Interleukin-2 variants and methods of use thereof

Abstract

The present invention relates to polypeptides that share a primary sequence with human IL-2, except for several mutated amino acids. A group of IL-2 variants includes mutations with impressive manufacturability, which preferentially promote the proliferation, survival, activation and / or function of immunosuppressive regulatory T cells (Treg:CD4+CD25+FoxP3+) relative to effector T cells and NK cells. Such IL-2 selective agents are also included, used alone or in combination with immunomodulators or disease tissue targeting antibodies, proteins or peptides, to treat Treg cell deficiency, various autoimmune disorders and inflammatory disorders, organ transplantation and graft-versus-host disease. On the other hand, the present invention relates to a pharmaceutical composition comprising the disclosed polypeptide. Finally, the present invention relates to the therapeutic use of the disclosed polypeptide and pharmaceutical composition, due to their selective regulatory effect on the immune system to diseases such as autoimmune disorders and inflammatory disorders.

Description

[0001] Related patent applications

[0002] 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 Art

[0003] Interleukin-2 (IL-2) is the first growth factor described 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 the ability to enhance immune responses in the context of T virus infection (Blattman, JN, et al. (2003) Nat Med 9, 540-7) or vaccines (Fishman, M., et al. (2008) J Immunother.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).

[0004] IL-2 has been used in cancer treatment. Recombinant human IL-2 is an effective immunotherapy for metastatic melanoma and renal cancer, with approximately 10% of patients experiencing durable responses. However, the short half-life and severe toxicity limit the optimal dosing of IL-2. In addition, IL-2 also binds with high affinity to its heterotrimeric receptor IL-2Rαβγ, preferentially amplifying immunosuppressive regulatory T cells (Tregs) that express high constitutive IL-2Rα levels. 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 could be used to treat autoimmune disorders and various inflammatory disorders.

[0005] Treg is the core of immune system homeostasis and plays a major role in maintaining peripheral immune tolerance by suppressing (self-reactive) effector T cells. Various autoimmune diseases and inflammatory diseases have been shown to have defects in Treg cell number or Treg function. Therefore, there is great interest in developing therapies that strengthen the number and / or function of Treg cells. A method of treating an autoimmune disease under study is to use low doses of IL-2 to target Treg cells, because the IL-2Rα constitutive levels of Treg cells are higher, so compared with many other immune cell types, Treg cells respond to lower concentrations of IL-2. (Klatzmann D, 2015 Nat Rev Immunol. 15: 283-94). Clinical trials of low-dose IL-2 in patients with various GvHD (Koreth, J., et al., 2011, N Engl J Med., 365: 2055-66) and HCV-related autoimmune vasculitis (Saadoum, D., et al., 2011, N Engl J Med., 365: 2067-77) have shown increased Treg levels and clinical efficacy. However, even these lower doses can lead to serious safety and tolerability issues. Therefore, there is a need for an effective autoimmune / inflammatory disease therapy that targets Treg cells more specifically than IL-2 and can enhance the number and function of Treg cells.

[0006] Recently found that IL-2 can be modified to selectively stimulate cytotoxic effector T cells or Treg cells. Various methods have resulted in the production of IL-2 variants with improved and selective immunostimulatory ability (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 No. 20050142106). In the present invention, IL-2 variants are designed to increase the ability of the molecule to carry out signal transduction mainly by high-affinity receptors (α chain, β chain and γ chain) rather than by medium-affinity receptors (β chain and γ chain). Basic concept is to promote signal transduction in Treg cells rather than signal transduction in effector T cells and NK cells, which is considered to be the reason for the toxic effects observed. Importantly, there is no IL-2 variant in the prior art disclosure that is related to the ability of stimulating natural regulatory T cells based on the reduction of IL-2 variants and has a therapeutic effect in vivo that is greater than natural IL-2. However, since the initial studies on IL-2 variants, research in the field has more fully established that Treg cells constitutively express high IL-2Rα (CD25) as well as IL-2Rβ and γ C, IL-2 variants that are selective agonists of IL-2Rαβγ should be selective for Treg cells.

[0007] In summary, IL-2 is a highly pleiotropic cytokine that is closely linked to the biological activities of different cell populations. This property makes IL-2 an important node in regulating immune responses, making IL-2 an attractive target for therapeutic and complex immunomodulation. In addition, receptor subunit-biased IL-2 variants can be prepared to achieve IL-2-mediated selective immunomodulation 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.

[0008] Disclosure of the Invention

[0009] In one aspect, the present invention relates to the generation of mutant variants of IL-2. These variants are characterized in that they have higher selectivity 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 disorders 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 different primary sequences corresponding to the binding of IL-2 to IL-2Rβ and / or γ. C The variants have amino acid substitutions at the interacting positions, 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 to IL-2Rα and the ability to bind to 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.

[0010] In one aspect, the present invention relates to the generation of mutant variants of IL-2. These variants have a combination of properties that include Treg selective activity, reduced aggregation, increased expression, improved manufacturability, and developability. These variants also provide improved protein biophysical properties and reduced immunogenicity risks associated with IL-2 molecules. 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 or more mutated amino acids.

[0011] In one aspect, the present invention relates to the generation of IL-2 mutant variants characterized by the deletion of a proposed bacterial toxin component.19 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. It is expected that the introduction of mutations to remove the key residue D20 or the two residues flanking the toxin-like domain will eliminate the toxic motif and prevent endothelial cell damage and significantly reduce VLS. Importantly, since this motif is located 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 activated 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 the treatment of Treg cell-deficient autoimmune disorders and various inflammatory disorders.

[0012] The present invention allows substantial improvements in the current strategies of immunomodulation based on IL-2 in the therapy of autoimmune disorders and various inflammatory disorders. In particular, the replacement of natural IL-2 by mutant variants described herein will result in selective stimulation of the CD25 bias of Treg cells. In various embodiments, IL-2 variants (or mutants) include sequences of IL-2 variants (or mutants) derived from the mature human IL-2 polypeptide sequence listed in SEQ ID NO:3. In various embodiments, IL-2 variants act as IL-2 agonists. In various embodiments, IL-2 variants act as IL-2 antagonists. In various embodiments, IL-2 variants include sequences listed in SEQ ID NO:4-43, 108-146, and 193-197.

[0013] The present invention allows substantial improvements in the current strategies of immunomodulation based on IL-2 in the therapy of Treg cell deficiency autoimmune disorders and various inflammatory disorders. In particular, the replacement of natural IL-2 by mutant variants described herein will result in selective stimulation of the CD25 bias of Treg cells, and it is expected that toxic motifs will be eliminated, as well as preventing endothelial cell damage and significantly reducing VLS. In various embodiments, IL-2 variants (or mutants) include sequences of IL-2 variants (or mutants) derived from the mature human IL-2 polypeptide sequence listed in SEQ ID NO:3. In various embodiments, IL-2 variants act as IL-2 agonists. In various embodiments, IL-2 variants act as IL-2 antagonists. In various embodiments, IL-2 variants include SEQ ID NO:5-14, 26-43, 108-111, 125-146 and 193-197.

[0014] On the other hand, the IL-2 variants of the present invention are 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 on the fusion molecule. Such polypeptides include IgG Fc or other polypeptides that bind to neonatal Fcγ / receptors, human serum albumin, or polypeptides that bind to proteins with extended serum half-lives, including IgG, non-IgG immunoglobulins, proteins, and non-protein agents that have increased in vivo half-life due to the presence of an IgG constant domain or portion thereof that binds to FcRn, which has one or more amino acid modifications that increase the affinity of the constant domain or fragment for FcRn. Such proteins and molecules with increased half-life have the advantage that smaller amounts and / or less frequent dosing are required for the treatment, prevention, or diagnosis of such molecules (see, e.g., U.S. Patent No. 7,658,921).

[0015] In various embodiments, the IL-2 variant can be linked to the N-terminus or the C-terminus of the heterologous protein.

[0016] 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 listed in SEQ ID NO: 44. In various embodiments, the Fc domain is an Fc domain having the amino acid sequence listed in SEQ ID NO: 45. In various embodiments, the Fc domain is an Fc domain having the amino acid sequence listed in SEQ ID NO: 46. In various embodiments, the Fc domain is an Fc domain having the amino acid sequence listed in SEQ ID NO: 47. In various embodiments, the Fc domain is derived from the human IgG2 heavy chain constant domain sequence. In various embodiments, the Fc domain is derived from the human IgG4 heavy chain constant domain sequence.

[0017] The term "Fc" refers to a molecule or sequence comprising the sequence of a non-antigen binding fragment of a complete antibody, whether in monomeric or multimeric form. The original immunoglobulin source of native Fc is preferably human origin, and can be any immunoglobulin disclosed in the art. Native Fc consists of monomeric polypeptides, which can be linked to dimers or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between the monomer subunits of native Fc molecules depends on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2) and is in the range of 1 to 4. An example of native Fc is a 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 monomers, dimers, and multimeric forms. The Fc domain contains binding sites for protein A, protein G, various Fc receptors, and complement proteins.

[0018] In various embodiments, the term "Fc variant" refers to a molecule or sequence that is modified from a native Fc but still contains a 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 interactions with salvage receptors and are hereby incorporated by reference. In addition, a 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 a selected host cell, (3) N-terminal heterogeneity when expressed in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0019] The term "Fc domain" includes natural Fc and Fc variant molecules and sequences as defined above. Like Fc variants and natural Fc, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from a complete antibody or expressed by a recombinant gene or produced by other means. In various embodiments, an "Fc domain" refers to a dimer of two Fc domain monomers (SEQ ID NO: 44), which typically include all or part of the hinge region. In various embodiments, the Fc domain can be mutated to lack effector functions. In various embodiments, each Fc domain monomer in the Fc domain comprises an amino acid substitution in the CH2 antibody constant domain to reduce the interaction or binding between the Fc domain and the Fc γ receptor. In various embodiments, each subunit of the Fc domain comprises two amino acid substitutions, which reduce binding and / or effector functions to activating Fc receptors, wherein the amino acid substitutions are L234A, L235A. In various embodiments, each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to an activating Fc receptor and / or effector function, wherein the amino acid substitutions are L234A, L235A, and G237A (SEQ ID NO: 45).

[0020] 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 comprises three amino acid substitutions that enhance binding to human FcRn, wherein the amino acid substitutions are M252Y, S254T, and T256E, disclosed in U.S. Patent Publication No. 7,658,921. In various embodiments, each subunit of the Fc domain comprises an amino acid substitution that enhances binding to human FcRn, wherein the amino acid substitution is N434A, disclosed in U.S. Patent Publication No. 7,371,826. In various embodiments, each subunit of the Fc domain comprises an amino acid substitution that enhances binding to human FcRn, wherein the amino acid substitutions are M428L and N434S, disclosed in U.S. Patent Publication No. 8,546,543. In various embodiments, half-life extension mutations can be combined with amino acid substitutions that reduce binding to activating Fc receptors and / or effector function.

[0021] In various embodiments, each of the two Fc domain monomers in the Fc domain comprises an amino acid substitution that promotes the heterodimerization of the two monomers. In various other embodiments, the 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" technology is also disclosed in U.S. Patent Publication No. 8,216,805. In yet another embodiment, one Fc domain monomer comprises a knob mutation T366W, and another Fc domain monomer comprises a hole mutation T366S, L358A, and Y407V. In various embodiments, two Cys residues (S354C on the "knob" side and Y349C on the "hole" side) that form a stabilized disulfide bridge are introduced. The use of heterodimer Fc can produce a monovalent IL-2 variant construct.

[0022] In various embodiments, the IL-2 variant Fc-fusion protein will be monomeric, i.e., contain only a single IL-2 mutein molecule. In such embodiments, the fusion protein is co-expressed with a heterodimeric Knob-Fc linked to the IL-2 variant and a matching heterodimeric Hole Fc. When a heterodimer of the two Fc-containing polypeptides forms, the resulting protein contains only a single IL-2 variant.

[0023] In various embodiments, the IL-2 variants are used to prepare the Fc-IL-2 fusion proteins set forth in SEQ ID NOs: 69-107, 147-189, and 198-208.

[0024] In various embodiments, the IL-2 variants of the present invention may be attached to antibodies that confer extended half-life to the fusion molecule, such as anti-keyhole limpet antibodies. Antibodies to KLH. These antibodies recognize foreign antigens and have a long half-life, but they have no biological function or damage in humans. IgG classes can be IgG, IgA, IgE, or subclasses (e.g., IgG1, IgG2, IgG3, IgA1, IgA2).

[0025] In various embodiments, the heterologous protein is attached to the IL-2 variant via a linker and / or hinge peptide. The linker or hinge peptide can be an artificial sequence of 5, 10, 15, 20, 30, 40 or more amino acids that is relatively free of secondary structure.

[0026] In various embodiments, the heterologous protein is attached to the IL-2 variant via a rigid linker peptide of between 10, 15, 20, 30, 40 or more amino acids that exhibits an α-helical conformation and can serve as a rigid spacer between protein domains.

[0027] On the other hand, IL-2 variants can be connected to various non-protein polymers in a 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, including but not limited to various polyols such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes. 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 than non-PEGylated proteins.

[0028] "Polyethylene glycol" or "PEG" means a polyalkylene glycol compound or a derivative thereof, with or without a coupling agent or derivatization with a coupling or activating moiety (e.g., with an aldehyde, hydroxysuccinimidyl, hydrazide, thiol, triflate, tresylate, aziridine, oxirane, orthopyridyldisulphide, vinylsulfone, iodoacetamide, or maleimide moiety). In various embodiments, PEG includes substantially linear, straight-chain PEG, branched PEG, or dendrimer PEG. PEG is a well-known water-soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pp. 138-161).

[0029] In various embodiments, the 11-2 variants can be non-covalently or covalently linked at the N-terminus or C-terminus to IgG Fc or other polypeptides that bind to neonatal Fcγ / receptors, human serum albumin, or polypeptides that bind to proteins with extended serum half-life, or various non-protein polymers.

[0030] In another aspect, the present disclosure provides pharmaceutical compositions comprising an isolated IL-2 variant admixed with a pharmaceutically acceptable carrier.

[0031] In another aspect, the IL-2 variant can be formulated for sustained release, such as by being covalently or non-covalently attached to a hydrogel, nanoparticle, or the like.

[0032] On the other hand, the present disclosure provides a method for treating an autoimmune disease in a subject, the method comprising administering a therapeutically effective amount of a pharmaceutical composition of the present invention to a subject in need thereof. In one embodiment, the subject is a human subject. The autoimmune disease associated with the present invention is a disease or disorder caused by and directed against an individual's own tissue, or a co-isolation system or manifestation thereof or a condition resulting therefrom. In various embodiments, autoimmune diseases include, but are not limited to, graft-versus-host disease (GvHD), immune-related adverse events (irAEs), 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 mellitus (type 1 diabetes), various dermatitis, Addison's disease, celiac disease, chronic fatigue syndrome, autoimmune hepatitis, autoimmune alopecia, ankylosing spondylitis, ulcerative colitis, Crohn's disease, and the like. disease, fibromyalgia, pemphigus vulgaris, Sjögren'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 patients with multiply transfused aplastic anemia), 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.

[0033] In another aspect, the present disclosure provides methods for treating an autoimmune disease in a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition of the present invention in combination with a second therapeutic agent capable of treating the autoimmune disease.

[0034] On the other hand, the present disclosure provides a method for treating an inflammatory disease in a subject, the method comprising administering a therapeutically effective amount of a pharmaceutical composition of the present invention to a subject in need thereof. In one embodiment, the subject is a human subject. In various embodiments, the inflammatory disease to be treated includes, but is not limited to, Crohn's disease, colitis, dermatitis, psoriasis, diverticulitis, hepatitis, irritable bowel syndrome (IBS), lupus erythematosus, 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-related periodic syndromes and chronic obstructive pulmonary disease.

[0035] In another aspect, the present disclosure provides methods for treating an inflammatory disease in a subject, the methods comprising administering a therapeutically effective amount of a pharmaceutical composition of the present invention in combination with a second therapeutic agent capable of treating the inflammatory disease.

[0036] In another aspect, the present disclosure provides a method for organ transplantation or related graft-versus-host disease in a subject, 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 transplant is an organ selected from the group consisting of heart, kidney, liver, lung, pancreas, intestine, and thymus, or a tissue selected from the group consisting of bone, tendon, cornea, skin, heart valve, nerve, and blood vessel.

[0037] 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.

[0038] In another aspect, the present disclosure provides the use of an IL-2 variant for the preparation of a medicament for the treatment of organ transplantation and GvHD.

[0039] 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.

[0040] 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 a nucleic acid described herein. In various embodiments, the vector is an expression vector. In another aspect, the present disclosure provides an isolated cell comprising a nucleic acid of the present disclosure. In various embodiments, the cell is a host cell comprising an expression vector of the present disclosure. In another aspect, a method for preparing an IL-2 variant is provided by culturing a host cell under conditions that promote expression of a protein or polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 depicts size exclusion chromatograms of exemplary IL-2 Fc fusion proteins A) P-0250, B) P-0318, C) P-0317, D) P-0447, and E) P-0511 following purification by Protein A. Figures 1D and 1E also illustrate SDS-PAGE of each sample in the absence (lane 2) and presence (lane 3) of a reducing agent.

[0043] Figure 2 depicts the differential effects of an Fc fusion protein of an IL-2 variant having an amino acid substitution of aspartic acid at position 20 (D20X) compared to the wild-type fusion protein (P-0250) on the induction of STAT5 phosphorylation in CD4+ Treg cells (A) and Tconv cells (B) in a human PBMC assay.

[0044] Figure 3 depicts the differential effects of the IL-2 variant Fc fusion protein P-0375(N88Q) on the induction of STAT5 phosphorylation in CD4+ Treg cells (A) and Tconv cells (B) compared to wild-type (P-0250) and benchmark protein in a human PBMC assay.

[0045] Figure 4 depicts the differential effects of Fc fusion proteins of IL-2 variants with amino acid substitutions at position 19 on STAT5 phosphorylation compared to wild-type (P-0250). In a human PBMC assay, the ability to induce STAT5 phosphorylation in CD4+ Treg cells (A and C) and Tconv cells (B and D) was determined by FACS analysis.

[0046] Figure 5 depicts the differential effects of Fc fusion proteins of IL-2 variants with single amino acid substitutions or combined mutations (P-0447) at position 19 or position 126 compared to wild type (P-0250) or benchmark-1 protein on STAT5 phosphorylation. The ability to induce STAT5 phosphorylation in CD4+ Treg cells (A, C, and E) and CD4+ Tconv cells (B, D, and F) was determined by FACS analysis.

[0047] Figure 6 depicts the different effects of Fc fusion proteins (P-0419, P-0464, P-0471, P-0474, P-0417, and P-0322) of IL-2 variants containing different combinations of dual amino acid substitutions on STAT5 phosphorylation compared to wild type (P-0250). The biological activities of P-0417 and P-0322 were also compared to their counterparts P-0373 and P-0363 with single amino acid substitutions, respectively. In human PBMC assays, the ability to induce STAT5 phosphorylation in CD4+ Treg cells (A and C) and Tconv cells (B and D) was determined by FACS analysis.

