IL2 mutant protein and application thereof

Abstract

Provided are an IL2 mutant protein (or "IL2 mutant"), an isolated polynucleotide encoding the IL2 mutant protein, an isolated vector containing the polynucleotide, a host cell containing the isolated polynucleotide or the isolated vector encoding the IL2 mutant protein, a fusion protein containing the IL2 mutant protein, a pharmaceutical composition containing the IL2 mutant protein, and a pharmaceutical composition containing the IL2 mutant protein. And the use of such molecules in IL2 immunotherapy.

Description

[0001] Cross-references

[0002] This application claims priority to PCT application No. PCT / CN2022 / 104035 filed on July 6, 2022. The contents of the prior application are considered part of this disclosure and are incorporated herein in their entirety.

[0003] Sequence Listing

[0004] The Sequence Listing entitled DCF230724WO-Seqlisting.xml, created on July 5, 2023 and having a size of 24,064 bytes, is hereby incorporated by reference in its entirety. Technical Field

[0005] The present disclosure relates to IL2 mutant proteins (or "IL2 mutants"), isolated vectors containing polynucleotides encoding IL2 mutant proteins, host cells containing isolated polynucleotides or isolated vectors encoding IL2 mutant proteins, fusion proteins containing IL2 mutant proteins, pharmaceutical compositions containing IL2 mutant proteins, and uses thereof. Background Art

[0006] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0007] Interleukin 2 (IL2), a member of the interleukin 2 cytokine subfamily, is a secreted cytokine produced by activated CD4+ and CD8+ T lymphocytes that is important for the proliferation of T and B lymphocytes. IL2 is also known as T cell growth factor (TCGF). Human IL2 (UniProt: P60568) consists of a 20 amino acid N-terminal signal peptide and a 133 amino acid polypeptide chain in the mature protein. IL2 plays a key function during immune homeostasis through its effects on regulatory T (Treg) cells and the optimization and fine-tuning of effector lymphocyte responses.

[0008] IL2 signals through a heterotrimeric receptor complex composed of different α, β, and γ chains (also known as CD25, CD122, and CD132, respectively). It is generally believed that IL2Rα (CD25) is not directly involved in information transduction, but once IL2Rα binds to IL2, it can increase the overall affinity of cytokines for medium-affinity receptors (IL2Rβγ heterodimers) from the low nanomolar range to the picomolar range (high-affinity receptors, IL2Rαβγ heterotrimers) (Tang et al., Cytokine:X1 [Cytokine:X1] (2019) 100001). T cells expressing IL2Rα include regulatory T cells (Treg cells), which are essential for curbing autoimmune inflammation. IL2Rβ and IL2Rγ are expressed on both cytotoxic effector T cells and Treg cells.

[0009] FDA approved IL2 for metastatic renal cell carcinoma (1992) and metastatic melanoma (1998) (Tang et al., Cytokine:X1 (2019) 100001). However, IL2 immunotherapy has not been widely adopted due to toxic adverse reactions when given at high doses (as required for anti-tumor immunotherapy) and its ability to stimulate both cytotoxic effector T cells and Treg cells. In anti-cancer IL2 immunotherapy, activation of Treg cells is an undesirable effect because Treg cells may weaken effector T cell responses against tumor antigens (Natalia et al., Trends Immunol. 2015 December; 36(12): 763-777).

[0010] Therefore, there is still a need for IL2 mutant proteins with reduced toxicity in IL2 immunotherapy. Summary of the invention

[0011] To achieve the above purpose, novel IL2 mutant proteins that reduce, disrupt or eliminate binding to IL2Rα are provided herein. In some embodiments, the IL2 mutant protein comprises an amino acid sequence comprising amino acid substitutions C125V or C125S as compared to the wild-type IL2 of SEQ ID NO: 1.

[0012] In some embodiments, the IL2 mutant protein has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO: 1, 2 or 3.

[0013] In some embodiments, the IL2 mutant protein comprises one or more amino acid substitutions selected from the group consisting of: K8, I28, R38, F42, K43, K49, E61, E62, E68, and L72 as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3.

[0014] In some embodiments, the IL2 mutant protein comprises one or more amino acid substitutions selected from the group consisting of K8R, I28T, R38D / R38A / R38Q / R38T / R38E / R38N, F42E / F42R / F42A / F42H / F42Q, K43E / K43G / K43S / K43A / K43R / K43P / K43Q, K49E, E61R, E62T / E62G, K64M, E68G / E68V / E68S / E68K / E68A, and L72S / L72K as compared to the amino acid sequence shown in SEQ ID NO:2 or 3.

[0015] In some embodiments, the IL2 mutant protein comprises one or more amino acid substitutions selected from the group consisting of I28T, R38D / R38A / R38Q / R38T / R38E / R38N, K43E / K43G / K43S / K43A / K43R / K43P / K43 Q, E61R and E68G / E68V / E68S / E68K / E68A as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3; or

[0016] R38D / R38A / R38Q / R38T / R38E / R38N, F42E / F42R / F42A / F42H / F42Q and K43E / K43G / K43S / K43A / K43R / K43P / K43Q; or

[0017] R38D / R38A / R38Q / R38T / R38E / R38N, F42E / F42R / F42A / F42H / F42Q, K43E / K43G / K43S / K43A / K43R / K43P / K43Q, K49E; or

[0018] R38D / R38A / R38Q / R38T / R38E / R38N, F42E / F42R / F42A / F42H / F42Q, K43E / K43G / K43S / K43A / K43R / K43P / K43Q, and E68G / E68V / E68S / E68K / E68A; or

[0019] F42E / F42R / F42A / F42H / F42Q and K43E / K43G / K43S / K43A / K43R / K43P / K43Q; or

[0020] or

[0021] R38D / R38A / R38Q / R38T / R38E / R38N, E62T / E62G, E68G / E68V / E68S / E68K / E68A, and L72S / L72K; or

[0022] R38D / R38A / R38Q / R38T / R38E / R38N, F42E / F42R / F42A / F42H / F42Q, K49E, K64M, K43E / K43G / K43S / K43A / K43R / K43P / K43Q and E68G / E68V / E68S / E68K / E68A.

[0023] In some embodiments, the IL2 mutant protein comprises an amino acid substitution as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3.

[0024] R38D, K43E / K43G and E61R;

[0025] R38A, F42E and K43G;

[0026] R38E, F42Q and K43Q;

[0027] R38A, F42E, K43G, E62T, E68G and L72S / L72K;

[0028] R38A, F42E, K43G, and E68G;

[0029] R38Q / R38T, F42R / F42A, K43S / K43A, and E68G / E68V;

[0030] R38A / R38T / R38E, E62T, E68S and L72K;

[0031] E62T / E62G, E68G and L72S;

[0032] R38E / R38N, K43R and I28T;

[0033] R38E / R38N, K43R, I28T and E68K;

[0034] F42H / F42Q and K43Q;

[0035] R38E, F42H / F42Q, K43Q and K49E;

[0036] R38D / R38E, F42Q, K43G / K43Q and K64M;

[0037] R38D / R38E, F42Q, K43G / K43Q, and K49E; or

[0038] R38A / R38T, F42E / F42A, K43P, E62G, E68A and K8R.

[0039] In some embodiments, the IL2 mutant protein comprises an amino acid substitution as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3.

[0040] R38D, K43E and E61R;

[0041] R38A, F42E and K43G;

[0042] R38E, F42Q and K43Q;

[0043] R38A, F42E, K43G, E62T, E68G and L72S;

[0044] R38A, F42E, K43G and E68G;

[0045] R38Q, F42R, K43S and E68V;

[0046] R38T, F42A, K43A and E68G;

[0047] R38E, E62T, E68S and L72K;

[0048] E62T, E68G and L72S;

[0049] I28T, R38E and K43R;

[0050] I28T, R38N, K43R and E68K;

[0051] F42H and K43Q;

[0052] R38E, F42Q, K43Q and K49E;

[0053] R38D, F42Q, K43G, and K64M; or

[0054] R38T, F42E, K43P, E62G, E68A and K8R.

[0055] In some embodiments, the IL2 mutein comprises amino acid substitutions N88D, V91D / V91A / V91E and / or I92T as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3.

[0056] In some embodiments, the IL2 mutant protein comprises an amino acid substitution as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3.

[0057] R38E, F42Q, K43Q, K49E and N88D;

[0058] R38E, F42Q, K43Q, K49E and V91D / V91A / V91E;

[0059] R38E, F42Q, K43Q, K49E and I92T;

[0060] R38E, F42Q, K43Q, K49E, N88D, and V91D / V91A / V91E;

[0061] R38E, F42Q, K43Q, K49E, V91D / V91A / V91E and I92T;

[0062] R38E, F42Q, K43Q, K49E, N88D, V91D / V91A / V91E and I92T;

[0063] I28T, R38E, K43R and N88D;

[0064] I28T, R38E, K43R and V91D / V91A / V91E;

[0065] I28T, R38E, K43R and I92T;

[0066] I28T, R38E, K43R, N88D and V91D / V91A / V91E;

[0067] I28T, R38E, K43R, V91D / V91A / V91E, and I92T; or

[0068] I28T, R38E, K43R, N88D, V91D / V91A / V91E and I92T.

