Interleukin 2 mutants and uses thereof

Abstract

A novel interleukin-2 (IL-2) mutein is provided. Also provided are fusion proteins and immunoconjugates comprising the IL-2 mutein, as well as nucleic acids encoding the IL-2 mutein, vectors comprising the nucleic acids, and host cells. Further provided are methods for preparing the IL-2 mutein, pharmaceutical compositions comprising the IL-2 mutein, and therapeutic uses of the mutein.

Description

TECHNICAL FIELD

[0001] The present application relates to novel interleukin 2 (IL-2) muteins and uses thereof. In particular, the present application relates to IL-2 muteins having improved properties compared to wild-type IL-2, such as improved drugability, reduced IL-2Rα receptor binding capacity and / or increased IL-2Rβ receptor binding capacity. The present application also provides fusion proteins, immunoconjugates comprising the IL-2 muteins, as well as nucleic acids encoding the IL-2 muteins, vectors and host cells comprising the nucleic acids. The present application further provides methods of making and screening the IL-2 muteins, pharmaceutical compositions comprising the IL-2 muteins and therapeutic uses of the muteins. BACKGROUND

[0002] Interleukin-2 (IL-2), also known as T-cell growth factor (TCGF), is a pleiotropic cytokine produced by T helper cells, especially CD4 + Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)). In most cases, IL-2 acts through three different receptors: interleukin 2 receptor alpha (IL-2Ra; CD25), interleukin 2 receptor beta (IL-2Rβ; CD122), and interleukin 2 receptor gamma (IL-2Rγ; CD132). IL-2Rβ and IL-2Rγ are essential for IL-2 signaling, whereas IL-2Ra (CD25) is not required for signaling but can confer high-affinity binding of IL-2 to the receptor (Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)). The trimeric receptor formed by the association of IL-2Ra, β, and γ (IL-2aβγ) is the high-affinity receptor for IL-2 (KD ~ 10 pM), the dimeric receptor composed of β and γ (IL-2βγ) is the intermediate-affinity receptor (KD ~ 1 nM), and the IL-2 receptor formed by the α subunit alone is the low-affinity receptor.

[0003] Interleukin 2 has four anti-parallel, amphipathic alpha helices, which form a quaternary structure essential for its function (Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)). In most cases, IL-2 acts through three different receptors: interleukin 2 receptor alpha (IL-2Ra; CD25), interleukin 2 receptor beta (IL-2Rβ; CD122), and interleukin 2 receptor gamma (IL-2Rγ; CD132). IL-2Rβ and IL-2Rγ are essential for IL-2 signaling, whereas IL-2Ra (CD25) is not required for signaling but can confer high-affinity binding of IL-2 to the receptor (Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)). The trimeric receptor formed by the association of IL-2Ra, β, and γ (IL-2aβγ) is the high-affinity receptor for IL-2 (KD ~ 10 pM), the dimeric receptor composed of β and γ (IL-2βγ) is the intermediate-affinity receptor (KD ~ 1 nM), and the IL-2 receptor formed by the α subunit alone is the low-affinity receptor.

[0004] express the dimeric or trimeric IL-2 receptor. The dimeric receptor is expressed on cytotoxic CD8 + T cells and natural killer cells (NK), while the trimeric receptor is mainly expressed on activated lymphocytes and CD4 + CD25 + FoxP3 + suppressor T cells (Treg) (Byman, O. and Sprent. J. Nat. Rev. Immunol. 12, 180-190 (2012)). Since resting effector T cells and NK cells do not have CD25 on their cell surface, they are relatively insensitive to IL-2. Whereas Treg cells consistently express the highest levels of CD25 in vivo, IL-2 will therefore normally preferentially stimulate Treg cell proliferation.

[0005] IL-2 mediates multiple effects in the immune response through binding to the IL-2 receptor on different cells. On the one hand, as an immune system stimulator, IL-2 can stimulate T cell proliferation and differentiation, induce cytotoxic T lymphocyte (CTL) generation, promote B cell proliferation and differentiation and immunoglobulin synthesis, and stimulate natural killer (NK) cell production, proliferation and activation, and has therefore been approved as an immunotherapeutic agent for the treatment of cancer and chronic viral infections. On the other hand, IL-2 can promote immunosuppressive CD4 + CD25 + the maintenance of regulatory T cells (i.e., Treg cells) (Fontenot et al., Nature Immunol 6, 1142-51 (2005); D'Cruz and Klein, Nature Immunol 6, 1152-59 (2005); Maloy and Powrie, Nature Immunol 6, 1171-72 (2005)), and mediates activation-induced cell death (AICD) and is involved in the establishment and maintenance of immune tolerance to self-antigens and tumor antigens (Lenardo et al., Nature 353:858 (1991)), thereby causing tumor tolerance due to AICD and immunosuppression due to activated Treg cells in patients. In addition, high-dose IL-2 can cause vascular leak syndrome (VLS) in patients. It has been shown that IL-2 induces pulmonary edema by direct binding to the IL-2 trimeric receptor (IL-2αβγ) on pulmonary endothelial cells (Krieg et al., Proc Nat Acad Sci USA 107, 11906-11 (2010)).

[0006] To overcome the above problems associated with IL-2 immunotherapy, it has been proposed to reduce the toxicity and / or to enhance the efficacy of IL-2 treatment by changing the selectivity or preference of IL-2 for different receptors. For example, it has been proposed to use a complex of a monoclonal antibody with IL-2 to induce a preferential expansion of CD122 high expressing CD122 but not CD25 cells, thereby enhancing the in vivo IL-2 treatment effect (Boyman et al., Science 311, 1924-1927 (2006)). Oliver AST et al. (US 2018 / 0142037) proposed to introduce a triple mutation F42A / Y45A / L72G at amino acid residue positions 42, 45 and 72 of IL-2 to reduce the affinity for the IL-2Ra receptor. Aron M. Levin et al. (Nature, Vol 484, p529-533, DOI: 10.1038 / nature10975) proposed an IL-2 mutant called "superkine" IL-2 H9 , which mutant comprises a quintuple mutation L80F / R81D / L85V / I86V / I92F with enhanced IL-2Rß binding, thereby improving the stimulation of CD25 - negative cells, but still maintains high binding to CD25. Rodrigo Vazquez-Lombardi et al. (Nature Communications, 8:15373, DOI: 10.1038 / ncomms15373) proposed a triple mutant human IL-2 mutein IL-2 3X , which protein has residue mutations R38D-K43E-E61R at amino acid residue positions 38, 43 and 61, respectively, resulting in that the mutein does not bind to IL-2Ra, but the mutein activates CD25 - cells weakly, and the activation bias towards CD25 + cells remains. In addition, Rodrigo Vazquez-Lombardi et al. also proposed to improve the pharmacodynamic properties of interleukin by preparing an interleukin 2-Fc fusion, but the expression of the fusion protein is low and prone to form aggregates.

[0007] In view of the role of IL-2 in immunomodulation and disease, there is still a need in the art to develop new IL-2 molecules with improved properties, in particular IL-2 molecules exhibiting advantageous production, purification, with improved pharmacodynamic properties. SUMMARY

[0009] The present application satisfies the above-mentioned needs by providing novel IL-2 muteins having improved drugability properties and / or improved IL-2 receptor selectivity / bias relative to wild-type IL-2.

[0010] Accordingly, in one aspect, the present application provides novel IL-2 muteins. In some embodiments, the IL-2 muteins of the present application have one or more of the following properties, preferably at least properties (i) and (ii):

[0011] (i) improved drugability, in particular improved expression levels and / or purification performance when expressed in mammalian cells,

[0012] (ii) reduced or abolished binding to IL-2Ra;

[0013] (iii) enhanced binding to IL-2Rb.

[0014] In some embodiments, the present application provides IL-2 muteins comprising mutations at the interface of IL-2 and IL-2Ra and having a shortened B’C’ loop sequence.

[0015] In addition, the present application provides fusion proteins and immunoconjugates comprising the IL-2 muteins, pharmaceutical compositions and combination products; nucleic acids encoding the IL-2 muteins, vectors and host cells comprising the same; and methods of producing the IL-2 muteins, fusion proteins and immunoconjugates of the present application.

[0016] Further, the present application also provides methods and uses of treating diseases and stimulating the immune system of a subject using the IL-2 muteins and fusion proteins and immunoconjugates of the present application.

[0017] The present application is further illustrated in the following drawings and specific embodiments. However, these drawings and specific embodiments should not be considered limiting the scope of the present application, and modifications readily occurring to those skilled in the art are to be embraced within the spirit of the present application and scope of the appended claims. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Crystal structure of IL-2 complexed with IL-2Ra (PDB: 1Z92) is shown.

[0019] Figure 2 shows the crystal structure of IL-2 (PBD: 2ERJ) (A) and the B’C’ loop structure superposition of human IL-2 and human IL-15 (B).

[0020] Figure 3 Primers used for construction of the mutational library IBYDL029 are shown.

[0021] Figure 4 IL-2 muteins selected and constructed from the mutational library IBYDL029 and their sequences are shown.

[0022] Figure 5A -B shows IL-2 mutant -FC fusion proteins activate p-STAT5 on CD8 + T cells (A) and CD25 - T cells (B). + CD25 + T cells (B).

[0023] Figure 6A -C shows IL-2 mutant -FC fusion protein Y092 (A) and the body weight change of the animals monitored after administration (B and C).

[0024] Figure 7A -C shows IL-2 mutant -FC fusion protein Y144 (A) and the body weight change of the animals monitored after administration (B and C).

[0025] Figure 8 The amino acid sequence of the wild-type IL-2 protein IL-2 WT (SEQ ID NO: 1) is shown along with the amino acid residue numbering and the sequence alignment with the mutein IL-2 3X .

[0026] Figure 9 The full sequence of the yeast display plasmid pYDC011 is shown. DETAILED DESCRIPTION

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. For the purposes of the present application, the following terms are defined below.

[0029] The term "about" when used in association with a numerical value means a numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0030] The term "and / or" should be understood to mean either one of the items or a combination of any two or more of the items.

[0031] As used herein, the term "comprising" or "including" means including stated elements, integers or steps, but does not exclude other elements, integers or steps. In this context, when used in the term "comprising" or "including", the phrase "consisting essentially of" does not mean excluding other elements, integers or steps, unless otherwise indicated. For example, when referring to an IL-2 mutein "comprising" or "including" a certain mutation or combination of mutations, it is also intended to encompass an IL-2 mutein having only the stated mutation or combination of mutations.

[0032] In the present context, wild-type "interleukin-2" or "IL-2" refers to the parent IL-2 protein, preferably a naturally occurring IL-2 protein, e.g. a naturally occurring IL-2 protein derived from human, mouse, rat, non-human primates, including both unprocessed (e.g. signal peptide not removed) and processed (e.g. signal peptide removed) forms, which is used as a template for introducing the mutations or combinations of mutations of the present application. One full-length naturally occurring human IL-2 sequence including the signal peptide is shown in SEQ ID NO: 2, and the sequence of the mature protein is shown in SEQ ID NO: 3. Furthermore, the expression also includes naturally occurring allelic variants and splice variants, isoforms, homologues, and species homologues of IL-2. The expression also includes variants of natural IL-2, e.g. which can have at least 95-99% or more identity to natural IL-2 or have no more than 1-10 or 1-5 amino acid mutations (especially conservative amino acid substitutions), and preferably have essentially the same IL-2R alpha binding affinity and / or IL2R beta binding affinity as the natural IL-2 protein. Thus, in some embodiments, the wild-type IL-2 can comprise amino acid mutations which do not affect its binding to the IL-2 receptor, e.g. a naturally occurring human IL-2 protein with the mutation C125S introduced at position 125 (uniprot: P60568) belongs to the wild-type IL-2 of the present application. An example of a wild-type human IL-2 protein comprising the C125S mutation is shown in SEQ ID NO: 1. In some embodiments, the wild-type IL-2 sequence can have at least 85%, 95%, even at least 96%, 97%, 98%, or 99% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or 2 or 3.

[0033] In the present context, amino acid mutations can be amino acid substitutions, deletions, insertions and additions. Any combination of substitutions, deletions, insertions and additions can be made to obtain a final mutein construct having desired properties, e.g. reduced IL-2Rα binding affinity and / or improved drugability. Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxy terminus of a polypeptide sequence, as well as deletions and insertions within a polypeptide sequence. For example, an alanine residue can be deleted at position 1 of full-length human IL-2, or one or several amino acids can be deleted in the B’C’ loop region to shorten the length of the loop region. In some embodiments, preferred amino acid mutations are amino acid substitutions, e.g. combinations of single amino acid substitutions or replacement of a sequence segment. For example, it can be a replacement of the B’C’ loop region sequence of wild-type IL-2, in whole or in part, with a different sequence, preferably to obtain a B’C’ loop region sequence of shortened length.

[0034] In the present application, when referring to amino acid positions in an IL-2 protein or IL2 sequence segment, the determination is made by reference to the amino acid sequence SEQ ID NO: 1 of the wild-type human IL-2 protein (also referred to as IL-2 WT ) (as indicated in Figure 8 ). Corresponding amino acid positions on other IL-2 proteins or polypeptides, including full-length sequences or truncated fragments, can be identified by amino acid sequence alignment with SEQ ID NO: 1. Thus, in the present application, unless otherwise specified, amino acid positions of an IL-2 protein or polypeptide are amino acid positions numbered according to SEQ ID NO: 1. For example, when referring to “F42”, this refers to the 42nd phenylalanine residue F of SEQ ID NO: 1, or the amino acid residue at the corresponding position on other IL-2 polypeptide sequences, as aligned. Sequence alignment for the purpose of amino acid position determination can be performed using the Basic Local Alignment Search Tool, available from https: / / blast.ncbi.nlm.nih.gov / Blast.cgi, with default parameters.

[0035] In this document, when referring to IL-2 muteins, single amino acid substitutions are described in the following manner: [original amino acid residue / position / substituted amino acid residue]. For example, a lysine at position 35 substituted for glutamic acid can be denoted as K35E. When there can be multiple alternative amino acid substitutions at a given position (e.g., D, E at position K35), the amino acid substitution can be denoted as: K35D / E. Correspondingly, individual single amino acid substitutions can be linked by a plus sign “+” or a minus sign “-” to indicate a combination of mutations at multiple given positions. For example, a combination of mutations at positions K35E, T37E, R38E, and F42A is denoted as: K35E+T37E+R38E+F42A, or K35E-T37E-R38E-F42A.

[0036] In this document, a "percentage of sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, wherein the sequence identity is determined over the entire length of a reference sequence (e.g., SEQ ID NO: 1). Methods of alignment of sequences for comparison are well known in the art. Algorithms for determining percent sequence identity include, for example, BLAST and BLAST 2.0 algorithms (see Altschul et al., Nuc. Acids Res. 25:3389-402, 1977 and Altschul et al. J. Mol. Biol. 215:403-10, 1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. For purposes of this application, percentage identity is determined using the Basic Local Alignment Search Tool available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi with default parameters.

[0037] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the biological function of the protein / polypeptide comprising the amino acid sequence. Conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis, for example. Typical conservative amino acid substitutions refer to the substitution of one amino acid for another amino acid having similar chemical properties (e.g., charge or hydrophobicity). Tables of conservative substitution of functionally similar amino acids are well known in the art. In the present application, conservative substitution residues are from the conservative substitution table X, especially the preferred conservative amino acid substitution residues in Table X.