[0048] Figure 7 depicts the differential effects of Fc fusion proteins of IL-2 variants with single amino acid substitutions or combined mutations (P-0447) at position 19 or position 126 (P-0303) on STAT5 phosphorylation compared to wild type (P-0250), as well as the differential effects of Fc fusion proteins of IL-2 variants containing different combinations of 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, the ability to induce STAT5 phosphorylation in CD4+ Treg cells (A and C) and CD4+ Tconv cells (B and D) was determined by FACS analysis.

[0049] Figure 8 depicts a comparison of the pSTAT5 stimulatory activity of IL-2 fusion proteins P-0250, P-0424, and P-447 with their respective counterparts P-0531, P-0491, and P-0511 containing an S125I substitution. In a human PBMC assay, the ability to induce STAT5 phosphorylation in CD4+ Treg cells (A, C, and E) and CD4+ Tconv cells (B, D, and F) was determined by FACS analysis.

[0050] Figure 9 depicts the differential effects of IL-2 variant Fc fusions (P-0511 and P-0512) on STAT5 phosphorylation in three CD4+ T cell subsets (A) CD4+FoxP3+CD25+ Treg cells, B) CD4+FoxP3-CD25+ activated Tconv cells, and C) CD4+FoxP3-CD25- naive Tconv cells) compared to wild type (P-0250) and three benchmark molecules. In a human PBMC assay, the ability to induce STAT5 phosphorylation was determined by FACS analysis.

[0051] Figure 10 depicts the differential effects of P-0511 and P-0512 on stimulating proliferation of A) CD8+ T cells and B) NK cells compared to wild type (P-0250) and a benchmark molecule. In the human PBMC assay, cell proliferation was determined by FACS analysis of CFSE dilution and expressed as the percentage of dividing cells.

[0052] Figures 11A-11D depict the differential effects of IL-2 variant Fc fusion P-0511 in inducing STAT5 phosphorylation in different cell types compared to the wild-type equivalent P-0531. In a human PBMC assay, the ability to induce STAT5 phosphorylation in A) CD4+ Treg cells, B) CD4+ Tconv cells, C) CD8+ T cells, and D) NK cells was determined by FACS analysis. Figure 11E Depicted are the binding strength of P-511 to IL-2Rβ and γc complexes compared to P-0531 and Benchmark-1 in ELISA assays.

[0053] Figure 12 depicts the proliferation and expansion of Treg cells in mice treated with Fc fusion proteins of IL-2 variants and benchmarks after a single subcutaneous injection. Blood was collected at the indicated time points for proliferation measurements and lymphocyte phenotyping. (A) Percentage of Treg cells positive for the proliferation marker Ki67; (B) Percentage of Treg cells in the total CD4+ T cell population; (C) Percentage of Treg cells in total blood lymphocytes. Data 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, compared to the PBS group at the respective time points.

[0054] Figure 13 depicts the proliferation of effector T cells and NK cells in mice treated with IL-2 mutant Fc fusion protein and benchmark after a single subcutaneous injection. Blood was collected at the indicated time points to measure lymphocyte proliferation. (A) Percentage of Ki67-positive CD4+T conventional (Tconv) cells; (B) Percentage of Ki67-positive CD8+T cells; (C) Percentage of Ki67-positive NK cells. Data 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, compared to the PBS group at the respective time points.

[0055] Figure 14 depicts the expansion of effector T cells and NK cells in mice treated with IL-2 mutant Fc fusion proteins and benchmarks after a single subcutaneous injection. (A) Percentage of CD4+ conventional T cells (Tconv) in total CD4+ T cells (A) and total blood lymphocytes (B). (C) Percentage of CD8+ T cells in total blood lymphocytes; (D) Percentage of NK cells in total blood lymphocytes. Data are expressed as mean ± SEM.

[0056] Figure 15 depicts the ratio of Treg to Tconv cells in mice treated with IL-2 mutant Fc fusion proteins and baseline, based on: A) percentage of Ki67 positive expression and B) cell number. Data were acquired using FACS and expressed 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 to the PBS group at the respective time points.

[0057] Figure 16 depicts the expression of CD25 and Foxp3 on Treg cells in mice treated with IL-2 mutant Fc fusion proteins and benchmarks after a single subcutaneous injection. A) Foxp3 and B) CD25 expression levels were analyzed by FACS analysis and expressed as mean fluorescence intensity (MFI). Data are expressed as mean ± SEM. ****p < 0.0001, compared to the PBS group at the respective time points.

[0058] Figure 17 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 phenotyping and measurement of the Ki67 proliferation marker. (A) Percentage of Ki67-positive Treg cells for proliferation marker; (B) Percentage of Treg cells in total CD4+ T cells; (C) Number of Treg cells per microliter of whole blood; (D) Change in the number of Treg cells in each group from baseline. Data 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, *p<0.05, compared with the PBS group at their respective time points.

[0059] Figure 18 depicts the dose-dependent effect of IL-2 variant Fc fusion protein P-0511 on the percentage of Treg cells (A), CD4+Tconv cells (B), CD8 T cells (C) and NK cells (D) in the total lymphocytes of mice after a single injection. Blood was collected at the indicated time points for lymphocyte phenotyping. The data were determined by FACS analysis and expressed as mean ± SEM. Statistical analysis was performed by one-way ANOVA test followed by Tukey's post hoc test. ****p<0.0001, **p<0.01, *p<0.05, compared with the PBS group at each time point.

[0060] Figure 19 depicts the dose-dependent increase in A) Treg to Tconv cell number ratio, B) CD25 expression on Treg cells, and C) Foxp3 expression on Treg cells 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 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.

[0061] Figure 20 depicts the sustained proliferation and expansion of Treg cells in mice that received repeated doses of Fc fusion proteins of IL-2 variants (P-0511 and P-0512) instead of wild type (P-0531) and benchmarks. The compound was administered sc once every three days (Q3D), and blood was collected 3 days after the first and third injections for lymphocyte phenotyping and measurement of the proliferation marker Ki67. (A) The percentage of Ki67-positive Treg cells; (B) The percentage of Treg cells in total CD4+T cells; (C) The percentage of Treg cells in total blood lymphocytes. The 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 their respective PBS groups.

[0062] Figure 21 depicts the continued increase in Treg cell counts in mice that received repeated doses of Fc fusion proteins of IL-2 variants (P-0511 and P-0512) instead of wild type (P-0531) and benchmarks. The compound was administered sc once every three days (Q3D), and blood was collected 3 days after the first and third injections for lymphocyte phenotyping and measurement of the proliferation marker Ki67. (A) The number of Treg cells per microliter of whole blood; (B) The change in the number of Tregs compared to the PBS control group. The 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 groups.

[0063] Figure 22 Depicts the maintenance of the ratio of Treg to Tconv that was elevated in mice receiving repeated doses of Fc fusion proteins of IL-2 variants (P-0511 and P-0512) instead of wild type (P-0531) and benchmark. The compound was administered sc once every three days (Q3D), and blood was collected 3 days after the first and third injections for Treg and Tconv cell phenotyping. The ratio was calculated based on Treg% and Tconv% in total CD4 cells. The 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, compared to the respective PBS groups.

[0064] Figure 23 depicts the keyhole P-0511 inhibits antigen-driven inflammation in a mouse model of delayed-type hypersensitivity (DTH) induced by hemocyanin (KLH) antigen. Mice were immunized with KLH on day 0 and re-challenged in the right ear on day 5. Starting on day -2, mice were treated with P-0511 Q3D or Q5D. For A) Q3D and B) Q5D dosing regimens, the change in ear thickness relative to baseline values ​​at different times after KLH challenge (Δ ear thickness) was used to illustrate the kinetics of the DTH response. Data are expressed 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 to the respective PBS groups at the respective time points.

[0065] Figure 24Depicted is the inhibition of antigen-driven inflammation by P-0511 compared to a benchmark molecule (Benchmark-1) in a mouse model of DTH induced by the KLH antigen. Mice were immunized with KLH on day 0 and re-challenged in the right ear on day 5. Starting on day -2, mice were treated with compound Q5D. The change in ear thickness relative to baseline values ​​(Δ ear thickness) at different times after KLH challenge was used to illustrate the kinetics of the DTH response. Data are expressed 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 to the respective PBS group at the respective time points.

[0066] Means for carrying out the present disclosure

[0067] The present invention relates to polypeptides that share a primary sequence with human IL-2, except for one to several mutated amino acids. One group of IL-2 variants comprises 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 used 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 comprises mutations that significantly reduce the ability of these polypeptides to stimulate Treg cells or Tconv cells, while having receptor binding activity and making them effective as IL-2 antagonists. On the other hand, 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, which is due to their selective regulatory effects on the immune system against diseases such as autoimmune disorders and inflammatory disorders.

[0068] definition

[0069] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. In various embodiments, "peptide", "polypeptide" and "protein" are amino acid chains whose α carbons are connected by peptide bonds. Therefore, the terminal amino acid at one end (amino terminal) of the chain has a free amino group, while the terminal amino acid at the other end (carboxyl terminal) of the chain has a free carboxyl group. As used herein, the term "amino terminal" (abbreviated as N-terminal) refers to the free α-amino group on the amino acid at the amino terminal of a peptide, or refers to the α-amino group (amino group when participating in a peptide bond) of the amino acid at any other position in the peptide. Similarly, the term "carboxyl terminal" refers to the free carboxyl group on the carboxyl terminal of a peptide, or the carboxyl group of the amino acid at any other position in the peptide. Peptide also includes substantially any polyamino acid, including but not limited to peptide mimetics (peptide mimetic), such as the amino acid connected by ether bonds rather than amide bonds.

[0070] The polypeptides of the present disclosure include polypeptides that have been modified in any manner and for any reason, for example, to: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or alter other physicochemical or functional properties.

[0071] As used herein, an amino acid "substitution" refers to the replacement of an amino acid at a specific position in a parent polypeptide sequence with a different amino acid. Amino acid substitutions can be produced using genetic 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 portions of a polypeptide outside of the domains forming intermolecular contacts). A "conservative amino acid substitution" refers to the replacement of an amino acid in a polypeptide with a functionally similar amino acid. The following six groups each contain amino acids that are conservative substitutions for each other:

[0072] 1) Alanine (A), serine (S), and threonine (T)

[0073] 2) Aspartic acid (D) and glutamic acid (E)

[0074] 3) Asparagine (N) and glutamine (Q)

[0075] 4) Arginine (R) and Lysine (K)

[0076] 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V)

[0077] 6) Phenylalanine (F), tyrosine (Y) and tryptophan (W)

[0078] A "non-conservative amino acid substitution" refers to a substitution of a member of one of these classes for a member from another class. When making such changes, according to various embodiments, the hydropathic index of the amino acid can be considered. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics. 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); glutamate (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5).

[0079] The importance of the hydropathic amino acid index in conferring interactive biological function on proteins is understood in the art (see, for example, Kyte et al., 1982, J. Mol. Biol. 157: 105-131). It is known that certain amino acids can be substituted with other amino acids having similar hydropathic indices or scores and still retain similar biological activity. When making changes based on the hydropathic index, in various embodiments, substitutions of amino acids whose hydropathic index is within ± 2 are included. In various embodiments, those within ± 1 are included, and in various embodiments, those within ± 0.5 are included.

[0080] It is also understood in the art that substitution of similar amino acids can be effectively made based on hydrophilicity, particularly when the resulting biologically functional protein or peptide is intended for use in immunological embodiments, as disclosed herein. In various embodiments, the greatest local average hydrophilicity of a protein (as determined by the hydrophilicity of its adjacent amino acids) correlates with its immunogenicity and antigenicity, i.e., with the biological properties of the protein.

[0081] 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, substitution of amino acids whose hydrophilicity values ​​are within ±2 is included, in various embodiments, those within ±1 are included, and in various embodiments, those within ±0.5 are included.

[0082] Exemplary amino acid substitutions are listed in Table 1.

[0083] Table 1

[0084]

[0085]

[0086] The skilled artisan will be able to determine suitable polypeptide variants as listed herein using known techniques. In various embodiments, one skilled in the art can identify suitable regions of the molecule that can be altered without destroying activity by targeting regions that are not considered important for activity. In other embodiments, one skilled in the art can identify residues and portions of the molecule that are conserved between similar polypeptides. In additional embodiments, even regions that may be important for biological activity or structure can undergo conservative amino acid substitutions without destroying biological activity or adversely affecting polypeptide structure.

[0087] In addition, one skilled in the art can review structure-function studies that identify residues in similar polypeptides that are important for activity or structure. Based on such comparisons, one can predict the importance of amino acid residues in a polypeptide that correspond to amino acid residues in similar polypeptides that are important for activity or structure. One skilled in the art can choose to substitute chemically similar amino acids for the amino acid residues predicted to be important.

[0088] Those skilled in the art can also analyze the three-dimensional structure and the amino acid sequence relevant to this structure in similar polypeptides.In view of such information, those skilled in the art can predict the arrangement of the amino acid residues of polypeptide in terms of its three-dimensional structure.In various embodiments, those skilled in the art can choose not to carry out radical changes (radical changes) to the amino acid residues predicted on the polypeptide surface, because such residues may participate in important interactions with other molecules.In addition, those skilled in the art can produce test variants comprising single amino acid substitutions at each desired amino acid residue.Then activity assays known to those skilled in the art can be used to screen variants.These variants can be used to collect information about suitable variants.For example, if people find that the change of specific amino acid residues causes destruction, undesirably reduced or inappropriate activity, then variants with such changes can be avoided.In other words, based on the information collected from such routine experiments, those skilled in the art can easily determine to avoid further substituted amino acids therein separately or in combination with other mutations.

[0089] 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. A fragment can also comprise one or more additional amino acids at either or both ends, 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).

[0090] As used herein, the terms "polypeptide variant," "hybrid polypeptide," and "polypeptide mutant" refer to a polypeptide comprising an amino acid sequence in which one or more amino acid residues are inserted into, deleted from, and / or substituted into an 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. Hybrids of the present disclosure include fusion proteins.

[0091] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, for example, by conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (eg, human serum albumin), phosphorylation, and glycosylation.

[0092] The term "% sequence identity" is used interchangeably with the term "% identity" in this article and refers 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 using a sequence alignment program. For example, as used herein, 80% identity determined by a defined algorithm means the same as 80% sequence identity, and means that a given sequence is at least 80% identical to another sequence of another length. In various embodiments, % identity is selected from, for example, 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 sequence identity to a given sequence. In various embodiments, % identity is, for example, in the range of 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%.

[0093] The term "% sequence homology" is used interchangeably with the term "% homology" herein and refers 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 compared using a sequence alignment program. For example, as used herein, 80% homology as determined by a defined algorithm means the same as 80% sequence homology, and thus homologs of a given sequence have a sequence homology greater than 80% relative to the length of the given sequence. In various embodiments, % homology is selected from, for example, 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 sequence homology to a given sequence. In various embodiments, % homology is, for example, in the range of 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%.

[0094] Exemplary computer programs that can be used to determine the identity between two sequences include, but are not limited to, a set 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 (with particular reference 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 amino acid sequences in GenBank protein sequences and other public databases, the BLASTP program is typically used for sequence searching. The BLASTX program is preferably used to search for nucleic acid sequences that have been translated in all reading frames for amino acid sequences in GenBank protein sequences and other public databases. Both BLASTP and BLASTX are run using the default parameters of an open gap penalty of 11.0 and an extended gap penalty of 1.0 and using the BLOSUM-62 matrix.

[0095] In addition to calculating 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 provides an indication of the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, a nucleic acid is considered similar to a reference sequence if, in a comparison of a test nucleic acid to a reference nucleic acid, the minimum total probability is, e.g., less than about 0.1, less than about 0.01, or less than about 0.001.

[0096] As used herein, the term "modification" refers to any manipulation of the peptide backbone (eg, amino acid sequence) or post-translational modification of the polypeptide (eg, glycosylation).

[0097] 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, the "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 technique is described, for example, in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001).

[0098] 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 peptide linkers. 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.

[0099] "Joint" refers to a molecule that connects two other molecules covalently or by ionic bonds, van der Waals forces or hydrogen bonds, for example, a nucleic acid molecule that hybridizes at the 5' end with a complementary sequence and at the 3' end with another complementary sequence to connect two non-complementary sequences. "Cleaving joint" refers to a joint that can be degraded or otherwise cut off to separate the two components connected by the cleavable joint. Cleaving joints are usually cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, etc. Cleaving joints can also be cleaved by environmental factors, such as changes in temperature, pH, salt concentration, etc.

[0100] As used herein, the term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides include, for example, (G4S) n 、(SG4) n or G4(SG4)n peptide linker. "n" is typically a number between 1 and 10, usually between 2 and 4.

[0101] "Pharmaceutical composition" refers to a composition suitable for pharmaceutical use in animals. A pharmaceutical composition comprises a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. "Pharmacologically effective amount" refers to the amount of an agent that effectively produces the expected pharmacological result. "Pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, vehicle, buffer and excipient, such as phosphate buffered saline solution, 5% dextrose aqueous solution, and emulsions, such as oil / water emulsions or water / oil emulsions, 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 salts" are salts of compounds that can be formulated for pharmaceutical use, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or salts of organic amines.

[0102] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of the disease in the individual being treated, and the clinical intervention can be performed for prevention or during the course of clinical pathology. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of the symptoms of the disease, disorder, or condition. Additionally, references herein to "treatment" include references to curative, palliative, and prophylactic treatments.

[0103] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or composition sufficient to treat a particular disorder, condition, or disease, such as to improve, alleviate, mitigate, and / or delay one or more symptoms thereof. With respect to cancer or other unwanted cell proliferation, an effective amount includes an amount sufficient to: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow, and preferably stop to some extent, the infiltration of cancer cells into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; and / or (vii) alleviate to some extent one or more symptoms associated with cancer. An effective amount can be administered in one or more administrations.

[0104] The phrases "administer" or "cause to be administered" refer to actions taken by a medical professional (e.g., a physician) or person controlling a patient's medical care to control and / or permit the administration of the agent / compound in question to a patient. Causing administration can include diagnosing and / or determining an appropriate treatment regimen, and / or prescribing a particular agent / compound to a patient. Such prescribing can include, for example, drafting a prescription form, annotating a medical record, etc. "Causing administration" is also contemplated when describing administration herein.

[0105] The terms "patient," "individual," and "subject" are used interchangeably and refer to mammals, preferably humans or non-human primates, but also domestic mammals (e.g., canines or felines), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., equines, bovines, swine, ovines). In various embodiments, a patient can be a human (e.g., an adult male, an adult female, an adolescent male, an adolescent female, a male child, a female child) under the care of a physician or other health worker in a hospital, psychiatric care facility, such as an outpatient clinic, or other clinical setting. In various embodiments, a patient can be an immunocompromised patient or a patient with a weakened immune system, including but not limited to patients with primary immunodeficiency, AIDS; cancer patients and transplant patients taking certain immunosuppressive drugs; and patients with genetic diseases that affect 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 high mutation rates (Lawrence et al., Nature, 499(7457):214-218, 2013).

