Novel interleukin-2 variant for cancer treatment

Abstract

The present invention relates to polypeptides that share the primary sequence with human IL-2 except for a number of mutated amino acids. A group of IL-2 variants contains mutations that substantially reduce the ability of these polypeptides to stimulate Treg cells and render them more effective in the treatment of tumors. Also included is the therapeutic use of these mutant variants alone, or in combination with vaccines, or TAA-targeted biologics, or immune checkpoint blockers, or as building blocks in bifunctional molecular constructs, for the treatment of diseases such as cancer or infections where Treg activity is not desired. 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 modulation of the immune system's action on diseases such as autoimmune and inflammatory disorders, cancer, and various infectious diseases.

Description

[0001] Related patent applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 947,806, filed Dec. 13, 2019, and U.S. Provisional Application No. 62 / 861,651, filed Jun. 14, 2019, each of which is incorporated herein by reference in its entirety. Background of the Invention

[0003] Interleukin-2 (IL-2) was the first described growth factor for T cells. Since its discovery, IL-2 has been shown to promote T cell proliferation and survival in vitro (Smith, K A. (1988) Science. 240, 1169-76) and to enhance the immune response in the context of T viral infection (Blattman, J N et al., (2003) Nat Med 9, 540-7) or vaccination (Fishman, M. et al., (2008) JImmunother. 31, 72-80, Kudo-Saito, C. et al., (2007) Cancer Immunol Immunother. 56, 1897-910; Lin, C T. et al., (2007) Immunol Lett. 114, 86-93).

[0004] IL-2 has been used in cancer treatment. Recombinant human IL-2 is an effective immunotherapy for metastatic melanoma and renal cell carcinoma, with durable responses in approximately 10% of patients. However, its short half-life and severe toxicity limit optimal dosing. In addition, IL-2 binds with high affinity to its heterotrimeric receptor IL-2Rαβγ, preferentially expanding immunosuppressive regulatory T cells (Tregs) that express high constitutive levels of IL-2Rα. Expansion of Tregs represents an undesirable effect of IL-2 on cancer immunotherapy. Thus, successful immunotherapy for cancer using IL-2 must address two fundamental important issues: 1) how to limit side effects while being active where needed; and 2) how to preferentially activate effector T cells while limiting Treg stimulation.

[0005] It has recently been found that IL-2 can be modified to selectively stimulate cytotoxic effector T cells. Various methods have led to the production of IL-2 variants with improved and selective immunostimulatory capabilities. Some of these IL-2 variants are designed to increase the ability of the molecule to signal primarily through the high-affinity receptor (α-chain, β-chain, and γ-chain) rather than through the intermediate-affinity receptor (β-chain and γ-chain). The basic idea is to promote signaling in T cells rather than in NK cells, where signaling in NK cells is thought to be the cause of the observed toxic effects. The following inventions are all within this series of work: U.S. Patent No. 7,186,804, U.S. Patent No. 7,105,653, U.S. Patent No. 6,955,807, U.S. Patent No. 5,229,109, U.S. Patent Application 20050142106. It is important to note that none of these inventions relate to IL-2 variants that have greater therapeutic efficacy in vivo than native IL-2.

[0006] In summary, IL-2 is a highly pleiotropic cytokine that is closely related to the biological activities of different cell populations. This property makes IL-2 an important node in the regulation of the immune response and makes IL-2 an attractive target for therapy and complex immunomodulation. In addition, receptor subunit-biased IL-2 variants can be prepared to achieve IL-2-mediated selective immunomodulation, preferentially amplifying and activating Teff cells to attack cancer cells while reducing the amplification and activation of Treg cells.

[0007] Disclosure of the invention

[0008] In one aspect, the present invention relates to the generation of IL-2 mutant variants that are characterized as selective agonists of IL-2 activity and have reduced or eliminated binding ability to IL-2Rα. In particular, these variants will provide a method to overcome the limitations observed in native IL-2 therapy, which stem from the proven ability of native IL-2 to expand native regulatory T cells in vivo. The present invention relates to polypeptides that share their primary sequence with human IL-2 except for a number of mutated amino acids. The introduced mutations significantly reduce the ability of these polypeptides to stimulate Treg cells and confer greater efficacy to IL-2. In addition, the introduced mutations are expected to reduce CD25-mediated VLS and CD25-mediated sink effect. The present invention relates to polypeptides that share their primary sequence with human IL-2 except for one to a number of mutated amino acids. The present invention also includes the therapeutic use of these mutant variants alone or in combination with vaccines, or immune checkpoint inhibitors, or tumor-associated antigen (TAA)-targeted biologics, or as part of a bifunctional fusion construct, for the treatment of diseases such as cancer or infection where regulatory T cell (Treg) activity is not desired.

[0009] In one aspect, the present invention relates to the generation of IL-2 mutant variants that are characterized as selective agonists of IL-2 activity and have an optimally regulated overall potency by reducing IL-2Rβγ interaction in addition to reduced or eliminated binding ability to IL-2Rα. The introduced mutations prevent overactivation of the pathway, reduce undesired "on-target" "off-tissue" toxicity, decrease potential absorption, reduce activation-induced cell exhaustion associated with lymphocyte overstimulation, mitigate receptor-mediated IL-2 internalization, and thus prolong the in vivo half-life and result in slow and persistent pharmacodynamics to improve biodistribution, bioavailability, function, and anti-tumor efficacy. The inventors also propose that using IL-2 variants with reduced or eliminated binding to IL-2Rα and attenuated IL-2Rβγ activity will facilitate the establishment of a stoichiometric balance between the cytokine and antibody arms that exhibit significantly different potencies and molecular weights to allow optimal dosing and maintenance of the function of each arm. The present invention relates to polypeptides that share their primary sequence with human IL-2 except for one to several mutated amino acids. The present invention also includes the therapeutic use of these mutant variants alone or in combination with vaccines, or immune checkpoint inhibitors, or tumor-associated antigen (TAA)-targeted biologics, or as part of a bifunctional fusion construct for treating diseases such as cancer or infection.

[0010] In one aspect, the present invention relates to the generation of IL-2 mutant variants that are characterized as selective agonists of IL-2 activity and have reduced IL-2Rβγ interaction in addition to reduced or eliminated binding ability to IL-2Rα. The introduced mutations provide prolonged and persistent pharmacodynamics and potentially pharmacokinetics. In addition, the introduced mutations reduce cell exhaustion and activation-induced cell death and enhance persistent lymphocyte responsiveness. Thus, the introduced mutations allow for less frequent dosing regimens and provide clinical dosing convenience. The cost of goods is also expected to be reduced. The present invention relates to polypeptides that share their primary sequence with human IL-2 except for one to several mutated amino acids. The present invention also includes the therapeutic use of these mutant variants alone or in combination with vaccines, or immune checkpoint inhibitors, or tumor-associated antigen (TAA)-targeted biologics, or as part of a bifunctional fusion construct for treating diseases such as cancer or infection.

[0011] In one aspect, the present invention relates to the generation of IL-2 mutant variants that are characterized as selective agonists of IL-2 activity, having eliminated binding to IL-2Rα and an unexpectedly high level of enhanced effector T cell and NK cell responses that cannot be matched by wild-type counterparts. The CD25-binding eliminating mutations are expected to reduce the sink to CD25 or CD25+ cells and thus increase the availability to IL-2Rβγ. Enriched receptor occupancy triggers a strong cytotoxic cell response and a strong tumor killing efficacy. The present invention relates to polypeptides that share their primary sequence with human IL-2 except for one to several mutated amino acids. The present invention also includes the therapeutic use of these mutant variants alone or in combination with vaccines, or immune checkpoint inhibitors, or tumor-associated antigen (TAA)-targeted biologics, or as part of a bifunctional fusion construct for treating diseases such as cancer or infections.

[0012] In one aspect of the present invention, the introduced mutations reduce the binding ability to IL-2Rα (CD25), but retain a low level of Treg response. The residual immunomodulatory Tregs provide immune balance to improve systemic tolerance and ensure that the immune balance does not overly bias towards cytotoxic effector cells. The finely tuned Treg response is set not to suffer from tumor killing efficacy, but strong enough to maintain peripheral tolerance. The present invention relates to polypeptides that share their primary sequence with human IL-2 except for one to several mutated amino acids. The present invention also includes the therapeutic use of these mutant variants alone or in combination with vaccines, or immune checkpoint inhibitors, or tumor-associated antigen (TAA)-targeted biologics, or as part of a bifunctional fusion construct for treating diseases such as cancer or infections.

[0013] In one aspect, the present invention relates to the generation of IL-2 mutant variants that have reduced aggregation, increased expression, improved manufacturability and developability, accompanied by a combination of properties including, for example, a substantially reduced ability to stimulate Treg cells, reduced receptor overactivation, reduced undesired "on-target" "off-tissue" toxicity, and extended pharmacodynamics to improve biodistribution, bioavailability, function, and anti-tumor efficacy. The present invention relates to polypeptides that share their primary sequence with human IL-2 except for one to several mutated amino acids. The present invention also includes the therapeutic use of these mutant variants alone or in combination with vaccines, or immune checkpoint inhibitors, or tumor-associated antigen (TAA)-targeted biologics, or as part of a bifunctional fusion construct for treating diseases such as cancer or infections.

[0014] In one aspect, the present invention relates to the generation of IL-2 mutant variants that are characterized by a reduction in severe toxicities such as the vascular leak syndrome (VLS) associated with high-dose IL-2 in the clinical treatment of renal cancer and melanoma. In particular, the introduced mutations substantially reduce the binding ability to IL-2Rα (CD25); thus, binding to CD25+ lung endothelial cells is weakened, and endothelial cell damage is expected to be prevented and VLS is significantly reduced. The present invention relates to polypeptides that share their primary sequence with human IL-2 except for one to several mutated amino acids. The present invention also includes the therapeutic use of these mutant variants alone or in combination with vaccines, or immune checkpoint modulators, or tumor-associated antigen (TAA)-targeted biologics, or as part of a bifunctional fusion construct for treating diseases such as cancer or infection to improve the safety profile.

[0015] The present invention allows for a substantial improvement in the current IL-2-based immunomodulatory strategies in cancer treatment. In particular, replacing native IL-2 with the mutant variants described herein will result in not preferentially stimulating Treg cells over cytotoxic effector cells, reducing the undesired "hit the target" "miss the tissue" toxicity, minimizing cell exhaustion associated with overstimulation, and improving pharmacodynamics and potentially pharmacokinetics. The mutations are expected to weaken the binding to CD25+ lung endothelial cells and thus reduce VLS. In various embodiments, the IL-2 variant (or mutant) comprises a sequence of an IL-2 variant (or mutant) derived from the mature human IL-2 polypeptide sequence listed in SEQ ID NO:3. In various embodiments, the IL-2 variant 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 NO:31-66, or SEQ ID NO:111-120, or amino acids 9-133, 10-133, and 11-113 of SEQ ID NO:47.

[0016] In another aspect, the IL-2 variant of the invention is attached to at least one heterologous protein. In various embodiments, the IL-2 variant is fused to at least one polypeptide that confers an extended half-life to the fusion molecule. Such polypeptides include IgG Fc or other polypeptides that bind to the neonatal Fc receptor, human serum albumin, or polypeptides that bind to proteins with an extended serum half-life. In various embodiments, the IL-2 variant is fused to an IgG Fc molecule. In various embodiments, the Fc domain is a human IgG Fc domain. In various embodiments, the Fc domain is derived from the human IgG1 heavy chain constant domain sequence set forth in SEQ ID NO:6. In various embodiments, the Fc domain is an Fc domain having the amino acid sequence set forth in SEQ ID NO:7. In various embodiments, the Fc domain is an Fc domain having the amino acid sequence set forth in SEQ ID NO:8. In various embodiments, the Fc domain is derived from the human IgG2 heavy chain constant domain sequence. In various embodiments, the Fc domain is derived from the human IgG4 heavy chain constant domain sequence.

[0017] In various embodiments, the IL-2 variant can be linked to the N-terminus or C-terminus of the IgG Fc region.

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

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

[0020] The term "Fc domain" includes native Fc and Fc variant molecules and sequences as defined above. Like Fc variants and native Fc, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from a full antibody or produced by recombinant gene expression or by other means. In various embodiments, the "Fc domain" refers to a dimer of two Fc domain monomers (SEQ ID NO:6), which typically includes all or part of the hinge region. In various embodiments, the Fc domain can be mutated to lack effector function. In various embodiments, each Fc domain monomer in the Fc domain contains amino acid substitutions in the CH2 antibody constant domain to reduce the interaction or binding between the Fc domain and Fcγ receptors. In various embodiments, each subunit of the Fc domain contains three amino acid substitutions that reduce binding to activating Fc receptors and / or effector function, wherein the amino acid substitutions are L234A, L235A, and G237A (SEQ ID NO:7). In various embodiments, each subunit of the Fc domain contains three amino acid substitutions that reduce binding to activating Fc receptors and / or effector function, wherein the amino acid substitutions are L234A, L235A, and P329G.

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

[0022] In various embodiments, the IL-2 variant Fc-fusion protein will be monomeric, i.e., contain only a single IL-2 mutant protein molecule. In such embodiments, the fusion protein is co-expressed as a heterodimeric Fc (e.g., Knob-Fc having the sequence listed in SEQ ID NO:9) linked to the IL-2 variant and a matching heterodimeric Fc (e.g., Hole-Fc having the sequence listed in SEQ ID NO:10). When the heterodimer of the two Fc-containing polypeptides forms, the resulting protein contains only the monovalent IL-2 variant. In various embodiments, the heterodimeric Fc domain used to prepare the monovalent IL-2 Fc fusion protein is a Knob Fc domain (SEQ ID NO:134) with reduced / eliminated effector function and extended half-life and a Hole-Fc domain (SEQ ID NO:135) with reduced / eliminated effector function and extended half-life.

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

[0024] In various embodiments, the IL-2 variant construct of the present invention contains a targeting moiety in the form of an antibody, antibody fragment, protein, or peptide that binds to a molecule enriched in cancer tissue, such as a tumor-associated antigen (TAA).

[0025] The TAA can be any molecule, macromolecule, molecular combination, etc. for which an immune response is desired. The TAA can be a protein comprising more than one polypeptide subunit. For example, the protein can be a dimer, trimer, or higher-order multimer. In various embodiments, two or more subunits of the protein can be linked by covalent bonds such as, for example, disulfide bonds. In various embodiments, the subunits of the protein can be held together by non-covalent interactions. Thus, the TAA can be any peptide, polypeptide, protein, nucleic acid, lipid, carbohydrate, or organic small molecule or any combination thereof that a person of ordinary skill in the art wishes to induce an immune response against. In various embodiments, the TAA is a peptide comprising from 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 that is typically administered to a subject by injection.

[0026] In various embodiments, a tumor-specific antibody or binding protein is used as a targeting moiety to direct the IL-2 variant to the site of the lesion, such as the tumor site, where they can stimulate a better anti-tumor immune response while avoiding the systemic toxicity of free cytokine therapy. For an IL-2 full agonist, the IL-2-IL-2R interaction, rather than antibody-antigen targeting, can determine the localization of the immunocytokine to IL-2 receptor-expressing cells rather than tumor cells at typical antibody doses. In various embodiments, using an IL-2 variant with reduced / abolished binding to IL-2Rα and attenuated potency in an antibody fusion protein helps to establish a stoichiometric balance between IL-2 and the targeting antibody to achieve an optimal dose at which the antibody can achieve sufficient target occupancy while the IL-2 moiety does not cause over-activation of the pathway. Using an IL-2 variant with reduced / abolished binding to IL-2Rα and attenuated potency in an IL-2 antibody fusion protein and further enhancing tumor targeting via the antibody minimizes peripheral activation and AICD, reduces antigen uptake, and promotes tumor targeting via the antibody arm.

[0027] In various embodiments, the IL-2 variant of the present invention can be attached to a targeting / bifunctional moiety that is an antibody, antibody fragment, protein, or peptide that targets an immune checkpoint modulator.

[0028] Numerous immune checkpoint protein antigens expressed on various immune cells have been reported, including, for example, SIRP (expressed on macrophages, monocytes, dendritic cells), CD47 (highly expressed on tumor cells and other cell types), VISTA (expressed on monocytes, dendritic cells, B cells, T cells), CD152 (expressed by activated CD8+ T cells, CD4+ T cells, and regulatory T cells), CD279 (expressed on tumor-infiltrating lymphocytes, expressed by activated T cells (both CD4 and CD8), regulatory T cells, activated B cells, activated NK cells, anergic T cells, monocytes, dendritic cells), CD274 (expressed on T cells, B cells, dendritic cells, macrophages, vascular endothelial cells, pancreatic islet cells), and CD223 (expressed by activated T cells, regulatory T cells, anergic T cells, NK cells, NKT cells, and plasmacytoid dendritic cells) (see, e.g., Pardoll, D., Nature Reviews Cancer, 12:252-264, 2012). Antibodies that bind to antigens identified as immune checkpoint proteins are known to those of skill in the art.For example, various anti-CD276 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20120294796 (Johnson et al.) and references cited therein); various anti-CD272 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20140017255 (Mataraza et al.) and references cited therein); various anti-CD152 / CTLA-4 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20130136749 (Korman et al.) and references cited therein); various anti-LAG-3 / CD223 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20110150892 (Thudium et al.) and references cited therein); various anti-CD279 (PD-1) antibodies have been described in the art (see, e.g., U.S. Patent No. 7,488,802 (Collins et al.) and references cited therein); various anti-CD274 (PD-L1) antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20130122014 (Korman et al.) and references cited therein); various anti-TIM-3 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20140044728 (Takayanagi et al.) and references cited therein); and various anti-B7-H4 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20110085970 (Terrett et al.) and references cited therein); and various anti-TIGIT antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20180169239A1 (Grogan) and references cited therein). Each of these references is hereby incorporated by reference in its entirety for the specific antibodies and sequences taught therein.

[0029] In various embodiments, the IL-2 variant can be fused to an antibody, antibody fragment, or protein or peptide that exhibits antigen binding to an immune checkpoint protein present on the surface of immune cells. In various embodiments, the immune checkpoint protein antigen is selected from the group consisting of, but 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.

[0030] In various embodiments, the antibody is an antagonistic FAP antibody or antibody fragment. In various embodiments, the antibody is a humanized antagonistic FAP antibody comprising the variable domain sequences set forth in SEQ ID NOs: 136 and 137. In various embodiments, the heterologous protein is an antibody or antibody fragment against an immune checkpoint modulator. In various embodiments, the antibody is an antagonistic PD-1 antibody or antibody fragment. In various embodiments, the antibody is an antagonistic PD-1 antibody comprising the variable domain sequences set forth in: SEQ ID NOs: 138 and 139, SEQ ID NOs: 140 and 141, SEQ ID NOs: 142 and 143, SEQ ID NOs: 144 and 145, or SEQ ID NOs: 146 and 147. In various embodiments, the antibody is an antagonistic human PD-L1 antibody comprising the variable domain sequences set forth in SEQ ID NOs: 148 and 149. In various embodiments, the antibody is an antagonistic CTLA-4 antibody comprising the variable domain sequences set forth in SEQ ID NOs: 150 and 151. In various embodiments, the heterologous protein is attached to the IL-2 variant via a linker and / or a hinge linker peptide. The linker or hinge linker can be an artificial sequence between 5, 10, 15, 20, 30, 40 or more amino acids that is relatively free of secondary structure.

[0031] In various embodiments, the heterologous protein is attached to the IL-2 variant via a rigid linker peptide between 10, 15, 20, 30, 40 or more amino acids, the rigid linker peptide exhibiting an α-helical conformation and serving as a rigid spacer between protein domains.

[0032] In another aspect, the IL-2 variant can be linked to various non-protein polymers, including but not limited to various polyols such as polyethylene glycol, polypropylene glycol or polyoxyalkylene, in the manner set forth in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 or 4,179,337. In various embodiments, amino acid substitutions can be made at different positions within the IL-2 variant to facilitate the addition of polymers such as PEG. In various embodiments, such PEGylated proteins can have an increased half-life and / or reduced immunogenicity compared to non-PEGylated proteins.

[0033] In various embodiments, the IL-2 variant can be non-covalently or covalently linked at the N-terminus or C-terminus to an IgG Fc or other polypeptide that binds to the neonatal Fcγ / receptor, human serum albumin, or a polypeptide that binds to a protein with an extended serum half-life or various non-protein polymers.

[0034] In another aspect, the present disclosure provides a pharmaceutical composition comprising an isolated IL-2 variant admixed with a pharmaceutically acceptable carrier.

[0035] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of the present invention. In one embodiment, the subject is a human subject. In various embodiments, the cancer is selected from, but not limited to, pancreatic cancer, gastric cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, or rhabdomyosarcoma.

[0036] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention in combination with a second therapy selected from the group consisting of: cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiotherapy, stem cell transplantation. In various embodiments, the combination therapy may comprise administering to the subject a therapeutically effective amount of an immunotherapy comprising, but not limited to, treatment with depleting antibodies against specific tumor antigens; treatment with antibody-drug conjugates; treatment with agonistic, antagonistic or blocking antibodies against co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CTLA-4, PD-1, PD-L1, OX-40, CD137, TIGIT, GITR, LAG3, TIM-3, CD47, SIRPα, ICOS and VISTA; treatment with bispecific T cell engaging antibodies such as blinatumomab; treatment involving administration of a biological response modifier such as 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; treatment with a therapeutic vaccine such as sipuleucel-T; treatment with a dendritic cell vaccine or a tumor antigen peptide vaccine; treatment with chimeric antigen receptor (CAR)-T cells; treatment with CAR-NK cells; treatment with tumor infiltrating lymphocytes (TIL); treatment with adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR transgenic); treatment with TALL-104 cells; and treatment with immune stimulants such as Toll-like receptor (TLR: TLR7, TLR8 and TLR9) agonists CpG and imiquimod; wherein the combination therapy provides increased effector cell killing of tumor cells, i.e., there is a synergistic effect between the IL-2 variant and the immunotherapy when co-administered.

[0037] In another aspect, the present disclosure provides the use of an IL-2 variant for the preparation of a medicament for treating cancer.

[0038] In another aspect, the present disclosure provides an isolated nucleic acid molecule comprising a polynucleotide encoding an IL-2 variant of the present disclosure. In another aspect, the present disclosure provides a vector comprising the nucleic acid described herein. In various embodiments, the vector is an expression vector. In another aspect, the present disclosure provides an isolated cell comprising the nucleic acid of the present disclosure. In various embodiments, the cell is a host cell comprising the expression vector of the present disclosure. In another aspect, a method for preparing an IL-2 variant is provided by culturing a host cell under conditions that promote the expression of a protein or polypeptide.

[0039] The present disclosure also provides the following items:

[0040] 1. An isolated interleukin-2 (IL-2) variant polypeptide, wherein the IL-2 variant polypeptide comprises an amino acid sequence in which one or more of the amino acid residues at positions R38, T41, F42, F44, E62, P65, E68, Y107 or S125 in SEQ ID NO: 3 are replaced by another amino acid, and wherein, compared to the polypeptide represented by SEQ ID NO: 3, the IL-2 variant polypeptide is unable to bind to IL-2Rα and no longer preferentially activates Treg cells, but still retains the ability to bind to and activate the IL-2Rβγ complex.

[0041] 2. An isolated IL-2 variant polypeptide, wherein the IL-2 variant polypeptide comprises an amino acid sequence in which one or more of the amino acid residues at positions R38, T41, F42, F44, E62, P65, E68, Y107 or S125 in SEQ ID NO: 3 are replaced by another amino acid, and wherein, compared to the polypeptide represented by SEQ ID NO: 3, the IL-2 variant polypeptide exhibits reduced binding to IL-2Rα and lower Treg activity, but still retains the ability to bind to and activate the IL-2Rβγ complex.

[0042] 3. An isolated IL-2 variant polypeptide, wherein the IL-2 variant polypeptide comprises an amino acid sequence in which one or more of the amino acid residues at positions R38, T41, F42, F44, E62, P65, E68, Y107 or S125 in SEQ ID NO: 3 are replaced by another amino acid, and wherein, compared to the polypeptide represented by SEQ ID NO: 3, the IL-2 variant polypeptide exhibits enhanced binding to IL-2Rα and increased Treg activity.

[0043] 4. An isolated IL-2 variant polypeptide, wherein the IL-2 variant polypeptide comprises an amino acid sequence in which one or more of the amino acid residues at positions L19, D20, S125 or Q126 in SEQ ID NO:3 are replaced by another amino acid, and wherein the IL-2 variant polypeptide exhibits a reduced ability to activate the IL-2Rβγ complex as compared to the polypeptide represented by SEQ ID NO:3.

[0044] 5. An isolated IL-2 variant polypeptide, wherein the IL-2 variant polypeptide comprises an amino acid sequence in which an amino acid residue at position S125 in SEQ ID NO:3 is replaced by another amino acid, and wherein the IL-2 variant polypeptide exhibits improved protein expression and purity as compared to the polypeptide represented by SEQ ID NO:3.

[0045] 6. The IL-2 variant polypeptide according to any one of items 1 to 5, wherein the amino acid substitution is selected from the group consisting of: substitution of L19D, L19H, L19N, L19P, L19Q, L19R, L19S, L19Y at position 19 of SEQ ID NO:3, substitution of R38A, R38F, R38G at position 38, substitution of T41A, T41G and T41V at position 41, substitution of F42A at position 42, substitution of F44G and F44V at position 44, substitution of E62A, E62F, E62H and E62L at position 62, substitution of P65A, P65E, P65G, P65H, P65K, P65N, P65Q, P65R at position 65, substitution of E68A, E68F, E68H, E68L and E68P at position 68, substitution of Y107G, Y107H, Y107L and Y107V at position 107, and substitution of S125I at position 125, substitution of Q126E at position 126, and deletion mutants of 5, 6, 7, 8, 9, 10 or 11 amino acids at the N-terminus of SEQ ID NO:3, or any combination of these substitutions or deletion mutants.

[0046] 7. The IL-2 variant polypeptide according to any one of items 1 to 6, wherein the IL-2 variant polypeptide comprises two amino acid substitutions at amino acid residues positions P65 and S125 in SEQ ID NO:3.

[0047] 8. The IL-2 variant polypeptide according to any one of items 1 to 6, wherein the IL-2 variant polypeptide comprises three amino acid substitutions at amino acid residues positions L19, P65 and S125 in SEQ ID NO:3.

[0048] 9. The IL-2 variant polypeptide according to any one of Items 1 to 8, wherein the IL-2 variant polypeptide comprises an amino acid sequence selected from the group consisting of the amino acid sequences listed in SEQ ID NOs: 31-66 and SEQ ID NOs: 111-120.

[0049] 10. An isolated IL-2 variant polypeptide, the isolated IL-2 variant polypeptide comprising an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, and wherein compared to the polypeptide represented by SEQ ID NO: 3, the IL-2 variant polypeptide no longer preferentially activates Tregs, but still retains the ability to activate the IL-2 receptor complex.

[0050] 11. An isolated fusion protein, the isolated fusion protein comprising 1) an IL-2 variant polypeptide according to any one of Items 1 to 10 and 2) a heterologous protein.

[0051] 12. The isolated fusion protein according to Item 11, wherein the IL-2 variant polypeptide is optionally fused to the C-terminal amino acid of the heterologous protein at its N-terminal amino acid in monomeric or dimeric form through a peptide linker.

[0052] 13. The isolated fusion protein according to Item 11, wherein the IL-2 variant polypeptide is optionally fused to the N-terminal amino acid of the heterologous protein at its C-terminal amino acid in monomeric or dimeric form through a peptide linker.

[0053] 14. The isolated fusion protein according to any one of Items 11 to 13, wherein the heterologous protein increases the circulating half-life of the IL-2 variant polypeptide.