[0069] In some embodiments, the IL2 mutant protein has at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in any one of SEQ ID NOs: 7-23.

[0070] In some embodiments, the IL2 mutant protein differs from the amino acid sequence shown in any one of SEQ ID NOs: 7-23 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid.

[0071] In some embodiments, the IL2 mutant protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 7-23.

[0072] In some embodiments, the IL2 mutein is capable of reduced affinity binding to the IL2Rβγ heterodimer.

[0073] In some embodiments, the IL2 mutein is capable of reducing affinity binding to the IL2Rαβγ heterotrimer by reducing or eliminating binding to IL2Rα activated by the IL2Rαβγ heterotrimer.

[0074] In some embodiments, the IL2 mutein is capable of reducing the immune response of regulatory T cells.

[0075] In some embodiments, the IL2 mutant protein binds to the IL2Rβγ heterodimer with a KD (affinity constant) greater than 0.1E-11M, greater than 9.9E-11M, or greater than 1E-10M.

[0076] In some embodiments, the IL2 mutant protein binds to IL2Rα with a KD (affinity constant) greater than 1.0E-12M.

[0077] In some embodiments, the IL2 mutein has improved thermal stability, wherein the Tm value is not less than 50°C, not less than 55°C, not less than 60°C, or not less than 65°C.

[0078] In one aspect, the disclosure also provides isolated polynucleotides encoding an IL2 mutant protein as described herein.

[0079] In one aspect, the present disclosure also provides an isolated vector comprising a polynucleotide as described herein.

[0080] In one aspect, the disclosure also provides a host cell comprising an isolated polynucleotide as described herein or an isolated vector as described herein. In some embodiments, the host cell comprises a HEK293 cell.

[0081] In one aspect, the disclosure also provides a fusion protein comprising an IL2 mutant protein as described herein. In some embodiments, the IL2 mutant protein is fused to an Fc region, optionally via a linker.

[0082] In one aspect, the present disclosure also provides a pharmaceutical composition comprising an IL2 mutant protein as described herein, or an isolated polynucleotide as described herein, or an isolated vector as described herein, or a host cell as described herein, or a fusion protein as described herein, and a pharmaceutically acceptable carrier.

[0083] In one aspect, the present disclosure also provides use of an IL2 mutant protein as described herein, or an isolated polynucleotide as described herein, or an isolated vector as described herein, or a host cell as described herein, or a fusion protein as described herein, or a pharmaceutical composition as described herein in the manufacture of a therapeutic agent for diagnosing, preventing or treating a disease, disorder or condition.

[0084] In one aspect, the present disclosure also provides a method for diagnosing, preventing or treating a disease, disorder or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an IL2 mutant protein as described herein, or an isolated polynucleotide as described herein, or an isolated vector as described herein, or a host cell as described herein, or a fusion protein as described herein, or a pharmaceutical composition as described herein.

[0085] In one aspect, the present disclosure also provides a method of enhancing an immune response to a disease, disorder or condition associated with IL2 in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an IL2 mutant protein as described herein, or an isolated polynucleotide as described herein, or an isolated vector as described herein, or a host cell as described herein, or a fusion protein as described herein, or a pharmaceutical composition as described herein.

[0086] In some embodiments, the disease, disorder or condition comprises a tumor.

[0087] In one aspect, the present disclosure also provides a method for reducing adverse reactions associated with IL2 in anti-tumor immunotherapy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the above-mentioned IL2 mutant protein, isolated polynucleotide, isolated vector, host cell, fusion protein or pharmaceutical composition.

[0088] In some embodiments, the subject is a mammal, including a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] The following is a brief description of the drawings, which are presented for the purpose of illustrating exemplary embodiments disclosed herein and not for the purpose of limiting the same.

[0090] Figure 1 The tetrameric structure of IL2-IL2Rα-IL2Rβ-γc (PDB ID: 2B5I) is shown.

[0091] Figure 2 The position of the C125 residue in the IL2 protein (PDB ID: 2B5I) is shown. The C125 residue is located in the hydrophobic core of the 4-helix bundle.

[0092] Figure 3 Purity analysis of IL2 mutants with C125 residue substitution is shown. Figure 3 A shows SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis of purified IL2-WT, IL2-C125S and IL2-C125V under reducing (reducing; R) and non-reducing (non-reducing; NR) conditions. IL2-WT refers to wild-type human IL2 protein, IL2-C125S refers to IL2-WT with C125S substitution, and IL2-C125V refers to IL2-WT with C125V substitution. Figure 3 B shows SEC-HPLC (size exclusion chromatography-high performance liquid chromatography; SEC-HPLC) analysis of purified IL2-WT, IL2-C125S and IL2-C125V.

[0093] Figure 4 The detection of IL2Rα binding affinity of IL2-WT and IL2 mutant with C125V substitution by octet rule is shown, and nm at the ordinate refers to the unit of binding size.

[0094] Figure 5 The detection of IL2Rβγ binding affinity of IL2-WT and IL2 mutant with C125V substitution by the octet rule is shown, and nm at the ordinate refers to the unit of binding size.

[0095] Figure 6 Detection of IL2Rα binding by the octet rule for the following IL2 variants is shown: ESE01.004, ESE01.006, ESE01.008, ESE01.010 ( Figure 6A), and ESE01.016, ESE01.017, ESE01.018, ESE01.019, ESE01.020, ESE01.042 and IL2-C125V ( Figure 6 B).

[0096] Figure 7 Detection of IL2Rβγ binding by the octet rule for the following IL2 variants is shown: ESE01.004, ESE01.006, ESE01.008, ESE01.010, ESE01.016, ESE01.017 ( Figure 7 A), and ESE01.018, ESE01.019, ESE01.020, ESE01.042 and IL2-C125V ( Figure 7 B).

[0097] Figure 8 The affinity detection of IL2Rβγ binding of IL2 variants is shown. The detection of IL2Rβγ binding of the following IL2 variants by the octet rule is shown in Figure 8 In A: ESE01.027, ESE01.028, ESE01.029, ESE01.030, ESE01.031, ESE01.032, ESE01.033 and ESE01.034, and the detection of IL2Rβγ binding to the following IL2 variants is shown Figure 8 In B: ESE01.035, ESE01.036, ESE01.037, ESE01.038, ESE01.039 and ESE01.040. BLI affinity assays for IL2Rβγ binding of the following IL2 variants are shown in Figure 8 In C: ESE01.027, ESE01.028, ESE01.032, ESE01.033, and ESE01.034 and ESE01.035, and BLI affinity assays for IL2Rβγ binding of the following IL2 variants are shown Figure 8 In D: ESE01.036, ESE01.037, ESE01.038, ESE01.039 and ESE01.040.

[0098] Fig. 9The binding ability of IL2 variants (ESE01.027, ESE01.028, ESE01.029, ESE01.030, ESE01.031, ESE01.032, ESE01.033, ESE01.034, ESE01.035, ESE01.036, ESE01.037, ESE01.038, ESE01.039, ESE01.040 and ESE01.042), ESE01.019, IL2-WT, IL2-C125V and blank control (unstained control) to IL2Rβγ by FACS is shown. DETAILED DESCRIPTION

[0099] The present disclosure will be explained in more detail below. This specification is not intended to be a list of all the different ways in which the present invention can be implemented or all the features that can be added to the present invention. For example, features described with respect to one embodiment can be incorporated into other embodiments, and features described with respect to a particular embodiment can be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art, without departing from the present invention, by considering the present disclosure. Therefore, the following description is intended to illustrate some specific embodiments of the present invention, rather than to exhaustively specify all permutations, combinations, and variations thereof.

[0100] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to the methods and materials described herein can be used in the testing practice of the present disclosure, preferred materials and methods are described herein. In describing and claiming the present disclosure, the following terms will be used.

[0101] The present disclosure provides examples of IL2 (interleukin 2) mutant proteins that reduce, disrupt or eliminate binding to IL2Rα (interleukin 2 receptor subunit α). The complete amino acid sequence of human IL2 (Protein Database No.: 2B5I) is shown in SEQ ID NO:1.

[0102] IL2 mutant protein (recombinant human IL2) is an effective anti-tumor immunotherapy, however, the short half-life and severe toxicity limit the optimal dose of IL2. Figure 1As shown in , once IL2Rα binds to IL2, IL2 can bind to the IL2Rαβγ heterotrimer (high affinity receptor) with greater affinity, which preferentially amplifies immunosuppressive regulatory T cells (Treg cells) expressing high constitutive levels of IL-2Rα. Regulatory T cells are central to immune homeostasis and play an important role in maintaining peripheral immune tolerance by attenuating (autoreactive) effector T cells. The expansion of Tregs represents an undesirable effect of IL2 on cancer immunotherapy.

