Anti-PD-1 / IL-2 fusion protein, pharmaceutical composition, and therapeutic application

Abstract

Provided herein are anti-PD-1 / IL-2 fusion proteins and pharmaceutical compositions thereof. Also provided herein are methods of using them to treat, prevent, or improve one or more symptoms of proliferative disorders.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Application No. 63 / 482,790, filed on 1 February 2023; its disclosure is incorporated herein by reference in its entirety.

[0002] (Field) Provided herein are anti-PD-1 / IL-2 fusion proteins and pharmaceutical compositions thereof. Also provided herein are methods of using them to treat, prevent, or improve one or more symptoms of proliferative disorders.

[0003] (Reference to the sequence list) This specification was filed with a sequence listing entitled 216A020WO01_SEQLIST_ST26.XML, which is 238,939 bytes in size and was created on January 31, 2024; its contents are incorporated herein by reference in their entirety. [Background technology]

[0004] (background) Abnormal regulation of the host immune system is one important immune resistance mechanism against cancer. (Hanahan and Weinberg, Cell 2011, 144, 646-74; Pardoll, Nat. Rev. Cancer 2012, 12, 252-64). One class of immunotherapy involves drugs that target specific checkpoint proteins that play a crucial role in regulating T cell activation and proliferation. (Waldman et al., Nat. Rev. Immunol. 2020, 20, 651-68). These proteins function as co-receptors on the surface of T cells to help regulate the T cell response after T cell activation. (Wolchok et al., Cancer J. 2010, 16, 311-7). The two most well-characterized checkpoint proteins are cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed cell death-1 (PD-1), both of which act as negative regulators of T cell activation. (Waldman et al., Nat. Rev. Immunol. 2020, 20, 651-68). T cell activation induces the expression of CTLA-4 and PD-1, thereby inhibiting T cell activation and proliferation. (Pardoll, Nat. Rev. Cancer 2012, 12, 252-64). Immune checkpoint blockade removes such inhibitory signals and triggers an anti-tumor immune response. (Ibid.; Sharma and Allison, Science 2015, 348, 56-61). The CTLA-4 blocking antibody ipilimumab was the first immune checkpoint inhibitor approved by the FDA for cancer treatment. (Ibid.). Since then, several anti-PD-1 antibodies have been approved for cancer treatment. Gong et al., J. Immunother. Cancer 2018, 6, 8. Immunotherapy is a major advance in cancer treatment, but up to 85 percent of patients whose cancer is treated with checkpoint inhibitors do not benefit. Haslam and Prasad, JAMA Netw. Open 2019, 2, e192535.

[0005] Interleukin-2 (IL-2) is a multifaceted cytokine that maintains immune homeostasis by regulating the proliferation, survival, and function of both immune effector (Teff) cells and regulatory T (Treg) cells. (Bluestone, N. Engl. J. Med. 2011, 365, 2129-31; Boyman et al., Nat. Rev. Immunol. 2012, 12, 180-90.) IL-2 promotes T cell proliferation, enhances natural killer (NK) cell lysis activity, induces differentiation of regulatory T (Treg) cells, and mediates activation-induced cell death. (Liao et al., Curr. Opin. Immunol. 2011, 23, 598-604.)

[0006] The interleukin-2 receptor (IL-2R) consists of three distinct interleukin-2 receptor chains: α chain (IL-2Rα or CD25), β chain (IL-2Rβ or CD122), and γ chain (IL-2Rγ, γ c It exists in three different forms generated from (or CD132). Wang et al., Science 2005, 310, 1159-63. IL-2 has low affinity (K d It binds to IL-2Rα at approximately 10 nM. Ibid. From the crystal structure of the IL-2 signaling complex, which consists of four elements, 15 amino acid residues on IL-2 (K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, K64, P65, E68, L72, and Y107) have been identified as interface residues between IL-2 and IL-2Rα. Stauber et al., Proc. Natl. Acad. Sci. USA 2006, 103, 2788-93. IL-2 has a moderate affinity (K) for the heterodimer complex of IL-2Rβ and IL-2Rγ expressed on memory T cells and NK cells. d It binds with a ≈1nM. (Wang et al., Science 2005, 310, 1159-63). IL-2 has a high affinity (K) for the heterotrimer complex of IL-2Rα, IL-2Rβ, and IL-2Rγ expressed on Treg cells. d They bind at approximately 10 pM. Same as above. IL-2 has a dissociation constant of approximately 100 nM (Kd ) binds to IL-2Rβ alone, but no binding affinity is detected for IL-2Rγ alone. Ibid. IL-2Rα alone has no signaling activity. Ibid. IL-2 transmits signals via the moderate-affinity heterodimer IL-2Rβ / γ complex or the high-affinity heterotrimer IL-2Rα / β / γ complex. Liao et al., Curr. Opin. Immunol. 2011, 23, 598-604. Binding of IL-2 to the moderate-affinity heterodimer IL-2Rβ / γ complex leads to activation and proliferation of immunostimulatory Teff cells, while binding of IL-2 to the high-affinity heterotrimer IL-2Rα / β / γ complex leads to activation and proliferation of immunosuppressive Treg cells. References: Malek et al., Immunity 2010, 33, 153-65; Bluestone, N. Engl. J. Med. 2011, 365, 2129-31; Boyman et al., Nat. Rev. Immunol. 2012, 12, 180-90; Spangler et al., Annu. Rev. Immunol. 2015, 33, 139-67. These two opposing functions of immunostimulation and immunosuppression present a major challenge in developing IL-2 as a safe and effective therapeutic agent. References: Skrombolas et al., Expert Rev. Clin. Immunol. 2014, 10, 207-17; Abbas et al., Sci. Immunol. 2018, 3, eaat1482.

[0007] Aldesleukin, a recombinant human IL-2, was approved by the FDA in 1992 for metastatic renal cell carcinoma and in 1998 for metastatic melanoma. (Rosenberg, J. Immunol. 2014, 192, 5451-8). 5-10% of patients with metastatic melanoma or renal cancer experience complete regression, and another 10% experience partial regression. (Atkins et al., J. Clin. Oncol. 1999, 17, 2105-16; Klapper et al., Cancer 2008, 113, 293-301). Approximately 70% of those who respond completely to IL-2 therapy do not recur. (Rosenberg, Sci. Transl. Med. 2012, 4, 127ps8). However, the success of IL-2 as cancer immunotherapy is hindered by its high toxicity and limited efficacy. One major limiting factor to its effectiveness as an anticancer agent is immunosuppression resulting from the preferential expression of Treg cells driven by IL-2. (Abbas et al., Sci. Immunol. 2018, 3, eaat1482). Furthermore, high-dose treatment schedules are required for IL-2 to be effective in cancer treatment. (Bluestone, N. Engl. J. Med. 2011, 365, 2129-31; Abbas et al., Sci. Immunol. 2018, 3, eaat1482). However, this dosing regimen causes vasoleakage syndrome, limiting the application of IL-2 in cancer treatment. (Abbas et al., Sci. Immunol. 2018, 3, eaat1482).

[0008] Despite advances in cancer treatment, cancer remains a major global public health problem. In 2021 alone, an estimated 1,898,160 new cancer cases were diagnosed in the United States, and 608,570 deaths were attributed to cancer (Cancer Facts & Figures 2021). Therefore, effective treatments for cancer are urgently needed. [Overview of the Initiative]

[0009] (Summary of this disclosure) Provided herein is an anti-PD-1 / IL-2 fusion protein comprising an IL-2 domain, an anti-PD-1 single-domain antibody (sdAb), and a crystalline fragment (Fc) domain; the anti-PD-1 sdAb comprises CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3.

[0010] Also provided herein is an anti-PD-1 / IL-2 fusion protein comprising an IL-2 domain, first and second anti-PD-1 sdAbs, and an Fc domain comprising first and second peptide chains; wherein the C-terminus of the first anti-PD-1 sdAb is directly or via a peptide linker to the N-terminus of the first peptide chain of the Fc domain, and the C-terminus of the first peptide chain of the Fc domain is directly or via a peptide linker to the N-terminus of the IL-2 domain; the C-terminus of the second anti-PD-1 sdAb is directly or via a peptide linker to the N-terminus of the second peptide chain of the Fc domain; and the anti-PD-1 sdAb comprises CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3.

[0011] Furthermore, provided herein are anti-PD-1 / IL-2 fusion proteins comprising first and second IL-2 domains, first and second anti-PD-1 sdAbs, and Fc domains comprising first and second peptide chains; the C-terminus of the first anti-PD-1 sdAb is directly or via a peptide linker to the N-terminus of the first peptide chain of the Fc domain, and the C-terminus of the first peptide chain of the Fc domain is directly or via a peptide linker to the N-terminus of the first IL-2 domain; the C-terminus of the second anti-PD-1 sdAb is directly or via a peptide linker to the N-terminus of the second peptide chain of the Fc domain, and the C-terminus of the second peptide chain of the Fc domain is directly or via a peptide linker to the N-terminus of the second IL-2 domain; and the anti-PD-1 sdAb is an anti-PD-1 / IL-2 fusion protein containing CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3).

[0012] Furthermore, provided herein are pharmaceutical compositions comprising the anti-PD-1 / IL-2 fusion protein and pharmaceutically acceptable excipients provided herein.

[0013] Provided herein are methods for treating, preventing, or improving one or more symptoms of a proliferative disorder in a subject, comprising administering a therapeutically effective amount of the anti-PD-1 / IL-2 fusion protein provided herein to the subject in need thereof.

[0014] Provided herein is a method for inhibiting cell growth, comprising contacting the subject with an effective amount of the anti-PD-1 / IL-2 fusion protein provided herein. [Brief explanation of the drawing]

[0015] (Brief explanation of the drawing) [Figure 1]Figure 1 shows exemplary fusion proteins: (i) an anti-PD-1 / IL-2 fusion protein comprising first and second IL-2 domains, first and second anti-PD-1 sdAbs, and Fc domains comprising first and second peptide chains; the C-terminus of the first anti-PD-1 sdAb is directly ligated to the N-terminus of the first peptide chain of the Fc domain, and the C-terminus of the first peptide chain of the Fc domain is ligated to the N-terminus of the first IL-2 domain via a peptide linker; and (ii) an anti-PD-1 / IL-2 fusion protein comprising an IL-2 domain, first and second anti-PD-1 The present invention relates to an anti-PD-1 / IL-2 fusion protein comprising an sdAb and an Fc domain containing first and second peptide chains; the C-terminus of the first anti-PD-1 sdAb is directly ligated to the N-terminus of the first peptide chain of the Fc domain, and the C-terminus of the first peptide chain of the Fc domain is ligated to the N-terminus of the IL-2 domain via a peptide linker; the C-terminus of the second anti-PD-1 sdAb is directly ligated to the N-terminus of the second peptide chain of the Fc domain; and the Fc domain has a knob-into-hole structure. [Modes for carrying out the invention]

[0016] (Detailed explanation) To facilitate understanding of the disclosures presented herein, several terms are defined below.

[0017] The nomenclature used herein, as well as the experimental procedures in biochemistry, biology, cell biology, immunology, molecular biology, and pharmacology described herein, are generally well known and commonly used in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by experts in the field to which this disclosure belongs.

[0018] The term “subject” refers to animals, including but not limited to primates (e.g., humans), cattle, pigs, sheep, goats, horses, dogs, cats, rabbits, rats, or mice. The terms “subject” and “patient” are used interchangeably herein, for example, with respect to mammalian subjects, for example, human subjects. In one embodiment, the subject is human.

[0019] The terms “to treat,” “to treat,” and “treatment” are intended to include reducing or neutralizing one or more of a disorder, disease, or illness, or symptoms associated with such disorder, disease, or illness; or reducing or eliminating the cause of such disorder, disease, or illness itself.

[0020] The terms “prevent,” “prevent,” and “prevention” are intended to include delaying and / or making impossible the onset of a disability, disease, or illness and / or its associated symptoms; preventing a subject from acquiring a disability, disease, or illness; or reducing the risk to a subject acquiring a disability, disease, or illness.

[0021] The terms "alleviate" and "alleviating" refer to reducing one or more symptoms (e.g., pain) of a disorder, disease, or illness. These terms may also refer to reducing the adverse effects associated with the active ingredient. Sometimes, the beneficial effects that a subject may obtain from a preventive or therapeutic agent do not result in a cure for the disorder, disease, or illness.

[0022] The terms “contact” or “contact” are intended to refer to bringing a therapeutic agent together with cells or tissues so that physiological and / or chemical effects occur as a result of such contact. Contact can occur in vitro, ex vivo, or in vivo. In one embodiment, a therapeutic agent is brought into contact with cells in cell culture (in vitro) to determine the effect of the therapeutic agent on the cells. In another embodiment, contact of a therapeutic agent with cells or tissues includes administering the therapeutic agent to a subject having cells or tissues to be contacted.

[0023] The terms “therapeutic effective dose” or “effective dose” are intended to include the amount of a compound (e.g., polypeptide or fusion protein) that, when administered, is sufficient to prevent the onset of the disorder, disease, or illness being treated, or to some extent to alleviate one or more of the symptoms of said disorder, disease, or illness. The terms “therapeutic effective dose” or “effective dose” also refer to the amount of a compound that is sufficient to induce a biological or medical response in a biomolecule (e.g., protein, enzyme, RNA, or DNA), cell, tissue, system, animal, or human, as determined by researchers, veterinarians, physicians, or clinicians.