[0054] 15. The isolated fusion protein according to any one of Items 11 to 13, wherein the heterologous protein enhances the expression level and overall purity of the IL-2 variant polypeptide.

[0055] 16. The isolated fusion protein according to any one of Items 11 to 13, wherein the heterologous protein serves as a marker or tag or targeting moiety.

[0056] 17. The isolated fusion protein according to any one of Items 11 to 16, wherein the heterologous protein is an Fc domain selected from the group consisting of: human IgG1 Fc domain, human IgG2 Fc domain, human IgG3 Fc domain, human IgG4 Fc domain, IgA Fc domain, IgD Fc domain, IgE Fc domain, IgG Fc domain and IgM Fc domain.

[0057] 18. The isolated fusion protein according to item 17, wherein the Fc domain is an Fc domain having silenced effector functions and / or having an extended half-life function.

[0058] 19. The isolated fusion protein according to any one of items 17 to 18, wherein the Fc domain is an Fc domain having an amino acid sequence selected from the group consisting of the amino acid sequences listed in SEQ ID NOs: 7-10 and 134-135.

[0059] 20. The isolated fusion protein according to any one of items 11 to 16, wherein the fusion protein comprises an amino acid sequence selected from the group consisting of the amino acid sequences listed in SEQ ID NOs: 67-107.

[0060] 21. The isolated fusion protein according to any one of items 11 to 16, wherein the heterologous protein is a targeting moiety targeting a tumor-associated antigen (TAA) in the form of: an antibody, an antibody heavy or light chain, an antibody fragment, a protein, and a peptide.

[0061] 22. The isolated fusion protein according to item 21, wherein the antibody or antibody fragment is selected from the group consisting of: a PD-1 antagonistic antibody; a PD-L1 antagonistic antibody; a TIGIT antagonistic antibody; a CTLA-4 antagonistic antibody; a CD20 antagonistic antibody; a Her-2 / neu antagonistic antibody; an EGFR antagonistic antibody; an FAP antagonistic antibody; an anti-integrin α4β7 anti-inflammatory antibody; a TNFα antagonistic antibody; and an agonistic CD40 antibody.

[0062] 23. The isolated fusion protein according to item 22, wherein the antibody is an antagonistic fibroblast activation protein (FAP) antibody or antibody fragment.

[0063] 24. The isolated fusion protein according to item 23, wherein the antibody is a humanized antagonistic FAP antibody comprising the heavy and light chain amino acid sequences listed in SEQ ID NOs: 136 and 137.

[0064] 25. The isolated fusion protein according to item 21, wherein the heterologous protein is an antibody or antibody fragment against an immune checkpoint regulator.

[0065] 26. The isolated fusion protein according to item 25, wherein the antibody is an antagonistic programmed death-1 (PD-1) antibody or antibody fragment.

[0066] 27. The isolated fusion protein according to item 26, wherein the antibody is an antagonistic humanized PD-1 antibody selected from antibodies comprising the heavy and light chain amino acid sequences listed in SEQ ID NO: 138 and 139; the heavy and light chain amino acid sequences listed in SEQ ID NO: 140 and 141; the heavy and light chain amino acid sequences listed in SEQ ID NO: 142 and 143; the heavy and light chain amino acid sequences listed in SEQ ID NO: 144 and 145; and the heavy and light chain amino acid sequences listed in SEQ ID NO: 146 and 147.

[0067] 28. The isolated fusion protein according to any one of items 11-27, wherein the IL-2 variant polypeptide is optionally fused to the C-terminal amino acid of the heterologous protein at its N-terminal amino acid in dimer or monomer form via a peptide linker.

[0068] 29. The isolated fusion protein according to item 28, wherein the IL-2 variant polypeptide is fused to the heterologous protein in dimer or monomer form via a peptide linker.

[0069] 30. The fusion protein according to item 29, wherein the peptide linker comprises between 1 and 40 amino acids.

[0070] 31. A pharmaceutical composition comprising the IL-2 variant polypeptide or the isolated fusion protein according to any one of items 1 to 30 mixed with a pharmaceutically acceptable carrier.

[0071] 32. A method of treating a disease or infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to item 31.

[0072] 33. The method according to item 32, wherein the disease is cancer.

[0073] 34. The method according to item 33, wherein the method further comprises administering a second therapeutic agent or therapy capable of treating the cancer in the subject.

[0074] 35. An isolated nucleic acid molecule encoding the IL-2 variant polypeptide or fusion protein according to any one of items 1 to 30.

[0075] 36. An expression vector comprising the nucleic acid molecule according to item 35.

[0076] 37. A host cell comprising the nucleic acid molecule according to item 36.

[0077] 38. A method for producing an IL-2 variant polypeptide or fusion protein according to any one of Items 1 to 30, the method comprising culturing a host cell according to Item 37 under conditions that promote the expression of the IL-2 variant polypeptide or fusion protein, and recovering the IL-2 variant polypeptide or fusion protein.

[0078] 39. An isolated IL-2 variant polypeptide or fusion protein, the isolated IL-2 variant polypeptide or fusion protein being produced by the method according to Item 38. Brief description of the drawings

[0080] Figure 1 depicts the purity of exemplary IL-2 variant Fc fusion proteins P-0635 (1A) and P-0704 (1B) determined by SDS-PAGE (under non-reducing conditions (lane 1) and reducing conditions (lane 2)) and the percentage of monomers evaluated by SEC-HPLC. P-0635 and P-0704 share the same amino acid substitution P65R in wild-type IL-2. P-0635 contains a bivalent IL-2 variant fused to a homodimeric Fc, while P-0704 contains a monovalent IL-2 variant fused to a knob-into-hole heterodimeric Fc.

[0081] Figure 2 depicts the size-exclusion chromatograms of exemplary IL-2 Fc fusion proteins P-0250 (2A), P-0318 (2B), P-0317 (2C), and P-0531 (2D) after protein A purification.

[0082] Figure 3 Depicts the effect of IL-2 valency on the binding strength of IL-2 Fc fusion proteins to IL-2Rα in ELISA. P-0531 and P-0689 share the same amino acid substitution S125I for improved developability in wild-type IL-2. P-0531 contains a bivalent IL-2 variant fused to a homodimeric Fc, while P-0689 contains a monovalent IL-2 fused to a knob-into-hole heterodimeric Fc.

[0083] Figure 4 depicts the effect of various mutations on the binding strength of IL-2 variant Fc fusions to IL-2Rα in ELISA. (4A) The IL-2 variant Fc fusion contains an amino acid substitution for T41; (4B) The IL-2 variant Fc fusion contains an amino acid substitution for Y107; (4C and 4D) The IL-2 variant Fc fusion contains an amino acid substitution for R38.

[0084] Figure 5 Depicts the effect of the IL-2 E68 substitution on the binding strength of IL-2 variant Fc fusions to IL-2Rα in ELISA.

[0085] Figure 6 Depicts the effect of IL-2 E62 substitution on the binding strength of IL-2 variant Fc fusions to IL-2Rα in ELISA.

[0086] Figure 7 depicts the effect of various IL-2 P65 substitutions on the binding strength of IL-2 variant Fc fusions to IL-2Rα in ELISA. (7A - 7B) IL-2 P65 substitution results in enhanced binding to IL-2Rα; (7C) IL-2 P65 substitution results in reduced binding to IL-2Rα; (7D) IL-2 P65 substitution results in complete loss of binding to IL-2Rα.

[0087] Figure 8 depicts the effect of combinations of IL-2 amino acid substitutions on the binding strength to IL-2Rα in ELISA. (8A) Effect of IL-2 F42A substitution on the binding strength to IL-2Rα; (8B) The combination of F42A and the CD25-disrupting substitution E62F results in complete loss of binding to IL-2Rα; (8C) The combination of F42A and the CD25-disrupting substitution P65H results in complete loss of binding to IL-2Rα.

[0088] Figure 9 Depicts the differential effects of IL-2 variant Fc fusion proteins on the dose-dependent induction of STAT5 phosphorylation in CD4+ Treg cells compared to wild-type fusion protein (P-0531) and a reference protein (P-0551) in a human PBMC assay. This set of IL-2 variants contains CD25-interfering mutations that result in enhanced, reduced, or eliminated binding to IL-2Rα.

[0089] Figure 10 Depicts the complete retention of binding of a set of IL-2 variant Fc fusion proteins to IL-2Rβγ in ELISA compared to wild-type IL-2 fusion protein P-0531 and reference protein P-0551. This set of IL-2 variants contains CD25-interfering mutations that result in enhanced, reduced, or eliminated binding to IL-2Rα.

[0090] Figure 11 depicts a set of IL-2 variant Fc fusion proteins that exhibit comparable activity in inducing Ki67 expression on CD8+ T cells (11A) and NK cells (11B) in human PBMCs. This set of IL-2 variants contains CD25-interfering mutations that result in enhanced, reduced, or eliminated binding to IL-2Rα. Wild-type IL-2 fusion protein P-0531 and reference protein P-0511 are included for comparison.

[0091] Figure 12Depicts the effect of IL-2 valency on the activity of inducing Ki67 expression on CD8+ T cells in human PBMCs. P-0531 and P-0689 are the bivalent and monovalent counterparts of wild-type IL-2Fc fusion proteins. P-0635 and P-0704 are the bivalent and monovalent equivalents of IL-2P65R Fc fusions.

[0092] Figure 13 depicts the effect of various amino acid substitutions or N-terminal deletions that modulate IL-2Rβ / γc on the activity of inducing pSTAT5 expression on CD4+ T cells compared to their wild-type counterparts. (13A) IL-2 mutants with an amino acid substitution at position D20; (13B-13C) IL-2 mutants with an amino acid substitution at position L19; (13D) IL-2 Q126E mutation; and (13E) IL-2 mutants with an N-terminal amino acid deletion.

[0093] Figure 14 depicts the effect of amino acid substitutions that disrupt IL-2Rβ or γc on the binding strength to IL-2Rβγ in ELISA (14A) and on the activity of inducing Ki67 expression on CD8+ T cells in human PBMCs (14B). P-0689 is a monovalent wild-type IL-2Fc fusion protein, and P-0704 is a monovalent IL-2P65R Fc fusion that can no longer bind to IL-2Rα but retains full affinity and functional activity for the dimeric IL-2Rβγ receptor.

[0094] Figure 15 depicts the effect of various amino acid changes that disrupt IL-2Rβ on the activity of IL-2 variant Fc fusions in inducing Ki67 expression on CD8+ T cells (15A), NK cells (15B), and CD4+ T cells (15C) in human PBMCs. P-0704 and a reference molecule (monomeric form of P-0551) were included for comparison.

[0095] Figure 16 depicts the time-dependent effect of P-0704 on the expansion of Treg cells (16A), CD8+ T cells (16B), and NK cells (16C) in peripheral blood after a single injection in Balb / C mice. P-0704 is a monovalent IL-2P65R Fc fusion; P-0689 (monovalent wild-type IL-2Fc fusion protein) was included for comparison. Blood was collected on days 3 and 5 for lymphocyte phenotyping by FACS analysis.

[0096] Figure 17 depicts the effect of the fusion forms on the dose-dependent induction of STAT5 phosphorylation on CD4+ Treg cells (17A), CD8+ T cells (17B), and NK cells (17C) in a human PBMC assay. P-0704 is a monovalent IL-2P65R Fc fusion, and P-0803 is an antibody fusion containing the same IL-2 moiety.

[0097] Figure 18 depicts the differential effects of IL-2 variant antibody fusions on the dose-dependent induction of STAT5 phosphorylation on CD4+ Treg cells (18A), CD8+ T cells (18B), and NK cells (18C) compared to the wild-type fusion protein (P-0837) in a human PBMC assay. P-0838 contains the IL-2P65Q mutation that significantly reduces the binding ability to IL-2Rα, and P-0782 has the IL-2P65R moiety with abolished binding to IL-2Rα.

[0098] Figure 19 depicts the effect of amino acid changes modulating IL-2Rβ on the activity of IL-2 variant antibody fusions in terms of stimulating STAT5 phosphorylation on CD8+ T cells (19A) and NK cells (19B) and inducing Ki67 expression on CD8+ T cells (19C) and NK cells (19D) in human PBMCs. All three compounds contain the P65R mutation in the IL-2 moiety, and P-0786 and P-0783 contain additional mutations L19Q and L19H disrupting IL-2Rβ, respectively.

[0099] Figure 20 depicts the effect of amino acid changes modulating IL-2Rβ on the activity of IL-2 variant antibody fusions in terms of stimulating STAT5 phosphorylation on CD4+ Treg cells (20A), CD8+ T cells (20B), and NK cells (20C) and inducing Ki67 expression on CD8+ T cells (20D) and NK cells (20E) in a human PBMC assay. All three compounds, P-0838, P-0790, and P-0787, contain the P65Q mutation in the IL-2 moiety, and P-0790 and P-0787 contain additional mutations L19Q and L19H modulating IL-2Rβ, respectively. P-0837 is the wild-type IL-2 fusion counterpart.

[0100] Figure 21Depicts the effect of amino acid changes regulating IL-2Rβ on the activity of IL-2 variant antibody fusions in terms of CTLL-2 cell proliferation. P-0782, P-0783, and P-0786 all contain the P65R mutation in the IL-2 portion; P-0786 and P-0783 contain additional mutations L19Q and L19H regulating IL-2Rβ, respectively. P-0837 is the wild-type IL-2 fusion counterpart.

[0101] Figure 22 depicts the minimal effect of the fusion of IL-2 variants on direct binding to the antibody arm (22A) and ligand competitive inhibition (22B) in ELISA, and similarly, analyzed by FACS, the IL-2 variant human PD-1 antibody IL-2 shows similar binding to cell surface-expressed PD1 as the parental antibody (Figure 22C). P-0795 is a human PD-1 antagonist antibody, and P-0803, P-0880, and P-0885 have monomeric IL-2 P65R variants covalently linked to the C-terminus of the heavy chain of P-0795. P-0803 and P-0885 share the same IL-2 P65R / S125I substitution but have different linkers ((G3S)2 and (G4S)3, respectively). P-0885 contains an additional L19Q mutation compared to P-0880. P-0704 and P-0859 are the Fc fusion counterparts of P-0880 and P-0885, respectively.

[0102] Figure 23 depicts the different effects of IL-2 variant antibody fusion proteins on the dose-dependent induction of STAT5 phosphorylation on CD4+ Treg cells (23A and 23B), CD8+ T cells (23C and 23D), and NK cells (23E and 23F) in human PBMC. P-0803 and P-0804 are IL-2 variant human PD-1 antibody fusion proteins containing the P65R and L19H / P65R mutations, respectively. P-0782 is an IL-2 P65R surrogate mouse PD-1 antibody fusion, and P-0783 contains an additional L19H mutation compared to P-0782.

[0103] Figure 24 depicts the size exclusion chromatograms of IL-2 variant human PD-1 antibody fusion proteins P-0840 (24A), P-0841 (24B), P-0803 (24C), and P-0880 (24D) after protein A purification.

[0104] Figure 25 depicts the effect of linker length of the IL-2 variant antibody fusion proteins on the dose-dependent induction of STAT5 phosphorylation on CD8+ T cells (25A and 25B) and NK cells (25C and 25D) in a human PBMC assay. Both P-0840 and P-0841 are IL-2L19Q / P65Q variant human PD-1 antibody fusion proteins; P-0840 contains a (G3S)2 linker, while P-0841 has a (G4S)3 linker. Similarly, P-0803 and P-0880 are IL-2P65R variant human PD-1 antibody fusion proteins; P-0803 contains a (G3S)2 linker, while P-0880 has a (G4S)3 linker.

[0105] Figure 26 depicts the effect of amino acid changes modulating IL-2Rβ on the activity of IL-2 variant human PD-1 antibody fusions in terms of stimulating STAT5 phosphorylation on CD8+ T cells (26A) and NK cells (26B) and inducing Ki67 expression on CD8+ T cells (26C) and NK cells (26D) in human PBMC. All three compounds P-0880, P-0885, and P-0882 contain the P65R mutation in the IL-2 portion, while P-0885 and P-0882 contain additional mutations L19Q and L19H modulating IL-2Rβ, respectively. P-0849 is the wild-type IL-2 fusion counterpart. All compounds have a (G4S)3 linker connecting the PD-1 antibody heavy chain and IL-2.

[0106] Figure 27 depicts the time-dependent effects of IL-2 variant replacement mouse PD-1 antibody fusion proteins P-0782, P-0838, P-0781 (benchmark), and P-0837 on Ki67 expression on CD8+ T cells (27A) and NK cells (27B) and on the time-dependent effects on CD8 cell (27C) and NK cell (27D) expansion after a single injection in C57BL6 mice. Cell expansion is represented as the fold change in cell number relative to baseline. P-0782 contains the P65R mutation in the IL-2 portion, P-0838 contains the P65Q mutation, P-0781 contains the benchmark IL-2 variant with abolished binding to IL-2Rα, and P-0837 is the wild-type IL-2 fusion counterpart.

[0107] Figure 28 depicts the time- and dose-dependent effects of the IL-2 variant substituted murine PD-1 antibody fusion protein P-0786 on Ki67 expression on CD8+ T cells (28A) and NK cells (28B), and on the cell expansion of CD8+ T cells (28C) and NK cells (28D) after a single injection in C57BL6 mice. Cell expansion is represented as the fold change in cell number relative to the baseline. P-0786 contains the L19Q / P65R mutation, which results in the elimination of binding to IL-2Rα and a reduction in overall potency. P-0837 (wild-type IL-2 fusion counterpart) was included for comparison.

[0108] Figure 29 depicts the time- and dose-dependent effects of the IL-2 variant substituted murine PD-1 antibody fusion protein P-0783 on Ki67 expression on CD8+ T cells (29A) and NK cells (29B), and on the cell expansion of CD8 cells (29C) and NK cells (29D) after a single injection in C57BL6 mice. Cell expansion is represented as the fold change in cell number relative to the baseline. P-0783 contains the L19H / P65R mutation, which results in the elimination of binding to IL-2Rα and a reduction in overall potency. P-0837 (wild-type IL-2 fusion counterpart) was included for comparison.

[0109] Figure 30 Depicts the body weight changes of C57BL / 6 mice treated with the IL-2 variant substituted murine PD-1 antibody fusion proteins P-0782, P-0786, and P-0783. All compounds contain the P65R mutation in the IL-2 portion, P-0781 contains the benchmark IL-2 variant that eliminates binding to IL-2Rα, and P-0786 and P-0783 contain additional mutations L19Q and L19H that disrupt IL-2Rβ, respectively. Data are represented as mean ± SEM.

[0110] Figure 31 depicts the anti-tumor efficacy (31A) and body weight changes (31B) of the IL-2 variant substituted murine PD-1 antibody fusion proteins in a subcutaneous B16F10 murine melanoma tumor model according to a Q7D repeated dosing regimen. All three antibody fusion proteins contain the IL-2P65Q mutation to attenuate binding to IL-2Rα; P-0790 and P-0787 contain additional L19Q and L19H mutations, respectively, to further modulate overall potency. Data are represented as mean ± SEM.

[0111] Figure 32 depicts the anti-tumor efficacy (32A) and body weight changes (32B) of two different doses of P-0787 in a subcutaneous B16F10 murine melanoma tumor model according to a Q7D repeated dosing regimen. P-0787 is an IL-2 variant-containing murine PD-1 antibody fusion protein with the L19H / P65Q mutation. Data are represented as mean ± SEM.

[0112] Figure 33 Depicts the anti-tumor efficacy of IL-2 variant-containing murine PD-1 antibody fusion proteins P-0782 and P-0786 in a subcutaneous B16F10 murine melanoma tumor model according to a Q7D repeated dosing regimen. P-0722 (murine PD-1 antibody replacement) was included for comparison. Both P-0782 and P-0786 contain the P65R mutation that abrogates IL-2Rα binding, while P-0786 contains an additional L19Q mutation to modulate overall potency. Data are represented as mean ± SEM.

[0113] Figure 34 depicts the dose-dependent inhibition of P-0790 on lung metastasis nodules in a murine B16F10 lung metastasis model. (34A) Mean lung nodule count; (34B) Lung pictures from representative animals in each group. P-0790 is an IL-2 L19Q / P65Q murine PD-1 antibody fusion protein with significantly impaired binding to IL-2Rα and modulated overall potency. Data are represented as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test. *p < 0.05.

[0114] Modes for carrying out the present disclosure

[0115] The present invention relates to polypeptides that share the primary sequence with human IL-2 except for one to several mutated amino acids. The IL-2 variants contain mutations that substantially reduce the ability of these polypeptides to stimulate Treg cells and render them more effective in tumor treatment. Also included are the therapeutic uses of these mutant variants alone or in combination with vaccines, or TAA-targeted biologics, or immune checkpoint blockers, or as building blocks in bifunctional molecular constructs for the treatment of diseases such as cancer or infections where the activity of regulatory T cells (Tregs) is not desired. In another aspect, the present invention relates to pharmaceutical compositions comprising the disclosed polypeptides. Finally, the present invention relates to the therapeutic uses of the disclosed polypeptides and pharmaceutical compositions due to their selective modulation of the immune system's action against cancer and other infectious diseases.

[0116] Definitions

[0117] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to polymers of amino acid residues. In various embodiments, a "peptide", "polypeptide", and "protein" is a chain of amino acids whose α-carbons are joined by peptide bonds. Thus, the terminal amino acid at one end (amino terminus) of the chain has a free amino group, while the terminal amino acid at the other end (carboxyl terminus) of the chain has a free carboxyl group. As used herein, the term "amino terminus" (abbreviated N-terminus) refers to the free α-amino group on the amino acid at the amino terminus of a peptide, or to the α-amino group of an amino acid at any other position in a peptide (the amino group when participating in a peptide bond). Similarly, the term "carboxyl terminus" refers to the free carboxyl group on the carboxyl terminus of a peptide, or to the carboxyl group of an amino acid at any other position in a peptide. Peptides also include substantially any polyamino acid, including but not limited to peptide mimetics, such as amino acids joined by ether bonds rather than amide bonds.

[0118] The polypeptides of the present disclosure include polypeptides that have been modified in any way and for any reason, e.g., to: (1) reduce sensitivity to proteolysis, (2) reduce sensitivity to oxidation, (3) alter the binding affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or alter other physicochemical or functional properties.

[0119] As used herein, an amino acid "substitution" refers to replacing an amino acid at a specific position in a parental polypeptide sequence in a polypeptide with a different amino acid. Amino acid substitutions can be made using genetic methods or chemical methods well known in the art. For example, single amino acid substitutions or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in a naturally occurring sequence (e.g., in a polypeptide portion other than a domain forming intermolecular contacts). A "conservative amino acid substitution" refers to an amino acid in a polypeptide being replaced with an amino acid that is functionally similar. The following six groups each contain amino acids that are conservative substitutions for one another:

[0120] 1) Alanine (A), serine (S), and threonine (T)

[0121] 2) Aspartic acid (D) and glutamic acid (E)

[0122] 3) Asparagine (N) and glutamine (Q)

[0123] 4) Arginine (R) and lysine (K)

[0124] 5) Isoleucine (I), leucine (L), methionine (M), and valine (V)

[0125] 6) Phenylalanine (F), tyrosine (Y), and tryptophan (W)

[0126] "Non-conservative amino acid substitution" means the substitution of a member of one of these categories with a member from another category. In making such a change, the hydropathic index of the amino acid can be considered according to various embodiments. Based on the hydrophobicity and charge characteristics of the amino acids, each amino acid has been assigned a hydropathic index. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0127] Those skilled in the art understand the importance of the hydrophilic amino acid index in conferring biological functions of protein interactions (see, e.g., Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known that certain amino acids can be substituted with other amino acids having similar hydropathic indices or scores and still retain similar biological activities. When making changes based on the hydropathic index, in various embodiments, substitutions of amino acids with hydropathic indices within ±2 are included. In various embodiments, those within ±1 are included, and in various embodiments, those within ±0.5 are included.

[0128] Those skilled in the art also understand that substitutions of similar amino acids can be effectively made based on hydrophilicity, especially when the resulting biofunctional protein or peptide is intended for use in immunological embodiments as disclosed herein. In various embodiments, the maximum local average hydrophilicity of the protein (as determined by the hydrophilicity of its neighboring amino acids) is related to its immunogenicity and antigenicity, i.e., to the biological properties of the protein.

[0129] The following hydrophilicity values are assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). When making changes based on similar hydrophilicity values, in various embodiments, substitutions of amino acids having hydrophilicity values within ±2 are included, in various embodiments, those within ±1, and in various embodiments, those within ±0.5.

[0130] Exemplary amino acid substitutions are listed in Table 1.

[0131] Table 1

[0132]

[0133]

[0134] One of ordinary skill in the art will be able to determine suitable polypeptide variants as listed herein using well-known techniques. In various embodiments, one of ordinary skill in the art can identify suitable regions in a molecule that can be altered without disrupting activity by targeting regions that are not thought to be important for activity. In other embodiments, one of ordinary skill in the art can identify residues and portions of the molecule that are conserved among similar polypeptides. In additional embodiments, even regions that may be important for biological activity or for structure can undergo conservative amino acid substitutions without disrupting biological activity or unduly affecting polypeptide structure.

[0135] In addition, one of ordinary skill in the art can review structure - function studies that identify residues important for activity or structure in similar polypeptides. In view of such comparisons, one of ordinary skill in the art can predict the importance of amino acid residues in a polypeptide corresponding to amino acid residues important for activity or structure in similar polypeptides. One of ordinary skill in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0136] One of ordinary skill in the art can also analyze the three-dimensional structure and amino acid sequence of similar polypeptides that are related to such structure. Given such information, one of ordinary skill in the art can predict the arrangement of the amino acid residues of the polypeptide in terms of its three-dimensional structure. In various embodiments, one of ordinary skill in the art can choose not to make radical changes to the amino acid residues predicted to be on the surface of the polypeptide, as such residues may be involved in important interactions with other molecules. In addition, one of ordinary skill in the art can generate test variants that contain a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to one of ordinary skill in the art. These variants can be used to gather information about suitable variants. For example, if one finds that a change in a particular amino acid residue results in disrupted, undesirably reduced, or inappropriate activity, variants having such a change can be avoided. In other words, based on the information collected from such routine experiments, one of ordinary skill in the art can readily determine the amino acids at which further substitutions, either alone or in combination with other mutations, should be avoided.

[0137] As used herein, the terms “polypeptide fragment” and “truncated polypeptide” refer to polypeptides that have an amino-terminal deletion and / or a carboxyl-terminal deletion as compared to the corresponding full-length protein. In various embodiments, the length of the fragment can be, for example, at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 amino acids. In various embodiments, the length of the fragment can also be, for example, at most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 450, at most 400, at most 350, at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 25, at most 10, or at most 5 amino acids. The fragment can also contain one or more additional amino acids at either or both of its termini, for example, sequences of amino acids from different naturally occurring proteins (e.g., Fc or leucine zipper domains) or artificial amino acid sequences (e.g., artificial linker sequences).

[0138] As used herein, the terms "polypeptide variant", "hybrid polypeptide", and "polypeptide mutant" refer to polypeptides that comprise an amino acid sequence in which one or more amino acid residues are inserted into, deleted from, and / or substituted into the amino acid sequence relative to another polypeptide sequence. In various embodiments, the number of amino acid residues to be inserted, deleted, or substituted can be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids in length. The hybrids of the present disclosure include fusion proteins.

[0139] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, such as conjugation to another chemical moiety (such as, for example, polyethylene glycol, albumin (such as, for example, human serum albumin)), phosphorylation, and glycosylation.