[0106] As used herein, the term "Fc domain" or "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region comprises 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 chain and a corresponding introduced "cavity" ("hole") in another chain; see U.S. Patent No. 5,821,333, which is expressly incorporated herein by reference). Such a variant CH3 domain can be used to promote the 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.

[0107] As used herein, the term "effector function" refers to those biological activities attributable to the Fc region of an immunoglobulin, which differs among immunoglobulin subtypes. 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, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0108] As used herein, the term "regulatory T cells" or "Treg cells" refers to a specialized type of CD4+ T cell that can suppress the response of other T cells (effector T cells). Treg cells are characterized by the expression of CD4, the α subunit of the IL-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.

[0109] As used herein, the term "conventional CD4+ T cells" means CD4+ T cells other than regulatory T cells.

[0110] As used herein, the term "selective activation of Treg cells" means the activation of Treg cells that are 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 distinguishing these cell types are described in the embodiments. Activation can include inducing IL-2 receptor signaling (as measured by, for example, detecting phosphorylated STAT5), inducing proliferation (as measured by, for example, detecting Ki-67) and / or raising the expression of activation markers (such as, for example, CD25) and the amplification of cell number.

[0111] As used herein, "specific binding" 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 to 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.

[0112] 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 (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y can generally be characterized by the dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (koff and kon, respectively). A specific method for measuring affinity is surface plasmon resonance (SPR).

[0113] 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.

[0114] 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; and mixtures and modifications thereof. Furthermore, unless expressly limited otherwise, the term "polymer" shall include all possible geometric configurations of the material. These configurations include, but are not limited to, isotactic, syndiotactic, and random symmetries.

[0115] "Polynucleotide" refers to a polymer comprising nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") and nucleic acid analogs. Nucleic acid analogs include those comprising the following: non-naturally occurring bases, nucleotides that engage with other nucleotides with connections other than naturally occurring phosphodiester bonds, or nucleotides that comprise bases attached by connections other than phosphodiester bonds. Therefore, nucleotide analogs include, for example and without limitation, phosphorothioate, phosphorodithioate, phosphotriester, phosphoramidate, boranophosphate, methylphosphonate, chiral methylphosphonate, 2-O-methyl ribonucleotide, peptide nucleic acid (PNA), etc. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" generally refers to large polynucleotides. The term "oligonucleotide" generally refers to short polynucleotides, generally not more than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (ie, A, T, G, C), this also includes RNA sequences (ie, A, U, G, C) in which "U" replaces "T."

[0116] Conventional notation is used herein 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 referred to as the 5'-direction. The direction of nucleotide addition from 5' to 3' of a nascent RNA transcript is referred to as the direction of transcription. The DNA strand having the same sequence as the mRNA is referred to as the "coding strand"; the sequence on the DNA strand having the same sequence as the mRNA transcribed from that DNA and located 5' to the 5'-end of the RNA transcript is referred to as the "upstream sequence"; the sequence on the DNA strand having the same sequence as the RNA and located 3' to the 3'-end of the coding RNA transcript is referred to as the "downstream sequence."

[0117] "Complementary" refers to the topological compatibility or matching 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 identical to 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.

[0118] "Specifically hybridizes to" or "specifically hybridizes" or "selectively hybridizes to" refers to the preferential binding, duplexing or hybridization of a nucleic acid molecule to a particular nucleotide sequence when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA under stringent conditions. The term "stringent conditions" refers to conditions under which a probe will preferentially hybridize to its target subsequence and to other sequences to a lesser extent or not at all. In the context of nucleic acid hybridization experiments such as Southern hybridization and Northern hybridization, "stringent hybridization" and "stringent hybridization wash conditions" are sequence-dependent and are different under different environmental parameters. An extensive guide to 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, NY; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3rd Suppl. ed., NY; and Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

[0119] Typically, highly stringent hybridization and washing conditions are selected to be about 5°C lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH conditions) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm of the specific probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids having more than about 100 complementary residues on filters in Southern or Northern blotting is hybridization at 42°C in 50% formalin with 1 mg of heparin overnight. An example of highly stringent washing conditions is 0.15 M NaCl at 72°C for about 15 minutes. An example of stringent washing conditions is 0.2x SSC washing at 65°C for 15 minutes. See Sambrook et al. for a description of SSC buffer. A low stringency wash can be performed to remove background probe signal, followed by a high stringency wash. An exemplary moderate stringency wash for, e.g., a duplex of more than about 100 nucleotides is 1 x SSC at 45° C. for 15 minutes. An exemplary low stringency wash for, e.g., a duplex of more than about 100 nucleotides is 4-6 x SSC at 40° C. for 15 minutes. Typically, a signal-to-noise ratio of 2x (or higher) compared to the signal-to-noise ratio observed for an unrelated probe in a particular hybridization assay indicates that specific hybridization has been detected.

[0120] " Primer " refers to the polynucleotide that can hybridize with the polynucleotide template specificity of appointment and provide the starting point for synthesizing complementary polynucleotide. When polynucleotide primer is placed under the condition of induction synthesis (that is, in the presence of nucleotide, complementary polynucleotide template and the agent such as DNA polymerase for polymerization), such synthesis occurs. Primer is normally single-stranded, but can be double-stranded. Primer is generally deoxyribonucleic acid, but many synthetic and naturally occurring primers can be used for many applications. Primer is complementary to template, and primer is designed to hybridize with template to be used as the site of synthesis initiation, but does not need to reflect the accurate sequence of template. In such case, the specific hybridization of primer and template depends on the stringency of hybridization conditions. Primer can be used as detectable part with for example color development, radioactivity or fluorescence part mark.

[0121] When used in relation to polynucleotides, "probe" refers to a polynucleotide that can specifically hybridize to a given sequence of another polynucleotide. The 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. The probe can be labeled with, for example, a colorimetric, radioactive or fluorescent moiety and used as a detectable moiety. In the case where the probe provides a starting point for synthesizing a complementary polynucleotide, the probe can also be a primer.

[0122] " vector " is the polynucleotide that can be used to another nucleic acid being connected thereto and is introduced into the cell.A type of vector is " plasmid ", and it refers to that other nucleic acid segment can be connected to linear or circular double-stranded DNA molecule wherein.Another type of vector is viral vector (for example, replication defective retrovirus, adenovirus and adeno-associated virus), and wherein other DNA segment can be introduced in the viral genome.Some vector can be autonomously replicated (for example, bacterial vector and additional mammalian vector (episomal mammalian vector) comprising bacterial replication origin) in the host cell having introduced them.Other vectors (for example, non-additional mammalian vector) are integrated into the genome of the host cell after being introduced into the host cell, and thereby replicate together with the host genome." expression vector " is a type of vector that can guide the expression of the polynucleotide of selection.

[0123] A "regulatory sequence" is a nucleic acid that affects the expression (e.g., the level, timing, or position of expression) of a nucleic acid to which it is operably linked. A regulatory sequence can, for example, exert its effect directly on the regulated nucleic acid, or exert its effect through the action of one or more other molecules (e.g., a polypeptide bound to the regulatory sequence and / or 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 in, for example, 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 (e.g., the level, timing, or position of expression) of the nucleotide sequence.

[0124] "Host cell" is a cell that can be used to express the polynucleotides of the present disclosure. The host cell can be a prokaryotic organism, such as Escherichia coli (E. coli), or the host cell can be a eukaryotic organism, such as a unicellular eukaryotic organism (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., a human cell, monkey cell, hamster cell, rat cell, mouse cell or insect cell) or a hybridoma. Generally, a host cell is a cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, and the nucleic acid can then be expressed in the host cell. The term "recombinant host cell" can be used to represent a host cell that has been transformed or transfected with a nucleic acid to be expressed. The host cell can also be a cell that comprises nucleic acid but does not express the nucleic acid at a desired level, unless a regulatory sequence is introduced into the host cell so that the regulatory sequence becomes operably connected with the nucleic acid. It should be understood that the term host cell refers not only to specific subject's cells, but also to the offspring or potential offspring of such a cell. Because certain modifications may occur in succeeding generations due to, for example, mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0125] The term "isolated molecule" (wherein the molecule is, for example, a polypeptide or polynucleotide) is a molecule that, by virtue of its origin or source of derivation, (1) is not associated with naturally associated components with which it accompanies it in its natural state, (2) is substantially free of other molecules from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cell from which it naturally originates would be "isolated" from its naturally associated components. A molecule may also be rendered substantially free of naturally associated components by isolation using purification techniques well known in the art. Molecular purity or homogeneity can be determined by a number of means well known in the art. For example, the purity of a polypeptide sample can be determined using polyacrylamide gel electrophoresis and staining the gel to visualize the polypeptide using techniques well known in the art. For some purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

[0126] A protein or polypeptide is "substantially pure," "substantially homogeneous," or "substantially purified" when at least about 60% to 75% of the sample represents a single species of polypeptide. The polypeptide or protein can be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80%, or 90% w / w of the protein sample, more typically about 95% and preferably will be greater than 99% pure. Protein purity or homogeneity can be indicated by many means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization of individual polypeptide bands by staining the gel with stains well known in the art. For some purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification.

[0127] 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 biotinyl moiety that can be detected by labeled avidin (e.g., streptavidin containing 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 a toxin. Various methods of 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; a predetermined polypeptide epitope recognized by a secondary reporter (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag); 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 analogs or homologs thereof. In various embodiments, the label is attached by spacer arms of various lengths to reduce potential steric hindrance.

[0128] As used herein, the term "heterologous" refers to a non-native or non-natural combination or state, which can be achieved, for example, by replacing existing native components or states with components or states derived from another source. Similarly, expression of a protein in an organism other than the organism in which the protein is naturally expressed constitutes a heterologous expression system and a heterologous protein.

[0129] It should be understood that aspects and embodiments of the present disclosure described herein include "consisting of" and / or "consisting essentially of" these aspects and embodiments.

[0130] Reference herein to "about" a value or parameter includes (and describes) variations with respect to that value or parameter itself. For example, reference to "about X" includes a description of "X."

[0131] As used herein and in the appended claims, the singular forms "a," "an," "or," and "the" include plural referents unless the context clearly dictates otherwise. It should be understood that aspects and variations of the present disclosure described herein include "consisting of" and / or "consisting essentially of" these aspects and variations.

[0132] IL-2

[0133] Interleukin-2 (IL-2) is a classic Th1 cytokine produced by T cells following activation via the T cell antigen receptor and the co-stimulatory molecule CD28. Regulation of IL-2 occurs through the activation of signaling pathways and transcription factors that act on the IL-2 promoter to produce new gene transcription, but also involves regulation of the stability of IL-2 mRNA. IL-2 binds to multi-chain receptors, including a highly regulated α chain and β and γ chains that mediate signaling via the Jak-STAT pathway. IL-2 delivers activation, growth, and differentiation signals to T cells, B cells, and NK cells. IL-2 is also important in mediating activation-induced cell death of T cells, a function that provides a key mechanism for terminating the immune response. A commercially available unglycosylated human recombinant IL-2 product, aldesleukin (available from Prometheus Laboratories Inc., San Diego Calif. under the trademark IL-2 is a cytokine derived from human interleukin-2 (derived from des-alanyl-1, serine-125) and has been approved for administration to patients suffering from metastatic renal cell carcinoma and metastatic melanoma. IL-2 has also been suggested for administration to patients suffering from or infected with hepatitis C virus (HCV), human immunodeficiency virus (HIV), acute myeloid leukemia, non-Hodgkin's 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.

[0134] As used herein, the terms "natural IL-2" and "natural 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 amino acid sequences of natural mammalian interleukin-2 of 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, natural IL-2 is an immature or precursor form of naturally occurring mammalian IL-2. In other embodiments, natural 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:

[0135]

[0136] In various embodiments, the IL-2 comprises the amino acid sequence of the wild-type sequence of the mature form of human IL-2 set forth in SEQ ID NO:3 comprising a cysteine ​​to serine substitution at position 125, but without altered IL-2 receptor binding compared to naturally occurring IL-2:

[0137]

[0138] IL-2 variants

[0139] The present invention relates to polypeptides having a common primary sequence with human IL-2 except for one to several mutated amino acids. A group of IL-2 variants comprises 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. Also included are such IL-2 selective agonists used alone or in combination with disease tissue targeting proteins or peptides to treat Treg cell deficiency autoimmune disorders and various inflammatory disorders. On the other hand, the present invention relates to pharmaceutical compositions comprising the disclosed polypeptides. Finally, the present invention relates to the therapeutic uses 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 or cancer and various infectious diseases.

[0140] The present invention relates to polypeptides having a length of 100 to 500 amino acids, preferably 140 residues, with an apparent molecular weight of at least 15 kD. These polypeptides retain a high sequence identity of greater than 90% with native IL-2. These polypeptides are mutated at certain positions to introduce amino acid residues that differ from the amino acid residues at the same positions in native IL-2.

[0141] The polypeptides of the present invention may be referred to as immunomodulatory polypeptides, IL-2 analogs, or IL-2 variants, among 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 primarily introduced into IL-2 positions corresponding to amino acids that interact with the receptor subunits β, γ, or βγ.

[0142] 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 differs from the native (or wild-type) IL-2 protein. In various embodiments, the IL-2 variant binds to the IL-2Rα polypeptide and acts as an IL-2 agonist or antagonist. In various embodiments, the IL-2 variant having agonist activity has superagonist activity. In various embodiments, the IL-2 variant can act as an IL-2 agonist or antagonist, regardless of its association with IL-2Rα. IL-2 agonists are exemplified by having comparable or increased biological activity compared to wild-type IL-2. IL-2 antagonists are exemplified by having reduced biological activity compared to wild-type IL-2 or by the ability to inhibit an IL-2-mediated response. In various embodiments, the sequence of the IL-2 variant has at least one amino acid change, such as a substitution or deletion, compared to the native IL-2 sequence, such changes resulting in IL-2 agonist or antagonist activity. In various embodiments, the IL-2 variants as Fc fusion proteins have the amino acid sequences set forth in SEQ ID NOs: 4-43, 108-146, and 193-197, with reduced binding to IL-2Rβ and / or γ C The combination of TNF-α and TNF-α enhances the selectivity of regulatory T cell (Treg) activation and proliferation.

[0143] Exemplary IL-2 variants are provided in Tables 2A-2F:

[0144] Table 2A

[0145] IL-2 single mutations targeting both the IL-2Rβ interface and the proposed toxicity motif

[0146]

[0147] Table 2B

[0148] IL-2 single mutations targeting the IL-2Rβ interface

[0149]

[0150] Table 2C

[0151] IL-2 single mutation targeting the γc receptor interface

[0152]

[0153] Table 2D

[0154] Combinations of IL-2 mutations targeting both the IL-2Rβ interface and proposed toxicity motifs

[0155]

[0156] Table 2E

[0157] Combinations of IL-2 mutations targeting the IL-2Rβ,γc interface and proposed toxicity motifs

[0158]

[0159] Table 2F

[0160] Single or combined IL-2 mutations that improve fusion protein manufacturability and target the IL-2Rβ and / orγc interface and proposed toxicity motifs

[0161]

[0162] The present invention also includes additional modifications to the above-mentioned IL-2 variant categories, in particular additional modifications to the IL-2 variants described in Table 2A-Table 2F. As will be appreciated by those skilled in the art, additional combination mutants combining the preferred mutations described in Table 2A-Table 2F can produce IL-2 agonists that are more selective for Treg cells. Any additional combination mutations are in line with 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 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 properties (phage libraries, gene expression libraries in yeast or bacteria). On the other hand, the present invention relates to a fusion protein comprising any of the immunomodulatory polypeptides described above coupled to a carrier protein. The carrier protein can be an Fc region of albumin or a human immunoglobulin.

[0163] Fc domain

[0164] The immunoglobulin of the IgG class is one of the most abundant proteins in human blood. Their circulation half-life can be as long as 21 days. It has been reported that the fusion protein of the Fc region of IgG is combined with the domain of another protein (such as various cytokines and receptors) (see, for example, Capon et al., Nature, 337:525-531, 1989; Chamow et al., Trends Biotechnol., 14:52-60, 1996); U.S. Patent Nos. 5,116,964 and 5,541,087). The prototype fusion protein is a homodimeric protein connected by the cysteine ​​residues in the hinge region of IgG Fc, producing a molecule similar to an IgG molecule, without a heavy chain variable region and CH1 domain and a light chain. The dimer nature of the fusion protein comprising the Fc domain may be advantageous in providing a higher order interaction (i.e., bivalent or bispecific binding) with other molecules. Due to structural homology, Fc fusion proteins exhibit in vivo pharmacokinetic profiles comparable to human IgG of similar isotype.

[0165] The term "Fc" refers to a molecule or sequence comprising the sequence of a non-antigen binding fragment of a complete antibody, whether in monomeric or multimeric form. The original immunoglobulin source of native Fc is preferably human origin, and can be any immunoglobulin, although IgG1 and IgG2 are preferred. Native Fc consists of monomeric polypeptides, which can be linked to dimers or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between the monomer subunits of native Fc molecules depends on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2) and is in the range of 1 to 4. An example of native Fc is a 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 monomers, dimers, and multimeric forms. The Fc domain contains binding sites for protein A, protein G, various Fc receptors, and complement proteins.

[0166] In various embodiments, the term "Fc variant" refers to a molecule or sequence that is modified from a native Fc but still contains a 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 interactions with salvage receptors and are hereby incorporated by reference. In addition, a 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 a selected host cell, (3) N-terminal heterogeneity when expressed in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0167] The term "Fc domain" includes natural Fc and Fc variant molecules and sequences as defined above. Like Fc variants and natural Fc, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from a complete antibody or expressed by a recombinant gene or produced by other means. In various embodiments, an "Fc domain" refers to a dimer of two Fc domain monomers (SEQ ID NO: 44), which typically include all or part of the hinge region. In various embodiments, the Fc domain can be mutated to lack effector functions. In various embodiments, each Fc domain monomer in the Fc domain comprises an amino acid substitution 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 comprises two amino acid substitutions that reduce binding and / or effector functions to activating Fc receptors, wherein the amino acid substitutions are L234A and L235A. In various embodiments, each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to an activating Fc receptor and / or effector function, wherein the amino acid substitutions are L234A, L235A, and G237A (SEQ ID NO: 45).

[0168] In various embodiments, each of the two Fc domain monomers in the Fc domain comprises an amino acid substitution that promotes the heterodimerization of the two monomers. In various other embodiments, the 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. "Knob-into-hole" technology is also disclosed in U.S. Patent Publication No. 8,216,805. In another embodiment, an Fc domain monomer comprises a knob mutation T366W, and another Fc domain monomer comprises a hole mutation T366S, L358A and Y407V. In various embodiments, two Cys residues (S354C on the "knob" side and Y349C on the "hole" side) that form a stabilized disulfide bridge are introduced. The use of heterodimer Fc can produce monovalent IL-2 variants.