[0103] IL2 mutants with improved and selective immunostimulatory capacity could promote the expansion and activity of effector T cells while minimizing Treg cells and reducing anti-inflammatory effects.

[0104] definition

[0105] The terms "mutated", "mutation", "mutant" and "mutant" are used interchangeably herein. Typically and preferably, the mutated amino acid or mutation is an exchange of one amino acid by one or more amino acids, an insertion, a deletion, or a combination thereof. Most preferably, the mutated amino acid or mutation is an exchange of a single amino acid by a different single amino acid.

[0106] As used herein, "IL2 mutant protein" or "IL2 variant" means a mutant protein derived from the sequence shown in SEQ ID NO: 1, one or more amino acid mutations, wherein the mutated amino acid or mutation is an exchange of one amino acid by one or more amino acids, insertion, deletion, or a combination thereof. Most preferably, the mutated amino acid or mutation is an exchange of a single amino acid by a different single amino acid. The IL2 mutant protein comprises at least any one of the sequences shown in SEQ ID NO: 1-3 and has one or more amino acids exchanged by different single amino acids.

[0107] As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length of at least two nucleotides, and includes, but is not limited to, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0108] As used herein, the term "vector" refers to a polynucleotide that can be engineered to contain one or more clones that can be propagated in a host cell. A vector may include one or more of the following components: an origin of replication, one or more regulatory sequences (e.g., promoters and / or enhancers) that regulate the expression of a polypeptide of interest, and / or one or more selectable marker genes (e.g., antibiotic resistance genes and genes that can be used for colorimetric assays, such as β-galactosidase).

[0109] As used herein, the term "host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. The host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells such as primate or non-primate cells.

[0110] As used herein, the term "fusion protein" refers to a protein consisting of at least two domains encoded by separate genes, which have been linked so that they are transcribed and translated as a single unit, thereby producing a single polypeptide. In some embodiments, the fusion protein can be produced in vivo, for example, as a result of a chromosomal rearrangement. In some embodiments, the fusion protein refers to an Fc fusion protein. In some embodiments, the fusion protein is constructed by connecting two protein domains with a peptide linker.

[0111] As used herein, the term "Fc region" includes natural and mutant protein forms of polypeptides derived from the Fc region of an antibody. Also included are truncated forms of such polypeptides containing hinge regions that promote dimerization. In some embodiments, the Fc region contains antibody CH2 and CH3 domains. While extending the serum half-life, fusion proteins containing Fc portions (and oligomers formed therefrom) provide the advantage of being easily purified by affinity chromatography on protein A or protein G columns. Preferred Fc regions are derived from human IgG, including IgG1, IgG2, IgG3, and IgG4. One of the functions of the Fc portion of an antibody is to communicate with the immune system when the antibody binds to its target.

[0112] As used herein, the term "subject" refers to humans and non-human animals. Non-human animals include all vertebrates such as mice, rats, rabbits, cats, dogs, pigs, monkeys, chimpanzees, gorillas, etc. Unless otherwise noted, the terms "patient" or "subject" are used interchangeably herein.

[0113] IL2 refers to interleukin 2. The terms "IL2" and "IL-2" are used interchangeably herein.

[0114] IL2Rα stands for interleukin 2 receptor subunit α, and most preferably, stands for human interleukin 2 receptor subunit α. The terms "IL2Rα", "IL-2Rα" and "CD25" are used interchangeably herein.

[0115] IL2Rβ stands for interleukin 2 receptor subunit β, and most preferably, stands for human interleukin 2 receptor subunit β. The terms "IL2Rβ", "IL-2Rβ" and "CD122" are used interchangeably herein.

[0116] IL2Rγ stands for interleukin 2 receptor subunit γ. Most preferably, it stands for human interleukin 2 receptor subunit γ or IL2RG. The terms "IL2Rγ", "IL-2Rγ", "IL2RG", "IL-2RG" and "CD132" are used interchangeably herein.

[0117] IL2Rβγ refers to a heterodimer of IL2Rβ and IL2Rγ. In some embodiments, IL2Rβγ is a medium affinity receptor for IL2. The terms "IL2Rβγ" and "IL-2Rβγ" are used interchangeably herein.

[0118] IL2Rαβγ refers to a heterotrimer of IL2Rα, IL2Rβ and IL2Rγ. In some embodiments, IL2Rαβγ is a high affinity receptor for IL2. The terms "IL2Rαβγ" and "IL-2Rαβγ" are used interchangeably herein.

[0119] IL2 mutant protein

[0120] Provided herein are novel IL2 muteins that reduce, disrupt or eliminate binding to IL2Rα. Further provided herein are IL2 muteins having a C125 residue substitution.

[0121] like Figure 2 As shown in , the C125 residue is located in the hydrophobic core of the 4-helix bundle. In order to reduce the IL2Rα binding affinity of the IL2 mutant protein, in some embodiments, the IL2 mutant protein comprises an amino acid sequence having an amino acid substitution C125V or C125S as compared to wild-type IL2.

[0122] In some embodiments, the wild-type IL2 is human IL2 of SEQ ID NO:1.

[0123] In some preferred embodiments, as compared to the wild-type IL2 of SEQ ID NO: 1, the IL2 mutant protein comprises an amino acid sequence comprising an amino acid substitution C125S.

[0124] In some embodiments, the IL2 mutein comprises the amino acid sequence shown in SEQ ID NO:2, and has one or more amino acid substitutions.

[0125] In some preferred embodiments, as compared to the wild-type IL2 of SEQ ID NO: 1, the IL2 mutant protein comprises an amino acid sequence comprising an amino acid substitution C125V.

[0126] Furthermore, in some embodiments, the IL2 mutant protein contains the amino acid sequence shown in SEQ ID NO: 3, and has one or more amino acid substitutions.

[0127] In some embodiments, the IL2 mutant protein has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO: 1, 2 or 3.

[0128] In some embodiments, the IL2 mutant protein contains one or more amino acid substitutions selected from the group consisting of: K8, I28, R38, F42, K43, K49, E61, E62, E68, and L72 as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3.

[0129] In some embodiments, the IL2 mutein is mutated at position K8, I28, R38, F42, K43, K49, E61, E62, K64, E68 and / or L72 compared to wild-type IL2.

[0130] In some preferred embodiments, as compared to wild-type IL2, the IL2 mutant protein further contains the following substitutions: K8R, I28T, R38D, R38A, R38Q, R38T, R38E, R38N, F42E, F42R, F42A, F42H, F42Q, K43E, K43G, K43S, K43A, K43R, K43P, K43Q, K49E, E61R, E62T, E62G, K64M, E68G, E68V, E68S, E68K, E68A, L72S, or L72K, or a combination thereof.

[0131] In the present disclosure, a substitution (e.g., K8R) means that the amino acid "K" is substituted with the amino acid "R" at position 8. Unless otherwise defined, the abbreviations of amino acids used herein have the same meanings as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Amino acids in the present disclosure are represented by standard single-letter codes according to standard IUPAC (International Union of Pure and Applied Chemistry) amino acid abbreviations.

[0132] The term "position" refers to the amino acid residue number counted from the N-terminus of the mutant protein, polypeptide or protein. When there is a signal peptide, the amino acid residue numbering can start from the first amino acid after the signal peptide, or from the first amino acid of the mature protein. Taking human IL2 as an example, human IL2 consists of a 20-amino acid N-terminal signal peptide and a 133-amino acid polypeptide chain (mature protein). K8R substitution refers to the substitution of amino acid "K" by amino acid "R" at position 8 (i.e., the 28th amino acid of human IL2 (SEQ ID NO: 1)).

[0133] In some embodiments, "positions" are defined by Kabat numbering. Kabat numbering is well known to those skilled in the art, see, for example, Kabat, EA et al. Sequences of Proteins of Immunological Interest, 1991.

[0134] In some embodiments, the amino acid may be substituted with an amino acid, such as R38D / R38A / R38Q / R38T / R38E / R38N, which means amino acid substitutions R38D, R38A, R38Q, R38T, R38E or R38N.

[0135] In some embodiments, the IL2 mutant protein contains one or more amino acid substitutions selected from the group consisting of K8R, I28T, R38D / R38A / R38Q / R38T / R38E / R38N, F42E / F42R / F42A / F42H / F42Q, K43E / K43G / K43S / K43A / K43R / K43P / K43Q, K49E, E61R, E62T / E62G, K64M, E68G / E68V / E68S / E68K / E68A, and L72S / L72K that reduce, disrupt or eliminate binding to IL2α as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3.

