Novel interleukin 2 and uses thereof

Abstract

To provide a novel interleukin-2 (IL-2) mutant protein, a fusion protein and an immunoconjugate comprising the IL-2 mutant protein, a nucleic acid encoding the IL-2 mutant protein, and a vector and a host cell comprising the nucleic acid, and further provide a method for preparing the IL-2 mutant protein, a pharmaceutical composition comprising the IL-2 mutant protein, and therapeutic use of the mutant protein.SOLUTION: An IL-2 mutant protein comprises, as compared to a wild-type IL-2, at least one mutation introducing one or more glycosylation motifs N-X-S / T at specific amino acid positions, wherein X is any amino acid other than proline, and preferably X is an amino acid same as the amino acid at the corresponding position in the wild-type IL-2 or a conservatively substituted residue thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to novel interleukin-2 (IL-2) muteins and uses thereof. In particular, the present invention relates to IL-2 muteins having improved properties compared to wild-type IL-2, such as improved drug formability, reduced IL-2Rα receptor binding ability, and / or increased IL-2Rβ receptor binding ability. The present invention also provides fusion proteins, immunoconjugates comprising said IL-2 muteins, and nucleic acids encoding said IL-2 muteins, vectors and host cells comprising said nucleic acids. The present invention further provides methods for preparing said IL-2 muteins, pharmaceutical compositions comprising said IL-2 muteins, and therapeutic uses of said muteins. [Background technology]

[0002] Interleukin-2 (IL-2), also known as T cell growth factor (TCGF), stimulates activated T cells, especially CD4 + It is a pluripotent cytokine mainly produced by T helper cells. In eukaryotic cells, human IL-2 (uniprot:P60568) is synthesized as a precursor polypeptide of 153 amino acids, and after removing the N-terminal 20 amino acids, mature secretory IL-2 is produced. The sequences of IL-2 of other species are also disclosed, see NCBI Ref Seq No. NP032392 (mouse), NP446288 (rat) or NP517425 (chimpanzee).

[0003] Interleukin-2 has four antiparallel amphipathic α-helices that 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 functions through three different receptors: interleukin-2 receptor α (IL-2Rα; CD25), interleukin-2 receptor β (IL-2Rβ; CD122), and interleukin-2 receptor γ (IL-2Rγ; CD132). IL-2Rβ and IL-2Rγ are important for IL-2 signaling, whereas IL-2Rα (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 combining IL-2R α, β, and γ (IL-2αβγ) is a high-affinity receptor for IL-2 (KD approximately 10 pM), the dimeric receptor consisting of β and γ (IL-2βγ) is an intermediate-affinity receptor (KD approximately 1 nM), and the IL-2 receptor formed only from the α subunit is a low-affinity receptor.

[0004] Immune cells express dimeric or trimeric IL-2 receptors. The dimeric receptor binds the cytotoxic CD8 + Expressed on T cells and natural killer (NK) cells, the trimeric receptor binds primarily to activated lymphocytes and CD4 + CD25 + FoxP3 + It is expressed on suppressive regulatory T cells (Tregs) (Byman, O. and Sprent. J. Nat. Rev. Immunol. 12, 180-190(2012)). Resting effector T cells and NK cells lack CD25 on their cell surface and are therefore relatively insensitive to IL-2. On the other hand, Treg cells consistently express the highest levels of CD25 in the body, and therefore IL-2 normally preferentially stimulates the proliferation of Treg cells.

[0005] IL-2 mediates multiple actions in immune responses through binding to IL-2 receptors on different cells. On the one hand, as an immune system stimulant, IL-2 stimulates the proliferation and differentiation of T cells, induces the production of cytotoxic T lymphocytes (CTLs), promotes the proliferation and differentiation of B cells and the synthesis of immunoglobulins, and stimulates the production, proliferation, and activation of natural killer (NK) cells, and is therefore approved for use as an immunotherapeutic agent in the treatment of cancer and lentiviral infections. On the other hand, IL-2 inhibits the immunosuppressive CD4 + CD25 + It can promote 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)), mediate activation-induced cell death (AICD), and participate in the establishment and maintenance of immune tolerance to self- and tumor antigens (Lenardo et al., Nature 353:858 (1991)), thus causing tumor tolerance by AICD and immune suppression by activated Treg cells in patients. Furthermore, high-dose IL-2 can cause vascular leak syndrome (VLS) in patients. IL-2 has been shown to induce pulmonary edema by directly 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-mentioned problems associated with IL-2 immunotherapy, it has been proposed to reduce the toxicity and / or increase the efficacy of IL-2 therapy by altering the selectivity or preference of IL-2 for various receptors. For example, conjugates of IL-2 monoclonal antibodies and IL-2 can be used to target IL-2 to cells expressing CD122, rather than CD25, thereby inhibiting the expression of CD122. highIt has been proposed that this induces preferential expansion of the IL-2 population and enhances the in vivo IL-2 therapeutic effect (Boyman et al., Science 311, 1924-1927(2006)). Oliver AST et al.(US2018 / 0142037) propose introducing the triple mutation F42A / Y45A / L72G at amino acid residue positions 42, 45 and 72 of IL-2 to reduce its affinity for the IL-2Rα receptor. Aron M. Levin et al.(Nature, Vol 484, p529-533, DOI:10.1038 / nature10975) have proposed the introduction of a triple mutation F42A / Y45A / L72G at amino acid residue positions 42, 45 and 72 of IL-2, which contains the quintuple mutations L80F / R81D / L85V / I86V / I92F, called "superkine". H9 proposed that this enhances IL-2Rβ binding and thereby CD25 - Rodrigo Vazquez-Lombardi et al. (Nature Communications, 8:15373, DOI: 10.1038 / ncomms15373) have developed a triple mutant human IL-2 mutein IL-2 3X The protein has residue mutations R38D-K43E-E61R at amino acid residue positions 38, 43 and 61, respectively, so that the mutant protein does not bind to IL-2Rα, but binds to CD25 - It has a weak effect on activating cells and CD25 + The bias of activation towards cells still exists. Rodrigo Vazquez-Lombardi et al. also proposed to improve the pharmacological properties of interleukins by preparing interleukin-2-Fc fusion proteins, but the expression level of these fusion proteins is low and they are prone to forming aggregates.

[0007] Given the role of IL-2 in immune regulation and disease, there remains a need in the art to develop new IL-2 molecules with improved properties, particularly IL-2 molecules that are advantageous for production, purification, and that exhibit improved pharmacological properties.

[0008] The present invention meets the above needs by providing novel IL-2 muteins that have improved druggability properties and / or improved IL-2 receptor selectivity / polarization compared to wild-type IL-2.

[0009] Thus, in one aspect, the invention provides novel IL-2 muteins. In some embodiments, the IL-2 muteins of the invention have one or more of the following properties:

[0010] (i) improved drug formability, particularly improved expression and / or purification when expressed in mammalian cells; (ii) reducing or eliminating binding to IL-2Rα; (iii) Enhanced binding to IL-2Rβ.

[0011] In some embodiments, the invention provides IL-2 muteins that contain a mutated glycosylation motif introduced into the binding interface between IL-2 and IL-2Rα. In other embodiments, the invention provides IL-2 muteins that contain deletions and / or substitutions in the B'C' loop region of IL-2 to have a truncated loop sequence. In yet other embodiments, the invention provides IL-2 muteins that have both a mutated glycosylation motif and a truncated B'C' loop sequence.

[0012] Furthermore, the present invention 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 said nucleic acids, as well as methods for producing the IL-2 muteins, fusion proteins and immunoconjugates of the present invention.

[0013] Furthermore, the present invention also provides methods for treating diseases using the IL-2 muteins and fusions and immunoconjugates of the present invention, as well as methods and uses for stimulating the immune system of a subject. In some embodiments, the methods of the present invention involve the induction of CD25 in a subject. - This results in potent activation and expansion of effector T cells and NK cells. In yet another embodiment, the method of the present invention can effectively reduce the immune downregulatory effect of IL-2 mediated by Treg cells.

[0014] The present invention will be further described below with reference to the drawings and embodiments. However, these drawings and embodiments should not be considered as limiting the scope of the present invention, and any modifications that may be easily conceived by those skilled in the art will fall within the spirit of the present invention and the scope of the appended claims. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows the crystal structures of IL-2 and IL-2Rα (PDB: 1Z92) (A) and a schematic diagram of the structure of the IL-2 glycosylation-modified protein (B). [Diagram 2] FIG. 2 shows the IL-2 crystal structure (PBD:2ERJ) (A) and the B'C' loop structure superpose of human and mouse IL-2 and human IL15 (B). [Diagram 3] FIG. 3 shows the sample HPLC purity detection profile after IL-2Rα purification. [Figure 4] FIG. 4 shows the sample HPLC purity detection profile after IL-2Rβ purification. [Diagram 5] Figure 5 shows the signal curves of several selected and constructed IL-2 mutant-FCs activating p-STAT5 on CD8+ CD25- / CD25+ T cells. [Figure 6]FIG. 6 shows the mature protein sequence (SEQ ID NO:26) of human interleukin-2 (IL-2) and its amino acid residue numbers, illustrating exemplary IL-2 glycosylation mutants and IL-2 chimeras and truncated B'C' loop mutants. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] 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. For purposes of the present invention, the following terms are defined:

[0017] The term "about" when used in conjunction with numerical values ​​is meant to include numerical values ​​in a range from 5% below the stated numerical value as a lower limit to 5% above the stated numerical value as an upper limit.

[0018] The term "and / or" should be understood to refer to any one of the selectable options or a combination of any two or more of the selectable options.

[0019] As used in this application, the term "comprises" or "comprises" means including the stated element, integer or step, but not excluding other elements, integers or steps. In this application, when the term "comprises" or "comprises", unless otherwise specified, it should include the case of consisting of the stated element, integer or step. For example, when referring to an IL-2 mutein that "comprises" or "comprises" a certain mutation or combination of mutations, it is also intended to encompass IL-2 muteins having only that mutation or combination of mutations.

[0020] As used herein, wild-type "interleukin-2" or "IL-2" refers to the parent IL-2 protein, preferably a naturally occurring IL-2 protein, which is the template into which a mutation or combination of mutations of the invention is introduced, e.g., native IL-2 protein from human, mouse, rat, non-human primate, including unprocessed (e.g., without removal of the signal peptide) and processed (e.g., with removal of the signal peptide) forms. The full-length native human IL-2 sequence, including the signal peptide, is shown in SEQ ID NO:29, and the sequence of the mature protein is shown in SEQ ID NO:30. Additionally, this term also includes naturally occurring IL-2 allelic and splice variants, isotypes, homologs, and species homologs. This expression also includes variants of native IL-2, for example said variants may have at least 95%-99% or more identity with native IL-2, or may have no more than 1-10 or no more than 1-5 amino acid mutations (particularly conservative amino acid substitutions) and have substantially the same IL-2Rα and / or IL2Rβ binding affinity as the native IL-2 protein. Thus, in some embodiments, wild-type IL-2 refers to a native human IL-2 protein that, compared to the native IL-2 protein, has an amino acid mutation that does not affect its binding to the IL-2 receptor, for example the mutation C125S introduced at position 125 (uniprot: P60568), which belongs to the wild-type IL-2 of the present invention. An example of a wild-type human IL-2 protein comprising the C125S mutation is shown in SEQ ID NO: 26. In some embodiments, the wild-type IL-2 sequence has at least 85%, 95%, or even at least 96%, 97%, 98%, or 99% or even higher amino acid sequence identity to the amino acid sequence of SEQ ID NO:26 or 29 or 30.

[0021] As used herein, amino acid mutations may be amino acid substitutions, deletions, insertions, and additions. Any combination of substitutions, deletions, insertions, and additions may be performed to obtain a final mutein construct with desired properties (e.g., reduced IL-2Rα binding affinity). Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxy termini of the polypeptide sequence. For example, an alanine residue may be deleted at full-length human IL-2 position 1. In some embodiments, the preferred amino acid mutation is an amino acid substitution. In other embodiments, the preferred amino acid mutation is an amino acid deletion. In some embodiments, mutations are introduced at specific mutated amino acid positions described herein to obtain an IL-2 mutein with an altered glycosylation motif. In some embodiments, mutations are introduced at specific mutated amino acid positions described herein to obtain an IL-2 mutein with a truncated B'C' loop sequence.