[0169] 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 comprises three amino acid substitutions that enhance binding to human FcRn, wherein the amino acid substitutions are M252Y, S254T, and T256E (SEQ ID NO: 156), disclosed in U.S. Patent Publication No. 7,658,921. In various embodiments, each subunit of the Fc domain comprises an amino acid substitution that enhances binding to human FcRn, wherein the amino acid substitution is N434A, disclosed in U.S. Patent Publication No. 7,371,826. In various embodiments, each subunit of the Fc domain comprises an amino acid substitution that enhances binding to human FcRn, wherein the amino acid substitutions are M428L and N434S, disclosed in U.S. Patent Publication No. 8,546,543. In various embodiments, half-life extension mutations can be combined with amino acid substitutions that reduce binding to activating Fc receptors and / or effector function.

[0170] In various embodiments, the Fc domain sequence used to prepare the IL-2 variant Fc fusion is the human IgG1-Fc domain sequence with reduced / eliminated effector function set forth in SEQ ID NO: 45:

[0171]

[0172] Wherein SEQ ID NO: 45 contains amino acid substitutions (underlined) that eliminate FcγR and C1q binding.

[0173] In various embodiments, the Fc domain sequence used to prepare the IL-2 variant Fc fusion is the IgG1-Fc domain with extended half-life and reduced / abolished effector function set forth in SEQ ID NO: 46.

[0174]

[0175] SEQ ID NO: 46 includes amino acid substitutions that abolish FcγR and C1q binding (underlined) and substitutions that extend the serum half-life of the fusion protein (bold).

[0176] In various embodiments, the Fc domain sequence used to prepare the IL-2 variant Fc fusion is an IgG1-Fc domain with reduced / eliminated effector function and extended half-life and has the amino acid sequence set forth in SEQ ID NO: 47

[0177]

[0178] SEQ ID NO: 47 includes amino acid substitutions that abolish FcγR and C1q binding (underlined) and substitutions that increase the serum half-life of the fusion protein (bold).

[0179] In various embodiments, the heterodimeric Fc domain sequence used to prepare the IL-2 variant Fc fusion is the Knob-Fc domain sequence with extended in vivo half-life set forth in SEQ ID NO: 212:

[0180]

[0181] SEQ ID NO: 212 includes amino acid substitutions that abolish FcγR and C1q binding (underlined) and amino acid substitutions that extend half-life (bold).

[0182] In various embodiments, the heterodimeric Fc domain sequence used to prepare the IL-2 variant Fc fusion is the Hole-Fc domain sequence with extended in vivo half-life set forth in SEQ ID NO: 213:

[0183]

[0184] SEQ ID NO: 213 includes amino acid substitutions that abolish FcγR and C1q binding (underlined) and amino acid substitutions that extend half-life (bold).

[0185] connector

[0186] In various embodiments, the heterologous protein is attached to the IL-2 variant via a linker and / or hinge peptide. The linker or hinge peptide can be an artificial sequence of 5, 10, 15, 20, 30, 40 or more amino acids that is relatively free of secondary structure or exhibits an alpha helical conformation.

[0187] Peptide linkers provide covalent attachment and additional structural and / or spatial flexibility between protein domains. As known in the art, peptide linkers contain flexible amino acid residues, such as glycine and serine. In various embodiments, the peptide linker can comprise 1-100 amino acids. In various embodiments, the spacer can comprise the motif GGGSGGGS (SEQ ID NO: 55). In other embodiments, the linker can comprise the motif (GGGGS) n, where n is an integer from 1 to 10. In other embodiments, the linker can also comprise amino acids other than glycine and serine. In another embodiment, the linker can comprise other protein motifs, including but not limited to sequences of α-helical conformations, 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 levels and aggregation tendencies. In another embodiment, the peptide linker can be a simple chemical bond, such as an amide bond (e.g., chemical conjugation by PEG).

[0188] Exemplary peptide linkers are provided in Table 3:

[0189] Table 3

[0190] Adapter 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

[0191] polynucleotides

[0192] In another aspect, the present disclosure provides isolated nucleic acid molecules comprising a polynucleotide encoding an IL-2, IL-2 variant, IL-2 fusion protein, or IL-2 variant fusion protein of the present disclosure. The subject nucleic acid may be single-stranded or double-stranded. Such nucleic acids may be DNA or RNA molecules. DNA includes, for example, cDNA, genomic DNA, synthetic DNA, DNA amplified by PCR, and combinations thereof. Genomic DNA encoding an IL-2 polypeptide can be obtained from genomic libraries, which 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 a T7 promoter and RNA polymerase. cDNA can be obtained from libraries prepared from mRNA isolated from various tissues expressing IL-2. DNA molecules of the present disclosure include full-length genes as well as polynucleotides and fragments thereof. Full-length genes may also include a sequence encoding an N-terminal signal sequence. Such nucleic acids can be used, for example, in methods for preparing novel IL-2 variants.

[0193] 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 joint described herein.

[0194] 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 known in the art and are selected to direct the expression of IL-2 variants. 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). Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription start and stop sequences, translation start and stop sequences, and enhancers or activation sequences. The present disclosure contemplates constitutive or inducible promoters known in the art. The promoter may be a naturally occurring promoter or a hybrid promoter combining elements of more than one promoter. The expression construct may be present on an episome in the cell, such as a plasmid, or the expression construct may be inserted into a chromosome. In various embodiments, the expression vector comprises a selective 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.

[0195] In another aspect of the present disclosure, the subject nucleic acid is provided in an expression vector comprising 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 to express a polypeptide from a polynucleotide sequence. Vectors suitable for expression in host cells 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 the 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 early promoter mediated by adenovirus or cytomegalovirus, the RSV promoter, the lac system, the trp system, the TAC or TRC system, the T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter region of lambda phage, the control region of the fd coat protein, the promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter of acid phosphatases such as PhoS, the promoter of yeast α-mating factor, the polyhedron promoter of the baculovirus system, and other sequences known to control gene expression in prokaryotes 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 the host cell to be transformed and / or the type of protein desired to be expressed. In addition, the copy number of the vector, the ability to control the copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. An exemplary expression vector suitable for expressing vIL-2 is pDSRa comprising the vIL-2 polynucleotide (described in WO 90 / 14363, incorporated herein by reference), and its derivatives, as well as any additional suitable vector known in the art or described below.

[0196] The recombinant nucleic acids of the present disclosure can be produced by ligating the cloned gene or a portion thereof into a vector suitable for expression in prokaryotes, eukaryotic cells (yeast, birds, insects, or mammals), 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 prokaryotes such as E. coli.

[0197] Some mammalian expression vectors include prokaryotic sequences that are beneficial to vector propagation in bacteria and one or more eukaryotic transcription units expressed in eukaryotic cells. PcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfecting eukaryotic cells. Some of these vectors are modified by sequences from bacterial plasmids such as pBR322 to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, viruses such as derivatives of bovine papillomavirus (BPV-1) or derivatives of 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 are found in the description of gene therapy delivery systems below. In some cases, it may be desirable to express recombinant polypeptides 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).

[0198] In various embodiments, vectors will be designed for producing 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 constructs can be used to cause expression of the subject IL-2 variants in cells proliferating in culture, e.g., to produce proteins, including fusion proteins or variant proteins, for purification.

[0199] The present disclosure also relates to host cells transfected with recombinant genes comprising nucleotide sequences encoding the amino acid sequences of one or more subject IL-2 variants. The host cells can be prokaryotic or eukaryotic cells. 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.

[0200] Accordingly, the present disclosure also relates to methods for producing 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 allow expression of the IL-2 variant to occur. The IL-2 variant can be secreted from cells containing the IL-2 variant and separated from a mixture of cells and culture medium containing the IL-2 variant. Alternatively, the IL-2 variant can be retained in the cytoplasm or retained in a membrane fraction, and harvested, lysed, and protein isolated. The cell culture comprises host cells, culture medium, and other by-products. Suitable culture media for cell culture are well known in the art.

[0201] 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, at one level, involve the crude fractionation of protein fractions and non-protein fractions. After the peptide or polypeptide is separated from other proteins, the peptide or polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). As used herein, the terms "isolated polypeptide" or "purified polypeptide" are intended to refer to a composition that can be separated from other components, wherein the polypeptide is purified to any degree relative to its naturally available state. Thus, a purified polypeptide also refers to a polypeptide that has been separated 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, the designation will refer to a peptide or polypeptide composition in which the polypeptide or peptide forms a majority component of the composition, such as about 50%, about 60%, about 70%, about 80%, about 85%, or about 90% or more of the protein in the composition.

[0202] Various techniques suitable for purification will be well known to those skilled in the art. These techniques include, for example, precipitation with ammonium sulfate, PEG, antibodies (immunoprecipitation), or 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 believed that the order in which the various purification steps are performed can be changed, or that certain steps can be omitted, and still result in a suitable method for preparing a substantially purified polypeptide.

[0203] Pharmaceutical composition

[0204] On the other hand, the present disclosure provides a pharmaceutical composition comprising an IL-2 variant or IL-2 variant fusion protein mixed 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, for example, Remington's Pharmaceutical Sciences, 18th edition, A.R. Gennaro, Mack Publishing Company, 1990). A pharmaceutically acceptable carrier can be included for changing, maintaining or maintaining, for example, pH, molar osmotic pressure concentration, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, the purpose of absorption or penetration of the composition. Such a pharmaceutical composition can affect the physical state, stability, in vivo release rate and in vivo clearance rate 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 borates, bicarbonates, Tris-HCl, citrates, phosphates, other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as PEG-100). agent) (such as caffeine, polyvinyl pyrrolidone, 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); colorants; flavorings and diluents; emulsifiers; hydrophilic polymers (such as polyvinyl pyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, propylene glycol); the present invention also provides a pharmaceutical composition comprising: ...

[0205] The main vehicle or carrier in the pharmaceutical composition can be aqueous or non-aqueous in nature. For example, suitable vehicles or carriers can be water for injection, physiological saline solution or artificial cerebrospinal fluid that may be supplemented with other materials common in the composition for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are other exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer of about pH 7.0-8.5 or acetate buffer of about pH 4.0-5.5, which can also include sorbitol or a suitable substitute for sorbitol. In one embodiment of the present disclosure, the composition with the desired degree of purity can be prepared by mixing with an optional formulation agent (formulation agent) (Remington's Pharmaceutical Sciences, the same) to store in the form of a lyophilized block or aqueous solution. In addition, the therapeutic composition can be formulated as a lyophilized product using suitable excipients such as sucrose. The best pharmaceutical composition will be determined by a person of ordinary skill in the art depending on, for example, the intended route of administration, delivery form and desired dosage.

[0206] When parenteral administration is envisioned, the therapeutic pharmaceutical composition can be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising 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, wherein the polypeptide is formulated as a sterile, isotonic solution suitable for storage. In various embodiments, pharmaceutical formulations suitable for injectable administration can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks solution, Ringer solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. In addition, suspensions of the active compound can be prepared as suitable oily injection suspensions. Optionally, the suspension may also contain a suitable stabilizer or an agent that increases the solubility of the compound and allows the preparation of a highly concentrated solution.

[0207] In various embodiments, the therapeutic pharmaceutical composition can be formulated as for targeted delivery using a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, pearls, 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, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. The targeting of liposomes can also be based on, for example, organ-specificity, cell-specificity, and organelle-specificity and known in the art.

[0208] In various embodiments, oral administration of the pharmaceutical composition is contemplated. Pharmaceutical compositions administered in this form may be formulated with or without carriers commonly used in the formulation 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 may be combined with one or more pharmaceutically acceptable carriers such as sodium citrate or dibasic calcium phosphate. 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, polyvinyl pyrrolidone, 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 retarding agents such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as, for example, cetyl alcohol and glyceryl 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 may also contain a buffer. Solid compositions of similar types can also be used as fillers in soft-filled and hard-filled gelatin capsules using excipients such as lactose (lactose) or lactose (milk sugar) and high molecular weight polyethylene glycol. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to active ingredients, liquid dosage forms can include 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-butylene glycol, oil (especially, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0209] In various embodiments, it is envisioned that the pharmaceutical composition is topically applied to the skin or to a mucous membrane. Topical preparations can also include one or more of the various agents known as skin or stratum corneum penetration enhancers. Examples of these agents are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methanol or isopropyl alcohol, dimethyl sulfoxide (DMSO) and azone. Other agents can also be included to make the preparation cosmetically acceptable. Examples of these agents are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers and surfactants. Keratin softeners (keratolytic agents) such as those known in the art can also be included. Examples are salicylic acid and sulphur. Formulations for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and with any preservatives, buffers, or propellants that may be required. 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, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0210] Other pharmaceutical compositions contemplated for use herein include formulations comprising the polypeptide in sustained or controlled delivery formulations. The techniques for formulating various other sustained or controlled delivery vehicles such as liposome vehicles, bioerodible microparticles or porous beads, and depot injections are also known to those skilled in the art.

[0211] The effective amount of the pharmaceutical composition to be used for treatment will depend on, for example, treatment background and treatment goal. It will be understood by those skilled in the art that the appropriate dosage level for treatment will therefore depend in part on the indication, route of administration, and patient's size (body weight, body surface or organ size) and condition (age and overall health) of the molecule being delivered, the polypeptide being used for and change. Accordingly, clinicians can adjust dosage and change route of administration to obtain optimal therapeutic effect. Typical dosage can depend on the factors mentioned above and in the range of from about 0.001mg / kg to about 100mg / kg or more. Peptide composition can preferably be injected or administered intravenously. Long-acting pharmaceutical composition can depend on the half-life and clearance rate of specific preparation and be administered once every three days to every four days, once a week or once every two weeks. The frequency of administration will depend on the pharmacokinetic parameters of the polypeptide in the preparation used. Generally, the composition is administered until the dosage that achieves the desired effect is reached. Therefore, the composition can be administered as a single dose or as multiple doses (with the same or different concentrations / dosages) over time or as a continuous infusion. Further improvement of appropriate dosage is routinely carried out. Appropriate dosages can be determined through use of appropriate dose-response data.

[0212] The route of administration of the pharmaceutical composition is according to known methods, such as oral; By intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal vein, intralesional approach, intramedullary, intrathecal, intraventricular, percutaneous, subcutaneous or intraperitoneal or intratumoral injection; And intranasal, intestinal, local, sublingual, urethra, vaginal or rectal mode; By sustained release system or by implant device. When desired, composition can be administered by bolus injection, or continuously administered by infusion, or administered by implant device. Alternatively or additionally, composition can be administered locally via the implantation of the film, sponge or another suitable material that has been adsorbed or encapsulated thereon by the desired molecule. When using implant device, the device can be implanted in any suitable tissue or organ, and the delivery of the desired molecule can be administered via diffusion, sustained release bolus or continuously administered.

[0213] Therapeutic uses

[0214] The present disclosure provides a method for treating an autoimmune disease in a subject, the method 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. An autoimmune disease associated with the present invention is a disease or disorder arising from and directed against an individual's own tissues, or a co-isolated system or manifestation thereof, or a condition resulting therefrom. 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 mellitus, Addison's disease, celiac disease, chronic fatigue syndrome, autoimmune hepatitis, autoimmune alopecia, ankylosing spondylitis, ulcerative colitis, Crohn's disease, fibromyalgia, pemphigus vulgaris, Sjögren's syndrome, Kawasaki disease, hyperthyroidism / Graves' disease, hypothyroidism / Hashimoto's disease, endometriosis These include: leukemia, scleroderma, pernicious anemia, Goodpasture's syndrome, Guillain-Barré syndrome, Wegener's disease, glomerulonephritis, aplastic anemia (including patients with multiply transfused aplastic anemia), 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.

[0215] On the other hand, the present disclosure provides a method for treating an inflammatory disease in a subject, the method 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. "Inflammatory disease" includes all diseases associated with acute inflammation or various inflammations. Acute inflammation is the body's initial response to harmful stimuli and is caused by the increased movement of plasma and white blood cells (such as, for example, granulocytes) from the blood to damaged tissues. Many biochemical events propagate and mature the inflammatory response, which involves the local vascular system, immune system, and various cells within the damaged tissue. Long-term inflammation is referred to as chronic inflammation, which results in progressive changes in the cell types present at the site of inflammation and is characterized by simultaneous destruction and healing of tissues from the inflammatory process. On the other hand, 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 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 erythematosus, 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.

[0216] 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 a pharmaceutical composition of the present invention. In one embodiment, the subject is a human subject. In various embodiments, the transplant is an organ transplant selected from the group consisting of heart, kidney, liver, lung, pancreas, intestine, and thymus, or a tissue transplant selected from the group consisting of bone, tendon, cornea, skin, heart valve, nerve, and blood vessels. As used herein, the term "graft-versus-host disease" or "GvHD" refers to a condition, both acute and chronic, caused by the effects of transplanted (graft) cells on host cells and tissues caused by GVH. In other words, the donor immune cells within the infused graft, or donor immune cells developed from stem cells, may perceive the patient's (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 common affected tissues are skin, intestine, and liver. In severe cases, GvHD can cause blistering of the skin or excessive diarrhea and weight loss. Prednisone and / or other immunosuppressive drugs are used to treat acute GVHD.

[0217] The therapeutically effective dose can be determined by EC 50 Initially estimated from cell culture assays. A dose can then be formulated in animal models to achieve an EC that includes the EC as determined in cell culture. 50 The circulating plasma concentration range of the drug can be used to more accurately determine the dosage that is useful in humans. The levels in plasma can be measured, for example, by HPLC. The exact composition, route of administration, and dosage can be selected by the individual physician in view of the subject's condition.

[0218] The dosage regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic response or a preventive response). For example, a single bolus injection can be administered, several divided doses (multiple or repeated or maintenance) can be administered over time, and the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic scenario. For ease of administration and consistency of dosage, it is particularly beneficial to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically dispersed units suitable as single doses for a mammalian subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. The specifications of the dosage unit form of the present disclosure will be primarily determined by the unique characteristics of the antibody and the specific therapeutic or preventive effect to be achieved.

[0219] Therefore, it will be understood by those skilled in the art that, based on the disclosure provided herein, dosage and administration regimens are adjusted according to methods well known in the therapeutic field. That is, the maximum tolerated dose can be easily determined, and the effective amount that provides a detectable therapeutic benefit to the subject can also be determined, and the time requirement for applying each agent to provide a detectable therapeutic benefit to the subject can also be determined. Therefore, although certain dosages and administration regimens are exemplified herein, these examples are by no means limiting the dosages and administration regimens that can be provided to the subject when practicing this disclosure.