[0140] The term "% sequence identity" is used interchangeably herein with the term "% identity" and refers to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% identity as determined by a defined algorithm means the same as 80% sequence identity and means that a given sequence is at least 80% identical to another sequence of another length. In various embodiments, the % identity is selected from, for example, sequence identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or greater with a given sequence. In various embodiments, the % identity is in the range of, for example, from about 60% to about 70%, from about 70% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, or from about 95% to about 99%.

[0141] The terms “% sequence homology” and “% homology” are used interchangeably herein and refer to the level of amino acid sequence homology between two or more peptide sequences or the level of nucleotide sequence homology between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% homology and 80% sequence homology as determined by a defined algorithm mean the same thing, and thus a homolog of a given sequence has a sequence homology greater than 80% relative to the length of the given sequence. In various embodiments, the % homology is selected from, for example, sequence homology of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% or greater with a given sequence. In various embodiments, the % homology is in the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95% or about 95% to about 99%.

[0142] Exemplary computer programs that can be used to determine identity between two sequences include, but are not limited to, a set of BLAST programs publicly available on the NCBI website on the Internet, such as BLASTN, BLASTX and TBLASTX, BLASTP and TBLASTN. See also Altschul et al., J. Mol. Biol. 215:403-10, 1990 (specifically refer to the published default settings, i.e., parameters w = 4, t = 17) and Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997. When evaluating a given amino acid sequence relative to amino acid sequences in GenBank protein sequences and other public databases, the BLASTP program is generally used for sequence retrieval. The BLASTX program is preferably used to retrieve nucleic acid sequences that have been translated in all reading frames against amino acid sequences in GenBank protein sequences and other public databases. Both BLASTP and BLASTX are run using default parameters of an open gap penalty of 11.0 and an extended gap penalty of 1.0 and using the BLOSUM-62 matrix.

[0143] In addition to calculating the percent sequence identity, the BLAST algorithm performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l Acad. Sci. USA, 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)), which gives an indication of the probability that a match between two nucleotide or amino acid sequences occurred by chance. For example, if in a comparison of a test nucleic acid to a reference nucleic acid, the minimum total probability is, for example, less than about 0.1, less than about 0.01, or less than about 0.001, then the nucleic acid is considered similar to the reference sequence.

[0144] 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).

[0145] As used herein, the term "knob-into-hole modification" refers to a modification within the interface between the CH3 domains of two immunoglobulin heavy chains. In one embodiment, a "knob-into-hole modification" comprises the amino acid substitution T366W and optionally the amino acid substitution S354C in one antibody heavy chain, and the amino acid substitutions T366S, L368A, Y407V and optionally Y349C in the other antibody heavy chain. The knob-into-hole technique is described, for example, in U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).

[0146] As used herein, the term "fusion protein" refers to a fusion polypeptide molecule comprising two or more genes that originally encoded different proteins, wherein the components of the fusion protein are directly linked to each other by a peptide bond or are linked to each other by a peptide linker. As used herein, the term "fusion" refers to components that are directly linked by a peptide bond or are linked via one or more peptide linkers.

[0147] A "linker" is a molecule that covalently or by ionic, van der Waals, or hydrogen bonds links two other molecules, such as a nucleic acid molecule that hybridizes to one complementary sequence at the 5' end and to another complementary sequence at the 3' end to link two non-complementary sequences. A "cleavable linker" is a linker that can be degraded or otherwise cleaved to separate two components that are linked by the cleavable linker. Cleavable linkers are typically cleaved by enzymes, usually peptidases, proteases, nucleases, lipases, etc. Cleavable linkers can also be cleaved by changes in environmental factors, such as, for example, temperature, pH, salt concentration, etc.

[0148] As used herein, the term "peptide linker" refers to a peptide comprising one or more amino acids, typically from about 2 to 20 amino acids. Peptide linkers are known in the art or are described herein. Suitable non-immunogenic linker peptides include, for example, (G4S) n , (SG4) n or G4(SG4)n peptide linkers. "n" is typically a number between 1 and 10, usually between 2 and 4.

[0149] "Pharmaceutical composition" means a composition suitable for pharmaceutical use in an animal. A pharmaceutical composition comprises a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. "Pharmacologically effective amount" means the amount of an agent effective to produce the desired pharmacological result. "Pharmaceutically acceptable carrier" means any standard pharmaceutical carrier, vehicle, buffer, and excipient, such as phosphate buffered saline solution, aqueous solution of 5% dextrose, and emulsions, such as oil / water emulsions or water / oil emulsions, and various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21st Edition 2005, Mack Publishing Co, Easton. "Pharmaceutically acceptable salt" is a salt of a compound that can be formulated for pharmaceutical use, including, for example, salts of metals (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or salts of organic amines.

[0150] As used herein, "treatment" (and its grammatical variations such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of a disease in an individual being treated, and the clinical intervention can be used prophylactically or during a clinical pathologic process. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, alleviating any direct or indirect pathologic consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or ameliorating the disease state, and relieving or improving the prognosis. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of symptoms of the disease, disorder, or condition. Additionally, reference herein to "treatment" includes reference to curative, palliative, and prophylactic treatment.

[0151] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or composition sufficient to treat a particular disorder, condition, or disease, such as to ameliorate, mitigate, relieve, and / or delay one or more of its symptoms. With reference to cancer or other unwanted cell proliferation, an effective amount includes an amount sufficient to: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) to some extent inhibit, retard, slow, and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., to some extent slow and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the onset and / or recurrence of a tumor; and / or (vii) to some extent relieve one or more cancer-related symptoms. An effective amount may be administered in one or more administrations.

[0152] The phrase "administer" or "cause to be administered" refers to the action of a medical professional (e.g., a physician) or a person controlling a patient's medical care to control and / or permit the administration of the agent / compound under discussion to the patient. Causing administration may include diagnosing and / or determining a suitable treatment regimen, and / or prescribing a particular agent / compound for the patient. Such prescribing may include, for example, drafting a prescription form, annotating medical records, etc. "Causing administration" is also contemplated when administration is described herein.

[0153] The terms "patient", "individual", and "subject" may be used interchangeably and refer to a mammal, preferably a human or non-human primate, but also to domestic mammals (e.g., canine or feline), laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig), and agricultural mammals (e.g., equine, bovine, swine, ovine). In various embodiments, the patient may be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other healthcare worker in a hospital, psychiatric care facility such as an outpatient clinic, or other clinical setting. In various embodiments, the patient may be an immunocompromised patient or a patient with a weakened immune system, including but not limited to patients with primary immunodeficiency, AIDS; cancer patients and transplant patients taking certain immunosuppressive drugs; and patients with genetic diseases affecting the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency). In various embodiments, the patient has an immunogenic cancer, including but not limited to bladder cancer, lung cancer, melanoma, and other cancers reported to have a high mutation rate (Lawrence et al., Nature, 499(7457):214 - 218, 2013).

[0154] The term "immunotherapy" refers to cancer treatment including but not limited to the following: treatment with depleting antibodies against specific tumor antigens; treatment with antibody-drug conjugates; treatment with agonistic, antagonistic or blocking antibodies against co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CTLA-4, PD-1, OX-40, CD137, GITR, LAG3, TIM-3, SIRP, CD47 and VISTA; treatment with bispecific T cell-engaging antibodies such as blinatumomab; treatment involving administration of biological response modifiers such as IL-2, IL-12, IL-15, IL-21, GM-CSF, IFN-α, IFN-β and IFN-γ; treatment with therapeutic vaccines such as sipuleucel-T; treatment with dendritic cell vaccines or tumor antigen peptide vaccines; treatment with chimeric antigen receptor (CAR)-T cells; treatment with CAR-NK cells; treatment with tumor-infiltrating lymphocytes (TIL); treatment with adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR transgenic); treatment with TALL-104 cells; and treatment with immune stimulants such as Toll-like receptor (TLR) agonists CpG and imiquimod.

[0155] "Tolerant or refractory cancer" refers to tumor cells or cancer that do not respond to a previous anti-cancer therapy, which includes, for example, chemotherapy, surgery, radiotherapy, stem cell transplantation and immunotherapy. Tumor cells can be tolerant or refractory at the start of treatment, or they can become tolerant or refractory during treatment. Refractory tumor cells include tumors that do not respond at the start of treatment, or tumors that initially respond to treatment for a short period but then fail to respond to treatment. Refractory tumor cells also include tumors that respond to treatment with an anti-cancer therapy but then fail to respond to subsequent rounds of therapy. For the purposes of the present invention, refractory tumor cells also include tumors that show suppression following treatment with an anti-cancer therapy but then recur up to 5 years, sometimes up to 10 years or longer, after treatment has stopped. The anti-cancer therapy can use a single chemotherapeutic agent, radiotherapy alone, targeted therapy alone, surgery alone or a combination thereof. For ease of description and not by way of limitation, it should be understood that refractory tumor cells are interchangeable with tolerant tumors.

[0156] As used herein, the term "Fc domain" or "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. The IgG Fc region includes the IgG CH2 and IgG CH3 domains. The CH3 region herein can be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced "protuberance" ("knob") in one of its chains and a corresponding introduced "cavity" ("hole") in the other of its chains; see U.S. Patent No. 5,821,333, which is hereby expressly incorporated by reference). Such variant CH3 domains can be used to facilitate heterodimerization of two different immunoglobulin heavy chains as described herein. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system.

[0157] As used herein, the term "effector function" refers to those biological activities attributable to the Fc region of an immunoglobulin, which vary with the immunoglobulin isotype. Examples of immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), cytokine secretion, antigen uptake by immune complex-mediated antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Effector function can also refer to similar immune responses elicited by effector immune cells such as CD8 cells and NK cells.

[0158] As used herein, the term "regulatory T cell" or "Treg cell" means a specialized type of CD4+ T cell that can inhibit the responses of other T cells (effector T cells). Treg cells are characterized by the expression of CD4, the α subunit of the interleukin-2 receptor (CD25), and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)), and play a key role in inducing and maintaining peripheral self-tolerance to antigens, including antigens expressed by tumors.

[0159] As used herein, the term "conventional CD4+ T cell" means a CD4+ T cell other than a regulatory T cell. Conventional CD4+ T cells express CD3 and CD4. Under naive and unstimulated conditions, they do not express the α subunit (CD25) of the interleukin-2 receptor, but express the βγ subunits of the interleukin-2 receptor.

[0160] The term "CD8 T cell" refers to a class of cytotoxic T lymphocytes characterized by the expression of CD3 and CD8. CD8 T cells mainly express the βγ subunits of the IL-2 receptor and play a key role in killing cancer cells, virus-infected cells, or otherwise damaged cells.

[0161] The term "NK cell" refers to a class of cytotoxic lymphocytes that are crucial for the innate immune system. NK cells mainly express the βγ subunits of the IL-2 receptor and provide a rapid response against virus-infected cells and tumor formation.

[0162] As used herein, "specifically binds" means that the binding to an antigen is selective and can be distinguished from unwanted or non-specific interactions. The ability of an immunoglobulin to bind a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art such as surface plasmon resonance (SPR) techniques.

[0163] As used herein, the term "affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (such as an antibody) and its binding partner (such as an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (koff and kon, respectively). A particular method for measuring affinity is surface plasmon resonance (SPR).

[0164] As used herein, the term "reduced binding" refers to a decrease in the affinity of the respective interaction, as measured, for example, by SPR. In contrast, "increased binding" refers to an increase in the binding affinity of the respective interaction.

[0165] As used herein, the term "polymer" generally includes, but is not limited to, homopolymers; copolymers, such as, for example, block, graft, random, and alternating copolymers; and terpolymers; as well as mixtures and modifications thereof. In addition, unless otherwise explicitly defined, the term "polymer" shall include all possible geometric configurations of the material. These configurations include, but are not limited to, isotactic, syndiotactic, and atactic symmetries.

[0166] "Polyethylene glycol" or "PEG" means a polyalkylene glycol compound or derivative thereof, with or without a coupling agent or derivatization, said derivatization bearing a coupling moiety or an activating moiety (e.g., bearing an aldehyde, hydroxysuccinimide, hydrazide, thiol, triflate, tresylate, aziridine, oxirane, orthopyridyldisulphide, vinyl sulfone, iodoacetamide or maleimide moiety). In various embodiments, PEG includes substantially linear, straight-chain PEG, branched PEG or dendritic PEG. PEG is a well-known water-soluble polymer, commercially available or preparable 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).

[0167] "Polynucleotide" refers to a polymer containing nucleotide units. Polynucleotides include naturally occurring nucleic acids such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") and nucleic acid analogs. Nucleic acid analogs include those containing: non-naturally occurring bases, nucleotides that engage with other nucleotides via linkages other than the naturally occurring phosphodiester bond, or nucleotides containing bases attached via linkages other than the phosphodiester bond. Thus, nucleotide analogs include, for example and without limitation, phosphorothioates, dithiophosphates, phosphorotriesters, phosphoramidates, organophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs), etc. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" generally refers to large polynucleotides. The term "oligonucleotide" generally refers to short polynucleotides, usually no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes the RNA sequence in which "U" replaces "T" (i.e., A, U, G, C).

[0168] This text uses conventional symbols to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is called the 5'-direction. The direction in which nucleotides are added to a nascent RNA transcript from 5' to 3' is called the transcription direction. The DNA strand having the same sequence as the mRNA is called the "coding strand"; the sequence at the 5' that is on the DNA strand having the same sequence as the mRNA transcribed from that DNA and that is 5' of the 5'-end of the RNA transcript is called the "upstream sequence"; the sequence at the 3' that is on the DNA strand having the same sequence as the RNA and that is 3' of the 3'-end encoding the RNA transcript is called the "downstream sequence".

[0169] "Complementary" refers to the topological compatibility or fitting together of the interaction surfaces of two polynucleotides. Thus, the two molecules can be described as complementary, and further, the contact surface features are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially the same as the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize to the second polynucleotide under stringent hybridization conditions.

[0170] "Specific hybridization with", "specific hybridization", or "selective hybridization with" means that when a particular nucleotide sequence is present in a complex mixture (e.g., total cellular) DNA or RNA, the nucleic acid molecule preferentially binds to, duplexes with, or hybridizes to that sequence under stringent conditions. The term "stringent conditions" refers to conditions under which a probe will preferentially hybridize to its target sequence and to a lesser extent or not at all to other sequences. In the context of nucleic acid hybridization experiments such as DNA hybridization and RNA hybridization, "stringent hybridization" and "stringent hybridization wash conditions" are sequence-dependent and vary under different environmental parameters. Extensive guidance on nucleic acid hybridization can be found in: Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, N.Y.; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3rd ed. Suppl., NY; and Ausubel et al., eds., current edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

[0171] Typically, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength and pH. The Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe (under defined ionic strength and pH conditions). Very stringent conditions are selected to be equal to the Tm of a particular probe. Examples of stringent hybridization conditions for hybridizing complementary nucleic acids having more than about 100 complementary residues to a filter in a Southern or Northern blot are hybridization overnight at 42°C in 50% formamide with 1 mg heparin. Examples of highly stringent wash conditions are washing at 72°C in 0.15 M NaCl for about 15 minutes. Examples of stringent wash conditions are washing at 65°C in 0.2x SSC for 15 minutes. See Sambrook et al. for a description of SSC buffer. Low stringency washes can be performed to remove background probe signal, followed by high stringency washes. An exemplary moderately stringent wash for a duplex of more than about 100 nucleotides, for example, is washing at 45°C in 1x SSC for 15 minutes. An exemplary low stringency wash for a duplex of more than about 100 nucleotides, for example, is washing at 40°C in 4 - 6x SSC for 15 minutes. Typically, in a particular hybridization assay, a signal-to-noise ratio of 2x (or higher) compared to the signal-to-noise ratio observed for an unrelated probe indicates specific hybridization has been detected.

[0172] A "primer" refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a starting point for the synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions that induce synthesis (i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as DNA polymerase). Primers are typically single-stranded, but can be double-stranded. Primers are typically deoxyribonucleic acids, but many synthetic and naturally occurring primers can be used for many applications. The primer is complementary to the template, and the primer is designed to hybridize to the template to serve as a site for the start of synthesis, but does not need to reflect the exact sequence of the template. In such cases, the specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, for example, chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.

[0173] When used in reference to polynucleotide usage, a "probe" refers to a polynucleotide that is capable of specifically hybridizing to a designated sequence of another polynucleotide. The probe specifically hybridizes to a target complementary polynucleotide, but does not need to reflect the exact complementary sequence of the template. In such cases, the specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. The probe can be labeled with, for example, chromogenic, radioactive, or fluorescent moieties and used as a detectable moiety. In cases where the probe provides a starting point for the synthesis of a complementary polynucleotide, the probe can also be a primer.

[0174] "Vector" is a polynucleotide that can be used to introduce another nucleic acid linked thereto into a cell. One type of vector is a "plasmid", which refers to a linear or circular double-stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), into which additional DNA segments can be introduced into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell and thus replicate along with the host genome. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide.

[0175] "Regulatory sequence" is a nucleic acid that affects the expression (e.g., level, timing, or location of expression) of a nucleic acid to which it is operably linked. A regulatory sequence can, for example, exert its effect directly on the nucleic acid being regulated or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and / or the nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Additional examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego; California. and Baron et al., 1995, Nucleic Acids Res. 23: 3605-06. A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression of the nucleotide sequence (e.g., level, timing, or location of expression).

[0176] "Host cell" is a cell that can be used to express the polynucleotides of the present disclosure. The host cell can be a prokaryote, such as Escherichia coli (E. coli), or the host cell can be a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., human cell, monkey cell, hamster cell, rat cell, mouse cell, or insect cell), or a hybridoma. Generally, the host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, which can then be expressed in the host cell. The phrase "recombinant host cell" can be used to denote a host cell that has been transformed or transfected with the nucleic acid to be expressed. The host cell can also be a cell that contains the nucleic acid but does not express the nucleic acid at a desired level, unless regulatory sequences are introduced into the host cell such that the regulatory sequences become operably linked to the nucleic acid. It should be understood that the term host cell refers not only to a particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations due to, for example, mutations or environmental influences, such progeny may not actually be identical to the parental cell, but are still included within the scope of the term as used herein.

[0177] The term "isolated molecule" (wherein the molecule is, for example, a polypeptide or a polynucleotide) is a molecule that by virtue of its origin or source from which it is derived (1) is not associated with the components that are naturally associated with it in its natural state, (2) is substantially free of other molecules from the same species, (3) is expressed by cells from a different species, or (4) does not exist in nature. Thus, a molecule that is chemically synthesized or expressed in a cell system different from the cell of its natural origin will be "isolated" from its naturally associated components. Molecules can also be isolated by using purification techniques well known in the art to render the molecule substantially free of naturally associated components. The purity or homogeneity of the molecule can be determined by many means well known in the art. For example, the purity of a polypeptide sample can be determined using techniques well known in the art using polyacrylamide gel electrophoresis and staining the gel to visualize the polypeptide. For certain purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification.

[0178] A protein or polypeptide is "substantially pure", "substantially homogeneous", or "substantially purified" when at least about 60% to 75% of the sample appears as a single species of polypeptide. The polypeptide or protein can be monomeric or polymeric. A substantially pure polypeptide or protein will generally comprise about 50%, 60%, 70%, 80%, or 90% w / w of the protein sample, more typically about 95% and preferably will be more than 99% pure. Protein purity or homogeneity can be indicated by many means well known in the art, such as polyacrylamide gel electrophoresis of the protein sample followed by visualization of individual polypeptide bands after staining the gel with a stain well known in the art. For some purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification.

[0179] As used herein, the term "label" or "labeled" refers to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable marker, such as a polypeptide incorporating a radiolabeled amino acid or attached to a biotinyl moiety that can be detected by labeled avidin (e.g., streptavidin incorporating a fluorescent marker or an enzyme activity that can be detected by optical or calorimetric methods). In another embodiment, the label or marker can be therapeutic, such as a drug conjugate or toxin. A variety of methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131I); fluorescent labels (such as FITC, rhodamine, lanthanide fluorophores); enzyme labels (such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a second reporter (such as leucine zipper pairing sequences, binding sites for second antibodies, metal-binding domains, epitope tags); magnetic agents, such as gadolinium chelates; toxins such as pertussis toxin, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogs or homologs. In various embodiments, the label is attached via spacer arms of various lengths to reduce potential steric hindrance.

[0180] As used herein, the term "heterologous" refers to a combination or state that is not natural or does not exist in nature, which combination or state can be achieved, for example, by replacing existing natural components or states with components or states from another source. Similarly, protein expression in an organism other than the organism in which the protein is naturally expressed constitutes a heterologous expression system and a heterologous protein.

[0181] It should be understood that the aspects and embodiments of the present disclosure described herein include "consisting of these aspects and embodiments" and / or "consisting essentially of these aspects and embodiments".

[0182] As used herein, the recitation of "about" a value or parameter includes (and describes) variations that are directed to that value or parameter itself. For example, the recitation of "about X" includes the description of "X".

[0183] Unless the context clearly dictates otherwise, the singular forms "a", "or", and "the" as used herein and in the appended claims include plural referents. It should be understood that the aspects and variations of the present disclosure described herein include "consisting of these aspects and variations" and / or "consisting essentially of these aspects and variations".

[0184] IL-2

[0185] Interleukin-2 (IL-2) is a classical Th1 cytokine that is produced by T cells upon activation through the T cell antigen receptor and the co-stimulatory molecule CD28. Regulation of IL-2 occurs through signal transduction pathways and activation of transcription factors that act on the IL-2 promoter to generate new gene transcription, but also involves regulation of the stability of IL-2 mRNA. IL-2 binds to a multi-chain receptor, including a highly regulated α-chain that mediates signal transduction through the Jak-STAT pathway, and β and γ chains. IL-2 delivers activation, growth, and differentiation signals to T cells, B cells, and NK cells. IL-2 is also important in mediating the cell death of activated-induced T cells, a function that provides a key mechanism for terminating the immune response. An unglycosylated recombinant human IL-2 product, aldesleukin (available from Prometheus Laboratories Inc., San Diego, Calif. as des-alanyl-1, serine-125 human interleukin-2 under the trademark ), has been approved for administration to patients with metastatic renal cell carcinoma and metastatic melanoma. IL-2 has also been proposed for use in patients with 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, short half-life and severe toxicity limit the optimal dosing of IL-2.

[0186] As used herein, the terms "native IL-2" and "native interleukin-2" in the context of a protein or polypeptide refer to any naturally occurring mammalian interleukin-2 amino acid sequence, including immature or precursor forms and mature forms. Non-limiting examples of GenBank accession numbers for the amino acid sequences of native mammalian interleukin-2 from various species include NP_032392.1 (Mus musculus, immature form), NP_001040595.1 (Macaca mulatta, immature form), NP_000577.2 (human, precursor form), CAA01199.1 (human, immature form), AAD48509.1 (human, immature form), and AAB20900.1 (human). In various embodiments of the present invention, native IL-2 is an immature or precursor form of naturally occurring mammalian IL-2. In other embodiments, native IL-2 is a mature form of naturally occurring mammalian IL-2. In various embodiments, native IL-2 is a precursor form of naturally occurring human IL-2. In various embodiments, native IL-2 is a mature form of naturally occurring human IL-2. In various embodiments, domain D2 of IL-2 is derived from the amino acid sequence of the human IL-2 precursor sequence set forth in SEQ ID NO:1:

[0187] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML

[0188] TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSET

[0189] TFMCEYADETATIVEFLNRWITFCQSIISTLT(SEQ ID NO:1).

[0190] In various embodiments, domain D2 of IL-2 comprises the amino acid sequence of the wild-type sequence of the mature form of human IL-2 having a cysteine to serine substitution at position 125 set forth in SEQ ID NO:3, but without altering IL-2 receptor binding as compared to naturally occurring IL-2:

[0191] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE

[0192] EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRW

[0193] ITFSQSIISTLT (SEQ ID NO:3).

[0194] IL-2 variants

[0195] The present invention relates to polypeptides that share the primary sequence with human IL-2 except for one to several mutated amino acids. A group of IL-2 variants contain mutations that substantially reduce the ability of these polypeptides to stimulate Treg cells and make them more effective in the treatment of tumors. Also included is the therapeutic use of these mutant variants alone or in combination with vaccines, or TAA-targeted biologics, or immune checkpoint blockers, or as building blocks in bifunctional molecular constructs for the treatment of diseases such as cancer or infections where unwanted regulatory T cell (Treg) activity is not desired. In another aspect, the present invention relates to pharmaceutical compositions comprising the disclosed polypeptides. Finally, the present invention relates to the therapeutic use of the disclosed polypeptides and pharmaceutical compositions due to their selective regulatory effects on the immune system against diseases such as autoimmune and inflammatory disorders or cancer and various infectious diseases.

[0196] The present invention relates to polypeptides having a length of 100 to 500 amino acids, preferably a size of 140 residues, and an apparent molecular weight of at least 15 kD. These polypeptides maintain a high sequence identity of more than 90% with native IL-2. In one region of their sequence, these polypeptides are mutated to introduce amino acid residues different from those at the same positions in native IL-2.

[0197] The polypeptides of the present invention may be referred to as immunomodulatory polypeptides, IL-2 analogs or IL-2 variants and other names. These polypeptides are designed based on the 3D structure of the IL-2 receptor complex (available in the PDB public database), and mutations are mainly introduced at the positions of IL-2 corresponding to the amino acids that interact with the IL-2 receptor subunit α.

[0198] In various embodiments, the IL-2 variant (or mutant) comprises a sequence derived from the mature human IL-2 polypeptide sequence as set forth in SEQ ID NO:3. In various embodiments, the IL-2 variant comprises an amino acid sequence that is different from the native (or wild-type) IL-2 protein. In various embodiments, the IL-2 variant interacts with the IL-2 receptor polypeptide and functions as an IL-2 agonist or antagonist. In various embodiments, the IL-2 variant having agonist activity has superagonist activity. In various embodiments, the IL-2 variant can function as an IL-2 agonist or antagonist independent of its association with IL-2Rα. IL-2 agonists are exemplified by comparable 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 the ability to inhibit IL-2-mediated responses. In various embodiments, compared to the native IL-2 sequence, the sequence of the IL-2 variant has at least one amino acid change, such as a substitution or deletion, such a change resulting in IL-2 agonist or antagonist activity. In various embodiments, the IL-2 variant has the amino acid sequence set forth in SEQ ID NOs: 31-66, which has reduced / abolished binding to IL-2Rα to selectively activate and proliferate effector T cells (Teff). In various embodiments, the IL-2 variant has the amino acid sequence set forth in SEQ ID NOs: 111-120, which in addition to mutations that result in reduced / abolished binding to IL-2Rα, also contains mutations that modulate IL-2Rβ or γc to selectively activate and proliferate effector T cells with reduced potency, thereby reducing IL-2Rβ- or γc-related toxicity, attenuating cellular exhaustion and improving durable pharmacodynamics. In various embodiments, the IL-2 variant has the amino acid sequence of any of the following: SEQ ID NO:189 (amino acids 462-586), SEQ ID NO:190 (amino acids 462-585), and SEQ ID NO:191 (amino acids 462-584), which in addition to mutations that result in reduced / abolished binding to IL-2Rα, also contains an N-terminal deletion to selectively activate and proliferate effector T cells with reduced potency. In various embodiments, the IL-2 variants having the amino acid sequences set forth in SEQ ID NOs: 31-66, 111-120 and amino acids 9-133, 10-133 and 11-133 of SEQ ID NO:47 also contain the S125I amino acid substitution to improve the developability profile of IL-2 and the corresponding fusion proteins.

[0199] Exemplary IL-2 variants having amino acid substitutions introduced at the interface with IL-2Rα are provided in Table 2.

[0200] Table 2

[0201] An IL-2 variant or fusion construct comprising a mutation of an amino acid that interacts with the receptor subunit α. All variants contain a substitution (S125I) that improves developability.