[0022] In the present invention, when referring to the amino acid positions of the IL-2 protein, the wild-type human IL-2 protein of SEQ ID NO: 26 (IL-2 WT The amino acid position of an IL-2 protein or polypeptide is determined by reference to the amino acid sequence (shown in FIG. 6) of the IL-2 protein or polypeptide (also referred to as F42). By performing an amino acid sequence alignment (e.g., using BLAST, the Basic Local Alignment Search Tool available at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp& PAGE_TYPE=BlastSearch&LINK_LOC=blasthome, alignment is performed using default parameters), corresponding amino acid positions on other IL-2 proteins or polypeptides (including full-length sequences or truncated fragments) are identified. Thus, in the present invention, an amino acid position in an IL-2 protein or polypeptide is an amino acid position according to SEQ ID NO:26, unless otherwise specified. For example, reference to "F42" refers to the phenylalanine residue F at position 42 of SEQ ID NO:26, or the amino acid residue aligned at the corresponding position on other IL-2 polypeptide sequences.

[0023] In the present specification, when referring to IL-2 muteins, the mutations are described as follows: Amino acid substitutions are represented as [original amino acid residue / position / substituted amino acid residue]. For example, the amino acid at position 35 is substituted with asparagine (N) and can be represented as 35N, and if the original amino acid residue at position 35 is lysine, it can also be represented as K35N. When the substituted residue is represented by X, for example 36X means that the amino acid at position 36 can be substituted with any residue, and when X has a specific residue, the position is substituted with a defined specific X residue. However, expressions indicating only the original residue and position, for example L36 and T37 in the mutant glycosylation motif K35N-L36-T37 of the present invention, mean that no mutation occurs at said positions 36 and 37, i.e. positions 36 and 37 retain the original residues L and T.

[0024] As used herein, "percent sequence identity" can be determined by comparing two optimally aligned sequences within a comparison window. Preferably, sequence identity is determined over the entire length of the reference sequence (e.g., SEQ ID NO: 26). Sequence alignment methods for comparison are well known in the art. Suitable 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 BLAST analysis is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). For the purposes of this application, percent identity is usually determined using the BLAST 2.0 algorithm set as default parameters.

[0025] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the biological function of a protein / polypeptide that contains the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A typical conservative amino acid substitution refers to the replacement of one amino acid with another amino acid that has similar chemical properties (e.g., charge or hydrophobicity). The following six pairs each contain amino acids that can be typically conservatively substituted for each other: 1) alanine (A), serine (S), threonine (T), 2) aspartic acid (D), glutamic acid (E), 3) asparagine (N), glutamine (Q), 4) arginine (R), lysine (K), 5) isoleucine (I), leucine (L), methionine (M), valine (V), and 6) phenylalanine (F), tyrosine (Y), tryptophan (W). For example, a wild-type IL-2 protein may have, or may only have, conservative amino acid substitutions relative to any of SEQ ID NOs: 26, 29 or 30. Further for example, a mutant IL-2 protein of the invention may have, or may only have conservative amino acid substitutions relative to an IL-2 mutant protein sequence specifically set forth herein (e.g., any of SEQ ID NOs: 31-50).

[0026] "Affinity" or "binding affinity" can be used to reflect the interbinding capacity of an interaction between members of a binding pair. The affinity of a molecule X for its binding partner Y is expressed as the equilibrium dissociation constant (K D ), and the equilibrium dissociation constant is given by the dissociation rate constant and the association rate constant (k dis and k on (wherein the avidity is the ratio of the avidity to the avidity of the antibody. The binding affinity is measured by routine methods known in the art. One particular method for measuring affinity is by Biolayer Interferometry (BLI) technology as described herein.

[0027] As used herein, an antibody binding molecule is a polypeptide molecule capable of specifically binding to an antigen, such as an immunoglobulin molecule, an antibody or an antibody fragment, such as an Fab fragment and an scFv fragment.

[0028] As used herein, antibody Fc fragment refers to the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region, and can include native sequence Fc fragments and variant Fc fragments. In one embodiment, the human IgG heavy chain Fc fragment extends from Cys226 or from Pro230 of the heavy chain to the carboxyl terminus. In other embodiments, the C-terminal lysine (Lys447) of the Fc-fragment may or may not be present. In other embodiments, the Fc fragment may include mutations, such as L234A / L235A mutations. Unless otherwise stated in this application, the numbering of amino acid residues in the Fc fragment is according to the EU numbering system, also referred to as the EU index. For example, see Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0029] Various aspects of the invention are described in further detail in the following sections.

[0030] 1. IL-2 Muteins of the Invention The present invention provides, in one embodiment, novel IL-2 muteins with improved druggability properties and / or improved IL-2 receptor selectivity / preference.

[0031] Advantageous Biological Properties of the IL-2 Muteins of the Invention IL-2 protein functions by inducing signal transduction by interacting with the IL-2 receptor. Wild-type IL-2 exhibits different affinities for different IL-2 receptors. IL-2β and IL-2γ receptors, which have low affinity for wild-type IL-2, are expressed by resting effector cells (CD8 + IL-2Rα, which has a high affinity for wild-type IL-2, was expressed on regulatory T cells (Treg) and activated effector cells. Due to its high affinity, wild-type IL-2 preferentially binds to IL-2Rα on the cell surface, recruits IL-2Rβγ, and releases downstream p-STAT5 signals via IL-2Rβγ to stimulate Treg and activated effector cells. Thus, without being bound by theory, reducing or eliminating the affinity of IL-2 for the IL-2Rα receptor may be a mechanism by which IL-2 can bind to CD25 + This would reduce the bias towards preferentially activating T cells and reduce the immune downregulatory effects of IL-2 mediated Treg cells. Without wishing to be bound by theory, maintaining or enhancing affinity for the IL-2Rβ receptor may promote CD8 + It preserves or enhances the activation and thereby immunostimulatory effects of IL-2 on effector cells such as cytotoxic T cells and NK cells.

[0032] The inventors have found that by introducing one or more specific N-glycosylation motifs into the binding interface between IL-2 and the IL-2Rα receptor, it is possible to improve the expression and / or purity of IL-2 muteins and / or reduce the binding of IL-2 muteins to IL-2Rα. Furthermore, the inventors have found that by replacing the B'C' loop sequence of IL-2 itself with a short B'C' loop sequence from another interleukin cytokine, such as IL-15, or by truncating the B'C' loop sequence of IL-2 itself, it is possible to increase the expression and / or purity of IL-2 and at the same time increase its affinity for IL-2Rβ.

[0033] Thus, the present invention provides IL-2 muteins having improved properties, which may have one or more improved properties compared to wild-type IL-2, for example selected from the group of: (i) improved expression and / or purity when expressed in mammalian cells, (ii) reduced or eliminated binding to the IL-2Rα receptor, and / or (iii) enhanced binding to the IL-2Rβ receptor.

[0034] In some embodiments, the IL-2 muteins of the invention have one or more improved properties compared to wild-type IL-2, for example selected from the group consisting of: improved IL-2 activity;

[0035] (1) reducing or eliminating binding affinity to the IL-2Rα receptor; (2) enhanced binding affinity to the IL-2Rβ receptor; (3) reduced binding affinity to the high-affinity IL-2R receptor (IL-2Rαβγ); (4) increased binding affinity to the intermediate affinity IL-2R receptor (IL-2Rβγ); (5) CD25 + Cells (especially activated CD8 + Activation of IL-2 signaling in T cells and Treg cells, particularly the reduction in the ability to activate STAT5 phosphorylation signals, (6)IL-2-mediated CD25 + Cells (especially activated CD8 + Decreased activation and proliferation of T cells and Treg cells, (7) reducing or eliminating the bias of IL-2 to preferentially stimulate the proliferation of Treg cells; (8) Reduction of the immune downregulatory effect of Treg cells under IL-2 induction, (9) In particular, CD25 - Cells (especially CD25 - Maintaining or enhancing activation of T effector cells and NK cells, (10) IL-2-mediated increased activation and proliferation of effector T cells and NK cells; (11) Improved immune stimulating effect (12) Improved antitumor effect.

[0036] In some embodiments, the IL-2 muteins of the invention have property (1) above, and preferably further have one or more, particularly all of the properties selected from the group consisting of (3) and (5)-(8), more preferably further have one or more, particularly all of the properties selected from the group consisting of (2) and (9)-(12). In some embodiments, the IL-2 muteins of the invention have property (2) above, and preferably further have one or more, particularly all of the properties selected from the group consisting of (9)-(12), more preferably further have one or more, particularly all of the properties selected from the group consisting of (1), (3) and (5)-(8).

[0037] In some preferred embodiments, the IL-2 muteins of the present invention further have one or more properties of reduced in vivo toxicity mediated by binding of IL-2 to the high affinity receptor IL-2Rαβγ compared to wild-type IL-2.

[0038] In some embodiments, the IL-2 muteins of the present invention have improved drug formability properties, e.g., when expressed in mammalian cells, such as H293T cells, preferably when expressed as an Fc fusion protein, have one or more properties selected from the group consisting of: (i) better expression levels than wild-type IL-2 protein, (ii) better stability than wild-type IL-2 protein, and (iii) easier purification to higher protein purity.

[0039] In some embodiments of the invention, the IL-2 muteins of the invention show increased expression levels compared to wild-type IL-2. In some embodiments of the invention, the increased expression occurs in a mammalian cell expression system. The expression level can be determined by any suitable method that allows quantitative or semi-quantitative analysis of the amount of recombinant IL-2 protein in the cell culture supernatant (preferably the supernatant after one-step affinity chromatography purification). For example, the amount of recombinant IL-2 protein in the sample can be evaluated by Western blot or ELISA. In some embodiments, the IL-2 muteins of the invention increase the expression amount in mammalian cells by at least 1.1-fold, or at least 1.5-fold, or at least 2-fold, 3-fold, or 4-fold or more compared to wild-type IL-2.

[0040] In some embodiments, the IL-2 mutein-Fc fusions of the invention exhibit better stability, e.g., less tendency to form aggregates, than wild-type IL-2 protein fusions, as shown by measuring the purity of the purified protein after Protein A affinity chromatography. In some embodiments, protein purity is detected by SEC-HPLC techniques. In some preferred embodiments, after primary Protein A affinity chromatography purification, the purity of the IL-2 mutein product of the invention can reach 70% or more, or 80% or more, or 90% or more.

[0041] In some embodiments, the IL-2 muteins of the invention are similar to wild-type IL-2 (e.g., the IL-2 shown in SEQ ID NO:26). WT ), the binding affinity to the IL-2Rα receptor is reduced by at least 5-fold, at least 10-fold, or at least 25-fold, in particular at least 30-fold, 50-fold or 100-fold or more. In a preferred embodiment, the mutants of the invention do not bind to IL-2 receptor alpha. The binding affinity is determined by the equilibrium dissociation constant (KD ) can be determined by measuring the monovalent binding affinity of an IL-2 mutein (e.g., in the form of an Fc fusion) to the receptor IL-2Rα or IL-2Rβ by biolayer interferometry (BLI) technology. In some embodiments, the monovalent binding affinity of an IL-2 mutein (e.g., in the form of an Fc fusion) to the receptor IL-2Rα or IL-2Rβ is measured by BLI technology.

[0042] In some embodiments, the IL-2 muteins of the invention are similar to wild-type IL-2 (e.g., the IL-2 shown in SEQ ID NO:26). WT The binding affinity to the IL-2Rβ receptor is enhanced by at least 5-fold, at least 10-fold, or at least 25-fold, in particular at least 30-fold, 50-fold, or 100-fold, and more preferably at least 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, or 550-fold or more, compared to the IL-2Rβ receptor. The binding affinity is determined by the equilibrium dissociation constant (K D ) can be determined by measuring the binding affinity K of the IL-2 mutein of the present invention to the IL-2Rβ receptor by biolayer interferometry (BLI) technology. D The value is less than 10.0E-07M in the form of an IL-2-Fc fusion protein in a BLI measurement (e.g., a BLI measurement described in the Examples), for example, 8.0E-07M to 1.0E-07M, for example, 4.0E-07M, 3.0E-07M, 2.0E-07M, 1.0E-07M, more preferably less than 10.0E-08M, for example less than 9.0E-10M.

[0043] In one embodiment, the IL-2 muteins of the invention reduce IL-2-mediated CD25 upregulation compared to wild-type IL-2. + This results in a decrease in cell activation and proliferation. + The cells are CD25 + CD8 + In another embodiment, the CD25 +The cells are Treg cells. In one embodiment, the IL-2 mutein inhibits CD25 in a STAT5 phosphorylation assay. + By detecting activation of STAT5 phosphorylation signaling in cells, we confirmed that IL-2 mutant proteins upregulate CD25 + The ability to activate cells is identified. For example, the half-maximal effective concentration (EC50) can be determined by analyzing STAT5 phosphorylation in cells by flow cytometry, as described in the examples of this application.