[0038] Table X

[0039] Original Residues Exemplary Substitutions Preferred conservative amino acid substitutions Ala (A) Val; Leu; lie Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Asp; Lys; Arg Gin Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gin (Q) Asn; Glu Asn Glu (E) Asp; Gin Asp Gly (G) Ala Ala His (H) Asn; Gin; Lys; Arg Arg lie (I) Leu; Val; Met; Ala; Phe; norleucine Leu Leu (L) norleucine; lie; Val; Met; Ala; Phe lie Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; lie Leu Phe (F) Trp; Leu; Val; lie; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) lie; Leu; Met; Phe; Ala; norleucine Leu

[0040] For example, a wild-type IL-2 protein can have conservative amino acid substitutions, or only conservative amino acid substitutions, relative to one of SEQ ID NOs: 1-3. For another example, a mutant IL-2 protein of the present application can have conservative amino acid substitutions, or only conservative amino acid substitutions, relative to an IL-2 mutant protein sequence specifically given herein, e.g., any one of SEQ ID NOs: 22-26.

[0041] “Affinity” or “binding affinity” can be used to reflect the intrinsic binding capacity of the interaction between members of a binding pair. The affinity of a molecule X for its binding partner Y can be represented by the equilibrium dissociation constant (K D ), which is the ratio of the dissociation rate constant and the association rate constant (k dis and k on , respectively). Binding affinity can be measured by common methods known in the art. One particular method for measuring affinity is the ForteBio affinity assay technique herein.

[0042] In the present context, an antigen binding molecule is a polypeptide molecule that can specifically bind to an antigen, e.g., an immunoglobulin molecule, an antibody or an antibody fragment, e.g., a Fab fragment and a scFv fragment.

[0043] In the present context, an antibody Fc fragment refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region, and can include native sequence Fc fragments and variant Fc fragments. Native sequence Fc fragments encompass various naturally occurring immunoglobulin Fc sequences, e.g., Fc regions of various Ig subtypes and allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389 / fimmu.2014.00520.). In one embodiment, a human IgG heavy chain Fc fragment extends from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. In another embodiment, the C-terminal lysine (Lys447) of the Fc-fragment can or can not be present. In further embodiments, the Fc fragment is a variant Fc fragment comprising mutations, e.g., comprising L234A-L235A mutations. Unless otherwise indicated herein, numbering of amino acid residues in the Fc fragment is according to the EU numbering system, also called the EU index, as described in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0044] Aspects of the application will be further detailed in the following subsections.

[0045] 1. IL-2 muteins of the application

[0046] The present application provides in one aspect novel IL-2 muteins having improved drugability properties and / or improved IL-2 receptor selectivity / preference.

[0047] Advantageous biological properties of IL-2 muteins of the invention

[0048] The present inventors have discovered that the binding of IL-2 muteins to IL-2Ra can be reduced or eliminated by introducing one or more specific mutations at the IL-2 binding interface with the IL-2Ra receptor. In addition, the present inventors have discovered that the expression and / or purity of IL-2 can be increased and / or the affinity for IL-2Rb can be enhanced by replacing the B'C' loop sequence of IL-2 itself with a short B'C' loop sequence from other interleukin cytokines, such as IL-15, or by truncating the B'C' loop sequence of IL-2 itself. The present inventors have further discovered that by combining the IL-2Ra binding interface mutations with the short B'C' loop mutations, IL-2 muteins can be provided having one or more of the improved properties selected from, for example: (i) improved expression and / or purity; (ii) reduced or eliminated binding to the IL-2Ra receptor; and / or (iii) enhanced binding to the IL-2Rb receptor.

[0049] Accordingly, the present invention provides IL-2 muteins having one or more of the above improved properties (i)-(iii), and in particular, IL-2 muteins having both improved properties (i) and (ii).

[0050] Improved drugability properties

[0051] In some embodiments, the IL-2 muteins of the present invention have improved drugability properties, for example, when expressed in mammalian cells, such as H293T or CHO cells, for example, as Fc fusion proteins, having one or more properties selected from: (i) improved expression levels over wild-type IL-2 protein; and (ii) ease of purification to higher protein purity. In some embodiments, the IL-2 muteins of the present invention also have storage stability, for example, a decrease in protein purity of no more than 5%, 2% or 1% as measured by SEC-HPLC, or a decrease in protein purity of no more than 5%, 3% or 2% as measured by CE-SDS, after 14 days of storage at 40°C in PBS buffer at pH 7.4.

[0052] In some embodiments of the application, the IL-2 muteins of the application exhibit increased expression levels compared to wild-type IL-2. In some embodiments of the application, the increased expression occurs in mammalian cell expression systems. Expression levels can be determined by any suitable method that allows for quantitative or semi-quantitative analysis of the amount of recombinant IL-2 protein in the cell culture supernatant, preferably after one-step affinity chromatography purification. For example, the amount of recombinant IL-2 protein in a sample can be assessed by Western blot or ELISA. In some embodiments, the IL-2 muteins of the application exhibit at least 1.1-fold, or at least 1.5-fold, or at least 2-fold, 3-fold or 4-fold, or at least 5, 6, 7, 8, or 9-fold, or even 10-fold or more increased expression in mammalian cells compared to wild-type IL-2.

[0053] In some embodiments, the IL-2 muteins of the application exhibit higher purity compared to wild-type IL-2 protein fusions, as shown by determining the purity of the purified protein after protein A affinity chromatography. In some embodiments, the purity of the protein is detected by SEC-HPLC techniques. In some preferred embodiments, the purity of the IL-2 mutein products of the application can reach 70%, or 80%, or 90% or more, preferably 92%, 93%, 94%, 95%, 98% or 99% or more, after one-step protein A affinity chromatography purification, following the IL-2 fusion protein purification method described, for example, in Example 2.

[0054] Improved IL-2 receptor selectivity / preference

[0055] IL-2 protein elicits signaling and functions by interacting with IL-2 receptors. Wild-type IL-2 exhibits different affinities for different IL-2 receptors. IL-2β and γ receptors, which exhibit lower affinity for wild-type IL-2, are expressed on resting effector cells, including CD8 + T cells and NK cells. IL-2Rα, which exhibits high affinity for wild-type IL-2, is expressed on regulatory T (Treg) cells and activated effector cells. Due to the high affinity, wild-type IL-2 preferentially binds to IL-2Rα on the cell surface, recruits IL-2Rβγ, and releases downstream p-STAT5 signaling through IL-2Rβγ to stimulate Treg cells and activated effector cells. Without being bound by theory, therefore, reducing or eliminating the affinity of IL-2 for IL-2Rα receptors will reduce the bias of IL-2 to preferentially activate CD25 + cells, reducing IL-2-mediated immune downregulation of Treg cells. Without being bound by theory, maintaining or enhancing the affinity for IL-2β receptors will preserve or enhance IL-2’s effect on effector cells, such as CD8 +Activation of T cells and NK cells and the resulting immunostimulatory effect of IL-2.

[0056] Thus, in some embodiments, the IL-2 muteins of the present application have, relative to wild-type IL-2, one or more improved properties selected from, for example:

[0057] (1) reduced or ablated binding affinity for the IL-2Ra receptor,

[0058] (2) increased binding affinity for the IL-2Rb receptor;

[0059] (3) reduced binding affinity for the high-affinity IL-2R receptor (IL-2Rabg);

[0060] (4) increased binding affinity for the intermediate-affinity IL-2R receptor (IL-2Rbg);

[0061] (5) reduced ability to activate IL-2 signaling, particularly the activation of STAT5 phosphorylation signaling, in CD25 + cells, particularly activated CD8 + T cells and Treg cells;

[0062] (6) resulting in reduced IL-2-mediated activation and proliferation of CD25 + cells, particularly activated CD8 + T cells and Treg cells;

[0063] (7) reduced or ablated bias of IL-2 to preferentially stimulate Treg cell proliferation;

[0064] (8) reduced IL-2-induced immunosuppressive effect of Treg cells;

[0065] (9) maintained or increased, particularly increased, activation of CD25 - cells, particularly CD25 - T effector cells and NK cells;

[0066] (10) resulting in increased IL-2-mediated activation and proliferation of effector T cells and NK cells.

[0067] In some embodiments, the IL-2 mutein of the application has the properties of (1) above, preferably further having one or more, especially all, of the properties selected from the group consisting of (3) and (5)-(8); more preferably further having one or more, especially all, of the properties selected from the group consisting of (2), (4) and (9)-(10). In some embodiments, the IL-2 mutein of the application has the properties of (2), (4) above, preferably further having one or more, especially all, of the properties selected from the group consisting of (9)-(10); more preferably further having one or more, especially all, of the properties selected from the group consisting of (1), (3) and (5)-(8).

[0068] In preferred embodiments, the IL-2 mutein of the application exhibits an anti-tumor effect in vivo, e.g., against colon cancer.

[0069] In some preferred embodiments, the IL-2 mutein of the application further has reduced in vivo toxicity mediated by IL-2 binding to high affinity receptor IL-2αβγ relative to wild-type IL-2.

[0070] In some preferred embodiments, the IL-2 mutein of the application is not significantly toxic upon administration to a subject, e.g., as reflected by a change in body weight of the subject after administration. For example, the body weight does not decrease by more than 15% or more than 10% or more than 5% after administration, e.g., after a period of administration of 20 days or more, relative to pre-administration.

[0071] In some embodiments, the IL-2 mutein of the application has reduced binding affinity for the IL-2Rα receptor by at least 5-fold, at least 10-fold, or at least 25-fold, especially at least 30-fold, 50-fold or 100-fold or more, relative to wild-type IL-2 (e.g., the IL-2 set forth in SEQ ID NO: 1 WT ). The binding affinity of the IL-2 mutein of the application, e.g., the IL-2 mutein of the application fused to an Fc fragment, for the receptor IL-2Rα receptor can be determined by the ForteBio affinity assay technique, e.g., as equilibrium dissociation constant (K D ).

[0072] In some embodiments, the IL-2 mutein of the application has increased binding affinity for the IL-2Rβ receptor, e.g., 5-10-fold or more, relative to wild-type IL-2 (e.g., the IL-2 set forth in SEQ ID NO: 1 WT ). The binding affinity of the IL-2 mutein of the application, e.g., the IL-2 mutein of the application fused to an Fc fragment, for the receptor IL-2Rβ receptor can be determined by the ForteBio affinity assay technique, e.g., as equilibrium dissociation constant (KD ) to determine binding affinity. In one embodiment, the bivalent binding affinity K D values of less than 10.0E-09 M, e.g., 1.0E-09 M to 7E-09 M, or e.g., less than 1.0E-09 M, e.g., 1.0E-10 M to 7.0E-10 M.

[0073] In one embodiment, the IL-2 muteins of the application result in reduced IL-2 mediated CD25 + cell activation and / or proliferation relative to wild-type IL-2. In one embodiment, the CD25+ cells are CD25 + CD8 + T cells. In another embodiment, the CD25 + cells are Treg cells. In one embodiment, the ability of the IL-2 muteins to activate CD25 + cells is identified by detecting activation of STAT5 phosphorylation signal by the IL-2 muteins in CD25 + cells in a STAT5 phosphorylation assay. For example, the half maximal effective concentration (EC50) can be determined by flow cytometry analysis of STAT5 phosphorylation in the cells, as described in the Examples of the application.

[0074] In one embodiment, the IL-2 muteins of the application result in maintained or enhanced IL-2 mediated CD25 - effector cell activation and / or proliferation relative to wild-type IL-2. In one embodiment, the CD25 - cells are CD8 + effector T cells or NK cells. In one embodiment, the ability of the IL-2 muteins to activate CD25 - cells is identified by detecting the EC50 value of the IL-2 muteins to activate STAT5 phosphorylation signal in CD25 - cells in a STAT5 phosphorylation assay. In one embodiment, the IL-2 muteins of the application have an improved ability to activate CD25 - cells relative to wild-type IL-2 protein (e.g., human IL-2 of SEQ ID NO: 1) by at least 1-fold, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, as determined in a STAT5 phosphorylation assay.

[0075] In one embodiment, the IL-2 mutein of the application removes or reduces the bias of IL-2 for preferential activation of CD25 + cells relative to wild-type IL-2. In one embodiment, the CD25 + cells are CD8 + T cells. In another embodiment, the CD25 + cells are Treg cells. In one embodiment, the ability of the IL-2 mutein to activate CD25 + cells is identified by measuring the EC50 values for activation of STAT5 phosphorylation signal by the IL-2 mutein in CD25 - cells and in CD25 + cells, respectively, in a STAT5 phosphorylation assay. For example, the bias of the IL-2 mutein for activation of CD25 - cells over CD25 - and CD25 + T cells is determined by calculating the ratio of the EC50 values for activation of STAT5 phosphorylation signal on CD25 + cells. Preferably, the bias of the mutein for CD25 + cells is reduced at least 10-fold, preferably at least 100-fold, 150-fold, 200-fold, 300-fold or more relative to the wild-type protein.

[0076] Muteins of the invention

[0077] IL-2 protein belongs to the short-chain type I cytokine family members with four alpha-helical bundle (A, B, C, D) structure. According to the analysis of crystal structure (PDB: 1Z92), IL-2 has the following amino acid sites for interaction with CD25 (i.e., IL-2Rα) in the region of amino acid residues 35-72: 35; 37; 38; 41; 42; 43; 45; 61; 62; 68; 72. The present inventors found that by introducing specific mutations at the CD25 interaction sites (i.e., sites 35; 37; 38; 41; 42; 43; 45; 61; 62; 68; 72) in the region of amino acid residues 35-72 of IL-2, the binding of IL-2 to IL-2Rα can be reduced or eliminated. In this context, the mutations at these sites in this region are referred to as “CD25 binding region” mutations.

[0078] In addition, by comparing the crystal structure of IL-2 monomer (PDB: 1M47) and complex (PDB: 2ERJ), the present inventors found that the B’C’ loop is missing in the crystal structure of IL-2 monomer, which is due to the fact that the B’C’ loop is very active in solution and cannot form a relatively stable conformation. By genetically engineering the B’C’ loop, such as sequence substitution or truncation, the stability of the B’C’ loop can be increased, which improves the drugability of IL-2 and / or the binding affinity to IL-2Rβ receptor. In this context, these sequence substitution or truncation mutations occurring in the B’C’ loop region are simply referred to as “B’C’ loop region mutations”.

[0079] The present inventors further found that the “CD25 binding region mutation” and the “B’C’ loop region mutation” can be combined to further improve the properties of IL-2. Thus, the present application provides an IL-2 mutein, wherein, compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the sequence of SEQ ID NO: 1), the mutein comprises (i) a “CD25 binding region mutation” and (ii) a “B’C’ loop region mutation”.

[0080] CD25 binding region mutations

[0081] In one aspect, the IL-2 mutein of the present application comprises one or more mutations in the CD25 binding region, preferably at positions 35, 37, 38, 41, 42, 43, 45, 61, 68 and 72, relative to wild-type IL-2, which eliminate or reduce the binding affinity to the IL-2Rα receptor.

[0082] In some embodiments, the CD25 binding region mutation of the present application comprises a combination of mutations selected from one of the following combinations (1)-(9):

[0083]

[0084] In preferred embodiments, the CD25 binding region mutation of the present application comprises a combination of mutations selected from one of the following combinations (1)-(9), in particular from one of combinations (1)-(6):

[0085]

[0086] In a preferred embodiment, the CD25 binding region mutation of the present application comprises or consists of the combination of mutations K35E + T37E + R38E + F42A.