[0024] The terms “medically acceptable carrier,” “medically acceptable excipient,” “physiologically acceptable carrier,” or “physiologically acceptable excipient” refer to medically acceptable materials, compositions, or vehicles, such as liquid or solid fillers, diluents, solvents, or encapsulating materials. In one embodiment, each component is “medically acceptable” in the sense that it is compatible with other components of a pharmaceutical formulation and is suitable for use in contact with the tissues or organs of a subject (e.g., human or animal) without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, and is commensurate with a reasonable benefit / risk ratio. For example, see Remington: The Science and Practice of Pharmacy, 23rd edition; edited by Adejare; Academic Press, 2020; Handbook of Pharmaceutical Excipients, 9th edition; edited by Sheskey et al.; Pharmaceutical Press, 2020; Handbook of Pharmaceutical Additives, 3rd edition; edited by Ash and Ash; Synapse Information Resources, 2007; Pharmaceutical Preformulation and Formulation, 1st edition; edited by Gibson; CRC Press, 2015.

[0025] The terms “about” or “approximately” mean an acceptable error in relation to a particular value as determined by those skilled in the art, which depends to some extent on the method by which the value is measured or determined. In some embodiments, the terms “about” or “approximately” mean within 1, 2, 3, or 4 standard deviations. In some embodiments, the terms “about” or “approximately” mean within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

[0026] When referring to a compound, the terms “substantially pure” and “substantially homogeneous” mean, but are not limited to, that the compound is sufficiently homogeneous to appear free of readily detectable impurities when determined by standard analytical methods used by those skilled in the art, including gel electrophoresis, high-performance liquid chromatography (HPLC), and mass spectrometry (MS); or that the compound is sufficiently pure to ensure that further purification does not alter its physical, chemical, biological, and / or pharmacological properties, such as enzymatic and biological activity, to a detectable degree. In some embodiments, “substantially pure” or “substantially homogeneous” refers to a group of molecules in which, when determined by standard analytical methods, at least about 50% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 98% by weight, at least about 99% by weight, or at least about 99.5% by weight of the molecule is a single compound.

[0027] (Anti-PD-1 / IL-2 fusion protein) In one embodiment, the herein provides an anti-PD-1 / IL-2 fusion protein comprising an IL-2 domain, an anti-PD-1 single-domain antibody (sdAb), and a crystalline fragment (Fc) domain; the anti-PD-1 sdAb is an anti-PD-1 / IL-2 fusion protein comprising complementarity-determining region (CRD) 1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3.

[0028] In another embodiment, provided herein is an anti-PD-1 / IL-2 fusion protein comprising one IL-2 domain, two anti-PD-1 sdAbs, and an Fc domain; wherein the anti-PD-1 sdAbs comprise CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3.

[0029] In one embodiment, the anti-PD-1 / IL-2 fusion protein provided herein comprises an IL-2 domain, first and second anti-PD-1 sdAbs, and an Fc domain comprising first and second peptide chains; wherein the C-terminus of the first anti-PD-1 sdAb is directly or via a peptide linker to the N-terminus of the first peptide chain of the Fc domain, and the C-terminus of the first peptide chain of the Fc domain is directly or via a peptide linker to the N-terminus of the IL-2 domain; and wherein the C-terminus of the second anti-PD-1 sdAb is directly or via a peptide linker to the N-terminus of the second peptide chain of the Fc domain.

[0030] In another embodiment, the anti-PD-1 / IL-2 fusion protein provided herein comprises an IL-2 domain, first and second anti-PD-1 sdAbs, and an Fc domain comprising first and second peptide chains; wherein the C-terminus of the first anti-PD-1 sdAb is directly ligated to the N-terminus of the first peptide chain of the Fc domain, and the C-terminus of the first peptide chain of the Fc domain is ligated to the N-terminus of the IL-2 domain via a peptide linker; and wherein the C-terminus of the second anti-PD-1 sdAb is directly ligated to the N-terminus of the second peptide chain of the Fc domain.

[0031] In yet another embodiment, the foregoing provides an anti-PD-1 / IL-2 fusion protein comprising two IL-2 domains, two anti-PD-1 sdAbs, and an Fc domain; wherein the anti-PD-1 sdAbs comprise CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3.

[0032] In one embodiment, the anti-PD-1 / IL-2 fusion protein provided herein comprises first and second IL-2 domains, first and second anti-PD-1 sdAbs, and an Fc domain comprising first and second peptide chains; wherein the C-terminus of the first anti-PD-1 sdAb is directly or via a peptide linker to the N-terminus of the first peptide chain of the Fc domain, and the C-terminus of the first peptide chain of the Fc domain is directly or via a peptide linker to the N-terminus of the first IL-2 domain; wherein the C-terminus of the second anti-PD-1 sdAb is directly or via a peptide linker to the N-terminus of the second peptide chain of the Fc domain, and the C-terminus of the second peptide chain of the Fc domain is directly or via a peptide linker to the N-terminus of the second IL-2 domain.

[0033] In another embodiment, the anti-PD-1 / IL-2 fusion protein provided herein comprises first and second IL-2 domains, first and second anti-PD-1 sdAbs, and an Fc domain comprising first and second peptide chains; wherein the C-terminus of the first anti-PD-1 sdAb is directly ligated to the N-terminus of the first peptide chain of the Fc domain, and the C-terminus of the first peptide chain of the Fc domain is ligated to the N-terminus of the first IL-2 domain via a peptide linker; wherein the C-terminus of the second anti-PD-1 sdAb is directly ligated to the N-terminus of the second peptide chain of the Fc domain, and the C-terminus of the second peptide chain of the Fc domain is ligated to the N-terminus of the second IL-2 domain via a peptide linker.

[0034] In one embodiment, each anti-PD-1 sdAb in the anti-PD-1 / IL-2 fusion protein provided herein independently comprises the amino acid sequence of SEQ ID NO: 4 or 5. In another embodiment, each anti-PD-1 sdAb in the anti-PD-1 / IL-2 fusion protein provided herein comprises the amino acid sequence of SEQ ID NO: 4. In yet another embodiment, each anti-PD-1 sdAb in the anti-PD-1 / IL-2 fusion protein provided herein comprises the amino acid sequence of SEQ ID NO: 5.

[0035] In certain embodiments, the anti-PD-1 sdAb is an anti-PD-1 V H H sdAb.

[0036] In one embodiment, provided herein is an anti-PD-1 / IL-2 fusion protein comprising an IL-2 domain, an anti-PD-1 V H H sdAb, and an Fc domain; wherein the anti-PD-1 V H H sdAb comprises CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3.

[0037] In another embodiment, provided herein is an anti-PD-1 / IL-2 fusion protein comprising one IL-2 domain, two anti-PD-1 V H H sdAbs, and an Fc domain; wherein the anti-PD-1 V H H sdAbs comprise CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3.

[0038] In one embodiment, the anti-PD-1 / IL-2 fusion protein provided herein comprises an IL-2 domain, first and second anti-PD-1 V H H sdAbs, and an Fc domain comprising first and second peptide chains; wherein the first anti-PD-1 V HThe C-terminus of H sdAb is directly or via a peptide linker attached to the N-terminus of the first peptide chain of the Fc domain, and the C-terminus of the first peptide chain of the Fc domain is directly or via a peptide linker attached to the N-terminus of the IL-2 domain; and here the second anti-PD-1 V H The C-terminus of H sdAb is directly or via a peptide linker attached to the N-terminus of the second peptide chain of the Fc domain.

[0039] In another embodiment, the anti-PD-1 / IL-2 fusion protein provided herein comprises an IL-2 domain, first and second anti-PD-1 V H The first anti-PD-1 V comprises H sdAb and an Fc domain containing first and second peptide chains; where the first anti-PD-1 V H The C-terminus of H sdAb is directly connected to the N-terminus of the first peptide chain of the Fc domain, and the C-terminus of the first peptide chain of the Fc domain is connected to the N-terminus of the IL-2 domain via a peptide linker; and here the second anti-PD-1 V H The C-terminus of H sdAb is directly attached to the N-terminus of the second peptide chain of the Fc domain.

[0040] In yet another embodiment, provided herein are two IL-2 domains, two anti-PD-1 V H An anti-PD-1 / IL-2 fusion protein comprising H sdAb and an Fc domain; said anti-PD-1 V H H sdAb is an anti-PD-1 / IL-2 fusion protein containing CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3).

[0041] In one embodiment, the anti-PD-1 / IL-2 fusion protein provided herein comprises first and second IL-2 domains, first and second anti-PD-1 V H The first anti-PD-1 V comprises H sdAb and an Fc domain containing first and second peptide chains; where the first anti-PD-1 V HThe C-terminus of H sdAb is directly or via a peptide linker attached to the N-terminus of the first peptide chain of the Fc domain, and the C-terminus of the first peptide chain of the Fc domain is directly or via a peptide linker attached to the N-terminus of the first IL-2 domain; and here the second anti-PD-1 V H The C-terminus of H sdAb is directly or via a peptide linker connected to the N-terminus of the second peptide chain of the Fc domain, and the C-terminus of the second peptide chain of the Fc domain is directly or via a peptide linker connected to the N-terminus of the second IL-2 domain.

[0042] In another embodiment, the anti-PD-1 / IL-2 fusion protein provided herein comprises first and second IL-2 domains, first and second anti-PD-1 V H The first anti-PD-1 V comprises H sdAb and an Fc domain containing first and second peptide chains; where the first anti-PD-1 V H The C-terminus of H sdAb is directly ligated to the N-terminus of the first peptide chain of the Fc domain, and the C-terminus of the first peptide chain of the Fc domain is ligated to the N-terminus of the first IL-2 domain via a peptide linker; and here the second anti-PD-1 V H The C-terminus of H sdAb is directly connected to the N-terminus of the second peptide chain of the Fc domain, and the C-terminus of the second peptide chain of the Fc domain is connected to the N-terminus of the second IL-2 domain via a peptide linker.

[0043] In one embodiment, each of the anti-PD-1 V in the anti-PD-1 / IL-2 fusion proteins provided herein H H sdAb independently comprises the amino acid sequence of SEQ ID NO: 4 or 5. In another embodiment, each anti-PD-1 V in the anti-PD-1 / IL-2 fusion protein provided herein H H contains the amino acid sequence of SEQ ID NO: 4. In another embodiment, each anti-PD-1 V in the anti-PD-1 / IL-2 fusion protein provided hereinH H sdAb contains the amino acid sequence of SEQ ID NO: 5.

[0044] In one embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein is an hIgG1 Fc domain or its mutaine or a fragment thereof. In another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein is an hIgG1 Fc having the N297A amino acid substitution. In yet another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein is an hIgG2 Fc domain or its mutaine or a fragment thereof. In yet another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein is an hIgG4 Fc domain or its mutaine or a fragment thereof.

[0045] In one embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein comprises one of the amino acid sequences of SEQ ID NOs. 162 to 175. In another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein comprises the amino acid sequence of SEQ ID NOs. 162. In another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein comprises the amino acid sequence of SEQ ID NOs. 163. In yet another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein comprises the amino acid sequence of SEQ ID NOs. 164. In yet another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein comprises the amino acid sequence of SEQ ID NOs. 165. In yet another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein comprises the amino acid sequence of SEQ ID NOs. 166. In yet another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein comprises the amino acid sequence of SEQ ID NOs. 167. In another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein includes the amino acid sequence of SEQ ID NO: 168. In yet another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein includes the amino acid sequence of SEQ ID NO: 169. In yet another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein includes the amino acid sequence of SEQ ID NO: 170. In yet another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein includes the amino acid sequence of SEQ ID NO: 171. In yet another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein includes the amino acid sequence of SEQ ID NO: 172. In yet another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein includes the amino acid sequence of SEQ ID NO: 173. In yet another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein includes the amino acid sequence of SEQ ID NO: 174.In another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein comprises the amino acid sequence of SEQ ID NO: 175.

[0046] In one embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein comprises a pair of chains in a knob-in-hole structure. Thus, in one embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein comprises the amino acid sequences of SEQ ID NOs. 165 and 166, 167 and 168, 169 and 170, 173 and 174, or 174 and 175 as a pair of chains in a knob-in-hole structure. In another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein comprises the amino acid sequences of SEQ ID NOs. 165 and 166 as a pair of chains in a knob-in-hole structure. In yet another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein comprises the amino acid sequences of SEQ ID NOs. 167 and 168 as a pair of chains in a knob-in-hole structure. In another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein comprises the amino acid sequences of SEQ ID NOs. 169 and 170 as a pair of chains in a knob-in-hole structure. In yet another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein comprises the amino acid sequences of SEQ ID NOs. 173 and 174 as a pair of chains in a knob-in-hole structure. In yet another embodiment, the Fc domain in the anti-PD-1 / IL-2 fusion protein provided herein comprises the amino acid sequences of SEQ ID NOs. 174 and 175 as a pair of chains in a knob-in-hole structure.

[0047] In one embodiment, each IL-2 domain in the anti-PD-1 / IL-2 fusion protein provided herein is independently IL-2 or its mutaine.

[0048] In one embodiment, the IL-2 mutein described herein is, as shown in SEQ ID NOs: 46, 47, 48, 49, 50, or 51, (i) an amino acid substitution at position L18, Q126, or S130; and / or (ii) a substitution of an amino acid residue at positions N29-L40 with a peptide containing the amino acid sequence of the IL-15 hinge or a fragment thereof ("IL-15 hinge fragment-containing peptide"); a disulfide bond formed between two amino acid residues at positions N30-L80; and / or Includes amino acid substitutions at positions K8, K9, Q13, E15, H16, L19, D20, M23, K32, P34, K35, L36, T37, R38, M39, L40, T41, F42, K43, F44, Y45, M46, P47, E61, E62, L63, K64, P65, L66, E67, E68, V69, L70, N71, L72, A73, Q74, K76, H79, R81, D84, S87, N88, V91, I92, E95, Y107, D109, T111, or S127.

[0049] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; and / or (ii) a substitution of an amino acid residue at positions N29-L40 with an IL-15 hinge fragment-containing peptide; a disulfide bond formed between two amino acid residues at positions N30-L80; an amino acid substitution at positions E15, H16, D20, K32, K76, S87, N88, or I92; and / or an amino acid substitution at positions R38-Y45.