[0044] In one embodiment, the IL-2 muteins of the invention reduce IL-2-mediated CD25 upregulation compared to wild-type IL-2. - This results in sustained or enhanced activation and proliferation of effector cells. In one embodiment, CD25 - The cells are CD8 + In one embodiment, the STAT5 phosphorylation assay is performed to measure CD25 - EC of IL-2 mutant proteins activating STAT5 phosphorylation signaling in cells 50 By detecting the value of CD25 - The ability of the IL-2 muteins to activate cells is identified. In one embodiment, the IL-2 muteins of the invention reduce the CD25 phosphorylation compared to wild-type IL-2 protein (e.g., human IL-2 of SEQ ID NO: 26) as measured in a STAT5 phosphorylation assay. + The ability to activate cells is increased by at least 1-fold, such as 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold.

[0045] In one embodiment, the IL-2 muteins of the invention have a CD25 + In one embodiment, the bias of IL-2 towards preferential activation of CD25 cells is eliminated or reduced. + The cells are CD25 + CD8 + In another embodiment, the CD25 +The cells are Treg cells. In one embodiment, the IL-2 variant binds to CD25 - The ability to activate cells was assessed by measuring STAT5 phosphorylation, and was compared with that of CD25 and CD31. - Cells and CD25 + EC of IL-2 mutant proteins that activate STAT5 phosphorylation signaling in cells 50 For example, CD25 + Activation bias of IL-2 mutant proteins towards CD25 cells - and CD25 + The ratio of EC50 values ​​that activate STAT5 phosphorylation signals on T cells is determined. Preferably, the mutant protein is CD25 + The bias for is reduced by at least 10-fold, preferably at least 100-fold, 150-fold, or 200-fold compared to the wild-type protein.

[0046] Mutant Proteins of the Present Invention Glycosylated mutant proteins In one embodiment, the invention provides an IL-2 mutein that contains a mutated glycosylation motif in the IL-2 and IL-2Ra binding interface.

[0047] As known in the art, polypeptides are typically glycosylated via N-linked or O-linked. N-linked glycosylation means that a carbohydrate moiety is attached to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine (NXS) and asparagine-X-threonine (NXT) are N-linked glycosylation motifs, where X is any amino acid except proline. The presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. Addition of N-linked glycosylation sites to a protein (e.g., IL-2) can be conveniently accomplished by altering the amino acid sequence to include one or more of the above tripeptide sequences. For example, N-linked glycosylation sites can be added by altering the codon for a single amino acid. For example, a codon encoding NXz (where z is any amino acid) can be altered to encode NXT (or NXS), or a codon encoding yXT / S can be altered to encode NXT / S. Alternatively, a codon encoding two amino acids may be altered simultaneously to introduce an N-linked glycosylation site (eg, a codon for yXz may be altered to encode NXT / S).

[0048] Herein, the glycosylation motif that appears due to the mutation introduced into the IL-2 protein can be described as a mutated glycosylation motif. For example, the mutated glycosylation motif K35N-L36-T37 is an N-linked glycosylation motif in which the lysine at position 35 is replaced by asparagine, and the residues at positions 36 and 37 remain unchanged. In a preferred embodiment of the present invention, the mutated glycosylation motif introduced is an N-linked glycosylation motif, NXS / T, where X is any amino acid except proline. In some embodiments, for example, X may be the same amino acid as the amino acid at the corresponding position in wild-type IL-2, or a conservative replacement residue thereof.

[0049] In some embodiments, the present invention provides an IL-2 antibody that is capable of inhibiting IL-2, as compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the sequence of SEQ ID NO:26). Provided are IL-2 muteins comprising at least one mutation introducing one or more glycosylation motifs NXS / T at an amino acid position selected from 35N-36X-37T / S, 38N-39X-40T / S, 41N-42X-43T / S, 43N-44X-45T / S, 45N-46X-47T / S, 62N-63X-64T / S, 68N-69X-70T / S, 72N-73X-74T / S, 74N-75X-76T / S, where X is any amino acid other than proline, and preferably X is the same amino acid as the amino acid at the corresponding position in wild-type IL-2, or a conservative replacement residue thereof, where the amino acid positions are numbered according to SEQ ID NO: 26. In some embodiments, the number of N-linked glycosylation sites introduced may be more than one, for example two glycosylation sites. Different glycosylation sites can confer different properties to IL-2, for example, some glycosylation sites can confer improved expression and / or purification properties, some glycosylation sites can improve IL-2 receptor selectivity. In yet other embodiments, the muteins of the invention may contain at least 1-30 amino acid residues, e.g., 1-20, 1-15, 1-10, or 1-5 different amino acid residues that differ from wild-type IL-2, in addition to the glycosylation motifs introduced by mutation. These different residues may be conservative substitutions or other mutations that confer other improved properties to IL-2.

[0050] Glycosylation mutations that improve druggability In some embodiments, the mutated glycosylation motif improves the druggability properties of the IL-2 protein, in particular facilitating expression and / or purification of the IL-2 protein.

[0051] In one embodiment, the mutated glycosylation motif that improves the drug formability is selected from the group consisting of 35N-36X-37T / S, 38N-39X-40T / S, and 74N-75X-76T / S. In one preferred embodiment, the mutated glycosylation motif is selected from the group consisting of (i) K35N-L36-T37, (ii) R38N-M39-L40S, and (iii) Q74N-S75-K76T. In one more preferred embodiment, the mutated glycosylation motif is K35N-L36-T37.

[0052] Thus, in some embodiments, the present invention provides an IL-2 mutein comprising a mutated glycosylation motif selected from the group consisting of 35N-36X-37T / S, 38N-39X-40T / S, and 74N-75X-76T / S, compared to wild-type IL-2, said mutein having improved druggability properties. In one embodiment, said mutations may facilitate expression and / or purification of the mutein when expressed in mammalian cells, preferably when expressing the IL-2 mutein in the form of an Fc-fusion protein. In yet another embodiment, said mutations may facilitate stability of IL-2, e.g., reduced tendency to aggregate formation during production, compared to wild-type IL-2, when expressed as an Fc-fusion protein. For example, after expression and one-step Protein A affinity purification, the mutein may have a higher purity than the wild-type protein. In one preferred embodiment, compared to wild-type IL-2, the mutant protein comprises a mutated glycosylation motif selected from the group consisting of: (i) K35N-L36-T37, (ii) R38N-M39-L40S, (iii) Q74N-S75-K76T, more preferably the mutant protein comprises a mutated glycosylation motif K35N-L36-T37.

[0053] In some embodiments, the mutated glycosylation motif is introduced into the IL-2 protein via the mutation K35N. In some embodiments, the invention provides an IL-2 mutein having a mature region having at least 90% identity in amino acid sequence to a wild-type IL-2 protein set forth in any of SEQ ID NOs: 26, 29 or 30, and having amino acid residue T37 and the mutation K35N.

[0054] In some embodiments, the mutated glycosylation motif introduces an IL-2 protein with paired mutations selected from R38N / L40S or Q74N / K76T. In some embodiments, the invention provides an IL-2 mutein having a mature region having at least 90% identity in amino acid sequence to a wild-type IL-2 protein set forth in any of SEQ ID NOs: 26, 29 or 30, and having paired mutations selected from R38N / L40S or Q74N / K76T.

[0055] In some embodiments, the mutant protein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 32 and 38. In further preferred embodiments, the mutant protein comprises the amino acid sequence SEQ ID NOs: 31, 32 and 38.

[0056] Glycosylation mutations that reduce IL-2Rα binding In some embodiments, the mutated glycosylation motif improves receptor selectivity of the IL-2 protein, in particular reducing binding of IL-2 to IL-2Rα.

[0057] In one embodiment, the mutated glycosylation motif that reduces binding of IL-2 to IL-2Rα is selected from 41N-42X-43T / S, 43N-44X-45T / S, 45N-46X-47T / S, 68N-69X-70T / S, 72N-73X-74T / S, preferably the glycosylation motif 43N-44X-45T / S, where the amino acid positions are numbered according to SEQ ID NO:26. In one preferred embodiment, the mutated glycosylation motif that reduces binding of IL-2 to IL-2Rα is selected from the group consisting of: (i) T41N-F42-K43S, (ii) K43N-F44-Y45T, (iii) Y45N-M46-P47S, (iv) E68N-V69-L70S, (v) L72N-A73-Q74T, more preferably K43N-F44-Y45T.

[0058] Thus, in some embodiments, the present invention provides IL-2 muteins comprising a mutated glycosylation motif compared to wild-type IL-2, said muteins comprising one or more mutated glycosylation motifs selected from the group consisting of 41N-42X-43T / S, 43N-44X-45T / S, 45N-46X-47T / S, 68N-69X-70T / S, 72N-73X-74T / S, preferably 43N-44X-45T / S, wherein the amino acid positions are numbered according to SEQ ID NO:26, and said muteins have reduced or eliminated IL-2Rα binding compared to wild-type IL-2.

[0059] In yet another embodiment, the present invention provides an IL-2 mutein comprising a mutated glycosylation motif compared to wild type IL-2, said mutein comprising one or more mutated glycosylation motifs selected from the group consisting of: (i) T41N-F42-K43S, (ii) K43N-F44-Y45T, (iii) Y45N-M46-P47S, (iv) E68N-V69-L70S, (v) L72N-A73-Q74T, more preferably said mutein comprises the mutated glycosylation motif K43N-F44-Y45T.

[0060] In some embodiments, the mutant glycosylation motif is introduced into the IL-2 protein by paired mutations selected from the group consisting of T41N / K43S, K43N / Y45T, Y45N / P47S, E68N / L70S, and L72N / Q74T. In some embodiments, the invention provides IL-2 muteins having a mature region having at least 85% or 90% identity in amino acid sequence to the wild-type IL-2 protein set forth in any of SEQ ID NOs: 26, 29 or 30, and having paired mutations selected from the group consisting of T41N / K43S, K43N / Y45T, Y45N / P47S, E68N / L70S, and L72N / Q74T, preferably having paired mutation K43N / Y45T. In some embodiments, the mutant protein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from SEQ ID NOs: 33, 34, 35, 37, and 39.

[0061] In some embodiments, the IL-2 mutein may comprise, in addition to the above mutated glycosylation motifs that reduce binding of IL-2 to IL-2Rα, (i) a mutated glycosylation motif selected from 35N-36X-37T / S, 38N-39X-40T / S, and 74N-75X-76T / S, and / or (ii) the mutation K35Q, which, compared to wild-type IL-2, has reduced or eliminated IL-2Rα binding and improved expression and / or purification properties (e.g., when expressed in mammalian cells in the form of an Fc fusion protein). In some preferred embodiments, the invention provides IL-2 muteins having a mature region having at least 85% or 90% identity in amino acid sequence to a wild-type IL-2 protein set forth in any of SEQ ID NOs: 26, 29 or 30, and having paired mutations selected from the group consisting of T41N / K43S, K43N / Y45T, Y45N / P47S, E68N / L70S, and L72N / Q74T, and having a mutation selected from K35N, R38N / L40S, Q74N / K76T or K35Q. In some embodiments, the mutein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from SEQ ID NOs: 45-47.

[0062] B'C' loop chimeric mutant proteins and truncation mutant proteins In one aspect, the present invention provides B'C' loop chimeric IL-2 muteins and truncated IL-2 muteins formed by introducing mutations into the B'C' loop region of IL-2. IL-2 protein is a member of the short chain type I cytokine family with a four alpha helix (A, B, C, D) structure. As used herein, the "B'C' loop region" or "B'C' loop sequence" are used interchangeably and refer to the linking sequence between the B helix and the C helix of the IL-2 protein. In one embodiment, the linking sequence is the sequence that links the residue at position 72 and the residue at position 84 of the IL-2 polypeptide according to SEQ ID NO: 26. In the wild type proteins of SEQ ID NOs: 26, 29 and 30, the linking sequence comprises a total of 11 amino acids, A73-R83.