[0202]

[0203]

[0204] A major aspect of the present invention is to improve the selectivity of IL-2 for cells expressing IL-2Rβγ (rather than IL-2Rα) over cells expressing IL-2Rαβγ relative to wild-type IL-2 for cancer treatment. One method used by the inventors was to generate highly selective IL-2-Fc fusion proteins by introducing mutations that disrupt CD25 into the cytokine component. The selection of mutations that disrupt CD25 was based on examination of the IL-2 / IL-2R co-crystal structure (PDB code 2B51). More than one amino acid substitution (including R38, T41, F42, F44, E62, P65, E68, and Y107) was introduced at one or two relevant residues at the interface with the IL-2 receptor α subunit, aiming to reduce or eliminate binding to IL-2Rα. These constructs also contain the S125I mutation for significantly improving developability. Additionally, the weakened binding of the IL-2 variant to IL-2Rα+ lung endothelial cells is expected to prevent endothelial cell damage and significantly reduce VLS. Furthermore, the weakened CD25 binding is also expected to reduce CD25 antigen uptake and enrich cytokine occupancy of cells expressing IL-2Rβγ, and thus enhance the in vivo response and tumor killing efficacy.

[0205] Since all targeted IL-2 residues (R38, T41, F42, F44, E62, P65, E68, and Y107) are at the interface with IL-2Rα and form hydrogen bonds / salt bridges or hydrophobic interactions with more than one IL-2Rα residue (Mathias Rickert et al., (2005) Science 308, 1477-80), it is inferred that the IL-2 variants and analogs listed in the table are expected to disrupt the interaction with IL-2Rα and generate IL-2 variants with reduced or eliminated binding to IL-2Rα. However, it was found that mutations at different sites and different substitutions at the same site can lead to significant differences in affecting IL-2Rα binding, which cannot be predicted by structure-based mutagenesis methods and some are particularly unexpected (see Example 4 and Example 5).

[0206] In addition, it is hypothesized that for optimal activity, substitutions that modulate IL-2Rβγ can be further introduced to attenuate the overall potency. Agonists that modulate the potency of IL-2Rβγ can prevent the overactivation of cytotoxic lymphocytes and minimize "on-target" but "off-tissue" toxicity. Additionally, overstimulation-induced cellular exhaustion and apoptosis can be minimized. Furthermore, attenuation of the cytokine signaling molecule binding affinity can reduce receptor-mediated internalization, decrease unwanted target uptake, and result in sustained receptor activation and durable pharmacodynamics and pharmacokinetics; thus, substitutions that modulate IL-2Rβγ can potentially reduce toxicity and improve pharmacokinetics, pharmacodynamics, and the therapeutic index.

[0207] Exemplary IL-2 variants with amino acid substitutions (including mutations that disrupt IL-2Rβ or γc in IL-2 variants with reduced / abolished binding to IL-2Rα) are provided in Table 3:

[0208] Table 3

[0209] Substitutions that disrupt IL-2Rβ or γc are introduced into IL-2 variants with reduced / abolished binding to IL-2Rα. All variants contain a substitution (S125I) that improves developability.

[0210]

[0211] The present invention also includes additional modifications to the above-mentioned classes of IL-2 variants and in particular those described in Tables 2 and 3, including deletions of 8, 9, or 10 N-terminal residues of the above-mentioned IL-2 variants in order to selectively activate and proliferate effector T cells with various levels of attenuated potency. Any additional combinatorial mutations are within the spirit and scope of the present invention, whether altering their affinity for specific components of the IL-2 receptor or improving their in vivo pharmacodynamics: increasing the half-life or reducing their internalization by T cells. These additional mutations can be obtained by rational design using bioinformatics tools or by using combinatorial molecular libraries of different natures (phage libraries, gene expression libraries in yeast or bacteria). In another aspect, the present invention relates to a fusion protein that comprises any of the above-described immunomodulatory polypeptides conjugated to a carrier protein. The carrier protein can be albumin or the Fc region of human immunoglobulin.

[0212] In various embodiments, an IL-2Rα Sushi having the amino acid sequence listed in SEQ ID NO: 170 is linked between IL-2 and an Fc domain using linkers of various lengths and compositions. The Fc domain can be at the N-terminus or C-terminus. IL-2-IL-2Rα Sushi-Fc fusion proteins having the amino acid sequences listed in SEQ ID NO: 171 - 172 are expected to have reduced binding to IL-2Rα to selectively activate and proliferate effector T cells.

[0213] In various embodiments, IL-2 and IL-2Rα Sushi form a non-covalent complex. IL-2 is fused to the N-terminus or C-terminus of the Hole-Fc chain (SEQ ID NO: 10), and IL-2Rα Sushi is fused to the N-terminus or C-terminus of the Knob-Fc chain (SEQ ID NO: 9). The non-covalent C-terminal IL-2-IL-2Rα Sushi-Fc fusion proteins have the amino acid sequences listed in SEQ ID NO: 173 - 174.

[0214] Table 4 IL-2 and IL-2Rα Sushi are covalently linked or non-covalently complexed as an Fc fusion protein

[0215]

[0216] Fc domain

[0217] Immunoglobulins of the IgG class are among the most abundant proteins in human blood. Their circulating half-life can be up to 21 days. Fusion proteins combining the Fc region of IgG with domains of another protein (such as various cytokines and receptors) have been reported (see, for example, Capon et al., Nature, 337:525 - 531, 1989; Chamow et al., Trends Biotechnol., 14:52 - 60, 1996); U.S. Pat. Nos. 5,116,964 and 5,541,087). The prototype fusion protein is a homodimeric protein linked by cysteine residues in the hinge region of IgG Fc, yielding a molecule similar to an IgG molecule without the heavy chain variable region and CH1 domain and the light chain. The dimeric nature of fusion proteins containing an Fc domain may be advantageous in providing higher-order interactions with other molecules (i.e., bivalent or bispecific binding). Due to structural homology, Fc fusion proteins exhibit in vivo pharmacokinetic profiles comparable to those of human IgG with similar isotypes.

[0218] The term "Fc" refers to a molecule or sequence comprising the sequence of the non-antigen-binding fragment of a full antibody, whether in monomeric or multimeric form. The original immunoglobulin source of native Fc is preferably of human origin and can be any immunoglobulin, although IgG1 and IgG2 are preferred. Native Fc consists of monomeric polypeptides that can be joined into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent associations. The number of intermolecular disulfide bonds between the monomeric subunits of a native Fc molecule ranges from 1 to 4 depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). An example of native Fc is the disulfide-bonded dimer produced by papain digestion of IgG (see Ellison et al. (1982), Nucleic Acids Res. 10:4071-9). As used herein, the term "native Fc" is a general term for monomeric, dimeric, and multimeric forms. The Fc domain contains binding sites for Protein A, Protein G, various Fc receptors, and complement proteins.

[0219] In various embodiments, the term "Fc variant" refers to a molecule or sequence that is modified from native Fc but still contains the binding site for the salvage receptor FcRn. International Applications WO 97 / 34631 (published September 25, 1997) and WO 96 / 32478 describe exemplary Fc variants and their interactions with the salvage receptor and are hereby incorporated by reference. Additionally, native Fc contains sites that can be removed because they provide structural features or biological activities not desired for the fusion molecules of the present invention. Thus, in various embodiments, the term "Fc variant" includes molecules or sequences lacking one or more native Fc sites or residues that affect or participate in (1) disulfide bond formation, (2) incompatibility with the selected host cell, (3) N-terminal heterogeneity upon expression in the selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor, or (7) antibody-dependent cell cytotoxicity (ADCC).

[0220] The term "Fc domain" includes native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from a full antibody or produced by recombinant gene expression or by other means. In various embodiments, the "Fc domain" refers to a dimer of two Fc domain monomers (SEQ ID NO:6), which typically includes all or part of the hinge region. In various embodiments, the Fc domain can be mutated to lack effector function. In various embodiments, each Fc domain monomer in the Fc domain contains amino acid substitutions in the CH2 antibody constant domain to reduce the interaction or binding between the Fc domain and Fcγ receptors. In various embodiments, each subunit of the Fc domain contains three amino acid substitutions that reduce binding to activating Fc receptors and / or effector function, wherein the amino acid substitutions are L234A, L235A, and G237A (SEQ ID NO:7).

[0221] In various embodiments, each of the two Fc domain monomers in the Fc domain contains an amino acid substitution that promotes heterodimerization of the two monomers. In various other embodiments, heterodimerization of the Fc domain monomers can be promoted by introducing different but compatible substitutions such as "knob-into-hole" residue pairs in the two Fc domain monomers. The "knob-into-hole" technique is also disclosed in U.S. Patent Publication No. 8,216,805. In yet another embodiment, one Fc domain monomer contains the knob mutation T366W, and the other Fc domain monomer contains the hole mutations T366S, L358A, and Y407V. In various embodiments, two Cys residues are introduced to form a stabilizing disulfide bridge (S354C on the "knob" side and Y349C on the "hole" side) (SEQ ID NO:9 and 10). The use of heterodimeric Fc can produce a monovalent IL-2 variant.

[0222] In various embodiments, the Fc domain sequence used to prepare the dimeric IL-2 variant Fc fusion is the human IgG1-Fc domain sequence set forth in SEQ ID NO:7:

[0223] DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY

[0224] VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

[0225] KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT

[0226] PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0227] (SEQ ID NO:7)

[0228] Among them, SEQ ID NO:7 contains amino acid substitutions (underlined) that eliminate FcγR and C1q binding.

[0229] In various embodiments, the Fc domain sequence used to prepare the dimeric IL-2Fc fusion protein is the IgG1-Fc domain sequence listed in SEQ ID NO:8:

[0230] DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY

[0231] VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

[0232] KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT

[0233] PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPG

[0234] (SEQ ID NO:106)

[0235] Among them, SEQ ID NO:8 contains amino acid substitutions (underlined) that eliminate FcγR and C1q binding and amino acid substitutions (bold) that extend the half-life.

[0236] In various embodiments, the heterodimeric Fc domain sequence used to prepare the monomeric IL-2 variant Fc fusion is the Knob-Fc domain sequence listed in SEQ ID NO:9:

[0237] DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY

[0238] VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

[0239] KAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTT

[0240] PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0241] (SEQ ID NO:9)

[0242] Among them, SEQ ID NO:9 contains amino acid substitutions (underlined) that eliminate FcγR and C1q binding.

[0243] In various embodiments, the heterodimeric Fc domain sequence used to prepare the IL-2 variant is the Hole-Fc domain sequence listed in SEQ ID NO:10:

[0244] DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY

[0245] VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

[0246] KAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTT

[0247] PPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0248] (SEQ ID NO:10)

[0249] Among them, SEQ ID NO:10 contains amino acid substitutions (underlined) that eliminate FcγR and C1q binding.

[0250] In various embodiments, the heterodimeric Fc domain for preparing the monomeric IL-2 variant Fc fusion protein is the Knob-Fc domain with reduced / eliminated effector function and extended half-life and has the amino acid sequence listed in SEQ ID NO:134:

[0251] DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY

[0252] VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

[0253] KAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTT

[0254] PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPG

[0255] (SEQ ID NO:134)

[0256] Wherein SEQ ID NO:134 contains amino acid substitutions (underlined) that eliminate FcγR and C1q binding and amino acid substitutions (bold) that extend the half-life.

[0257] In various embodiments, the heterodimeric Fc domain for preparing the monomeric IL-2 variant Fc fusion protein is the Hole-Fc domain with reduced / eliminated effector function and extended half-life and has the amino acid sequence set forth in SEQ ID NO:135:

[0258] DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY

[0259] VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

[0260] KAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTT

[0261] PPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPG

[0262] (SEQ ID NO:135)

[0263] Among them, SEQ ID NO: 135 contains amino acid substitutions (underlined) that eliminate FcγR and C1q binding and amino acid substitutions (bold) that extend the half-life.

[0264] Antibodies as targeting moieties

[0265] In various embodiments, the IL-2 variant constructs of the invention comprise targeting moieties in the form of antibodies, antibody fragments, proteins or peptides that bind to molecules enriched in cancer tissue, such as tumor-associated antigens (TAAs).

[0266] A TAA can be any molecule, macromolecule, molecular combination, etc. for which an immune response is desired. A TAA can be a protein comprising more than one polypeptide subunit. A TAA can be a protein comprising more than one polypeptide subunit. For example, the protein can be a dimer, trimer or higher-order multimer. In various embodiments, two or more subunits of the protein can be linked by covalent bonds such as, for example, disulfide bonds. In various embodiments, the subunits of the protein can be held together by non-covalent interactions. Thus, a TAA can be any peptide, polypeptide, protein, nucleic acid, lipid, carbohydrate or organic small molecule or any combination thereof that a person skilled in the art wishes to induce an immune response against. In various embodiments, the TAA is a peptide comprising: 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 that is typically administered to a subject by injection. In various embodiments, after administration, a tumor-specific antibody or binding protein is used as a targeting moiety to direct the IL-2 variant to the site of the lesion, such as the cancer site, where the active domain can be released and interact with its cognate receptor on the diseased cells.

[0267] Any of the foregoing markers can be used as a TAA target for the IL-2 variants of the invention. In various embodiments, one or more TAAs, TAA variants or TAA mutants contemplated for use in the IL-2 variant constructs and methods of the present disclosure are selected from or derived from the list provided in Table 5.

[0268] Table 5

[0269]

[0270]

[0271]

[0272] In various embodiments, the IL-2 variants of the present invention can be attached to a targeting / bifunctional moiety that is an antibody, antibody fragment, protein, or peptide targeting an immune checkpoint modulator.

[0273] Numerous immune checkpoint protein antigens expressed on various immune cells have been reported, including, for example, SIRP (expressed on macrophages, monocytes, dendritic cells), CD47 (highly expressed on tumor cells and other cell types), VISTA (expressed on monocytes, dendritic cells, B cells, T cells), CD152 (expressed by activated CD8+ T cells, CD4+ T cells, and regulatory T cells), CD279 (expressed on tumor-infiltrating lymphocytes, expressed by activated T cells (both CD4 and CD8), regulatory T cells, activated B cells, activated NK cells, invariant T cells, monocytes, dendritic cells), CD274 (expressed on T cells, B cells, dendritic cells, macrophages, vascular endothelial cells, pancreatic islet cells), and CD223 (expressed by activated T cells, regulatory T cells, invariant T cells, NK cells, NKT cells, and plasmacytoid dendritic cells) (see, e.g., Pardoll, D., Nature Reviews Cancer, 12:252-264, 2012). Antibodies that bind to antigens determined to be immune checkpoint proteins are known to those of skill in the art. For example, various anti-CD276 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20120294796 (Johnson et al.) and references cited therein); various anti-CD272 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20140017255 (Mataraza et al.) and references cited therein); various anti-CD152 / CTLA-4 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20130136749 (Korman et al.) and references cited therein); various anti-LAG-3 / CD223 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20110150892 (Thudium et al.) and references cited therein); various anti-CD279 (PD-1) antibodies have been described in the art (see, e.g., U.S. Patent No. 7,488,802 (Collins et al.) and references cited therein); various anti-CD274 (PD-L1) antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20130122014 (Korman et al.) and references cited therein); various anti-TIM-3 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20140044728 (Takayanagi et al.) and references cited therein); and various anti-B7-H4 antibodies have been described in the art (see, e.g., U.S. Patent Publication No. 20110085970 (Terrett et al.) and references cited therein).Each of these references is hereby incorporated by reference in its entirety for the specific antibodies and sequences taught therein.

[0274] In various embodiments, the IL-2 fusion partner can be an antibody, antibody fragment, or protein or peptide that exhibits antigen binding to an immune checkpoint protein present on the surface of immune cells. In various embodiments, the immune checkpoint protein antigen is selected from the group consisting of but not limited to PD1 (CD279), PDL-1 (CD274), CD276, CD272, CD152 (CTLA-4), CD223, CD279, CD274, CD40, SIRPα, CD47, OX-40, GITR, ICOS, CD27, 4-1BB, TIM-3, B7-H3, B7-H4, TIGIT, and VISTA.

[0275] In various embodiments, the antibody is an antagonistic FAP antibody or antibody fragment. In various embodiments, the antibody is a humanized antagonistic FAP antibody comprising the variable domain sequences listed in SEQ ID NOs: 136 and 137. In various embodiments, the heterologous protein is an antibody or antibody fragment against an immune checkpoint modulator. In various embodiments, the antibody is an antagonistic human TIGIT antibody. In various embodiments, the antibody is an antagonistic PD-1 antibody or antibody fragment. In various embodiments, the antibody is an antagonistic PD-1 antibody comprising the variable domain sequences listed in: SEQ ID NOs: 138 and 139, SEQ ID NOs: 140 and 141, SEQ ID NOs: 142 and 143, SEQ ID NOs: 144 and 145, or SEQ ID NOs: 146 and 147. In various embodiments, the antibody is an antagonistic human PD-L1 antibody comprising the variable domain sequences listed in SEQ ID NOs: 148 and 149. In various embodiments, the antibody is an antagonistic human CTLA-4 antibody comprising the variable domain sequences listed in SEQ ID NOs: 150 and 151. In various embodiments, exemplary bifunctional IL-2-PD1 antibody fusion proteins are listed in Table 12.

[0276] Bifunctional IL-2 variant-PD-1 antibody fusion protein

[0277] In various embodiments, immune checkpoint blocking antibodies that bypass immunosuppressive effects in the tumor microenvironment or immune stimulatory antibodies that enhance existing responses are used to construct IL-2 antibody fusion proteins. On T cells that are infiltrated in the tumor microenvironment and have exhausted in the face of tumor antigens, the expression levels of negative immune checkpoints are particularly increased. In various embodiments, tethering an IL-2 variant to an antibody that targets an immune checkpoint is expected to direct IL-2 to exhausted T cells and make the tumor microenvironment immunologically hot. In various embodiments, a bifunctional IL-2 variant checkpoint inhibitor antibody fusion protein can preferentially deliver IL-2 in cis to cells that express checkpoint inhibitors, such as exhausted T cells that are infiltrated in the tumor microenvironment and have experienced tumor antigens, to promote selective signaling and enhance activity at the desired tumor site. In various embodiments, the bifunctional IL-2 variant checkpoint inhibitor antibody fusion protein provides synergy by removing negative regulation and revitalizing T cell function as well as expanding the number of Teff cells to further enhance the anti-tumor activity of the immune system.

[0278] In various embodiments, the bifunctional IL-2 variant checkpoint inhibitor antibody fusion protein reduces the systemic exposure and off-target toxicity of IL-2. In various embodiments, using an IL-2 variant with both reduced / eliminated binding to IL-2Rα and attenuated / modulated IL-2Rβγ activity helps to establish a stoichiometric balance between cytokine IL-2 activity and antibody activity. The attenuated IL-2 activity variant and having sufficient antibody targeting or cis-activation at exhausted Teff cells will allow for optimal dosing and maintenance of the function of each arm. Additionally, the attenuated IL-2 activity variant fused to an antibody is expected to minimize peripheral activation, reduce T cell AICD, mitigate antigen uptake, and promote tumor killing via the antibody targeting moiety against tumor and / or immune cell sites.

[0279] In various embodiments, the IL-2 variant of the present invention can be attached to a checkpoint inhibitor, which is an antibody, antibody fragment, protein, or peptide that targets an immune checkpoint regulator. In various embodiments, the immune checkpoint inhibitor is an antagonist PD-1 antibody. In various embodiments, the PD-1 antibody comprises the variable domain sequences listed in: SEQ ID NO:138 and 139, SEQ ID NO:140 and 141, SEQ ID NO:142 and 143, SEQ ID NO:144 and 145, or SEQ ID NO:146 and 147. In various embodiments, exemplary bifunctional IL-2-PD1 antibody fusion proteins are listed in Table 12.

[0280] Linker

[0281] In various embodiments, the heterologous protein is attached to the IL-2 variant via a linker and / or a hinge linker peptide. The linker or hinge linker can be an artificial sequence between 5, 10, 15, 20, 30, 40 or more amino acids that is relatively devoid of secondary structure or exhibits an α-helical conformation.

[0282] The peptide linker provides a covalent connection between protein domains and additional structural and / or spatial flexibility. As is known in the art, peptide linkers contain flexible amino acid residues such as glycine and serine. In various embodiments, the peptide linker can contain 1 - 100 amino acids. In various embodiments, the spacer can contain the motif GGGSGGGS (SEQ ID NO:18). In other embodiments, the linker can contain the motif GGGGS (SEQ ID NO:21)n, where n is an integer from 1 to 10. In other embodiments, the linker can also contain amino acids other than glycine and serine. In another embodiment, the linker can contain other protein motifs, including but not limited to sequences with an α-helical conformation such as AEAAAKEAAAKEAAAKA (SEQ ID NO:16). In various embodiments, the linker length and composition can be adjusted to optimize activity or developability, including but not limited to expression level and aggregation propensity. In another embodiment, the peptide linker can be a simple chemical bond, such as an amide bond (e.g., chemical conjugation via PEG).

[0283] Exemplary peptide linkers are provided in Table 6:

[0284] Table 6

[0285]

[0286]

[0287] Polynucleotide

[0288] In another aspect, the present disclosure provides isolated nucleic acid molecules that comprise polynucleotides encoding the IL-2, IL-2 variants, IL-2 fusion proteins, or IL-2 variant fusion proteins of the present disclosure. The subject nucleic acids can be single-stranded or double-stranded. Such nucleic acids can be DNA or RNA molecules. DNA includes, for example, cDNA, genomic DNA, synthetic DNA, DNA amplified by PCR, and combinations thereof. Genomic DNA encoding the IL-2 polypeptide is obtained from genomic libraries that are available for many species. Synthetic DNA is obtainable by chemically synthesizing overlapping oligonucleotide fragments and then assembling the fragments to reconstruct part or all of the coding region and flanking sequences. RNA can be obtained from prokaryotic expression vectors that direct high-level synthesis of mRNA, such as vectors that use the T7 promoter and RNA polymerase. cDNA is obtained from libraries prepared from mRNA isolated from various tissues that express IL-2. The DNA molecules of the present disclosure include full-length genes as well as polynucleotides and fragments thereof. The full-length genes can also include sequences encoding an N-terminal signal sequence. Such nucleic acids can be used, for example, in methods for preparing novel IL-2 variants.

[0289] In various embodiments, the isolated nucleic acid molecule comprises the polynucleotides 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.

[0290] In various embodiments, the recombinant nucleic acids of the present disclosure can be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory sequences are recognized in the art and are selected to direct the expression of the IL-2 variants. Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). Generally, the one or more regulatory nucleotide sequences can include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. The present disclosure contemplates constitutive or inducible promoters known in the art. The promoter can be a naturally occurring promoter or a hybrid promoter that combines elements of more than one promoter. The expression construct can be present on an episome such as a plasmid in a cell, or the expression construct can be inserted into a chromosome. In various embodiments, the expression vector contains a selectable marker gene to allow selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.

[0291] In another aspect of the present disclosure, the subject nucleic acid is provided in an expression vector, the expression vector comprising a nucleotide sequence encoding an IL-2 variant and operably linked to at least one regulatory sequence. The term "expression vector" refers to a plasmid, phage, virus, or vector used for expressing a polypeptide from a polynucleotide sequence. Vectors suitable for expression in host cells are readily available and nucleic acid molecules are inserted into the vectors using standard recombinant DNA techniques. Such vectors can include a variety of expression control sequences that, when operably linked to a DNA sequence, control the expression of that DNA sequence and can be used in these vectors to express the DNA sequence encoding the IL-2 variant. Such useful expression control sequences include, for example, the early and late promoters of SV40, the tet promoter, the adenovirus- or cytomegalovirus-mediated early promoters, the RSV promoter, the lac system, the trp system, the TAC or TRC systems, the T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of lambda phage, the control region of the fd coat protein, the promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter of acid phosphatase such as PhoS, the promoter of yeast a-mating factor, the polyhedron promoter of the baculovirus system, and other sequences known to control the gene expression of prokaryotic cells, eukaryotic cells (yeast, avian, insect, or mammalian), or their viruses and various combinations thereof. It should be understood that the design of the expression vector may depend on factors such as the choice of host cell to be transformed and / or the type of protein desired to be expressed. In addition, consideration should also be given to the copy number of the vector, the ability to control that copy number, and the expression of any other proteins encoded by the vector such as antibiotic markers. Exemplary expression vectors suitable for expressing IL-2 are pDSRa (described in WO 90 / 14363, incorporated herein by reference) and its derivatives that contain the IL-2 polynucleotide, and any other suitable vectors known in the art or described hereinafter.

[0292] The recombinant nucleic acids of the present disclosure can be produced by ligating a cloned gene or a portion thereof to a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, avian, insect, or mammalian), or both. Expression vectors for producing recombinant IL-2 polypeptides include plasmids and other vectors. For example, suitable vectors include plasmids of the following types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotic cells such as Escherichia coli.

[0293] Some mammalian expression vectors contain both prokaryotic sequences that facilitate the replication of the vector in bacteria and one or more eukaryotic transcription units for expression in eukaryotic cells. Examples of mammalian expression vectors suitable for transfection of eukaryotic cells are vectors derived from pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg. Some of these vectors are modified with sequences from bacterial plasmids such as pBR322 to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Optionally, derivatives of viruses such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived, and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found in the description of gene therapy delivery systems below. The various methods employed in the preparation of plasmids and the transformation of host organisms are well known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells and for general recombinant procedures, see Chapters 16 and 17 of Molecular Cloning A Laboratory Manual, 2nd edition (Cold Spring Harbor Laboratory Press, 1989) by Sambrook, Fritsch, and Maniatis. In some instances, it may be desirable to express recombinant polypeptides by using a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as pBlueBac III containing B-gal).

[0294] In various embodiments, the vector will be designed to produce the subject IL-2 variants in CHO cells, such as the Pcmv-Script vector (Stratagene, La Jolla, Calif.), the pcDNA4 vector (Invitrogen, Carlsbad, Calif.), and the pCI-neo vector (Promega, Madison, Wis.). It will be apparent that the subject gene constructs can be used to cause expression of the subject IL-2 variants in cells growing in culture, for example to produce proteins for purification, including fusion proteins or variant proteins.

[0295] The present disclosure also relates to host cells transfected with a recombinant gene comprising a nucleotide sequence encoding an amino acid sequence of one or more of the subject IL-2 variants. The host cell can be a prokaryotic cell or a eukaryotic cell. For example, the IL-2 variants of the present disclosure can be expressed in bacterial cells such as Escherichia coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.

[0296] Accordingly, the present disclosure also relates to methods of producing the subject IL-2 variants. For example, host cells transfected with an expression vector encoding an IL-2 variant can be cultured under appropriate conditions that permit expression of the IL-2 variant. The IL-2 variant can be secreted from the cells containing the IL-2 variant and isolated from a mixture of the cells containing the IL-2 variant and the culture medium. Optionally, the IL-2 variant can be retained in the cytoplasm or in the membrane fraction, and the cells can be harvested, lysed, and the protein isolated. Cell cultures include host cells, culture media, and other by-products. Suitable culture media for cell culture are well known in the art.

[0297] The polypeptides and proteins of the present disclosure can be purified according to protein purification techniques well known to those skilled in the art. These techniques involve at one level a crude fractionation of protein fractions and non-protein fractions. After separating the peptide or polypeptide from other proteins, chromatographic and electrophoretic techniques can be utilized to further purify the peptide or polypeptide of interest to achieve partial or complete purification (or purification to homogeneity). As used herein, the terms "isolated polypeptide" or "purified polypeptide" are intended to refer to a composition separable from other components, wherein the polypeptide is purified to any degree relative to its naturally occurring state. Thus, a purified polypeptide also refers to a polypeptide removed from the environment in which it may naturally occur. Generally, "purified" will refer to a polypeptide composition that has undergone fractionation to remove various other components, and the polypeptide composition substantially retains its expressed biological activity. When the term "substantially purified" is used, this designation will refer to a peptide or polypeptide composition in which the polypeptide or peptide forms the majority of the components of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 85%, or about 90% or more of the proteins in the composition.

