 variant

[0173] The present invention relates to polypeptides that share the primary sequence with human IL-2 except for one to several mutated amino acids. A group of IL-2 variants contain mutations that preferentially promote the proliferation, survival, activation, and / or function of immunosuppressive regulatory T cells (T CD4+CD25+FoxP3+) relative to effector T cells and NK cells. Also included is the therapeutic use of such IL-2 selective agonists alone or in combination with disease tissue-targeting proteins or peptides to treat Treg cell-deficient autoimmune disorders and various inflammatory disorders. In another aspect, the present invention relates to pharmaceutical compositions comprising the disclosed polypeptides. Finally, the present invention relates to the therapeutic use of the disclosed polypeptides and pharmaceutical compositions due to their selective regulatory effects on the immune system against diseases such as autoimmune and inflammatory disorders or cancer and various infectious diseases.

[0174] The present invention relates to polypeptides having a length of 100 to 500 amino acids, preferably a size of 140 residues, and an apparent molecular weight of at least 15 kD. These polypeptides maintain a high sequence identity of more than 90% with native IL-2. At these positions, these polypeptides are mutated to introduce amino acid residues different from those at the same positions in native IL-2.

[0175] The polypeptides of the present invention may be referred to as immunomodulatory polypeptides, IL-2 analogs, IL-2 variants, and other names. These polypeptides are designed based on the 3D structure of the IL-2 receptor complex (available in the PDB public database), and mutations are mainly introduced at the IL-2 positions corresponding to the amino acids that interact with the receptor subunits β or γ or βγ.

[0176] In various embodiments, the IL-2 variant (or mutant) comprises a sequence derived from the mature human IL-2 polypeptide sequence as set forth in SEQ ID NO:3. In various embodiments, the IL-2 variant comprises an amino acid sequence that is different from the native (or wild-type) IL-2 protein. In various embodiments, the IL-2 variant binds to the IL-2Rα polypeptide and functions as an IL-2 agonist or antagonist. In various embodiments, the IL-2 variant with agonist activity has superagonist activity. In various embodiments, the IL-2 variant can function as an IL-2 agonist or antagonist independent of its association with IL-2Rα. IL-2 agonists are exemplified by comparable or increased bioactivity compared to wild-type IL-2. IL-2 antagonists are exemplified by decreased bioactivity compared to wild-type IL-2 or by the ability to inhibit IL-2-mediated responses. In various embodiments, compared to the native IL-2 sequence, the sequence of the IL-2 variant has at least one amino acid change, such as a substitution or deletion, and such a change results in IL-2 agonist or antagonist activity. In various embodiments, the IL-2 variant as an Fc fusion protein has the amino acid sequences listed in SEQ ID NOs: 4-43, 108-146, and 193-197, with reduced binding to IL-2Rβ and / or γ C and enhanced selectivity for activating and proliferating regulatory T cells (Tregs).

[0177] Exemplary IL-2 variants are provided in Tables 2A - 2F:

[0178] Table 2A IL - 2 single mutations targeting both the IL - 2Rβ interface and the proposed toxicity motif

[0179]

[0180] Table 2B IL-2 single mutations targeting the IL-2Rβ interface

[0181]

[0182] Table 2C IL - 2 single mutations targeting the γc receptor interface

[0183]

[0184] Table 2D IL-2 mutation combinations targeting both the IL-2Rβ interface and the proposed toxicity motif

[0185]

[0186] Table 2E IL - 2 mutation combinations targeting the IL - 2Rβ, γc interfaces and the proposed toxicity motif

[0187]

[0188]

[0189] Table 2F

[0190] Improving the manufacturability of the fusion protein and targeting the IL-2Rβ and / or γc interfaces and single IL-2 mutations or combinations of IL-2 mutations of the proposed toxicity motifs

[0191]

[0192] The present invention also includes additional modifications to the above-mentioned classes of IL-2 variants, particularly additional modifications to the IL-2 variants described in Tables 2A-2F. As can be understood by a person skilled in the art, additional combinatorial mutants that combine the preferred mutations described in Tables 2A-2F can produce more Treg cell-selective IL-2 agonists. Any additional combinatorial mutations are within the spirit and scope of the present invention, whether increasing their affinity for specific components of the IL-2 receptor or improving their in vivo pharmacodynamics: increasing the half-life or reducing their internalization by T cells. These additional mutations can be obtained by rational design using bioinformatics tools or by using combinatorial molecular libraries of different natures (phage libraries, gene expression libraries in yeast or bacteria). In another aspect, the present invention relates to a fusion protein comprising any of the above-described immunomodulatory polypeptides conjugated to a carrier protein. The carrier protein can be albumin or the Fc region of human immunoglobulin.

[0193] Fc domain

[0194] Immunoglobulins of the IgG class are among the most abundant proteins in human blood. Their circulatory half-life can be up to 21 days. Fusion proteins combining the Fc region of IgG with domains of another protein (such as various cytokines and receptors) have been reported (see, for example, Capon et al., Nature, 337:525-531, 1989; Chamow et al., Trends Biotechnol., 14:52-60, 1996); U.S. Patents Nos. 5,116,964 and 5,541,087). The prototype fusion protein is a homodimeric protein linked by a cysteine residue in the hinge region of IgG Fc, producing a molecule similar to an IgG molecule without the heavy chain variable region and CH1 domain and the light chain. The dimeric nature of fusion proteins containing the Fc domain may be advantageous in providing higher-order interactions with other molecules (i.e., bivalent or bispecific binding). Due to structural homology, Fc fusion proteins exhibit in vivo pharmacokinetic profiles comparable to those of human IgG with similar isotypes.

[0195] The term "Fc" refers to a molecule or sequence that comprises the sequence of a non-antigen-binding fragment of a full antibody, whether in monomeric or multimeric form. The original immunoglobulin source of native Fc is preferably of human origin and can be any immunoglobulin, although IgG1 and IgG2 are preferred. Native Fc consists of monomeric polypeptides that can be joined into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent associations. The number of intermolecular disulfide bonds between the monomeric subunits of a native Fc molecule ranges from 1 to 4 depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). An example of native Fc is the disulfide-bonded dimer produced by papain digestion of IgG (see Ellison et al. (1982), Nucleic Acids Res. 10:4071-9). The term "native Fc" as used herein is a collective term for monomeric, dimeric, and multimeric forms. The Fc domain contains binding sites for protein A, protein G, various Fc receptors, and complement proteins.

[0196] In various embodiments, the term "Fc variant" refers to a molecule or sequence that is modified from native Fc but still contains the binding site for the salvage receptor FcRn. International applications WO 97 / 34631 (published September 25, 1997) and WO 96 / 32478 describe exemplary Fc variants and their interaction with the salvage receptor, which are hereby incorporated by reference. In addition, native Fc contains sites that can be removed because they provide structural features or biological activities that are not required for the fusion molecules of the present invention. Thus, in various embodiments, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues that affect or participate in (1) disulfide bond formation, (2) incompatibility with the selected host cell, (3) N-terminal heterogeneity upon expression in the selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor, or (7) antibody-dependent cell cytotoxicity (ADCC).

[0197] The term "Fc domain" includes native Fc and Fc variant molecules and sequences as defined above. Like Fc variants and native Fc, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from a full antibody or produced by recombinant gene expression or by other means. In various embodiments, the "Fc domain" refers to a dimer of two Fc domain monomers (SEQ ID NO:44), which typically includes all or part of the hinge region. In various embodiments, the Fc domain can be mutated to lack effector function. In various embodiments, each Fc domain monomer in the Fc domain contains amino acid substitutions in the CH2 antibody constant domain to reduce the interaction or binding between the Fc domain and the Fcγ receptor. In various embodiments, each subunit of the Fc domain contains two amino acid substitutions that reduce binding to activating Fc receptors and / or effector function, wherein the amino acid substitutions are L234A and L235A. In various embodiments, each subunit of the Fc domain contains three amino acid substitutions that reduce binding to activating Fc receptors and / or effector function, wherein the amino acid substitutions are L234A, L235A, and G237A (SEQ ID NO:45).

[0198] In various embodiments, each of the two Fc domain monomers in the Fc domain contains an amino acid substitution that promotes heterodimerization of the two monomers. In various other embodiments, heterodimerization of the Fc domain monomers can be promoted by introducing different but compatible substitutions such as "knob-into-hole" residue pairs in the two Fc domain monomers. The "knob-into-hole" technique is also disclosed in U.S. Patent Publication No. 8,216,805. In yet another embodiment, one Fc domain monomer contains the knob mutation T366W, and the other Fc domain monomer contains the hole mutations T366S, L358A, and Y407V. In various embodiments, two Cys residues are introduced that form a stabilizing disulfide bridge (S354C on the "knob" side and Y349C on the "hole" side). The use of heterodimeric Fc can produce a monovalent IL-2 variant.

[0199] In various embodiments, the Fc domain can be mutated to further extend the in vivo half-life. In various embodiments, each subunit of the Fc domain contains three amino acid substitutions that enhance binding to human FcRn, wherein the amino acid substitutions are M252Y, S254T, and T256E (SEQ ID NO: 156), as disclosed in U.S. Patent Publication No. 7,658,921. In various embodiments, each subunit of the Fc domain contains one amino acid substitution that enhances binding to human FcRn, wherein the amino acid substitution is N434A, as disclosed in U.S. Patent Publication No. 7,371,826. In various embodiments, each subunit of the Fc domain contains one amino acid substitution that enhances binding to human FcRn, wherein the amino acid substitutions are M428L and N434S, as disclosed in U.S. Patent Publication No. 8,546,543. In various embodiments, the half-life extending mutations can be combined with amino acid substitutions that reduce binding to activating Fc receptors and / or effector functions.

[0200] In various embodiments, the Fc domain sequence for preparing the IL-2 variant Fc fusion is the human IgG1-Fc domain sequence listed in SEQ ID NO: 45 with reduced / eliminated effector functions:

[0201]

[0202] wherein SEQ ID NO: 45 contains amino acid substitutions (underlined) that eliminate FcγR and C1q binding.

[0203] In various embodiments, the Fc domain sequence for preparing the IL-2 variant Fc fusion is the IgG1-Fc domain with extended half-life and reduced / eliminated effector functions listed in SEQ ID NO: 46

[0204]

[0205] wherein SEQ ID NO: 46 contains amino acid substitutions (underlined) that eliminate FcγR and C1q binding and substitutions (bold) that extend the serum half-life of the fusion protein.

[0206] In various embodiments, the Fc domain sequence for preparing the IL-2 variant Fc fusion is the IgG1-Fc domain with reduced / eliminated effector functions and extended half-life and has the amino acid sequence listed in SEQ ID NO: 47

[0207]

[0208] Among them, SEQ ID NO:47 contains amino acid substitutions (underlined) that eliminate FcγR and C1q binding and substitutions (bold) that extend the serum half-life of the fusion protein.

[0209] In various embodiments, the heterodimeric Fc domain sequence used to prepare the IL-2 variant Fc fusion is the Knob-Fc domain sequence with an extended in vivo half-life listed in SEQ ID NO:212:

[0210]

[0211] Among them, SEQ ID NO:212 contains amino acid substitutions (underlined) that eliminate FcγR and C1q binding and amino acid substitutions (bold) that extend the half-life.

[0212] In various embodiments, the heterodimeric Fc domain sequence used to prepare the IL-2 variant Fc fusion is the Hole-Fc domain sequence with an extended in vivo half-life listed in SEQ ID NO:213:

[0213]

[0214] Among them, SEQ ID NO:213 contains amino acid substitutions (underlined) that eliminate FcγR and C1q binding and amino acid substitutions (bold) that extend the half-life.

[0215] Linker

[0216] In various embodiments, the heterologous protein is attached to the IL-2 variant via a linker and / or a hinge linker peptide. The linker or hinge linker can be an artificial sequence between 5, 10, 15, 20, 30, 40 or more amino acids that is relatively free of secondary structure or exhibits an α-helical conformation.

[0217] Peptide linkers provide covalent connections and additional structural and / or spatial flexibility between protein domains. As is known in the art, peptide linkers contain flexible amino acid residues such as glycine and serine. In various embodiments, the peptide linker can contain 1-100 amino acids. In various embodiments, the spacer can contain the motif GGGSGGGS (SEQ ID NO:55). In other embodiments, the linker can contain the motif (GGGGGS)n, where n is an integer from 1 to 10. In other embodiments, the linker can also contain amino acids other than glycine and serine. In another embodiment, the linker can contain other protein motifs, including but not limited to sequences in an α-helical conformation, such as AEAAAKEAAAKEAAAKA (SEQ ID NO:53). In various embodiments, the linker length and composition can be adjusted to optimize activity or developability, including but not limited to expression level and aggregation propensity. In another embodiment, the peptide linker can be a simple chemical bond, such as an amide bond (e.g., by chemical conjugation of PEG).