[0063] In some embodiments, the introduced mutations comprise a truncated B'C' loop region (i.e. a shortening of the length of the linking sequence between amino acid residues aa72 and aa84) of the mutein compared to wild-type IL-2 (preferably human IL-2, more preferably an IL-2 comprising the sequence of SEQ ID NO:26), preferably said truncated loop region is less than 10, 9, 8, 7, 6 or 5 amino acids in length, preferably 7 amino acids in length, wherein the amino acid residues are numbered according to SEQ ID NO:26.

[0064] In some embodiments, the IL-2 mutein of the invention is a B'C' loop chimeric mutein. The mutein comprises a substitution of aa73 to aa83 sequence compared to wild type IL-2, e.g., a substitution of a short B'C' loop sequence from another four-helix short chain cytokine family member. From other four-helix short chain cytokine IL family members such as IL-15, IL-4, IL-21, or IL family members from non-human species (e.g., mouse), short B'C' loops suitable for substituting wild type IL-2 were identified via superposition of crystal structures. In one preferred embodiment, the sequence for substitution is the B'C' loop sequence from interleukin IL-15 (particularly human IL-15). Preferably, the sequence of residues 73-83 in wild type IL-2 is replaced with the sequence SGDASIH.

[0065] In some embodiments, the IL-2 muteins of the invention are B'C' loop truncation muteins. The muteins comprise a truncation of the sequence aa73 to aa83, e.g., a truncation of 1, 2, 3 or 4 amino acids from the C-terminus, compared to wild-type IL-2. Preferably, the truncated loop region (i.e., the linking sequence between positions 72 and 84) has the sequence A(Q / G)S(K / A)N(F / I)H, preferably the truncated loop region has the sequence AQSKNFH or AGSKNFH.

[0066] In one embodiment, substitution or truncation of the B'C' loop can increase the stability of the B'C' loop, thereby increasing the stability of IL-2 and / or affinity for IL-2Rβ. Thus, in one embodiment, the invention provides an IL-2 mutein having increased stability and / or increased IL-2Rβ binding affinity compared to wild-type IL-2, said mutein comprising the above-mentioned B'C' loop chimeric mutation or B'C' loop truncation mutation, in particular the alternative loop sequence SGDASIH or the truncated loop sequence AQSKNFH or AGSKNFH located between positions 72 and 84.

[0067] In one embodiment, the chimeric or truncated B'C' loop mutation not only confers increased IL-2Rβ binding but may also facilitate expression and / or purification of IL-2 protein, particularly in mammalian cell expression systems. Thus, in one embodiment, the present invention provides an IL-2 mutein having enhanced IL-2Rβ binding and / or improved expression and / or purification properties compared to wild-type IL-2. Said IL-2 mutein comprises the aforementioned B'C' loop chimeric or B'C' loop truncated mutation, particularly the alternative loop sequence SGDASIH or the truncated loop sequence AQSKNFH or AGSKNFH located between positions 72 and 84.

[0068] In some preferred embodiments, the invention provides an IL-2 mutein having a mature region having at least 85% or 90% identity in amino acid sequence to a wild-type IL-2 protein set forth in any of SEQ ID NOs: 26, 29 or 30, and comprising a linking sequence selected from the group consisting of SGDASIH, AQSKNFH, AGSKNFH, AQSANFH, and AQSANOH between amino acid positions 72 and 84. In some embodiments, the mutein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-44, preferably SEQ ID NOs: 40-42, more preferably SEQ ID NO: 40 or 41.

[0069] Combinatorial mutant proteins In one embodiment, the present invention provides IL-2 muteins comprising combination mutations. In one embodiment, the glycosylation mutations introduced into the IL-2 and IL-Rα binding interface may be combined with each other or with B'C' loop mutations, preferably with the B'C' loop mutations described herein. In another embodiment, the B'C' loop mutations of the present invention may also be combined with glycosylation mutations introduced into the IL-2 and IL-Rα binding interface, preferably with the glycosylation mutations described herein. In one preferred embodiment, the B'C' loop mutations may be combined with glycosylation mutations introduced into the IL-2 and IL-Rα binding interface to provide improved properties selected from the group consisting of: (i) reduced (or eliminated) IL-2Rα binding, (ii) enhanced IL-2Rα binding, and (iii) improved expression levels and purification.

[0070] Thus, in one embodiment, the invention provides an IL-2 mutein comprising, as compared to wild-type IL-2 (preferably human IL-2, more preferably an IL-2 comprising the sequence of SEQ ID NO:26), a combination mutation: (i) a mutated glycosylation motif selected from the group consisting of 41N-42X-43T / S, 43N-44X-45T / S, 45N-46X-47T / S, 68N-69X-70T / S, 72N-73X-74T / S, and (ii) a truncated B'C' loop region sequence selected from SGDASIH and A(Q / G)S(K / A)N(F / I)H, located between amino acid positions aa72 to aa84, wherein the amino acid positions are numbered according to SEQ ID NO:26.

[0071] In some preferred embodiments, the present invention provides IL-2 muteins having a mature region having at least 85% or 90% identity in amino acid sequence to a wild-type IL-2 protein set forth in any of SEQ ID NOs: 26, 29 or 30, comprising a linking sequence between amino acid positions 72 and 84 selected from the group consisting of SGDASIH, AQSKNFH, AGSKNFH, AQSANFH, and AQSANOH, and having paired mutations selected from the group consisting of T41N / K43S, K43N / Y45T, Y45N / P47S, E68N / L70S, and L72N / Q74T. In some preferred embodiments, the invention provides an IL-2 mutein having a mature region having at least 85% or 90% identity in amino acid sequence to a wild-type IL-2 protein set forth in any of SEQ ID NOs: 26, 29 or 30, comprising a linking sequence selected from the group consisting of SGDASIH, AQSKNFH, or AGSKNFH between amino acid positions 72 and 84, and having the paired mutations K43N / Y45T. In some embodiments, the mutein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from SEQ ID NOs: 48, 49 or 50, preferably SEQ ID NOs: 48 or 49. In some embodiments, the mutein consists of SEQ ID NOs: 48, 49 or 50.

[0072] In some embodiments, the combination mutation is CD25 + Reduced bias of IL-2 to preferentially stimulate p-STATA5 signaling in T cells and CD25 - Thus, in one embodiment, the present invention provides a combination mutation: (i) the mutated glycosylation motif K43N-F44-Y45T at amino acid positions 43-45 and the replacement sequence SGDASIH between amino acid positions aa72-aa84, or (ii) comprising a mutated glycosylation motif K43N-F44-Y45T at amino acid positions 43 to 45 and a truncation sequence AQSKNFH between amino acid positions aa72 to aa84, The mutant protein inhibits CD25 as compared to wild-type IL-2. + Reduced bias to preferentially stimulate p-STATA5 signaling in T cells and CD25 - The ability to stimulate signal transduction in T cells is enhanced. Preferably, the mutein comprises the sequence of SEQ ID NO: 48 or 49, or a sequence having at least 95%, 96% or more identity thereto. More preferably, the mutein consists of the sequence of SEQ ID NO: 48 or 49.

[0073] Other mutations In addition to the mutations in the above regions and positions, the IL-2 muteins of the present invention may have one or more mutations in other regions or positions, so long as they retain one or more of the above beneficial properties of the IL-2 muteins of the present invention. For example, the IL-2 muteins of the present invention may use 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, for example, U.S. Patent No. 4,518,584). Those skilled in the art will know how to determine additional mutations that can be incorporated into the IL-2 muteins of the present invention.

[0074] The difference in sequence between the IL-2 mutein and the wild-type protein can be expressed by sequence identity or by the number of different amino acids between them. In one embodiment, the IL-2 mutein and the wild-type protein have at least 85%, 86%, 87%, 88%, 89% identity, preferably 90% or more identity, preferably 95%, but preferably 97% or less, more preferably 96% or less identity. In another embodiment, the IL-2 mutein and the wild-type protein may have 15 or less mutations, for example 1-10 or 1-5 mutations, in addition to the above glycosylation mutations or B'C' loop mutations or combination mutations thereof of the present invention. In one embodiment, the remaining mutations may be conservative substitutions.

[0075] 2. Fusion Proteins and Immunoconjugates The invention also provides fusion proteins comprising the IL-2 muteins of the invention. In a preferred embodiment, the IL-muteins of the invention are fused to another polypeptide, such as albumin, more preferably an antibody Fc fragment, which can confer improved pharmacokinetic properties. In one embodiment, the Fc fragment comprises a mutation that reduces or eliminates effector function, such as the L234A / L235A mutation or L234A / L235E / G237A, which reduces binding to Fcγ receptors. Preferably, the fusion protein comprising Fc has an increased serum half-life. In a preferred embodiment, the fusion protein comprising Fc simultaneously has a reduced or eliminated effector function mediated by the Fc region, such as an ADCC or ADCP or CDC effector function.

[0076] In one embodiment, the present invention also provides an IL-2 mutein-Fc fusion protein, in which the Fc fragment contains an effector function such as ADCC. As reported in the literature (Rodrigo Vazquez-Lombardi et al., cited above), wild-type IL-2, when fused to Fc, can deplete Treg cells by Fc-mediated immune effector functions (particularly mediated by binding to FcγR), improving the treatment of tumors. Therefore, it is also contemplated by the present invention to fuse an IL-2 mutein of the present invention, with improved production characteristics such as expression and / or purification, to an Fc fragment that retains immune effector functions. In one embodiment, the fusion protein contains the mutations K35N or K35Q, or the paired mutations R38N / L40S or Q74N / K76T. In another embodiment, the fusion protein contains the alternative sequence SGDASIH or the truncation sequence A(Q / G)S(K / A)N(F / I)H between amino acid positions aa72 to aa84. In one embodiment, the fusion protein comprises 90%-99% or more identity to amino acid sequence SEQ ID NO:7, 8, 14, 20-22. In another embodiment, the fusion protein comprises 0-10 or 0-5 or less amino acid mutations to amino acid sequence SEQ ID NO:12.

[0077] In some embodiments, the IL-2 mutein is fused to the Fc region by a linker. - The linker can be selected to enhance the activation of the Fc fusion protein to T cells. In one embodiment, the linker is GSGS, more preferably 2x(G4S).

[0078] In some embodiments, the Fc fusion protein comprises a sequence having 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: 3-13 and 16-25. In some embodiments, the Fc fusion protein consists of the sequence of SEQ ID NOs: 3-13 and 16-25.

[0079] The present invention also provides an immunoconjugate comprising an IL-2 mutein of the present invention and an antigen-binding molecule. Preferably, the antigen-binding molecule is an immunoglobulin molecule, particularly an IgG molecule, or an antibody or antibody fragment, particularly a Fab molecule and an scFv molecule. In some embodiments, the antigen-binding molecule specifically binds to an antigen present on tumor cells or in the tumor environment, for example, an antigen selected from the group consisting of fibroblast activation protein (FAP), A1 domain of tenascin-C (TNC A1), A2 domain of tenascin-C (TNC A2), ectodomain B of fibronectin (Extra Domain B, EDB), carcinoembryonic antigen (CEA), melanoma-associated chondroitin sulfate proteoglycan (MCSP). Thus, the immunoconjugate of the present invention can target tumor cells or the tumor environment after administration to a subject, providing further therapeutic advantages, such as the possibility of treatment at a lower dose and thus reduced side effects, enhanced antitumor effects, etc.

[0080] In the fusion proteins and immunoconjugates of the invention, the IL-2 muteins of the invention can be linked to another molecule or antigen-binding molecule directly or via a linker, and in some embodiments, a proteolytic cleavage site is included between the two.

[0081] 3. Polynucleotides, Vectors and Hosts The present invention provides nucleic acids encoding any of the above IL-2 muteins or fusions or complexes. Polynucleotide sequences encoding the muteins of the present invention can be generated by de novo solid-phase DNA synthesis or PCR mutagenesis of existing sequences encoding wild-type IL-2 using methods well known in the art. Additionally, the polynucleotides and nucleic acids of the present invention can include a segment encoding a secretory signal peptide and be operably linked to the segment encoding the mutein of the present invention to direct the secretory expression of the mutein of the present invention.

[0082] The present invention also provides a vector comprising the nucleic acid of the present invention.In one embodiment, the vector is an expression vector, for example, a eukaryotic expression vector.Non-limiting examples of vectors include virus, plasmid, cosmid, lambda phage or yeast artificial chromosome (YAC).In a preferred embodiment, the expression vector of the present invention is pYDO_017 expression vector.