[0087] In preferred embodiments, the CD25 binding region mutation of the present application results in abrogated CD25 binding, e.g. less than limit of detection of CD25 binding using ForteBio affinity assay.

[0088] For CD25 binding region mutations suitable for use in the present application, reference can also be made to the co-pending application PCT / CN2019 / 107055 of the present applicant. This application is incorporated herein in its entirety by reference.

[0089] B'C' loop region mutations

[0090] IL-2 protein belongs to the short-chain type I cytokine family members with four alpha-helical bundle (A, B, C, D) structure. In the present context, the terms “B’C’ Loop” or “B’C’ loop region” or “B’C’ loop sequence” are used interchangeably to refer to the connecting sequence between the B helix and the C helix of an IL-2 protein. The B’C’ loop sequence of an IL-2 protein can be determined by crystal structure analysis of IL-2, e.g. PDB: 2ERJ. For the purpose of the present application, the B’C’ loop sequence refers to the sequence of residues connecting position 72 and position 84 in the IL-2 polypeptide according to the numbering of SEQ ID NO: 1. In the wild-type IL-2 protein of SEQ ID NO: 1, 2 and 3, the connecting sequence comprises a total of 11 amino acid residues A73-R83.

[0091] In the present context, the terms “shortened loop region” or “shortened B’C’ loop region” refer to a mutant protein having a B’C’ loop sequence of reduced length relative to the wild-type IL-2 protein, i.e. the connecting sequence between amino acid residues aa72 and aa84 is shortened according to the numbering of SEQ ID NO: 1. The “shortened loop region” can be achieved by substitution or truncation of the loop sequence. The substitution or truncation can occur at any region or portion of the B’C’ loop sequence. For example, the substitution or truncation can be substitution of the loop region A73-R83 sequence or truncation of one or more amino acid residues from the C-terminus of this sequence. For another example, the substitution or truncation can be substitution of the loop region A74-R83 sequence or truncation of one or more amino acid residues from the C-terminus of this sequence. After the substitution or truncation is made, if necessary, further single amino acid substitutions can be introduced into the loop region sequence, e.g. amino acid substitutions for abrogating glycosylation, and / or back mutations, to further improve the performance of the mutant protein, e.g. drugability. Thus, in the present context, the shortened B’C’ loop region after mutation can be described by the sequence connecting the residue at position 72 and the residue at position 84 after the mutation is introduced.

[0092] In one aspect, the IL-2 mutein of the application comprises a B’C’ loop region mutation relative to wild-type IL-2, preferably the mutation results in a B’C’ loop region with increased stability; more preferably, the mutation results in an IL-2 mutein of the application with improved drugability, e.g., increased expression level and / or purity.

[0093] In some embodiments, the introduced mutation results in a mutein comprising a shortened B’C’ loop region (i.e., the length of the connecting sequence between amino acid residues aa72 and aa84 is shortened) relative to wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the sequence of SEQ ID NO: 1), preferably the shortened loop region has a length of less than 10, 9, 8, 7, 6 or 5 amino acids, and preferably 7 amino acids, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0094] In this context, B’C’ loop region mutations suitable for use in the application include truncations and substitutions of the B’C’ loop region. In one embodiment, the mutation comprises a truncation or substitution of amino acid residues aa73 to aa83 of the B’C’ loop region, e.g., a truncation to A(Q / G)S(K / A / D)N(F / I)H, or a substitution to SGDASIH. In another embodiment, the mutation comprises a truncation or substitution of amino acid residues aa74 to aa83 of the B’C’ loop region, e.g., a truncation to (Q / G)S(K / A / D)N(F / I)H, or a substitution to GDASIH.

[0095] In some embodiments, the IL-2 mutein of the application comprises a B’C’ loop chimeric mutation. Relative to wild-type IL-2, the mutein comprises a substitution of all or part of the sequence connecting aa72 to aa84, e.g., a substitution to a short B’C’ loop sequence from a member of the other four-helix short-chain cytokine IL family. Suitable short B’C’ loops for substitution into wild-type IL-2 can be identified by superpose of crystal structures from members of the other four-helix short-chain cytokine IL family, e.g., IL-15, IL-4, IL-21, or from members of the IL family from non-human species (e.g., mouse). In one embodiment, the sequence for substitution is a B’C’ loop sequence from interleukin IL-15 (especially human IL-15). In one embodiment, the substitution comprises a substitution of amino acid residues aa73 to aa83 of the B’C’ loop region. In another embodiment, the substitution comprises a substitution of amino acid residues aa74 to aa83 of the B’C’ loop region. Preferably, upon substitution, the IL-2 mutein of the application has a B’C’ loop sequence (i.e., the sequence connecting aa72 to aa84) selected from the group consisting of SGDASIH or AGDASIH.

[0096] In some embodiments, the IL-2 muteins of the application comprise a B’C’ loop truncation mutation. The muteins comprise a truncation of the sequence linking aa72 to aa84 relative to wild-type IL-2. In one embodiment, the truncation comprises a truncation of B’C’ loop region amino acid residues aa73 to aa83. In another embodiment, the truncation comprises a truncation of B’C’ loop region amino acid residues aa74 to aa83. For example, 1, 2, 3, or 4 amino acids can be truncated from the C-terminus. Preferably,

[0097] Following truncation, the B’C’ loop region of the IL-2 muteins of the application has the sequence A(Q / G)S(K / A / D)N(F / I)H. Preferably,

[0098] Following truncation, the IL-2 muteins of the application have a B’C’ loop sequence (i.e., the sequence linking aa72 to aa84) selected from the group consisting of:

[0099] B'C' loop sequences AQSKNFH AQSANFH AQSDNFH AGSKNFH AQSANFH AQSANIH

[0100] In one preferred embodiment, the IL-2 muteins of the application comprise a B’C’ loop region sequence selected from the group consisting of: AQSKNFH; AQSANFH; AQSDNFH; or SGDASIH or AGDASIH.

[0101] For B’C’ loop mutations suitable for use in the application, see also co-pending application PCT / CN2019 / 107054 of the present applicant. This application is incorporated by reference in its entirety.

[0102] Preferred exemplary combinations of mutations

[0103] In some preferred embodiments, the B’C’ loop mutations of the application are combined with the CD25 binding region mutations of the application to provide improved properties selected from two or all three of: (i) reduced (or eliminated) IL-2Rα binding; (ii) enhanced IL-2Rα binding, and (ii) improved expression levels and purity.

[0104] In some embodiments, the application provides IL-2 muteins, wherein the muteins comprise, relative to wild-type IL-2:

[0105] (i) a combination of mutations selected from one of the following combinations (1)-(9), especially one of combinations (1)-(6):

[0106] Combination Mutations 1 K35E + T37D + R38W + F42Q + Y45K + E61K + E68R 2 K35E + T37E + R38E + F42A 3 K35D + R38E + T41E + K43E 4 K35D + T37E + R38D + K43Y + Y45K + L72F 5 K35E + R38E + T41E + K43Y + Y45K + L72F 6 K35D + T37E + R38D + T41E + K43E + L72F 7 K35D + T37E + R38D + K43E + L72F 8 K35E + T37D + R38D + K43E + L72F 9 K35E + R38D + T41E + K43E + E61K + L72F

[0107] and (ii) a B’C’ loop region sequence selected from the group consisting of:

[0108] B'C' loop sequences SGDASIH AGDASIH AQSKNFH AQSANFH AQSDNFH AGSKNFH AQSANFH AQSANIH

[0109] In particular, the B'C loop sequence is selected from the group consisting of: AQSKNFH; AQSANFH; AQSDNFH; or SGDASIH; or AGDASIH.

[0110] Preferably, the present application provides an IL-2 mutein, wherein the mutein comprises, relative to wild-type IL-2:

[0111] (i) the mutation combination K35E + T37E + R38E + F42A; and

[0112] (ii) a B'C loop sequence selected from the group consisting of: AQSKNFH; AQSANFH; AQSDNFH; SGDASIH; AGDASIH.

[0113] Thus, in one embodiment, the present application provides an IL-2 mutein having a mature region that is at least 85% or 90% identical in amino acid sequence to the mature region of a wild-type IL-2 protein set out in one of SEQ ID NOs: 1-3, and comprising a linking sequence selected from the group consisting of: AGDASIH, SGDASIH; AQSKNFH; AQSANFH; AQSDNFH; AGSKNFH; AQSANFH; and AQSANIH; between amino acid positions 72 and 84, and having the mutation combination: K35E + T37E + R38E + F42A.

[0114] In some preferred embodiments, the present application provides an IL-2 mutein comprising an amino acid sequence that is at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identical to an amino acid sequence selected from one of SEQ ID NOs: 22, 23, 24, 25, and 26. In some embodiments, the mutein comprises or consists of the amino acid sequence of SEQ ID NOs: 22, 23, 24, 25, and 26.

[0115] Preferably, the combination of mutations results in an IL-2 that has a reduced bias for stimulating p-STATA5 signalling in CD25 + T cells, and has an enhanced ability to stimulate signalling in CD25 - T cells.

[0116] Other mutations

[0117] In addition to the mutations in the "CD45 binding" region and the "B'C' loop region" described above, the IL-2 muteins of the application can also have one or more mutations in other regions or positions, so long as they retain one or more of the beneficial properties of the IL-2 muteins of the application described above. For example, the IL-2 muteins of the application can also comprise a substitution at position 125, such as C125S, C125A, C125T, or C125V, to provide additional advantages such as improved expression or homogeneity or stability (see, e.g., U.S. Patent No. 4,518,584). For another example, the IL-2 muteins of the application can also comprise a substitution at position 3, such as T3A, to remove O-glycosylation of the N-terminus of IL2. For yet another example, the IL-2 muteins of the application can also comprise a substitution at position 76, such as K76D / A, to enhance T cell activation activity. Those skilled in the art know how to determine additional mutations that can be incorporated into the IL-2 muteins of the application.

[0118] The sequence difference between the IL-2 muteins and the wild-type protein can be expressed in terms of sequence identity, or in terms of the number of amino acids that differ between the two. In one embodiment, the IL-2 muteins have at least 85%, 86%, 87%, 88%, 89% identity, preferably 90% or more identity, preferably 95%, but preferably no more than 97%, more preferably no more than 96% identity with the wild-type protein. In another embodiment, the IL-2 muteins can have no more than 15, for example 1-10, or 1-5 mutations, for example, 0, 1, 2, 3, 4 mutations, in addition to the CD25 region mutations and the B'C' loop region mutations described above in the application. In one embodiment, the remaining mutations can be conservative substitutions. In one embodiment, the remaining mutations occur outside of the CD25 region and the B'C' loop region.

[0119] 2. Fusion proteins and immunoconjugates

[0120] The application also provides fusion proteins comprising the IL-2 muteins of the application. In a preferred embodiment, the IL-2 muteins of the application are fused to another polypeptide that can confer improved pharmacokinetic properties, such as albumin, more preferably an antibody Fc fragment. In one embodiment, the Fc fragment comprises mutations that reduce or eliminate effector function, such as the L234A / L235A mutations or L234A / L235E / G237A that reduce binding to Fcy receptors. Preferably, the fusion proteins comprising Fc have increased serum half-life. In a preferred embodiment, the fusion proteins comprising Fc also have reduced effector function mediated by the Fc region, such as reduced or eliminated ADCC or ADCP or CDC effector function.

[0121] In some embodiments, the Fc fragment fused to the IL-2 mutein is a human IgG Fc, e.g., a human IgGl Fc, a human IgG2 Fc, or a human IgG4 Fc. In one embodiment, the Fc fragment comprises the amino acid sequence of SEQ ID NO: 7 or at least 90% identity, e.g., 95%, 96%, 97%, 99% or more, thereto.

[0122] In some embodiments, the IL-2 mutein is fused to the Fc via a linker. In some embodiments, the linker can be selected to improve the Fc fusion protein for CD25 - activation of T cells. In one embodiment, the linker is GSGS, more preferably 2x(G4S).

[0123] In some embodiments, the Fc fusion protein comprises at least 85%, at least 95%, or at least 96% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-19. In some embodiments, the Fc fusion protein consists of the sequence of SEQ ID NOs: 15-19.

[0124] The present application also provides immunoconjugates comprising the IL2 muteins of the present application and an antigen binding molecule. Preferably, the antigen binding molecule is an immunoglobulin molecule, in particular an IgG molecule, or an antibody or antibody fragment, in particular a Fab molecule and a scFv molecule. In some embodiments, the antigen binding molecule specifically binds to an antigen presented on a tumor cell or in the tumor environment, e.g., an antigen selected from the group consisting of Fibroblast Activation Protein (FAP), Al domain of Tenascin C (TNC Al), A2 domain of Tenascin C (TNC A2), Extra Domain B of Fibronectin (EDB), Carcinoembryonic Antigen (CEA), and Melanoma- associated Chondroitin Sulfate Proteoglycan (MCSP). Thereby, the immunoconjugates of the present application can target tumor cells or the tumor environment upon administration to a subject, thereby providing further therapeutic benefits, e.g., the possibility to treat at lower doses and the resulting lower side effects; enhanced anti-tumor effects; etc.

[0125] In the fusion proteins and immunoconjugates of the present application, the IL-2 muteins of the present application can be linked to another molecule or antigen binding molecule, either directly or via a linker, and in some embodiments, a proteolytic cleavage site is comprised between the two.

[0126] 3. Polynucleotides, vectors and hosts

[0127] The present application provides nucleic acids encoding any of the above IL-2 muteins or fusions or conjugates. The polynucleotide sequences encoding the muteins of the present application can be generated by de novo solid phase DNA synthesis or by PCR mutagenesis of an existing sequence encoding wild-type IL-2 using methods well known in the art. In addition, the polynucleotides and nucleic acids of the present application can comprise a segment encoding a secretion signal peptide operably linked to a segment encoding a mutein of the present application, such that secretory expression of the mutein of the present application can be directed.

[0128] The present application also provides vectors comprising the nucleic acids of the present application. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs). In a preferred embodiment, the expression vector of the present application is a pCDNA3.1 expression vector.

[0129] The present application also provides host cells comprising the nucleic acid or the vector. Host cells suitable for replication and support of expression of mutant IL-2 proteins or fusions or immunoconjugates are well known in the art. Such cells can be transfected or transduced with particular expression vectors, and large quantities of vector-containing cells can be grown for seeding large scale fermentors to obtain sufficient quantities of IL-2 mutants or fusions or immunoconjugates for clinical applications. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell (e.g., a CHO cell or a 293 cell). For example, polypeptides can be produced in bacteria, particularly when glycosylation is not desired. Following expression, the polypeptide can be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, lower eukaryotes such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding polypeptides, including fungal and yeast strains in which the glycosylation pathway has been "humanized", resulting in production of polypeptides with partially or fully human glycosylation pattern. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006). Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as described, e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr- CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 and Sp2 / 0. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphocytic cell (e.g., YO, NS0, Sp20 cell).

[0130] 4. Method of manufacture

[0131] In another aspect, the present application provides a method of making an IL-2 mutein or fusion or conjugate of the present application, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the protein or fusion or conjugate, under conditions suitable for expression of the IL-2 mutein or fusion or conjugate, as provided herein above, and optionally recovering the protein or fusion or conjugate from the host cell (or host cell culture medium).