[0218] Exemplary peptide linkers are provided in Table 3:

[0219] Table 3

[0220] Linker sequence SEQ ID NO: GGGSGGGSGGGS 48 GGGS 49 GSSGGSGGSGGSG 50 GSSGT 51 GGGGSGGGGSGGGS 52 AEAAAKEAAAKEAAAKA 53 GGGGSGGGGSGGGGSGGGGS 54 GGGSGGGS 55 GS 56 GGS 57 GGGGS 58 GGSG 59 SGGG 60 GSGS 61 GSGSGS 62 GSGSGSGS 63 GSGSGSGSGS 64 GSGSGSGSGSGS 65 GGGGSGGGGS 66 GGGGSGGGGSGGGGS 67

[0221] Polynucleotide

[0222] In another aspect, the present disclosure provides isolated nucleic acid molecules that comprise polynucleotides encoding the IL-2, IL-2 variants, IL-2 fusion proteins, or IL-2 variant fusion proteins of the present disclosure. The subject nucleic acids can be single-stranded or double-stranded. Such nucleic acids can be DNA or RNA molecules. DNA includes, for example, cDNA, genomic DNA, synthetic DNA, DNA amplified by PCR, and combinations thereof. Genomic DNA encoding the IL-2 polypeptide is obtained from genomic libraries that are available for many species. Synthetic DNA can be obtained by chemically synthesizing overlapping oligonucleotide fragments and then assembling the fragments to reconstruct part or all of the coding region and flanking sequences. RNA can be obtained from prokaryotic expression vectors that direct high-level synthesis of mRNA, such as vectors using the T7 promoter and RNA polymerase. cDNA is obtained from libraries prepared from mRNA isolated from various tissues expressing IL-2. The DNA molecules of the present disclosure include full-length genes as well as polynucleotides and fragments thereof. Full-length genes can also contain sequences encoding an N-terminal signal sequence. Such nucleic acids can be used, for example, in methods for preparing novel IL-2 variants.

[0223] In various embodiments, the isolated nucleic acid molecule comprises a polynucleotide described herein and further comprises a polynucleotide encoding at least one heterologous protein described herein. In various embodiments, the nucleic acid molecule further comprises a polynucleotide encoding a linker or hinge linker described herein.

[0224] In various embodiments, the recombinant nucleic acids of the present disclosure can be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory sequences are recognized in the art and are selected to direct the expression of the IL-2 variant. Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). Generally, the one or more regulatory nucleotide sequences can include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. The present disclosure contemplates constitutive or inducible promoters known in the art. The promoter can be a naturally occurring promoter or a hybrid promoter that combines elements of more than one promoter. The expression construct can be present on an episome such as a plasmid in a cell, or the expression construct can be inserted into a chromosome. In various embodiments, the expression vector contains a selectable marker gene to allow selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.

[0225] In another aspect of the present disclosure, the subject nucleic acid is provided in an expression vector that comprises a nucleotide sequence encoding an IL-2 variant and operably linked to at least one regulatory sequence. The term "expression vector" refers to a plasmid, phage, virus, or vector used for expressing a polypeptide from a polynucleotide sequence. Vectors suitable for expression in a host cell are readily available and utilize standard recombinant DNA techniques to insert nucleic acid molecules into the vector. Such vectors can include a variety of expression control sequences that, when operably linked to a DNA sequence, control the expression of that DNA sequence and can be used in these vectors to express the DNA sequence encoding the IL-2 variant. Such useful expression control sequences include, for example, the early and late promoters of SV40, the tet promoter, the adenovirus- or cytomegalovirus-mediated early promoters, the RSV promoter, the lac system, the trp system, the TAC or TRC systems, the T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage λ, the control region of the fd coat protein, the promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter of acid phosphatase such as PhoS, the promoter of yeast a-mating factor, the polyhedron promoter of the baculovirus system, and other sequences known to control gene expression in prokaryotic or eukaryotic cells or their viruses and various combinations thereof. It should be understood that the design of the expression vector may depend on factors such as the choice of host cell to be transformed and / or the type of protein desired to be expressed. In addition, consideration should also be given to 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. Exemplary expression vectors suitable for expressing vIL-2 are pDSRa (described in WO 90 / 14363, incorporated herein by reference) that contains the vIL-2 polynucleotide and its derivatives, and any other suitable vectors known in the art or described hereinafter.

[0226] The recombinant nucleic acids of the present disclosure can be produced by ligating a cloned gene or a portion thereof to a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, avian, insect, or mammalian), or both. Expression vectors for producing recombinant IL-2 polypeptides include plasmids and other vectors. For example, suitable vectors include the following types of plasmids: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotic cells such as Escherichia coli.

[0227] Some mammalian expression vectors contain both prokaryotic sequences that facilitate the replication of the vector in bacteria and one or more eukaryotic transcription units for expression in eukaryotic cells. Examples of mammalian expression vectors suitable for transfection of eukaryotic cells are vectors derived from pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg. Some of these vectors are modified with sequences from bacterial plasmids such as pBR322 to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Optionally, derivatives of viruses such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived, and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found in the description of gene therapy delivery systems below. The various methods employed in the preparation of plasmids and the transformation of host organisms are well known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells and for general recombinant procedures, see Chapters 16 and 17 of Molecular Cloning A Laboratory Manual, 2nd edition (Cold Spring Harbor Laboratory Press, 1989) by Sambrook, Fritsch, and Maniatis. In some instances, it may be desirable to express recombinant polypeptides by using a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as pBlueBac III containing B-gal).

[0228] In various embodiments, the vector will be designed to produce the subject IL-2 variants in CHO cells, such as the Pcmv-Script vector (Stratagene, La Jolla, Calif.), the pcDNA4 vector (Invitrogen, Carlsbad, Calif.), and the pCI-neo vector (Promega, Madison, Wis.). It will be apparent that the subject gene construct can be used to cause expression of the subject IL-2 variants in cells growing in culture, for example to produce proteins for purification, including fusion proteins or variant proteins.

[0229] The present disclosure also relates to host cells transfected with a recombinant gene comprising a nucleotide sequence encoding an amino acid sequence of one or more of the subject IL-2 variants. The host cell can be a prokaryotic cell or a eukaryotic cell. For example, the IL-2 variants of the present disclosure can be expressed in bacterial cells such as Escherichia coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.

[0230] Accordingly, the present disclosure also relates to methods for producing the subject IL-2 variants. For example, host cells transfected with an expression vector encoding an IL-2 variant can be cultured under appropriate conditions that permit expression of the IL-2 variant. The IL-2 variant can be secreted from the cells containing the IL-2 variant and isolated from a mixture of the cells containing the IL-2 variant and the culture medium. Optionally, the IL-2 variant can be retained in the cytoplasm or retained in the membrane fraction, and the cells can be harvested, lysed, and the protein isolated. Cell cultures include host cells, culture media, and other by-products. Suitable culture media for cell culture are well known in the art.

[0231] The polypeptides and proteins of the present disclosure can be purified according to protein purification techniques well known to those skilled in the art. These techniques involve, at one level, a crude fractionation of protein fractions and non-protein fractions. After separating the peptide or polypeptide from other proteins, chromatographic and electrophoretic techniques can be utilized to further purify the peptide or polypeptide of interest to achieve partial or complete purification (or purification to homogeneity). As used herein, the term "isolated polypeptide" or "purified polypeptide" is intended to refer to a composition separable from other components, wherein the polypeptide is purified to any degree relative to its naturally occurring state. Thus, a purified polypeptide also refers to a polypeptide removed from the environment in which it may naturally occur. Generally, "purified" will refer to a polypeptide composition that has undergone fractionation to remove various other components, and the polypeptide composition substantially retains its expressed biological activity. When the term "substantially purified" is used, this designation will refer to a peptide or polypeptide composition in which the polypeptide or peptide forms the majority of the components of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 85%, or about 90% or more of the proteins in the composition.

[0232] The various techniques applicable to purification will be well known to those skilled in the art. These techniques include, for example, precipitation with ammonium sulfate, PEG, antibodies (immunoprecipitation), etc., or precipitation by heat denaturation followed by centrifugation; chromatography, such as affinity chromatography (protein A column), ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, hydroxyapatite chromatography, hydrophobic interaction chromatography; isoelectric focusing; gel electrophoresis; and combinations of these techniques. As is generally known in the art, it is considered that the order in which the various purification steps are carried out can be varied, or certain steps can be omitted, and still a suitable method for preparing substantially purified polypeptides is obtained.

[0233] Pharmaceutical composition

[0234] In another aspect, the present disclosure provides a pharmaceutical composition comprising an IL-2 variant or an IL-2 variant fusion protein admixed with a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are well known and understood by those of ordinary skill in the art and have been widely described (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, ed., Mack Publishing Company, 1990). Pharmaceutically acceptable carriers can be included for the purpose of modifying, maintaining or preserving, e.g., pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, absorption or penetration of the composition. Such pharmaceutical compositions can affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the polypeptide. Suitable pharmaceutically acceptable carriers include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose or dextrin); proteins (such as serum albumin, gelatin or immunoglobulins); coloring agents; flavoring agents and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides (preferably sodium chloride or potassium chloride), mannitol and sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants).

[0235] The major vehicle or carrier in a pharmaceutical composition can be substantially aqueous or non-aqueous. For example, suitable vehicles or carriers can be water for injection, saline solution, or artificial cerebrospinal fluid, which may be supplemented with other materials commonly found in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are additional exemplary vehicles. Other exemplary pharmaceutical compositions contain a Tris buffer at about pH 7.0 - 8.5 or an acetate buffer at about pH 4.0 - 5.5, which may also contain sorbitol or a suitable alternative to sorbitol. In one embodiment of the present disclosure, a composition can be prepared by mixing a selected composition having a desired degree of purity with an optional formulation agent (Remington's Pharmaceutical Sciences, supra) for storage in the form of a lyophilized cake or an aqueous solution. Additionally, a therapeutic composition can be formulated as a lyophilized product using a suitable excipient such as sucrose. The optimal pharmaceutical composition will be determined by one of ordinary skill in the art depending on, for example, the intended route of administration, delivery form, and desired dose.

[0236] When parenteral administration is contemplated, the therapeutic pharmaceutical composition can be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired IL-2 polypeptide or IL-2 polypeptide fusion protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the polypeptide is formulated as a sterile, isotonic solution for suitable storage. In various embodiments, a pharmaceutical preparation suitable for injectable administration can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or physiological buffered saline. An aqueous injectable suspension can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, a suspension of the active compound can be prepared as a suitable oily injectable suspension. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compound and allow the preparation of highly concentrated solutions.

[0237] In various embodiments, a therapeutic pharmaceutical composition can be formulated for targeted delivery using colloidal dispersion systems. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, and lipid-based systems include oil-in-water emulsions, micelles, mixed micelles, and liposomes. Examples of lipids useful in liposome production include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Exemplary phospholipids include lecithin, dipalmitoyl phosphatidylcholine, and distearoyl phosphatidylcholine. Targeting of liposomes can also be based on, for example, organ specificity, cell specificity, and organelle specificity and is known in the art.

[0238] In various embodiments, oral administration of the pharmaceutical composition is contemplated. The pharmaceutical composition administered in this form can be formulated with or without the carriers commonly used in the compounding of solid dosage forms such as tablets and capsules. In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), one or more therapeutic compounds of the present disclosure can be admixed with one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or gum arabic; (3) humectants, such as glycerol; (4) disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution blockers, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical composition can also contain buffering agents. Similar types of solid compositions can also be used as fillers in soft-fill and hard-fill gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifying agents such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (particularly, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan and mixtures thereof. In addition to the inert diluent, the oral compositions can also contain adjuvants such as wetting agents, emulsifying agents, and suspending agents, sweetening agents, flavoring agents, coloring agents, perfuming agents, and preservatives.

[0239] In various embodiments, topical administration of the pharmaceutical composition to the skin or to mucous membranes is contemplated. The topical formulation may also contain one or more of a variety of agents known to be effective as skin or stratum corneum penetration enhancers. Examples of such agents are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methanol or isopropanol, dimethyl sulfoxide, and azone. Additional agents may also be included to make the formulation cosmetically acceptable. Examples of such agents are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surfactants. Keratolytic agents, such as those known in the art, may also be included. Examples are salicylic acid and sulfur. Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound can be mixed with a pharmaceutically acceptable carrier and with any preservatives, buffers, or propellants as may be required, under sterile conditions. In addition to the subject compounds of the present disclosure (e.g., IL-2 variants), ointments, pastes, creams, and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, and zinc oxide or mixtures thereof.