[0083] The invention also provides a host cell comprising said nucleic acid or said vector. Suitable host cells for replicating and supporting the expression of mutant IL-2 proteins or fusions or immunoconjugates are well known in the art. Such cells can be transfected or transduced with a particular expression vector, and many cells containing the vector can be cultured to inoculate large fermenters to obtain sufficient quantities of IL-2 mutants or fusions or immunoconjugates for clinical use. In one embodiment, the host cell is a eukaryotic cell. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells). For example, the polypeptide can be produced in bacteria, especially if glycosylation is not required. After expression, the polypeptide can be separated from the bacterial cell paste in a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungal and yeast strains in which the glycosylation pathway is already "humanized", resulting in the production of polypeptides with a partially or completely human glycosylation mode. See Gerngross, NatBiotech 22, 1409-1414 (2004) and Li et al., NatBiotech 24, 210-215 (2006).Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (e.g., 293 or 293T cells described in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), dog kidney cells (MDCK), buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (HepG2), mouse mammary tumor (MMT060562), TRI cells (e.g., Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other mammalian host cell lines that can be used include Chinese hamster ovary (CHO) cells, such as dhfr-CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)), 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 lymphocyte (e.g., YO, NS0, Sp20 cell).

[0084] 4. Preparation method In yet another aspect, the invention provides a method for preparing an IL-2 mutein or fusion or complex of the invention, said method comprising culturing a host cell comprising a nucleic acid encoding the IL-2 mutein or fusion or complex under conditions suitable for expression of said protein or fusion or complex, and optionally recovering said protein or fusion or complex from the host cell (or host cell medium).

[0085] 5. Measurement Method A variety of assays known in the art can be used to identify, screen, or characterize the physical / chemical properties and / or biological activity of the IL-2 muteins of the invention.

[0086] In one embodiment, the binding activity of the IL-2 mutein of the present invention to the IL-2 receptor can be measured. For example, binding to human IL-2R α or β protein can be measured using methods known in the art, such as ELISA, Western blot, and the like, or the exemplary methods disclosed in the examples of the present application. For example, cells transfected to express the mutein on the cell surface, such as yeast display cells, can be reacted with labeled (e.g., biotin-labeled) IL-2R α or β protein using flow cytometry. Alternatively, binding kinetics (e.g., K D Binding of a mutant protein containing a β-terminal ...

[0087] In yet another embodiment, the ability of an IL-2 mutein to bind to the IL-2 receptor can be measured indirectly by measuring signaling and / or immunostimulatory effects that occur downstream of receptor binding.

[0088] Thus, in some embodiments, a measurement method is further provided for identifying mutant IL-2 proteins having biological activity. Biological activity includes, for example, the ability to induce proliferation of IL-2 receptor-bearing T and / or NK cells and / or Treg cells, the ability to induce IL-2 signaling in IL-2 receptor-bearing T and / or NK cells and / or Treg cells, the ability to induce apoptosis in T cells, the induction of tumor regression and / or improved survival, and reduced in vivo toxicity properties such as reduced vascular permeability. The present invention also provides mutant IL-2 proteins having such biological activity in vivo and / or in vitro.

[0089] To determine the biological activity of IL-2, methods such as those known in the art can be used. For example, a suitable measurement technique for testing the ability of the IL-2 mutant protein of the present invention to stimulate NK cell IFN-γ production includes incubating cultured NK cells with the mutant IL-2 protein or fusion or immunoconjugate of the present invention, and then measuring the IFN-γ concentration in the medium by ELISA. IL-2 signaling induces several signaling pathways, involving JAK (Janus kinase) and STAT (signal transducer and activator of transcription) signaling molecules.

[0090] The interaction of IL-2 with the receptor β and γ subunits results in phosphorylation of the receptor and JAK1 and JAK3 (which bind to the β and γ subunits, respectively). STAT5 then binds to the phosphorylated receptor and is itself phosphorylated on critical tyrosine residues. This results in receptor dissociation, STAT5 dimerization, and shifting of STAT5 from the STAT5 dimer to the nucleus, which promotes the transcription of target genes. Thus, the ability of mutant IL-2 polypeptides to induce signal transduction through 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 the mutant IL-2 polypeptides or fusions or immune complexes of the present invention, and the level of phosphorylated STAT5 can be determined by flow cytometry.

[0091] Furthermore, T cell or NK cell response to IL-2 proliferation is measured by incubating T cells or NK cells isolated from blood with the mutant IL-2 polypeptide or immune complex of the present invention and then measuring the ATP content in the lysate of treated cells. Prior to treatment, T cells can be pre-stimulated with plant lectin (PHA-M). This measurement technique allows for sensitive quantification of viable cell numbers, and many suitable alternative measurement techniques (e.g., [3H]-thymidine incorporation measurement technique, cell titration GloATP measurement technique, AlamarBlue measurement technique, WST-1 measurement technique, MTT measurement technique) are also known in the art.

[0092] Furthermore, the effect of mutant IL-2 on tumor growth and survival can be evaluated in various animal tumor models known in the art. For example, xenografts of human cancer cell lines can be implanted in immunodeficient mice and treated with mutant IL-2 polypeptides or fusions or immunoconjugates of the present invention. The toxicity of mutant IL-2 polypeptides, fusions and immunoconjugates of the present invention in vivo can be measured based on mortality, life-stage observations (visible symptoms of adverse effects such as behavior, weight, temperature, etc.), and clinical and anatomic pathology (such as blood chemistry measurements and / or histopathology analysis). For example, vascular permeability induced by IL-2 treatment can be examined using vascular exudation reporter molecules in vascular permeability pretreatment animal models. Preferably, the vascular exudation reporter molecules are large enough to reveal the permeability of the IL-2 wild-type form for pretreatment.

[0093] Furthermore, for the IL-2 glycosylation muteins of the present invention, the presence, absence, or degree of glycosylation can be determined by any method known to one of skill in the art and can include semi-qualitative measurements of molecular weight (MW) offsets, such as those observed from Western blots or Coomassie stained SDS-PAGE gels, and quantitative measurements can include the use of mass spectrometry techniques and observing a MW offset corresponding to the addition of asparagine-linked glycosylation, or a mass offset associated with the removal of asparagine-linked glycosylation by an enzyme such as peptide-N-glycine glycyrrhizinase F (PNGase-F; Sigma Aldrich, St. Louis, MO).

[0094] 6. Screening Method In yet another aspect, the present invention provides methods for obtaining IL-2 muteins with improved properties.

[0095] In one embodiment, the present invention provides a method for producing a medicament for the treatment of a cancer, comprising: - engineering one or more (e.g. two or three) glycosylation motifs NXS / T (wherein X can be any amino acid, except P (proline)) in the binding interface between IL-2 and IL-2Ra, preferably introducing glycosylation motifs in the regions selected from aa35-40, aa41-47, aa62-64, aa68-74, aa74-76 of IL-2; - expressing the modified IL-2 mutein in mammalian cells (e.g. HEK293 or CHO cells), for example in the form of an Fc fusion (e.g. FcLALA fusion). For designing the site for introducing a glycosylation motif, N-glycosylation prediction tools can be used to select potential mutable sites to promote N-linked glycosylation, for example by identifying residues that can be mutated to form a canonical NxT / S glycosylation site (where N is asparagine and x is any amino acid except proline). Furthermore, using structure-based methods, an amino acid in IL-2 that is 3.6 Å away from IL-2Rα and whose side chain is exposed to solution can be identified as a candidate amino acid for mutation to asparagine. In some preferred embodiments, the introduced glycosylation motif mutation is selected from the group consisting of K35N-L36-T37, R38N-M39-L40S, T41N-F42-K43S, K43N-F44-Y45T, Y45N-M46-P47S, E62N-L63-K64T, E68N-V69-L70S, L72N-A73-Q74T, Q74N-S75-K76T.

[0096] In another embodiment, the present invention provides a method for obtaining an IL-2 mutein, comprising the steps of: - introducing deletions and / or substitutions into the B'C'loop region (aa73-83) of IL-2 to form a truncated loop region, preferably substituting the B'C'loop sequence of another four-helix short chain cytokine family member such as IL15 to form a B'C'loop chimera, or truncating the B'C'loop of IL-2 to form a B'C'loop truncation, preferably the truncated loop region is less than 10, 9, 8, preferably equal to 7 amino acids in length, preferably truncating 1, 2, 3 or 4 amino acids from the C-terminus of the loop region, preferably the truncated loop region has the sequence A(Q / G)S(K / A)N(F / I)H, or SGDASIH; - expressing the modified IL-2 mutein in mammalian cells (e.g. HEK293 or CHO cells), for example in the form of an Fc fusion (e.g. an FcLALA fusion).

[0097] In one embodiment, the method further comprises identifying IL-2 muteins with improved druggability (e.g., expression level and / or product stability and / or homogeneity, e.g., one-step Fc affinity chromatography purity) after protein expression and purification. In one preferred embodiment, glycosylation motif mutations are introduced in the region aa35-40 or aa74-76 of IL-2 to improve the druggability of the mutein. Preferably, the introduced glycosylation motif mutations are selected from the group consisting of K35N-L36-T37, R38N-M39-L40S, and Q74N-S75-K76T. In another preferred embodiment, the druggability of the mutein is improved by replacing the B'C'loop loop with a loop sequence of a truncated loop such as IL15, or by truncating the B'C'loop loop. Preferably, the truncated loop sequence is selected from A(Q / G)S(K / A)NFH, or SGDASIH. In a further preferred embodiment, the method includes introducing other site mutations, in addition to the glycosylation mutations, to improve the druggability of the mutein, e.g., K35Q. As will be apparent to one of skill in the art, these mutations can be combined with mutations that confer other improved properties to obtain IL-2 muteins with multiple improved properties.

[0098] In one embodiment, the method further comprises identifying IL-2 muteins with reduced (preferably eliminated) IL-2Ra binding ability compared to wild type IL-2. In one embodiment, the binding ability of the IL-2 muteins to IL-2Ra is determined by measuring affinity KD values, for example by biolayer interferometry techniques. In yet another embodiment, the binding ability is measured by measuring the affinity KD value of CD25. + The activation of T cells is determined by determining the effectiveness of the IL-2 mutein on T cells. In one embodiment, the IL-2 mutein inhibits CD25 T cells compared to wild-type IL-2, as determined, for example, by measuring activation of p-STAT5 signaling in cells. +The IL-2 mutants exhibit reduced efficacy in T cell activation. Preferably, mutations are introduced into the regions of IL-2, aa41-47 or aa68-70 or aa72-74, to form potential N-linked glycosylation sites, and then detect whether the mutations result in reduced or eliminated binding of IL-2 to IL-2Rα. Preferably, the introduced glycosylation motif mutations are selected from the group consisting of T41N-F42-K43S, K43N-F44-Y45T, Y45N-M46-P47S, E68N-V69-L70S, L72N-A73-Q74T. As will be apparent to those skilled in the art, these glycosylation mutations can be combined with mutations that confer other modification properties to obtain IL-2 muteins with multiple modification properties.

[0099] In one embodiment, the method further comprises identifying an IL-2 mutein having enhanced IL-2Rβ binding relative to wild-type IL-2. In one embodiment, the ability of the IL-2 mutein to bind to IL-2Rβ is determined by measuring affinity KD values, for example by biolayer interferometry. In yet another embodiment, binding ability is measured by measuring affinity KD values, for example by biolayer interferometry, for CD25. - The activation of T cells is determined by determining the effectiveness of the IL-2 mutein on T cells. In one embodiment, the IL-2 mutein inhibits CD25 T cells compared to wild-type IL-2, as determined, for example, by measuring activation of p-STAT5 signaling in cells. - The glycosylation mutations show enhanced efficacy of T cell activation. In one preferred embodiment, the B'C'loop loop is replaced with a shortened loop sequence such as IL15 or a truncated B'C'loop loop to enhance binding to IL-2Rβ. Preferably, the truncated loop sequence is selected from A(Q / G)S(K / A)NFH, or SGDASIH. As will be apparent to those skilled in the art, these glycosylation mutations can be combined with mutations that confer other modification properties to obtain IL-2 muteins with multiple modification properties.

[0100] In yet another embodiment, the method comprises introducing a combination of mutations improving drug form, mutations decreasing IL2Ra binding, and / or mutations enhancing IL2Rβ binding, and / or mutations conferring other modified properties to obtain an IL-2 mutein with multiple modified properties. In one preferred embodiment, glycosylation mutations, such as regions aa41-47 and aa68-74, and truncation and / or substitution mutations shortening the length of the B'C'loop loop region, are introduced in combination. In one preferred embodiment, the method comprises identifying an IL-2 mutein that exhibits reduced IL-2Ra binding and enhanced IL-2Rβ binding relative to wild-type IL-2, and optionally also has improved drug form (e.g. improved expression and / or purity, and / or product stability and / or homogeneity).