[0050] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions at positions L18, Q126, or S130; and (ii) substitutions between an amino acid residue at positions N29-L40 and an IL-15 hinge fragment-containing peptide; a disulfide bond formed between two amino acid residues at positions N30-L80; or amino acid substitutions at positions E15, H16, D20, K32, R38, L40, F42, K76, S87, N88, or I92.

[0051] In another embodiment, the IL-2 muteins described herein include (i) an amino acid substitution at position L18 or Q126; and (ii) a substitution of an amino acid residue at positions N29 to L40 with an IL-15 hinge fragment-containing peptide; or an amino acid substitution at position I92.

[0052] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions at positions L18 and Q126; and (ii) substitution of amino acid residues at positions N29-L40 with IL-15 hinge fragment-containing peptides.

[0053] In yet another embodiment, the IL-2 muteins described herein include (i) an amino acid substitution at position Q126; and (ii) a substitution of an amino acid residue at positions N29-L40 with an IL-15 hinge fragment-containing peptide; and an amino acid substitution at position I92.

[0054] In one embodiment, the amino acid residue at position E15 is one of the 20 natural amino acids other than E (i.e., Ala(A), Cys(C), Asp(D), Glu(E), Phe(F), Gly(G), His(H), Ile(I), Lys(K), Leu(L), Met(M), Asn(N), Pro(P), Gln(Q), Arg(R), Ser(S), Thr(T), Val(V), Trp(W), and Tyr(Y)). In one embodiment, the amino acid residue at position E15 is K.

[0055] In one embodiment, the amino acid residue at position H16 is one of 20 natural amino acids other than H. In one embodiment, the amino acid residue at position H16 is E, F, I, or V. In one embodiment, the amino acid residue at position H16 is E, I, or V. In one embodiment, the amino acid residue at position H16 is E. In one embodiment, the amino acid residue at position H16 is F. In one embodiment, the amino acid residue at position H16 is I. In one embodiment, the amino acid residue at position H16 is V.

[0056] In one embodiment, the amino acid residue at position L18 is one of the 20 natural amino acids other than L. In one embodiment, the amino acid residue at position L18 is C or S. In one embodiment, the amino acid residue at position L18 is C. In one embodiment, the amino acid residue at position L18 is C, and the amino acid residue at position C125 is C. In one embodiment, the amino acid residue at position L18 is S.

[0057] In one embodiment, the amino acid residue at position D20 is one of 20 natural amino acids other than D. In one embodiment, the amino acid residue at position D20 is A, E, K, or T. In one embodiment, the amino acid residue at position D20 is A. In one embodiment, the amino acid residue at position D20 is E. In one embodiment, the amino acid residue at position D20 is K. In one embodiment, the amino acid residue at position D20 is T.

[0058] In one embodiment, the amino acid residue at position K32 is one of the 20 natural amino acids other than K. In one embodiment, the amino acid residue at position K32 is D, E, or Q. In one embodiment, the amino acid residue at position K32 is D. In one embodiment, the amino acid residue at position K32 is E. In one embodiment, the amino acid residue at position K32 is Q.

[0059] In one embodiment, the amino acid residue at position R38 is one of the 20 natural amino acids other than R. In one embodiment, the amino acid residue at position R38 is E or N. In one embodiment, the amino acid residue at position R38 is E. In one embodiment, the amino acid residue at position R38 is N.

[0060] In one embodiment, the amino acid residue at position L40 is one of the 20 natural amino acids other than L. In one embodiment, the amino acid residue at position L40 is S or T. In one embodiment, the amino acid residue at position L40 is S. In one embodiment, the amino acid residue at position L40 is T.

[0061] In one embodiment, the amino acid residue at position F42 is one of the 20 natural amino acids other than F. In one embodiment, the amino acid residue at position F42 is A, C, K, or N. In one embodiment, the amino acid residue at position F42 is A or K. In one embodiment, the amino acid residue at position F42 is A. In one embodiment, the amino acid residue at position F42 is C. In one embodiment, the amino acid residue at position F42 is K. In one embodiment, the amino acid residue at position F42 is N.

[0062] In one embodiment, the amino acid residue at position K76 is one of the 20 natural amino acids other than K. In one embodiment, the amino acid residue at position K76 is D, E, or Q. In one embodiment, the amino acid residue at position K76 is D. In one embodiment, the amino acid residue at position K76 is E. In one embodiment, the amino acid residue at position K76 is Q.

[0063] In one embodiment, the amino acid residue at position S87 is one of 20 natural amino acids other than S. In one embodiment, the amino acid residue at position S87 is D or E. In one embodiment, the amino acid residue at position S87 is D. In one embodiment, the amino acid residue at position S87 is E.

[0064] In one embodiment, the amino acid residue at position N88 is one of 20 natural amino acids other than N. In another embodiment, the amino acid residue at position N88 is A.

[0065] In one embodiment, the amino acid residue at position I92 is one of the 20 natural amino acids other than I. In one embodiment, the amino acid residue at position I92 is A, D, E, or G. In one embodiment, the amino acid residue at position I92 is A. In one embodiment, the amino acid residue at position I92 is D. In one embodiment, the amino acid residue at position I92 is E. In one embodiment, the amino acid residue at position I92 is G.

[0066] In one embodiment, the amino acid residue at position Q126 is one of the 20 natural amino acids other than Q. In one embodiment, the amino acid residue at position Q126 is E, K, R, S, or T. In one embodiment, the amino acid residue at position Q126 is E, K, or R. In one embodiment, the amino acid residue at position Q126 is E. In one embodiment, the amino acid residue at position Q126 is K. In one embodiment, the amino acid residue at position Q126 is R. In one embodiment, the amino acid residue at position Q126 is S. In one embodiment, the amino acid residue at position Q126 is T.

[0067] In one embodiment, the amino acid residue at position S130 is one of the 20 natural amino acids other than S. In one embodiment, the amino acid residue at position S130 is A, E, Q, or R. In one embodiment, the amino acid residue at position S130 is A. In one embodiment, the amino acid residue at position S130 is E. In one embodiment, the amino acid residue at position S130 is Q. In one embodiment, the amino acid residue at position S130 is R.

[0068] In one embodiment, the IL-2 mutein described herein is (i) an amino acid substitution of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and / or (ii) a substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide; each independently at positions K35, R Disulfide bonds formed between two different amino acid residues at 38, F42, Y45, E62, V69, or L72; and / or K8D, K8E, K8Q, K9D, K9E, K9Q, Q13E, Q13N, E15K, E15Q, E15V, H16E, H16F, H16I, H16V, L19S, D20A, D20E, D20K, D20T, M23K, K32D, K32E, K32Q, P34N, K35N , L36S, L36T, T37N, R38E, R38N, M39N, L40S, L40T, T41N, F42A, F42C, F42K, F42N, K43N, F44N, Y45N, M46S, M46T, P4 7S, P47T, E61N, E62N, L63S, L63T, K64N, P65N, L66N, E67S, E67T, E68N, V69C, V69N, L70S, L70T, N71S, N71T, L72N, Includes amino acid substitutions of A73S, A73T, Q74S, Q74T, K76D, K76E, K76Q, H79D, H79E, H79Q, R81D, R81E, R81Q, D84T, S87D, S87E, N88A, V91I, I92A, I92D, I92E, I92G, I92L, E95K, E95N, E95Q, Y107N, D109N, T111S, S127A, S127E, S127F, or S127W.

[0069] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and / or (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide; each independently at positions K35, R38, Disulfide bonds formed between two different amino acid residues in F42, Y45, E62, V69, or L72; and / or K8D, K8E, K8Q, K9D, K9E, K9Q, Q13E, Q13N, E15K, E15Q, E15V, H16E, H16F, H16I, H16V, L19S, D20A, D20E, D20K, D20T, M23K, K32D, K32E, K32Q, P34N, K35N, L 36S, L36T, T37N, R38E, R38N, M39N, L40S, L40T, T41N, F42A, F42C, F42K, F42N, K43N, F44N, Y45N, M46S, M46T, P47 S, P47T, E61N, E62N, L63S, L63T, K64N, P65N, L66N, E67S, E67T, E68N, V69C, V69N, L70S, L70T, N71S, N71T, L72N, A Includes amino acid substitutions of 73S, A73T, Q74S, Q74T, K76D, K76E, K76Q, H79D, H79E, H79Q, R81D, R81E, R81Q, D84T, S87D, S87E, N88A, V91I, I92A, I92D, I92E, I92G, I92L, E95K, E95N, E95Q, Y107N, D109N, T111S, S127A, S127E, S127F, or S127W.

[0070] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide; Includes a disulfide bond formed between F42C and V69C; or an amino acid substitution of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, R38E, R38N, L40S, L40T, F42A, F42K, F42N, K76D, K76E, K76Q, S87D, S87E, N88A, I92A, I92D, I92E, or I92G.

[0071] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide; Includes a disulfide bond formed between F42C and V69C; or an amino acid substitution of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, R38E, R38N, L40S, L40T, F42A, F42K, F42N, K76D, K76E, K76Q, S87D, S87E, N88A, I92A, I92D, I92E, or I92G.

[0072] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, S130A, S130E, S130Q, or S130R; and (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide; Includes a disulfide bond formed between F42C and V69C; or an amino acid substitution of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, R38E, R38N, L40S, L40T, F42A, F42K, F42N, K76D, K76E, K76Q, S87D, S87E, N88A, I92A, I92D, I92E, or I92G.

[0073] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, S130A, S130E, S130Q, or S130R; and (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide; Includes a disulfide bond formed between F42C and V69C; or an amino acid substitution of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, R38E, R38N, L40S, L40T, F42A, F42K, F42N, K76D, K76E, K76Q, S87D, S87E, N88A, I92A, I92D, I92E, or I92G.

[0074] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, S130A, S130E, S130Q, or S130R; and (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide; Includes a disulfide bond formed between F42C and V69C; or an amino acid substitution of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, R38E, R38N, L40S, L40T, F42A, F42K, F42N, K76D, K76E, K76Q, S87D, S87E, N88A, I92A, I92D, I92E, or I92G.

[0075] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, or S130R; and (ii) substitutions of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide; a disulfide bond formed between F42C and V69C; or amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, R38E, R38N, L40S, L40T, F42A, F42K, F42N, K76D, K76E, K76Q, S87D, S87E, N88A, I92A, I92D, I92E, or I92G.

[0076] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitution of L18S or Q126E; and (ii) substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide; or amino acid substitution of I92A.

[0077] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18S and Q126E; and (ii) substitution of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide.

[0078] In yet another embodiment, the IL-2 muteins described herein include (i) an amino acid substitution of Q126E; and (ii) a substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide; and an amino acid substitution of I92A.

[0079] In one embodiment, the IL-15 hinge fragment-containing peptide comprises the amino acid sequence of SEQ ID NO: 85 or 86. In another embodiment, the IL-15 hinge fragment-containing peptide comprises the amino acid sequence of SEQ ID NO: 85. In yet another embodiment, the IL-15 hinge fragment-containing peptide comprises the amino acid sequence of SEQ ID NO: 86.

[0080] In one embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, or S130R; and (ii) substitutions of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; a disulfide bond formed between F42C and V69C; or amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, R38E, R38N, L40T, F42A, F42K, K76E, S87D, N88A, I92A, I92D, I92E, or I92G.

[0081] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, or S130R; and (ii) substitutions of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; a disulfide bond formed between F42C and V69C; or amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, R38E, R38N, L40T, F42A, F42K, K76E, S87D, N88A, I92A, I92D, I92E, or I92G.

[0082] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, or S130R; and (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85; a disulfide bond formed between F42C and V69C; or amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, R38E, R38N, L40T, F42A, F42K, K76E, S87D, N88A, I92A, I92D, I92E, or I92G.

[0083] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, or S130R; and (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85; a disulfide bond formed between F42C and V69C; or amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, R38E, R38N, L40T, F42A, F42K, K76E, S87D, N88A, I92A, I92D, I92E, or I92G.

[0084] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, or S130R; and (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 86; a disulfide bond formed between F42C and V69C; or amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, R38E, R38N, L40T, F42A, F42K, K76E, S87D, N88A, I92A, I92D, I92E, or I92G.

[0085] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, or S130R; and (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 86; a disulfide bond formed between F42C and V69C; or amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, R38E, R38N, L40T, F42A, F42K, K76E, S87D, N88A, I92A, I92D, I92E, or I92G.

[0086] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitution of L18S or Q126E; and (ii) substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85; or amino acid substitution of I92A.

[0087] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitution of L18S or Q126E; and (ii) substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 86; or amino acid substitution of I92A.

[0088] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18S and Q126E; and (ii) substitution of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85.

[0089] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18S and Q126E; and (ii) substitution of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 86.

[0090] In another embodiment, the IL-2 muteins described herein include (i) an amino acid substitution of Q126E; and (ii) a substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85; and an amino acid substitution of I92A.

[0091] In yet another embodiment, the IL-2 muteins described herein include (i) an amino acid substitution of Q126E; and (ii) a substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 86; and an amino acid substitution of I92A.

[0092] In one embodiment, the IL-2 muteins described herein are (i) amino acid substitutions at positions L18, Q126, or S130; and (ii) amino acid substitutions at positions K8, K9, Q13, E15, H16, L19, D20, M23, K32, P34, K35, L36, T37, R38, M39, L40, T41, F42, K43, Includes amino acid substitutions in F44, Y45, M46, P47, E61, E62, L63, K64, P65, L66, E67, E68, V69, L70, N71, L72, A73, Q74, K76, H79, R81, D84, S87, N88, V91, I92, E95, Y107, D109, T111, or S127.

