Novel interleukin-2 variants and their bifunctional fusion molecules

Abstract

The present invention relates to bifunctional fusion molecules for the treatment of autoimmune diseases and various inflammatory diseases, cancer, or cancer metastasis. The bifunctional fusion molecules comprise various IL-2 variants containing mutations that preferentially promote the proliferation, survival, activation, and / or function of immunosuppressive regulatory T cells (T CD4+CD25+FoxP3+) over effector T cells and NK cells, or IL-2 variants containing mutations that substantially reduce the ability of these polypeptides to stimulate Treg cells, making them more effective in treating tumors. Furthermore, the bifunctional fusion molecules include biologics that target diseased tissue or tumor-associated antigens (TAA). In another aspect, the present invention relates to pharmaceutical compositions comprising the disclosed polypeptides. Finally, the present invention relates to the therapeutic use of the disclosed polypeptides and pharmaceutical compositions due to their selective regulatory effect on the immune system against diseases such as autoimmune diseases, inflammatory diseases, cancer, and various infectious diseases.

Description

[Background technology]

[0001] Related patent applications This application claims the benefits of U.S. Provisional Patent Application No. 62 / 861,484, filed on 14 June 2019, each of which is incorporated herein by reference in its entirety.

[0002] Interleukin-2 (IL-2) was the first growth factor described in relation to T cells. Since its discovery, IL-2 has been shown to promote T cell proliferation and survival in vitro (Smith, KA. (1988), Science., vol. 240, pp. 1169-76), and has demonstrated the ability to boost the immune response in relation to T cells and viral infections (T viral infection) (Blattman, JN et al. (2003), Nat Med, vol. 9, pp. 540-7) and vaccines (Fishman, M. et al. (2008), J Immunother., vol. 31, pp. 72-80, Kudo-Saito, C. et al. (2007), Cancer Immunol Immunother., vol. 56, pp. 1897-910; Lin, CT. et al. (2007), Immunol Lett., vol. 114, pp. 86-93).

[0003] IL-2 is used in cancer treatment. Recombinant human IL-2 is a highly effective immunotherapy for metastatic melanoma and renal cell carcinoma, with a sustained response observed in approximately 10% of patients. However, its short half-life and high toxicity limit the optimal dose of IL-2. Furthermore, because IL-2 binds with greater affinity to its heterotrimeric receptor, IL-2Rαβγ, it leads to the preferential proliferation of immunosuppressive regulatory T cells (Tregs) that constitutively express high levels of IL-2Rα. Treg proliferation is an undesirable effect of IL-2 in cancer immunotherapy. However, the ability of IL-2 to stimulate Treg cells even at low doses suggests potential applications in the treatment of autoimmune diseases and various inflammatory diseases.

[0004] Treg cells are central to immune system homeostasis and play a major role in maintaining peripheral immune tolerance by suppressing (autoreactive) effector T cells. Deficiencies in Treg cell number or function have been shown in several autoimmune and inflammatory diseases. As a result, there is great interest in developing therapies that enhance the number and / or function of Treg cells. One therapeutic approach under consideration for autoimmune diseases is the use of low doses of IL-2 to target Treg cells, because Treg cells respond better to lower concentrations of IL-2 than many other immune cell types due to their constitutively high IL-2Rα levels (Klatzmann D, 2015, Nat Rev Immunol., vol. 15: pp. 283-94). Clinical trials of low-dose IL-2 treatment in various GVHD patients (Koreth, J. et al., 2011, N Engl J Med., Vol. 365: pp. 2055-66) and HCV-associated autoimmune vasculitis patients (Saadoum, D. et al., 2011, N Engl J Med., Vol. 365: pp. 2067-77) showed increased Treg levels and indications of clinical efficacy. However, even at these low doses, serious safety and tolerability issues arose. Therefore, there is a need for effective treatments for autoimmune / inflammatory diseases that target Treg cells more specifically than IL-2 and can enhance the number and function of Treg cells.

[0005] More recently, it has been discovered that IL-2 can be modified to selectively stimulate either cytotoxic effector T cells or Treg cells. Through various approaches, IL-2 variants with improved selective immunostimulatory ability have been generated. Some of these IL-2 variants are designed to enhance the ability to signal primarily through high-affinity receptors (α, β, and γ chains) while not enhancing the ability to signal through intermediate-affinity receptors (β and γ chains). The basic idea is to promote signaling in T cells without promoting signaling in NK cells, which is thought to be the cause of the observed toxicity. Inventions in this field include: U.S. Patent Nos. 7,186,804, 7,105,653, 6,955,807, 5,229,109, and U.S. Patent Application Publication No. 20050142106. It is important to note that none of these inventions relate to IL-2 variants that have higher therapeutic efficacy in vivo than native IL-2, based on a reduced ability to stimulate native regulatory T cells. However, since the initial research on IL-2 variants, research in this field has shown that Treg cells can stimulate IL-2Rβ and γ C It has been more fully established that these variants, along with the constitutive expression of high levels of IL-2Rα(CD25), must be selective to Treg cells as IL-2Rαβγ-selective agonists.

[0006] In summary, IL-2 is a highly multifaceted cytokine deeply involved in the biological activity of various cell populations. This characteristic makes IL-2 a crucial intersection in the regulation of immune responses and an attractive target for various therapeutic approaches and complex immunomodulations. Furthermore, IL-2 variants biased towards receptor subunits can be constructed to achieve IL-2-mediated selective immunomodulation, promoting the proliferation and activity of regulatory T cells (Tregs) while minimizing cytotoxic T effector (Teff) cells and reducing levels of pro-inflammatory signaling molecules. Conversely, IL-2 variants biased towards receptor subunits can be constructed to achieve IL-2-mediated selective immunomodulation, preferentially promoting the proliferation and activation of Teff cells that attack cancer cells while reducing the proliferation and activation of Treg cells. [Overview of the Initiative]

[0007] In one embodiment, the present invention relates to the creation of mutant IL-2 variants. These variants are characterized by enhanced selectivity to stimulate Treg (T CD4+CD25+FoxP3+) cells more than cytotoxic effector lymphocytes, including CD8+ T cells and NK cells. Specifically, these variants provide a practical solution for improving IL-2 therapy in autoimmune and inflammatory disorders. The present invention relates to polypeptides that share the primary sequence of human IL-2 with the exception of one to several mutated amino acids. These variants have their IL-2Rβ and / or γ CThese variants have amino acid substitutions that reduce affinity for IL-2Rβγ receptor complexes, resulting in reduced affinity for IL-2Rβγ receptor complexes and a reduced or absent ability to activate IL-2Rβγ-expressing cells, while retaining the ability to bind to IL-2Rα and to bind to and activate the IL-2Rαβγ receptor complex. The present invention also includes using these mutant variants therapeutically for the treatment of autoimmune diseases and various inflammatory diseases, either alone, in combination with disease tissue-targeted biologics, or as part of a bifunctional molecular construct using disease tissue-targeted biologics.

[0008] In one embodiment, the present invention relates to a proposed "component similar to that of a bacterial toxin." 19 The invention relates to the creation of mutant IL-2 variants characterized by the removal of the "LDL" motif (Baluna R, Rizo et al., Proc Natl Acad Sci, 1999; Vol. 96: pp. 3957-62). This "toxic motif" is partially involved in the direct vascular toxicity of IL-2. Mutations introduced to remove D20 or two adjacent residues, which are critically important residues of the toxin-like domain, are expected to remove the toxic motif, prevent endothelial cell damage, and significantly reduce VLS. Importantly, since this motif is located at the contact surface with IL-2Rβ, amino acid substitutions to this motif will reduce its affinity for IL-2Rβ, and the resulting molecule is expected to have two beneficial properties, including selectivity for activated Treg cells and reduced endothelial damage. The present invention relates to a polypeptide that shares its primary sequence with human IL-2, except for a few mutated amino acids. The present invention also includes using these mutant variants therapeutically for the treatment of autoimmune diseases and various inflammatory diseases, either alone, in combination with disease tissue-targeted biologics, or as part of a bifunctional molecular construct using disease tissue-targeted biologics.

[0009] In one aspect, the present invention relates to the generation of mutant variants of IL-2 that are selective agonists of IL-2 activity with reduced or abolished binding ability to IL-2Rα. Specifically, these variants will provide a way to overcome the limitations observed with native IL-2 in the treatment by native IL-2, which has a proven ability to expand native regulatory T cells in vivo. The present invention relates to polypeptides that share the primary sequence of human IL-2, except for a few mutated amino acids. The introduced mutations substantially reduce the ability of these polypeptides to stimulate Treg cells and confer higher efficacy by IL-2. The present invention also relates to the use of these mutant variants for the treatment of diseases such as cancer or infectious diseases where the activity of regulatory T cells (Treg) is undesirable, either alone or in combination with vaccines, immune checkpoint inhibitors, tumor-associated antigen (TAA) target biologics, or as part of a bifunctional molecular construct using a disease tissue target biologic to improve the safety profile and increase the efficacy in treatment.

[0010] In one aspect, the present invention relates to the generation of mutant variants of IL-2 that are characterized by reducing severe toxicities such as vascular leak syndrome (VLS) associated with high-dose clinical IL-2 for the treatment of renal cancer and melanoma. Specifically, the introduced mutations are expected to significantly reduce the binding ability to IL-2Rα (CD25), and as a result, impair the binding to CD25+ lung endothelial cells, prevent endothelial cell damage, and significantly reduce VLS. The present invention relates to polypeptides that share the primary sequence of human IL-2, except for a few mutated amino acids. The present invention also relates to the use of these mutant variants for the treatment of diseases such as cancer or infectious diseases, either alone or in combination with vaccines, immune checkpoint regulators, tumor-associated antigen (TAA) target biologics, or as part of a bifunctional molecular construct using a disease tissue target biologic to improve the safety profile and increase the efficacy in treatment.

[0011] The present invention enables a substantial improvement of current IL-2-based immunomodulatory strategies in the treatment of autoimmune and various inflammatory disorders. Specifically, replacement of native IL-2 with the mutant variants described herein results in a CD25-biased selective stimulation of Treg cells. In various embodiments, the IL-2 variant (or mutant) comprises the sequence of an IL-2 variant (or mutant) derived from the sequence of the mature human IL-2 polypeptide set forth in SEQ ID NO: 3. In various embodiments, the IL-2 variant functions as an IL-2 agonist. In various embodiments, the IL-2 variant functions as an IL-2 antagonist. In various embodiments, the IL-2 variant comprises the sequences set forth in SEQ ID NOs: 4-43, 113- to 151, 208-212, and 275-292.

[0012] The present invention enables a substantial improvement of current IL-2-based immunomodulatory strategies in the treatment of autoimmune and various inflammatory disorders. Specifically, replacement of native IL-2 with the mutant variants described herein will result in a CD25-biased selective stimulation of Treg cells, removing the toxic motif, preventing endothelial cell damage, and is expected to significantly reduce VLS. In various embodiments, the IL-2 variant (or mutant) comprises the sequence of an IL-2 variant (or mutant) derived from the sequence of the mature human IL-2 polypeptide set forth in SEQ ID NO: 3. In various embodiments, the IL-2 variant functions as an IL-2 agonist. In various embodiments, the IL-2 variant functions as an IL-2 antagonist. In various embodiments, the IL-2 variant comprises SEQ ID NOs: 5-14, 26-43, 113-116, 130-151, 208-212, and 275-292.

[0013] The present invention enables substantial improvements to current IL-2-based immunomodulatory strategies in cancer treatment. Specifically, substitution of native IL-2 with the mutant variants described herein is expected to result in directed preferential stimulation of cytotoxic effector cells, weakening their binding to CD25+ pulmonary endothelial cells and consequently reducing VLS. In various embodiments, the IL-2 variant (or mutant) comprises a sequence of the IL-2 variant (or mutant) derived from the sequence of the mature human IL-2 polypeptide described in SEQ ID NO: 3. In various embodiments, the IL-2 variant functions as an IL-2 agonist. In various embodiments, the IL-2 variant functions as an IL-2 antagonist. In various embodiments, the IL-2 variant comprises SEQ ID NOs. 220-234 and 293-299.

[0014] The present invention also includes using these mutant variants to enhance efficacy in the treatment of autoimmune diseases and various inflammatory diseases, cancer, or cancer metastasis, either alone, in combination with disease tissue-targeted biologics, or as part of a bifunctional molecular construct using disease tissue-targeted biologics.

[0015] In another embodiment, the IL-2 variant of the present invention is attached to at least one heterologous protein. In various embodiments, the IL-2 variant is fused with at least one polypeptide that gives the fusion molecule an extended half-life. Such polypeptides include other polypeptides that bind to IgG Fc or neonatal Fcγ / receptors, human serum albumin, or polypeptides that bind to proteins with an extended serum half-life. In various embodiments, the IL-2 variant is fused with 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 described in SEQ ID NO: 44. In various embodiments, the Fc domain is an Fc domain having the amino acid sequence described in SEQ ID NO: 45. 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.

[0016] In various embodiments, the IL-2 variant can be ligated to the N-terminus or C-terminus of the IgG Fc region.

[0017] The term "Fc" refers to a molecule or sequence containing a sequence of non-antigen-binding fragments of a full-length antibody, which may be in monomeric or polymeric form. The original immunoglobulin source for native Fc is preferably human, but may be any immunoglobulin disclosed in the art. Native Fc consists of monomeric polypeptides that can be linked by covalent (i.e., disulfide) and non-covalent bonds to form dimeric or polymeric forms. The number of intermolecular disulfide bonds between monomeric subunits of a native Fc molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of native Fc is a disulfide-bonded dimer resulting from the papain degradation of IgG (Ellison et al., (1982), Nucleic Acids Res., Vol. 10: pp. 4071-9). The term "native Fc," as used herein, refers to the monomeric, dimeric, and polymeric forms of Fc. It includes Fc domains containing 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 has been modified from native Fc but still contains a binding site to the salvage receptor FcRn. International Publication No. 97 / 34631 (published September 25, 1997) and International Publication No. 96 / 32458 describe exemplary Fc variants and their interactions with salvage receptors, which are incorporated herein by reference. Furthermore, native Fc may include sites that confer structural features or biological activity not required for the fusion molecule of the present invention and may therefore be removed. In other words, in various embodiments, the term “Fc variant” includes molecules or sequences lacking one or more native Fc sites or residues that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with selected host cells, (3) heterogeneity of the N-terminus after expression in selected host cells, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cell-mediated cytotoxicity (ADCC).

[0019] The term “Fc domain” encompasses the molecules and sequences of native Fc and Fc variants as defined above. Similar to Fc variants and native Fc, the term “Fc domain” encompasses molecules in monomeric or multimeric form, digested from full-length antibodies or produced by recombinant gene expression or other means. In various embodiments, “Fc domain” refers to a dimer consisting of two Fc domain monomers (SEQ ID NO: 44), typically containing all or part of the hinge region. In various embodiments, the Fc domain may be mutated to lack effector function. In various embodiments, each of the Fc domain monomers of the Fc domain contains amino acid substitutions in the CH2 antibody constant domain to reduce interaction or binding between the Fc domain and the Fcγ receptor. In various embodiments, each subunit of the Fc domain contains two amino acid substitutions (L234A and L235A) to reduce binding to the activating Fc receptor and / or effector function. In various embodiments, each subunit of the Fc domain contains three amino acid substitutions (L234A, L235A, and G237A) that reduce binding to the activating Fc receptor and / or effector function (SEQ ID NO: 45).

[0020] In various embodiments, the two Fc domain monomers of the Fc domain each contain amino acid substitutions that promote heterodimerization of these two monomers. In various other embodiments, heterodimerization of the Fc domain monomers can be promoted by introducing different but compatible substitutions (such as a "knob-into-hole" residue pair) into the two Fc domain monomers. This "knob-into-hole" technique is also disclosed in U.S. Patent No. 8,216,805. In yet another embodiment, one Fc domain monomer contains the knob-type mutation T366W, and the other Fc domain monomer contains the hole-type mutations T366S, L358A, and Y407V. In various embodiments, two Cy residues that form a stabilizing disulfide bridge (S354C on one chain and Y349C on the corresponding chain) are introduced (SEQ ID NOs: 46 and 47). By using the heterodimer Fc, monovalent IL-2 variant constructs can be obtained.

[0021] In various embodiments, the IL-2 variant Fc fusion protein is monomeric, i.e., contains only one IL-2 mutant protein molecule. In such embodiments, the fusion protein consists of a heterodimer Fc linked to the IL-2 variant (e.g., Hole-Fc having the sequence described in SEQ ID NO: 47) and a corresponding heterodimer Fc (e.g., Knob-Fc having the sequence described in SEQ ID NO: 46). When heterodimers of the two Fc-containing polypeptides are formed, the resulting protein contains one IL-2 variant.

[0022] In various embodiments, the Fc domain may be mutated to further extend its in vivo half-life. In various embodiments, each subunit of the Fc domain contains three amino acid substitutions (M252Y, S254T, and T256E) that enhance binding to human FcRn, as disclosed in U.S. Patent No. 7,658,921 (SEQ ID NO: 251). In various embodiments, each subunit of the Fc domain contains one amino acid substitution (N434A) that enhances binding to human FcRn, as disclosed in U.S. Patent No. 7,371,826 (SEQ ID NO: 252). In various embodiments, each subunit of the Fc domain contains one amino acid substitution (M428L and N434S) that enhances binding to human FcRn, as disclosed in U.S. Patent No. 8,546,543. In various embodiments, IL-2 variants are used to produce the Fc-IL-2 fusion proteins described in SEQ ID NOs. 71-112, 152-194, 213-219, and 235-249.

[0023] In various embodiments, the IL-2 variant of the present invention can be attached to antibodies that provide an extended half-life to the fusion molecule, such as anti-keyhole limpet hemocyanin (KLH) antibodies. Such antibodies recognize foreign antigens and provide a longer half-life, but have no biological function or harm in humans. The IgG class can be IgG, IgA, IgE, or subclasses (e.g., IgG1, IgG2, IgG3, IgA1, IgA2).

[0024] In various embodiments, the IL-2 variant of the present invention can bind to a targeting / dual-function moiety which is an antibody, antibody fragment, protein, or peptide that targets molecules enriched in target tissue, or to a targeting / dual-function moiety which exhibits binding to disease cells or the disease microenvironment such as inflammatory tissue targets, TNF, TNF receptor, IL-6, IL-6 receptor, integrin α4β7, β7, MAdCAM-1, BLYS, TSLP, APRIL, or autoimmune or inflammatory modulators (Table 1).

[0025] In various embodiments, IL-2 variants are used to produce the dual-functional fusion constructs described in Sequence IDs 200-207, 253-274, and 307-312.

[0026] Any of the aforementioned proteins highly expressed in various inflammatory tissues or immune cells can be used as autoimmune / inflammatory disease targets for the IL-2 variants of the present invention. In various embodiments, one or more autoimmune / inflammatory disease targets, their variants or variants / isoforms intended for use in the IL-2 variant constructs and methods of this disclosure are selected from or derived from the list shown in Table 1. TIFF0007850417000001.tif144170TIFF0007850417000002.tif172170

[0027] In various embodiments, the IL-2 variant constructs of the present invention include a targeting moiety in the form of a peptide that binds to cancer tissue-rich molecules such as antibodies, antibody fragments, bispecific antibodies, proteins, or tumor-associated antigens (TAAs).

[0028] A TAA can be any molecule, macromolecule, or combination of molecules against which an immune response is desired. A TAA can be a protein comprising two or more polypeptide subunits. For example, a protein can be a dimer, trimer, or higher-order multimer. In various embodiments, two or more subunits of a protein can be linked by covalent bonds, such as disulfide bonds. In various embodiments, subunits of a protein can be held together by non-covalent interactions. Thus, a TAA can be any peptide, polypeptide, protein, nucleic acid, lipid, carbohydrate, or small organic molecule, or any combination thereof, against which a person skilled in the art would like to induce an immune response. In various embodiments, TAA is a peptide containing about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 amino acids. In various embodiments, the peptide, polypeptide, or protein is a molecule commonly administered to a subject by injection. In various embodiments, tumor-specific antibodies or binding proteins, after administration, function as targeting moieties to guide IL-2 variants to disease sites such as cancer sites, where their active domains are released and can interact with their cognitive receptors on diseased cells.

[0029] Any of the aforementioned markers can be used as the TAA target for the IL-2 variant of the present invention. In various embodiments, one or more TAAs, TAA variants, or TAA variants intended for use in the IL-2 variant constructs and methods of this disclosure are selected from or derived from the list shown in Table 2. TIFF0007850417000003.tif255170TIFF0007850417000004.tif204170

[0030] In various embodiments, the IL-2 variant of the present invention can bind to a targeting / dual-functional moiety, which is an antibody, antibody fragment, bispecific antibody, protein, or peptide that targets an immune checkpoint regulator.

[0031] Numerous immune checkpoint protein antigens have been reported to be expressed on various immune cells, including, for example, SIRP (expressed in macrophages, monocytes, and dendritic cells), CD47 (highly expressed in tumor cells and other cell types), VISTA (expressed in monocytes, dendritic cells, B cells, and T cells), CD152 (expressed in activated CD8+ T cells, CD4+ T cells, and regulatory T cells), CD279 (expressed in tumor-infiltrating lymphocytes, activated T cells (both CD4 and CD8), regulatory T cells, activated B cells, activated NK cells, allergic T cells, monocytes, and dendritic cells), CD274 (expressed in T cells, B cells, dendritic cells, macrophages, vascular endothelial cells, and pancreatic islet cells), and CD223 (expressed in activated T cells, regulatory T cells, allergic T cells, NK cells, NKT cells, and plasmacytoid dendritic cells) (e.g., Pardoll, D., Nature Reviews). This includes Cancer, 12:252-264, 2012). Antibodies that bind to antigens that have been identified as immune checkpoint proteins are known to those skilled in the art. For example, various anti-CD276 antibodies have been described in the art (see, for example, U.S. Patent Application Publication No. 20120294796 (Johnson et al) and the references cited therein). Various anti-CD272 antibodies have been described in the art (see, for example, U.S. Patent Application Publication No. 20140017255 (Mataraza et al) and the references cited therein). Various anti-CD152 / CTLA-4 antibodies have been described in the art (see, for example, U.S. Patent Application Publication No. 20130136749 (Korman et al) and the references cited therein). Various anti-LAG-3 / CD223 antibodies have been described in the art (see, for example, U.S. Patent Application Publication No. 20110150892 (Thudium et al) and the references cited therein). Various anti-CD279 / PD-1 antibodies have been described in the art (see, for example, U.S. Patent No. 7,488,802 (Collins et al) and the references cited herein). Various anti-PD-L1 antibodies have been described in the art (see, for example, U.S. Patent Application Publication No. 20130122014 (Korman et al) and the references cited herein).Various anti-TIM-3 antibodies have been described in the art (see, for example, U.S. Patent Application Publication No. 20140044728 (Takayanagi et al) and the references cited herein). Various anti-B7-H4 antibodies have been described in the art (see, for example, U.S. Patent Application Publication No. 20110085970 (Terrett et al) and the references cited herein). Each of these documents is incorporated herein by reference in its entirety with respect to the specific antibodies and sequences taught therein.

[0032] In various embodiments, the IL-2 fusion partner may be an antibody, antibody fragment, bispecific antibody, or protein or peptide that binds to an immune checkpoint protein antigen present on the surface of immune cells. In various embodiments, the immune checkpoint protein antigen may be selected from the group consisting of, but is not limited to, CD279 (PD-1), CD274 (PDL-1), CD276, CD272, CD152, CD223 (LAG-3), CD40, SIRPα, CD47, OX-40, GITR, ICOS, CD27, 4-1BB, TIM-3, B7-H3, B7-H4, TIGIT, and VISTA.

[0033] In various embodiments, heterologous proteins are bound to IL-2 variants by linker and / or hinge-linker peptides. The linker or hinge-linker may be an artificial sequence consisting of 5, 10, 15, 20, 30, 40 (or any number in between) or more amino acids, with relatively few secondary structures.

[0034] In various embodiments, heterologous proteins are attached to the IL-2 variant by rigid linker peptides consisting of 10, 15, 20, 30, 40 (or more) amino acids, which exhibit an α-helix higher-order structure and can act as rigid spacers between protein domains.

[0035] In another embodiment, the IL-2 variant can be linked to various non-proteinaceous polymers, including but not limited to various polyols such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, by the methods described in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192, or 4,179,337. In various embodiments, amino acid substitutions may be made at various positions within the IL-2 variant to facilitate the addition of polymers such as PEG. In various embodiments, such PEGylated proteins may have a longer half-life and / or reduced immunogenicity than non-PEGylated proteins.

[0036] "Polyethylene glycol" or "PEG" means a polyalkylene glycol compound or a derivative thereof, with or without derivatization by a conjugating agent or a conjugated or activated moiety (e.g., an aldehyde moiety, a hydroxysuccinimidyl moiety, a hydrazide moiety, a thiol moiety, a triflate moiety, a toresylate moiety, an azirdine moiety, an oxirane moiety, an orthopyridyl disulfide moiety, a vinyl sulfone moiety, an iodoacetamide moiety, or a maleimide moiety). In various embodiments, PEG encompasses substantially linear straight-chain PEG, branched-chain PEG, or dendritic 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).

[0037] In various embodiments, the Il-2 variant can be covalently or non-covalently linked at its N-terminus or C-terminus to other polypeptides that bind to IgG Fc or neonatal Fcγ / receptors, human serum albumin, or polypeptides that bind to proteins with a long half-life in the blood, or to various non-proteinogenic polymers.

[0038] In another embodiment, the disclosure provides a pharmaceutical composition comprising an IL-2 variant mixed with a pharmaceutically acceptable carrier.

[0039] In another embodiment, the present disclosure provides a method for treating an autoimmune disease in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a subject in need thereof. In one embodiment, the subject is a human subject. An autoimmune disease according to the present invention is a disease or disorder originating from or against the tissues of an individual, or a co-segregate or manifestation thereof, or a condition arising therefrom. In various embodiments, autoimmune diseases include arthritis (including rheumatoid arthritis and reactive arthritis), systemic lupus erythematosus (SLE), graft-versus-host disease (GvHD), 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, scleroderma, pernicious anemia, Goodpasture syndrome, Guillain-Barré syndrome, and Wegener's disease. Examples of diseases that may be considered include, but are not limited to, glomerulonephritis, aplastic anemia (including patients with aplastic anemia due to frequent transfusions), paroxysmal nocturnal hemoglobinuria, myelodysplastic syndrome, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, Evans 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.

[0040] In another embodiment, the Disclosure provides a method for treating an autoimmune disease in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention in combination with a second therapeutic agent capable of treating the autoimmune disease.