[0220] It should be noted that dosage values ​​may vary with the type and severity of the condition to be alleviated and may 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 or supervising the administration of the composition, and that the dosage ranges listed herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. In addition, the dosage regimen of the compositions of the present disclosure may 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 specific antibody used. Thus, the dosage regimen may vary widely but can be routinely determined using standard methods. For example, the dosage may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or experimental values. Thus, the present disclosure includes intra-subject dose-escalation as determined by a skilled person. Determining appropriate dosages and regimens is well known in the relevant art and will be understood to be within the skill of the art once the teachings disclosed herein are provided.

[0221] Depending on the factors described above, exemplary, non-limiting daily dosing ranges for a therapeutically or prophylactically effective amount of an IL-2 variant or IL-2 variant fusion protein of the present disclosure can be in the range of 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 the desired suppression of disease symptoms occurs. In various embodiments, the dosage will be in the range of 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 10mg / kg, 0.0001mg / kg to 5mg / kg, 0.0001mg / kg to 4mg / kg, 0.0001mg / kg to 3mg / kg, 0.0001mg / kg to 2mg / kg, 0.0001mg / kg to 1mg / kg, 0.001mg / kg to 50mg / kg, 0.001mg / kg to 40mg / kg, 0.001mg / kg to 30mg / kg, 0.001mg / kg to 20mg / kg, 0.001mg / kg to 10mg / kg, 0.001mg / kg to 5mg / kg, 0.001mg / kg to 4mg / kg, 0.001mg / kg to 3mg / kg, 0.001mg / kg to 2mg / kg, 0.001mg / kg to 1mg / kg, 0.010mg / kg to 50mg / kg, 0.010mg / kg to 40mg / kg, 0.010mg / kg to 30mg / kg, 0.010mg / kg to 20mg / kg, 0.010mg / kg to 10mg / kg, 0.010mg / kg to 5mg / kg, 0.010mg / kg to 4mg / kg kg, 0.010mg / kg to 3mg / kg, 0.010mg / kg to 2mg / kg, 0.010mg / kg to 1mg / kg, 0.1mg / kg to 50mg / kg, 0.1mg / kg to 40mg / kg, 0.1mg / kg to 30mg / kg, 0.1mg / kg to 20mg / kg, 0.1mg / kg to 10mg / kg, 0.1mg / kg to 5mg / kg, 0.1mg / kg to 4mg / kg, 0.1mg / kg to 3mg / kg, 0.1mg / kg to 2mg / kg, 0.In the scope of 1mg / kg to 1mg / kg, 1mg / kg to 50mg / kg, 1mg / kg to 40mg / kg, 1mg / kg to 30mg / kg, 1mg / kg to 20mg / kg, 1mg / kg to 10mg / kg, 1mg / kg to 5mg / kg, 1mg / kg to 4mg / kg, 1mg / kg to 3mg / kg, 1mg / kg to 2mg / kg or 1mg / kg to 1mg / kg body weight etc., can be used based on the quantity described above.Therefore, one or more dosages of about 0.5mg / kg, 2.0mg / kg, 5.0mg / kg or 10mg / kg (or any combination thereof) can be used to the patient.It should be noted that dosage value can be along with the type and severity change of the situation to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the composition, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0222] Toxicity and therapeutic index of the pharmaceutical compositions of the present disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD 50 (a dose lethal to 50% of the population) and ED 50 (the dose that is 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 that exhibit large therapeutic indices are generally preferred.

[0223] The dosing frequency of the IL-2 variant or IL-2 variant fusion protein pharmaceutical composition depends on the nature of the therapy and the specific 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.

[0224] Combination therapy

[0225] As used herein, the terms "co-administration," "co-administered," and "in combination with" in reference to an IL-2 variant or IL-2 variant fusion protein of the present disclosure and one or more other therapeutic agents are intended to mean, and do mean and include, the following: simultaneous administration of such combination of an IL-2 variant or IL-2 variant fusion protein of the present disclosure and one or more therapeutic agents to a subject in need of treatment, wherein such components are formulated together into a single dosage form that releases said components to said subject at substantially the same time; substantially simultaneous administration of such combination of an IL-2 variant or IL-2 variant fusion protein of the present disclosure and one or more therapeutic agents to a subject in need of treatment, wherein such components are formulated separately from one another into separate dosage forms that are taken by said subject at substantially the same time, whereupon said components are released to said subject at substantially the same time; Such combinations of a variant fusion protein and one or more therapeutic agents are administered sequentially to a subject in need of treatment, wherein such components are formulated separately from one another into separate dosage forms that are taken by the subject at successive times with significant time intervals between each administration, whereby the components are released to the subject at substantially different times; and such combinations of an IL-2 variant or 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, sequentially, and / or overlappingly at the same and / or different times, whereby each part may be administered by the same or different routes.

[0226] 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 a pharmaceutical composition of the present invention in combination with a second therapeutic agent capable of treating the 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 to other lymphocyte surface markers (e.g., CD40, α-4 integrin) or antibodies to cytokines), other fusion proteins (e.g., CTLA-4-Ig (ORENCIA.RTM.), TNFR-Ig ), TNF-α blockers such as and Cyclophosphamide (CTX) ), methotrexate (MTX) (ie ), rituximab, belimumab (i.e. ), anti-IL-6 antibodies (such as Sarilumab), or anti-IL-6 receptor antibodies (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 in immunosuppression, or any other biological agents that target any inflammatory cytokine, nonsteroidal anti-inflammatory drugs / Cox-2 inhibitors, hydroxychloroquine, sulfasalazine (sulphasalazo) pryine), gold salts, etanercept, infliximab, mycophenolate mofetil, basiliximab, atacicept, rituximab, cyclophosphamide, interferon beta-1a, interferon beta-1b, glatiramer acetate, mitoxantrone hydrochloride, anakinra and / or other biological agents and / or intravenous immunoglobulin (IVIG). Non-limiting examples of such known therapeutic agents include interferons, such as interferon-beta-1a ( and ) and IFN-β-1b Peptide Glatiramer Acetate Natalizumab and the cytotoxic agent mitoxantrone

[0227] 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 a pharmaceutical composition of the present invention in combination with a second therapeutic agent, the second therapeutic agent being 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 MMPs); inhibiting or reducing the activity of the following cells: 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 MMPs); -23, IL-22, IL-21 and MMP); inhibit or reduce Th1 and / or Th17 pathways; inhibit or reduce cytokine production and / or secretion by the following cells: 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); inhibit or reduce the proliferation of the following cells: 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 nonsteroidal 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, fen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, and tiaprofenic; pyrazoles such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone, and trimethazone. Mixtures of these nonsteroidal 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-methyldexamethasone, 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 acetate (fluprednylidene), flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, flurandrenolone, fludrocortisone, diflurosone diacetate, fluradrenoloneacetonide, medrysone, amcinafel, amcinafide, betamethasone and its ester balance, cloprednisone, cloprednisone acetate, clocortelone, clescinolone, dichlorisone, diflurprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortisone ciproate, hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone, and mixtures thereof.

[0228] In various embodiments, the combination therapy includes the simultaneous administration of an IL-2 variant and a second composition in the same pharmaceutical composition or in separate pharmaceutical compositions. In various embodiments, the IL-2 variant composition and the second composition are administered sequentially, i.e., the IL-2 variant composition is administered before or after the second composition is administered. In various embodiments, the administration of the IL-2 variant composition and the second composition is simultaneous, i.e., the administration time periods of the IL-2 variant composition and the second composition overlap. In various embodiments, the administration of the IL-2 variant composition and the second composition is non-simultaneous. For example, in various embodiments, the administration of the IL-2 variant composition is terminated and the second composition is then administered. In various embodiments, the administration of the second composition is terminated and the IL-2 variant composition is then administered.

[0229] 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.

[0230] Example 1

[0231] Design of IL-2 variants that selectively target Treg cells

[0232] In one aspect, the present invention relates to one or more mutations that reduce the affinity of IL-2 for IL-2Rβ and / or γc receptor subunits. In the case of weakened IL-2Rβγ interaction, the enhanced IL-2 sensitivity of Tregs conferred by IL-2Rα expression can lead to a significant growth advantage of this cell subset. Therefore, in autoimmune and inflammatory diseases, these mutants can be used as Treg promoters.

[0233] 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 contained 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 participates in an extensive hydrogen bond network with the side chains of the receptor subunits at the IL-2Rβ interface. Similarly, N88 is an energy hotspot for IL-2 / IL-2Rβ interactions, participating in key hydrogen bonds with the receptor chain. Q126 is essential for γc interactions, and Q22 is similarly located at the γc interface. The inventors hypothesize that mutations at the above-mentioned sites or adjacent residues may result in defects in their ability to interact with the IL-2 intermediate affinity receptor IL-2Rβγ.

[0234] Interestingly, the proposed ' 19 The LDL' motif (Baluna R, Rizo et al., Proc Natl Acad Sci 1999;96:3957-62) overlaps with the IL-2Rβ interface. This 'toxicity motif' is partially responsible for the direct vascular toxicity of IL-2. Therefore, replacement of the key toxicity motif residue D20 or introduction of mutations that replace flanking residues L19 and L21 with non-aliphatic residues is also expected to eliminate the toxicity motif and prevent endothelial cell damage, and also significantly reduce VLS.

[0235] In the present invention, 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, Q126 26Y, Q125E, S125K, S125H, S125W, S125I, Q22N, Q22H, Q22K, Q22Y, Q22I, D20I / N88G, D20I / N88R, D20T / N88R, D20I / N 88I, 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, L A panel of IL-2 variants (SEQ ID NOs: 4-43, 108-146, and 193-197) comprising 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) were expressed as C-terminal fusions to an Fc homodimer via a "GGGSGGGS" linker (SEQ ID NO: 55). IL-2 variants with D20I, D20I / N88G, D20E, or L19N amino acid substitutions were also expressed as N-terminal fusions to the Fc homodimer via a rigid "AEAAAKEAAAKEAAAKA" linker (SEQ ID NO: 53). Collectively, the sequences of these IL-2 variant Fc fusion constructs are listed in SEQ ID NOs: 73-107, 147-189, and 198-211.Constructs with wild-type IL-2 in the same Fc fusion format (both C-terminally and N-terminally) were also prepared (SEQ ID NOs: 71 and 72).

[0236] All of the above IL-2 variant Fc fusion molecules are designed to provide growth advantages to cells that highly express IL-2Rα, resulting in Treg cells proliferating in preference to other lymphocytes (including CD4+ conventional T cells, CD8+ T cells, and NK cells). In addition, mutations at positions 19, 20, or 21 are expected to eliminate toxic motifs that cause vascular toxicity, so the resulting molecules may 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 regulating the level of damage to the activation of effector T cells and NK cells, while maintaining a sufficiently high Treg cell activation potential, and thereby maximizing the window of selective targeting of Treg subsets.

[0237] Furthermore, a single point mutation has been identified that results in improved protein stability, higher expression levels, and lower aggregation tendency. The combination of reduced aggregation and Treg-targeted mutations results in superior protein developability and Treg cell growth advantages.

[0238] Example 2

[0239] Construction and production of IL-2Fc fusion constructs

[0240] 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 a 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.

[0241] The constructs were generated by co-transfecting HEK293-F cells grown in suspension with mammalian expression vectors using polyethyleneimine (PEI, 25,000 MW linear, Polysciences). If two or more expression vectors were used, the vectors were transfected at a 1:1 ratio. For transfection, HEK293 cells were grown in serum-free FreeStyle TM HEK293 cells were cultured in 1000 ml shake flasks (working volume 330 mL) at 0.8 × 10 6The density of individual cells / ml was inoculated and transfected after 24 hours. A total of 330 μg of DNA expression vector was mixed with 16.7ml Opti-mem culture medium (ThermoFisher). After adding 0.33mg PEI diluted in 16.7ml Opti-mem culture medium, the mixture was vortexed for 15sec, and incubated at room temperature for 10min. The DNA / PEI solution was then added to the cells and incubated at 37°C with 8% CO2 in an incubator. At the 4th day, sodium butyrate (Millipore Sigma) was added to the cells at a final concentration of 2mg / L to help maintain protein expression. After cultivating for 6 days, supernatant was collected and used for purification by centrifuging at 2200rpm for 20min. The solution was sterile filtered (0.22 μm filter, Corning). Protein A affinity chromatography was used to purify the secreted protein from the cell culture supernatant.

[0242] For affinity chromatography, each supernatant was loaded onto a HiTrap MabSelectSure column (CV = 5 mL, GE Healthcare) equilibrated with 25 ml of phosphate buffered saline pH 7.2 (ThermoFisher). Unbound protein was removed by washing with 5 column volumes of PBS pH 7.2 and the target protein was eluted with 25 mM sodium citrate, 25 mM sodium chloride, pH 3.2. The protein solution was neutralized 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), was also used to refine the protein A material as needed. The target protein was purified by HPLC. The cells were concentrated using an Ultra-15 concentrator 10 kDa NMWC (Merck Millipore Ltd.).

[0243] The purity and molecular weight of the purified constructs were analyzed by SDS-PAGE and staining with Coomassie Brilliant Blue (ImperialR Stain) in the presence and absence of reducing agents. Precast gel system (4%-12% or 8%-16% Bis-Tris, ThermoFisher). The protein concentration of the purified protein samples was determined by measuring the UV absorbance at 280 nm (Nanodrop spectrophotometer, ThermoFisher) divided by the molar extinction coefficient calculated based on the amino acid sequence. The aggregate content of the constructs was analyzed on an Agilent 1200 high performance liquid chromatography (HPLC) system. The samples were injected onto an AdvanceBio size exclusion column ( 4.6 x 150 mm, 2.7 μm, LC column, Agilent).

[0244] Notably, the expression profiles and aggregation propensities of IL-2 variant Fc fusions differed significantly between constructs with different mutation sites or mutants sharing the same mutation site but with different residue substitutions.

[0245] Example 3

[0246] A single amino acid substitution in IL-2 leads to a general improvement in the developability of fusion compounds

[0247] When applied to IL-2, the engineering methods for finding mutation combinations that produce variant proteins with desired biological properties have encountered major challenges. As known in the art, naturally occurring IL-2 proteins are often less stable and prone to aggregation. This has been demonstrated in our experiments, with wild-type IL-2 Fc fusion protein (P-0250) expressed at low levels (transiently expressed at about 3 mg / L in HEK-293F cells) and having a high aggregation tendency, as illustrated by the SEC chromatogram depicted in Figure 1A. Engineering efforts have been bogged down, as amino acid substitutions for the desired biologically active IL-2 typically result in mutant proteins that are even more unstable. The IL-2 variants in current work have a large portion expressed at very low levels, and some variants are significantly more susceptible 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.

[0248] It was also observed that the expression profiles and aggregation propensities of IL-2 variant Fc fusions differed significantly between constructs with different mutation sites or mutants that shared the same mutation site but with different residue substitutions. This observation is exemplified by P-0317 (SEQ ID NO: 94) and P-0318 (SEQ ID NO: 95). The two variant fusions shared the same mutation site 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. Both variant fusions were expressed at similar low levels. As can be seen in Figure 1B, P-0318 was very prone to aggregation: 65% high molecular weight material, which makes the expected peak appear as a minor substance in the chromatogram and is 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 tendency of the resulting fusion protein. However, IL-2 fusion proteins P-0254 (SEQ ID NO: 71) and P-0324 (SEQ ID NO: 96), generated by either the N88R single mutation or the D20T / N88R double mutation, exhibited aggregation propensity of 30%-40% aggregates, respectively. Therefore, the contribution of a single amino acid substitution to protein stability appears to be context-dependent.

[0249] 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. Therefore, it is highly desirable to find residue substitutions that can generally enhance protein developability, including improved stability, higher expression levels, and lower aggregation propensity.

[0250] The amino acid substitution at position 125 was initially intended to modulate IL-2 selectivity because this residue is adjacent to Q126, which is essential for γc interaction. Naturally occurring IL-2 contains an unpaired cysteine ​​at position 125, which is replaced by serine in proleukin, and in the present invention, S125 is considered to be the wild-type IL-2 residue. IL-2 containing an alanine substitution at position 125 is also widely used. Since serine or alanine substitutions for cysteine ​​at position 125 retain all biological activity, bulky charged residues 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 order to interfere with the interaction between Q126 and γc, thereby achieving altered biological activity. With the exception of P-0471 (SEQ ID NO: 178), all resulting fusion molecules expressed at too low a level to be characterized. In contrast, when P-0471 was compared to its S125 counterpart (P-0372), P-0471 was expressed at significantly higher levels (19.3 mg / L vs. 4.0 mg / L titer) with a greatly reduced tendency to aggregate (1% vs. 21.7% aggregation). Developability, especially the impressive improvement in product purity, prompted us to evaluate whether this improvement in position 125 isoleucine substitution could be reproduced in different mutational backgrounds.

[0251] Therefore, S125I substitution is introduced into many IL-2 variant Fc fusion molecules. The construct containing Ile-125 substitution in IL-2 uses the same carrier as its Ser-125 counterpart and is expressed under the same culture conditions and purified using MabSelectSure. Expression levels (mg / L) and the purity assessed by SEC chromatography with the aggregation % of exemplary molecules are summarized in Table 4. The two molecules in the same row of Table 4 have the same other amino acid substitutions, and the difference is only that residue 125 is serine or isoleucine. For example, the SEC chromatogram and SDS-PAGE diagram of P-0447 (SEQ ID NO: 168) and its Ile-125 counterpart P-0511 (SEQ ID NO: 198) are further illustrated in Figure 1 D and Figure 1 E. It is clear from Table 4 that the isoleucine substitution at position 125 causes expression levels to increase to 4 to 11 times, and aggregation tendency is consistently low.

[0252] Table 4

[0253] 125I substitution reduces aggregation and increases expression of various IL-2 fusion proteins

[0254] It is obvious from the present invention that the isoleucine at position 125 replaces and causes the general improvement of the developability of IL-2 fusion construct, and retains biological activity completely. The Ile at position 125 of IL-2 variants with different mutation backgrounds replaces and all causes 4 to 11 times of expression level to improve, and aggregation tendency is uniformly low in wild-type IL-2 and Fc fusion forms. The purity of the protein A purified material assessed by aggregation % of exemplary molecules by expression level (mg / L) and SEC chromatography is summarized in Table 4. This discovery is especially valuable because the fact that the wild-type IL-2 that changes critical stability usually causes even more unstable mutant protein hinders the engineering of IL-2 for obtaining the desired biological property. The inherent challenge of IL-2 engineering can be alleviated by replacing with isoleucine single amino acid at position 125. In a word, the isoleucine at position 125 of IL-2 or IL-2 variant replaces and significantly improves the developability spectrum of protein, which is demonstrated by the protein expression increase of IL-2 construct and the substantial reduction of aggregation tendency.

[0255] Example 4

[0256] Identification of single amino acid substitution IL-2 variants exhibiting distinct selectivity for Treg lymphocytes

[0257] 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 are intended to reduce the signal transduction ability of IL-2 through the medium-affinity IL-2Rβγ complex and to confer signal transduction specificity from 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. It is known that after IL-2 binds to the transmembrane IL-2 receptor, STAT5 participates in downstream signaling cascades. In FACS analysis, fresh human peripheral blood mononuclear cells (PBMCs) were used to measure the phosphorylation of STAT5 in defined lymphocyte subsets, and forkhead transcription factor FOXP3 was used to identify Treg populations.