[0136] Furthermore, in some embodiments, the IL2 mutant protein contains one or more amino acid substitutions selected from the group consisting of I28T, R38D / R38A / R38Q / R38T / R38E / R38N, K43E / K43G / K43S / K43A / K43R / K43P / K43Q, E61R and E68G / E68V / E68S / E68K / E68A as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3; or

[0137] R38D / R38A / R38Q / R38T / R38E / R38N, F42E / F42R / F42A / F42H / F42Q and K43E / K43G / K43S / K43A / K43R / K43P / K43Q; or

[0138] R38D / R38A / R38Q / R38T / R38E / R38N, F42E / F42R / F42A / F42H / F42Q, K43E / K43G / K43S / K43A / K43R / K43P / K43Q, K49E; or

[0139] R38D / R38A / R38Q / R38T / R38E / R38N, F42E / F42R / F42A / F42H / F42Q, K43E / K43G / K43S / K43A / K43R / K43P / K43Q, and E68G / E68V / E68S / E68K / E68A; or

[0140] F42E / F42R / F42A / F42H / F42Q and K43E / K43G / K43S / K43A / K43R / K43P / K43Q; or

[0141] or

[0142] R38D / R38A / R38Q / R38T / R38E / R38N, E62T / E62G, E68G / E68V / E68S / E68K / E68A, and L72S / L72K; or

[0143] R38D / R38A / R38Q / R38T / R38E / R38N, F42E / F42R / F42A / F42H / F42Q, K49E, K64M, K43E / K43G / K43S / K43A / K43R / K43P / K43Q and E68G / E68V / E68S / E68K / E68A.

[0144] In some embodiments, the IL2 mutant protein contains an amino acid substitution as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3.

[0145] R38D, K43E / K43G and E61R;

[0146] R38A, F42E and K43G;

[0147] R38E, F42Q and K43Q;

[0148] R38A, F42E, K43G, E62T, E68G and L72S / L72K;

[0149] R38A, F42E, K43G, and E68G;

[0150] R38Q / R38T, F42R / F42A, K43S / K43A, and E68G / E68V;

[0151] R38A / R38T / R38E, E62T, E68S and L72K;

[0152] E62T / E62G, E68G and L72S;

[0153] R38E / R38N, K43R and I28T;

[0154] R38E / R38N, K43R, I28T and E68K;

[0155] F42H / F42Q and K43Q;

[0156] R38E, F42H / F42Q, K43Q and K49E;

[0157] R38D / R38E, F42Q, K43G / K43Q and K64M;

[0158] R38D / R38E, F42Q, K43G / K43Q, and K49E; or

[0159] R38A / R38T, F42E / F42A, K43P, E62G, E68A and K8R.

[0160] In some embodiments, the IL2 mutant protein contains an amino acid substitution as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3.

[0161] R38D, K43E and E61R;

[0162] R38A, F42E and K43G;

[0163] R38E, F42Q and K43Q;

[0164] R38A, F42E, K43G, E62T, E68G and L72S;

[0165] R38A, F42E, K43G and E68G;

[0166] R38Q, F42R, K43S and E68V;

[0167] R38T, F42A, K43A and E68G;

[0168] R38E, E62T, E68S and L72K;

[0169] E62T, E68G and L72S;

[0170] I28T, R38E and K43R;

[0171] I28T, R38N, K43R and E68K;

[0172] F42H and K43Q;

[0173] R38E, F42Q, K43Q and K49E;

[0174] R38D, F42Q, K43G, and K64M; or

[0175] R38T, F42E, K43P, E62G, E68A and K8R.

[0176] In some embodiments, provided herein are IL2 mutant proteins with enhanced binding affinity to IL2Rβγ. In some embodiments, the IL2 mutant protein contains amino acid substitutions N88D, V91D / V91A / V91E and / or I92T as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3.

[0177] In some embodiments, the IL2 mutein contains amino acid substitutions R38E, F42Q, K43Q, K49E, and N88D as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3;

[0178] R38E, F42Q, K43Q, K49E and V91D / V91A / V91E;

[0179] R38E, F42Q, K43Q, K49E and I92T;

[0180] R38E, F42Q, K43Q, K49E, N88D, and V91D / V91A / V91E;

[0181] R38E, F42Q, K43Q, K49E, V91D / V91A / V91E and I92T;

[0182] R38E, F42Q, K43Q, K49E, N88D, V91D / V91A / V91E and I92T;

[0183] I28T, R38E, K43R and N88D;

[0184] I28T, R38E, K43R and V91D / V91A / V91E;

[0185] I28T, R38E, K43R and I92T;

[0186] I28T, R38E, K43R, N88D and V91D / V91A / V91E;

[0187] I28T, R38E, K43R, V91D / V91A / V91E, and I92T; or

[0188] I28T, R38E, K43R, N88D, V91D / V91A / V91E and I92T.

[0189] In some embodiments, the IL2 mutein contains a mutation that reduces, disrupts, or eliminates binding to IL2Rα. In some embodiments, the IL2 mutein contains a mutation that reduces binding to IL2Rβγ.

[0190] In some embodiments, the IL2 mutant protein has a percent identity of at least 90%, 95%, 96%, 97%, 98%, 99% to the amino acid sequence shown in any one of SEQ ID NOs: 7-23.

[0191] Methods on how to determine the percent identity between two amino acid sequences are known in the art, including but not limited to BLAST (Basic Local Alignment Search Tool) at NCBI.

[0192] In some embodiments, the IL2 mutant protein differs from the amino acid sequence shown in any one of SEQ ID NOs: 7-23 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid.

[0193] Furthermore, in some embodiments, the IL2 mutant protein contains the amino acid sequence shown in any one of SEQ ID NOs: 7-23.

[0194] In some embodiments, the IL2 mutein is derived from human IL2. In some embodiments, the IL2 mutein is derived from a non-human vertebrate, including but not limited to mice, rats, rabbits, cats, dogs, pigs, monkeys, chimpanzees, and gorillas.

[0195] Characteristics of IL2 mutant proteins

[0196] The IL2 mutant protein provided herein can reduce, destroy or eliminate affinity binding with IL2Rα. At the same time, the IL2 mutant protein provided herein can reduce affinity binding with IL2Rβγ heterodimer.

[0197] In some embodiments, the IL2 muteins provided herein can reduce, disrupt or eliminate affinity binding to the IL2Rαβγ heterotrimer.

[0198] In some embodiments, the IL2 mutant protein can enhance the immune response by selectively activating effector T cells, and can promote the expansion and activity of effector T cells, for example, by increasing the proliferation of CD8+ effector T cells.

[0199] In some embodiments, the IL2 mutant protein can reduce the activation of Treg cells or the number of Treg cells. Therefore, the IL2 mutant protein provided herein reduces the toxic adverse reactions in IL2 immunotherapy.

[0200] In some embodiments, the IL2 muteins improve selective immunostimulatory capacity and reduce anti-inflammatory effects mediated by Treg cells.

[0201] In some embodiments, IL2 muteins with improved and selective immunostimulatory capacity can promote the expansion and activity of effector T cells while minimizing Treg cells and reducing anti-inflammatory effects.

[0202] In some embodiments, the IL2 mutant protein is capable of reducing affinity binding to the IL2Rαβγ heterotrimer by reducing or eliminating binding to IL2Rα.

[0203] In some embodiments, the IL2 mutein is capable of reducing the immune response of regulatory T cells activated by the IL2Rαβγ heterotrimer.

[0204] In some embodiments, the affinity constant (KD = koff / kon, or KD = Kd / Ka) of the IL2 mutant protein binding to the IL2Rβγ heterodimer is greater than 0.1E-11 M, greater than 9.9E-11 M, greater than 1E-10 M, or greater than 9E-10 M. Kd / koff is the dissociation constant, and Ka / kon is the association constant.

[0205] In some embodiments, the IL2 mutein binds to IL2Rα with a KD greater than 1.0E-12M, greater than 1.0E-11M, or greater than 1.0E-10M.

[0206] In some embodiments, the IL2 mutant protein has improved thermal stability, wherein the Tm value is not less than 50° C., not less than 55° C., not less than 60° C., or not less than 65° C. In some embodiments, the Tm of the IL2 mutant protein is greater than 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., or 70° C.

[0207] In some embodiments, the IL2 mutant protein is expressed in mammalian cells (including but not limited to HEK293 cells). In some embodiments, the expression level of the IL2 mutant protein is greater than 70 mg / L, greater than 80 mg / L, greater than 85 mg / L, greater than 100 mg / L, greater than 105 mg / L, greater than 110 mg / L, greater than 115 mg / L, greater than 120 mg / L or greater than 125 mg / L.

[0208] In some embodiments, the monomer percentage of the IL2 mutant protein is greater than 50%, greater than 60%, greater than 65%, greater than 68%, greater than 70%, greater than 71% or greater than 72%. The monomer percentage can be detected using any method known in the art such as SEC-HPLC.

[0209] Polynucleotides, vectors, host cells and fusion proteins

[0210] The present disclosure also provides an isolated polynucleotide encoding the IL2 mutant protein as described above, which can reduce, destroy or eliminate affinity binding to IL2Rα. In some embodiments, the IL2 mutant protein can reduce affinity binding to IL2Rβγ heterodimer.