[0093] In another embodiment, the IL-2 muteins described herein are (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) K8D, K8E, K8Q, K9D, K9E, K9Q, Q13E, Q13N, E15K , E15Q, E15V, H16E, H16F, H16I, H16V, L19S, D20A, D20E, D20K, D20T, M23K, K32D, K32E, K32Q, P34N, K35N, L36S, L36T, T37N, R38E, R38N, M39N, L40S, L40T, T41N, F42A, F42C, F42K, F42N, K 43N, F44N, Y45N, M46S, M46T, P47S, P47T, E61N, E62N, L63S, L63T, K64N, P65N, L66N, E67S, E6 7T, E68N, V69C, V69N, L70S, L70T, N71S, N71T, L72N, A73S, A73T, Q74S, Q74T, K76D, K76E, K76 Includes amino acid substitutions of Q, H79D, H79E, H79Q, R81D, R81E, R81Q, D84T, S87D, S87E, N88A, V91I, I92A, I92D, I92E, I92G, I92L, E95K, E95N, E95Q, Y107N, D109N, T111S, S127A, S127E, S127F, or S127W.

[0094] In another embodiment, the IL-2 muteins described herein are (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) K8D, K8E, K8Q, K9D, K9E, K9Q, Q13E, Q13N, E15K, E1 5Q, E15V, H16E, H16F, H16I, H16V, L19S, D20A, D20E, D20K, D20T, M23K, K32D, K32E, K32Q, P3 4N, K35N, L36S, L36T, T37N, R38E, R38N, M39N, L40S, L40T, T41N, F42A, F42C, F42K, F42N, K43 N, F44N, Y45N, M46S, M46T, P47S, P47T, E61N, E62N, L63S, L63T, K64N, P65N, L66N, E67S, E67 T, E68N, V69C, V69N, L70S, L70T, N71S, N71T, L72N, A73S, A73T, Q74S, Q74T, K76D, K76E, K76Q Includes amino acid substitutions of H79D, H79E, H79Q, R81D, R81E, R81Q, D84T, S87D, S87E, N88A, V91I, I92A, I92D, I92E, I92G, I92L, E95K, E95N, E95Q, Y107N, D109N, T111S, S127A, S127E, S127F, or S127W.

[0095] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions at positions L18, Q126, or S130; and (ii) amino acid substitutions at positions E15, H16, D20, K32, R38, L40, F42, K76, S87, N88, or I92.

[0096] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, R38E, R38N, L40S, L40T, F42A, F42K, F42N, K76D, K76E, K76Q, S87D, S87E, N88A, I92A, I92D, I92E, or I92G.

[0097] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, R38E, R38N, L40S, L40T, F42A, F42K, F42N, K76D, K76E, K76Q, S87D, S87E, N88A, I92A, I92D, I92E, or I92G.

[0098] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, R38E, R38N, L40T, F42A, F42K, K76E, S87D, N88A, I92A, I92D, I92E, or I92G.

[0099] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, R38E, R38N, L40T, F42A, F42K, K76E, S87D, N88A, I92A, I92D, I92E, or I92G.

[0100] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, or S130R; and (ii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, R38E, R38N, L40T, F42A, F42K, K76E, S87D, N88A, I92A, I92D, I92E, or I92G.

[0101] In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, or S130R; and (ii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, R38E, R38N, L40T, F42A, F42K, K76E, S87D, N88A, I92A, I92D, I92E, or I92G.

[0102] In one embodiment, the IL-2 mutein described herein includes an amino acid substitution at position L18. In another embodiment, the IL-2 mutein described herein includes an amino acid substitution at L18C or L18S. In yet another embodiment, the IL-2 mutein described herein includes an amino acid substitution at L18C. In yet another embodiment, the IL-2 mutein described herein includes an amino acid substitution at L18S.

[0103] In one embodiment, the IL-2 mutein described herein includes an amino acid substitution at position Q126. In another embodiment, the IL-2 mutein described herein includes an amino acid substitution of Q126E, Q126K, Q126R, Q126S, or Q126T. In yet another embodiment, the IL-2 mutein described herein includes an amino acid substitution of Q126E. In yet another embodiment, the IL-2 mutein described herein includes an amino acid substitution of Q126K. In yet another embodiment, the IL-2 mutein described herein includes an amino acid substitution of Q126R. In yet another embodiment, the IL-2 mutein described herein includes an amino acid substitution of Q126S. In yet another embodiment, the IL-2 mutein described herein includes an amino acid substitution of Q126T.

[0104] In one embodiment, the IL-2 mutein described herein includes an amino acid substitution at position S130. In another embodiment, the IL-2 mutein described herein includes an amino acid substitution at S130A, S130E, S130Q, or S130R. In yet another embodiment, the IL-2 mutein described herein includes an amino acid substitution at S130A. In yet another embodiment, the IL-2 mutein described herein includes an amino acid substitution at S130E. In yet another embodiment, the IL-2 mutein described herein includes an amino acid substitution at S130Q. In yet another embodiment, the IL-2 mutein described herein includes an amino acid substitution at S130R.

[0105] In one embodiment, the IL-2 mutein described herein includes amino acid substitutions at positions L18 and S126. In another embodiment, the IL-2 mutein described herein includes amino acid substitutions of L18C or L18S and amino acid substitutions of Q126E, Q126K, Q126R, Q126S, or Q126T. In yet another embodiment, the IL-2 mutein described herein includes amino acid substitutions of L18C and amino acid substitutions of Q126E, Q126K, Q126R, Q126S, or Q126T. In yet another embodiment, the IL-2 mutein described herein includes amino acid substitutions of L18C and Q126E. In yet another embodiment, the IL-2 mutein described herein includes amino acid substitutions of L18C and Q126K. In yet another embodiment, the IL-2 mutein described herein includes amino acid substitutions of L18C and Q126R. In yet another embodiment, the IL-2 mutein described herein includes the amino acid substitutions L18C and Q126S. In yet another embodiment, the IL-2 mutein described herein includes the amino acid substitutions L18C and Q126T.

[0106] In one embodiment, the IL-2 mutein described herein includes the amino acid substitution of L18S and the amino acid substitution of Q126E, Q126K, Q126R, Q126S, or Q126T. In another embodiment, the IL-2 mutein described herein includes the amino acid substitution of L18S and Q126E. In yet another embodiment, the IL-2 mutein described herein includes the amino acid substitution of L18S and Q126K. In yet another embodiment, the IL-2 mutein described herein includes the amino acid substitution of L18S and Q126R. In yet another embodiment, the IL-2 mutein described herein includes the amino acid substitution of L18S and Q126S. In yet another embodiment, the IL-2 mutein described herein includes the amino acid substitution of L18S and Q126T.

[0107] In one embodiment, the IL-2 mutein described herein includes amino acid substitutions at positions S126 and S130. In another embodiment, the IL-2 mutein described herein includes amino acid substitutions of Q126E, Q126K, Q126R, Q126S, or Q126T and amino acid substitutions of S130A, S130E, S130Q, or S130R. In yet another embodiment, the IL-2 mutein described herein includes amino acid substitutions of Q126E, Q126K, Q126R, Q126S, or Q126T and amino acid substitutions of S130E or S130R.

[0108] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution at position E15. In another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution of E15K. In yet another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution of E15K.

[0109] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution at position H16. In another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution of H16E, H16F, H16I, or H16V. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of H16E, H16F, H16I, or H16V. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of H16E, H16I, or H16V. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of H16E. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of H16F. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of H16I.In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of H16V.

[0110] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution at position D20. In another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution of D20A, D20E, D20K, or D20T. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of D20A, D20E, D20K, or D20T. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of D20A. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of D20E. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of D20K. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of D20T.

[0111] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution at position K32. In another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution of K32E. In yet another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution of K32E.

[0112] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution at position R38. In another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution of R38E or R38N. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of R38E or R38N. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of R38E. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of R38N.

[0113] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution at position L40. In another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution of L40S or L40T. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of L40S or L40T. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of L40S. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of L40T.

[0114] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution at position F42. In another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution of F42A, F42C, or F42K. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of F42A, F42C, or F42K. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of F42A or F42K. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of F42A. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of F42C. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of F42K.

[0115] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution at position K76. In another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution of K76E. In yet another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution of K76E.

[0116] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution at position S87. In another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution of S87D. In yet another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution of S87D.

[0117] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution at position N88. In another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution of N88A. In yet another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution of N88A.

[0118] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution at position I92. In another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution of I92A, I92D, I92E, or I92G. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of I92A, I92D, I92E, or I92G. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of I92A. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of I92D. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of I92E. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of I92G.

[0119] In one embodiment, the IL-2 mutein described herein includes amino acid substitutions at positions I92 and S126. In another embodiment, the IL-2 mutein described herein includes amino acid substitutions of I92A, I92D, I92E, or I92G and amino acid substitutions of Q126E, Q126K, Q126R, Q126S, or Q126T. In yet another embodiment, the IL-2 mutein described herein includes amino acid substitutions of I92A or I92G and amino acid substitutions of Q126E, Q126K, Q126R, Q126S, or Q126T. In yet another embodiment, the IL-2 mutein described herein includes amino acid substitutions of I92A and Q126E. In yet another embodiment, the IL-2 mutein described herein includes amino acid substitutions of I92A and Q126K. In yet another embodiment, the IL-2 mutein described herein includes the amino acid substitutions of I92A and Q126R. In yet another embodiment, the IL-2 mutein described herein includes the amino acid substitutions of I92A and Q126S. In yet another embodiment, the IL-2 mutein described herein includes the amino acid substitutions of I92A and Q126T.

[0120] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution at positions R38 and L40. In another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution at positions E15, R38, and L40. In yet another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution at positions R38, L40, and K76. In yet another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution at positions E15, R38, L40, and K76.

[0121] In one embodiment, the IL-2 muteins described herein include (i) amino acid substitutions at positions L18, Q126, or S130; and (ii) amino acid substitutions of R38N and L40S or L40T. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38N, and L40S or L40T. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38N, and L40S or L40T. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of R38N, L40S or L40T, and K76E. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38N, L40S or L40T, and K76E.

[0122] In one embodiment, the IL-2 muteins described herein include (i) amino acid substitutions at positions L18, Q126, or S130; and (ii) amino acid substitutions of R38N and L40S. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38N, and L40S. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38N, and L40S. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of R38N, L40S, and K76E. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38N, L40S, and K76E.

[0123] In one embodiment, the IL-2 muteins described herein include (i) amino acid substitutions at positions L18, Q126, or S130; and (ii) amino acid substitutions of R38N and L40T. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38N, and L40T. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38N, and L40T. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of R38N, L40T, and K76E. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38N, L40T, and K76E.

[0124] In one embodiment, the IL-2 mutein described herein is glycosylated. In another embodiment, the IL-2 mutein described herein is N-glycosylated. In yet another embodiment, the IL-2 mutein described herein is glycosylated with nitrogen in the side chain of an asparagine residue. In yet another embodiment, the IL-2 mutein described herein comprises (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution of N-glycosylated R38N.

[0125] In one embodiment, the IL-2 mutein described herein is an N-glycosylated IL-2 mutein comprising (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution at positions R38 and L40. In another embodiment, the IL-2 mutein described herein is an N-glycosylated IL-2 mutein comprising (i) an amino acid substitution at position L18, Q126, or S130; (ii) an amino acid substitution at positions E15, R38, and L40; and (iii) optionally an amino acid substitution at position H16, D20, K76, S87, N88, or I92. In yet another embodiment, the IL-2 mutein described herein is an N-glycosylated IL-2 mutein comprising (i) an amino acid substitution at position L18, Q126, or S130; (ii) an amino acid substitution at positions R38, L40, and K76; and (iii) optionally an amino acid substitution at position E15, H16, D20, S87, N88, or I92. In yet another embodiment, the IL-2 mutein described herein is an N-glycosylated IL-2 mutein comprising (i) an amino acid substitution at position L18, Q126, or S130; (ii) an amino acid substitution at positions E15, R38, L40, and K76; and (iii) optionally an amino acid substitution at position H16, D20, S87, N88, or I92.

[0126] In one embodiment, the IL-2 mutein described herein is an N-glycosylated IL-2 mutein comprising (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution of R38N and L40S or L40T. In another embodiment, the IL-2 mutein described herein is an N-glycosylated IL-2 mutein comprising (i) an amino acid substitution of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution of E15K, R38N, and L40S or L40T. In another embodiment, the IL-2 mutein described herein is an N-glycosylated IL-2 mutein comprising (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38N, and L40S or L40T. In another embodiment, the IL-2 mutein described herein is an N-glycosylated IL-2 mutein comprising (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of R38N, L40S or L40T, and K76E. In yet another embodiment, the IL-2 muteins described herein are N-glycosylated IL-2 muteins comprising (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38N, L40S or L40T, and K76E.

[0127] In one embodiment, the IL-2 mutein described herein is an N-glycosylated IL-2 mutein comprising (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution at R38N and L40S. In another embodiment, the IL-2 mutein described herein is an N-glycosylated IL-2 mutein comprising (i) an amino acid substitution at L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution at E15K, R38N, and L40S. In another embodiment, the IL-2 mutein described herein is an N-glycosylated IL-2 mutein comprising (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38N, and L40S. In another embodiment, the IL-2 mutein described herein is an N-glycosylated IL-2 mutein comprising (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of R38N, L40S, and K76E. In yet another embodiment, the IL-2 muteins described herein are N-glycosylated IL-2 muteins comprising (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38N, L40S, and K76E.

[0128] In one embodiment, the IL-2 mutein described herein is an N-glycosylated IL-2 mutein comprising (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution at R38N and L40T. In another embodiment, the IL-2 mutein described herein is an N-glycosylated IL-2 mutein comprising (i) an amino acid substitution at L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution at E15K, R38N, and L40T. In another embodiment, the IL-2 mutein described herein is an N-glycosylated IL-2 mutein comprising (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38N, and L40T. In another embodiment, the IL-2 mutein described herein is an N-glycosylated IL-2 mutein comprising (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of R38N, L40T, and K76E. In yet another embodiment, the IL-2 muteins described herein are N-glycosylated IL-2 muteins comprising (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38N, L40T, and K76E.