[0240] Additional pharmaceutical compositions contemplated for use herein include those that contain polypeptides in sustained release or controlled release formulations. Techniques for formulating various other sustained release or controlled release vehicles such as liposomal carriers, bioerodible microparticles or porous beads, and depot injections are also known to those of skill in the art.

[0241] The effective amount of the pharmaceutical composition to be used for treatment will depend, for example, on the treatment context and the treatment objective. Those skilled in the art will understand that the appropriate dosage level for treatment will thus vary in part depending on the molecule being delivered, the indication for which the polypeptide is used, the route of administration, and the size (body weight, body surface or organ size) and condition (age and general health) of the patient. Accordingly, the clinician can adjust the dosage and change the route of administration to obtain the optimal therapeutic effect. Typical dosages can range from about 0.001 mg / kg to up to about 100 mg / kg or more depending on the factors mentioned above. The polypeptide composition can preferably be administered by injection or intravenously. The long-acting pharmaceutical composition can be administered once every three to four days, once a week or once every two weeks depending on the half-life and clearance rate of the particular formulation. The dosing frequency will depend on the pharmacokinetic parameters of the polypeptide in the formulation used. Generally, the composition is administered until a dose is reached that achieves the desired effect. Thus, the composition can be administered as a single dose or as multiple doses over time (at the same or different concentrations / doses) or as a continuous infusion. Further refinement of the appropriate dosage is routinely carried out. The appropriate dosage can be determined by using appropriate dose-response data.

[0242] The route of administration of the pharmaceutical composition is according to known methods, for example oral; by intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional routes, intramedullary, intrathecal, intraventricular, percutaneous, subcutaneous or intraperitoneal or intratumoral injection; and intranasal, enteral, topical, sublingual, urethral, vaginal or rectal modalities; by sustained release systems or by implant devices. When desired, the composition can be administered by bolus injection, or continuously by infusion, or by implant device. Optionally or additionally, the composition can be administered locally by implantation of a membrane, sponge or another suitable material to which the desired molecule has been adsorbed or encapsulated. When using an implant device, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be by diffusion administration, sustained release bolus or continuous administration.

[0243] Therapeutic use

[0244] The present disclosure provides methods for treating autoimmune diseases in a subject, the methods comprising administering to the subject a therapeutically effective amount (as a monotherapy or in a combination therapy regimen) of an IL-2 variant or IL-2 variant fusion protein of the present disclosure in a pharmaceutically acceptable carrier. Autoimmune diseases associated with the present invention are diseases or disorders or their co-segregating lines or manifestations or conditions arising from and directed against an individual's own tissues. In various embodiments, autoimmune diseases include, but are not limited to, arthritis (including rheumatoid arthritis, reactive arthritis), systemic lupus erythematosus (SLE), psoriasis, and inflammatory bowel disease (IBD), encephalomyelitis, uveitis, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes, Addison's disease, celiac disease, chronic fatigue syndrome, autoimmune hepatitis, alopecia areata, ankylosing spondylitis, ulcerative colitis, Crohn's disease, fibromyalgia, pemphigus vulgaris, Sjogren's syndrome, Kawasaki disease, hyperthyroidism / Graves' disease, hypothyroidism / Hashimoto's disease, endometriosis, scleroderma, pernicious anemia, Goodpasture's syndrome, Guillain-Barré syndrome, Wegener's disease, glomerulonephritis, aplastic anemia (including aplastic anemia patients who have received multiple blood transfusions), paroxysmal nocturnal hemoglobinuria, myelodysplastic syndrome, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, Evan's syndrome, factor VIII inhibitor syndrome, systemic vasculitis, dermatomyositis, polymyositis, and rheumatic fever, autoimmune lymphoproliferative syndrome (ALPS), autoimmune bullous pemphigoid, Parkinson's disease, sarcoidosis, vitiligo, primary biliary cirrhosis, and autoimmune myocarditis.

[0245] In another aspect, the present disclosure provides methods for treating an inflammatory disease in a subject, the methods comprising administering to the subject a therapeutically effective amount (as a single therapy or in a combination therapy regimen) of an IL-2 variant or an IL-2 variant fusion protein of the present disclosure in a pharmaceutically acceptable carrier. "Inflammatory disease" includes all diseases associated with acute inflammation or various inflammations. Acute inflammation is the initial response of the body to a harmful stimulus and is caused by an increased movement of plasma and white blood cells (such as, for example, granulocytes) from the blood into the damaged tissue. Many biochemical events propagate and mature the inflammatory response, which involves the local vascular system, the immune system, and various cells within the damaged tissue. Chronic inflammation, which is long-term inflammation, results in a progressive change in the cell types present at the site of inflammation and is characterized by the simultaneous destruction and healing of tissue from the inflammatory process. In another aspect, the present disclosure provides methods for treating an inflammatory disease in a subject, the methods comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention. In one embodiment, the subject is a human subject. In various embodiments, the inflammatory diseases to be treated include, but are not limited to, Crohn's disease, colitis, dermatitis, psoriasis, diverticulitis, hepatitis, irritable bowel syndrome (IBS), lupus, nephritis, Parkinson's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behçet's syndrome, and indeterminate colitis multiple sclerosis (MS), Alzheimer's disease, arthritis, rheumatoid arthritis, asthma, and various cardiovascular diseases such as atherosclerosis and vasculitis. In various embodiments, the inflammatory disease is selected from the group consisting of rheumatoid arthritis, diabetes, gout, cryopyrin-associated periodic syndromes, and chronic obstructive pulmonary disease.

[0246] In another aspect, the present disclosure provides methods for organ transplantation or related graft-versus-host disease in a subject, the methods comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of the present invention. In one embodiment, the subject is a human subject. In various embodiments, the transplantation is an organ transplantation selected from the group consisting of heart, kidney, liver, lung, pancreas, intestine, and thymus, or a tissue transplantation selected from the group consisting of bone, tendon, cornea, skin, heart valve, nerve, and blood vessel. As used herein, the term "graft-versus-host disease" or "GvHD" refers to a condition, including acute and chronic, caused by the GVH-induced action of transplanted (graft) cells on host cells and tissues. In other words, donor immune cells within the infused graft or donor immune cells developed from stem cells may recognize the patient (host) cells as foreign and mount an immune response against them. Specifically, acute graft-versus-host disease (GvHD) is a disorder caused by donor immune cells in patients who have undergone allogeneic bone marrow or blood cell transplantation. The most commonly affected tissues are the skin, intestine, and liver. In severe cases, GvHD can cause blistering of the skin or excessive diarrhea and wasting. Prednisone and / or other immunosuppressive drugs are used to treat acute graft-versus-host disease.

[0247] The therapeutically effective dose can be initially evaluated by determining the EC 50 from cell culture assays. Then, the dose can be formulated in an animal model to achieve a circulating plasma concentration range that includes the EC 50 as determined in cell culture. Such information can be used to more precisely determine the dose useful in humans. The levels in plasma can be measured, for example, by HPLC. The exact composition, route of administration, and dose can be selected by the individual physician in view of the condition of the subject.

[0248] The dosage regimen can be adjusted to provide the optimum desired response (e.g., a therapeutic response or a prophylactic response). For example, a single bolus can be administered, several divided doses (multiple or repeated or maintenance) can be administered over time, and the dose can be proportionally decreased or increased as indicated by the exigencies of the therapeutic situation. To facilitate administration and dosage uniformity, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as a single dose for the mammalian subject to be treated; each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present disclosure will be determined primarily by the unique characteristics of the antibody and the particular therapeutic or prophylactic effect to be achieved.

[0249] Accordingly, those skilled in the art will understand that, based on the disclosure provided herein, the dosage and administration regimen are adjusted according to methods well known in the art of therapy. That is, the maximum tolerable dose can be readily determined, and the effective amount that provides a detectable therapeutic benefit to a subject can also be determined, as can the time requirements for administering each agent to provide a detectable therapeutic benefit to a subject. Accordingly, although certain dosages and administration regimens are exemplified herein, these examples are in no way limiting the dosages and administration regimens that can be provided to a subject in practicing the present disclosure.

[0250] It should be noted that the dosage values can vary with the type and severity of the condition to be alleviated and can include a single dose or more than one dose. It should also be understood that, for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering the composition or supervising the administration of the composition, and the dosage ranges listed herein are merely exemplary and are not intended to limit the scope of the claimed composition or practice. Additionally, the dosage regimen of the compositions of the present disclosure can be based on a number of factors, including the type of disease, the age, weight, sex, medical condition, severity of the condition, route of administration, and the particular antibody used of the subject. Accordingly, the dosage regimen can vary widely but can be routinely determined using standard methods. For example, the dosage can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which can include clinical effects such as toxic effects and / or experimental values. Accordingly, the present disclosure includes intra-subject dose-escalation as determined by those skilled in the art. Determining the appropriate dosage and regimen is well known in the relevant art and will be understood to be within the grasp of those skilled in the art once the teachings disclosed herein are provided.

[0251] Depending on the factors described above, an exemplary, non-limiting daily dosage range of a therapeutically or prophylactically effective amount of an IL-2 variant or IL-2 variant fusion protein of the present disclosure can range from about 0.1 μg / kg to 100 mg / kg or more. For repeated administration over several days or longer, depending on the condition, treatment will generally continue until suppression of the desired disease symptoms occurs. In various embodiments, the dosage will be in the range from about 0.0001 mg / kg to 100 mg / kg, 0.0001 mg / kg to 90 mg / kg, 0.0001 mg / kg to 80 mg / kg, 0.0001 mg / kg to 70 mg / kg, 0.0001 mg / kg to 60 mg / kg, 0.0001 mg / kg to 50 mg / kg, 0.0001 mg / kg to 40 mg / kg, 0.0001 mg / kg to 30 mg / kg, 0.0001 mg / kg to 20 mg / kg, 0.0001 mg / kg to 10 mg / kg, 0.0001 mg / kg to 5 mg / kg, 0.0001 mg / kg to 4 mg / kg, 0.0001 mg / kg to 3 mg / kg, 0.0001 mg / kg to 2 mg / kg, 0.0001 mg / kg to 1 mg / kg, 0.001 mg / kg to 50 mg / kg, 0.001 mg / kg to 40 mg / kg, 0.001 mg / kg to 30 mg / kg, 0.001 mg / kg to 20 mg / kg, 0.001 mg / kg to 10 mg / kg, 0.001 mg / kg to 5 mg / kg, 0.001 mg / kg to 4 mg / kg, 0.001 mg / kg to 3 mg / kg, 0.001 mg / kg to 2 mg / kg, 0.001 mg / kg to 1 mg / kg, 0.010 mg / kg to 50 mg / kg, 0.010 mg / kg to 40 mg / kg, 0.010 mg / kg to 30 mg / kg, 0.010 mg / kg to 20 mg / kg, 0.010 mg / kg to 10 mg / kg, 0.010 mg / kg to 5 mg / kg, 0.010 mg / kg to 4 mg / kg, 0.010 mg / kg to 3 mg / kg, 0.010 mg / kg to 2 mg / kg, 0.010 mg / kg to 1 mg / kg, 0.1 mg / kg to 50 mg / kg, 0.1 mg / kg to 40 mg / kg, 0.1 mg / kg to 30 mg / kg, 0.1 mg / kg to 20 mg / kg, 0.1 mg / kg to 10 mg / kg, 0.1 mg / kg to 5 mg / kg, 0.1 mg / kg to 4 mg / kg, 0.1 mg / kg to 3 mg / kg, 0.1 mg / kg to 2 mg / kg, 0.In the ranges of 1 mg / kg to 1 mg / kg, 1 mg / kg to 50 mg / kg, 1 mg / kg to 40 mg / kg, 1 mg / kg to 30 mg / kg, 1 mg / kg to 20 mg / kg, 1 mg / kg to 10 mg / kg, 1 mg / kg to 5 mg / kg, 1 mg / kg to 4 mg / kg, 1 mg / kg to 3 mg / kg, 1 mg / kg to 2 mg / kg, or 1 mg / kg to 1 mg / kg body weight, etc., administration can be based on the amounts described above. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof) can be administered to a patient. It should be noted that the dose values can vary with the type and severity of the condition to be alleviated. It should also be understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering the composition or supervising the administration of the composition, and the dosage ranges listed herein are merely exemplary and are not intended to limit the scope of the claimed composition or its practice.

[0252] The toxicity and therapeutic index of the pharmaceutical compositions of the present disclosure can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, e.g., determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic dose and the therapeutically effective dose is the therapeutic index, and the therapeutic index can be expressed as the ratio LD 50 / ED 50 . Compositions showing a large therapeutic index are generally preferred.