[0211] The polynucleotide is a nucleic acid sequence of DNA, RNA, a DNA / RNA hybrid, or a modification thereof. In some embodiments, the polynucleotide is a nucleic acid sequence of DNA. In some embodiments, the polynucleotide is a nucleic acid sequence of RNA. The DNA or RNA encoding the IL2 mutant protein can be easily isolated and sequenced using known procedures known in the art. The encoding DNA or RNA can also be obtained by synthetic methods.

[0212] In one aspect, the disclosure also provides an isolated vector containing a polynucleotide as described herein. The isolated polynucleotide provided can be inserted into a vector for further cloning (amplification of DNA) or for expression using recombinant techniques known in the art.

[0213] Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (eg, SV40, CMV, EF-1α), and a transcription termination sequence.

[0214] In some embodiments, the vector provided herein comprises at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, parvoviruses (e.g., SV40), lambda phages, and M13 phages, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p1 5TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0215] In some embodiments, a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which can involve the use of a non-pathogenic (defective), replication-competent virus, or a replication-defective virus can be used.

[0216] In one aspect, the present disclosure also provides a host cell comprising an isolated polynucleotide as described herein or an isolated vector as described herein.

[0217] The vector containing the polynucleotide sequence encoding IL2 mutant protein can be introduced into the host cell for cloning or gene expression. Suitable host cells for cloning or expressing the above polynucleotides (nucleic acid sequences of DNA, RNA or DNA / RNA hybrids) in the vector herein are the above-mentioned prokaryotes, yeast or higher eukaryotic cells.

[0218] In some implementations, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding IL2 muteins. Among lower eukaryotic host microorganisms, Saccharomyces cerevisiae or common baker's yeast is most commonly used. However, many other genera, species and strains are commonly available and can be used herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0219] Suitable host cells for expressing the IL2 muteins provided herein are derived from multicellular organisms. However, vertebrate cells have been of the greatest interest, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. [Annals of the New York Academy of Sciences] 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma line (Hep G2).

[0220] In some embodiments, the host cell is a mammalian culture cell line, such as CHO, BHK, NS0, 293, and derivatives thereof.

[0221] In some embodiments, the host cell comprises a HEK293 cell.

[0222] The vector can be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., using recombinant viruses). Therefore, the non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0223] In one aspect, the present disclosure also provides a fusion protein comprising an IL2 mutant protein as described herein. In some embodiments, the IL2 mutant protein is fused to an Fc region and / or a linker.

[0224] In some embodiments, the Fc region is functional and likely mediates effector functions including ADCC (antibody-dependent cell-mediated cytotoxicity) and / or phagocytosis.

[0225] In some embodiments, the Fc region is derived from human IgG, including IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc region is anti-hlgG Fc capture (AHC).

[0226] In some embodiments, the Fc region prolongs the half-life (t 1 / 2 ).

[0227] In some embodiments, the fusion protein contains a linker between the Fc region and the IL2 mutein. Many different linker polypeptides are known in the art and can be used in the context of an IL2 mutein Fc fusion protein.

[0228] In some embodiments, the IL-2 mutein Fc fusion protein contains one or more copies of a peptide consisting of GSGS (SEQ ID NO: 5) between the Fc region and the IL2 mutein.

[0229] In some embodiments, the linker is glycosylated when expressed in appropriate cells, and such glycosylation can help stabilize the protein in solution and / or stabilize the protein when administered in vivo. Thus, in certain embodiments, the IL2 mutein fusion protein comprises a glycosylated linker between the Fc region and the IL2 mutein region.

[0230] Pharmaceutical composition

[0231] Provided herein is a pharmaceutical composition comprising the above-mentioned IL2 mutant protein, isolated polynucleotide, isolated vector, host cell or fusion protein, and a pharmaceutically acceptable carrier. The pharmaceutical composition can be formulated in any manner known in the art.

[0232] Compositions containing one or more of any of the IL2 muteins, isolated polynucleotides, isolated vectors, host cells, or fusion proteins described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound to allow for ease of administration and uniformity of dosage).

[0233] The pharmaceutical composition is formulated to be compatible with its intended route of administration (eg, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).

[0234] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, sterile diluents (e.g., sterile water or saline), fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents, antibacterial or antifungal agents (e.g., benzyl alcohol or methyl parabens, phenol, ascorbic acid, thimerosal and the like), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetates, citrates or phosphates), and isotonic agents (e.g., sugars (e.g., dextrose), polyols (e.g., mannitol or sorbitol) or salts (e.g., sodium chloride)), or any combination thereof. Liposomal suspensions may also be used as pharmaceutically acceptable carriers.

[0235] The preparation of the composition can be prepared and packaged in an ampoule, a disposable syringe or a multiple dose vial. Where necessary (e.g., in an injectable formulation), suitable fluidity can be maintained by, for example, using a coating (such as lecithin or a surfactant). The absorption of the antibody or its Fab can be extended by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microcapsule delivery systems, which can include biodegradable biocompatible polymers (e.g., ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid).

[0236] In some embodiments, the pharmaceutical composition is formulated as an injectable composition. The injectable pharmaceutical composition can be prepared in any known form, such as a liquid solution, a suspension, an emulsion, or a solid form suitable for producing a liquid solution, a suspension or an emulsion. Injectable formulations may include sterile and / or non-pyrogenic solutions ready for injection, sterile dry soluble products (such as lyophilized powders, including subcutaneous injection tablets) ready for combination with solvents before use, sterile suspensions ready for injection, sterile dry insoluble products ready for combination with vehicles before use, and sterile and / or non-pyrogenic emulsions. The solution may be aqueous or non-aqueous.

[0237] In some embodiments, a sterile lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment as disclosed herein in a suitable solvent. The solvent may contain excipients that improve the stability of other pharmacological components of the powder or the reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents.

[0238] Data obtained from cell culture experiments and animal studies can be used to formulate appropriate doses of any given agent for use in a subject. A therapeutically effective amount of a therapeutic agent (i.e., an IL2 mutant protein, an isolated polynucleotide, an isolated vector, a host cell, a fusion protein, or a pharmaceutical composition) will be an amount that treats a disease in a subject, reduces the severity, frequency, and / or duration of one or more symptoms of a disease in a subject. Effectiveness and administration can be determined by a health care professional or a veterinary professional using methods known in the art and by observing one or more symptoms of a subject's disease.

[0239] Exemplary dosages of therapeutic agent (i.e., IL2 mutant protein, isolated polynucleotide, isolated vector, host cell, fusion protein or pharmaceutical composition) per kilogram of subject weight (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; or about 1 μg / kg to about 50 μg / kg). In addition, it should be understood that the specific dosage level for any particular subject will depend on a variety of factors, including the activity of the specific compound employed, the age, weight, general health, sex and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody or antibody fragment in vivo.

[0240] Use in the manufacture of therapeutic agents

[0241] The present disclosure also provides use of the IL2 mutant protein as described above in the manufacture of a therapeutic agent for diagnosing, preventing or treating a disease, disorder or condition.

[0242] The present disclosure also provides the use of the above-mentioned isolated polynucleotide in the manufacture of a therapeutic agent for diagnosing, preventing or treating a disease, disorder or condition.

[0243] The present disclosure also provides use of the above-mentioned isolated vector in the manufacture of a therapeutic agent for diagnosing, preventing or treating a disease, disorder or condition.

[0244] The present disclosure also provides use of the above host cells in the manufacture of a therapeutic agent for diagnosing, preventing or treating a disease, disorder or condition.

[0245] The present disclosure also provides use of the above fusion protein in the manufacture of a therapeutic agent for diagnosing, preventing or treating a disease, disorder or condition.

[0246] In some embodiments, the therapeutic agent is a drug or a pharmaceutical substance.

[0247] In some embodiments, the disease, disorder or condition is a disease associated with IL2. Provided IL2 mutant proteins reduce toxic adverse reactions in IL2 immunotherapy.

[0248] In some embodiments, the disease, disorder or condition comprises a tumor, which may be, without limitation, a hematological tumor or a solid tumor. In some embodiments, the tumor may be malignant or benign. In some embodiments, the disease, disorder or condition is cancer. In some embodiments, at least one cell of the tumor expresses IL2.

[0249] In some embodiments, the therapeutic agent is used to diagnose, prevent or treat a disease, disorder or condition in a mammal (e.g., a human). In some embodiments, the therapeutic agent is used to diagnose, prevent or treat a disease, disorder or condition in a non-human vertebrate, including a mouse, rat, rabbit, cat, dog, pig, monkey, chimpanzee or gorilla.

[0250] Treatment

[0251] The above-mentioned IL2 mutant proteins, isolated polynucleotides, isolated vectors, host cells or fusion proteins can be used for various therapeutic purposes.