[0132] 5. Assays

[0133] The IL-2 muteins provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by a variety of assays known in the art.

[0134] In one aspect, the IL-2 muteins of the present application can be tested for their binding activity to IL-2 receptors. For example, binding to human IL-2Ra or β proteins can be determined by methods known in the art, such as ELISA, Western blotting, etc., or the exemplary methods disclosed in the Examples herein. For example, the assay can be performed using flow cytometry, wherein cells transfected to express the mutein on the cell surface, e.g., yeast display cells, are reacted with labeled (e.g., biotin-labeled) IL-2Ra or β proteins. Alternatively, the binding of the mutein to the receptor, including binding kinetics (e.g., Kd values), can be determined using recombinant mutein-Fc fusions in ForteBio assays. D In another aspect, the ability of the IL-2 muteins to bind to IL-2 receptors can be indirectly measured by determining signaling and / or immune activation effects that occur downstream of receptor binding.

[0135] In another aspect, the ability of the IL-2 muteins to bind to IL-2 receptors can be indirectly measured by determining signaling and / or immune activation effects that occur downstream of receptor binding.

[0136] Thus, in some embodiments, assays are provided for identifying mutant IL-2 proteins having biological activities. Biological activities can include, for example, the ability to induce proliferation of T and / or NK cells and / or Treg cells having IL-2 receptors, the ability to induce IL-2 signaling in T and / or NK cells and / or Treg cells having IL-2 receptors, the ability to reduce induction of apoptosis in T cells, the ability to induce tumor regression and / or improved survival, and reduced in vivo toxic properties, e.g., reduced vascular permeability. The present application also provides mutant IL-2 proteins having such biological activities in vivo and / or in vitro.

[0137] A variety of methods known in the art can be used to measure the biological activity of IL-2. For example, a suitable assay for testing the ability of an IL-2 mutein of the application to stimulate NK cells to produce IFN-γ can comprise the steps of incubating cultured NK cells with a mutant IL-2 protein or fusion or immunoconjugate of the application, and subsequently measuring the concentration of IFN-γ in the culture medium by ELISA. IL-2 signaling induces several signaling pathways, and involves JAK (Janus kinase) and STAT (signal transducer and activator of transcription) signaling molecules.

[0138] The interaction of IL-2 with the receptor β and γ subunits leads to phosphorylation of the receptor as well as JAK1 and JAK3 (which bind to the β and γ subunits, respectively). STAT5 then binds to the phosphorylated receptor and phosphorylates itself on a very important tyrosine residue. This leads to dissociation of STAT5 from the receptor, dimerization of STAT5, and translocation of the STAT5 dimers to the nucleus, where they promote transcription of target genes. Thus, the ability of a mutant IL-2 polypeptide to induce signaling via the IL-2 receptor can be assessed, for example, by measuring the phosphorylation of STAT5. Details of this method are disclosed in the Examples. For example, PBMCs can be treated with a mutant IL-2 polypeptide or fusion or immunoconjugate of the application, and the level of phosphorylated STAT5 can be determined by flow cytometry.

[0139] Further, the effect of a mutant IL-2 on tumor growth and survival can be assessed in a variety of animal tumor models known in the art. For example, xenografts of a cancer cell line can be implanted into immunodeficient mice, and treated with a mutant IL-2 polypeptide or fusion or immunoconjugate of the application. The in vivo anti-tumor effects of a mutant IL-2 polypeptide, fusion and immunoconjugate of the application can be detected based on tumor inhibition rates (e.g., calculated relative to an isotype control antibody). In addition, the toxicity of a mutant IL-2 polypeptide, fusion and immunoconjugate of the application in vivo can be determined based on changes in the body weight of the animals (e.g., changes in absolute body weight or percent body weight change relative to pre-dosing). The in vivo toxicity can also be determined based on mortality, life span observations (observable signs of adverse effects, e.g., behavior, body weight, body temperature), and clinical and anatomic pathology (e.g., measurement of blood chemistry values and / or histopathological analysis).

[0140] In yet another aspect, the drugability of the mutant proteins of the application can be characterized by methods known in the art, for example, expression level and product purity. For the determination of expression level, when the mutant protein is expressed in the culture supernatant from the cultured cells, the protein content of the centrifugally collected cell culture can be determined. Alternatively, the determination can be performed after one-step purification of the collected cell culture, for example, after one-step affinity chromatography purification. For the determination of product purity, the purity can be determined after one-step affinity chromatography purification of the harvested culture supernatant of the production cells to detect the purification performance of the mutant protein. Preferably, the mutant protein of the application has a significantly higher purity than the wild-type protein after the one-step affinity chromatography purification, indicating that the mutant protein of the application has a better purification performance. The method for determining purity can be any conventional method known in the art, including but not limited to SEC-HPLC method.

[0141] In yet another aspect, the storage stability of the mutant IL-2 proteins of the application can also be detected by methods known in the art. In the present application, a "stable" antibody refers to an antibody formulated in a buffer that retains an acceptable degree of physical stability and / or chemical stability after storage under a specific condition. In one embodiment, the buffer is PBS buffer at pH 7.4. In another embodiment, the buffer is histidine buffer at pH 6.5. In one embodiment, the stability of the antibody is detected after storage at 40°C for a period of time, for example, 2 weeks or more. The stability of the antibody can be determined by detecting the degree of purity reduction of the stored antibody by SEC-HPLC method or CE-SDS method.

[0142] 6. Screening method

[0143] In yet another aspect, the present application provides methods for obtaining IL-2 mutant proteins with improved properties and IL-2 mutant proteins obtained by the methods.

[0144] In one embodiment, the method of the present application comprises the following steps:

[0145] (1) introducing one or more mutations at the binding interface of IL-2 and IL-2Ra by mutation, and shortening the sequence of the B'C' loop region of IL-2 by mutation,

[0146] Preferably, the combination of CD25 binding region mutations as described before and / or the chimeric or truncated mutation of the B'C' loop sequence as described before is introduced;

[0147] (2) expressing the IL-2 mutant protein, for example, in the form of an Fc fusion (for example, Fc LALA fusion), in mammalian cells (for example, HEK293 or CHO cells);

[0148] - identifying muteins having one or more improved properties: (i) expression level and / or purity of the protein after purification (e.g. purity after one-step affinity chromatography, e.g. as measured by SEC-HPLC); (ii) reduced IL2Ra binding; (iii) enhanced IL2R beta binding.

[0149] In one embodiment, the method comprises: prior to performing the combination of mutations of step (1), identifying an IL-2 mutation that improves drugability (e.g. expression level and / or product stability and / or homogeneity, e.g. one-step Fc affinity chromatography purity). In a preferred embodiment, drugability of the mutein is improved by replacing or truncating the B’C’ loop to a shortened B’C’ loop.

[0150] In one embodiment, the method comprises: prior to performing the combination of mutations of step (1), identifying an IL-2 mutation that confers reduced (preferably abolished) IL-2Ra binding ability relative to wild-type IL-2. In one embodiment, CD25 binding affinity is reduced or abolished by performing an amino acid substitution or combination of substitutions at a site in the CD25 binding region.

[0151] As will be apparent to the skilled person, these mutations can be combined with mutations conferring further improved drugability or other improved properties to obtain IL-2 muteins having multiple improved properties.

[0152] In one embodiment, a (e.g. known) CD25 region mutation that results in reduced IL2Ra binding is combined with a (e.g. known) truncation and / or replacement mutation of the B’C’ loop region length that improves drugability, introduced into an IL-2 protein, and the properties are identified. In a preferred embodiment, the identification comprises reduced IL-2Ra binding and improved drugability (e.g. improved expression and / or purity, and / or product stability and / or homogeneity) relative to wild-type IL-2, optionally substantially unaltered or weakened or enhanced IL-2R beta binding affinity.

[0153] In some embodiments, the parent wild-type IL-2 protein used as a template for mutation is preferably at least 85%, or at least 90% or 95% identical to SEQ ID NO: 1, more preferably a human-derived IL-2 protein.

[0154] 7. Pharmaceutical compositions and pharmaceutical formulations

[0155] The present application also includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising an IL-2 mutein or fusion or immunoconjugate thereof and compositions comprising a polynucleotide encoding an IL-2 mutein or fusion or immunoconjugate thereof. These compositions can also optionally comprise suitable pharmaceutical adjuvants, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, as known in the art.

[0156] Pharmaceutically acceptable carriers suitable for use in the present application can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. See also "Handbook of Pharmaceutical Excipients," Fifth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago for a list of pharmaceutical excipients and their uses. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.

[0157] Pharmaceutical formulations comprising the IL-2 mutein, fusion or immunoconjugate of the present application can be prepared by mixing such protein having the desired degree of purity with one or more optional pharmaceutically acceptable adjuvants (Remington's Pharmaceutical Sciences, 16thEdition, Osol, A. Ed. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution. Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulation including a histidine-acetate buffer. In addition, sustained release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the protein, which matrices are in the form of shaped articles, e.g., films or microcapsules.

[0158] In one embodiment, the pharmaceutical composition of the application comprises a buffer at pH 6-8, such as a PBS buffer or a histidine buffer. In one embodiment, the PBS buffer is, for example, a PBS buffer at about pH 7.4. In one embodiment, the histidine buffer is a histidine buffer at about pH 6.5, for example, comprising 10 mM histidine, 5% sorbitol, 0.02% polysorbate 80. The pharmaceutical composition of the application preferably maintains storage stability in said buffer.

[0159] The pharmaceutical composition or formulation of the application can also comprise one or more other active ingredients required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it is desirable to further provide other anti-cancer active ingredients, such as chemotherapeutic agents, immune checkpoint inhibitors. Said active ingredients are suitably combined in amounts that are effective for the purpose intended.

[0160] 8. Combination products

[0161] In one aspect, the application also provides combination products comprising a mutein of the application or a fusion or immunoconjugate thereof, and one or more other therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective active agents, etc.). The combination products of the application can be used in the therapeutic methods of the application.

[0162] In some embodiments, the application provides combination products wherein the other therapeutic agent is, for example, a therapeutic agent such as an antibody effective to stimulate the immune response, thereby further enhancing, stimulating or upregulating the immune response in a subject.

[0163] In some embodiments, the combination product is used to prevent or treat cancer. In some embodiments, the cancer is, for example, a gastrointestinal cancer, such as rectal cancer, colon cancer, colorectal cancer. In some embodiments, the combination product is used to prevent or treat an infection, such as a bacterial infection, a viral infection, a fungal infection, a protozoal infection, etc.

[0164] 9. Therapeutic methods and uses

[0165] In the present context, the terms "individual" or "subject" are used interchangeably and refer to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the subject is a human.

[0166] As used herein, the term "treatment" refers to clinical intervention designed to alter the natural course of the disease in an individual receiving treatment. Desired effects of treatment include, but are not limited to, preventing occurrence or reoccurrence of disease, alleviating symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis.

[0167] In one aspect, the present application provides a method of stimulating the immune system of a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising an IL-2 mutein or fusion or immunoconjugate of the present application. The IL-2 mutein of the present application has high activity and selectivity for CD25 - CD122 + effector cells (cytotoxic CD8 + T cells and NK cells) and has reduced stimulation of CD25 + Treg cells. Thus, the IL-2 mutein of the present application can be used at low doses to stimulate the immune system of a subject.

[0168] Thus, in some embodiments, the present application relates to a method of enhancing the immune response of the body in a subject, the method comprising administering to the subject an effective amount of any of the IL-2 muteins, or fusions or immunoconjugates thereof, described herein. In some embodiments, the IL-2 mutein, or fusion or immunoconjugate thereof, of the present application is administered to a subject carrying a tumor, stimulating an anti-tumor immune response. In other embodiments, the antibody, or antigen binding portion thereof, of the present application is administered to a subject carrying an infection, stimulating an anti-infective immune response.

[0169] In another aspect, the present application relates to a method of treating a disease, such as cancer, in a subject, the method comprising administering to the subject an effective amount of any of the IL-2 muteins, or fusions or immunoconjugates thereof, described herein. The cancer can be in an early, intermediate or advanced stage or be metastatic. In some embodiments, the cancer can be, for example, a gastrointestinal cancer, such as rectal cancer, colon cancer, colorectal cancer.

[0170] In another aspect, the present application relates to a method of treating an infectious disease, such as a chronic infection, in a subject, the method comprising administering to the subject an effective amount of any of the IL-2 muteins, or fragments thereof, described herein, or an immunoconjugate, multispecific antibody, or pharmaceutical composition comprising the antibody or fragment. In one embodiment, the infection is a viral infection.

[0171] The mutant proteins of the application (and pharmaceutical compositions comprising the same or fusions or immunoconjugates thereof, and optionally additional therapeutic agents) can be administered by any suitable method, including parenterally, intrapulmonary, and intranasally, and, if local treatment is desired, intralesionally. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Depending on whether the medication is short- or long-term, it can be administered by any suitable route, for example by injection, for example intravenously or subcutaneously. Various dosing schedules are contemplated herein, including, but not limited to, single dosing or multiple dosing at multiple time points, bolus dosing, and pulse infusion.

[0172] For the prevention or treatment of disease, the appropriate dosage of the mutant proteins of the application (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

[0173] In yet another aspect, the application also provides the use of the IL-2 mutant proteins, compositions, immunoconjugates, fusions of the application in the manufacture of a medicament for the aforementioned methods (e.g., for therapy).

[0174] The following examples are set forth to assist in understanding the application. No limitation on the scope of the application is intended by the examples that follow. Examples

[0175] Example 1: Design and construction of the interleukin 2 point mutation library

[0176] Design of the interleukin 2 point mutation library

[0177] According to the crystal structure of the complex of interleukin 2 (IL-2) and its alpha receptor CD25 (IL-2Rα) (PDB: 1Z92) (as shown in Figure 1 , the IL-2 residues at the interaction sites were mutated according to Table 1. The original amino acid at each site accounted for 50% and the rest 50% was equally divided by the “mutated amino acids” in Table 1. The theoretical diversity of the library designed for the mutual binding sites of IL-2 and IL-2Rα was 3x8x8x9x6x6x3x6x6x5x6≈2.0x10 8 , the library was named IBYDL029 (Innoventbio Yeast Display Library).

[0178] Table 1. Mutation site table of IBYDL029 library

[0179] Site Amino acid residue Mutated amino acid Diversity 35 Lys (K) D, E 3 37 Thr (T) D, E, R, K, F, Y, W 8 38 Arg (R) D, E, F, Y, W, A, V 8 41 Thr (T) K, R, M, F, Y, W, Q, E 9 42 Phe (F) K, R, A, E, Q 6 43 Lys (K) E, D, F, Y, W 6 45 Tyr (Y) R, K 3 61 Glu (E) R, K, W, Y, L 6 62 Glu (E) R, K, W, Y, L 6 68 Glu (E) R, K, W, Y 5 72 Leu (L) R, K, F, Y, W 6

[0180] Construction of interleukin-2 point mutation library

[0181] Wild-type IL-2 (uniprot: P60568, aa21-153, C125S, referred to as IL-2 WT ) into the yeast display plasmid pYDC011 (the complete sequence of the plasmid is shown in Figure 9 ) between the two BamHI restriction sites. WT The sequence of is shown in SEQ ID NO: 1 in this application, and a C125S mutation was introduced at position 125 of the sequence to prevent the formation of disulfide-bridged IL-2 dimers. The specific steps of plasmid construction are as follows:

[0182] 1. Use primers AMP0210 and AMP0211 to express IL-2 WT Gene was used as template for amplification (primer sequences see Figure 3 );

[0183] 2. Plasmid pYDC011 was digested with BamHI (New England Biolab, Cat. No. R3136V) and then recovered from gel (QIAGEN Gel Extraction Kit, Cat. 28704);

[0184] 3. The amplified products and enzyme digestion products were recovered by 1% agarose gel;

[0185] 4. After recovery, perform in vitro homologous recombination using the One Step Cloning Kit (Vazyme Catalog No.: C113-02);

[0186] 5. The recombinant product was transformed into E. coli Top10 competent cells (Tian Gen, Cat. No.: CB104-02), spread on LB plates containing ampicillin resistance, and cultured at 37°C overnight;

[0187] 6. After sequencing and verification of the grown monoclonal colony, the correct plasmid was named pYDC035.