[0041] In another embodiment, the present disclosure provides a method for treating an inflammatory disease in a subject, comprising administering a therapeutically effective amount of the 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 diseases of the subject 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, vacant colitis, Behçet's syndrome and unclassifiable 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 mellitus, gout, cryopyrin-associated periodic syndromes, and chronic obstructive pulmonary disease.

[0042] In another embodiment, the present disclosure provides a method for treating an inflammatory disease in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention in combination with a second therapeutic agent capable of treating an inflammatory disease.

[0043] In another embodiment, the present disclosure provides a method for organ transplantation or related graft-versus-host disease in a subject, comprising administering a therapeutically effective amount of the 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 transplantation is selected from heart, kidney, liver, lung, pancreas, intestine, and thymus organ transplantation, or from bone tissue transplantation, tendon tissue transplantation, corneal tissue transplantation, skin tissue transplantation, heart valve tissue transplantation, nerve tissue transplantation, and venous tissue transplantation.

[0044] In another embodiment, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the 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 cancer is selected from pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, and rhabdomyosarcoma.

[0045] In another embodiment, the Disclosure provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the Invention in combination with a second therapeutic method selected from the group consisting of: cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiotherapy, and stem cell transplantation. In various embodiments, the combination therapy may include administering a therapeutically effective amount of immunotherapy to the subject, where the immunotherapy is a depleting antibody against a specific tumor antigen. Therapy using antibodies; therapy using antibody-drug conjugates; therapy using agonist antibodies, antagonist antibodies, or inhibitory antibodies against costimulatory or coinhibitory molecules (immune checkpoints) such as CTLA-4, PD-1, PD-L1, OX-40, CD137, GITR, LAG3, TIM-3, CD40, CD47, SIRPα, ICOS, Siglec8, Siglec9, Siglec15, TIGIT, and VISTA; therapy using bispecific T cell-inducing antibodies (BiTE®) such as blinatumomab; therapy using biological response modifiers such as the TNF family, IL-1, IL-4, IL-7, IL-12, IL-15, IL-17, IL-21, IL-22, GM-CSF, IFN-α, IFN-β, and IFN-γ Treatments include, but are not limited to, those using: IL-2 variants; therapeutic vaccines such as cyproisel T; dendritic cell vaccines or tumor antigen peptide vaccines; chimeric antigen receptor (CAR)-T cells; CAR-NK cells; tumor-infiltrating lymphocytes (TILs); adoptive transplant antitumor T cells (grown in vitro and / or TCR transgenic); TALL-104 cells; and immunostimulants such as Toll-like receptor (TLR: TLR7, TLR8, and TLR9) agonists CpG and imiquimod. The above combination therapies increase the killing of tumor cells by effector cells, meaning that a synergistic effect exists between IL-2 variants and immunotherapy when administered simultaneously.

[0046] In another embodiment, the present disclosure provides the use of IL-2 variants for preparing pharmaceuticals for the treatment of autoimmune diseases.

[0047] In another embodiment, the present disclosure provides the use of IL-2 variants for preparing pharmaceuticals for the treatment of organ transplantation and GVHD.

[0048] In another embodiment, the present disclosure provides the use of IL-2 variants for preparing pharmaceuticals for the treatment of inflammatory disorders.

[0049] In another embodiment, the present disclosure provides the use of IL-2 variants for preparing pharmaceuticals for the treatment of cancer.

[0050] In another embodiment, the Disclosure provides an isolated nucleic acid molecule comprising a polynucleotide encoding an IL-2 variant of the Disclosure. In another embodiment, the Disclosure provides a vector comprising the nucleic acid described herein. In various embodiments, the vector is an expression vector. In another embodiment, the Disclosure provides an isolated cell comprising the nucleic acid of the Disclosure. In various embodiments, the cell is a host cell comprising the expression vector of the Disclosure. In another embodiment, a method for producing an IL-2 variant is provided by culturing the host cell under conditions that promote the expression of the protein or polypeptide described above. [Brief explanation of the drawing]

[0051] [Figure 1] Figure 1 shows 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 after purification of protein A. Figures 1D and 1E show SDS-PAGE of each sample in the absence (lane 2) and presence (lane 3) of a reducing agent. [Figure 2]Figure 2 shows the difference in the effect of an IL-2 variant Fc fusion protein with an aspartic acid amino acid substitution (D20X) at position 20 on inducing STAT5 phosphorylation in CD4+ Treg (A) cells compared to Tconv (B) cells, compared to a wild-type fusion protein (P-0250) in a human PBMC assay. [Figure 3] Figure 3 shows the difference in the effect of the IL-2 variant P-0375(N88Q) Fc fusion protein on inducing STAT5 phosphorylation in CD4+ Treg(A) cells compared to wild-type (P-0250) and reference proteins in a human PBMC assay. [Figure 4] Figure 4 shows the difference in the effect of Fc fusion proteins of IL-2 variants with an amino acid substitution at position 19 on STAT5 phosphorylation compared to the wild type (P-0250). The ability to induce STAT5 phosphorylation in CD4+ Treg (A and C) and Tconv (B and D) cells was revealed by FACS analysis in human PBMC assays. [Figure 5] Figure 5 shows the differences in the effect of Fc fusion proteins on STAT5 phosphorylation by IL-2 variants with individual amino acid substitutions at either position 19 (P-0372) or position 126 (P-0303), and a combined mutant (P-0419), compared to the wild-type (P-0250) or reference protein. The ability to induce STAT5 phosphorylation in CD4+Treg (A, C, and E) and CD4+Tconv (B, D, and F) cells was revealed by FACS analysis. [Figure 6]Figure 6 shows the differences in the effects of IL-2 variant Fc fusion proteins (P-0419, P-0464, P-0471, P-0474, P-0417, and P-0322) with different combinations of double amino acid substitutions on STAT5 phosphorylation, compared to the wild type (P-0250). Furthermore, the biological activity of P-0417 and P-0322 was compared with their single-amino acid substitution counterparts, P-0373 and P-0363, respectively. The ability to induce STAT5 phosphorylation in CD4+Treg (A and C) and CD4+Tconv (B and D) cells was revealed by FACS analysis in human PBMC assays. Figures 6E and 6F show the differences in the effects of further IL-2 variant Fc fusion proteins, P-0860 and P-0859, on STAT5 phosphorylation in CD4+Treg cells. [Figure 7] Figure 7 shows the differences in the effects of IL-2 variant Fc fusion proteins with a single amino acid substitution at position 19 (P-0424) or position 126 (P-0303), or a combination mutant (P-0447), on STAT5 phosphorylation compared to the wild type (P-0250), as well as the differences in the effects of IL-2 variant Fc fusion proteins with various combination amino acid substitutions (P-0419, P-0447, P-0448, and P-0449) on STAT5 phosphorylation compared to the wild type (P-0250) and reference Fc fusion proteins. The ability to induce STAT5 phosphorylation in CD4+Treg (A and C) and CD4+Tconv (B and D) cells was revealed by FACS analysis in human PBMC assays. [Figure 8] Figure 8 shows the pSTAT5 stimulating activity of the IL-2 fusion proteins P-0250, P-0424, and P-447 compared to their respective S125I-substituted counterparts, P-0531, P-0491, and P-0511. Their ability to induce STAT5 phosphorylation in CD4+ Treg (A, C, and E) and CD4+ Tconv (B, D, and F) cells was revealed by FACS analysis in human PBMC assays. [Figure 9]Figure 9 shows the differences in the effect of IL-2 variant Fc fusions (P-0511 and P-0512) on STAT5 phosphorylation compared to wild-type (P-0250) and three reference molecules in three subsets of CD4+ T cells: A) CD4+FoxP3+CD25+ Treg cells, B) CD4+FoxP3-CD25+ activated Tconv cells, and C) CD4+FoxP3-CD25- naive Tconv cells. The ability to induce STAT5 phosphorylation was revealed by FACS analysis in a human PBMC assay. [Figure 10] Figure 10 shows the differences in the effects of P-0511 and P-0512 on stimulating the proliferation of A) CD8+ T cells and B) NK cells, compared to wild-type (P-0250) and the reference molecule. Cell proliferation was measured in a human PBMC assay by FACS analysis of CFSE dilution and expressed as the percentage of dividing cells. [Figure 11] Figure 11 shows the differences in the effect of IL-2 variant Fc fusion P-0511 on inducing STAT5 phosphorylation compared to wild-type equivalent P-0531 in various cell types. The ability to induce STAT5 phosphorylation in A) CD4+ Treg, B) CD4+ Tconv, C) CD8+ T cells, and D) CD56+ NK cells was revealed by FACS analysis in human PBMC assays. Figure 11E shows the binding strength of P-511 to the IL-2Rβ-γc complex compared to P-0531 and reference 1 in ELISA assays. [Figure 12] Figure 12 shows the proliferation and expansion of Treg cells in mice administered with IL-2 variant Fc fusion protein and reference after a single subcutaneous injection. Blood was collected at specified time points for the measurement of proliferation and lymphocyte phenotype. (A) Percentage of Ki67-positive Treg cells, (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 using one-way ANOVA followed by Tukey's post-hoc test. For each time point relative to the PBS group, ****p<0.0001; ***p<0.001. [Figure 13] Figure 13 shows the proliferation of effector T cells and NK cells in mice administered with IL-2 mutant Fc fusion protein and a baseline after a single subcutaneous injection. Blood was collected at specified time points to measure lymphocyte proliferation. (A) Percentage of Ki67-positive CD4+ conventional T (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 using one-way ANOVA followed by Tukey's post-hoc test. ****p<0.0001; ***p<0.001 for the PBS group at each time point. [Figure 14] Figure 14 shows the proliferation of effector T cells and NK cells in mice administered with IL-2 mutant Fc fusion protein and a baseline after a single subcutaneous injection. (A-B) Percentage of conventional CD4+ 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. [Figure 15] Figure 15 shows A) the percentage of Ki67-positive expression and B) the ratio of Treg cells to Tconv cells based on cell number in mice treated with IL-2 mutant Fc fusion protein and control cells. Data were obtained using FACS. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. At each time point, p<0.0001 and p<0.05 were observed for the PBS group. [Figure 16] Figure 16 shows the expression of CD25 and Foxp3 on Treg cells in mice administered with IL-2 mutant Fc fusion protein and a reference after a single subcutaneous injection. A) Foxp3 and B) CD25 expression levels were analyzed by FACS and expressed as mean fluorescence intensity (MFI). Data are expressed as mean ± SEM. At each time point, ****p < 0.0001 compared to the PBS group. [Figure 17]Figure 17 shows the proliferation of Treg cells and the dose-dependent increase in proliferation in mice after a single injection of the IL-2 variant Fc fusion protein P-0511. Blood was collected at specified time points for lymphocyte phenotyping and measurement of the Ki67 proliferation marker. A) Percentage of Ki67-positive Treg cells, B) Percentage of Treg cells among all CD4+ T cells, C) Number of Treg cells per microliter of whole blood, D) Multiplier change in the number of Treg cells from baseline for each group. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. For each time point relative to the PBS group, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. [Figure 18] Figure 18 shows the dose-dependent effect of a single injection of the IL-2 variant Fc fusion protein P-0511 on the percentage proportions of Treg cells (A), CD4+ Tconv cells (B), CD8 T cells (C), and NK cells (D) to total lymphocytes in mice. Blood was collected at specified time points for lymphocyte phenotyping. Data were measured by FACS. Values ​​are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. At each time point, relative to the PBS group, ****p<0.0001, **p<0.01, and *p<0.05 were observed. [Figure 19] Figure 19 shows the dose-dependent increase in A) the ratio of Treg cells to Tconv cells, B) CD25 expression on Treg cells, and C) Foxp3 expression on Treg cells in mice after a single injection of P-0511. Data were measured by FACS. Values ​​are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. At each time point, relative to the PBS group, ****p<0.0001, ***p<0.001, and **p<0.01 were observed. [Figure 20]Figure 20 shows the proliferation and persistence of Treg cells in mice that received repeated administration of Fc fusion proteins of IL-2 variants (P-0511 and P-0512), not wild-type (P-0531) or reference. The compound was administered subcutaneously once every 3 days (Q3D), and blood was collected for lymphocyte phenotyping and measurement of the proliferation marker Ki67 on day 3 after the first and third injections. A) Percentage of Ki67-positive Treg cells, B) Percentage of Treg cells among total CD4+ T cells, C) Percentage of Treg cells among total blood lymphocytes. Data were measured by FACS. Expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Compared to the PBS group, ****p<0.0001 and *p<0.05, respectively. [Figure 21] Figure 21 shows the sustained increase in Treg cell count in mice that received repeated administration of Fc fusion proteins of IL-2 variants (P-0511 and P-0512), neither wild-type (P-0531) nor control. The compound was administered subcutaneously once every 3 days (Q3D), and blood was collected for lymphocyte phenotyping and measurement of the proliferation marker Ki67 on day 3 after the first and third injections. A) Number of Treg cells per microliter of whole blood, B) Calculated change in Treg count compared to the PBS control group. Data were measured by FACS. Expressed as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post-hoc test. Compared to each PBS group, ****p<0.0001, ***p<0.001, **p<0.01. [Figure 22]Figure 22 shows the retention of the elevated ratio of Tregs to Tconvs in mice that received repeated doses of Fc fusion proteins of IL-2 variants (P-0511 and P-0512), neither wild-type (P-0531) nor reference. The compound was administered subcutaneously once every 3 days (Q3D), and blood was collected for Treg and Tconv cell phenotyping on day 3 after the first and third injections. The ratios were calculated based on %Treg and %Tconv in total CD4 cells. Data were measured by FACS. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Compared to each PBS group, ****p<0.0001. [Figure 23] Figure 23 shows the suppression of antigen-induced inflammation by P-0511 in a mouse model of delayed-type hypersensitivity (DTH) induced by keyhole limpet hemocyanin (KLH) antigen. Mice were immunized with KLH on day 0 and re-challenged in the right ear on day 5. From day 2, mice were administered P-0511 in Q3D or Q5D. The kinetics of the DTH response, using the change in ear thickness (Δear thickness) relative to baseline at various time points after KLH challenge, are shown for A) Q3D and B) Q5D administration schedules. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Compared to each PBS group at each time point, ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05. [Figure 24] Figure 24 shows the suppression of antigen-induced inflammation by P-0511 compared to reference 1 in a mouse model of KLH antigen-induced DTH. Mice were immunized with KLH on day 0 and re-challenged in the right ear on day 5. Mice were administered the compound via Q5D from day 2. Kinetics of the DTH response are shown using the change in ear thickness (Δear thickness) relative to baseline at various time points after KLH challenge. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey post-hoc test. Compared to each PBS group at each time point, ****p<0.0001, **p<0.01, and *p<0.05. [Figure 25] Figure 25 shows the difference in the effect of P-0573 on Ki67 expression in A) CD4+ T cells, B) CD8+ T cells, and C) NK cells compared to wild-type (P-0531) and reference 4. The dose-dependent increase in the percentage of Ki67 expression was revealed by FACS analysis in human PBMC assays. [Figure 26] Figure 26 shows the differences in the effects of various IL-2 variant bifunctional constructs on STAT5 phosphorylation compared to the Treg-selective IL-2 variant Fc fusion protein P-0511 and / or the corresponding antibody fusion protein P-0536. Dose-dependent induction of STAT5 phosphorylation in CD4+ Treg (A, C, and E) and CD4+ Tconv (B, D, and F) cells was revealed by FACS analysis in human PBMC assays. [Figure 27] Figure 27 shows the proliferation and growth of Treg cells in mice after a single subcutaneous injection of either an IL-2 variant bifunctional construct (P-0536, P-0546, P-0559, or P-0560) or the Treg-selective IL-2 variant Fc fusion protein P-0511. Blood was collected at specified time points for proliferation measurement and lymphocyte phenotyping. (A) Percentage of Ki67-positive Treg cells, (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. [Figure 28] Figure 28 shows the proliferation of effector T cells and NK cells in mice after a single subcutaneous injection of either an IL-2 variant bifunctional construct (P-0536, P-0546, P-0559, or P-0560) or the Treg-selective IL-2 variant Fc fusion protein P-0511. Blood was collected at specified time points for the measurement of lymphocyte proliferation. (A) Percentage of Ki67-positive CD4+Foxp3-Tconv cells, (B) Percentage of Ki67-positive CD4+CD25+Foxp3-Teff cells, (C) Percentage of Ki67-positive CD8+ T cells, (D) Percentage of Ki67-positive NK cells. Data are expressed as mean ± SEM. [Figure 29] Figure 29 shows the proliferation of effector T cells and NK cells in mice after a single subcutaneous injection of either an IL-2 variant bifunctional construct (P-0536, P-0546, P-0559, or P-0560) or the Treg-selective IL-2 variant Fc fusion protein P-0511. (A) Percentage of CD4+Foxp3- Tconv cells in total blood lymphocytes, (B) Percentage of CD4+CD25+Foxp3-Teff cells in total blood lymphocytes, (C) Percentage of CD8+ T cells in total blood lymphocytes, (D) Percentage of Ki67-positive NK cells in total blood lymphocytes. Data are expressed as mean ± SEM. [Figure 30] Figure 30 shows A) the percentage of Ki67-positive expression and B) the ratio of Tregs to Tconv cells based on cell number in mice administered with either an IL-2 variant bifunctional construct (P-0536, P-0546, P-0559, or P-0560) or the Treg-selective IL-2 variant Fc fusion protein P-0511. Data were obtained using FACS. Values ​​are expressed as mean ± SEM. [Figure 31] Figure 31 shows the expression of CD25 and Foxp3 in Treg cells in mice treated with either a bifunctional IL-2 variant construct (P-0536, P-0546, P-0559, or P-0560) or the Treg-selective IL-2 variant Fc fusion protein P-0511. A) Foxp3 and B) CD25 expression levels were analyzed by FACS and are expressed as mean fluorescence intensity (MFI). Data are expressed as mean ± SEM. [Modes for carrying out the invention]

[0052] This invention relates to polypeptides that share the primary sequence with human IL-2, except for a few mutated amino acids. One panel of IL-2 variants includes mutations that preferentially promote the proliferation, survival, activation, and / or function of immunosuppressive regulatory T cells (T CD4+CD25+FoxP3+) more than effector T cells or NK cells. It also encompasses therapeutic applications of such IL-2 selective agonists, used alone, in combination with disease-tissue-targeting proteins or peptides, or as components of bifunctional molecular constructs, for the treatment of autoimmune diseases and various inflammatory diseases. Another panel of IL-2 variants includes mutations that substantially reduce the ability of these polypeptides to stimulate Treg cells, making them more effective in treating tumors. It also encompasses therapeutic applications of these mutated variants, used alone, in combination with vaccines, TAA-targeting biologics, or immune checkpoint blockers, or as components of bifunctional molecular constructs, for the treatment of diseases such as cancer and infections where regulatory T cell (Treg) activity is undesirable. In another embodiment, the present invention relates to pharmaceutical compositions comprising the polypeptides of the present disclosure. Finally, the present invention relates to therapeutic uses of the polypeptides and pharmaceutical compositions of the present disclosure by selectively modulating the immune system against autoimmune and inflammatory disorders or diseases such as cancer and various infectious diseases.

[0053] definition In this specification, the terms “polypeptide,” “peptide,” and “protein” are used synonymously and refer to polymers of amino acid residues. In various embodiments, “peptide,” “polypeptide,” and “protein” are amino acid chains in which α-carbons are linked together by peptide bonds. The terminal amino acid at one end of the chain (amino-terminus) has a free amino group, while the terminal amino acid at the other end of the chain (carboxy-terminus) has a free carboxyl group. As used herein, the term “amino-terminus” (abbreviated as N-terminus) refers to the free α-amino group on the amino acid at the amino-terminus of a peptide, or to the α-amino group (imino group if participating in a peptide bond) of any other amino acid within the peptide. Similarly, the term “carboxy-terminus” refers to the free carboxyl group at the carboxy-terminus of a peptide, or to the carboxyl group of any other amino acid within the peptide. Peptides also include substantially any polyamino acids, including, but not limited to, peptide mimetic compounds, such as those in which amino acids are linked by ethers rather than amide bonds.

[0054] The polypeptides of this disclosure also include polypeptides that have been modified in any way and for any reason, such as (1) reduced sensitivity to proteolysis, (2) reduced sensitivity to oxidation, (3) alteration of binding affinity for the purpose of protein complex formation, (4) alteration of binding affinity, and (5) conferring or altering other physicochemical or functional properties.

[0055] As used herein, an amino acid "substitution" refers to the substitution of a specific amino acid at a particular position within a parent polypeptide sequence with a different amino acid within the polypeptide. Amino acid substitutions can be produced using genetic or chemical methods well known in the art. For example, a single amino acid substitution (e.g., a conserved amino acid substitution) or multiple amino acid substitutions may be made in the natural sequence (e.g., in the polypeptide portion outside of domains forming intermolecular contacts). A "conserved amino acid substitution" refers to the substitution of an amino acid within a polypeptide with a functionally similar amino acid. The following six groups each contain amino acids that are conserved substitutions of each other. 1) Alanine (A), serine (S), and threonine (T) 2) Aspartic acid (D) and glutamic acid (E) 3) Asparagine (N) and glutamine (Q) 4) Arginine (R) and Lysine (K) 5) Isoleucine (I), leucine (L), methionine (M), and valine (V) 6) Phenylalanine (F), tyrosine (Y), and tryptophan (W)

[0056] A "non-conservative amino acid substitution" refers to the substitution of a component of one class with a component of another class. In various embodiments, the hydrophobicity and hydrophilicity index of amino acids may be considered when making such changes. Each amino acid is assigned a hydrophobicity and hydrophilicity index based on its hydrophobicity and charge properties. The hydrophobic and hydrophilic indices for each are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0057] The importance of amino acid hydrophobicity and hydrophilicity indices in conferring interactive biological functions to proteins is well understood in the art (see, for example, Kyte et al., 1982, J.Mol.Biol., 157:105-131). It is known that similar biological activity can be maintained by substituting a particular amino acid with another amino acid having a similar hydrophobicity or hydrophilicity score. When inducing changes based on hydrophobicity and hydrophilicity indices, various embodiments include substitutions of amino acids whose hydrophobicity and hydrophilicity indices are within ±2. Various embodiments include substitutions within ±1, and various embodiments include substitutions within ±0.5.

[0058] Furthermore, it is understood in the art that substitutions of similar amino acids can be efficiently performed based on hydrophilicity, especially when the biologically functional proteins or peptides thus produced are intended for use in the immunological embodiments disclosed herein. In various embodiments, the maximum local mean hydrophilicity of a protein, controlled by the hydrophilicity of adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with the biological properties of the protein.

[0059] The following hydrophilic 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 similar changes based on hydrophilicity values ​​occur, various embodiments include substitutions between amino acids with hydrophilicity values ​​of ±2 or less, substitutions of ±1 or less, and substitutions of ±0.5 or less.

[0060] Examples of amino acid substitutions are shown in Table 3. TIFF0007850417000005.tif183170

[0061] Those skilled in the art can determine preferred variants of the polypeptides described herein using well-known methods. In various embodiments, those skilled in the art can identify preferred regions of the molecule that can be altered without disrupting activity by targeting regions not considered important for activity. In other embodiments, those skilled in the art can identify residues and portions of the molecule that are conserved among similar polypeptides. In further embodiments, even regions that may be important for biological activity or structure can be subjected to conserved amino acid substitutions without disrupting biological activity or adversely affecting the polypeptide structure.

[0062] Furthermore, those skilled in the art can investigate structural-functional studies to identify residues within similar polypeptides that are important for their activity or structure. Taking such comparisons into account, those skilled in the art can predict the importance of amino acid residues within a polypeptide that correspond to amino acid residues that are important for the activity or structure of similar polypeptides. Those skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0063] Furthermore, those skilled in the art can analyze the three-dimensional structure and amino acid sequence in relation to the structure of a similar polypeptide. Based on such information, they can predict the arrangement of amino acid residues in the polypeptide with respect to its three-dimensional structure. In various embodiments, those skilled in the art can select amino acid residues predicted to be present on the polypeptide surface in a way that avoids fundamental changes, as such residues may be involved in important interactions with other molecules. Furthermore, those skilled in the art can prepare test variants containing a single amino acid substitution for each of the desired amino acid residues. These variants can then be screened using activity quantification methods known to those skilled in the art. Using such variants, information on suitable variants can be gathered. For example, if it is found that a change to a particular amino acid residue results in disruption of activity, an undesirable reduction of activity, or inappropriate activity, variants with such changes can be avoided. In other words, based on information gathered from such conventional experiments, those skilled in the art can easily identify amino acids, alone or in combination with other mutations, that should be avoided for further substitution.

[0064] The terms “polypeptide fragment” and “cleaved polypeptide,” as used herein, refer to polypeptides having deletions at the amino and / or carboxyl terminals compared to the corresponding full-length protein. In various embodiments, the fragments may have amino acid lengths of, for example, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1000 or more. In various embodiments, the fragments may also have amino acid lengths of, for example, 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 450 or less, 400 or less, 350 or less, 300 or less, 250 or less, 200 or less, 150 or less, 100 or less, 50 or less, 25 or less, 10 or less, or 5 or less. The fragment may further contain one or more additional amino acids at one or both of its ends, for example, amino acid sequences derived from different natural proteins (e.g., Fc or leucine zipper domain) or artificial amino acid sequences (e.g., artificial linker sequences).

[0065] The terms “polypeptide variant,” “hybrid polypeptide,” and “polypeptide variant,” as used herein, refer to a polypeptide comprising an amino acid sequence in which, compared to another polypeptide sequence, one or more amino acid residues are inserted into the amino acid sequence, one or more amino acid residues are deleted from the amino acid sequence, and / or one or more amino acid residues are substituted in the amino acid sequence. In various embodiments, the number of inserted, deleted, or substituted amino acid residues 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 acid lengths. The hybrids of this disclosure encompass fusion proteins.

[0066] A "derivative" of a polypeptide is a chemically modified polypeptide, such as one that has been attached to another chemical moiety, such as polyethylene glycol or albumin (e.g., human serum albumin), or that has been phosphorylated or glycosylated.

[0067] In this specification, the term "% sequence identity" is used synonymously with the term "% identity" and refers to the value of amino acid sequence identity between two or more peptide sequences, or the value of nucleotide sequence identity between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same as 80% sequence identity measured by a specified algorithm, meaning that a given sequence has at least 80% identity with respect to another sequence of a different 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 more sequence identity with respect to a given sequence. In various embodiments, the percentage identity is, for example, within 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%.

[0068] In this specification, the term "% sequence homology" is used synonymously with the term "% homology" and refers to the value of amino acid sequence homology between two or more peptide sequences, or the value of nucleotide sequence homology between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% homology means the same as 80% sequence homology measured by a specified algorithm, i.e., homologs of a given sequence have more than 80% sequence homology over a certain 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 more, with respect to a given sequence. In various embodiments, the percentage 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%.