[0258] Briefly, human PBMCs were isolated from buffy coats of healthy donors by Ficoll-Hypaque centrifugation. PBMCs were starved in serum-free MACS buffer at 4°C for 1 hour. 2 × 10 5Each PBMC was treated with a serial dilution of the test compound for 30 min. By incubating with 1X Foxp3 fixation / permeabilization working solution for 30 minutes and washing with 1X permeabilization buffer, the cells were fixed and permeabilized with a Foxp3 / transcription factor staining buffer set (EBIO). In addition, the cells were fixed with Cytofix buffer and permeabilized with Perm buffer III (BD Biosciences) and then washed. After blocking Fc receptors by adding human TruStainFcX (1:50 dilution), the cells were stained at room temperature for 45 minutes with a mixture of anti-CD25-PE, anti-FOXP3-APC, anti-pSTAT5-FITC and anti-CD4-PerCP-Cy5.5 antibodies at a concentration recommended by the manufacturer. The cells were collected by centrifugation, washed, resuspended in FACS buffer, and analyzed by flow cytometry. The flow cytometry data of Treg and CD4 conventional T cell subsets were gated as CD4+Foxp3+ / CD25 高 and CD4+Foxp3- / CD25 低 Data are expressed as the percentage of pStat5-positive cells in the gated population.

[0259] Fig. 2 shows the dose response effect of the exemplary Fc fusion protein of IL-2 variants on STAT5 phosphorylation in CD4 positive Treg cells and Tconv cells compared with wild-type fusion protein.Wild-type IL-2 Fc fusion protein (P-0250) induces 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 is about 250 times higher than in CD4+Tconv cells, which is consistent with the higher expression levels of high-affinity trimeric receptors in Treg cells.

[0260] Various substitutions P-0364 (D20E), P-0363 (D20T), P-0365 (D20N), P-0366 (D20Q) and P-0367 (D20S) of aspartic acid at position 20 demonstrate the ability to induce STAT5 phosphorylation in Treg cells, while this activity is significantly reduced or eliminated in CD4+Tconv cells (Fig. 2A and Fig. 2B). These variants are IL-2 agents that activate Treg cells for the potential Treg bias for the treatment of autoimmune diseases. In addition, it is expected that mutations at the key residue D20 of the proposed toxin-like motif eliminate the toxic motif and prevent endothelial cell damage. Therefore, it is expected that these variants have Treg selection activity and have an improved safety profile for VLS. In addition, P-0368 does not show biological activity (Fig. 2A and Fig. 2B).

[0261] Figure 3 shows the ability of IL-2 variant P-0375 (N88Q) to induce STAT5 phosphorylation in CD4+ Treg cells and CD4+ Tconv cells compared to benchmark-1 and benchmark-2 compounds containing V91K and N88R mutations, respectively. The activity profile of the N88Q variant was similar to that of benchmark-2.

[0262] Figure 4 shows the biological activity of IL-2 variants containing various mutations at position 19 compared to the wild type. Variants P-0372 (L19Y), P-0373 (L19N), P-0374 (L19R), P-0423 (L19Q), P-0424 (L19H), and P-0427 (L19S) exhibited similar activity to the wild type in inducing STAT5 phosphorylation in Treg cells (Figures 4A and 4C). Variants P-0372, P-0374, P-0423, and P-0427 also substantially retained biological activity against CD4+ Tconv cells (Figures 4B and 4D), while variants P-0373 and P-0424 had reduced activity against CD4+ Tconv. Mutant P-0425 (L19D) showed a slightly reduced ability to induce STAT5 phosphorylation in Treg cells, while this activity was significantly impaired on CD4+Tconv cells (Figures 4C and 4D). The selective activation of Treg cells relative to CD4+Tconv cells demonstrated by mutants P-0373, P-0424, and P-0425, in particular the wide window of selective targeting of Treg subpopulations by P-0373 and P-0425, make 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.

[0263] Example 5

[0264] Combinations of IL-2Rβ and γc-targeted amino acid substitutions in IL-2 with distinct selectivity for Treg lymphocytes

[0265] In Example 4, it was demonstrated that targeted mutagenesis aimed at reducing the affinity of IL-2 for either the IL-2Rβ or γc receptor subunits can generate IL-2 mutants with different selectivity for Treg lymphocytes. It was then reasoned that modulating the affinity of IL-2 for both the IL-2Rβ and γc receptor subunits by combining one amino acid substitution targeting the β receptor with another targeting the γ receptor could generate the desired potency and selectivity window for Treg lymphocytes.

[0266] This basic principle is illustrated in Figure 5. Figures 5A and 5B 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, Figures 5C and 5D show the STAT5 phosphorylation activity of P-0303 (Q126E), which contains an amino acid substitution targeted to interfere with the interaction with the gamma receptor. The data show that each single amino acid substitution minimally affects pSTAT5 activation potency, but also shows only a slightly improved selectivity window for Treg lymphocyte subsets relative to the wild type. As shown in Figures 5E and 5F, the selective activation window for Treg cells is significantly expanded by combining the L19Y and Q126E mutations in P-0419. Treg activation potency is largely retained in P-0419, and the activity spectrum of the P-0419 variant is 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 predicted to have an improved safety profile and reduced VLS.

[0267] However, combining an amino acid substitution targeting beta receptors and another substitution targeting gamma receptors may not always produce desired efficacy and selectivity window. It is necessary to carry out appropriate activity regulation for each aspect. The four IL-2 variants in Fig. 6 A and Fig. 6 B have the same L19Y substitution targeting beta receptors, and the other mutations designed to target gamma receptors are respectively Q126E in P-0419, Q126K in P-0464, S125I in P-0471 and Q22K in P-0474. Although all mutants retain the efficacy (Fig. 6 A) of the STAT5 phosphorylation in comparable induction Treg cells, this activity change to CD4+Tconv cells is significant (Fig. 6 B), demonstrating the different abilities of regulating Treg activation selectivity via combined amino acid substitutions.

[0268] However, by combining the Q126E substitutions into the specific IL-2 variants that have shown a bias towards Treg subgroups to compound other receptors, it is possible to significantly reduce or eliminate the activity for Treg cells. As shown in (Fig. 6 C and Fig. 6 D), two variants P-0373 (L19N) and P-0363 (D20T) have shown some or significant selectivity windows (Fig. 6 C and Fig. 6 D) for the bias towards Treg cells. Their respective counterparts P-0417 and P-0322 with other Q126E substitutions show significantly reduced Treg cell activation efficacy. Therefore, it is crucial to find the correct residue substitution combination so that activity is adjusted to the efficacy and bias specificity of the expectation for Treg cells.

[0269] Additional variants containing double amino acid substitutions at positions L19 and Q126, including P-0447 (L19H / Q126E), P-0448 (L19Q / Q126E), and P-0449 (L19S / Q126E), were evaluated, and their activities are 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, exhibited robust biological activity in stimulating STAT5 phosphorylation in Treg cells, while this activity on Tconv cells was almost completely abolished (Figures 7A and 7B). In another study evaluating P-0419, P-0447, P-0448, and P-0449 compared to two benchmark compounds, all four variants demonstrated significant potential to induce STAT5 phosphorylation in Treg cells, while this activity on CD4+ Tconv cells was essentially abolished (Figures 7C and 7D). P-0419 had an activity profile comparable to benchmark-1, as similarly demonstrated in Figures 5E and 5F, while P-0447, P-0448, and P-0449 were comparable to benchmark-3 in terms of potency and selectivity window against Treg cells.

[0270] All of these mutants are potential Treg-biased IL-2 agents that activate Treg cells for the treatment of autoimmune diseases. In addition, these mutants are also expected to have an improved safety profile and reduced VLS due to the elimination of potentially toxic motifs.

[0271] Example 6

[0272] IL-2 variants with isoleucine substitution at position 125 retain full biological activity

[0273] In Example 3, it was shown that substitution of isoleucine at position 125 resulted in a general improvement in the developability of IL-2 fusion constructs. In order for the S125I substitution to become a viable approach to alleviate the developability challenges of IL-2 engineering, it was important to demonstrate that this amino acid substitution did not impair the biological activity of the resulting fusion protein compared to its Ser-125 counterpart.

[0274] The S125I substitution was then introduced into wild-type IL-2 or IL-2 variants that already contained one or two mutations targeting receptor subunits β, γ, or βγ. The resulting IL-2 variants containing isoleucine at position 125 were tested for their ability to stimulate STAT5 phosphorylation in Treg cells and Tconv cells and compared with their respective counterparts containing serine at position 125. Table 5 lists the potency and selectivity of 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 that position 125 is serine or isoleucine. The data show that the S125I substitution fully retains or slightly improves the biological activity of the various tested IL-2 variants without changing Treg specificity.

[0275] Table 5

[0276] IL-2 variants containing the S125I substitution retain biological activity and Treg selectivity

[0277]

[0278] 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 FIG8 . P-0250 is a wild-type IL-2 Fc fusion molecule, P-0424 contains a single 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 FIG8 . As shown in FIG8A-8F , the S125I substitution slightly increased the potency of the three test compounds, while the Treg selectivity of P-0531 and P-0491 was unchanged; for P-0511, the S125I substitution further expanded the Treg selectivity window.

[0279] Therefore, the data show that the S125I substitution in IL-2 retains the IL-2 activity spectrum of the IL-2 fusion protein in different mutation backgrounds. In short, the isoleucine substitution at position 125 of IL-2 leads to the general developability improvement (increased product yield, reduced aggregation, reduced immunogenicity potential) of IL-2, IL-2 fusions, IL-2 variants and IL-2 variant fusions, as well as the complete retention of biological activity and selectivity. This specific amino acid substitution represents a feasible mitigation strategy for solving the inherent IL-2 engineering challenges.

[0280] Example 7

[0281] Effects of IL-2 variants on CD25+CD4+ T cells, CD8 cytotoxic T cells, and NK cells

[0282] In addition to evaluating the differential stimulation of CD4+ Tregs (CD4+ / Foxp3+ / CD25+) by the two variants P-0511 and P-0512, the two variants P-0511 and P-0512 were compared with wild-type IL-2 (P-0250) and three IL-2 benchmark molecules containing V91K, N88R, and N88D, respectively. 高 ) and Tconv(CD4+ / Foxp3- / CD25 低 ) cells, we further determined the ability of the two variants, P-0511 and P-0512, to stimulate other effector T cells and NK cells, including CD4-positive Teff (CD4+ / Foxp3- / CD25+), CD8 cytotoxic effector T cells, and NK cells.

[0283] The IL-2 variants of the present invention have weakened IL-2Rβγ interactions, and the clear growth advantage of Treg versus CD4+Tconv is conferred by high constitutive IL-2Rα (CD25) expression in Treg. Following immune stimulation, CD25 expression in CD4+T effector cells can be induced. Therefore, it is expected to confirm that the IL-2 variants retain Treg specificity relative to other CD25+ lymphocyte subsets. Exemplary lymphocyte subsets with moderate to high CD25 expression levels include CD4+ effector T cells (Teff).

[0284] After treating human PBMC cells with serial dilutions of the test compounds, fixing and permeabilizing, washing, and staining with a mixture of anti-CD25-PE, anti-FOXP3-APC, anti-pSTAT5-FITC, and anti-CD4-PerCP-Cy5.5 antibodies, flow cytometry analysis was performed for Treg cells, CD4 effector cells, and CD4 naive T cell subsets gated as CD4+ / Foxp3+ / CD25+, CD4+ / Foxp3- / CD25+, and CD4+ / Foxp3- / CD25- groups, respectively. Data are presented as the percentage of pSTAT5-positive cells in the gated populations and are illustrated in FIG9 . P-0512 had comparable activity profiles 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 for both Treg cells versus 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 is clearly demonstrated in Figures 9A and 9B that IL-2 variants with reduced IL-2Rβγ interaction, particularly P-0511, preferentially activate Tregs over Teffs.

[0285] 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 labeling carboxyfluorescein diacetate succinimidyl ester (CFSE) method was used. Briefly, human PBMC (1×10 5 10A and 10B are used to analyze the cells of the NK cells. The cells were labeled with CFSE (cells / well), plated on 96-well plates, and incubated with increasing concentrations of different IL-2 compounds. The cells were then collected after 5 or 7 days of incubation, and NK cells were stained with anti-CD56-APC antibodies or CD8+T cells were stained with anti-CD8-APC antibodies and analyzed by flow cytometry. The data are expressed as the percentage of cells that split, and CD8+T cell proliferation is shown in Figure 10A and NK cell proliferation is shown in Figure 10B.

[0286] As expected, all IL-2 variants demonstrated reduced stimulation of both CD8+T cells and NK cells compared to wild-type IL-2 fusion molecule P-0250. Consistent with the observations (Fig. 9) in STAT5 phosphorylation assays, P-0512 had an activity profile comparable to benchmark-1 for the efficacy of both lymphocyte subsets, and P-0511 was comparable to benchmark-3, while benchmark-2 showed much weaker efficacy.

[0287] The STAT5 phosphorylation activity of P-0511 on other responder cells besides CD4+ T cell subsets, including CD8+ T cells and NK cells, was compared with that of 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 was almost completely abolished on CD4+ Tconv cells (Figure 11B), CD8+ T cells (Figure 11C), and NK cells (Figure 11D). The IL-2 receptors expressed on CD4+ Tconv cells, CD8+ T cells, and NK cells are primarily dimeric IL-2Rs, including IL-2Rβ and γc. To confirm that the significantly attenuated pSTAT5 signaling on CD8+ T cells and NK cells by P-0511 was due to impaired interaction with IL-2Rβ and γc, an ELISA assay was developed.

[0288] 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), a 3-fold serial dilution of IL-2Fc fusion protein starting at 100 nM or 270 nM was added to each well at 100 μl / well. After incubation at room temperature for 1 hour, 1 μg / ml of biotinylated mouse anti-human IL-2 Ab (BD BioSciences) was added to each well, followed by incubation with 1 μg / ml of HRP-avidin (ThermoFisher) for 1 hour. After each step, the wells were thoroughly aspirated and washed three times with PBS / 0.05% Tween-20. Finally, 100 μl of TMB substrate was added to each well; the plate was developed for 10 minutes at room temperature in the dark, 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 the Figure 11E Shown in.

[0289] like Figure 11E As shown in Figure 2, the developability-improved wild-type IL-2 fusion protein P-0531 binds to IL-2 with subnanomolar affinity (EC 50 = 0.06 nM) binds to the IL-2 dimer receptor complex; the benchmark-1 molecule shows reduced binding (EC 50=1.6nM), which is consistent with its corresponding weakened effect of stimulating STAT5 phosphorylation in CD8+ T cells and NK cells (Figure 10A-Figure B). In contrast, P-0511 did not show appreciable binding to the IL-2Rβ and γc complexes, indicating that the two IL-2 mutations of P-0511 at the interface of both β and γc receptor subunits significantly impaired its interaction with the complex. The binding of P-0511 to the dimeric IL-2 receptor complex was almost eliminated, and surprisingly, P-0511 showed only slightly reduced activity against Treg compared to the wild-type IL-2 fusion. P-0511 exemplifies an IL-2 variant with desired efficacy and selectivity window for Treg lymphocytes.

[0290] In summary, a series of IL-2 variants listed in Table 2A-Table 2F are constructed, expressed, and tested in vitro assays. The biological activity of exemplary IL-2 variants to Treg and other lymphocyte subsets (including CD4+Tconv cells, CD4+Teff cells, CD8+T cells and NK cells) is shown in Figure 2-Figure 11. Many variants retain the high efficacy for Treg cells, and the activity reduction or elimination for Tconv cells and other lymphocyte subsets. Some variants have an activity spectrum similar to benchmark 1, while other variants are similar to the activity characteristics of benchmark-2 or benchmark-3. In addition, most IL-2 variants are eliminated from the proposed toxin-like motif, which is intended to reduce VLS. Importantly, the incorporation of S125I amino acid substitutions produces an IL-2 variant fusion with excellent developability spectrum, while retaining the biological activity and selectivity to Treg cells. These variants are potentially IL-2 agents with improved safety profiles for the treatment of Treg biased in autoimmune diseases.

[0291] Example 8

[0292] Fc fusion proteins of IL-2 variants preferentially proliferate and expand Treg cells in mice

[0293] IL-2 variant Fc fusion proteins were administered to mice, and their ability to proliferate and expand regulatory T cells (CD4+CD25+FoxP3+ T cells) preferentially over effector T cells and NK cells was determined in vivo.

[0294] Female C57 / BL6 mice (7 weeks old) are received from Charles River laboratory and adapted to at least 7 days in the institution and then studied. At day 0, every kind of test compound or IL-2 benchmark compound of vehicle (PBS), 0.3mg / kg is administered subcutaneously to mice. On the 3rd day, 5th day and 7th day after treatment, peripheral blood samples are collected in the pipe treated with heparin. Each group comprises 6 mice, and collects baseline blood 2 days (-2 days) before treatment. After erythrocyte lysis, total survival mononuclear blood cells are counted by trypan blue dead cell exclusion method, and proceed to intracellular staining, so that immune cell phenotype and Ki67 proliferation marker are analyzed by flow cytometry. The cells were stained with two sets of antibodies listed below: 1) for CD4+ regulatory T cells (Treg), 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).

[0295] All IL-2 compounds tested stimulated Treg cell proliferation and amplification, as shown by increased Ki67-positive Treg cells and elevated Treg cells accounting for the percentage of total CD4+T cells or total lymphocytes (Figure 12A-Figure 12C). This effect was observed 3 days after injection and continued to the 5th or 7th day after a single injection. In the IL-2 variants that induce Treg phosphorylation, in contrast to the in vitro observations that always show the highest efficacy with benchmark-1, in mice, all three tested variants P-0511, P-0512, and P-0514 showed stronger in vivo efficacy than the benchmark molecules to stimulate Treg cell proliferation and amplification. P-0511, P-0512, and P-0514 showed considerable activity. The relative in vivo efficacy ranking between the three benchmarks was consistent with the in vitro human PBMC cell assay, i.e., benchmark-1 had the highest efficacy, followed by benchmark-3. Benchmark-2 was much weaker in terms of proliferation and amplification of Treg cells. (Figure 12A-Figure 12C).

[0296] For T effector cells and NK cells, benchmark-1 showed strong Ki67 stimulation of cytotoxic CD8 T cells and NK cells, while benchmarks-2 and -3 showed low effects on CD8 T cells and NK cells (Figures 13A-13C). Variant P-0514 showed similar Ki67 stimulation to CD8+ T cells as benchmark-1, while variants P-0511 and P-0512 showed mild Ki67 stimulation to CD8 T cells and NK cells as benchmarks-2 and benchmarks-3 (Figures 13A-13C). The data show that variants P-0511 and P-0512 exhibited superior biological activity and selectivity to Tregs than benchmarks 1 and 2. Benchmark-3 was ineffective in stimulating and expanding both Treg cells and effector cells.