[0188] According to existing literature, IL-2 mutant IL-2 3X It does not bind to IL-2Rα, but maintains its binding affinity to IL-2Rβ (Rodrigo Vazquez-Lombardi et al., Nature Communications, 8:15373, DOI:10.1038 / ncomms15373). 3X Displayed on the yeast surface, used as a control. 3XThe sequence of the protein is shown in SEQ ID NO: 4. WT The same also contains the C125S mutation.

[0189] According to the library construction scheme in Table 1, the required primers (such as Figure 3 shown) and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0190] Amplification of IBYDL029 library DNA: 1. Using pYDC035 as a template, primers AMP0191 and AMP0200 amplified fragment 029-F. 2. Using pYDC035 as a template, primers AMP0201 and AMP0199 amplified fragment 029-R. 3. Gel-recovered fragments 029-F and 029-R were used as PCR templates, and primers AMP0191 and AMP0199 amplified the full-length fragment 029.

[0191] 100 μg of plasmid pYDC011 was digested with BamHI and recovered using a QIAGEN PCR Purification Kit (Cat. 28104) to obtain sufficient linearized plasmid. The linearized plasmid was mixed with library DNA at a ratio of 4 μg:12 μg. This mixture was then electroporated into the EBY100 yeast strain according to existing methods (Lorenzo Benatuil et al., An improved yeast transformation method for the generation of very large human antibody libraries. Protein Engineering, Design & Selection, vol. 23, no. 4, pp. 155–159, 2010). After electroporation, the library was serially diluted and plated on SD-Trp (TAKARA, Cat. No. 630309) plates. The number of colonies that grew was counted, yielding a library diversity of 4.2 × 10 IBYDL029. 8 , which is greater than the theoretical diversity of the library.

[0192] Example 2: Screening and identification of IL-2 point mutation library

[0193] Preparation and biotin labeling of IL-2Rα and IL-2Rβ proteins

[0194] Construction and transfection of expression plasmids

[0195] IL-2 receptor IL-2Ra (Uiprot: P01589, aa22-217) and IL-2Rβ (Uiprot: P14784, aa27-240) were tagged with an avi tag (GLNDIFEAQKIEWHE, the tag peptide can be biotinylated by BirA enzyme) and 6 histidine tags (HHHHHH) at the C-terminus of the sequence, respectively, and were constructed into pTT5 vector (Addgene) for expression of IL-2Ra and IL-2Rβ proteins. The sequences of the receptors constructed are shown in SEQ ID NO: 5 and 6.

[0196] The expression plasmid vectors constructed above were used to transiently transfect cultured HEK293-F (Invitrogen, Cat# R79007) cells using chemical transfection reagent polyethylenimine (PEI, Polysciences, Cat# 23966) according to the manufacturer's protocol.

[0197] Protein expression and purification

[0198] The cell culture supernatant of IL-2Ra and IL-2Rβ proteins was centrifuged at 4500 rpm for 30 min, and the cells were discarded. The supernatant was filtered with a 0.22 μm filter and further purified. Briefly, the nickel column (5 mL Histrap excel, GE, 17-3712-06) used for purification was soaked with 0.1 M NaOH for 2 h, then rinsed with 5-10 column volumes of ultrapure water to remove the alkali solution. Before purification, the purification column was equilibrated with 5 column volumes of binding buffer (20 mM Tris pH 7.4, 300 mM NaCl); the cell supernatant was passed through the equilibrated column; the column was washed with 10 column volumes of washing buffer (20 mM Tris pH 7.4, 300 mM NaCl, 10 mM imidazole) to remove non-specifically bound impurities; then the target protein was eluted with 3-5 column volumes of elution buffer (20 mM Tris pH 7.4, 300 mM NaCl, 100 mM imidazole). The collected protein was ultrafiltrated and concentrated and exchanged into PBS (Gibco, 70011-044), and then further separated and purified with superdex200 increase (GE, 10 / 300 GL, 10245605), and the monomer elution peak was collected. The column was equilibrated and eluted with PBS (Gibco, 70011-044). 100 μg of the purified protein sample was used to determine the protein purity using gel filtration chromatography column SW3000 (TOSOH Cat# 18675).

[0199] Biotinylation of IL-2Ra and IL-2Rβ proteins

[0200] IL-2Rα and IL-2Rβ proteins were labeled with biotin using an enzymatic method as follows: An appropriate amount of IL-2Rα and IL-2Rβ protein solution expressed and purified as above was taken, 1 / 10 (m / m) mass of His-BirA protein (uniprot: P06709) was added, and a final concentration of 2 mM ATP (sigma catalog number: A2383-10G), 5 mM MgCl2, 0.5 mM D-biotin (AVIDITY catalog number: K0717) was added; incubated at 30°C for 1 h, purified by Superdex 200 increase (GE, 10 / 300 GL, 10245605) to remove excess biotin and His-BirA; the purified sample was verified by Fortebio's Streptavidin (SA) sensor (PALL, 18-5019) to confirm successful biotin labeling. The biotin-labeled IL-2Rα and IL-2Rβ IH proteins obtained in this example are referred to as IL-2Rα-Biotin and IL-2Rβ IH-Biotin, respectively.

[0201] Screening of IL-2 mutant library mutant and staining identification

[0202] Screening of IL-2 that does not bind to IL-2Rα but binds to IL-2Rβ mutant

[0203] From the yeast-based IL-2 mutant display library IBYDL029, 2.0 x 1010 9 yeast cells were taken for culture and induction, where the diversity of the library was 2.0 x 1010 8 Due to the large diversity of the IBYDL029 library, the first round of screening used the MACS system of Miltenyi for magnetic bead cell sorting. First, 2 x 1010 9Yeast cells were incubated at room temperature for 30 minutes in FACS wash buffer (1× PBS containing 1% bovine serum albumin) containing 500 nM biotinylated IL-2Rβ (Acro Biosystems, labeled with EZ-Link Sulfo-NHS-LC-Biotin, referred to as IL-2Rβ-Biotin). Wash once with 50 ml of ice-cold FACS wash buffer, resuspend the cells in 10 ml of the same wash buffer, and incubate at 4°C for 15 minutes with 40 μl of streptavidin microbeads (Miltenyi biotec, Cat. No. 130-090-485). After centrifugation at 3000 rpm for 3 minutes, the supernatant was discarded and the cells were resuspended in 10 ml of FACS wash buffer. The cell solution was then applied to a Miltenyi LS column. After loading, the column was washed three times with 3 ml of FACS wash buffer. The Miltenyi LS column was removed from the magnetic area, eluted with 5 ml of growth medium, and the eluted yeast cells were collected and grown overnight at 30°C.

[0204] The library cells obtained after one round of screening were induced at 20°C for 24 hours to express IL-2 mutant , and the second round of sorting was performed using flow cytometry. Briefly, 3×10 7 Yeast cells were washed three times with FACS buffer and incubated in FACS buffer containing IL-2Rβ-biotin (300 nM) and anti-Flag antibody at room temperature for 30 minutes. After washing twice with FACS wash buffer, cells were mixed with FACS wash buffer containing SA-PE (phycoerythrin-conjugated streptavidin, eBioscience, Cat. No. 12-4317-87) and goat anti-mouse IgG conjugated to Alex Flour-647 (Thermo Fisher, Cat. No. A21235) and incubated at 4°C in the dark for 15 minutes. Cells were washed twice with pre-chilled FACS wash buffer, resuspended in 1 mL of buffer, and transferred to a filter-lined separation tube. Cells were sorted using MoFlo_XDP and grown overnight at 30°C. The third round of sorting was similar to the second round. After three rounds of screening, single clones were selected and sent for sequencing.

[0205] After three rounds of screening using IL-2Rβ-Biotin, 53 mutant sequences were obtained from the library IBYDL029.

[0206] IL-2 mutant Staining identification

[0207] Yeast cells containing a single mutant sequence after sequencing were induced by shaking at 20°C for 24 hours to express IL-2 mutant; respectively with its receptor IL-2Rα-Biotin, IL-2RβIH-Biotin staining, the specific steps are as follows:

[0208] I. IL-2 mutant Yeast cells displayed with IL-2Rα-Biotin staining analysis:

[0209] 1. Each sample takes 1 x 10 6 cells, centrifugal discard supernatant with FACS buffer wash once and then used;

[0210] 2. Add 100 μL containing 50 nM IL-2Rα-Biotin and Anti Flag antibody FACS buffer room temperature for 30 minutes;

[0211] 3. Pre-cooled FACS buffer 3000 rpm 4 ℃ centrifugal 3 min wash twice;

[0212] 4. Add 100 μL containing SA-PE, goat anti-mouse conjugated Alex Flour-647 FACS buffer, ice light incubation for 20 min;

[0213] 5. After washing twice with pre-cooled FACS buffer, resuspend the cells with 100 μL buffer, and analyze the IL-2 mutant binding level of IL-2Rα by flow cytometry (BD, ACCURI C6).

[0214] II. IL-2 mutant Yeast cells displayed with IL-2RβIH-Biotin staining analysis:

[0215] 1. Each sample takes 1 x 10 6 cells, centrifugal discard supernatant with FACS buffer wash once and then used;

[0216] 2. Add 100 μL containing IL-2RβIH-Biotin (30 nM-100 nM) and Anti Flag antibody FACS buffer room temperature for 30 minutes;

[0217] 3. The same as steps 3-5 above, analyze the IL-2 mutant binding level of IL-2Rβ.

[0218] From the results of flow cytometry: 53 IL-2 mutant and IL-2Rα binding average fluorescence signal intensity of IL-2 3X close, that is, none of the combination; IL-2Rβ binding average fluorescence signal intensity is stronger than IL-2 3X .

[0219] IL-2 mutant Expression of FC fusion protein and determination of its affinity to receptors

[0220] Construction of expression plasmid

[0221] IL-2 mutant The sequence was linked to FcLALA through two GGGGS residues and constructed into the pCDNA3.1 (Addgene) vector for expressing IL-2 mutant -FC fusion protein.

[0222] In addition, as a control, IL-2 WT ,IL-2 3X The gene sequence was linked to FcLALA through two GGGGS residues and constructed into pCDNA3.1 for expression of IL-2 WT -FC and IL-2 3X -FC fusion protein. The Fc used in this example and subsequent examples refers to the Fc of human IgG1 with mutations L234A and L235A (abbreviated as FcLALA, SEQ ID NO: 7).

[0223] Expression and purification of IL-2 and FC fusion protein

[0224] HEK293 cells were transfected with the vector encoding the fusion protein gene using chemical transfection. Transient transfection of cultured HEK293 cells was performed using the chemical transfection reagent PEI according to the manufacturer's protocol. Plasmid DNA and transfection reagent were prepared in a clean bench. 3 mL of Opti-MEM medium (Gibco catalog number: 31985-070) was added to a 50 mL centrifuge tube, followed by 30 μg of the corresponding plasmid DNA. The Opti-MEM medium containing the plasmid was filtered through a 0.22 μm filter. 90 μg of PEI (1 g / L) was then added and allowed to stand for 20 minutes. The DNA / PEI mixture was gently poured into 27 mL of HEK293 cells and mixed thoroughly. After incubation at 37°C, 8% CO₂ for 20 hours, VPA was added to a final concentration of 2 mM and 2% (v / v) Feed was added. Culture was continued for 6 days.

[0225] After cell culture, the cells were centrifuged at 13,000 rpm for 20 minutes, and the supernatant was collected and purified using prepacked Hitrap MabSelect Sure columns. The procedure was as follows: Before purification, the column was equilibrated with 5 column volumes of equilibration buffer (20 mM Tris, 150 mM NaCl, pH 7.2). The collected supernatant was passed through the column, and the column was then washed with 10 column volumes of equilibration buffer to remove nonspecifically bound proteins. The column was then rinsed with 5 column volumes of elution buffer (1 M sodium citrate, pH 3.5), and the eluate was collected. 80 μL of Tris (2 M Tris) was added per 1 mL of eluate, and the eluate was exchanged into PBS using an ultrafiltration concentrator. The concentration was then determined. 100 μg of the purified protein was adjusted to 1 mg / mL, and the purity of the IL-2-Fc dimer was determined using gel filtration chromatography. The results are shown in Table 2. Figure 4 The sequences of the IL-2 mutants of the present invention listed in Table 2 are shown.

[0226] Table 2. Expression level and purity of IL-2-FC in 293 cells

[0227] IL-2 Expression (mg / L) Purity (SEC-HPLC) IL-2 WT ]]> 18 45％ Mutant IL-2 3X ]] 34 70％ Mutant Y29A1 No expression N.D Mutant Y29A2 37 79％ Mutant Y29A5 12 73％ Mutant Y29A6 1 37％ Mutant Y29B2 77 88％ Mutant Y29C5 10 60％ Mutant Y29D2 17 80％ Mutant Y29D6 23 60％ Mutant Y30B1 20 70％ Mutant Y30B4 No expression N.D Mutant Y30D4 No expression N.D Mutant Y30E1 60 78％

[0228] IL-2 mutant Determination of affinity of -FC to its receptor

[0229] The IL-2 of the present invention was determined by biointerferometry (ForteBio) mutant The equilibrium dissociation constant (K) of FC and its receptor D ).

[0230] ForteBio affinity assay was performed according to the existing method (Estep, P et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013. 5 (2): p. 270-8). Briefly, the candidate IL-2 mutant -FC affinity to IL-2Rα and IL-2Rβ: the sensor was equilibrated offline in the assay buffer for 20 minutes, and then tested online for 120 seconds to establish a baseline; human biotinylated IL-2Rα or IL-2Rβ was loaded onto the SA sensor (PALL, 18-5019) for ForteBio affinity measurement; the sensor loaded with IL-2Rα-Biotin or IL-2RβIH-Biotin was placed in a 100 nM IL-2 mutant- FC in solution until plateau, after which the sensor was transferred to assay buffer for at least 2 minutes for on- and off-rate measurements. Kinetics were analyzed using a 1 : 1 binding model.

[0231] In experiments performed as described above for the assay, IL-2 mutant - FC to its receptor K D Values are shown in Table 3. As a control, IL-2 WT - FC to IL-2 3X - FC fusion protein to its receptor K D Values are also shown in Table 3.