[0069] Examples of computer programs that can be used to verify the identity between two sequences include, but are not limited to, a set of BLAST programs publicly available on the NCBI website, such as BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN. See also Altschul et al., J.Mol.Biol., vol. 215: pp. 403-10, 1990 (particularly in relation to the published initial settings, i.e., parameter w=4, parameter t=17), and Altschul et al., Nucleic Acids Res., vol. 25: pp. 3389-3402, 1997. When evaluating a given amino acid sequence by comparing it with amino acid sequences in GenBank Protein Sequences or other publicly available databases, sequence searches are typically performed using the BLASTP program. The BLASTX program is preferred for searching for nucleic acid sequences translated in all read frames against amino acid sequences in GenBank Protein Sequences and other publicly available databases. Using initial parameters of open gap penalty = 11.0 and gap extension penalty = 1.0, both BLASTP and BLASTX are performed, utilizing the BLOSUM-62 matrix.

[0070] In addition to calculating percent sequence identity, the BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA, Vol. 90: pp. 5873–5787, 1993). One of the similarity measures provided by the BLAST algorithm is the smallest sum probability (P(N)), which indicates the probability that a match occurs by chance between two nucleotide sequences or two amino acid sequences. For example, if the smallest sum probability when comparing a test nucleic acid to a reference nucleic acid is, for example, less than approximately 0.1, less than approximately 0.01, or less than approximately 0.001, the nucleic acid is considered similar to the reference sequence.

[0071] As used herein, the term "modification" refers to any manipulation of the peptide backbone (e.g., amino acid sequence) or post-translational modification of a polypeptide (e.g., glycosylation).

[0072] The term “knob-into-hole modification,” as used herein, refers to a modification within the interface between two immunoglobulin heavy chains in the CH3 domain. In one embodiment, the “knob-into-hole modification” includes, in one antibody heavy chain, the amino acid substitution T366W and optionally the amino acid substitution S354C, and in the other antibody heavy chain, the amino acid substitutions T366S, L368A, Y407V and optionally Y349C. The knob-into-hole technique has been described in U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al., Prot Eng, Vol. 9, pp. 617-621 (1996); and Carter, J Immunol Meth, Vol. 248, pp. 7-15 (2001), among others.

[0073] The term "fusion protein," as used herein, refers to a fusion polypeptide molecule containing two or more genes that originally encoded separate proteins, wherein its components are linked to one another by peptide bonds, either directly or via peptide linkers. The term "fused," as used herein, refers to components being linked by peptide bonds, either directly or via one or more peptide linkers.

[0074] A "linker" is a molecule that connects two other molecules via covalent bonds, ionic bonds, van der Waals bonds, or hydrogen bonds. For example, a nucleic acid molecule that links two non-complementary sequences by hybridizing to the 5' end of one complementary sequence and then to the 3' end of another complementary sequence. A "cleavable linker" refers to a linker that can be cleaved by degradation or other means to separate the two components linked by the linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, and lipases. Cleavable linkers can also be cleaved by environmental factors, such as changes in temperature, pH, and salt concentration.

[0075] The term "peptide linker," as used herein, refers to a peptide containing one or more amino acids, typically 2 to 20 amino acids. Peptide linkers are either known in the art or described herein. Suitable non-immunogenic linker peptides include (G4S) n Peptide linker, (SG4) n Examples include peptide linkers, or G4(SG4)n peptide linkers. "n" is usually a number from 1 to 10, typically from 2 to 4.

[0076] "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 agent effective in obtaining the intended pharmacological effect. "Pharmacologically acceptable carrier" refers to any standard pharmaceutical carrier, solvent, buffer, and pharmaceutical additive, such as phosphate-buffered saline, 5% glucose aqueous solution, emulsions such as oil-in-water or water-in-oil emulsions, and various types of wetting agents and / or auxiliaries. Suitable pharmaceutical carriers and formulations are listed in Remington's Pharmaceutical Sciences, 21st edition, 2005, Mack Publishing Co., Easton. "Pharmacologically acceptable salt" refers to a salt that can be incorporated into a compound for pharmaceutical use, such as metal salts (sodium salts, potassium salts, magnesium salts, calcium salts, etc.), ammonia salts, and organic amine salts.

[0077] As used herein, “treatment” (and its grammatical variations such as “to treat” and “to treat”) refers to a clinical intervention to alter the natural course of a disease in an individual receiving treatment, which may be performed preventively or during the course of clinicopathology. Desired therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, symptom relief, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, improvement or mitigation of the condition, and remission or improved prognosis. As used herein, “mitigating” a disease, disorder, or condition means reducing the severity and / or frequency of the symptoms of the disease, disorder, or condition. Furthermore, as used herein, “treatment” encompasses descriptions of curative, symptomatic, and preventive treatments.

[0078] The terms “effective dose” or “therapeutic effective dose,” as used herein, refer to an amount of a compound or composition sufficient to treat a particular disorder, condition, or disease, such as improving, alleviating, reducing, and / or delaying one or more of its symptoms. With respect to cancer or the growth of other undesirable cells, an effective dose includes an amount sufficient to: (i) reduce the number of cancer cells; (ii) reduce the size of the tumor; (iii) inhibit, delay, slow to some extent, preferably stop, cancer cell infiltration into peripheral organs; (iv) inhibit tumor metastasis (i.e., slow to some extent, preferably stop); (v) inhibit tumor growth; (vi) suppress or delay tumor development and / or recurrence; and / or (vii) reduce to some extent one or more of the symptoms associated with cancer. An effective dose may be administered in one or more doses.

[0079] The expressions “to administer” or “cause to be administered” refer to an act performed by a healthcare professional (e.g., a physician) or a person responsible for the patient’s medical care who manages and / or authorizes the administration of the drug / compound in question to a patient. Causing administration may include making a diagnosis and / or determining an appropriate treatment regimen and / or prescribing a specific drug / compound to a patient. Such prescribing may include, for example, a draft prescription form or annotations in a medical record. Where administration is described herein, “cause to be administered” is also intended.

[0080] The terms “patient,” “individual,” and “subject” may be used synonymously and refer to mammals, preferably humans or non-human primates, but may also refer to domesticated mammals (e.g., dogs or cats), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., horses, cattle, pigs, sheep). In various embodiments, a patient may be a human being (e.g., adult male, adult female, adolescent male, adolescent female, boy, girl) receiving treatment from a physician or other healthcare professional in a hospital, psychiatric medical facility, as an outpatient, or in other clinical settings. In various embodiments, a patient may also be an immunocompromised or immune-impaired patient, such as, but not limited to, patients with primary immunodeficiency, AIDS; cancer patients and transplant patients taking certain immunosuppressants; and patients with genetic diseases affecting the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency). In various embodiments, the patient has immunogenic cancer, including, but is not limited to, bladder cancer, lung cancer, melanoma, and other cancers reported to have a high mutation rate (Lawrence et al., Nature, Vol. 499 (No. 7457): pp. 214-218, 2013).

[0081] The term "immunotherapy" refers to cancer treatment, including, for example, treatment using depletion antibodies against specific tumor antigens; treatment using antibody-drug conjugates; treatment using agonist antibodies, antagonist antibodies, or inhibitory antibodies against costimulatory or coinhibitory molecules (immune checkpoints) such as CTLA-4, PD-1, OX-40, CD137, GITR, LAG3, TIM-3, SIRP, CD40, CD47, Siglec8, Siglec9, Siglec15, TIGIT, and VISTA; and treatment using bispecific T-cell inducing antibodies (BiTE®) such as blinatumomab: IL-2, IL-12, IL-15, IL-21, GM-CSF, IFN-α, IFN Treatments include, but are not limited to, those involving the administration of biological response modifiers such as β-β and IFN-γ; treatments using therapeutic vaccines such as cyproisel T; treatments using Calmette-Guérin bacillus (BCG); treatments using dendritic cell vaccines or tumor antigen peptide vaccines; treatments using chimeric antigen receptor (CAR)-T cells; treatments using CAR-NK cells; treatments using tumor-infiltrating lymphocytes (TILs); treatments using adoptive transplant antitumor T cells (T cells grown in vitro and / or TCR transgenic); treatments using TALL-104 cells; and treatments using immunostimulants such as Toll-like receptor (TLR) agonists CpG and imiquimod.

[0082] "Resistant or refractory cancer" refers to tumor cells or cancer that do not respond to conventional anti-cancer treatments, including chemotherapy, surgery, radiation therapy, stem cell transplantation, and immunotherapy. Tumor cells may be resistant or refractory from the start of treatment, or they may become resistant or refractory during treatment. Refractory tumor cells include tumors that do not respond to the start of treatment, or that show a short initial response but do not respond to treatment. Refractory tumor cells also include tumors that respond to anti-cancer therapy but do not respond to subsequent treatment. For the purposes of this invention, refractory tumor cells also include tumors that appeared to be inhibited by anti-cancer therapy but recur within 5 years, in some cases within 10 years, or longer, after discontinuation of treatment. Anti-cancer treatments may include chemotherapy alone, radiation alone, targeted therapy alone, immunotherapy alone, surgery alone, or a combination of these. For the sake of simplicity and not to limit the scope of explanation, it should be understood that the above-mentioned refractory tumor cells are interchangeable with resistant tumors.

[0083] The terms “Fc domain” or “Fc region,” as used herein, are used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. These terms encompass both native and variant Fc regions. The IgG Fc region includes the IgG CH2 and IgG CH3 domains. The CH3 region may, as used herein, be either the native CH3 domain or a variant CH3 domain (e.g., a CH3 domain in which a “knob” is introduced on one chain and a “hole” corresponding to the other chain; see U.S. Patent No. 5,821,333, as expressly incorporated herein by reference). Such variant CH3 domains may facilitate heterodimerization of two non-identical immunoglobulin heavy chains as described herein. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering scheme.

[0084] As used herein, the term "effector function" refers to the biological activity resulting from the Fc region of an immunoglobulin, which varies depending on the immunoglobulin isotype. Examples of effector functions of immunoglobulins include complement-dependent cell-mediated cytotoxicity (CDC) through binding to C1q, binding to Fc receptors, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), cytokine secretion, antigen uptake via immune complexes by antigen-presenting cells, decreased expression of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0085] As used herein, the terms “regulatory T cells” or “Treg cells” refer to a specific type of CD4+ T cell capable of suppressing 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 forkheadbox P3 (FOXP3) (Sakaguchi, Annu Rev Immunol, Vol. 22, pp. 531-62 (2004)), and play a crucial role in inducing and maintaining peripheral self-tolerance to antigens, including those expressed by tumors.

[0086] The term "conventional CD4+ T cell," as used herein, refers to CD4+ T cells other than regulatory T cells.

[0087] The term “selective activation of Treg cells,” as used herein, means the activation of Treg cells without the activation of other T cell subsets (such as CD4+ helper T cells, CD8+ cytotoxic T cells, or NK T cells) or natural killer (NK) cells. Methods for identifying these cell types are described in the examples. Activation may include induction of IL-2 receptor signaling (measured by detection of phosphorylated STAT5a, for example), induction of proliferation (measured by detection of Ki-67, for example), and / or upregulation of the expression of activation markers (e.g., CD25).

[0088] As used herein, “specific binding” means that binding is selective to the antigen and can be distinguished from undesirable or nonspecific interactions. The ability of an immunoglobulin to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or by other methods well known to those skilled in the art, such as surface plasmon resonance (SPR).

[0089] The terms "affinity" or "binding affinity," as used herein, refer to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Generally, the affinity of molecule X for its partner Y can be expressed by the dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Surface plasmon resonance (SPR) is a specific method for measuring affinity.

[0090] The term "decreased binding affinity," as used herein, refers to a decrease in affinity for each interaction, as measured by methods such as SPR. Conversely, "increased binding affinity" refers to an increase in binding affinity for each interaction.

[0091] The term "polymer," as used herein, typically includes, but is not limited to, homopolymers; copolymers such as block copolymers, graft copolymers, random copolymers, and alternating copolymers; as well as terpolymers; and mixtures and modifications thereof. Furthermore, unless otherwise specified, the term "polymer" encompasses all possible geometric configurations of the substance. These configurations include, but are not limited to, isotactic symmetry, syndiotactic symmetry, and random symmetry.

[0092] A "polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include not only natural nucleic acids such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), but also nucleic acid analogs. Nucleic acid analogs include those containing non-natural bases, which are nucleotides that participate in bonding with nucleotides other than natural phosphodiester bonds, and those containing bases added through bonding other than phosphodiester bonds. In other words, nucleic acid analogs include, but are not limited to, phosphorothioates, phosphorodithioates, phosphorotryesters, phosphoramidates, boranophosphates, methylphosphonic acid, chiral-methylphosphonic acid, 2-O-methylribonucleotides, and peptide-nucleic acid (PNA). Such polynucleotides can be synthesized using automated DNA synthesizers, etc. The term "nucleic acid" typically refers to large polynucleotides. The term "oligonucleotide" typically refers to short polynucleotides, generally about 50 nucleotides or less. It should be understood that when a nucleotide sequence is represented as a DNA sequence (i.e., A, T, G, C), the RNA sequence in which "U" is replaced by "T" (i.e., A, U, G, C) is also included.

[0093] This specification describes polynucleotide sequences using conventional notation. The left end of a single-stranded polynucleotide sequence is the 5' end; the left-handed direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The addition of nucleotides from the 5' direction to the 3' direction to a nascent RNA transcript is referred to as the transcription direction. The DNA strand having the same sequence as mRNA is called the "coding strand"; the sequence on the DNA strand having the same sequence as the mRNA transcribed from DNA and located on the 5' side of the 5' end of the RNA transcript is called the "upstream sequence"; and the sequence on the DNA strand having the same sequence as RNA and located on the 3' side of the 3' end of the coding RNA transcript is called the "downstream sequence".

[0094] "Complementary" refers to topological compatibility, that is, the agreement between the interacting surfaces of two polynucleotides. In other words, these two molecules can be described as complementary, and furthermore, their 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 is hybridizable to the second polynucleotide under stringent hybridization conditions.

[0095] "Specific hybridization" or "selective hybridization" refers to the preferential binding, double-stranding, or hybridization of a nucleic acid molecule to a specific nucleotide sequence under stringent conditions, when that sequence is present in a mixture of (e.g., whole-cell) DNA or RNA. The term "stringent conditions" refers to conditions under which a probe will preferentially hybridize to its target substance but less to other sequences, or not hybridize at all. In relation to nucleic acid hybridization experiments such as Southern and Northern hybridization, "stringent hybridization" and "stringent hybridization washing conditions" are sequence-dependent and will differ under different environmental parameters. Detailed guidelines for 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, New York; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3rd edition, New York; and Ausubel et al. (eds.), Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, New York.

[0096] Typically, highly stringent hybridization and highly stringent washing conditions are selected to be approximately 5°C lower than the melting temperature (Tm) of a particular sequence at specified ionic strength and pH. Tm is the temperature at which 50% of the target sequence hybridizes to a perfect match probe (under specified ionic strength and pH). Very stringent conditions are selected to be the same as the Tm of a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids with more than approximately 100 complementary residues on a filter in Southern or Northern blotting is 50% formalin + 1 mg heparin, 42°C, and overnight hybridization. An example of highly stringent washing conditions is 0.15 M NaCl, 72°C, and approximately 15 minutes. An example of stringent washing conditions is 0.2 × SSC washing, 65°C, and 15 minutes. For a description of SSC buffers, see Sambrook et al. A less stringent wash before a highly stringent wash can remove background probe signals. For example, a moderately stringent wash for double helix of over approximately 100 nucleotides is 1×SSC, 45°C, 15 minutes. A less stringent wash for double helix of over approximately 100 nucleotides is 4–6×SSC, 40°C, 15 minutes. Typically, in a particular hybridization assay, a signal-to-noise ratio (or more) that is twice (or greater) the signal-to-noise ratio observed with an unrelated probe indicates the detection of a specific hybridization.

[0097] A "primer" refers to a polynucleotide capable of specifically hybridizing to a designated polynucleotide template, thereby providing a synthesis initiation site for a complementary polynucleotide. Such synthesis occurs when a polynucleotide primer is placed under conditions that induce synthesis, i.e., in the presence of nucleotides, a complementary polynucleotide template, and a polymerization agent such as DNA polymerase. Primers are typically single-stranded, but may also be double-stranded. Primers are typically deoxyribonucleic acid, but a wide variety of synthetic and natural primers are useful in many applications. Primers are complementary to the template and are designed to hybridize to the template to act as a synthesis initiation site, but they do not need to strictly reflect the template sequence. In such cases, the specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with chromogenic, radioactive, or fluorescent moieties and used as detectable portions.

[0098] When used to refer to a polynucleotide, a "probe" is a polynucleotide capable of specifically hybridizing to a specified sequence of another polynucleotide. While a probe specifically hybridizes to the complementary polynucleotide of a target, it does not need to strictly reflect the complementary sequence of the template. In such cases, the specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. Probes can be labeled with chromogenic, radioactive, or fluorescent moieties, and these can be used as detectable regions. If the probe provides a synthesis starting point for the complementary polynucleotide, it can also be used as a primer.

[0099] A "vector" is a polynucleotide that can be used to introduce another nucleic acid, to which it is ligated, into a cell. One type of vector is a "plasmid," which is a linear or circular double-stranded DNA molecule to which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-deficient retrovirus, replication-deficient adenovirus, and replication-deficient adeno-associated virus), into which additional DNA segments can be introduced. Certain vectors can autonomously replicate within the host cell into which they are introduced (e.g., bacterial vectors containing bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are replicated together with the host genome by being incorporated into the host cell's genome after introduction into the host cell. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide.

[0100] A “regulatory sequence” is a nucleic acid that affects the expression of the nucleic acid to which it is functionally linked (e.g., expression level, timing, or site of expression). Regulatory sequences can exert their effects, for example, directly on the controlled nucleic acid or through the action of one or more other molecules (e.g., the regulatory sequence and / or polypeptides that bind to the nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, California, and Baron et al., 1995, Nucleic Acids Res., Vol. 23, pp. 3605-06. When a regulatory sequence affects the expression of a nucleotide sequence (e.g., expression level, timing, or site of expression), the nucleotide sequence is “functionally linked” to the regulatory sequence.

[0101] A “host cell” is a cell that can be used for the expression of the polynucleotides of this disclosure. The host cell may be a prokaryote, for example, *E. coli*, or it may be a eukaryote, for example, a single-celled eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., human, monkey, hamster, rat, mouse, or insect cell), or a hybridoma. Typically, the host cell is a cultured cell capable of transformation or transfection with polypeptide-coding nucleic acids, enabling the expression of these polypeptide-coding nucleic acids. The term “recombinant host cell” may be used to describe a host cell that has been transformed with or transfected with the nucleic acid of interest. The host cell may also be a cell that contains the nucleic acid but does not express it at the desired level, unless a regulatory sequence has been introduced into the host cell to functionally link with the nucleic acid. The term “host cell” is understood to refer not only to a specific target cell but also to the offspring and potential offspring of such a cell. Because certain modifications may occur in later generations due to mutations or environmental influences, such offspring may not actually be identical to the parent cells; however, even in such cases, they are still included within the scope of the terms used herein.

[0102] The term "isolated molecule" (where the molecule is a polypeptide or polynucleotide, etc.) is defined, based on its origin or source of origin, as a molecule that (1) is not accompanied by its naturally occurring confoundings, (2) substantially does not contain other molecules from the same species, (3) is expressed by cells of a different species, or (4) does not occur naturally. In other words, a chemically synthesized molecule, or a molecule expressed in a cell line different from the cells in which it naturally occurs, is "isolated" from its naturally occurring confoundings. A molecule may be made substantially free of naturally occurring confoundings by isolation using purification methods known in the art. The purity or homogeneity of a molecule can be evaluated by several means known in the art. For example, the purity of a polypeptide sample can be evaluated by visualizing the polypeptide by polyacrylamide gel electrophoresis and gel staining using methods known in the art. For a particular purpose, higher resolution may be obtained by using HPLC or other purification methods known in the art.

[0103] A protein or polypeptide is considered "substantially pure," "substantially homogeneous," or "substantially purified" if at least about 60% to 75% of the sample exhibits a single polypeptide species. This polypeptide or protein may be monomeric or polymeric. Typically, substantially pure polypeptides or proteins constitute about 50%, 60%, 70%, 80%, or 90% (W / W), usually about 95%, of a protein sample, and it is preferable to be over 99% pure. The purity or homogeneity of a protein can be demonstrated by several means known in the art, such as visualizing single polypeptide bands by gel staining using staining methods well known in the art after subjecting the protein sample to polyacrylamide gel electrophoresis. For certain purposes, higher resolution may be obtained by using HPLC or other purification methods well known in the art.

[0104] As used herein, the term "label" or "labeling" refers to incorporating another molecule into an antibody. In one embodiment, the label is a detectable marker, for example, incorporation of a radioactively labeled amino acid, or addition of a biotinyl moiety to a polypeptide that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzyme activity detectable by optical or calorimetric methods). In another embodiment, the label or marker can be a therapeutic label or marker, for example, a drug conjugate or a toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to: 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 phosphors, rhodamine phosphors, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites to secondary antibodies, metal-binding domains, epitope labeling), magnetic drugs (e.g., gadolinium chelates), toxins (e.g., pertussis toxin, taxol), Cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues or homologues. In various embodiments, the labeling is added by spacer arms of varying lengths to suppress any steric hindrance.

[0105] As used herein, the term "heterogeneous" refers to a composition or state that is not natural or does not exist in nature, for example, a composition or state that can be achieved by replacing an existing natural composition or state with a composition or state from another source. Similarly, the expression of a protein in an organism other than the organism in which it is naturally expressed constitutes a heterogeneous expression system and a heterogeneous protein.

[0106] The aspects and embodiments of the Disclosure described herein are understood to encompass "consisting of" and / or "essentially consisting of" the aspects and embodiments.

[0107] In this specification, any mention of a value or parameter "about" includes (and describes) the variation of that value or parameter itself. For example, a mention of "about X" includes the mention of "X".

[0108] Where used herein and in the appended claims, singular nouns such as “a,” “or,” and “the” are plural unless otherwise explicitly indicated by the context. The aspects and variations of the disclosure described herein are understood to include “consisting of” and / or “essentially consisting of” the aspects and variations.

[0109] IL-2 Interleukin-2 (IL-2) is a classic Th1 cytokine produced by post-activation T cells via the T cell antigen receptor and the co-stimulating molecule CD28. IL-2 regulation occurs through activation of signaling pathways and transcription factors that act on the IL-2 promoter to revitalize gene transcription, but also involves regulation of IL-2 mRNA stability. IL-2 binds to a highly regulated multi-chain receptor containing α, β, and γ chains, mediating signaling via the Jak-STAT pathway. IL-2 delivers activation, proliferation, and differentiation signals to T cells, B cells, and NK cells. IL-2 also plays a crucial role in mediating activation-induced cell death of T cells, a function that provides essential mechanisms for terminating the immune response. Aldesleukin, a commercially available non-glycosylated human recombinant IL-2 product (available from Prometheus Laboratories Inc. in San Diego, California, under the trademark PROLEUKIN® as des-alanil-1, serine-125 human interleukin-2), is approved for administration to patients with metastatic renal cell carcinoma and metastatic melanoma. IL-2 has also been suggested for use in patients with hepatitis C virus (HCV), human immunodeficiency virus (HIV), acute myeloid leukemia, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, juvenile rheumatoid arthritis, atopic dermatitis, breast cancer, and bladder cancer. Unfortunately, its short half-life and high toxicity limit the optimal dose of IL-2.

[0110] As used herein, the terms “native IL-2” and “native interleukin-2” refer to any natural mammalian interleukin-2 amino acid sequence, encompassing immature or precursor forms and mature forms, within the context of proteins or polypeptides. Non-limiting examples of GenBank accession numbers for amino acid sequences of various native mammalian interleukin-2 species include NP_032392.1 (mouse (Mus musculus), immature form), NP_001040595.1 (rhesus monkey (Macaca mulatta), immature form), NP_000577.2 (human, precursor form), CAA01199.1 (human, immature form), AAD48509.1 (human, immature form), and AAB20900.1 (human). In various embodiments of the present invention, native IL-2 is an immature or precursor form of natural mammalian IL-2. In other embodiments, native IL-2 is the mature form of natural mammalian IL-2. In various embodiments, native IL-2 is the precursor form of natural human IL-2. In various embodiments, native IL-2 is the mature form of natural human IL-2. In various embodiments, the IL-2-based domain D2 is derived from the amino acid sequence of the human IL-2 precursor sequence described in Sequence ID No. 1 below: MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(Sequence ID 1)

[0111] In various embodiments, the IL-2-based domain D2 includes the amino acid sequence of the mature wild-type human IL-2 sequence described in Sequence ID No. 3 below, which includes a cysteine-to-serine substitution at position 125, but does not alter the binding affinity to the IL-2 receptor compared to natural IL-2: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 3)

[0112] IL-2 variant This invention relates to polypeptides that share the primary sequence with human IL-2, except for a few mutated amino acids (including amino acid substitutions, deletions, and insertions). One panel of IL-2 variants includes mutations that preferentially promote the proliferation, survival, activation, and / or function of immunosuppressive regulatory T cells (T CD4+CD25+FoxP3+) more than effector T cells or NK cells. It also encompasses therapeutic applications of such IL-2 selective agonists, used alone, in combination with disease-targeting proteins or peptides, or as components of bifunctional molecules, for the treatment of autoimmune diseases and various inflammatory diseases. Another panel of IL-2 variants includes mutations that substantially reduce the ability of these polypeptides to stimulate Treg cells, making them more effective in treating tumors. It also encompasses therapeutic applications of these mutated variants, used alone, in combination with vaccines, TAA-targeted biologics, or immune checkpoint blockers, or as components of bifunctional molecular constructs, for the treatment of diseases such as cancer and infections where regulatory T cell (Treg) activity is undesirable. In another embodiment, the present invention relates to pharmaceutical compositions comprising the polypeptides of the present disclosure. Finally, the present invention relates to therapeutic applications of the polypeptides and pharmaceutical compositions of the present disclosure by selectively modulating the immune system against autoimmune and inflammatory diseases or diseases such as cancer and various infectious diseases.

[0113] The present invention relates to polypeptides with an apparent molecular weight of at least 15 kD, a length of 100 to 500 amino acids, and preferably a size of 140 residues. These polypeptides maintain a high sequence identity of over 90% with native IL-2. At these positions, these polypeptides undergo mutations that introduce amino acid residues different from those at the same positions in native IL-2.

[0114] The polypeptides of the present invention may also be referred to by other names, such as immunomodulatory polypeptides, IL-2 analogs, or IL-2 variants. These polypeptides are designed based on the three-dimensional structure of the IL-2 receptor complex (available in the PDB public database), and primarily involve mutations introduced at IL-2 positions corresponding to amino acids that interact with receptor subunits β, γ, or βγ.