[0129] In one embodiment, the N-glycosylated IL-2 mutein provided herein comprises one glycan. In another embodiment, the N-glycosylated IL-2 mutein provided herein comprises one glycan bonded to the nitrogen in the side chain of an asparagine residue. In yet another embodiment, the N-glycosylated IL-2 mutein provided herein comprises one glycan bonded to the nitrogen in the side chain of an asparagine residue at position R38N.

[0130] In one embodiment, the N-glycosylated IL-2 mutein provided herein comprises two glycans. In another embodiment, the N-glycosylated IL-2 mutein provided herein comprises two glycans, each of which at least one glycan is bound to a nitrogen in the side chain of an asparagine residue. In yet another embodiment, the N-glycosylated IL-2 mutein provided herein comprises two glycans, each of which is bound to a nitrogen in the side chain of an asparagine residue.

[0131] In one embodiment, the N-glycosylated IL-2 mutein provided herein comprises three glycans. In one embodiment, the glycan is an N-glycan.

[0132] In one embodiment, the N-glycan on N-glycosylated IL-2 mutein provided herein is of the oligomannose type. In another embodiment, the N-glycan on N-glycosylated IL-2 mutein provided herein is of the complex type. In yet another embodiment, the N-glycan on N-glycosylated IL-2 mutein provided herein is of the hydride type.

[0133] In one embodiment, the N-glycan on N-glycosylated IL-2 mutein provided herein is a bibranched complex. In another embodiment, the N-glycan on N-glycosylated IL-2 mutein provided herein is a tertiary complex. In yet another embodiment, the N-glycan on N-glycosylated IL-2 mutein provided herein is a tetrabranched complex.

[0134] In one embodiment, the N-glycan on N-glycosylated IL-2 mutein provided herein is one of the glycans described in the literature of Szabo et al., J. Proteome. Res. 2018, 17, 1559-1574, the disclosure of which is fully incorporated herein by reference.

[0135] In one embodiment, the IL-2 mutein described herein includes (i) amino acid substitutions at positions L18, Q126, or S130; and (ii) amino acid substitutions at positions R38 and F42. In another embodiment, the IL-2 mutein described herein includes (i) amino acid substitutions at L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions at positions K32, R38, and F42. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions at positions K32, R38, and F42. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions at positions R38, F42, and K76. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions at positions E15, K32, R38, and F42. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions at positions E15, R38, F42, and K76.

[0136] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; and (ii) an amino acid substitution of R38E and F42A or F42K. In another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) an amino acid substitution of K32E, R38E, and F42A or F42K. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of K32E, R38E, and F42A or F42K. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of R38E, F42A or F42K, and K76E. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, K32E, R38E, and F42A or F42K. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38E, F42A or F42K, and K76E.

[0137] In one embodiment, the IL-2 muteins described herein include (i) amino acid substitutions at positions L18, Q126, or S130; and (ii) amino acid substitutions at positions K32, R38, and F42. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of K32E, R38E, and F42A or F42K. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of K32E, R38E, and F42A or F42K. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of K32E, R38E, and F42A. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of K32E, R38E, and F42K.

[0138] In one embodiment, the IL-2 muteins described herein include (i) amino acid substitutions at positions L18, Q126, or S130; and (ii) amino acid substitutions at positions R38, F42, and K76. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of R38E, F42A, or F42K, and K76E. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of R38E, F42A, or F42K, and K76E. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of R38E, F42A, and K76E. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of R38E, F42K, and K76E.

[0139] In one embodiment, the IL-2 muteins described herein include (i) amino acid substitutions at positions L18, Q126, or S130; and (ii) amino acid substitutions at positions E15, K32, R38, and F42. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, K32E, R38E, and F42A or F42K. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, K32E, R38E, and F42A or F42K. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, K32E, R38E, and F42A. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, K32E, R38E, and F42K.

[0140] In one embodiment, the IL-2 muteins described herein include (i) amino acid substitutions at positions L18, Q126, or S130; and (ii) amino acid substitutions at positions E15, R38, F42, and K76. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38E, F42A, or F42K, and K76E. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38E, F42A, or F42K, and K76E. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) amino acid substitutions of E15K, R38E, F42A, and K76E. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (b) amino acid substitutions of E15K, R38E, F42K, and K76E.

[0141] In one embodiment, the IL-2 mutein described herein comprises (i) an amino acid substitution at position L18, Q126, or S130; and (ii) a disulfide bond formed between two different amino acid residues at positions N30 to L80. In another embodiment, the IL-2 mutein described herein comprises (i) an amino acid substitution at position L18, Q126, or S130; and (ii) a disulfide bond formed between two different amino acid residues at positions K35 to L72. In yet another embodiment, the IL-2 mutein described herein comprises (i) an amino acid substitution at position L18, Q126, or S130; and (ii) a disulfide bond formed between two different amino acid residues at positions K35 to L69.

[0142] In one embodiment, the IL-2 mutein described herein comprises (i) an amino acid substitution at position L18, Q126, or S130; and (ii) a disulfide bond formed independently between two different amino acid residues at K35, R38, F42, Y45, E62, V69, or L72. In another embodiment, the IL-2 mutein described herein comprises (i) an amino acid substitution at position L18, Q126, or S130; and (ii) a disulfide bond formed between an amino acid residue at position K35, R38, F42, or Y45 and an amino acid residue at position E62, V69, or L72. In yet another embodiment, the IL-2 mutein described herein comprises (i) an amino acid substitution at position L18, Q126, or S130; and (ii) a disulfide bond formed between an amino acid residue at position F42 and an amino acid residue at position E62, V69, or L72. In yet another embodiment, the IL-2 mutein described herein comprises (i) an amino acid substitution at position L18, Q126, or S130; and (ii) a disulfide bond formed between an amino acid residue at position K35, R38, F42, or Y45 and an amino acid residue at position V69.

[0143] In one embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) disulfide bonds formed between K35C and L72C, R38C and L72C, F42C and V69C, Y42C and L72C, or Y45C and E62C. In another embodiment, the IL-2 mutein described herein includes (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) a disulfide bond formed between K35C and L72C, R38C and L72C, F42C and V69C, Y42C and L72C, or Y45C and E62C. In yet another embodiment, the IL-2 domain in the fusion protein includes (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) a disulfide bond formed between K35C and L72C. In another embodiment, the IL-2 domain in the fusion protein includes (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) a disulfide bond formed between R38C and L72C. In yet another embodiment, the IL-2 domain in the fusion protein includes (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) a disulfide bond formed between F42C and V69C. In another embodiment, the IL-2 domain in the fusion protein includes (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) a disulfide bond formed between Y42C and L72C.In yet another embodiment, the IL-2 domain in the fusion protein includes (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; and (ii) a disulfide bond formed between Y45C and E62C.

[0144] In one embodiment, the IL-2 mutein described herein is (i) an amino acid substitution at position L18, Q126, or S130; and (ii) a disulfide bond formed between two different amino acid residues at positions N30 to L80; and (iii) positions K8, K9, Q13, E15, H16, L19, D20, M23, K32, P34, K35, L36, T37, R Includes amino acid substitutions in 38, M39, L40, T41, F42, K43, F44, Y45, M46, P47, E61, E62, L63, K64, P65, L66, E67, E68, V69, L70, N71, L72, A73, Q74, K76, H79, R81, D84, S87, N88, V91, I92, E95, Y107, D109, T111, or S127.

[0145] In another embodiment, the IL-2 muteins described herein are (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) independently between two different amino acid residues at positions K35, R38, F42, Y45, E62, V69, or L72 (iii) disulfide bonds formed; and (iii) K8D, K8E, K8Q, K9D, K9E, K9Q, Q13E, Q13N, E15K, E15Q, E15V, H16E, H16F, H16I, H16V, L19S, D20A, D20E, D20K, D20T, M23K, K32D, K32E, K32Q, P34N, K35N, L36S, L36T, T37N, R38E, R38N , M39N, L40S, L40T, T41N, F42A, F42C, F42K, F42N, K43N, F44N, Y45N, M46S, M46T, P47S, P47T, E61N, E62 N, L63S, L63T, K64N, P65N, L66N, E67S, E67T, E68N, V69N, L70S, L70T, N71S, N71T, L72N, A73S, A73T, Q7 Includes amino acid substitutions of 4S, Q74T, K76D, K76E, K76Q, H79D, H79E, H79Q, R81D, R81E, R81Q, D84T, S87D, S87E, N88A, V91I, I92A, I92D, I92E, I92G, E95K, E95N, E95Q, Y107N, D109N, T111S, S127A, S127E, S127F, or S127W.

[0146] In another embodiment, the IL-2 muteins described herein are (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) each independently formed between two different amino acid residues at positions K35, R38, F42, Y45, E62, V69, or L72 disulfide bonds formed; and (iii) K8D, K8E, K8Q, K9D, K9E, K9Q, Q13E, Q13N, E15K, E15Q, E15V, H16E, H16F, H16I, H16V, L19S, D20A, D20E, D20K, D20T, M23K, K32D, K32E, K32Q, P34N, K35N, L36S, L36T, T37N, R38E, R38N, M39N, L40S, L40T, T41N, F42A, F42C, F42K, F42N, K43N, F44N, Y45N, M46S, M46T, P47S, P47T, E61N, E62 N, L63S, L63T, K64N, P65N, L66N, E67S, E67T, E68N, V69N, L70S, L70T, N71S, N71T, L72N, A73S, A73T, Q7 Includes amino acid substitutions of 4S, Q74T, K76D, K76E, K76Q, H79D, H79E, H79Q, R81D, R81E, R81Q, D84T, S87D, S87E, N88A, V91I, I92A, I92D, I92E, I92G, E95K, E95N, E95Q, Y107N, D109N, T111S, S127A, S127E, S127F, or S127W.

[0147] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) disulfide bonds formed between F42C and V69C; and (iii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, K76E, S87D, N88A, I92A, I92D, I92E, or I92G.

[0148] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) disulfide bonds formed between F42C and V69C; and (iii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, K32E, K76E, S87D, N88A, I92A, I92D, I92E, or I92G.

[0149] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) disulfide bonds formed between F42C and V69C; and (iii) amino acid substitutions of E15K, H16E, H16I, H16V, D20A, D20E, D20K, D20T, K32E, K76E, S87D, N88A, I92A, I92D, I92E, or I92G.

[0150] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) disulfide bonds formed between F42C and V69C; and (iii) amino acid substitutions of E15K, H16E, H16I, H16V, D20A, D20E, D20K, D20T, K32E, K76E, S87D, N88A, I92A, I92D, I92E, or I92G.

[0151] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) disulfide bonds formed between F42C and V69C; and (iii) amino acid substitutions of E15K, K32E, or K76E.

[0152] In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) disulfide bonds formed between F42C and V69C; and (iii) amino acid substitutions of E15K, K32E, or K76E.

[0153] In one embodiment, the IL-2 mutein described herein comprises (i) an amino acid substitution at position L18, Q126, or S130; and (ii) a disulfide bond formed between F42C and V69C. In another embodiment, the IL-2 mutein described herein comprises (i) an amino acid substitution at position L18, Q126, or S130; (ii) a disulfide bond formed between F42C and V69C; an amino acid substitution at position K32 or K76; and (iii) optionally, an amino acid substitution at position E15. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) a disulfide bond formed between F42C and V69C; and (iii) an amino acid substitution of K32E. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) a disulfide bond formed between F42C and V69C; and (iii) an amino acid substitution of K76E. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) disulfide bonds formed between F42C and V69C; and (iii) amino acid substitutions of E15K and K32E. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) disulfide bonds formed between F42C and V69C; and (iii) amino acid substitutions of E15K and K76E.

[0154] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; and (ii) a substitution of an amino acid residue at positions N29-L40 with an IL-15 hinge fragment-containing peptide. In another embodiment, the IL-2 mutein described herein includes a substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86. In yet another embodiment, the IL-2 mutein described herein includes a substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85. In yet another embodiment, the IL-2 mutein described herein includes a substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 86.

[0155] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; (ii) a substitution of an amino acid residue at positions N29-L40 with an IL-15 hinge fragment-containing peptide; and (iii) an amino acid substitution at position K8, K9, Q13, E15, H16, L19, D20, M23, E61, E62, L63, K64, P65, L66, E67, E68, V69, L70, N71, L72, A73, Q74, K76, H79, R81, D84, S87, N88, V91, I92, E95, Y107, D109, T111, or S127.

[0156] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitution of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with IL-15 hinge fragment-containing peptides; and (iii) K8D, K8E, K8Q, K9D, K9E, K9Q, Q13E, Q13N, E15K, E15Q, E15V, H16D, H16E, H16F, H16I, H16N, H16Q, H16V, L19S, D20A, D20E, D20K , D20T, M23K, E61N, E62N, L63S, L63T, K64N, P65N, L66N, E67S, E67T, E68N, V69N, L70 S, L70T, N71S, N71T, L72N, A73S, A73T, Q74S, Q74T, K76D, K76E, K76Q, H79D, H79E, H7 Includes amino acid substitutions of 9Q, R81D, R81E, R81Q, D84T, S87D, S87E, N88A, V91I, I92A, I92D, I92E, I92G, I92L, E95K, E95N, E95Q, Y107N, D109N, T111S, S127A, S127E, S127F, or S127W.

[0157] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitution of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide; and (iii) K8D, K8E, K8Q, K9D, K9E, K9Q, Q13E, Q13N, E15K, E15Q, E15V, H16D, H16E, H16F, H16I, H16N, H16Q, H16V, L19S, D20A, D20E, D20K, D20T, M23K, E61N, E62N, L63S, L63T, K64N, P65N, L66N, E67S, E67T, E68N, V69N, L70 S, L70T, N71S, N71T, L72N, A73S, A73T, Q74S, Q74T, K76D, K76E, K76Q, H79D, H79E, H7 Includes amino acid substitutions of 9Q, R81D, R81E, R81Q, D84T, S87D, S87E, N88A, V91I, I92A, I92D, I92E, I92G, I92L, E95K, E95N, E95Q, Y107N, D109N, T111S, S127A, S127E, S127F, or S127W.