[0232] Table 3. IL-2 mutant - FC to its receptor K D Values

[0233] IL-2 IL-2Rα avidity IL-2Rβ avidity IL-2 WT ]]> 1.0E-08 4.1E-08 IL-2 3X ]]> N.B 1.6E-08 Y29A2 Very weak 4.8E-10 Y29A5 N.B 3.4E-10 Y29A6 N.B 1.8E-09 Y29B2 N.B 3.4E-10 Y29C5 N.B 4.2E-10 Y29D2 Very weak 8.5E-11 Y29D6 N.B 1.9E-10 Y30B1 N.B 1.9E-10 Y30E1 N.B 5.9E-09

[0234] N.B. none binding

[0235] From the affinity data, it can be seen that all the above mutants obtained from the IBYDL029 library block the binding of IL-2Ra while maintaining the binding to IL-2Rb.

[0236] Example 3: Construction, screening and identification of IL-2 chimeric and truncated mutants

[0237] Design of IL-2 B’C’ loop chimeras and truncations

[0238] B’C’ loop: connecting sequence between B helix and C helix of IL-2 Figure 2A ), including 11 amino acids from A73-R83.

[0239] By comparing the crystal structure of IL-2 monomer (PDB: 1M47) and complex (PDB: 2ERJ), we found that the B’C’ loop is missing in the crystal structure of IL-2 monomer, which is due to the fact that the B’C’ loop is very active in solution and cannot form a relatively stable conformation.

[0240] By genetically engineering the B’C’ loop, we increase the stability of the B’C’ loop, and thus the stability of IL-2 and its affinity to IL-2R. Therefore we aligned the crystal structure of human IL15 (PDB: 2Z3Q) and found that its B’C’ loop is shorter and more stable Figure 2B). Therefore we designed an IL-2 chimeric molecule (L017) and 4 truncation molecules (L057~L060) (see Table 4).

[0241] Table 4. IL-2 B’C’ loop optimized sequences

[0242] Name B'C' loop sequence L 001 (IL-2 WT )]]> AQSKNFHLRPR L 017 (IL-2 hyb15BCL )]]> SGDASIH L 057 (IL-2 truncate1 )]]> AQSKNFH L 058 (IL-2 truncate2 )]]> AGSKNFH L 059 (IL-2 truncate3 )]]> AQSANFH L 060 (IL-2 truncate4 )]]> AQSANIH

[0243] Construction of expression plasmids

[0244] Wild type IL-2 (uniprot: P60568, aa21-153, C125S, referred to as IL-2 WT ), and IL-2 mutants IL-2 3X (R38D, K43E, E61R), B’C’ loop chimeras and truncations, linked to human IgG1 Fc (L234A, L235A, referred to as FcLALA, SEQ ID NO: 7) via a GSGS linker sequence, and constructed into a pTT5 vector for expression of the following proteins:

[0245] Protein name Structure SEQ ID NOs Y001 IL-2 WT -GSGS-FcLALA SEQ ID NO: 8 Y002 IL-2. 3X -GSGS-FcLALA SEQ ID NO: 9 Y017 IL-2 hyb15BCL -GSGS-FcLALA SEQ ID NO: 10 Y057 IL-2 truncate1 -GSGS-FcLALA SEQ ID NO: 11 Y058 IL-2 truncate2 -GSGS-FcLALA SEQ ID NO: 12 Y059 IL-2 truncate3 -GSGS-FcLALA SEQ ID NO: 13 Y060 IL-2 truncate4 -GSGS-FcLALA SEQ ID NO: 14

[0246] B’C’ loop chimeras (Y017) or truncations (Y057) were combined with library- screened mutant Y30E1 (K35E, T37E, R38E, F42A), linked to FcLALA via two GGGGS, and constructed into a pCDNA3.1 vector for expression of the following proteins. Among them, Y092 has a chimeric B’C’ loop sequence AGDASIH, and removes the potential N-glycosylation modification brought by the NLS at amino acid residues 71-73 in Y017; Y093 and Y094 introduce further amino acid substitutions K76A or K76D in the truncated loop sequence on the basis of Y089, aiming to improve the T cell activation activity of the B’C’ loop truncation; and Y144 increases T3A on the basis of Y092, aiming to remove the O-glycosylation modification at the N-terminus of IL2.

[0247] Protein name Structure SEQ ID NOs Y089 IL-2 .Y30E1.truncate1 -2*(G4S)-FcLALA SEQ ID NO: 15 Y092 <![CDATA[IL-2 .Y30E1.15BCL -2*(G4S)-FcLALA]]> SEQ ID NO: 16 Y093 IL-2 .Y30E1.truncate1.K76A -2*(G4S)-FcLALA]]> SEQ ID NO: 17 Y094 IL-2 .Y30E1.truncate1.K76D -2*(G4S)-FcLALA]]> SEQ ID NO: 18 Y144 IL-2 .Y30E1.15BCL.T3A -2*(G4S)-FcLALA SEQ ID NO: 19

[0248] In addition, IL-2 WT and IL-2 3X were also linked to FcLALA via two GGGGS, and constructed into a pCDNA3.1 vector for expression of the following proteins:

[0249] Protein name Structure SEQ ID NOs Y040 IL-2 .3X -2*(G4S)-FcLALA SEQ ID NO: 20 Y045 IL-2 WT -2*(G4S)-FcLALA SEQ ID NO: 21

[0250] The specific sequence information of the above protein molecules is shown in the sequence listing.

[0251] Expression and purification of IL-2 fusion proteins

[0252] The above protein molecules were expressed in 293 cells and CHO cells, respectively. The expression in HEK293 cells was performed according to the method for IL-2-Fc fusion protein expression in Example 2. The expression in CHO cells was performed as follows.

[0253] ExpiCHO cells (Invitrogen) were passaged according to the required cell volume, and the cell density was adjusted to 3.5 x 10 6 cells / ml one day before transfection. The cell density was detected on the day of transfection (about 8-10 x 10 6 cells / ml), and the viability reached more than 95%, and the cell density was adjusted to 6 x 10 TM cells / ml with ExpiCHO 6 Expression Medium (Gibco, Cat. No. A29100-01). The final volume was 8% (v / v) OptiPRO TM SFM (Gibco, Cat. No. 12309-019) was used as the transfection buffer, and the corresponding amount (0.8 μg / mL of cells) of plasmid was added, mixed, filtered with a 0.22 μm filter to remove bacteria, and ExpiFectamine TM CHO Transfection Kit (Gibco, Cat. No. A29130) reagent was added at a ratio of 3.2 μL / mL of cells. The complex of transfection reagent and plasmid DNA was incubated at room temperature for 1-5 min, then slowly added to the cells, and after 18 h of incubation at 37°C, 8% CO2, 0.6% (v / v) Enhancer and 30% (v / v) Feed were added, and the incubation was continued for 6 days.

[0254] After cell culture, the cell culture fluid was centrifuged at 13000 rpm for 20 min, and the supernatant was collected and purified using a pre-packed column Hitrap Mabselect Sure (GE, 11-0034-95). The operation was as follows: before purification, the filler column was equilibrated with 5 times the column volume of equilibration buffer (20 mM Tris, 150 mM NaCl, pH 7.2); the collected supernatant was passed through the column, and the filler column was washed with 10 times the column volume of equilibration buffer to remove non-specifically bound proteins; the filler was flushed with 5 times the column volume of elution buffer (100 mM sodium citrate, pH 3.5), and the eluate was collected. 80 μL of Tris (2 M Tris) was added to each 1 ml of eluate, which was concentrated using an ultrafiltration concentration tube (MILLIPORE, item number: UFC901096) to exchange into PBS buffer (Gibco, item number: 70011-044), and the concentration was determined. 100 μg of the purified protein was taken, the concentration was adjusted to 1 mg / mL, and the protein purity was determined using a gel filtration chromatography column SW3000 (TOSOH item number: 18675).

[0255] The fusion proteins of the B’C’loop chimeras (Y017) and truncations (Y057 / 058 / 059) showed a great improvement in both expression level in HEK293 cells and one-step affinity chromatography purity compared to the fusion protein of wild-type IL-2 (Y001). The results are shown in Table 5 below.

[0256] Table 5. Expression level and purity of IL-2 mutants in HEK293

[0257] Protein name Expression (mg / L) Purity (SEC-HPLC) Y001 16.35 44.74％ Y002 23.92 69.85％ Y017 54.47 93.45％ Y057 52.36 92.77％ Y058 49.86 99.09％ Y059 36.52 86.95％ Y060 21.20 66.33％

[0258] The fusion proteins comprising the B’C’loop chimeras (Y092 / 144) and truncations (Y089 / 093 / 094) and further mutations Y30E1 showed a great improvement in both expression level in CHO cells and one-step affinity chromatography purity compared to the fusion protein of wild-type IL-2 (Y045). The results are shown in Table 6 below.

[0259] Table 6. Expression level and purity of IL-2 mutants in CHO

[0260] Protein name Expression (mg / L) Purity (SEC-HPLC) Y040 20.28 40.75％ Y045 2.44 50.85％ Y089 249.6 99.11％ Y092 118.8 99.07％ Y093 95.2 98.98％ Y094 142 99.02％ Y144 114 97.78％

[0261] Affinity determination of IL-2 mutant Fc fusion proteins to their receptors

[0262] The affinity K D values of the following mutant proteins were determined according to the ForteBio affinity determination method described in Example 2. The results are shown in Table 7 below.

[0263] Table 7. K D values of IL-2 mutants with IL-2Ra and IL-2R

[0264] IL-2 mutant ]]> IL-2Rα avidity IL-2Rβ avidity Y30E1 N.B. 6.45E-09 Y089 N.B. 4.53E-09 Y093 N.B. 1.96E-09 Y094 N.B. 3.35E-09 Y092 N.B. 3.27E-09 Y144 N.B. 1.55E-09

[0265] N.B. none binding

[0266] From the above data, it can be seen that: 1) Y30E1 and other point mutation molecules obtained by screening the yeast library can block the binding of IL-2Ra; 2) B’C’ loop chimeric molecules and truncated molecules not only increase the expression and purity of the molecules, but also increase the affinity of the molecules to IL-2R; 3) the combination of Y30E1 and B’C’ loop mutant molecules can not only block IL-2Ra, but also increase the expression and purity of the molecules, and improve the binding activity to IL-2R.

[0267] Example 4: In vitro functional experiment of IL-2 mutant

[0268] IL-2 WT IL-2 has higher affinity to IL-2Ra than to IL-2R and IL-2Ry, and can preferentially bind to IL-2Ra on the cell surface, then recruit IL-2Rby, and release downstream p-STAT5 signal through IL-2Rby to stimulate T cell and NK cell proliferation. Since Treg cells have IL-2Ra on their surface, and effector T cells and NK cells do not have IL-2Ra on their surface, under normal circumstances, IL-2 WT will preferentially stimulate Treg cell proliferation, down-regulating immune response. IL-2 mutant does not bind to IL-2Ra, eliminating the preference for stimulating Treg cell proliferation, and stimulating T cell and NK cell proliferation, effectively increasing the number of effector T cells and NK cells, and improving the anti-tumor effect.

[0269] In this example, the activation of p-STAT5 signal of primary human CD8 mutant T cells by each IL-2 + -FC was detected to verify the removal of the activation bias of CD25 + cells by each mutant, and to screen mutants with strong activation effect on CD25 - cells. The specific steps are as follows:

[0270] 1. Revive PBMC cells:

[0271] a) Take PBMC cells (Allcells, PB005F, 100M) from liquid nitrogen and quickly place them in a 37°C water bath to revive PBMC cells;

[0272] b) Add 10 mL pre-warmed X-VIVO 15 medium (Lonza Cat. No. 04-418Q) with 5% human AB serum (GemCell Cat. No. 100-512) and 1% DNAse (STEMCELL Cat. No. 07900) to the flask.

[0273] c) Add 20 mL of medium to resuspend the cells and incubate in a 37°C carbon dioxide incubator overnight.

[0274] c) Add 20 mL of medium to resuspend the cells and incubate in a 37°C carbon dioxide incubator overnight.

[0275] 2. Purification of human CD8 + T cells:

[0276] a) Aspirate the cell suspension from step 1 and centrifuge to discard the supernatant.

[0277] b) Add 1 mL Robosep buffer (STEMCELL Cat. No. 20104) with 100 μL human AB serum and 100 μL human CD8 + T cell purification kit (Invitrogen Cat. No. 11348D) negative selection antibody cocktail to resuspend the cells.

[0278] c) Mix well and incubate at 4°C for 20 minutes, shaking every 5 minutes.

[0279] d) After incubation, add 10 mL Robosep buffer and centrifuge to wash twice.

[0280] e) Meanwhile, take 1 mL magnetic microspheres (human CD8 + T cell purification kit) and add 7 mL Robosep buffer to the magnetic stand for 1 minute to discard the supernatant and pre-wash the magnetic microspheres.

[0281] f) Add 1 mL Robosep buffer to resuspend the microspheres and cells respectively, mix well and incubate at room temperature for 30 minutes.

[0282] g) After incubation, add 6 mL Robosep buffer to the magnetic stand for 1 minute and collect the supernatant.

[0283] h) Collect the supernatant again and place it on the magnetic stand for 1 minute.

[0284] i) Centrifuge to discard the supernatant, resuspend with pre-warmed T medium and adjust the density to 1 x 10 6 / mL.

[0285] j) Take 1 / 3 of the cells for later CD25 expression stimulation and incubate the remaining cells in a 37°C carbon dioxide incubator overnight.

[0286] 3. Stimulation of CD8 + T cells expressing CD25:

[0287] a) Take 1 / 3 of CD8 + T cells purified in Step 2, add magnetic microspheres coated with anti-human CD3 / CD28 antibody (GIBCO Cat. No. 11131D) at a ratio of 3:1 (cells to microspheres);

[0288] b) Incubate at 37°C in a CO2incubator for 3 days;

[0289] c) Wash 2 times with 10 mL of medium;

[0290] d) Adjust cell density to 1 x 10 6 / mL with medium and incubate at 37°C in a CO2incubator for 2 days.

[0291] 4. Detection of cell purity and expression level:

[0292] a) Use anti-human CD8-PE (Invitrogen Cat. No. 12-0086-42), anti-human CD25-PE (eBioscience Cat. No. 12-0259-42), and isotype control antibody (BD Cat. No. 556653) to detect CD8 and CD25 on cells;

[0293] b) Cells in Step 2 are CD8 + CD25 - T cells, and cells in Step 3 are CD8 + CD25 + T cells.

[0294] 5. Detection of EC mutant of each IL-2 + -FC on CD8 - CD25 50 T cell activation of p-STAT5 signal:

[0295] a) Take CD8 + CD25 - T cells and seed 1 x 10 5 cells per well in a 96-well U-bottom plate (Costar Cat. No. CLS3799-50EA);

[0296] b) Add 100 μL of each IL-2 mutant -FC, commercial IL-2 (R&D Cat. No. 202-IL-500), IL-2 WT -FC, IL-2 3X-FC, the highest concentration was 266.7 nM, followed by 4-fold serial dilutions for a total of 12 gradients, and incubated in a 37°C incubator for 20 min;

[0297] c) Add 55.5 μL of 4.2% formaldehyde solution and fix at room temperature for 10 minutes;

[0298] d) Centrifuge and discard the supernatant. Add 200 μL of ice-cold methanol (Fisher Catalog No.: A452-4) to resuspend the cells and incubate at 4°C for 30 minutes.

[0299] e) Centrifuge and discard the supernatant. Wash three times with 200 μL staining buffer (BD Catalog No. 554657).