[0115] In various embodiments, the IL-2 variant (or mutant) contains a sequence derived from the sequence of the mature human IL-2 polypeptide described in SEQ ID NO: 3. In various embodiments, the IL-2 variant contains an amino acid sequence different from that of the native (or wild-type) IL-2 protein. In various embodiments, the IL-2 variant binds to the IL-2Rα polypeptide and functions as an IL-2 agonist or antagonist. In various embodiments, an IL-2 variant having agonist activity has superagonist activity. In various embodiments, the IL-2 variant can function as an IL-2 agonist or antagonist independently of its binding to IL-2Rα. IL-2 agonists are exemplified by equivalent or increased biological activity compared to wild-type IL-2. IL-2 antagonists are exemplified by decreased biological activity compared to wild-type IL-2, or by their ability to inhibit IL-2-mediated reactions. In various embodiments, the sequence of the IL-2 variant has at least one amino acid change, e.g., substitution or deletion, compared to the native IL-2 sequence, and such change results in either IL-2 agonist activity or IL-2 antagonist activity. In various embodiments, the IL-2 variant as an Fc fusion protein has the amino acid sequences described in SEQ ID NOs: 4-43, 113-151, 208-212, and 275-292, and includes IL-2Rβ and / or γ C The binding affinity to IL-2Rα is reduced, and the selectivity for the activation and proliferation of regulatory T cells (Tregs) is enhanced. In various embodiments, the IL-2 variant as an Fc fusion protein has the amino acid sequences described in SEQ ID NOs. 220-232 and 293-299, which reduce / eliminate binding to IL-2Rα and selectively activate and proliferate effector T cells (Teffs).

[0116] In various embodiments, IL-2RαSushi having the amino acid sequence described in SEQ ID NO: 68 was ligated between IL-2 and the Fc domain using linkers of varying lengths and compositions. The Fc domain can be located at the N-terminus or C-terminus. The IL-2-IL-2RαSushi-Fc fusion protein has the amino acid sequence described in SEQ ID NOs: 69-70 and is expected to have reduced binding to IL-2Rα, selectively activating and proliferating effector T cells.

[0117] In various embodiments, IL-2 and IL-2RαSushi form a non-covalent complex. IL-2 is fused to either the N-terminus or C-terminus of the Hole-Fc chain (SEQ ID NO: 47), and IL-2RαSushi is fused to either the N-terminus or C-terminus of the Knob-Fc chain (SEQ ID NO: 46). The non-covalent C-terminal IL-2-IL-2RαSushi-Fc fusion protein has the amino acid sequence described in SEQ ID NOs: 196-197.

[0118] Exemplary IL-2 variants are shown in Tables 4A-4H. TIFF0007850417000006.tif161170TIFF0007850417000007.tif88170TIFF000 7850417000008.tif171170TIFF0007850417000009.tif65170TIFF00078504170 00010.tif113170TIFF0007850417000011.tif230170TIFF0007850417000012. tif169170TIFF0007850417000013.tif67170TIFF0007850417000014.tif76170

[0119] The present invention also encompasses further modifications to the above-described classes of IL-2 variants, particularly those described in Tables 4A-4F and 4H. As will be understood by those skilled in the art, further combinational variants combining the preferred mutations described in Tables 4A-4E and 4H may result in more Treg cell-selective IL-2 agonists. Further combinational variants combining the preferred mutations described in Table 4F may result in more Teff cell-selective IL-2 agonists. Any further combinational variants, whether increasing their affinity for specific components of the IL-2 receptor, fine-tuning their activity to desired potency, signaling intensity, and specificity, or improving their in vivo pharmacodynamic properties (extending their half-life or reducing their internalization by T cells), are included in the spirit and scope of the present invention. These additional mutations may be obtained by rational design using bioinformatics tools or by using combinatorial molecular libraries of varying properties (phage libraries, yeast or bacterial gene expression libraries). In another embodiment, the present invention relates to a fusion protein comprising one of the above-mentioned immunomodulatory polypeptides conjugated to a carrier protein. The carrier protein may be the Fc region of albumin or human immunoglobulin. In another embodiment, the present invention relates to a fusion protein conjugated to a targeting / dual-function site. The targeting / dual-function site may be an antibody, antibody fragment, protein, or peptide.

[0120] FC Domain IgG class immunoglobulins are the most abundant proteins in human blood. Their circulating half-life can reach as long as 21 days. Fusion proteins have been reported in which the Fc region of IgG is combined with the domains of other proteins, such as various cytokines and receptors (see, e.g., Capon et al., Nature, Vol. 337: pp. 525-531, 1989; Chamow et al., Trends Biotechnol., Vol. 14: pp. 52-60, 1996); U.S. Patents No. 5,116,964 and 5,541,087). The prototype of a fusion protein is a homodimeric protein, which resembles an IgG molecule but lacks the variable region of the heavy chain and the CH1 domain and light chain, linked via a cysteine ​​residue in the hinge region of the Fc of IgG. The dimeric properties of fusion proteins containing the Fc domain can be advantageous in achieving higher-order interactions (i.e., bivalent or bispecific binding) with other molecules. Due to structural homology, Fc fusion proteins exhibit a comparable in vivo pharmacokinetic profile to human IgG with a similar isotype.

[0121] The term "Fc" refers to a molecule or sequence containing a sequence of non-antigen-binding fragments of a full-length antibody, which may be in monomeric or polymeric form. The original immunoglobulin source for native Fc is preferably human, and may be any immunoglobulin, but IgG1 and IgG2 are preferred. Native Fc consists of monomeric polypeptides that can be linked by covalent (i.e., disulfide) and non-covalent bonds to form dimeric or polymeric forms. The number of intermolecular disulfide bonds between monomeric subunits of a native Fc molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of native Fc is a disulfide-bonded dimer resulting from the papain degradation of IgG (Ellison et al., (1982), Nucleic Acids Res., Vol. 10: pp. 4071-9). The term "native Fc," as used herein, refers to the monomeric, dimeric, and polymeric forms of Fc. It includes Fc domains containing binding sites for protein A, protein G, various Fc receptors, and complement proteins.

[0122] In various embodiments, the term “Fc variant” refers to a molecule or sequence that has been modified from native Fc but still contains a binding site to the salvage receptor FcRn. International Publication No. 97 / 34631 (published September 25, 1997) and International Publication No. 96 / 32478 describe exemplary Fc variants and their interactions with salvage receptors, which are incorporated herein by reference. Furthermore, native Fc may include sites that confer structural features or biological activity not required for the fusion molecule of the present invention and may therefore be removed. In other words, in various embodiments, the term “Fc variant” includes molecules or sequences lacking one or more native Fc sites or residues that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with selected host cells, (3) heterogeneity of the N-terminus after expression in selected host cells, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cell-mediated cytotoxicity (ADCC).

[0123] The term “Fc domain” encompasses the molecules and sequences of native Fc and Fc variants as defined above. Similar to Fc variants and native Fc, the term “Fc domain” encompasses molecules in monomeric or multimeric form, digested from full-length antibodies or produced by recombinant gene expression or other means. In various embodiments, “Fc domain” refers to a dimer consisting of two Fc domain monomers (SEQ ID NO: 44), typically containing all or part of the hinge region. In various embodiments, the Fc domain may be mutated to lack effector function. In various embodiments, each Fc domain monomer of the Fc domain contains amino acid substitutions in the CH2 antibody constant domain to reduce interaction or binding between the Fc domain and the Fcγ receptor. In various embodiments, each subunit of the Fc domain contains three amino acid substitutions (L234A, L235A, and G237A) to reduce binding to the activating Fc receptor and / or effector function (SEQ ID NO: 45).

[0124] In various embodiments, the two Fc domain monomers of the Fc domain each contain amino acid substitutions that promote heterodimerization of these two monomers. In various other embodiments, heterodimerization of the Fc domain monomers can be promoted by introducing different but compatible substitutions (such as a "knob-into-hole" residue pair) into the two Fc domain monomers. This "knob-into-hole" technique is also disclosed in U.S. Patent No. 8,216,805. In yet another embodiment, one Fc domain monomer contains the knob-type mutation T366W, and the other Fc domain monomer contains the hole-type mutations T366S, L358A, and Y407V. In various embodiments, two Cy residues that form a stabilizing disulfide bridge (S354C on one strand and Y349C on the matching strand) have been introduced (SEQ ID NOs. 46 and 47). By using heterodimer Fc, monovalent IL-2 variants can be obtained.

[0125] In various embodiments, the Fc domain sequence used to produce the IL-2 variant is the human IgG1-Fc domain sequence described in Sequence ID No. 45 below: DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence No. 45) Sequence ID No. 45 contains amino acid substitutions (underlined) that break the FcγR bond and the C1q bond.

[0126] In various embodiments, the heterodimeric Fc domain sequence used to prepare the IL-2 variant is the Knob-Fc domain sequence described in Sequence ID No. 46 below: DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVCTLPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence No. 46) Sequence ID No. 46 contains amino acid substitutions (underlined) that break the FcγR bond and the C1q bond.

[0127] In various embodiments, the heterodimeric Fc domain sequence used to prepare the IL-2 variant is the Hole-Fc domain sequence described in Sequence ID No. 47 below: DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 47) Sequence ID No. 47 contains amino acid substitutions (underlined and bolded) that break the FcγR bond or the C1q bond.

[0128] In various embodiments, the Fc domain sequence used to create the IL-2 variant is the human IgG1-Fc domain sequence described in Sequence ID No. 251 below, which has an extended half-life and reduced / absent effector function: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 251) Sequence ID No. 251 contains amino acid substitutions that break FcγR and C1q binding (underlined) and substitutions that extend the blood half-life of the fusion protein (bold).

[0129] In various embodiments, the Fc domain sequence used to produce IL-2 variants is an IgG1-Fc domain having the amino acid sequence described in Sequence ID No. 252 below, in which effector function is reduced / absent and half-life is extended: DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPG (Sequence ID 252) Sequence ID No. 252 contains amino acid substitutions that break FcγR and C1q binding (underlined) and substitutions that extend the blood half-life of the fusion protein (bold).

[0130] Linker In various embodiments, heterologous proteins are attached to IL-2 variants by linker peptides and / or hinge linker peptides. The linker or hinge linker may be an artificial sequence consisting of 5, 10, 15, 20, 30, 40 (or any number in between) or more amino acids, which have relatively few secondary structures or exhibit an α-helix higher-order structure.

[0131] Peptide linkers provide covalent bonds and additional structural and / or spatial mobility between protein domains. As is well known in the art, peptide linkers include mobile amino acid residues such as glycine and serine. In various embodiments, peptide linkers may contain 1 to 100 amino acids. In various embodiments, the spacer may include the motif GGGSGGGS (SEQ ID NO: 55). In other embodiments, the linker may include the (GGGGS)(SEQ ID NO: 58)n motif, where n is an integer from 1 to 10. In other embodiments, the linker may also include amino acids other than glycine and serine. In yet another embodiment, the linker may include other protein motifs, such as, but are not limited to, sequences of α-helix higher-order structures, such as AEAAAKEAAAKEAAAKA (SEQ ID NO: 53). In various embodiments, the length and composition of the linker may be adjusted to optimize activity or development suitability, including, but not limited to, expression level and aggregation tendency. In another embodiment, the peptide linker may be a simple chemical bond, such as an amide bond (e.g., a chemical bond of PEG).

[0132] Exemplary peptide linkers are shown in Table 5. TIFF0007850417000015.tif124170

[0133] Polynucleotides In another embodiment, the Disclosure provides isolated nucleic acid molecules comprising polynucleotides encoding IL-2, IL-2 variants, IL-2 fusion proteins, or IL-2 variant fusion proteins of the Disclosure. The nucleic acids of the subject may be single-stranded or double-stranded. Such nucleic acids may be DNA molecules or RNA molecules. Examples of DNA include cDNA, genomic DNA, synthetic DNA, PCR-amplified DNA, and combinations thereof. Genomic DNA encoding IL-2 polypeptides can be obtained from genome libraries corresponding to many species. Synthetic DNA can be obtained by chemically synthesizing duplicate oligonucleotide fragments, assembling these fragments, and reconstructing some or all of the coding region and adjacent sequences. RNA can be obtained from RNA polymerase and prokaryotic expression vectors that direct high levels of mRNA synthesis, such as vectors using a T7 promoter. cDNA can be obtained from libraries prepared from mRNA isolated from various tissues expressing IL-2. The DNA molecules of the Disclosure include not only full-length genes but also polynucleotides and their fragments. Full-length genes may also include sequences encoding N-terminal signal sequences. Such nucleic acids can be used in methods for creating novel IL-2 variants, among other applications.

[0134] In various embodiments, the isolated nucleic acid molecule comprises a polynucleotide described herein, and further comprises a polynucleotide encoding at least one heterologous protein described herein. In various embodiments, the nucleic acid molecule further comprises a polynucleotide encoding a linker or hinge linker described herein.

[0135] In various embodiments, the recombinant nucleic acids of this disclosure may be functionally ligated to one or more regulatory nucleotide sequences within an expression construct. The regulatory sequences are those approved in the art and selected to direct the expression of an IL-2 variant. That is, the term regulatory sequence encompasses promoters, enhancers, and other expression regulatory elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, California (1990). Typically, 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 termination sequences, translation start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art are contemplated in this disclosure. The promoter may be a natural promoter or a hybrid promoter combining two or more promoter elements. The expression construct may be intracellular or episomal, such as a plasmid, or it may be inserted into a chromosome. In various embodiments, the expression vector includes a selection marker gene to enable the selection of transformed host cells. The selection marker gene is well known in the art and varies depending on the host cell used.

[0136] In another aspect of this disclosure, the nucleic acid of the subject is provided in an expression vector containing a nucleotide sequence functionally ligated to at least one regulatory sequence encoding an IL-2 variant. The term “expression vector” refers to a plasmid, phage, virus, or vector for expressing a polypeptide from a polynucleotide sequence. Vectors suitable for expression in host cells are readily available, and insertion of nucleic acid molecules into the vector is performed using standard recombinant DNA techniques. Such vectors may contain a variety of regulatory sequences that can be used in these vectors to express a DNA sequence encoding an IL-2 variant, which, when functionally ligated, modulates the expression of the DNA sequence. Such useful regulatory sequences include, for example, the early and late promoters of SV40, the tet promoter, the pre-early promoter of adenovirus or cytomegalovirus, the RSV promoter, the lac system, the trp system, the TAC system or TRC system, the T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of λ phage, regulatory regions for fd coat proteins, promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, promoters for acid phosphatases (e.g., PhoS), promoters for yeast α-conjugation factors, polyhedron promoters of baculovirus systems, and other sequences known to regulate the expression of genes in prokaryotic or eukaryotic cells or their viruses, as well as various combinations thereof. It will be understood that the design of expression vectors may differ depending on factors such as the selection of the host cell to be transformed and / or the type of protein to be expressed. Furthermore, the copy number of the vector, its ability to regulate copy number, and the expression of any other proteins encoded by the vector (such as antibiotic markers) should also be considered.Examples of suitable expression vectors for vIL-2 include pDSRa (described in International Publication No. 90 / 14363, incorporated herein by reference) and its derivatives, which contain vIL-2 polynucleotide and any additional suitable vectors known in the art or described below.

[0137] The recombinant nucleic acid of this disclosure can be produced by ligating the cloned gene or a portion thereof into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast cells, avian cells, insect cells, or mammalian cells), or both. The expression solvent for producing the recombinant IL-2 polypeptide includes plasmids and other vectors. Suitable vectors include, for example, the following plasmids for expression in prokaryotic cells such as E. coli: pBR322 plasmid, pEMBL plasmid, pEX plasmid, pBTac plasmid, and pUC plasmid.

[0138] Some mammalian expression vectors contain both prokaryotic sequences that promote vector growth in bacteria and one or more eukaryotic transcription units expressed in eukaryotic cells. Examples of mammalian expression vectors suitable for eukaryotic cell transfection include pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg. Some of these vectors have been modified with sequences derived from bacterial plasmids, such as pBR322, to promote replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, for transient protein expression in eukaryotic cells, derivatives of viruses such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, derived from pREP, and p205) can be used. Examples of other viral (including retrovirus) expression systems can be found in the description of gene therapy delivery systems below. Various methods used in plasmid preparation and host organism transformation are well known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, as well as general recombination methods, see Chapters 16 and 17 of Molecular Cloning: A Laboratory Manual, 2nd edition, Sambrook, Fritsch, and Maniatis (eds.) (Cold Spring Harbor Laboratory Press, 1989). In some cases, it may be desirable to express recombinant polypeptides using baculovirus expression systems. 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 B-gal-containing pBlueBacIII).

[0139] In various embodiments, vectors have been designed to produce the subject IL-2 variant in CHO cells, such as the Pcmv-Script vector (Stratagene, La Jolla, California), the pcDNA4 vector (Invitrogen, Carlsbad, California), and the pCI-neo vector (Promega, Madison, Wisconsin). As will be described later, the subject gene construct can be used to induce expression of the subject IL-2 variant in cells grown in culture medium, thereby producing, for example, a protein (including a fusion protein or variant protein) which can then be purified.

[0140] This disclosure also relates to host cells transfected with recombinant genes comprising nucleotide sequences encoding amino acid sequences of one or more subject IL-2 variants. The host cells may be any prokaryotic or eukaryotic cells. For example, the IL-2 variants of this disclosure may 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.

[0141] In response to this, the disclosure further relates to a method for producing the subject IL-2 variant. For example, the IL-2 variant can be expressed by culturing host cells transfected with an expression vector encoding the IL-2 variant under appropriate conditions. After secretion, the IL-2 variant can be isolated from a mixture of cells and culture medium containing the IL-2 variant. Alternatively, the IL-2 variant may be retained in the cytoplasm or membrane fraction, the cells may be recovered and lysed, and the protein isolated. The cell culture medium includes host cells, culture medium, and other by-products. Suitable culture media for cell culture are well known in the art.

[0142] The polypeptides and proteins of this disclosure can be purified according to protein purification methods well known to those skilled in the art. These methods involve crude fractionation of proteinaceous and nonproteinaceous fractions at a certain level. After separating the peptide polypeptide from other proteins, the peptide or polypeptide of interest can be further purified by chromatography and electrophoresis to achieve partial or complete purification (i.e., purification to homogeneity). The terms “isolated polypeptide” or “purified polypeptide,” as used herein, are intended to refer to a composition that can be isolated from other components, in which the polypeptide has been purified to any degree relative to its naturally occurring state. A purified polypeptide also refers to a polypeptide that has been isolated from the environment in which it may occur naturally. Generally, “purified” refers to a polypeptide composition that has been fractionated and various other components removed, substantially retaining its expressed biological activity. When the term "substantially purified" is used, this designation refers to a peptide composition or polypeptide composition in which polypeptides or peptides form the main components of the composition, for example, constituting about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, or about 90% or more of the proteins in the composition.

[0143] Various methods suitable for purification are well known to those skilled in the art. These methods include, for example, precipitation using ammonium sulfate, PEG, antibodies (immunoprecipitation), or precipitation by thermal denaturation followed by centrifugation; chromatography such as affinity chromatography (protein A column), ion exchange chromatography, gel filtration chromatography, reversed-phase chromatography, hydroxyapatite chromatography, and hydrophobic interaction chromatography; isoelectric focusing; gel electrophoresis; and combinations of these methods. As is well known in the art, it is considered that even if the order in which various purification steps are performed is changed, or even if certain steps are omitted, a method suitable for preparing substantially purified polypeptides can still be obtained.

[0144] Pharmaceutical composition In another embodiment, the Disclosure provides a pharmaceutical composition comprising an IL-2 variant or an IL-2 variant fusion protein mixed with a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are well known and understood by those skilled in the art and have been extensively described (see, for example, Remington's Pharmaceutical Sciences, 18th edition, ARGennaro (ed.), Mack Press, 1990). The pharmaceutically acceptable carrier may be included, for example, for the purpose of altering, maintaining, or preserving the composition's pH, molar osmotic concentration, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate, release rate, adsorption, or permeability. Such a pharmaceutical composition may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the polypeptide.Suitable pharmaceutically acceptable carriers include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffering agents (such as borates, bicarbonates, tris-HCl, citrates, phosphates, and other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants; flavorings and diluting agents. Agent; emulsifier; hydrophilic polymer (e.g., polyvinylpyrrolidone); low molecular weight polypeptide; salt-forming counterion (e.g., sodium); preservative (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvent (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohol (e.g., mannitol or sorbitol); suspending agent; surfactant or wetting agent (e.g., Pluronic acid, PEG, sorbitan ester, polysorbate, e.g., polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapal); stabilizer (e.g., sucrose or sorbitol); isotonic enhancing agent (e.g., alkali metal halides (preferably sodium chloride or potassium chloride), mannitol, sorbitol); delivery carrier (delivery Examples include, but are not limited to, vehicles; diluents; excipients; and / or pharmaceutical adjuvant.

[0145] The primary solvent or carrier in the pharmaceutical composition may be essentially aqueous or non-aqueous. For example, a suitable solvent or carrier may be distilled water for injection, physiological saline, or artificial cerebrospinal fluid, and other substances commonly found in parenteral compositions may be added. Further exemplary solvents include neutral buffered saline or saline mixed with serum albumin. Other exemplary pharmaceutical compositions include Tris buffer at approximately pH 7.0–8.5, or acetate buffer solution at approximately pH 4.0–5.5, the buffer of which may further contain sorbitol or a suitable substitute. In one embodiment of this disclosure, a selected composition having a desired purity may be mixed with an optional formulation agent (Remington's Pharmaceutical Sciences, above) to prepare the composition for storage in the form of a lyophilized cake or aqueous solution. Furthermore, the therapeutic composition may be formulated as a lyophilized product with appropriate pharmaceutical additives such as sucrose. The optimal pharmaceutical composition is determined by those skilled in the art based on the intended route of administration, mode of delivery, and target dose, etc.

[0146] When parenteral administration is intended, the therapeutic pharmaceutical composition may be in the form of a pyrogenically free aqueous solution acceptable for parenteral administration, containing the desired IL-2 polypeptide or IL-2 polypeptide fusion protein in a pharmaceutically acceptable solvent. A particularly preferred solvent for parenteral injection is sterile distilled water, in which the polypeptide is formulated as a properly preserved sterile isotonic solution. In various embodiments, a pharmaceutical formulation suitable for injection may be formulated in an aqueous solution, but it is preferable to be formulated in a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or physiological buffered saline. The aqueous injection suspension may contain a substance that increases the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Furthermore, the suspension of the active compound may be prepared as a suitable oily injection suspension. Optionally, the suspension may contain a suitable stabilizer, i.e., an agent that increases the solubility of the compound, thereby enabling the preparation of a highly concentrated solution.

[0147] In various embodiments, therapeutic pharmaceutical compositions can be formulated for targeted delivery using colloidal dispersion systems. Examples of colloidal dispersion systems include polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Examples of lipids useful for liposome formation include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine, as well as sphingolipids, cerebrosides, and gangliosides. Exemplary phospholipids include egg yolk phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. Liposome targeting is also possible, for example, based on organ specificity, cell specificity, and organelle specificity, and is known in the art.

[0148] In various embodiments, the pharmaceutical composition is intended to be administered orally. The pharmaceutical composition administered in this manner may be formulated with or without carriers, which are commonly used in the preparation of solid dosage forms such as tablets and capsules. In solid dosage forms for oral administration (capsules, tablets, pills, sugars, powders, granules, etc.), one or more therapeutic compounds of the present disclosure may be mixed with one or more pharmaceutically acceptable carriers, for example, sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or bulking agents such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants such as glycerol; (4) disintegrants such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents such as paraffin. (6) absorption enhancers such as quaternary ammonium compounds; (7) wetting agents such as cetyl alcohol and glycerol monostearate; (8) adsorbents such as kaolin clay and bentonite clay; (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 contain buffering agents. Similar types of solid compositions may be used as fillers in soft gelatin capsules and hard gelatin capsules, using pharmaceutical additives such as lactose and high molecular weight polyethylene glycol. Examples of liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, aqueous solutions, suspensions, syrups, and elixirs.In addition to the active ingredient, the liquid dosage form may contain inert excipients commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol sorbitan fatty acid esters, and mixtures thereof. In addition to inert excipients, the oral composition may contain auxiliary agents such as wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, colorants, fragrances, and preservatives.

[0149] In various embodiments, topical administration of the pharmaceutical composition to the skin or mucous membranes is intended. Topical formulations may further contain one or more of various agents known to be effective as skin penetration enhancers or stratum corneum penetration enhancers. Examples of these include 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl alcohol, isopropyl alcohol, dimethyl sulfoxide, and azone. Additional agents may be further included to make the formulation cosmetically acceptable. Examples of these include fats, waxes, oils, pigments, fragrances, preservatives, stabilizers, and surfactants. Keratinophages, such as those known in the art, may also be included. Examples include salicylic acid and sulfur. Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and, if necessary, with any preservatives, buffers, or propellants. In addition to the compounds of the subject matter of this disclosure (e.g., IL-2 variants), ointments, pastes, creams, and gels may contain pharmaceutical additives such as animal fats, vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0150] Further pharmaceutical compositions intended for use herein include formulations comprising polypeptides in sustained delivery or controlled delivery formulations. Methods for formulating various other sustained delivery or controlled delivery means, such as liposome carriers, biodegradable microparticles or porous beads, and depot formulations, are also known to those skilled in the art.

[0151] The effective dose of a pharmaceutical composition used for therapeutic purposes will depend, for example, on the nature and purpose of the treatment. Therefore, those skilled in the art will understand that the appropriate dose level for treatment will vary, in part, depending on the molecules being delivered, the indications for which the polypeptide is used, the route of administration, and the patient's body size (weight, body surface or organ size) and condition (age and general health). For this reason, clinicians can adjust the dose setting and the route of administration to obtain the optimal therapeutic effect. Typical doses can range from about 0.001 mg / kg to about 100 mg / kg or higher, depending on the factors mentioned above. Preferably, the polypeptide composition may be injected or administered intravenously. A sustained-release pharmaceutical composition may be administered every 3-4 days, weekly, or bi-weekly, depending on the half-life and clearance rate of the particular formulation. The frequency of administration will depend on the pharmacokinetic parameters of the polypeptide in the formulation used. Typically, the composition is administered until a dose is reached that achieves the desired effect. Therefore, the composition may be administered as a single dose, as multiple doses over a long period (at the same or different concentrations per dose), or as a continuous intravenous infusion. Further improvements to the appropriate dosage will be made periodically. The appropriate dosage may be confirmed using appropriate dose-response data.