[0298] A variety of techniques suitable for purification will be well known to those skilled in the art. These techniques include, for example, precipitation with ammonium sulfate, PEG, antibodies (immunoprecipitation), etc. or precipitation by heat denaturation followed by centrifugation; chromatography, such as affinity chromatography (Protein A column), ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, hydroxyapatite chromatography, hydrophobic interaction chromatography; isoelectric focusing; gel electrophoresis; and combinations of these techniques. As is generally known in the art, it is considered that the order in which the various purification steps are carried out can be varied, or certain steps can be omitted, and still a suitable method for preparing a substantially purified polypeptide is obtained.

[0299] Pharmaceutical composition

[0300] In another aspect, the present disclosure provides a pharmaceutical composition comprising an IL-2 variant or an IL-2 variant fusion protein admixed with a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are well known and understood by those of ordinary skill in the art and have been widely described (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, ed., Mack Publishing Company, 1990). Pharmaceutically acceptable carriers may be included for the purpose of modifying, maintaining, or preserving, for example, pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, absorption, or penetration of the composition. Such pharmaceutical compositions can affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the polypeptide. Suitable pharmaceutically acceptable carriers include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, 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); coloring agents; flavoring agents, and diluents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (sucrose or sorbitol); tonicity enhancers (such as alkali metal halides (preferably sodium chloride or potassium chloride), mannitol, and sorbitol); delivery vehicles; diluents; excipients; and / or pharmaceutical adjuvants).

[0301] The primary vehicle or carrier in a pharmaceutical composition can be substantially aqueous or non-aqueous. For example, suitable vehicles or carriers can be water for injection, saline solution, or artificial cerebrospinal fluid, which may be supplemented with other materials commonly found in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are additional exemplary vehicles. Other exemplary pharmaceutical compositions contain a Tris buffer at about pH 7.0 - 8.5 or an acetate buffer at about pH 4.0 - 5.5, which may also contain sorbitol or a suitable alternative to sorbitol. In one embodiment of the present disclosure, a composition can be prepared by mixing a selected composition having a desired degree of purity with an optional formulation agent (Remington's Pharmaceutical Sciences, supra) for storage in the form of a lyophilized cake or an aqueous solution. Additionally, a therapeutic composition can be formulated as a lyophilized product using a suitable excipient such as sucrose. The optimal pharmaceutical composition will be determined by one of ordinary skill in the art depending on, for example, the intended route of administration, delivery form, and desired dose.

[0302] When parenteral administration is contemplated, the therapeutic pharmaceutical composition can be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired IL-2 polypeptide or IL-2 polypeptide fusion protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the polypeptide is formulated as a sterile, isotonic solution for suitable storage. In various embodiments, a pharmaceutical formulation suitable for injectable administration can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or physiological buffered saline. An aqueous injectable suspension can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, a suspension of the active compound can be prepared as a suitable oily injectable suspension. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compound and permit the preparation of highly concentrated solutions.

[0303] In various embodiments, a therapeutic pharmaceutical composition can be formulated for targeted delivery using colloidal dispersion systems. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, and lipid-based systems include oil-in-water emulsions, micelles, mixed micelles, and liposomes. Examples of lipids useful in liposome production include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Exemplary phospholipids include lecithin, dipalmitoyl phosphatidylcholine, and distearoyl phosphatidylcholine. Targeting of liposomes can also be based on, for example, organ specificity, cell specificity, and organelle specificity and is known in the art.

[0304] In various embodiments, oral administration of the pharmaceutical composition is contemplated. The pharmaceutical composition administered in this form can be formulated with or without the carriers commonly used in the compounding of solid dosage forms such as tablets and capsules. In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), one or more of the therapeutic compounds of the present disclosure can be mixed with one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or gum arabic; (3) humectants, such as glycerol; (4) disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders, such as paraffin wax; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glyceryl monostearate; (8) absorbents, such as kaolin and bentonite; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical composition can also contain buffering agents. Similar types of solid compositions can also be used as fillers in soft and hard gelatin capsules filled with excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifying agents such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (particularly, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan and mixtures thereof. In addition to the inert diluent, the oral compositions can also contain adjuvants such as wetting agents, emulsifying agents, and suspending agents, sweetening agents, flavoring agents, coloring agents, perfuming agents, and preservatives.

[0305] In various embodiments, topical administration of a pharmaceutical composition to the skin or to mucous membranes is contemplated. The topical formulation may also include one or more of a variety of agents known to be effective as skin or stratum corneum penetration enhancers. Examples of such agents are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methanol or isopropanol, dimethyl sulfoxide, and azone. Additional agents may also be included to make the formulation cosmetically acceptable. Examples of such agents are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surfactants. A keratolytic agent may also be included, such as those known in the art. Examples are salicylic acid and sulfur. Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound can be mixed with a pharmaceutically acceptable carrier and with any preservatives, buffers, or propellants as may be required, under sterile conditions. In addition to the subject compounds of the present disclosure (e.g., IL-2 variants), ointments, pastes, creams, and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, gum tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0306] Additional pharmaceutical compositions contemplated for use herein include formulations that include a polypeptide in a sustained release or controlled release formulation. In various embodiments, the pharmaceutical composition can be formulated in nanoparticles, formulated as a sustained release hydrogel, or incorporated into an oncolytic virus. Such nanoparticle approaches include, for example, encapsulation in nanoparticles comprising a polymer having a hydrophobic backbone and hydrophilic side chains as a drug carrier, encapsulation in microparticles, insertion into liposomes as an emulsion, and conjugation with other molecules. Examples of nanoparticles include mucoadhesive nanoparticles coated with chitosan and carbopol (Takeuchi et al., Adv. Drug Deliv. Rev. 47(1):39-54, 2001) and nanoparticles comprising a combination of charged polyesters, poly(2-sulfoethyl vinyl alcohol), and poly(D,L-lactic-co-glycolic acid) (Jung et al., Eur. J. Pharm. Biopharm. 50(1):147-160, 2000). Albumin-based nanoparticle compositions have been developed as drug delivery systems for delivering hydrophobic drugs such as taxanes. See, for example, U.S. Patent Nos. 5,916,596, 6,506,405, 6,749,868, 6,537,579, 7,820,788, and 7,923,536. (Albumin-stabilized paclitaxel nanoparticle formulation) was approved in the United States in 2005 and subsequently approved in many other countries for the treatment of metastatic breast cancer.

[0307] Techniques for formulating various other sustained or controlled delivery vehicles such as liposomal carriers, bioerodible microparticles or porous beads, and depot injections are also known to those skilled in the art.

[0308] The effective amount of the pharmaceutical composition to be used therapeutically will depend, for example, on the therapeutic context and the therapeutic objective. Those skilled in the art will understand that the appropriate dosage level for treatment will thus vary in part depending on the molecule being delivered, the indication for which the polypeptide is used, the route of administration, and the size (body weight, body surface or organ size) and condition (age and general health) of the patient. Accordingly, the clinician can adjust the dosage and change the route of administration to obtain the best therapeutic effect. Typical dosages can range from about 0.001 mg / kg to up to about 100 mg / kg or more depending on the factors mentioned above. The polypeptide composition can preferably be administered by injection or intravenously. The long-acting pharmaceutical composition can be administered once every three to four days, once a week or once every two weeks depending on the half-life and clearance rate of the particular formulation. The dosing frequency will depend on the pharmacokinetic parameters of the polypeptide in the formulation used. Generally, the composition is administered until a dose is reached that achieves the desired effect. Thus, the composition can be administered as a single dose or as multiple doses over time (at the same or different concentrations / doses) or as a continuous infusion. Further refinement of the appropriate dosage is routinely carried out. The appropriate dosage can be determined by using appropriate dose-response data.

[0309] The route of administration of the pharmaceutical composition is according to known methods, for example, orally; by intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional routes, intramedullary, intrathecal, intraventricular, percutaneous, subcutaneous or intraperitoneal or intratumoral injection; and intranasal, enteral, topical, sublingual, urethral, vaginal or rectal routes; by sustained release systems or by implant devices. When desired, the composition can be administered by bolus injection, or continuously by infusion, or by implant device. Optionally or additionally, the composition can be administered locally by implantation of a membrane, sponge or another suitable material to which the desired molecule has been adsorbed or encapsulated. When using an implant device, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be by diffusion administration, slow release bolus or continuous administration.

[0310] Therapeutic use

[0311] In one aspect, the present disclosure provides a method for treating cancer cells in a subject, the method comprising administering to the subject a therapeutically effective amount (as a single therapy or in a combination therapy regimen) of an IL-2 variant or an IL-2 variant fusion protein of the present disclosure in a pharmaceutically acceptable carrier, wherein such administration inhibits the growth and / or proliferation of cancer cells. In particular, the IL-2 variant or IL-2 variant fusion protein of the present disclosure is useful in treating disorders characterized by cancer. Such disorders include, but are not limited to, solid tumors such as breast cancer, respiratory cancer, brain cancer, cancers of the reproductive organs, cancers of the digestive tract, cancers of the urinary tract, eye cancer, liver cancer, skin cancer, head and neck cancer, thyroid cancer, parathyroid cancer, 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 in situ. Examples of respiratory cancer include, but are not limited to, small cell lung cancer and non-small cell lung cancer, as well as bronchial adenoma and pleuropulmonary blastoma. Examples of brain cancer include, but are not limited to, brainstem and hypothalamic gliomas, cerebellar and cerebral astrocytomas, medulloblastoma, ependymoma, and neuroectodermal and pineal tumors. Tumors of the male / male reproductive organs include, but are not limited to, prostate cancer and testicular cancer. Tumors of the female / female reproductive organs include, but are not limited to, endometrial cancer, cervical cancer, ovarian cancer, vaginal cancer, and vulvar cancer, as well as sarcoma of the uterus. Tumors of the digestive tract include, but are not limited to, anal cancer, colon cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, pancreatic cancer, rectal cancer, small intestine cancer, and salivary gland cancer. Tumors of the urinary tract include, but are not limited to, bladder cancer, penile cancer, kidney cancer, renal pelvis cancer, ureteral cancer, and urethral cancer. Eye cancer includes, but is not limited to, intraocular melanoma and retinoblastoma. Examples of liver cancer include, but are not limited to, hepatocellular carcinoma (with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular cholangiocarcinoma. Skin cancer includes, but is not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer. Head and neck cancer includes, but is not limited to, nasopharyngeal cancer and cancers of the lip and oral cavity. Lymphoma includes, but is not limited to, AIDS-related lymphoma, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and central nervous system lymphoma. Sarcoma includes, but is not limited to, soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Leukemia includes, but is not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, various lymphocytic leukemias, various myelocytic leukemias, and hairy cell leukemia.In various embodiments, the cancer will be a cancer having high expression of TGF-β family members such as activin A, myostatin, TGF-β, and GDF15, such as pancreatic cancer, gastric cancer, ovarian cancer, colorectal cancer, melanoma leukemia, lung cancer, prostate cancer, brain cancer, bladder cancer, and head and neck cancer.

[0312] A "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a therapeutic agent that will reduce one or more symptoms of a disorder being treated to some extent.

[0313] The therapeutically effective dose can be initially evaluated by determining the EC 50 from cell culture assays. Then, the dose can be formulated in an animal model to achieve a circulating plasma concentration range that includes the EC as determined in cell culture 50 . Such information can be used to more precisely determine the dose useful in humans. The levels in plasma can be measured, for example, by HPLC. The exact composition, route of administration, and dose can be selected by the individual physician in view of the condition of the subject.

[0314] The dosage regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic response or a prophylactic response). For example, a single bolus can be administered, several divided doses (multiple or repeated or maintenance) can be administered over time, and the dose can be proportionally decreased or increased as indicated by the exigencies of the treatment situation. To facilitate administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as a single dose for the mammalian subject to be treated; each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present disclosure will be determined primarily by the unique characteristics of the antibody and the particular therapeutic or prophylactic effect to be achieved.

[0315] Thus, those skilled in the art will understand that, based on the disclosure provided herein, the dosage and dosing regimen are adjusted according to methods well known in the art of therapy. That is, the maximum tolerable dose can be readily determined, and the effective amount that provides a detectable therapeutic benefit to the subject can also be determined, as can the time requirements for administering each agent to provide a detectable therapeutic benefit to the subject. Thus, although certain doses and dosing regimens are exemplified herein, these examples are in no way limiting as to the doses and dosing regimens that can be provided to a subject in practicing the present disclosure.

[0316] It should be noted that the dosage value can vary with the type and severity of the condition to be alleviated and can include a single dose or more than one dose. It should also be understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering the composition or supervising the administration of the composition, and the dosage ranges listed herein are only exemplary and are not intended to limit the scope of the claimed composition or practice. Additionally, the dosage regimen of the compositions of the present disclosure can be based on multiple factors, including the type of disease, the age, weight, gender, medical condition, severity of the condition, route of administration, and the specific antibody used of the subject. Thus, the dosage regimen can vary widely but can be routinely determined using standard methods. For example, the dosage can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which can include clinical effects such as toxic effects and / or experimental values. Accordingly, the present disclosure includes intra-subject dose-escalation as determined by a person skilled in the art. Determining the appropriate dosage and regimen is well known in the relevant art and will be understood to be within the grasp of a person skilled in the art once the teachings disclosed herein are provided.

[0317] Exemplary, non-limiting daily dosage ranges for a therapeutically effective amount or prophylactically effective amount of an IL-2 variant or IL-2 variant fusion protein of the present disclosure can be from 0.001 to 100 mg / kg body weight, from 0.001 to 90 mg / kg body weight, from 0.001 to 80 mg / kg body weight, from 0.001 to 70 mg / kg body weight, from 0.001 to 60 mg / kg body weight, from 0.001 to 50 mg / kg body weight, from 0.001 to 40 mg / kg body weight, from 0.001 to 30 mg / kg body weight, from 0.001 to 20 mg / kg body weight, from 0.001 to 10 mg / kg body weight, from 0.001 to 5 mg / kg body weight, from 0.001 to 4 mg / kg body weight, from 0.001 to 3 mg / kg body weight, from 0.001 to 2 mg / kg body weight, from 0.001 to 1 mg / kg body weight, from 0.010 to 50 mg / kg body weight, from 0.010 to 40 mg / kg body weight, from 0.010 to 30 mg / kg body weight, from 0.010 to 20 mg / kg body weight, from 0.010 to 10 mg / kg body weight, from 0.010 to 5 mg / kg body weight, from 0.010 to 4 mg / kg body weight, from 0.010 to 3 mg / kg body weight, from 0.010 to 2 mg / kg body weight, from 0.010 to 1 mg / kg body weight, from 0.1 to 50 mg / kg body weight, from 0.1 to 40 mg / kg body weight, from 0.1 to 30 mg / kg body weight, from 0.1 to 20 mg / kg body weight, from 0.1 to 10 mg / kg body weight, from 0.1 to 5 mg / kg body weight, from 0.1 to 4 mg / kg body weight, from 0.1 to 3 mg / kg body weight, from 0.1 to 2 mg / kg body weight, from 0.1 to 1 mg / kg body weight, from 1 to 50 mg / kg body weight, from 1 to 40 mg / kg body weight, from 1 to 30 mg / kg body weight, from 1 to 20 mg / kg body weight, from 1 to 10 mg / kg body weight, from 1 to 5 mg / kg body weight, from 1 to 4 mg / kg body weight, from 1 to 3 mg / kg body weight, from 1 to 2 mg / kg body weight, or from 1 to 1 mg / kg body weight. It should be noted that the dosage values can vary with the type and severity of the condition to be alleviated. It should also be understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering the composition or supervising the administration of the composition, and the dosage ranges listed herein are merely exemplary and are not intended to limit the scope of the claimed composition or its practice.

[0318] The toxicity and therapeutic index of the pharmaceutical compositions of the present disclosure can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, e.g., determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic dose and the therapeutically effective dose is the therapeutic index, and the therapeutic index can be expressed as the ratio LD50 / ED 50 Compositions that exhibit a large therapeutic index are generally preferred.

[0319] The dosing frequency of administration of an IL-2 variant or IL-2 variant fusion protein pharmaceutical composition depends on the nature of the therapy and the particular disease being treated. The subject can be treated at regular intervals, such as twice weekly, weekly, or monthly, until the desired therapeutic outcome is achieved. Exemplary dosing frequencies include, but are not limited to: once weekly without interruption; once every 2 weeks; once every 3 weeks; once weekly without interruption for 2 weeks, then once monthly; once weekly without interruption for 3 weeks, then once monthly; once monthly; once every two months; once every 3 months; once every 4 months; once every 5 months; or once every 6 months, or once a year.

[0320] Combination therapy

[0321] As used herein, when referring to an IL-2 variant or IL-2 variant fusion protein of the present disclosure and one or more other therapeutic agents, the terms "co-administration", "co-administered", and "in combination with" are intended to mean, and do in fact mean and include the following: such combinations of an IL-2 variant or IL-2 variant fusion protein of the present disclosure and one or more therapeutic agents are administered simultaneously to a subject in need of treatment, wherein such components are formulated together into a single dosage form that releases the components to the subject at substantially the same time; such combinations of an IL-2 variant or IL-2 variant fusion protein of the present disclosure and one or more therapeutic agents are administered to a subject in need of treatment substantially simultaneously, wherein such components are formulated separately into separate dosage forms that are taken by the subject at substantially the same time, whereupon the components are released to the subject at substantially the same time; such combinations of an IL-2 variant or IL-2 variant fusion protein of the present disclosure and one or more therapeutic agents are administered to a subject in need of treatment sequentially, wherein such components are formulated separately into separate dosage forms that are taken by the subject at consecutive times with a significant time interval between each administration, whereupon the components are released to the subject at substantially different times; and, such combinations of an IL-2 variant or IL-2 variant fusion protein of the present disclosure and one or more therapeutic agents are administered to a subject in need of treatment sequentially, wherein such components are formulated together into a single dosage form that releases the components in a controlled manner, whereupon the components are released to the subject simultaneously, continuously, and / or overlappingly at the same and / or different times, wherein each portion can be administered by the same or different routes.

[0322] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention in combination with a second therapy, the second therapy including but 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 for prophylactic cancer prevention, prevention of cancer recurrence and metastasis after surgery, and as an adjunct to other conventional cancer therapies. The present disclosure recognizes that the effectiveness of conventional cancer therapies (e.g., chemotherapy, radiotherapy, phototherapy, immunotherapy, and surgery) can be enhanced by the use of the combination methods described herein.

[0323] A large number of conventional compounds have been shown to have antineoplastic activity. These compounds have been used as agents in chemotherapy to shrink solid tumors, prevent metastasis and further growth, or reduce the number of malignant T cells in leukemia or myeloid malignancies. Although chemotherapy is effective in treating various types of malignancies, many antineoplastic compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, the treatments can work synergistically and allow for a reduction in the dose of each treatment, thereby reducing the harmful side effects produced by each compound at higher doses. In other cases, malignancies that are refractory to treatment can respond to combination therapies of two or more different treatments.

[0324] In various embodiments, a second anti-cancer agent, such as a chemotherapeutic agent, will be administered to the patient. A list of exemplary chemotherapeutic agents includes but is not limited to, daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, mechlorethamine, 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, oxaliplatin, pentostatin, cladribine, cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethylstilbestrol (DES), fludarabine, ifosfamide, hydroxyureataxanes (such as paclitaxel and docetaxel) and / or anthracyclines, and combinations of agents, such as but not limited to DA-EPOCH, CHOP, CVP or FOLFOX. In various embodiments, the dose of such chemotherapeutic agents includes but is not limited to about 10 mg / m 2 、20 mg / m 2 、30 mg / m 2, 40 mg / m 2 , 50 mg / m 2 , 60 mg / m 2 , 75 mg / m 2 , 80 mg / m 2 , 90 mg / m 2 , 100 mg / m 2 , 120 mg / m 2 , 150 mg / m 2 , 175 mg / m 2 , 200 mg / m 2 , 210 mg / m 2 , 220 mg / m 2 , 230 mg / m 2 , 240 mg / m 2 , 250 mg / m 2 , 260 mg / m 2 and 300 mg / m 2 any one of.

[0325] In various embodiments, the combination treatment methods of the present disclosure may further include administering to a subject a therapeutically effective amount of an immunotherapy, which includes but is not limited to, treatment with depleting antibodies against specific tumor antigens; treatment with antibody-drug conjugates; treatment with agonistic, antagonistic or blocking antibodies against co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CTLA-4, PD-1, OX-40, CD137, GITR, LAG3, TIM-3, SIRP, CD47, CD40, TIGIT and VISTA; treatment with bispecific T cell engaging antibodies such as blinatumomab; treatment involving administration of biological response modifiers such as IL-12, IL-15, IL-21, GM-CSF, IFN-α, IFN-β and IFN-γ; treatment with therapeutic vaccines such as sipuleucel-T; treatment with dendritic cell vaccines or tumor antigen peptide vaccines; treatment with chimeric antigen receptor (CAR)-T cells; treatment with CAR-NK cells; treatment with tumor-infiltrating lymphocytes (TIL); treatment with adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR transgenic); treatment with TALL-104 cells; and treatment with immune stimulants such as Toll-like receptor (TLR) agonists CpG and imiquimod; wherein the combination therapy provides increased effector cell killing of tumor cells, i.e., there is a synergistic effect between the IL-2 variant and the immunotherapy when co-administered.

[0326] In various embodiments, the combination therapy comprises administering an IL-2 variant and a second agent composition either simultaneously in the same pharmaceutical composition or in separate pharmaceutical compositions. In various embodiments, the IL-2 variant composition and the second agent composition are administered sequentially, i.e., the IL-2 variant composition is administered either before or after the second agent composition. In various embodiments, the administration of the IL-2 variant composition and the second agent composition is simultaneous, i.e., the administration time periods of the IL-2 variant composition and the second agent composition overlap with each other. In various embodiments, the administration of the IL-2 variant composition and the second agent composition is non-simultaneous. For example, in various embodiments, the administration of the IL-2 variant composition is terminated and then the second agent composition is administered. In various embodiments, the administration of the second agent composition is terminated and then the IL-2 variant composition is administered.

[0327] The following examples are provided to more fully illustrate the disclosure but should not be construed as limiting the scope of the disclosure.

[0328] Example 1

[0329] Construction and production of IL-2Fc fusion constructs

[0330] All genes were codon-optimized for expression in mammalian cells, synthesized and subcloned into a recipient mammalian expression vector (GenScript). Protein expression was driven by the CMV promoter and a synthetic SV40 polyadenylation (polyA) signal sequence was present at the 3' end of the CDS. A leader sequence was engineered at the N-terminus of the construct to ensure proper signaling and processing for secretion.

[0331] Constructs were produced by co-transfecting suspension-grown HEK293-F cells with the mammalian expression vector using polyethylenimine (PEI, 25,000 MW linear, Polysciences). If there were two or more expression vectors, the vectors were transfected at a 1:1 ratio. For transfection, HEK293 cells were cultured in serum-free FreeStyle TM 293 expression medium (ThermoFisher). For production in 1000 ml shake flasks (working volume 330 mL), HEK293 cells were seeded at 0.8×10 6Inoculate at a density of cells / ml and perform transfection after 24 hours. Mix a total of 330 μg of the DNA expression vector with 16.7 ml of Opti-mem medium (ThermoFisher). After adding 0.33 mg of PEI diluted in 16.7 ml of Opti-mem medium, vortex the mixture for 15 sec and then incubate at room temperature for 10 min. Then add the DNA / PEI solution to the cells and incubate in an incubator with 8% CO2 at 37°C. On the 4th day, add sodium butyrate (Millipore Sigma) to the cells at a final concentration of 2 mM to help maintain protein expression. After culturing for 6 days, collect the supernatant for purification by centrifuging at 2200 rpm for 20 min. Sterile filter the solution (0.22 μm filter, Corning). Purify the secreted protein from the cell culture supernatant using protein A affinity chromatography.

[0332] Optionally, generate the construct in ExpiCHO cells (ThermoFisher) according to the manufacturer's instructions.

[0333] For affinity chromatography, load each supernatant onto a HiTrap MabSelectSure column (CV = 5 mL, GE Healthcare) equilibrated with 25 ml of phosphate-buffered saline pH 7.2 (ThermoFisher). Remove unbound proteins by washing with 5 column volumes of PBS pH 7.2 and elute the target protein with 25 mM sodium citrate, 25 mM sodium chloride, pH 3.2. Neutralize the protein solution by adding 3% of 1 M Tris pH 10.2. Ion exchange chromatography or mixed-mode chromatography, including but not limited to CaptoMMC (GE Healthcare), ceramic hydroxyapatite or ceramic fluorapatite (Bio-Rad), is also used as needed to refine the protein A material. The target protein is concentrated using an Ultra-15 concentrator 10KDa NMWC (Merck Millipore Ltd.).

[0334] Analyze the purity and molecular weight of the purified construct by SDS-PAGE and staining with Coomassie (ImperialR Stain) in the presence and absence of a reducing agent. Use according to the manufacturer's instructions Precast gel system (4%-12% or 8%-16% Bis-Tris, ThermoFisher). The protein concentration of the purified protein sample was determined by measuring the UV absorbance at 280 nm (Nanodrop spectrophotometer, ThermoFisher) and dividing it by the molar extinction coefficient calculated based on the amino acid sequence. The aggregate content of the construct was analyzed on an Agilent 1200 high performance liquid chromatography (HPLC) system. At 25 °C, using 150 mM sodium phosphate pH 7.0 as the mobile phase, the sample was injected onto an AdvanceBio size exclusion column ( 4.6 x 150 mm, 2.7 μm, LC column, Agilent).

[0335] SDS-PAGE and size exclusion chromatography analysis of the Protein A purified exemplary IL-2 variant Fc fusion constructs P-0635 and P-0704 are shown in Figure 1. P-0635 (SEQ ID NO:85; Figure 1A) and P-0704 (SEQ ID NO:96 and 10; Figure 1B) share the same amino acid substitution P65R in IL-2. P-0635 contains a bivalent IL-2 variant fused to a homodimeric Fc, while P-0704 contains a monovalent IL-2 variant fused to a knob-into-hole heterodimeric Fc. SDS-PAGE analysis demonstrated that both molecules exhibited high protein purity, and the samples run under reducing conditions (lane 2) showed the expected MW for the homodimeric Fc chain of P-0635 and the heterodimeric Fc chain of P-0704. Size exclusion chromatography analysis showed that after the initial Protein A capture step, both molecules exhibited a low aggregation propensity and less than 5% aggregation.

[0336] Example 2

[0337] Single amino acid substitutions in IL-2 result in a general improvement in the developability of the fusion compounds

[0338] When applied to IL-2, engineering approaches to find mutant combinations that produce variant proteins with desired biological properties have encountered significant challenges. It is known in the art that naturally occurring IL-2 proteins tend to be very unstable and prone to aggregation. This was demonstrated in our experiments where the wild-type IL-2Fc fusion protein (P-0250) was expressed at low levels (transiently expressed at approximately 3 mg / L in HEK-293F cells), had a high tendency to aggregate, as illustrated by the SEC chromatogram depicted in Figure 2A. Engineering efforts have been hampered because amino acid substitutions in IL-2 aimed at achieving desired bioactivity often result in mutant proteins that are even more unstable. A large portion of the IL-2 variants in the early stages of the current work were expressed at extremely low levels, and some variants were significantly more prone to aggregation, as illustrated by the SEC chromatogram of P-0318 (IL-2D20I / N88I Fc fusion) depicted in Figure 2B. This is problematic for the preparation and storage of therapeutic agents.