[0297] In all IL-2 variant treatment groups, the percentage of CD4+ T conventional cells decreased due to the increase in Treg populations (Figure 14A). No significant expansion of CD4+ Tconv cells, CD8 T cells, or NK cells was observed in mice treated with any of the Treg-biased IL-2 variants (P-0511, P-0512, and P-0514) or the three benchmarks (Figures 14B-14D).

[0298] All three variants, P-0511, P-0512, and P-0514, also exhibited the most beneficial Treg / Tconv ratios compared to the three benchmarks at all measured time points, both in terms of Ki67 stimulation and cell expansion based on cytometry ( FIG. 15A and FIG. 15B ).

[0299] Three days after injection, all tested IL-2 compounds caused increased Foxp3 expression on Treg cells ( FIG. 16A ), and all three variants showed relatively higher expression of CD25 and Foxp3 markers than the three benchmarks ( FIG. 16A and FIG. 16B ), indicating better Treg activation and functionality.

[0300] Body weight was monitored before and during treatment. No significant weight changes were observed (data not shown).

[0301] Overall, the data show that variants P-0511, P-0512, and P-0514 exhibit the ability to promote the activation, proliferation, and expansion of immunosuppressive Treg cells without affecting CD4+ conventional cells, cytotoxic effector T cells, and NK cells. The data also demonstrate that these three variants outperform the benchmark molecule in both efficacy and selectivity for Treg proliferation and expansion. These variants can be used as therapeutic agents to combat autoimmune and inflammatory diseases and organ transplant rejection.

[0302] Example 9

[0303] Dose-response pharmacodynamic study of IL-2 variant Fc fusion protein in mice after single injection

[0304] After a 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 / group), peripheral blood was collected on day -2 as a baseline and on days 3, 5, and 7 after administration. On day 7, mice were sacrificed and spleens harvested. Blood lymphocyte phenotyping, proliferation, and expansion were measured by flow cytometry using fresh whole blood at each time point.

[0305] There were no significant changes in body or spleen weights in any of the treatment groups (data not shown).

[0306] As illustrated in Figure 17, in mice treated with P-0511 at 1mg / kg, 0.3mg / kg or 0.1mg / kg dosing levels, it was observed that the dose-dependent increase of Treg cell proliferation was observed, as reflected by the increase in Ki67 positive cell percentage (Figure 17A). The treatment with 0.03mg / kg had minimal effect. With three higher dose levels, the stimulation of Ki67 expression in Treg cells reached a peak on the 3rd day, and was stable until the 5th day, and then declined. As a result, P-0511 treatment caused Treg to account for the percentage of total CD4+T cells (Figure 17B), the absolute number of Treg cells (Figure 17C) and the change multiple (Figure 17D) of cell count from baseline to increase in a dose-dependent manner. The increase in Treg cell amplification followed a kinetic pattern (Figure 17) similar to that of proliferation / activation Ki67 markers, i.e., reached a peak on the 3rd day, and further extended to the 5th day. Larger Treg amplitudes and durations were stimulated with 1mg / kg administration, and the signal continued to the 5th day.

[0307] Treatment with P-0511 also resulted in a dose-dependent and statistically significant increase in the percentage of Tregs in total lymphocytes (Figure 18A), while no statistically significant changes in the percentage of CD4+Tconv cells (Figure 18B), CD8Teff cells (Figure 18C), or NK cells (Figure 18D) in total lymphocytes were observed. At the peak, Tregs accounted for 4.5% of total lymphocytes, compared to 3.1% at a 0.3 mg / kg dose and 1.4% at a 0.1 mg / kg dose. In the vehicle control group, Tregs accounted for 0.5% of total lymphocytes (Figure 18A).

[0308] The Treg / Tconv ratio was calculated based on cell counts ( Figure 19AThe peak value of Treg / Tconv ratio was 0.27 at 1 mg / kg treatment, 0.18 at 0.3 mg / kg treatment, and 0.06 at 0.1 mg / kg treatment, compared with 0.027 at no treatment ( Figure 19A ), indicating that P-0511 preferentially expands Treg cells over Tconv cells. In addition, the expression of Treg cell functional 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 weakened to a lower level on day 5.

[0309] Overall, the data demonstrate that P-0511 exhibits potent and preferential Treg activation and expansion in a dose-dependent manner. Careful consideration is required to achieve the optimal dosing strategy to achieve maximum efficacy in promoting the activation, proliferation, and expansion of immunosuppressive Treg cells while sparing cytotoxic effector T cells and NK cells.

[0310] Example 10

[0311] Pharmacodynamics of IL-2 variant Fc fusion proteins after repeated administration in mice

[0312] Female Balb / C mice (7 weeks old) were adapted to the institution for 5-7 days and then studied. On the 0th day, the 3rd day and the 6th day, mice (n=5 / group) were subcutaneously administered with vehicle (PBS), 0.3mg / kg of P-0511, P-0512, P-0531 or benchmark-1 compound. On the 3rd day and the 9th day, three days after the first injection and multiple (3) injections, peripheral blood was collected. Based on earlier in vivo experiments, it is expected that the activation, proliferation and amplification of Treg cells will reach a peak on the 3rd day, and therefore data collection and analysis will be performed 3 days after injection. The changes in blood lymphocyte activation, proliferation and amplification were measured by flow cytometry. P-0531 is the S125I equivalent of wild-type IL-2 fusion protein. Benchmark-1 contains V91K mutations.

[0313] Three days after a single subcutaneous administration of IL-2 fusion protein, nearly 90% of Treg cells showed positive Ki67 expression in all test groups, and after receiving 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, Treg cells, expressed as a percentage of Treg to total CD4 T cells or total lymphocytes, decreased significantly to near control levels, while in mice treated with P-0511 and P-0512, Treg cells remained at significantly high levels after three consecutive Q3D treatments compared to one treatment (Figure 20B-Figure 20C). The data suggest that wild-type IL-2 or benchmark-1 can accelerate the depletion of Treg cells or promote Treg desensitization due to their greater potency in stimulating Tregs. Additional explanations could also include differences in half-life or "receptor silencing" on non-lymphocytes, leading to altered drug exposure of non-Treg-selective or less Treg-selective wild-type IL-2 or beta-1.

[0314] Treg cell counts and fold change relative to PBS control (Figure 21A-21B) and Treg / Tconv ratio ( Figure 22 ), similar observations were obtained. Compared with P-0531 and Benchmark-1, P-0511 and P-0512 showed better ability to maintain the Treg pool and maintain Treg selectivity.

[0315] Overall, the data suggest that P-0511 and P-0512 are superior IL-2 molecules, demonstrating preferential and sustained in vivo Treg expansion after multiple doses. Adjusting the dosing regimen of IL-2 variant Fc fusions, such as dosing amount and frequency, can further optimize the desired potency and selectivity for Tregs relative to proinflammatory immune activation.

[0316] Example 11

[0317] Inhibition of antigen-driven inflammation in a mouse model of delayed-type hypersensitivity (DTH) by IL-2 variant Fc fusion protein

[0318] The ability of Treg cells induced by IL-2 variants to suppress T cell antigen-driven inflammation in vivo was assessed in a delayed-type hypersensitivity (DTH) model. Female Balb / C mice (7 weeks old) were adapted to the institution for 7 days and randomly divided into groups. Subcutaneous administration of vehicle (PBS), 0.1 mg / kg or 0.3 mg / kg of P-0511 was started on day -2, and three injections were given every 3 days (Q3D) or two injections every 5 days (Q5D). Then on day 0, 100 grams of keyhole granules in 200 μl saline were subcutaneously administered. Mice were sensitized with hemocyanin (KLH). For Q3D dosing, two more subcutaneous injections of PBS or P-0511 (0.1 mg / kg or 0.3 mg / kg) were administered on day 1 and day 4; for Q5D dosing, an additional sc 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 saline) in the right ear. On day 5 before the KLH challenge and on day 6 to day 8 after the KLH challenge (corresponding to 24 hours, 48 ​​hours and 72 hours), the thickness of the right ear was measured using a caliper. One group of mice also received 5 mg / kg dexamethasone ip treatment every day from day 5 to day 8 as a positive control.

[0319] In Figure 23, the kinetics of the DTH response are illustrated using the changes in ear thickness relative to baseline values ​​([Delta] Ear Thickness) at different times after KLH challenge.

[0320] After subcutaneous KLH antigen sensitization, significant ear inflammation and swelling peaked 24 hours after intradermal KLH challenge in the auricle, and middle ear swelling was prolonged for 72 hours in the PBS group. Significantly, the immunosuppressive steroid dexamethasone was effective in inhibiting the KLH-induced inflammatory response, with daily administration of 5 mg / kg for 4 consecutive days achieving ~85% inhibition 72 hours after KLH challenge. In mice treated with 0.3 mg / kg of P-0511 Q3D or Q5D at all time points after KLH challenge, inhibition of antigen-driven inflammation by Treg cells induced by P-0511 was also evident (Figures 23A-23B). Similar trends in reducing DTH inflammatory responses were observed for both 0.1 mg / kg doses of Q3D and Q5D, but the effects did not reach statistical significance at most time points. Both the Q3D and Q5D dosing regimens were effective.

[0321] In another study, the dose-dependent response of P-0511 (0.1 mg / kg, 0.3 mg / kg and 1 mg / kg, Q5D) to inhibit KLH-induced DTH was determined and compared with Benchmark-1 (0.3 mg / kg, Q5D). Figure 24 As described in

[15] , P-0511 exhibited dose-dependent inhibition of ear inflammation. Mice receiving 1 mg / kg of P-0511 exhibited strong tolerance to KLH-induced DTH, and minimal ear swelling was observed after KLH challenge. Moderate and mild inhibition 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).

[0322] In summary, in the DTH model, Treg cells induced by P-0511 administration are effective in suppressing T cell antigen-driven inflammation. In addition, Treg suppression is sustained after KLH challenge without repeated administration. It is also obvious from the examples that it is crucial to adjust the dosing regimen to achieve optimal efficacy.

[0323] Example 12

[0324] Pharmacodynamics and pharmacokinetic effects of P-0511 in cynomolgus monkeys

[0325] The PK / PD properties of IL-2 variant Fc fusion protein P-0511 were evaluated in cynomolgus monkeys according to the Q14D×3 dosing schedule. Cynomolgus monkeys were acclimated for 3-4 weeks and randomly divided into 4 groups (n = 3-4 / group), followed by a baseline week before dosing. On days 1, 15, and 29, one group received subcutaneous administration of vehicle (PBS), and the other three groups received subcutaneous administration of P-0511 at 100 μg / kg, 30 μg / kg, or 10 μg / kg.

[0326] 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 Tregs, 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) divided into 5 parts: neutrophils, lymphocytes, monocytes, eosinophils, and basophils.

[0327] Mouse anti-human IL-2 mAb (BD Pharmingen) was used to coat 96-well plates to capture P-0511. The PK characteristics of P-0511 were assessed in cynomolgus monkey plasma samples by measuring the complete P-0511 of full length. Goat anti-human Fc polyclonal-HRP (ThermoFisher) was used to detect P-0511, and the plasma concentration of P-0511 was subsequently quantified. In addition to the plasma samples collected at -7 days, -3 days, 2 days, 4 days, 6 days, 8 days, 11 days, 14 days, 18 days, 21 days, 28 days, 32 days, 35 days, 43 days, 50 days, and 57 days, three plasma samples were collected at 0.5 hours, 3 hours, and 6 hours after using P-0511 for the first time on the 1st day.

[0328] 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, gamma-glutamyltransferase, albumin, total bilirubin, creatinine, blood urea nitrogen, and C-reactive protein.

[0329] In addition, the body weight and temperature of each animal were monitored once or twice a week throughout the study.

[0330] All articles and methods disclosed and claimed herein can be prepared and performed without undue experimentation in view of the present disclosure. Although the articles and methods of the present disclosure have been described according to 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 existing or developed later, 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 technicians in the field to which the present disclosure belongs. For all purposes, all patents, patent applications, and publications are incorporated herein by reference in their entirety, and to the extent that each individual publication is individually and specifically indicated to be incorporated herein by reference in its entirety for any and all purposes. The present disclosure exemplarily described herein can be suitably practiced under the condition that any one or more elements not specifically disclosed herein do not exist. It will be understood, therefore, that although the present disclosure has been particularly disclosed by preferred embodiments and optional features, those skilled in the art may seek modifications and variations of the concepts disclosed herein, and that such modifications and variations are considered to be within the scope of the present disclosure as defined by the appended claims.

[0331] Sequence Listing

[0332] 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 code for amino acids as set forth in 37 CFR 1.822.

[0333] SEQ ID NO: 1 is the amino acid sequence of human IL-2 precursor.

[0334] SEQ ID NO: 2 is the naturally occurring amino acid sequence of the mature form of human IL-2.

[0335] SEQ ID NO: 3 is the wild-type amino acid sequence of the mature form of human IL-2.

[0336] SEQ ID NOs: 4-43, 108-146, and 193-197 are the amino acid sequences of various IL-2 variants.

[0337] SEQ ID NO: 44 is the human IgG1-Fc amino acid sequence.

[0338] SEQ ID NO: 45 is the amino acid sequence of human IgG1-Fc with reduced / eliminated effector function.

[0339] SEQ ID NOs: 46 and 47 and 212-213 are the amino acid sequences of human IgGl-Fc with reduced / abolished effector function and extended half-life.

[0340] SEQ ID NOs: 48-67 are the amino acid sequences of various peptide linker sequences.

[0341] SEQ ID NO: 68 is the amino acid sequence of the human IL-2 receptor α Sushi domain.

[0342] SEQ ID NOs: 69-107, 147-189, and 198-211 are the amino acid sequences of various Fc-IL-2 fusion proteins.

[0343] SEQ ID NOs: 190-192 are the amino acid sequences of benchmark Fc-IL-2 variant fusion proteins.

[0344] SEQ ID NOs: 214-222 are the nucleotide sequences of various Fc-IL-2 fusion proteins.

[0345] Sequence Listing

[0346] Human IL-2 precursor sequence

[0347]

[0348] The naturally occurring sequence of the mature form of human IL-2

[0349]

[0350] Human IL-2 mature wild-type sequence

[0351]

[0352] IL-2 N88R variant sequence

[0353]

[0354] IL-2 D20T variant sequence

[0355]

[0356] IL-2 D20E variant sequence

[0357]

[0358] IL-2 D20N variant sequence

[0359]

[0360] IL-2 D20Q variant sequence

[0361]

[0362] IL-2 D20S variant sequence

[0363]

[0364] IL-2 D20Y variant sequence

[0365]

[0366] IL-2 D20I variant sequence

[0367]

[0368] IL-2 L19Y variant sequence

[0369]

[0370] IL-2 L19N variant sequence

[0371]

[0372] IL-2 L19R variant sequence

[0373]

[0374] IL-2 N88G variant sequence

[0375]

[0376] IL-2 N88I variant sequence

[0377]

[0378] IL-2 N88Q variant sequence

[0379]

[0380] IL-2 N88E variant sequence

[0381]

[0382] IL-2 N88T variant sequence

[0383]

[0384] IL-2 N88M variant sequence

[0385]

[0386] IL-2 Q126E variant sequence

[0387]

[0388] IL-2 Q126L variant sequence

[0389]

[0390] IL-2 Q126N variant sequence

[0391]

[0392] IL-2 Q126D variant sequence

[0393]

[0394] IL-2 Q126M variant sequence

[0395]

[0396] IL-2 D20I / N88G variant sequence

[0397]

[0398] IL-2 D20I / N88R variant sequence

[0399]

[0400] IL-2 D20T / N88R variant sequence

[0401]

[0402] IL-2 D20I / N88I variant sequence

[0403]

[0404] IL-2 D20T / Q126E variant sequence

[0405]

[0406] IL-2 D20T / N88R / Q126E variant sequence

[0407]

[0408] IL-2 D20T / Q126L variant sequence

[0409]

[0410] IL-2 D20T / N88R / Q126L variant sequence

[0411]

[0412] IL-2 L19N / Q126E variant sequence

[0413]

[0414] IL-2 L19R / Q126E variant sequence

[0415]

[0416] IL-2 L19Y / Q126E variant sequence

[0417]

[0418] IL-2 L19Q variant sequence

[0419]

[0420] IL-2 L19H variant sequence

[0421]

[0422] IL-2 L19D variant sequence

[0423]

[0424] IL-2 L19P variant sequence

[0425]

[0426] IL-2 D20T / S125I / Q126K variant sequence

[0427]

[0428] IL-2 L19N / S125I / Q126K variant sequence

[0429]

[0430] IL-2 L19R / S125I / Q126K variant sequence

[0431]

[0432] Human IgG1-Fc

[0433]

[0434]

[0435] Human IgG1-Fc with reduced / ablated effector function

[0436]

[0437] Human IgG1-Fc with reduced / eliminated effector function and extended half-life

[0438]

[0439] Human IgG1-Fc with reduced / eliminated effector function and extended half-life

[0440]

[0441] Peptide linker sequence GGGSGGGSGGGS (SEQ ID NO: 48)

[0442] Peptide linker sequence GGGS (SEQ ID NO: 49)

[0443] Peptide linker sequence GSSGGSGGSGGSG (SEQ ID NO: 50)

[0444] Peptide linker sequence GSSGT (SEQ ID NO: 51)

[0445] Peptide linker sequence GGGGSGGGGSGGGS (SEQ ID NO: 52)

[0446] Peptide linker sequence AEAAAKEAAAKEAAAKA (SEQ ID NO: 53)

[0447] Peptide linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 54)

[0448] Peptide linker sequence GGGSGGGS (SEQ ID NO: 55)

[0449] Peptide linker sequence GS (SEQ ID NO: 56)

[0450] Peptide linker sequence GGS (SEQ ID NO: 57)

[0451] Peptide linker sequence GGGGS (SEQ ID NO: 58)

[0452] Peptide linker sequence GGSG (SEQ ID NO: 59)

[0453] Peptide linker sequence SGGG (SEQ ID NO: 60)

[0454] Peptide linker sequence GSGS (SEQ ID NO: 61)

[0455] Peptide linker sequence GSGSGS (SEQ ID NO: 62)

[0456] Peptide linker sequence GSGSGSGS (SEQ ID NO: 63)

[0457] Peptide linker sequence GSGSGSGSGS (SEQ ID NO: 64)

[0458] Peptide linker sequence GSGSGSGSGSGS (SEQ ID NO: 65)

[0459] Peptide linker sequence GGGGSGGGGS (SEQ ID NO: 66)

[0460] Peptide linker sequence GGGGSGGGGSGGGGS (SEQ ID NO: 67)

[0461] Human IL-2Rα sushi domain sequence

[0462]

[0463]

[0464] P-0250

[0465]

[0466] P-0305

[0467]

[0468] P-0254

[0469]