[0152] The route of administration of the pharmaceutical composition is, in accordance with known methods, for example, by injection via oral, intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intramedullary, intrathecal, intraventricular, percutaneous, subcutaneous, or intraperitoneal or intratumoral route. Alternatively, it may be administered by means of intranasal, enteral, topical, sublingual, urethral, ​​vaginal, or rectal means, a sustained-release system, or an implantable device. If desired, the composition may be administered by bolus, continuous infusion, or by an implantable device. Alternatively or additionally, the composition may be administered topically by implantation of a membrane, sponge, or other suitable material on which the target molecule is adsorbed or encapsulated. When an implantable device is used, the device can be implanted in any suitable tissue or organ, and the delivery of the target molecule may be via diffusion, sustained-release bolus, or continuous administration.

[0153] therapeutic use This disclosure provides a method for treating an autoimmune disease in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of the IL-2 variant or IL-2 variant fusion protein of this disclosure in a pharmaceutically acceptable carrier. An autoimmune disease according to the present invention is a disease or disorder originating from or against the tissues of an individual, or a co-segregation or manifestation thereof, or a condition arising therefrom. In various embodiments, autoimmune diseases include arthritis (including rheumatoid arthritis and 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, scleroderma, pernicious anemia, Goodpasture syndrome, Guillain-Barré syndrome, and Wegener's disease. Examples of diseases that may be considered include, but are not limited to, glomerulonephritis, aplastic anemia (including patients with aplastic anemia due to frequent transfusions), paroxysmal nocturnal hemoglobinuria, myelodysplastic syndrome, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, Evans 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.

[0154] In another embodiment, the Disclosure provides a method for treating an inflammatory disease in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of the IL-2 variant or IL-2 variant fusion protein of the Disclosure in a pharmaceutically acceptable carrier. “Inflammatory disease” includes all diseases associated with acute or various types of inflammation. Acute inflammation is the body’s initial response to a harmful stimulus, resulting from increased migration of plasma and leukocytes (e.g., granulocytes) from the blood to the injured tissue. Numerous biochemical events propagate and mature the inflammatory response, involving the local vascular system, immune system, and various cells within the injured tissue. Prolonged inflammation is called various types of inflammation, characterized by a gradual change in the types of cells present at the site of inflammation and simultaneous tissue destruction and healing due to the inflammatory process. In another embodiment, the Disclosure provides the above method for treating an inflammatory disease in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the Disclosure to a subject in need. 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 erythematous, nephritis, Parkinson's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, vacuum colitis, Behçet's syndrome and unclassifiable 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 mellitus, gout, cryopyrin-associated periodic syndromes, and chronic obstructive pulmonary disease.

[0155] In another embodiment, the Disclosure provides a method for organ transplantation or related graft-versus-host disease in a subject, comprising administering a therapeutically effective amount of the 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 transplantation is selected from heart, kidney, liver, lung, pancreas, intestine, and thymus organ transplantation, or from bone, tendon, corneal, skin, heart valve, nerve, and venous tissue transplantation. As used herein, the terms “graft-versus-host disease” or “GVHD” refer to conditions, including acute and chronic, resulting from the effects of transplant (graft) cells on host cells and tissues due to GVH. In other words, donor immune cells, which originate from donor immune cells or stem cells injected into the graft, may recognize patient (host) cells as foreign and attack them using an immune response. Acute graft-versus-host disease (GVHD) is a disorder specifically caused by the donor's immune cells in patients who have received allogeneic bone marrow or blood cell transplants. The most vulnerable tissues are the skin, intestines, and liver. In severe cases, GVHD can cause skin blisters or excessive diarrhea and weakness. Prednisone and / or other immunosuppressants are used to treat acute graft-versus-host disease.

[0156] In another embodiment, the Disclosure provides a method for treating cancer cells in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of the IL-2 variant or IL-2 variant fusion protein of the Disclosure in a pharmaceutically acceptable carrier, wherein such administration inhibits the growth and / or proliferation of cancer cells. Specifically, the IL-2 variant or IL-2 variant fusion protein of the Disclosure is useful for treating disorders characterized as cancer. Such disorders include, but are not limited to, solid tumors such as cancers of the breast, respiratory tract, brain, genital tract, gastrointestinal tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid gland and their distant metastases, lymphoma, sarcoma, multiple myeloma, and leukemia. Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma. Examples of respiratory cancers include, but are not limited to, small cell and non-small cell lung cancer, as well as bronchial adenoma and pleuroblastoma. Examples of brain tumors include, but are not limited to, brainstem and pituitary glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, as well as neuroectoderm and pineal gland tumors. Examples of male reproductive organ tumors include, but are not limited to, prostate cancer and testicular cancer. Examples of female reproductive organ tumors include, but are not limited to, endometrial cancer, cervical cancer, ovarian cancer, vaginal cancer, and vulvar cancer, as well as uterine sarcoma. Examples of gastrointestinal tumors include, but are not limited to, anal cancer, colon cancer, colorectal cancer, esophageal cancer, gallbladder cancer, stomach cancer, pancreatic cancer, rectal cancer, small intestine cancer, and salivary gland cancer. Examples of urinary tract tumors include, but are not limited to, bladder cancer, penile cancer, kidney cancer, renal pelvis cancer, ureteral cancer, and urethral cancer. Examples of eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma. Examples of liver cancers include, but are not limited to, hepatocellular carcinoma (hepatocellular carcinoma with or without lamellar variants), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular carcinoma.Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell carcinoma, and non-melanoma skin cancer. Head and neck cancers include, but are not limited to, nasopharyngeal cancer, as well as lip and oral cancers. Lymphomas include, but are not limited to, AIDS-associated lymphoma, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and central nervous system lymphomas. Sarcomas include, but are not limited to, soft tissue sarcomas, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, various lymphocytic leukemias, various myeloid leukemias, and pilaris cell leukemia. In various embodiments, the cancers are those with high expression of TGF-β family members such as activin A, myostatin, TGF-β, and GDF15, for example, pancreatic cancer, gastric cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, lung cancer, prostate cancer, brain cancer, bladder cancer, and head and neck cancer.

[0157] The term "therapeutic dose" or "therapeutic dosage" refers to the amount of medication to be administered that will alleviate, to some extent, one or more symptoms of the disorder being treated.

[0158] The effective therapeutic dose is determined first from the EC cell culture assay. 50 This can be estimated by determining the EC. Then, in an animal model, the EC determined in cell culture 50 The dosage can be determined to achieve a circulating plasma concentration range that includes [specific component]. Using such information, a more accurate dose useful in humans can be determined. Plasma levels can be measured, for example, by HPLC. The exact composition, route of administration, and dosage may be selected by individual physicians, taking into account the patient's condition.

[0159] The administration plan can be adjusted to obtain the optimal desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus dose may be administered, followed by several divided doses (multiple doses, repeated doses, or maintenance doses) over a long period, with the dose increasing or decreasing in proportion to the needs of the treatment situation. For ease of administration and dose uniformity, it is particularly advantageous to formulate parenteral compositions in drug units. As used herein, a drug unit refers to a physically separated unit suitable as a uniform dose for the mammalian subject being treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in combination with the necessary pharmaceutical carrier. The specifications of the drug unit in this disclosure are determined primarily by the unique characteristics of the antibody and the specific therapeutic or prophylactic effect to be achieved.

[0160] In other words, as will be understood by those skilled in the art, based on the disclosures provided herein, doses and administration plans are adjusted according to methods well known in the therapeutic field. That is, the maximum tolerated dose can be easily established, the effective dose to give a detectable therapeutic effect to the subject can be determined, and similarly, the time requirements for administering each drug to give a detectable therapeutic effect to the subject can be determined. Thus, although certain doses and administration plans are illustrated herein, these examples do not in any way limit the doses and administration plans that may be given to a subject when implementing this disclosure.

[0161] Furthermore, dose values ​​will vary depending on the type and severity of the condition to be improved, and may include single doses or repeated doses. It should also be understood that for any particular subject, a specific administration plan should be adjusted over a long period according to the individual needs and the professional judgment of the person managing or supervising the administration of the composition, and that the dose ranges described herein are for illustrative purposes only and are not intended to limit the scope or practice of the claimed composition. Moreover, administration plans using the compositions of this disclosure may be based on a variety of factors, including the type of disease, the subject's age, weight, sex, medical condition, severity of the condition, route of administration, and the specific antibody used. That is, administration plans can be diverse but can be routinely determined using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include toxic effects and / or clinical effects such as laboratory values. In other words, this disclosure includes intra-subject dose escalation as required by those skilled in the art. Determining appropriate doses and administration plans is well known in the relevant art and should be understood as achievable by those skilled in the art if the teachings disclosed herein are provided.

[0162] The exemplary, non-limiting daily dose range for a therapeutic or prophylactic effective dose of the IL-2 variant or IL-2 variant fusion protein of this disclosure is 0.001–100 mg / kg, 0.001–90 mg / kg, 0.001–80 mg / kg, 0.001–70 mg / kg, 0.001–60 mg / kg, 0.001–50 mg / kg, 0.001– 40mg / kg, 0.001~30mg / kg, 0.001~20mg / kg, 0.001~10mg / kg, 0.001~5mg / kg, 0.001~4mg / kg, 0.0 01~3mg / kg, 0.001~2mg / kg, 0.001~1mg / kg, 0.010~50mg / kg, 0.010~40mg / kg, 0.010~30mg / kg, 0 .010~20mg / kg, 0.010~10mg / kg, 0.010~5mg / kg, 0.010~4mg / kg, 0.010~3mg / kg, 0.010~2mg / kg, 0.010~1mg / kg, 0.1~50mg / kg, 0.1~40mg / kg, 0.1~30mg / kg, 0.1~20mg / kg, 0.1~10mg / kg, 0.1~5m The dosage can be g / kg, 0.1-4 mg / kg, 0.1-3 mg / kg, 0.1-2 mg / kg, 0.1-1 mg / kg, 1-50 mg / kg, 1-40 mg / kg, 1-30 mg / kg, 1-20 mg / kg, 1-10 mg / kg, 1-5 mg / kg, 1-4 mg / kg, 1-3 mg / kg, 1-2 mg / kg, or 1-1 mg / kg body weight. The dosage may vary depending on the type and severity of the condition to be improved. Furthermore, it should be understood that for any particular subject, the specific administration plan should be adjusted over a long period according to the individual needs and the professional judgment of the person managing or supervising the administration of the composition. Additionally, the dosage ranges described herein are for illustrative purposes only and are not intended to limit the scope or practice of the claimed composition.

[0163] The toxicity and therapeutic index of the pharmaceutical compositions disclosed herein is LD50. 50 (A lethal dose for 50% of the population) or ED 50This can be determined using standard pharmaceutical methods in cell culture media or experimental animals to find the dose that is effective in treating 50% of the population. The dose ratio between the toxic dose and the therapeutic dose is the therapeutic index, or LD. 50 / ED 50 It can be expressed as a ratio. Compositions exhibiting a large therapeutic index are generally preferred.

[0164] The frequency of administration of IL-2 variants or IL-2 variant fusion protein pharmaceutical compositions will depend on the nature of the treatment and the specific disease being treated. Patients may be treated at regular intervals, such as twice a week, weekly, or monthly, until the desired treatment outcome is achieved. Exemplary administration frequencies include, but are not limited to, weekly, bi-weekly, tri-weekly, weekly for two weeks followed by monthly, weekly for three weeks followed by monthly, monthly, every two months, every three months, every four months, every five months, every six months, or annually, without interruption.

[0165] Combination therapy As used herein, the terms “concurrent administration,” “concurrently administered,” and “in combination with” referring to an IL-2 variant or IL-2 variant fusion protein of the Disclosure and one or more other therapeutic agents are intended to mean and encompass the following: concurrent administration of such combination of an IL-2 variant or IL-2 variant fusion protein of the Disclosure and a therapeutic agent to a subject in need of treatment, wherein such components are combined into a single dosage form and each component is released to the subject substantially simultaneously; substantial concurrent administration of such combination of an IL-2 variant or IL-2 variant fusion protein of the Disclosure and a therapeutic agent to a subject in need of treatment, wherein such components are formulated separately into separate dosage forms and are ingested substantially simultaneously by the subject, and each component is substantially Substantially simultaneous administration, in which the components are released simultaneously to the subject; sequential administration of such combination of the IL-2 variant or IL-2 variant fusion protein of the present disclosure and a therapeutic agent to a subject in need of treatment, wherein the components are formulated separately to form separate dosage forms, and when they are ingested by the subject in a continuous period of time with a considerable time interval between each administration, the components are released to the subject at substantially different times; and sequential administration of such combination of the IL-2 variant or IL-2 variant fusion protein of the present disclosure and a therapeutic agent to a subject in need of treatment, wherein the components are combined to form a single dosage form, and when the components are released in a sustained-release manner, they are released simultaneously and / or at different times simultaneously, sequentially and / or overlappingly to the subject, and each portion may be administered via the same or different routes.

[0166] In another embodiment, the present disclosure provides a method for treating an autoimmune disease in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention in combination with a second therapeutic agent capable of treating the autoimmune disease. In various embodiments, the second therapeutic agent is an immunosuppressant such as corticosteroids, cyclosporine, cyclophosphamide, prednisone, azathioprine, methotrexate, rapamycin, or tacrolimus; a biological agent such as a TNF-α blocker or antagonist; an immunosuppressive agent agents) (e.g., antibodies against other lymphocyte surface markers (e.g., CD40, α-4 integrin) or cytokines), other fusion proteins (e.g., CTLA-4-Ig (Orencia RTM), TNFR-Ig (Enbrel®)), Enbrel®, TNFα blockers such as Remicade®, Cimzia®, Humira®, cyclophosphamide (CTX) (i.e., Endoxan®, Cytoxan®, Neosar®, Procitox®, REVIMMUNE®), methotrexate (MTX) (i.e., Rheumatrex®, Trexol®), belimumab (i.e., Benlysta®), Alternatively, the following may be selected: other immunosuppressants (e.g., cyclosporine A, FK506-like compounds, rapamycin compounds, or steroids), antiproliferative agents, cytotoxic agents, or other compounds that may aid in immunosuppression, or other biological agents that target inflammatory cytokines, nonsteroidal anti-inflammatory drugs / Cox-2 inhibitors, hydroxychloroquine, sulfasazoprine, gold salts, etanercept, infiximab, mycophenolate mofetil, basiliximab, atacicept, rituximab, cytoxane, interferon beta-1a, interferon beta-1b, glatiramer acetate, mitoxantrone hydrochloride, anakinra, and / or other biological agents and / or intravenous immunoglobulins (IVIGs).Non-exclusive examples of such known therapeutic agents include interferons such as IFN-β-1a (REBIF®, Avonex®, and CINNOVEX®) and IFN-β-1b (BETASERON®, EXTAVIA®, Betaferon®, and ZIFERON®); polypeptides such as glatiramer acetate (Copaxone®); natalizumab (Tysabri®); and the cytotoxic agent mitoxantrone (Novantron®).

[0167] In another embodiment, the present disclosure relates to a method for treating an inflammatory disease in a control, comprising: inhibiting or reducing the differentiation of other cells that secrete or induce other cells to secrete inflammatory molecules, including Th1, Th17, Th22, and / or not limited to Th1, Th17, Th22, Th17 The present invention provides a method comprising administering a second therapeutic agent in combination with a second therapeutic agent that can inhibit or reduce the pathway; inhibit or reduce cytokine production and / or secretion by other cells that secrete or cause other cells to secrete inflammatory molecules, including Th1, Th17, Th22, and / or not limited to Th1, Th17, Th22, and / or not limited to Th1, Th17, Th22, and / or cause other cells to secrete inflammatory molecules, including Th1, Th17, Th22, and / or cause other cells to secrete inflammatory molecules, including Th1, Th17, Th22, and / or cause other cells to secrete inflammatory molecules, including Th1, Th17, Th22, and / or cause other cells to secrete inflammatory molecules, orIn various embodiments, the second therapeutic agent is, but is not limited to, oxicams such as piroxicam, isoxicam, tenoxicam, sudoxicam salicylate, such as aspirin, disalside, benolilate, tolilate, sapphin, sorprin, diflunisal, and fendosal; acetic acid derivatives such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, flofenac, thiopinac, zidomethacin, acematacin, fentiazac, zomepirac, clamdanac, oxepinac, felbumac, ketorolac, etc.; and fenamet, e.g. Nonsteroidal anti-inflammatory drugs (NSAIDs) include, for example, mefenum, meclofenum, flufenum, niflum, and tolfenam; propionic acid derivatives such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indopropfen, pirprofen, carprofen, oxaprozin, pranoprofen, microprofen, thioxaprofen, suprofen, aluminoprofen, and tiaprefenic; and pyrazoles such as phenylbutazone, oxyfenbutazone, feprazone, azapropazone, and trimethazone. Mixtures of these NSAIDs can also be used.In various embodiments, the second therapeutic agent is, but is not limited to, hydrocortisone, hydroxyltriamcinolone, α-methyldexamethasone, dexamethasone phosphate, beclomethasone dipropionate, clobetasol valerate, desonide, desoxymethasone, desoxycorticosterone acetate, dexamethasone, dichlorizone, diflorasone diacetate, diflucotron valerate, fluadrenolone, fluchlorone acetonide, fludrocortisone, flumethasone pivalate, phyosinolone acetonide, fluocinonide, flucortin butyrate, fluocortron, fluprednilidene acetate, flulantrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, These are steroidal anti-inflammatory drugs containing corticosteroids, such as cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolon, fludrocortisone, diflurosone diacetate, fluradrenolon acetonide, medrizone, amsinafel, amsinafid, betamethasone and its esters in balance, chloroprednisone, chlorprednisone acetate, crocorterone, cresinolone, dichlorizone, diflurprednate, flulchloride, flunisolide, fluoromethalone, fluperolon, flufrednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone, and mixtures thereof.

[0168] In another embodiment, the Disclosure provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the Invention in combination with a second treatment method, which includes but is not limited to immunotherapy, cytotoxic chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiotherapy, and stem cell transplantation. For example, such a method can be used prophylactically, for the prevention of cancer, and for the prevention of cancer recurrence and metastasis after surgery, as an adjunct to other conventional cancer treatments. As permitted by the Disclosure, the effectiveness of conventional cancer treatments (e.g., chemotherapy, radiotherapy, phototherapy, immunotherapy, and surgery) can be enhanced through the use of the combination methods described herein.

[0169] A wide range of conventional compounds have been shown to possess antitumor activity. These compounds are used as drugs in chemotherapy to regress solid tumors, inhibit metastasis and further growth, or reduce the number of malignant T cells in leukemic or myeloid malignancies. While chemotherapy has been effective in treating various types of malignancies, many antitumor compounds induce undesirable side effects. It has been shown that when two or more different therapies are combined, they may work synergistically, allowing for dose reductions of each therapy and thereby reducing the adverse side effects that each compound may exhibit at higher doses. In other cases, treatment-resistant malignancies may respond to combination therapy of two or more different therapies.

[0170] In various embodiments, a second anticancer agent, such as a chemotherapeutic agent, is administered to the patient. A list of exemplary chemotherapeutic agents includes daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, bendamustine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin, carboplatin, and oxaliplatin. Examples of such chemotherapeutic agents include, but are not limited to, tin, pentostatin, cladribine, cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethylstilbestrol (DES), fludarabine, ifosfamide, hydroxyurea, taxanes (such as paclitaxel and docetaxel), and / or anthracycline antibiotics, as well as combinations of agents such as DA-EPOCH, CHOP, CVP, or FOLFOX. In various embodiments, the dosage of such chemotherapeutic agents is approximately 10 mg / m². 2 , 20 mg / m² 2 , 30 mg / m² 2 , 40 mg / m² 2 50 mg / m² 2 , 60 mg / m² 2 75 mg / m² 2 , 80 mg / m² 2 90 mg / m² 2 , 100 mg / m² 2 , 120 mg / m² 2 , 150 mg / m² 2 , 175 mg / m² 2 , 200 mg / m² 2 , 210 mg / m² 2 , 220 mg / m² 2 , 230 mg / m² 2 , 240 mg / m² 2 , 250 mg / m² 2 , 260 mg / m² 2 , and 300 mg / m² 2This includes, but is not limited to, any of the following:

[0171] In various embodiments, the combination therapy of the present disclosure may further include administering a therapeutically effective dose of immunotherapy to a target, wherein the immunotherapy may include depleting antibodies against a specific tumor antigen. Therapy using antibodies; therapy using antibody-drug conjugates; therapy using agonist antibodies, antagonist antibodies, or inhibitory antibodies against costimulatory or coinhibitory molecules (immune checkpoints) such as CTLA-4, PD-1, OX-40, CD137, GITR, LAG3, TIM-3, SIRP, CD47, CD40, Siglec8, Siglec9, Siglec15, TIGIT, and VISTA; therapy using bispecific T cell inducing antibodies (BiTE®) such as blinatumomab; therapy including administration of biological response modifiers such as IL-12, IL-15, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ; therapy using therapeutic vaccines such as Ciproisel T; therapy using Calmette-Guérin bacilli (BCG); dendritic cell vaccine Treatments include, but are not limited to, those using tin or tumor antigen peptide vaccines; treatments using T cells, chimeric antigen receptor (CAR)-T cells, or iPS-induced T cells or iPS-induced CAR-T cells; treatments using NK cells, CAR-NK cells, or iPS-induced NK cells or iPS-induced CAR-NK cells; treatments using tumor-infiltrating lymphocytes (TILs); treatments using adoptive transplant antitumor T cells (grown in vitro and / or TCR transgenic); treatments using TALL-104 cells; and treatments using immunostimulants such as the Toll-like receptor (TLR) agonist CpG and imiquimod; the above combination therapies increase the killing of tumor cells by effector cells, meaning that a synergistic effect exists between IL-2 variants and immunotherapy when administered simultaneously.

[0172] In various embodiments, combination therapy involves simultaneously administering an IL-2 variant and a second drug composition, either in the same pharmaceutical composition or in separate pharmaceutical compositions. In various embodiments, the IL-2 variant composition and the second drug composition are administered sequentially, i.e., the IL-2 variant composition is administered before or after the administration of the second drug composition. In various embodiments, the administration of the IL-2 variant composition and the second drug composition is simultaneous, i.e., the administration periods of the IL-2 variant composition and the second drug composition overlap. In various embodiments, the administration of the IL-2 variant composition and the second drug composition is not simultaneous. For example, in various embodiments, the administration of the IL-2 variant composition is completed before the administration of the second drug composition. In various embodiments, the administration of the second drug composition is completed before the administration of the IL-2 variant composition. [Examples]

[0173] The following embodiments are provided to illustrate the disclosure more fully and are not intended to limit the scope of the disclosure.

[0174] Example 1 Design of IL-2 variants that selectively target Treg cells In one embodiment, the present invention targets one or more mutations that weaken the affinity of IL-2 to IL-2Rβ and / or γc receptor subunits. In a weakened IL-2Rβγ interaction, the enhanced IL-2 sensitivity of Tregs mediated by IL-2Rα expression can result in a clear proliferative advantage for this cell subset. Consequently, these variants may function as Treg promoters in autoimmune and inflammatory diseases.

[0175] The variants were computer-designed based on the structure of human IL-2 reported in the Protein Data Bank (PDB code 2B5I). A panel of variants was designed containing 1 to 3 mutations (introducing conserved and non-conserved amino acid substitutions) at or near the interface with the IL-2Rβ or γc receptor subunit. For example, D20 is involved in an extensive network of hydrogen bonds to the receptor subunit side chain at the IL-2Rβ interface. Similarly, N88 is a high-energy hotspot for IL-2 / IL-2Rβ interactions and is involved in critical hydrogen bonding with the receptor chain. Q126 is essential for γc interactions, and similarly, Q22 is located at the γc interface. The inventors of this invention hypothesized that mutations at the above sites or adjacent residues could result in defects in its ability to interact with IL-2Rβγ, the IL-2 intermediate affinity receptor.

[0176] Interestingly, the proposed " 19 The "LDL" motif (Baluna R, Rizo et al., Proc Natl Acad Sci, 1999;96:3957-62) partially coincides with the IL-2Rβ contact surface. This "toxic motif" is partly responsible for the direct vascular toxicity of IL-2. Consequently, mutations introduced to replace the critical toxic motif residue D20, or to replace the adjacent residues L19 and L21 with non-aliphatic residues, were also expected to remove the toxic motif, prevent endothelial cell damage, and significantly reduce VLS.

[0177] In this invention, the following 1 to 3 amino acid substitutions (D20T, D20E, D20N, D20Q, D20S, D20Y, D20I, L19Y, L19N, L19R, L19Q, L19H, L19D, L19P, L19S, L21S, L21N, L21R, N88R, N88G, N88I, N88Q, N88E, N88T, N88M, Q126E, Q126L, Q126N, Q126D, Q126M, Q126K, Q126H, Q126Y, Q126R, Q126S, Q126T, Q125E, S125K, S125H, S125W, S125 I, Q22N, Q22H, Q22K, Q22Y, Q22I, D20I / N88G, D20I / N88R, D20T / N88R, D20 I / N88I, D20T / Q126E, D20T / N88R / Q126E, D20T / Q126L, D20T / N88R / Q126L , L19N / Q126E, L19R / Q126E, L19Y / Q126E, L19H / Q126E, L19Q / Q126E, L19S / Q126E, L19Y / Q126K, L19Y / Q126H, L19Y / Q126Y, L19Y / S125E, L19Y / S125K , L19Y / S125H, L19Y / S125W, L19Y / S125I, L19Y / Q22N, L19Y / Q22H, L19Y / Q22K, L19Y / Q22Y, L19Y / Q22I, L19H / Q126K, L19H / S125I, L19D / S125I, D20E / S25I, D20T / S125I, and L19Y / S125I / Q126E, L19H / S125I / Q126E, L19H / S125I / Q126K, L19Q / S125I / Q126E, L19Q / Q126K, L19Q / S125I / Q126K, D20T In addition to / S125I / Q126K, L19N / S125I / Q126K, L19N / S125I / Q126E, L19R / S125I / Q126K, L19D / S125I / Q126E, D20E / S125I / Q126E, L19H / S125I / Q126D, L19H / S125I / Q126D, L19H / S125I / Q126H, L19H / S125I / Q126N, L19H / S125I / Q126R, L19H / S125I / Q126S, L19H / S125I / Q126T and L19H / S125I / Q126E,A panel of IL-2 variants with various N-terminal deletions (sequence numbers 288-291) (sequence numbers 4-43, 113-151, 208-212, 275-292) were expressed as C-terminal fusions to Fc homodimers via the "GGGSGGGS" linker (sequence number 55). Furthermore, IL-2 variants with D20I, D20I / N88G, D20E, or L19N amino acid substitutions were expressed as N-terminal fusions to Fc homodimers via the rigid "AEAAAKEAAAKEAAAKA" linker (sequence number 53). In summary, the sequences of these IL-2 variant Fc fusion constructs are listed in sequence numbers 73-112, 152-194, 213-219, and 299-305. Constructs containing wild-type IL-2 with the same Fc fusion form (both C-terminal and N-terminal) were also constructed (sequence numbers 71 and 72). ,

[0178] All of the above IL-2 variant Fc fusion molecules are designed to confer a proliferation advantage to cells highly expressing IL-2Rα, leading to a preference for Treg cells over other lymphocytes, including CD4+ conventional T cells, CD8+ T cells, and NK cells. Furthermore, mutations at positions 19, 20, or 21 are expected to eliminate toxic motifs that cause vascular toxicity, and as a result, the resulting molecule may possess two beneficial properties, including improved selectivity for Treg activation and reduced endothelial cell damage. Nevertheless, the optimal mutation or combination of mutations is crucial for tuning the level of impairment to maintain sufficiently high potency while maximizing the window for selective targeting of Treg subsets.