[0339] It was also observed that the expression profiles and aggregation tendencies of IL-2 variant Fc fusions were significantly different between constructs with different mutant sites or mutants that shared the same mutant site but had different residue substitutions. This observation was illustrated by P-0317 (IL-2D20I / N88R Fc fusion) and P-0318 (IL-2D20I / N88I Fc fusion). The two variant fusions shared the same mutant sites at residues 20 and 88 and differed by only one amino acid, and were expressed at similar low levels. As can be observed in Figure 2B, P-0318 was very prone to aggregation and contained 65% high molecular weight material, which made the expected peak a minor species in the chromatogram and was marked with an arrow. In contrast, P-0317 was relatively pure, with 7.5% aggregates (Figure 2C). It can be inferred that the N88R mutation can reduce the aggregation tendency of the resulting fusion protein. However, the fusion proteins P-0254 and P-0324 produced by IL-2 with the N88R single mutation or the D20T / N88R double mutation, respectively, had an aggregation tendency of 30%-40% aggregates. Thus, the contribution of a single amino acid substitution to protein stability appears to be context-dependent.

[0340] The fact that amino acid substitutions in IL-2 often result in less stable proteins is further complicated by the unpredictable contribution of different residue substitutions to protein stability. Thus, it is highly desirable to find residue substitutions that can generally enhance protein developability, including improved stability, higher expression levels, and lower aggregation tendencies.

[0341] The amino acid substitution at position 125 was initially aimed at modulating IL-2 selectivity as this residue is adjacent to Q126, which is essential for γc interaction. Native IL-2 contains an unpaired cysteine at position 125, which was replaced by serine in Proleukin. IL-2 containing an alanine substitution at position 125 has also been widely used. Since the serine or alanine substitution for cysteine at position 125 retained all biological activity, bulky charged or hydrophobic residues including Glu, Lys, Try, His, and Ile were introduced at position 125, aiming to interfere with the interaction of Q126 with γc and thus achieve altered biological activity. All of the resulting fusion molecules, except for the fusion molecule containing Ile125 (P-0531), expressed at too low levels to be characterized. When compared to its S125 counterpart (P-0250), P-0531 expressed at a significantly higher level (29.5 mg / L vs. 3.1 mg / L titer) and had a substantially reduced tendency to aggregate (0.7% vs. 25.7% aggregation). The impressive improvement in developability, particularly with respect to product purity, prompted us to evaluate whether this improvement with the isoleucine substitution at position 125 could be reproduced in different mutant backgrounds.

[0342] Therefore, the S125I substitution was introduced into a number of IL-2 variant Fc fusion molecules. Constructs containing an Ile substitution (125I) at amino acid position 125 of IL-2 were expressed using the same vector as their Ser-125 counterparts and under the same culture conditions, and were purified using MabSelectSure. The expression levels (mg / L) and purity evaluated as % aggregation of exemplary molecules by SEC chromatography are summarized in Table 7. The two molecules in the same row of Table 7 share the same other amino acid substitutions and differ only in having serine or isoleucine at residue 125. The SEC profile of the S125I equivalent of wild-type IL-2Fc fusion, P-0531 (SEQ ID NO:68), is further shown in Figure 2D. It is clear from Table 7 that the isoleucine substitution at position 125 led to a 4- to 11-fold increase in expression level and consistently low aggregation tendency.

[0343] Table 7 The S125I substitution reduces aggregation and increases expression of various IL-2 fusion proteins

[0344]

[0345] It is apparent from the present invention that isoleucine at position 125 results in a general improvement in the developability of the IL-2 fusion construct. This finding is particularly valuable because the fact that altering the critically stable wild-type IL-2 typically results in an even more unstable mutant protein has hampered the engineering of IL-2 for desired biological properties. The inherent challenge of IL-2 engineering can be alleviated by a single amino acid substitution with isoleucine at position 125.

[0346] Example 3

[0347] Designing IL-2 constructs to improve selectivity for effector T cells and NK cells

[0348] A major aspect of the present invention is to improve the selectivity of IL-2 for cells expressing IL-2Rβγ (rather than IL-2Rα) over cells expressing IL-2Rαβγ relative to wild-type IL-2 for cancer treatment. One approach used by the inventors was to generate highly selective IL-2-Fc fusion proteins by introducing mutations that disrupt CD25 into the cytokine moiety. The selection of mutations that disrupt CD25 was based on examination of the IL-2 / IL-2R co-crystal structure (PDB code 2B51). More than one amino acid substitution (including R38, T41, F42, F44, E62, P65, E68, and Y107) was introduced at one or two relevant residues at the interface with the α subunit of the IL-2 receptor, aiming to reduce or eliminate binding to IL-2Rα. These constructs also contain the S125I mutation for significant improvement in developability. Additionally, the attenuation of binding of the IL-2 variant to IL-2Rα+ lung endothelial cells is expected to prevent endothelial cell damage and significantly reduce VLS. Furthermore, the attenuation of CD25 binding is also expected to reduce CD25 antigen uptake and enrich cytokine occupancy of cells expressing IL-2Rβγ, and thus enhance the in vivo response and tumor killing efficacy.

[0349] Table 3 summarizes a group of IL-2 mutant proteins expressed as C-terminal fusions to an Fc homodimer or Fc heterodimer. A group of IL-2 variants (SEQ ID NO: 31 - 66) containing one or two amino acid substitutions at residues at the interface with the α subunit of the IL-2 receptor were fused via a "GGGSGGGS" linker (SEQ ID NO: 18) to the C-terminus of an Fc homodimer to form bivalent IL-2 fusions (SEQ ID NO: 69 - 95) or to the C-terminus of an Fc heterodimer to form monovalent IL-2 fusions (SEQ ID NO: 96 - 106).

[0350] Example 4

[0351] Effect of IL-2 mutations introduced at the interface with IL-2Rα on binding to receptor subunit α

[0352] A set of IL-2 mutant proteins were expressed as C-terminal fusions to Fc homodimers or Fc heterodimers and screened for binding to IL-2Rα in an enzyme-linked immunosorbent assay (ELISA). Briefly, IL-2Rα-ECD (SEQ ID NO:5) was coated onto the wells of a Nunc Maxisorp 96-well microplate at 0.1 μg / well. After incubation overnight at 4 °C and blocking with superblock (ThermoFisher), 3-fold serial dilutions of IL-2Fc fusion proteins starting at 100 nM were added to each well at 100 μl / well. After incubation for 1 h at room temperature, 100 μl / well of goat anti-human IgG Fc-HRP (1:5000 diluted in diluent) was added to each well and incubated for 1 h at room temperature. After each step, the wells were aspirated thoroughly and washed three times with PBS / 0.05% Tween-20. Finally, 100 μl of TMB substrate was added to each well; the plate was developed at room temperature in the dark for 10 min and 100 μl / well of stop solution (2N sulfuric acid, Ricca Chemical) was added. Absorbance at 450 nm was determined and curves were fitted using Prism software (GraphPad).

[0353] First, the CD25 binding of wild-type IL-2Fc fusion proteins P-0531 and the S125I equivalent of P-0689 was tested. P-0531 contains a bivalent IL-2 moiety (SEQ ID NO:68) fused to an Fc homodimer, and P-0689 (SEQ ID NO:107+10) is the monovalent counterpart of P-0531. As Figure 3 shown, the 2-fold difference in binding EC 50 between P-0531 and P-0689 (0.21 nM and 0.51 nM, respectively) is consistent with the IL-2 valence difference.

[0354] Since all targeted IL-2 residues R38, T41, F42, F44, E62, P65, E68 and Y107 are located at the interface with IL-2Rα and form hydrogen bonds / salt bridges or hydrophobic interactions with more than one IL-2Rα residue (Mathias Rickert et al., (2005) Science 308, 1477-80), it was deduced that amino acid substitutions at these sites were expected to disrupt the interaction with IL-2Rα and generate IL-2 variants with reduced or abolished binding to IL-2Rα. However, the binding data revealed that the effects of different IL-2 mutations on IL-2Rα binding were significantly different.

[0355] As shown in Figure 4, various substituted IL-2 homodimer Fc fusions containing at position T41 (exemplified by P-0603, P-0604, and P-0605 in Figure 4A) or Y107 (exemplified by P-0610, P-0611, and P-0612 in Figure 4B) completely retain the binding strength to IL-2Rα. The data indicate that residues T41 and Y107 may not be functionally important, although they are at the interface of IL-2Rα and interact with multiple IL-2Rα residues.

[0356] Residue R38 is considered an energetic hot spot for IL-2 / IL-2Rα interaction and is involved in key hydrogen bonds; multiple engineering efforts (e.g., Keith M. Heaton et al., (1993) Cancer Res. 53. 2597-2602, and Peisheng Hu et al., (2003) Blood 101:4853-4861) have shown that various substitutions at R38 result in disruption of the interaction with IL-2Rα. Thus, quite unexpectedly, it was observed that various mutations exemplified by P-0602 (R38A), P-0614 (R38F), and P-0615 (R38G) did not result in or only resulted in a minimal decrease (up to 3-fold) in the binding strength to IL-2Rα. The binding data are shown in Figure 4C-4D .

[0357] Similarly, residue E68 is involved in more than one hydrogen bond with IL-2Rα interface residues, but various substitutions of amino acid properties at E68 (exemplified by E68A (P-0628), E68F (P-0629), E68H (P-0630), and E68L (P-0631)) did not result in any decrease in binding to IL-2Rα. Interestingly, P-0629 and P-0630 actually showed a 3-fold and 14-fold increase in binding to IL-2Rα, respectively ( Figure 5 ).

[0358] In summary, replacement of IL-2 residues T41, R38, E68, and Y107 generally does not disrupt the IL-2Rα interaction, and the resulting IL-2 homodimer Fc fusions retain full or near-full binding to IL-2Rα. The ELISA binding EC for various IL-2 mutant proteins normalized to that of P-0531 is summarized in Table 8 50 ELISA binding EC of various IL-2 mutant proteins normalized 50 .

[0359] Table 8

[0360] Such substitutions generally do not disrupt the IL-2Rα interaction and the resulting IL-2 variants retain the IL-2 residues that are fully bound to IL-2Rα

[0361]

[0362] In contrast, amino acid substitutions at residue E62 (exemplified by P-0624 (E62A), P-0625 (E62F), P-0626 (E62H), and P-0627 (E62L)) all result in reduced binding to IL-2Rα, indicating that E62 is indeed an energetic hot spot for the IL-2 / IL-2Rα interaction. As Figure 6 shown, while the E62H and E62L substitutions only result in a slight 2- to 3-fold reduction in binding to IL-2Rα, the E62A and E62F mutations appear to cause a substantial disruption in the interaction with this IL-2R subunit, resulting in 60-fold and 150-fold reductions in binding to IL-2Rα, respectively. Additionally, the IL-2F42A mutation (P-0613) is well documented in the literature to disrupt the interaction with the receptor α, as shown in Figure 8A, where the binding to IL-2Rα is reduced 15-fold.

[0363] In summary, F42 and E62 are IL-2 residues such that their substitution generally disrupts the IL-2Rα interaction and the resulting IL-2 variants exhibit reduced binding to IL-2Rα. The ELISA binding EC 50 for P-0531 ELISA binding EC 50 was normalized and is shown in Table 9.

[0364] Table 9

[0365] IL-2 residues whose substitution generally disrupts the IL-2Rα interaction and the resulting IL-2 variants have reduced binding to IL-2Rα

[0366]

[0367] Example 5

[0368] Amino acid substitutions at residue P65 have unexpected manifold effects on binding to the receptor subunit α

[0369] The IL-2 residue P65 makes van der Waals interactions with several key IL-2Rα interface residues (including R36 and L42), but does not form salt bridges or hydrogen bonds with IL-2Rα. It is thus speculated that P65 substitution may only result in a slight disruption of the interaction with this IL-2R subunit and may cause a minor impact on the binding to IL-2Rα. However, the effect of P65 substitution on the IL-2Rα interaction is in sharp contrast to the hypothesis and unexpectedly multifaceted, including complete retention / enhancement, reduction, or complete elimination of the binding to IL-2Rα.

[0370] More than one substitution was introduced at P65, exemplified by P65G, P65E, P65A, P65H, P65N, P65Q, P65R, P65K, and the resulting IL-2 mutant proteins were expressed as C-terminal fusions to Fc homodimers or Fc heterodimers. This set of IL-2 mutant proteins was then screened in an ELISA for binding to CD25. The binding data are shown in Figure 7 and the ELISA binding ECs for P-0531 or P-0689 are summarized in Table 10. 50 ELISA binding ECs of IL-2 mutant proteins normalized to match the valency of each construct 50 .

[0371] Table 10 Substitution of P65 results in unexpected multifaceted effects on IL-2Rα binding

[0372]

[0373]

[0374] As shown in Figures 7A and 7B, the P65G (P-0608), P65E (P-0633), P65A (P-0706) mutations do not seem to cause any disruption in the interaction with the IL-2Rα subunit; on the contrary, the binding strength to IL-2Rα is enhanced by 18-fold, 10-fold, and 10-fold, respectively, when compared to their wild-type counterparts.

[0375] Another set of IL-2 mutant protein Fc fusions, P-0634, P-0708, and P-0709, contain P65 mutations that result in a significant disruption of the interaction between IL-2 and the IL-2Rα subunit. As shown in Figure 7C and summarized in Table 9, P65N (P-0708) results in a slight 8.6-fold reduction in the binding to IL-2Rα, while the P65H (P-0634) and P65H (P-0709) substitutions result in more pronounced effects, as demonstrated by 23-fold and 43-fold reductions in IL-2Rα binding, respectively.

[0376] Another class of IL-2 P65 substitutions for P65R and P65K appears to cause substantial disruption to the IL-2 and IL-R2Rα interaction and abolishes the binding of P-0635, P-0704, and P-0707 to IL-2Rα (Figure 7D). P-0635 and P-0704 are the bivalent and monovalent counterparts of an IL-2Fc fusion containing the P65R substitution, and P-0707 contains the P65K amino acid replacement. Figure 7D shows that all three IL-2 mutant protein Fc fusions show minimal signal at IL-2Rα concentrations up to 100 nM, comparable to a benchmark molecule containing the triple CD25-disrupting mutation F42A / Y45A / L72G, which exhibits abolished binding (Christian Klein et al. OncoImmunology (2017), 6:3, e1277306).

[0377] As summarized in Figures 7A - 7C and Tables 9 and 10, substitution of residue P65 results in unexpected and multifaceted effects on IL-2Rα binding. Importantly, its substitution can either fully preserve / enhance, reduce, or completely abolish the binding of the resulting IL-2 variant to IL-2Rα. As will be understood by those skilled in the art, this level of activity change resulting from a single amino acid change cannot be predicted by structure-based mutagenesis methods. Nor did the prior art anticipate or teach the complete abolition of IL-2Rα binding, as the mutation of P65 only alters a limited portion of the van der Waals interaction surface.

[0378] Example 6

[0379] Amino acid substitution combinations that modulate the binding of IL-2 to the receptor subunit α

[0380] As will be understood by those skilled in the art, the mutations disclosed in the present invention can optionally and independently be combined in any way to optimally modulate the binding of IL-2 to the receptor subunit α. Here, we demonstrate the design of IL-2 compounds that are unable to bind to IL-2Rα by combining two amino acid substitutions that disrupt IL-2Rα.

[0381] P-0613 contains the F42A mutation, which results in a 15-fold decrease in binding to IL-2Rα (Figure 8A). P-0625 and P-0634 contain the E62F and P65H substitutions, respectively, with 150-fold and 23-fold decreases in binding to IL-2Rα. Both the F42A and E62F double-mutation combination in P-0702 and the F42A and P65H double-mutation combination in P-0703 result in abolished binding to IL-2Rα (Figure 8B and Figure 8C). As expected, P-0766 containing the F42 / E62A double-amino acid change and P-0767 with the F42A / E62H double substitution are unable to bind to IL-2Rα (data not shown).

[0382] In addition to being an effective method for designing IL-2 mutant proteins with abolished binding to IL-2Rα, amino acid combinations can also be used to modulate the level of binding activity. One example shown here is P-0765, which combines a CD25-disrupting mutation F42A and a CD25-enhancing substitution P65A, and has a slightly 6.8-fold decrease in the binding strength to IL-2Rα compared to its wild-type counterpart P-0689 (data not shown), which is consistent with the combination of single mutations. The ELISA binding EC for P-0689 is summarized in Table 11 50 ELISA binding EC of normalized IL-2 mutant proteins 50 .

[0383] Table 11 Effect of combinations of amino acid substitutions at the CD25 interface on binding to IL-2Rα

[0384]

[0385] In summary, amino acid substitution combinations are a versatile method for modulating the binding of IL-2 to the receptor subunit α. It can achieve complete elimination of IL-2Rα binding by combining two CD25-disrupting residues, or it can be used to modulate IL-2Rα binding at different levels of attenuation.

[0386] Example 7

[0387] In ex vivo functional assays, modulation of IL-2Rα binding strength correlates with the IL-2 potency to stimulate Treg cells

[0388] Subsequently, the ability of a panel of IL-2 variant Fc fusion proteins to differentially stimulate STAT5 phosphorylation in CD4+ Treg cells compared to the wild-type fusion protein P-0531 and the benchmark molecule P-0551 (SEQ ID NO:95) was examined. STAT5 is known to be involved in the downstream signaling cascade following IL-2 binding to the transmembrane IL-2 receptor. In FACS analysis, phosphorylation of STAT5 in lymphocyte subsets was measured using fresh human peripheral blood mononuclear cells (PBMC), and the Treg population was identified using the forkhead transcription factor FOXP3.

[0389] Purified PBMC were starved for 1 hour in serum-free MACS buffer at 4°C. Then, 2 × 10 5 PBMC were treated with serial dilutions of the test compound for 30 min at 37°C. Cells were fixed and permeabilized with the Foxp3 / Transcription Factor Staining Buffer Set (eBioscience) by incubating with 1× Foxp3 Fixation / Permeabilization Working Solution for 30 min and washing with 1× Permeabilization Buffer. Cells were additionally fixed with Cytofix Buffer and permeabilized with Perm Buffer III (BD Biosciences), and then washed. After blocking Fc receptors by adding Human TruStain FcX (1:50 dilution), cells were stained with a mixture of anti-CD25-PE antibody, anti-FOXP3-APC antibody, anti-pSTAT5-FITC antibody, and anti-CD4-PerCP-Cy5.5 antibody at the concentrations recommended by the manufacturer for 45 min at room temperature. Cells were collected by centrifugation, washed, resuspended in FACS buffer, and analyzed by flow cytometry. Flow cytometry data for the Treg cell subset were gated as the CD4+ / Foxp3+ / CD25 高 population. Data are represented as the percentage of pStat5-positive cells in the gated population.

[0390] This panel of IL-2 variant Fc fusions contained amino acid substitutions that conferred: enhanced binding to IL-2Rα (P-0608), reduced binding to IL-2Rα (P-0626, P-0634, and P-0624), or abolished binding to IL-2Rα (P-0635). Additionally, P-0626, P-0634, and P-0624 exhibited different levels of attenuation of IL-2Rα binding strength; the binding reductions for P-0626, P-0634, and P-0624 were 2.6-fold, 23-fold, and 60-fold, respectively. The trends and levels of IL-2Rα binding modulation were reflected by the different potencies of the various IL-2 variant Fc fusions to stimulate STAT5 phosphorylation in CD4+ Treg cells ( Figure 9)。In terms of stimulating Treg STAT5 phosphorylation, P-0608 with enhanced IL-2Rα binding correspondingly showed a higher potency trend than P-0531. P-0626, P-0624, and P-0634 all showed reduced pSTAT5 potency, consistent with their lower IL-2Rα binding strength. Their retained, albeit lower, binding to IL-2Rα still led to more effective Treg activation than P-0635 and the benchmark P-0551, which had eliminated IL-2Rα binding. P-0635 and P-0551 had a comparable 5-log right shift in the potency to induce pSTAT5 in Treg cells, and this low level of Treg signaling may be caused by the activation of IL-Rβγ expressed on Treg cells. Thus, at concentrations where CD8+ T cells and NK cells are also activated, the mutants are expected to achieve the desired Treg-activating properties. It was surprisingly observed that complete elimination of IL-2Rα binding led to a more than 5-log reduction in Treg potency ( Figure 9 )。

[0391] Example 8

[0392] Effect of IL-2 mutations introduced at the IL-2Rα interface on the interaction with IL-2Rβγ

[0393] To investigate whether the IL-2 mutations introduced at the IL-2Rα interface would affect the interaction of IL-2 with IL-2Rβγ, binding to IL-2Rβγ was evaluated in the same set of IL-2 variant Fc fusion protein ELISAs as in Example 7.

[0394] Briefly, the recombinant IL-2Rβγ heterodimer containing IL-2Rβ ECD (SEQ ID NO: 109) fused to the N-terminus of the Fc hole chain (SEQ ID NO: 10) and γc ECD (SEQ ID NO: 110) fused to the N-terminus of the Fc knob chain (SEQ ID NO: 9) was coated onto the wells of a Nunc Maxisorp 96-well microplate at 2 μg / well. After incubation overnight at 4°C and blocking with 1% BSA, 3-fold serial dilutions of the IL-2Fc fusion protein starting from 10 nM were added to each well at 100 μl / well. After incubation for 1 hour at room temperature, biotinylated mouse anti-human IL-2 clone B33-2 (BD Biosiences) at 0.5 μg / ml was added to each well at 100 μl / well and incubated for 1 hour at room temperature. Subsequently, streptavidin-HRP (diluted 1:5000 in diluent) at 100 μl / well was added to each well and incubated for 40 min at room temperature. After each step, the wells were aspirated thoroughly and washed three times with PBS / 0.05% Tween-20. Finally, 100 μl of TMB substrate was added to each well; the plate was developed for 10 minutes at room temperature in the dark and 100 μl / well of stop solution (2N sulfuric acid, Ricca Chemical) was added. The absorbance at 450 nm was determined and the curve was fitted using Prism software (GraphPad).

[0395] As Figure 10 shown, compared to the wild-type IL-2 fusion P-0531, all of the exemplary IL-2 variant Fc fusions containing mutations that confer enhanced, reduced, or abolished binding to IL-2Rα exhibited unaltered binding to IL-2Rβγ. The data confirm that the tested IL-2 mutations introduced at the IL-2Rα interface indeed only interfere with CD25 binding without affecting the interaction with IL-2Rβγ.

[0396] This set of exemplary IL-2 variant Fc fusion proteins was further characterized by flow cytometry for their ability to induce Ki67 expression on human CD8+ T cells and NK cells. Freshly isolated NK cells and CD8+ T cells do not express CD25 or express very low levels of CD25, and IL-2 signaling is mainly mediated via the intermediate-affinity receptor subunits βγ. Ki67 is a nuclear protein that serves as a marker of cell proliferation.

[0397] Briefly, human PBMCs were isolated from the buffy coat of healthy donors by Ficoll-Hypaque centrifugation. The purified human PBMCs were treated with serially diluted IL-2 variant Fc fusion compounds and incubated at 37 °C for 5 days. On day 5, the cells were washed once with FACS buffer (1% FBS / PBS) and first stained with an Fc blocker and surface marker antibodies, anti-human CD56-FITC antibody, and anti-human CD8-APC antibody. After incubation for 30 minutes and washing, the cell pellet was completely resuspended with 200 μl / well of 1× Foxp3 fixation and permeabilization working solution and incubated at room temperature in the dark for 30 minutes. After centrifugation, 200 μl of 1× permeabilization buffer was added to each well for another wash. The cell pellet was resuspended in permeabilization buffer containing anti-human Ki67-PE antibody (1:25 dilution). After incubation at room temperature for 30 minutes, the cells were collected and washed, resuspended in FACS buffer, and analyzed by flow cytometry. Data are presented as the percentage of Ki-67 positive cells in gated populations.

[0398] The dose-dependent increase in Ki-67 expression on CD8+ T cells and NK cells in response to the IL-2 variant Fc fusion proteins is shown in FIGS. 11A and 11B compared to P-0531 and P-0551. The introduction of mutations that disrupt CD25 results in Fc fusion constructs with potency comparable to the wild-type IL-2 bivalent fusion protein P-0531.

[0399] In addition, the induction of Ki-67 expression on human CD8+ T cells by P-0689 and P-0704 (the monovalent counterparts of P-0531 and P-0635, respectively) was characterized. As Figure 12 shown, P-0689 (wild-type IL-2) and P-0704 containing the P65R mutation that abrogates binding to IL-2Rα showed a dose-dependent increase in Ki-67 expression on CD8+ T cells with equivalent potency. The combined ex vivo functional data further confirmed that IL-2 mutations introduced at the IL-2Rα interface have minimal or no effect on the interaction with IL-2Rβγ. In addition, the potency differences between P-0531 and P-0689 and between P-0635 and P-0704 are consistent with their respective IL-2 valency differences.

[0400] Example 9

[0401] Substitutions that disrupt IL-2Rβ or γc were introduced into IL-2 variants with reduced binding to IL-2Rα for overall potency attenuation

[0402] Full IL-2 agonists may lead to over-activation of pathways and undesirable "on-target" "off-tissue" toxicities. This may be particularly true for IL-2Rβγ-selective full agonists; due to enhanced selectivity and reduced CD25 engagement, IL-2Rβγ-selective full agonists can potentiate the apparent in vivo responses of CD4+, CD8+ effector T cells and NK cells. Thus, acute toxicity may be observed with significant weight loss. In addition, over-stimulation-induced cell exhaustion or death may cause loss of in vivo responses after repeated dosing. It is hypothesized that lower overall potency can prevent pathway over-activation and reduce unwanted target engagement; thus, toxicity may potentially be reduced and pharmacokinetics and pharmacodynamics improved. For optimal activity, substitutions that modulate IL-2Rβγ to attenuate overall potency are thus introduced into IL-2 variants with reduced / abolished binding to IL-2Rα. Reduced binding affinity for IL-2Rβγ will also reduce receptor-mediated IL-2 internalization, resulting in slower but sustained receptor activation and durable pharmacodynamics compared to wild-type IL-2.

[0403] The selection of mutations that disrupt IL-2Rβ or γc was based on examination of the IL-2 / IL-2R co-crystal structure (PDB code 2B51). Substitution of residues at or near the interface that makes direct contact with IL-2Rβ or γc can lead to reduced binding to IL-2Rβγ and thus modulate the overall potency of the activation pathway. For example, D20 is involved in an extensive hydrogen bond network with the side chains of receptor subunits at the IL-2Rβ interface. Similarly, N88 is an energetic hot spot for the IL-2 / IL-2Rβ interaction, participating in key hydrogen bonds with the receptor chain. Q126 is essential for γc interaction; however, amino acid substitutions at energetic hot spots may lead to substantial reduction in activity, presenting suboptimal potency, as exemplified by the various mutations (D20E, D20T, D20N, D20Q, D20S) at the D20 position in Figure 13A. All mutations were introduced into IL-2 in P-0250 (SEQ ID:67) and expressed as IL-2 variant Fc fusion proteins. As shown in Figure 13A, most mutations at D20 led to a substantial reduction or elimination of activity that stimulates pSTAT5 expression in CD4+ Tconv cells expressing only the IL-2Rβγ subunits. Similarly, mutations at position N88 also led to mostly eliminated activity for CD4+ Tconv cell activation (data not shown).

[0404] Therefore, amino acid substitutions were introduced at position L19 (this residue only has van der Waals interactions with IL-2Rβ), and the resulting mutants only regulate rather than eliminate the functional activity of IL-2. Figures 13B and 13C show that IL-2 variants containing various mutations at position 19 exhibit a range of potencies to induce STAT5 phosphorylation on CD4+ Tconv cells. Compared with the wild type, the L19Y, L19R, and L19Q mutations result in a mild decrease in activity, while the L19N and L19H mutations moderately decrease activity. For L19D, such activity is significantly impaired. The different levels of potency reduction achieved by mutating position L19 contribute to fine-tuning of activity to obtain the optimal potency with reduced toxicity and improved in vivo pharmacokinetics and pharmacodynamics.