[0252] In one aspect, the present disclosure also provides a method for diagnosing, preventing or treating a disease, disorder or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the above-mentioned IL2 mutant protein, isolated polynucleotide, isolated vector, host cell, fusion protein or pharmaceutical composition.

[0253] In one aspect, the present disclosure also provides a method for enhancing the immune response to an IL2-related disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the above-mentioned IL2 mutant protein, isolated polynucleotide, isolated vector, host cell, fusion protein or pharmaceutical composition.

[0254] In some embodiments, the disease, disorder or condition comprises a tumor. In some embodiments, the disease, disorder or condition comprises cancer. Without limitation, the method of treating reduces the rate at which the volume of a tumor in a subject increases over time, reduces the risk of metastasis, or reduces the risk of additional metastasis in a subject. In some embodiments, treatment can stagnate, slow, postpone or inhibit the progression of cancer. In some embodiments, treatment can result in a reduction in the number, severity and / or duration of one or more symptoms of cancer in a subject.

[0255] In one aspect, the present disclosure also provides a method for reducing adverse reactions associated with IL2 in anti-tumor immunotherapy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an IL2 mutant protein, isolated polynucleotide, isolated vector, host cell, or fusion protein as described herein.

[0256] In some embodiments, the IL2-related adverse reaction is caused by Treg cells activated by IL2, IL2-like, or a therapeutic agent (eg, IL2 mutein).

[0257] In some embodiments, the subject in need is identified or diagnosed as having cancer, such as breast cancer, carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer or hematological malignancies. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung carcinoma (NSCLC), small cell lung carcinoma (SCLC), bladder cancer or metastatic hormone-resistant prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC) or colorectal cancer.

[0258] As used herein, "effective amount" means an amount or dosage sufficient to produce a beneficial or desired result (including stagnation, slowing, delaying or inhibiting the progression of a disease). The effective amount will vary according to, for example, the age and weight of the subject to whom the IL2 mutant protein, the polynucleotide encoding the IL2 mutant protein, the vector comprising the polynucleotide, the fusion protein and / or the pharmaceutical composition is to be administered, the severity of the symptoms, and the route of administration, and thus administration can be determined on an individual basis. The effective amount can be administered in one or more administrations.

[0259] The typical daily dose of the therapeutic agent (for example, IL2 mutant protein or fusion protein) of an effective amount is 0.01mg / kg to 100mg / kg. In some embodiments, the dosage can be less than 100mg / kg, 10mg / kg, 9mg / kg, 8mg / kg, 7mg / kg, 6mg / kg, 5mg / kg, 4mg / kg, 3mg / kg, 2mg / kg, 1mg / kg, 0.5mg / kg or 0.1mg / kg. In some embodiments, the dosage can be greater than 10mg / kg, 9mg / kg, 8mg / kg, 7mg / kg, 6mg / kg, 5mg / kg, 4mg / kg, 3mg / kg, 2mg / kg, 1mg / kg, 0.5mg / kg, 0.1mg / kg, 0.05mg / kg or 0.01mg / kg. In some embodiments, the dosage is about 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0260] In any of the methods described herein, the above-described IL2 mutant protein, isolated polynucleotide, isolated vector, host cell, fusion protein or pharmaceutical composition is administered to the subject, and optionally at least one other therapeutic agent (pharmacologically active substance). The subject includes a mammal, and in some embodiments, the subject includes a human. In some embodiments, the subject is a human.

[0261] Example

[0262] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0263] Example 1: General method

[0264] Gene synthesis

[0265] All IL2 variants used in the embodiments were fused to the N-terminus of human IgG1-LALA (huIgG1 Fc-LALA, SEQ ID NO: 4) via a short linker GSGS (SEQ ID NO: 5).

[0266] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:4).

[0267] The desired gene segment was synthesized from synthetic oligonucleotides and cloned into the pcDNA3.4 vector. Plasmid DNA was purified from the transformed bacteria and the concentration was determined by UV spectroscopy. The DNA sequence of the gene segment was confirmed by DNA sequencing.

[0268] Protein production

[0269] HEK293 cells grown in a suspension of 30 ml scale were transfected by electroporation using the corresponding expression vector. Conditioned medium was collected by centrifugation 4 days after transfection. Protein A was used to purify IL2 variants by an affinity step. The resin-bound protein was eluted by 0.05M citrate buffer (pH 3.4) and immediately neutralized by Tris buffer (pH 9.0). The purified protein was dialyzed against phosphate buffered saline. The concentration of the protein was determined by UV spectroscopy (ultraviolet spectroscopy). Protein purity was evaluated by SDS-PAGE and SEC-HPLC (Agilent).

[0270] Protein thermal stability assessment

[0271] The thermal stability of the purified variants was assessed using a thermal shift assay. The protein was mixed with a freshly diluted protein thermal shift dye (Thermo Fisher Scientific). The mixture was then transferred to a 384-well plate and loaded onto a QuantStudio real-time PCR system (Thermo Fisher Scientific) for Tm measurement. The temperature from 25°C to 99°C was scanned with a heating rate of 1.6°C / second and a holding time of 2min. The melting curve obtained was analyzed using protein thermal shift software (Thermo Fisher Scientific) to obtain a Tm value.

[0272] Measurement of IL2 variant binding to IL2R

[0273] The affinity of IL2 variants to IL-2Rβγ heterodimers and IL-2Rα subunits was determined by bio-layer interferometry (Bio-Layer Interferometry; BLI) technology (Octet Red 96 system). In brief, IL2 variants at a concentration of 50nM were loaded and fixed on an AHC (anti-hIgG Fc capture) sensor until the signal reached 0.6nm. The sensor was immersed in a buffer until the signal returned to the baseline. Subsequently, human IL-2Rα subunits with HIS tags or human IL-2Rβγ heterodimers with HIS tags were applied to the sensor as analytes in a concentration range of 100nM to 3.125nM (1:2 dilution) in a kinetic buffer (Pall, USA) at 30°C. The following conditions were applied: 90s for association, 300s for dissociation, and 6x5s for regeneration using a regeneration solution (10mM glycine, pH 1.5). The obtained curve was fitted with a 1:1 binding model.

[0274] Flow cytometry (FCM)

[0275] Raji cells overexpressing IL2Rβγ suspended in FACS buffer (buffered saline solution containing fetal bovine serum and 0.09% sodium azide) were stained on ice for 1 h with serial dilutions (3-fold dilutions starting from 1000 nM) of IL2 variants, then washed three times. Subsequently, the cells were incubated with secondary antibody Alexa Fluor 647 goat anti-human IgG Fc (Southern Biotech, catalog number: 2048-31) for 30 min, then washed three times. Flow cytometry was performed on FACSCELESTA (BD Biosciences), and MFI (mean fluorescence intensity; MFI) was calculated accordingly.

[0276] Example 2: Construction and screening of IL2 mutation library

[0277] The overall structure of the IL2-IL2Rα-IL2Rβ-γc tetramer (Protein Data Bank ID: 2B5I) is presented in Figure 1 Analysis of the interface between IL-2 and IL-2Rα revealed that several residues on IL-2 are involved in electrostatic and hydrophobic interactions with IL-2Rα. Six of them (R38, F42, K43, E62, E68, L72) were selected for mutation using a library approach to disrupt the interaction with IL-2Rα.

[0278] Primers were designed to randomize residues at these selected positions, and these primers were used to construct a site-directed mutagenesis library by overlapping polymerase chain reaction (PCR). The PCR products were transformed into yeast together with the linearized pYD1 vector. Yeast cells from the IL-2 library were labeled with soluble IL-2Rα and biotinylated soluble IL-2Rβγ tagged with human IgG1-Fc. The cells were washed and labeled with streptavidin conjugated to R-phycoerythrin (PE) and mouse anti-human IgG Fc antibody conjugated to FITC. The cells were then sorted on an S3e cell sorter (Bio-Rad) to isolate strains that abolished binding to soluble IL-2Rα and comparable binding to soluble IL-2Rβγ relative to human wild-type IL-2. Three rounds of sorting were performed by flow cytometry, in which the sorted library was amplified and surface expression was induced between each sort. Extract the DNA from the strain of sorting, and mix it with the DNA fragment of wild-type IL2 as template, for random mutagenesis by error-prone PCR.Control the error rate by using different amount of template in PCR system.Amplify the PCR product obtained by PCR for adding homology arms.The final PCR product is transformed into yeast together with linearized pYD1 vector.Mark yeast cells from new IL2 library as mentioned above.Then on S3e cell sorter (Bio-Rad), cells are sorted.

[0279] Strains that abolished binding to soluble IL-2Rα and reduced binding to soluble IL-2Rβγ were isolated relative to human wild-type IL-2. Four rounds of sorting were performed by flow cytometry, in which the sorted library was amplified and surface expression was induced between each sorting. After the fourth sorting, the sorted sample pool was plated for strain picking. The picked strains were screened by FACS to confirm binding to IL-2Rα and IL-2Rβγ. Finally, a total of 14 individual strains were lysed as templates and the DNA was amplified by PCR. The sequence of the IL2 mutant was determined by DNA sequencing.