[0158] In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with IL-15 hinge fragment-containing peptides; and (iii) K8D, K8E, K8Q, K9D, K9E, K9Q, Q13E, Q13N, E15K, E15Q, E15V, H16D, H16E, H16F, H16I, H16N, H16Q, H16V, L19S, D20A, D20E, D20K, D20T, M23K, E61N, E62N, L63S, L63T, K64N, P65N, L66N, E67S, E67T, E68N, V69N, L70 S, L70T, N71S, N71T, L72N, A73S, A73T, Q74S, Q74T, K76D, K76E, K76Q, H79D, H79E, H7 Includes amino acid substitutions of 9Q, R81D, R81E, R81Q, D84T, S87D, S87E, N88A, V91I, I92A, I92D, I92E, I92G, I92L, E95K, E95N, E95Q, Y107N, D109N, T111S, S127A, S127E, S127F, or S127W.

[0159] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; (ii) a substitution of an amino acid residue at positions N29 to L40 with an IL-15 hinge fragment-containing peptide; and (iii) an amino acid substitution at position E15, H16, D20, S87, N88, or I92.

[0160] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide; and (iii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, S87D, N88A, I92A, I92D, I92E, or I92G.

[0161] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide; and (iii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, S87D, N88A, I92A, I92D, I92E, or I92G.

[0162] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide; and (iii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, S87D, N88A, I92A, I92D, I92E, or I92G.

[0163] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, S87D, N88A, I92A, I92D, I92E, or I92G.

[0164] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, S87D, N88A, I92A, I92D, I92E, or I92G.

[0165] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, S87D, N88A, I92A, I92D, I92E, or I92G.

[0166] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85; and (iii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, S87D, N88A, I92A, I92D, I92E, or I92G.

[0167] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85; and (iii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, S87D, N88A, I92A, I92D, I92E, or I92G.

[0168] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85; and (iii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, S87D, N88A, I92A, I92D, I92E, or I92G.

[0169] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 86; and (iii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, S87D, N88A, I92A, I92D, I92E, or I92G.

[0170] In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 86; and (iii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, S87D, N88A, I92A, I92D, I92E, or I92G.

[0171] In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 86; and (iii) amino acid substitutions of E15K, H16E, H16F, H16I, H16V, D20A, D20E, D20K, D20T, S87D, N88A, I92A, I92D, I92E, or I92G.

[0172] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; (ii) a substitution of an amino acid residue at position N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) an amino acid substitution at E15. In another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) a substitution of an amino acid residue at position N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) an amino acid substitution at E15K. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitution of E15K.

[0173] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; (ii) a substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) an amino acid substitution at H16. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitutions of H16E, H16F, H16I, or H16V. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitutions of H16E, H16F, H16I, or H16V.

[0174] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; (ii) a substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) an amino acid substitution at D20. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitutions of D20A, D20E, D20K, or D20T. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitutions of D20A, D20E, D20K, or D20T.

[0175] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; (ii) a substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) an amino acid substitution at S87. In another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) a substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) an amino acid substitution at S87D. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitution of S87D.

[0176] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; (ii) a substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) an amino acid substitution at N88. In another embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) a substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) an amino acid substitution at N88A. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitution of N88A.

[0177] In one embodiment, the IL-2 mutein described herein includes (i) an amino acid substitution at position L18, Q126, or S130; (ii) a substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) an amino acid substitution at I92. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitutions of I92A, I92D, I92E, or I92G. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitutions of I92A, I92D, I92E, or I92G. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitutions of I92A, I92D, I92E, or I92G.In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitution of I92A. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitution of I92A. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitution of I92A. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitutions of I92D.In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitution of I92D. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitution of I92D. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitution of I92E. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitution of I92E.In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitution of I92E. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitutions of I92G. In another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18C, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitution of an amino acid residue at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitution of I92G. In yet another embodiment, the IL-2 muteins described herein include (i) amino acid substitutions of L18S, Q126E, Q126K, Q126R, Q126S, Q126T, S130A, S130E, S130Q, or S130R; (ii) substitutions of amino acid residues at positions N29-L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86; and (iii) amino acid substitutions of I92G.

[0178] In one embodiment, each IL-2 domain in the anti-PD-1 / IL-2 fusion protein provided herein independently comprises one amino acid sequence from SEQ ID NOs. 46-83 and 87-156. In another embodiment, each IL-2 domain in the anti-PD-1 / IL-2 fusion protein provided herein independently comprises one amino acid sequence from SEQ ID NOs. 46-51. In yet another embodiment, each IL-2 domain in the anti-PD-1 / IL-2 fusion protein provided herein independently comprises an IL-2 mutain comprising one amino acid sequence from SEQ ID NOs. 52-83 and 87-156. In yet another embodiment, each IL-2 domain in the anti-PD-1 / IL-2 fusion protein provided herein independently comprises an IL-2 mutain comprising one amino acid sequence from SEQ ID NOs. 52-83 and 87-155. In yet another embodiment, each IL-2 domain in the anti-PD-1 / IL-2 fusion protein provided herein is independently an IL-2 mutein containing any one amino acid sequence of SEQ ID NOs. 52 to 83. In yet another embodiment, each IL-2 domain in the anti-PD-1 / IL-2 fusion protein provided herein is independently an IL-2 mutein containing any one amino acid sequence of SEQ ID NOs. 87 to 155. In yet another embodiment, each IL-2 domain in the anti-PD-1 / IL-2 fusion protein provided herein is independently an IL-2 mutein containing the amino acid sequence of SEQ ID NO. 155.

[0179] In one embodiment, the IL-2 mutein described herein has a reduced binding affinity to IL-2Rα compared to wild-type IL-2. In one embodiment, the binding affinity of the IL-2 mutein described herein to IL-2Rα is its K d The reciprocal of that K a It is measured by [method].

[0180] In one embodiment, the IL-2 mutein described herein is a K of wild-type IL-2 against IL-2Rα. dK for IL-2Rα is approximately 2 times, 5 times, 10 times, 100 times, 200 times, or 1,000 times higher than [the other factor]. d It has. In one embodiment, the IL-2 mutein described herein is K of wild-type IL-2 against IL-2Rα d K for IL-2Rα is more than twice as high as d It has. In another embodiment, the IL-2 mutein described herein is K of wild-type IL-2 against IL-2Rα d K for IL-2Rα is approximately 5 times higher than d It has. In another embodiment, the IL-2 mutein described herein has K of wild-type IL-2 relative to IL-2Rα. d K for IL-2Rα is more than 10 times higher than d It has. In another embodiment, the IL-2 mutein described herein has K of wild-type IL-2 relative to IL-2Rα. d K for IL-2Rα is more than 100 times higher than d It has. In another embodiment, the IL-2 mutein described herein has K of wild-type IL-2 relative to IL-2Rα. d K for IL-2Rα is approximately 200 times higher than that. d It has. In another embodiment, the IL-2 mutein described herein has K of wild-type IL-2 relative to IL-2Rα. d K for IL-2Rα is approximately 500 times higher than that. d It has. In yet another embodiment, the IL-2 mutein described herein is K of wild-type IL-2 against IL-2Rα. d K for IL-2Rα is approximately 1,000 times higher than that. d It has.

[0181] In one embodiment, the IL-2 mutein described herein has a K content of approximately 20 nM or more, approximately 50 nM or more, approximately 100 nM or more, approximately 1 μM or more, approximately 10 μM or more, approximately 100 μM or more, approximately 200 μM or more, approximately 500 μM or more, or approximately 1 mM or more for IL-2Rα. dIt has. In one embodiment, the IL-2 mutain described herein has a K of about 20 nM or more relative to IL-2Rα. d It has. In one embodiment, the IL-2 mutain described herein has a K relative to IL-2Rα of about 50 nM or more. d It has. In one embodiment, the IL-2 mutain described herein has a K to IL-2Rα of about 100 nM or more. d It has. In one embodiment, the IL-2 mutein described herein has a K of about 1 μM or more relative to IL-2Rα. d It has. In one embodiment, the IL-2 mutein described herein has a K for IL-2Rα of about 10 μM or more. d It has. In one embodiment, the IL-2 mutein described herein has a K for IL-2Rα of about 100 μM or more. d It has. In one embodiment, the IL-2 mutain described herein has a K for IL-2Rα of about 200 μM or more. d It has. In one embodiment, the IL-2 mutein described herein has a K for IL-2Rα of about 500 μM or more. d It has. In one embodiment, the IL-2 mutain described herein has a K of about 1 mM or more relative to IL-2Rα. d It has. In one embodiment, the IL-2 mutein described herein does not have measurable binding to IL-2Rα. In one embodiment, the IL-2 mutein described herein does not have detectable binding to IL-2Rα when measured by surface plasmon resonance (SPR). In one embodiment, the IL-2 mutein described herein does not have detectable binding to IL-2Rα when measured by biolayer interferometry (BLI).

[0182] In one embodiment, the IL-2 mutein described herein has a reduced binding affinity to the IL-2Rβ / γ complex compared to wild-type IL-2. In one embodiment, the binding affinity of the IL-2 mutein described herein to the IL-2Rβ / γ complex is its Kd The reciprocal of that K a It is measured by [method].

[0183] In one embodiment, the IL-2 mutein described herein is the K of wild-type IL-2 to the IL-2Rβ / γ complex. d K for IL-2Rβ / γ complex is approximately 5 times, 10 times, 20 times, 50 times, or 100 times higher than that. d It has. In one embodiment, the IL-2 mutein described herein has the K of wild-type IL-2 to the IL-2Rβ / γ complex. d K for IL-2Rβ / γ complex is approximately 5 times higher than d It has. In another embodiment, the IL-2 mutein described herein has the K of wild-type IL-2 to the IL-2Rβ / γ complex. d K for IL-2Rβ / γ complex is approximately 10 times higher than d It has. In another embodiment, the IL-2 mutein described herein has the K of wild-type IL-2 to the IL-2Rβ / γ complex. d K for IL-2Rβ / γ complex is approximately 20 times higher than d It has. In another embodiment, the IL-2 mutein described herein has the K of wild-type IL-2 to the IL-2Rβ / γ complex. d K for IL-2Rβ / γ complex is approximately 50 times higher than d It has. In yet another embodiment, the IL-2 mutein described herein has the K of wild-type IL-2 to the IL-2Rβ / γ complex. d K for IL-2Rβ / γ complex is approximately 100 times higher than that. d It has.

[0184] In one embodiment, the IL-2 mutein described herein has a K2 concentration of approximately 5 nM or more, approximately 10 nM or more, approximately 20 nM or more, approximately 50 nM or more, approximately 100 nM or more, or approximately 200 nM or more for the IL-2Rβ / γ complex. dIt has. In one embodiment, the IL-2 mutein described herein has a K for the IL-2Rβ / γ complex of about 5 nM or more. d It has. In one embodiment, the IL-2 mutein described herein has a K for the IL-2Rβ / γ complex of about 10 nM or more. d It has. In one embodiment, the IL-2 mutein described herein has a K for the IL-2Rβ / γ complex of about 20 nM or more. d It has. In one embodiment, the IL-2 mutein described herein has a K for the IL-2Rβ / γ complex of about 50 nM or more. d It has. In one embodiment, the IL-2 mutein described herein has a K for the IL-2Rβ / γ complex of about 100 nM or more. d It has. In one embodiment, the IL-2 mutein described herein has a K for the IL-2Rβ / γ complex of about 200 nM or more. d It has.

[0185] In one embodiment, IL-2Rα is human IL-2Rα. In another embodiment, human IL-2Rα has the amino acid sequence of SEQ ID NO: 157.

[0186] In one embodiment, IL-2Rβ is human IL-2Rβ. In another embodiment, human IL-2Rβ has the amino acid sequence of SEQ ID NO: 158.

[0187] In one embodiment, IL-2Rγ is human IL-2Rγ. In another embodiment, human IL-2Rγ has the amino acid sequence of SEQ ID NO: 159.

[0188] In one embodiment, the K of IL-2 relative to IL-2Rα d This is determined by surface plasmon resonance (SPR) spectroscopy. In another embodiment, the K of IL-2 relative to IL-2Rα d This is determined by the BIACORE® assay. In another embodiment, the K of IL-2 relative to IL-2Rα dis determined by the Biolayer Interference (BLI) method. In yet another embodiment, the K of IL-2 for IL-2Rα d is determined by the OCTET® assay.

[0189] In one embodiment, the K of IL-2 for IL-2Rβ d is determined by the SPR method. In another embodiment, the K of IL-2 for IL-2Rβ d is determined by the BIACORE® assay. In yet another embodiment, the K of IL-2 for IL-2Rβ d is determined by BLI. In yet another embodiment, the K of IL-2 for IL-2Rβ d is determined by the OCTET® assay.

[0190] In one embodiment, the K of IL-2 for the IL-2Rβ / γ complex d is determined by the SPR method. In another embodiment, the K of IL-2 for the IL-2Rβ / γ complex d is determined by the BIACORE® assay. In yet another embodiment, the K of IL-2 for the IL-2Rβ / γ complex d is determined by BLI. In yet another embodiment, the K of IL-2 for the IL-2Rβ / γ complex d is determined by the OCTET® assay.

[0191] In one embodiment, the K of IL-2 for the IL-2Rα / β / γ complex d is determined by the SPR method. In another embodiment, the K of IL-2 for the IL-2Rα / β / γ complex d is determined by the BIACORE® assay. In yet another embodiment, the K of IL-2 for the IL-2Rα / β / γ complex d is determined by BLI. In yet another embodiment, the K of IL-2 for the IL-2Rα / β / γ complex d is determined by the OCTET® assay.