[0405] In addition, IL-2 variants containing amino acid alterations at Q126 (this residue is essential for γc interaction) were similarly prepared. The functional activity of the IL-2 Q126E Fc fusion protein to induce STAT5 phosphorylation on CD4+ Tconv cells is shown in Figure 13D. Compared with its wild-type counterpart, Q126E results in a slightly decreased activity.

[0406] Furthermore, since the amino acids at the N-terminus of IL-2 are mainly involved in the interaction with IL-2Rβγ, N-terminal amino acid deletions are considered a different method to regulate the overall potency. Therefore, N-terminal deletion mutants (5, 7, 9, or 11 N-terminal amino acid deletions) based on the IL-2 variant containing L19H / S125I / Q126E were constructed and assayed in the human PBMC assay. As the parental molecule, the IL-2 L19H / S125I / Q126E variant retains full binding to IL-2Rα but reduced binding to IL-2Rβγ, so it can only be reliably assayed in Treg cells, which can still dissect the impact of the mutations on the overall potency. The Fc IL-2 variant containing an 11-aa deletion did not produce sufficient material for characterization. As depicted in Figure 13E, while the 5-aa deletion and 7-aa deletion fully retained potency, the 9-aa deletion led to a 25-fold loss of activity (18 pM vs. 0.74 pM). Therefore, it is expected that various IL-2 variants with different potencies can be further regulated by amino acid deletions of 7, 8, 9, or 10 amino acids at the N-terminus to obtain the desired activity profile.

[0407] The mutations L19H, L19Q, L19Y that disrupt IL-2Rβ and the mutation Q126E that disrupts γc were introduced into P-0704, generating P-0731, P-0759, P-0761, and P-0732, respectively. P-0704 contains the P65R amino acid substitution that results in a complete loss of binding to IL-2Rα. Compared to P-0704, P-0731, P-0759, P-0761, and P-0732 were evaluated for binding to IL-2Rβγ in ELISA and for induction of Ki-67 expression on human CD8+ T cells, CD4+ T cells, and NK cells by flow cytometry.

[0408] As shown in Figure 14A, compared to P-0689 and P-0704, all of the exemplary IL-2 variant Fc fusions showed varying levels of reduced binding to IL-2Rβγ. Due to the weak binding of IL-2 to the receptor subunits β or γ and the high dissociation rate, the binding activity of each individual subunit could not be reliably evaluated by ELISA (data not shown). However, the reduced binding to the IL-2Rβγ heterodimer was expected to be attributed to amino acid alterations that disrupted the interaction with the corresponding β or γ receptor subunit.

[0409] The potency reduction of the activity of inducing Ki67 expression on human CD8+ T cells in human PBMCs caused by the substitution L19H that disrupts IL-2Rβ in P-0731 and the mutation Q126E that disrupts γc in P-0732 was evaluated. The S125I equivalent of wild-type IL-2 monomer Fc fusion, P-0689, and P-0704 that lost binding to IL-2Rα but completely retained the affinity and functional activity for the dimeric IL-2Rβγ receptor were included for comparison. As shown in Figure 14B, all of the monomeric IL-2Fc fusion proteins induced an increase in the percentage of Ki-67 positive CD8+ T cells in a dose-dependent manner; compared to P-0704, P-0731 exhibited a potency reduction of approximately 30-fold. P-0732 exhibited the lowest potency, with an EC reduced by more than 100-fold compared to P-0704. 50 。

[0410] The dose-dependent increases in the proliferation of human CD8+ T cells, NK cells, and CD4+ T cells by P-0731, P-0759, and P-0761 are shown in FIGS. 15A, 15B, and 15C, respectively. The IL-2 variant Fc fusion proteins P-0731, P-0759, and P-0761 all contain, in addition to the substitution P65R that abrogates IL-2Rα binding in P-0704, mutations at position L19 that disrupt IL-2Rβ. Compared to P-0704, all variants showed a decreased potency as expected in the proliferation of human CD8+ T cells, NK cells, and CD4+ T cells. P-0759 (L19Q) and P-0761 (L19Y) showed a slightly decreased potency of 3- to 5-fold, while the L19H mutation in P-0731 resulted in a more profound 30-fold decrease in potency. The levels of potency attenuation by the L19Q and L19H substitutions followed the same trend in all evaluated cell subsets and were consistent with the levels of decreased activity in inducing pSTAT5 expression on CD4+ Tconv cells (FIGS. 13B and 13C) and the levels of decreased binding to recombinant IL-2Rβγ protein (FIG. 14A). The reference molecule showed comparable but slightly lower potency in inducing cell proliferation compared to P-0704.

[0411] In summary, in addition to introducing substitutions in IL-2 that disrupt CD25 to inhibit the unwanted expansion of immunosuppressive Tregs, substitutions that disrupt IL-2Rβγ or N-terminal deletions can be further introduced to attenuate the overall potency to obtain optimal activity. The lower potency can prevent overactivation of the pathway and reduce unwanted target uptake; thus, toxicity can potentially be reduced and pharmacokinetics and pharmacodynamics can be improved.

[0412] Example 10

[0413] Pharmacodynamic effects of IL-2 variant Fc fusion proteins after single injection in mice

[0414] The time course of cell expansion of different lymphocyte subsets after treatment with P-0704 (SEQ ID NOs: 96 and 10), a C-terminal monovalent IL-2 variant Fc fusion protein with abrogated binding to IL-2Rα, was performed in Balb / C mice after single injection. The effect on the expansion of peripheral blood lymphocytes was monitored over time. In addition, the immunopharmacodynamic profile of P-0704 was compared with that of the wild-type IL-2 counterpart P-0689 (SEQ ID NOs: 107 and 10).

[0415] Before the study, seven-week-old female Balb / c mice were received from Charles River Laboratories and acclimatized in-house for at least 7 days. The vehicle and P-0704 and P-0689 at a single dose of 0.6 mg / kg were administered intraperitoneally (i.p.) to the mice on day 0. Blood samples were drawn on days 3 and 5 after injection. Each group consisted of 4 mice.

[0416] Heparinized whole blood was used for immunophenotyping. After lysing red blood cells using BD pharm lysis buffer, total viable mononuclear blood cells were counted by trypan blue dead cell exclusion and intracellular staining for Ki67 was continued. The cell pellet was resuspended completely with 200 μl / well of 1× Foxp3 fixation / permeabilization working solution and incubated at room temperature in the dark for 30 minutes. After centrifugation, 200 μl of 1× permeabilization buffer was added to each well for another wash. After blocking Fc receptors with purified anti-mouse CD16 / CD32 antibody (1:50 dilution), the cells were stained with APC-cy7 CD3, BV510 CD4, FITC Foxp3, PE Ki67, APC CD335, and Percpcy 5.5CD8 (1:50 dilution). After 30 minutes of incubation, the cells were collected and washed, resuspended in FACS buffer, and analyzed by flow cytometry.

[0417] As shown in Figure 16A, wild-type IL-2 in P-0689 led to a strong expansion of Treg cells (a 6-fold increase in cell number) that peaked on day 3, which is considered undesirable for cancer treatment, while P-0704 had no Treg expansion on day 3 and only minimal Treg cell expansion on day 5. In contrast, P-0704 increased the percentage of CD8+ T cells in the total CD3+ lymphocyte population on day 3 and continued to enhance the CD8 population from 19% (baseline) to 67% on day 5 (Figure 16B). Conversely, CD8+ T cell expansion by P-0689 was minimal (Figure 16B). For NK cells, a 5.4-fold increase in cell number was observed on day 3 by P-0704, and the cells continued to expand and resulted in a 64-fold increase in cell number on day 5. P-0689 increased NK cell number by 7.8-fold on day 3, but this effect rapidly waned and returned to baseline on day 5 (Figure 16C).

[0418] In summary, P-0704 demonstrated nearly abolished Treg expansion and significantly enhanced CD8 cell and NK cell expansion, which was distinct from the cell expansion profile of P-0689. This observation was consistent with the large difference in IL-2Rα subunit binding ability and subsequent Treg cell responsiveness. Additionally, as an IL-2Rβγ selective full agonist, P-0704 could enhance the apparent in vivo responses of CD8+ effector T cells and NK cells due to enhanced selectivity and reduced CD25 uptake.

[0419] Example 11

[0420] Construction, Expression, and Purification of IL-2-Antibody Fusion Proteins

[0421] In this example, various IL-2-antibody fusion proteins were prepared and evaluated. Tethering an IL-2 variant to an antibody targeting an immune checkpoint was expected to direct IL-2 to exhausted T cells and make the tumor microenvironment immunologically "hot". This strategy also reduced the systemic exposure and off-target toxicity of IL-2. Bifunctional fusion proteins of immune checkpoint inhibitors and IL-2 variants were also expected to provide synergy by removing negative regulation and functionally and numerically rejuvenating T cells. Immune checkpoint-blocking antibody-cytokine fusion proteins were expected to further enhance the anti-tumor activity of the immune system. The inventors proposed that using an IL-2 variant with reduced or abolished binding to IL-2Rα and attenuated IL-2Rβγ activity would help establish a stoichiometric balance between the cytokine and the antibody arm, which exhibited significantly different potencies and molecular weights, to allow optimal dosing and maintenance of the function of each arm. Additionally, attenuated cytokine activity was expected to minimize peripheral activation, reduce antigen uptake, and promote tumor targeting via the antibody arm.

[0422] For checkpoint inhibitor targets expressed on cytotoxic T cells or other lymphocyte subsets that also express IL-2Rβγ, such as PD-1, an IL-2-PD-1 antibody fusion protein was expected to preferentially deliver the IL-2 variant in cis to PD-1+ cells, such as activated and exhausted CD8+ T cells in the tumor microenvironment, to promote selective signaling.

[0423] In accordance with this concept, various IL-2-antibody fusion proteins were constructed.

[0424] To prepare the IL-2-antibody fusion protein, the CH1-CH2-CH3 domains (antibody residues 118-447 based on EU numbering) of the heavy chain of the antibody listed above were replaced with the IgG1 sequence listed in SEQ ID NO:162, which contains the L234A, L235A, G237A mutations to eliminate binding to FcγR and C1q, but retain FcRn binding or PK. The IL-2 variant peptide was fused to the C-terminus of the Fc domain via a peptide linker with the sequence listed in Table 6. Optionally, to express a monovalent IL-2 variant, the CH1-CH2-CH3 domains of the heavy chain of the antibody listed above were replaced with the heterodimeric chains listed in SEQ ID NOs:163-164. The IL-2 variant peptide was fused to the C-terminus of the knob-containing heterodimeric heavy chain engineered using the knob-into-holes technology via a peptide linker with the sequence listed in Table 6. Half-life extension mutations, such as N434A, can be further introduced into the homodimeric or heterodimeric Fc chains. Exemplary IL-2 PD-1 antagonist antibody fusion proteins are listed in Table 12. Additionally, P-0844 is a benchmark IL-2 variant PD-1 antagonist antibody fusion protein containing SEQ ID NOs:182-184.

[0425] Table 12 Exemplary IL-2 variant antagonist PD-1 antibody fusion proteins

[0426]

[0427] Gene synthesis, expression vector construction, and protein production, purification, and characterization were performed according to the same procedures detailed in Example 1.

[0428] Similarly, murine alternative PD-1 IL-2 variant fusion proteins were generated for in vivo tumor models in immunocompetent mice. The alternative anti-mouse PD-1 antibody contains SEQ ID NOs:185-187, which carry Fc mutations for removing effector functions and for heterodimerization; the IL-2 variant was fused to the C-terminus of anti-mouse PD-1 HC chain 2 (SEQ ID NO:186) via a (G4S)3 linker (SEQ ID NO:15). Table 13 lists the IL-2 variants in each exemplary murine alternative PD1-IL-2 variant fusion protein:

[0429] Table 13 Exemplary murine alternative PD-1 IL-2 variant fusion proteins

[0430]

[0431] Example 12

[0432] In an ex vivo functional assay, the IL-2 variant antibody fusion protein completely retained the IL-2 potency and activity profile

[0433] The alternative murine PD-1 antagonist antibodies (SEQ ID NO: 185 - 187) in this study do not cross-react with human antigens; thus, it was used as a non-functional antibody in human cells to evaluate the effect of the antibody fusion form on the potency and activity profile of the IL-2 variant to stimulate lymphocyte subsets and cause their proliferation.

[0434] The effect of the antibody fusion form was illustrated by the comparison of P-0782 with its Fc-fused counterpart P-0704. Both P-0782 and P-0704 contain the monomeric IL-2 P65R variant, which is linked via a flexible (G3S)2 linker (SEQ ID NO: 18) to the C-terminus of the heterodimeric Fc domain. The P65R substitution in IL-2 abolishes binding to IL-2Rα (Figure 7D). As Figure 17A-17C depicted, P-0782 and P-0704 were equally effective in inducing dose-dependent STAT5 phosphorylation in CD4+ Treg cells (Figure 17A), CD8+ T cells (Figure 17B), and NK cells (Figure 17C). The data confirmed that the IL-2 portion fused to the antibody retained its activity as completely as in its corresponding Fc fusion protein.

[0435] In addition, the activities of three IL-2 variant murine PD1 antibody fusion proteins, P-0837, P-0838, and P-0782, to stimulate pSTAT5 in human PBMCs were compared. The IL-2 mutations in P-0838 and P-0782 were P65Q and P65R, respectively. P-0837 contains the wild-type IL-2 portion (SEQ ID NO: 4). Compared with the wild type, P65Q reduced the IL-2Rα binding strength by 43-fold (Table 10), and P65R abolished binding to IL-2Rα. The findings of the IL-2 Fc fusion molecules in the previous examples were confirmed, Figure 18A-18C showing that the IL-2 mutations introduced at the IL-2Rα interface indeed only interfere with CD25 and do not affect the interaction with IL-2Rβγ. Since naive CD8+ T cells and NK cells in human PBMCs do not express CD25 or express very low levels of CD25, all three molecules showed the same potency in the dose-dependent stimulation of pSTAT5 expression on these two lymphocyte subsets (Figure 18B and Figure 18C). In contrast, Treg cells constitutively express high levels of CD25, and thus P-0838 and P-0782 showed a significantly reduced response compared to the wild-type counterpart P-0837 in stimulating pSTAT5 expression in Treg cells (Figure 18A); for P-0837, P-0838, and P-0782, EC50 They were 0.45 pM, 0.36 nM (800 - fold weaker than P - 0837), and 4.5 nM (10,000 - fold weaker than P - 0837), respectively. The mutant P - 0838 with reduced CD25 binding and the mutant P - 0782 with abolished CD25 binding both retained similar potency to their wild - type counterparts in stimulating CD8 cells and NK cells. Additionally, abolishing IL - 2Rα binding in P - 0782 led to an EC 50 ratio of approximately 1, indicating no preferential stimulation of Treg cells over cytotoxic effector cells (the EC 50 of 4.5 nM for Treg cells compared to an EC 50 of 4.6 nM for CD8+ T cells). The significantly weakened but still present IL - 2Rα binding in P - 0838 led to a ~13 - fold enhanced pSTAT5 responsiveness in Treg cells compared to CD8+ T cells (0.36 nM for Treg cells compared to 4.6 nM for CD8+ T cells).

[0436] In in vitro functional assays of IL - 2 antibody fusion forms, IL - 2 variants with reduced potency due to mutations L19Q or L19H disrupting IL - 2Rβ in addition to reduced binding to IL - 2Rα were also evaluated. Compared to P - 0782, P - 0786 contains an additional L19Q substitution, and P - 0783 contains L19H. The Fc counterparts of P - 0782, P - 0786, and P - 0783 are P - 0704, P - 0759, and P - 0731, respectively.

[0437] Phosphorylation of STAT5 on human CD8+ T cells and NK cells induced by P - 0782, P - 0786, and P - 0783 in a dose - dependent manner is shown in Figures 19A and 19B, respectively; a dose - dependent increase in the proliferation of the same lymphocyte subsets is depicted in Figures 19C and 19D, respectively. Compared to P - 0782, P - 0786 showed a slightly reduced potency of 2 - 3 - fold in inducing STAT5 phosphorylation on CD8+ T cells (Figure 19A) and NK cells (Figure 19B), while the L19H mutation in P - 0783 led to a more profound 20 - 30 - fold reduction in potency (Figure 19A and Figure 19B). Similar levels of reduced potency were observed for the dose - dependent increase in Ki67 on CD8+ T cells (Figure 19C) and NK cells (Figure 19D). The levels of reduced potency of the antibody fusion proteins P - 0782, P - 0786, and P - 0783 followed the same trend as their corresponding Fc fusion proteins P - 0704, P - 0759, and P - 0731 (Figure 15A and Figure 15B).

[0438] In the context of the P65Q mutation in the IL-2 antibody fusion format, the potency attenuation by mutations disrupting IL-2Rβ was also evaluated. L19Q and L19H were introduced into P-0838 to prepare P-0790 and P-0787, respectively. Figures 20A, 20B, and 20C show their activities stimulating STAT5 phosphorylation on Treg cells, CD8+ T cells, and NK cells. Figures 20D and 20E show the dose-dependent increase in the proliferation marker Ki67 on CD8+ T cells and NK cells. The level of potency attenuation follows the same trend as observed for the Ab fusion substituting P65R based on elimination of IL-Rα.

[0439] The CTLL-2 proliferation activities of P-0782, P-0786, P-0783, and P-0837 were further evaluated, and P-0837 contains S125I equivalent wild-type IL-2. CTLL-2 cells are cytotoxic T cells derived from C57BL / 6 mice expressing α, β, and γ receptor subunits. Briefly, CTLL2 cells were harvested, washed, and resuspended in medium without IL-2 (RPMI1640, 10% FCS, 2 mM glutamine) and incubated for two hours (IL-2 starvation). After starvation, 50 μl of CTLL-2 cells resuspended at 50,000 cells / ml in fresh medium without IL-2 were transferred to a 96-well U-bottom plate. 50 μl of serially diluted IL-2 antibody fusion was added to the wells to make a final volume of 100 μl / well. The samples were incubated for 2 days, and proliferation was evaluated using CellTiter-Glo according to the manufacturer's instructions, and the luminescence signal was measured. As Figure 21 depicted, the level of potency attenuation by L19Q and L19h was also maintained in mouse cells. Since the IL-Rα subunit is expressed on CTLL-2 cells, similar to Treg cells, P-0837 containing wild-type IL-2 exhibited a significant growth advantage over P-0782.

[0440] In summary, in in vitro functional assays, the IL-2 variants substituting the mouse PD-1 antibody fusion protein format completely retained the potency and activity profiles observed in their Fc fusion equivalents.

[0441] Example 13

[0442] In vitro characterization of IL-2 variant human PD-1 antibody fusion proteins

[0443] P-0795 is a human PD-1 antagonist antibody comprising SEQ ID NO:140 as the heavy chain and SEQ ID NO:141 as the light chain. P-0803 (SEQ ID NO:166, 169 and 141) is an immunoconjugate of P-0795 and has an IL-2 variant fused to the C-terminus of the knob-containing heterodimeric heavy chain. The IL-2 variant in P-0803 contains the mutation P65R that eliminates IL-2Rα binding and the substitution S125I that improves developability. The function of the antibody arms in the antibody fusion protein exemplified by P-0803 was assayed for both direct binding and ligand competitive inhibition in an ELISA format.

[0444] For direct binding, huPD-1-His was used as the coating antigen and the same ELISA protocol as in Example 4 was followed. For the ligand (PD-L1) competitive inhibition ELISA, a similar ELISA protocol was used with some modifications. Briefly, the plates were coated overnight at 4 °C with 0.2 μg / well of human PD1-Fc protein. After washing and blocking with 2% BSA, 0.5 μg / mL of biotinylated human PDL1-Fc was mixed with serial dilutions of P-0795 or P-0803 at 1:1 (v / v); 100 μL of the mixture was added to each well and incubated at 37 °C for 1 hour. Streptavidin-HRP was added as the secondary antibody.

[0445] As depicted in Figure 22A, P-0803 and P-0795 have the same binding strength to PD-1 (EC 50 = 0.6 nM). P-0803 is also equally effective as P-0795 in blocking the binding of human PD-1 to PD-L1 immobilized on the surface (IC 50 = 2.1 nM; Figure 22B). These data together confirm that the antibody arms in the IL-2 antibody fusion are fully functional.

[0446] Similarly, by FACS analysis, the IL-2 variant human PD-1 antibody IL-2 showed similar binding to cell surface-expressed PD1 as the parental antibody (Figure 22C). P-0795 is an antagonist human PD-1 antibody, and both P-0880 and P-0885 contain a monovalent IL-2 attached to the C-terminus of P-0795 via a (G4S)3 linker. P-0880 contains the P65R / S125I substitution, while P-0885 contains the L19Q / P65R / S125I mutation. P-0704 and P-0759 are the Fc fusion counterparts of P-0880 and P-0885, respectively. As expected, due to the lack of a PD-1 targeting arm, P-0704 and P-0759 did not bind to PD-1 expressing cells.

[0447] Since the PD-1 antibody binds to the checkpoint inhibitor PD-1, it is expected that the immunoconjugate can preferentially deliver the IL-2 variant in cis to PD-1+ cells, such as activated and exhausted CD8+ T cells in the tumor microenvironment, to promote selective signaling. In PBMCs from healthy individuals, naive CD8+ T cells and NK cells are generally PD-1 negative, while Tregs express low constitutive levels of PD-1. Thus, the IL-2huPD-1Ab fusion proteins P-0803 and P-0804 were observed to be more than 15-fold more potent than their non-PD-1 targeting equivalents P-0782 and P-0783, respectively, in stimulating pSTAT5 in PD-1 positive T cells (Figure 23A and Figure 23B); however, for PD-1 negative cells, the potency differences were minimal or slight ( Figure 23C-23F ). In naive, unstimulated CD8 cells and NK cells, the huPD-1Ab fusion proteins also showed a trend of increased potency compared to their non-PD-1 targeting counterparts ( Figure 23C-23F ).

[0448] Higher PD-1 expression levels on T cells are expected to be more likely to be targeted by the antibody fusion proteins for selective signaling. Thus, in the tumor microenvironment, the IL-2PD-1 antibody fusion proteins will preferentially bind to Teff rather than Tregs.

[0449] In addition, the effect of the length of the linker connecting the antibody knob heavy chain and the IL-2 variant on the protein expression profile and activity was explored. P-0840 (SEQ ID NOs: 168, 169, and 141) and P-0841 (SEQ ID NOs: 178, 175, and 141) are two IL-2P-0795 fusion proteins that differ only in the linker length. P-0840 contains a (G3S)2 linker (SEQ ID NO: 18), while P-0841 has a (G4S)3 linker (SEQ ID NO: 15). As shown in FIGS. 24A and 24B, Protein A-purified P-0841 produced by transient expression in ExpiCHO showed significantly fewer low molecular weight impurities (3% vs. 16%) than P-0840 produced by the same production and purification process. Similar differences in impurity content (11% vs. 2.7%; FIGS. 24C and 24D) were observed for P-0803 and P-0880 (SEQ ID NOs: 177, 175, and 141), which have (G3S)2 and (G4S)3 linkers, respectively, and are identical in other sequences.

[0450] Although the slightly longer linkers in P-0841 and P-0880 led to improved purity compared to their respective shorter linker counterparts, the effect on the biological activity of the IL-2 portion was minimal or slightly enhanced, as exemplified by the pSTAT5 stimulation potency on cytotoxic lymphocytes (FIG. 25). Due to the favorable impact on the developability profile of the fusion protein, the longer linker is more preferred over the shorter linker without causing other negative effects.

[0451] Constructed were some P-0795 fusion proteins P-0880, P-0882 (SEQ ID NO: 176, 175, and 141), and P-0885 (SEQ ID NO: 179, 175, and 141) of IL-2 variants with the C-terminus of a knob-containing heterodimeric heavy chain fused via a (G4S)3 linker. As shown in Figure 22C, compared to the hPD1 antibody alone, in the IL-2 variant huPD-1 antibody fusion proteins with longer linkers, the binding to cell surface-expressed PD-1 was unchanged. They were further tested in an ex vivo functional assay to study the potency of IL-2 in stimulating pSTAT5 and inducing Ki67 expression in CD8+ T cells and NK cells (Figure 26). All three constructs contained the mutation P65R that eliminates IL-2Rα, while P-0882 and P-0885 contained additional L19H and L19Q mutations, respectively, to modulate the overall potency. The wild-type IL-2 counterpart P-0849 was included in the assay for comparison. The ex vivo functional activities were summarized in Table 14. The levels of potency attenuation by P-0885 and P-0882 followed the same trend in the evaluated cell subsets compared to P-0880, and were consistent with the reduced levels observed for P-0759 and P-0731 compared to P-0704 (their respective Fc fusion proteins; Figures 15A and 15B), and for P-0786 and P-0783 compared to P-0782 (their respective mouse PD1 antibody fusion proteins; Figure 19). As expected, the wild-type IL-2 fusion showed activity comparable to P-0880 against CD8+ T cells and NK cells.

[0452] Table 14 Activity of IL-2 variant human PD-1 antibody fusion proteins

[0453]

[0454] Example 14

[0455] Pharmacodynamic effects of IL-2 variant replacement mouse PD-1 antibody fusion proteins in C57BL6 mice

[0456] The pharmacodynamic effects of IL-2 variant mouse PD-1 antibody fusion proteins were evaluated after a single injection in C57BL6 mice. Female C57BL6 mice at 7 weeks of age were received from Charles River Laboratories and acclimated in-house for at least 7 days prior to the study. Vehicle and each IL-2 mouse PD-1 antibody fusion protein at a single dose were administered intraperitoneally to the mice at time 0. Blood samples were drawn on days 3, 5, 7, and 10 after injection. Each group included 5 mice. Heparinized whole blood was used for the immunophenotyping analysis described in Example 10.

[0457] P-0782 contains the IL-2 P65R moiety that eliminates IL-2Rα binding, P-0838 contains the IL-2 P65Q moiety that reduces IL-2Rα binding, while P-0837 contains wild-type IL-2. P-0781, a corresponding murine PD-1 antibody fusion protein with a reference IL-2 variant (SEQ ID NO:188) that has completely lost IL-2Rα binding, was included for comparison.

[0458] After a single injection at 2 mg / kg, for all compounds tested on CD8 cells and NK cells, Ki67 stimulation on CD8 cells and NK cells reached maximal levels (Figures 27A - 27B). For each compound, the peak Ki67 expression signal on CD8+ T cells reached maximal levels and peaked on day 3. For P-0782, P-0838, and the reference P-0781, the signal persisted until day 7 and waned by day 10. In contrast, for wild-type P-0837, the Ki67 signal waned at an accelerated rate (Figure 27A). Similar Ki67 induction was observed on NK cells for all compounds tested (Figure 27B).

[0459] Notably, CD8 cell and NK cell expansion showed a huge difference among the compounds tested. P-0782 with a mutation that eliminates IL-2Rα binding showed strong expansion of CD8+ T cells (Figure 27C) and NK cells (Figure 27D). Expansion of both lymphocyte subsets began on day 3, persisted, and peaked on day 7, with a 68-fold increase in CD8+ T cells and a 182-fold increase in NK cell numbers. P-0838 with a mutation that reduces IL-2Rα binding ability showed CD8 cell and NK cell expansion similar to or slightly stronger than that of the WT antibody fusion. For the reference P-0781, expansion of both lymphocyte subsets was moderate compared to P-0780 and P-0838. In sharp contrast, wild-type P-0837 peaked in cell expansion of both lymphocyte subsets on day 5, with a significantly reduced maximal signal (a 3.9-fold increase in CD8+ T cells and a 6.8-fold increase in NK cells; Figures 27C and 27D).