[0300] f) Add 200 μL of permeabilization / fixation buffer (BD Catalog No.: 51-2091KZ) containing anti-p-STAT5-AlexFlour647 (BD Catalog No.: 562076, 1:200 dilution) and incubate at room temperature in the dark for 3 hours;

[0301] g) Wash cells three times with staining buffer, resuspend cells in 100 μL of staining buffer, and perform flow cytometry analysis.

[0302] h) The EC curve of p-STAT5 signal was prepared with the concentration of IL-2 molecules as the horizontal axis and the intermediate fluorescence value of AlexFlour647 as the vertical axis. 50 Value, the result is Figure 5A and shown in Table 8.

[0303] 6. Detection of IL-2 mutant -FC vs. CD8 + CD25 + T cell activation of ECs by p-STAT5 signaling 50 :

[0304] a) Take CD8 + CD25 + T cells were collected at 1 × 10 per well 5 Cells were plated in 96-well U-bottom culture plates;

[0305] b) Prepare ECs for p-STAT5 signaling in the same manner as in steps 5bh. 50 Value, the result is Figure 5B and shown in Table 8.

[0306] Table 8. IL-2 mutants activate CD25 + / - EC50 and ratio of p-STAT5 signaling in T cells

[0307]

[0308] The experimental results show that: 1) the EC50 of Y089, Y092, Y093 and Y094 of the B'C' loop mutants of Y30E1 in activating p-STAT5 signal in CD25-CD8+ T cells are lower than Y30E1, which shows that the B'C' loop optimization can improve the activation of the molecule on CD25-CD8+ T cells, which is consistent with the IL-2Rβ affinity data; 2) from the results of CD25-EC50 / CD25+EC50 ratio, compared with Y045 (IL-2 WT-Fc), the mutant proteins Y30E1, Y089, Y092, Y093 and Y094 significantly reduced the bias of activation on CD25+ cells, which shows that Y30E1 mutant safely blocks the binding of CD25.

[0309] Example 5: Stability of IL-2 mutants

[0310] The stabilities of Y089, Y092 and Y094 stored in PBS buffer (pH 7.4, Gibco, Cat No. 10010-023) or histidine buffer (10 mM histidine, 5% sorbitol, 0.02% polysorbate 80, hydrochloric acid to adjust pH 6.5) were evaluated for 7 days and 14 days respectively in a constant temperature incubator (Zhicheng SHP-150) at 40°C. The detection indexes are the purity of IL-2 mutant proteins detected by SEC-HPLC and CE-SDS respectively.

[0311] Table 9a. Stability results of IL-2 mutants at 40°C environment detected by SEC-HPLC

[0312] Sample name / buffer Purity at 40°C / 0 days Purity at 40°C / 7 days Purity at 40°C / 14 days Y089 / PBS 99.80％ 99.41％ 99.32％ Y089 / histidine 99.64％ 99.23％ 99.23％ Y092 / PBS 99.57％ 99.63％ 99.55％ Y092 / histidine 99.51％ 97.49％ 97.55％ Y094 / PBS 99.62％ 99.47％ 99.33％ Y094 / histidine 99.58％ 99.22％ 98.96％

[0313] Table 9b. Stability results of IL-2 mutants at 40°C environment detected by CE-SDS

[0314]

[0315] The results show that Y089, Y092 and Y094 all show excellent stability after being placed in PBS buffer and histidine buffer for 14 days.

[0316] Example 6: Anti-tumor efficacy of IL-2 mutant molecules in vivo

[0317] In order to prove the in vivo efficacy of IL-2 mutant molecules, the anti-tumor efficacy of IL-2 mutant molecules (Y092) of the application was determined by inoculating Balb / c mice with CT26 cells (mouse colon cancer cell line, ATCC). SPF grade female Balb / c mice (18-20 g, purchased from Zhejiang Vintoon Lihua Experimental Animal Technology Co., Ltd.) were used in the experiment, and the qualified certificate number was NO. 1811230011.

[0318] CT26 cells were routinely subcultured for subsequent in vivo experiments. Cells were collected by centrifugation, and CT26 cells were dispersed in PBS (1x) to prepare a cell suspension with a cell concentration of 2.5x10 6 A CT26 tumor-bearing mouse model was established by subcutaneously inoculating 0.2 ml of the cell suspension into the right flank region of a Balb / c mouse on day 0.

[0319] The tumor volume of each mouse was measured 7 days after tumor cell inoculation, and the mice were grouped (6 mice per group). The dosing amounts and methods are shown in Table 10.

[0320] Table 10: Grouping, dosing amounts, and methods for in vivo experiments

[0321] Group Dose Frequency Route of administration h-IgG* 0.5 mg / kg Q7D x 2 i.p. Y092 0.004 mg / kg Q7D x 2 i.p. Y092 0.02 mg / kg Q7D x 2 i.p. Y092 0.1 mg / kg Q7D x 2 i.p. Y092 0.5 mg / kg Q7D x 2 i.p.

[0322] *: h-IgG is an isotype control antibody purchased from Equitech-Bio, batch number 161206-0656.

[0323] h-IgG and Y092 were used at concentrations of 10 mg / ml and 6.4 mg / ml, respectively, and were administered every 7 days for a total of 2 times (Q7Dx2). The mice were monitored for tumor volume and body weight 2-3 times per week, as shown in Tables 5, 6, and 7, and the monitoring was continued until day 21. Figure 6A 、 6B The relative tumor inhibition rate (TGI%) was calculated on day 21 after inoculation, using the following formula: TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume before administration of the control group). Tumor volume measurement: The maximum long axis (L) and the maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated using the following formula: V = L * W 2 / 2. The body weight was measured using an electronic balance.

[0324] The tumor inhibition rate results are shown in Table 11: on day 21 after inoculation, the single-agent inhibition rates of Y092, 0.004 mg / kg, Y092, 0.02 mg / kg, Y092, 0.1 mg / kg, and Y092, 0.5 mg / kg were 3.7%, 16.2%, 43.8%, and 53.5%, respectively, as compared with h-IgG. The results show that the modified IL2 molecule (Y092) has an anti-tumor effect and has a dose effect. The results of the detection of the body weight of the mice Figures 6B-6C ) show that a decrease of more than 10% in body weight occurred in the high-dose group (Y092, 0.5 mg / kg) during the period from inoculation to day 21, while the mice in the other groups did not experience a decrease of more than 5% in body weight. No deaths occurred in the mice of the administration groups.

[0325] Table 11. Tumor inhibition rate at day 21

[0326]

[0327]

[0328] Example 7: Anti-tumor efficacy of IL-2 mutant molecules in vivo

[0329] We further mutated Y092 (T3A) to reduce the glycosylation of the molecule. To demonstrate the in vivo efficacy of the IL-2 mutant molecules, we used C57 mice inoculated with MC38 cells (a mouse colon cancer cell line, ATCC) to determine the anti-tumor efficacy of the IL-2 mutant molecules (Y144) of the present application. SPF grade female C57 mice (15-18 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used in the experiment, and the license number was NO. 1100111911070497.

[0330] The MC38 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, resuspended in PBS (1x) to prepare a cell suspension with a concentration of 5x10 6 On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of C57 mice to establish a MC38 tumor-bearing mouse model.

[0331] The tumor volume of each mouse was measured 7 days after tumor cell inoculation, and the mice were grouped (6 mice per group). The dosages and administration methods are shown in Table 12.

[0332] Table 12: Grouping, dosages and administration methods of in vivo experiments

[0333] Group Dose Frequency Route of administration h-IgG* 1 mg / kg Q7D x 3 i.p. Y144 0.5 mg / kg Q7D x 3 i.p.

[0334] *: h-IgG is an isotype control antibody purchased from Equitech-Bio, batch number 161206-0656.

[0335] The concentrations of h-IgG and Y144 used were 10 mg / ml and 0.5 mg / ml, respectively, and the administration was performed every 7 days for a total of 3 times (Q7Dx3). The mice were administered on days 7, 14 and 21 after MC38 cell inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Table 13. Figure 7A, 7B and 7C, monitoring was ended after 24 days. The relative tumor growth inhibition (TGI%) was calculated at day 24 after inoculation, according to the following formula: TGI% = 100% * (tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - tumor volume of control group before administration). Tumor volume measurement: the maximum long axis (L) and the maximum wide axis (W) of the tumor were measured by vernier caliper, and the tumor volume was calculated according to the following formula: V = L * W 2 Body weight measurement: the body weight of the mice was measured by electronic balance.

[0336] The tumor growth inhibition results showed that the single-agent inhibition rate of Y144 was 30.05% at day 24 after inoculation, compared with the h-IgG, 1 mg / kg group. The results of the detection of the body weight of the mice Figure 7C ) showed that there was no significant difference in the body weight of the mice at day 24 after inoculation.

[0337] SEQUENCE LISTING

[0338]

[0339]

[0340]