[0253] The dosing frequency of administration of the IL-2 variant or IL-2 variant fusion protein pharmaceutical composition depends on the nature of the therapy and the particular disease being treated. The subject can be treated at regular intervals, such as twice a week, weekly, or monthly, until the desired therapeutic outcome is achieved. Exemplary dosing frequencies include, but are not limited to: once a week without interruption; once every 2 weeks; once every 3 weeks; once a week without interruption for 2 weeks, then once a month; once a week without interruption for 3 weeks, then once a month; once a month; once every two months; once every 3 months; once every 4 months; once every 5 months; or once every 6 months, or once a year.

[0254] Combination therapy

[0255] As used herein, when referring to an IL-2 variant or an IL-2 variant fusion protein of the present disclosure and one or more other therapeutic agents, the terms "co-administration", "co-administered", and "in combination with" are intended to mean, and indeed do mean and include the following: such combinations of an IL-2 variant or an IL-2 variant fusion protein of the present disclosure and one or more therapeutic agents are administered simultaneously to a subject in need of treatment, wherein such components are formulated together into a single dosage form that releases the components to the subject at substantially the same time; such combinations of an IL-2 variant or an IL-2 variant fusion protein of the present disclosure and one or more therapeutic agents are administered substantially simultaneously to a subject in need of treatment, wherein such components are formulated separately into separate dosage forms that are taken by the subject at substantially the same time, whereby the components are released to the subject at substantially the same time; such combinations of an IL-2 variant or an IL-2 variant fusion protein of the present disclosure and one or more therapeutic agents are administered sequentially to a subject in need of treatment, wherein such components are formulated separately into separate dosage forms that are taken by the subject at successive times with a significant time interval between each administration, whereby the components are released to the subject at substantially different times; and, such combinations of an IL-2 variant or an IL-2 variant fusion protein of the present disclosure and one or more therapeutic agents are administered sequentially to a subject in need of treatment, wherein such components are formulated together into a single dosage form that releases the components in a controlled manner, whereby the components are released to the subject simultaneously, continuously, and / or overlappingly at the same and / or different times, wherein each portion may be administered by the same or different routes.

[0256] In another aspect, the present disclosure provides methods for treating an autoimmune disease in a subject, the methods comprising administering a therapeutically effective amount of the pharmaceutical composition of the invention in combination with a second therapeutic agent capable of treating an autoimmune disease. In various embodiments, the second therapeutic agent is selected from the group consisting of immunosuppressants such as corticosteroids, cyclosporine, cyclophosphamide, prednisone, azathioprine, methotrexate, rapamycin, tacrolimus, biologics such as TNF-α blockers or antagonists, IL-10 agonists or long half-life extended IL-10 (PEGylated, antibody or Fc fusion IL-10); immunosuppressive agents (e.g., antibodies against other lymphocyte surface markers (e.g., CD40, α-4 integrin) or antibodies against cytokines), other fusion proteins (e.g., CTLA-4-Ig (ORENCIA.RTM.), TNFR-Ig ), TNF-α blockers such as and cyclophosphamide (CTX) (i.e., ), methotrexate (MTX) (i.e., ), rituximab, belimumab (i.e., ), anti-IL-6 antibody (such as Sarilumab), or anti-IL-6 receptor antibody (such as Tocilizumab), or other immunosuppressive drugs (e.g., cyclosporine A, FK506-like compounds, rapamycin compounds or steroids), antiproliferative agents, cytotoxic agents, or other compounds that can assist immunosuppression, or any other biologic agent targeting any inflammatory cytokine, non-steroidal anti-inflammatory drugs / Cox-2 inhibitors, hydroxychloroquine, sulphasalazopryine, gold salts, etanercept, infliximab, mycophenolate mofetil, basiliximab, atacicept, rituximab, cyclophosphamide, interferon β-1a, interferon β-1b, glatiramer acetate, mitoxantrone hydrochloride, anakinra and / or other biologics and / or intravenous immunoglobulin (IVIG). Non-limiting examples of such known therapeutic agents include interferons, such as interferon-β-1a( and ) and IFN-β-1b the polypeptide glatiramer acetate Natalizumab and the cytotoxic agent mitoxantrone

[0257] In another aspect, the present disclosure provides a method for treating an inflammatory disease in a subject, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of the invention in combination with a second therapeutic agent that is capable of inhibiting or reducing the differentiation of Th1, Th17, Th22, and / or other cells (secreting or causing other cells to secrete inflammatory molecules, including but not limited to IL-1β, TNF-α, TGF-β, IFN-γ, IL-17, IL-6, IL-23, IL-22, IL-21, and MMP); inhibiting or reducing the activity of Th1, Th17, Th22, and / or other cells (secreting or causing other cells to secrete inflammatory molecules, including but not limited to IL-1β, TNF-α, TGF-β, IFN-γ, IL-17, IL-6, IL-23, IL-22, IL-21, and MMP); inhibiting or reducing the Th1 and / or Th17 pathways; inhibiting or reducing the cytokine production and / or secretion of Th1, Th17, Th22, and / or other cells (secreting or causing other cells to secrete inflammatory molecules, including but not limited to IL-1β, TNF-α, TGF-β, IFN-γ, IL-17, IL-6, IL-23, IL-22, IL-21, and MMP); inhibiting or reducing the proliferation of Th1, Th17, Th22, and / or other cells (secreting or causing other cells to secrete inflammatory molecules, including but not limited to IL-1β, TNF-α, TGF-β, IFN-γ, IL-17, IL-6, IL-23, IL-22, IL-21, and MMP). In various embodiments, the second therapeutic agent is a non-steroidal anti-inflammatory agent, including but not limited to, oxicams, such as piroxicam, isoxicam, tenoxicam, sudoxicam; salicylates, such as aspirin, disalcid, benorylate, trilisate, safapryn, solprin, diflunisal, and fendosal; acetic acid derivatives, such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acematacin, fentiazac, zomepirac, clmdanac,Oxepinac, felbmac, and ketorolac; fenamates such as mefenamic acid, meclofenamic acid, flufenamic acid, niflumic acid, and tolfenamic acid; propionic acid derivatives such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indopropfen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, and tiaprofenic acid; pyrazoles such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone, and trimethazone. Mixtures of these non-steroidal anti-inflammatory agents may also be used. In various embodiments, the second therapeutic agent is a steroidal anti-inflammatory drug including, but not limited to, corticosteroids such as hydrocortisone, hydroxyl-triamcinolone, alpha-methyl dexamethasone, dexamethasone phosphate, beclomethasone dipropionates, clobetasol valerate, desonide, desoxymethasone, desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorolone acetonide, fludrocortisone, flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortine butylesters, fluocortolone,Fluprednidene (fluprednylidene), flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, flurandrenolone, fludrocortisone, diflurosone diacetate, fluradrenolone acetonide, medrysone, amcinafel, amcinafide, betamethasone and its esters balance, chloroprednisone, chloroprednisone acetate, clocortelone, clescinolone, dichlorisone, diflurprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone, and mixtures thereof.

[0258] In various embodiments, the combination therapy includes co - administering an IL - 2 variant and a second agent composition in the same pharmaceutical composition or in separate pharmaceutical compositions. In various embodiments, the IL - 2 variant composition and the second agent composition are administered sequentially, i.e., the IL - 2 variant composition is administered before or after the second agent composition. In various embodiments, the administration of the IL - 2 variant composition and the second agent composition is simultaneous, i.e., the administration time periods of the IL - 2 variant composition and the second agent composition overlap with each other. In various embodiments, the administration of the IL - 2 variant composition and the second agent composition is non - simultaneous. For example, in various embodiments, the administration of the IL - 2 variant composition is terminated and then the second agent composition is administered. In various embodiments, the administration of the second agent composition is terminated and then the IL - 2 variant composition is administered.

[0259] The following examples are provided to more fully illustrate the present disclosure but should not be construed as limiting the scope of the present disclosure.

[0260] Example 1

[0261] Design of IL-2 variants selectively targeting Treg cells

[0262] In one aspect, the present invention relates to one or more mutations that reduce the affinity of IL-2 for the IL-2Rβ and / or γc receptor subunits. In the case of reduced IL-2Rβγ interaction, the enhanced IL-2 sensitivity of Tregs conferred by IL-2Rα expression can result in a significant growth advantage for this cell subset. Thus, in autoimmune and inflammatory diseases, these mutants can be used as Treg promoters.

[0263] These variants were computationally designed based on the structure of human IL-2 reported in the protein data bank (PDB code 2B5I). A set of variants was designed that contain 1 to 3 mutations (introducing conservative and non-conservative amino acid substitutions) in residues at or near the interface that makes direct contact with the IL-2Rβ or γc receptor subunits. For example, D20 is involved in an extensive hydrogen bond network with the side chains of receptor subunits at the IL-2Rβ interface. Similarly, N88 is an energy hot spot for the IL-2 / IL-2Rβ interaction and is involved in key hydrogen bonds with the receptor chain. Q126 is essential for γc interaction, and Q22 is similarly located at the γc interface. The inventors hypothesized that mutations at or near the above sites could result in a defect in their ability to interact with the IL-2 intermediate affinity receptor IL-2Rβγ.

[0264] Interestingly, the proposed ‘ 19 LDL’ motif (Baluna R, Rizo et al., Proc Natl Acad Sci 1999; 96:3957 - 62), similar to a bacterial toxin component, overlaps with the IL-2Rβ interface. This ‘toxic motif’ is partly responsible for the direct vascular toxicity of IL-2. Thus, replacement of the key toxic motif residue D20 or introduction of mutations that substitute non-aliphatic residues for the flanking residues L19 and L21 are also expected to abrogate the toxic motif and prevent endothelial cell damage, as well as significantly reduce VLS.

[0265] In the present invention, a group of IL-2 variants (SEQ ID NO: 4-43, 108-146, and 193-197) having the following 1-3 amino acid substitutions (D20T, D20E, D20N, D20Q, D20S, D20Y, D20I, L19Y, L19N, L19R, L19Q, L19H, L19D, L19P, L19S, L21S, L21N, L21R, N88R, N88G, N88I, N88Q, N88E, N88T, N88M, Q126E, Q126L, Q126N, Q126D, Q126M, Q126K, Q126H, Q126Y, Q125E, S125K, S125H, S125W, S125I, Q22N, Q22H, Q22K, Q22Y, Q22I, D20I / N88G, D20I / N88R, D20T / N88R, D20I / N88I, D20T / Q126E, D20T / N88R / Q126E, D20T / Q126L, D20T / N88R / Q126L, L19N / Q126E, L19R / Q126E, L19Y / Q126E, L19H / Q126E, L19Q / Q126E, L19S / Q126E, L19Y / Q126K, L19Y / Q126H, L19Y / Q126Y, L19Y / S125E, L19Y / S125K, L19Y / S125H, L19Y / S125W, L19Y / S125I, L19Y / Q22N, L19Y / Q22H, L19Y / Q22K, L19Y / Q22Y, L19Y / Q22I, L19H / Q126K, L19H / S125I, L19D / S125I, D20E / S25I, D20T / S125I, L19Y / S125I / Q126E, L19H / S125I / Q126E, L19H / S125I / Q126K, L19Q / S125I / Q126E, L19Q / Q126K, L19Q / S125I / Q126K, D20T / S125I / Q126K, L19N / S125I / Q126K, and L19R / S125I / Q126K) are expressed as C-terminal fusions with an Fc homodimer via a "GGGSGGGS" linker (SEQ ID NO: 55). IL-2 variants having D20I, D20I / N88G, D20E, or L19N amino acid substitutions are also expressed as N-terminal fusions with an Fc homodimer via a rigid "AEAAAKEAAAKEAAAKA" linker (SEQ ID NO: 53). Overall, the sequences of these IL-2 variant Fc fusion constructs are listed as SEQ ID NO: 73-107, 147-189, and 198-211.Constructs of wild - type IL - 2 in the same Fc - fusion form (both C - terminus and N - terminus) were also prepared (SEQ ID NO:71 and 72).

[0266] All of the above IL - 2 variant Fc - fusion molecules were designed to provide a growth advantage to cells highly expressing IL - 2Rα, resulting in preferential proliferation of Treg cells over other lymphocytes, including CD4 + conventional T cells, CD8 + T cells, and NK cells. Additionally, mutations at positions 19, 20, or 21 were expected to eliminate the toxic motifs that cause vascular toxicity, so the resulting molecules could have two beneficial properties, including enhanced selectivity for Treg activation and reduced endothelial cell damage. Nevertheless, the optimal mutation or combination of mutations is crucial for modulating the level of damage to effector T cells and NK cell activation while maintaining a high enough Treg cell activation potential and thus maximizing the window for selective targeting of the Treg subset.