[0460] It is possible that mutations that abrogate CD25 binding may provide the advantage of reducing the CD25 sink effect and thus increasing the availability of IL-2Rβγ. Enriched receptor occupancy triggers strong cytotoxic cell expansion. Mutants with residual CD25 binding activity may still have a sink effect, resulting in activity against CD8 cells and NK cells similar to that of the wild type. In summary, compared to P-0838 and P-0837, P-0782 shows a distinct cell expansion profile. Compared to any of the compounds tested, P-0782 demonstrates significant proliferation and expansion of both CD8+ T cells and NK cells and is higher than the benchmark compound P-0781. As an IL-2Rβγ selective full agonist, P-0782 and P-0781 can enhance the significant in vivo responses of CD8+ effector T cells and NK cells due to enhanced selectivity and reduced CD25 sink. Although P-0838 does not show strong CD8 cell and NK cell expansion compared to the wild type, the introduced mutations that reduce the ability to bind to IL-2Rα (CD25) are expected to provide benefits in reducing VLS. In addition, the residual immunomodulatory Treg response can provide immune balance to enhance systemic tolerance and ensure that the immune balance does not overly bias towards cytotoxic effector cells. The Treg response can be finely tuned to avoid tumor killing efficacy but be strong enough to maintain peripheral tolerance.

[0461] The pharmacodynamics of bifunctional PD1 antibody fusion proteins with IL-2 variants were also tested, where the IL-2 variants contain mutations that not only abrogate the ability to bind to IL-2Rα but also reduce IL-2Rβγ interaction. Both P-0786 and P-0783 are IL-2 potency-attenuated counterparts of P-0782, introducing different mutations, L19Q and L19H, that modulate IL-2Rβ, respectively. Figures 19C and 19D show the in vitro potency differences of these three compounds in stimulating Ki67 expression. The effects of P-0786 and P-0783 on the proliferation and expansion of CD8+ cells and NK cells at two different dose levels are shown in Figures 28 and 29. As shown in Figure 28A, the lower potency compound P-0786 induced a peak Ki67 signal on CD8+ T cells on day 5, rather than day 3 as observed for wild type P-0837. Both P-0783 and P-0837 maximized the increase in Ki67 on NK cells (Figure 28B), which is consistent with the concept that NK cells are more responsive to IL-2 than CD8+ T cells.

[0462] Compared with the wild-type fusion, the pharmacodynamic effect of the PD1 antibody fusion of the attenuated IL-2 variant was significantly improved. Figures 28C and 28D show a significantly prolonged and enhanced dose-response effect of P-0786 on cell expansion compared with the wild type. The increase in CD8+ T cell and NK cell expansion was delayed but sustained and persistent. The response from the 2 mg / kg dose group peaked on day 7 and did not return to baseline on day 10, while the response from the 5 mg / kg dose group increased sharply and continuously and did not peak on day 10 after dosing. In contrast, the CD8 cell and NK cell expansion in the wild-type fusion group was marginal, peaked on day 5, and returned to baseline on day 7 (Figures 28C and 28D).

[0463] P-0783, which contains an even weaker IL-2 agonist, showed a similar delayed but sustained and persistent effect of inducing Ki67 expression (Figures 29A and 29B) and expansion (Figures 29C and 29D) of CD8+ cells and NK cells in a dose-dependent manner as P-0786. For each compound, the date when the cell number peaked and the fold change in cell number increase are summarized in Table 15.

[0464] Table 15 Peak peripheral cell number and fold change relative to baseline after treatment

[0465]

[0466] In addition, as shown in Figure 30 , the potency level and the corresponding cytotoxic lymphocyte expansion were related to the toxicity reflected by mouse weight loss. As an IL-2Rβγ selective full agonist, P-0782 caused a significant increase in both the number of CD8+ T cells and NK cells and led to the greatest weight loss; the attenuated agonists P-0786 and P-0783 showed improved tolerance in vivo. P-0783 had a slight advantage over P-0786 in terms of tolerance, which was consistent with the fact that P-0783 was a weaker agonist than P-0786.

[0467] In summary, P-0782 showed a strong pharmacodynamic effect in promoting the proliferation and expansion of CD8+ T cells and NK cells. P-0786 and P-0783 showed weaker but more sustained signals. The potency ranking of these three compounds was generally consistent between in vitro and in vivo. In addition, compared with the full agonist P-0782, the compounds P-0786 and P-0783 with attenuated potency showed improved pharmacodynamics and tolerance in vivo.

[0468] Example 15

[0469] In Vivo Efficacy of PD1 Antibody IL-2 Variant Fusion Protein in a Syngeneic Mouse Tumor Model

[0470] The anti-tumor efficacy of an IL-2 variant murine PD-1 antibody fusion protein was tested in a subcutaneous B16F10 melanoma mouse tumor model. Female C57BL / 6 mice (7 weeks old) were randomly assigned to treatment groups by body weight after a 4 - 7-day acclimation (n = 10 / group). Third-generation B16F10 cells (5 × 10 5 cells / mouse) were inoculated subcutaneously (s.c.) into the right flank of the mice on day - 1. Mice were administered the test compound (Q7D) intraperitoneally (i.p.) three times on days 0, 7, and 14. All mice were closely monitored and body weight was measured three times a week. Tumors were measured three times a week using standard calipers, and tumor size was calculated by the standard formula length × (width)w 2 × 0.5 in mm 3 . When the tumor size exceeded the limit of 1500 mm 3 , the mice were euthanized.

[0471] Three antibody fusion proteins, P-0838, P-0790, and P-0787, were administered at 3 mg / kg and twice at a Q7D dose. All three fusion proteins contained the IL-2P65Q mutation to attenuate binding to IL-2Rα; P-0790 and P-0787 contained additional L19Q and L19H mutations, respectively, to further reduce IL-2Rβγ activity and thus modulate overall potency. As shown in Figure 31A, all compounds demonstrated strong single-dose anti-tumor efficacy, with tumor growth inhibition of 78%, 64%, and 57% for P-0787, P-0790, and P-0838, respectively. The level of tumor suppression efficacy was correlated with the in vitro potency attenuation from P-0838 to P-0790 and to P-0787.

[0472] Similar to what was observed in Figure 30 , Figure 31B depicts that the full IL-2 agonist P-0838 had the earliest and highest toxicity, as reflected by the maximum body weight loss; and the attenuated agonists P-0790 and P-0787 showed improved tolerance in vivo. P-0787 had some advantages over P-0790 in terms of tolerance, which is consistent with the fact that P-0783 is a weaker agonist than P-0786. Overall, the data support that IL-2Rβγ-selective and attenuated mutants exhibit good tumor killing efficacy and improved tolerance.

[0473] For P-0787, the effect of dose on in vivo tumor suppression and tolerance was further investigated. As observed in Figure 32A, as the dose increased from 3 mg / kg to 5 mg / kg, P-0787 showed similar effective anti-tumor effects. The increased dose did not result in significant weight loss (Figure 32B), indicating that the weak agonist contributed to high doses and increased tolerance.

[0474] For P-0782 and P-0786 administered at 1.5 mg / kg and 2 doses Q7D, strong tumor growth inhibition was also observed ( Figure 33 ). In the B16F10 syngeneic model, the surrogate murine PD-1 antibody P-0722 did not show anti-tumor effects, while P-0782 and P-0786 showed comparable stronger tumor growth inhibition, although P-0786 is a weakened counterpart of P-0782.

[0475] Finally, the IL-2 variant antibody fusion protein P-0790 was tested in the murine B16F10 lung metastasis model. Briefly, 3×10 5 murine melanoma cells were injected intravenously into female B57BL6 mice (10 - 12 weeks old). Three Q7D treatments were initiated via intraperitoneal injection on the next day (day 1). Treatment groups (n = 5 animals / group) included P-0790 at 0.3 mg / kg, 1 mg / kg, and 3 mg / kg and the corresponding antibody P-0722 at 3 mg / kg. Vehicle (PBS) was included as a negative control. On day 24, all mice were sacrificed for tissue harvest. Lung tumor nodules were counted, and the anti-metastatic effect was represented by the different number of tumor nodules between the treatment groups and the vehicle control.

[0476] P-0790 is an IL-2L19Q / P65Q PD-1 antibody fusion protein with significantly impaired binding to IL-2Rα and modulated overall potency. Similarly, the surrogate murine PD-1 antibody P-0722 was ineffective in inhibiting B16F10 tumor cell metastasis, while dose-dependent inhibition of lung metastasis nodules was observed for P-0790. Figure 34A shows the mean lung nodule count, and Figure 34B shows pictures of the lungs from representative animals of each group. Data are represented as mean ± SEM.

[0477] In summary, various IL-2 variant murine PD-1 antibody fusion proteins demonstrated strong single-dose anti-tumor effects. The attenuated IL-2 agonists showed effective tumor growth inhibition and improved tolerance, which allowed higher doses for enhanced efficacy.

[0478] Example 16

[0479] Pharmacodynamics / Pharmacokinetics and Safety Evaluation of Selected IL-2 Variant PD-1 Antibody Fusion Protein in Cynomolgus Monkeys

[0480] The PK / PD characteristics and safety of the selected IL-2 variant PD-1 antibody fusion protein will be evaluated in cynomolgus monkeys. Cynomolgus monkeys without drugs will be acclimated and trained for 2 - 3 weeks and randomly divided into groups of one monkey each, followed by a pre-dose baseline week. On Day +1, one group will receive intravenous administration of vehicle (PBS), and the other groups will be intravenously dosed with different test compounds.

[0481] Blood will be collected on Days - 3, 2, 4, 6, 8, 10, 12, and 15. Peripheral blood mononuclear cells (PBMCs) will be isolated from monkey whole blood and used for FACS immunophenotyping of peripheral blood Tregs, non-regulatory CD4+ T cells, CD8+ T cells, CD8+ central memory cells, CD8+ effector memory cells, CD8+ T naive cells, and NK cells to determine pharmacodynamics. Cell activation and proliferation will also be monitored by measuring CD25 and Ki67. Whole blood will also be used for complete blood count (CBC), which is divided into 5 parts: neutrophils, lymphocytes, monocytes, eosinophils, and basophils.

[0482] The PK characteristics of the selected IL-2 variant PD-1 antibody fusion protein will be evaluated in cynomolgus monkey plasma samples by measuring the full-length intact molecule using a 96-well plate coated with mouse anti-human IL-2 Ab (BD Pharmingen) to capture the fusion protein. Mouse anti-human IL-2-biotin (in-house) will be used for detection, and subsequently, the plasma concentration of the test compound will be quantified. In addition to plasma samples collected on Days - 3, 2, 3, 4, 5, 6, 8, 10, 15, and four additional plasma samples will be collected at 10 minutes, 1 hour, 4 hours, and 8 hours on Day 1 after administration of the selected IL-2 variant PD-1 antibody fusion protein.

[0483] Plasma samples from Days - 7, 8, and 15 will also be used to evaluate the following clinical chemistry parameters: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ-glutamyltransferase, albumin, total bilirubin, creatinine, blood urea nitrogen, and C-reactive protein.

[0484] In addition, the body weight of each animal will be monitored weekly throughout the study. Body temperature and blood pressure will be monitored on Day - 1 (before dosing) and 6 hours, 24 hours, 96 hours, and 168 hours after drug administration.

[0485] All of the articles and methods disclosed and claimed herein can be made and executed in view of the present disclosure without undue experimentation. Although the articles and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the articles and methods without departing from the spirit and scope of the present disclosure. It will be apparent to those skilled in the art that all such variations and equivalents, whether existing or later developed, are considered to be within the spirit and scope of the present disclosure as defined by the appended claims. All patents, patent applications, and publications mentioned in the specification indicate the level of ordinary skill in the art to which the present disclosure pertains. For all purposes, all patents, patent applications, and publications are hereby incorporated by reference in their entirety, and to the extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety for any and all purposes. The present disclosure, as exemplified herein, can be suitably practiced in the absence of any one or more of the elements not specifically disclosed herein. Accordingly, it is to be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, those skilled in the art may seek alterations and variations of the concepts disclosed herein, and such alterations and variations are considered to be within the scope of the present disclosure as defined by the appended claims.

[0486] Sequence Listing

[0487] The nucleic acid and amino acid sequences listed in the attached Sequence Listing are shown using the standard letter abbreviations for nucleotide bases and the single-letter codes for amino acids as defined in 37 C.F.R. 1.822.

[0488] SEQ ID NO:1 is the amino acid sequence of human IL-2 precursor.

[0489] SEQ ID NO:2 is the naturally occurring amino acid sequence of the mature form of human IL-2.

[0490] SEQ ID NO:3 is the wild-type amino acid sequence of the mature form of human IL-2.

[0491] SEQ ID NO:4 is the amino acid sequence of the mature form of human IL-2 containing the S125I substitution for improving the developability profile of the fusion protein.

[0492] SEQ ID NO:5 is the amino acid sequence of the extracellular domain of human IL-2Rα.

[0493] SEQ ID NO:6 is the amino acid sequence of human IgG1-Fc.

[0494] SEQ ID NO:7 is the human IgG1-Fc sequence with reduced / abolished effector function.

[0495] SEQ ID NO:8 is a human IgG1-Fc sequence with reduced / abolished effector function and an extended half-life.

[0496] SEQ ID NO:9 is a Knob-Fc amino acid sequence with reduced / abolished effector function.

[0497] SEQ ID NO:10 is a Hole-Fc amino acid sequence with reduced / abolished effector function.

[0498] SEQ ID NO:11 - 30 are the amino acid sequences of various peptide linker sequences.

[0499] SEQ ID NO:31 - 66 are the amino acid sequences of various IL-2 variants with amino acid substitutions introduced at the interface with the α subunit of the IL-2 receptor.

[0500] SEQ ID NO:67 - 107 are the amino acid sequences of various IL-2 variant Fc fusion proteins.

[0501] SEQ ID NO:108 is the amino acid sequence of a reference IL-2 variant Fc fusion protein.

[0502] SEQ ID NO:109 is the amino acid sequence of the extracellular domain of human IL-2Rβ.

[0503] SEQ ID NO:110 is the amino acid sequence of the extracellular domain of human γc.

[0504] SEQ ID NO:111 - 120 are the amino acid sequences of various IL-2 variants.

[0505] SEQ ID NO:121 - 133 are the amino acid sequences of various IL-2 variant Fc fusion proteins.

[0506] SEQ ID NO:134 is a Knob-Fc amino acid sequence with reduced / abolished effector function and an extended half-life.

[0507] SEQ ID NO:135 is a Hole-Fc amino acid sequence with reduced / abolished effector function and an extended half-life.

[0508] SEQ ID NO:136 - 137 are the amino acid sequences of the heavy and light chains of a humanized anti-FAP antibody.

[0509] SEQ ID NO:138 - 139 are the amino acid sequences of the heavy and light chains of a human PD-1 antagonist antibody.

[0510] SEQ ID NO: 140 - 141 are the amino acid sequences of the heavy and light chains of a PD-1 antagonist antibody.

[0511] SEQ ID NO: 142 - 143 are the amino acid sequences of the heavy and light chains of a PD-1 antagonist antibody.

[0512] SEQ ID NO: 144 - 145 are the amino acid sequences of the heavy and light chains of a PD-1 antagonist antibody.

[0513] SEQ ID NO: 146 - 147 are the amino acid sequences of the heavy and light chains of a PD-1 antagonist antibody.

[0514] SEQ ID NO: 148 - 149 are the amino acid sequences of the heavy and light chains of a PD-L1 antagonist antibody.

[0515] SEQ ID NO: 150 - 151 are the amino acid sequences of the heavy and light chains of a CTLA-4 antagonist antibody.

[0516] SEQ ID NO: 152 - 153 are the amino acid sequences of the heavy and light chains of a CD40 agonist antibody.

[0517] SEQ ID NO: 154 - 155 are the amino acid sequences of the heavy and light chains of a fibronectin antagonist antibody.

[0518] SEQ ID NO: 156 - 157 are the amino acid sequences of the heavy and light chains of a CD20 antagonist antibody.

[0519] SEQ ID NO: 158 - 159 are the amino acid sequences of the heavy and light chains of a Her-2 / neu antagonist antibody.

[0520] SEQ ID NO: 160 - 161 are the amino acid sequences of the heavy and light chains of an EGFR antagonist antibody.

[0521] SEQ ID NO: 162 is the amino acid sequence of the human IgG1 CH1CH2CH3 domain sequence with reduced / abolished Fc effector function.

[0522] SEQ ID NO: 163 is the amino acid sequence of the knob chain of the human IgG1 CH1CH2CH3 domain with reduced / abolished Fc effector function.

[0523] SEQ ID NO: 164 is the amino acid sequence of the hole chain of the human IgG1 CH1CH2CH3 domain with reduced / abolished Fc effector function.

[0524] SEQ ID NO: 165 - 169 are the amino acid sequences of various IL-2 variant antibody fusion proteins.

[0525] SEQ ID NO: 170 is the amino acid sequence of the human IL-2 receptor α Sushi domain.

[0526] SEQ ID NO: 171 - 174 are the amino acid sequences of the IL-2 and IL-2R Sushi Fc fusion proteins.

[0527] SEQ ID NO: 175 - 181 are the amino acid sequences of the knob chains of various IL-2 variant human PD-1 antagonist antibody fusion proteins.

[0528] SEQ ID NO: 182 - 184 are the amino acid sequences of the benchmark IL-2 variant antibody fusion proteins.

[0529] SEQ ID NO: 185 - 187 are the amino acid sequences of an alternative anti-mouse PD-1 antibody with a heterodimeric heavy chain.

[0530] SEQ ID NO: 188 is the amino acid sequence of the benchmark IL-2 variant.

[0531] SEQ ID NO: 189 - 191 are the amino acid sequences of the knob chains of various IL-2 variant human PD-1 antagonist antibody fusion proteins.

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[0540]

[0541]

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[0545]

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[0547]

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[0549]

[0550]

[0551]

[0552]

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[0561]

[0562] Sequence Listing <110> KeyGen Biotech Corporation <120> Novel Interleukin-2 Variant for Cancer Treatment <130> CACCG1.0004WO <160> 191 <170> PatentIn version 3.5 <210> 1 <211> 153 <212> PRT <213> Homo sapiens <400> 1 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser Thr Leu Thr 145 150 <210> 2 <211> 133 <212> PRT <213> Homo sapiens <400> 2 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 3 <211> 133 <212> PRT <213> Homo sapiens <400> 3 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 4 <211> 133 <212> PRT <213> Artificial <220> <223> Human IL-2 S125I variant sequence <400> 4 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 5 <211> 219 <212> PRT <213> Homo sapiens <400> 5 Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys 1 5 10 15 Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg 20 25 30 Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly 35 40 45 Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser 50 55 60 Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln 65 70 75 80 Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp 85 90 95 Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn 100 105 110 Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr 115 120 125 Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu 130 135 140 Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln 145 150 155 160 Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln Phe Pro Gly Glu Glu 165 170 175 Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu Ser Glu Thr Ser Cys 180 185 190 Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr Glu Met Ala Ala Thr 195 200 205 Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln 210 215 <210> 6 <211> 226 <212> PRT <213> Homo sapiens <400> 6 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 7 <211> 226 <212> PRT <213> Artificial <220> <223> Human IgG1-Fc with reduced / ablated effector function <400> 7 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 8 <211> 226 <212> PRT <213> Artificial <220> <223> Human IgG1-Fc with reduced / abolished effector function and extended half-life <400> 8 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 9 <211> 226 <212> PRT <213> Artificial <220> <223> Human IgG Knob-Fc with reduced / ablated effector function <400> 9 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 10 <211> 226 <212> PRT <213> Artificial <220> Human IgG Hole-Fc with reduced / abrogated effector function <400> 10 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 11 <211> 12 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 11 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 <210> 12 <211> 4 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 12 Gly Gly Gly Ser 1 <210> 13 <211> 13 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 13 Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 10 <210> 14 <211> 5 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 14 Gly Ser Ser Gly Thr 1 5 <210> 15 <211> 15 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 15 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 16 <211> 17 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 16 Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 15 Ala <210> 17 <211> 20 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 17 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 18 <211> 8 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 18 Gly Gly Gly Ser Gly Gly Gly Ser 1 5 <210> 19 <211> 5 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 19 Gly Ser Gly Ser Thr 1 5 <210> 20 <211> 4 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 20 Gly Gly Ser Ser 1 <210> 21 <211> 5 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 21 Gly Gly Gly Gly Ser 1 5 <210> 22 <211> 4 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 22 Gly Gly Ser Gly 1 <210> 23 <211> 4 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 23 Ser Gly Gly Gly 1 <210> 24 <211> 4 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 24 Gly Ser Gly Ser 1 <210> 25 <211> 6 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 25 Gly Ser Gly Ser Gly Ser 1 5 <210> 26 <211> 8 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 26 Gly Ser Gly Ser Gly Ser Gly Ser 1 5 <210> 27 <211> 10 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 27 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 1 5 10 <210> 28 <211> 12 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 28 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 1 5 10 <210> 29 <211> 10 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 29 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 30 <211> 15 <212> PRT <213> Artificial <220> <223> Peptide linker <400> 30 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Gly Ser 1 5 10 15 <210> 31 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 F42A / S125I variant sequence <400> 31 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 32 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 R38F / S125I variant sequence <400> 32 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 33 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 R38G / S125I variant sequence <400> 33 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Gly Met Leu Thr Ala Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 34 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 R38A / S125I variant sequence <400> 34 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Ala Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 35 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 T41A / S125I variant sequence <400> 35 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Ala Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 36 <211> 133 <212> PRT <213> Artificial <220> <223> APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLGFKFYMPKKATELKHLQCLEEELKP LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIIS TLT <400> 36 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Gly Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 37 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 T41V / S125I variant sequence <400> 37 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Val Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 38 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 F44G / S125I variant sequence <400> 38 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Gly Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 39 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 F44V / S125I variant sequence <400> 39 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Val Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 40 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 E62A / S125I variant sequence <400> 40 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Ala Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 41 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 E62F / S125I variant sequence <400> 41 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Phe Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 42 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 E62H / S125I variant sequence <400> 42 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu His Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 43 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 E62L / S125I variant sequence <400> 43 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Leu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 44 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 P65G / S125I variant sequence <400> 44 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Gly Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 45 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 P65E / S125I variant sequence <400> 45 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Glu Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 46 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 P65H / S125I variant sequence <400> 46 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 His Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 47 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 P65R / S125I variant sequence <400> 47 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Arg Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 48 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 P65A / S125I variant sequence <400> 48 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Ala Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 49 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 P65K / S125I variant sequence <400> 49 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Lys Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 50 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 P65N / S125I variant sequence <400> 50 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Asn Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 51 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 P65Q / S125I variant sequence <400> 51 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Gln Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 52 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 E68A / S125I variant sequence <400> 52 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Ala Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 53 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 E68F / S125I variant sequence <400> 53 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Phe Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 54 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 E68H / S125I variant sequence <400> 54 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu His Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 55 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 E68L / S125I variant sequence <400> 55 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Leu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 56 <211> 133 <212> PRT <213> Artificial <220> <223> IL-2 E68P / S125I variant sequence <400> 56 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Pro Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 57 <211> 133 <212> PRT <213> A...

Claims

1. An isolated interleukin-2 (IL-2) variant polypeptide, wherein the amino acid differences between the IL-2 variant polypeptide and SEQ ID NO: 3 are at amino acid residue positions L19, P65, and S125, wherein the substitution at position P65 is P65R, the substitution at position S125 is S125I, the substitution at position L19 is selected from L19H and L19Q, and wherein the IL-2 variant polypeptide exhibits reduced binding to IL-2Rα and lower Treg activity compared to the polypeptide represented by SEQ ID NO: 3, but still retains the ability to bind and activate the IL-2Rβγ complex.

2. The IL-2 variant polypeptide according to claim 1, wherein the amino acid differences between the IL-2 variant polypeptide and SEQ ID NO: 3 are three amino acid substitutions L19H, P65R, and S125I.

3. The IL-2 variant polypeptide according to claim 1, wherein the amino acid differences between the IL-2 variant polypeptide and SEQ ID NO: 3 are three amino acid substitutions L19Q, P65R, and S125I.

4. The IL-2 variant polypeptide according to any one of claims 1 to 3, wherein the IL-2 variant polypeptide is composed of an amino acid sequence selected from the group consisting of the amino acid sequences listed in SEQ ID NOs: 111 - 112.

5. An isolated fusion protein, the isolated fusion protein comprising 1) an IL-2 variant polypeptide according to any one of claims 1 to 4 and 2) a heterologous protein, wherein the isolated fusion protein is in monomeric or dimeric form.

6. The isolated fusion protein according to claim 5, wherein the IL-2 variant polypeptide is optionally fused to the C-terminal amino acid of the heterologous protein at its N-terminal amino acid through a peptide linker, or wherein the IL-2 variant polypeptide is optionally fused to the N-terminal amino acid of the heterologous protein at its C-terminal amino acid through a peptide linker.

7. The isolated fusion protein according to any one of claims 5 to 6, wherein the heterologous protein increases the circulating half-life of the IL-2 variant polypeptide.

8. The isolated fusion protein according to any one of claims 5 to 6, wherein the heterologous protein is a targeting moiety in the form of an antibody that targets a tumor-associated antigen (TAA).

9. The isolated fusion protein according to any one of claims 5 to 6, wherein the heterologous protein is a targeting moiety in the form of an antibody heavy chain or light chain that targets a tumor-associated antigen (TAA).

10. The isolated fusion protein according to any one of claims 5 to 6, wherein the heterologous protein is a targeting moiety in the form of an antibody fragment that targets a tumor-associated antigen (TAA).

11. The isolated fusion protein according to any one of claims 5 to 6, wherein the heterologous protein is a targeting moiety in the form of a protein that targets a tumor-associated antigen (TAA).

12. The isolated fusion protein according to any one of claims 5 to 6, wherein the heterologous protein is a targeting moiety in the form of a peptide presenting a tumor-associated antigen (TAA).

13. The isolated fusion protein according to any one of claims 5 to 6, wherein the heterologous protein is an antagonistic programmed death-1 (PD-1) antibody or antibody fragment.

14. The isolated fusion protein according to claim 13, wherein the antibody is an antagonistic humanized PD-1 antibody selected from antibodies comprising the heavy and light chain amino acid sequences listed in SEQ ID NOs: 138 and 139; the heavy and light chain amino acid sequences listed in SEQ ID NOs: 140 and 141; the heavy and light chain amino acid sequences listed in SEQ ID NOs: 142 and 143; the heavy and light chain amino acid sequences listed in SEQ ID NOs: 144 and 145; and the heavy and light chain amino acid sequences listed in SEQ ID NOs: 146 and 147.

15. A pharmaceutical composition comprising the IL-2 variant polypeptide according to any one of claims 1 to 4 or the isolated fusion protein according to any one of claims 5 to 14 mixed with a pharmaceutically acceptable carrier.

16. Use of the pharmaceutical composition according to claim 15 in the preparation of a medicament for treating cancer in a subject, wherein a therapeutically effective amount of the pharmaceutical composition is administered to the subject when the medicament is used.

17. An isolated nucleic acid molecule encoding the IL-2 variant polypeptide according to any one of claims 1 to 4 or the fusion protein according to any one of claims 5 to 14.

18. An expression vector comprising the isolated nucleic acid molecule of claim 17.

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

20. A method for producing the IL-2 variant polypeptide according to any one of claims 1 to 4 or the fusion protein according to any one of claims 5 to 14, the method comprising culturing the host cell according to claim 19 under conditions that promote the expression of the IL-2 variant polypeptide or fusion protein, and recovering the IL-2 variant polypeptide or fusion protein.

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

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