[0341] SEQUENCE LISTING <110> Sinobiological Pharmaceuticals (Suzhou) Co., Ltd. <120> Interleukin 2 mutants and uses thereof <130> PF 210146 PCT <160> 26 <170> PatentIn version 3.3 <210> 1 <211> 133 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 1 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gin Met lie Leu Asn Gly lie Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gin Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gin Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu lie Ser Asn lie Asn Val lie Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr lie Val Glu Phe Leu Asn Arg Trp lie Thr Phe Ser Gin Ser lie 115 120 125 lie Ser Thr Leu Thr 130 <210> 2 <211> 153 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 2 Met Tyr Arg Met Gin Leu Leu Ser Cys lie Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser Thr Leu Thr 145 150 <210> 3 <211> 133 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 3 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 4 <211> 133 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 4 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 5 <211> 217 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 5 Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys 1 5 10 15 Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg 20 25 30 Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly 35 40 45 Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser 50 55 60 Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln 65 70 75 80 Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp 85 90 95 Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn 100 105 110 Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr 115 120 125 Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu 130 135 140 Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gin Pro Gin 145 150 155 160 Leu lie Cys Thr Gly Glu Met Glu Thr Ser Gin Phe Pro Gly Glu Glu 165 170 175 Lys Pro Gin Ala Ser Pro Glu Gly Arg Pro Glu Ser Glu Thr Ser Cys 180 185 190 Leu Val Thr Thr Gly Leu Asn Asp lie Phe Glu Ala Gin Lys lie Glu 195 200 205 Trp His Glu His His His His His His His 210 215 <210> 6 <211> 235 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 6 Ala Val Asn Gly Thr Ser Gin Phe Thr Cys Phe Tyr Asn Ser Arg Ala 1 5 10 15 Asn lie Ser Cys Val Trp Ser Gin Asp Gly Ala Leu Gin Asp Thr Ser 20 25 30 Cys Gin Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gin Thr Cys 35 40 45 Glu Leu Leu Pro Val Ser Gin Ala Ser Trp Ala Cys Asn Leu lie Leu 50 55 60 Gly Ala Pro Asp Ser Gin Lys Leu Thr Thr Val Asp lie Val Thr Leu 65 70 75 80 Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala lie Gin 85 90 95 Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro lie Ser Leu 100 105 110 Gln Val Val His Val Glu Thr His Arg Cys Asn lie Ser Trp Glu lie 115 120 125 Ser Gin Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu Ala Arg 130 135 140 Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu 145 150 155 160 Lys Gin Lys Gin Glu Trp lie Cys Leu Glu Thr Leu Thr Pro Asp Thr 165 170 175 Gln Tyr Glu Phe Gin Val Arg Val Lys Pro Leu Gin Gly Glu Phe Thr 180 185 190 Thr Trp Ser Pro Trp Ser Gin Pro Leu Ala Phe Arg Thr Lys Pro Ala 195 200 205 Ala Leu Gly Lys Asp Thr Gly Leu Asn Asp lie Phe Glu Ala Gin Lys 210 215 220 Ile Glu Trp His Glu His His His His His His His 225 230 235 <210> 7 <211> 227 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 7 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 8 <211> 364 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 8 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu Gin Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gin Met lie Leu Asn Gly lie Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gin Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gin Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu lie Ser Asn lie Asn Val lie Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr lie Val Glu Phe Leu Asn Arg Trp lie Thr Phe Ser Gin Ser lie 115 120 125 lie Ser Thr Leu Thr Gly Ser Gly Ser Asp Lys Thr His Thr Cys Pro 130 135 140 Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe 145 150 155 160 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 165 170 175 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 180 185 190 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 195 200 205 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 210 215 220 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 225 230 235 240 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 245 250 255 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 260 265 270 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 275 280 285 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 290 295 300 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 305 310 315 320 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin 325 330 335 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 340 345 350 Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 355 360 <210> 9 <211> 364 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 9 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu Gin Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gin Met lie Leu Asn Gly lie Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gin Cys Leu Glu Arg Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gin Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu lie Ser Asn lie Asn Val lie Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr lie Val Glu Phe Leu Asn Arg Trp lie Thr Phe Ser Gin Ser lie 115 120 125 lie Ser Thr Leu Thr Gly Ser Gly Ser Asp Lys Thr His Thr Cys Pro 130 135 140 Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe 145 150 155 160 Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val 165 170 175 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 180 185 190 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 195 200 205 Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 210 215 220 Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 225 230 235 240 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 245 250 255 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 260 265 270 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 275 280 285 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 290 295 300 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 305 310 315 320 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 325 330 335 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 340 345 350 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 355 360 <210> 10 <211> 360 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 10 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu Gin Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gin Met lie Leu Asn Gly lie Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gin Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ser Gly Asp Ala Ser lie His Asp 65 70 75 80 Leu lie Ser Asn lie Asn Val lie Val Leu Glu Leu Lys Gly Ser Glu 85 90 95 Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr lie Val Glu 100 105 110 Phe Leu Asn Arg Trp lie Thr Phe Ser Gin Ser lie lie Ser Thr Leu 115 120 125 Thr Gly Ser Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 130 135 140 Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 145 150 155 160 Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val 165 170 175 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 180 185 190 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin 195 200 205 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin 210 215 220 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 225 230 235 240 Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro 245 250 255 Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 260 265 270 Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 275 280 285 Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr 290 295 300 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 305 310 315 320 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe 325 330 335 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys 340 345 350 Ser Leu Ser Leu Ser Pro Gly Lys 355 360 <210> 11 <211> 360 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 11 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu Gin Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gin Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gin Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gin Ser Lys Asn Phe His Asp 65 70 75 80 Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu 85 90 95 Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu 100 105 110 Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu 115 120 125 Thr Gly Ser Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 130 135 140 Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 145 150 155 160 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 165 170 175 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 180 185 190 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 195 200 205 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 210 215 220 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 225 230 235 240 Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro 245 250 255 Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 260 265 270 Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 275 280 285 Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr 290 295 300 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 305 310 315 320 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe 325 330 335 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys 340 345 350 Ser Leu Ser Leu Ser Pro Gly Lys 355 360 <210> 12 <211> 360 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 12 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu Gin Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gin Met lie Leu Asn Gly lie Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gin Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gly Ser Lys Asn Phe His Asp 65 70 75 80 Leu lie Ser Asn lie Asn Val lie Val Leu Glu Leu Lys Gly Ser Glu 85 90 95 Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr lie Val Glu 100 105 110 Phe Leu Asn Arg Trp lie Thr Phe Ser Gin Ser lie lie Ser Thr Leu 115 120 125 Thr Gly Ser Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 130 135 140 Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 145 150 155 160 Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val 165 170 175 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 180 185 190 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin 195 200 205 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin 210 215 220 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 225 230 235 240 Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro 245 250 255 Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 260 265 270 Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 275 280 285 Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr 290 295 300 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 305 310 315 320 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe 325 330 335 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys 340 345 350 Ser Leu Ser Leu Ser Pro Gly Lys 355 360 <210> 13 <211> 360 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 13 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu Gin Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gin Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gin Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gin Ser Ala Asn Phe His Asp 65 70 75 80 Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu 85 90 95 Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu 100 105 110 Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu 115 120 125 Thr Gly Ser Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 130 135 140 Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 145 150 155 160 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 165 170 175 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 180 185 190 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 195 200 205 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 210 215 220 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 225 230 235 240 Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro 245 250 255 Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 260 265 270 Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 275 280 285 Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr 290 295 300 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 305 310 315 320 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe 325 330 335 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys 340 345 350 Ser Leu Ser Leu Ser Pro Gly Lys 355 360 <210> 14 <211> 360 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 14 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu Gin Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gin Met lie Leu Asn Gly lie Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gin Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gin Ser Ala Asn lie His Asp 65 70 75 80 Leu lie Ser Asn lie Asn Val lie Val Leu Glu Leu Lys Gly Ser Glu 85 90 95 Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr lie Val Glu 100 105 110 Phe Leu Asn Arg Trp lie Thr Phe Ser Gin Ser lie lie Ser Thr Leu 115 120 125 Thr Gly Ser Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 130 135 140 Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 145 150 155 160 Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val 165 170 175 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 180 185 190 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin 195 200 205 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin 210 215 220 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 225 230 235 240 Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro 245 250 255 Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 260 265 270 Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 275 280 285 Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr 290 295 300 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 305 310 315 320 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe 325 330 335 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys 340 345 350 Ser Leu Ser Leu Ser Pro Gly Lys 355 360 <210> 15 <211> 366 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 15 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu Gin Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gin Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Glu Leu Glu Glu Met Leu Thr Ala Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gin Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gin Ser Lys Asn Phe His Asp 65 70 75 80 Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu 85 90 95 Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu 100 105 110 Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu 115 120 125 Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr 130 135 140 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 145 150 155 160 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 165 170 175 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 180 185 190 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 195 200 205 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 210 215 220 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 225 230 235 240 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser 245 250 255 Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro 260 265 270 Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val 275 280 285 Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly 290 295 300 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 305 310 315 320 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 325 330 335 Gln Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 340 345 350 Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 355 360 365 <210> 16 <211> 366 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 16 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu Gin Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gin Met lie Leu Asn Gly lie Asn Asn Tyr Lys 20 25 30 Asn Pro Glu Leu Glu Glu Met Leu Thr Ala Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gin Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gly Asp Ala Ser lie His Asp 65 70 75 80 Leu lie Ser Asn lie Asn Val lie Val Leu Glu Leu Lys Gly Ser Glu 85 90 95 Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr lie Val Glu 100 105 110 Phe Leu Asn Arg Trp lie Thr Phe Ser Gin Ser lie lie Ser Thr Leu 115 120 125 Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr 130 135 140 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 145 150 155 160 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 165 170 175 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 180 185 190 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 195 200 205 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 210 215 220 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 225 230 235 240 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 245 250 255 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 260 265 270 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 275 280 285 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 290 295 300 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 305 310 315 320 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 325 330 335 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 340 345 350 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 355 360 365 <210> 17 <211> 366 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 17 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Glu Leu Glu Glu Met Leu Thr Ala Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Ala Asn Phe His Asp 65 70 75 80 Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu 85 90 95 Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu 100 105 110 Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu 115 120 125 Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr 130 135 140 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 145 150 155 160 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 165 170 175 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 180 185 190 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 195 200 205 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 210 215 220 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 225 230 235 240 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser 245 250 255 Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro 260 265 270 Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val 275 280 285 Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly 290 295 300 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 305 310 315 320 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 325 330 335 Gln Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 340 345 350 Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 355 360 365 <210> 18 <211> 366 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 18 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu Gin Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gin Met lie Leu Asn Gly lie Asn Asn Tyr Lys 20 25 30 Asn Pro Glu Leu Glu Glu Met Leu Thr Ala Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gin Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gin Ser Asp Asn Phe His Asp 65 70 75 80 Leu lie Ser Asn lie Asn Val lie Val Leu Glu Leu Lys Gly Ser Glu 85 90 95 Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr lie Val Glu 100 105 110 Phe Leu Asn Arg Trp lie Thr Phe Ser Gin Ser lie lie Ser Thr Leu 115 120 125 Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr 130 135 140 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 145 150 155 160 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 165 170 175 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 180 185 190 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 195 200 205 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 210 215 220 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 225 230 235 240 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 245 250 255 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 260 265 270 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 275 280 285 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 290 295 300 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 305 310 315 320 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 325 330 335 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 340 345 350 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 355 360 365 <210> 19 <211> 366 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 19 Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Glu Leu Glu Glu Met Leu Thr Ala Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gly Asp Ala Ser Ile His Asp 65 70 75 80 Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu 85 90 95 Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu 100 105 110 Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu 115 120 125 Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr 130 135 140 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 145 150 155 160 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 165 170 175 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 180 185 190 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 195 200 205 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 210 215 220 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 225 230 235 240 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 245 250 255 Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro 260 265 270 Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val 275 280 285 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 290 295 300 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 305 310 315 320 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 325 330 335 Gln Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 340 345 350 Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gin Lys 355 360 365 <210> 20 <211> 370 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 20 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu Gin Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gin Met lie Leu Asn Gly lie Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gin Cys Leu Glu Arg Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gin Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu lie Ser Asn lie Asn Val lie Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr lie Val Glu Phe Leu Asn Arg Trp lie Thr Phe Ser Gin Ser lie 115 120 125 lie Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 130 135 140 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 145 150 155 160 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 165 170 175 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 180 185 190 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 195 200 205 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 210 215 220 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 225 230 235 240 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 245 250 255 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 260 265 270 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 275 280 285 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 290 295 300 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 305 310 315 320 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 325 330 335 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 340 345 350 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 355 360 365 Gly Lys 370 <210> 21 <211> 370 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 21 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 130 135 140 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 145 150 155 160 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 165 170 175 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 180 185 190 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 195 200 205 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 210 215 220 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys ​​​​​​​​​​225 230 235 240 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 245 250 255 Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr 260 265 270 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu 275 280 285 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 290 295 300 Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 305 310 315 320 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 325 330 335 Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His 340 345 350 Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 355 360 365 Gly Lys 370 <210> 22 <211> 129 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 22 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Glu Leu Glu Glu Met Leu Thr Ala Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Asp 65 70 75 80 Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu 85 90 95 Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu 100 105 110 Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu 115 120 125 Thr <210> 23 <211> 129 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 23 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Glu Leu Glu Glu Met Leu Thr Ala Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gly Asp Ala Ser Ile His Asp 65 70 75 80 Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu 85 90 95 Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu 100 105 110 Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu 115 120 125 Thr <210> 24 <211> 129 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 24 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Glu Leu Glu Glu Met Leu Thr Ala Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Ala Asn Phe His Asp 65 70 75 80 Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu 85 90 95 Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu 100 105 110 Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu 115 120 125 Thr <210> 25 <211> 129 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 25 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Glu Leu Glu Glu Met Leu Thr Ala Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Asp Asn Phe His Asp 65 70 75 80 Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu 85 90 95 Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu 100 105 110 Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu 115 120 125 Thr <210> 26 <211> 129 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 26 Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Glu Leu Glu Glu Met Leu Thr Ala Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gly Asp Ala Ser Ile His Asp 65 70 75 80 Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu 85 90 95 Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu 100 105 110 Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu 115 120 125 Thr

Claims

1. An IL-2 mutant protein, wherein the mutant protein, compared to wild-type IL-2, comprises a mutation: (i) mutations in the binding interface between IL-2 and CD25; and (ii) a shortened B'C' loop region, wherein the B'C' loop region is a sequence connecting amino acid residues aa72 and aa84, Wherein the amino acid positions are numbered according to SEQ ID NO: 1, wherein the mutation (i) is the mutation combination K35E+T37E+R38E+F42A; wherein said mutation (ii) is: (a) substitution of aa74 to aa83 of the B'C' loop region with GDASIH; or (b) Truncation of the sequence from aa74 to aa83 of the B'C' loop region to a sequence selected from the group consisting of QSKNFH, QSANFH, and QSDNFH.

2. The IL-2 mutein of claim 1, wherein the mutation (ii) is a substitution of aa74 to aa83 in the B'C' loop region to the sequence GDASIH.

3. An IL-2 mutant protein, wherein the mutant protein, relative to wild-type IL-2, comprises: (i) mutation combination K35E+T37E+R38E+F42A; and (ii) a shortened B'C' loop sequence selected from the group consisting of: -AQSKNFH; -AQSANFH; -AQSDNFH; or -AGDASIH, Wherein the amino acid positions are numbered according to SEQ ID NO: 1, The B'C' loop region is a sequence connecting amino acid residues aa72 and aa84.

4. An IL-2 mutant protein, wherein the mutant protein, relative to wild-type IL-2, comprises: (i) mutation combination K35E+T37E+R38E+F42A; (ii) shortened B'C' loop sequence AGDASIH: and (iii) mutation T3A, Wherein the amino acid positions are numbered according to SEQ ID NO: 1, The B'C' loop region is a sequence connecting amino acid residues aa72 and aa84.

5. The IL-2 mutant protein of claim 1, wherein the mutant protein comprises an amino acid sequence selected from SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO:

26.

6. The IL-2 mutant protein according to any one of claims 1 to 5, wherein compared to wild-type IL-2, the mutant protein has at least one, two or all three properties selected from the group consisting of: - reduced or abolished IL-2Rα binding, - enhanced IL-2Rβ binding; and - Improved expression and / or purity when expressed as an Fc fusion protein in mammalian cells.

7. The IL-2 mutant protein according to any one of claims 1 to 5, wherein the mutant protein has one or more of the following properties compared to wild-type IL-2: - have reduced binding affinity for the high-affinity IL-2R receptor IL-2Rαβγ; - have increased binding affinity for the intermediate-affinity IL-2R receptor IL-2Rβγ; - Reduce activation of CD25+ cells; - Reduced stimulation of IL-2-mediated signaling in CD25+ cells; - Eliminate or reduce the bias of IL-2 to preferentially activate CD25+ cells; - Reduce the downregulation of immune responses induced by IL-2 via Treg cells; - Maintain or enhance activation of CD25- cells; and - Leads to increased proliferation and activation of CD25- effector T cells and NK cells.

8. The IL-2 mutant protein according to any one of claims 1 to 5, wherein the mutant protein has one or more of the following properties: - Anti-tumor effects in vivo; - no significant toxicity upon in vivo administration; and - Storage stable.

9. The IL-2 mutant protein according to any one of claims 1 to 5, wherein the wild-type IL-2 is human IL-2 or consists of the amino acid sequence of SEQ ID NO:

1.

10. An IL-2 mutein fusion protein comprising the IL-2 mutein according to any one of claims 1 to 9.

11. The fusion protein of claim 10, wherein the IL-2 mutein is fused to an Fc antibody fragment.

12. The fusion protein of claim 11, wherein the IL-2 mutein is fused to Fc via a linker. The fusion protein of claim 12 , wherein the linker is GSGS or 2x(G4S). The fusion protein of claim 11 , wherein the Fc is human IgG1 Fc. The fusion protein of claim 11 , wherein the Fc comprises a mutation that reduces or eliminates binding of the Fc to FcγR. The fusion protein of claim 15 , wherein the mutation is L234A+L235A.

17. The fusion protein of claim 10, wherein the fusion protein consists of an amino acid sequence selected from the group consisting of: SEQ ID NO:15-19.

18. An immunoconjugate comprising the IL-2 mutein according to any one of claims 1 to 9 and an antigen binding molecule.

19. The immunoconjugate of claim 18, wherein the antigen binding molecule is an immunoglobulin molecule.

20. The immunoconjugate of claim 19, wherein the immunoglobulin molecule is an IgG molecule.

21. The immunoconjugate of claim 18, wherein the antigen binding molecule is an antibody or antibody fragment.

22. The immunoconjugate of claim 18, wherein the antigen binding molecules are Fab molecules and scFv molecules.

23. The immunoconjugate of claim 18, wherein the antigen binding molecule specifically binds to an antigen presented on a tumor cell or in the tumor environment.

24. The immunoconjugate of claim 23, wherein the antigen is selected from the group consisting of fibroblast activation protein (FAP), the A1 domain of tenascin C (TNC A1), the A2 domain of tenascin C (TNC A2), the extradomain B (EDB) of fibronectin, carcinoembryonic antigen (CEA), and melanoma-associated chondroitin sulfate proteoglycan (MCSP).

25. An isolated polynucleotide encoding the IL-2 mutein of any one of claims 1 to 9, the fusion protein of any one of claims 10 to 17, or the immunoconjugate of any one of claims 18 to 24.

26. An expression vector comprising the polynucleotide of claim 25.

27. A host cell comprising the polynucleotide of claim 25 or the vector of claim 26.

28. The host cell of claim 27, wherein the host cell is a mammalian cell or a yeast.

29. The host cell of claim 27, wherein the host cell is a HEK293 cell or a CHO cell.

30. A method for producing an IL-2 mutein or a fusion protein or immunoconjugate thereof, comprising culturing the host cell according to any one of claims 27 to 29 under conditions suitable for expressing the IL-2 mutein or fusion protein or conjugate.

31. A pharmaceutical composition comprising the IL-2 mutant protein according to any one of claims 1 to 9, the fusion protein according to any one of claims 10 to 17, or the immunoconjugate according to any one of claims 18 to 24, and a pharmaceutically acceptable carrier.

32. The pharmaceutical composition of claim 31, wherein the pharmaceutical composition comprises a phosphate buffer or a histidine buffer.

33. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition has a pH of 6.0-7.

6.

34. Use of an IL-2 mutein fusion protein in the preparation of a medicament, wherein the IL-2 mutein fusion protein comprises the IL-2 mutein of claim 4 fused to an Fc antibody fragment, and wherein the medicament is for treating colon cancer or infection.

35. A method for obtaining an IL-2 mutant protein, comprising the following steps: (a) introducing the mutation combination K35E+T37E+R38E+F42A at the binding interface of IL-2 and IL-2Rα, and shortening the B'C' loop sequence by mutation in the IL-2. The B'C' loop region is mutated as follows: (a) Replacement of aa74 to aa83 of the B'C' loop region with the B'C' loop sequence of IL15 GDASIH; or (b) truncation of the sequence from aa74 to aa83 of the B'C' loop region to a sequence selected from the group consisting of: QSKNFH, QSANFH or QSDNFH, (b) expressing the IL-2 mutant protein in mammalian cells as an Fc fusion protein; (c) Identifying mutant proteins with one or more of the following improved properties: (i) improved expression and / or protein purity after purification; (ii) reduced IL-2Rα binding; (iii) enhanced IL-2Rβ binding.

36. The method of claim 35, wherein step (a) comprises introducing into IL-2: (i) mutation combination K35E+T37E+R38E+F42A; and (ii) a shortened B'C' loop sequence selected from the group consisting of: AQSKNFH; AQSANFH; AQSDNFH; or AGDASIH.

37. The method of claim 35, wherein step (a) comprises introducing into IL-2: (i) mutation combination K35E+T37E+R38E+F42A; (ii) a shortened B'C' loop sequence AGDASIH; and (iii) T3A mutation.

38. A method for modifying the B'C' loop region of an IL-2 protein, the method comprising: (a) substitution of aa74 to aa83 in the B'C' loop region with GDASIH; or (b) Truncation of aa74 to aa83 of the B'C' loop region to a sequence selected from the group consisting of QSKNFH, QSANFH, or QSDNFH.

39. An IL-2 protein modified by the method of claim 38.

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