[0267] In addition, single - point mutations have been identified that result in improved protein stability, higher expression levels, and lower aggregation propensity. The combination of reduced aggregation and Treg - targeting mutations leads to excellent protein developability and Treg cell growth advantage.

[0268] Example 2

[0269] Construction and production of IL - 2Fc - fusion constructs

[0270] All genes were codon - optimized for expression in mammalian cells, synthesized, and subcloned into a recipient mammalian expression vector (GenScript). Protein expression was driven by the CMV promoter, and a synthetic SV40 polyadenylation (polyA) signal sequence was present at the 3' end of the CDS. A leader sequence was engineered at the N - terminus of the construct to ensure proper signaling and processing for secretion.

[0271] Constructs were produced by co - transfecting suspension - growing HEK293 - F cells with the mammalian expression vector using polyethyleneimine (PEI, 25,000 MW linear, Polysciences). If there were two or more expression vectors, the vectors were transfected at a 1:1 ratio. For transfection, HEK293 cells were cultured in serum - free FreeStyle TM 293 expression medium (ThermoFisher). For production in 1000 ml shake flasks (working volume 330 mL), HEK293 cells were seeded at 0.8×10 6Inoculate at a density of cells / ml and perform transfection after 24 hours. Mix a total of 330 μg of DNA expression vector with 16.7 ml of Opti-mem medium (ThermoFisher). After adding 0.33 mg of PEI diluted in 16.7 ml of Opti-mem medium, vortex the mixture for 15 sec and then incubate at room temperature for 10 min. Then add the DNA / PEI solution to the cells and incubate in an incubator with 8% CO2 at 37 °C. On the 4th day, add sodium butyrate (Millipore Sigma) to the cells at a final concentration of 2 mg / L to help maintain protein expression. After culturing for 6 days, collect the supernatant for purification by centrifuging at 2200 rpm for 20 min. Sterile filter the solution (0.22 μm filter, Corning). Purify the secreted protein from the cell culture supernatant using protein A affinity chromatography.

[0272] For affinity chromatography, load each supernatant onto a HiTrap MabSelectSure column (CV = 5 mL, GE Healthcare) equilibrated with 25 ml of phosphate-buffered saline pH 7.2 (ThermoFisher). Remove unbound proteins by washing with 5 column volumes of PBS pH 7.2 and elute the target protein with 25 mM sodium citrate, 25 mM sodium chloride, pH 3.2. Neutralize the protein solution by adding 3% of 1 M Tris pH 10.2. Ion exchange chromatography or mixed-mode chromatography, including but not limited to CaptoMMC (GE Healthcare), ceramic hydroxyapatite or ceramic fluorapatite (Bio-Rad), is also used as needed to refine the protein A material. The target protein is concentrated with an Ultra-15 concentrator 10KDa NMWC (Merck Millipore Ltd.).

[0273] Analyze the purity and molecular weight of the purified construct by SDS-PAGE and staining with Coomassie (ImperialR Stain) in the presence and absence of a reducing agent. Use according to the manufacturer's instructions Precast gel system (4%-12% or 8%-16% Bis-Tris, ThermoFisher). The protein concentration of the purified protein sample was determined by measuring the UV absorbance at 280 nm (Nanodrop spectrophotometer, ThermoFisher) and dividing by the molar extinction coefficient calculated based on the amino acid sequence. The aggregate content of the construct was analyzed on an Agilent 1200 high performance liquid chromatography (HPLC) system. At 25 °C, using 150 mM sodium phosphate pH 7.0 as the mobile phase, the sample was injected onto an AdvanceBio size exclusion column ( 4.6 x 150 mm, 2.7 μm, LC column, Agilent).

[0274] Notably, the expression profiles and aggregation propensities of IL-2 variant Fc fusions were significantly different between constructs with different mutation sites or mutants sharing the same mutation site but with different residue substitutions.

[0275] Example 3

[0276] Single amino acid substitutions in IL-2 led to a general improvement in the developability of the fusion compounds

[0277] When applied to IL-2, engineering approaches to find mutation combinations that produce variant proteins with desired biological properties have faced significant challenges. It is known in the art that naturally occurring IL-2 proteins tend to be less stable and prone to aggregation. This was demonstrated in our experiments where wild-type IL-2Fc fusion protein (P-0250) was expressed at low levels (transiently expressed at approximately 3 mg / L in HEK-293F cells) and had a high aggregation propensity, as illustrated by the SEC chromatogram depicted in Figure 1A Engineering efforts were stymied because amino acid substitutions in IL-2 aimed at desired bioactivity often resulted in mutant proteins that were even less stable. A large portion of the IL-2 variants in the current work were expressed at extremely low levels, and some variants were significantly more prone to aggregation, as illustrated by the SEC chromatogram of P-0318 (SEQ ID NO:95) depicted in Figure 1B This is problematic for the preparation and storage of therapeutic agents.

[0278] It was also observed that the expression profiles and aggregation propensities of IL-2 variant Fc fusions were significantly different between constructs with different mutation sites or mutants sharing the same mutation site but with different residue substitutions. This observation was illustrated by Examples P-0317 (SEQ ID NO:94) and P-0318 (SEQ ID NO:95). The two variant fusions shared the same mutation sites at residues 20 and 88 and differed by only one amino acid. P-0317 contained the amino acid substitutions D20I and N88R, while P-0318 contained the D20I and N88I mutations. The two variant fusions were expressed at similar low levels. As observed in Figure 1B , P-0318 was highly prone to aggregation: 65% of the high molecular weight material, which made the expected peak a minor species in the chromatogram and was marked with an arrow. In contrast, P-0317 was relatively pure, with 7.5% aggregates ( Figure 1C ). It can be inferred that the N88R mutation can reduce the aggregation propensity of the resulting fusion protein. However, the fusion proteins P-0254 (SEQ ID NO:71) and P-0324 (SEQ ID NO:96) generated by the IL-2 with the N88R single mutation or the D20T / N88R double mutation, respectively, had an aggregation propensity of 30%-40% aggregates. Thus, the contribution of a single amino acid substitution to protein stability appears to depend on the background.

[0279] The fact that amino acid substitutions in IL-2 generally result in less stable proteins is further complicated by the unpredictable contribution of different residue substitutions to protein stability. Thus, it is highly desirable to find residue substitutions that can generally enhance protein developability, including improved stability, higher expression levels, and lower aggregation propensities.

[0280] The amino acid substitution at position 125 was initially designed to modulate IL-2 selectivity because this residue is adjacent to Q126, which is essential for γc interaction. Native IL-2 contains an unpaired cysteine at position 125, which is replaced by serine in Proleukin, and in the present invention, S125 is considered the wild-type IL-2 residue. IL-2 containing an alanine substitution at position 125 has also been widely used. Since the serine or alanine substitution for cysteine at position 125 retains all biological activity, bulky charged or hydrophobic residues including Glu, Lys, Try, His, and Ile were introduced at position 125 of P-0372 (SEQ ID NO:79) to replace Ser in an attempt to interfere with the interaction between Q126 and γc, thereby achieving altered biological activity. Except for P-0471 (SEQ ID NO:178), all of the resulting fusion molecules had too low expression levels to be characterized. In contrast, when P-0471 was compared to its S125 counterpart (P-0372), P-0471 was expressed at a significantly higher level (19.3 mg / L vs. 4.0 mg / L titer) and had a substantially reduced aggregation propensity (1% vs. 21.7% aggregation). The impressive improvement in developability, especially with respect to product purity, prompted us to evaluate whether this enhancement by the isoleucine substitution at position 125 could be reproduced in different mutant backgrounds.

[0281] Therefore, the S125I substitution was introduced into many IL-2 variant Fc fusion molecules. Constructs containing the Ile-125 substitution in IL-2 were expressed using the same vector and under the same culture conditions as their Ser-125 counterparts and were purified using MabSelectSure. The expression levels (mg / L) and purity evaluated as the aggregation % of exemplary molecules by SEC chromatography are summarized in Table 4. The two molecules in the same row of Table 4 share the same other amino acid substitutions and differ only in having serine or isoleucine at residue 125. For example, the SEC chromatograms and SDS-PAGE gels of P-0447 (SEQ ID NO:168) and its Ile-125 counterpart P-0511 (SEQ ID NO:198) are further illustrated in Figure 1D and Figure 1E It is clear from Table 4 that the isoleucine substitution at position 125 results in a 4- to 11-fold increase in expression level and consistently low aggregation propensity.

[0282] Table 4 125I substitution reduces aggregation and increases expression of various IL - 2 fusion proteins

[0283]

[0284] It is apparent from the present invention that the isoleucine substitution at position 125 results in a general improvement in the developability of the IL-2 fusion construct and fully retains the biological activity. The Ile substitution at position 125 of IL-2 variants with different mutant backgrounds in wild-type IL-2 and Fc fusion forms all led to a 4- to 11-fold increase in expression levels and consistently low aggregation propensity. The expression levels (mg / L) and the purity of the protein A-purified material evaluated by SEC chromatography as the aggregation % of exemplary molecules are summarized in Table 4. This finding is particularly valuable because the fact that altering the critically stable wild-type IL-2 usually results in even more unstable mutant proteins has hampered the engineering of IL-2 for desired biological properties. The inherent challenges of IL-2 engineering can be alleviated by a single amino acid substitution with isoleucine at position 125. In summary, the isoleucine substitution at position 125 of IL-2 or an IL-2 variant significantly improves the developability profile of the protein, as demonstrated by increased protein expression and a substantial reduction in the aggregation propensity of the IL-2 construct.

[0285] Example 4

[0286] Identification of IL-2 variants with single amino acid substitutions that exhibit differential selectivity for Treg lymphocytes

[0287] Single amino acid substitutions were introduced into IL-2 at positions corresponding to amino acids that interact with receptor subunits β or γ or βγ. These substitutions were designed to reduce the signaling capacity of IL-2 through the intermediate affinity IL-2Rβγ complex and confer signaling specificity from the high affinity IL-2Rαβγ. The ability of IL-2 variants containing single amino acid substitutions to differentially stimulate STAT5 phosphorylation in CD4-positive Treg cells and Tconv cells was examined. STAT5 is known to be involved in the downstream signaling cascade following IL-2 binding to the transmembrane IL-2 receptor. In FACS analysis, phosphorylation of STAT5 in defined lymphocyte subsets was measured using fresh human peripheral blood mononuclear cells (PBMC), and the forkhead transcription factor FOXP3 was used to identify the Treg population.

[0288] Briefly, human PBMC were isolated from the buffy coat of healthy donors by Ficoll-Hypaque centrifugation. The PBMC were starved in serum-free MACS buffer for 1 hour at 4 °C. Then, 2×10 5PBMCs were treated with serial dilutions of the test compound for 30 min. Cells were fixed and permeabilized with the Foxp3 / Transcription Factor Staining Buffer Set (eBioscience) by incubating with 1X Foxp3 Fixation / Permeabilization Working Solution for 30 min and washing with 1X Permeabilization Buffer. Cells were also fixed with Cytofix Buffer and permeabilized with Perm Buffer III (BD Biosciences) and then washed. After blocking Fc receptors by adding Human TruStain FcX (1:50 dilution), cells were stained with a mixture of anti-CD25-PE, anti-FOXP3-APC, anti-pSTAT5-FITC, and anti-CD4-PerCP-Cy5.5 antibodies at the manufacturer's recommended concentrations for 45 min at room temperature. Cells were collected by centrifugation, washed, resuspended in FACS buffer, and analyzed by flow cytometry. Flow cytometry data for Treg and CD4 conventional T cell subsets were gated as CD4+Foxp3+ / CD25 高 and CD4+Foxp3- / CD25 低 groups. Data are represented as the percentage of pStat5-positive cells in the gated population.

[0289] Figures 2A - 2B The dose-response effects of exemplary Fc fusion proteins of IL-2 variants on STAT5 phosphorylation in CD4-positive Treg cells and Tconv cells are shown compared to wild-type fusion proteins. The wild-type IL-2 Fc fusion protein (P-0250) induced STAT5 phosphorylation in both Treg cells and Teff cells with EC50 values of 0.1 pM and 25.4 pM, respectively. The potency of wild-type IL-2 in Treg cells was approximately 250-fold higher than in CD4+ Tconv cells, consistent with the higher expression level of the high-affinity trimeric receptor in Treg cells.