[0192] In one embodiment, the IL-2 mutein described herein contains an amino acid sequence that is approximately 80% or more identical to the amino acid sequence of SEQ ID NO: 46, 47, 48, 49, 50, or 51, approximately 85% or more, approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, or approximately 99% or more. In one embodiment, the IL-2 mutein described herein contains an amino acid sequence that is approximately 80% or more identical to the amino acid sequence of SEQ ID NO: 46, 47, 48, 49, 50, or 51. In one embodiment, the IL-2 mutein described herein contains an amino acid sequence that is approximately 85% or more identical to the amino acid sequence of SEQ ID NO: 46, 47, 48, 49, 50, or 51. In one embodiment, the IL-2 mutein described herein contains an amino acid sequence that is approximately 90% or more identical to the amino acid sequence of SEQ ID NO: 46, 47, 48, 49, 50, or 51. In one embodiment, the IL-2 mutein described herein contains an amino acid sequence that is approximately 91% or more identical to the amino acid sequence of SEQ ID NO: 46, 47, 48, 49, 50, or 51. In one embodiment, the IL-2 mutein described herein contains an amino acid sequence that is approximately 92% or more identical to the amino acid sequence of SEQ ID NO: 46, 47, 48, 49, 50, or 51. In one embodiment, the IL-2 mutein described herein contains an amino acid sequence that is approximately 93% or more identical to the amino acid sequence of SEQ ID NO: 46, 47, 48, 49, 50, or 51. In one embodiment, the IL-2 mutein described herein contains an amino acid sequence that is approximately 94% or more identical to the amino acid sequence of SEQ ID NO: 46, 47, 48, 49, 50, or 51. In one embodiment, the IL-2 mutein described herein contains an amino acid sequence that is approximately 95% or more identical to the amino acid sequence of SEQ ID NO: 46, 47, 48, 49, 50, or 51. In one embodiment, the IL-2 mutein described herein contains an amino acid sequence that is approximately 96% or more identical to the amino acid sequence of SEQ ID NO: 46, 47, 48, 49, 50, or 51. In one embodiment, the IL-2 mutein described herein contains an amino acid sequence that is approximately 97% or more identical to the amino acid sequence of SEQ ID NO: 46, 47, 48, 49, 50, or 51.In one embodiment, the IL-2 mutein described herein comprises an amino acid sequence that is approximately 98% or more identical to the amino acid sequence of SEQ ID NOs. 46, 47, 48, 49, 50, or 51. In one embodiment, the IL-2 mutein described herein comprises an amino acid sequence that is approximately 99% or more identical to the amino acid sequence of SEQ ID NOs. 46, 47, 48, 49, 50, or 51.

[0193] In one embodiment, the IL-2 domain in the anti-PD-1 / IL-2 fusion protein provided herein is the IL-2 mutain disclosed in CN 111018961 A, US 2018 / 0326010 A1, WO 2020 / 005819 A1, WO 2021 / 102063 A1, WO 2021 / 222150 A2, and WO 2022 / 212614 A1, the respective disclosures of which are fully incorporated herein by reference.

[0194] In one embodiment, the IL-2 mutain described herein further comprises one or more additional substitutions, deletions, and / or insertions; and / or one or more additional post-translational modifications.

[0195] In one embodiment, each peptide linker in the fusion protein provided herein independently contains GSG or one amino acid sequence from SEQ ID NOs. 6 to 45. In another embodiment, each peptide linker in the fusion protein provided herein independently contains GSG or the amino acid sequence of SEQ ID NOs. 6, 7, or 8. In yet another embodiment, each peptide linker in the fusion protein provided herein independently contains the amino acid sequence of SEQ ID NOs. 9, 10, 11, or 12. In yet another embodiment, each peptide linker in the fusion protein provided herein independently contains the amino acid sequence of SEQ ID NOs. 13, 14, 15, or 16. In yet another embodiment, each peptide linker in the fusion protein provided herein independently contains the amino acid sequence of SEQ ID NOs. 17, 18, 19, or 20. In yet another embodiment, each peptide linker in the fusion protein provided herein independently contains the amino acid sequence of SEQ ID NOs. 21, 22, 23, or 24. In another embodiment, each peptide linker in the fusion protein provided herein independently includes the amino acid sequence of SEQ ID NO: 25, 26, 27, or 28. In yet another embodiment, each peptide linker in the fusion protein provided herein independently includes the amino acid sequence of SEQ ID NO: 29, 30, 31, or 32. In yet another embodiment, each peptide linker in the fusion protein provided herein independently includes the amino acid sequence of SEQ ID NO: 33, 34, 35, or 36. In yet another embodiment, each peptide linker in the fusion protein provided herein independently includes the amino acid sequence of SEQ ID NO: 37. In yet another embodiment, each peptide linker in the fusion protein provided herein independently includes the amino acid sequence of SEQ ID NO: 38 or 39. In yet another embodiment, each peptide linker in the fusion protein provided herein independently includes the amino acid sequence of SEQ ID NO: 40 or 41. In yet another embodiment, each peptide linker in the fusion protein provided herein independently includes the amino acid sequence of SEQ ID NO: 42 or 43.In yet another embodiment, each peptide linker in the fusion protein provided herein independently comprises the amino acid sequence of SEQ ID NO: 44 or 45.

[0196] In one embodiment, the present invention provides an anti-PD-1 / IL-2 fusion protein comprising the amino acid sequence of SEQ ID NO: 160 and / or 161. In another embodiment, the present invention provides an anti-PD-1 / IL-2 fusion protein comprising the amino acid sequence of SEQ ID NO: 160. In yet another embodiment, the present invention provides an anti-PD-1 / IL-2 fusion protein comprising the amino acid sequence of SEQ ID NO: 161. In yet another embodiment, the present invention provides an anti-PD-1 / IL-2 fusion protein comprising the amino acid sequences of SEQ ID NO: 160 and 161.

[0197] (Pharmaceutical composition) In one embodiment, the foregoing provides a pharmaceutical composition comprising an anti-PD-1 / IL-2 fusion protein and a pharmaceutically acceptable excipient.

[0198] In one embodiment, the pharmaceutical composition is formulated as a single dosage form.

[0199] In one embodiment, the pharmaceutical composition provided herein is a solid formulation. In another embodiment, the pharmaceutical composition provided herein is a lyophilized solid formulation. In yet another embodiment, the pharmaceutical composition provided herein is a solution. In yet another embodiment, the pharmaceutical composition provided herein is an aqueous solution.

[0200] In one embodiment, the pharmaceutical composition provided herein is formulated in a dosage form for parenteral administration. In another embodiment, the pharmaceutical composition provided herein is formulated in a dosage form for intravenous administration. In yet another embodiment, the pharmaceutical composition provided herein is formulated in a dosage form for intramuscular administration. In yet another embodiment, the pharmaceutical composition provided herein is formulated in a dosage form for subcutaneous administration. In yet another embodiment, the pharmaceutical composition provided herein is formulated in a dosage form for intratumoral administration.

[0201] (How to use) In one embodiment, the foregoing provides a method for treating, preventing, or improving a proliferative disorder in a subject, comprising administering a therapeutically effective amount of the anti-PD-1 / IL-2 fusion protein provided herein to the subject in need thereof.

[0202] In one embodiment, the proliferative disorder is cancer. In another embodiment, the proliferative disorder is colon cancer or colorectal cancer.

[0203] In one embodiment, cancer is refractory and / or recurrent. In one embodiment, cancer is refractory. In one embodiment, cancer is recurrent. In one embodiment, cancer is metastatic. In one embodiment, cancer is resectable. In one embodiment, cancer is unresectable. In one embodiment, cancer is metastatic.

[0204] In one embodiment, cancer is drug-resistant. In one embodiment, cancer is multidrug-resistant. In one embodiment, cancer is resistant to chemotherapy. In one embodiment, cancer is resistant to immunotherapy. In one embodiment, cancer is resistant to standard cancer treatment.

[0205] In one embodiment, the subject is a mammal. In another embodiment, the subject is a human.

[0206] In another embodiment, a method for inhibiting cell growth is provided herein, comprising contacting the cells with an effective amount of the anti-PD-1 / IL-2 fusion protein provided herein. In one embodiment, the cells are cancer cells. In one embodiment, the cells are human cancer cells. In one embodiment, the cells are metastatic cancer cells.

[0207] In one embodiment, the therapeutically effective dose of the anti-PD-1 / IL-2 fusion protein provided herein is in the range of approximately 0.001 mg per kg of body weight per month (mg / kg per month) to 100 mg per kg of body weight per day (mg / kg per day), approximately 0.01 mg / kg per month to approximately 75 mg / kg per day, approximately 0.1 mg / kg per month to approximately 50 mg / kg per day, approximately 0.5 mg / kg per month to approximately 25 mg / kg per day, or approximately 1 mg / kg to approximately 20 mg / kg per day, which can be administered as a single or multiple dose. Within this range, the dosage can be approximately 0.005 mg / kg per month to approximately 0.05 mg / kg per day, approximately 0.05 mg / kg per month to approximately 0.5 mg / kg per day, approximately 0.5 mg / kg per month to approximately 5.0 mg / kg per day, approximately 1 mg / kg per month to approximately 15 mg / kg per day, approximately 1 mg / kg per month to approximately 20 mg / kg per day, or approximately 1 mg / kg per month to approximately 50 mg / kg per day.

[0208] This disclosure will be further understood by the following non-limiting embodiments. [Examples]

[0209] (Examples) (Example 1) (Cloning, expression, and purification of fusion proteins) The amino acid sequence of human IL-2 (hIL-2) was obtained from UNIPROT (hIL-2: P60568, 21-153aa). Mutant hIL-2 was generated by introducing mutations that attenuate CD25 and CD122 / CD132 binding. Mutant hIL-2, a human anti-PD-1 single-domain antibody, and the deoxyoligonucleotide (DNA) sequences encoding the Fc domain peptide chain were codon-optimized for CHO cell expression.

[0210] (i) hIL-2 mutain, (ii) human anti-PD-1 single-domain antibody, (iii) Fc domain peptide chain, and (iv) DNA sequence encoding the peptide linker were seamlessly assembled into a single molecule by homology assembly cloning using a commercially available kit. Oligonucleotides of each fusion protein were inserted into the UCOE® expression vector CET1019-AS-Puro for CHO expression.

[0211] Each fusion protein produced in CHO cells was purified using a two-step purification process: Protein A affinity chromatography using Protein A resin (e.g., AMSPHERE® A3 or MABSELECT® SURE®) and ion exchange chromatography (e.g., CAPTO® Q IMPRES or CAPTO® S IMPACT) or hydrophobic interaction chromatography (e.g., phenyl HP).

[0212] (Example 2) (Binding test of IL-2 mutein to CD122 and CD122 / CD132) The interactions between wild-type hIL-2 and hIL-2 mutein and CD25, CD122, or CD122 / CD132 heterodimers were characterized using OCTET® RED96. CD122 and AVITAG CD122 / CD132 heterodimers were purchased from ACROBIOSYSTEMS. Protein samples were diluted in OCTET® kinetic buffer. Briefly, the hIL-2 receptor was loaded into a biosensor. This biosensor was then immersed in a solution containing 10–2,000 nM wild-type hIL-2 and IL-2 mutein. Association and dissociation sensorgrams were fitted using global or local fitting with OCTET® data analysis HT software. The results are summarized in Table 1. All tested hIL-2 muteins were found to have lost or significantly reduced CD25 binding. Table 1. Interactions between wild-type hIL-2 and hIL-2 mutein with CD122 or CD122 / CD132 [Table 1]

[0213] (Example 3) (Antitumor activity of anti-PD-1 / IL-2 fusion protein in xenograft mouse models) MC38 cells are cultured and maintained in DMEM medium supplemented with 10% fetal bovine serum, GLUTAMAX®, non-essential amino acids (NEAAs), sodium pyruvate, and penicillin / streptomycin. Cells are trypsinized, washed in medium, and counted. Cells are diluted in PBS and 5 × 10⁶ cells are added to PBS (50 μL). 5The cells are subcutaneously injected into anesthetized C57BL / 6 mice using an 18-gauge needle. A stock solution of anti-PD-1 / IL-2 fusion protein is diluted in PBS on the day of administration, and the mice are intraperitoneally administered either PBS (control) or PBS containing anti-PD-1 / IL-2 fusion protein twice a week for two weeks. Tumor size (length (L) and width (W)) is measured twice a week using a digital caliper, and tumor volume (L×W×W) / 2 is calculated.

[0214] (Example 4.) (Antitumor activity of anti-PD-1 / IL-2 fusion protein in a xenograft model using PD-1 knock-in mice) MC38 cells or B16F10 cells are cultured and maintained in DMEM medium supplemented with 10% fetal bovine serum, GLUTAMAX®, non-essential amino acids (NEAAs), sodium pyruvate, and penicillin / streptomycin. Cells are trypsinized, washed with medium, and counted. Cells are diluted with PBS and 5 × 10⁶ cells are added to PBS (50 μL). 5 The cells are subcutaneously injected into anesthetized C57BL / 6 mice with human PD-1 knock-in using an 18-gauge needle. A stock solution of anti-PD-1 / IL-2 fusion protein is diluted in PBS on the day of administration, and the mice are intraperitoneally administered either PBS (control) or anti-PD-1 / IL-2 fusion protein in PBS twice a week for two weeks. Tumor size (length (L) and width (W)) is measured twice a week using a digital caliper, and tumor volume (L×W×W) / 2 is calculated.