[0101] In some embodiments, the parent wild-type IL-2 protein used as a mutation template preferably has at least 85%, or at least 90% or 95% identity to SEQ ID NO:26, and is more preferably a human-derived IL-2 protein.

[0102] 7. Pharmaceutical Compositions and Drug Formulations The present invention also includes compositions (including pharmaceutical compositions or drug formulations) comprising an IL-2 mutein or a fusion or immunoconjugate thereof, and compositions comprising a polynucleotide encoding an IL-2 mutein or a fusion or immunoconjugate thereof. These compositions may also contain suitable pharmaceutical additives, such as pharma- ceutically acceptable carriers, pharmaceutical excipients including buffers, as known in the art, as needed.

[0103] Pharmaceutically acceptable carriers suitable for the present invention may be sterile liquids such as water, oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., derived from petroleum, animals or plants, or synthetically. When the pharmaceutical composition is administered intravenously, water is the preferred vector. In addition, physiological saline solutions, aqueous dextrose and glycerin solutions can be used as liquid vectors, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene, diol, water, ethanol, etc. For the use of excipients and their applications, reference is made to "Handbook of Pharmaceutical Excipients", 5th edition, RC Rowe, PJ Eskey, SCOwen, Pharmaceutical Press, London, Chicago. If necessary, the composition may further contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. Oral formulations can include standard vectors such as medicinal mannitol, lactose, starch, magnesium stearate, saccharin, etc.

[0104] A drug formulation comprising the present invention is prepared by mixing the IL-2 mutein, fusion or immunoconjugate of the present invention having the desired purity with any one or more pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution. For a description of an exemplary lyophilized antibody formulation, see U.S. Pat. No. 6,267,958. For an aqueous antibody formulation, see U.S. Pat. No. 6,171,586 and WO2006 / 044908, the latter formulation containing a histidine-acetate buffer. A sustained release formulation can also be prepared. Suitable examples of sustained release formulations include semipermeable matrices of hydrophobic solid polymers containing the protein, said matrices having a certain form such as a film or a microcapsule.

[0105] The pharmaceutical composition or formulation of the present invention may further comprise one or more other active ingredients, which are necessary for the treatment of a specific indication and preferably include active complementary active ingredients that do not negatively affect each other. For example, it is preferable to further provide other anti-cancer active ingredients, such as chemotherapeutic agents, PD-1 axis binding antagonists (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies or anti-PD-L2 antibodies). The active ingredients are present in an appropriate combination form in an amount effective for the intended use.

[0106] Thus, in one embodiment, the composition further comprises a second therapeutic agent. For example, the second therapeutic agent may be an immune checkpoint inhibitor. For example, the second therapeutic agent may be selected from one or more of, for example, but not limited to, anti-CTLA-4 antibodies, anti-CD47 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CD40 antibodies, anti-OX40 (also called CD134, TNFRSF4, ACT35 and / or TXGP1L) antibodies, anti-LAG-3 antibodies, anti-CD73 antibodies, anti-CD137 antibodies, anti-CD27 antibodies, anti-CSF-1R antibodies, TLR agonists or small molecule antagonists of IDO or TGFβ. Preferably, the second therapeutic agent is a PD-1 antagonist, particularly anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG-3, anti-CD47. In addition to immunotherapeutic drugs, the second therapeutic agent may be other radiotherapy or chemotherapy drugs.

[0107] 8. Combination Products In one embodiment, the present invention further provides a combination product comprising the mutein of the present invention or a fusion or immunoconjugate thereof and one or more other therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, etc.). The combination product of the present invention can be used in the treatment method of the present invention.

[0108] In some embodiments, the invention provides a combination product, wherein the other therapeutic agent is a therapeutic agent, such as an antibody, that is effective to stimulate an immune response to further enhance, stimulate or upregulate the immune response of a subject, in some embodiments, the other antibody is, for example, an anti-PD-1 antibody or an anti-PD-L1 antibody or an anti-PD-L2 antibody or an anti-LAG-3 antibody or an anti-CTLA-4 antibody or an anti-TIM-3 antibody.

[0109] In some embodiments, the combination product is used to prevent or treat a tumor. In some embodiments, the tumor is a cancer, for example, a gastrointestinal cancer, such as gastric cancer, rectal cancer, colon cancer, colorectal cancer, or a skin cancer, such as malignant melanoma, or a renal cell carcinoma, bladder cancer, non-small cell lung cancer, etc. In some embodiments, the combination product is used to prevent or treat an infection, such as, for example, a bacterial infection, a viral infection, a fungal infection, a protozoan infection, etc.

[0110] 9. Treatment methods and uses In this application, the terms "individual" or "subject" can be used interchangeably and refer to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, goats, 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.

[0111] As used herein, the term "treatment" refers to a clinical intervention that seeks to alter the natural course of a disease in the individual being treated. Desired therapeutic effects include, but are not limited to, preventing the appearance or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or ameliorating the disease state, and alleviating or improving prognosis.

[0112] In one embodiment, the present invention provides a method of stimulating the immune system of a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an IL-2 mutein or fusion or immunoconjugate of the present invention. The IL-2 mutein of the present invention binds to CD25 - CD122 + Effector cells (cytotoxic CD8 + It has high activity and selectivity against CD25 + The IL-2 muteins of the present invention therefore can be used in lower doses to stimulate the immune system of a subject.

[0113] Thus, in some embodiments, the present invention relates to a method of enhancing the body's immune response in a subject, the method comprising administering to the subject an effective amount of any IL-2 mutein, or fusion or immunoconjugate thereof, as described herein. In some embodiments, the IL-2 mutein or fusion or immunoconjugate of the present invention is administered to a subject bearing a tumor to stimulate an anti-tumor immune response. In another embodiment, an antibody or antigen-binding portion thereof of the present invention is administered to a subject bearing an infection to stimulate an anti-infection immune response. In one embodiment, the IL-2 mutein of the present invention can be used in combination with a Treg-depleting antibody (e.g., FcγR-mediated Treg depletion) to further reduce immune suppression by Treg. In one embodiment, the IL-2 mutein of the present invention can be administered in combination with an immune checkpoint inhibitor, e.g., in combination with anti-PD-1 and anti-CTLA-4, to enhance the cancer immunotherapy effect.

[0114] In another aspect, the present invention relates to a method of treating a disease in a subject, such as tumors and cancer and infectious diseases, said method comprising administering to said subject an effective amount of any of the IL-2 muteins, or fusions or immunoconjugates thereof described herein.

[0115] The cancer may be early stage, mid stage or late stage cancer or metastatic cancer. In some embodiments, the tumor or tumor cell may be selected from colorectal tumor, ovarian tumor, pancreatic tumor, lung tumor, lung tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma and head and neck tumor. In some embodiments, the cancer may be selected from colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma and head and neck cancer. In some embodiments, the tumor is melanoma, renal cell carcinoma, colorectal cancer, bladder cancer, non-small cell lung cancer.

[0116] In another aspect, the invention relates to a method for treating an infectious disease, such as a chronic infection, in a subject, said method comprising administering to said subject an effective amount of any IL-2 mutein or fragment thereof as described herein, or an immunoconjugate, multispecific antibody, or pharmaceutical composition comprising said antibody or fragment. In one embodiment, said infection is a viral infection.

[0117] In some embodiments, in addition to the IL-2 mutein or fusion or conjugate thereof of the invention, the method of the invention further comprises administering to the subject one or more therapies (e.g., a therapy and / or other therapeutic agents) in combination. In some embodiments, the therapy comprises surgery and / or radiation therapy. In some embodiments, the method of the invention further comprises administering at least one other immunostimulatory antibody, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CD43 antibody, and / or an anti-CTLA-4 antibody, which may be, for example, a fully humanized antibody, a chimeric antibody, or a humanized antibody.

[0118] In some embodiments, the anti-PD-1 antibody is selected from IBI308 (Cindilimab, WO2017 / 025016A1), MDX-1106 (nivolumab, OPDIVO), Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA), CT-011 (Pidilizumab). In some embodiments, the anti-PD-1 antibody is MDX-1106. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4). Additionally, in some embodiments, the IL-2 mutein or fragment thereof, alone or in combination with a PD-1 antagonist, can be administered in combination with one or more other therapies, e.g., therapeutic regimens and / or other therapeutic agents. In some embodiments, the treatment includes surgery (e.g., tumor resection), radiation therapy (e.g., external particle beam therapy such as three-dimensional conformal radiation therapy with a set irradiation field), localized radiation (e.g., radiation to a predetermined target or organ), or focused radiation.

[0119] In some embodiments, the present disclosure provides a method for treating a disease (e.g., a tumor) comprising administering to a subject a mutein of the present invention and a CTLA-4 antagonistic antibody. The anti-CTLA-4 antibody can be, for example, YERVOY. (R) (ipilimumab or antibody 10D1, described in PCT Publication No. WO 01 / 14424), tremelimumab (formerly known as ticilimumab, CP-675,206), and anti-CTLA-4 antibodies described in WO 98 / 42752, WO 00 / 37504, U.S. Pat. No. 6,207,156, Hurwitz et al. (1998) Proc. Natl. Acad. Sci. USA 95(17):10067-10071, Camacho et al. (2004) J. Clin. Oncology 22(145): Abstract No. 2505 (antibody CP-675206), and Mokyr et al. (1998) Cancer Res. 58:5301-5304.

[0120] In some embodiments, the present disclosure provides a method for treating a disease (e.g., a tumor), comprising administering to a subject an anti-mutant protein and an anti-LAG-3 antagonist antibody of the present invention. The anti-LAG3 antibody can be selected from, for example, the antibodies 25F7, 26H10, 25E3, 8B7, 11F2, or 17E5 described in U.S. Patent Application Nos. US2011 / 0150892 and WO2014 / 008218, or antibodies comprising the CDRs or variable regions of these antibodies, BMS-986016, IMP731 described in US2011 / 007023.

[0121] In some embodiments, the IL-2 muteins of the invention may be administered in combination with chemotherapy or chemotherapeutic agents. In some embodiments, the IL-2 muteins of the invention may be administered in combination with radiation therapy or radiation therapy agents. In some embodiments, the IL-2 muteins of the invention may be administered in combination with targeted therapy or targeted therapeutic agents. In some embodiments, the IL-2 muteins of the invention may be administered in combination with immunotherapy or immunotherapeutic agents, such as monoclonal antibodies.

[0122] The mutations of the present invention (and pharmaceutical compositions or fusions or immunoconjugates thereof, or any other therapeutic agent) may be administered by any suitable method, including parenteral, pulmonary, or intranasal administration, and may be administered intralesionally for localized treatment. Parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Depending on the duration of administration, any suitable route may be used, including injection, such as intravenous or subcutaneous injection. In the present invention, administration at various frequencies, including but not limited to a single dose or multiple doses at multiple times, bolus administration, or pulse therapy, may be used.

[0123] When the mutant protein of the present invention is used for the prevention or treatment of a disease, its appropriate dosage (when administered alone or in combination with one or more other therapeutic agents) is determined by the type of disease to be treated, the type of antibody, the severity and course of the disease, whether it is for prophylactic or therapeutic purposes, previous treatment history, the patient's clinical history, the response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient as a single treatment or over a series of treatments.

[0124] In yet another aspect, the invention provides the use of an IL-2 mutein, composition, immunoconjugate or fusion of the invention in the preparation of a medicament for the above-mentioned methods (eg, for treatment).

[0125] The following examples are set forth to aid in the understanding of the present invention, and should not be construed as limiting the scope of protection of the present invention in any manner.

[0126] Example 1. Design of Interleukin-2 Mutants · Design of Interleukin-2 Glycosylated Protein According to the crystal structure of interleukin-2 (abbreviated as IL-2) and its α receptor CD25 (abbreviated as IL-2Rα) in the PDB database (PDB:1Z92) (Figure 1A), by artificially modifying the binding interface between IL-2 and IL-2Ra by site-specific mutation of amino acids, polysaccharide chains are formed on the surface of IL-2 by post-translational modification of cells during the expression process of IL-2 in HEK293 or CHO cells, and the binding of IL-2 to IL-2R is inhibited (as shown in the structural diagram in Figure 1B).