[0290] Various substitutions of aspartic acid at position 20, P-0364 (D20E), P-0363 (D20T), P-0365 (D20N), P-0366 (D20Q), and P-0367 (D20S), demonstrated the ability to induce STAT5 phosphorylation in Treg cells, while this activity was substantially reduced or eliminated in CD4+ Tconv cells ( Figure 2A and Figure 2B ). These variants are potential Treg-biased IL-2 agents for activating Treg cells for the treatment of autoimmune diseases. In addition, mutations at the key residue D20 of the proposed toxin-like motif are expected to eliminate this toxic motif and prevent endothelial cell damage. Thus, these variants are expected to have Treg-selective activity with an improved safety profile for VLS. In addition, P-0368 did not show biological activity ( Figure 2A andFigure 2B )。

[0291] Figures 3A - 3B Shows the ability of the IL-2 variant P-0375 (N88Q) to induce STAT5 phosphorylation in CD4-positive Treg cells and CD4+ Tconv cells compared to the reference-1 and reference-2 compounds containing the V91K and N88R mutations, respectively. The activity profile of the N88Q variant is similar to that of reference-2.

[0292] Figures 4A - 4D Shows the biological activity of IL-2 variants containing various mutations at position 19 compared to wild-type. Variants P-0372 (L19Y), P-0373 (L19N), P-0374 (L19R), P-0423 (L19Q), P-0424 (L19H), and P-0427 (L19S) showed similar activity to wild-type in inducing STAT5 phosphorylation in Treg cells ( Figure 4A and Figure 4C ). Variants P-0372, P-0374, P-0423, and P-0427 also substantially retained their biological activity against CD4+ Tconv cells ( Figure 4B and Figure 4D ), while the activity of variants P-0373 and P-0424 against CD4+ Tconv was reduced. Mutant P-0425 (L19D) showed a slightly reduced ability to induce STAT5 phosphorylation in Treg cells, while this activity against CD4+ Tconv cells was significantly impaired ( Figure 4C and Figure 4D ). The selective activation of Treg cells over CD4+ Tconv cells demonstrated by mutants P-0373, P-0424, and P-0425, especially the broad window of selective targeting of the Treg subset by P-0373 and P-0425, makes them potential Treg-biased IL-2 agents for activating Treg cells for the treatment of autoimmune diseases. Importantly, L19 is part of the proposed toxin-like motif and mutations at this site are also expected to have improved safety profiles and reduced VLS.

[0293] Example 5

[0294] Combinations of IL-2Rβ and γc targeting amino acid substitutions with different selectivities for Treg lymphocytes in IL-2

[0295] In Example 4, it was shown that directed mutations aimed at attenuating the affinity of IL-2 for the IL-2Rβ or γc receptor subunits can generate IL-2 mutants with different selectivities for Treg lymphocytes. It was then inferred that modulating the affinity of IL-2 for both the IL-2Rβ and γc receptor subunits via the combination of one amino acid substitution targeting the β receptor and another amino acid substitution targeting the γ receptor can generate the desired potency and selectivity window for Treg lymphocytes.

[0296] This principle is demonstrated Figures 5A - 5F in Figure 5A and Figure 5B which show the effect of the IL-2Rβ-targeted variant P-0372 (L19Y) on STAT5 phosphorylation in Treg cells and CD4+ Tconv cells compared to the wild-type IL-2 fusion protein P-0250. Similarly, Figure 5C and Figure 5D show the STAT5 phosphorylation activity of P-0303 (Q126E) containing an amino acid substitution targeted to interfere with interaction with the γ receptor. The data indicate that each single amino acid substitution minimally affects pSTAT5 activation potency but also shows only a slightly improved selectivity window for the Treg lymphocyte subset relative to the wild-type. As Figure 5E and Figure 5F shown, the selective activation window for Treg cells was significantly expanded in P-0419 by combining the L19Y and Q126E mutations. The Treg activation potency was mostly retained in P-0419, and the activity profile of the P-0419 variant was very comparable to that of the benchmark-1 molecule containing the V91K mutation. This strategy is particularly attractive because 19L is also part of the proposed toxin-like motif and mutations at this site are also expected to have improved safety characteristics and reduced VLS.

[0297] However, combining one amino acid substitution targeting the β receptor and another substitution targeting the γ receptor may not always generate the desired potency and selectivity window. Appropriate modulation of activity is required for each aspect. Figure 6A and Figure 6B The four IL-2 variants in Figure 6A share the same L19Y substitution targeting the β receptor, and the additional mutations designed to target the γc receptor are Q126E in P-0419, Q126K in P-0464, S125I in P-0471, and Q22K in P-0474. Although all mutants retained comparable potency to induce STAT5 phosphorylation in Treg cells ( Figure 6A ), the variation in this activity towards CD4+ Tconv cells was significant ( Figure 6B) shows different abilities to modulate Treg activation selectivity via combinatorial amino acid substitutions.

[0298] However, compounding additional receptor attenuation by combining the Q126E substitution into an IL-2 variant that already exhibits specificity biased towards a Treg subset can result in a significant reduction or elimination of activity against Treg cells. As shown in ( Figure 6C and Figure 6D ), two variants, P-0373 (L19N) and P-0363 (D20T), have shown some or significant selectivity windows biased towards Treg cells ( Figure 6C and Figure 6D ). Their respective counterparts, P-0417 and P-0322, with an additional Q126E substitution, show significantly reduced Treg cell activation potency. Thus, finding the correct combination of residue substitutions to modulate activity to the desired potency and bias specificity against Treg cells is crucial.

[0299] Additional variants containing dual amino acid substitutions at positions L19 and Q126 were evaluated, including P-0447 (L19H / Q126E), P-0448 (L19Q / Q126E), and P-0449 (L19S / Q126E), and the activity is shown in Figures 7A - 7D Compared to the IL-2 variants P-0424 (L19H) and P-0303 (Q126E), each containing a single amino acid substitution, the variant P-0447 (L19H, Q126E), containing a combination of two amino acid substitutions, shows robust bioactivity in stimulating STAT5 phosphorylation in Treg cells, while this activity against Tconv cells is almost completely eliminated ( Figure 7A and Figure 7B ). In another study evaluating the comparison of P-0419, P-0447, P-0448, and P-0449 with two reference compounds, all four variants show significant potential to induce STAT5 phosphorylation in Treg cells, while this activity against CD4+ Tconv cells is essentially eliminated ( Figure 7C and Figure 7D ). P-0419 has an activity profile comparable to that of reference-1, which is similarly shown in Figure 5E and Figure 5F , while P-0447, P-0448, and P-0449 are comparable to reference-3 in terms of potency and selectivity window against Treg cells.

[0300] All of these mutants are potential Treg-biased IL-2 agents for activating Treg cells for the treatment of autoimmune diseases. In addition, due to the elimination of potential toxic motifs, these mutants are also expected to have an improved safety profile and reduced VLS.

[0301] Example 6

[0302] The IL-2 variant with an isoleucine substitution at position 125 retains all of its biological activity

[0303] As shown in Example 3, the isoleucine substitution at position 125 led to a general improvement in the developability of the IL-2 fusion construct. To make the S125I substitution a viable approach to alleviating the developability challenges of IL-2 engineering, it was important to demonstrate that this amino acid substitution did not compromise the biological activity of the resulting fusion protein compared to its Ser-125 counterpart.

[0304] The S125I substitution was then introduced into wild-type IL-2 or IL-2 variants that already contained 1 or 2 mutations targeting the receptor subunits β or γ or βγ. The ability of the resulting IL-2 variants containing isoleucine at position 125 to stimulate STAT5 phosphorylation in Treg cells and Tconv cells was tested and compared to their respective counterparts with serine at position 125. Table 5 lists the potency and selectivity of the IL-2 variants for Treg cells. The two molecules in the same row of Table 5 share the same other amino acid substitutions and differ only in having serine or isoleucine at position 125. The data show that the S125I substitution fully retains or slightly enhances the biological activity of the various tested IL-2 variants without altering Treg specificity.

[0305] Table 5 IL-2 variants containing the S125I substitution retain bioactivity and Treg selectivity

[0306]

[0307] Data from three exemplary constructs P-0250, P-0424, and P-0447 and their S125I equivalents P-0531, P-0491, and P-0511 are shown in Figure 8A - Figure 8F P-0250 is a wild-type IL-2Fc fusion molecule, P-0424 contains one amino acid substitution L19H, and P-0447 contains two amino acid substitutions L19H / Q126E. Their dose-dependent effects on STAT5 phosphorylation in Treg cells and CD4+ Tconv cells are illustrated in Figure 8A - Figure 8F As Figure 8A - Figure 8FAs shown, the S125I substitution slightly increased the potency of the three test compounds without altering the Treg selectivity of P-0531 and P-0491; for P-0511, the S125I substitution further widened the Treg selectivity window.

[0308] Thus, the data demonstrate that the S125I substitution in IL-2 retains the IL-2 activity profile of IL-2 fusion proteins with different mutant backgrounds. In summary, the isoleucine substitution at position 125 of IL-2 results in general developability improvements (increased product yields, reduced aggregation, lower immunogenic potential) of IL-2, IL-2 fusions, IL-2 variants, and IL-2 variant fusions, as well as complete retention of biological activity and selectivity. This specific amino acid substitution represents a viable mitigation strategy to address the inherent challenges of IL-2 engineering.

[0309] Example 7

[0310] Effects of IL-2 variants on CD25+CD4+ T cells, CD8 cytotoxic T cells, and NK cells

[0311] Compared to wild-type IL-2 (P-0250) and three IL-2 benchmark molecules containing V91K, N88R, and N88D, respectively, in addition to evaluating the ability of two variants, P-0511 and P-0512, to differentially stimulate STAT5 phosphorylation in CD4-positive Tregs (CD4+ / Foxp3+ / CD25 高 ) and Tconvs (CD4+ / Foxp3- / CD25 低 ) cells, the ability of the two variants, P-0511 and P-0512, to stimulate other effector T cells and NK cells (including CD4-positive Teffs (CD4+ / Foxp3- / CD25+), CD8 cytotoxic effector T cells, and NK cells) was further determined.

[0312] The IL-2 variants of the present invention have a weakened IL-2Rβγ interaction, and the significant growth advantage of Tregs over CD4+ Tconvs is conferred by high constitutive IL-2Rα (CD25) expression in Tregs. After immune stimulation, CD25 expression in CD4+ T effector cells can be induced. Therefore, it is desirable to confirm that the IL-2 variants retain Treg specificity relative to other CD25+ lymphocyte subsets. Exemplary lymphocyte subsets with medium to high CD25 expression levels include CD4+ effector T cells (Teffs).

[0313] After treating human PBMC cells with serial dilutions of test compounds, fixing and permeabilizing, washing, and staining with a mixture of anti-CD25-PE antibody, anti-FOXP3-APC antibody, anti-pSTAT5-FITC antibody, and anti-CD4-PerCP-Cy5.5 antibody, flow cytometry analysis gated for Treg cells, CD4 effector cells, and CD4 naive T cell subsets into CD4+ / Foxp3+ / CD25+, CD4+ / Foxp3- / CD25+, and CD4+ / Foxp3- / CD25- groups, respectively. Data are represented as the percentage of pSTAT5-positive cells in the gated populations and are described in Figure 9A - Figure 9C . P-0512 had a comparable activity profile to Benchmark-1 against all three T cell subsets, while P-511 was superior to both Benchmark-2 and Benchmark-3 in terms of potency and selectivity window against Treg cells versus both Teff cells and naive CD4 T cells. Benchmark-2 showed much weaker potency in activating each of the three subsets. Although CD25 is expressed at moderate to high levels on Teff, it was clearly demonstrated in Figure 9A and Figure 9B that IL-2 variants with attenuated IL-2Rβγ interaction, especially P-0511, preferentially activated Tregs over Teffs.

[0314] In addition, the ability of P-0511 and P-0512 to stimulate the proliferation of NK cells and CD8+ T cells was tested compared to wild-type and benchmark molecules. The intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE) method was utilized. Briefly, human PBMCs (1×10 5 cells / well) were labeled with CFSE, plated in 96-well plates, and incubated with increasing concentrations of different IL-2 compounds. Cells were then harvested after 5 or 7 days of incubation and stained for NK cells with anti-CD56-APC antibody or for CD8+ T cells with anti-CD8-APC antibody and analyzed by flow cytometry. Data are represented as the percentage of dividing cells and are shown for CD8+ T cell proliferation in Figure 10A and for NK cell proliferation in Figure 10B .

[0315] As expected, all IL-2 variants showed attenuated potency in stimulating both CD8+ T cells and NK cells compared to the wild-type IL-2 fusion molecule P-0250. Consistent with the observations in the STAT5 phosphorylation assay ( Figure 9A - Figure 9C ), P-0512 had an activity profile comparable to Benchmark-1 and P-0511 was comparable to Benchmark-3 in terms of potency against the two lymphocyte subsets, while Benchmark-2 showed much weaker potency.