[0280] Example 3: Generation and evaluation of IL2 variants with C125 substitutions

[0281] Sequence analysis showed an unpaired cysteine ​​residue at position 125 in human IL2 (Uniprot: P60568). In the available literature (Weiger et al., Eur J Biochem 180, 295-300 (1989), PN 4518584), this residue was mutated to serine before further engineering. However, based on structural analysis of IL2 (Protein Data Bank No.: 2B5I), the C125 residue is located in the hydrophobic core of the 4-helix bundle ( Figure 1 It was hypothesized that replacing C125 with a hydrophobic residue with a small side chain (e.g., alanine (Ala, A), valine (Val, V), leucine (Leu, L), and isoleucine (Ile, I)) would improve the stability of the protein.

[0282] Therefore, human wild-type IL2 (IL2-WT, SEQ ID NO: 1), IL2-C125S (SEQ ID NO: 2) and IL2-C125V (SEQ ID NO: 3) were individually fused to an Fc tag (human IgG1-LALA), expressed and evaluated for various properties. Consistent with the hypothesis, SDS-PAGE ( Figure 3 A) and SEC-HPLC( Figure 3 B) The results show that the IL2-C125S mutant (which replaces the hydrophobic core of the 4-helix bundle with a hydrophilic serine residue) tends to form soluble aggregates, while both wild-type IL2 and the IL2-C125V variant exist mainly as monomers ( Figure 3 A, 3B).

[0283] In addition, as compared with wild-type IL2 (IL2-WT) and IL2-C125S variants, IL2-C125V variants expressed much higher expression levels (Table 1). In addition, IL2-C125V variants also showed higher Tm1 in thermal stability tests (Table 1), indicating that the C125V substitution did improve the stability of IL2 protein.

[0284] Table 1. Summary of properties of IL2 mutants with C125 residue substitution *N / A means not applicable.

[0285] More importantly, compared with wild-type IL2, the C125V substitution had a significant effect on the binding to IL2Rα ( Figure 4 , Table 2) or IL2Rβγ( Figure 5 , Table 3) had no negative effect.

[0286] Table 2. IL2Rα binding affinity of IL2 mutants with C125 residue substitution

[0287] Variants KD(M) IL2-C125V 3.65E-08 IL2-WT 3.36E-08

[0288] Table 3. IL2Rβγ binding affinity of IL2 mutants with C125 residue substitution

[0289] Variants KD(M) IL2-C125V <1.0E-12 IL2-WT <1.0E-12

[0290] Based on these data, C125V-substituted IL2 was used as the default template for further engineering.

[0291] Example 4: Generation and evaluation of IL2 variants that disrupt IL2Rα binding

[0292] After obtaining the sequence of the selected clones that disrupted IL2Rα binding and altered IL2Rβγ binding, further engineering and validation were divided into two steps. The first step aimed to identify IL2 variants that abolished IL2Rα binding and had the desired druggability; this variant was then used as a template for the second step, which introduced substitutions of residues involved in IL2Rβγ binding to generate a series of mutations with different degrees of IL2Rβγ binding.

[0293] Therefore, in order to identify IL2 variants that abolish IL2Rα binding, the sequences of 14 strains selected from the yeast display library were further analyzed in the IL2-IL2Rα-IL2Rβ-γc tetramer structure. Some of these sequences contain residue substitutions that affect the interaction with IL2Rβγ binding. These residues were backmutated in this step and studied in the second step. Thus, the sequences of 14 new IL2 variants (IL2Rα non-binders) were generated (Table 4-1).

[0294] Table 4-1. Potential IL2Rα non-binder mutations

[0295]

[0296] * Positions are defined with reference to SEQ ID NO: 1. The sequence of library number IL2-C125V is shown in SEQ ID NO:3.

[0297] IL2 variants that disrupted IL2Rα binding were expressed in HEK293 cells, and eventually 9 of them expressed different expression levels and monomer percentages (Table 4-2). The reported IL2-3X variant (SEQ ID NO: 6) that abolished IL2Rα binding but had normal IL2Rβγ binding was generated as a reference.

[0298] Table 4-2. Summary of properties of potential IL2Rα non-binders

[0299]

[0300]

[0301] *All variants contain the C125V mutation.

[0302] Compared with IL2-3X, variants ESE01.019 (SEQ ID NO: 7) and ESE01.020 (SEQ ID NO: 8) showed higher monomer percentages in SEC-HPLC and higher thermal stability in thermal shift assays (Table 4-2). ESE01.019 also showed high expression levels, with 251 mg of purified protein from 1 liter of culture medium (Table 4-2).

[0303] All nine variants as well as IL2-3X showed abrogated IL2Rα binding in affinity assays ( Figure 6 A and Figure 6 B). Compared with IL2-C125V and IL2-3X, ESE01.019 also showed equivalent IL2Rβγ binding ability ( Figure 7 A and Figure 7 B, Table 5).

[0304] According to the structural analysis of the IL2-IL2Rα interface, the K49 residue of IL2 replaced by a glutamic acid residue in ESE01.019 is not involved in the direct interaction with IL2Rα. Therefore, a new variant ESE01.042 (derived from ESE01.019 with a backmutated K49 residue) was generated for characterization. In fact, the ESE01.042 variant showed abolished IL2Rα binding ability in affinity detection tests ( Figure 6 A and Figure 6 B) and equivalent IL2Rβγ binding ability compared with IL2-C125V, IL2-3X and ESE01.019 ( Figure 7 A and Figure 7 B. Table 5). ESE01.042 also showed comparable expression levels and monomer percentages to ESE01.019 in SEC-HPLC analysis (Table 4-2).

[0305] Table 5. IL2Rβγ binding affinity of IL2 variants

[0306]

[0307] Example 5: Generation and evaluation of IL2 variants that abolish IL2Rα binding and reduce IL2Rβγ binding

[0308] Since the ESE01.019 variant showed promising functional properties - no detectable IL2Rα binding and comparable IL2Rβγ binding - and good protein properties, it was selected as a template for further engineering of IL2Rβγ binding manipulation. The substitution of three residues, N88D, V91D / A / E and I92T, was identified in the above library screening and combined with each other to be introduced into the sequence of the ESE01.019 variant. A total of 14 new IL2 variants were designed, expressed, and purified in one step by protein A for evaluation of protein properties and IL2 receptor binding. The sequences of the new series of IL2 variants are shown in Table 6.

[0309] Table 6. Mutations in IL2 variants *Positions are defined with reference to SEQ ID NO: 1.

[0310] A new series of IL2 variants showed different levels of IL2Rβγ binding ability ( Figure 8 A and Figure 8 B). To further measure the IL2Rβγ binding capacity of IL2 variants with significantly reduced binding capacity, the maximum concentration of HIS-tagged human IL-2Rβγ heterodimer was increased from 100 nM to 1000 nM in the BLI affinity detection assay ( Figure 8 C and Figure 8 D) The affinities of a series of IL2 variants are listed in Table 7-1.

[0311] Except ESE01.030, the yields of these variants were all above 130 mg / L. The molecular purity of the variants varied from 79.8% to 97.8%, as evaluated by the percentage of monomers in SEC-HPLC. Tm measurements using DSF (differential scanning fluorometry; DSF) showed that all new variants had good thermal stability, with Tm1 above 67°C (Table 7-2).

[0312] Table 7-1. IL2Rβγ binding affinity of IL2 variants

[0313]

[0314]

[0315] Table 7-2. Summary of properties of IL2 variants

[0316]

[0317] All 15 variants (ESE01.027, ESE01.028, ESE01.029, ESE01.030, ESE01.031, ESE01.032, ESE01.033, ESE01.034, ESE01.035, ESE01.036, ESE01.037, ESE01.038, ESE01.039, ESE01.040, and ESE01.042) along with IL2-WT, IL2-C125V, and ESE01.019 were compared for their ability to bind to IL2Rβγ by FACS using Raji cells overexpressing the receptor ( Fig. 9 Consistently, binding to cell surface IL2Rβγ was reduced to varying degrees in this series of IL2 variants compared to IL2-WT (SEQ ID NO: 1) and ESE01.019, as expected.

[0318] The above amino acid sequences in the present disclosure are shown in Table 8.

[0319] Table 8. Amino acid sequence

[0320]

[0321]

[0322]

[0323] Although specific embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are not or may not currently be foreseen by the applicant or others skilled in the art may occur. Therefore, the appended claims as filed and as they may be amended are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

1. An IL2 mutant protein, wherein the IL2 mutant protein that reduces or eliminates binding to IL2Rα as compared to the wild-type IL2 of SEQ ID NO: 1 comprises an amino acid sequence comprising an amino acid substitution C125V or C125S.

2. The IL2 mutant protein of claim 1, wherein as compared to SEQ ID NO: 2 or 3, the IL2 mutant protein comprises one or more mutations selected from the group consisting of: K8, I28, R38, F42, K43, K49, E61, E62, K64, E68 and L72.