[0179] Example 2 Design of IL-2 constructs to improve selectivity for effective T cells and NK cells Another aspect of the present invention is to improve the selectivity of IL-2 for cancer treatment, compared to wild-type IL-2, for cells expressing IL-2Rβγ (not IL-2Rα) rather than cells expressing IL-2Rαβγ. One approach is to reduce or eliminate the binding of IL-2 to IL-2Rα, thereby reducing the stimulation of Treg cells. The amino acids R38, F42, and P65, which interact with IL-2Rα, were mutated to reduce or eliminate binding to IL-2Rα. Furthermore, it is expected that IL-2 variants will prevent endothelial cell damage and significantly reduce VLS by impairing the binding of IL-2Rα+ pulmonary endothelial cells. IL-2 variants with the amino acid substitutions listed in Table 4F (SEQ ID NOs: 220-234 and 293-299) were expressed as C-terminal fusions (SEQ ID NOs: 235-249) to Fc homodimers via the "GGGSGGGS" linker (SEQ ID NO: 55).

[0180] Alternatively, a construct with improved selectivity for cells expressing IL-2Rαβγ compared to cells expressing IL-2Rαβγ can be achieved by creating an IL-2 / IL-2Rα complex Fc fusion. The basis for the improved selectivity is that the IL-2 / IL-2Rα complex fusion can form a high-affinity complex without requiring binding to IL-2Rα bound to the cell. The complexation of IL-2 and IL-2Rα can be covalent or non-covalent. In the case of covalent complexation, IL-2RαSushi (SEQ ID NO: 68) was fused between both the Fc polypeptide (SEQ ID NO: 45) and IL-2 (SEQ ID NO: 3) by a flexible linker (SEQ ID NO: 45). IL-2 may be at either the N-terminus (SEQ ID NO: 69) or the C-terminus (SEQ ID NO: 70). Non-covalent complexation was achieved by fusing IL-2 to either the N-terminus or C-terminus of the Hole-Fc chain (SEQ ID NO: 47) and IL-2RαSushi to either the N-terminus or C-terminus of the Knob-Fc chain (SEQ ID NO: 46). Co-expression of the two resulting polypeptides (SEQ ID NOs: 196 and 197 for C-terminal fusion) yields a heterodimeric Fc fusion protein (P-0482) in which IL-2 is non-covalently complexed with IL-2RαSushi on the opposite chain.

[0181] Example 3 Construction and fabrication of IL-2 Fc fusion constructs All genes were codon-optimized for expression in mammalian cells, synthesized, and subcloned into recipient mammalian expression vectors (GenScript). Protein expression is driven by a CMV promoter, and a synthetic SV40 poly(A) signaling sequence is present at the 3' end of the CDS. A leader sequence is designed at the N-terminus of the construct to ensure proper signaling and processing for secretion.

[0182] Constructs were produced by co-transfecting HEK293-F cells growing in suspension with the above mammalian expression vector using polyethyleneimine (PEI, molecular weight 25,000, linear, Polysciences). When two or more expression vectors were available, the vectors were transfected in a 1:1 ratio. For transfection, HEK293 cells were cultured in serum-free FreeStyle® 293 Expression Medium (Thermo Fisher Scientific). When producing in a 1000 ml shaking flask (working volume 330 mL), HEK293 cells were 0.8 × 10⁶ 24 hours before transfection. 6 Cells were seeded at a density of cells / ml. A total of 330 μg of DNA expression vector was mixed with 16.7 ml of Opti-mem medium (Thermo Fisher Scientific). After adding 0.33 mg of PEI diluted in 16.7 ml of Opti-mem medium, the mixture was vortexed for 15 seconds and then incubated at room temperature for 10 minutes. Subsequently, this DNA / PEI solution was added to the cells and incubated at 37°C in an 8% CO2 incubator. On day 4, sodium butyrate (MilliporeSigma) was added to the cells at a final concentration of 2 mg / L to assist in maintaining protein expression. After 6 days of culture, the supernatant was collected and purified by centrifugation at 2200 rpm for 20 minutes. This solution was filtered through a sterile filter (0.22 μm filter, Corning). Secretory proteins were purified from the cell culture supernatant using protein A affinity chromatography.

[0183] Alternatively, constructs were prepared using ExpiCHO cells (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0184] In affinity chromatography, each supernatant was placed on a HiTrap MabSelectSure column (CV=5mL, GE Healthcare) equilibrated with 25 ml of phosphate-buffered saline, pH 7.2 (Thermo Fisher Scientific). Unbound proteins were removed by washing with 5 column volumes of PBS, pH 7.2, and the target proteins were eluted with 25 mM sodium citrate and 25 mM sodium chloride, pH 3.2. The protein solution was neutralized by adding 3% 1 M Tris pH 10.2. The Protein A material was polished using ion exchange chromatography or mixed-mode chromatography (including, but not limited to, CaptoMMC (GE Healthcare), ceramic hydroxyapatite, or ceramic fluoroapatite (Bio-Rad)) as needed. The target proteins were concentrated in an Amicon® Ultra-15 Concentrator 10 kDa NMWC (Merck Millipore).

[0185] The purity and molecular weight of the purified construct were determined by SDS-PAGE and Coomassie (Imperial) with or without a reducing agent. R Stain (Imperial R Analysis was performed by staining with Stain. NuPAGE® Pre-Cast gel system (4-12% or 8-16% Bis-Tris, Thermo Fisher Scientific) was used according to the manufacturer's instructions. The protein concentration of the purified protein sample was determined by measuring the UV absorbance at 280 nm (Nanodrop spectrophotometer, Thermo Fisher Scientific) and dividing it by the molar extinction coefficient calculated based on the amino acid sequence. The aggregate content of the construct was analyzed using an Agilent 1200 high-performance liquid chromatography (HPLC) system. The sample was injected into an AdvanceBio size exclusion column (300 Å, 4.6 × 150 mm, 2.7 μm, LC column, Agilent) with 150 mM sodium phosphate, pH 7.0 as the mobile phase at 25°C.

[0186] It is noteworthy that the expression profiles and aggregation tendencies of IL-2 variant Fc fusions differ significantly between constructs with different mutation sites, or between mutants that share the same mutation site but have different residue substitutions.

[0187] Example 4 Single amino acid substitutions in IL-2 result in a universal improvement in the suitability of fusion compound development. Genetic engineering approaches to find mutation combinations that yield variant proteins with desired biological properties have faced significant challenges when applied to IL-2. Naturally occurring IL-2 proteins are known in this field to be relatively unstable and prone to aggregation. This was demonstrated in experiments where wild-type IL-2 Fc fusion protein (P-0250) was expressed at low levels (transiently at approximately 3 mg / L in HEK-293F cells) with a high tendency to aggregate, as exemplified by the SEC chromatogram shown in Figure 1A. Amino acid substitutions in IL-2 aimed at achieving desired biological activity typically resulted in even less stable mutant proteins, thus faltering genetic engineering efforts. Most of the IL-2 variants in this study were expressed at extremely low levels, and some variants were significantly more prone to aggregation, as exemplified by the SEC chromatogram of P-0318 (SEQ ID NO: 97) shown in Figure 1B. This poses a problem for the manufacture and storage of therapeutic drugs.

[0188] Furthermore, the expression profiles and aggregation tendencies of IL-2 variant fusions were observed to differ significantly between variants with different mutation sites or those sharing the same mutation site but with different residue substitutions. This observation is exemplified by P-0317 (SEQ ID NO: 96) and P-0318. Both variant fusions share the same mutation site at residues 20 and 88, differing in only one amino acid. P-0317 has the D20I and N88R amino acid substitutions, while P-0318 contains the D20I and N88I mutations. Both variant fusions were expressed at similarly low levels. As seen in Figure 1B, P-0318 was highly prone to aggregation, containing 65% high molecular weight species, with the expected peak in the chromatogram being the minor species, indicated by the arrow. In contrast, P-0317 was relatively pure with 7.5% aggregates (Figure 1C). It is inferred that the N88R mutation may reduce the aggregation tendency of the resulting fusion protein. However, P-0254 (SEQ ID NO: 73) and P-0324 (SEQ ID NO: 98) were fusion proteins resulting from IL-2 with a single N88R mutation or a D20T / N88R double mutation, respectively, and were prone to aggregation, with 30-40% aggregates. This suggests that the contribution of individual amino acid substitutions to protein stability appears to be context-dependent.

[0189] The unpredictable contribution of different residue substitutions to protein stability further exacerbates the fact that amino acid substitutions for IL-2 typically result in low-stability proteins. Therefore, finding residue substitutions (one or more) that can universally enhance protein development suitability, including improved stability, higher expression levels, and a lower tendency to aggregate, is highly desirable.

[0190] The amino acid substitution at position 125 was initially intended to modulate IL-2 selectivity, as the residue is located very close to Q126, which is essential for γc interaction. Naturally occurring IL-2 contains an unpaired cysteine ​​at position 125. In proleukin, it was serine instead. In this invention, S125 is considered the wild-type IL-2 residue. IL-2 containing an alanine substitution at position 125 is also widely used. Since the substitution of serine or alanine for cysteine ​​at position 125 completely preserved biological activity, bulky charged or hydrophobic residues, including Glu, Lys, Try, His, and Ile, were introduced at position 125 to substituted Ser in P-0372 (SEQ ID NO: 81) in order to interfere with the interaction between Q126 and γc and alter biological activity. With the exception of P-0471 (SEQ ID NO: 183), all of the resulting fusion molecules had expression levels too low to evaluate their properties. On the contrary, P-0471 was expressed at a significantly higher level (19.3 mg / L vs. 4.0 mg / L titer) compared to its S125 counterpart (P-0372), and its tendency to aggregate was greatly reduced (1% vs. 21.7% aggregation). Given the remarkable improvement in development suitability, particularly the purity of the product, we decided to evaluate whether such improvements due to the isoleucine substitution at position 125 could be reproduced under different mutational conditions.

[0191] Therefore, the S125I substitution was introduced into numerous IL-2 variant Fc fusion molecules. Constructs with the Ile125 substitution in IL-2 were expressed using the same vector and culture conditions as their Ser125 counterparts and purified using MabSelectSure. Table 6 summarizes the expression levels in mg / L and purity in aggregate % as assessed by SEC chromatography for exemplary molecules. Two molecules in the same row of Table 6 share the same other amino acid substitutions (one or more) and differ only in residue 125, either serine or isoleucine. For example, SEC chromatograms and SDS-PAGE images of P-0447 (SEQ ID NO: 173) and its Ile125 counterpart P-0511 (SEQ ID NO: 213) are further shown in Figures 1D and 1E. It is clear from Table 6 that isoleucine substitution at position 125 results in a 4- to 11-fold increase in expression levels and uniformly lower aggregate fluorescence. TIFF0007850417000016.tif82170

[0192] It is evident from this invention that an isoleucine substitution at position 125 resulted in a universal improvement in the developmental suitability of the IL-2 fusion construct. This finding is particularly beneficial because the fact that modifying the slightly stable wild-type IL-2 typically results in a less stable mutant protein has hindered the genetic engineering of IL-2 for desired biological properties. The problems associated with the genetic engineering of IL-2 can be mitigated by a single amino acid substitution at the isoleucine position 125.

[0193] Example 5 Identification of single-amino acid substitution IL-2 variants showing differences in selectivity for Treg lymphocytes. Single amino acid substitutions were introduced into IL-2 at positions corresponding to amino acids that interact with receptor subunits β, γ, or βγ. These substitutions aimed to reduce IL-2 signaling capacity through the moderate-affinity IL-2Rβγ complex and confer signaling specificity from the high-affinity IL-2Rαβγ complex. The ability of IL-2 variants, including single amino acid substitutions, to stimulate STAT5 phosphorylation in CD4-positive Treg and Tconv cells was investigated. STAT5 is known to be involved in the downstream signaling cascade when IL-2 binds to the transmembrane IL-2 receptor. STAT5 phosphorylation in defined lymphocyte subpopulations was measured using fresh human peripheral blood mononuclear cells (PBMCs), and Treg populations were identified using the forkhead transcription factor FOXP3 in FACS analysis.

[0194] In short, human PBMCs were isolated from the buffy coat of healthy donors by Ficoll-Hypaque centrifugation. The PBMCs were starved in serum-free MACS buffer at 4°C for 1 hour. Then, 2 × 10⁻⁶ ¹ 5 Each PBMC was treated with a serial dilution of the test compound at 37°C for 30 minutes. The cells were fixed and permeabilized using Foxp3 / Transcription Factor Staining Buffer Set (EBIO) by incubation in 1× Foxp3 fixation / permeabilization working solution for 30 minutes and washing with 1× permeabilization buffer. The cells were further fixed with Cytofix buffer, permeabilized with Perm Buffer III (BD Biosciences), and then washed. After blocking the Fc receptor by adding human TruStain FcX (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 concentrations recommended by the manufacturer. The cells were harvested by centrifugation, washed, resuspended in FACS buffer, and analyzed by flow cytometry. Treg and CD4+ conventional T cell subsets were analyzed using CD4+ / Foxp3+ / CD25 highand CD4+ / Foxp3- / CD25 low The groups were gated. The data is expressed as the percentage of pStat5-positive cells in the gated population.

[0195] Figure 2 shows the dose-response effect of an exemplary IL-2 variant Fc fusion protein on STAT5 phosphorylation in CD4-positive Treg and Tconv cells compared to wild-type fusion protein. Wild-type IL-2 Fc fusion protein (P-0250) induced STAT5 phosphorylation in both Treg and Teff cells, with EC50 values ​​of 0.1 pM and 25.4 pM, respectively. The potency of wild-type IL-2 was approximately 250-fold greater in Treg cells than in CD4+ Tconv cells, consistent with the higher expression levels of the high-affinity trimer receptor in Treg cells.

[0196] Various substitutions of aspartate at position 20, P-0364(D20E), P-0363(D20T), P-0365(D20N), P-0366(D20Q), and P-0367(D20S), showed the ability to induce STAT5 phosphorylation in Treg cells, while such activity was largely reduced or absent in CD4+ Tconv cells (Figures 2A and 2B). These variants may be Treg-biased IL-2 agents that activate Treg cells for the treatment of autoimmune diseases. Furthermore, mutations at D20, a key residue of the proposed toxin-like motif, are expected to remove the toxic motif and prevent endothelial cell damage. Therefore, these variants are expected to have Treg-selective activity with an improved VLS safety profile. In addition, P-0368 showed no biological activity (Figures 2A and 2B).

[0197] Figure 3 shows the ability of the IL-2 variant P-0375(N88Q) to induce STAT5 phosphorylation in CD4-positive Treg and CD4+Tconv cells compared to reference 1 and venture-Q-2 compounds, respectively, which possess the V91K and N88R mutations. The activity profile of the N88Q variant was similar to that of reference 1.

[0198] Figure 4 shows the biological activity of IL-2 variants with 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) showed 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 largely retained their biological activity in CD4+ Tconv cells (Figures 4B and 4D), while such activity was reduced in variants P-0373 and P-0424 in CD4+ Tconv cells. The mutant P-0425 (L19D) showed slightly reduced potency in inducing STAT5 phosphorylation in Treg cells, while such activity was significantly impaired in CD4+ Tconv cells (Figures 4C and 4D). The demonstrated selective activation of Treg cells against CD4+ Tconv cells by mutants P-0373, P-0424, and P-0425, particularly the broad window for selective targeting of Treg subsets by P-0373 and P-0425, suggests that these mutants are 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 improve the safety profile by reducing VLS.

[0199] Example 6 Differential selectivity for Treg lymphocytes: IL-2Rβ and γ in IL-2 c Combinations of amino acid substitutions that target Example 4 demonstrated that directed mutations aimed at weakening the affinity of IL-2 to either the IL-2Rβ or γc receptor subunit can result in IL-2 variants with differing selectivity for Treg lymphocytes. It was then hypothesized that modifying the affinity of IL-2 to both the IL-2Rβ and γc receptor subunits by combining one or more amino acid substitutions targeting the β receptor with other substitutions targeting the γ receptor could yield desired potency and selectivity windows for Treg lymphocytes.

[0200] Such theoretical justification is demonstrated in Figure 5. Figures 5A and 5B show the effect of the IL-2Rβ target variant P-0372(L19Y) on STAT5 phosphorylation in Treg 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) with amino acid substitutions aimed at disrupting the interaction with the γ receptor. The data suggest that each single amino acid substitution not only has a minimal effect on pSTAT5 activation potency but also shows only a slight improvement in the selection window for Treg lymphocyte subsets compared to the wild type. The window of selective activation of Treg cells was greatly broadened by combining the L19Y and Q126E mutations in P-0419, as shown in Figures 5E and 5F. The efficacy of Treg activation was primarily secured by P-0419, and the activity profile of the P-0419 variant was very comparable to that of a reference single molecule containing the V91K mutation. Since 19L is also part of the proposed toxin-like motif, mutations at this site are expected to improve the safety profile by reducing VLS, making this strategy particularly attractive.

[0201] Combining one amino acid substitution targeting the β receptor with another substitution targeting the γ receptor does not necessarily yield the desired potency and selectivity window. Appropriate activity adjustments are required for each embodiment. The four IL-2 variants in Figures 6A and 6B share the same L19Y substitution targeting the β receptor, with additional mutations designed to target the γc receptor being Q126E in P-0419, Q126K in P-0464, S125I in P-0471, and Q22K in P-0474, respectively. All variants retained comparable potency in inducing STAT5 phosphorylation in Treg cells (Figure 6A), while such activity differed significantly in CD4+ Tconv cells (Figure 6B), demonstrating differences in the ability to modulate the selectivity of Treg activation through the combination of amino acid substitutions.

[0202] Adding further receptor attenuation by combining Q126E substitution with IL-2 variants that already exhibit biased specificity for Treg subsets can significantly reduce Treg cell specificity. While seemingly undesirable, this actually generates Treg-selective IL-2 variants with a broad potency range. As shown in Figures 6C and 6D, both variants P-0373(L19N) and P-0363(D20T) already exhibited somewhat or significantly biased selection windows for Treg cells (Figures 6C and 6D). Their respective counterparts with additional Q126E substitutions, P-0417 and P-0322, showed a marked decrease in potency in Treg cell activation. Similarly, P-0860 (with the IL-2 L19D / S125I / Q125E mutation) and P-0859 (L19N / S125I / Q125E) showed different levels of attenuation in Treg cell activation (Figure 6E). Compared to P-0511 (with the IL-2 L19H / S125I / Q125E mutation), substitution of L19D for L19H resulted in an 8500-fold decrease in Treg cell responsiveness (6226 pM vs. 0.74 pM).

[0203] Furthermore, data from Figure 6E suggested that weaker compounds induced lower signal transduction amplitudes. The maximum possible effect of STAT5 phosphorylation by P-0860 was significantly lower than that achievable by P-0511, while P-0859 moderately reduced signal transduction intensity. Such compounds could function as partial agonists. Additional partial agonists with different signal transduction intensities may be generated by optimal combinations of amino acid substitutions that allow for fine-tuning of signal transduction amplitude. Therefore, it is crucial to find appropriate combinations of residue substitutions to modulate activity to desired potency, signal transduction intensity, and biased specificity for Treg cells.

[0204] Furthermore, attenuation of Treg cell efficacy and selectivity can also be achieved through amino acid deletion. N-terminal deletions of 5, 7, or 9 amino acids were introduced into P-0511 to create P-0862, P-0863, and P-0864, ​​respectively. As shown in Figure 6F, deletions of 5 and 7 amino acids completely preserved efficacy, while deletion of 9 amino acids resulted in a 25-fold decrease in activity (18 pM vs. 0.74 pM). It is expected that various IL-2 variants with different efficacy, signaling intensity, and specificity for Treg cells can be further tuned to desired activity profiles using N-terminal deletions of 8–10 amino acids.

[0205] Further variants with double amino acid substitutions at sites L19 and Q126, including P-0447(L19H / Q126E), P-0448(L19Q / Q126E), and P-0449(L19S / Q126E), were evaluated, and their activity is shown in Figures 7A-7D. Compared to IL-2 variants P-0424(L19H) and P-0303(Q126E), each containing a single amino acid substitution, variant P-0447(L19H,Q126E), containing a combination of two amino acid substitutions, showed strong robust biological activity in STAT5 phosphorylation stimulation in Treg cells, while such activity was almost completely abolished in Tconv cells (Figures 7A and 7B). In another study evaluating P-0419, P-0447, P-0448, and P-0449 compared to two reference compounds, all four variants showed significant efficacy in inducing STAT5 phosphorylation in Treg cells, while such activity was largely absent in CD4+ Tconv cells (Figures 7C and 7D). P-0419 had an activity profile comparable to reference 1, which was similarly demonstrated in Figures 5E and 5F. P-0447, P-0448, and P-0449, on the other hand, were comparable to reference 2 in terms of potency and selectivity window for Treg cells.

[0206] All of these variants are potentially Treg-biased IL-2 agents that activate Tregs to treat autoimmune diseases. Furthermore, these variants are also expected to have reduced VLS and improved safety profiles by eliminating potentially toxic motifs.

[0207] Example 7 IL-2 variants with an isoleucine substitution at position 125 retain full biological activity. Example 3 demonstrated that isoleucine substitution at position 125 leads to a universal improvement in the developmental suitability of IL-2 fusion constructs. To make S125I substitution a viable approach and mitigate the challenges of developmental suitability of IL-2 gene manipulation, it is important to demonstrate that such amino acid substitutions do not impair the biological activity of the resulting fusion protein compared to its Ser125 counterpart.

[0208] Therefore, the S125I substitution was introduced into wild-type IL-2 or IL-2 variants that already possessed 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 and Tconv cells compared to their respective serine counterparts at position 125. Table 7 lists the potency and selectivity of the IL-2 variants in Treg cells. Two molecules in the same row of Table 7 differ only in serine or isoleucine at position 125, sharing the same amino acid substitution otherwise. The data showed that the S125I substitution fully preserved or slightly enhanced the biological activity of the various IL-2 variants tested without altering Treg specificity. TIFF0007850417000017.tif87170

[0209] Data from three exemplary compositions, P-0250, P-0424, and P-0447, as well as their S125I equivalents, P-0531, P-0491, and P-0511, respectively, are shown in Figure 8. P-0250 is a wild-type IL-2 Fc fusion molecule, P-0424 contains one amino acid substitution L19H, and P-0447 contains two amino acid substitutions L19H / Q126E. Their dose-dependent effects on STAT5 phosphorylation in Treg and CD4+ Tconv cells are shown in Figure 8. As shown in Figures 8A–8F, the S125I substitution slightly increased the potency of the three compounds tested without altering the Treg selectivity of P-0531 and P-0491. In the case of P-0511, the S125I substitution further broadened the Treg selectivity window.

[0210] Thus, the data showed that the S125I substitution of IL-2 preserved the IL-2 activity profile of IL-2 fusion proteins under different mutational conditions. In summary, the isoleucine substitution at position 125 of IL-2 resulted in improved universal development potential (increased yield, reduced aggregation, and decreased potential immunogenicity) of IL-2, IL-2 fusions, IL-2 variants, and IL-2 variant fusions, as well as complete preservation of biological activity and selectivity. This particular amino acid substitution represents a viable mitigation strategy for addressing the challenges associated with IL-2 genetic engineering.

[0211] Example 8 Effects of IL-2 variants on CD25+CD4+ T cells, CD8 cytotoxic T cells, and NK cells Two variants, P-0511 and P-0512, Tconv(CD4+ / Foxp3- / CD25 low ) Compared to cells, CD4-positive Treg (CD4+ / Foxp3+ / CD25 high In addition to evaluating the differences in their ability to stimulate Stat5 phosphorylation, we further investigated their ability to stimulate other effector T and NK cells, such as CD4-positive Teff (CD4+ / Foxp3- / CD25+), CD8 cytotoxic T effector, and NK cells, compared to wild-type IL-2 (P-0250) including V91K, N88R, and N88D, and three IL-2 reference molecules, respectively.

[0212] The IL-2 variants of the present invention weaken IL-2Rβγ interaction, and the significant proliferative advantage of Tregs compared to CD4+ Tconv cells induced by these variants was conferred by the high constitutive IL-2Rα (CD25) expression of Tregs. CD25 expression can be induced in CD4+ T effector cells after immune stimulation. Therefore, it is desirable to confirm that the IL-2 variants retain specificity to Tregs more than other CD25+ lymphocyte subsets. CD4+ effector T cells (Teffs) are an exemplary lymphocyte subset that expresses CD25 at moderate to high levels.

[0213] Human PBMC cells were treated with serial dilutions of the test compounds, fixed, permeabilized, washed, and stained with a mixture of anti-CD25-PE, anti-FOXP3-APC, anti-pSTAT5-FITC, and anti-CD4-PerCP-Cy5.5 antibodies. Flow cytometry analysis was then performed, gated for Treg, CD4 effector, and CD4 naive T cell subsets into CD4+ / Foxp3+ / CD25+, CD4+ / Foxp3- / CD25+, and CD4+ / Foxp3- / CD25- groups, respectively. Data are expressed as the percentage of pStat5-positive cells in the gated population and are shown in Figure 9. P-0512 showed an activity profile comparable to criterion 1 for all three T cell subsets, while P-511 was superior to both criteria 2 and 3 in terms of potency and selectivity window against Treg cells compared to both Teff and naive CD4 T cells. Criterion 2 showed significantly weaker potency in the activation of each of the three subsets. Despite moderate to high levels of CD25 expression in Teff, Treg was preferentially activated over Teff by IL-2 variants with attenuated IL-2Rβγ interaction, particularly P-0511, as clearly shown in Figures 9A and 9B.

[0214] Furthermore, P-0511 and P-0512 were tested for their ability to stimulate the proliferation of NK and CD8+ T cells compared to wild-type and reference molecules. The intracellular fluorescent labeling carboxyfluorosein diacetate succinimidyl (CFSE) method was used. In short, human PBMCs (1 × 10⁻¹⁰) 5 Cells (per well) were labeled with CFSE, seeded in 96-well plates, and incubated with increasing concentrations of various IL-2 compounds. Cells were then harvested after 5 or 7 days of incubation, stained with anti-CD56-APC antibody for NK cells and anti-CD8-APC antibody for CD8+ T cells, and analyzed by flow cytometry. Data are expressed as the percentage of divided cells, with CD8+ T cell proliferation shown in Figure 10A and NK cell proliferation shown in Figure 10B.