[0316] The STAT5 phosphorylation activity of P-0511 on other responder cells other than CD4+ T cell subsets (including CD8+ T cells and NK cells) was compared with P-0531 (the S125I equivalent of wild-type P-0250). Similar to P-0531 ( Figure 11A ), P-0511 exhibited significant activity in stimulating STAT5 phosphorylation in Treg cells, while this activity on CD4+ Tconv cells ( Figure 11B ), CD8+ T cells ( Figure 11C ) and NK cells ( Figure 11D ) was almost completely abolished. The IL-2 receptors expressed on CD4+ Tconv cells, CD8+ T cells and NK cells are mainly the dimer IL-2R, including IL-2Rβ and γc. To confirm that the significantly attenuated pSTAT5 signaling of P-0511 on CD8+ T cells and NK cells is due to impaired interaction with IL-2Rβ and γc, an ELISA assay was developed.

[0317] Briefly, the non-covalent complex of IL-2Rβ-ECD (NP_000869) and γc-ECD (NP_000197) was coated onto the wells of a Nunc Maxisorp 96-well microplate at 2 μg / well via a heterodimeric Fc chain. After incubation overnight at 4 °C and blocking with superblock (ThermoFisher), 3-fold serial dilutions of IL-2Fc fusion protein starting from 100 nM or 270 nM were added to each well at 100 μl / well. After incubation at room temperature for 1 hour, 1 μg / ml biotinylated mouse anti-human IL-2 Ab (BD BioSciences) was added to each well, and then incubated with 1 μg / ml HRP-avidin (ThermoFisher) for 1 hour. After each step, the wells were aspirated thoroughly and washed three times with PBS / 0.05% Tween-20. Finally, 100 μl TMB substrate was added to each well; the plate was developed at room temperature in the dark for 10 minutes, and 100 μl / well of stop solution (2N sulfuric acid, Ricca Chemical) was added. The absorbance at 450 nm was determined; the curve was fitted using Prism software (GraphPad) and shown in Figure 11E .

[0318] As shown in Figure 11E , the developability-improved wild-type IL-2 fusion protein P-0531 binds to the IL-2 dimer receptor complex with sub-nanomolar affinity (EC 50 = 0.06 nM); the reference-1 molecule showed reduced binding (EC 50= 1.6 nM), which is consistent with its correspondingly diminished potency to stimulate STAT5 phosphorylation in CD8+ T cells and NK cells ( Figure 10A - Figure 10B ). In contrast, P-0511 did not show detectable binding to the IL-2Rβ and γc complex, indicating that the two IL-2 mutations at the interface with both the β and γc receptor subunits significantly impaired its interaction with the complex. Binding of P-0511 to the dimeric IL-2 receptor complex was almost abolished, and surprisingly, P-0511 exhibited only slightly reduced activity against Tregs compared to the wild-type IL-2 fusion. P-0511 exemplifies an IL-2 variant with a desired potency and selectivity window against Treg lymphocytes.

[0319] In summary, a series of IL-2 variants listed in Tables 2A - 2F were constructed, expressed, and tested in in vitro assays. The Figure 2A - Figure 11E biological activities of exemplary IL-2 variants against Tregs and other lymphocyte subsets, including CD4+ Tconv cells, CD4+ Teff cells, CD8+ T cells, and NK cells, are shown. Many variants retained high potency against Treg cells, with reduced or abolished activity against Tconv cells and other lymphocyte subsets. Some variants had an activity profile similar to Benchmark 1, while other variants resembled the activity signatures of Benchmark-2 or Benchmark-3. Additionally, most IL-2 variants had the proposed toxin-like motifs eliminated, aiming to reduce VLS. Importantly, the incorporation of the S125I amino acid substitution yielded IL-2 variant fusions with an excellent developability profile while retaining biological activity and selectivity against Treg cells. These variants are potentially Treg-biased IL-2 agents for treating autoimmune diseases with an improved safety profile.

[0320] Example 8

[0321] Fc fusion proteins of IL-2 variants preferentially proliferate and expand Treg cells in mice

[0322] IL-2 variant Fc fusion proteins were administered to mice, and their ability to preferentially proliferate and expand regulatory T cells (CD4+CD25+FoxP3+ T cells) over effector T cells and NK cells was determined in vivo.

[0323] Female C57 / BL6 mice (7 weeks old) were received from Charles River Laboratories and acclimated in-house for at least 7 days before the study. On day 0, mice were administered subcutaneously with vehicle (PBS), each test compound at 0.3 mg / kg, or an IL-2 reference compound. On days 3, 5, and 7 after treatment, peripheral blood samples were collected into heparinized tubes. Each group consisted of 6 mice, and baseline blood was collected 2 days before treatment (day -2). After red blood cell lysis, total viable mononuclear blood cells were counted by trypan blue dead cell exclusion, and intracellular staining was continued for flow cytometry analysis of immune cell phenotypes and Ki67 proliferation marker. Cells were stained separately with two sets of antibodies listed below: 1) For CD4+ regulatory T cells (Tregs), anti-mouse Foxp3-FITC, Ki67-PE, anti-mouse CD25-APC, and anti-mouse CD4-Percpcy5.5 (1:50 dilution); 2) For CD8+ T cells and NK cells, anti-mouse CD3-FITC, Ki67-PE, anti-mouse CD335-APC, and anti-mouse CD8-Percpcy5.5 (1:50 dilution).

[0324] All tested IL-2 compounds stimulated Treg cell proliferation and expansion, as demonstrated by increased Ki67-positive Tregs and elevated percentages of Tregs among total CD4+ T cells or total lymphocytes ( Figure 12A - Figure 12C ). This effect was observed 3 days after injection and persisted until day 5 or 7 after a single injection. Among the IL-2 variants that induced Treg phosphorylation, in contrast to the in vitro observation where reference-1 always showed the highest potency, in mice, all three tested variants P-0511, P-0512, and P-0514 demonstrated stronger in vivo efficacy in stimulating Treg cell proliferation and expansion than the reference molecule. P-0511, P-0512, and P-0514 exhibited comparable activities. The relative in vivo potency ranking among the three references was consistent with the in vitro human PBMC cell assay, i.e., reference-1 had the highest potency, followed by reference-3. Reference-2 was much weaker in proliferating and expanding Treg cells. ( Figure 12A - Figure 12C ).

[0325] For effector T cells and NK cells, reference-1 showed strong Ki67 stimulation in cytotoxic CD8 T cells and NK cells, while reference-2 and -3 showed low effects on CD8 T cells and NK cells ( Figure 13A - Figure 13C ). Variant P-0514 showed similar Ki67 stimulation in CD8+ T cells as reference-1, while variants P-0511 and P-0512 showed mild Ki67 stimulation in CD8 T cells and NK cells similar to reference-2 and reference-3Figure 13A - Figure 13C ) Data indicate that variants P-0511 and P-0512 exhibit superior bioactivity and selectivity for Tregs over Benchmark 1 and Benchmark 2. Benchmark-3 was ineffective at stimulating and expanding both Treg cells and effector cells.

[0326] In all IL-2 variant treatment groups, the percentage of CD4+ T conventional cells decreased due to an increase in the Treg population ( Figure 14A ). No significant expansion of CD4+ Tconv cells, CD8 T cells, or NK cells was observed in mice treated with any Treg-biased IL-2 variant (P-0511, P-0512, and P-0514) or the three benchmarks ( Figure 14B - Figure 14D ).

[0327] At all measured time points, all three variants, P-0511, P-0512, and P-0514, also exhibited the most beneficial Treg / Tconv ratio in terms of both Ki67 stimulation and cell expansion based on cell counts compared to the three benchmarks ( Figure 15A and Figure 15B ).

[0328] Three days after injection, all tested IL-2 compounds caused an increase in Foxp3 expression on Treg cells ( Figure 16A ), and all three variants showed relatively higher expression of the CD25 and Foxp3 markers compared to the three benchmarks ( Figure 16A and Figure 16B ), indicating better Treg activation and functionality.

[0329] Body weight was monitored before and during treatment. No significant weight changes were observed (data not shown).

[0330] Overall, the data demonstrate that variants P-0511, P-0512, and P-0514 exhibit the ability to promote the activation, proliferation, and expansion of immunosuppressive Treg cells while not affecting CD4+ conventional cells, cytotoxic effector T cells, and NK cells. The data also demonstrate that these three variants are superior to the benchmark molecules in terms of both efficacy and selectivity for Treg proliferation and expansion. These variants can be used as therapeutic agents to combat autoimmune diseases, inflammatory diseases, and organ transplant rejection.

[0331] Example 9

[0332] Pharmacodynamic study of the dose response of IL-2 variant Fc fusion proteins after single injection in mice

[0333] After single subcutaneous administration of vehicle (PBS) or P-0511 (1 mg / kg, 0.3 mg / kg, 0.1 mg / kg or 0.03 mg / kg) to female Balb / C mice (n = 5 mice / group), peripheral blood was collected on day -2 as baseline and on days 3, 5 and 7 after dosing. On day 7, the mice were sacrificed and spleens were harvested. Blood lymphocyte phenotypic analysis, proliferation and expansion were measured by flow cytometry using fresh whole blood at each time point.

[0334] There were no significant changes in body weight or spleen weight in any treatment group (data not shown).

[0335] As Figure 17A - Figure 17D described, a dose-dependent increase in Treg cell proliferation was observed in mice treated with P-0511 at dosing levels of 1 mg / kg, 0.3 mg / kg or 0.1 mg / kg, as reflected by an increase in the percentage of Ki67-positive cells ( Figure 17A ). Treatment at 0.03 mg / kg had minimal effect. At the three higher dose levels, the stimulation of Ki67 expression in Treg cells peaked on day 3 and stabilized until day 5, then declined. As a result, P-0511 treatment led to a dose-dependent increase in the percentage of Tregs among total CD4+ T cells ( Figure 17B ), the absolute number of Treg cells ( Figure 17C ) and the fold change in cell count from baseline ( Figure 17D ). The increase in Treg cell expansion followed a kinetic pattern similar to that of the proliferation / activation Ki67 marker ( Figure 17A - Figure 17D ), i.e., peaking on day 3 and extending further to day 5. Treatment at 1 mg / kg stimulated a greater magnitude and duration of Tregs, and the signal persisted until day 5.

[0336] Treatment with P-0511 also led to a dose-dependent and statistically significant increase in the percentage of Tregs among total lymphocytes ( Figure 18A ), while no statistically significant changes were observed in the percentage of CD4+ Tconv cells ( Figure 18B ), CD8Teff cells ( Figure 18C ) or NK cells ( Figure 18D ) among total lymphocytes. At a single dose of 1 mg / kg, at the peak, Tregs accounted for 4.5% of total lymphocytes, compared to 3.1% at a dose of 0.3 mg / kg and 1.4% at a dose of 0.1 mg / kg. In the vehicle control group, Tregs accounted for 0.5% of total lymphocytes ( Figure 18A ).

[0337] The Treg / Tconv ratio was calculated based on cell countsFigure 19A )。The peak of the Treg / Tconv ratio was 0.27 at the 1 mg / kg treatment, 0.18 at the 0.3 mg / kg treatment, and 0.06 at the 0.1 mg / kg treatment, compared to 0.027 without treatment( Figure 19A ), indicating that P-0511 preferentially expands Treg cells over Tconv cells. In addition, the expression of Treg cell function markers, including CD25( Figure 19B ) and FoxP3( Figure 19C ), increased in a dose-dependent manner. The increase in the mean fluorescence intensity (MFI) of CD25 and FoxP3 peaked on day 3 and decreased to a lower level on day 5.

[0338] Overall, the data demonstrated that P-0511 exhibited effective and preferential Treg activation and expansion in a dose-dependent manner. Careful consideration is required to achieve the optimal dosing strategy to achieve the maximum potency in promoting the activation, proliferation, and expansion of immunosuppressive Treg cells while not affecting cytotoxic effector T cells and NK cells.

[0339] Example 10

[0340] Pharmacodynamic study after repeated administration of an IL-2 variant Fc fusion protein in mice

[0341] Female Balb / C mice (7 weeks old) were acclimatized in the facility for 5 - 7 days and then studied. On days 0, 3, and 6, mice (n = 5 per group) were administered subcutaneously with vehicle (PBS), 0.3 mg / kg of P-0511, P-0512, P-0531, or reference-1 compound. On days 3 and 9, peripheral blood was collected three days after the first injection and three days after multiple (3 times) injections, respectively. Based on earlier in vivo experiments, it was expected that the activation, proliferation, and expansion of Treg cells would peak on day 3, and thus data collection and analysis were chosen to be performed three days after injection. Changes in the activation, proliferation, and expansion of blood lymphocytes were measured by flow cytometry. P-0531 is the S125I equivalent of the wild-type IL-2 fusion protein. Reference-1 contains the V91K mutation.