3. The IL2 mutant protein of claim 1 or 2, wherein the IL2 mutant protein comprises one or more amino acid substitutions selected from the group consisting of K8R, I28T, R38D / R38A / R38Q / R38T / R38E / R38N, F42E / F42R / F42A / F42H / F42Q, K43E / K43G / K43S / K43A / K43R / K43P / K43Q, K49E, E61R, E62T / E62G, K64M, E68G / E68V / E68S / E68K / E68A, and L72S / L72K as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3.

4. The IL2 mutant protein of any one of claims 1 to 3, wherein the IL2 mutant protein comprises one or more amino acid substitutions selected from the group consisting of I28T, R38D / R38A / R38Q / R38T / R38E / R38N, K43E / K43G / K43S / K43A / K43R / K43P / K43Q, E61R and E68G / E68V / E68S / E68K / E68A as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3; or R38D / R38A / R38Q / R38T / R38E / R38N, F42E / F42R / F42A / F42H / F42Q and K43E / K43G / K43S / K43A / K43R / K43P / K43Q; or R38D / R38A / R38Q / R38T / R38E / R38N, F42E / F42R / F42A / F42H / F42Q, K43E / K43G / K43S / K43A / K43R / K43P / K43Q, K49E; or R38D / R38A / R38Q / R38T / R38E / R38N, F42E / F42R / F42A / F42H / F42Q, K43E / K43G / K43S / K43A / K43R / K43P / K43Q, and E68G / E68V / E68S / E68K / E68A; or F42E / F42R / F42A / F42H / F42Q and K43E / K43G / K43S / K43A / K43R / K43P / K43Q; or or R38D / R38A / R38Q / R38T / R38E / R38N, E62T / E62G, E68G / E68V / E68S / E68K / E68A, and L72S / L72K; or R38D / R38A / R38Q / R38T / R38E / R38N, F42E / F42R / F42A / F42H / F42Q, K49E, K64M, K43E / K43G / K43S / K43A / K43R / K43P / K43Q and E68G / E68V / E68S / E68K / E68A.

5. The IL2 mutant protein of any one of claims 1 to 4, wherein the IL2 mutant protein comprises amino acid substitutions R38D, K43E / K43G and E61R as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3; R38A, F42E and K43G; R38E, F42Q and K43Q; R38A, F42E, K43G, E62T, E68G and L72S / L72K; R38A, F42E, K43G and E68G; R38Q / R38T, F42R / F42A, K43S / K43A, and E68G / E68V; R38A / R38T / R38E, E62T, E68S and L72K; E62T / E62G, E68G and L72S; R38E / R38N, K43R and I28T; R38E / R38N, K43R, I28T and E68K; F42H / F42Q and K43Q; R38E, F42H / F42Q, K43Q and K49E; R38D / R38E, F42Q, K43G / K43Q and K64M; R38D / R38E, F42Q, K43G / K43Q, and K49E; or R38A / R38T, F42E / F42A, K43P, E62G, E68A and K8R.

6. The IL2 mutant protein of any one of claims 1 to 6, wherein the IL2 mutant protein comprises amino acid substitutions R38D, K43E and E61R as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3; R38A, F42E and K43G; R38E, F42Q and K43Q; R38A, F42E, K43G, E62T, E68G and L72S; R38A, F42E, K43G and E68G; R38Q, F42R, K43S and E68V; R38T, F42A, K43A and E68G; R38E, E62T, E68S and L72K; E62T, E68G and L72S; I28T, R38E and K43R; I28T, R38N, K43R and E68K; F42H and K43Q; R38E, F42Q, K43Q and K49E; R38D, F42Q, K43G, and K64M; or R38T, F42E, K43P, E62G, E68A and K8R.

7. The IL2 mutant protein of any one of claims 1 to 6, wherein the IL2 mutant protein comprises amino acid substitutions N88D, V91D / V91A / V91E and / or I92T as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3.

8. The IL2 mutant protein of any one of claims 1 to 7, wherein the IL2 mutant protein comprises an amino acid substitution as compared to the amino acid sequence shown in SEQ ID NO: 2 or 3. R38E, F42Q, K43Q, K49E and N88D; R38E, F42Q, K43Q, K49E and V91D / V91A / V91E; R38E, F42Q, K43Q, K49E and I92T; R38E, F42Q, K43Q, K49E, N88D, and V91D / V91A / V91E; R38E, F42Q, K43Q, K49E, V91D / V91A / V91E and I92T; R38E, F42Q, K43Q, K49E, N88D, V91D / V91A / V91E and I92T; I28T, R38E, K43R and N88D; I28T, R38E, K43R and V91D / V91A / V91E; I28T, R38E, K43R and I92T; I28T, R38E, K43R, N88D and V91D / V91A / V91E; I28T, R38E, K43R, V91D / V91A / V91E, and I92T; or I28T, R38E, K43R, N88D, V91D / V91A / V91E and I92T.

9. The IL2 mutant protein of any one of claims 1-8, wherein the IL2 mutant protein has at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in any one of SEQ ID NOs: 7-23.

10. The IL2 mutant protein of any one of claims 1-9, wherein the IL2 mutant protein differs from the amino acid sequence shown in any one of SEQ ID NOs: 7-23 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid.

11. The IL2 mutant protein according to any one of claims 1 to 10, wherein the IL2 mutant protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 7 to 23.

12. The IL2 mutant protein according to any one of claims 1 to 11, wherein the IL2 mutant protein is capable of reducing affinity binding to the IL2Rβγ heterodimer.

13. The IL2 mutant protein according to any one of claims 1 to 12, wherein the IL2 mutant protein is capable of reducing affinity binding to the IL2Rαβγ heterotrimer by reducing or eliminating binding to IL2Rα. 14 . The IL2 mutant protein according to claim 1 , wherein the IL2 mutant protein is capable of reducing the immune response of regulatory T cells activated by IL2Rαβγ heterotrimers.

15. The IL2 mutant protein according to any one of claims 1 to 14, wherein the KD (affinity constant) of the IL2 mutant protein binding to the IL2Rβγ heterodimer is greater than 0.1E-11M, greater than 9.9E-11M or greater than 1E-10M.

16. The IL2 mutant protein according to any one of claims 1 to 15, wherein the KD (affinity constant) of the IL2 mutant protein binding to IL2Rα is greater than 1.0E-12M.

17. The IL2 mutant protein of any one of claims 1-16, wherein the IL2 mutant protein has improved thermal stability, wherein the Tm value is not less than 50°C, not less than 55°C, not less than 60°C or not less than 65°C.

18. An isolated polynucleotide encoding the IL2 mutant protein according to any one of claims 1 to 17.

19. An isolated vector comprising the isolated polynucleotide of claim 18.

20. A host cell comprising the isolated polynucleotide of claim 18 or the isolated vector of claim 19, wherein the host cell comprises a HEK293 cell.

21. A fusion protein comprising the IL2 mutant protein according to any one of claims 1 to 17, wherein the IL2 mutant protein is fused to an Fc region, and optionally, is fused to the Fc region via a linker.

22. A pharmaceutical composition comprising the IL2 mutant protein according to any one of claims 1 to 17, or the isolated polynucleotide according to claim 18, or the isolated vector according to claim 19, or the host cell according to claim 20, or the fusion protein according to claim 21, and a pharmaceutically acceptable carrier.

23. Use of the IL2 mutant protein of any one of claims 1 to 17, or the isolated polynucleotide of claim 18, or the isolated vector of claim 19, or the host cell of claim 20, or the fusion protein of claim 21, or the pharmaceutical composition of claim 22 in the manufacture of a therapeutic agent for diagnosing, preventing or treating a disease, disorder or condition.

24. A method of diagnosing, preventing or treating a disease, disorder or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an IL2 mutant protein according to any one of claims 1-17, an isolated polynucleotide according to claim 18, an isolated vector according to claim 19, a host cell according to claim 20, a fusion protein according to claim 21, or a pharmaceutical composition according to claim 22.

25. A method of enhancing an immune response to a disease, disorder or condition associated with IL2 in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an IL2 mutant protein as described in any one of claims 1-17, an isolated polynucleotide as described in claim 18, an isolated vector as described in claim 19, a host cell as described in claim 20, a fusion protein as described in claim 21, or a pharmaceutical composition as described in claim 22.

26. The method or use of any one of claims 23-25, wherein the disease, disorder or condition comprises a tumor.

27. A method for reducing IL2-related adverse reactions in anti-tumor immunotherapy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an IL2 mutant protein as described in any one of claims 1-17, an isolated polynucleotide as described in claim 18, an isolated vector as described in claim 19, a host cell as described in claim 20, a fusion protein as described in claim 21, or a pharmaceutical composition as described in claim 22.

28. The method of any one of claims 24-27, wherein the subject is a mammal, including a human.

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