[0215] As expected, all IL-2 variants showed reduced potency in stimulation of both CD8+ T and NK cells compared to the wild-type IL-2 fusion molecule P-0250. Combined with observations in the STAT5 phosphorylation assay (Figure 9), P-0512 had an activity profile equivalent to criterion 1, P-511 was equivalent to criterion 3 in terms of potency against both lymphocyte subsets, while criterion 2 showed significantly weaker potency.

[0216] The Stat5 phosphorylation activity of P-0511 in responder cells other than CD4+ T cell subsets, including CD8+ T and NK cells, was compared with that of P-0531, the S125I equivalent of wild-type P-0250. P-0511 showed strong activity in stimulating STAT5 phosphorylation in Treg cells, similar to the P-0531 wild-type fusion (Figure 11A), whereas such activity was almost completely abolished in CD4+ Tconv (Figure 11B), CD8+ T (Figure 11C), and NK (Figure 11D) cells. The IL-2 receptor expressed in CD4+ Tconv, CD8+ T, and NK cells is primarily the dimerized IL-2R containing IL-2Rβ and γc. An ELISA assay was developed to confirm that the significant reduction in pSTAT5 signaling by P-0511 in CD8+ T and NK cells was due to impaired interaction with IL-2Rβ and γc.

[0217] In short, non-covalent complexes of IL-2Rβ-ECD (NP_000869) and γc-ECD (NP_000197) via heterodimer Fc chains were coated at 2 μg / well in the wells of a Nunc Maxisorp 96-well microplate. After incubation overnight at 4°C and blocking with Superblock (Thermo Fisher Scientific), 3-fold serial dilutions of IL-2 Fc fusion protein starting at either 100 or 270 nM were added to each well at 100 μl / well. After incubation at room temperature for 1 hour, biotin mouse anti-human IL-2Ab (BD Biosciences) was added to each well at 1 μg / ml, followed by incubation with 1 μg / ml HRP-avidin (Thermo Fisher Scientific) for 1 hour. The wells were thoroughly aspirated and washed three times with PBS / 0.05% Tween-20 after each step. Finally, 100 μl of TMB substrate was added to each well, and the plate was allowed to develop color at room temperature in the dark for 10 minutes. Then, 100 μl / well of stop solution (2N sulfuric acid, Ricca Chemical) was added. Absorbance was measured at 450 nm, and the curve was fitted using prism software (GraphPad), as shown in Figure 11E.

[0218] As shown in Figure 11E, the wild-type IL-2 fusion protein P-0531, with improved development compatibility, binds to the IL-2 dimer receptor complex with sub-nanomole affinity (EC 50 (=0.06nM), and one reference molecule showed weakened binding (EC 50(=1.6nM), which was consistent with the reduced potency observed in response to its reduced binding upon stimulation of STAT5 phosphorylation in CD8+ T and NK cells (Figure 10A-B). In contrast, P-0511 showed no apparent binding to the IL-2Rβ-γc complex, indicating that the two IL-2 mutations in P-511 at the interface with both β and γc receptor subunits dramatically weakened its interaction with the complex. It is surprising that P-0511 only slightly reduced its activity against Tregs compared to wild-type IL-2 fusions, given the virtually complete absence of binding to the dimeric IL-2 receptor complex. P-0511 exemplifies an IL-2 variant with the desired potency and selectivity window against Treg lymphocytes.

[0219] In summary, the spectra of IL-2 variants listed in Tables 4A-4H were constructed, expressed, and tested in in vitro assays. The biological activity of exemplary IL-2 variants in Treg cells compared to other lymphocyte subsets, including CD4+ Tconv, CD4+ Teff, CD8+ T, and NK cells, is shown in Figures 2-11. Many variants retained high potency against Treg cells, while their activity against Tconv cells and other lymphocyte subsets was reduced or absent. Some variants had activity profiles similar to criterion 1, while others resembled the activity characteristics of criterion 2 or 3. Furthermore, the majority of IL-2 variants had the proposed toxin-like motif removed to reduce VLS. Importantly, by incorporating the S125I amino acid substitution, IL-2 variant fusions with excellent developmental suitability profiles were obtained while retaining biological activity and selectivity in Treg cells. These variants have the potential to become Treg-biased IL-2 agents for the treatment of autoimmune diseases with improved safety profiles.

[0220] Example 9 The IL-2 variant Fc fusion protein preferentially proliferates and expands Treg cells in mice. We administered an IL-2 variant Fc fusion protein to mice and measured its ability to preferentially proliferate regulatory T cells (CD4+CD25+FoxP3+T cells) over effector T cells and NK cells in vivo.

[0221] Female C57 / BL6 mice (7 weeks old) were received from Charles River Laboratory and acclimatized in-house for at least 7 days prior to the study. On day 0, mice were subcutaneously administered a vehicle (PBS), 0.3 mg / kg of each test compound, or an IL-2 reference compound. Peripheral blood samples were collected in heparinized tubes on days 3, 5, and 7 post-administration. Each group consisted of 6 mice, and baseline blood was collected 2 days prior to administration (-2 days). After erythrocyte lysis, all viable mononuclear blood cells were counted using trypan blue dead cell exclusion, and intracellular staining for immunocytophenotyping and Ki67 proliferation markers was performed using flow cytometry. Cells were stained separately with the two antibody panels described below. 1) For CD4+ T regulatory cells (Treg), use anti-mouse Foxp3-FITC, Ki67-PE, anti-mouse CD25-APC, and anti-mouse CD4-Percpcy 5.5 (1:50 dilution); 2) For CD8+ T and NK cells, use anti-mouse CD3-FITC, Ki67-PE, anti-mouse CD335-APC, and anti-mouse CD8-Percpcy 5.5 (1:50 dilution).

[0222] All IL-2 compounds tested stimulated Treg cell proliferation and growth, as indicated by an increase in Ki67-positive Treg cells and an increase in the percentage of Tregs relative to total CD4+ T cells or total lymphocytes (Figures 12A-12C). The effect was observed 3 days after injection and persisted up to 5 or 7 days after a single injection. In contrast to in vitro observations where criterion 1 consistently showed the highest potency among the IL-2 variants in inducing Treg phosphorylation, in mice, all three tested variants, P-0511, P-0512, and P-0514, showed stronger in vivo efficacy than the criterion in stimulating Treg cell proliferation and growth. P-0511, P-0512, and P-0514 showed comparable activity. The relative ranking of in vivo potency among the three criteria was consistent with the in vitro human PBMC cell assay, namely criterion 1 compound being the most potent, followed by criterion 3. Criterion 2 is characterized by significantly weaker Treg cell proliferation (Figures 12A-12C).

[0223] In T effector and NK cells, criterion 1 showed strong Ki67 stimulation to cytotoxic CD8 T cells and NK cells, while criteria 2 and 3 showed low efficacy to CD8 T cells and NK cells (Figures 13A-13C). Variant P-0514 showed Ki67 stimulation in CD8+ T cells similar to criterion 1, while variants P-0511 and P-0512 showed mild Ki67 stimulation to CD8 T cells and NK cells, similar to criteria 2 and 3 (Figures 13A-13C). The data suggest that variants P-0511 and P-0512 exhibit superior biological activity and selectivity for Tregs compared to criteria 1 and 2. Criterion 3 was not effective in stimulating or promoting the proliferation of both Tregs and effector cells.

[0224] The percentage of conventional CD4+ T cells decreased in all IL-2 variant treatment groups due to the increase in the Treg population (Figure 14A). Significant proliferation of CD4+ T conv cells, CD8 T cells, or NK cells was not observed in mice treated with any or all of the Treg-biased IL-2 variants (P-0511, P-0512, and P-0514).

[0225] Furthermore, compared to the three baselines, all three variants, P-0511, P-0512, and P-0514, exhibited the most beneficial Treg / Tconv ratio in terms of both Ki67 stimulation and cell number-based cell proliferation at all measured time points (Figures 15A and 15B).

[0226] Foxp3 expression was increased in Treg cells by all IL-2 compounds tested 3 days after injection (Figure 16A), and all three variants showed relatively high expression of CD25 and Foxp3 markers above the three baselines (Figures 16A and 16B), suggesting superior Treg activation and functionality.

[0227] Body weight was monitored before and during administration. No significant changes in body weight were observed (data not shown).

[0228] Overall, the data showed that variants P-0511, P-0512, and P-0514 have the ability to promote the activation, proliferation, and growth of immunosuppressive Treg cells while preserving conventional CD4+ cells, cytotoxic effector T cells, and NK cells. The data also supported the superiority of these three variants compared to the reference molecule in terms of both efficacy and selectivity for Treg proliferation and growth. These variants may be useful as therapeutic agents to combat autoimmune and inflammatory diseases, as well as organ transplant rejection.

[0229] Example 10 Dose-response pharmacodynamic studies using IL-2 variant Fc fusion protein in mice after single injection Female Balb / C mice (n=5 / group) were administered a single subcutaneous dose of vehicle (PBS) or P-0511 (1, 0.3, 0.1, or 0.03 mg / kg). Peripheral blood was collected at baseline (day 2) and at post-administration (days 3, 5, and 7). On day 7, the mice were sacrificed and the spleen was collected. The phenotype, proliferation, and growth of hematopoietic lymphocytes were elucidated by flow cytometry using fresh whole blood at each time point.

[0230] There were no significant changes in body weight or spleen weight in any of the treatment groups (data not shown).

[0231] As shown in Figure 17, a dose-dependent increase in Treg cell proliferation, reflected by an increase in the percentage of Ki67-positive cells (Figure 17A), was observed in mice administered P-0511 at dose levels of 1, 0.3, or 0.1 mg / kg. Administration at 0.03 mg / kg had minimal effect. Stimulation of Ki67 expression in Treg cells peaked at the three high dose levels on day 3, plateaued until day 5, and then declined. Consequently, P-0511 treatment resulted in dose-dependent increases in the percentage of Tregs relative to total CD4+ T cells (Figure 17B), absolute Treg cell count (Figure 17C), and multiplier changes in cell count from baseline (Figure 17D). The increase in Treg cell proliferation followed a kinetic pattern similar to that of the proliferation / activated Ki67 marker (Figure 17), namely reaching a peak on day 3 and extending to a peak until day 5. Administration at 1 mg / kg stimulated Tregs more significantly and for a longer period, with the signaling lasting up to day 5.

[0232] Administration of P-0511 also resulted in a dose-dependent and statistically significant increase in the percentage of Treg cells relative to total lymphocytes (Figure 18A), while there were no statistically significant changes in the percentages of CD4+ Tconv cells (Figure 18B), CD8 Teff cells (Figure 18C), and NK cells (Figure 18D) relative to total lymphocytes. At peak levels, Treg cells accounted for 4.5% of total lymphocytes with a single dose of 1 mg / kg, compared to 3.1% with 0.3 mg / kg and 1.4% with 0.1 mg / kg. In the vehicle control group, Treg cells accounted for 0.5% of total lymphocytes (Figure 18A).

[0233] The most beneficial Treg / Tconv ratio was calculated based on cell number (Figure 19A). The Treg / Tconv ratio peaked at 0.27 with 1 mg / kg administration, 0.18 with 0.3 mg / kg, and 0.06 with 0.1 mg / kg, compared to 0.027 in the untreated group (Figure 19A), suggesting that P-0511 preferentially proliferates Treg cells over Tconv cells. Furthermore, the expression of Treg cell function markers, including CD25 (Figure 19B) and FoxP3 (Figure 19C), increased in a dose-dependent manner. The increase in mean fluorescence intensity (MFI) of CD25 and FoxP3 peaked on day 3 and decreased to lower levels on day 5.

[0234] Overall, the data showed that P-0511 exhibits dose-dependent potent and preferential activation and proliferation of Treg cells. Careful consideration is needed to achieve the optimal drug dosing strategy to maximize efficacy in promoting the activation, proliferation, and growth of immunosuppressive Treg cells while preserving cytotoxic effector T cells and NK cells.

[0235] Example 11 Pharmacodynamic studies in mice after repeated administration of IL-2 variant Fc fusion protein Female Balb / C mice (7 weeks old) were acclimated in the facility for 5 - 7 days before the study. Vehicle (PBS), 0.3 mg / kg of P-0511, P-0512, P-0531, or reference 1 compound was subcutaneously administered to the mice (n = 5 / group) on days 0, 3, and 6. Peripheral blood was collected on days 3 and 9, 3 days after the first injection and 3 days after each of the multiple (3 times) injections. Based on previous in vivo experiments, since the activation, proliferation, and expansion of Treg cells were expected to peak on day 3, day 3 after injection was selected for data collection and analysis. Changes in the activation, proliferation, and expansion of blood lymphocytes were measured by flow cytometry. P-0531 is the S125I equivalent of the wild-type IL-2 fusion protein. Reference 1 contains the V91K mutation.

[0236] Three days after a single subcutaneous administration of the IL-2 fusion protein, nearly 90% of Treg cells showed positive Ki67 expression in all groups tested, and Ki67-positive cells remained significantly high even after the third administration of all compounds tested (Figure 20A). Interestingly, compared to a single administration, after three consecutive Q3D administrations, Treg cells, expressed as %Treg relative to total CD4 T cells or total lymphocytes, significantly decreased to near control levels in mice administered P-0531 and reference 1, while remaining at significantly higher levels in mice administered P-0511 and P-0512 (Figures 20B - 20C). The data suggest that wild-type IL-2 or reference 1 may accelerate Treg cell exhaustion or promote Treg desensitization due to a more potent efficacy against Treg stimulation. As additional explanations, differences in the half-life of non-lymphocytes or "receptor sinks" leading to changes in drug exposure of wild-type IL-2 or reference 1 with no or low Treg selectivity can be cited.

[0237] Similar observations were also obtained for Treg cell numbers and fold changes compared to the PBS control (Figures 21A - 21B), and the Treg / Tconv ratio (Figure 22). P-0511 and P-0512 showed excellent functions of maintaining the Treg pool and maintaining Treg selectivity compared to P-0531 and reference 1.

[0238] Overall, data showed that P-0511 and P-0512 are excellent IL-2 molecules that exhibit preferential and sustained in vivo Treg proliferation after multiple administrations. By adjusting the dosing schedule of the IL-2 variant Fc fusion, such as dose and frequency, the desired efficacy and selectivity for Tregs can be further optimized over pro-inflammatory immune activation.

[0239] Example 12 Suppression of antigen-induced inflammation by an IL-2 variant Fc fusion protein in a delayed-type hypersensitivity (DTH) mouse model The ability of Treg cells induced by an IL-2 variant to suppress T cell antigen-induced inflammation in vivo was evaluated in a delayed-type hypersensitivity (DTH) model. Female Balb / C mice (7 weeks old) were acclimated in the facility for 7 days and randomly assigned to groups. Subcutaneous administration of vehicle (PBS), 0.1 mg / kg or 0.3 mg / kg of P-0511 was initiated on day -2, injected 3 times at 3-day intervals (Q3D), or 2 times at 5-day intervals (Q5D). Then, the mice were sensitized on day 0 by subcutaneous administration of 100 μg of keyhole limpet hemocyanin (KLH) in 200 μl of saline. For Q3D administration, an additional 2 subcutaneous injections of PBS or P-0511 (0.1 or 0.3 mg / kg) were given on days 1 and 4, and for Q5D administration, an additional 1 subcutaneous injection of PBS, 0.1 or 0.3 mg / kg of P-0511 was given on day 3. On day 5, the mice were given an intradermal challenge of KLH (5 μg in 10 μl saline) in the right ear. The thickness of the right ear was measured daily from day 6 to day 9, corresponding to 5 days before the KLH challenge and 24 hours, 48 hours, 72 hours, and 96 hours after the KLH challenge. Also, one group of mice received intraperitoneal administration of 5 mg / kg of dexamethasone daily from day 5 to day 8 as a positive control.

[0240] Figure 23 shows the kinetics of the DTH response using changes in ear thickness (amount of change in ear thickness) relative to baseline values ​​at various time points after the KLH challenge.

[0241] Significant ear inflammation and swelling peaked 24 hours after intradermal KLH challenge of the auricle following subcutaneous KLH antigen sensitization, and ear swelling lasted for 72 hours in the PBS group. Dexamethasone, an immunosuppressive steroid, demonstrated potency in suppressing KLH-induced inflammatory responses, achieving approximately 85% suppression 72 hours after KLH challenge when administered at 5 mg / kg for 4 consecutive days. Suppression of antigen-induced inflammation by Treg cells induced by P-0511 was evident at all time points after KLH challenge in mice administered 0.3 mg / kg of P-0511 in either Q3D or Q5D (Figures 23A-23B). At a dose of 0.1 mg / kg, a similar trend of reducing the DTH inflammatory response was observed in both Q3D and Q5D regimens, but the effect was not statistically significant at most time points. Both Q3D and Q5D dosage schedules were effective.

[0242] Another study revealed the dose-dependent effects of P-0511 (0.1, 0.3, and 1 mg / kg, Q5D) on the suppression of KLH-induced DTH, compared to reference level 1 (0.3 mg / kg, Q5D). As shown in Figure 24, P-0511 demonstrated dose-dependent suppression of ear inflammation. Mice administered 1 mg / kg of P-0511 showed strong resistance to KLH-induced DTH, and minimal ear swelling was observed after KLH challenge. Moderate and mild suppressive effects were observed with 0.3 mg / kg and 0.1 mg / kg of P-0511, respectively. Reference level 1 showed mild suppression of ear swelling, and the effect of reference level 0.3 mg / kg was similar to that achieved with 0.1 mg / kg of P-0511 (Figure 24).

[0243] In summary, Treg cells induced by P-0511 administration were effective in suppressing T cell antigen-induced inflammation in a DTH model. Furthermore, Treg suppression was maintained without repeated administration after KLH challenge. This case study also highlighted the importance of adjusting the drug regimen to achieve optimal efficacy.

[0244] Example 13 IL-2 variants showed reduced / absence of binding to IL-2 receptor subunit α for cancer indications. P-0613 and P-0573 are two exemplary IL-2 variant Fc fusion proteins. P-0613 contains the F42A amino acid substitution, and P-0573 contains the R38A / P65G dual amino acid modification. F42, R38, and P65 are all at the interface with IL-2Rα and form either hydrophobic interactions or salt bridges with multiple IL-2Rα residues (Mathias Rickert, et al. (2005) Science 308, 1477-80). Mutations in these residues are expected to break the interaction with IL-2Rα, resulting in IL-2 variants with reduced or absent binding to IL-2Rα. Furthermore, both P-0613 and P-0573 contain the S125I substitution, which has been shown to significantly improve the developmental potential profile of IL-2 Fc fusion molecules with fully retained biological activity. The binding activity of P-0613 and P-0573 to IL-2Rα was determined by enzyme-linked immunosorbent assay (ELISA) compared with P-0531 and reference 4. P-0531 is the S125I equivalent of the wild-type IL-2 Fc fusion protein, while reference 4 contains the triple mutant F42A / Y45A / L72G, which disrupts IL-2Rα.

[0245] In short, IL-2Rα-ECD (SinoBiological) was coated at 1 μg / well in a Nunc Maxisorp 96-well microplate. After incubation at 4°C overnight and blocking with Superblock (Thermo Fisher Scientific), 3-fold serial dilutions of IL-2 Fc fusion protein, starting at 100 nM, were added to each well at 100 μl / well. After incubation at room temperature for 1 hour, goat anti-human IgG Fc-HRP (diluted 1:5000 with diluent) was added to each well at 100 μl / well, and incubated at room temperature for 1 hour. The wells were thoroughly aspirated and washed three times with PBS / 0.05% Tween-20 after each step. Finally, 100 μl of TMB substrate was added to each well, the plate was allowed to develop color at room temperature in the dark for 10 minutes, and 100 μl / well of stop solution (2N sulfuric acid, Ricca Chemical) was added. Absorbance was measured at 450 nm, and the curve was fitted using prism software (GraphPad).

[0246] Example 14 IL-2 variant Fc fusion protein with reduced Treg activation in in vitro functional assays for cancer indications. Next, exemplary IL-2 variant Fc fusion proteins were characterized using functional assays with fresh human peripheral blood mononuclear cells (PBMCs). P-0573 and P-0613 were compared to P-0531 and reference 4 to examine differences in their ability to stimulate STAT5 phosphorylation in CD4+ Treg cells, CD4+ Tconv cells, CD8+ T cells, and NK cells. STAT5 is known to be involved in the downstream signaling cascade in the binding of IL-2 to the transmembrane IL-2 receptor. STAT5 phosphorylation in lymphocyte subpopulations was measured using fresh human peripheral blood mononuclear cells (PBMCs), and Treg populations were identified by FACS analysis using the forkhead transcription factor FOXP3.

[0247] The exemplary IL-2 variant Fc fusion protein P-0573 was characterized by flow cytometry for its induction of Ki-67 expression. The dose-dependent increase in Ki67 expression in human CD4+ T cells, CD8+ T cells, and NK cells in response to P-0573 was compared to that of P-0531 and criterion 4.

[0248] As shown in Figures 25B and 25C, P-0573 and criterion 4 were equally effective in inducing Ki67 expression in NK and CD8+ T cells. In CD4+ T cells (Figure 25A), P-0573 showed a substantial decrease in potency compared to wild-type P-0531, but higher potency than criterion 4. This is likely due to residual binding of P-0573 to IL-2Rα, which, although at a reduced level, still preferentially activated Tregs. To achieve the desired characteristic of Tregs that are activated only at concentrations when CD8C T and NK cells are also activated, more mutations that disrupt the binding of IL-2 to IL-2Rα can be incorporated and combined, such as, but are not limited to, the mutations listed in Table 4F.

[0249] Example 15 Production of a bifunctional IL-2 variant fusion protein The use of recombinant antibody-cytokine fusion proteins (immunocytokines) shows promise in increasing the therapeutic index of cytokines by targeting them to the disease site. Delivering IL-2 variants that preferentially proliferate Treg cells at the intended treatment site may further enhance existing therapeutic responses to various autoimmune and inflammatory diseases.

[0250] In line with this concept, antibody-IL-2 fusion proteins were constructed based on IL-2 variants with biased selectivity for Treg lymphocyte subsets. Exemplary targets include, but are not limited to, integrins α4β7, β7, MAdCAM-1, BAFF, TNFα, and IL-6Rα. As will be understood by those skilled in the art, bifunctional fusion proteins can be constructed using any IL-2 variant with biased selectivity for Treg disclosed herein as a component to enhance or augment antibody-based therapies for autoimmune diseases or inflammatory conditions.

[0251] This invention discloses a wide variety of Treg-selective IL-2 variants with broad levels of potency. The inventors of this invention suggest that the use of attenuated IL-2 variants is likely to facilitate the establishment of a stoichiometric balance between the cytokine arm and the antibody arm. Furthermore, attenuation of cytokine activity is expected to minimize peripheral activation, reduce antigen sink, and facilitate targeting of diseased tissue via the antibody arm.

[0252] Exemplary bifunctional constructs based on anti-inflammatory antibodies and Treg cell-selective IL-2 variants are shown in Table 8. TIFF0007850417000018.tif164169

[0253] In addition to antibodies, the IL-2 variant of the present invention can be bound to proteins that function as targeting moieties, such as TACI. TACI, a transmembrane activator and CAML interactor, is a membrane-bound receptor and a member of the tumor necrosis factor receptor (TNFR) family (von Bullow and Bram, Science 228:138 (1997); Bram and von Bulow, U.S. Patent No. 5,969,102 (1999)). TACI has an extracellular domain containing two cysteine-rich pseudorepeats (SEQ ID NO: 313), a transmembrane domain, and a cytoplasmic domain that interacts with calcium modulators and cyclophylline ligands (CAML). The TACI receptor binds to subsets of B cells and T cells. It binds to two members of the tumor necrosis factor (TNF) ligand family. One ligand is called BAFF or BlyS, and the other is called APRIL.

[0254] Along the same concept of antibody-IL-2 fusion, a TACI-IL-2 fusion molecule based on an IL-2 variant with biased selectivity for the Treg lymphocyte subset was constructed. As can be understood by those skilled in the art, any IL-2 variant and construct (summarized in Table 4) with biased selectivity for Treg disclosed in the present invention can be used as components to construct a bifunctional fusion protein to enhance or augment antibody-based therapy for autoimmune diseases or inflammatory conditions. TACI can be the mature form of the entire mature extracellular domain (amino acids 30 to 165 of SEQ ID NO: 313) or any functional fragment thereof (e.g., SEQ ID NO: 314). To facilitate expression / purification and increase the in vivo half-life, an Fc domain is linked between TACI and the IL-2 variant. The Fc domain is a homodimer or heterodimer with reduced / lost functional activity and / or further extended half-life. TACI can be located at either the N-terminus or the C-terminus of the Fc domain, and the same applies to the IL-2 variant. The linker can be any of flexible or rigid 1 to 100 amino acids, natural or mutated immunoglobulin hinge sequences, and linker peptides (SEQ ID NOs: 48 to 67) listed in Table 5.

[0255] Exemplary bifunctional constructs based on TACI are shown in Table 9. TIFF0007850417000019.tif65170

[0256] Similarly, an IL-2 variant genetically engineered to preferentially proliferate and activate Teff cells while reducing the proliferation and activation of Treg cells can be used as a component to construct a bifunctional fusion protein to enhance cancer therapy. In addition to an antibody targeting the tumor, an immune checkpoint blockade antibody that avoids the immunosuppressive effect in the tumor microenvironment or an immune-stimulating antibody that enhances the existing response can be fused to the IL-2 variant to achieve further enhancement of the immune system's activity against the tumor.

[0257] Examples of immune checkpoint blocking antibodies include, but are not limited to, the PD-1 / PD-L1 blocking antibody JS-001, the anti-CTLA4 antibody ipilimumab, and the agonist CD40 antibody RO7009789. Examples of tumor antigen-targeting antibodies include, but are not limited to, L19 against the extradomain of fibronectin, rituximab against CD20, herceptin against Her-2, and cetuximab against EGFR.

[0258] Example 16 Confirmation of Treg cell selectivity by IL-2 variants in various bifunctional constructs. We investigated the differences in the ability of several exemplary IL-2 variant tocilizumab bifunctional fusion proteins to stimulate STAT5 phosphorylation in CD4-positive Treg and Tconv cells. STAT5 phosphorylation in the defined lymphocyte subpopulations was revealed by FACS analysis using fresh human PBMCs, as described in the example above.