[0215] (Example 5) (Antitumor activity of anti-PD-1 / IL-2 fusion protein in xenograft mouse models) CT26 mouse cells are cultured and maintained in RPMI medium supplemented with 10% fetal bovine serum, GLUTAMAX®, and penicillin / streptomycin. Cells are trypsinized, washed with medium, and counted. Cells are diluted with PBS and 1 × 10⁶ cells are added to PBS (100 μL). 6Individual cells are subcutaneously injected into anesthetized BALB / c mice using an 18-gauge needle. The stock solution of the anti-PD-1 / IL-2 fusion protein is diluted in PBS on the day of administration, and the mice are intraperitoneally administered PBS (control) or the anti-PD-1 / IL-2 fusion protein in PBS twice a week for 2 weeks. Tumor size (length (L) and width (W)) is measured twice a week using a digital caliper, and the tumor volume (L × W × W) / 2 is calculated.

[0216] (Example 6) (Antitumor activity of anti-PD-1 / IL-2 fusion protein in xenograft mouse model) HT-29 cells are cultured and maintained in McCoys 5a medium supplemented with 10% fetal bovine serum and penicillin / streptomycin. The cells are trypsinized, washed with medium, counted, and washed with PBS. The cell suspension (1×10 6 cells in PBS (100 μL)) is subcutaneously injected into anesthetized NCG mice using a 27-gauge needle. Six days later, human PBMC (1×10 7 cells in PBS (100 μL)) are injected into the tail vein of each mouse. The stock solution of the anti-PD-1 / IL-2 fusion protein is diluted in PBS on the day of administration, and the mice are intraperitoneally administered PBS (control) or the anti-PD-1 / IL-2 fusion protein in PBS twice a week for 2 weeks. Tumor size (length (L) and width (W)) is measured twice a week using a digital caliper, and the tumor volume (L × W × W) / 2 is calculated.

[0217] (Example 7) (Effect of anti-PD-1 / IL-2 fusion protein on STAT5 signaling) This study evaluates the effect of anti-PD-1 / IL-2 fusion proteins on pSTAT5 signaling via their IL-2 domain in CD3 T cells. Briefly, 100,000 CD3 T cells are treated with anti-PD-1 / IL-2 fusion proteins in a Hanks equilibrium salt solution containing 10 mM HEPES at 37°C and 5% CO2 for 30 minutes. Phosphorylated STAT5 is measured using a phosph-STAT5 (Tyr694) HTRF assay. The signal ratio at 665 nm / 620 nm is multiplied by 1,000, and the data is analyzed using global fitting to determine the EC (Emission Control Value). 50 Determine the value.

[0218] (Example 8) (Binding study of anti-PD-1 / IL-2 fusion protein to human PD-1 and IL-2 receptors) Anti-PD-1 / IL-2 fusion protein A1 was generated by co-expression of SEQ ID NOs. 160 and 161 in CHO cells. The interaction between anti-PD-1 / IL-2 fusion protein A1 and human PD-1 or IL-2 receptors was investigated using OCTET® RED96. For human PD-1 binding, anti-PD-1 / IL-2 fusion protein A1 was captured using an anti-human Fc(AHC) biosensor. The coated biosensor was associated with His-tagged human PD-1 protein at predetermined concentrations and serial dilutions. For human IL-2 receptor binding, human IL-2Rα or human IL-2Rβγ(His) protein was captured using an anti-penta-HIS(HIS1K) biosensor. The coated biosensor was associated with anti-PD-1 / IL-2 fusion protein A1 at predetermined concentrations and serial dilutions.

[0219] His-tagged IL-2Rα (CD25) protein, His-tagged and Twin Strep-tagged human IL-2Rβ and IL-2Rγ heterodimer proteins, His-tagged human PD-1, and recombinant human IL-2 (rhIL-2) protein were purchased from ACROBiosystems. The association and dissociation sensorgrams were fitted using global or local fitting with OCTET® data analysis HT software. The results are summarized in Table 2. Table 2. Interactions between fusion proteins A1 and rhIL-2 with human IL-2 receptor and human PD-1. [Table 2]

[0220] The sequences described herein are provided in the sequence listing below. (Sequence Listing) [Table 3] TIFF2026509975000004.tif224170TIFF2026509975000005.tif230170TIFF2026509975000006.tif229170TIFF2026509975000007.t if229170TIFF2026509975000008.tif229170TIFF2026509975000009.tif230170TIFF2026509975000010.tif229170TIFF20265099750 00011.tif230170TIFF2026509975000012.tif230170TIFF2026509975000013.tif229170TIFF2026509975000014.tif230170TIFF202 6509975000015.tif230170TIFF2026509975000016.tif228170TIFF2026509975000017.tif230170TIFF2026509975000018.tif97170* * * * *

[0221] The above embodiments are provided to give a complete disclosure and explanation to those skilled in the art of the methods for producing and using the claimed embodiments, and are not intended to limit the scope of what is disclosed herein. Modifications that are obvious to those skilled in the art are intended to be within the scope of the following claims. All publications, patents, and patent applications referenced herein are incorporated herein by reference as if each such publication, patent, or patent application were specifically and individually incorporated herein by reference.

Claims

1. A fusion protein comprising an IL-2 domain, an anti-PD-1 single-domain antibody (sdAb), and a crystalline fragment (Fc) domain; wherein the anti-PD-1 sdAb comprises CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO:

3.

2. A fusion protein according to claim 1, comprising an IL-2 domain, first and second anti-PD-1 sdAbs, and an Fc domain comprising first and second peptide chains; wherein the C-terminus of the first anti-PD-1 sdAb is directly or via a peptide linker to the N-terminus of the first peptide chain of the Fc domain, and the C-terminus of the first peptide chain of the Fc domain is directly or via a peptide linker to the N-terminus of the IL-2 domain; and the C-terminus of the second anti-PD-1 sdAb is directly or via a peptide linker to the N-terminus of the second peptide chain of the Fc domain.

3. A fusion protein according to claim 1, comprising first and second IL-2 domains, first and second anti-PD-1 sdAbs, and an Fc domain comprising first and second peptide chains; wherein the C-terminus of the first anti-PD-1 sdAb is directly or via a peptide linker to the N-terminus of the first peptide chain of the Fc domain, and the C-terminus of the first peptide chain of the Fc domain is directly or via a peptide linker to the N-terminus of the first IL-2 domain; and the C-terminus of the second anti-PD-1 sdAb is directly or via a peptide linker to the N-terminus of the second peptide chain of the Fc domain, and the C-terminus of the second peptide chain of the Fc domain is directly or via a peptide linker to the N-terminus of the second IL-2 domain.

4. The aforementioned anti-PD-1 sdAb is anti-PD-1 V H A fusion protein according to any one of claims 1 to 3, wherein the fusion protein is H sdAb.

5. The fusion protein according to any one of claims 1 to 4, wherein each anti-PD-1 sdAb independently comprises the amino acid sequence of SEQ ID NO: 4 or 5.

6. A fusion protein according to any one of claims 1 to 5, wherein each anti-PD-1 sdAb contains the amino acid sequence of SEQ ID NO:

4.

7. A fusion protein according to any one of claims 1 to 5, wherein each anti-PD-1 sdAb contains the amino acid sequence of SEQ ID NO:

5.

8. The fusion protein according to any one of claims 1 to 7, wherein each anti-PD-1 sdAb is independently human or humanized anti-PD-1 sdAb.

9. The fusion protein according to any one of claims 1 to 8, wherein the Fc domain is an hIgG1 Fc domain, an hIgG2 Fc domain, an hIgG4 Fc domain, or a mutain thereof.

10. The fusion protein according to any one of claims 1 to 9, wherein the Fc domain is an hIgG4 Fc domain or its mutain.

11. The fusion protein according to any one of claims 1 to 10, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO: 173, 174, or 175.

12. The fusion protein according to any one of claims 1 to 11, wherein the Fc domain comprises the amino acid sequence of SEQ ID NOs. 173 and 174 or 174 and 175.

13. The fusion protein according to any one of claims 1 to 12, wherein the Fc domain has a knob-in-hole structure.

14. The fusion protein according to any one of claims 1 to 13, wherein each IL-2 domain is an IL-2 mutain comprising: (i) an amino acid substitution at position L18, Q126, or S130; or (ii) a substitution between an amino acid residue at positions N29 to L40 and an IL-15 hinge fragment-containing peptide; a disulfide bond formed between two amino acid residues at positions N30 to L80; or an amino acid substitution at position E15, H16, D20, K32, R38, M39, L40, T41, F42, K43, F44, Y45, K76, S87, N88, or I92.

15. The fusion protein according to any one of claims 1 to 14, wherein each IL-2 domain is independently an IL-2 mutein containing an amino acid substitution at position L18.

16. The fusion protein according to any one of claims 1 to 15, wherein each IL-2 domain is independently an IL-2 mutein containing the L18C or L18S amino acid substitution.

17. The fusion protein according to any one of claims 1 to 16, wherein each IL-2 domain is independently an IL-2 mutein containing the L18S amino acid substitution.

18. The fusion protein according to any one of claims 1 to 17, wherein each IL-2 domain is independently an IL-2 mutein containing an amino acid substitution at position Q126.

19. The fusion protein according to any one of claims 1 to 18, wherein each IL-2 domain is independently an IL-2 mutein containing the amino acid substitutions Q126E, Q126K, Q126R, Q126S, or Q126T.

20. The fusion protein according to any one of claims 1 to 19, wherein each IL-2 domain is independently an IL-2 mutein containing the Q126E amino acid substitution.

21. The fusion protein according to any one of claims 1 to 20, wherein each IL-2 domain is independently an IL-2 mutein containing an amino acid substitution at position S130.

22. The fusion protein according to any one of claims 1 to 21, wherein each IL-2 domain is independently an IL-2 mutein containing the amino acid substitutions S130A, S130E, S130Q, or S130R.

23. The fusion protein according to any one of claims 1 to 22, wherein each IL-2 domain is independently an IL-2 mutein comprising (i) Q126E or Q126T; (ii) L18C and Q126E, Q126K, Q126R, or Q126S; (iii) L18S and Q126E; (iv) Q126T and S130R; or (v) L18C, Q126E, and S130E.

24. The fusion protein according to any one of claims 1 to 23, wherein each IL-2 domain is independently an IL-2 mutein containing the amino acid substitutions L18S and Q126E.

25. The fusion protein according to any one of claims 1 to 24, wherein each IL-2 domain is an IL-2 mutein comprising the substitution of an amino acid residue at positions N29 to L40 with an IL-15 hinge fragment-containing peptide.

26. The fusion protein according to any one of claims 1 to 25, wherein each IL-2 domain is an IL-2 mutein comprising the substitution of an amino acid residue at positions N29 to L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86.

27. The fusion protein according to any one of claims 1 to 26, wherein each IL-2 domain is an IL-2 mutein comprising (i) amino acid substitutions of L18S and Q126E and (ii) substitution of an amino acid residue at positions N29 to L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO: 85 or 86.

28. The fusion protein according to any one of claims 1 to 27, wherein each IL-2 domain is an IL-2 mutein comprising (i) amino acid substitutions of L18S and Q126E and (ii) substitutions of amino acid residues at positions N29 to L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO:

85.

29. The fusion protein according to any one of claims 1 to 27, wherein each IL-2 domain is an IL-2 mutein comprising (i) amino acid substitutions of L18S and Q126E and (ii) substitutions of amino acid residues at positions N29 to L40 having the amino acid sequence of SEQ ID NO: 84 with an IL-15 hinge fragment-containing peptide having the amino acid sequence of SEQ ID NO:

86.

30. The fusion protein according to any one of claims 1 to 13, wherein each IL-2 domain independently comprises one amino acid sequence from among SEQ ID NOs: 46-83 and 87-156.

31. The fusion protein according to any one of claims 1 to 13 and 30, wherein each IL-2 domain is independently an IL-2 mutein containing one amino acid sequence from sequence numbers 52 to 83 and 87 to 155.

32. The fusion protein according to any one of claims 1 to 13, 30, and 31, wherein the IL-2 domain comprises one amino acid sequence from any one of sequence numbers 87 to 155.

33. The fusion protein according to any one of claims 1 to 12 and 30 to 32, wherein the IL-2 domain comprises the amino acid sequence of SEQ ID NO:

155.

34. The fusion protein according to any one of claims 1 to 33, wherein each peptide linker independently comprises one amino acid sequence of GSG or SEQ ID NOs. 6 to 45.

35. A fusion protein according to any one of claims 1 to 34, wherein each peptide linker independently comprises the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16.

36. A fusion protein according to any one of claims 1 to 35, wherein each peptide linker contains the amino acid sequence of SEQ ID NO:

15.

37. The fusion protein according to claim 1, wherein the fusion protein comprises the amino acid sequences of SEQ ID NOs. 160 and 161.

38. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 37 and a pharmaceutically acceptable excipient.

39. The pharmaceutical composition according to claim 38, wherein the pharmaceutical composition is a single dosage form.

40. The pharmaceutical composition according to claim 38 or 39, wherein the pharmaceutical composition is a solid.

41. The pharmaceutical composition according to claim 38 or 39, wherein the pharmaceutical composition is in parenteral dosage form.

42. The pharmaceutical composition according to claim 41, wherein the pharmaceutical composition is in the form of an intravenous dosage.

43. The pharmaceutical composition according to any one of claims 38, 39, 41, and 42, wherein the pharmaceutical composition is a solution.

44. A method for treating, preventing, or improving one or more symptoms of a proliferative disorder in a subject, comprising administering a therapeutically effective amount of a fusion protein according to any one of claims 1 to 37 or a pharmaceutical composition according to any one of claims 38 to 43 to a subject in need thereof.

45. The method according to claim 44, wherein the proliferative disease is cancer.

46. The method according to claim 44 or 45, wherein the proliferative disease is metastatic cancer.

47. A method for inhibiting cell growth, comprising contacting the cells with an effective amount of the fusion protein described in any one of claims 1 to 37 or the pharmaceutical composition described in any one of claims 38 to 43.

48. The method according to claim 47, wherein the cells are cancer cells.

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