[0470] P-0363

[0471]

[0472] P-0364

[0473]

[0474] P-0365

[0475]

[0476] P-0366

[0477]

[0478] P-0367

[0479]

[0480] P-0368

[0481]

[0482] P-0252

[0483]

[0484] P-0372

[0485]

[0486]

[0487] P-0373

[0488]

[0489] P-0374

[0490]

[0491] P-0253

[0492]

[0493] P-0302

[0494]

[0495] P-0375

[0496]

[0497] P-0376

[0498]

[0499] P-0377

[0500]

[0501] P-0378

[0502]

[0503] P-0303

[0504]

[0505] P-0304

[0506]

[0507] P-0369

[0508]

[0509]

[0510] P-0370

[0511]

[0512] P-0371

[0513]

[0514] P-0251

[0515]

[0516] P-0317

[0517]

[0518] P-0318

[0519]

[0520] P-0324

[0521]

[0522] P-0322

[0523]

[0524] P-0323

[0525]

[0526] P-0325

[0527]

[0528] P-0326

[0529]

[0530] P-0417

[0531]

[0532]

[0533] P-0418

[0534]

[0535] P-0419

[0536]

[0537] P-0416

[0538]

[0539] P-0412

[0540]

[0541] P-0306

[0542]

[0543] P-0319

[0544]

[0545] IL-2 L19S variant sequence

[0546]

[0547] IL-2 L21S variant sequence

[0548]

[0549] IL-2 L21N variant sequence

[0550]

[0551] IL-2 L21R variant sequence

[0552]

[0553] IL-2 Q126K variant sequence

[0554]

[0555] IL-2 Q126H variant sequence

[0556]

[0557] IL-2 Q126Y variant sequence

[0558]

[0559] IL-2 S125E variant sequence

[0560]

[0561] IL-2 S125K variant sequence

[0562]

[0563] IL-2 S125H variant sequence

[0564]

[0565] IL-2 S125W variant sequence

[0566]

[0567] IL-2 S125I variant sequence

[0568]

[0569] IL-2 Q22N variant sequence

[0570]

[0571] IL-2 Q22H variant sequence

[0572]

[0573] IL-2 Q22K variant sequence

[0574]

[0575] IL-2 Q22Y variant sequence

[0576]

[0577] IL-2 Q22I variant sequence

[0578]

[0579] IL-2 L19H / Q126E variant sequence

[0580]

[0581] IL-2 L19Q / Q126E variant sequence

[0582]

[0583] IL-2 L19S / Q126E variant sequence

[0584]

[0585] IL-2 L19Y / Q126K variant sequence

[0586]

[0587] IL-2 L19Y / Q126H variant sequence

[0588]

[0589] IL-2 L19Y / Q126Y variant sequence

[0590]

[0591] IL-2 L19Y / S125E variant sequence

[0592]

[0593] IL-2 L19Y / S125K variant sequence

[0594]

[0595] IL-2 L19Y / S125H variant sequence

[0596]

[0597] IL-2 L19Y / S125W variant sequence

[0598]

[0599] IL-2 L19Y / S125I variant sequence

[0600]

[0601] IL-2 L19Y / Q22N variant sequence

[0602]

[0603] IL-2 L19Y / Q22H variant sequence

[0604]

[0605] IL-2 L19Y / Q22K variant sequence

[0606]

[0607] IL-2 L19Y / Q22Y variant sequence

[0608]

[0609] IL-2 L19Y / Q22I variant sequence

[0610]

[0611] IL-2 L19H / Q126K variant sequence

[0612]

[0613] IL-2 L19H / S125I variant sequence

[0614]

[0615] IL-2 L19D / S125I variant sequence

[0616]

[0617] IL-2 D20E / S125I variant sequence

[0618]

[0619] IL-2 D20T / S125I variant sequence

[0620]

[0621] IL-2 L19Y / S125I / Q126E variant sequence

[0622]

[0623] P-0423

[0624]

[0625] P-0424

[0626]

[0627]

[0628] P-0425

[0629]

[0630] P-0426

[0631]

[0632] P-0427

[0633]

[0634] P-0428

[0635]

[0636] P-0429

[0637]

[0638] P-0430

[0639]

[0640] P-0497

[0641]

[0642] P-0498

[0643]

[0644] P-0499

[0645]

[0646] P-0500

[0647]

[0648] P-0501

[0649]

[0650]

[0651] P-0502

[0652]

[0653] P-0503

[0654]

[0655] P-0531

[0656]

[0657] P-0505

[0658]

[0659] P-0506

[0660]

[0661] P-0507

[0662]

[0663] P-0508

[0664]

[0665] P-0509

[0666]

[0667] P-0447

[0668]

[0669] P-0448

[0670]

[0671] P-0449

[0672]

[0673]

[0674] P-0464

[0675]

[0676] P-0465

[0677]

[0678] P-0466

[0679]

[0680] P-0467

[0681]

[0682] P-0468

[0683]

[0684] P-0469

[0685]

[0686] P-0470

[0687]

[0688] P-0471

[0689]

[0690] P-0472

[0691]

[0692] P-0473

[0693]

[0694] P-0474

[0695]

[0696]

[0697] P-0475

[0698]

[0699] P-0476

[0700]

[0701] P-0480

[0702]

[0703] P-0491

[0704]

[0705] P-0492

[0706]

[0707] P-0493

[0708]

[0709] P-0494

[0710]

[0711] P-0495

[0712]

[0713] P-0496 (Benchmark-2)

[0714]

[0715] Benchmark-1

[0716]

[0717] Benchmark-3

[0718]

[0719]

[0720] IL-2 L19H / S125I / Q126E variant sequence

[0721]

[0722] IL-2 L19H / S125I / Q126K variant sequence

[0723]

[0724] IL-2 L19Q / Q126K variant sequence

[0725]

[0726] IL-2 L19Q / S125I / Q126E variant sequence

[0727]

[0728] IL-2 L19Q / S125I / Q126K variant sequence

[0729]

[0730] P-0511

[0731]

[0732] P-0512

[0733]

[0734]

[0735] P-0513

[0736]

[0737] P-0514

[0738]

[0739] P-0515

[0740]

[0741] P-0582

[0742]

[0743] P-0583

[0744]

[0745] P-0584

[0746]

[0747] P-0585

[0748]

[0749] P-0586

[0750]

[0751] P-0616

[0752]

[0753] P-0672Knob-Fc chain

[0754]

[0755] P-0673Knob-Fc chain

[0756]

[0757]

[0758] P-0674

[0759]

[0760] Knob-Fc domain with extended in vivo half-life

[0761]

[0762] Hole-Fc domain with extended in vivo half-life

[0763]

[0764] P-0511

[0765]

[0766] P-0512

[0767]

[0768]

[0769] P-0514

[0770]

[0771] P-0582

[0772]

[0773]

[0774] P-0583

[0775]

[0776] P-0584

[0777]

[0778] P-0585

[0779]

[0780]

[0781] P-0586

[0782]

[0783] P-0616

[0784] Sequence Listing <110> Co-Gene <120> Interleukin-2 variants and methods of use thereof <130> CACCG1.0003WO <160> 222 <170> PatentIn version 3.5 <210> 1 <211> 153 <212> PRT <213> Homo sapiens <400> 1 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser Thr Leu Thr 145 150 <210> 2 <211> 133 <212> PRT <213> Homo sapiens <400> 2 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 3 <211> 133 <212> PRT <213> Homo sapiens <400> 3 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 [[ID=Y]]Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 4 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 N88R change sequence <400> 4 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Arg Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 5 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 D20T change sequence <400> 5 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Thr Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 6 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 D20E change sequence <400> 6 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Glu Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 7 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 D20N change sequence <400> 7 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asn Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 8 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 D20Q change sequence <400> 8 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Gln Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 9 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 D20S change sequence <400> 9 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Ser Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 10 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 D20Y change sequence <400> 10 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Tyr Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 11 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 D20I change sequence <400> 11 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Ile Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 12 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 L19Y change sequence <400> 12 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Tyr Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 13 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 L19N change sequence <400> 13 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Asn Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 14 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 L19R change sequence <400> 14 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Arg Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 15 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 N88G change sequence <400> 15 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Gly Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 16 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 N88I change sequence <400> 16 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Ile Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 17 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 N88Q change <400> 17 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Gln Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 18 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 N88E change sequence <400> 18 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Glu Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 19 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 N88T change sequence <400> 19 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Thr Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 20 <211> 132 <212> PRT <213> artificial <220> <223> IL-2 N88M change sequence <400> 20 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Met Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leo 130 <210> 21 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 Q126E change sequence <400> 21 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Glu Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 22 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 Q126L change sequence <400> 22 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Leu Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 23 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 Q126N change sequence <400> 23 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Asn Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 24 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 Q126D change sequence <400> 24 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Asp Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 25 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 Q126M change sequence <400> 25 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Met Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 26 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 D20I / N88G change sequence <400> 26 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Ile Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Gly Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 27 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 D20I / N88R change sequence <400> 27 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Ile Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Arg Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 28 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 D20T / N88R change sequence <400> 28 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Thr Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Arg Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 29 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 D20I / N88I change sequence <400> 29 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Ile Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Ile Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 30 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 D20T / Q126E change sequence <400> 30 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Thr Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Glu Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 31 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 D20T / N88R / Q126E change <400> 31 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Thr Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Arg Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Glu Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 32 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 D20T / Q126L change sequence <400> 32 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Thr Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Leu Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 33 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 D20T / N88R / Q126L change <400> 33 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Thr Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Arg Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Leu Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 34 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 L19N / Q126E change sequence <400> 34 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Asn Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Glu Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 35 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 L19R / Q126E change sequence <400> 35 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Arg Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Glu Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 36 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 L19Y / Q126E change sequence <400> 36 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Tyr Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Glu Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 37 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 L19Q change sequence <400> 37 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Gln Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 38 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 L19H change sequence <400> 38 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu His Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 39 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 L19D change sequence <400> 39 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Asp Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 40 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 L19P change sequence <400> 40 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Pro Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 41 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 D20T / S125I / Q126K change <400> 41 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Thr Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Lys Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 42 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 L19N / S125I / Q126K change <400> 42 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Asn Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Lys Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 43 <211> 133 <212> PRT <213> artificial <220> <223> IL-2 L19R / S125I / Q126K change sequence <400> 43 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Arg Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Lys Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 44 <211> 226 <212> PRT <213> Homo sapiens <400> 44 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 45 <211> 226 <212> PRT <213> Artificial <220> <223> Human IgG1-Fc with reduced / ablated effector function <400> 45 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 46 <211> 226 <212> PRT <213> Artificial <220> <223> Human IgG1-Fc with reduced / eliminated effector function and extended half-life <400> 46 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 47 <211> 226 <212> PRT <213> Artificial <220> <223> Human IgG1-Fc with reduced / eliminated effector function and extended half-life <400> 47 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 48 <211> 12 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 48 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 <210> 49 <211> 4 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 49 Gly Gly Gly Ser 1 <210> 50 <211> 13 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 50 Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 10 <210> 51 <211> 5 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 51 Gly Ser Ser Gly Thr 1 5 <210> 52 <211> 14 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 52 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 <210> 53 <211> 17 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 53 Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 15 Ala <210> 54 <211> 20 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 54 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 55 <211> 8 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 55 Gly Gly Gly Ser Gly Gly Gly Ser 1 5 <210> 56 <211> 2 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 56 Gly Ser 1 <210> 57 <211> 3 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 57 Gly Gly Ser 1 <210> 58 <211> 5 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 58 Gly Gly Gly Gly Ser 1 5 <210> 59 <211> 4 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 59 Gly Gly Ser Gly 1 <210> 60 <211> 4 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 60 Ser Gly Gly Gly 1 <210> 61 <211> 4 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 61 Gly Ser Gly Ser 1 <210> 62 <211> 6 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 62 Gly Ser Gly Ser Gly Ser 1 5 <210> 63 <211> 8 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 63 Gly Ser Gly Ser Gly Ser Gly Ser 1 5 <210> 64 <211> 10 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 64 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 1 5 10 <210> 65 <211> 12 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 65 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 1 5 10 <210> 66 <211> 10 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 66 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 67 <211> 15 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 67 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 68 <211> 165 <212> PRT <213> Homo sapiens <400> 68 Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys 1 5 10 15 Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg 20 25 30 Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly 35 40 45 Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser 50 55 60 Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln 65 70 75 80 Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp 85 90 95 Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn 100 105 110 Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr 115 120 125 Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu 130 135 140 Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln 145 150 155 160 Leu Ile Cys Thr Gly 165 <210> 69 <211> 367 <212> PRT <213> Artificial <220> <223> P-0250 <400> 69 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 70 <211> 371 <212> PRT <213> artificial <220> <223> P-0305 <400> 70 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 130 135 140 Glu Ala Ala Ala Lys Ala Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 145 150 155 160 Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 165 170 175 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 180 185 190 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 195 200 205 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 210 215 220 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 225 230 235 240 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 245 250 255 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 260 265 270 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 275 280 285 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 290 295 300 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 305 310 315 320 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 325 330 335 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 340 345 350 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 355 360 365 Ser Pro Gly 370 <210> 71 <211> 367 <212> PRT <213> Artificial <220> <223> P-0254 <400> 71 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly<00035​​​​​​​​​​​​​​Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Arg Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 72 <211> 367 <212> PRT <213> Artificial <220> <223> P-0363 <400> 72 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Thr Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 73 <211> 367 <212> PRT <213> Artificial <220> <223> P-0364 <400> 73 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Glu Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 74 <211> 367 <212> PRT <213> Artificial <220> <223> P-0365 <400> 74 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asn Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 75 <211> 367 <212> PRT <213> Artificial <220> <223> P-0366 <400> 75 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Gln Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 76 <211> 367 <212> PRT <213> Artificial <220> <223> P-0367 <400> 76 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val<s 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Ser Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile<{ 305 310 31{5 320 Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 77 <211> 367 <212> PRT <213> Artificial <220> <223> P-0368 <400> 77 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Tyr Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 78 <211> 367 <212> PRT <213> Artificial <220> <223> P-0252 <400> 78 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Ile Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr[[ID=$$]] 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr<00039!0>355 360 365 <210> 79[[ID=3?]] <211> 367 <212> PRT <213> Artificial <220> <223> P-0372 <400> 79 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Tyr Asp Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 80 <211> 367 <212> PRT <213> Artificial <220> <223> P-0373 <400> 80 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His<00(03976>35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val ]>50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Asn Asp Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320<00么04011>Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 81 <211> 367 <212> PRT <213> Artificial <220> <223> P-0374 <400> 81 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Arg Asp Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 82 <211> 367 <212> PRT <213> Artificial <220> <223> P-0253 <400> 82 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Gly Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 83 <211> 367 <212> PRT <213> Artificial <220> <223> P-0302 <400> 83 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Ile Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 84 <211> 367 <212> PRT <213> Artificial <220> <223> P-0375 <400> 84 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Gln Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 85 <211> 367 <212> PRT <213> Artificial <220> <223> P-0376 <400> 85 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Glu Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 86 <211> 367 <212> PRT <213> Artificial <220> <223> P-0377 <400> 86[[ID=;30]] Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Thr Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 87 <211> 367 <212> PRT <213> Artificial <220> <223> P-0378 <400> 87 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Met Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 88 <211> 367 <212> PRT <213> Artificial <220> <223> P-0303 <400> 88 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45<00…​​​​His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Glu Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 89 <211> 367 <212> PRT <213> Artificial <220> <223> P-0304 <400> 89 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg 260 265 270 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Ser Leu Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 90 <211> 367 <212> PRT <213> Artificial <220> <223> P-0369 <400> 90 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gl...

Claims

1. An isolated interleukin-2 (IL-2) variant polypeptide, wherein 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 compared to the polypeptide represented by SEQ ID NO: 3, wherein the IL-2 variant polypeptide is selected from the group consisting of: an IL-2 variant polypeptide comprising only three amino acid substitutions L19H, S125I and Q126E to SEQ ID NO: 3, and an IL-2 variant polypeptide comprising only three amino acid substitutions L19H, S125I and Q126K to SEQ ID NO:

3.

2. The IL-2 variant polypeptide 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: 193-194.

3. The IL-2 variant polypeptide according to claim 2, wherein the IL-2 variant polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

193.

4. The IL-2 variant polypeptide according to claim 2, wherein the IL-2 variant polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

194.

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

5.

6. The IL-2 variant polypeptide according to any one of claims 3-4, wherein stimulation with the IL-2 variant polypeptide results in higher stimulation of regulatory T cells (Treg) relative to stimulation of resting conventional T cells (Tconv).

7. An isolated fusion protein, comprising an IL-2 variant polypeptide fused to an Fc domain, the IL-2 variant polypeptide being selected from the group consisting of an IL-2 variant polypeptide consisting of the amino acid sequence listed in SEQ ID NO: 193 and an IL-2 variant polypeptide consisting of the amino acid sequence listed in SEQ ID NO: 194, the Fc domain consisting of the amino acid sequence listed in SEQ ID NO: 45; wherein the IL-2 variant polypeptide is fused to the Fc domain via a peptide linker consisting of the amino acid sequence listed in SEQ ID NO: 55, wherein the isolated fusion protein comprises, from N-terminus to C-terminus: the Fc domain, the peptide linker and the IL-2 variant polypeptide.

8. The isolated fusion protein of claim 7, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:

198.

9. The isolated fusion protein of claim 7, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:

199.

10. The isolated fusion protein of claim 8, wherein the isolated fusion protein comprises two polypeptide chains, wherein each of the polypeptide chains consists of the amino acid sequence set forth in SEQ ID NO:

198.

11. The isolated fusion protein of claim 8, wherein the isolated fusion protein comprises two polypeptide chains linked by at least one interchain disulfide bond.

12. The isolated fusion protein of claim 11, wherein the isolated fusion protein comprises two polypeptide chains linked by two interchain disulfide bonds.

13. The isolated fusion protein of claim 9, wherein the isolated fusion protein comprises two polypeptide chains, wherein each of the polypeptide chains consists of the amino acid sequence set forth in SEQ ID NO:

199.

14. The isolated fusion protein of claim 9, wherein the isolated fusion protein comprises two polypeptide chains linked by at least one interchain disulfide bond.

15. The isolated fusion protein of claim 14, wherein the isolated fusion protein comprises two polypeptide chains linked by two interchain disulfide bonds.

16. A pharmaceutical composition comprising the IL-variant polypeptide according to any one of claims 1 to 6 or the isolated fusion protein according to any one of claims 7 to 15 in admixture with a pharmaceutically acceptable carrier.

17. An isolated nucleic acid molecule encoding the IL-2 variant polypeptide according to any one of claims 1 to 6 or the fusion protein according to any one of claims 7 to 15.

18. An expression vector comprising the nucleic acid molecule according to claim 17.

19. A host cell comprising the nucleic acid molecule according to claim 17 or the expression vector according to claim 18.

20. A method for producing an IL-2 variant polypeptide according to any one of claims 1 to 6 or a fusion protein according to any one of claims 7 to 15, the method comprising culturing the host cell according to claim 19 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.

21. An isolated IL-2 variant polypeptide or fusion protein produced by the method according to claim 20.

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