[0127] IL-2 glycosylation site design: An amino acid in IL-2 that is 3.6 Å away from IL-2Ra and whose side chain is exposed to the solution was found and mutated to asparagine, and then the third amino acid was mutated to serine or threonine to form an NXS / T motif (X can be any amino acid except P) (see Table 1). [Table 1]

[0128] Design of IL-2 B'C'loop chimeras and truncations B'C' loop: a linking sequence between the B helix and the C helix of IL-2 (FIG. 2A), containing a total of 11 amino acids from A73 to R83.

[0129] Comparing the crystal structure of the IL-2 monomer (PDB:1M47) with that of the complex (PDB:2ERJ), the B'C' loop is missing in the crystal structure of the IL-2 monomer because the B'C' loop is highly active in solution and cannot form a relatively stable conformation.

[0130] By genetically manipulating the B'C'loop, the stability of the B'C'loop is increased, and the stability of IL-2 and its affinity with IL-2Rb are increased. Therefore, we compared the human IL15 crystal structure (PDB: 2Z3Q) and found that the B'C'loop is short and stable (Figure 2.B). Therefore, we designed one IL-2 chimeric molecule (L017) and four truncated molecules (L057-L060) (see Table 2). [Table 2]

[0131] Example 2: Expression and purification of IL-2 mutant-Fc fusion proteins and IL-2 receptor Construction of expression plasmids Wild type IL-2(uniprot:P60568,aa21-153,C125S,IL-2 WT) and the IL-2 mutant IL-2 3X (R38D,K43E,E61R),IL-2 glycans The B'C'loop chimera and truncated fragments were linked to human IgG1 Fc (L234A, L235A, FcLALA, abbreviated as SEQ ID NO: 28) via a GSGS linking sequence, constructed on a pTT5 vector, and the following proteins were expressed. [Table 3]

[0132] IL-2 WT , IL-2 3X and L011 (IL-2 glycan5 ) was linked to FcLALA via two GGGGS residues and constructed on the pCDNA3.1 vector to express the following proteins: [Table 4]

[0133] L011(IL-2 glycan5 ) was added with another glycosylation site or a K35Q mutation site (the K35Q mutation was designed based on the aforementioned mutant protein Y007 and the protein 3D structure), linked to FcLALA through a GSGS linking sequence, and constructed on a pTT5 vector to express the following proteins: [Table 5]

[0134] The B'C'loop chimera (L017) and truncated (L057 / 058) were combined with glycosylated IL-2 (L011) and linked to FcLALA via two GGGGS, and constructed on the pCDNA3.1 vector to express the following proteins: [Table 6]

[0135] Specific sequence information of the above protein molecules is shown in the sequence listing.

[0136] Wild-type IL-2 used to construct the above molecules WT The sequence of IL-23X is shown in SEQ ID NO: 26, which has a C125S mutation at position 125 to avoid disulfide-bridged IL-2 dimer formation. IL-23X is an IL-2 mutant reported in a previous publication (Rodrigo Vazquez-Lombardi et al., Nature Communications, 8:15373, DOI: 10.1038 / ncomms15373), which also contains the C125S mutation as in IL-2WT, and contains the mutations R38D, K43E, and E61R, and its sequence is shown in SEQ ID NO: 27. According to the publication, IL-2 3X does not bind to IL-2Rα, and maintains binding affinity to IL-2Rβ equivalent to that of wild-type IL-2.

[0137] Expression and purification of IL-2 fusion proteins Expi293 cells (Invitrogen) in the designated transfection volume were subcultured to a cell density of 1.5 × 10 the day before transfection. 6 On the day of transfection, the cell density should be approximately 3 × 10 6 The transfection concentration is 1000 cells / ml. 1 / 10 (v / v) of the final volume of Opti-MEM medium (Gibco product number: 31985-070) is used as the transfection buffer, and the expression plasmid constructed above is added and mixed uniformly, and then filtered through a 0.22 μm filter for preparation for use. An appropriate amount of polyethyleneimine (PEI) (Polysciences, 23966) is added to the plasmid from the previous step (the ratio of plasmid to PEI is 1:3), mixed uniformly, and then incubated at room temperature for 10 min to obtain a DNA / PEI mixture. The DNA / PEI mixture is gently poured onto the HEK293 cells, mixed uniformly, and incubated at 37°C, 8% CO 2After culturing for 24 h under these conditions, VPAVPA (Sigma, product number: P4543-100G) was added to a final concentration of 2 mM and 2% (v / v) feed (1 g / L Phytone Peptone + 1 g / L Difco Select Phytone), and culturing was continued for 6 d.

[0138] The cell culture was centrifuged at 13000 rpm for 20 min, the supernatant was collected, and the supernatant was purified using a pre-column Hitrap Mabselect Sure (GE, 11-0034-95). The procedure is as follows: before purification, the packed column was equilibrated with 5 column volumes of equilibration solution (20 mM Tris, 150 mM NaCl, pH 7.2), the collected supernatant was passed through the column, the packed column was washed with 10 column volumes of equilibration solution to remove non-specific binding proteins, the packing was washed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), and the eluate was collected. 80 μL Tris (2 M Tris) was added per 1 ml of eluate, and the eluate was exchanged into PBS buffer (Gibco, product number: 70011-044) using an ultrafiltration concentration tube (MILLIPORE, product number: UFC901096), and the concentration was measured. The concentration of 100 μg of purified protein was adjusted to 1 mg / mL, and the purity of the protein was measured using a gel filtration column SW3000 (TOSOH product number: 18675).

[0139] Glycosylation mutants Y007, Y008 and Y014 significantly improved protein expression and purity compared to Y001 by mutating one or two surface amino acids. Y048, Y049 and Y050, which added one glycosylation site or one K35Q mutation site based on Y011, significantly improved the druggability of the molecule by increasing the expression level from 7.77mg / L to more than 50mg / L (Y048 and Y049) or 40mg / L (Y050) and the purity from 31.35% to more than 80%.

[0140] The B'C'loop chimera (Y017) and truncated versions (Y057 / 058 / 059) showed significantly improved expression levels and one-step affinity chromatography purity compared to Y001.

[0141] When the B'C'loop optimized sequence was combined with the glycosylation mutation L011, Y056, Y081 and Y082 were improved in both expression amount and purity compared to the mutant protein Y011 with L011 (Table 3). [Table 7]

[0142] Expression and purification of IL-2 receptor Human IL-2 receptor a (Uiprot: P01589, aa22-217) and b (Uiprot: P14784, aa27-240) were constructed on pTT5 vectors by linking an avi tag (polypeptide: GLNDIFEAQKIEWHE, which can catalyze biotinylation by BirA enzyme) and six histidine tags (HHHHHH) to the C-terminus of the sequence. The plasmid transfection 293F cells (Invitrogen) method is the same as the expression method of IL-2Fc fusion protein.

[0143] Before purification, the collected medium was centrifuged at 4500 rpm for 30 min to discard the cells. The supernatant was then filtered through a 0.22 μl filter. The nickel column (5 ml Histrap excel, GE, 17-3712-06) used for purification was immersed in 0.1 M NaOH for 2 h, and then washed with 5-10 column volumes of ultrapure water to remove the alkaline 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, 10 column volumes of washing buffer (20 mM Tris 7.4, 300 mM NaCl, 10 mM imidazole) was passed through the column to remove non-specifically bound heteroproteins, and then the target protein was eluted with 3-5 column volumes of elution buffer (20 mM Tris 7.4, 300 mM NaCl, 100 mM imidazole). After exchanging the recovered protein ultrafiltration concentrate into PBS (Gibco, 70011-044), superdex200 increase (GE, 10 / 300GL, 10245605) was further isolated and purified, and the elution peak of the monomer was collected, and the equilibration and elution buffer of the column was PBS (Gibco, 70011-044). The protein purity of 100 μg of the purified protein sample was measured using a gel filter column SW3000 (TOSOH product number: 18675) (FIGS. 3 and 4).

[0144] Example 3. IL-2 mutant Fc fusion protein (abbreviation: IL-2 mutant -FC) and its receptor affinity measurement IL-2 of the present invention binds to human IL-2Rα and IL-2Rβ mutant The equilibrium dissociation constant (KD) of -FC was measured using BiolayerInterferometry (BLI) technology. The BLI affinity measurement was performed using a conventional method (Estep, P et al., High throughput solution based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2):p270-8).

[0145] Half an hour before the start of the experiment, prepare an appropriate amount of AHC (ForteBio, 18-5060) (for positive control detection) sensors according to the quantity of the sample, and immerse them in SD buffer (PBS 1×, BSA 0.1%, Tween-20 0.05%).

[0146] SD buffer, IL-2 mutant -FC, 100 μl each of IL-2 receptor α or β are added to a 96-well black polystyrene half-volume microplate (Greiner, 675076). Place the plate corresponding to the position of the sample and determine the position of the sensor. Set the instrument parameters as follows. Execute steps Baseline, Loading~1 nm, Baseline, Association, Dissociation, determine the execution time of each step based on the binding and dissociation rates of the sample, the rotation speed is 400 rpm, and the temperature is 30 °C. Analyze the K D value using ForteBio analysis software.

Table 8

[0147] From the above affinity data, the following can be seen. 1) Y009, Y010, Y011, Y013 and Y015 can block the binding of IL-2Rα (Table 4a), 2) The B’C’ loop chimeric molecules and cleavage molecules not only increase the expression level of the molecules, but also increase the affinity between the molecules and IL-2R (R) (Table 4b), 3) The combination of IL-2 glycosylation and B’C’ loop modification, Y056 and Y081, compared with Y045 (IL-2 WT -2*(G4S)-FcLALA), Y040 (IL-2. 3X -2*(G4S)-FcLALA) and Y038 (IL-2. glycan5 -2*(G4S)-FcLALA), while inhibiting the binding to IL2Rα, also enhanced the affinity for IL2R (R) binding.

[0148] Example 4: IL-2 mutant -FC in vitro functional test IL-2 for IL-2Rα WT The affinity of IL-2Rα is higher than that of IL-2Rβ and IL-2Rγ, and it preferentially binds to IL-2Rα on the cell surface, recruits IL-2Rβγ, and releases downstream p-STAT5 signaling via IL-2Rβγ, stimulating the proliferation of T cells and NK cells. Treg cells have IL-2Rα on their surface, but effector T cells and NK cells lack IL-2Rα, so they do not express IL-2Rα under normal conditions. WT Preferentially stimulates Treg cell proliferation and downregulates immune responses. IL-2 mutant does not bind to IL-2Rα, eliminating the preference for preferentially stimulating the proliferation of Treg cells, while stimulating the proliferation of T cells and NK cells, effectively increasing the number of effector T cells and NK cells, and enhancing the anti-tumor effect.

[0149] In this example, each IL-2 mutant - Primary human CD8 FCs + By detecting activation of T cell p-STAT5 signaling, CD25 + Verify the removal of cell activation bias and CD25 - We screened for mutants with strong cell activation effects. The specific steps are as follows.

[0150] 1. Resuscitation of PBMC cells a) Remove PBMC cells (Allcells product number: PB005F, 100 ml) from liquid nitrogen and quickly place in a 37°C water bath to resuscitate the PBMC cells. b) The cells were added to 10 mL of prewarmed X-VIVO15 (Lonza product number: 04-418Q) medium containing 5% human AB serum (GemCell product number: 100-512) and 1‰ DNA enzyme (STRMCELL product number: 07900) and washed by centrifugation at 400G for 10 minutes at 25°C (all subsequent centrifugations were under these conditions). c) 20 mL of medium was added to resuspend the cells, and the cells were cultured overnight in a carbon dioxide incubator at 37°C.

[0151] 2. Human CD8 + T cell purification: a) The cell suspension from step 1 was aspirated, centrifuged, and the supernatant was discarded. b) 1 mL of Robosep buffer (STEMCELL product number: 20104) and 100 μL of human AB serum and human CD8 + The cells were resuspended in 100 μL of a T cell purification kit (Invitrogen product number: 11348D) with negative screening of the antibody mixture. c) After mixing uniformly, the mixture was incubated at 4°C for 20 minutes and shaken every 5 minutes. d) After incubation, 10 mL of Robosep buffer was added and centrifuged to wash twice. e) At the same time, 1 mL of magnetic microspheres (human CD8 + A T cell purification kit was prepared, 7 mL of Robosep buffer was added, the mixture was placed in a magnetic stand for 1 min, the supernatant was discarded, and the magnetic microspheres were pre-washed. f) 1 mL of Robosep buffer was added to resuspend the microspheres and cells, respectively, and mixed uniformly, followed by rotating incubation at room temperature for 30 min. g) After incubation, 6 mL of Robosep buffer was added, placed on the magnetic stand for 1 min, and the supernatant was collected. h) The collected fluid was placed on the magnetic stand again for 1 minute and the supernatant was collected. i) Centrifuge, discard the supernatant, resuspend in prewarmed T medium, and adjust the density to 1 x 10 6 / mL. j) One-third of the cells were prepared to stimulate the expression of CD25, and the remaining cells were cultured overnight in a 37°C carbon dioxide incubator.