[0259] Figure 26 shows the dose-response effects of exemplary tocilizumab IL-2 variant bifunctional fusions on STAT5 phosphorylation in CD4+ Treg and Tconv cells. P-0536 (SEQ ID NOs. 253 and 255) and P-0546 (SEQ ID NOs. 253, 254, and 256) both contain IL-2 with the L19H / S125I / Q126E mutation; P-0536 contains a divalent IL-2 variant at the C-terminus of the tocilizumab heavy chain, while P-0546 contains a monomeric IL-2 variant linked to a knob-containing heterodimeric heavy chain. P-0559 (SEQ ID NOs. 253 and 265) and P-560 (SEQ ID NOs. 253, 254, and 266) are bifunctional counterparts of divalent and monovalent IL-2 variants, respectively, containing IL-2 with the D20Q / S125I mutation. For comparison, we included P-0511, an IL-2 variant Fc fusion protein containing the L19H / S125I / Q126E mutation in IL-2.

[0260] Figure 3 shows the ability of the IL-2 variant P-0375(N88Q) to induce STAT5 phosphorylation in CD4-positive Treg and CD4+Tconv cells, compared to reference 1 and reference 2 compounds, respectively, which possess the V91K and N88R mutations. The activity profile of the N88Q variant was similar to that of reference 2.

[0261] P-0536 and P-0546 exhibited similar activity profiles to P-0511 in inducing STAT5 phosphorylation in Treg and Tconv cells (Figures 26A and 26B). As expected, the dimerized bifunctional fusion P-0536 showed slightly higher activity than its monomeric counterpart P-0546, which is likely due to the avidity effect. These results suggest that the antibody IL-2 variant fusion retained the activity and selectivity of its Fc fusion counterpart.

[0262] Furthermore, P-0559 and P-0560 demonstrated selective activation of Treg cells compared to CD4+ Tconv cells (Figures 26A and 26B). Compared to P-0511, P-0559 showed reduced potency in inducing STAT5 phosphorylation in Treg cells, but such activity was essentially abolished in CD4+ Tconv cells, resulting in a broad window of selectivity. Interestingly, the dimerized bifunctional fusion P-0559 showed reduced Treg-inducing potency than its monomeric equivalent P-0560 (EC 50 These are 146 nM and 12.1 pM, respectively.

[0263] Further IL-2 variant tocilizumab bifunctional fusion proteins, exemplified by P-0588(D20S / S125I), P-0589(D20N / S125I), and P-0590(L19N / S125I / Q126E), exhibited activity profiles similar to P-0559, namely reduced potency in inducing STAT5 phosphorylation in Treg cells but a wider selectivity window than in conventional CD4+ T cells (Figures 26C and 26D). Furthermore, P-0590 induced lower signaling amplitudes and exhibited similar partial agonist properties to its Fc fusion counterpart, P-0859 (Figure 6E).

[0264] Furthermore, attenuation of efficacy and selectivity for Treg cells can also be achieved by various amino acid substitutions at the Q126 position, which is essential for γc interaction. Several IL-2 variant totocilizumab bifunctional fusion proteins were constructed and evaluated by pSTAT assay. All of these compounds share the same L19H / S125 mutation as P-0536, and the substitutions at the Q126 position are Q126D, Q126H, Q126N, Q126R, Q126S, and Q126T in P-0694, P-0695, P-0697, P-0698, P-0699, and P-0700, respectively, while P-0536 contains the Q126E substitution. As shown in Figures 26E and 26F, all other substitutions tested, except for Q126D in P-0694, neither impaired protein activity nor improved Treg selectivity. Similar to P-0559, P-0694 showed reduced potency in inducing STAT5 phosphorylation in Treg cells, but such activity was essentially abolished in CD4+ Tconv cells, resulting in a broad selection window (Figures 26E and 26F). Consequently, Q126D substitutions, in combination with the various Rβ disruption mutations disclosed herein, allowed for further fine-tuning of activity to achieve desired potency, signaling intensity, and biased specificity to Treg cells.

[0265] Example 17 In mice, the bifunctional construct of the IL-2 variant preferentially promotes the proliferation of Treg cells. The IL-2 variant tocilizumab bifunctional proteins P-0536, P-0546, P-0559, and P-0560 were administered to mice along with the IL-2 variant Fc fusion protein P-0511. The ability of these proteins to preferentially proliferate and increase regulatory T cells (CD4+CD25+FoxP3+ T cells) compared to effector T cells and NK cells was demonstrated in vivo. Since tocilizumab does not exhibit species cross-reactivity with mouse IL-6Rα, this in vivo experiment aimed to determine the phenotypic response of cells to IL-2 variants with different mutations and valencies in the context of the bifunctional construct.

[0266] Female C57 / BL6 mice (7 weeks old) were received from Charles River Laboratory and acclimatized in the facility for at least 7 days prior to the study. On day 0, the mice were subcutaneously administered the vehicle (PBS) and each test compound at 15 nmol / kg. Peripheral blood samples were collected in heparin-treated tubes on days 2, 4, and 8 post-treatment. Five mice were placed in each group, and baseline blood was collected 3 days prior to treatment (-3 days).

[0267] All IL-2 compounds tested stimulated Treg cell proliferation and growth, as indicated by an increase in Ki67-positive Treg cells and an increase in the proportion of Treg cells to total CD4+ T cells or total lymphocytes (Figures 27A-27C). Increased Ki67 expression was observed 2 days post-injection and peaked 4 days post-single injection. At dose concentrations of 15 nmol / kg or 1.2-2.7 mg / kg depending on the molecular weight of each compound, Ki67 expression in Treg cells increased to 100% on 4 days with all IL-2 compounds tested. Treg cell proliferation was not observed on 2 days but became marked on 4 days. After IL-2 compound stimulation, Treg cells accounted for up to 40% of the CD4+ T cell subpopulation and 12% of total lymphocytes. In contrast to in vitro observations, the two monomeric IL-2 variant bifunctional molecules, P-0546 and P-0560, showed the highest potency in Treg cell proliferation and proliferation induction. However, the relative ranking of in vivo potency among P-0546 and P-0560, as well as their dimer counterparts P-0536 and P-559, was consistent with the in vitro human PBMC cell assay. Furthermore, the IL-2 variant Fc fusion P-0511 showed comparable in vivo Treg stimulating effect to P-0560, despite being more than 100-fold potent in the in vitro cell assay.

[0268] All tested IL-2 compounds also showed Ki67 stimulation to CD4+Tconv (CD4+Foxp3-), activated CD4+T (CD4+CD25+Foxp3-), CD8+T, and NK cells (Figures 28A-28D). The relative potency ranking among the different compounds followed the same trend observed in Treg cells of the same group in the administered mice. No significant proliferation of CD4+Tconv cells, CD8 T cells, or NK cells was observed in mice administered any of the tested IL-2 compounds (Figures 29A, 29C-D). Since IL-2Rα expression was induced in CD4+T cells, there was a slight increase in activation of this T cell subset in response to the most potent compounds, P-0546 and P-0560 (Figure 29B).

[0269] Furthermore, all IL-2 variant bifunctional compounds also showed beneficial Treg / Tconv ratios in terms of both Ki67 stimulation and cell number-based cell proliferation (Figures 30A and 30B). They also showed high expression of CD25 and Foxp3 and CD25 markers (Figures 31A and 31B). Among the four bifunctional compounds, P-0546 and P-0560 consistently showed the highest potency in stimulating Treg cell proliferation and growth, the most beneficial Treg / Tconv ratio based on cell number, and the highest CD25 expression in Treg cells, suggesting superior Treg activation and functionality. These compounds may be useful as therapeutic agents to combat autoimmune and inflammatory diseases, as well as organ transplant rejection.

[0270] All articles and methods disclosed and claimed herein can be manufactured and performed without any unnecessary experimentation by reference to this disclosure. Although the articles and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications to the articles and methods may be made without departing from the spirit and scope of this disclosure. All such modifications and equivalents, whether existing or subsequently developed, which are apparent to those skilled in the art, are deemed to be included in the spirit and scope of this disclosure as defined by the appended claims. All patents, patent applications, and publications cited herein represent the level of those skilled in the art in which this disclosure pertains. All patents, patent applications, and publications cited herein are incorporated herein by reference for all purposes to the same extent that each individual publication is explicitly and individually indicated as being incorporated herein by reference for all purposes. Disclosures described herein by example may be preferably carried out in the absence of any elements not specifically disclosed herein. In other words, although this disclosure is specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the concepts disclosed herein may be made by those skilled in the art, and that such modifications and variations shall be deemed to fall within the scope of this disclosure as defined by the appended claims.

[0271] Sequence List In the nucleic acid and amino acid sequences listed in the attached sequence listing, nucleotide bases are indicated using standard abbreviations and amino acids are indicated using single-letter notation, as required by §1.822 of the U.S. Patent Act Enforcement Rules. Sequence ID 1 is the amino acid sequence of the human IL-2 precursor. Sequence ID 2 is a naturally occurring amino acid sequence of mature human IL-2. Sequence ID 3 is the wild-type amino acid sequence for mature human IL-2. Sequence IDs 4-43, 113-151, 208-212, and 275-292 are amino acid sequences of various IL-2 variants for preferential Treg activation. Sequence ID 44 is the amino acid sequence of human IgG1-Fc. Sequence ID 45 is the sequence of human IgG1-Fc with reduced / absent effector function. Sequence ID 46 is the amino acid sequence of Knob-Fc. Sequence ID 47 is the amino acid sequence of Hole-Fc. Sequence IDs 48-67 are amino acid sequences of various peptide linker sequences. Sequence ID 68 is the amino acid sequence of the alpha-Sushi domain of the human IL-2 receptor. Sequence IDs 69-70 and 196-197 are the amino acid sequences of the IL-2 and IL-2RSushiFc fusion proteins. Sequence IDs 71 and 72 are the amino acid sequences of the wild-type IL-2 Fc fusion protein. Sequence IDs 73-112, 152-194, 213-219, and 300-306 are amino acid sequences of various IL-2 Fc fusion proteins for preferential Treg activation. Sequence IDs 195 and 198-199 are amino acid sequences of the reference Fc-IL-2 variant fusion protein for preferential Treg activation. Sequence IDs 200-207 are the amino acid sequences of various antibody IL-2 variant fusion constructs. Sequence IDs 220-234 and 293-299 are amino acid sequences of various IL-2 variants that reduce Treg activation. Sequence IDs 235-249 are amino acid sequences of various Fc-IL-2 fusion proteins that reduce Treg activation. Sequence ID 250 is the amino acid sequence of a reference Fc-IL-2 variant fusion protein that reduces Treg activation. Sequence numbers 251-252 are sequences of human IgG1-Fc with reduced / absent effector function and extended half-life. Sequence IDs 253-268 are the amino acid sequences of various tocilizumab-IL-2 variant bifunctional constructs. Sequence IDs 269-274 and 307-312 are amino acid sequences of various belimumab-IL-2 variant bifunctional constructs. Sequence ID 313 is the amino acid sequence of the TACI extracellular domain. Sequence ID 314 is the amino acid sequence of a functional TACI ECD fragment. Sequence IDs 315-320 are amino acid sequences of various TACI-IL-2 variant bifunctional constructs.

[0272] Sequence List Sequence of human IL-2 precursor MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(Sequence ID 1) Naturally occurring sequences of mature human IL-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(Sequence ID 2) Human IL-2 mature wild-type sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 3) Sequence of the IL-2 N88R variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 4) IL-2 D20T variant array APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 5) Sequence of IL-2 D20E variant APTSSSTKKTQLQLEHLLLELQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 6) Sequence of IL-2 D20N variant APTSSSTKKTQLQLEHLLLNLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 7) Sequence of the IL-2 D20Q variant APTSSSTKKTQLQLEHLLLQLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 8) IL-2 D20S variant array APTSSSTKKTQLQLEHLLLSLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 9) IL-2 D20Y variant sequence APTSSSTKKTQLQLEHLLLYLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 10) Sequence of IL-2 D20I variant APTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 11) Sequence of the IL-2 L19Y variant APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 12) Sequence of the IL-2 L19N variant APTSSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 13) Sequence of the IL-2 L19R variant APTSSSTKKTQLQLEHLLRDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 14) Sequence of the IL-2 N88G variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 15) Sequence of the IL-2 N88I variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISIINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 16) Sequence of the IL-2 N88Q variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISQINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 17) Sequence of the IL-2 N88E variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISEINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 18) IL-2 N88T variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 19) Sequence of the IL-2 N88M variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISMINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 20) Sequence of the IL-2 Q126E variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT(Sequence ID 21) Sequence of the IL-2 Q126L variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSLSIISTLT(Sequence ID 22) Sequence of the IL-2 Q126N variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSNSIISTLT(Sequence ID 23) Sequence of the IL-2 Q126D variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSDSIISTLT(Sequence ID 24) Sequence of the IL-2 Q126M variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSMSIISTLT(Sequence ID 25) Sequence of IL-2 D20I / N88G variant APTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 26) Sequence of IL-2 D20I / N88R variant APTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 27) IL-2 D20T / N88R variant arrangement APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 28) Sequence of IL-2 D20I / N88I variants APTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISIINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 29) IL-2 D20T / Q126E variant arrangement APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT(Sequence ID 30) IL-2 D20T / N88R / Q126E variant arrangement APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT(Sequence ID 31) IL-2 D20T / Q126L variant arrangement APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSLSIISTLT(Sequence ID 32) IL-2 D20T / N88R / Q126L variant arrangement APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSLSIISTLT(Sequence ID 33) Sequence of the IL-2 L19N / Q126E variant APTSSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT(Sequence ID 34) Sequence of IL-2 L19R / Q126E variant APTSSSTKKTQLQLEHLLRDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT(Sequence ID 35) Sequence of the IL-2 L19Y / Q126E variant APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT(Sequence ID 36) Sequence of the IL-2 L19Q variant APTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 37) Sequence of the IL-2 L19H variant APTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 38) Sequence of the IL-2 L19D variant APTSSSTKKTQLQLEHLLDDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 39) Sequence of the IL-2 L19P variant APTSSSTKKTQLQLEHLLPDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 40) IL-2 D20T / S125I / Q126K variant arrangement APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT(Sequence ID 41) Sequence of IL-2 L19N / S125I / Q126K variants APTSSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT(Sequence ID 42) IL-2 L19R / S125I / Q126K variant arrangement APTSSSTKKTQLQLEHLLRDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT(Sequence ID 43) Human IgG1-Fc DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 44) Human IgG1-Fc with reduced / absent effector function DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(Sequence ID 45) Knob-FC DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(Sequence ID 46) Hole-Fc DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(Sequence ID 47) Peptide linker sequence GGGSGGGSGGGS (Sequence ID 48) Peptide linker sequence GGGS (SEQ ID NO: 49) Peptide linker sequence: GSSGGSGGSGGSG (SEQ ID NO: 50) Peptide linker sequence GSSGT (SEQ ID NO: 51) Peptide linker sequence: GGGGSGGGGSGGGS (Sequence ID 52) Peptide linker sequence AEAAAKEAAAKEAAAKA (SEQ ID NO: 53) Peptide linker sequence: GGGGSGGGGSGGGGSGGGGS (Sequence ID 54) Peptide linker sequence GGGSGGGS (SEQ ID NO: 55) Peptide linker sequence GS (SEQ ID NO: 56) Peptide linker sequence GGS (SEQ ID NO: 57) Peptide linker sequence GGGGS (SEQ ID NO: 58) Peptide linker sequence GGSG (SEQ ID NO: 59) Peptide linker sequence SGGG (SEQ ID NO: 60) Peptide linker sequence GSGS (SEQ ID NO: 61) Peptide linker sequence GSGSGS (SEQ ID NO: 62) Peptide linker sequence GSGSGSGS (SEQ ID NO: 63) Peptide linker sequence GSGSGSGSGS (Sequence ID 64) Peptide linker sequence GSGSGSGSGSGS (Sequence ID 65) Peptide linker sequence GGGGSGGGGS (Sequence ID 66) Peptide linker sequence: GGGGSGGGGSGGGGS (Sequence ID 67) Sequence of the human IL-2Rαsushi domain ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG (Sequence ID 68) P-0327 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(SEQ ID NO: 69) P-0422 (Sequence ID 70) P-0250 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 71) P-0305 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTAEAAAKEAAAKEAAAKACPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(Sequence ID 72) P-0254 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID: 73) P-0363 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 74) P-0364 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLELQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 75) P-0365 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLNLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 76) P-0366 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLQLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 77) P-0367 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLSLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 78) P-0368 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLYLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 79) P-0252 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence No. 80) P-0372 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 81) P-0373 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 82) P-0374 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLRDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 83) P-0253 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 84) P-0302 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISIINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 85) P-0375 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISQINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 86) P-0376 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISEINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 87) P-0377 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 88) P-0378 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISMINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 89) P-0303 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT(Sequence No. 90) P-0304 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSLSIISTLT(Sequence ID 91) P-0369 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSNSIISTLT(Sequence No. 92) P-0370 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSDSIISTLT(Sequence No. 93) P-0371 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSMSIISTLT(Sequence No. 94) P-0251 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 95) P-0317 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence No. 96) P-0318 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISIINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 97) P-0324 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 98) P-0322 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT(Sequence No. 99) P-0323 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSLSIISTLT(Sequence ID 100) P-0325 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT(Sequence No. 101) P-0326 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSLSIISTLT(Sequence No. 102) P-0417 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT(Sequence No. 103) P-0418 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLRDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT(Sequence No. 104) P-0419 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT(Sequence No. 105) P-0416 APTSSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTAEAAAKEAAAKEAAAKACPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(Sequence ID 106) P-0412 APTSSSTKKTQLQLEHLLLELQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTAEAAAKEAAAKEAAAKACPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(Sequence ID 107) P-0306 APTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTAEAAAKEAAAKEAAAKACPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(Sequence ID 108) P-0319 APTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTAEAAAKEAAAKEAAAKACPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(Sequence ID 109) P-0582 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT(Sequence No. 110) P-0583 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT(Sequence No. 111) P-0584 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLRDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT(Sequence No. 112) Sequence of IL-2 L19S variant APTSSSTKKTQLQLEHLLSDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 113) Sequence of IL-2 L21S variant APTSSSTKKTQLQLEHLLLDSQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 114) Sequence of the IL-2 L21N variant APTSSSTKKTQLQLEHLLLDNQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 115) Sequence of the IL-2 L21R variant APTSSSTKKTQLQLEHLLLDRQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 116) Sequence of the IL-2 Q126K variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSKSIISTLT(Sequence ID 117) Sequence of the IL-2 Q126H variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSHSIISTLT(Sequence ID 118) Sequence of the IL-2 Q126Y variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSYSIISTLT(Sequence ID 119) IL-2 S125E variant array APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFEQSIISTLT(Sequence No. 120) IL-2 S125K variant array APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFKQSIISTLT(Sequence ID 121) IL-2 S125H variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFHQSIISTLT(Sequence ID 122) IL-2 S125W variant array APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFWQSIISTLT(Sequence ID 123) IL-2 S125I variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(Sequence ID 124) Sequence of the IL-2 Q22N variant APTSSSTKKTQLQLEHLLLDLNMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 125) Sequence of the IL-2 Q22H variant APTSSSTKKTQLQLEHLLLDLHMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 126) Sequence of the IL-2 Q22K variant APTSSSTKKTQLQLEHLLLDLKMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 127) Sequence of the IL-2 Q22Y variant APTSSSTKKTQLQLEHLLLDLYMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 128) Sequence of the IL-2 Q22I variant APTSSSTKKTQLQLEHLLLDLIMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 129) Sequence of the IL-2 L19H / Q126E variant APTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT(Sequence No. 130) Sequence of IL-2 L19Q / Q126E variant APTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT(Sequence ID 131) Sequence of IL-2 L19S / Q126E variant APTSSSTKKTQLQLEHLLSDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT(Sequence ID 132) Sequence of the IL-2 L19Y / Q126K variant APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSKSIISTLT(Sequence ID 133) Sequence of the IL-2 L19Y / Q126H variant APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSHSIISTLT(Sequence ID 134) Sequence of the IL-2 L19Y / Q126Y variant APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSYSIISTLT(Sequence ID 135) IL-2 L19Y / S125E variant arrangement APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFEQSIISTLT(Sequence ID 136) IL-2 L19Y / S125K variant arrangement APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFKQSIISTLT(Sequence ID 137) IL-2 L19Y / S125H variant arrangement APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFHQSIISTLT(Sequence ID 138) IL-2 L19Y / S125W variant arrangement APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFWQSIISTLT(Sequence ID 139) IL-2 L19Y / S125I variant arrangement APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(Sequence ID 140) Sequence of the IL-2 L19Y / Q22N variant APTSSSTKKTQLQLEHLLYDLNMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 141) Sequence of the IL-2 L19Y / Q22H variant APTSSSTKKTQLQLEHLLYDLHMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 142) Sequence of the IL-2 L19Y / Q22K variant APTSSSTKKTQLQLEHLLYDLKMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 143) Sequence of IL-2 L19Y / Q22Y variant APTSSSTKKTQLQLEHLLYDLYMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 144) Sequence of the IL-2 L19Y / Q22I variant APTSSSTKKTQLQLEHLLYDLIMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 145) Sequence of IL-2 L19H / Q126K variant APTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSKSIISTLT(Sequence ID 146) Sequence of IL-2 L19H / S125I variant APTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(Sequence ID 147) Sequence of IL-2 L19D / S125I variant APTSSSTKKTQLQLEHLLDDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(Sequence ID 148) IL-2 D20E / S125I variant arrangement APTSSSTKKTQLQLEHLLLELQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(Sequence ID 149) IL-2 D20T / S125I variant array APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(Sequence No. 150) Sequence of IL-2 L19Y / S125I / Q126E variants APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLT(Sequence ID 151) P-0423 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence No. 152) P-0424 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence No. 153) P-0425 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLDDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 154) P-0426 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLPDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence No. 155) P-0427 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLSDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence No. 156) P-0428 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDSQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence No. 157) P-0429 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDNQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence No. 158) P-0430 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDRQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence No. 159) P-0497 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSKSIISTLT(Sequence No. 160) P-0498 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSHSIISTLT(Sequence No. 161) P-0499 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSYSIISTLT(Sequence No. 162) P-0500 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFEQSIISTLT(Sequence No. 163) P-0501 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFKQSIISTLT(Sequence No. 164) P-0502 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFHQSIISTLT(Sequence No. 165) P-0503 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFWQSIISTLT(Sequence No. 166) P-0531 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(Sequence No. 167) P-0505 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLNMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 168) P-0506 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLHMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence No. 169) P-0507 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLKMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence No. 170) P-0508 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLYMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 171) P-0509 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLIMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence No. 172) P-0447 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT(Sequence No. 173) P-0448 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT(Sequence No. 174) P-0449 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLSDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT(Sequence No. 175) P-0464 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSKSIISTLT(Sequence No. 176) P-0465 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSHSIISTLT(Sequence No. 177) P-0466 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSYSIISTLT(Sequence No. 178) P-0467 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFEQSIISTLT(Sequence No. 179) P-0468 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFKQSIISTLT(Sequence No. 180) P-0469 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFHQSIISTLT(Sequence ID 181) P-0470 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFWQSIISTLT(Sequence No. 182) P-0471 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(Sequence ID 183) P-0472 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLNMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence ID 184) P-0473 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLHMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence No. 185) P-0474 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLKMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence No. 186) P-0475 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLYMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence No. 187) P-0476 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLIMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(Sequence No. 188) P-0480 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSKSIISTLT(Sequence No. 189) P-0491 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFI...

Claims

1. 1) an isolated bifunctional fusion protein comprising an IL-2 variant polypeptide and 2) a heterologous protein, The IL-2 variant polypeptide is selected from the group consisting of an IL-2 variant polypeptide having the amino acid sequence shown in SEQ ID NO: 3 having amino acid substitutions L19H, S125I, and Q126E, and an IL-2 variant polypeptide having the amino acid sequence shown in SEQ ID NO: 3 having amino acid substitutions D20Q and S125I. The heterologous protein comprises a functional portion that targets molecules concentrated in the target tissue, and the heterologous protein is an IL-6R antibody or antibody fragment. An isolated, dual-functional fusion protein characterized by the following:

2. The isolated fusion protein according to claim 1, wherein the IL-2 variant polypeptide is fused to the C-terminal amino acid of the heterologous protein at its N-terminal amino acid, either in monomeric or dimeric form.

3. The isolated fusion protein according to claim 2, wherein the IL-2 variant polypeptide is fused to the C-terminal amino acid of the heterologous protein at its N-terminal amino acid using a peptide linker, either in monomeric or dimeric form.

4. The isolated fusion protein according to claim 1, wherein the IL-2 variant polypeptide is fused to the N-terminal amino acid of the heterologous protein at its C-terminal amino acid, either in monomeric or dimeric form.

5. The isolated fusion protein according to claim 4, wherein the IL-2 variant polypeptide is fused to the N-terminal amino acid of the heterologous protein at its C-terminal amino acid using a peptide linker, either in monomeric or dimeric form.

6. 1) an isolated bifunctional fusion protein comprising an IL-2 variant polypeptide and 2) a heterologous protein, The IL-2 variant polypeptide is selected from the group consisting of an IL-2 variant polypeptide having the amino acid sequence shown in SEQ ID NO: 3 having amino acid substitutions L19H, S125I, and Q126E, and an IL-2 variant polypeptide having the amino acid sequence shown in SEQ ID NO: 3 having amino acid substitutions D20Q and S125I. The isolated bifunctional fusion protein is selected from the group consisting of: isolated bifunctional fusion proteins comprising the amino acid sequences shown in SEQ ID NOs. 253 and 255; isolated bifunctional fusion proteins comprising the amino acid sequences shown in SEQ ID NOs. 253, 254, and 256; isolated bifunctional fusion proteins comprising the amino acid sequences shown in SEQ ID NOs. 253 and 265; and isolated bifunctional fusion proteins comprising the amino acid sequences shown in SEQ ID NOs. 253, 254, and 266. An isolated, dual-functional fusion protein characterized by the following:

7. A pharmaceutical composition comprising an isolated fusion protein according to any one of claims 1 to 6, mixed with a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7 for treating an autoimmune disease in a subject.

9. A pharmaceutical composition according to claim 7 for treating an inflammatory disease in a subject.

10. An isolated nucleic acid molecule encoding a fusion protein according to any one of claims 1 to 6.

11. An expression vector comprising the nucleic acid molecule described in claim 10.

12. A host cell comprising the nucleic acid molecule according to claim 10 or the expression vector according to claim 11.

13. A method for producing a fusion protein according to any one of claims 1 to 6, comprising culturing the host cells according to claim 12 under conditions that promote the expression of the fusion protein and recovering the fusion protein.

14. An isolated protein prepared using the method described in claim 13.

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