[0342] Three days after a single subcutaneous administration of the IL-2 fusion protein, in all test groups, nearly 90% of Treg cells showed positive Ki67 expression, and after the third dose of all test compounds, Ki67-positive cells remained significantly high( Figure 20A)。Interestingly, in mice treated with P-0531 and Benchmark-1, the percentage of Treg cells, expressed as the percentage of Treg in total CD4 T cells or total lymphocytes, significantly decreased to near control levels, while in mice treated with P-0511 and P-0512, after three consecutive Q3D treatments compared to a single treatment, Treg cells remained at significantly high levels( Figure 20B - Figure 20C )。The data indicate that wild-type IL-2 or Benchmark-1 can accelerate the depletion of Treg cells or desensitize Treg due to stronger potency in Treg stimulation. Additional explanations may also include differences in half-life or "receptor sink" on non-lymphocytes, resulting in altered drug exposure of wild-type IL-2 or Benchmark-1 with non-Treg selectivity or lower Treg selectivity.

[0343] Similar observations were also obtained for Treg cell counts and fold change relative to the PBS control( Figure 21A - Figure 21B ) and the Treg / Tconv ratio( Figure 22 )。Compared to P-0531 and Benchmark-1, P-0511 and P-0512 demonstrated a better ability to maintain the Treg pool and maintain Treg selectivity.

[0344] Overall, the data demonstrate that P-0511 and P-0512 are better IL-2 molecules, showing preferential and sustained in vivo Treg expansion after multiple doses. Modulating the dosing regimen of the IL-2 variant Fc fusion, such as the dose and frequency of administration, can further optimize the potency and selectivity for Treg relative to pro-inflammatory immune activation.

[0345] Example 11

[0346] Inhibition of antigen-driven inflammation in the delayed-type hypersensitivity (DTH) mouse model by IL-2 variant Fc fusion proteins

[0347] The ability of Treg cells induced by IL-2 variants to inhibit T cell antigen-driven inflammation in vivo was evaluated in the delayed-type hypersensitivity (DTH) model. Female Balb / C mice (7 weeks old) were acclimated in the facility for 7 days and randomly grouped. Starting on day -2, vehicle (PBS), 0.1 mg / kg or 0.3 mg / kg of P-0511 was administered subcutaneously and given three injections every 3 days (Q3D) or two injections every 5 days (Q5D). Then on day 0, 100 μg of keyhole Mice were sensitized with keyhole limpet hemocyanin (KLH). For Q3D dosing, two additional subcutaneous injections of PBS or P-0511 (0.1 mg / kg or 0.3 mg / kg) were administered on days 1 and 4; for Q5D dosing, one additional s.c. injection of PBS, 0.1 mg / kg or 0.3 mg / kg of P-0511 was administered on day 3. On day 5, mice received an intradermal KLH challenge (5 μg in 10 μl of saline) in the right ear. The thickness of the right ear was measured using calipers on day 5 before KLH challenge and on days 6 to 8 after KLH challenge (corresponding to 24 hours, 48 hours, and 72 hours). A group of mice also received i.p. treatment with 5 mg / kg of dexamethasone daily from day 5 to day 8 as a positive control.

[0348] In Figure 23A to Figure 23B , the kinetics of the DTH response was illustrated using the change in ear thickness relative to the baseline value (Δ ear thickness) at different times after KLH challenge.

[0349] After subcutaneous KLH antigen sensitization, obvious ear inflammation and swelling peaked 24 hours after intradermal KLH challenge in the auricle, and ear swelling in the PBS group lasted for 72 hours. Apparently, the immunosuppressive steroid dexamethasone was effective in suppressing the KLH-induced inflammatory response, achieving ~85% inhibition 72 hours after KLH challenge when administered at 5 mg / kg daily for 4 consecutive days. At all time points after KLH challenge, the suppression of antigen-driven inflammation by Treg cells induced by P-0511 was also obvious in mice treated with 0.3 mg / kg of P-0511 Q3D or Q5D ( Figure 23A - Figure 23B ). A similar trend of reducing the DTH inflammatory response was observed for both Q3D and Q5D administrations at a dose of 0.1 mg / kg, but the effect was not statistically significant at most time points. Both Q3D and Q5D dosing regimens were effective.

[0350] In another study, the dose-dependent response of P-0511 (0.1 mg / kg, 0.3 mg / kg, and 1 mg / kg, Q5D) on the inhibition of KLH-induced DTH was determined and compared with Benchmark-1 (0.3 mg / kg, Q5D). As Figure 24 illustrated, P-0511 demonstrated dose-dependent inhibition of ear inflammation. Mice receiving 1 mg / kg of P-0511 showed strong tolerance to KLH-induced DTH, and minimal ear swelling was observed after KLH challenge. Moderate and mild inhibitions were observed for 0.3 mg / kg and 0.1 mg / kg of P-0511, respectively. Benchmark-1 showed mild inhibition of ear swelling, and the effect of 0.3 mg / kg of Benchmark-1 was similar to that achieved by 0.1 mg / kg of P-0511 (Figure 24 )。

[0351] In summary, in the DTH model, Treg cells induced by administration of P-0511 were effective in suppressing T cell antigen-driven inflammation. In addition, Treg suppression persisted after KLH challenge without repeated dosing. It was also evident from the examples that it was crucial to adjust the dosing regimen to achieve optimal efficacy.

[0352] Example 12

[0353] Pharmacodynamic / pharmacokinetic effects of P-0511 in cynomolgus monkeys

[0354] According to the Q14D×3 dosing regimen, the PK / PD characteristics of the IL-2 variant Fc fusion protein P-0511 in cynomolgus monkeys were evaluated. Cynomolgus monkeys naive to the drug were acclimated for 3 - 4 weeks and randomly divided into 4 groups (n = 3 - 4 animals / group), followed by a pre-dose baseline week. On days 1, 15, and 29, one group received subcutaneous administration of vehicle (PBS), and the other three groups were dosed subcutaneously with P-0511 at 100 μg / kg, 30 μg / kg, or 10 μg / kg.

[0355] Blood was collected on days -7, -3, 2, 4, 6, 8, 11, 14, 18, 21, 28, 32, 35, 43, 50, and 57. Whole blood was used for FACS immunophenotyping of naive and memory peripheral blood Treg, non-regulatory CD4 T cells, CD8 T cells, and NK cells to determine pharmacodynamics. Cell activation and proliferation were also monitored by measuring CD69 and Ki67. Whole blood was also used for a complete blood count (CBC), which was divided into 5 parts: neutrophils, lymphocytes, monocytes, eosinophils, and basophils.

[0356] A 96-well plate was coated with mouse anti-human IL-2 mAb (BD Pharmingen) to capture P-0511, and the PK properties of P-0511 were evaluated in cynomolgus monkey plasma samples by measuring the full-length intact P-0511. P-0511 was detected using goat anti-human Fc polyclonal-HRP (ThermoFisher), and the plasma concentration of P-0511 was subsequently quantified. In addition to plasma samples collected on days -7, -3, 2, 4, 6, 8, 11, 14, 18, 21, 28, 32, 35, 43, 50, and 57, three plasma samples were also collected at 0.5 h, 3 h, and 6 h after the first administration of P-0511 on day 1.

[0357] Plasma samples from days -8, 8, 21, 35, 43, and 57 were also used to evaluate the following clinical chemistry parameters: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ-glutamyl transferase, albumin, total bilirubin, creatinine, blood urea nitrogen, and C-reactive protein.

[0358] In addition, the body weight and body temperature of each animal were monitored once or twice a week throughout the study.

[0359] All articles and methods disclosed and claimed herein can be made and executed in view of the present disclosure without undue experimentation. Although the articles and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the articles and methods without departing from the spirit and scope of the present disclosure. It will be apparent to those skilled in the art that all such variations and equivalents, whether now existing or later developed, are considered to be within the spirit and scope of the present disclosure as defined by the appended claims. All patents, patent applications, and publications mentioned in the specification indicate the level of ordinary skill in the art to which the present disclosure pertains. For all purposes, all patents, patent applications, and publications are hereby incorporated by reference in their entirety, and to the extent that each individual publication is specifically and individually indicated to be incorporated by reference in its entirety for any and all purposes. The present disclosure, as exemplified herein, can be suitably practiced in the absence of any one or more of the elements not specifically disclosed herein. Accordingly, it is to be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, those skilled in the art may seek variations and changes of the concepts disclosed herein, and such variations and changes are considered to be within the scope of the present disclosure as defined by the appended claims.

[0360] Sequence Listing

[0361] The nucleic acid and amino acid sequences listed in the accompanying Sequence Listing are shown using the standard letter abbreviations for nucleotide bases and the single-letter codes for amino acids as defined in 37 C.F.R. 1.822.

[0362] SEQ ID NO:1 is the amino acid sequence of human IL-2 precursor.

[0363] SEQ ID NO:2 is the amino acid sequence of the naturally occurring mature form of human IL-2.

[0364] SEQ ID NO:3 is the wild-type amino acid sequence of the mature form of human IL-2.

[0365] SEQ ID NO:4-43, 108-146 and 193-197 are the amino acid sequences of various IL-2 variants.

[0366] SEQ ID NO:44 is the amino acid sequence of human IgG1-Fc.

[0367] SEQ ID NO:45 is the amino acid sequence of human IgG1-Fc with reduced / abolished effector function.

[0368] SEQ ID NO:46 and 47 and 212-213 are the amino acid sequences of human IgG1-Fc with reduced / abolished effector function and extended half-life.

[0369] SEQ ID NO:48-67 are the amino acid sequences of various peptide linker sequences.

[0370] SEQ ID NO:68 is the amino acid sequence of the human IL-2 receptor α Sushi domain.

[0371] SEQ ID NO:69-107, 147-189 and 198-211 are the amino acid sequences of various Fc-IL-2 fusion proteins.

[0372] SEQ ID NO:190-192 is the amino acid sequence of a reference Fc-IL-2 variant fusion protein.

[0373] SEQ ID NO:214-222 are the nucleotide sequences of various Fc-IL-2 fusion proteins.

[0374] Sequence Listing

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

Claims

1. An isolated fusion protein, the isolated fusion protein comprising 1) an IL-2 variant polypeptide and 2) an Fc domain, wherein the fusion protein is in the form of a dimer, and wherein each IL-2 variant polypeptide of the fusion protein is fused to the Fc domain via a peptide linker.

2. The isolated fusion protein according to claim 1, wherein the IL-2 variant polypeptide comprises an amino acid sequence selected from the group consisting of the amino acid sequences listed in SEQ ID NOs: 34-36, 42-43, 125, 127-110, 141, 146, 193-194, and 196-197, and wherein the Fc domain is an Fc domain having an amino acid sequence selected from the group consisting of the amino acid sequences listed in SEQ ID NOs: 44-47 and 212-213, and wherein the IL-2 variant polypeptide is fused to the Fc domain via a peptide linker, and wherein the peptide linker comprises between 1 and 40 amino acids.

3. The isolated fusion protein according to claim 2, wherein the IL-2 variant polypeptide comprises the amino acid sequence listed in SEQ ID NO:

193.

4. The isolated fusion protein according to claim 2, wherein the IL-2 variant polypeptide comprises the amino acid sequence listed in SEQ ID NO:

194.

5. The isolated fusion protein according to any one of claims 3-4, wherein stimulation with the IL-2 variant polypeptide results in a regulatory T cell (Treg) / naive conventional T cell (Tconv) ratio of at least 2.

5.

6. The isolated fusion protein according to any one of claims 3-4, wherein stimulation with the IL-2 variant polypeptide results in a higher stimulation of Tregs relative to the stimulation of Tconvs.

7. The isolated fusion protein according to claim 1, wherein the fusion protein is selected from the group consisting of: a fusion protein wherein the IL-2 variant polypeptide is fused to the C-terminal amino acid of the Fc domain at its N-terminal amino acid, or a fusion protein wherein the IL-2 variant polypeptide is fused to the N-terminal amino acid of the Fc domain at its C-terminal amino acid.

8. The isolated fusion protein according to claim 1, wherein the fusion protein comprises an amino acid sequence selected from the group consisting of the amino acid sequences listed in SEQ ID NOs: 101-103, 168-173, 184, 189, 198-199, and 204-211.

9. The isolated fusion protein according to claim 8, wherein the fusion protein comprises the amino acid sequence listed in SEQ ID NO:

198.

10. The isolated fusion protein according to claim 8, wherein the fusion protein comprises the amino acid sequence listed in SEQ ID NO: 199.

Images