[0152] 3. CD8 + T cells were stimulated to express CD25: a) CD8 purified in step 2 of 1 / 3 + T cells were prepared and anti-human CD3 / CD28 magnetic microspheres (GIBCO product number: 11131D) were added at a cell to microsphere ratio of 3:1. b) Place in a carbon dioxide incubator at 37°C for 3 minutes. c) 10 mL of medium was added and washed twice. d) Add medium to bring the cell density to 1×10 6 The culture was adjusted to 1 mL / mL and cultured statically in a carbon dioxide incubator at 37°C for 2 d.

[0153] 4. Determination of cell purity and expression levels: a) Cellular CD8 and CD25 were detected using anti-human CD8-PE (Invitrogen product number: 12-0086-42), anti-human CD25-PE (eBioscience product number: 12-0259-42), and isotype control antibody (BD product number: 556653). b) In step 2, the cells are CD8 + CD25 - T cells, and in step 3 the cells are CD8 + CD25 + It was T cells.

[0154] 5. Each IL-2 mutant -CD8 by FC + CD25 - EC of p-STAT5 signaling activation in T cells 50 Detection of: a) CD8 + CD25 - Prepare T cells at 1 x 10 per well. 5 The cells were plated in 96-well U-bottom culture plates (Costar product number: CLS3799-50EA). b) 100 μL of each IL-2 mutant -FC, commercially available IL-2 (R&D product number: 202-IL-500), IL-2 WT -FC, IL-2 3X -FC was added and diluted in a 4-fold concentration gradient starting from a maximum concentration of 266.7 nM for a total of 12 steps, and incubated in an incubator at 37°C for 20 min. c) 55.5 μL of 4.2% formaldehyde solution was added and the sections were fixed at room temperature for 10 minutes. d) The supernatant was centrifuged and the cells were resuspended by adding 200 μL of ice-cold methanol (Fisher product number: A452-4) and incubated in a 4° C. refrigerator for 30 min. e) The supernatant was centrifuged and washed 3 times with 200 μL staining buffer (BD product number: 554657). f) 200 μL of membrane rupture / fixation buffer (BD product number: 51-2091KZ) containing anti-p-STAT5-AlexFlour647 (BD product number: 562076, diluted 1:200) was added and incubated at room temperature in the dark for 3 hours. g) Wash three times with staining buffer, resuspend the cells in 100 μL of staining buffer, and perform flow cytometry detection. h) EC50 of p-STAT5 signal with IL-2 molecule concentration on the horizontal axis and AlexFlour647 mean fluorescence on the vertical axis. 50 The results of creating the values ​​are shown in Figure 5 and Table 5.

[0155] 6. Each IL-2 mutant -CD8 by FC + CD25 + EC of p-STAT5 signaling activation in T cells 50 Detection of: a) CD8 + CD25 + Prepare T cells at 1 x 10 per well. 5 The cells were plated in 96-well U-bottom culture plates. b) EC of p-STAT5 signal was measured in the same manner as in step 5. 50 The values ​​were generated and the results are shown in FIG. [Table 9]

[0156] Experimental results (comparison with the same donor): 1) Y001(IL-2 WT -GSGS-FcLALA) and Y045 (IL-2 W T-2*(G4S)-FcLALA), Y002(IL-2. 3X-GSGS-FcLALA) and Y040 (IL-2. 3X -2*(G4S)-FcLALA), Y011(IL-2. glycan5 -GSGS-FcLALA) and Y038 (IL-2. glycan5 The long linkage sequence (GGGGSGGGGS) was more effective than the short linkage sequence (GSGS) in detecting CD25. - CD8 + It was found to be effective in activating T cells (Figure 5A). 2) After chimerizing the B'C' loop of human IL-15, CD25 was induced by Y017 (IL-2hyb15BCL-GSGS-FcLALA) - CD8 + T cell activation (EC 50 The value is 0.9902) for Y001 (EC 50 The value was 10.79 times higher than that of CD25 (10.69) (Figure 5A). + CD8 + T cell activation (EC 50 The value is 0.0018) for Y001 (EC 50 value corresponds to 0.0020) (Figure 5B). 3) After an increase of one N-glycan at the IL-2 interface (Y038), CD25 - CD8 + T cell activation (EC of Y038) 50 The value was 369.0) for wild-type IL-2 (Y045, EC 50 The value was 11.63 times lower than the IL-2 value reported in the literature (31.73). 3X (Y040). When the B'C' loop of human IL-15 was chimerized to this (Y056, EC 50 value is 8.571), CD25 - CD8 + T cell activation was 3.7-fold improved compared to Y045 and 43.05-fold improved compared to Y038 (Figure 5C). 4) When comparing CD8+ T cells before and after stimulation from the same donor, Y056 and Y081 were CD25 - CD8 + Enhances T cell activation and CD25 +It was shown that the IL-14-dependent agonist activation of the IL-14-dependent agonist decreased the polarization of the IL-14-dependent agonist activation of the cells (Fig. 5D, E and Table 5). [Table 10] JPEG0007681073000011.jpg209150 JPEG0007681073000012.jpg203150 JPEG0007681073000013.jpg202150 JPEG0007681073000014.jpg206150 JPEG0007681073000015.jpg154150

Claims

1. comprises a truncated B'C' loop region sequence selected from the group consisting of SGDASIH and A(Q / G)S(K / A)N(F / I)H, located between amino acid residues aa72 and aa84, as compared to wild-type human IL-2, wherein the amino acid residues are numbered according to SEQ ID NO:26; wild-type human IL-2 comprises the sequence of SEQ ID NO: 26, 29 or 30; An IL-2 mutein having at least 90% identity with wild-type human IL-2.

2. 2. The IL-2 mutein of claim 1, comprising, as compared to wild-type IL-2, a combination mutation: (i) a mutated glycosylation motif selected from 41N-42X-43T / S, 43N-44X-45T / S, 45N-46X-47T / S, 68N-69X-70T / S, and 72N-73X-74T / S, where X is any amino acid except proline, and (ii) a truncated B'C' loop region sequence selected from the group consisting of SGDASIH and A(Q / G)S(K / A)N(F / I)H, located between amino acid positions aa72 to aa84, wherein the amino acid positions are numbered according to SEQ ID NO:

26.

3. The truncated B'C' loop region sequence between amino acid positions aa72 to aa84 (i)SGDASIH, (ii) AQSKNFH, and (iii) AGSKNFH The mutant protein of claim 1 or 2, selected from the group consisting of:

4. A mutant protein according to any one of claims 1 to 3, which when expressed in mammalian cells in the form of an Fc fusion protein has enhanced IL-2Rβ binding and improved expression yield and / or purity compared to wild-type IL-2.

5. Compared to wild-type IL-2, it has the following properties: - eliminated or reduced binding affinity to the IL-2Rα receptor, - enhanced binding affinity to the IL-2Rβ receptor, - reduced binding affinity to the high affinity IL-2R receptor (IL-2Rαβγ), - increased binding affinity to the medium affinity IL-2R receptor (IL-2Rβγ), - Decreased CD25 ＋ CD8 ＋ Activation of T cells, - Decreased CD25 ＋ CD8 ＋ Stimulatory effect on IL-2-mediated signaling in T cells; - CD25 eliminated or reduced ＋ Bias to preferentially activate Treg cells; - Downregulation of immune responses by reduced IL-2-induced Treg cells; - maintained or enhanced CD25 － Activation effect on cells, - increased proliferation and activation of IL-2-stimulated effector T cells and NK cells, and - Improved anti-tumor effect The mutant protein according to any one of claims 1 to 4, having one or more of the following:

6. When expressed in mammalian cells in the form of an Fc fusion protein, it has the following properties compared to wild-type IL-2: - improved expression levels compared to the wild-type IL-2 protein, and - higher purity after one-step Protein A affinity chromatography purification.

7. CD25 compared to wild-type IL-2 ＋ Reduced bias to preferentially stimulate p-STATA5 signaling in T cells and CD25 － 7. An IL-2 mutein according to any one of claims 1 to 6, which has an enhanced ability to stimulate signalling in T cells.

8. Combination mutations: (i) the mutated glycosylation motif K43N-F44-Y45T at amino acid positions 43-45 and the replacement sequence SGDASIH between amino acid positions aa72-aa84, or (ii) an IL-2 mutein according to any one of claims 1 to 7, comprising a mutated glycosylation motif K43N-F44-Y45T at amino acid positions 43 to 45 and a truncation sequence AQSKNFH between amino acid positions aa72 to aa84.

9. 9. The IL-2 mutein of any one of claims 1 to 8, wherein the wild-type IL-2 comprises the sequence of SEQ ID NO:

26.

10. 10. The mutant protein of any one of claims 1 to 9, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-44 and 48-50, or a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity thereto.

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

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

13. The fusion protein of claim 12, wherein the IL-2 mutein is fused to Fc via a linker, said linker being GSGS or 2x(G4S).

14. The fusion protein of claim 11, comprising a sequence having at least 85%, at least 95%, or at least 96% identity to an amino acid sequence selected from SEQ ID NOs: 12 and 19-25.

15. 10. An immune complex comprising an IL-2 mutein according to any one of claims 1 to 9 and an antigen-binding molecule, preferably the antigen-binding molecule being an immunoglobulin molecule, in particular an IgG molecule, or an antibody or antibody fragment, in particular a Fab molecule and an scFv molecule.

16. An isolated polynucleotide encoding an IL-2 mutein according to any one of claims 1 to 10, a fusion protein according to any one of claims 11 to 14, or an immunoconjugate according to claim 15.

17. An expression vector comprising the polynucleotide of claim 16.

18. A host cell comprising the polynucleotide of claim 16 or the vector of claim 17.

19. A method for producing an IL-2 mutein or a fusion or immunoconjugate thereof, comprising culturing a host cell according to claim 18 under conditions suitable for expression of the IL-2 mutein or the fusion or immunoconjugate.

20. A pharmaceutical composition comprising an IL-2 mutein according to any one of claims 1 to 10, a fusion protein according to any one of claims 11 to 14, or an immunoconjugate according to claim 15, and a pharma- ceutically acceptable carrier.

21. 20. Use of an IL-2 mutein according to any one of claims 1 to 10, a fusion protein according to any one of claims 11 to 14, an immunoconjugate according to claim 15, or a pharmaceutical composition according to claim 20, in the preparation of a medicament for treating a disease in a subject, wherein the disease is cancer, preferably selected from the group consisting of colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma and head and neck cancer.

22. Use of an IL-2 mutein according to any one of claims 1 to 10, a fusion protein according to any one of claims 11 to 14, or an immunoconjugate according to claim 15 in the preparation of a pharmaceutical composition for stimulating the immune system of a subject.

23. A method for obtaining an IL-2 mutein, comprising the steps of: - truncating the B'C' loop region of the wild type IL-2 protein to have a truncated B'C' loop region sequence selected from the group consisting of SGDASIH or A(Q / G)S(K / A)N(F / I)H, located between amino acid residues aa72 to aa84, or - introducing one or more glycosylation motifs N-X-S / T into the wild-type IL-2 protein by mutation in the binding interface between IL-2 and IL-2Rα, wherein X may be any amino acid except P (proline), and truncating the B'C' loop region of the wild-type IL-2 protein to have a truncated B'C' loop region sequence selected from the group consisting of SGDASIH or A(Q / G)S(K / A)N(F / I)H, located between amino acid residues aa72 to aa84; - expressing said IL-2 mutein in the form of an Fc fusion in a mammalian cell, And -(i) improved expression and / or purity, and enhanced IL2Rβ binding, or (ii) improved expression and / or purity, reduced IL2Rα binding, and enhanced IL2Rβ binding identifying mutant proteins having improved properties of Including, The method, wherein the wild-type IL-2 comprises the sequence of SEQ ID NO: 26, 29 or 30.

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